WO2022127834A1 - 一种补体抑制剂的开发和应用 - Google Patents

一种补体抑制剂的开发和应用 Download PDF

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WO2022127834A1
WO2022127834A1 PCT/CN2021/138447 CN2021138447W WO2022127834A1 WO 2022127834 A1 WO2022127834 A1 WO 2022127834A1 CN 2021138447 W CN2021138447 W CN 2021138447W WO 2022127834 A1 WO2022127834 A1 WO 2022127834A1
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seq
variant
variable region
chain variable
nucleotide sequence
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French (fr)
Chinese (zh)
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徐刚
陈博
王常玉
张国惠
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Keymed Biosciences Co Ltd
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Keymed Biosciences Co Ltd
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Priority to CN202180085328.6A priority Critical patent/CN116648464A/zh
Priority to CA3202507A priority patent/CA3202507A1/en
Priority to EP21905760.1A priority patent/EP4265641A1/en
Priority to JP2023537034A priority patent/JP2023554081A/ja
Priority to AU2021398697A priority patent/AU2021398697A1/en
Priority to KR1020237023581A priority patent/KR20230118941A/ko
Priority to IL303794A priority patent/IL303794A/en
Priority to US18/267,680 priority patent/US20240067751A1/en
Publication of WO2022127834A1 publication Critical patent/WO2022127834A1/zh
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Definitions

  • the present disclosure relates to a complement inhibitor and applications thereof, in particular to MASP-2 inhibitory antibodies and applications thereof.
  • MASP-2 in the blood recognizes the molecules mannose-binding lectin (MBL), collectin 11 (CL-K1) and ficolin (Ficolin- 1, Ficolin-2 and Ficolin-3) bind and form a complex, when MBL, CL-K1 or ficolin bind to pathogen-associated molecular patterns (PAMPs such as D-mannose, N-acetyl-D-glucosamine or acetyl) ), MASP-1 and MASP-2 are sequentially activated, the activated MASP-2 cleaves C4 and C2, and the fragment C4b formed by cleavage interacts with C2a to generate C3 convertase (C4b2a), C3 convertase (C4b2a) activates C3, This results in the production of C5 convertase (C4b2a3b) and the formation of a membrane attack complex (C5b-9) capable of causing microbial lysis.
  • MBL mannose-binding lectin
  • C3 and C4 are covalently deposited on the surface of exogenous targets, which are recognized by complement receptors on various phagocytes and engulf the corresponding pathogens.
  • the lectin pathway plays an important role in the body's innate immunity against pathogenic microorganisms such as bacteria, yeast, and viruses.
  • IgA nephropathy IgAN
  • ischemia-reperfusion injury transplant rejection
  • rheumatoid arthritis myocardial infarction
  • revascularization after stroke ARDS
  • septicemia Shock capillary leakage after thermal burns
  • inflammation after cardiopulmonary bypass multiple sclerosis
  • myasthenia gravis and Alzheimer's disease.
  • the present disclosure relates to anti-MASP-2 inhibitory antibodies for inhibiting the adverse effects of MASP-2-dependent complement activation.
  • the present disclosure provides an antibody or antigen-binding portion thereof that binds MASP-2.
  • the present disclosure provides nucleic acids encoding the aforementioned MASP-2-binding antibodies, or antigen-binding portions thereof, or bispecific antibodies, or antigen-binding portions thereof.
  • the present disclosure provides vectors comprising the aforementioned nucleic acids.
  • the present disclosure provides cells comprising the aforementioned vectors.
  • the present disclosure provides an antibody, or antigen-binding portion thereof, encoding nucleic acid, vector, cell, composition, or kit comprising the foregoing.
  • the present disclosure provides a method of treating a disease associated with MASP-2-dependent complement activation, comprising the step of administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of any of the preceding aspects, Nucleic acids, vectors, cells and/or compositions.
  • the present disclosure provides an antibody or antigen-binding fragment, nucleic acid, vector, cell, and/or pharmaceutical composition of any of the preceding aspects associated with MASP-2-dependent complement activation in preparation for use in the treatment of a subject Use in medicines or kits for diseases.
  • Figure 4 shows inhibition of C3b deposition by hybridoma clone chimeric antibodies.
  • Figure 5 shows ELISA detection of candidate chimeric antibody binding to different species of MASP-2 D4-6.
  • Figure 6 shows inhibition of C4b deposition by phage display library Fab candidate clones.
  • Figure 7 shows the inhibition of C4b deposition after purification of Fab candidate clones of the phage library display library.
  • Figure 8 shows inhibition of C4b deposition following mutation of the 28O14 clone.
  • Ser102 represents the wild-type antibody, and its 102-position is serine.
  • Gly102, Tyr102, Ala102, Glu102, Gln102, Val102, Leu102 and His102 represent mutant antibodies whose 102-position was mutated from serine to glycine, tyrosine, alanine, glutamic acid, glutamine, valine, Leucine and Histidine.
  • Figures 9A,B show inhibition of C4b deposition by humanized MASP-2 antibodies.
  • FIGS 10A, B, C show inhibition of C3b deposition by humanized MASP-2 antibodies.
  • Figure 11 shows ELISA detection of humanized MASP-2 antibody binding to different species antigens.
  • antibodies eg, monoclonal antibodies
  • antigen-binding fragments thereof that specifically bind MASP-2.
  • monoclonal anti-MASP-2 antibodies that specifically bind MASP-2, wherein the anti-MASP-2 antibodies comprise variants of the parent antibody.
  • antibodies that specifically bind MASP-2 eg, human MASP-2).
  • anti-MASP-2 antibodies comprising modifications in one or more amino acid residues (eg, 5-13 amino acid substitutions in the framework regions of the heavy chain variable regions), in contrast to Compared to the modified parent antibody, it retains its affinity for the antigen.
  • MASP-2 refers to any MASP-2 molecule known to those skilled in the art.
  • the MASP-2 may be of mammalian origin, eg, the MASP-2 may be of human origin.
  • 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.
  • MASP-2 inhibitory antibodies 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 more than 80%, or more than 90%, or more than 95%.
  • lectin pathway refers to complement activation that occurs through the specific binding of serum and non-serum carbohydrate-binding proteins, including mannan-binding lectin (MBL) and ficolin.
  • the phrase "substantially identical" can be understood as exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, Antibody chains of 97%, 98%, 99% or more sequence identity.
  • nucleic acid sequences the term is to be understood as exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 A nucleotide sequence of %, 99% or greater sequence identity.
  • sequence identity has an art-recognized meaning, and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along regions of the molecule. While many methods exist for measuring the identity between two polynucleotides or polypeptides, the term “identity” is well known to the skilled artisan (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ).
  • substitutional variant is one in which at least one amino acid residue in the native sequence has been removed and a different amino acid inserted in its same position.
  • the substitutions can be single, wherein only one amino acid is substituted in the molecule, or multiple, wherein the same molecule has two or more amino acids substituted. Multiple substitutions can be made at consecutive sites.
  • one amino acid may be substituted by multiple residues, wherein such variants include both substitutions and insertions.
  • An “insertional” variant is one in which one or more amino acids are inserted into an amino acid immediately adjacent to a specific position in a native sequence. Immediately adjacent to an amino acid means attachment to the alpha-carboxyl or alpha-amino functional group of the amino acid.
  • a “deletion” variant is one in which one or more amino acids in the native amino acid sequence have been removed. Typically, deletion variants have one or two amino acids deleted in a specific region of their molecule.
  • variable domains of antibodies refers to certain portions of related molecules that differ widely in sequence between antibodies and are used for the specific recognition and binding of a particular antibody against its specific target. However, the variability is not evenly distributed throughout the variable domains of antibodies. Variability is concentrated in three segments called complementarity determining regions (CDRs; ie CDR1, CDR2 and CDR3) or hypervariable regions, all located within the variable domains of light and heavy chains. The more conserved portions of the variable domains are referred to as framework (FR) regions or framework sequences.
  • CDRs complementarity determining regions
  • FR framework regions
  • Each variable domain of native heavy and light chains includes four FR regions, predominantly in a beta-sheet configuration, linked by three CDRs that form loops that connect the beta-sheet structure and Partial ⁇ -sheet structures are formed in some cases.
  • the CDRs of each chain are usually linked in proximity by FR regions and, with the aid of CDRs from other chains, contribute to the formation of antibody target binding sites (epitopes or determinants).
  • the numbering of immunoglobulin amino acid residues is according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated.
  • a CDR can have the ability to specifically bind to the cognate epitope.
  • 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 which comprises at least a portion of the variable region (eg, one or more of the variable regions of said antibody that binds an antigen) CDRs and/or one or more antibody binding sites), and thus retain binding specificity and at least part of the specific binding capacity of the full-length antibody.
  • an antigen-binding fragment refers to an antibody fragment comprising an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived.
  • Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, eg, 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, For example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov).
  • the fragments may comprise multiple chains linked together, eg, 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 the antibody framework (eg, by substituting the corresponding region), results in an antibody that immunospecifically binds (ie, exhibits a Ka of at least or at least about 107-108 M -1 ) to an antigen .
  • a "functional fragment” or “analog of an anti-MASP-2 antibody” is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction.
  • functional fragments generally have the same meaning as "antibody fragments” and, in the case of antibodies, may refer to fragments that prevent or substantially reduce the ability of the receptor to bind a ligand or initiate signal transduction, eg, Fv, Fab , F(ab') 2 , and so on.
  • "Fv" fragments consist of a dimer ( VH - VL dimer) formed by non-covalent association of the variable domains of a heavy chain and the variable domains of a light chain. In this configuration, the three CDRs of each variable domain interact to define the target binding site on the surface of the VH - VL dimer, as is the case with intact antibodies. The six CDRs collectively confer the target-binding specificity of the intact antibody. However, even a single variable domain (or half of an Fv that includes only 3 target-specific CDRs) can still have the ability to recognize and bind targets.
  • BsAb Bispecific antibody
  • a bispecific antibody and/or antigen-binding molecule Contains two antigen binding sites, each of which is specific for a different antigenic determinant.
  • 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.
  • monoclonal antibody refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to other antibody molecules. This property is in contrast to that of polyclonal populations of antibodies, which comprise antibodies with 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).
  • monoclonal antibodies can be prepared by immortalizing B cells, eg, by fusion with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV.
  • Recombinant techniques can also be used to prepare antibodies from clonal populations of host cells in vitro by transforming the host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.
  • hybridomas refers to a cell or cell line (usually myeloma or lymphoma cells) produced by fusing antibody-producing lymphocytes and non-antibody-producing cancer cells.
  • hybridomas can proliferate and provide continuous supply to produce specific monoclonal antibodies. Methods for generating hybridomas are known in the art.
  • hybridomas When referring to the term “hybridoma” or “hybridoma cell”, it also includes subclones and progeny cells of hybridomas.
  • a full-length antibody is one that has two full-length heavy chains (eg, VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region antibodies, such as those naturally produced by antibody-secreting B cells as well as those produced synthetically with the same domains.
  • chimeric antibody refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody of antibodies.
  • Humanized antibodies refer to non-human (eg, mouse) forms of antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) containing minimal sequence derived from non-human immunoglobulins.
  • the humanized antibody is a human immunoglobulin (recipient antibody) in which the complementarity determining region (CDR) residues of the recipient antibody are derived from a non-human species with the desired specificity, affinity and capacity ( donor antibody) such as mouse, rat or rabbit CDR residue substitutions.
  • CDR complementarity determining region
  • telomeres can be mutated amino acid residues within the CDR1, CDR2 and/or CDR3 regions of VH and/or VL, thereby improving one or more binding properties (eg, affinity) of the antibody .
  • PCR-mediated mutagenesis can be performed to introduce mutations whose effect on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions.
  • CDR refers to a complementarity-determining region
  • each of the heavy and light chains of antibody molecules is known to have 3 CDRs.
  • the CDRs are also referred to as hypervariable regions, and are present in the variable regions of each heavy and light chain of antibodies, with sites of very high variability in the primary structure of the CDRs.
  • the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 derived from the amino terminal of the amino terminal sequence of the heavy chain
  • CDRs of the light chain are represented by CDR1, CDR2, and CDR3 derived from the amino terminal of the amino terminal sequence of the light chain.
  • epitopic determinants refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants typically comprise chemically active surface profiles of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • an antibody that immunospecifically binds (or specifically binds) an antigen has an affinity constant Ka of about or 1 ⁇ 10 7 M -1 or 1 ⁇ 10 8 M -1 or greater (or 1 ⁇ 10-7 M or 1 ⁇ 10 ⁇ 8 M or lower dissociation constant (Kd)) binds to the antigen.
  • Affinity constants can be determined by standard kinetic methods of antibody responses, eg, 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 also U.S. Pat. No. 7,229,619 describing exemplary SPR and ITC methods for calculating binding affinity of antibodies No). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see, Malmqvist (2000) Biochem. Soc. Trans. 27:335).
  • nucleic acid molecules refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including usually linked together by phosphodiester bonds Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleic acid molecule is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.
  • operably linked in reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other.
  • a promoter can be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.
  • amino acids with basic side chains eg, lysine, arginine, histidine
  • amino acids with acidic side chains eg, aspartic acid, glutamic acid
  • amino acids with uncharged polar side chains amino acids e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • amino acids with non-polar side chains e.g. alanine, valine
  • leucine, isoleucine, proline, phenylalanine, methionine amino acids with beta branched side chains
  • the predicted non-essential amino acid residue in the anti-MASP-2 antibody is preferably replaced by another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of nucleotides and amino acids that do not abolish antigen binding are well known in the art (for example, 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)).
  • mutations can be introduced randomly along all or a portion of the anti-MASP-2 antibody coding sequence, eg, by saturation mutagenesis, and the resulting modified anti-MASP-2 antibody can be screened for improved binding activity .
  • expression refers to the process by which a polypeptide is produced by transcription and translation of a polynucleotide. Expression levels of a polypeptide can be assessed using any method known in the art, including, for example, methods that determine the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining followed by gel electrophoresis, Lowry protein assay, and Bradford protein assay.
  • a vector also includes a "viral vector” or “viral vector.”
  • a viral vector is an engineered virus that is operably linked to a foreign gene to transfer (either as a vehicle or shuttle) the foreign gene into a cell.
  • an "expression vector” includes a vector capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally, one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are typically 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 an appropriate host cell, results in the expression of cloned DNA. Appropriate expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and/or prokaryotic cells as well as those that remain episomal or that integrate into the host cell genome.
  • treating an individual with a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged following treatment.
  • treatment includes prevention, treatment and/or cure.
  • Prevention refers to preventing an underlying disease and/or preventing the worsening of symptoms or the development of a disease.
  • Treatment also includes any provided antibodies or antigen-binding fragments thereof and any pharmaceutical uses of the compositions provided herein.
  • therapeutic effect refers to an effect resulting from treatment of an individual that alters, generally ameliorates or ameliorates the symptoms of a disease or condition, or cures the disease or condition.
  • a “therapeutically effective amount” or “therapeutically effective dose” refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, retard or partially retard the symptoms of a disease or disorder.
  • a prophylactically effective amount or “prophylactically effective dose” refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, eg, prevent or delay a disease or symptom occurrence or recurrence, and reduce the likelihood of occurrence or recurrence of disease or symptoms.
  • a fully prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.
  • An antibody or antigen-binding portion thereof comprising a heavy chain CDR1 selected from the amino acid sequence SEQ ID NO. 7, 26, 36, 65, 94, 126, 136, 149 or any variant thereof, selected from Heavy chain CDR2 of sequence SEQ ID NO. 8, 17, 27, 37, 46, 66, 85, 95, 104, 113, 127, 137, 150, 165, 183, 192 or any variant thereof, selected from the amino acid sequence Heavy chain CDR3 of SEQ ID NO. 9, 18, 28, 38, 47, 60, 67, 79, 86, 96, 105, 114, 128, 138, 141, 151 or any variant thereof; and/or selected from Light chain CDR1 of amino acid sequence SEQ ID NO.
  • a light chain CDR3 selected from the amino acid sequence SEQ ID NO. 14, 23, 33, 43, 52, 72, 82, 91, 101, 110, 133, 146, 156, 174, any variant thereof.
  • An antibody or antigen-binding portion thereof comprising a combination of CDRs of heavy and light chains selected from the group consisting of:
  • (6) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 17 and 18, and/or comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 21, 57 and 23 respectively;
  • (7) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 17 and 60, and/or comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 50, 51 and 52 respectively;
  • (9) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 17 and 47, and/or comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 50, 51 and 52 respectively;
  • (10) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 17 and 79, and/or respectively comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 50, 51 and 82;
  • (11) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 36, 85 and 86, and/or respectively comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 41, 42 and 43;
  • (12) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 36, 85 and 86, and/or respectively comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 41, 42 and 91;
  • (17) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 126, 127, 128, and/or comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 131, 132, 133, respectively;
  • (20) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 149, 150, 151, and/or respectively comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 154, 155, 156;
  • (21) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 17, 9, and/or comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 161, 162, 14, respectively;
  • (22) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 165 and 9, and/or respectively comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 144, 168 and 14;
  • (23) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 17, and 9, and/or respectively comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 144, 173 and 174;
  • (24) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 136, 183, 138, and/or respectively comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 216, 132, 133;
  • (26) respectively comprise the heavy chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 7, 17 and 141, and/or respectively comprise the light chain CDR1, CDR2 and CDR3 sequences of SEQ ID NOs: 201, 202 and 146;
  • the antibody or antigen-binding portion thereof comprising an amino acid sequence selected from the group consisting of amino acid sequences SEQ ID NO. 119, 124, 134, 139, 147, 157, 163, 169, 175, 177, 179, 181, 184, 186, 188, 190, 193, 195, 197, or the heavy chain variable region of any variant thereof, and / or selected from the amino acid sequence SEQ ID NO. light chain variable regions of 166, 171, 199, 203, 205, 208, 210, 212, 214 or any variants thereof;
  • the antibody or antigen-binding portion thereof comprises a heavy chain variable region of amino acid sequence SEQ ID NO. 5, or any variant thereof, and a light chain of amino acid sequence SEQ ID NO. 10, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 15, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 19, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 24, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 29, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 34, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 39, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 44, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 48, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises a heavy chain variable region of amino acid sequence SEQ ID NO. 53 or any variant thereof, and a light chain of amino acid sequence SEQ ID NO. 55 or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises a heavy chain variable region of amino acid sequence SEQ ID NO. 58 or any variant thereof, and a light chain of amino acid sequence SEQ ID NO. 61 or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 63, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 68, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises a heavy chain variable region of amino acid sequence SEQ ID NO. 77, or any variant thereof, and a light chain of amino acid sequence SEQ ID NO. 80, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 83, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 87, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 83, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 89, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 92, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 97, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 102, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 106, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 111 or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 115 or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 119 or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 121 or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 124, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 129, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 134, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 129, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 139, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 142, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 147 or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 152 or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 157, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 159, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises a heavy chain variable region of amino acid sequence SEQ ID NO. 163, or any variant thereof, and a light chain of amino acid sequence SEQ ID NO. 166, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 169 or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 171 or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 181 or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 214 or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises a heavy chain variable region of amino acid sequence SEQ ID NO. 184 or any variant thereof, and a light chain of amino acid sequence SEQ ID NO. 214 or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 188 or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 214 or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 190, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 214, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 193 or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 214 or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 197, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 214, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 175, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 199, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 175, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 203, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises a heavy chain variable region of amino acid sequence SEQ ID NO. 175 or any variant thereof, and a light chain of amino acid sequence SEQ ID NO. 205 or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises a heavy chain variable region of amino acid sequence SEQ ID NO. 177 or any variant thereof, and a light chain of amino acid sequence SEQ ID NO. 205 or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 177, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 199, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 177, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 203, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 179, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 199, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 179, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 203, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 179, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 205, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 175, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 208, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises a heavy chain variable region of amino acid sequence SEQ ID NO. 175 or any variant thereof, and a light chain of amino acid sequence SEQ ID NO. 210 or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 175, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 212, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 177, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 208, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 177, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 210, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 177, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 212, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 179, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 208, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 179, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 210, or any variant thereof variable region;
  • the antibody or antigen-binding portion thereof comprises the heavy chain variable region of amino acid sequence SEQ ID NO. 179, or any variant thereof, and the light chain of amino acid sequence SEQ ID NO. 212, or any variant thereof variable region.
  • the present disclosure provides a bispecific or multispecific molecule comprising the antibody or antigen-binding portion thereof of any of the preceding aspects.
  • the antibody or antigen-binding portion thereof is humanized.
  • the present disclosure provides nucleic acid molecules encoding antibodies, or antigen-binding portions thereof, or bispecific or multispecific molecules according to any of the preceding aspects.
  • the nucleic acid comprises a nucleic acid selected from the group consisting of SEQ ID NO. 140, 148, 158, 164, 170, 176, 178, 180, 182, 185, 187, 189, 191, 194, 196, 198, or any variant thereof, an antibody heavy chain variable region nucleic acid sequence, and/or an alternative 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 of the antibody light chain variable region nucleic acid sequence;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence of SEQ ID NO: 6 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 11 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 16, or any variant thereof, and the nucleotide sequence of SEQ ID NO: 20, or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence of SEQ ID NO:25 or any variant thereof, and the nucleotide sequence of SEQ ID NO:30 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence of SEQ ID NO:35 or any variant thereof, and the nucleotide sequence of SEQ ID NO:40 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence of SEQ ID NO:45 or any variant thereof, and the nucleotide sequence of SEQ ID NO:49 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence of SEQ ID NO:54 or any variant thereof, and the nucleotide sequence of SEQ ID NO:56 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence of SEQ ID NO:59 or any variant thereof, and the nucleotide sequence of SEQ ID NO:62 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence of SEQ ID NO:64 or any variant thereof, and the nucleotide sequence of SEQ ID NO:69 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence of SEQ ID NO:78 or any variant thereof, and the nucleotide sequence of SEQ ID NO:81 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence of SEQ ID NO: 84 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 88 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence of SEQ ID NO:84 or any variant thereof, and the nucleotide sequence of SEQ ID NO:90 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence of SEQ ID NO:93 or any variant thereof, and the nucleotide sequence of SEQ ID NO:98 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 120 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 122 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 125, or any variant thereof, and the nucleotide sequence of SEQ ID NO: 130, or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 140 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 143 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 148 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 153 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 170, or any variant thereof, and the nucleotide sequence of SEQ ID NO: 172, or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 185 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 215 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence of SEQ ID NO: 187 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 215 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 189 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 215 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 191 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 215 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 194 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 215 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 198 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 215 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 176 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 200 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of the nucleotide sequence of SEQ ID NO: 176 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 206 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 178, or any variant thereof, and the nucleotide sequence of SEQ ID NO: 206, or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 178, or any variant thereof, and the nucleotide sequence of SEQ ID NO: 200, or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 178 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 204 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 180, or any variant thereof, and the nucleotide sequence of SEQ ID NO: 200, or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 180, or any variant thereof, and the nucleotide sequence of SEQ ID NO: 204, or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 180 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 206 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 176 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 211 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 176, or any variant thereof, and the nucleotide sequence of SEQ ID NO: 213, or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 178 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 209 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 178 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 211 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 178 or any variant thereof, and the nucleotide sequence of SEQ ID NO: 213 or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 180, or any variant thereof, and the nucleotide sequence of SEQ ID NO: 209, or any variant thereof The nucleotide sequence of the light chain variable region;
  • the nucleic acid comprises the heavy chain variable region nucleotide sequence of nucleotide sequence SEQ ID NO: 180, or any variant thereof, and the nucleotide sequence of SEQ ID NO: 211, or any variant thereof The nucleotide sequence of the light chain variable region;
  • the present disclosure provides an antibody or antigen-binding portion thereof that binds MASP-2 having at least greater than 60%, 65%, 70%, 75%, 80% of the antibody or antigen-binding portion thereof of any of the preceding aspects , 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity.
  • the present disclosure provides nucleic acid molecules encoding, or having at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90% thereof, an antibody or antigen-binding portion thereof according to any of the preceding aspects , 95%, 96%, 97%, 98%, 99% or higher sequence identity of nucleic acid molecules.
  • the present disclosure provides a vector comprising the nucleic acid of any of the preceding aspects.
  • the present disclosure provides cells comprising the vector of any of the preceding aspects.
  • the present disclosure is a composition comprising the aforementioned antibody or antigen-binding portion thereof, bispecific or multispecific molecule, nucleic acid, vector and/or cell.
  • the antibodies of the present disclosure are useful as therapeutic or diagnostic tools in various diseases in which MASP-2 is unfavorably expressed or found.
  • a method of treating a disease associated with MASP-2-dependent complement activation with a MASP-2 inhibitor of the present disclosure comprises the step of administering to a subject a therapeutically effective amount of an antibody or antigen-binding fragment or nucleic acid thereof of any of the preceding aspects Molecules or carriers or cells or pharmaceutical compositions.
  • MASP-2-dependent complement activation has been implicated in contributing to the pathogenesis of many acute and chronic disease states, including MASP-2-dependent complement-mediated vascular conditions, ischemia-reperfusion injury, atherosclerosis, inflammatory gastrointestinal Tract disorders, pulmonary conditions, extracorporeal reperfusion procedures, skeletal muscle conditions, renal conditions, skin conditions, organ or tissue transplantation, neurological conditions or injuries, blood disorders, genitourinary tract conditions, diabetes, chemotherapy or radiation therapy, malignancy , endocrine disorders, coagulation disorders, or ophthalmic conditions. Accordingly, the MASP-2 inhibitory antibodies of the present disclosure can be used to treat the above-mentioned diseases and conditions.
  • the clinical condition of a preferred embodiment of the present disclosure is a chronic inflammatory disease.
  • a chronic inflammatory disease can be, for example, an autoimmune inflammatory condition.
  • Autoimmune inflammatory conditions can be loosely categorized into those diseases that are primarily localized to specific organs or tissues and those that affect the entire body.
  • organ-specific disorders include multiple sclerosis (myelin coating the neurites), type 1 diabetes (pancreas), Hashimoto's thyroiditis (thyroid), pernicious anemia (stomach) ), Addison's disease (adrenal glands), myasthenia gravis (acetylcholine receptors at neuromuscular junctions), rheumatoid arthritis (lining of joints), uveitis (eyes), psoriasis (skin), Guillain-Barré syndrome (nerve cells) and Graves' disease (thyroid).
  • Systemic autoimmune diseases include systemic lupus erythematosus, glomeronephritis, and dermatomyositis.
  • the preferred clinical condition is selected from rheumatoid arthritis or systemic lupus erythematosus.
  • autoimmune disorders include asthma, eczema, atopic dermatitis, contact dermatitis, other eczematous dermatitis, seborrheic dermatitis, rhinitis, lichen planus, Pemplugus, bullous pemphigoid Sores, epidermolysis bullosa, urticaria, angioedema, vasculitis, erythema, cutaneous eosinophilia, localized alopecia, arteriosclerosis, primary biliary cirrhosis, and nephrotic syndrome.
  • Associated diseases include enteritis, such as coeliac diseasease, proctitis, eosinophilic gastroenteritis, mastocytosis, inflammatory bowel disease, Chrohn's disease and ulcerative colitis, and food-related allergies .
  • the clinical condition is characterized by massive cell loss, eg, due to programmed cell death or necrosis. Necrosis or programmed cell death can be induced by a variety of factors.
  • the clinical condition is ischemic/reperfusion injury.
  • Ischemia can be caused by a variety of reasons, such as after stroke, myocardial infarction, major surgery, or organ transplantation.
  • the clinical condition may be ischemic/reperfusion injury caused by, for example, stroke, myocardial infarction, major surgery, or organ transplantation.
  • the clinical condition may be ischemic/reperfusion injury, which is the result of PTCA (percutaneous transluminal coronary angioplasty) or CABG (coronary artery bypass grafting).
  • the clinical condition may be ischemic/reperfusion injury, which is the result of acute myocardial infarction or cerebral ischemia.
  • MASP-2 inhibitor-containing drug will generally vary depending on the age, weight, height, sex, general disease condition, and medical history of the subject.
  • MASP-2 inhibitors such as MASP-2 antibodies
  • the composition comprises a combination of an anti-MASP-2 antibody and a MASP-2 inhibitory peptide.
  • Compositions and methods comprising MASP-2 inhibitors may optionally include one or more additional therapeutic agents that enhance the activity of the MASP-2 inhibitor or provide related therapeutic functions in an additive or synergistic manner.
  • one or more MASP-2 inhibitors can be used in combination with one or more anti-inflammatory and/or analgesic drugs. The amount and selection of additional drugs can be determined to achieve the desired therapeutic result.
  • Suitable anti-inflammatory and/or analgesic drugs include: serotonin receptor antagonists; serotonin receptor agonists; histamine receptor antagonists; bradykinin receptor antagonists; kallikrein inhibitors; Kinin receptor antagonists, including neurokinin 1 and neurokinin 2 receptor subtype antagonists; calcitonin gene-related peptide (CGRP) receptor antagonists; interleukin receptor antagonists; arachidonic acid metabolites Inhibitors of enzymes active in synthetic pathways, including phospholipase inhibitors, including PLA2 isoform inhibitors and PLC ⁇ isoform inhibitors, cyclooxygenase (COX) inhibitors (which can be COX-1, COX -2 inhibitors or non-selective COX-1 and COX-2 inhibitors), lipoxygenase inhibitors; prostanoid receptor antagonists, including eicosanoids EP-1 and EP-4 receptors Body subtype antagonists and thromboxane receptor subtype antagonists; leukotriene receptor antagonists
  • anti-restenotic drugs include: antiplatelet drugs, including thrombin inhibitors and receptor antagonists, adenosine diphosphate (ADP) receptor antagonists (also known as purinoceptor 1 receptor antagonists), thromboxane inhibitors agents and receptor antagonists and platelet membrane glycoprotein receptor antagonists; inhibitors of cell adhesion molecules, including selectin inhibitors and integrin free radicals; antichemotactic agents; interleukin receptor antagonists; and intracellular signaling Transduction inhibitors, including protein kinase C (PKC) inhibitors and protein tyrosine phosphatases, modulators of intracellular protein tyrosine kinase inhibitors, inhibitors of the src homology 2 (SH2) domain, and calcium channels antagonist.
  • PLC protein kinase C
  • SH2 src homology 2
  • Biological inhibitors of complement include soluble complement factor 1 (sCR1). This is a naturally occurring inhibitor found on the outer membrane of human cells. Other membrane inhibitors include DAF, MCP and CD59.
  • sCR1 soluble complement factor 1
  • sCR1 has been shown to be effective in xenografts where the complement system (both alternative and classical) induces hyperreactive rejection syndrome within minutes of perfusion of blood in the newly transplanted organ. The use of sCR1 protects and prolongs the survival time of transplanted organs, suggesting a complement pathway in the mechanism of organ survival.
  • complement inhibitors suitable for use in combination with the compositions of the present disclosure also include, for example, the monoclonal antibody (MoAb) Connecticut being developed by Alexion Pharmaceuticals, Inc. New Haven, and the anti-properdin monoclonal antibody (MoAb).
  • 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 chondroprotective agents A promoter of cartilage anabolism and/or one or more inhibitors of cartilage catabolism, suitably including both an anabolic agent and a catabolic inhibitory agent administered concurrently.
  • one or more chondroprotective agents can include one or more chondroprotective agents A promoter of cartilage anabolism and/or one or more inhibitors of cartilage catabolism, suitably including both an anabolic agent and a catabolic inhibitory agent administered concurrently.
  • Suitable anabolic chondroprotective agents include interleukin (IL) receptor agonists, including IL-4, IL-10, IL-13, rhIL-4, rhIL-10 and rhIL-13 as well as chimeric IL-4, IL-10 or IL-13; transforming growth factor beta superfamily agonists (including TGF-beta, TGF-beta1, TGF-beta2, TGF-beta3), bone-forming 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- ⁇ and BMP, and chimeric TGF- ⁇ and BMP; insulin-like growth factors, including IGF-1; and fibroblast growth factors, including bFGF.
  • IL interleukin
  • the antibody is administered in a single dose or in multiple doses.
  • the aforementioned antibodies are administered in multiple doses according to the present disclosure, preferably 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 The aforementioned antibodies are administered in at least 10 doses and preferably at most 30 doses, 25 doses, 20 doses, 15 doses or 10 doses. Doses of the aforementioned antibodies are preferably administered at intervals of at least 7 days, at least 10 days, at least 14 days, or at least 20 days. Doses of the aforementioned antibodies are preferably administered at intervals of 7 to 30 days, 10 to 20 days, and preferably about 14 days.
  • the present disclosure provides an antibody or antigen-binding fragment or nucleic acid molecule or vector or cell or pharmaceutical composition of any of the preceding aspects prepared for use in the treatment of a disease associated with MASP-2-dependent complement activation in a subject use in medicines.
  • the present disclosure provides a kit comprising the aforementioned antibodies or antigen-binding portions thereof, bispecific or multispecific molecules, nucleic acid molecules, vectors, cells, and/or compositions.
  • Kits of the present disclosure include antibodies of the present disclosure, fragments thereof, homologues, derivatives thereof, and the like, such as labeled or cytotoxic conjugates, as well as instructions for use of the antibodies, conjugates that kill specific types of cells, and the like .
  • the instructions may include instructions for using the antibody, conjugate, etc. in vitro, in vivo or ex vivo.
  • Antibodies can be in liquid form or solid, usually lyophilized.
  • the kit may contain other suitable reagents, such as buffers, reconstitution solutions, and other necessary components for the intended use. Packaged reagent combinations in predetermined quantities are contemplated along with instructions for their use, eg, for therapeutic use or for performing diagnostic assays.
  • the kit may include a substrate and cofactors required by the enzyme (eg, a substrate precursor that provides a detectable chromophore or fluorophore).
  • a substrate precursor that provides a detectable chromophore or fluorophore e.g. a substrate precursor that provides a detectable chromophore or fluorophore.
  • 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 can be varied to provide concentrates of reagent solutions, which provides user flexibility, space savings, reagent savings, and the like.
  • These reagents can also be provided in dry powder form, usually lyophilized, including excipients which, when dissolved, provide a solution of the reagents of appropriate concentrations.
  • the present disclosure provides the aforementioned antibodies or antigen-binding portions, bispecific or multispecific molecules, nucleic acid molecules, vectors, cells and/or compositions thereof in the manufacture of antibodies for the diagnosis, treatment or prevention of MASP- Use in a medicament or a kit for a disease associated with 2-dependent complement activation.
  • antibodies of the present disclosure can also be used in immunoassays, purification methods, and other methods using immunoglobulins or fragments thereof. Such uses are well known in the art.
  • compositions comprising the anti-MASP-2 antibodies of the present disclosure or fragments thereof, the antibodies being conveniently combined with a pharmaceutically acceptable carrier, diluent or excipient, as is routine in the art .
  • the term "pharmaceutical composition” refers to a formulation of various preparations. Formulations containing a therapeutically effective amount of the multivalent antibody are in sterile liquid solutions, liquid suspensions, or lyophilized forms, optionally containing stabilizers or excipients.
  • therapeutic agents according to the foregoing embodiments will be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents that are incorporated into the formulation to provide improved transfer, delivery, tolerability, and the like.
  • suitable pharmaceutically acceptable carriers, excipients, and other agents that are incorporated into the formulation to provide improved transfer, delivery, tolerability, and the like.
  • suitable formulations can be found in the pharmacopoeia known to all medicinal chemists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, Pa. (1975)), especially Chapter 87 in Blaug, Seymour.
  • Such formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (eg, Lipofectin TM ), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycols (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycols. Any of the foregoing mixtures may be suitable for use in treatment or therapy according to the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible and tolerated by the route of administration.
  • MASP-2 polypeptides can be isolated by standard techniques such as immunoaffinity, chromatography or immunoprecipitation using antibodies specific for MASP-2.
  • Antibodies to the MASP-2 protein (or fragments thereof) can be used to detect the protein in biological samples.
  • MASP-2 can be detected in a biological sample as part of a clinical testing procedure, eg, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (ie, physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase;
  • suitable prosthetic complexes include streptavidin/biotin and avidin/ Biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine aminofluorescein, dansyl chloride, or phycoerythrin;
  • luminescent materials include Mino;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include125I, 131I , 35S , or3H .
  • antibodies according to the present disclosure can be used as reagents for detecting the presence of MASP-2 or protein fragments thereof in a sample.
  • the antibody comprises a detectable label.
  • the antibody is a polyclonal antibody, or more preferably a monoclonal antibody. Whole antibodies or fragments thereof (eg Fab, scFv or F(ab') 2 ) are used.
  • labeling in reference to an antibody is intended to include direct labeling of the antibody by conjugating (ie, physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reaction with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody, and end-labeling the antibody with biotin to enable detection with fluorescently-labeled streptavidin.
  • bio sample is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present in a subject.
  • biological sample is used to include blood and fractions or components in blood, including serum, plasma, or lymph.
  • the detection methods of the foregoing embodiments can be used to detect analyte mRNA, protein or genomic DNA in biological samples in vitro and in vivo.
  • in vitro detection techniques for analyte mRNA include Norhtern hybridization and in situ hybridization.
  • Analyte protein in vitro detection techniques 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", Vol. 42, JRCrowther (ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T.
  • in vivo detection techniques for analyte proteins include introducing into a subject a labeled anti-analyte protein antibody.
  • an antibody can be labeled with a radiolabel, and the presence and location of the radiolabel in a subject can then be detected by standard imaging techniques.
  • compositions suitable for administration 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.
  • compositions typically comprise the antibody and a pharmaceutically acceptable carrier.
  • antibody fragments When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the target protein binding domain may be preferred.
  • peptide molecules can be designed that retain the ability to bind target protein sequences. Such peptides can be chemically synthesized and/or produced by recombinant DNA techniques (see, eg, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).
  • the term "pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration .
  • Suitable pharmaceutically acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard bibliography in the art, which is incorporated herein by reference.
  • Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
  • Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. In addition to any conventional media or reagents that are incompatible with the antibody, its use in compositions is contemplated.
  • compositions of the preceding embodiments are formulated to be compatible with their intended route of administration.
  • routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal administration.
  • Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: sterile injectable diluents such as water, saline solutions, fixed oils, polyethylene glycols, glycerol, 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 to adjust osmotic pressure, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (herein water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable pharmaceutically acceptable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that it is easy to inject. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi.
  • isotonic agents such as sugars, polyols (such as mannitol, sorbitol), sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the foregoing compositions an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • the compounds are delivered as an aerosol spray from a pressurized container or dispenser containing a gas of a suitable propellant, such as carbon dioxide, or a nebulizer.
  • a gas of a suitable propellant such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the permeation barrier are used in the formulation.
  • penetrants are generally known in the art and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • one or more of the foregoing antibodies may be formulated into an ointment, ointment, gel, or cream as generally known in the art.
  • the compounds can also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas.
  • suppositories eg, with conventional suppository bases such as cocoa butter or other glycerides
  • retention enemas e.g., retention enemas.
  • the aforementioned antibodies may be prepared with a carrier that will prevent their rapid elimination from the body, such as a sustained/controlled release formulation, including implants and microencapsulated delivery systems.
  • a sustained/controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art.
  • Dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier
  • One or more of the foregoing antibodies are dictated by and directly dependent upon the unique characteristics of the antibody and the particular therapeutic effect to be achieved, and the limitations inherent in the art of formulation of such antibodies for the treatment of individuals.
  • compositions can be placed in a container, pack, or dispenser with instructions for administration.
  • compositions described herein may also contain more than one of the foregoing antibodies, preferably those having complementary activities but not negatively affecting each other, depending on the particular condition to be treated.
  • the composition may, for example, comprise an agent that enhances its function, such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent.
  • agents that enhances its function such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent.
  • Such molecules are suitably combined in amounts effective for the intended purpose. For example, it can be combined in a kit, and can also be combined in use.
  • one or more of the foregoing antibodies may be administered in combination therapy, ie, with other agents such as therapeutic agents, which are useful in the treatment of pathological conditions or disorders, such as various forms of autoimmune diseases and inflammatory diseases disease) combined.
  • agents such as therapeutic agents, which are useful in the treatment of pathological conditions or disorders, such as various forms of autoimmune diseases and inflammatory diseases disease
  • combination refers to the administration of the agents substantially simultaneously, 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.
  • “combination” can also be the simultaneous inclusion of an antibody of the present disclosure and other therapeutic agents in a kit.
  • a combination therapy can comprise one or more antibodies described herein with one or more additional therapeutic agents (eg, one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors) , enzyme inhibitors, and/or cytotoxins or cytostatics, as described in more detail below) are co-formulated and/or co-administered.
  • additional therapeutic agents eg, one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors
  • enzyme inhibitors e.g., enzyme inhibitors, and/or cytotoxins or cytostatics
  • the experiments of the present disclosure demonstrate that the various monoclonal antibodies and their variants that can bind to MASP-2 with high affinity and inhibit lectin-mediated complement activation and their variants can specifically recognize human, monkey, mouse and rat MASP-2.
  • the enzymatic domain of 2, CCP1-CCP2-SP can effectively inhibit the production of C4b and C3 convertase C4b2a at low concentrations, and its blocking activity is 50-200 times higher than that of known antibodies.
  • the results of the pre-toxicology study in rhesus monkeys showed that the animals tolerated the high-dose novel MASP-2 antibody well without any toxic reaction, and its efficacy and safety were better than similar antibodies.
  • MBL in plasma After MBL in plasma recognizes the repetitive N-galactosamine or mannose structure, it can sequentially activate the serine proteases MASP-1 and MASP-2, the activated MASP-2 cleaves the complement components C4 and C2, and the formed C4b is deposited on the well plate , can be recognized by anti-C4b antibody; C3 convertase C4b2a formed by C4 and C2 cleavage products, after cleaving complement component C3, the formed C3b is deposited on the well plate and can be recognized by anti-C3b antibody. Therefore, the inhibitory activity of MASP-2 antibody on MASP-2 in serum can be shown by detecting the production of C4b or C3b in the lectin activation reaction.
  • 6-week-old BALB/c female mice were selected and immunized subcutaneously or by footpad using human or cynomolgus monkey MASP-2 D4-6 recombinant protein mixed with an equal volume of Freund's adjuvant. Two weeks after the initial immunization, booster immunization was performed. When the serum titer of the immunized mice reached 1:100,000, a shock immunization was performed. Three days later, the spleen and peripheral lymph nodes of the mice were collected and B cells were isolated.
  • B cells and SP2/0 cells were fused, screened in DMEM complete medium containing HAT, and the hybridoma cells were cultured in 5% CO 2 at 37° C. for 5-7 days.
  • DMEM complete medium containing HAT fetal calf serum
  • a total of 778 monoclonal antibodies can cross-recognize human or cynomolgus MASP-2.
  • the supernatants were harvested and assayed by C4 cleavage activity, of which 61 clones had significant MASP-2 inhibitory activity (as shown in Figure 2).
  • RNA of candidate hybridoma monoclonal cells was extracted by TRNzol lysis method, and then reverse-transcribed to synthesize single-stranded cDNA, which was used as a template to amplify the variable region sequence of the antibody in the hybridoma monoclonal cells.
  • variable region of the MASP-2 antibody is as follows:
  • 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.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.12, 13, 14.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.7, 17, 18.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.21, 22, 23.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.26, 27, 28.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.31, 32, 33.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.36, 37, 38.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.41, 42, 43.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.7, 46, 47.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.50, 51, 52.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.7, 17, 18.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.21, 57, 23.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.7, 17, 60.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.50, 51, 52.
  • amino acid sequence of heavy chain VH is shown in SEQ ID NO.63
  • its encoding nucleic acid is shown in SEQ ID NO.64
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.65, 66, 67.
  • 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
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.70, 71, and 72, respectively.
  • amino acid sequence of heavy chain VH is shown in SEQ ID NO.73
  • its encoding nucleic acid is shown in SEQ ID NO.74
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.7, 17, 47.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.50, 51, 52.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.7, 17, 79.
  • 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.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.36, 85, 86.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.41, 42, 43.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.36, 85, 86.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.41, 42, 91.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.94, 95, 96.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.99, 100, 101.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.7, 104, 105.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.108, 109, 110.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.26, 113, 114.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.117, 118, 33.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.94, 95, 96.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.123, 100, 101.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.131, 132, 133.
  • the light chain variable region of the above antibody was cloned into a vector containing human light chain constant region
  • the heavy chain variable region was cloned into a vector containing human heavy chain constant region
  • the plasmid was co-transfected into CHO-S cells
  • the complete IgG antibody After expression, it was secreted into the medium, and after 7 days of expression at 37° C. 5% CO 2 125 rpm, the supernatant was collected, purified by Protein A chromatography to obtain chimeric IgG, and the concentration of IgG was determined by spectrophotometry.
  • the control antibody 17D20_3521N11 (OMS646) was synthesized according to the document US20120282263.
  • the inhibitory test of C3 convertase activity was carried out on the screened dominant chimeric antibodies, and the GraphPad Prism 5 software was used to calculate the results after collecting the data. The results are shown in Figure 4.
  • the dominant chimeras 10L3, 3B9, 16A17, 4F18, 25L15, 28O14 and 1B2 showed extremely strong inhibitory activity, which was significantly stronger than that of the control antibody.
  • Example 3 Part of the immunized mouse B cells obtained in Example 3 was taken, total RNA was extracted, and the antibody cDNA library was obtained by reverse transcription using light and heavy chain specific primers respectively.
  • the antibody variable region fragments were specifically amplified with variable region primers, and cloned into phage plasmids by enzyme digestion to construct an antibody Fab phage display library based on M13 phage, with a library capacity of 1 ⁇ 10 10 .
  • variable region sequence of the Fab antibody was obtained by Sanger sequencing, as follows:
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.136, 137, 138.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.131, 132, 133.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.7, 17, 141.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.144, 145, 146.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.149, 150, 151.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.154, 155, 156.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.7, 17, 9.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.161, 162, 14.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.7, 165, 9.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.144, 168, 14.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.7, 17, 9.
  • 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
  • its CDR1, CDR2 and CDR3 are respectively shown in SEQ ID NO.144, 173, 174.
  • the mouse anti-28O14, 3H6 and 16B11 with high affinity and strong activity were selected for humanization of the variable region sequence.
  • the 28O14 heavy chain CDR3 glycosylation site NYS was cloned for site-directed mutagenesis, and the mutant was expressed as Fab in E.coli. prokaryotes.
  • the S-A mutant (Ala102) mutating serine to alanine did not affect MASP-2 binding activity (as shown in Figure 8).
  • the humanized sequence is as follows:
  • h16B11 H1 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 respectively shown in SEQ ID NO.7, 17, 141.
  • h16B11 H2 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.
  • h16B11 H3 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.
  • h3H6 H1 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 respectively shown in SEQ ID NO.136, 183, 138.
  • h3H6 H2 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.
  • h3H6 H3 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.
  • h3H6 H4 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.
  • 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.
  • amino acid sequence of h28O14 H2 is shown in SEQ ID NO.193
  • its encoding nucleic acid is shown in SEQ ID NO.194
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.126, 192, 128 respectively.
  • amino acid sequence of h28O14 H3 is shown in SEQ ID NO.195
  • its encoding nucleic acid is shown in SEQ ID NO.196
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.126, 192, 128 respectively.
  • amino acid sequence of h16B11 K1 is shown in SEQ ID NO.199
  • its encoding nucleic acid is shown in SEQ ID NO.200
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.201, 202, 146 respectively.
  • amino acid sequence of h16B11 K2 is shown in SEQ ID NO.203
  • its encoding nucleic acid is shown in SEQ ID NO.204
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.201, 202, 146 respectively.
  • amino acid sequence of h16B11 K3 is shown in SEQ ID NO.205
  • its encoding nucleic acid is shown in SEQ ID NO.206
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.201, 207, 146 respectively.
  • 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.
  • amino acid sequence of h16B11 K5 is shown in SEQ ID NO.210
  • its encoding nucleic acid is shown in SEQ ID NO.211
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.201, 202, 146 respectively.
  • amino acid sequence of h16B11 K6 is shown in SEQ ID NO.212
  • its encoding nucleic acid is shown in SEQ ID NO.213
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.201, 207, 146 respectively.
  • amino acid sequence of h3H6 K1 is shown in SEQ ID NO.214
  • its encoding nucleic acid is shown in SEQ ID NO.215
  • its CDR1, CDR2 and CDR3 are shown in SEQ ID NO.216, 132, 133 respectively.
  • the MASP-2 humanized antibody binds to human, cynomolgus monkey, rat, and mouse MASP-2 with comparable affinities.
  • STX2 (Sino Biological, Cat. No.: EG40019-G) as the template
  • STX2A Arg23-Lys319
  • STX2B Al20-Asp89
  • the N-terminus of STX2A protein is fused with a twin strep tag to facilitate purification, and an enterokinase cleavage site DDDDK is added between the two.
  • the constructed STX2 expression plasmid was transformed into TG1 E.
  • coli competent cells single clones were picked into bacterial medium, IPTG was added to induce culture overnight, the cells were collected, PPB and 5mM MgSO4 solution were added successively, the bacterial cell wall was lysed, and centrifugation was performed.
  • the protein expression solution in the periplasmic cavity was collected, purified by Strep-TactinX (Sino HaiGene, Cat. No.: C0402) to obtain the Shiga toxin STX2 protein, and EK enterokinase was added to the protein collection solution, incubated at 25°C overnight, and the twin strep tag was removed.
  • mice weighing about 18 g were selected and divided into 4 dose groups of MASP-2 antibody 0 mg/kg, 3/mg/kg, 10 mg/kg and 30 mg/kg, with 7 mice in each group.
  • the anti-MASP-2 antibody h16B11H2K4 (100 ⁇ l) was injected into the tail vein 2 hours in advance, and LPS (5 ⁇ g/only, L6136-Sigma) and STX2 (10 ng/only) were injected intraperitoneally. Mice were monitored for survival 60 hours after dosing and sacrificed when they reached the lethargic stage of HUS pathology. The results are shown in Figure 12, the anti-MASP-2 antibody can significantly prolong the survival time of mice in a dose-dependent manner.

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WO2024140939A3 (zh) * 2022-12-29 2024-08-22 苏州创胜医药集团有限公司 含有治疗性抗体的药物制剂及其用途

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