Igm proteases

Abstract

The present disclosure relates to proteases that exhibit activity against immunoglobulins, particularly IgM antibodies, and uses of the proteases for analysis of IgM, for generation of IgM antibody fragments, diagnostics related to IgM antibodies, and for treating and / or preventing diseases mediated by IgM antibodies.

Description

IGM PROTEASESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 580,130, filed September 1, 2023, which is incorporated by reference herein.REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

[0002] The Sequence Listing concurrently submitted herewith as file name CX7- 246W01_ST26.xml, created on August 30, 2024 with a file size of 109,358 bytes, is part of the specification and is hereby incorporated by reference herein in its entirety.TECHNICAL FIELD

[0003] The present disclosure relates to polypeptides that have protease activity against immunoglobulins, particularly IgM antibodies, and use of the proteases for diagnostic and therapeutic purposes.BACKGROUND

[0004] Enzymatic cleavage of immunoglobulins is an adaptation of some pathogenic bacteria to evade immune surveillance during infection. For example, IdeS is a cysteine protease produced by Streptococcus pyogenes having high degree of specificity for IgG. IdeS cleaves in the lower hinge region of the heavy chains of human IgG resulting in generation of Fc and F(ab’)2 fragments. IdeZ is another cysteine protease that specifically cleaves IgG and is produced by Streptococcus zooepidemicus. Similar to IdeS protease, IdeZ cleaves below the hinge region of IgG to form Fc and F(ab')2 fragments.

[0005] These immunoglobulin specific proteases have found utility in methods for production of Fab, F(ab')2 and Fc fragments and for the analysis / detection of IgG. IdeS and IdeZ proteases have also been suggested a potential therapeutics for treatment of diseases associated with abnormal IgG activity, such acute transplant rejection, Goodpasture's syndrome, relapsed systemic lupus, immune thrombocytopenic purpura (ITP), and Guillain-Barre syndrome (GBS). Moreover, IdeS and IdeZ have also been proposed as adjunctive therapy where attenuating IgG levels is warranted, for example, for reducing adverse immune response in organ transplantation and viral vector based gene therapy.

[0006] IdeS and IdeZ are specific to IgG antibodies. A protease from Streptococcus suis serotype 2, also referred to as IdeSsuis, has been identified that specifically cleaves IgM antibodies (see, e.g.,Seele et al., J Bacteriol., 2013, 195(5):930-940). However, IdeSsuis is host specific, specifically cleaving porcine IgM but not IgM from six other species, including human IgM. Desirable are other proteases that could be used for other classes of antibodies, particularly human IgM, not only as tools for research and diagnostics but also as potential therapeutics in treating disease or conditions mediated by other types of antibodies.SUMMARY

[0007] The present disclosure provides proteases that cleave antibodies, in particular IgM antibodies. In some embodiments, the protease comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 16, 18, 26, 30, 42, 46, 50, or 36, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 5, 8, 9, 10, 11, or 12, wherein the protease preferentially cleaves IgM compared to IgG.

[0008] In some embodiments, the protease comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 16, 18, 26, 30, 42, 46, 50, or 36.

[0009] In some embodiments, the protease comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 2, 4, 5, 8, 9, 10, 11, or 12.

[0010] In some embodiments, the protease comprises an amino acid sequence comprising SEQ ID NO: 16, 18, 26, 30, 42, 46, 50, or 36. In some embodiments, the protease comprises an amino acid sequence comprising SEQ ID NO: 2, 4, 5, 8, 9, 10, 11, or 12.

[0011] In some embodiments, the protease further comprises a fusion polypeptide. In some embodiments, the protease comprises an amino acid sequence comprising SEQ ID NO: 28, 32, 44, 48, 52, 24, or 40.

[0012] In some embodiments, the protease cleaves IgM in the CH2-CH3 linker region, near the N- terminus of the CH3 domain, thereby generating antibody fragments F(ab)2 and Fc of IgM. In some embodiments, the IgM antibody is a human IgM antibody. In some embodiments, the protease cleaves IgM antibody at cleavage site MCVPDQDTJ.AIRVFAI.

[0013] In another aspect, the present disclosure provides a recombinant polynucleotide comprising a polynucleotide sequence encoding a protease described herein.

[0014] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to SEQ ID NO: 17, 25, 29, 41, 45, 49, 15, or 35.

[0015] In some embodiments, the polynucleotide sequence encoding the protease is codon optimized. In some embodiments, the polynucleotide sequence is codon optimized for expression in a bacterial cell, fungal cell, insect cell, or mammalian cell.

[0016] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising SEQ ID NO: 17, 25, 29, 41, 45, 49, 15, or 35.

[0017] In another aspect, the present disclosure provides an expression vector comprising a recombinant polynucleotide encoding a protease that cleaves IgM. In some embodiments, the expression vector further comprises a control sequence operably linked to the recombinant polynucleotide. In some embodiments, the control sequence comprises a promoter, particularly a heterologous promoter.

[0018] In a further aspect, the present disclosure provides a host cell comprising an expression vector encoding the protease polypeptide. In some embodiments, the host cell comprises a bacterial cell, fungal cell, insect cell, or mammalian cell.

[0019] In some embodiments, the host cells are used to produce the protease polypeptides. In some embodiments, a method of producing a protease polypeptide comprises culturing the host cell comprising an expression vector encoding a protease described herein under suitable culture conditions such that the encoded protease is produced. In some embodiments, the method further comprises isolating the protease from the media and / or host cell. In some embodiments, the method further comprises purifying the protease.

[0020] In some embodiments, the protease is provided as a composition. In some embodiments, the composition comprises a protease, and an IgM antibody, particularly a human IgM antibody. In some embodiments, the composition further comprises a buffer and / or a preservative. In some embodiments, the protease in the composition is provided in solution, lyophilized, or immobilized on a substrate.

[0021] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a protease that cleaves IgM, and a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the pharmaceutical composition is suitable for parenteral injection or infusion to a human.

[0022] In some embodiments, the protease is used in a method of cleaving IgM antibody, the method comprising contacting an IgM antibody with a protease described herein under suitable conditions such that the IgM antibody is cleaved. In some embodiments, the method is used to prepare F(ab)2 and Fc fragments of IgM antibody.

[0023] In some embodiments, the protease is used for treating a disease or condition associated with abnormal IgM antibody activity. In some embodiments, a method for treating a disease or condition associated with abnormal IgM antibody activity comprises administering to a subject in need thereof an effective amount of a protease described herein.

[0024] In some embodiments, the subject for treatment has IgM nephropathy or Waldenstrom’s disease.

[0025] In some embodiments, the subject for treatment has an autoimmune disease or condition mediated by IgM antibody. In some embodiments, the autoimmune disease or condition is rheumatoid arthritis, bullous pemphigoid, or autoimmune neuropathy.

[0026] In some embodiments, the protease is used for reducing or preventing IgM-mediated immune response to gene therapy. In some embodiments, a method of reducing or preventing IgM-mediated immune response to gene therapy, comprises administering adjunctively to a subject being treated with a gene therapy vector an effective amount of a protease described herein.

[0027] In some embodiments, the subject for adjunctive treatment with protease is being treated with a virus-based gene therapy vector, including, among others, adeno-associated virus (AAV), adenovirus, retrovirus, and lentivirus gene therapy vectors.

[0028] In some embodiments, the protease is used in the preparation of a medicament for treating a disease or condition associated with abnormal IgM activity or for reducing or preventing IgM- mediated immune response to gene therapy. In some embodiments, for the forgoing use, the disease or condition associated with autoreactive IgM is selected from IgM nephropathy, Waldenstrom’s disease, rheumatoid arthritis, bullous pemphigoid, and autoimmune neuropathy. In some embodiments, for the foregoing use, the gene therapy is based on viral-based gene therapy vectors, for example, adeno-associated virus (AAV), adenovirus, retrovirus, and lentivirus gene therapy vectors.BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 depicts a representative Western blot that shows the proteolysis of human IgM heavy chain by E. coli lysate containing IgM proteases with indicated SEQ ID numbers. Human IgM (0. 1 mg / mL) was treated with 45 pL lysate in 50 pL reaction for 20 h.

[0030] FIG. 2 depicts a Coomassie Blue-stained SDS polyacrylamide gel that shows the proteolysis of human IgM heavy chain by E. coli lysate containing IgM proteases. Human IgM (0. 1 mg / mL) was treated with 5 pL in 50 pL reaction for 1 h.

[0031] FIG. 3 depicts a representative Western blot that shows lack of proteolysis of human IgM heavy chain by E. coli lysate containing IgG-degrading enzymes. Human IgM (0.1 mg / mL) was treated with 45 pL lysate in 50 pL reaction for 20 h.

[0032] FIG. 4 depicts a Coomassie Blue-stained SDS polyacrylamide gel that shows purified IgM proteases and cleavage of human IgM heavy chain by these purified proteins. Purified protein at 500 ug / mL was incubated withoutor with (“+”) human IgM (0.1 mg / mL) for 1 h.

[0033] FIG. 5 depicts a Coomassie Blue-stained SDS polyacrylamide gel that shows proteolysis of human IgM heavy chain by purified proteins. Human IgM (0.1 mg / mL) was incubated with 50 ug / mL or 5 ug / mL purified proteins for 1 h.

[0034] FIG. 6 shows a comparison of cleavage site of IdeSsuis and the confirmed cleavage site for four immunoglobulin-degrading enzymes including those from Lachnoanaerobaculum sp. ICM7 (SEQ ID NO: 48), Mediterraneibacter massiliensis (SEQ ID NO: 52), Lachnospiraceae bacterium (SEQ ID NO: 24), and Oribacterium sinus (SEQ ID NO: 40). The upper sequence is that of heavy chain of human IgM from CHI to CH4 (SEQ ID NO: 53) while the lower sequence is that of heavy chain of porcine IgM (SEQ ID NO: 54). The amino acid residues identical in the alignment are shown with an “*”.DETAILED DESCRIPTION

[0035] The present disclosure provides proteases that cleave IgM antibodies. In some embodiments, the protease preferentially cleaves IgM compared to IgG antibodies. In some embodiments, the proteases cleave IgM in the linker region of CH2-CH3 domain of IgM. In some embodiments, the proteases find use for generating fragments of IgM, diagnostics for IgM antibodies, treating diseases or conditions associated with abnormal IgM activity or IgM pathology, and reducing or preventing IgM mediated immune response in gene therapy.Abbreviations and Definitions

[0036] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well-known and commonly employed in the art.

[0037] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary, depending upon the context they are used by those of skill in the art. Accordingly, the terms defined immediately below are more fully described by reference to the application as a whole.

[0038] As used herein, the singular “a,” “an,” and “the” include the plural references, unless the context clearly indicates otherwise.

[0039] In addition, the term “comprising” and its cognates are used in their inclusive sense (i.e., equivalent to the term “including” and its corresponding cognates).

[0040] It is to be further understood that where description of embodiments use the term “comprising” and its cognates, the embodiments can also be described using language “consisting essentially of’ or “consisting of.”

[0041] Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

[0042] The term “about” means an acceptable error for a particular value. In some instances, “about” means within 0.05%, 0.5%, 1.0%, or 2.0%, of a given value range. In some instances, “about” means within 1, 2, 3, or 4 standard deviations of a given value. In some instances, “about” encompasses values that are within 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% of a given value.

[0043] ‘ ‘EC” number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

[0044] “ATCC” refers to the American Type Culture Collection whose biorepository collection includes genes and strains.

[0045] “NCBI” refers to National Center for Biological Information and the sequence databases provided therein.

[0046] ‘ ‘Protease,” “proteinase,” and “peptidase” refer to enzymes that hydrolyze proteins, polypeptides, and / or oligopeptides. Proteases, proteinases, and peptidases breakdown proteins, polypeptides, or oligonucleotides into smaller peptides or single amino acids.

[0047] “Proteolysis” or “proteolytic activity” refers to the breakdown (e.g., through hydrolysis) of proteins and / or polypeptides into smaller peptides and / or amino acids.

[0048] ‘ ‘Protein,” “polypeptide,” and “peptide” are used interchangeably to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0049] ‘ ‘Amino acids” are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission. The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glycine (Gly or G), glutamate (Glu or E), glutamine (Gin or Q), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V). When the three-letter abbreviations are used, unless specifically preceded by an “L” or a “D” or clear from the context in which the abbreviation is used, the amino acid may be in either the L- or D-configuration about a-carbon (Ca). For example, whereas “Ala” designates alanine without specifying the configuration about the a carbon, “D-Ala” and “L-Ala” designate D-alanine and L-alanine, respectively. When the one-letter abbreviations are used, upper case letters designate amino acids in the L-configuration about the a-carbon and lower case letters designate amino acids in the D-configuration about the a-carbon. For example, “A” designates L-alanine and “a” designates D- alanine. When polypeptide sequences are presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequences are presented in the amino (N) to carboxy (C) direction in accordance with common convention.

[0050] ‘ ‘Fusion protein,” and “chimeric protein” and “chimera” refer to hybrid proteins created through the joining of two or more polynucleotides that originally encode separate proteins. In some embodiments, fusion proteins are created by recombinant technology (e.g., molecular biology techniques known in the art).

[0051] ‘ ‘Mature protein” or “mature polypeptide” refers to the final processed biological protein or polypeptide or product.

[0052] ‘ ‘Pro-protein,” “pro-polypeptide,” or “pro-peptide” refers to a precursor protein, polypeptide, or peptide that is processed by post-translational modification, to form a biologically active protein,polypeptide, or peptide. In some embodiments, the post translational modification is a cleavage reaction to form the protein, polypeptide, or peptide. “Pro-enzyme” refers to a precursor polypeptide that is processed by post-translational modification, in particular a cleavage reaction, to form an active enzyme.

[0053] ‘ ‘Pre-pro-protein,” “pre-pro-polypeptide,” or “pre-pro-peptide” refers to a precursor protein, polypeptide, or peptide that includes a signal sequence and which can be processed by posttranslational modification, in particular a cleavage reaction, to generate a pro-protein, propolypeptide, or pro-peptide. Generally, a cleavage reaction removes a signal sequence to generate a pro-protein, pro-polypeptide, or pro-peptide. “Pre-pro-enzyme” refers to a precursor protein, polypeptide, or peptide that is processed by post-translational modification, in particular a cleavage reaction that removes a signal sequence, to form a pro-enzyme.

[0054] ‘ ‘Full-length” in context of a protein or polypeptide refers to the protein or polypeptide which is not processed to alter the amino acid sequence of the entire protein or polypeptide. For example, a full-length protein is the entire protein encoded in the corresponding mRNA.

[0055] “Polynucleotide” is used herein to denote a polymer comprising at least two nucleotides where the nucleotides are either deoxyribonucleotides or ribonucleotides or mixtures of deoxyribonucleotides and ribonucleotides. In some embodiments, the abbreviations used for the genetically encoding nucleosides are conventional and are as follow: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2 ’-deoxyribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2’ -deoxyribonucleosides on an individual basis or on an aggregate basis. When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5 ’ to 3 ’ direction in accordance with common convention, and the phosphates are not indicated. The term “DNA” refers to deoxyribonucleic acid. The term “RNA” refers to ribonucleic acid.

[0056] “Engineered,” “recombinant,” “non-naturally occurring,” and “variant,” when used with reference to a cell, a polynucleotide or a polypeptide refers to a material or a material corresponding to the natural or native form of the material that has been modified in a manner that would not otherwise exist in nature or is identical thereto but produced or derived from synthetic materials and / or by manipulation using recombinant techniques.

[0057] “Wild-type” and “naturally-occurring” refer to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.

[0058] “Coding sequence” refers to that part of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.

[0059] ‘ ‘Percent (%) sequence identity” is used herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Those of skill in the art appreciate that there are many established algorithms available to align two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection, as known in the art. Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to the BLAST and BLAST 2.0 algorithms, which are described by Altschul et al. (See, Altschul et al., J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., 1977, Nucleic Acids Res., 1977, 3389-3402, respectively). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (See, Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) andN (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89: 10915). Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

[0060] ‘ ‘Reference sequence” refers to a defined sequence used as a basis for a sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotide or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length or the full length of the nucleic acid or polypeptide. Since two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences, sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity. In some embodiments, a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence.

[0061] “Corresponding to”, “reference to” or “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of a protease describe herein, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.

[0062] ‘ ‘Conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acids having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basis side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0063] “Non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0064] ‘ ‘Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide. Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference enzyme while retaining enzymatic activity and / or retaining the improved properties of an engineered enzyme. Deletions can be directed to the internal portions and / or terminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous.

[0065] ‘ ‘Insertion” refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

[0066] ‘ ‘Functional fragment” or a “biologically active fragment” used interchangeably herein refers to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion(s) and / or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full-length protease of the present disclosure) and that retains substantially all of the activity of the full-length polypeptide.

[0067] ‘ ‘Isolated polypeptide” refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides). The term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis). The protease polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the protease polypeptides can be an isolated polypeptide.

[0068] “Substantially pure polypeptide” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. Generally, a substantially pure protease comprises about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated protease polypeptides are substantially pure polypeptide compositions.

[0069] “Hybridization stringency” relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2001; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, 2003). The term “moderately stringent hybridization” refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target-polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in 0.2xSSPE, 0.2% SDS, at 42°C. “High stringency hybridization” refers generally to conditions that are about 10 °C or less from the thermal melting temperature Tm as determined under the solution condition for a defined polynucleotide sequence. In some embodiments, a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65 °C (i.e., if a hybrid is not stable in 0.018M NaCl at 65 °C, it will not be stable under high stringency conditions, as contemplated herein). High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in O. l xSSPE, and 0.1% SDS at 65 °C. Another high stringency condition is hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0.1% (w:v) SDS at 65 °C and washing in O.lx SSC containing 0.1% SDS at 65 °C. Other high stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.

[0070] ‘ ‘Codon optimized” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is more efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding the protease polypeptide may be codon optimized for optimal production from the host organism selected for expression.

[0071] ‘ ‘Control sequence” refers herein to include all components, which are necessary or advantageous for the expression of a polynucleotide and / or polypeptide of the present application. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, pro-peptide sequence, promoter sequence, signal peptide sequence, initiation sequence and transcription terminator. In some embodiments, the control sequences include a promoter, and transcriptional and translational stop signals.

[0072] “Operably linked” or “operatively linked” is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide, and where appropriate the encoded polypeptide of interest.

[0073] ‘ ‘Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of a polynucleotide of interest. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0074] ‘ ‘Suitable reaction conditions” refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co-solvents, etc.) under which a protease polypeptide of the present application is capable of converting a substrate to the desired product compound. Exemplary “suitable reaction conditions” are provided in the present application and illustrated by the Examples. “Substrate” in the context of an enzymatic conversion reaction process refers to the compound or molecule acted on by the protease polypeptide. “Product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the protease on a substrate.

[0075] “Culturing” refers to the growing of a population of cells under any suitable conditions (e.g., using a liquid, gel or solid medium).

[0076] “Vector” refers to a polynucleotide construct for introducing a polynucleotide sequence into a cell. In some embodiments, the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polynucleotide of interest and where appropriate the encoded polypeptide.

[0077] “Expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0078] ‘ ‘Produces” refers to the production of proteins and / or other compounds by cells. It is intended that the term encompass any step involved in the production of polypeptides including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0079] “Heterologous” refers to the relationship between two or more nucleic acid or protein sequences (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) that are derived from different sources and are not associated in nature.

[0080] “Host cell” and “host strain” refer to suitable hosts for expression vectors comprising polynucleotide provided herein (e.g., the polynucleotides encoding a protease). In some embodiments, the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques as known in the art.

[0081] “Therapeutic” refers to a compound administered to a subject who shows signs or symptoms of pathology having beneficial or desirable medical effects.

[0082] ‘ ‘Effective amount” means an amount sufficient to produce the desired result. One of general skill in the art may determine what the effective amount by using routine experimentation.

[0083] “Subject” encompasses mammals such as humans, non-human primates, livestock, companion animals (e.g., cats, dogs, rodents), and laboratory animals (e.g., rodents and lagomorphs). It is intended that the term encompass females as well as males.

[0084] ‘ ‘Patient” means any subject that is being assessed for, treated for, or is experiencing disease.

[0085] “Composition” and “formulation” encompass products comprising at least a protease of the present disclosure, or a recombinant polynucleotide encoding the protease intended for any suitable use (e.g., pharmaceutical compositions, etc.).

[0086] “Administration” and “administering” a composition mean providing a composition of the present disclosure to a subject (e.g., to a subject suffering from the effects of IgM nephropathy).

[0087] ‘ ‘Pharmaceutical composition” refers to a composition suitable for pharmaceutical use in a mammalian subject (e.g., human) comprising a pharmaceutically effective amount of a protease polypeptide.

[0088] “Pharmaceutically acceptable” means a material that can be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the components in which it is contained and that possesses the desired biological activity.

[0089] ‘ ‘Carrier” when used in reference to a pharmaceutical composition means any of the standard pharmaceutical carrier, buffers, and excipients, such as stabilizers, preservatives, and adjuvants.

[0090] “Excipient” refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API; e.g., the protease polypeptides of the present disclosure). Excipients are typically included for formulation and / or administration purposes.

[0091] “Therapeutically effective amount” when used in reference to symptoms of disease / condition refers to the amount and / or concentration of a compound (e.g., protease polypeptides) that ameliorates, attenuates, or eliminates one or more symptom of a disease / condition or prevents ordelays the onset of symptom(s). A “therapeutically effective amount” when used in reference to a disease / condition refers to the amount and / or concentration of a composition (e.g., protease polypeptides) that ameliorates, attenuates, or eliminates the disease / condition. In some embodiments, the term is use in reference to the amount of a composition that elicits the biological (e.g., medical) response by a tissue, system, or animal subject that is sought by the researcher, physician, veterinarian, or other clinician.

[0092] "Treating" or "treatment" of a disease, disorder, or syndrome, as used herein, includes (i) preventing the disease, disorder, or syndrome from occurring in a subject, i.e., causing the clinical symptoms of the disease, disorder, or syndrome not to develop in an animal that may be exposed to or predisposed to the disease, disorder, or syndrome but does not yet experience or display symptoms of the disease, disorder, or syndrome; (ii) inhibiting the disease, disorder, or syndrome, i.e., arresting its development; and (iii) relieving the disease, disorder, or syndrome, i.e., causing regression of the disease, disorder, or syndrome. As such, the terms “treating,” “treat” and “treatment” encompass preventative (e.g., prophylactic), as well as palliative treatment. As is known in the art, adjustments for systemic versus localized delivery, age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable by one of ordinary skill in the art.Engineered Protease Polypeptides

[0093] In one aspect, the present disclosure provides polypeptides having protease activity against immunoglobulins. In particular, the proteases preferentially cleave IgM antibodies, including human IgM antibodies. This contrasts with IdeS and IdeZ proteases, which cleave IgG antibodies and do not act on IgM antibodies.

[0094] In some embodiments, the protease comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 16, 18, 26, 30, 42, 46, 50, or 36, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 5, 8, 9, 10, 11, or 12. In some embodiments, the protease preferentially cleaves IgM compared to IgG.

[0095] In some embodiments, the protease comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 16.

[0096] In some embodiments, the protease comprises an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ IDNO: 18.

[0097] In some embodiments, the protease comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 26.

[0098] In some embodiments, the protease comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 30.

[0099] In some embodiments, the protease comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 42.

[0100] In some embodiments, the protease comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 46.

[0101] In some embodiments, the protease comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 50.

[0102] In some embodiments, the protease comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 36.

[0103] In some embodiments, the protease comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 2.

[0104] In some embodiments, the protease comprises an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ IDNO: 4.

[0105] In some embodiments, the protease comprises an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ IDNO: 5.

[0106] In some embodiments, the protease comprises an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ IDNO: 8.

[0107] In some embodiments, the protease comprises an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ IDNO: 9.

[0108] In some embodiments, the protease comprises an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ IDNO: 10.

[0109] In some embodiments, the protease comprises an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ IDNO: 11.

[0110] In some embodiments, the protease comprises an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ IDNO: 12.[oni] In some embodiments, the amino acid sequence of the protease comprises SEQ ID NO: 16, 18, 26, 30, 42, 46, 50, or 36. In some of embodiments, the amino acid sequence of the protease has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 insertions, substitutions, and / or deletions. In some embodiments, the amino acid sequence of the protease has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the protease has 1, 2, 3, 4, up to 5 insertions, substitutions, and / or deletions. In some embodiments, the amino acid sequence of the protease has 1, 2, 3, 4, up to 5 substitutions. In some embodiments, the amino acid substitutions comprises non-conservativeand / or conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions.

[0112] In some embodiments, the amino acid sequence of the protease comprises SEQ ID NO: 2, 4,5, 8, 9, 10, 11, or 12. In some embodiments, the amino acid sequence of the protease has 1, 2, 3, 4, 5,6, 7, 8, 9, or up to 10 insertions, substitutions, and / or deletions. In some embodiments, the amino acid sequence of the protease has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the amino acid sequence of the protease has 1, 2, 3, 4, up to 5 insertions, substitutions, and / or deletions. In some embodiments, the amino acid sequence of the protease has 1, 2, 3, 4, up to 5 substitutions. In some embodiments, the amino acid substitutions comprises non-conservative and / or conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions.

[0113] In some embodiments, the amino acid insertions, substitutions, and / or deletions can be based on alignment of the amino acid sequences of the IgM proteases described herein, and changing the amino acid residue present in one IgM protease parent sequence to a different amino acid residue (or insertion or deletion) in the corresponding amino acid residue position present in the amino acid sequence of another IgM protease, thereby generating an amino acid difference relative to the parent amino acid sequence.

[0114] In some embodiments, the protease cleaves IgM within the CH2-CH3 linker region, near the N-terminus of the CH3 domain, to generate F(ab)2 and Fc fragments of IgM. In some embodiments, the protease has the following cleavage site on the IgM antibody:MCVPDQDT^AIRVFAI (SEQ ID NO: 55)

[0115] In some embodiments, the protease comprises a full length protein, for example, with the native signal sequence, such as provided in SEQ ID NO: 2, 4, 5, 8, 9, 10, 11, or 12. In some embodiments, the signal sequence present in the full length protease polypeptide is replaced with a compatible signal sequence or signal peptide. In some embodiments, the signal sequence is a heterologous bacterial signal sequence. In some embodiments, the signal sequence is a heterologous Sec or Tat signal sequence (see, e.g., Roland Freudl, Microbial Cell Factories, 2018, 17(52): 1-10).

[0116] In some embodiments, the protease includes a signal sequence functional in fungal, insect, or mammalian cells, wherein the signal sequence allows secretion of the protease polypeptide. Exemplary signal sequence include, among others, yeast alpha mating signal sequence, human betagalactosidase signal sequence, Drosophila Bip signal sequence (Bip-sp), and honeybee melittin signal sequence (HBM-sp).

[0117] In some embodiments, the protease is provided as a pre-pro-polypeptide, where the signal sequence is a native or heterologous signal sequence. In some embodiments, the pre-pro-polypeptide is capable of being processed by removal of the signal sequence to form the pro-polypeptide.

[0118] In some embodiments, the protease is provided as a pro-polypeptide, where the pro- polypeptide can be further processed to a mature for of the protease. In some embodiments, the proregion is a heterologous pro-region, such as yeast alpha-mating factor pro-region.

[0119] In some embodiments, the protease is processed to or expressed as a form lacking the signal sequence. In some embodiments, the protease is processed or expressed as a mature form of the protease, such as described herein.

[0120] In some embodiments, the protease is expressed to include a fusion protein or fusion polypeptide. In some embodiments, the protease described herein can be fused to a variety of polypeptide sequences, such as, by way of example and not limitation, polypeptide tags that can be used for detection and / or purification, or another enzyme or protein. In some embodiments, the fusion polypeptide of the protease comprises a glycine-histidine or histidine-tag (His-tag). In some embodiments, the fusion polypeptide of the protease comprises an epitope tag, such as c-myc, FLAG, V5, or hemagglutinin (HA). In some embodiments, the fusion polypeptide of the protease comprises a GST, SUMO, Strep, MBP, or GFP tag. In some embodiments, the fusion is to the amino (N-) terminus of the protease polypeptide. In some embodiments, the fusion is to the carboxy (C-) terminus of the protease polypeptide.

[0121] In some embodiments, the protease comprises a fusion polypeptide comprising an amino acid sequence comprising SEQ ID NO: 20, 28, 32, 44, 48, 52, 24, or 40.

[0122] In some embodiments, the protease is provided as a purified preparation or composition for example, as a full length polypeptide, a polypeptide lacking the signal sequence, or fused to a fusion polypeptide / protein.

[0123] In some embodiments, the present disclosure further provides a functional or biologically active fragment of proteases described herein. In some embodiments, functional or biologically active fragments have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of the protease from which is derived (i.e., the parent protease). In some embodiments, a functional or biologically active fragment comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the protease. In some embodiments the functional fragment is truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, and less than 50 amino acids.

[0124] In some embodiments, the protease described herein is provided in solution, as a lyophilizate, or immobilized on a substrate. In some embodiments, the substrate is a solid substrate or a membrane or particles. The enzyme can be entrapped in matrixes or membranes. In some embodiments, matrices include polymeric materials such as calcium-alginate, agar, k-carrageenin, polyacrylamide, and collagen, or solid matrices, such as activated carbon, porous ceramic, and diatomaceous earth. In some embodiments, the matrix is a particle, a membrane, or a fiber. Types of membranes include, among others, nylon, cellulose, polysulfone, or polyacrylate.

[0125] In some embodiments, the enzyme is immobilized on the surface of a support material. In some embodiments, the enzyme is adsorbed on the support material. In some embodiments, the enzyme is immobilized on the support material by covalent attachment. Support materials include, among others, inorganic materials, such as alumina, silica, porous glass, ceramics, diatomaceous earth, clay, and bentonite, or organic materials, such as cellulose (CMC, DEAE-cellulose), starch, activated carbon, polyacrylamide, polystyrene, polyacrylate, and ion-exchange resins, such as Amberlite, Sephadex, and Dowex.Polynucleotides Encoding Protease Polypeptides, Expression Vectors and Host Cells

[0126] In another aspect, the present disclosure provides polynucleotides encoding the proteases described herein. In some embodiments, the polynucleotides are operatively linked to one or more heterologous or homologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the protease. Expression constructs containing a heterologous polynucleotide encoding the protease can be introduced into appropriate host cells to express the encoded protease polypeptide.

[0127] As will be apparent to the skilled artisan, availability of a protein sequence and the knowledge of the codons corresponding to the various amino acids provide a description of all the polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons, allows an extremely large number of nucleic acids to be made, all of which encode the protease polypeptides. Thus, having knowledge of a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the protein. In this regard, the present invention specifically contemplates each and every possible variation of polynucleotides that could be made encoding the proteases described herein by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any protease polypeptide described herein, including the proteases provided in Table 1.1.

[0128] As discussed above, it is to be understood that the present disclosure provides recombinant polynucleotides encoding each and every proteases described herein, or a biologically or functionally active fragment thereof, or a full length or mature polypeptide or fusion protein thereof.

[0129] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 16, 18, 26, 30, 42, 46, 50, or 36, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 5, 8, 9, 10, 11, or 12.

[0130] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 16.

[0131] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 18.

[0132] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 26.

[0133] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 30.

[0134] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 42.

[0135] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 46.

[0136] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 50.

[0137] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 36.

[0138] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 2.

[0139] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 4.

[0140] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 5.

[0141] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 8.

[0142] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 9.

[0143] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 10.

[0144] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 11.

[0145] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the protease comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the reference sequence corresponding to SEQ ID NO: 12.

[0146] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to SEQ ID NO: 17, 25, 29, 41, 45, 49, 15, or 35.

[0147] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence codon-optimized for expression of the encoded protease polypeptide. In some embodiments, the polynucleotide sequence is codon-optimized for expression in bacterial cells. In some embodiments, the polynucleotide sequence is codon optimized for expression in mammalian cells.

[0148] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising SEQ ID NO: 17, 25, 29, 41, 45, 49, 15, or 35.

[0149] In some embodiments, the recombinant polynucleotide hybridizes under highly stringent conditions to a reference polynucleotide sequence described herein encoding a protease described herein, or a reverse complement thereof. In some embodiments, the reference polynucleotide sequence corresponds to SEQ ID NO: 17, 25, 29, 41, 45, 49, 15, or 35, or a reverse complement thereof. In some embodiments, the recombinant polynucleotide encodes a protease that specifically cleaves IgM and hybridizes under highly stringent conditions to a reverse complement of a reference polynucleotide sequence corresponding to SEQ ID NO: 17, 25, 29, 41, 45, 49, 15, or 35.

[0150] In some embodiments, the polynucleotide that hybridizes under highly stringent conditions comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polynucleotide sequence corresponding to SEQ ID NO: 17, 25, 29, 41, 45, 49, 15, or 35.

[0151] In some embodiments, a recombinant polynucleotide encoding any of the protease provided herein is manipulated in a variety of ways to provide for expression of the protease polypeptide. In some embodiments, the recombinant polynucleotide encoding the polypeptides are provided asexpression vectors where one or more control sequences is operably linked to the recombinant polynucleotide to regulate the expression of the polynucleotide and / or encoded polypeptide. The techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are known in the art.

[0152] In some embodiments, the control sequences include, among others, promoter sequences, Kozak sequence, leader sequences, polyadenylation sequences, pro-peptide sequences, signal peptide sequences, regulatory elements, and transcription terminators. As known in the art, suitable promoters can be selected based on the host cells used. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure, include, but are not limited to promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA, 1978, 75:3727-3731), as well as the tac promoter (See e.g., DeBoer et al., Proc. Natl Acad. Sci. USA, 1983, 80: 21-25). Exemplary promoters for filamentous fungal host cells, include, but are not limited to promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha- amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GALI), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde- 3 -phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3 -phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (See e.g., Romanos et al., Yeast, 1992, 8:423-488). Exemplary promoters for use in mammalian cells include, but are not limited to, those from cytomegalovirus (CMV), chicken [3-actin promoter fused with the CMV enhancer, Simian vacuolating virus 40 (SV40), from Homo sapiens phosphoglycerate kinase, beta actin, elongation factor- la or glyceraldehyde-3 -phosphate dehydrogenase, or from Gallus [3-actin.

[0153] In some embodiments, the control sequence is also a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments,the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the leucine decarboxylase polypeptide. Any suitable terminator which is functional in the host cell of choice finds use in the present invention. For bacterial expression, the transcription terminators can be a Rho-dependent terminators that rely on a Rho transcription factor, or a Rho-independent, or intrinsic terminators, which do not require a transcription factor. Exemplary bacterial transcription terminators are described in Peters et al., J Mol Biol., 2011, 412(5):793-813. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (See e.g., Romanos et al., supra). Exemplary terminators for mammalian cells include, but are not limited to those from cytomegalovirus (CMV), Simian virus 40 (SV40), from Homo sapiens growth hormone hGH, from bovine growth hormone BGH, and from human or rabbit beta globulin.

[0154] In some embodiments, the control sequence is also a suitable leader sequence (i.e., a nontranslated region of an mRNA that is important fortranslation by the host cell). In some embodiments, the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the protease polypeptide. Any suitable leader sequence that is functional in the host cell of choice find use in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3 -phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). Suitable leaders for mammalian host cells include but are not limited to the 5 -UTR element present in orthopoxvirus mRNA.

[0155] In some embodiments, the control sequence comprises a 3 ’ untranslated nucleic acid region and polyadenylation tail nucleic acid sequence, sequences operably linked to the 3' terminus of the protein coding nucleic acid sequence which mediate binding to proteins involved in mRNA trafficking and translation and mRNA half-life. Any polyadenylation sequence and 3’ UTR which is functional in the host cell of choice may be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to those from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (See e.g., Guo and Sherman, Mol. Cell. Biol., 1995, 15:5983-5990). Useful polyadenylation and 3’ UTR sequences for mammalian host cells include, but are not limited to, the 3 '-UTRs of a- and [3-globin mRNAs that harbor several sequence elements that increase the stability and translation of mRNA.

[0156] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region which directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered polypeptide(s). Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to those obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alphaamylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (See e.g., Simonen and Palva, Microbiol. Rev., 1993, 57: 109-137). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for mammalian host cells include but are not limited to, those from the genes for immunoglobulin gamma (IgG) and the signal peptide in a human secreted protein, such as human beta-galactosidase polypeptide.

[0157] In some embodiments, the control sequence is a regulatory sequence that facilitates the regulation of the expression of the recombinant polynucleotide and / or encoded polypeptide. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to theADH2 system or GALI system. In filamentous fungi, suitable regulatory sequences include, but are not limited to the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.

[0158] In another aspect, the present disclosure provides an expression vector comprising a recombinant polynucleotide encoding a protease polypeptide, where the recombinant polynucleotide is operably or operatively linked to a control sequence, such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced. The recombinant expression vector may be any suitable vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and bring about the expression of the protease polynucleotide sequence. The choice of the vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

[0159] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some alternative embodiments, the vector may be one which, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.

[0160] In some embodiment, recombinant polynucleotides may be provided on a non-replicating expression vector or plasmid. In some embodiments, the non-replicating expression vector or plasmid can be based on viral vectors defective in replication (see, e.g., Travieso et al., npj Vaccines, 2022, Vol. 7, Article 75).

[0161] In some embodiments, the expression vector contains one or more selectable markers, which permit easy selection of transformed cells. A “selectable marker” is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Examples of bacterial selectable markers include, but are not limited to the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5 '-phosphate decarboxylase; e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.

[0162] In another aspect, the present disclosure provides a host cell comprising at least one recombinant polynucleotide encoding a protease polypeptide of the present disclosure, the recombinant polynucleotide (s) being operatively linked to one or more control sequences for expression of the protease polypeptide. In some embodiments, the host cells suitable for use in expressing the polypeptides encoded by the expression vectors is a prokaryotic cell or eukaryotic cell, known in the art and include but are not limited to, bacterial cells, such as E. coli, Vibrio fluvialis, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal (e.g., mammalian) cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110 (AfhuA) and BL21).

[0163] In another aspect, the present disclosure provides a method of producing the protease polypeptides, where the method comprises culturing a host cell comprising an expression vector capable of expressing or producing the protease polypeptide under suitable culture conditions such that the protease polypeptide is expressed or produced. In some embodiments, the method comprises isolating the protease from the culture medium and / or host cell, as described herein. In some further embodiments, the method further comprises purifying the expressed protease polypeptide. In some embodiments, any of the protease polypeptide expressed in a host cell is recovered and purified from the cells and / or the culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme or detergent treatment, sonication, filtration, saltingout, precipitation, ultra-centrifugation, and chromatography, such as described herein.

[0164] Chromatographic techniques for isolation / purification of the protease polypeptides include, among others, reverse phase chromatography, high-performance liquid chromatography, ionexchange chromatography, hydrophobic -interaction chromatography, size -exclusion chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying the protease depends, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art. In some embodiments, affinity techniques may be used to isolate the protease. For affinity chromatography purification, an antibody that specifically binds protease polypeptide may be used. In some embodiments, an affinity tag, e.g., His-tag, can be introduced into the protease polypeptide for purposes of isolation / purification.

[0165] Appropriate culture media and growth conditions for the above-described host cells are well known in the art. Polynucleotides for expression of the proteases may be introduced into cells byvarious methods known in the art. Techniques include, among others, electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion.

[0166] In some embodiments, the polynucleotides encoding the protease polypeptide can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, polynucleotide fragments can be individually synthesized, then joined (e.g., by enzymatic or chemical litigation methods, or polymerase mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method (See e.g., Beaucage et al., Tetra. Lett., 1981, 22: 1859-69; and Matthes et al., EMBO J., 1984, 3:801-05), as it is typically practiced in automated synthetic methods.

[0167] In some embodiments, a method for preparing the protease can comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequence of a protease, such as described in Table 1.1, and (b) expressing the engineered protease encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide can optionally have one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the substitutions can be conservative or non-conservative substitutions. The expressed polypeptide can be assessed for the desired property, e.g., protease activity on IgM.Compositions

[0168] In a further aspect, the present disclosure provides compositions comprising a protease described herein. The composition may vary depending on the form and intended use.

[0169] In some embodiments, the composition comprises a protease described herein, and at least a buffer (e.g., Tris or PBS). In some embodiments, the composition further comprises a salt, such as NaCl or KC1. In some embodiments, the composition further comprises an IgM antibody. In some embodiments, the IgM antibody is a human IgM antibody. In some embodiments, the IgM is prepared from a serum or blood sample.

[0170] In some embodiments, the protease is provided as a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a protease described herein, and a pharmaceutically acceptable excipient and / or carrier. In some embodiments, the pharmaceutically composition is formulated for administration by any suitable route, such as intradermal, intravenous, intraperitoneal, subcutaneous, intramuscular, and / or intranasal administration. In some embodiments, the protease is provided in a form suitable for injection or infusion.

[0171] Depending on the mode of administration, the pharmaceutical composition comprising a therapeutically effective amount of a protease can be in the form of a solid, semi- solid, powder, or liquid. In some embodiments, the compositions include other pharmaceutically acceptable components such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, fillers, and other ingredients. Details on techniques for formulation and administration are known in the art, such as described in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J., 1991) and Remington: The Science and Practice of Pharmacy, A. Adejare ed., 23 Ed., Academic Press, 2020).

[0172] In some embodiments, the excipients and / or carriers may include, but not limited to, sugars (e.g., lactose, sucrose, mannitol, and / or sorbitol), starches, cellulose (e.g., methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxy-methylcellulose), gums (e.g., arabic, tragacanth, guar, etc.), and / or proteins (e.g., gelatin, collagen, etc.). In some embodiments, the excipient and / or carrier, includes diluents such as water, saline, glycerol and ethanol. In some embodiments, the excipients or carriers comprise pharmaceutically acceptable salts, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, and benzoates. Additional components may include coloring and or sweetening agents (e.g., glucose, sucrose, and mannitol) and lubricating agents (e.g., magnesium stearate), as well as enteric coatings (e.g., methacrylate polymers, hydroxyl propyl methyl cellulose phthalate, and / or any other suitable enteric coating known in the art). In some embodiments, disintegrating or solubilizing agents are included (e.g., cross-linked polyvinyl pyrrolidone, agar, alginic acid or salts thereof, such as sodium alginate). In some embodiments, the protease polypeptide is combined with various additional components, including but not limited to preservatives, suspending agents, thickening agents, wetting agents, alcohols, fatty acids, and / or emulsifiers, particularly in liquid formulations.

[0173] In some embodiments, the protease polypeptide is encapsulated. In some alternative embodiments, the protease polypeptide is encapsulated microparticles or nanoparticles (e.g., nanotubes, nanotubules, nanocapsules, or microcapsules, microspheres, liposomes, etc.).

[0174] In some embodiments, the protease is modified by glycosylation, chemical crosslinking reagents, pegylation (i.e., modified with polyethylene glycol (PEG) or activated PEG, etc.) or othercompounds (see e.g., Ikeda, Amino Acids, 2005, 29:283-287; US Pat. Nos. 7,531,341, 7,534,595, and 7,560,263; US Pat. Publication Nos. 2013 / 0039898, 2012 / 0177722, etc.).Uses and Methods

[0175] In another aspect, the proteases are used for cleaving IgM antibodies, such as in a diagnostic assay for IgM antibodies. In some embodiments, a method for cleaving IgM antibody in vitro comprises contacting a protease described herein with an IgM antibody under conditions suitable for proteolysis of the IgM antibody. In some embodiments, the IgM antibody is a human IgM antibody. In some embodiments, the IgM antibody is prepared from serum, blood, mucus, or lymph of a patient. In some embodiments, in a diagnostic assay, the sample of IgM antibody treated with the protease is analyzed for cleaved products, for example by SDS polyacrylamide gel electrophoresis (PAGE), mass spectrometry, or immunoassay.

[0176] In some embodiments, the proteases are used as therapeutics for treating a disease or condition associated with abnormal IgM activity. In some embodiments, a method for treating a disease or condition associated with abnormal IgM antibody activity comprises administering to a subject in need thereof an effective amount of a protease described herein. In some embodiments, an effective amount of the protease is administered to lower levels of IgM in serum, blood, mucus, and / or lymph.

[0177] In some embodiments, the disease or condition associated with abnormal IgM antibody activity is IgM nephropathy or Waldenstrom’s disease.

[0178] In some embodiments, the disease or condition associated with abnormal IgM antibody activity is autoimmune disease characterized by presence of autoreactive IgM. In some embodiments, the autoreactive IgM is present in the serum or blood of the subject. In some embodiments, the autoimmune disease is rheumatoid arthritis, bullous pemphigoid, or autoimmune neuropathy.

[0179] In some embodiments, the protease is used for reducing or preventing IgM mediated response to gene therapy. In some embodiments, a method of reducing or preventing IgM-mediated immune response to gene therapy comprises administering adjunctively to a subject being treated with a gene therapy vector an effective amount of a protease described herein.

[0180] In some embodiments, the protease is administered prior to, concurrently with, or subsequent to administration of the gene therapy vector.

[0181] In some embodiments, the protease is administered adjunctively with a viral-based gene therapy vector. In some embodiments, the viral-based gene therapy vector, includes, among others, those based on adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus.

[0182] In some embodiments, the protease described herein is used for the preparation of a medicament for treating a disease or condition associated with abnormal IgM activity or for reducing or preventing IgM-mediated immune response to gene therapy.

[0183] In some embodiments, the protease described herein is used for the treatment of a disease or condition associated with abnormal IgM activity or for reducing or preventing IgM-mediated immune response to gene therapy.

[0184] In some embodiments, for the use in the foregoing embodiments, the disease or condition associated with abnormal IgM activity is selected from IgM nephropathy or Waldenstrom’s disease, rheumatoid arthritis, bullous pemphigoid, and autoimmune neuropathy.

[0185] In some embodiments, for use in reducing or preventing IgM-mediated immune response to gene therapy, the gene therapy is virus-based gene therapy vectors, such as adenovirus, adeno- associated virus (AAV), retrovirus, and lentivirus based vectors.EXAMPLES

[0186] The following Examples, including experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the present invention.

[0187] In the experimental disclosure below, the following abbreviations apply where applicable: ppm (parts per million); M (molar); mM (millimolar), uM and pM (micromolar); nM (nanomolar); mol (moles); gm and g (gram); mg (milligrams); ug and pg (micrograms); L and 1 (liter); ml and m (milliliter); pl and pL (microliter); cm (centimeters); mm (millimeters); um and pm (micrometers); sec. (seconds); min(s) (minute(s)); h(s) and hr(s) (hour(s)); U (units); MW (molecular weight); D (daltons); rpm (rotations per minute); °C (degrees Centigrade); DNA (deoxyribonucleic acid); and RNA (ribonucleic acid).Example 1Gene Acquisition and Construction of Expression Vectors

[0188] Bacterial immunoglobulin degrading enzyme genes were obtained from public databases and truncated to the ranges specified in Table 1.1. These truncated amino acid sequences were used to perform multiple sequence alignment and the percent sequence identity matrix is shown in Table 1.2. The genes were codon optimized for expression in E. coli, synthesized and cloned into the E. coli expression vector pCKl 10900 vector system (See e.g., US Pat. Appln. Publn. 2006 / 0195947, which is hereby incorporated by reference herein) with or without a C-terminal 6xHis tag as specified in Table 1.1. Each plasmid construct was transformed into an E. coli strain derived from W3110. A negative control expressing a non-protease protein was also included.Example 2High-throughput Growth and Lysis of Escherichia coli

[0189] Transformed E. coli cells were selected by plating onto LB agar plates containing 1% glucose with 30 pg / mL chloramphenicol. After overnight incubation at 37 °C, colonies were individually picked into 96-well shallow flat bottom plates (NUNC™, Thermo-Scientific) filled with 180 pl / well LB media supplemented with 1% glucose and 30 pg / mL chloramphenicol. The cultures were grown overnight for 18-20 h in a shaker (200 rpm, 30 °C, and 85% relative humidity; Kuhner).

[0190] Overnight growth samples (20 pL) were transferred into Costar 96-well deep plates filled with 380 pL of Terrific Broth supplemented with 30 pg / mL chloramphenicol. The plates were incubated for approximately 2 h in a shaker (250 rpm, 30 °C, and 85% relative humidity; Kuhner). The cells were then induced with 40 pL of 10 mM IPTG in sterile water and incubated overnight for 20-24 h in a shaker (250 rpm, 30 °C, and 85% relative humidity; Kuhner). The cells were pelleted (3220 x g for 15 min, 4 °C), the supernatants were discarded, and the cells were frozen at -80 °C.

[0191] Lysis buffer (400 pL: IX PBS, pH 7.2, 1 mg / ml lysozyme and 0.5 mg / ml polymyxin B sulfate) was added to the cell pellets and the mixture was agitated for 1.5 h at room temperature, then centrifuged (3220 x g for 15 min, 4 °C). The supernatant containing immunoglobulin degrading enzymes, were subsequently used in various assays described herein.Example 3Analysis of Clarified E. coli Lysate for Activity on Human IgM

[0192] IgM degradation activity was assessed by following the disappearance of the human IgM heavy (p) chain by Western blot or Coomassie blue-stained SDS polyacrylamide gel electrophoresis. Reactions were started by mixing 5 pL of IgM from human serum (1 mg / mL; Cat. No. 18260, Sigma, St. Louis, MO) with 45 pL of lysate. Reactions were incubated at 37 °C for 20 h and stopped by diluting 8x into denaturing SDS sample buffer containing iodoacetamide (final concentration 20 mM). Samples were heated for 5 minutes at 95 °C and 10 pL was analyzed by SDS polyacrylamide gel electrophoresis (4-12% Invitrogen™ Nupage™ (Thermo Fisher, Waltham, MA) Bis-Tris Midi gel; 200V for 35 minutes). Proteins were transferred onto PVDF membrane. Membranes were processed following manufacturer protocol (SuperSignal™ West HisProbe Kit, Thermo Scientific™ 15168), except that anti-human IgM (p-chain specific)-peroxidase antibody (A6907 Sigma, St. Louis, MO) was used.

[0193] For Coomassie blue-stained analysis, a similar protocol was used except that 5 pL of lysate was added to 40 pL of IX PBS and then 5 pL of IgM, incubated for 1 h, diluted 2x (into final concentrations of 20 mM iodoacetamide, lx denaturing SDS sample buffer), and Coomassie Blue stained instead of transferring to a membrane.

[0194] FIG. 1 shows a representative Western blot, where the IgM heavy chain was fully cleaved by lysate expressing SEQ ID NO: 18 / 20, 46 / 48, 16 / 24, and 36 / 40. Lysate expressing SEQ ID NO: 26 / 28, 30 / 32, 42 / 44, and 50 / 52 showed low levels of activity. Ladder sizes (kDa) are depicted. SEQ ID NO: 42 has been previously shown to be inactive on human IgM (Seele et al., J Bact., 2013, 195(5):930- 40), however the conditions tested here differ in that the protein was expressed in E. coli lysate, reactions were incubated longer, purified IgM was used (potentially higher IgM concentration), and analysis was under reducing conditions.

[0195] FIG. 2 shows a Coomassie Blue-stained SDS polyacrylamide gel, where lower amounts of lysate (5 pL, rather than 45 pL) were incubated for a shorter period (1 h rather than 20 h) than in FIG. 1. Under these conditions, intact IgM heavy chain was detectable by Coomassie Blue stain over the background bands from E. coli lysate. Bands from the overexpressed immunoglobulin degrading enzymes (35-48 kDa) were additionally present in this analysis (FIG. 2). Enzymes that fully cleavedthe IgM heavy chain in FIG. 1 also showed significant cleavage of the heavy chain under the conditions in FIG. 2. Enzymes that showed low levels of degradation in FIG. 1 did not significantly reduce intact IgM heavy chain under the conditions in FIG. 2, including SEQ ID NO: 42.

[0196] FIG. 3 shows a representative Western blot analyzing IgM heavy chain degradation by previously reported IgG-degrading enzymes. Consistent with prior reports, these enzymes did not cleave human IgM heavy chain. This image was compiled from a single western blot that contained a larger set of samples, including samples in FIGS. 1 and 2 which did show IgM heavy chain degradation.

[0197] All enzymes in FIGS. 1-3 were tested for protease activity on human IgG (data not shown) and enzymes that cleaved IgM (FIGS. 1 and 2) did not cleave IgG.Example 4Shake Flask Expression, Purification, and Characterization of Immunoglobulin Degrading Enzymes

[0198] A single colony of E. coli containing a plasmid encoding a polypeptide of interest was inoculated into 5 mL Luria Bertani media with 1% glucose and 30 pg / mL chloramphenicol. Cells were grown overnight (at least 16 h) in an incubator at 30 °C with shaking at 250 rpm. The culture was diluted into 250 mL Terrific Broth (12 g / L bacto-tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 65 mM potassium phosphate, pH 7, 1 mM MgSOft with 30 pg / mL chloramphenicol in a 1 liter flask and allowed to grow at 30 °C. Expression of the genes were induced by addition of IPTG to a final concentration of 1 mM when the OD600 of the culture was 0.6 to 0.8. Incubation was then continued overnight (18-20 h). Cells were harvested by centrifugation (5500 rpm, 10 min, 4 °C) and the supernatant discarded. The cells were resuspended in 30 mL of lysis buffer #2 (20 mM sodium phosphate, 300 mM NaCl, 25 mM imidazole, pH 7.4) and passed through a MICROFLUDIZER® high pressure homogenizer (Microfluidics, Westwood, MA) with standard E. coli lysis settings and maintained at 4 °C.

[0199] The lysate was pelleted (10,000 rpm, 60 min, 4 °C) and passed through His GraviTrap™ column (Cytiva, Marlborough, MA) that had been previously equilibrated in lysis buffer #2. Columns were washed with 15 mL lysis buffer #2 and bound proteins eluted with 20 mM sodium phosphate, 300 mM NaCl, 300 mM imidazole, pH 7.4. Elution fractions were exchanged into IX PBS, pH 7.2 using PD 10 Desalting column (Cytiva, Marlborough, MA), following manufacturer protocol. SEQ ID NO: 20 and 32 did not yield significant A280 after His purification and were concentrated using 10k MWCO spin unit.

[0200] The purified variants were assayed for activity on human IgM in a similar manner as described in Example 3 for Coomassie blue-stained analysis, except instead of using cell lysates, purified enzymes were used to final concentration in reaction of 5, 50, or 500 ug / mL.

[0201] FIG. 4 shows a Coomassie Blue-stained SDS polyacrylamide gel, where 500 ug / mL purified proteins were incubated without or with 0.1 mg / mL human IgM for 1 hr. In the lanes without IgM, the bands from the purified proteins are visible. SEQ ID NO: 20 and 32 were not overexpressed and / or highly soluble, and concentrations are estimates due to presence of significant host cell proteins. The remaining proteins were visibly purified from host cell proteins, with overexpressed bands visible. In the lanes with IgM, additional bands from IgM are present and the disappearance of the intact heavy chain (p) can be followed. Most purified proteins led to disappearance of the intact heavy chain, with the exception of SEQ ID NO: 28 and 44, consistent with the low level of IgM cleavage by these two polypeptides in lysate (see FIG. 1).

[0202] FIG. 5 shows a Coomassie Blue-stained SDS polyacrylamide gel, where lower concentrations of purified proteins (5 and 50 ug / mL) were incubated with 0.1 mg / mL human IgM for 1 hr. In this figure, the intact heavy chain band is more prominent over background bands from the purified proteins. Apparent complete cleavage of the heavy chain occurs with 5 ug / mL of SEQ ID NO: 48 and 24, and 50 ug / mL of SEQ ID NO: 40. SEQ ID NO: 20, 32, and 52 show moderate levels of IgM heavy chain cleavage.Example 5Determining Protease Cleavage Site on IgM Antibodies

[0203] For determining the cleavage site, IgM antibody (pooled human IgM) was cleaved with protease of SEQ ID NO: 24, 40, 48, or 52, and the cleavage products separated by SDS-PAGE. The separated proteins were transferred to PVDF membrane (0.22 pm) and stained with Coomassie Blue. The protein bands of interest were cut out from the membrane, placed in a special glass block and sealed. The N-terminal sequence was determined by automated Edman degradation (PPSQ-31 A automatic protein sequencer, Shimadzu, Japan), where analysis of phenylthiohydantoin (PTH) derivatives were determined by RP-HPLC. Products of ~40 kD for SEQ ID NO: 24, 40, and 48, and products of ~35 kD for SEQ ID NO: 24, 40, 48, and 52 resulted in the same N-terminal sequence of AIRVFAI. This sequence is within the IgM constant region CH3, indicating that the cleavage site is as follows:MCVPDQDT( AIRVFAI (SEQ ID NO: 55)

[0204] While the invention has been described with reference to the specific embodiments, various changes can be made and equivalents can be substituted to adapt to a particular situation, material,composition of mater, process, process step or steps, thereby achieving benefits of the invention without departing from the scope of what is claimed.

[0205] For all purposes, each and every publication and patent document cited in this disclosure is incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an indication that any such document is pertinent prior art, nor does it constitute an admission as to its contents or date.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A protease that cleaves IgM antibody, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%.94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 16, 18, 26, 30, 42, 46, 50, or 36, or to a reference sequence corresponding to SEQ ID NO: 2, 4, 5, 8, 9, 10, 11, or 12, wherein the protease preferentially cleaves IgM compared to IgG.

2. The protease of claim 1, comprising an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ IDNO: 16.

3. The protease of claim 1, comprising an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ IDNO: 18.

4. The protease of claim 1, comprising an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ IDNO: 26.

5. The protease of claim 1, comprising an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ IDNO: 30.

6. The protease of claim 1, comprising an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ IDNO: 42.

7. The protease of claim 1, comprising an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ IDNO: 46.

8. The protease of claim 1, comprising an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ IDNO: 50.

9. The protease of claim 1, comprising an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ IDNO: 36.

10. The protease of claim 1, comprising an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ IDNO: 2.

11. The protease of claim 1, comprising an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ IDNO: 4.

12. The protease of claim 1, comprising an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ IDNO: 5.

13. The protease of claim 1, comprising an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ IDNO: 8.

14. The protease of claim 1, comprising an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ IDNO: 9.

15. The protease of claim 1, comprising an amino acid sequence having at least 70%,75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ IDNO: 10.

16. The protease of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 11.

17. The protease of claim 1, comprising an amino acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 12.

18. The protease of claim 1, wherein the amino acid sequence of the protease comprises SEQ ID NO: 16, 18, 26, 30, 42, 46, 50, or 36.

19. The protease of claim 1, wherein the amino acid sequence of the protease comprises SEQ ID NO: 2, 4, 5, 8, 9, 10, 11, or 12.

20. The protease of any one of claims 1-19, further comprising a fusion polypeptide.

21. The protease of claim 20, wherein the amino acid sequence comprises SEQ ID NO: 20, 28, 32, 44, 48, 52, 24, or 40.

22. The protease of any one of claims 1-21, wherein the protease cleaves IgM at CH2- CH3 linker region at the amino terminal region of CH3.

23. The protease of claim 22, wherein the protease is characterized by a cleavage site of MCVPDQDTJ.AIRVFAI (SEQ ID NO: 55).

24. The protease of any one of claims 1-23, wherein the protease is purified.

25. A recombinant polynucleotide comprising a polynucleotide sequence encoding a protease of any one of claims 1-23.

26. The recombinant polynucleotide of claim 25, wherein the polynucleotide sequence comprises at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference polynucleotide sequence corresponding to SEQ ID NO: 17, 25, 29, 41, 45, 49, 15, or 35.

27. The recombinant polynucleotide of claim 25 or 26, wherein the polynucleotidesequence is codon optimized for expression of the encoded protease polypeptide.

28. The recombinant polynucleotide of claim 25, wherein the polynucleotide sequence comprises SEQ ID NO: 17, 25, 29, 41, 45, 49, 15, or 35.

29. An expression vector comprising a recombinant polynucleotide of any one of claims 25-28.

30. The expression vector of claim 29, further comprising a control sequence operably linked to the recombinant polynucleotide.

31. The expression vector of claim 29 or 30, wherein control sequence comprises a promoter.

32. A host cell comprising an expression vector of any one of claims 29-31.

33. The host cell of claim 32, comprising a bacterial cell, fungal cell, insect cell, or mammalian cell.

34. The host cell of claim 32, comprising a bacterial cell.

35. A method of producing a protease of any one of claims 1-23, comprising culturing the host cell of any one of claims 32-34 under suitable culture conditions such that the encoded protease is produced.

36. The method of claim 35, further comprising isolating the protease from the media and / or host cell.

37. The method of claim 35 or 36, further comprising purifying the protease.

38. A composition comprising a protease of any one of claims 1-24.

39. The composition of claim 38, further comprising an IgM antibody.

40. The composition of claim 39, wherein the IgM antibody is a human IgM antibody.

41. The composition of any one of claims 38-40, further comprising a buffer and / or preservative.

42. The composition of any one of claims 38-41, wherein the polypeptide is provided in solution, as a lyophilizate, or immobilized on a substrate.

43. A pharmaceutical composition comprising the protease of any one of claims 1-24, and a pharmaceutically acceptable carrier and / or excipient.

44. The pharmaceutical composition of claim 43, wherein the composition is suitable for parenteral injection or infusion to a human.

45. An in vitro method of cleaving IgM antibody, comprising contacting an IgM antibody with a protease of any one of claims 1-24 under suitable conditions such that the IgM is cleaved.

46. The method of claim 45, wherein the IgM is human IgM.

47. The method of claim 45 or 46, wherein the IgM is cleaved to produce F(ab)2 and Fc fragment of IgM.

48. A method for treating a disease or condition associated with abnormal IgM antibody activity, comprising administering to a subject in need thereof an effective amount of the protease of any one of claims 1-24.

49. The method of claim 48, wherein the disease or condition associated with abnormal IgM antibody activity is IgM nephropathy or Waldenstrom’s disease.

50. The method of claim 48, wherein disease or condition associated with abnormal IgM antibody activity is an autoimmune disease or condition characterized by presence of autoreactive IgM.

51. The method of claim 50, wherein the autoimmune disease is rheumatoid arthritis, bullous pemphigoid, or autoimmune neuropathy.

52. A method of reducing or preventing IgM-mediated immune response to gene therapy, comprising administering adjunctively to a subject being treated with a gene therapy vector an effective amount of a protease of any one of claims 1-24.

53. The method of claim 52, wherein the protease is administered prior to, concurrently with, or subsequent to administration of the gene therapy vector.

54. The method of claim 52 or 53, wherein the gene therapy vector is viral based gene therapy vector.

55. The method of claim 54, wherein the viral-based gene therapy is adeno-associatedvirus (AAV), adenovirus, retrovirus, and lentivirus gene therapy vectors.

56. Use of the protease of any one of claims 1-24 for the treatment of a disease or condition associated with abnormal IgM activity or for reducing or preventing IgM -mediated immune response to gene therapy.

57. Use of the protease of any one of claims 1-24 in the preparation of a medicament for treating a disease or condition associated with abnormal IgM activity or for reducing or preventing IgM-mediated immune response to gene therapy.

58. The use of claim 56 or 57, wherein the disease or condition is selected from IgM nephropathy, Waldenstrom’s disease, rheumatoid arthritis, bullous pemphigoid, and autoimmune neuropathy.

59. The use of claim 56 or 57, wherein the gene therapy is with viral vector based gene therapy.

60. The use of claim 59, wherein the viral -based gene therapy is adeno-associated virus (AAV), adenovirus, retrovirus, or lentivirus gene therapy vectors.

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