Iga protease polypeptide agents

Abstract

The present disclosure provides certain IgA protease polypeptide agents, and uses therefore, including, in some embodiments cleaving IgAl present in deposits in vivo.

Description

IGA PROTEASE POLYPEPTIDE AGENTSCross-Reference to Related Applications

[0001] The present application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 424,585, filed on November 11, 2022, the entirety of which is incorporated herein by reference.Background

[0002] Deposition of immunoglobulin Al (IgAl) in human tissues and organs is a characteristic of many human diseases including, but not limited to, IgA nephropathy (IgAN), dermatitis herpetiformis, and Henoch-Schoenlein purpura. IgAN is the most common form of glomerulonephritis throughout the world. IgAN is characterized by the presence of potentially injurious mesangial deposits containing complexes of IgAl in all kidney glomeruli. Clinical findings of IgAN include proteinuria, hematuria, and hypertension that may evolve into end-stage renal disease. Kidney failure is a lethal problem unless addressed with kidney dialysis, and ultimately with transplantation of a healthy donor kidney into the patient.Summary

[0003] The present disclosure provides certain IgA protease polypeptide agents, and uses therefore, including, in some embodiments use to cleave IgAl present in deposits, e.g, in vivo.

[0004] Among other things, the present disclosure surprisingly demonstrates that relatively small polypeptides (e.g.. truncation variant(s) of a reference IgA protease polypeptide), referred to herein as '‘subdomain” agents (or ‘'SD-IgAPP” agents) which include one or more sequence elements (e.g., “subdomains”) found in a reference IgA protease (e.g., a wild type IgA protease and / or another reference IgA protease polypeptide as described herein) but lack one or more other sequence elements found in such reference IgA protease polypeptide, can retain IgA proteolytic activity (e.g., that is reasonably comparable to such activity observed with the relevant reference IgA protease). In some embodiments, a provided SD-IgAPP agent may show IgAl cleavage activity (e.g., at least comparable to that of a relevant reference IgA protease).

[0005] In some embodiments, a provided SD-IgAPP agent may show IgA2 cleavage activity (e.g.. at least comparable to that of a relevant reference IgA protease). In some embodiments, a provided SD-IgAPP agent may show reduced IgA2 cleavage (e.g., relative to a relevant reference IgA protease).

[0006] The present disclosure provides various technologies related to such SD- IgAPP agents including, for example, the agents themselves, nucleic acids that encode them (and / or that are complementary thereto), conjugates associated with or fusion proteins that include these agents, cells that include and / or express either of the foregoing, compositions (e.g., pharmaceutical compositions) that include and / or deliver any of the foregoing, and / or methods of making, using, and / or characterizing any of the foregoing.

[0007] In some embodiments, a SD-IgAPP agent as described herein may be or comprise a polypeptide that is, or that shares significant sequence identity with, a fragment of an M64 IgA protease, e.g.. a Clostridium ramosum IgA protease (e.g., a C. ramosum IgA protease having an amino acid sequence as set forth in SEQ ID NO: 1 and / or SEQ ID NO: 2, see Figure 2).

[0008] Advantages of certain embodiments of provided technologies may include, among other things, reduced immunogenicity, ease of manufacture, increased bioavailability, and / or increased half-life of a SD-IgAPP agent (e.g., relative to an appropriate reference IgA protease such as for example, a C. ramosum IgA protease, e.g., having an amino acid sequence as set forth in SEQ ID NO: 1 and / or SEQ ID NO: 2, see Figure 2) in some embodiments while maintaining at least reasonably comparable IgA cleavage (e.g., IgAl cleavage) activity relative to such reference IgA protease. In some embodiments, a provided SD-IgAPP agent may show increased IgA cleavage (e.g., increased IgAl cleavage activity ) relative to such reference. In some particular embodiments, such comparable or increased IgA cleavage is or comprises cleavage of IgA deposit(s) (e.g., IgAl deposit(s)) in vivo).

[0009] In some embodiments, one or more of immunogenicity , bioavailability , half- life and / or IgAl and / or IgA2 cleavage is assessed in vivo (e.g., when a SD-IgAPP agent is administered or otherwise delivered to an organism such as a mammal and. in particular, a human being).

[0010] In some embodiments, a proteolytically active (i.e., with respect to IgA, e.g, to IgAl) SD-IgAPP agent according to the present disclosure is provided. In some embodiments, a proteolytically active (i.e.. with respect to IgA, e.g.. to IgA included in an aggregate or deposit, e.g., an in vivo deposit) SD-IgAPP agent according to the present disclosure is provided. In some embodiments, such a proteolytically active SD-IgAPP agent is useful in treatment of one or more IgA deposition diseases such as. for example (but not limited to), IgA nephropathy.

[0011] In some embodiments, an IgA protease polypeptide agent has an amino acid sequence that is or comprises an element having the general formula: Y1- N-M1-M2-M3-C-Y2wherein each of Y1, N, M1, M2, M3, C, and Y2comprises or consists of a consecutive sequence of amino acids, wherein M1substantially comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 7, wherein M2comprises or consists of a HEX1X2H motif, wherein X1and X2are amino acids, wherein M3substantially comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 9, wherein the polypeptide’s full amino acid sequence does not comprise or consist of the amino acid sequence of SEQ ID NO: 1 or 2, and wherein Y1, N, C, and Y2are optional.

[0012] In some aspects, a nucleic acid molecule encoding a provided SD-IgAPP agent is provided. In some embodiments, such nucleic acid molecule may be included in a vector (e.g., a DNA plasmid, a viral vector, etc.). In some embodiments, such nucleic acid may be associated with one or more expression control sequences (e.g.. a promoter, an enhancer and / or binding site for a transcriptional regulator such as a transcriptional activator or transcriptional repressor, a transcription termination signal, one or more splice acceptor or donor sites, a translational start site, etc. ). In some embodiments, an encoding nucleic acid may be included in a vector (e.g., a plasmid or portion, such as a fragment, e.g., a cleavage fragment or an amplicon, thereof) amenable to in vitro transcription. In some embodiments, an encoding nucleic acid may be included in a vector amenable to expression (e.g., by transcription, translation, or both) in vitro (e.g., in vitro cell culture). In some such embodiments, an encoding nucleic acid may be included in a vector amenable to expression (e.g., by transcription, translation, or both) in vivo (e.g., in a mammalian cell or organism and, in some embodiments specifically in a human). For example, in some embodiments, anencoding nucleic acid may be included in a viral construct, or in an RNA construct (e.g., as may have been produced by in vitro transcription) amenable to inclusion in a pharmaceutical composition for administration to and expression in a mammalian subject such as a human subject.

[0013] In some aspects, a pharmaceutical composition that comprises or delivers (e.g.. upon administration to a subject) a SD-IgAPP agent (e.g. a proteolytically active SD- IgAPP agent) as described herein is provided; in some embodiments such pharmaceutical composition is for the treatment of a disease, disorder or condition associated with IgA deposition.

[0014] In some aspects, a method for treatment of a disease characterized by IgA deposits using an IgA proteolytic active SD-IgAPP agent according to the present disclosure is provided. In some embodiments, use of SD-IgAPP agents to treat IgA nephropathy, dermatitis herpetiformis (DH). and / or Henoch-Schoenlein purpura (HS) is disclosed.

[0015] In some aspect of the disclosure, methods comprising a step of administering to an individual having deposits of IgA, such as e.g., IgAl deposition, composition that comprises or delivers a SD-IgAPP agent of the disclosure are provided.

[0016] In some embodiments, a SD-IgAPP agent is characterized in that it cleaves both IgAl and IgA2. In some embodiments, a SD-IgAPP agent is characterized in that it specifically cleaves IgAl. In some embodiments a SD-IgAPP agent is characterized in that it preferentially cleaves IgAl relative to IgA2; in some such embodiments, such SD-IgAPP is characterized in that no detectable IgA2 cleavage is detected under conditions where robust (e.g., substantially comparable to that achieved by an appropriate reference IgAl protease, such as a full-length M64 IgAl protease) IgAl cleavage is observed.Brief Description of the Drawings

[0017] Figure 1 shows hinge peptide of IgAl / 2 and the IgAl protease family members that cleave it. ‘Lollipops’ indicate positions of O-glycans.

[0018] Figure 2 depicts a representation of the structure of a full length, wild type C. ramosum IgA protease. As can be seen, each of three different domains is depicted: an N-terminal domain (corresponding to amino acid residues 31-313 of SEQ ID NO: 1); a middle domain (corresponding to amino acid residues 314-807 of SEQ ID NO: 1); and a C-terminal domain (corresponding to amino acid resides 808-1234 of SEQ ID NO: 1). Residue numbering is that of the Cr-IgA protease set forth in SEQ ID NO: 1; residues 1-30 of SEQ ID NO: 1 represent a pro-peptide that is cleaved from the mature protease. Structural and functional studies described herein indicate that the middle domain is the only domain necessary for IgA protease activity.

[0019] Figures 3A-D show Coomassie stained SDS-gels showing expression of exemplary A) Full-length, B) N-terminal domain, C) middle domain, and D) C-terminal domain constructs of Cr-IgAP.

[0020] Figures 4A-C show cry stal structure of the C. ramosum IgAP Δ15 middle domain (Cr-Δ15 MD) and first C-terminal domain (CTD1). A) full Cr-Δ15 MD-CTD1, black shows Cr-Δ15 MD and grey shows CTD1; B) 1.60 A structure of active site; and C) loop containing D520 and E521 bridges the active site to a nearby pocket where the second zinc- binding site is located.

[0021] Figure 5 depicts a Coomassie stained SDS-gel showing IgAl digestion by a Cr-MD IgAPP and a reference Haemophilus Influenzae (HIN) IgAP. Lane A: IgAl alone; Lane B: HIN-IgAP alone; Lane C: HIN-IgAP + IgAl; Lane D: Cr-IgAP NTD alone; Lane E; Cr-IgAP NTD + IgAl (1 hour at 37°C); Lane F: Cr-IgAP NTD + IgAl (O / N at 37°C); Lane G: Cr-IgAP MD alone; Lane H: Cr-IgAP MD + IgAl (1 hour at 37°C).

[0022] Figures 6A-H show sequence alignment. A middle domain sequence (Cr- MD), as set forth in SEQ ID NO: 4, was used as a query .

[0023] Figures 7A-C show a mass spectrometry analysis of synthetically generated unlabeled hinge peptides in the presence of a full-length (Cr-FL) IgA protease as set forth in SEQ ID NO: 2 or a middle domain (Cr-MD) of IgA protease as set forth in SEQ ID NO: 4. Alpha-Cyano-4-hydroxy cinnamic acid (HCCA) was used as a matrix for the analysis. A)-C) represent three different MR ranges of the analysis. HCCA matrix alone (sample blank) is shown in black (lower spectra). Peptide (isolated IgA peptide) alone, sample digest with Cr- MD, and Sample digest with Cr-FL spectra are identical, showing no peptide cleavage uponenzyme treatment. Theoretical masses of peptides are: Intact - 798.95 g / mol, N-terminal fragment - 414.5 g / mol, C-terminal fragment -402.5 g / mol.

[0024] Figures 8A-D depict SDS-gels showing A) IgAl digestion by Cr-MD or Cr- FL; B) Cr-NTD-MD; C) Cr-Δ15 MD or Cr-MD; and D) Cr-MD or a reference Haemophilus influenzae IgAP.

[0025] Figures 9A-B show SDS-gels showing IgA2 digestion by A) Cr-MD or Cr- FL; or B) Cr-NTD+MD.

[0026] Figures 10A-C show SDS-gels showing IgGl digestion by A) Cr-MD or Cr- FL; B) IgG2 digestion by Cr-MD or Cr-FL; or C) Cr-NTD+MD.

[0027] Figures 11A-C show a gel-based kinetic assay for IgAPPs. A) gel showing IgAl or cleaved IgAl fragments; B) percentage of IgAl heavy chain cleaved over time; and C) Michaelis-Menten curve for Cr-IgAP peptides. C. ramosum N-terminal domain in conjunction with the middle domain (NM; dotted line), middle domain (MD; dashed line), and N-lerminally truncated middle domain (Δ15 MD; solid line).

[0028] Figures 12A-B show Western blots of A) IgAl cleaved by Cr-Δ15 MD IgAP (40nM) in HEPES Buffer and B) IgAl cleaved by Cr- Δ15 MD IgAP (40nM) in pooled human serum with myeloma IgA addition.

[0029] Figures 13A-C show Western Blots A) visualizing endogenous levels of IgAl in pooled normal and IgAN patient sera, B) IgAl digestion by Cr-Δ15 MD IgAP (top three gels), Cr-full length IgAP (middle three gels), and H. influenzae IgAP (bottom three gels) (all 40nM). and C) IgAl digestion by Cr-Δ15 MD IgAP (400nM).Definitions

[0030] Agent. In general, the term “agent”, as used herein, is used to refer to an entity (e.g., for example, a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc, or complex, combination, mixture or system [e.g., cell, tissue, organism] thereof), or phenomenon (e.g.. heat, electric current or field, magnetic force or field, etc). In appropriate circumstances, as will be clear from context to those skilled in the art, the termmay be utilized to refer to an entity that is or comprises a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as context will make clear, the term may be used to refer to a natural product in that it is found in and / or is obtained from nature (e.g., a naturally occurring IgA protease such as a naturally-occurring M64 IgA protease such as a C. ramosum IgA protease). In some embodiments, an agent may be utilized in isolated or pure form; in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided and / or utilized as collections or libraries, for example that may be screened to identify or characterize active agents within them.

[0031] Amino acid: As used herein, the term "amino acid” refers to a compound, substance, or moiety that can be or is incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally- occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. As used herein, the term “standard amino acid” refers to any of the twenty L-amino acids commonly found in naturally occurring peptides. “Nonstandard amino acid” refers to any amino acid, other than the standard amino acids, regardless of whether it is or can be found in a natural source. In some embodiments, an amino acid, including a carboxy - and / or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared to the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and / or the hydroxyl group) as compared to the general structure. In some embodiments, such modification may, for example, alter the stability or the circulating half- life of a polypeptide containing the modified amino acid as compared to one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared to one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term “amino acid” may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide, e.g., an amino acid residue within a polypeptide.

[0032] Antibody: As used herein, the term "antibody" refers to an immunoglobulin molecule, or fragment thereof, that binds specifically to an epitope (e.g., of an antigen). Naturally-occurring human antibodies ty pically include two identical heavy chains and two identical light chains, each of which includes a variable domain and a constant domain. The constant domain defines the antibody isotype (IgG, IgA, IgM, IgE, etc). Those skilled in the art are aware that different animal species utilize different antibody structures in nature. For example, camelid antibodies are single chain antibodies. Those skilled in the art will further be aware that antibody variable domains are typically characterized by framework region (FR) sequences and complement-determining-region (CDR) sequences. Each variable domain typically includes three CDRs - CDR1, CDR2, and CDR3, which together contribute to specificity and / or affinity of epitope binding. In some embodiments, antibodies may be produced in or by organisms. In some embodiments, antibodies may be produced in or by cells in vitro (e.g., by hybridomas and / or by engineered cells). In some embodiments, antibody fragment(s) (e.g., as may be produced by cleavage or recombinantly) may be generated and / or utilized in accordance with the present disclosure. Those skilled in the art are aware that a variety of technologies have been developed to incorporate binding features (e.g., one or more CDRs and / or FR sequences, and in particular sets of 3 CDRs, optionally together with FR sequences) into new contexts. In some embodiments, CDR sequences may be maintained and other elements changed - e.g., as is done in humanization. Alternatively or additionally, in some embodiments, variable regions may be associated with alternative constant regions (e.g., with constant regions from a different organism and / or that include one or more particular sequence features or elements desired, for example, to impart a particular attribute to the antibody agent). Those skilled in the art are further aware of a variety of technologies commonly utilized to associate binding features of two or more different antibodies with one another - e.g., in a single multispecific (most commonly bi-specific) agent.

[0033] Associated: Two events or entities are 'associated" with one another, as that term is used herein, if the presence, level, degree, type and / or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and / or form correlates with incidence of, susceptibility to, severity of. stage of, etc. the disease, disorder, or condition (e.g., across a relevantpopulation). In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and / or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0034] Combination therapy: As used herein, the term “combination therapy” refers to a clinical intervention in which a subject is exposed to two or more therapeutic regimens (e.g. two or more therapeutic agents or modalities). In some embodiments, the two or more therapeutic regimens may be administered simultaneously. In some embodiments, the two or more therapeutic regimens may be administered sequentially (e.g., a first regimen administered prior to administration of any dose or episode of a second regimen). In some embodiments, two or more therapeutic regimens are administered in overlapping dosing regimens. In some embodiments, administration of a combination therapy may involve administration of one or more therapeutic agents or modalities to a subject receiving the other agent(s) or modality. In some embodiments, combination therapy does not necessarily require that individual agents be administered together in a single composition (or even necessarily at the same time). In some embodiments, two or more therapeutic agents or modalities of a combination therapy are administered to a subject separately, e.g., in separate compositions, via separate administration routes (e.g., one agent orally and another agent intravenously), and / or at different time points. In some embodiments, two or more therapeutic agents may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity), via the same administration route, and / or at the same time.

[0035] Comparable, reasonable comparable and substantially comparable. As used herein, the term “comparable”, “reasonable comparable” and “substantially comparable refers” to two or more agents, entities, situations, sets of conditions, that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantiallyidentical features and one or a small number of varied features. Those of ordinary' skill in the art will understand, in context, what degree of identity’ is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, to be considered comparable. For example, those of ordinary' skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by' or indicative of the variation in those features that are varied. For example, in some embodiments, SD-IgAPP retain IgAl proteolytic activity that is reasonably comparable to such activity observed with a relevant reference IgA protease as exemplified, for example, in Example 3 and shown in Figure 5, demonstrating that SD-IgAPP cleaves the hinge region of IgAl comparably to a relevant reference IgA protease. In some embodiments, a provided SD-IgAPP agent cleaves more IgA (e.g., IgAl) than a reference IgA protease (e.g., full length IgA protease). In some embodiments, a SD-IgAPP agent cleaves at least 80% IgA (e.g., IgAl). at least 85%. at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% IgA (e.g., IgA), such as 100% IgA (e.g., IgA) comparable to a reference IgA protease (e.g., full length IgA protease).

[0036] Complexed: The term "complex’" as used herein typically refers to a physical association between or among two or more distinct chemical entities. Typically, such association in a “complex” is non-covalent.

[0037] Corresponding to. As used herein in the context of polypeptides, nucleic acids, and chemical compounds, the term “corresponding to”, designates the position / identity of a structural element, e.g., of an amino acid residue, a nucleotide residue, or a chemical moiety, in a compound or composition through comparison with an appropriate reference compound or composition. For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as “corresponding to” a residue in an appropriate reference polymer. For example, those of ordinary' skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190thamino acid in a particular amino acid chainbut rather corresponds to the residue found at position 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify “corresponding” amino acids. Those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH / GLSEARCH, Genoogle, HMMER, HHpred / HHsearch, IDF, Infernal, KLAST, USEARCH, parasail. PSI-BLAST. PSI-Search, ScalaBLAST. Sequilab, SAM, S SEARCH, SWAPHI. SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify “corresponding” residues in polypeptides and / or nucleic acids in accordance with the present disclosure.

[0038] Dosing regimen: Those skilled in the art will appreciate that the term “dosing regimen” may be used to refer to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

[0039] Engineered: In general, the term “engineered” refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and / or when a particular residue in a polynucleotide is non-naturally occurring and / or is caused through action of the hand of man to be linked with an entity ormoiety with which it is not linked in nature. For example, in some embodiments described and / or utilized herein, an engineered polynucleotide comprises a regulatory sequence that is found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, is linked by the hand of man so that it is operatively associated with the second coding sequence. Comparably, a polypeptide may be considered to be “engineered” if encoded by or expressed from an engineered polynucleotide, and / or if produced other than natural expression in a cell. Analogously, a cell or organism is considered to be “engineered” if it has been subjected to a manipulation, so that its genetic, epigenetic, and / or phenotypic identity is altered relative to an appropriate reference cell such as an otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or comprises a genetic manipulation, so that its genetic information is altered (e.g., new genetic material not previously present has been introduced, for example by transformation, mating, somatic hybridization, transfection, transduction, or other mechanism, or previously present genetic material is altered or removed, for example bysubstitution or deletion mutation, or by mating protocols). In some embodiments, an engineered cell is one that has been manipulated so that it contains and / or expresses a particular agent of interest (e.g., a protein, a nucleic acid, and / or a particular form thereof) in an altered amount and / or according to altered timing relative to such an appropriate reference cell. As is common practice and is understood by those in the art, progeny of an engineered polynucleotide or cell are typically still referred to as “engineered” even though the actual manipulation was performed on a prior entity.

[0040] Host cell, as used herein, refers to a cell into which an exogenous nucleic acid (e.g., an engineered nucleic acid) has been introduced and / or which otherwise expresses or comprises an engineered polypeptide. Persons of skill upon reading this disclosure will understand that such terms refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. In some embodiments, a host cell may be a prokaryotic cell; in other embodiments, a host cell may be a eukaryotic cell. In some particular embodiments, a host cell is a bacterial cell (e.g., an E. coll, Bacillus spp., Slreptomyces spp., etc. cell), a mycobacterial cell, a fungal or yeast (e.g., an S cerevisiae, S. pombe, P. pastoris. P. methanolica, etc. cell), a plant cell, an insectcell (e.g., an SF-9, SF-21, Trichoplusia ni, etc. cell; in some embodiments, a baculovirus- infected cell), a non-human animal cell (e.g., a mouse or other rodent, etc., cell), a human cell, or a cell fusion such as, for example, a hybridoma or quadroma. In some embodiments, a host cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, a host cell is a eukary otic cell, for example selected the following cells: CHO (e.g., CHO KI, DXB- 1 1 CHO, Veggie-CHO), COS (e.g., COS-7) cells, CV1 cells. Daudi cells, epidermal (e.g., A431) cells, Jurkat cells, kidney (e.g.. HEK293. 293 EBN A, MSR 293. MDCK, HaK, BHK [e.g., BHK21]) cells, HeLa cells, HepG2 cells, WI38 cells, MRC 5 cells, Colo205 cells, HB 8065 cells, HL-60 cells, U937 cells, 3T3 cells, L cell cells, Cl 27 cells, SP2 / 0 cells, NS-0 cells, MMT 060562 cells, Sertoli cellal, BRL 3 A cells, HT1080 cells, myeloma cells, tumor cells. Vero cells, or other cells (e.g, cells from a tissue sample or cell lines).

[0041] Initiation: As used herein, the term "initiation" when applied to a dosing regimen can be used to refer to a first administration of a pharmaceutical agent or modality to a subject who has not previously received the pharmaceutical agent or modality, or who is initiating anew course of the pharmaceutical agent or modality. Alternatively, as will be clear from context, in some embodiments, the term "initiation" may be used to refer to administration of a particular unit dose of a pharmaceutical agent, or a particular course of treatment, during therapy of a patient.

[0042] IgA protease: As used herein, the term "IgA protease" refers to a polypeptide that cleaves IgA molecules. In some embodiments, an IgA protease cleaves IgAl; in some embodiments, an IgA protease cleaves IgA2; in some embodiments, an IgA protease cleaves both IgAl and IgA2. In some embodiments, an IgA protease shows preferential cleavage of IgAl relative to IgA2. In some embodiments, an IgA protease shows preferential cleavage of IgA2 relative to IgAl. In some embodiments, an IgA protease cleaves IgAl and IgA2 comparably. In some embodiments, an IgA protease cleaves an IgA with preferential specificity relative to an otherwise comparable IgA having a different hinge sequence. Those skilled in the art are aware of or are able to readily ascertain (e.g., by amino acid sequence and / or activity assessments) a number of IgA proteases that occur in nature. Table 2 provides exemplary amino acid sequences of certain known IgA proteases. As indicated, Mistry D, Stockley RA. IgAl protease. Int J Biochem Cell Biol. 2006;38(8): 1244-8 and D. Judy Shon, Angel Kuo, Michael J. Ferracane, Stacy A. Malaker; Classification, structural biology, and applications of mucin domain-targeting proteases. Biochem J 30 April 2021;478 (8): 1585-1603 recognize various “types” of IgA proteases, based on sequence comparisons and / or mechanistic similarities. See also figure 1.

[0043] IgA protease polypeptide: The term “IgA protease polypeptide” as used herein refers to a polypeptide that (a) shows IgA protease activity; and (b) shares at least one characteristic sequence with and / or shows an overall degree of identity with, a reference IgA protease (and / or with a fragment thereof, particularly with a fragment that is or comprises a characteristic sequence thereof); in many embodiments, an IgA protease polypeptide (a) shows IgA protease activity; and (bl) shares at least one characteristic sequence with a reference IgA protease (or fragment thereof) and (b2) shows an overall degree of sequence identity with such reference IgA protease (or fragment thereof). Table 2 presents amino acid sequence(s) of certain known IgA proteases (e.g., reference IgA proteases). Table 2 presents certain sequences that may, in some embodiments, be considered characteristic sequences of an IgA protease polypeptide relevant to the present disclosure. In some embodiments, an IgA protease polypeptide shows at least 70% sequence identity with a reference IgA protease (or fragment thereol). In some embodiments, an IgA protease polypeptide shares at least one characteristic sequence element with an IgA protease polypeptide and shows at least 60% sequence identity with such reference IgA protease (or fragment thereol). In some embodiments, an IgA protease polypeptide is a SD-IgAPP in that it is fewer than about 1000 amino acids long, and in many embodiments is fewer than about 750. or than about 700, or than about 650, or than about 600, or than about 550, or than about 500, or than about 490, or than about 480, or than about 400, or than about 350 amino acids long. In some embodiments, a SD-IgAPP is a middle domain of a reference IgA protease (or fragment thereof). In some embodiments, an IgA protease polypeptide that is such a SD-IgAPP, shares a characteristic sequence with an IgA protease and shows at least about 40% overall sequence identity' with a fragment of such reference IgA protease. In some embodiments, a SD-IgAPP have at least 80 % (e.g., at least 85%, at least 90, at least 95%) identity to SEQ ID NO:4 or SEQ ID NO: 22. In many embodiments, SD-IgAPP agents provided herein are IgA proteases, in that they cleave at least IgAl. In some embodiments, SD-IgAPP agent(s) provided cleave IgAl and IgA2; in many embodiments, SD-IgAPP agent(s) provided cleave IgAl at least as efficiently and / or effectively as they cleave IgA2.

[0044] Pharmaceutical composition. As used herein, the term “pharmaceutical composition” refers to an active agent, formulated together with one or morepharmaceutically acceptable carriers. In some embodiments, active agent is present in a unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, or to other mucosal surfaces.

[0045] Polypeptide. As used herein refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and / or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D-amino acids. L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g.. modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation. etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and / or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In someembodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term "polypeptide’' may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and / or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and / or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and / or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and / or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the poly peptide of interest is a derivative of its parent polypeptide.

[0046] Preferential cleavage. The term “preferential cleavage”, when used herein with reference to an agent having a cleavage activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities or states. For example, in some embodiments, a SD-IgAPP agent is said to preferentially cleave a target (e.g, a specific IgA) if it preferentially cleaves that target in the presence of one or more competing alternative targets (e.g., cleaves substantially only IgAl in the presence of IgA2) and / or it is observed to act on the preferential target under conditions (e.g, enzyme target concentration, temperature, ionic strength, etc.) that are reasonably comparable. In some embodiments, a SD-IgAPP is characterized in that it cleaves at least 20% more (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90% more) IgAl than IgA2.

[0047] Source: The term “source” as used herein, typically refers to a context in which an agent of interest (e.g., that may be or comprise a carbohydrate, a lipid, a nucleicacid, a metal, polypeptide, a small molecule, or a combination thereof) may be found in nature, or from which such agent can be or has been obtained (e.g, isolated). In some embodiments, a source may be or comprise a biological source (e.g. an organism, tissue, or cell, or sample thereof); in some embodiments a source may be an environmental source. In some embodiments, a source may be or comprise a primary sample from an organism (e.g., which may be or comprise a tissue or fluid of such organism, and / or may be or comprise cell(s) of such organism). In some embodiments, an organism may be or comprise a prokaryotic organism (e.g., a bacterium) or a eukaryotic organism (e.g, a fungus or yeast, an insect, a mammal, a plant, a reptile, etc.). In some embodiments, an infectious agent such as a virus may be considered an organism for purposes of this disclosure, and in particular with respect to being a source. In some embodiments, a source may be or comprise an engineered source, such as a cell line or culture, an in vitro system, etc.

[0048] Specific binding: As used herein, the term ‘‘specific binding’' refers to an ability to discriminate between possible binding partners in the environment in which binding occurs. A binding agent that interacts with one particular target when other potential targets are present is said to "bind specifically" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining degree and / or rate of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or determining degree and / or rate of dissociation of a binding agent- partner complex; in some embodiments, specific binding is assessed by detecting or determining ability of the binding agent to compete with an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations of one or both binding partners. In some embodiments, two agents that bind specifically to one another are said to form a complex with one another.

[0049] Specificity. As is known in the art, “specificity” of binding is a measure of the ability of a particular ligand to distinguish its binding partner from other potential binding partners.

[0050] Substantially. As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological andchemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially"’ is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0051] Therapeutically effective amount. As used herein, the term “therapeutically effective amount” refers to an amount that produces a desired effect (e.g., a desired biological, clinical, or pharmacological effect) in a subject or population to which it is administered. In some embodiments, the term refers to an amount statistically likely to achieve the desired effect when administered to a subject in accordance with a particular dosing regimen (e.g., a therapeutic dosing regimen). In some embodiments, the term refers to an amount sufficient to produce the effect in at least a significant percentage (e.g., at least about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more) of a population that is suffering from and / or susceptible to a disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is one that reduces the incidence and / or severity of, and / or delays onset of, one or more symptoms of the disease, disorder, and / or condition. Those of ordinary skill in the art wall appreciate that the term “therapeutically effective amount” does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be an amount that provides a particular desired response in a significant number of subjects when administered to patients in need of such treatment, e.g., in at least about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more patients within a treated patient population. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount sufficient to induce a desired effect as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine). Those of ordinary skill in the art will appreciate that, in some embodiments, a therapeutically effective amount of a particular agent or therapy may be formulated and / or administered in a single dose. In some embodiments, a therapeutically effective agent may be formulated and / or administered in a plurality of doses, for example, as part of a dosing regimen.

[0052] Treatment. As used herein, "treatment" refers to a reduction of undesirable or abnormal IgA (e.g., IgAl) in tissues (e.g., kidney, skin, blood vessels etc) and / or in the circulation. Treatment can refer to a decrease in IgAl deposition of at least about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and up to and including at least about 100% relative to the amount of IgAl deposition in a control individual not administered the SD-IgAPP of the disclosure. Preferably, treatment refers to an increase in IgAl clearance of at least about 25% relative to the amount of IgAl in tissues and / or in the circulation in the same individual prior to administration of the isolated polypeptide of the disclosure. Reduction of abnormal IgAl in the circulation may prevent it to be deposited in tissues including kidney and skin of at least about 5% in response to the administration of an IgA protease polypeptide agent of the disclosure compared to the amount of IgAl in the circulation in the same individual prior to administration of the IgA protease polypeptide agent of the disclosure. For example, "treatment" can refer to an increase in IgAl clearance of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and up to and including at least about 100% relative to the amount of IgAl present in the circulation and / or present in tissue(s) measured in the same individual prior to the administration of the SD-IgAPP of the disclosure.

[0053] The term "unit dose" when used in reference to a therapeutic composition of the present disclosure refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier or vehicle.Detailed Description of Certain EmbodimentsImmunoglobin A (IgA)

[0054] Immunoglobin A (IgA) is the major class of immunoglobulin found in human mucosal secretions. IgA is a polymeric antibody, typically containing two copies of IgA that are assembled with one joining-chain to form a dimeric IgA. The dimeric IgA immunoglobulin protein reaches the fluids of the gastrointestinal and respiratory tract by binding to another polypeptide chain, termed the "‘polymeric immunoglobulin receptor’, which is produced by mucosal epithelial cells. Once dimeric IgA antibodies bind to this receptor, they are transported by an endocytic transport pathway to the apical surface of the epithelial cell and released into the mucosal fluid space as secretory IgA (slgA).

[0055] IgA includes two isotypes: IgAl and IgA2. The main differences between the isotypes IgAl and IgA2 lie in the hinge region of the heavy polypeptide chain; a 13-amino acid deletion characterizes the IgA2 hinge region. IgAl and IgA2 are also found in human blood plasma and serum.IgA proteases

[0056] IgA proteases are bacterial enzy mes that cleave human IgA molecules at their hinge region. Some IgA proteases cleave both IgAl and IgA2, whereas some IgAl proteases preferentially or only cleave one relative to the other. For example, certain IgA proteases cleave IgAl at a site in its hinge region that is mutated in or absent from a corresponding IgA2 hinge. Cleavage in the hinge region of monomeric IgAl yields two intact Fab regions and an intact Fc, and these otherwise unmodified fragments retain most of the biological properties that they have in the intact IgAl protein.

[0057] IgA proteases are expressed in gram negative and gram positive bacteria as a single-chain precursor that traverses the bacterial membrane. IgA proteases of gram negative bacteria undergo auto-catalytic cleavage releasing an N-terminal soluble IgA mature protease.

[0058] Several classes of IgA proteases have been defined, by amino acid sequence comparison (within each classes) and / or mechanistic similarity. Such classes include, for example: IgA Cysteine proteases, IgA Metalloproteases, IgA M26 Zinc-Metalloproteases, IgA S6 Serine proteases, and IgA M64 Metalloproteases. IgA Cysteine proteases of Prevotella ssp. and IgA Metalloproteases of Capnocytophaga ssp. (human) cleave peptide bond P223-S224 of IgA. M26 Zinc-Metalloproteases of the genera Streptococcus and Gemella cleave peptide bond P227-T228 of IgAl. Table 2 provides exemplary IgA proteases amino acid sequences.

[0059] M64 Metalloproteases from Clostridium ramosum is the only known enzyme reported to cleave both IgAl and IgA2. This protease cleaves IgA at the hinge bond P221- V222 (in the canonical numbering system); this bond is present in both IgAl and IgA2 immunoglobulins. The metallotype and serine-type IgAl proteinases have entirely different primary structures but are reported to share a stringent selectivity for human IgAl as substrate, due to their IgA cleavage site.

[0060] The C. ramosum IgA M64 protease is a metalloendopeptidase with a putative extended zinc-binding motif. The primary structure of the IgA M64 protease shows no significant overall similarity to any other known metalloendopeptidase, including any IgAl proteinase belonging to this class of proteolytic enzymes. To date only the primary sequence and identification of various motifs were know n for Clostridium ramosum IgA M64 protease. The domain architecture of Clostridium ramosum IgA M64 protease has not previously been described.

[0061] SEQ ID NO: 1 (Table 2) provides an exemplary amino acid sequence of a C. ramosum IgA protease containing a 30 amino acid signal peptide. SEQ ID NO: 2 (Table 2) provides an exemplary amino acid sequence of a mature soluble C. ramosum IgA protease, without the 30 amino acid signal peptide. In many embodiments of the present disclosure, the C. ramosum IgA protease of SEQ ID NO: 2 is a useful reference protease relative to which provided SD-IgAPP agent(s) may be assessed or compared.

[0062] It should be noted that IgA proteases are also found in gram-positive bacteria. Naturally-occurring gram-positive IgA proteases typically lack an autocatalytic secretion mechanism; in some embodiments, if such proteases are to be utilized, it may be desirable to employ technologies, such as recombinant technologies, to facilitate their isolation (e.g., by tagging them with an epitope tag).Subdomain IgA protease polypeptide (SD-IgAPP) Agents

[0063] The present disclosure provides IgA protease polypeptide agents that effectively cleave at least IgAl and have certain useful characteristics and advantages relative to other IgA protease polypeptides, including specifically to certain naturally occurring IgA proteases such as the C. ramosum IgA protease whose mature amino acid sequence is set forth in SEQ ID NO: 2.

[0064] In particular, the present disclosure surprisingly demonstrates that SD-IgAPP agents can be effective in cleaving IgA.

[0065] Among other things, the present disclosure determines a domain structure for certain M64 IgA proteases, specifically including the C. ramosum IgA protease and, moreover, surprisingly demonstrates that various sequences, including two full domains,found therein are not necessary for useful IgA protease (and specifically for useful IgAl protease) activity.

[0066] That is, the present disclosure describes architecture studies performed with an M64 IgA protease (e.g., a representative M64 IgA protease; specifically, in the present examples, a C. ramosum IgA protease). The present disclosure teaches that N-terminal and / or C-terminal domain sequences may be dispensable (e.g., not required for IgA protease activity ). Thus, among other things, the present disclosure specifically provides SD-IgAPP agents that are M64 IgAPP subdomain agents.

[0067] The present disclosure defines a “middle domain” section present in various IgA proteases, and specifically in various M64 IgA proteases (see, for example, SEQ ID NO: 4 and Figures 6A-H) that are sufficient for IgA protease activity. As exemplified, in some embodiments, such middle domain retains IgA protease activity comparable to that of the relevant full-length IgA protease in that it cleaves IgAl. In some embodiments, such middle domain demonstrated increased IgA cleavage (e.g., IgAl cleavage activity) relative to such reference protease. In some embodiments, such middle domain retains IgA protease activity comparable to that of the relevant full-length IgA protease in that it cleaves IgA2. In some embodiments, such middle domain shows reduced IgA2 cleavage (e.g.. relative to a relevant reference IgA protease).

[0068] In some embodiments, an IgA protease polypeptide agent according to the present disclosure is a polypeptide that is an M64 IgA protease polypeptide agent in that it shares certain sequence feature(s) (e.g., one or more characteristic sequence elements and / or an overall percent identity over at least one significant (e.g., greater than about 200, 250, 300, 350, 400, 410, 420, 430, 440, 450, 460 , 470, 480, 490, or more amino acids) stretch, but that is shorter than full-length M64 IgA protease, such as shorter than full-length C. ramosum M64 IgA protease. In some embodiments of the foregoing, a stretch comprises amino acids 314-807 of the amino acid sequence as set forth in SEQ ID NO: 1 . In some embodiments, a stretch comprises amino acids 329-807 of the amino acid sequence as set forth in SEQ ID NO: 1. In one embodiment of the foregoing, a stretch comprises amino acids 331-632 of the amino acid sequence as set forth in SEQ ID NO: 1. In another embodiment of the foregoing, a stretch comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to amino acids 314-807 of the amino acid sequence as setforth in SEQ ID NO: 1. In some embodiments, a stretch comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to amino acids 329-807 of the amino acid sequence as set forth in SEQ ID NO: 1. In another embodiment of the foregoing, the stretch comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to amino acids 331-632 of the amino acid sequence as set forth in SEQ ID NO: 1. The present disclosure provides useful agents that are or comprise an IgA protease polypeptide whose amino acid sequence differs from a relevant reference IgA protease (e.g., SEQ ID NO: 2, Table 2), or fragment thereof, for example, by one or more sequence modifications selected from disclosure deletions, insertions, inversions, substitutions, truncations, and combinations thereof. Typically, such sequence modification is or comprises at least one deletion or truncation. The present disclosure has identified that the N-terminal domain tolerates significant structural alterations, such as deletion of or parts of the N-terminal domain and that the C- terminal domain tolerates significant structural alterations, such as deletion of or parts of the C-terminal domain. Alterations or ablations of such domains are found to have little or no deleterious effects on IgA protease polypeptide agent activity. In many embodiments, such sequence modification is or comprises a C-terminal truncation. In many embodiments, such sequence modification is or comprises an N-terminal truncation. In many embodiments, such sequence modification is or comprises both a C-terminal truncation and an N-terminal truncation.

[0069] In many embodiments, a relevant reference IgA protease is an M64 IgA protease. In many such embodiments, a provided IgA protease polypeptide (e.g., SD-IgAPP agent) lacks C-terminal and / or N-terminal sequences relative to such reference. In some embodiments, C-terminal amino acids of a relevant reference IgA protease are residues corresponding to amino acids 808-1234 of SEQ ID NO: 1; in some embodiments, N-terminal amino acids of a relevant reference IgA protease are residues corresponding to amino acids 31-313 of SEQ ID NO: 1 and / or residues corresponding to amino acids 1-313 of SEQ ID NO: 1. In some embodiments, N-terminal amino acids of a relevant reference IgA protease are residues corresponding to amino acids 31-330 of SEQ ID NO: 1 and / or residues corresponding to amino acids 1-330 of SEQ ID NO: 1. In some embodiments, N-terminal amino acids of a relevant reference IgA protease are residues corresponding to amino acids 31-328 of SEQ ID NO: 1 and / or residues corresponding to amino acids 1-328 of SEQ ID NO:1. In some embodiments of the disclosure, IgA protease polypeptide agents are provided that are truncation variant(s) of such regions. In some embodiments, IgA protease polypeptide agents are truncations variant(s) of a reference IgA protease polypeptide.

[0070] In many embodiments, such modifications (e.g., truncations) improve the desirability of the IgA protease as a therapeutic agent (e.g., by reducing immunogenicity, increasing enzymatic activity, increasing ability to reduce deposits of IgAl, increasing bioavailability, and / or increasing half-life of the IgAl protease polypeptide in a subject, etc.).

[0071] In some embodiments, a middle domain present in various M64 IgA proteases can be sufficient for IgA protease activity. In some embodiments, a SD-lgAPP agent may only comprise one domain of an IgA protease, such as the middle domain (M1-M2-M3) of an IgA M64 protease.

[0072] In some embodiments, an IgA protease polypeptide agent according to the present disclosure (e.g., an SD-lgAPP agent) has an amino acid sequence that is or comprises an element having the general formula Y1- N-M1-M2-M3-C-Y2

[0073] wherein each of Y1, N, M1, M2, M3, C and Y2comprises or consists of a consecutive sequence of amino acids, wherein M1substantially comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 7, wherein M2comprises or consists of a HEX 1 X?H motif, wherein X1and X2are amino acids, wherein M3substantially comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 9, wherein the polypeptide's full amino acid sequence does not comprise or consist of the amino acid sequence of SEQ ID NO: 1 or 2, and wherein Y1, N, C, and Y2are optional.

[0074] In some embodiments, M1- M2-M3represents the middle domain of an IgA protease, such as the middle domain of a M64 protease, such as a middle domain of a C. ramosum IgA M64 protease, wherein M2comprises a HEX1X2H motif supporting proteolytic activity of the IgA protease. M1-M2-M3can be any consecutive sequence of amino acids of a middle domain of an IgA protease as long as the amino acid sequence contains the HEX1X2H motif. In some embodiments, M1-M2-M3is the middle domain of an IgA protease (e.g.,middle domain of a M64 protease, such as a C. ramosum IgA M64 protease). In some embodiments, N represents the N-terminal domain of an IgA protease, such as the N-terminal domain of M64 protease, such as the N-terminal domain of a C. ramosum IgA protease. In some embodiments, C represents the C-terminal domain of an IgA protease, such as the C- terminal domain of a M64 protease, such as the C-terminal domain of the C. ramosum IgA protease.

[0075] In some embodiments, M2comprises or consists of the HEX1X2H motif, wherein X1and X2can be any amino acid that can be incorporated into the polypeptide chain. X1or X2can be a naturally-occurring amino acid or a non-natural amino acid. In some embodiments, M2consists of the HEX1X2H motif. In some embodiments, X1is an amino acid with hydrophobic side chains. In some embodiments, X1is selected from the group consisting of alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan. In some embodiments, X1is phenylalanine or leucine. In some embodiments, X2is glycine. In some embodiments, X1is phenylalanine and X2is glycine, e.g.. wherein the amino acid sequences is the sequence of SEQ ID NO: 8. In some embodiments, X1is leucine and X2is glycine.

[0076] In some embodiments, M2comprises the HEX1X2H motif and about 1 to about 100 additional amino acid residues. The additional amino acid residues may in some embodiments be inserted between the HEX1X2H motif and M1and / or the HEX1X2H motif and M3, thereby separating M1with M2and / or M2with M3. In some embodiments M2comprises the HEX1X2H motif and about 1 to about 100 additional amino acid residues, such as 2 to 50 additional amino acid residues, such as 10 to 40 additional amino acid residues, or such as 20 to 30 additional amino acid residues.

[0077] In some embodiments, M1and M2are separated by one or more amino acids. In some embodiments. M1and M2are separated by at the most 20 amino acids, such as 10 amino acids, such as 5 amino acids. In some embodiments, M2and M3are separated by one or more amino acids. In some embodiments, M2and M3are separated by at the most 20 amino acids, such as 10 amino acids, or such as 5 amino acids.

[0078] In some embodiments, a M1amino acid sequence is at least 75% identical to SEQ ID NO: 7, such as at least 80% identical to SEQ ID NO: 7, such as at least 85% identical to SEQ ID NO: 7, such as at least 90% identical to SEQ ID NO: 7, such as at least 91%identical to SEQ ID NO: 7, such as at least 92% identical to SEQ ID NO: 7, such as at least 93% identical to SEQ ID NO: 7. such as at least 94% identical to SEQ ID NO: 7. such as at least 95% identical to SEQ ID NO: 7, such as at least 96% identical to SEQ ID NO: 7, such as at least 97% identical to SEQ ID NO: 7, such as at least 98% identical to SEQ ID NO: 7, such as at least 99% identical to SEQ ID NO: 7, or such as 100% identical to SEQ ID NO: 7. In some embodiment, M1is or comprises at least a part of SEQ ID NO: 7. In some embodiments, a M1amino acid sequence is at least 75% identical to SEQ ID NO: 25, such as at least 80% identical to SEQ ID NO: 25, such as at least 85% identical to SEQ ID NO: 25, such as at least 90% identical to SEQ ID NO: 25, such as at least 91% identical to SEQ ID NO: 25, such as at least 92% identical to SEQ ID NO: 25, such as at least 93% identical to SEQ ID NO: 25, such as at least 94% identical to SEQ ID NO: 25, such as at least 95% identical to SEQ ID NO: 25, such as at least 96% identical to SEQ ID NO: 25, such as at least 97% identical to SEQ ID NO: 25, such as at least 98% identical to SEQ ID NO: 25, such as at least 99% identical to SEQ ID NO: 25, or such as 100% identical to SEQ ID NO: 25. In some embodiment. Mj is or comprises at least a part of SEQ ID NO: 25. In some embodiments, a Mj amino acid sequence is at least 75% identical to SEQ ID NO: 28, such as at least 80% identical to SEQ ID NO: 28, such as at least 85% identical to SEQ ID NO: 28, such as at least 90% identical to SEQ ID NO: 28, such as at least 91% identical to SEQ ID NO: 28, such as at least 92% identical to SEQ ID NO: 28, such as at least 93% identical to SEQ ID NO: 28, such as at least 94% identical to SEQ ID NO: 28. such as at least 95% identical to SEQ ID NO: 28, such as at least 96% identical to SEQ ID NO: 28, such as at least 97% identical to SEQ ID NO: 28, such as at least 98% identical to SEQ ID NO: 28, such as at least 99% identical to SEQ ID NO: 28, or such as 100% identical to SEQ ID NO: 28. In some embodiment, M1is or comprises at least a part of SEQ ID NO: 28.

[0079] In some embodiments, a M3amino acid sequence is at least 75% identical to SEQ ID NO: 9, such as at least 80% identical to SEQ ID NO: 9, such as at least 85% identical to SEQ ID NO: 9, such as at least 90% identical to SEQ ID NO: 9, such as at least 91% identical to SEQ ID NO: 9, such as at least 92% identical to SEQ ID NO: 9, such as at least 93% identical to SEQ ID NO: 9, such as at least 94% identical to SEQ ID NO: 9, such as at least 95% identical to SEQ ID NO: 9, such as at least 96% identical to SEQ ID NO: 9, such as at least 97% identical to SEQ ID NO: 9, such as at least 98% identical to SEQ ID NO: 9, such as at least 99% identical to SEQ ID NO: 9. or such as 100% identical to SEQ ID NO: 9.In some embodiments, a M3amino acid sequence is at least 75% identical to SEQ ID NO: 26, such as at least 80% identical to SEQ ID NO: 26, such as at least 85% identical to SEQ ID NO: 26, such as at least 90% identical to SEQ ID NO: 26, such as at least 91% identical to SEQ ID NO: 26, such as at least 92% identical to SEQ ID NO: 26, such as at least 93% identical to SEQ ID NO: 26, such as at least 94% identical to SEQ ID NO: 26, such as at least 95% identical to SEQ ID NO: 26, such as at least 96% identical to SEQ ID NO: 26, such as at least 97% identical to SEQ ID NO: 26, such as at least 98% identical to SEQ ID NO: 26, such as at least 99% identical to SEQ ID NO: 26, or such as 100% identical to SEQ ID NO: 26. In some embodiments, a M3amino acid sequence comprises a DEY motif. In some embodiments, the DEY motif present within a M3amino acid sequence and a M2amino acid sequence are separated by about 3 to about 9 amino acids, such as about 4 to about 8, such as about 5 to about 7, such as 6 amino acids. Without being bound to a particular theory, a M3amino acid sequence may comprise one or more cysteine amino acids (e.g., cysteine amino acids corresponding to position 63, 73 and / or 76 in SEQ ID NO: 9) that are necessary' or sufficient to coordinate a metal (e.g., zinc ion) thereby inactivating the SD-IgAPP. In some embodiments, one or more of the cytosine amino acids may be substituted or removed thereby reducing or abolishing the ability of SD-IgAPP to coordinate the metal (e g., zinc ion) and inactive the SD-IgAPP. In some embodiments, one or more cysteine amino acids at position 63, 73 and / or 76 in SEQ ID NO: 9 (corresponding to amino acid position 616, 626 and 629 in SEQ ID NO: 1) are substituted or removed (See Example 2 and Figure 4). In some embodiment, a M3amino acid sequence is or comprises at least of a part of SEQ ID NO: 9.

[0080] In some embodiments, M1-M2-M3corresponds to the middle domain of an IgA protease, such as a M64 protease (e.g., C. ramosum IgA M64 protease of SEQ ID NO: 1 or 2). In some embodiments, IgA protease polypeptide agents (e.g., SD-IgAPP agents) have an amino acid sequence that comprises a part of an amino acid sequence of SEQ ID NO: 4, such as about 200 to about 302, such as about 250 to about 302 amino acids, such as at least 200 amino acids, such as at least 250 amino acids, or such as at least 300 amino acids of SEQ ID NO: 4. In some embodiments, IgA protease polypeptide agents (e.g, SD-IgAPP agents) have an amino acid sequence that comprises a part of an amino acid sequence of SEQ ID NO: 27, such as about 200 to about 450, such as about 250 to about 400 amino acids, such as at least 250 amino acids, such as at least 300 amino acids, such as at least 350 amino acids of SEQ ID NO: 27. In some embodiments. IgA protease polypeptide agents (e.g, SD-IgAPPagents) have an amino acid sequence that comprises a part of an amino acid sequence of SEQ ID NO: 22. such as about 200 to about 450, such as about 250 to about 400 amino acids, such as at least 250 amino acids, such as at least 300 amino acids, such as at least 350 amino acids of SEQ ID NO: 22. In some embodiments, IgA protease polypeptide agents (e.g., SD-IgAPP agents) have an amino acid sequence that is or comprises an amino acid sequence having at least 45 % overall sequence identity with SEQ ID NO: 4. In some embodiments, IgA protease polypeptide agents (e.g., SD-IgAPP agents) have an amino acid sequence that is or comprises an amino acid sequence having at least 45 % overall sequence identity with SEQ ID NO: 27. In some embodiments, IgA protease polypeptide agents (e.g., SD-IgAPP agents) have an amino acid sequence that is or comprises an amino acid sequence having at least 45 % overall sequence identity with SEQ ID NO: 22. SEQ ID NO: 4. SEQ ID NO: 27. and SEQ ID NO 22 are consensus sequences for a C. ramosum IgA M64 protease middle domain. In some embodiments, an IgA protease polypeptide agent (e.g., a SD-IgAPP agent) has an amino acid sequence that shares at least 50%, such as at least 55%, such as at least 60%, such as at least 65%, such as at least 70%, or such as at least 75% sequence identity with SEQ ID NO: 4. In some embodiments, the polypeptide sequence comprises or consists of an amino acid sequence having at least 75% identity to SEQ ID NO: 4, such as at least 80% identity to SEQ ID NO: 4, such as at least 85% identity7to SEQ ID NO: 4, such as at least 90% identity to SEQ ID NO: 4, such as at least 91% identity to SEQ ID NO: 4, such as at least 92% identity to SEQ ID NO: 4, such as at least 93% identity to SEQ ID NO: 4. such as at least 94% identity' to SEQ ID NO: 4, such as at least 95% identity to SEQ ID NO: 4, such as at least 96% identity to SEQ ID NO: 4, such as at least 97% identity to SEQ ID NO: 4, such as at least 98% identity' to SEQ ID NO: 4. such as at least 99% identity to SEQ ID NO: 4, or such as 100% identity to SEQ ID NO: 4. In some embodiments, M1-M2-M3(i.e. middle domain of an IgA protease) comprises a HEXXH motif and a DEY motif. In some embodiments, M1- M2-M3comprises a HEFGH motif and a DEY motif. In some embodiments, M1-M2-M3comprises an amino acid sequence that shares at least 80% identity to SEQ ID NO: 27, such as at least 85% identity to SEQ ID NO: 27, such as at least 90% identity' to SEQ ID NO: 27, such as at least 91% identity to SEQ ID NO: 27, such as at least 92% identity to SEQ ID NO: 27, such as at least 93% identity to SEQ ID NO: 27, such as at least 94% identity to SEQ ID NO: 27, such as at least 95% identity to SEQ ID NO: 27, such as at least 96% identity7to SEQ ID NO: 27, such as at least 97% identity to SEQ ID NO: 27, such as at least 98% identity to SEQ ID NO: 27, such as at least 99% identity to SEQ ID NO: 27. or such as 100% identity toSEQ ID NO: 27. In some embodiments, M1-M2-M3comprises an amino acid sequence that shares at least 80% identity- to SEQ ID NO: 22, such as at least 85% identity to SEQ ID NO: 22, such as at least 90% identity to SEQ ID NO: 22, such as at least 91% identity to SEQ ID NO: 22, such as at least 92% identity to SEQ ID NO: 22, such as at least 93% identity to SEQ ID NO: 22, such as at least 94% identity to SEQ ID NO: 22, such as at least 95% identity- to SEQ ID NO: 22, such as at least 96% identity to SEQ ID NO: 22. such as at least 97% identity to SEQ ID NO: 22, such as at least 98% identity to SEQ ID NO: 22, such as at least 99% identity to SEQ ID NO: 22, or such as 100% identity to SEQ ID NO: 22.

[0081] In some embodiments, a SD-IgAPP agent provided by the disclosure has an amino acid sequence that includes M1-M2-M3(e.g., corresponding to a "middle domain” of an IgA protease such as an M64 IgA protease as described herein) further comprises an N- terminal domain (N) or part thereof.

[0082] In some embodiments, N comprises or consists of a consecutive sequence of amino acids (e.g.. as may be found in a reference IgA protease polypeptide) that also includes M1-Mz-Ms

[0083] In some embodiments, N may be or comprise one or more amino acids that are found N-terminal to M1-M2-M3in a reference IgA protease (e.g., a reference M64 IgA protease such as the C. ramosum IgA protease). In some embodiments, N includes all, or substantially- all, of the amino acids found N-terminal of M1-M2-M3in such reference IgA protease (e.g., in the mature form of such reference IgA protease). In some embodiments, N includes less than all of the amino acids found N-terminal to M1-M2-M3in such reference (mature) IgA protease; in some such embodiments, N includes those amino acids found most proximal to M1-M2-M3in such reference (mature) IgA protease. In some embodiments, N comprises amino acids 314-330 of the amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, N comprises amino acids 329-330 of the amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, N comprises an amino acid sequence comprising amino acids 200-313 of the amino acid sequence as set forth in SEQ ID NO: 1 or parts thereof.

[0084] In some embodiments, where a provided IgA protease polypeptide agent includes N and N corresponds to a part of or all of the N-terminal portion of a reference IgA protease such as a reference M64 IgA protease (e.g.. the C. ramosum IgA protease), Nincludes one or more sequence variations (i.e., modifications) relative to such N-terminal portion, while maintaining sufficient overall sequence identity- and / or character that one skilled in the art will appreciate that it reasonably corresponds to part of such N-terminal portion.

[0085] In some embodiments, a N amino acid sequence is at least 70% identical to SEQ ID NO: 5, such as least 75% identical, such as least 80% identical, such as least 85% identical, such as least 90% identical, such as least 91% identical, such as least 92% identical, such as least 93% identical, such as least 94% identical, such as least 95% identical, such as least 96% identical, such as least 97% identical, such as least 98% identical, such as least 99% identical, or such as least 100% identical to SEQ ID NO: 5. In some embodiments, a N amino acids 1-328 of the amino acid sequence as set forth in SEQ ID NO: 1, or fragments thereof.

[0086] In some embodiments, an amino acid sequence of a SD-IgAPP agent provided by the present disclosure comprises or consists of an amino acid sequence having at least 75% identity to SEQ ID NO: 3, such as at least 80% identity to SEQ ID NO: 3, such as at least 85% identity- to SEQ ID NO: 3, such as at least 90% identity to SEQ ID NO: 3, such as at least 91% identity to SEQ ID NO: 3, such as at least 92% identity to SEQ ID NO: 3. such as at least 93% identity to SEQ ID NO: 3, such as at least 94% identity to SEQ ID NO: 3, such as at least 95% identity7to SEQ ID NO: 3, such as at least 96% identity to SEQ ID NO: 3, such as at least 97% identity to SEQ ID NO: 3, such as at least 98% identity to SEQ ID NO: 3, such as at least 99% identity to SEQ ID NO: 3, or such as 100% identity- to SEQ ID NO: 3.

[0087] In some embodiments, C may be or comprise one or more amino acids that are found C-terminal to M1-M2-M3in a reference IgA protease (e.g., a reference M64 IgA protease such as the C. ramosum IgA protease). In some embodiments. C includes all, or substantially all, of the amino acids found C-terminal of M1-M2-M3in such reference IgA protease (e.g., in the mature form of such reference IgA protease). In some embodiments, C includes less than all of the amino acids found C-terminal to M1-M2-M3in such reference (mature) IgA protease; in some such embodiments, C includes those amino acids found most proximal to M1-M2-M3in such reference (mature) IgA protease. In some embodiments, C comprises amino acids 633-807 of the amino acid sequence as set forth in SEQ ID NO: 1. Insome embodiments, C comprises an amino acid sequence comprising amino acids 808-1000 of the amino acid sequence as set forth in SEQ ID NO: 1 or parts thereof.

[0088] In some embodiments, where a provided IgA protease polypeptide agent includes C and C corresponds to part or all of the C-terminal portion of a reference IgA protease such as a reference M64 IgA protease (e.g, the C. ramosum IgA protease), C includes one or more sequence variations (i.e.. modifications) relative to such C-terminal portion, while maintaining sufficient overall sequence identity and / or character that one skilled in the art will appreciate that it reasonably corresponds to part of such C-terminal portion.

[0089] In some embodiments, a C amino acid sequence is at least 70% identical to SEQ ID NO: 6, such as least 75% identical, such as least 80% identical, such as least 85% identical, such as least 90% identical, such as least 91% identical, such as least 92% identical, such as least 93% identical, such as least 94% identical, such as least 95% identical, such as least 96% identical, such as least 97% identical, such as least 98% identical, such as least 99% identical, or such as least 100% identical to SEQ ID NO:6.

[0090] In some embodiments, a amino acid sequence of a SD-IgAPP agent provided by the present disclosure comprises or consists of an amino acid sequence having at least 75% identity to SEQ ID NO: 10, such as at least 80% identity to SEQ ID NO: 10, such as at least 85% identity to SEQ ID NO: 10, such as at least 90% identity to SEQ ID NO: 10, such as at least 91% identity to SEQ ID NO: 10, such as at least 92% identity to SEQ ID NO: 10, such as at least 93% identity to SEQ ID NO: 10, such as at least 94% identity to SEQ ID NO: 10, such as at least 95% identity to SEQ ID NO: 10, such as at least 96% identity7to SEQ ID NO: 10, such as at least 97% identity to SEQ ID NO: 10, such as at least 98% identity to SEQ ID NO: 10, such as at least 99% identity to SEQ ID NO: 10, or such as 100% identity to SEQ ID NO: 10.

[0091] In some embodiments, a provided SD-IgAPP agent includes a middle domain M1-M2-M3as described above, and further comprises an N-terminal domain (N) and a C- terminal domain (C) as described above.

[0092] In some embodiments, a provided SD-IgAPP agent has an amino acid sequence that comprises or consists of a sequence at least 75% identity to a correspondingportion of SEQ ID NO: 2, such as at least 80% identity to such portion of SEQ ID NO: 2, such as at least 85% identity to such portion of SEQ ID NO: 2, such as at least 90% identity to such portion of SEQ ID NO: 2, such as at least 91% identity to such portion of SEQ ID NO: 2, such as at least 92% identity to SEQ ID NO: 2, such as at least 93% identity to such portion of SEQ ID NO: 2, such as at least 94% identity to such portion of SEQ ID NO: 2, such as at least 95% identity to such portion of SEQ ID NO: 2, such as at least 96% identity to such portion of SEQ ID NO: 2, such as at least 97% identity to such portion of SEQ ID NO: 2, such as at least 98% identity to such portion of SEQ ID NO: 2, such as at least 99% identity to such portion of SEQ ID NO: 2, or such as 100% identity to such portion of SEQ ID NO: 2.

[0093] In some embodiments, IgA protease polypeptide agents (such as SD-IgAPP agents) correspond to a fragment of an IgA protease, such as an M64 IgA protease and / or a C. ramosum IgA protease. That is, in some embodiments, a provided agent has an amino acid sequence that shows high identity (e.g., at least 70 % or more, and in some embodiments at least 75 % or more, such as at least 80% or more, such as at least 85% or more, such as at least 90% or more, such as at least 91% or more, such as at least 92% or more, such as at least 93% or more, such as at least 94% or more, such as at least 95% or more, such as at least 96% or more, such as at least 97% or more, or such as at least 98% or more, such as at least 99% or more) with a corresponding fragment of SEQ ID NO: 2. In some such embodiments, one skilled in the art will recognize that a provided SD-IgAPP agent corresponds to a fragment that lacks amino acid residues from C-terminal to residue 808 of SEQ ID NO: 1, such as lacking from C-terminal to residue 810, such as lacking from C- terminal to residue 820, such as lacking from C-terminal to residue 830, such as lacking from C-terminal to residue 840, such as lacking from C-terminal to residue 850, such as lacking from C-terminal to residue 860, such as lacking from C-terminal to residue 870, such as lacking from C-terminal to residue 880, such as lacking from C-terminal to residue 890, such as lacking from C-terminal to residue 900, such as lacking from C-terminal to residue 910, such as lacking from C-terminal to residue 920, such as lacking from C-terminal to residue 930, such as lacking from C-terminal to residue 940, such as lacking from C-terminal to residue 950, such as lacking from C-terminal to residue 1000, such as lacking from C- terminal to residue 1050, such as lacking from C-terminal to residue 1100, such as lackingfrom C-terminal to residue 1150, such as lacking from C-terminal to residue 1200, or such as lacking from C-terminal to residue 1230 of SEQ ID NO: 1.

[0094] In some embodiments, one skilled in the art will recognize that a provided SD- IgAPP agent corresponds to a fragment that lacks amino acids 1-30 of SEQ ID NO: 1, such as lacking amino acids 1-50, such as lacking amino acids 1-60, such as lacking amino acids 1- 70, such as lacking amino acids 1-80, such as lacking amino acids 1-90, such as lacking amino acids 1-100, such as lacking amino acids 1-150, such as lacking amino acids 1-200, such as lacking amino acids 1-250, such as lacking amino acids 1-300, or such as lacking amino acids 1-313. or such as lacking amino acids 1-328 of SEQ ID NO: 1.

[0095] In some such embodiments, one skilled in the art will recognize that a provided SD-IgAPP agent corresponds to a fragment that lacks amino acids at both the C- terminus and N-terminus, such as lacking any of the combinations described herein above.

[0096] In some embodiments, a provided IgA protease polypeptide agent (e g., an SD-IgAPP agent) has an amino acid sequence that is not longer than 600, such as 550, such as 500. In some embodiments, a SD-IgAPP agent according to any of the foregoing embodiments comprises of at the most 500 amino acids, such of at the most 494 amino acids. In some embodiments, a SD-IgAPP agent according to any of the foregoing embodiments comprises of at the most 500 amino acids, such as at the most 400 amino acids, such of at the most 300 amino acids.

[0097] As described above, the present disclosure reveals, among other things, that the N-terminal domain and C-terminal domain (in particular, amino acid residues 1 to 313, or 1 to 328, and 808-1234 of SEQ ID NO: 1) of a C. ramosum IgA protease polypeptide can tolerate alteration and / or deletion.

[0098] In some embodiments, the IgA protease polypeptide agent comprises any one of the foregoing sequences but does not comprise full-length protease, such as full-length wild-type IgA protease.

[0099] Those skilled in the art are aware of a variety of technologies to compare and assess polypeptide sequence and structural relationships (e.g., to identify and / or assess IgA protease polypeptide sequences that may be variants or alternatives to exemplified sequencesherein and useful in accordance with the present disclosure). For example, sequence alignment programs such as BLAST ( available, for example, on the website of the National Center for Biotechnology Information) or Clustal W (available for example, at the website whose address is "www" followed immediately by ".clustal.org" and at the website whose address is "www" followed immediately by ".ebi.ac.uk / Tools / clustalw2 / index.html" (a website hosted by the European Bioinformatics Institute at the European Molecular Biology Laboratory) can be used to identify regions of sequence similarity between or among two or more polypeptides. BLAST can also be used to compare two or more nucleotide sequences, as can Sequencher, a software program available from GeneCodes (Ann Arbor, MI).

[0100] For example, sequence alignments can be performed between C. ramosum IgA protease polypeptides (using, for example, any of SEQ ID NOs 1-13 summarized in Table 2) and IgA protease polypeptides produced by other bacterial species and / or strains. For example, regions in other IgA proteases that bear sequence similarity to the region defined by- amino acid residues 1 to 313 and 808-1234 of SEQ ID NO: 1 (or subregions thereof) are likely regions that tolerate alteration and / or deletion in such other IgAl proteases.

[0101] Furthermore, those skilled in the art are aware of functional and / or physical similarities between or among different amino acids that may permit them to be substituted for one another with low risk of significantly disrupting structure and / or function of the polypeptide. For example, in some embodiments, amino acids are sometimes classified as acidic, neutral, or basic, or as polar or non-polar, or as having bulky- vs small side chains. Substitution of similar residues can be referred to as ‘“homologous” substitution, and features taking account for such homologous substitution (the exact features of which can often be defined by a user) are often built into sequence comparison software. Those skilled in the art will appreciate that, in some embodiments, sequence identity7can be assessed independent of homologous substitution - i.e.. so that a homologous residue would not be “counted” as a non-identical residue. Thus, any reference to sequence identity in the present disclosure can, in some embodiments, be considered to be identity independent of homology.

[0102] Those skilled in the art are also aware that sequence comparison technologies typically can account for gaps or other changes in relative positioning between or among sequence elements.

[0103] In some embodiments, a SD-IgAPP agent as provided by this disclosure comprising a Ml -M2 -M3 middle domain (e.g., corresponding to a "‘middle domain"’ of an IgA protease such as an M64 IgA protease as described herein) further comprises an N- terminal domain (N) or part thereof (e.g., N-terminal domain of an IgA protease such as a M64 IgA protease). In some embodiments, such SD-IgAPP agent is characterized in that it demonstrates greater preferential cleavage of IgAl relative to IgA2 (e.g., showing restricted IgA2 cleavage). In some embodiments, such SD-IgAPP when incubated in the presence of IgAl or IgA2 in HEPES buffer, pH 7.5 at 37°C for 15 to 60 minutes demonstrates higher cleavage of IgAl comparably to a SD-IgAPP consisting of the middle domain only.

[0104] In some embodiments, a SD-IgAPP agent is not a dimeric protein. In some embodiments, a SD-IgAPP agent is less than 300 kDa, such as less than 290 kDa, such as less than 280 kDa, such as less than 270 kDa, such as less than 260 kDa, such as less than 250 kDa, such as less than 240 kDa, such as less than 230 kDa, such as less than 220 kDa, such as less than 210 kDa, such as less than 200 kDa, such as less than 190 kDa, such as less than 180 kDa, such as less than 170 kDa, such as less than 160 kDa, such as less than 150 kDa, such as less than 140 kDa, such as less than 130 kDa, such as less than 120 kDa, such as less than 110 kDa, such as less than 100 kDa, such as less than 90 kDa, such as less than 80 kDa, such as less than 70 kDa, such as less than 60 kDa. In some embodiments, a SD-IgAPP agent comprises a stretch of ammo acids that is less than amin acids 31-1203 of the amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, a SD-IgAPP agent comprises an alanine residue at position 31 of SEQ ID NO: 1. In some embodiments, a SD-IgAPP agent does not comprises a A31G amino acid substitution of SEQ ID NO: 31.Heterologous Moieties:

[0105] In some embodiment, a provided SD-IgAPP agent may include one or more moieties that is not found in a reference IgA protease. In some embodiments, such heterologous moiety(ies) is or comprises a polypeptide moiety that is fused with IgA protease sequences. In some such embodiments, a polypeptide moiety is fused at an N-terminus or C- terminus of such IgA protease sequences (e.g., as represented herein by Yj or Y2in the general formula).

[0106] In some embodiments, a fused polypeptide moiety may act as a ‘‘tag” - e.g., useful for detection and / or purification, for example via association with a binding agentthereto. In some embodiments, fusing a tag to an IgA protease polypeptide agent of the present disclosure may aid in purification and / or detection of the polypeptide agent, and / or may provide a means by which the IgA protease polypeptide agent can form a complex with a ligand, such as an anti-tag antibody, e.g., for therapeutic purposes.

[0107] Those skilled in the art are aware of technologies by which a tag can be incorporated into or otherwise associated with an IgA protease polypeptide comprising a tag. For example, in some embodiments, a tag may be or comprise a peptide, which can be chemically ligated to an IgA protease polypeptide or can be incorporated into it (e.g., through recombinant techniques known in the art). In alternative embodiments, a tag may be or comprise a non-peptide moiety (e.g., biotin / avidin etc.); typically, such tags are associated by an IgA protease polypeptide by chemical conjugation.

[0108] In some embodiments, a tag may be removably associated with an IgA protease polypeptide agent. For example, a tag (whether a peptide or non-peptide tag) may- be associated via a cleavable bond (e.g., by a chemically cleavable and / or a protease- cleavable bond). For example, in one particular embodiment, a nucleotide sequence encoding a tag can be linked (e.g, by ligation, amplification, or other means) in frame to a sequence encoding an IgA protease polypeptide, for example upstream of DNA sequence encoding an IgA protease auto-catalytic cleavage site such that, upon cleavage of the IgA protease precursor polypeptide, a soluble IgA protease polypeptide comprising a tag is generated (e.g., that is or can be secreted from a producing cell).

[0109] In some embodiments, a tag may be or comprise a moiety that is specifically bound by an antibody. In some embodiments, such a tag may be or comprise a known moiety- for which useful antibodies are readily available. Alternatively or additionally, in some embodiments, a tag may be or comprise a metal-binding moiety- (e.g, a so-called His tag, which permits isolation, for example via a metal-chelating resin or bead, for example nickel-NTA beads).

[0110] In some embodiments, a tag comprises a (directly or indirectly) detectable entity, such as. for example, a chromogen, an enzyme (e.g., that may catalyze a reaction that results in a color change, in fluorescence, in luminescence, etc.) a fluorophore, a lumisescent entity-, radioisotope, etc. Particular detectable entities that will be familiar to those skilled in the art include, for example, biotin / strepavidin, fluorescent dyes (e.g., fluorescein, Texas Red,rhodamine, green fluorescent protein, and the like), radiolabels (e.g., 3H, 1251, 35S, 14C, or 32P). enzymes (e.g., horse radish peroxidase, alkaline phosphatase, glucose oxidase, and others commonly used in an ELISA), and calorimetric labels such as colloidal gold. Exemplary patents teaching the use of such detectable markers include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, the entireties of which are incorporated herein by reference.

[0111] In some embodiments, an SD-IgAPP agent according to the present disclosure may be obtained or manufactured by producing (e.g., by expressing it from a nucleic acid such as an engineered nucleic acid) a polypeptide of having a desired amino acid sequence (e.g., as described herein), and subsequently and optionally modifying such polypeptide.

[0112] Nucleic acid sequences (e.g., genes encoding various IgA proteases are known (see, for example, Table 2), and many such sequences have been deposited in the GenBank database at the National Center for Biotechnology Information. Alternatively or additionally, nucleic acid sequences encoding desired polypeptides can be determined or designed, including for example, taking into consideration features such as codon optimization preferences of a particular expression system, sequence elements that may impact stability and / or expressibility of a nucleic acid. etc.

[0113] Genetic material containing genes encoding such IgA protease polypeptides, or fragments thereof, are often publicly available through genetic repositories and / or through laboratories who have published gene sequence information for such IgA proteases. Such genetic material can be manipulated using, for example, molecular biology techniques, to generate nucleic acids encoding provided IgA protease polypeptide agents (e.g.. SD-IgAPP agents) as described herein.

[0114] Those skilled in the art are familiar with a variety of technologies useful and appropriate for production of SD-IgAPP agents. For example, in some embodiments, an SD- IgAPP agent may be recombinantly produced (e.g., expressed from a recombinant nucleic acid) in an expression system (such as, for example, an in vitro expression systems or a cellular system, e.g., that may utilize cells such as bacterial, insect, mammalian, and / or yeast cells). In some embodiments, an IgA protease polypeptide may be modified after expression in an expression system. In some embodiments, an IgA protease polypeptide may bemodified after isolation and / or purification. For example, in some embodiments, a tag (particularly a non-peptide tag), may be linked to the polypeptide.

[0115] In some embodiments, a provided SD-IgAPP agent may include a moiety that is fused to the N-terminal end of the IgA protease sequence. In some embodiments, the moiety is an Fc fusion. In some embodiments, a SD-IgAPP agent comprises a Fc region. In some embodiments, the moiety is albumin (e.g., human albumin). In some embodiments, such moiety may increase the half-life of the SD-IgAPP agent. In some embodiments, such moiety7may stabilize the SD-IgAPP agent.

[0116] In some embodiments, a stabilizing moiety suitable is an Fc domain. In some embodiments, an Fc domain is derived from a human immunoglobulin. In some embodiments, an Fc domain is from a human IgG constant region. It is understood, however, that the Fe domain may be derived from an immunoglobulin of another mammalian species, including for example, a rodent (e.g., a mouse, rat, rabbit, guinea pig) or non- human primate (e g., chimpanzee, macaque) species. In some embodiments, an Fc domain or portion thereof may be derived from an immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and an immunoglobulin isotypc, including IgGl, IgG2, IgG3, and IgG4.IgA protease activity)

[0117] SD-IgAPP agents provided by the present disclosure are characterized by an ability to cleave IgA (e.g., human IgAl and / or human IgA2). In some embodiments, a provided SD-IgAPP agent cleaves in the hinge region of IgA. In some embodiments, a SD- IgAPP agent cleaves in the hinge region of IgAl. In some embodiments, a SD-IgAPP agent cleaves in the hinge region of IgA2. In some embodiments, a SD-IgAPP agent accord to the present disclosure cleaves in the hinge region of IgAl and IgA2.

[0118] In many embodiments, a provided SD-IgAPP agent cleaves IgAl, in many such embodiments, preferentially relative to IgA2. In some embodiments, a provided SD- IgAPP agent demonstrates proteolytic activity against IgA, such as IgAl and / or IgA2, to a comparable (e.g., the same degree) as a reference IgA protease (See Examples 6 and 7). In some embodiments, a provided SD-IgAPP agent demonstrates greater proteolytic activity7than a relevant reference IgA protease (e.g., when assessed under comparable conditions, e.g., side-by-side). In some embodiments a provided SD-IgAPP agent demonstrates greaterpreference for IgAl (e.g., relative to IgA2) as compared with a relevant reference IgA protease (e.g., when assessed under comparable conditions, e.g, side-by-side). In some embodiments, a SD-IgAPP comprising a M1-M2-M3middle domain and an N-terminal domain (N) or part thereof demonstrates greater preference for IgAl (e.g., relative to IgA2) as compared with a relevant reference IgA protease. In some embodiments, a provided SD- IgAPP agent comprising a middle domain and an N-terminal domain or part thereof demonstrates greater proteolytic activity for IgAl compared to IgA2.

[0119] In some embodiments, a provided SD-IgAPP agent cleaves at V222-P223 in the IgAl and / or IgA2 hinge. In some embodiments, a provided SD-IgAPP agent cleaves at a site corresponding to V222-P223 in the IgAl hinge (according to canonical numbering).

[0120] Those skilled in the art will be aware of a variety of assays and technologies that may be useful to assess IgA proteolytic activity. In some embodiments, IgA proteolytic activity is assessed as described in Plaut & BachovchinAfeA Enzymol. 244: 137, 1994 (entitled “IgA-specific prolyl endopeptidases: serine type” and incorporated herein.

[0121] In some embodiments, proteolytic activity of an IgA protease polypeptide (e.g., an SD-IgAPP agent and / or of a reference IgA protease) may be assessed for the protease polypeptide in purified or pure form; in some embodiments, such proteolytic activity may be assessed in a complex context - e.g., in the context of a system (or extract or other fraction thereof, such as for example a cell [e.g., a bacterial cell or mammalian cell, optionally an engineered cell such as a cell engineered to express the protease polypeptide] extract or fraction thereof) in which the protease polypeptide was produced.

[0122] In many embodiments, proteolytic activity is assessed with respect to cleavage of human IgA (e.g., human IgAl).

[0123] In some embodiments, an IgA protease polypeptide agent (e.g., SD-IgAPP agent) is considered to have sufficient activity to be used as therapeutic agent if it has at least one unit activity, with one unit activity being equal to one microgram of human IgA cleaved per minute per mg of protease at 37°C.

[0124] Examples included herein (e.g.. Example 2) document usefulness of SD- IgAPP agents as described herein. For instance, Example 2 demonstrates that the middledomain (MD) of Cr-IgA protease retained full proteolytic capacity and cleaved the hinge region of human IgAl to the same degree as the full-length Cr-IgA protease (Figure 4).

[0125] Among other things, provided SD-IgAPP are useful in in vivo and in vitro methods of cleaving IgA, such as IgAl and / or IgA2.

[0126] In some embodiments, a method of cleaving IgA comprises contacting IgA (e.g., IgA in a sample) with an IgA protease polypeptide agent according to the present disclosure or a pharmaceutical composition according to the present disclosure, whereby the IgA protease polypeptide agent cleaves IgA.Characterization of IgA protease polypeptide agents:

[0127] In many embodiments, an IgA protease polypeptide agent according to the present disclosure cleaves IgA (e.g., cleaves IgAl, e.g., has IgAl cleavage activity that is reasonably or substantially comparable to that of a reference IgA protease such as a reference M64 IgA protease such as the C. ramosum M64 IgA protease of SEQ ID NO: 2).

[0128] In some embodiments, SD-IgAPP cleavage activity is comparable to a reference IgA protease. In some embodiments, SD-IgAPP IgA (e.g., IgAl) cleavage activity is measured. A variety of methods are available for measuring IgA (e.g., IgAl) cleavage activity. IgA (e.g., IgAl) cleavage activity' can be measured, for example, using cleavage gel assays (e.g., SDS PAGE) (see e.g., Example 3 herein) or Mass Spec (see e.g., Example 5 herein). In some embodiments, a method of cleaving IgA comprises contacting IgA (e.g., IgA in a sample) with an IgA protease polypeptide agent according to the present disclosure and hereafter separate the generated products be e.g., an SDS-PAGE gel.

[0129] In some embodiments, a provided IgA protease polypeptide agent (e.g, a provided SD-IgAPP agent) with a structure that is or comprises M1-M3has IgA cleavage activity characterized in that it cleaves the V222-P223 site in the IgA hinge.

[0130] As noted herein, in some embodiments, a provided IgA protease polypeptide agent (e.g. a provided SD-IgAPP agent) may show sufficient structural and functional similarity to a known reference M64 IgA protease (e.g., to the C. ramosum IgA M64 protease whose mature amino acid sequence is set forth in SEQ ID NO: 2) that one skilled in the art will appreciate its proper classification as an "M64 IgA protease polypeptide” as describedherein, but may nonetheless include certain amino acid sequence difference(s) and / or other modifications (e.g. attachment of pendant groups such as glycans, PEG moieties, etc., fusion with tag or other functional peptides - while preferably still preserving attribute(s) (such as smaller size than full-length) and / or benefit(s) as described herein relative to such reference IgA protease.

[0131] For example, in some embodiments, a provided IgA protease polypeptide agent (e.g., SD-IgAPP agent) may show significant sequence identity to a middle domain of C. ramosum IgA M64 protease, but may have one or a small number of modifications relative to such middle domain of C. ramosum IgA M64 protease, which modification(s) may be, for example, one or more alterations of amino acid sequence (such as, e.g.. truncations, substitutions, deletions, insertions, and combinations thereof).

[0132] In some embodiments, SD-IgAPP agents as described herein, and / or compositions that include them, may are characterized by one or more features or attributes (e.g., one or more physical and / or functional parameters) that, for example, may make them particularly useful in context(s) of interest (e.g., in therapeutic context(s) and / or in diagnostic and / or research contexts).

[0133] Relevant physical parameters may include, for example, size and / or stability. Relevant functional parameters may include, for example, protease cleavage ability and / or selectivity, toxicity, pharmacokinetic and / or pharmacodynamics performance, etc.

[0134] For example, in some embodiments, a provided SD-IgAPP agent may be characterized in that its size makes it particularly amenable to administration. A less space- filling protein may be more readily delivered than a bulky one. Alternatively or additionally , a shorter polypeptide may be more amenable to deliver)’ via administration of a nucleic acid, as shorter nucleic acids may be more readily incorporated into, for example, viral vectors or lipid-based delivery systems.

[0135] In some embodiments, a provided SD-IgAPP agent is characterized in that, when it is delivered to a cell or organism (e.g.. via administration of a composition that includes or encodes it), it displays an acceptable level of off-target and / or otherwise undesirable effects. Those skilled in the art will be aware of what level might be “acceptable” in a given context and / or in a particular assay. In some embodiments, suchlevel may be less than that observed when a relevant reference IgA protease polypeptide is delivered.

[0136] In some embodiments, a provided SD-IgAPP agents is characterized in that, when it is delivered to a cell or organism (e.g.. via administration of a composition that includes or encodes it), it displays an acceptable level of toxicity (e.g, is relatively non-toxic, e.g, relative to a reference IgA polypeptide).

[0137] In some embodiments, a provided SD-IgAPP agents is characterized in that, when it is delivered to a cell or organism (e.g.. via administration of a composition that includes or encodes it), it displays an acceptable level of immunogenicity (e.g. is relatively non-immunogenic, e g, relative to a reference IgA polypeptide).

[0138] In some embodiments, a provided SD-IgAPP agents is characterized by ease of manufacture, and / or by stability (e.g. under relevant reference conditions).

[0139] In some embodiments, a provided SD-IgAPP agents is characterized in that, when it is delivered to a cell or organism (e.g.. via administration of a composition that includes or encodes it), it displays a high level of bioavailability (e.g. relative to a reference IgA polypeptide).

[0140] In some embodiments, a provided SD-IgAPP agents is characterized in that, when it is delivered to a cell or organism (e.g. via administration of a composition that includes or encodes it), it displays a high half-life level of immunogenicity (e.g, relative to a reference IgA polypeptide).

[0141] Among other things, the present disclosure provides nucleic acid molecules that encode a SD-IgAPP agent as described herein.

[0142] In some embodiments, a provided nucleic acid is found in or isolated from a naturally occurring cell (e.g. a source cell). In some embodiments, a provided nucleic acid is engineered (e.g., designed and / or generated by the hand of man).

[0143] Those skilled in the art will be aware that, as described herein, certain IgA proteases are naturally expressed by microbial cells (e.g., bacterial cells); such cells may be considered '‘source cells” for purposes of the present disclosure. These bacteria may include,but are not limited to Clostridium ramosum, Haemophilus influenzae type 1 and 2, Neisseria meningitidis type 1 and 2, Neissseria gonorrhoeae, Neisseria lactamica, Prevotella melaninogenica, Streptococcus mitis biovar I, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus sanguis, and Ureaplasma urealyticum. In some embodiments, source cells are C. ramosum cells.

[0144] In some embodiments, a provided nucleic acid may have a nucleotide sequence that is codon optimized, e.g., for expression in a particular host cell of interest.Those skilled in the art are familiar with preferred codons for use in various different cell types of interest. In general, as is understood, codon optimization typically involves selecting a nucleic acid sequence for optimal expression in a host cell of interest by utilizing codons that are more frequently or most frequently used in genes of that host cell while maintaining native desired ammo acid sequence.

[0145] Various species exhibit particular preference for certain codons that encode a particular amino acid. Without wishing to be bound by any particular theory, codon preference (differences in codon usage between organisms) often correlates with mRNA translation efficiency in the relevant host cell, which is in turn believed to be dependent on, among other things, the properties of codons being translated and / or availability of particular transfer RNAs (tRNAs). The predominance of particular tRNAs in a cell may generally be a reflection of the codons used most frequently in polypeptide synthesis in that cell.Accordingly, genes may be tailored for optimal gene expression in a given cell type (e.g., organism) through codon optimization. Codon usage tables are available, for example, at the "Codon Usage Database" available at kazusa.orjp / codon / and these tables may be adapted in a number of ways. Computer algorithms for codon optimizing a particular sequence for expression in a particular subject or its cells, such as Gene Forge (Aptagen; Jacobus, PA), are also available.

[0146] In some embodiments, a provided nucleic acid is codon optimized for expression in bacterial (e.g., E. coll) cells. In some embodiments, a provided nucleic acid is codon optimized for expression in insect cells (e.g., via a baculovirus expression system). In some embodiments, a provided polynucleotide is codon optimized for expression in mammalian cells (e.g., CHO cells or other common mammalian production cells).

[0147] In some embodiments, a provided nucleic acid may be or comprise DNA, RNA, or a combination thereof. In some embodiments, a provided nucleic acid may be single stranded, double stranded, or may have some single stranded and some double- stranded regions.

[0148] In some embodiments, a provided nucleic acid may be used as a template, e.g., for primer extension, including for amplification (e.g., by polymerase chain reaction [“PCR”]), and / or for transcription (e.g., for in vitro transcription such as may be utilized to produce an RNA transcript for delivery to a cell that will express it and / or for in vivo transcription, such as by a cell into which such provided nucleic acid has been introduced). Vector constructs:

[0149] In some embodiments, a provided nucleic acid may be incorporated into a vector. In some embodiments, such vector may include one or more expression control elements (e.g., one or more of promoters, transcriptional regulator binding sites such as enhancers or repressor sites, transcriptional terminators, splice donor and / or acceptor sites, translation start sites, polyA tails, etc.), or a complement thereof. Alternatively or additionally, in some embodiments, such vector may include a replication site such as an origin of replication or a primer landing site, etc., or a complement thereof.

[0150] In some embodiments, a vector may include an insertion site, such as an integration site or a polycloning site (e.g., that may include recognition sequences for multiple restriction enzymes) so that the vector is adapted to receive heterologous (e.g., “payload”) nucleic acid(s), e.g., such as a provided nucleic acid encoding an SD-IgAPP agent, and, in some embodiments to permit, facilitate, or achieve expression and / or replication or other duplication thereof.

[0151] In some embodiments, a vector may include one or more detection and / or one or more selection markers, as is known in the art, e.g., to facilitate identification of cells that have received the vector.

[0152] Those skilled in the art are aware of a variety of vector systems, including vector systems particularly useful and / or adapted for introduction into certain cells (e.g., microbial, such as bacterial or yeast, cells, insect cells, mammalian cells, etc.), and / or forexpression (e.g., of heterologous nucleic acids) therein. For example, those skilled in the art are aware of a variety of plasmids, phage, viral vectors, etc., useful for delivering payload nucleic acids (e.g., encoding an SD-IgAPP agent as described herein) into particular cell(s) of interest in vitro and / or in vivo, and / or for expressing such payload nucleic acids in such cells.

[0153] In some particular embodiments, a provided nucleic acid encoding an SD- IgAPP agent as described herein is incorporated into a plasmid, for example, useful for introduction into and / or expression by, a bacterial cell (e.g., an E. coli cell), and / or insect cell, and / or by a yeast cell (e.g., an S. cerevisiae cell), and / or by a mammalian cell (e.g., a CHO cell, a BHK cell, a HEK293 cell, an NSO cell, etc.).

[0154] In some embodiments, a provided nucleic acid encoding an SD-IgAPP agent as described herein is incorporated into a viral vector such as, for example, a phage vector, a baculovirus vector, an AAV vector, etc.

[0155] In some embodiments, a provided nucleic acid encoding an SD-IgAPP agent as described herein is an RNA, e.g., an mRNA (e.g., an RNA encoding the SD-IgAPP without intervening intron sequences): in some such embodiments, such provided nucleic acid is produced by in vitro transcription and / or is associated with a delivery system (e.g., a lipid-based delivery system, such as a lipid nanoparticle {“LNP”}) as is known in the art.Pharmaceutical compositions

[0156] In some embodiments, a provided SD-IgAPP agent may be useful in medicine, e.g.. to treat a disease, disorder or condition as described herein (e.g., a disease, disorder or condition associated with IgA deposition).

[0157] The present disclosure provides pharmaceutical compositions that, when administered to a subject (e.g., a human subject), and particularly when administered to a subject suffering from a disease, disorder or condition associated with IgA deposition, deliver an SD-IgAPP agent as described herein to such subject. Thus, in some embodiments, the present disclosure provides pharmaceutical compositions that comprise or delivery an SD- IgAPP agent as described herein,

[0158] In some embodiments, a provided pharmaceutical composition comprises an SD-IgAPP agent (e.g., a “pro’" form or a “mature’" form thereol)- In some embodiments, aprovided pharmaceutical composition comprises a nucleic acid encoding an SD-IgAPP agent (e.g., a “pro” form or a “mature” form thereof); in some such embodiments, such nucleic acid is or comprises single stranded DNA (e.g., as in certain viral vectors); in some embodiments, such nucleic acid is or comprises double stranded DNA (e.g., as in certain viral vectors and / or certain plasmids); in some embodiments, such nucleic acid is or comprises RNA (e.g., as in certain viral vectors and / or as in mRNA therapeutics), etc.

[0159] Typically, a pharmaceutical composition includes an active agent (e.g., an SD- IgAPP agent as described herein, or a nucleic acid that encodes it) in combination with one or more pharmaceutically acceptable carriers or excipients such as, for example one or more buffers, diluents, fillers, salts, solubilizers, stabilizers, and / or other materials as is known in the art. Those skilled in the art will be aware of a variety of carrier components appropriate to a particular active type (e.g., polypeptide versus nucleic acid, viral vector vs plasmid versus RNA, etc.) and / or route of administration (e.g., parenteral, enteral, etc.)

[0160] In some embodiments, a provided pharmaceutical composition may comprise or deliver an SD-IgAPP agent in a form complexed with an antibody agent (i.e., in an immuno-complex). Without wishing to be bound by any particular theory, it is proposed that such an immuno-complex may be particularly useful for treatment of diseases characterized by IgAl deposition in the kidney since a large immuno-complex is believed to lodge in the renal glomerulus upon administration.

[0161] In some embodiments, a pharmaceutical composition, or set thereof, may comprise or deliver two or more different SD-IgAPP agents, so that such agents may be administered in combination (e.g., substantially simultaneously or sequentially) to subject(s).

[0162] In some embodiments, a pharmaceutical composition may contain one or more agents that, for example, may improve stability of the composition and / or its active agent (e.g., to particular storage conditions and / or period(s) of time), facilitate delivery of the composition and / or its active agent, and / or otherwise enhance effectiveness (and / or reduce one or more undesirable side effects) of the active agent or composition once administered.

[0163] Alternatively or additionally, in some embodiments, a provided pharmaceutical composition may comprise or deliver another active agent in addition to an SD-IgAPP agent as described herein.Uses and Methods

[0164] In some embodiments, a provided SD-IgAPP agent may be useful as a reagent (e.g., a biotech reagent). In some embodiments, the present disclosure provides technologies (e.g.. compositions, mixtures, methods, and / or systems, etc.) comprising a SD-IgAPP.Among other things, the present disclosure provides SD-IgAPP compositions (e.g., compositions comprising one or more SD-IgAPPs), as well as SD-IgAPP reaction mixtures (e.g., comprising SD-IgAPP and IgA). Compositions, mixtures, methods and kits according to the present disclosure can be used in a number of applications, such as therapeutic and / or non-therapeutic applications. In some embodiments, methods using SD-IgAPPs are useful in therapeutic and / or non-therapeutic applications. In some embodiments, compositions, mixtures, methods, kits and uses described herein may be used for non-therapeutic purposes, such as diagnostic purposes. In some embodiments, a SD-IgAPP is used in a in vitro assay. In some embodiments, a SD IgAPP is used in an in vitro cleavage assay.

[0165] In some embodiments, the present disclosure provides methods of cleaving IgA, the methods comprise: (a), contacting a sample comprising IgA with an IgA protease polypeptide agent as provided herein whereby the IgA protease polypeptide agent cleaves IgA. In some embodiments, an IgA is an IgAl. In some embodiments, an SD-IgAPP agent cleaves the IgA comparably to a relevant reference IgA protease.Host cells

[0166] Among other things, the present disclosure provides host cells that express or otherwise comprise (e.g.. have expressed) a SD-IgAPP agent as described herein and / or a nucleic acid molecule encoding such agent.

[0167] In some embodiments, the host cell expresses a SD-IgAPP agent according to the present disclosure and / or a nucleic acid molecule encoding SD-IgAPP agent according to the present disclosure.

[0168] In some embodiments, a host cell is a bacterial cell (e.g.. an E. coli cell), an insect cell, a mammalian cell (e.g.. a rodent cell or a primate cell such as a human cell, non- limiting examples hereof are e.g.. a CHO cell, a BHK cell, a HEK293 cell, an NS0 cell, ete.), a plant cell, or a yeast cell (e.g., an S. cerevisicte cell).A variety of bacteria produce IgA proteases and / or homologues of IgA proteases that may be useful (e.g., as reference IgA protease polypeptides) in the present disclosure. These bacteria include, but are not limited to Clostridium ramosum, Haemophilus influenzae type 1 and 2, Neisseria meningitidis type 1 and 2, Neissseria gonorrhoeae. Neisseria lactamica, Prevotella melaninogenica, Streptococcus mitis biovar I, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus sanguis, and Ureaplasma urealyticum.

[0169] In some embodiments, a host cell comprises a vector construct as described herein (e.g, a vector construct that includes and / or expresses a nucleic acid encoding an SD- IgAPP agent as described herein). Those skilled in the art are aware of a variety of technologies useful for introducing appropriate vector construct(s) into relevant host cells.

[0170] To give but a few examples, in some embodiments, vector constructs may be introduced to appropriate bacterial cells by infection using bacteriophage vector particles such as lambda or Ml 3, or by any of a number of transformation methods for plasmid vectors or for bacteriophage DNA. For example, standard calcium-chloride-mediated bacterial transformation is still commonly used to introduce naked DNA to bacteria (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), but electroporation may also be used (Ausubel et al.. Current Protocols in Molecular Biology, (1988), (John Wiley & Sons, Inc., NY, NY)). Methods of transforming bacterial host cells with e.g., vectors expressing a SD-IgAPP according to the present disclosure, inducing expression of proteins encoded by such expression vectors, and isolation and purification of such proteins are known in the art. See, for example, U.S. Pat. No.7,407,653 and Sambrook et al. (2001), the contents of both of which are incorporated by reference in their entirety.Use of host cells

[0171] Those skilled in the art, reading the present disclosure, will recognize a variety of uses for provided host cells as described herein.

[0172] Among other things, provided host cells may be useful to produce a provided SD-IgAPP agent, and / or a nucleic acid encoding it. In some embodiments, the present disclosure provides methods of producing a SD-IgAPP agent, or a nucleic acid that encodes it, by cultivating a cell or organism (e.g., culturing a cell) that contains and / or expresses (e.g., a cell that secretes a SD-IgAPP agent) it.

[0173] Thus, among other things, in some embodiments, the present disclosure provides populations of cells (e.g. a cell culture) in which an SD-IgAPP agent, or nucleic acid that encodes it, is present at or above a particular desired level. In some embodiments, such population of cells expresses or has expressed the SD-IgAPP agent; in some embodiments, such population of cells has secreted the SD-IgAPP agent. In some embodiments, the present disclosure provides a cell culture, and / or a medium therefrom, comprising an SD-IgAPP agent at or above a desired level (e.g., a desired concentration). In some embodiments, a desired level (e.g., of an SD-IgAPP agent and / or of a nucleic acid encoding an SD-IgAPP agent) is a level appropriate for manufacture of a pharmaceutical composition that comprises or delivers such SD-IgAPP agent.In some embodiments, an SD-IgAPP agent, and / or a nucleic acid encoding it, may be produced by culturing host cells that comprise and / or express such SD-IgAPP agent and / or nucleic acid encoding it. In some embodiments, a step of inducing expression of such SD- IgAPP agent is performed. Alternatively or additionally, in some embodiments, cell(s) are separated from medium and SD-IgAPP agent is isolated from the medium and / or cell(s) are lysed and SD-IgAPP agent and / or nucleic acid encoding it is isolated thereafter. Those skilled in the art are familiar with a variety of technologies for isolating components from cell media and / or from lysed cells.Diseases, Disorders, and Conditions

[0174] Those skilled in the art, reading the present disclosure, will appreciate that provided SD-IgAPP agents (and / or nucleic acid(s) encoding such and / or compositions that comprise and / or deliver either), may be useful in medicine, and particularly in treatment of certain diseases, disorders and conditions such as one or more diseases, disorders or conditions associated with IgA deposits.

[0175] Immunoglobulin Al (IgAl) deposition in human tissues and organs is a characteristic of several human diseases, including IgA nephropathy, dermatitis herpetiformis (DH), and Henoch-Schoenlein purpura (HS). IgAl deposition is responsible for a variety of clinical manifestations such as renal failure, skin blistering, rash, arthritis, gastrointestinal bleeding and abdominal pain.

[0176] Available treatment options for patients that present with abnormal IgAl deposition include administration of corticosteroids that have immunosuppressive and anti-inflammatory properties, dietary fish oil supplements that reduce renal inflammation, and angiotensin converting enzyme inhibitors that reduce the risk of progressive renal disease and renal failure. Such treatments do not directly act on IgAl deposits in tissue or organs and do not address the IgAl deposit removal.

[0177] The present disclosure incorporates the identification of a middle domain of an IgA protease, such as an IgA M64 protease (e.g., IgA M64 C. ramosum) in the design of novel SD-IgAPP agents that have therapeutic value. In some embodiments of the present disclosure, SD-IgAPP agents, and / or nucleic acids that encode them and / or compositions that comprise and / or deliver either of the foregoing, are used to treat diseases characterized by IgAl deposition. In some embodiments, SD-IgAPP agents, and / or nucleic acids that encode them and / or compositions that comprise and / or deliver either of the foregoing, comprise a middle domain of an IgA protease, such as an IgA M64 protease (e.g., IgA M64 C. ramosum), and, optionally, further comprises an N-terminal domain of an IgA protease or part thereof (e.g., IgA M64 C. ramosum) and / or a C-terminal domain of an IgA protease or part thereof (e.g., IgA M64 C. ramosum). Such embodiments can demonstrate greater preference for IgAl (e.g., relative to IgA2) as compared with a relevant reference IgA protease and may therefore be particular useful in treating IgAl deposition diseases, such as but not limited to IgA nephropathy.

[0178] In some embodiments, a provided SD-IgAPP is delivered (e.g., by administration of a pharmaceutical composition as described herein that comprises or delivers such agent, or a nucleic acid that encodes it) to a subject suffering from or susceptible to a disease, disorder or condition associated with IgA deposition as described herein, or otherwise displaying abnormal IgA deposit(s) (e.g., in kidneys, blood vessels, skin, or elsewhere).

[0179] In some embodiments, SD-IgAPP is delivered to individuals having an IgA deposition disease. In some embodiments, the individual is a human. In some embodiments, the IgA is IgAl. Is some embodiments, the IgAl is human IgAl.

[0180] In some embodiments, the disclosure provides a method for treating IgA nephropathy by delivering to a patient in need of such treatment a SD-IgAPP according to the present disclosure. IgA nephropathy is a disease of the kidney. The disease is considered to be an immune-complex-mediated glomerulonephritis, which is characterized by granulardeposition of IgAl in the glomerular mesangial areas. Nephropathy results and is defined by proliferative changes in the glomerular mesangial cells. IgA nephropathy is one of the most common types of chronic glomerulonephritis and a frequent cause of end-stage renal disease.

[0181] In some embodiments, the disclosure provides a method for treating dermatitis herpetiformis (DH) by administering to a patient in need of such treatment a SD-IgAPP according to the present disclosure. Dermatitis herpetiformis is a chronic blistering skin disease associated with deposits of IgAl at the dermal -epi dermal junction (Hall, RP & T.J. Lawley, J. Immunol. (1985) 135(3): 1760-5). DH patients have granular IgAl deposits and often have an associated gluten-sensitive enteropathy (GSE).

[0182] In some embodiments, the disclosure provides a method for treating Henoch- Schoenlein purpura (HS) by administering to a patient in need of such treatment a SD-IgAPP according to the present disclosure. Henoch-Schoenlein purpura is a skin, blood vessel and kidney disease. HSP is characterized by deposition of IgAl containing immune complexes in tissue. The disease is diagnosed by observing evidence of IgAl deposition in the skin tissue or kidney via immunofluorescence microscopy. The clinical manifestations typically include rash; arthralgias; abdominal pain; and renal disease.

[0183] In some embodiments, SD-IgAPP is delivered to an individual after kidney transplantation. In some embodiments, SD-IgAPP is delivered to an individual after kidney transplantation to prevent IgA (e.g., IgAl) deposition in the transplanted kidney. Without wishing to be bound by any particular theory, it is proposed that delivery of an SD-IgAPP to an individual having kidney transplantation preserves the function of the donor kidney, which may avoid a further kidney transplantation.Administration

[0184] In some embodiments of the disclosure, provided are methods comprising a step of delivering to an individual suffering from or susceptible to abnormal IgA deposits (e.g., IgAl deposits) an SD-IgAPP agent as described herein. In some embodiments, SD- IgAPP agent is delivered in an amount effective to reduce IgAl deposits. In some embodiments, the individual has deposits of human IgAl.

[0185] Deliver}' of an SD-IgAPP agent can be achieved e.g., by administration of a pharmaceutical composition as described herein, such as a pharmaceutical composition that comprises the SD-IgAPP agent or a nucleic acid that encodes it, for example via oral ingestion, inhalation, topical application or parenteral administration (e.g., cutaneous, subcutaneous, intraperitoneal, intramuscular or intravenous injection). In many embodiments, administration is by intravenous or intramuscular injection. In some embodiments, administration may be by local administration, e.g., to a site of IgA deposition. In some embodiments, local administration may be or comprise topical administration (e.g., to the skin) or parenteral administration (e.g., by injection to a site of deposition such as to the kidney).

[0186] The compositions containing the therapeutic polypeptide agent of the present disclosure can be administered intravenously, as by injection of a unit dose, for example.

[0187] Those skilled in the art will be aware of typical guiding principles for formulation of pharmaceutical compositions for administration by a particular route. For example, pharmaceutical compositions for parenteral injection typically comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol and the like), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil) and injectable organic esters such as ethyl oleate. Desired fluidity may be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and / or by the use of surfactants. In some embodiments, pharmaceutical compositions formulated for parenteral administration may contain components such as preservatives, wetting agents, emulsifying agents and dispersing agents, and / or one or more components that reduce immunogenicity, such as be shielding an immunogenic determinant (e.g., an epitope) of an administered composition or of an active agent (e.g., an SD-IgAPP agent) that it comprises or delivers. In some embodiments, it may be desirable to include one or more agents such as paraben, chlorobutanol, phenol sorbic acid and the like, which may have antimicrobial properties. Alternatively or additionally, in some embodiments, it may be desirable to include one or more isotonic agents such as sugars, sodium chloride and the like. In someembodiments, particularly if prolonged absorption of or from an injectable pharmaceutical composition is desired, one or more agents such as aluminum monostearate and gelatin, which can delay absorption, may be included. Alternatively or additionally, in some embodiments an injectable depot formulation may be generated by forming microencapsule matrices of a biodegradable polymer such as a polylactide-polyglycolide, a poly(orthoester) and / or a poly(anhydride). Those skilled in the art are aware that adjustments to ratio of active agent to polymer and / or nature of the particular polymer employed can adjust the rate of release of the active agent from such a depot formulation. Alternatively or additionally, in some embodiments, a depot injectable formulation can be prepared by entrapping an active agent in liposomes or microemulsions which are compatible with body tissues. Injectable injectable formulations may be sterilized, for example, by filtration through a bacterial- retaining filter or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable mediajust prior to use.

[0188] In some embodiments, deliver}' of an SD-IgAPP agent can be achieved e.g., by administration of a pharmaceutical composition as described herein, such as a pharmaceutical composition that comprises the SD-IgAPP agent or a nucleic acid that encodes it, may be oral, rectal, ophthalmic (including intravitreal or intracameral). nasal, topical (including buccal and sublingual), intrauterine, vaginal or parenteral (including subcutaneous, intraperitoneal, intramuscular, intravenous, intradermal, intracranial, intratracheal, and epidural). Those skilled in the art will be aware of typical guiding principles for formulation of pharmaceutical compositions for administration by such routes. For example, such techniques may include the step of bringing into association the SD-IgAPP agent or a nucleic acid that encodes it and the pharmaceutical carrier(s) or excipient(s). In some embodiments, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0189] Topical administration, in which a composition is brought in contact with tissue(s), may be suitable for dermatitis herpetiformis. "Contacting" is meant to include not only topical application, but also those modes of delivery that introduce a composition or agent into tissues, or into cells of the tissues.Dosing Regimens:

[0190] In some embodiments, polypeptide agents (SD-IgAPP agents) and / or nucleic acid molecules and / or pharmaceutical compositions thereof are administered in a single dose in the range of 100 pg -10 mg / kg body weight. In some embodiments, a single dose is in the range of 1 pg-100 pg / kg body weight. This dosage may be, for example, as considered appropriate by the treating physician.

[0191] Amounts of IgA protease polypeptide agents (SD-IgAPP agents) administered in a single dose may depend on the nature and / or severity of the condition being treated and / or on the nature of prior treatments that the patient has undergone. In some embodiments, the attending phy sician decides the amount of IgA protease polypeptide agent with which to treat each individual patient. In some embodiments, the attending physician initially administers low doses of IgA protease polypeptide agent(s) of the present disclosure and observe the patient's response. In some embodiments, larger doses are administered until an optimal therapeutic effect is obtained for the patient, after which dosage is not increased further.Combination therapies:

[0192] According to the present disclosure, SD-IgAPP agents may be administered in combination with one or more other pharmaceutical agents. For example, SD-IgAPP agent may be administered in combination with one or more other therapeutic agents for IgAl deposition diseases (such as agents that ameliorate symptoms of IgAl deposition diseases), and / or in combination with one or more other pharmaceutical agents.

[0193] In some embodiments, pharmaceutical compositions are administered via a single dose regimen. In some embodiments, pharmaceutical compositions are administered via a multi-dose regimen. In some embodiments, pharmaceutical compositions are administered via a single dose regimen with one or more cycle(s) of doses.

[0194] In some embodiments, SD-IgAPP agents are administered with one or more immunomodulators. In some embodiments, a pharmaceutical agent is an immune stimulatory agent. In some embodiments, a pharmaceutical agent is an immune inhibitory agent. In some embodiments, a pharmaceutical agent suppress an adaptive immune response. In some embodiments, SD-IgAPP agents are administered with an agent that prevent anti-drugantibodies (AD As). In some embodiments, SD-IgAPP agents are administered with nanoparticles encapsulating rapamycin (e.g., ImmTOR® or SVP-rapamycin). In some embodiments, SD-IgAPP agents are administered with IL-2 and nanoparticles encapsulating rapamycin (e.g., ImmTOR-IL™).Exemplification

[0195] The present disclosure will be better understood in connection with the following examples, which are intended as an illustration only and not limiting of the scope of the disclosure. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and such changes and modifications including, without limitation, those relating to the chemical structures, substituents, derivatives, compositions and / or methods of the disclosure may be made without departing from the spirit of the disclosure and the scope of the appended claims.Example 1: Structure, production and purification of certain peptides (e.g., exemplary IgA protease polypeptides).

[0196] The present Example documents Cr-IgA protease polypeptide structure and domain architecture.

[0197] A predicted structure of C. ramosum IgA protease (Cr-IgAP) (corresponding to amino acid residues 31-1195 in SEQ ID NO: 1 corresponding to SEQ ID NO: 2), along with its domain architecture, is depicted in Figure 2. The N-terminal domain (corresponding to amino acid residues 31-313 in SEQ ID NO: 1) is rendered in blue, the middle domain (corresponding to amino acid residues 314-807 in SEQ ID NO: I) is rendered in green and the C-terminal domain (corresponding to amino acid residues 808-1195 in SEQ ID NO: 1) is rendered in yellow, orange and red.

[0198] Soluble polypeptides representing each of the follow ing domains were generated as soluble proteins: i) full-length structure (FL); ii) N-terminal domain (NTD); iii) middle domain (MD, which includes an HEXXH motif); and iv) C-terminal domain (CTD, which includes an IgG-like CBDX domain).

[0199] Specifically, codon-optimized nucleotide sequences were developed that encoded each of these domains; these codon-optimized sequences were cloned into plasmid vectors carrying N-terminus His6-tagged Small Ubiquitin-like Modifier (SUMO) domain sequences, so that domain-fusion proteins were generated. These plasmids were transformed into BL21 (DE3) E. coli cells. Saturated overnight cultures of these BL21 (DE3) cells were grown and used (50mL) to inoculate IL of ZYP-5052 auto induction media. The cells were grown for 2 days at 14°C, while being shaken at 180 rpm. Cells were harvested by centrifugation and the pellet was stored at -80C until purification. The resultant cell pellet was solubilized in 25mM HEPES, pH 7.5 + 0.5M NaCl + lOmM imidazole + ImM TCEP (Buffer A) and passaged through a French Press twice. The resultant solution was clarified by centrifugation and the clarified lysate incubated with with NiNTA resin. The NiNTA resin was washed with IL 25mM HEPES, pH 7.5 + 0.1% (v / v) IgePal CA630 + lOmM imidazole + ImM TCEP followed by IL Buffer A.

[0200] Expressed fusion proteins were eluted with Buffer A supplemented with 0.3M imidazole, and were cut with SUMO protease overnight (Cth) so that the relevant domain was released from the N-terminal SUMO sequences. The protein digest containing the protein of interest, the SUMO fusion, and SUMO protease, was dialyzed into 25mM HEPES, pH 7.5 + 0.5M NaCl + ImM TCEP twice overnight. The protein solution was incubated with NiNTA resin and loaded into a chromatography column to remove the SUMO fusion and SUMO protease from the protein digest. The NiNTA flowthrough was concentrated to <lmL and injected onto S75 gel filtration column equilibrated in 25mM HEPES, pH 7.5 + ImM TCEP. The purified IgA protease was concentrated and stored at -80°C.

[0201] Figure 3 shows SDS-gels of the expression of the full-length (Figure 3A), N- terminal domain (NTD) (Figure 3B), middle domain (MD) (Figure 3C), and C-terminal domain (CTD) (Figure 3D) Cr-IgAP polypeptides. Figure 3 depicts that the CTD undergoes proteolysis into two components consistent with extended nature of its predicted structure (Figure 3D).Example 2: Structure of an exemplary Cr-IgAPP

[0202] The present Example documents Δ 15 middle domain-C -terminal domain I329'876(Δ15 MD+CTD1) IgA protease polypeptide structure (SEQ ID NO: 30). The present example demonstrates the location of the active site is within the MD.

[0203] The crystal structure of Al 5 MD+CTD1 Cr- IgAP shows a conserved M64 fold with additional C-terminal beta-sheet domains. The two metal-binding sites are conserved, and residues involved in those sites are rendered as cylinders.

[0204] The most C-terminal beta-sheet-containing domain (CTD1; gray) is required for crystallization but is not present in the most-active, minimized Δ15 MD construct (black) (Figure 4A). The 1.60 A structure (depicted with 2Fo-Fc density at 1.3 o; fine mesh) shows a somewhat-flexible active site whereby the active-site zinc is coordinated by members of the HEXXH motif and downstream D520 (Figure 4B). The location of metal ions were confirmed by anomalous difference peaks (depicted at 5.0 o; thick chicken wire). The loop containing D520 and E521 bridges the active site to a nearby pocket where the second zinc- binding site is located (maps are contoured at the same o values) (Figure 4C).

[0205] The present example demonstrates the structure of Al 5 MD+CTD1 IgAPP. The Al 5 MD+CTD1 illustrates the location of the active site and the coordination of a catalytically Zn ion by the HEXXH motif along with a secondary Zn ion bound near the active site and coordinated by 3 cysteine residues and 1 glutamate’.Example 3: Assessment of protease activity

[0206] The present Example demonstrates, surprisingly, that the middle domain (MD) of Cr-IgA protease retained full proteolytic capacity and cleaved the hinge region of IgAl to the same degree as reference HIN-IgA protease (Figure 5).Materials and Methods

[0207] IgAl from human myeloma plasma was purchased from Athens Research and Technology was incubated with 20 pg / ml protease or a domain thereof for one hour at 37 °C or O / N at 37 °C to cleave the IgAl heavy polypeptide chain, thereby measuring enzy me activity7. Products of IgAl hydrolysis were separated on SDS / PAGE gels. The Fc cleavagefragments were detected by their size comparison with MW standards run on the same gel. IgAl control is a purified human myeloma protein; MW are molecular weight standards.Results

[0208] The present Example shows that MD (SEQ ID NO: 22) demonstrates proteolytic activity against IgAl (Figure 5). Specifically, the present Example demonstrates that MD cleaves the hinge region of IgAl, thus showing that MD is sufficient for the protease to express proteolytic activity against IgAl. In other words, the N-terminal domain and the C- terminal domain of Cr-IgA protease may be dispensable for the protease to cleave IgAl. Note that the extent of digestion of IgAl by a reference IgAP (H. Influenzae IgAP) and MD observed in Figure 5 are the same. Thus, this Example identifies a C. ramosum IgA protease middle domain that demonstrates proteolytic activity towards IgA, such as IgAl and / or IgA2 comparable to that of full-length C. ramosum IgA protease.

[0209] The architecture studies suggest that up to 22% of the amino acids can be removed from the N-terminal end, and up to 31 % of the amino acids can be removed from the C-terminal end of C. ramosum, together removing up to 53% amino acids of C. ramosum IgA protease. Percentage calculation is based on the length of the mature secreted form of the polypeptide, which lacks the N-terminal signal sequence. Thus, structural architecture analysis studies described herein reveal that the C-terminal domain (corresponding approximately to amino acids 808-1234 of SEQ ID NO: 1) is dispensable. Furthermore, the present studies also reveal that the N-terminal domain corresponding to the amino acids 31- 313 of SEQ ID NO: 1 is dispensable. The present disclosure therefore surprisingly demonstrates that both the C-terminal domain (corresponding approximately to amino acids 808-1234 of SEQ ID NO: 1) and the N-terminal domain (corresponding approximately to amino acids 31-313 of SEQ ID NO: 1) are dispensable.Example 4: Production and purification of NTD-MD and NTD-MD-CTD1 / 2

[0210] A construct containing both the NTD and MD (Cr-NTD-MD) was synthesized (SEQ ID NO: 11). A construct containing the NTD, MD and the first A of the C-terminal domain (NTD-MD-CTD1 / 2) will be generated. NTD-MD-CTD1 / 2 is resistant to proteolytic degradation and contains a folded carbohydrate binding domain (CBD). NTD-MD-CTD1 / 2 is generated to examine if the CBD domain contributes to IgA substrate selectivity by bindingto glycans on IgAl or IgA2. Expression and purification of both constructs (i.e., NTD-MD and NTD-MD-CTD1 / 2) will be performed.Example 5: IgAl cleavage assessment by mass spectrometry

[0211] The present Example demonstrates that neither the full length (FL) Cr-IgA protease nor the middle domain (MD, SEQ ID NO: 22) of Cr-IgA protease cleaves the IgA hinge in isolation when assessed by MS as described in this Example. However, Cr-MD cleaves the IgA hinge in the context of the IgAl structure (Example 3 and Figure 5).Material and methods

[0212] The ability of FL and MD to cleave the synthetic hinge peptide in isolation via mass spectrometry (MS) was tested. The following synthetically generated unlabeled hinge peptide was generated VPCPVPST. The chemical agent Alpha-Cyano-4-hydroxy cinnamic acid (HCCA) was used as a matrix, allowing the sample to ionize and be characterized by MALDI-TOF MS. HCCA alone is used as a blank sample. 10 nM FL and MD. in HCCA matrix, were added to synthetic generated hinge peptides (15 uM) and incubated at 37°C for >12h.Results

[0213] Figures 7A-C demonstrate different mass regimes of the entire MS data set. Different spectra of the HCCA matrix alone (blank), untreated isolated hinge peptide, isolated hinge peptide with MD, isolated hinge peptide with FL are shown in Figure 7A-C. The spectra for the untreated isolated hinge peptide with and without MD or FL are identical showing that MD and FL are not characterized to cleave the isolated synthetic IgAl / 2 hinge peptide. Thus, neither the full-length (FL) protease nor the MD construct exhibited any proteolytic activity against hinge peptides encompassing the know n IgAl / 2 cleavage sequence (VPCP^VPST) as assessed by mass spectrometry approaches (Figures 7A-C).Example 6: IgAl cleavage activity

[0214] The present Example demonstrates that truncated IgAP peptides, including Cr- Δ15 MD IgAPP and Cr-MD IgAPP, show improved IgAl cleavage activity compared to Cr- FL IgAPP and Cr-NTD-MD IgAPP.Gel-based cleavage assayMaterial and methods

[0215] IgA2 cleavage activity was characterized using a cleavage gel assay for the following IgAPP and control IgAP:• Full length314195Cr-IgA protease (Cr-FL);• N-terminal domain-middle31'807domain of Cr-IgA protease (Cr-NTD-MD):• Middle domain314'807of Cr-IgA protease (Cr-MD);• Δ15 Middle domain329'807Cr-IgA protease (Cr-Δ15 MD); and• Haemophilus influenzae IgA protease (HIN IgAP).

[0216] 40 nM of enzyme construct (Cr-Δ15 MD, Cr-MD, Cr-FL and Cr-NTD-MD) were incubated with ~1.3 uM IgAl obtained from Athens Biotech, Athens GA at 37C in 25 mM HEPES, pH 7.5. Samples of the assay mix were quenched by immersion in 6x SDS loading buffer at various time points between 15 min and 1 hour. The cleavage products were visualized by standard SDS-gel electrophoresis.Results

[0217] The results confirm that the middle domain of C. ramosum (Cr-MD) is proteolytic active and is capable of cleaving IgAl and (e.g., especially after being contacted for more than 15 minutes) (Figures 8 A). Quantitative comparisons between the IgA protease peptides show that, under these reaction conditions, IgAPP Cr-Δ15 MD and IgAPP Cr- MD show a high IgA cleaving rate comparable to Cr-FL IgAPP and Cr-NTD-MD IgAPP. Additionally, the most truncated IgAPP (Cr-Δ15 MD) cleaves IgA faster than the Cr-FL IgA protease (Figure 8A), the Cr-NTD-MD (Figure 8B), and the Cr-MD (Figure 8C). The Cr-Al 5 MD has comparable IgAl cleavage activity to the reference Haemophilus influenzae IgAP (Figure 8D).Gel-based Kinetic AssaysMaterials and methods

[0218] 10 pM IgAl was incubated with 0.5 nM C. ramosum Δ15 MD IgAP (Cr-Δ15MD). Samples were taken at an hour and quenched by the addition of SDS-PAGE loading dye and heating at 95°C. The initial steady-state rate at a single concentration of IgAl was measured via a discontinuous gel-based assay.Results

[0219] The appearance of the larger cleaved heavy chain fragment (arrow) was quantified using densitometry and normalized to the intensity of the light chain (bottom band) in each lane to account for loading and concentration differences (Figure 11 A). Initial rates were then extracted by applying linear regression to the band intensity over time, which was converted to the percentage / molanty of IgAl cleaved with a standard curve (standard curv e is not shown) (Figure 11B).

[0220] The enzymes were also assessed at longer incubation times whereby the percentage of IgAl cleaved exceeded the typical 10% for initial rates. Even at high concentrations of product, the enzymes did not deviate from linearity . The assay was also verified in human serum and was shown to behave the same as in buffer (25 mM HEPES, pH 7.5).

[0221] This assay was repeated using seven different concentrations of IgAl at a fixed enzyme concentration to obtain a Michaelis-Menten curve (Figure 11C).

[0222] Vmax and Km values for C. ramosum N-terminal domain in conjunction with the middle domain (Cr-NTD-MD), the middle domain (Cr-MD) and N-terminally truncated Δ15 middle domain (Cr-Δ15 MD) IgAPP are shown in the table below.Table 1

[0223] These results show that the most-truncated construct, Al 5 MD IgA protease (Al 5 MD329-807), has the highest activity out of all the truncated IgAPP for all IgAl concentrations.Example 7: IgA2 cleavage activity

[0224] The present Example demonstrates that Cr-MD is capable of cleaving IgA2 (e.g., especially after Cr-MD and IgA2 are being contacted for more than 15 minutes).Material and methods

[0225] IgAl cleavage activity was characterized using a cleavage gel assay for the following IgAPP:• Full length31 1195Cr-IgA protease (Cr-FL);• N-terminal domain-middle domain31-807of Cr-IgA protease (Cr-NTD-MD); and• Middle domain314-807of Cr-IgA protease (Cr-MD).

[0226] 40 nM of enzyme construct (Cr-MD, Cr-FL and Cr-NTD-MD) were incubated with ~1.3 uM IgA2 obtained from Athens Biotech, Athens GA at 37C in 25 mM HEPES, pH 7.5. Samples of the assay mix were quenched by immersion in 6x SDS loading buffer at various time points between 15 min and 1 hour. The cleavage products were visualized by standard SDS-gel electrophoresis.Results

[0227] Qualitative comparisons show that the tested C. ramosum IgAP polypeptides are capable of cleaving IgA2 to varying degrees. Similar to the observations for IgAl cleavage (Figures 8A-D), the more truncated IgAPP (Cr-MD) shows higher IgA2 activity7comparable to Cr-FL IgAPP (Figure 9A) and Cr-NTD-MD IgAPP (Figure 9B). Figure 8Band Figure 9B furthermore show that when Cr-MD is complexed with Cr-NTD it specifically cleaves IgAl to a larger extent than IgA2, hereby showing selectivity for IgAl.Example 8: IgGl and IgG2 cleavage activity

[0228] The present Example demonstrated that none of the tested IgAPP, including Cr-MD IgAPP, are capable of cleaving IgGl and IgG2.Material and Methods

[0229] IgGl and IgG2 cleavage activity were characterized using a cleavage gel assay for the following IgAPP:• Full length314195Cr-IgA protease (Cr-FL);• N-terminal domain-middle31'807domain of Cr-IgA protease (Cr-NTD-MD); and• Middle domain314'807of Cr-IgA protease (Cr-MD).

[0230] 1.3 uM IgGl or IgG2 w as incubated with 40 nM IgAPP at 37°C. Samples were taken at an hour and quenched by the addition of SDS-PAGE loading dye and heating at 95°C.Results

[0231] The C. ramosum full length (Cr-FL), middle domain (Cr-MD), and N-terminal domain coupled with the middle domain (Cr-NTD-MD) IgAPP do not cleave IgGl or IgG2, the main subclasses and antibodies in the blood, at prolonged incubations in standard assay (Figures 10A-C).Example 9: Co-crystal complexes

[0232] Co-crystal complexes of CR-IgAP with intact IgAl and IgA2, as well as Fc and Fab fragments, will be generated. Co-crystals with inactive HAXXH variants of the MD- CR-IgAP construct may aid in designing a proteolytically active SD-IgAPP having selectivity for only IgAl.Example 10: Therapeutic effect

[0233] The therapeutic effect of SD-IgAPP agents for the treatment of IgA Nephropathy, dermatitis herpetiformis or Henoch-Schoenlein purpura can be tested in amouse model for IgA Nephropathy, dermatitis herpetiformis or Henoch-Schoenlein purpura, respectively. Useful mouse models have been described, for example, Lamm ME, Emancipator SN, Robinson JK, Yamashita M, Fujioka H, Qiu J, Plaut AG. Microbial IgA protease removes IgA immune complexes from mouse glomeruli in vivo: potential therapy for IgA nephropathy. Am J Pathol. 2008 Jan;172(l):31-6. Doi: 10.2353 / ajpath.2008.070131. Epub 2007 Dec 28. PMID: 18165266; PMCID: PMC2189629.Example 11: BLAST DATA and alignment data

[0234] The BLASTP program and nr-clustered(experimental) database were used to generated BLAST and alignment data for the middle domain of C. ramosum IgA protease (SEQ ID NO: 22). Figure 6A-H shows the alignment data and BLAST results.

[0235] The data define a conserved domain within predicted M64 IgA proteases found in other bacteria. Bacteria with a conserved CR-like middle domain structure were found to have at least 50% sequence coverage with the middle domain of C. ramosum IgA protease (SEQ ID NO: 4). Exemplary species having at least 50% coverage are Clostridales, Eubacterium, Caprococcus, Roseburia, Dorea, Oscillibacter, and Anaerotruncus. All belonging to the Firmicutes that have a larger middle domain. Moreover, the analysis reveals that various other proteins, including some proteins of unknown functions, share relevant sequences. The present disclosure therefore teaches that these proteins may act as IgA proteases (e.g., as M64-type IgA proteases); such proteins (and variants that preserve or otherwise include an M64 domain characteristic sequence as defined herein) may be useful to cleave IgA as described herein.

[0236] In some embodiments, the present disclosure teaches that polypeptides including a characteristic sequence of SEQ ID NO: 4 may be considered IgA protease polypeptides as described herein. Alternatively or additionally, in some embodiments, polypeptides comprising SEQ ID NO: 7, and / or SEQ ID NO: 8, and / or SEQ ID NO:9 may be considered IgA protease polypeptides as described herein.Example 12: ELISA based assays

[0237] ELISA based-assays can be used to assess kinetics of cleavage of IgAl and / or IgA2 - e.g.. by IgA protease polypeptide(s) as described herein.

[0238] Alternatively or additionally, binding of synthetic peptides and / or one or both of IgAl and Ig2 molecules to inactive (e.g., HAXXH variant(s) of exemplified IgA protease polypeptides) will be tested via surface plasmon resonance studies.

[0239] In some embodiments, such kinetic studies and / or binding studies will distinguish between structural elements that contribute to and / or are necessary for catalysis and those that may be required for IgA selectivity.

[0240] Such kinetic and / or other binding studies may reveal or confirm one or more features (e.g., amino acid sequence element(s)) that may be necessary and / or sufficient to characterize a useful IgA protease polypeptide as described herein.Example 13: IgAP polypeptide activity in blood

[0241] The present Example demonstrates that Δ15 MD IgAPP cleaves IgAl in HEPES Buffer and pooled human sera with myeloma IgAl addition. Δ15 MD IgAPP IgAl cleavage activity is at least maintained in human serum, or improved, compared to IgAl cleavage activity in HEPES buffer.Material and Methods

[0242] 70 pM IgAl was incubated with 40 nM Δ15 MD IgAPP at 37°C in 25 rnMHEPES, pH 7.5 or pooled human serum collected off the clot. Samples were taken at various time points, diluted to a sample concentration of 2.33 pM IgAl (1:30 dilution), and quenched by the addition of SDS-PAGE loading dye and heating at 95°C. The Western blots were transferred and blocked with standard procedures using a PVDF membrane, incubated with a mouse anti -human IgAl Fc antibody conjugated with HRP. and developed with ECL.Results

[0243] Δ15 MD IgAPP can fully cleave exogenously added myeloma IgAl in the context of buffer (Figure 12A) and human serum (Figure 12B). The results suggest that Δ15 MD IgAPP is more active in serum than in buffer, as Al 5 MD IgAPP fully cleaves endogenous IgAl as seen by the disappearance of all IgAl heavy chains despite trace amounts of endogenous IgAl being present in the serum sample (Figure 12B).Example 14: IgA Polypeptide activity cleavage activity of IgAl from IgAN patient sera

[0244] The present Example demonstrates that Al 5 MD IgAP can cleave IgAl from IgAN patients.IgAl visualization

[0245] Endogenous levels of IgAl were visualized in:• Sera from patients having IgA Nephropathy (IgAN): IgAN 1 - IgAN 6• Pooled sera from individuals not having IgAN: Normal serum

[0246] Human serum was diluted 1 :30 for the Western blot samples. Western blots were processed as previously described (Example 13). Myeloma IgAl and IgA2 were added as the positive and negative controls, respectively. Low levels of endogenous IgAl were seen in the pooled normal serum and each IgAN patient serum (IgAN 1 - 6) (Figure 13 A), suggesting that the mouse anti-human IgAl Fc monoclonal conjugated with HRP was sufficient to visualize cleavage of endogenous IgAl with longer exposures.IgAl from IgAN patient seraMaterial and methods

[0247] 40 nM Δ15 MD329’807Cr-IgAPP (Cr-Δ15 MD), full length Cr-IgA protease(Cr-FL) ox Haemophilus influenzae IgA protease, or 400 nM Δ15 MD329’807Cr-IgAP (Cr-Δ15 MD), was incubated with IgAl from IgAN patient serum at 37°C. Samples were taken at various time points, diluted 1 :30, and quenched by the addition of SDS-PAGE loading dye and heating at 95°C. Western blots were processed as previously described (Example 13).Results

[0248] Δ15 MD Cr-IgAPP (top three gels), Cr-FL IgAPP (middle three gels), and H. influenzae IgAP (bottom three gels) are all able to cleave endogenous IgAl from IgAN patient sera (Figures 13A-C). Δ15 MD Cr-IgAPP w as able to cleave endogenous IgAl from all six IgAN patients, in particular after being contacted for more than one hour. H. influenzaeIgAP is relatively inactive in IgAN patient #2 serum while the C. ramosum IgAP full length and Al 5 MD Cr-IgAPP still retain IgAl cleavage activity.

[0249] To verify that Al 5 MD Cr-IgAPP was able to fully cleave endogenous IgAl from all six IgAN patient samples, especially from patients #5 and #6 which were shown to be more resistant than others in Figure 13B, the enzyme concentration was increased by a factor of ten. The results showed that all endogenous IgAl was cleaved after 24 hours. Thus, Al 5 MD IgAPP can indeed cleave IgAl from IgAN patients.Sequence Listing

[0250] Amino acid sequences of certain IgA protease polypeptides (e.g, full-length Cr-IgA protease, or a fragment thereof such as a subdomain described herein), as well as nucleotide sequences encoding them, are listed in the sequence listing in Table 2. An amino acid sequences of a mature form of IgA protease polypeptide from C. ramosum is provided as amino acid residues 31-1234 of SEQ ID NO: 1; residues 1-30 comprise an N-terminal signal peptide that is cleaved during processing, generating SEQ ID NO: 2. That is, SEQ ID NO: 1 is a pro form of a wild type C. ramosum IgA protease, and SEQ ID NO: 2 is a mature wild type C. ramosum IgA protease. Table 2 describes certain CR-IgAPP candidates, such as C. ramosum IgAPP (Genbank Accession, AY028440).Table 2: Amino acid sequences and nucleotide sequences. Amino acids in bold and underscore indicate a HEXXH domain.Equivalents

[0251] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. The scope of the present disclosure is not intended to be limited to the above Description.

Claims

ClaimsWe claim:

1. An IgA protease polypeptide agent that has an amino acid sequence that comprises an element having the general formulaY1- N-M1-M2-M3-C-Y2wherein each of Y1, N, M1, M2, M3, C, and Y2comprises or consists of a consecutive sequence of amino acids, wherein M1substantially comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 7, wherein M2comprises or consists of a HEX1X2H motif, wherein X1 and X2are amino acids, wherein M3substantially comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 9, wherein the polypeptide’s full amino acid sequence does not comprise or consist of the amino acid sequence of SEQ ID NO: 1 or 2, and wherein Y1, N, C, and Y2are optional.

2. The polypeptide agent of claim 1, wherein M2consists of the HEX1X2H motif.

3. The polypeptide agent of claim 1 or 2, wherein X1is an amino acid with hydrophobic side chains.

4. The polypeptide agent of claim 1, 2 or 3, wherein X1is selected from the group consisting of alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan.

5. The polypeptide agent of claim 4, wherein X1is phenylalanine or leucine.

6. The polypeptide agent of any one of the preceding claims, wherein X2is glycine.

7. The polypeptide agent of any one of the preceding claims, wherein X1is phenylalanine and X2is glycine.

8. The polypeptide agent of any one of claims 1-6, wherein X1is leucine and X2is glycine.

9. The polypeptide agent of any one of the preceding claims, wherein the M1amino acid sequence is at least 80% identical to SEQ ID NO: 7.

10. The polypeptide agent of any one of the preceding claims, wherein the M1amino acid sequence is at least 90% identical to SEQ ID NO: 7.

11. The polypeptide agent of any one of the preceding claims, wherein the M1amino acid sequence is 100% identical to SEQ ID NO: 7.

12. The polypeptide agent of any one of the preceding claims, wherein the M3amino acid sequence is at least 80% identical to SEQ ID NO: 9.

13. The polypeptide agent of any one of the preceding claims, wherein the M3amino acid sequence is at least 90% identical to SEQ ID NO: 9.

14. The polypeptide agent of any one of the preceding claims, wherein the M3amino acid sequence is 100% identical to SEQ ID NO: 9.

15. The polypeptide agent of claim 1 comprising or consisting of an amino acid sequence having at least 70% identity to SEQ ID NO: 4.

16. The polypeptide agent of any one of the preceding claims, wherein the polypeptide comprises the amino acid sequence N.

17. The polypeptide agent of claim 16, wherein the N amino acid sequence comprises amino acids 299-300 of the amino acid sequence as set forth in SEQ ID NO: 5.

18. The polypeptide agent of any one of the preceding claims, wherein the polypeptide comprises the amino acid sequence C.

19. The polypeptide agent of claim 18, wherein the C amino acid sequence comprises amino acids 1-175 of the amino acid sequence as set forth in SEQ ID NO: 6.

20. The polypeptide agent of claim 1 comprising or consisting of an amino acid sequence having at least 70% identity to SEQ ID NO: 27.

21. The polypeptide agent of claim 16, wherein the N amino acid sequence comprises amino acids 284-300 of the amino acid sequence as set forth in SEQ ID NO: 5.

22. The polypeptide agent of claim 1 comprising or consisting of an amino acid sequence having at least 70% identity to SEQ ID NO: 22.

23. The polypeptide agent of claim 16, wherein the N amino acid sequence is at least 70% identical to SEQ ID NO: 5.

24. The polypeptide agent of claim 16, wherein the N amino acid sequence is at least 80% identical to SEQ ID NO: 5.

25. The polypeptide agent of claim 16, wherein the N amino acid sequence is at least 90% identical to SEQ ID NO: 5.

26. The polypeptide agent of claim 16, wherein the N amino acid sequence is 100% identical to SEQ ID NO: 5.

27. The polypeptide agent of claim 1 comprising or consisting of an amino acid sequence having at least 70% identity to SEQ ID NO: 3.

28. The polypeptide agent of claim 22, wherein the C amino acid sequence is at least 70% identical to SEQ ID NO: 6.

29. The polypeptide agent of claim 18, wherein the C amino acid sequence is at least 80% identical to SEQ ID NO: 6.

30. The polypeptide agent of claim 18, wherein the C amino acid sequence is at least 90% identical to SEQ ID NO: 6.

31. The polypeptide agent of claim 18, wherein the C amino acid sequence is 100% identical to SEQ ID NO: 6.

32. The polypeptide agent of any one of the preceding claims, wherein the polypeptide sequence comprises Y1or Y2.

33. The polypeptide agent of claim 32, wherein Y1or Y2is a tag.

34. The polypeptide agent of claim 33, wherein the tag specifically binds to a protein ligand.

35. The polypeptide agent of claim 34, wherein the protein ligand is an antibody.

36. The polypeptide agent of claim 35, wherein the antibody is an anti-IgAl.

37. The polypeptide agent of claim 36, wherein the tag binds to an antibody and forms a complex.

38. The polypeptide agent of claim 33, wherein the tag is selected from the group consisting of c-myc, HA; VSV-G; HSV; FLAG; V5; and HIS.

39. The polypeptide agent of any one of the preceding claims, wherein the polypeptide agent cleaves at the hinge region of IgA.

40. The polypeptide agent of any one of the preceding claims, wherein the polypeptide agent cleaves at the hinge region of IgAl.

41. The polypeptide agent of any one of the preceding claims, wherein the polypeptide agent cleaves at the hinge region of IgA2.

42. The polypeptide agent of any one of the preceding claims, wherein the IgA protease polypeptide sequence is fewer than 1000 amino acids long.

43. The polypeptide agent of claim 42, wherein the polypeptide sequence consists of at the most 750 amino acids long.

44. The polypeptide agent of claim 42, wherein the polypeptide sequence consists of at the most 600 amino acids long.

45. The polypeptide agent of claim 42, wherein the polypeptide sequence consists of at the most 500 amino acids long.

46. The polypeptide agent of claim 42, wherein the polypeptide sequence consists of at the most 400 amino acids long.

47. The polypeptide agent of claim 42, wherein the polypeptide sequence consists of at the most 350 amino acids long.

48. The polypeptide agent of any one of the preceding claims, wherein the polypeptide agent is fused to an immunoglobulin or a fragment thereof.

49. A nucleic acid molecule comprising a DNA sequence encoding the IgA protease polypeptide agent according to any one of the preceding claims.

50. A vector comprising the nucleic acids of claim 49.

51. The vector of claim 50, wherein the vector is a plasmid.

52. A pharmaceutical composition for the treatment of IgA deposits, comprising the IgA protease polypeptide agent of any one of claims 1-48 and / or the nucleic acid molecule of claim 49 and a pharmaceutically acceptable carrier.

53. The pharmaceutical composition of claim 52, wherein said composition is mixed with an antibody.

54. The pharmaceutical composition of claim 52 or 53, wherein said IgA protease polypeptide agent is fused to an immunoglobulin or a fragment thereof.

55. The pharmaceutical composition of any one of claims 52-54, for the treatment of a disease that is renal failure, skin blistering, rash, arthritis, gastrointestinal bleeding or abdominal pain.

56. A host cell that contains and / or expresses an IgA protease polypeptide agent of any one of claims 1-48 and / or the nucleic acid molecule of claim 49.

57. A method for treatment of a disease characterized by IgA deposition, comprising administering to a patient a therapeutically effective amount of i) the IgA protease polypeptide agent of any one of claims 1-48; ii) the nucleic acid molecule of claim 50; or iii) the pharmaceutical composition of any one of claims 52-55.

58. The method of claim 57, wherein the IgA deposition is IgAl deposition.

59. The method of claim 57, wherein the individual is suffering from a condition selected from the group consisting of IgAl nephropathy, dermatitis herpetiformis, and Henoch- Schoenlein purpura.

60. The method of claim 57, wherein the individual is suffering from IgAl nephropathy.

61. The method of any one of claims 57-60, wherein the polypeptide agent is administered in an amount effective such that IgA deposits are reduced.

62. The method of any one of claims 57-60, wherein the IgA protease polypeptide agent, nucleic acid molecule, or pharmaceutical composition is administered in combination with a second therapy.

63. A method comprising a step of administering to an individual having IgA depositions an IgA protease polypeptide agent of any one of claims 1-48, the nucleic acid molecule of claim 44 or the pharmaceutical composition of any one of claims 52-55 such that IgA deposits are reduced.

64. The method of claim 63, wherein the polypeptide agent, nucleic acid molecule, or pharmaceutical composition is administered in combination with a second therapy.

65. The method of claim 63, wherein the polypeptide agent, nucleic acid molecule, or pharmaceutical composition is administered by oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection.

66. The method of claim 64, wherein the individual receives the polypeptide agent, nucleic acid molecule, or pharmaceutical composition and the second therapy at the same time.

67. The method of claim 64, wherein the individual does not receive the polypeptide agent and the second therapy at the same time.

68. The method of claim 64, wherein the second therapy is a pharmaceutical agent.

69. The method of claim 68, wherein the therapy is selected from the group consisting of corticosteroids, dietary fish oil supplements, angiotensin converting enzyme inhibitors, and combinations thereof.

70. The method of any one of claims 63-69, wherein the IgA depositions are IgAl depositions.

71. The method of any one of claims 63-69, wherein the individual is a human.

72. A method comprising a step of administering to an individual receiving a therapy that treats a disease, disorder or condition associated with IgA deposition an IgA protease polypeptide agent of any one of claims 1-48.

73. The method of claim 72, wherein the individual is a human.

74. A method of producing an IgA protease polypeptide agent of any one of claims 1-48, wherein the method comprises the steps of- providing a host cell expressing an IgA protease polypeptide agent of any one of claims 1-48;- culturing the host cell under conditions allowing the host cell to produce the polypeptide agent; and- optionally lysing the cell and recover and / or isolate the polypeptide agent.

75. A method of cleaving IgA, the method comprising a. contacting a sample comprising IgA with an IgA protease polypeptide agent of any one of claims 1-48 or the pharmaceutical composition of any one of claims 52-55 whereby the IgA protease polypeptide agent cleaves IgA.

76. The method according to claim 75, wherein IgA is an IgAl.

77. The method according to claim 75 or 76, wherein the IgA protease polypeptide agent cleaves the IgA comparably to a relevant reference IgA protease.

78. A method for cleaving IgA in an individual comprising administering to an individual: a. the IgA protease polypeptide agent of any one of claims 1-48; b. the nucleic acid molecule of claim 49; or c. the pharmaceutical composition of any one of claims 52-55.

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