IgA protease polypeptide agents

Abstract

The present disclosure provides certain IgA protease polypeptide agents, and in some embodiments, uses thereof, including cleaving IgA1 present in deposits in vivo. Among other things, the present disclosure surprisingly provides that relatively small polypeptides (e.g., truncated variant(s) of a reference IgA protease polypeptide), referred to herein as "subdomain" agents (or "SD-IgAPP" agents), contain 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 described herein), but lack one or more other sequence elements found in the reference IgA protease, and can retain IgA proteolytic activity (e.g., reasonably comparable to such activity observed with the appropriate reference IgA protease).

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 424,585, filed November 11, 2022, which is incorporated herein by reference in its entirety. (Background technology)

[0002] Deposition of immunoglobulin A1 (IgA1) in human tissues and organs is a hallmark of many human diseases, including, but not limited to, IgA nephropathy (IgAN), dermatitis herpetiformis, and Henoch-Schönlein purpura. IgAN is the most common form of glomerulonephritis worldwide. It is characterized by the presence of potentially damaging mesangial deposits containing IgA1 complexes in all renal glomeruli. Clinical manifestations of IgAN include proteinuria, hematuria, and hypertension, which can progress to end-stage renal disease. Renal failure is fatal unless addressed with renal dialysis and, ultimately, transplantation of healthy donor kidneys into the patient. Summary of the Invention

[0003] The present disclosure provides certain IgA protease polypeptide agents and, in some embodiments, uses thereof, including use to cleave IgA1 present in deposits, for example, in vivo.

[0004] Among other things, the present disclosure surprisingly provides that relatively small polypeptides (e.g., truncated variant(s) of a reference IgA protease polypeptide), referred to herein as "subdomain" agents (or "SD-IgAPP" agents), which comprise 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 described herein), but lack one or more other sequence elements found in those reference IgA proteases, can retain IgA proteolytic activity (e.g., reasonably comparable to such activity observed with an appropriate reference IgA protease). In some embodiments, provided SD-IgAPP agents can exhibit IgA1 cleavage activity (e.g., at least comparable to that of an appropriate reference IgA protease).

[0005] In some embodiments, provided SD-IgAPP agents may exhibit IgA2 cleavage activity (e.g., at least comparable to that of a suitable reference IgA protease). In some embodiments, provided SD-IgAPP agents may exhibit reduced IgA2 cleavage (e.g., compared to a suitable reference IgA protease).

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

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

[0008] Advantages of certain embodiments of the provided technology may include, among other things, reduced immunogenicity, ease of manufacture, increased bioavailability, and / or increased half-life of the SD-IgAPP agent (e.g., compared to a suitable reference IgA protease, e.g., the IgA protease of C. ramosum having the amino acid sequence set forth in SEQ ID NO:1 and / or SEQ ID NO:2; see FIG. 2 ), and in some embodiments, maintain at least reasonably equivalent IgA cleavage (e.g., IgA1 cleavage) activity compared to such reference IgA protease. In some embodiments, provided SD-IgAPP agents may exhibit increased IgA cleavage (e.g., increased IgA1 cleavage activity) compared to such reference. In some particular embodiments, such equivalent or increased IgA cleavage is or includes cleavage of in vivo IgA deposit(s) (e.g., IgA1 deposit(s)).

[0009] In some embodiments, one or more of immunogenicity, bioavailability, half-life and / or IgA1 and / or IgA2 cleavage are assessed in vivo (e.g., when the SD-IgAPP agent is administered or otherwise delivered to an organism, such as a mammal, particularly a human).

[0010] In some embodiments, SD-IgAPP agents are provided that have proteolytic activity (i.e., against IgA, e.g., IgA1) according to the present disclosure. In some embodiments, SD-IgAPP agents are provided that are proteolytically active (i.e., against IgA, e.g., against IgA contained in aggregates or deposits, e.g., in vivo deposits). In some embodiments, such proteolytically active SD-IgAPP agents are useful for treating one or more IgA deposition diseases, such as (but not limited to) IgA nephropathy.

[0011] In some embodiments, an IgA protease polypeptide agent has an amino acid sequence that is or includes an element having the following general formula: Y1-N-M1-M2-M3-C-Y2 (wherein Y1, N, M1, M2, M3, C, and Y2 each comprise or consist of a contiguous amino acid sequence, M1 substantially comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 7, M2 comprises or consists of a HEX1X2H motif, X1 and X2 are amino acids, M3 substantially comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 9, the complete amino acid sequence of the polypeptide does not comprise or consist of the amino acid sequence of SEQ ID NO: 1 or 2, and Y1, N, C, and Y2 are optional.)

[0012] In some aspects, nucleic acid molecules encoding the provided SD-IgAPP agents are provided. In some embodiments, such nucleic acid molecules may be included in a vector (e.g., a DNA plasmid, a viral vector, etc.). In some embodiments, such nucleic acids may be associated with one or more expression control sequences (e.g., a promoter, an enhancer, and / or binding sites for transcriptional regulators such as transcriptional activators or repressors, a transcription termination signal, one or more splice acceptor or donor sites, a translation initiation site, etc.). In some embodiments, the encoding nucleic acid may be included in a vector suitable for in vitro transcription (e.g., a plasmid or a portion, e.g., a fragment, e.g., a truncated fragment or amplicon thereof, etc.). In some embodiments, the encoding nucleic acid may be included in a vector suitable for expression (e.g., by transcription, translation, or both) in vitro (e.g., in in vitro cell culture). In some such embodiments, the encoding nucleic acid may be included in a vector suitable for expression (e.g., by transcription, translation, or both) in vivo (e.g., in a mammalian cell or organism, in some embodiments, particularly a human). For example, in some embodiments, the encoding nucleic acid may be included in a viral construct, or an RNA construct (e.g., which may be generated by in vitro transcription), suitable for inclusion in a pharmaceutical composition for administration to and expression in a mammalian subject, such as a human subject.

[0013] In some aspects, pharmaceutical compositions are provided that comprise or deliver (e.g., upon administration to a subject) an SD-IgAPP agent (e.g., an SD-IgAPP agent with proteolytic activity) described herein. In some embodiments, such pharmaceutical compositions are for the treatment of a disease, disorder, or condition associated with IgA deposition.

[0014] In some aspects, methods for treating diseases characterized by IgA deposition using an SD-IgAPP agent with IgA proteolytic activity according to the present disclosure are provided. In some embodiments, the use of an SD-IgAPP agent to treat IgA nephropathy, dermatitis herpetiformis (DH), and / or Henoch-Schönlein purpura (HS) is disclosed.

[0015] In some embodiments of the present disclosure, an individual with IgA deposits, e.g., IgA1 deposits, In one embodiment, a method is provided comprising administering a composition comprising or delivering an SD-IgAPP agent of the present disclosure to a subject.

[0016] In some embodiments, an SD-IgAPP agent is characterized by cleaving both IgA1 and IgA2. In some embodiments, an SD-IgAPP agent is characterized by specifically cleaving IgA1. In some embodiments, an SD-IgAPP agent is characterized by preferentially cleaving IgA1 over IgA2, and in some such embodiments, such an SD-IgAPP is characterized by no detectable IgA2 cleavage under conditions in which robust IgA1 cleavage is observed (e.g., substantially comparable to that achieved by a suitable reference IgA1 protease, such as full-length M64IgA1 protease). [Brief explanation of the drawings]

[0017] [Figure 1] The hinge peptide of IgA1 / 2 and the IgA1 protease family members that cleave it are shown. The "lollipop" indicates the location of the O-glycan.

[0018] [Figure 2]1 depicts the structure of the full-length C. ramosum wild-type IgA protease. As can be seen, each of the three distinct domains is depicted: the N-terminal domain (corresponding to amino acid residues 31-313 of SEQ ID NO:1), the middle domain (corresponding to amino acid residues 314-807 of SEQ ID NO:1), and the C-terminal domain (corresponding to amino acid residues 808-1234 of SEQ ID NO:1). The residue numbering is that of the Cr-IgA protease represented by SEQ ID NO:1, with residues 1-30 of SEQ ID NO:1 representing the propeptide that is cleaved from the mature protease. Structural and functional studies described herein demonstrate that the middle domain is the only domain required for IgA protease activity.

[0019] [Figure 3A] 1 shows a Coomassie stained SDS gel showing expression of an exemplary Cr-IgAP full-length construct. [Figure 3B] 1 shows a Coomassie stained SDS gel showing the expression of an exemplary Cr-IgAP N-terminal domain construct. [Figure 3C] 1 shows a Coomassie stained SDS gel showing expression of an exemplary Cr-IgAP intermediate domain construct. [Figure 3D] 1 shows a Coomassie-stained SDS gel showing the expression of an exemplary Cr-IgAP C-terminal domain construct.

[0020] [Figure 4A] Crystal structures of the C. ramosum IgAP Δ15 intermediate domain (Cr-Δ15 MD) and first C-terminal domain (CTD1) are shown. The complete Cr-Δ15 MD-CTD is shown. Black indicates the Cr-Δ15 MD, and gray indicates CTD1. [Figure 4B] The crystal structures of the C. ramosum IgAP Δ15 middle domain (Cr-Δ15 MD) and the first C-terminal domain (CTD1) are shown. The 1.60 Å structure of the active site. [Figure 4C]Crystal structures of the C. ramosum IgAP Δ15 intermediate domain (Cr-Δ15 MD) and first C-terminal domain (CTD1) are shown. The 1.60 Å structure of the active site is shown. A loop containing D520 and E521 bridges the active site to a nearby pocket where the second zinc-binding site is located.

[0021] [Figure 5] Figure 1 shows a Coomassie-stained SDS gel showing IgA1 digestion by Cr-MD IgAPP and reference Haemophilus influenzae (HIN) IgAP. Lane A: IgA1 alone; Lane B: HIN-IgAP alone; Lane C: HIN-IgAP + IgA1; Lane D: Cr-IgAP NTD alone; Lane E: Cr-IgAP NTD + IgA1 (1 h at 37°C); Lane F: Cr-IgAP NTD + IgA1 (overnight at 37°C); Lane G: Cr-IgAP MD alone; Lane H: Cr-IgAP MD + IgA1 (1 h at 37°C).

[0022] [Figure 6A-1] The sequence alignment is shown in Figure 1. The intermediate domain sequence (Cr-MD) represented by SEQ ID NO: 4 was used as a query. [Figure 6A-2] Same as above [Figure 6A-3] Same as above [Figure 6A-4] Same as above [Figure 6B-1] The sequence alignment is shown in Figure 1. The intermediate domain sequence (Cr-MD) represented by SEQ ID NO: 4 was used as a query. [Figure 6B-2] Same as above [Figure 6B-3] Same as above [Figure 6B-4] Same as above [Figure 6C-1] The sequence alignment is shown in Figure 1. The intermediate domain sequence (Cr-MD) represented by SEQ ID NO: 4 was used as a query. [Figure 6C-2] Same as above [Figure 6C-3] Same as above [Figure 6C-4] Same as above [Figure 6D-1]The sequence alignment is shown in Figure 1. The intermediate domain sequence (Cr-MD) represented by SEQ ID NO: 4 was used as a query. [Figure 6D-2] Same as above [Figure 6D-3] Same as above [Figure 6D-4] Same as above [Figure 6E] The sequence alignment is shown in Figure 1. The intermediate domain sequence (Cr-MD) represented by SEQ ID NO: 4 was used as a query. [Figure 6F-1] The sequence alignment is shown in Figure 1. The intermediate domain sequence (Cr-MD) represented by SEQ ID NO: 4 was used as a query. [Figure 6F-2] Same as above [Figure 6F-3] Same as above [Figure 6F-4] Same as above [Figure 6G-1] The sequence alignment is shown in Figure 1. The intermediate domain sequence (Cr-MD) represented by SEQ ID NO: 4 was used as a query. [Figure 6G-2] Same as above [Figure 6G-3] Same as above [Figure 6G-4] Same as above [Figure 6H-1] The sequence alignment is shown in Figure 1. The intermediate domain sequence (Cr-MD) represented by SEQ ID NO: 4 was used as a query. [Figure 6H-2] Same as above [Figure 6H-3] Same as above [Figure 6H-4] Same as above

[0023] [Figure 7A]Mass spectrometry analysis of unlabeled hinge peptides synthetically produced in the presence of full-length (Cr-FL) IgA protease as set forth in SEQ ID NO:2 or the middle domain of IgA protease as set forth in SEQ ID NO:4 (Cr-MD) is shown. Alpha-cyano-4-hydroxycinnamic acid (HCCA) was used as the matrix for the analysis. Three different MR ranges of the analysis are represented. HCCA matrix alone (sample blank) is shown in black (lower spectrum). The spectra of the peptide alone (isolated IgA peptide), sample digested with Cr-MD, and sample digested with Cr-FL are identical, indicating no peptide cleavage due to enzymatic treatment. The theoretical masses of the peptides are: intact - 798.95 g / mol, N-terminal fragment - 414.5 g / mol, and C-terminal fragment - 402.5 g / mol. [Figure 7B] Mass spectrometry analysis of synthetically produced unlabeled hinge peptides in the presence of full-length (Cr-FL) IgA protease as set forth in SEQ ID NO:2 or the middle domain of IgA protease as set forth in SEQ ID NO:4 (Cr-MD) is shown. Alpha-cyano-4-hydroxycinnamic acid (HCCA) was used as the matrix for the analysis. Three different MR ranges of the analysis are represented. HCCA matrix alone (sample blank) is shown in black (lower spectrum). The spectra of the peptide (isolated IgA peptide) alone, sample digested with Cr-MD, and sample digested with Cr-FL are identical, indicating no peptide cleavage due to enzymatic treatment. [Figure 7C] Mass spectrometry analysis of synthetically produced unlabeled hinge peptides in the presence of full-length (Cr-FL) IgA protease as set forth in SEQ ID NO:2 or the middle domain of IgA protease as set forth in SEQ ID NO:4 (Cr-MD) is shown. Alpha-cyano-4-hydroxycinnamic acid (HCCA) was used as the matrix for the analysis. Three different MR ranges of the analysis are represented. HCCA matrix alone (sample blank) is shown in black (lower spectrum). The spectra of the peptide (isolated IgA peptide) alone, sample digested with Cr-MD, and sample digested with Cr-FL are identical, indicating no peptide cleavage due to enzymatic treatment.

[0024] [Figure 8A] 1 depicts an SDS gel showing IgA1 digestion by Cr-MD or Cr-FL. [Figure 8B] 1 depicts an SDS gel showing IgA1 digestion with Cr-NTD-MD. [Figure 8C] 1 depicts an SDS gel showing IgA1 digestion by Cr-Δ15 MD or Cr-MD. [Figure 8D] 1 depicts an SDS gel showing IgA1 digestion by Cr-MD or reference Haemophilus influenzae IgAP.

[0025] [Figure 9A] 1 shows an SDS gel showing IgA2 digestion by Cr-MD or Cr-FL. [Figure 9B] 1 shows an SDS gel showing IgA2 digestion by Cr-NTD+MD.

[0026] [Figure 10A] 1 shows an SDS gel showing IgG1 digestion with Cr-MD or Cr-FL. [Figure 10B] 1 shows an SDS gel showing IgG2 digestion by Cr-MD or Cr-FL. [Figure 10C] An SDS gel showing digestion with Cr-NTD+MD is shown.

[0027] [Figure 11A] Figure 1 shows a gel-based kinetic assay of IgAPP. Gels showing IgA1 or cleaved IgA1 fragments. [Figure 11B] Figure 1 shows a gel-based kinetic assay of IgAPP: Percentage of IgA1 heavy chain cleaved over time. [Figure 11C] Gel-based kinetic assay of IgAPP. Michaelis-Menten curves for the Cr-IgAP peptide, containing the C. ramosum N-terminal domain in combination with the middle domain (NM, dotted line), the middle domain (MD, dashed line), and the N-terminal truncated middle domain (Δ15 MD, solid line).

[0028] [Figure 12A] Western blot of IgA1 cleaved by Cr-Δ15 MD IgAP (40 nM) in HEPES buffer is shown. [Figure 12B] Western blot of IgA1 cleaved by Cr-Δ15 MD IgAP (40 nM) in pooled human serum spiked with myeloma IgA is shown.

[0029] [Figure 13A] Western blot visualizing endogenous levels of IgA1 in pooled normal serum and serum from IgAN patients is shown. [Figure 13B] Western blots visualizing IgA1 digestion by Cr-Δ15 MD IgAP (top three gels), Cr-full-length IgAP (middle three gels), and H. influenzae IgAP (bottom three gels) (all 40 nM) are shown. [Figure 13C] Western blot visualizing IgA1 digestion by Cr-Δ15 MD IgAP (400 nM) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0030] definition Agent (Agent): Generally, as used herein, the term "agent" is used to refer to an entity (e.g., a lipid, metal, nucleic acid, polypeptide, polysaccharide, small molecule, etc., or a complex, combination, mixture, or system thereof (e.g., a cell, tissue, organism)) or a phenomenon (e.g., heat, an electric current or electric field, a magnetic force or field, etc.). Under appropriate circumstances, as will be clear from the context to one of skill in the art, the term may be used to refer to an entity that is or includes a cell or organism, or a fraction, extract, or component thereof. Alternatively or additionally, as will be clear from the context, the term may be used to refer to a natural product that occurs in and / or is obtained from nature (e.g., a naturally occurring IgA protease, such as a naturally occurring M64 IgA protease, such as the IgA protease of C. ramosum). In some embodiments, an agent may be utilized in isolated or pure form, and in some embodiments, an agent may be utilized in crude form. In some embodiments, potential agents may be provided as a collection or library that can be screened to identify or characterize active agents among them.

[0031] Amino acid: As used herein, the term "amino acid" refers to any compound, substance, or entity that can be incorporated into a polypeptide chain, for example, by the formation of one or more peptide bonds, or that is a polypeptide. In some embodiments, an amino acid has the general structure HN-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 20 L-amino acids commonly found in naturally occurring peptides. A "non-standard amino acid" refers to any amino acid other than a standard amino acid, regardless of whether it is found in or may be found in a natural source. In some embodiments, amino acids comprising the carboxy and / or amino termini in a polypeptide may contain structural modifications compared to the general structures above. For example, in some embodiments, an amino acid may be modified relative to the general structure by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and / or substitution (e.g., of an amino group, a carboxylic acid group, one or more protons, and / or a hydroxyl group). In some embodiments, such modifications may alter the stability or circulating half-life of a polypeptide comprising the modified amino acid, for example, compared to a polypeptide comprising an otherwise identical, unmodified amino acid. In some embodiments, such modifications do not significantly alter the relevant activity of a polypeptide comprising the modified amino acid, compared to a polypeptide comprising an otherwise identical, unmodified amino acid. As will be clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid. In some embodiments, the term 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 specifically binds to an epitope (e.g., of an antigen). Naturally occurring human antibodies typically contain two identical heavy chains and two identical light chains, each of which contains a variable domain and a constant domain. The constant domain defines the antibody isotype (IgG, IgA, IgM, IgE, etc.). Those skilled in the art will recognize that different antibody structures are naturally utilized by different animal species. For example, camelid antibodies are single-chain antibodies. Those skilled in the art will further recognize that antibody variable domains are typically characterized by framework region (FR) sequences and complement-determining region (CDR) sequences. Each variable domain typically contains three CDRs, i.e., CDR1, CDR2, and CDR3, which together contribute to the specificity and / or affinity of epitope binding. In some embodiments, antibodies may be produced in or by an organism. In some embodiments, antibodies may be produced intracellularly or by cells in vitro (e.g., by hybridomas and / or engineered cells). In some embodiments, antibody fragment(s) (e.g., those that may be produced by cleavage or recombinantly) may be generated and / or utilized in accordance with the present disclosure. Those of skill in the art will recognize that a variety of techniques have been developed to incorporate binding characteristics (e.g., one or more CDR and / or FR sequences, particularly a set of three CDRs, optionally together with FR sequences) into new contexts. In some embodiments, CDR sequences may be maintained and other elements may be altered, as occurs, for example, in humanization. Alternatively or additionally, in some embodiments, the variable region may be combined with an alternative constant region (e.g., a constant region from a different organism and / or one that contains one or more particular sequence features or elements desired, for example, to confer particular attributes to the antibody agent). Those of skill in the art will further recognize a variety of techniques commonly utilized to combine the binding characteristics of two or more different antibodies together, for example, in a single multispecific (most commonly bispecific) agent.

[0033] Associated: As used herein, the term "associated" between two events or entities refers to the presence, level, degree, and / or form of one being correlated with that of the other. For example, a particular entity (e.g., a polypeptide, genetic signature, metabolite, microorganism, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with the occurrence, susceptibility, severity, stage, etc. of the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact, directly or indirectly, such that they are in physical proximity and / or remain in physical proximity to one another. In some embodiments, two or more entities that physically associate with one another are covalently bound to one another; in some embodiments, two or more entities that physically associate with one another are not covalently bound to one another, but are non-covalently bound by, for example, hydrogen bonds, van der Waals interactions, 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) simultaneously. 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 is administered before the administration of any dose or episode of the second regimen). In some embodiments, the two or more therapeutic regimens are administered in overlapping dosing regimens. In some embodiments, administration of the combination therapy may include administering one or more therapeutic agents or modalities to a subject receiving other agent(s) or modality. In some embodiments, combination therapy does not necessarily require that the individual agents be administered together in a single composition (or necessarily simultaneously). In some embodiments, the two or more therapeutic agents or modalities of the combination therapy are administered separately to the subject, e.g., via separate routes of administration (e.g., one agent orally and the other intravenously) and / or in separate compositions at different times. In some embodiments, two or more therapeutic agents may be administered together via the same route of administration and / or simultaneously, in a combination composition or compound (e.g., as part of a single chemical complex or covalent entity).

[0035] Equivalent, Reasonably Equivalent, and Substantially Equivalent: As used herein, the terms "equivalent," "reasonably equivalent," and "substantially equivalent" refer to two or more agents, entities, circumstances, or sets of conditions that may not be identical to one another, but are sufficiently similar to be comparable, so that one of skill in the art would understand that conclusions can be reasonably drawn based on observed differences or similarities. In some embodiments, equivalent sets of conditions, circumstances, individuals, or populations are characterized by multiple substantially identical characteristics and one or a few diverse characteristics. One of skill in the art will understand the degree of identity required for two or more such agents, substances, circumstances, sets of conditions, etc. to be considered equivalent in any given situation in context. For example, one of skill in the art will understand that sets of circumstances, individuals, or populations are equivalent to one another when they are characterized by a sufficient number and type of substantially identical characteristics to warrant a reasonable conclusion that differences in results obtained under or with different sets of circumstances, individuals, or populations, or in observed phenomena, are caused by or indicate variations in the diverse characteristics. For example, in some embodiments, SD-IgAPP retains IgA1 proteolytic activity that is reasonably equivalent to the activity observed with a suitable reference IgA protease, e.g., as exemplified in Example 3, which demonstrates that SD-IgAPP cleaves the hinge region of IgA1 equivalent to the suitable reference IgA protease, and shown in Figure 5. In some embodiments, provided SD-IgAPP agents cleave more IgA (e.g., IgA1) than the reference IgA protease (e.g., full-length IgA protease). In some embodiments, the SD-IgAPP agent cleaves at least 80% of IgA (e.g., IgA1), 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% of IgA (e.g., IgA), e.g., 100% of IgA (e.g., IgA) equivalently to a reference IgA protease (e.g., full-length IgA protease).

[0036] Complexation: As used herein, the term "complex" typically refers to a physical association between two or more distinct chemical entities. Typically, such association in a "complex" is non-covalent.

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

[0038] Dosage regimen: Those skilled in the art will understand that the term "dosage regimen" can refer to a set of unit doses (typically more than one) administered individually to a subject, typically spaced apart. In some embodiments, a given therapeutic agent has a recommended dosing regimen that can include one or more doses. In some embodiments, a dosing regimen includes multiple doses, each separated in time from the other doses. In some embodiments, the individual doses are separated from each other by periods of equal length, and in some embodiments, a dosing regimen includes multiple doses and at least two different periods separating the individual doses. In some embodiments, all doses within a dosing regimen are the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen includes a first dose at a first dosage amount, followed by one or more additional doses at a second dosage amount that is different from the first dosage amount. In some embodiments, a dosing regimen includes a first dose at a first dosage amount, followed by one or more additional doses at a second dosage amount that is the same as the first dosage amount. In some embodiments, the dosing regimen correlates with a desired or beneficial outcome when administered across a relevant population (ie, is a therapeutic dosing regimen).

[0039] Engineered: In general, the term "engineered" refers to aspects that have been manipulated by human hands. For example, a polynucleotide is considered "engineered" when two or more sequences that are not naturally linked together in that order are manipulated by the hand of man so that they are directly linked to one another in the engineered polynucleotide, and / or when certain residues within the polynucleotide are caused through the action of man to be linked to entities or moieties that do not occur in nature and / or are not naturally linked. For example, in some embodiments used and / or utilized herein, an engineered polynucleotide includes regulatory sequences found in nature in operative association with a first coding sequence but not in operative association with a second coding sequence, and which are linked by the hand of man so as to be operably associated with the second coding sequence. Similarly, a polypeptide can be considered "engineered" when it is encoded by or expressed from an engineered polynucleotide and / or produced other than by natural expression in a cell. Similarly, a cell or organism is considered "engineered" when, upon being subjected to a manipulation, its genetic, epigenetic, and / or phenotypic identity is altered relative to an otherwise identical cell that has not been so manipulated. In some embodiments, the manipulation is or includes a genetic manipulation that alters its genetic information (e.g., new genetic material not previously present is introduced, e.g., by transformation, mating, somatic hybridization, transfection, transduction, or other mechanisms, or previously present genetic material is altered or removed, e.g., by substitution or deletion mutations, or mating protocols). In some embodiments, an engineered cell is one that has been engineered to contain and / or express a particular agent of interest (e.g., a protein, nucleic acid, and / or a particular form thereof) in an altered amount and / or according to an altered timing relative to such an appropriate reference cell.As is common practice and understood by those of skill in the art, the progeny of an engineered polynucleotide or cell are typically still referred to as "engineered," despite the actual manipulation performed on the earlier entity.

[0040] Host cell: As used herein, refers to a cell into which exogenous nucleic acid (e.g., an engineered nucleic acid) has been introduced and / or which otherwise expresses or contains an engineered polypeptide. Those skilled in the art will understand, upon reading this disclosure, 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 subsequent generations, either due to mutation or environmental influences, such progeny may not actually 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 certain embodiments, host cells include bacterial cells (e.g., cells of E. coli, Bacillus species, Streptomyces species, etc.), mycobacterial cells, fungi or yeast (e.g., cells of S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., cells of SF-9, SF-21, Trichoplusia ni, etc., in some embodiments, baculovirus-infected cells), non-human animal cells (e.g., cells of a mouse or other rodent), human cells, or cell fusions, e.g., hybridomas or quadromas, etc. In some embodiments, the host cells are human, monkey, ape, hamster, rat, or mouse cells.In some embodiments, the host cell is a eukaryotic cell, e.g., selected from the following cells: CHO (e.g., CHO K1, DXB-1 1CHO, Vegge-CHO), COS (e.g., COS-7) cells, CV1 cells, Daudi cells, epidermal (e.g., A431) cells, Jurkat cells, kidney (e.g., HEK293, 293EBNA, MSR293, MDCK, HaK, BHK [e.g., BHK21]) cells, HeLa cells, HepG2 cells, WI38 cells, MRC 5 cells, Colo205 cells, HB8065 cells, HL-60 cells, U937 cells, 3T3 cells, L-cell cells, C127 cells, SP2 / 0 cells, NS-0 cells, MMT 060562 cells, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, Vero cells, or other cells (eg, cells derived from a tissue sample or cell line).

[0041] Initiation: As used herein, the term "initiation" when applied to a dosing regimen can refer to the first administration of a pharmaceutical agent or modality to a subject who has not previously received the pharmaceutical agent or modality, or to a subject who is initiating or beginning a new course of the pharmaceutical agent or modality. Alternatively, as is clear from the context, in some embodiments, the term "initiation" can be used to refer to the administration of a particular unit dose of a pharmaceutical agent or a particular course of treatment during the treatment 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 IgA1. In some embodiments, an IgA protease cleaves IgA2. In some embodiments, an IgA protease cleaves both IgA1 and IgA2. In some embodiments, an IgA protease exhibits preferential cleavage of IgA1 over IgA2. In some embodiments, an IgA protease exhibits preferential cleavage of IgA2 over IgA1. In some embodiments, an IgA protease cleaves IgA1 and IgA2 equally. In some embodiments, an IgA protease cleaves IgA with preferential specificity compared to an otherwise equivalent IgA with a different hinge sequence. Those skilled in the art are aware of or can readily identify (e.g., by amino acid sequence and / or activity assessment) several naturally occurring IgA proteases. Table 2 provides exemplary amino acid sequences of certain known IgA proteases. As indicated, Mistry D, Stockley RA. IgA1 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 comparison and / or mechanistic similarities. See Figure 1.

[0043] IgA protease polypeptide: As used herein, the term "IgA protease polypeptide" refers to a polypeptide that (a) exhibits IgA protease activity and (b) shares at least one signature sequence with a reference IgA protease (and / or a fragment thereof, particularly a fragment that is or includes the signature sequence) and / or exhibits overall identity with the reference IgA protease; in many embodiments, an IgA protease polypeptide (a) exhibits IgA protease activity, (b1) shares at least one signature sequence with a reference IgA protease (or a fragment thereof), and (b2) exhibits overall sequence identity with such reference IgA protease (or a fragment thereof). Table 2 sets forth the amino acid sequence(s) of particular known IgA proteases (e.g., reference IgA proteases). Table 2 presents, in some embodiments, particular sequences that may be considered signature sequences of IgA protease polypeptides relevant to the present disclosure. In some embodiments, the IgA protease polypeptide exhibits at least 70% sequence identity with a reference IgA protease (or fragment thereof). In some embodiments, the IgA protease polypeptide shares at least one characteristic sequence element with the IgA protease polypeptide and exhibits at least 60% sequence identity with such reference IgA protease (or fragment thereof). In some embodiments, the IgA protease polypeptide is less than about 1000 amino acids in length, and in many embodiments is an SD-IgAPP in that it has a length of less than about 750, or less than about 700, or less than about 650, or less than about 600, or less than about 550, or less than about 500, or less than about 490, or less than about 480, or less than about 400, or less than about 350 amino acids. In some embodiments, the SD-IgAPP is the middle domain of the reference IgA protease (or fragment thereof). In some embodiments, such SD-IgAPP IgA protease polypeptides share a characteristic sequence with an IgA protease and exhibit at least about 40% overall sequence identity with a fragment of such a reference IgA protease.In some embodiments, SD-IgAPP has 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, the SD-IgAPP agents provided herein are IgA proteases, in that they cleave at least IgA1. In some embodiments, the SD-IgAPP agent(s) effect cleavage of IgA1 and IgA2, and in many embodiments, the SD-IgAPP agent(s) cleave IgA1 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 with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dosage suitable for administration in a treatment regimen that exhibits a statistically significant likelihood of achieving a predetermined therapeutic effect when administered to an appropriate population. In some embodiments, the pharmaceutical composition may be specially formulated for administration in solid or liquid form, including oral administration, e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or sustained-release formulation; topical application, e.g., as a cream, ointment, or controlled-release patch or spray applied to the skin, lungs, or oral cavity; vaginal or rectal administration, e.g., as a pessary, cream, or foam; sublingual administration; ocular administration; transdermal administration; or those suitable for nasal, pulmonary, and other mucosal surface administration.

[0045] Polypeptide: As used herein, refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has a naturally occurring amino acid sequence. In some embodiments, a polypeptide has a non-naturally occurring amino acid sequence. In some embodiments, a polypeptide has an engineered amino acid sequence, in that it has been designed and / or generated through the act of man. In some embodiments, a polypeptide can comprise or consist of natural amino acids, unnatural amino acids, or both. In some embodiments, a polypeptide can comprise or consist of only natural amino acids or only unnatural amino acids. In some embodiments, a polypeptide can comprise D-amino acids, L-amino acids, or both. In some embodiments, a polypeptide can comprise only D-amino acids. In some embodiments, a polypeptide can comprise only L-amino acids. In some embodiments, a polypeptide can comprise one or more pendant groups or other modifications, e.g., modification of or attachment to one or more amino acid side chains, at the N-terminus of the polypeptide, the C-terminus of the polypeptide, or any combination thereof. In some embodiments, such pendant groups or modifications can 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 include a cyclic portion. In some embodiments, a polypeptide is not cyclic and / or does not include a cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or include a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity, or structure, and in such cases, it is used herein to refer to polypeptides that share the appropriate activity or structure and can therefore be considered members of the same class or family of polypeptides. For each such class, the specification provides, and / or one of skill in the art will be aware of, exemplary polypeptides within the class whose amino acid sequence and / or function are known.In some embodiments, such exemplary polypeptides are reference polypeptides of a class or family of polypeptides. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of the class, and in some embodiments, with all polypeptides in the class, share common sequence motifs (e.g., characteristic sequence elements), and / or share common activity (in some embodiments, at a similar level or within a specified range). In some embodiments, a suitable polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide may comprise or consist of multiple fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to each other than that found in the polypeptide of interest (e.g., a fragment directly linked to the parent may be spatially separated in the polypeptide of interest, or vice versa, and / or the fragments may be present in a different order in the polypeptide of interest than in the parent), and thus the polypeptide of interest is a derivative of that parent polypeptide.

[0046] Preferential cleavage: As used herein with respect to an agent having cleavage activity, the term "preferential cleavage" will be understood by those skilled in the art to mean that the agent discriminates between potential target entities or conditions. For example, in some embodiments, an 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 IgA1 in the presence of IgA2) and / or is observed to act on the preferential target under reasonably equivalent conditions (e.g., enzyme target concentration, temperature, ionic strength, etc.). In some embodiments, the SD-IgAPP is characterized by cleaving at least 20% more IgA1 (e.g., at least 30%, 40%, 50%, 60%, 70%, 80%, 90% more) than IgA2.

[0047] Source: As used herein, the term "source" generally refers to an environment in which an agent of interest (e.g., a carbohydrate, lipid, nucleic acid, metal, polypeptide, small molecule, or combination thereof) may be naturally found, or from which such agent may be obtained or has been obtained (e.g., isolated). In some embodiments, a source may be or include a biological source (e.g., an organism, tissue, or cell, or a sample thereof). In some embodiments, a source may be an environmental source. In some embodiments, a source may be or include a primary sample from an organism (e.g., may be or include tissue or bodily fluids of such an organism, and / or may be or include cell(s) of such an organism). In some embodiments, an organism may be or include 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, particularly with respect to being a source. In some embodiments, the source may be or may include an engineered source, such as a cell line or culture, an in vitro system, or the like.

[0048] Specific binding: As used herein, the term "specific binding" refers to the ability to distinguish between possible binding partners in the environment in which the binding occurs. A binding agent that interacts with one specific target in the presence of other potential targets is said to "specifically bind" to that interacting target. In some embodiments, specific binding is assessed by detecting or measuring the degree and / or rate of association between the binding agent and its partner; in some embodiments, specific binding is assessed by detecting or measuring the degree and / or rate of dissociation of a binding agent-partner complex; in some embodiments, specific binding is assessed by detecting or measuring the ability of a binding agent to compete with an alternative interaction of its partner with another entity. In some embodiments, specific binding is assessed by performing such detection or measurement over a range of concentrations of one or both binding partners. In some embodiments, two agents that specifically bind to each other are said to form a complex with each other.

[0049] Specificity: As known in the art, the "specificity" of binding is the degree to which a particular ligand is able 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 the entire or nearly entire extent or degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical phenomena, if they exist at all, rarely go to completion and / or rarely proceed perfectly, or rarely achieve or avoid absolute results. Thus, the term "substantially" is 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 that is statistically likely to achieve a desired effect when administered to a subject according to a particular dosing regimen (e.g., a therapeutic dosing regimen). In some embodiments, the term refers to an amount sufficient to produce an effect in at least a significant proportion (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 suffering from and / or susceptible to a disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is an amount that reduces the incidence and / or severity of and / or delays the onset of one or more symptoms of a disease, disorder, and / or condition. Those skilled in the art will understand that the term "therapeutically effective amount" does not actually require achieving successful treatment in a particular individual. Rather, a therapeutically effective amount may be an amount that, when administered to a patient in need of such treatment, provides a particular desired response in a significant number of subjects, e.g., at least about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more patients within the patient population being treated. In some embodiments, reference to a therapeutically effective amount may be a reference to an amount sufficient to elicit a desired effect as measured in one or more particular tissues (e.g., tissues affected by a disease, disorder, or condition) or bodily fluids (e.g., blood, saliva, serum, sweat, tears, urine). One of skill in the art will understand 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 multiple doses, e.g., as part of a dosing regimen.

[0052] Treatment: As used herein, "treatment" refers to a reduction in undesired or abnormal IgA (e.g., IgA1) in tissues (e.g., kidneys, skin, blood vessels, etc.) and / or circulation. Treatment can refer to a reduction in IgA1 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%, compared to the amount of IgA1 deposition in a control individual not administered an SD-IgAPP of the present disclosure. Preferably, treatment refers to an increase in IgA1 clearance of at least about 25% compared to the amount of IgA1 in tissues and / or circulation in the same individual prior to administration of an isolated polypeptide of the present disclosure. A reduction in abnormal IgA1 in the circulation can prevent deposition of IgA1 in tissues, including the kidney and skin, by at least about 5% in response to administration of an IgA protease polypeptide agent of the disclosure, compared to the amount of IgA1 in the circulation in the same individual before administration of an IgA protease polypeptide agent of the disclosure. For example, "treatment" can refer to an increase in IgA 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%.

[0053] When used in connection with therapeutic compositions of the present disclosure, the term "unit dosage form" refers to physically discrete units suited as unitary dosages for subjects, each unit containing a predetermined quantity of active material calculated to produce a desired therapeutic effect in combination with the required diluent, i.e., carrier or vehicle.

[0054] Detailed Description of Specific Embodiments Immunoglobulin A (IgA) Immunoglobulin A (IgA) is the major class of immunoglobulin found in human mucosal secretions. IgA is a polymeric antibody, typically containing two copies of IgA assembled in a single binding chain to form dimeric IgA. Dimeric IgA immunoglobulin proteins reach the fluids of the gastrointestinal and respiratory tracts by binding to another polypeptide chain called the "polymeric immunoglobulin receptor," which is produced by mucosal epithelial cells. Upon binding to this receptor, dimeric IgA antibodies are transported to the apical surface of epithelial cells by an intracellular transport pathway and released into the mucosal fluid space as secretory IgA (sIgA).

[0055] IgA includes two isotypes, IgA1 and IgA2. The main difference between isotypes IgA1 and IgA2 is in the hinge region of the heavy polypeptide chain. A deletion of 13 amino acids characterizes the IgA2 hinge region. IgA1 and IgA2 are also found in human plasma and serum.

[0056] IgA protease IgA protease is a bacterial enzyme that cleaves human IgA molecules at the hinge region. Some IgA proteases cleave both IgA1 and IgA2, while some IgA1 proteases preferentially cleave one over the other, or only one. For example, certain IgA proteases cleave IgA1 at a hinge region that is mutated or absent in the corresponding IgA2 hinge. Cleavage of monomeric IgA1 at the hinge region yields two intact Fab regions and an intact Fc; these otherwise unmodified fragments retain most of the biological properties of the intact IgA1 protein.

[0057] IgA proteases are expressed in both Gram-negative and Gram-positive bacteria as single-chain precursors that cross the bacterial membrane. Gram-negative IgA proteases undergo autocatalytic cleavage to release the N-terminal soluble IgA mature protease.

[0058] Several classes of IgA proteases have been defined by comparison of amino acid sequences (within each class) and / or mechanistic similarities. Examples of such classes include IgA cysteine ​​proteases, IgA metalloproteases, IgA M26 zinc metalloproteases, IgA S6 serine proteases, and IgA M64 metalloproteases. The IgA cysteine ​​proteases of Prevotella species and the IgA metalloproteases of Capnocytophaga species (humans) cleave the peptide bond P223-S224 of IgA. The M26 zinc metalloproteases of Streptococcus and Gemella cleave the peptide bond P227-T228 of IgA1. Table 2 shows examples of amino acid sequences of IgA proteases.

[0059] The M64 metalloprotease from Clostridium ramosum is the only known enzyme reported to cleave both IgA1 and IgA2. This protease cleaves IgA at the hinge bond P221-V222 (in the standard numbering system), which is present in both IgA1 and IgA2 immunoglobulins. Metallo- and serine-type IgA1 proteinases, although quite different in primary structure, are reported to share strict selectivity for human IgA1 as a substrate due to their IgA cleavage sites.

[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 other known metalloendopeptidases, including any IgA1 proteinases, belonging to this class of proteolytic enzymes. To date, only the primary sequence and identification of various motifs are known for the Clostridium ramosum IgA M64 protease. The domain structure of the Clostridium ramosum IgA M64 protease has not yet been described.

[0061] SEQ ID NO:1 (Table 2) provides an exemplary amino acid sequence of a C. ramosum IgA protease that includes 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 that does not have 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 against which the provided SD-IgAPP agent(s) can be evaluated or compared.

[0062] It should also 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, when such proteases are utilized, it may be desirable to use techniques such as recombinant techniques to facilitate their isolation (e.g., by tagging them with epitope tags).

[0063] Subdomain IgA protease polypeptide (SD-IgAPP) agents The present invention provides IgA protease polypeptide agents that effectively cleave at least IgA1 and have certain useful properties and advantages compared to other IgA protease polypeptides, including, in particular, certain naturally occurring IgA proteases, such as the IgA protease of C. ramosum, 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 the domain structure for certain M64 IgA proteases, specifically including the IgA protease of C. ramosum, and further demonstrates, surprisingly, that various sequences, including the two complete domains found therein, are not required for useful IgA protease activity, particularly useful IgA1 protease activity.

[0066] That is, the present disclosure describes structural studies performed with M64 IgA proteases (e.g., a representative M64 IgA protease, specifically, in this example, the IgA protease of C. ramosum). The present disclosure teaches that N-terminal and / or C-terminal domain sequences may be unnecessary (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 an "intermediate domain" section that is present in various IgA proteases, particularly various M64 IgA proteases (see, e.g., SEQ ID NO: 4 and Figures 6A-H), and is sufficient for IgA protease activity. As exemplified, in some embodiments, such intermediate domains retain IgA protease activity equivalent to that of an appropriate full-length IgA protease in cleaving IgA1. In some embodiments, such intermediate domains exhibit increased IgA cleavage (e.g., IgA1 cleavage activity) compared to such reference proteases. In some embodiments, such intermediate domains retain IgA protease activity equivalent to that of an appropriate full-length IgA protease in cleaving IgA2. In some embodiments, such intermediate domains exhibit reduced IgA2 cleavage (e.g., compared to an appropriate 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 particular sequence feature(s) (e.g., one or more distinctive sequence elements) and / or overall percent identity over at least one significant (e.g., about 200, 250, 300, 350, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or more amino acids) stretch, but is shorter than the full-length M64 IgA protease, e.g., the full-length C. ramosum M64 IgA protease. In some of the foregoing embodiments, the stretch comprises amino acids 314-807 of the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, the stretch comprises amino acids 329-807 of the amino acid sequence set forth in SEQ ID NO:1. In one of the foregoing embodiments, the stretch comprises amino acids 331-632 of the amino acid sequence set forth in SEQ ID NO:1. In another embodiment of the foregoing, the interval 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 set forth in SEQ ID NO: 1. In some embodiments, the interval 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 set forth in SEQ ID NO: 1. In another embodiment of the foregoing, the interval 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 set forth in SEQ ID NO: 1. The present disclosure provides useful agents that are or comprise IgA protease polypeptides having an amino acid sequence that differs from a suitable reference IgA protease (e.g., SEQ ID NO: 2, Table 2) or a fragment thereof by, for example, one or more sequence modifications selected from the disclosed deletions, insertions, inversions, substitutions, truncations, and combinations thereof. Typically, such sequence modifications are or comprise at least one deletion or truncation.The present disclosure confirms that the N-terminal domain tolerates significant structural changes, such as deletion of the N-terminal domain or portions thereof, and that the C-terminal domain tolerates significant structural changes, such as deletion of the C-terminal domain or portions thereof. Alterations or truncations of such domains have been found to have little or no adverse effect on IgA protease polypeptide agent activity. In many embodiments, such sequence modifications are or include C-terminal truncations. In many embodiments, such sequence modifications are or include N-terminal truncations. In many embodiments, such sequence modifications are or include both C- and N-terminal truncations.

[0069] In many embodiments, a suitable reference IgA protease is M64 IgA protease. In many such embodiments, provided IgA protease polypeptides (e.g., SD-IgAPP agents) lack C-terminal and / or N-terminal sequences relative to such references. In some embodiments, the C-terminal amino acids of a suitable reference IgA protease are residues corresponding to amino acids 808-1234 of SEQ ID NO:1, and in some embodiments, the N-terminal amino acids of a suitable 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, the N-terminal amino acids of a suitable 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, the N-terminal amino acids of a suitable 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 present disclosure, IgA protease polypeptide agents are provided that are truncation variant(s) of such regions, hi some embodiments, an IgA protease polypeptide agent is a truncation 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 the ability to reduce IgA deposition, increasing bioavailability, and / or increasing the longevity of the IgA protease polypeptide in a subject, etc.).

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

[0072] In some embodiments, an IgA protease polypeptide agent (e.g., an SD-IgAPP agent) according to the present disclosure has an amino acid sequence that is or includes an element having the following general formula: Y1-N-M1-M2-M3-C-Y2 (wherein Y1, N, M1, M2, M3, C, and Y2 each comprise or consist of a consecutive sequence of amino acids, M1 comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 7, M2 comprises or consists of a HEX1X2H motif, X1 and X2 are amino acids, M3 substantially comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 9, the complete amino acid sequence of the polypeptide does not comprise or consist of the amino acid sequence of SEQ ID NO: 1 or 2, and Y1, N, C, and Y2 are optional.)

[0073] In some embodiments, M1-M2-M3 represent the intermediate domain of an IgA protease, e.g., the intermediate domain of an M64 protease, e.g., the intermediate domain of the C. ramosum IgA M64 protease, where M2 contains a HEX1X2H motif that supports the proteolytic activity of the IgA protease. M1-M2-M3 can be any contiguous sequence of amino acids in the intermediate domain of an IgA protease, so long as the amino acid sequence contains the HEX1X2H motif. In some embodiments, M1-M2-M3 are the intermediate domain of an IgA protease (e.g., the intermediate domain of an M64 protease, such as the C. ramosum IgA M64 protease). In some embodiments, N represents the N-terminal domain of an IgA protease, e.g., the N-terminal domain of an M64 protease, e.g., the N-terminal domain of the C. ramosum IgA protease. In some embodiments, C represents the C-terminal domain of an IgA protease, eg, the C-terminal domain of an M64 protease, eg, the C-terminal domain of the IgA protease of C. ramosum.

[0074] In some embodiments, M2 comprises or consists of a HEX1X2H motif, and X1 and X2 can be any amino acid that can be incorporated into a polypeptide chain. X1 or X2 can be a naturally occurring or non-naturally occurring amino acid. In some embodiments, M2 consists of a HEX1X2H motif. In some embodiments, X1 is an amino acid having a hydrophobic side chain. In some embodiments, X1 is selected from the group consisting of alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan. In some embodiments, X1 is phenylalanine or leucine. In some embodiments, X2 is glycine. In some embodiments, X1 is phenylalanine and X2 is glycine, e.g., the amino acid sequence is the sequence of SEQ ID NO: 8. In some embodiments, X1 is leucine and X2 is glycine.

[0075] In some embodiments, M2 comprises a 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 M1, and / or between the HEX1X2H motif and M3, thereby separating M1 from M2 and / or separating M2 from M3. In some embodiments, M2 comprises a HEX1X2H motif and about 1 to about 100 additional amino acid residues, e.g., 2 to 50 additional amino acid residues, e.g., 10 to 40 additional amino acid residues, or 20 to 30 additional amino acid residues.

[0076] In some embodiments, M1 and M2 are separated by one or more amino acids. In some embodiments, M1 and M2 are separated by up to 20 amino acids, e.g., 10 amino acids, e.g., 5 amino acids. In some embodiments, M2 and M3 are separated by one or more amino acids. In some embodiments, M2 and M3 are separated by up to 20 amino acids, e.g., 10 amino acids, or e.g., 5 amino acids.

[0077] In some embodiments, the M1 amino acid sequence is at least 75% identical to SEQ ID NO:7, such as at least 80% identical to SEQ ID NO:7, for example, at least 85% identical to SEQ ID NO:7, for example, at least 90% identical to SEQ ID NO:7, for example, at least 91% identical to SEQ ID NO:7, for example, at least 92% identical to SEQ ID NO:7, for example, at least 93% identical to SEQ ID NO:7, for example, at least 94% identical to SEQ ID NO:7, for example, at least 95% identical to SEQ ID NO:7, for example, at least 96% identical to SEQ ID NO:7, for example, at least 97% identical to SEQ ID NO:7, for example, at least 98% identical to SEQ ID NO:7, for example, at least 99% identical to SEQ ID NO:7, or e.g., 100% identical to SEQ ID NO:7. In some embodiments, M1 is or comprises at least a portion of SEQ ID NO:7. In some embodiments, the M1 amino acid sequence is at least 75% identical to SEQ ID NO:25, such as at least 80% identical to SEQ ID NO:25, for example, at least 85% identical to SEQ ID NO:25, for example, at least 90% identical to SEQ ID NO:25, for example, at least 91% identical to SEQ ID NO:25, for example, at least 92% identical to SEQ ID NO:25, for example, at least 93% identical to SEQ ID NO:25, for example, at least 94% identical to SEQ ID NO:25, for example, at least 95% identical to SEQ ID NO:25, for example, at least 96% identical to SEQ ID NO:25, for example, at least 97% identical to SEQ ID NO:25, for example, at least 98% identical to SEQ ID NO:25, for example, at least 99% identical to SEQ ID NO:25, or for example, 100% identical to SEQ ID NO:25. In some embodiments, M1 is or comprises at least a portion of SEQ ID NO:25.In some embodiments, the M1 amino acid sequence is at least 75% identical to SEQ ID NO: 28, such as at least 80% identical to SEQ ID NO: 28, for example, at least 85% identical to SEQ ID NO: 28, for example, at least 90% identical to SEQ ID NO: 28, for example, at least 91% identical to SEQ ID NO: 28, for example, at least 92% identical to SEQ ID NO: 28, for example, at least 93% identical to SEQ ID NO: 28, for example, at least 94% identical to SEQ ID NO: 28, for example, at least 95% identical to SEQ ID NO: 28, for example, at least 96% identical to SEQ ID NO: 28, for example, at least 97% identical to SEQ ID NO: 28, for example, at least 98% identical to SEQ ID NO: 28, for example, at least 99% identical to SEQ ID NO: 28, or for example, 100% identical to SEQ ID NO: 28. In some embodiments, M1 is or comprises at least a portion of SEQ ID NO: 28.

[0078] In some embodiments, the M3 amino acid sequence is at least 75% identical to SEQ ID NO:9, such as at least 80% identical to SEQ ID NO:9, for example, at least 85% identical to SEQ ID NO:9, for example, at least 90% identical to SEQ ID NO:9, for example, at least 91% identical to SEQ ID NO:9, for example, at least 92% identical to SEQ ID NO:9, for example, at least 93% identical to SEQ ID NO:9, for example, at least 94% identical to SEQ ID NO:9, for example, at least 95% identical to SEQ ID NO:9, for example, at least 96% identical to SEQ ID NO:9, for example, at least 97% identical to SEQ ID NO:9, for example, at least 98% identical to SEQ ID NO:9, for example, at least 99% identical to SEQ ID NO:9, or, for example, 100% identical to SEQ ID NO:9. In some embodiments, the M3 amino acid sequence is at least 75% identical to SEQ ID NO: 26, such as at least 80% identical to SEQ ID NO: 26, for example, at least 85% identical to SEQ ID NO: 26, for example, at least 90% identical to SEQ ID NO: 26, for example, at least 91% identical to SEQ ID NO: 26, for example, at least 92% identical to SEQ ID NO: 26, for example, at least 93% identical to SEQ ID NO: 26, for example, at least 94% identical to SEQ ID NO: 26, for example, at least 95% identical to SEQ ID NO: 26, for example, at least 96% identical to SEQ ID NO: 26, for example, at least 97% identical to SEQ ID NO: 26, for example, at least 98% identical to SEQ ID NO: 26, for example, at least 99% identical to SEQ ID NO: 26, or for example, 100% identical to SEQ ID NO: 26. In some embodiments, the M3 amino acid sequence comprises a DEY motif. In some embodiments, the DEY motifs present in the M3 amino acid sequence and the M2 amino acid sequence are separated by about 3 to about 9 amino acids, for example, about 4 to about 8 amino acids, for example, about 5 to about 7 amino acids, for example, 6 amino acids.Without being bound by theory, the M3 amino acid sequence may contain one or more cysteine ​​amino acids (e.g., cysteine ​​amino acids corresponding to positions 63, 73, and / or 76 of SEQ ID NO:9) necessary or sufficient to coordinate metals (e.g., zinc ions) and thereby inactivate SD-IgAPP. In some embodiments, one or more cytosine amino acids may be substituted or removed, thereby reducing or abolishing the ability of SD-IgAPP to coordinate metals (e.g., zinc ions) and rendering SD-IgAPP inactive. In some embodiments, one or more cysteine ​​amino acids at positions 63, 73, and / or 76 of SEQ ID NO:9 (corresponding to amino acid positions 616, 626, and 629 of SEQ ID NO:1) are substituted or removed (see Example 2 and Figure 4). In some embodiments, the M3 amino acid sequence is or includes at least a portion of SEQ ID NO:9.

[0079] In some embodiments, M1-M2-M3 correspond to the middle domain of an IgA protease, such as the M64 protease (e.g., the C. ramosum IgA M64 protease of SEQ ID NO: 1 or 2). In some embodiments, an IgA protease polypeptide agent (e.g., an SD-IgAPP agent) has an amino acid sequence comprising a portion of the amino acid sequence of SEQ ID NO: 4, e.g., about 200 to about 302, e.g., about 250 to about 302, amino acids, e.g., at least 200 amino acids, e.g., at least 250 amino acids, or e.g., at least 300 amino acids of SEQ ID NO: 4. In some embodiments, an IgA protease polypeptide agent (e.g., an SD-IgAPP agent) has an amino acid sequence that includes a portion of the amino acid sequence of SEQ ID NO: 27, e.g., an amino acid sequence that includes about 200 to about 450, e.g., about 250 to about 400 amino acids, e.g., at least 250 amino acids, e.g., at least 300 amino acids, e.g., at least 350 amino acids, of SEQ ID NO: 27. In some embodiments, an IgA protease polypeptide agent (e.g., an SD-IgAPP agent) has an amino acid sequence that includes a portion of the amino acid sequence of SEQ ID NO: 22, e.g., an amino acid sequence that includes about 200 to about 450, e.g., about 250 to about 400 amino acids, e.g., at least 250 amino acids, e.g., at least 300 amino acids, e.g., at least 350 amino acids, of SEQ ID NO: 22. In some embodiments, an IgA protease polypeptide agent (e.g., an SD-IgAPP agent) has an amino acid sequence that is or includes an amino acid sequence having at least 45% overall sequence identity to SEQ ID NO: 4. In some embodiments, an IgA protease polypeptide agent (e.g., an SD-IgAPP agent) has an amino acid sequence that is or includes an amino acid sequence having at least 45% overall sequence identity to SEQ ID NO: 27. In some embodiments, an IgA protease polypeptide agent (e.g., an SD-IgAPP agent) has an amino acid sequence that is or includes an amino acid sequence that has at least 45% overall sequence identity to SEQ ID NO: 22. SEQ ID NO: 4, SEQ ID NO: 27, and SEQ ID NO: 22 are consensus sequences for the C. ramosum IgA M64 protease middle domain.In some embodiments, an IgA protease polypeptide agent (e.g., an SD-IgAPP agent) has an amino acid sequence that shares at least 50%, e.g., at least 55%, e.g., at least 60%, e.g., at least 65%, e.g., at least 70%, e.g., at least 75% 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, for example, at least 85% identity to SEQ ID NO:4, for example, at least 90% identity to SEQ ID NO:4, for example, at least 91% identity to SEQ ID NO:4, for example, at least 92% identity to SEQ ID NO:4, for example, at least 93% identity to SEQ ID NO:4, for example, at least 94% identity to SEQ ID NO:4, for example, at least 95% identity to SEQ ID NO:4, for example, at least 96% identity to SEQ ID NO:4, for example, at least 97% identity to SEQ ID NO:4, for example, at least 98% identity to SEQ ID NO:4, for example, at least 99% identity to SEQ ID NO:4, or for example, 100% identity to SEQ ID NO:4. In some embodiments, M1-M2-M3 (i.e., the middle domain of an IgA protease) comprises a HEKXH motif and a DEY motif. In some embodiments, M1-M2-M3 comprises a HEFGH motif and a DEY motif.In some embodiments, M1-M2-M3 comprise an amino acid sequence sharing at least 80% identity to SEQ ID NO:27, such as at least 85% identity to SEQ ID NO:27, for example, at least 90% identity to SEQ ID NO:27, for example, at least 91% identity to SEQ ID NO:27, for example, at least 92% identity to SEQ ID NO:27, for example, at least 93% identity to SEQ ID NO:27, for example, at least 94% identity to SEQ ID NO:27, for example, at least 95% identity to SEQ ID NO:27, for example, at least 96% identity to SEQ ID NO:27, for example, at least 97% identity to SEQ ID NO:27, for example, at least 98% identity to SEQ ID NO:27, for example, at least 99% identity to SEQ ID NO:27, or e.g., 100% identity to SEQ ID NO:27. In some embodiments, M1-M2-M3 comprise 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, for example, at least 90% identity to SEQ ID NO:22, for example, at least 91% identity to SEQ ID NO:22, for example, at least 92% identity to SEQ ID NO:22, for example, at least 93% identity to SEQ ID NO:22, for example, at least 94% identity to SEQ ID NO:22, for example, at least 95% identity to SEQ ID NO:22, for example, at least 96% identity to SEQ ID NO:22, for example, at least 97% identity to SEQ ID NO:22, for example, at least 98% identity to SEQ ID NO:22, for example, at least 99% identity to SEQ ID NO:22, or, for example, 100% identity to SEQ ID NO:22.

[0080] In some embodiments, SD-IgAPP agents provided by the present disclosure have an amino acid sequence that includes M1-M2-M3 (e.g., corresponding to the "middle domain" of an IgA protease such as the M64 IgA protease described herein) and further includes the N-terminal domain (N) or a portion thereof.

[0081] In some embodiments, N comprises or consists of a contiguous amino acid sequence that also includes M1-M2-M3 (eg, as may be found in a reference IgA protease polypeptide).

[0082] In some embodiments, N can be or include one or more amino acids found at the N-terminus of M1-M2-M3 of a reference IgA protease (e.g., a reference M64 IgA protease, such as the IgA protease of C. ramosum). In some embodiments, N includes all or substantially all of the amino acids found at the N-terminus of M1-M2-M3 of such reference IgA protease (e.g., in the mature form of such reference IgA protease). In some embodiments, N includes fewer than all of the amino acids found at the N-terminus of M1-M2-M3 in such reference (mature) IgA protease, and in some such embodiments, N includes the amino acids found most proximal to M1-M2-M3 in such reference (mature) IgA protease. In some embodiments, N includes amino acids 314-330 of the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, N includes amino acids 329-330 of the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, N comprises an amino acid sequence comprising amino acids 200 to 313 of the amino acid sequence set forth in SEQ ID NO:1, or a portion thereof.

[0083] In some embodiments, when a provided IgA protease polypeptide agent comprises N, and N corresponds to part or all of the N-terminal portion of a reference IgA protease, such as the reference M64 IgA protease (e.g., the IgA protease of C. ramosum), N contains one or more sequence variations (i.e., modifications) compared to such N-terminal portion, but maintains sufficient overall sequence identity and / or characteristics that one of skill in the art would understand that the sequence reasonably corresponds to part of such N-terminal portion.

[0084] In some embodiments, the N amino acid sequence is at least 70% identical to SEQ ID NO: 5, such as at least 75% identical, for example, at least 80% identical, for example, at least 85% identical, for example, at least 90% identical, for example, at least 91% identical, for example, at least 92% identical, for example, at least 93% identical, for example, at least 94% identical, for example, at least 95% identical, for example, at least 96% identical, for example, at least 97% identical, for example, at least 98% identical, for example, at least 99% identical, or at least 100% identical to SEQ ID NO: 5. In some embodiments, the N amino acids 1 to 328 of the amino acid sequence set forth in SEQ ID NO: 1, or a fragment thereof, are included.

[0085] In some embodiments, the amino acid sequence of an 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, at least 80% identity to SEQ ID NO:3, such as at least 85% identity to SEQ ID NO:3, for example at least 90% identity to SEQ ID NO:3, for example at least 91% identity to SEQ ID NO:3, such as at least 92% identity to SEQ ID NO:3, for example at least 93% identity to SEQ ID NO:3, for example at least 94% identity to SEQ ID NO:3, for example at least 95% identity to SEQ ID NO:3, for example at least 96% identity to SEQ ID NO:3, such as at least 97% identity to SEQ ID NO:3, for example at least 98% identity to SEQ ID NO:3, for example at least 99% identity to SEQ ID NO:3, or for example 100% identity to SEQ ID NO:3.

[0086] In some embodiments, C can be or include one or more amino acids found at the C-terminus of M1-M2-M3 of a reference IgA protease (e.g., a reference M64 IgA protease, such as the IgA protease of C. ramosum). In some embodiments, C includes all or substantially all of the amino acids found at the C-terminus of M1-M2-M3 of such reference IgA protease (e.g., in the mature form of such reference IgA protease). In some embodiments, C includes fewer than all of the amino acids found at the C-terminus of M1-M2-M3 in such reference (mature) IgA protease, and in some such embodiments, C includes the amino acids found most proximal to M1-M2-M3 in such reference (mature) IgA protease. In some embodiments, C includes amino acids 633 to 807 of the amino acid sequence set forth in SEQ ID NO:1. In some embodiments, C comprises an amino acid sequence including amino acids 808 to 1000 of the amino acid sequence set forth in SEQ ID NO:1, or a part thereof.

[0087] In some embodiments, when a provided IgA protease polypeptide agent comprises 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 IgA protease of C. ramosum), C contains one or more sequence variations (i.e., modifications) compared to such C-terminal portion, but maintains sufficient overall sequence identity and / or characteristics that one of skill in the art would understand that the sequence reasonably corresponds to part of such C-terminal portion.

[0088] In some embodiments, the C amino acid sequence is at least 70% identical to SEQ ID NO:6, such as at least 75% identical, for example, at least 80% identical, for example, at least 85% identical, for example, at least 90% identical, for example, at least 91% identical, such as at least 92% identical, for example, at least 93% identical, for example, at least 94% identical, for example, at least 95% identical, for example, at least 96% identical, for example, at least 97% identical, for example, at least 98% identical, for example, at least 99% identical, or at least 100% identical to SEQ ID NO:6.

[0089] In some embodiments, the amino acid sequence of an 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, for example at least 85% identity to SEQ ID NO:10, for example at least 90% identity to SEQ ID NO:10, for example at least 91% identity to SEQ ID NO:10, for example at least 92% identity to SEQ ID NO:10, for example at least 93% identity to SEQ ID NO:10, for example at least 94% identity to SEQ ID NO:10, for example at least 95% identity to SEQ ID NO:10, for example at least 96% identity to SEQ ID NO:10, for example at least 97% identity to SEQ ID NO:10, for example at least 98% identity to SEQ ID NO:10, for example at least 99% identity to SEQ ID NO:10, or for example 100% identity to SEQ ID NO:10.

[0090] In some embodiments, provided SD-IgAPP agents comprise the intermediate domains M1-M2-M3 described above, and further comprise the N-terminal domain (N) and the C-terminal domain (C) described above.

[0091] In some embodiments, the provided SD-IgAPP agents have an amino acid sequence that comprises or consists of a sequence having at least 75% identity to the corresponding portion of SEQ ID NO:2, such as at least 80% identity to such portion of SEQ ID NO:2, for example at least 85% identity to such portion of SEQ ID NO:2, for example at least 90% identity to such portion of SEQ ID NO:2, for example at least 91% identity to such portion of SEQ ID NO:2, such as at least 92% identity to such portion of SEQ ID NO:2, for example at least 93% identity to such portion of SEQ ID NO:2, for example at least 94% identity to such portion of SEQ ID NO:2, for example 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, for example at least 97% identity to such portion of SEQ ID NO:2, for example at least 98% identity to such portion of SEQ ID NO:2, for example at least 99% identity to such portion of SEQ ID NO:2, or for example 100% identity to such portion of SEQ ID NO:2.

[0092] In some embodiments, an IgA protease polypeptide agent (such as an SD-IgAPP agent) corresponds to a fragment of an IgA protease, such as M64 IgA protease and / or the IgA protease of C. ramosum. That is, in some embodiments, provided agents exhibit high identity (e.g., at least 70% or more, in some embodiments at least 75% or more, e.g., at least 80% or more, at least 85% or more, e.g., at least 90% or more, e.g., at least 91% or more, e.g., at least 92% or more, e.g., at least 93% or more, e.g., at least 94% or more, e.g., at least 95% or more, e.g., at least 96% or more, e.g., at least 97% or more, or e.g., at least 98% or more, e.g., at least 99% or more) to the corresponding fragment of SEQ ID NO:2. In some such embodiments, one skilled in the art will understand that the provided SD-IgAPP agent is a fragment lacking amino acid residues from the C-terminus to residue 808 of SEQ ID NO:1, e.g., a deletion from the C-terminus to residue 810 of SEQ ID NO:1, for example, a deletion from the C-terminus to residue 820, for example, a deletion from the C-terminus to residue 830, for example, a deletion from the C-terminus to residue 840, for example, a deletion from the C-terminus to residue 850, for example, a deletion from the C-terminus to residue 860, for example, a deletion from the C-terminus to residue 870, for example, a deletion from the C-terminus to residue 880, for example, a deletion from the C-terminus to residue 890, for example, a deletion from the C-terminus to residue 900 of SEQ ID NO:1. It will be appreciated that a deletion from the C-terminus to residue 900, for example, a deletion from the C-terminus to residue 910, for example, a deletion from the C-terminus to residue 920, for example, a deletion from the C-terminus to residue 930, for example, a deletion from the C-terminus to residue 940, for example, a deletion from the C-terminus to residue 950, for example, a deletion from the C-terminus to residue 1000, for example, a deletion from the C-terminus to residue 1050, for example, a deletion from the C-terminus to residue 1100, for example, a deletion from the C-terminus to residue 1150, for example, a deletion from the C-terminus to residue 1200, or for example, a deletion from the C-terminus to residue 1230.

[0093] In some embodiments, one of skill in the art will recognize that the provided SD-IgAPP agents correspond to a fragment of SEQ ID NO: 1 lacking amino acids 1-30, for example, a fragment of SEQ ID NO: 1 having a deletion of amino acids 1-50, for example a deletion of amino acids 1-60, for example a deletion of amino acids 1-70, for example a deletion of amino acids 1-80, for example a deletion of amino acids 1-90, for example a deletion of amino acids 1-100, for example a deletion of amino acids 1-150, a deletion of SEQ ID NO: 1-200, for example a deletion of amino acids 1-250, for example a deletion of amino acids 1-300, or a deletion of amino acids 1-313, or a deletion of amino acids 1-328.

[0094] In some such embodiments, one of skill in the art will recognize that the provided SD-IgAPP agent corresponds to a fragment lacking amino acids at both the C-terminus and the N-terminus, e.g., lacking any of the combinations described above herein.

[0095] In some embodiments, provided IgA protease polypeptide agents (e.g., SD-IgAPP agents) have an amino acid sequence of 600 or less, e.g., 550 or less, e.g., 500 or less. In some embodiments, an SD-IgAPP agent according to any of the foregoing embodiments includes a maximum of 500 amino acids, e.g., a maximum of 494 amino acids. In some embodiments, an SD-IgAPP agent according to any of the foregoing embodiments includes a maximum of 500 amino acids, a maximum of 400 amino acids, e.g., a maximum of 300 amino acids.

[0096] As noted above, the present disclosure allows for alterations and / or deletions, particularly in the N-terminal and C-terminal domains of the C. ramosum IgA protease polypeptide (particularly amino acid residues 1-313, or 1-328, and 808-1234 of SEQ ID NO: 1).

[0097] In some embodiments, the IgA protease polypeptide agent comprises any one of the foregoing sequences, but does not comprise a full-length protease, such as a full-length wild-type IgA protease.

[0098] Those skilled in the art are aware of various techniques for comparing and evaluating polypeptide sequences and structural relationships (e.g., for identifying and / or evaluating IgA protease polypeptide sequences that may be variants or alternatives to the sequences exemplified herein and are useful in accordance with the present disclosure). For example, BLAST (available, for example, at 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" or 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 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).

[0099] For example, sequence alignments can be performed between the IgA protease polypeptides of C. ramosum (e.g., using 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 have sequence similarity to the region defined by amino acid residues 1-313 and 808-1234 of SEQ ID NO: 1 (or subregions thereof) are regions that are likely to be tolerant to alteration and / or deletion in such other IgA proteases.

[0100] Furthermore, those skilled in the art recognize functional and / or physical similarities between different amino acids, which may allow substitution of one amino acid for another with reduced risk of significantly disrupting the structure and / or function of a polypeptide. For example, in some embodiments, amino acids may be classified as acidic, neutral, or basic, or as polar or nonpolar, or as having bulky versus small side chains. Substitutions with similar residues may be referred to as "homologous" substitutions, and the characteristics that account for such homologous substitutions (the exact characteristics of which can often be user-defined) are often built into sequence comparison software. Those skilled in the art will understand that in some embodiments, sequence identity may be assessed independently of homologous substitutions, i.e., homologous residues are not "counted" as non-identical residues. Thus, any reference to sequence identity in the present disclosure may, in some embodiments, be considered identity independent of homology.

[0101] Those skilled in the art also recognize that sequence comparison techniques can typically take into account gaps or other variations in the relative positioning between sequence elements.

[0102] In some embodiments, SD-IgAPP agents provided by the present disclosure that include an M1-M2-M3 intermediate domain (e.g., corresponding to the "intermediate domain" of an IgA protease, such as the M64 IgA protease described herein) further include an N-terminal domain (N) or a portion thereof (e.g., the N-terminal domain of an IgA protease, such as the M64 IgA protease). In some embodiments, such SD-IgAPP agents are characterized by exhibiting preferential cleavage of IgA1 over IgA2 (e.g., exhibiting limited IgA2 cleavage). In some embodiments, such SD-IgAPP agents exhibit enhanced cleavage of IgA1 compared to SD-IgAPPs consisting of only the intermediate domain when incubated in HEPES buffer (pH 7.5) at 37°C for 15-60 minutes in the presence of IgA1 or IgA2.

[0103] In some embodiments, the SD-IgAPP agent is not a dimeric protein. In some embodiments, the SD-IgAPP agent is less than 300 kDa, for example, less than 290 kDa, for example, less than 280 kDa, for example, less than 270 kDa, for example, less than 260 kDa, for example, less than 250 kDa, for example, less than 240 kDa, for example, less than 230 kDa, for example, less than 220 kDa, for example, less than 210 kDa, for example, less than 200 kDa, for example, 190 kDa. For example, less than 180 kDa, for example, less than 170 kDa, for example, less than 160 kDa, for example, less than 150 kDa, for example, less than 140 kDa, for example, less than 130 kDa, for example, less than 120 kDa, for example, less than 110 kDa, for example, less than 100 kDa, for example, less than 90 kDa, for example, less than 80 kDa, for example, less than 70 kDa, for example, less than 60 kDa. In some embodiments, the SD-IgAPP agent comprises a section of amino acids 31 to less than 1203 of the amino acid sequence set forth in SEQ ID NO: 1. In some embodiments, the SD-IgAPP agent comprises an alanine residue at position 31 of SEQ ID NO: 1. In some embodiments, the SD-IgAPP agent does not comprise the A31G amino acid substitution of SEQ ID NO: 31.

[0104] Dissimilar parts: In some embodiments, provided SD-IgAPP agents may include one or more moieties not found in the reference IgA protease. In some embodiments, such heterologous moiety(s) is or includes a polypeptide moiety fused to an IgA protease sequence. In some such embodiments, the polypeptide moiety is fused at the N-terminus or C-terminus of such IgA protease sequence (e.g., represented herein in the general formula as Y1 or Y2).

[0105] In some embodiments, the fused polypeptide portion may be useful, e.g., for detection and / or purification, and may function as a "tag," e.g., via association with a binding agent therefor. In some embodiments, fusing a tag to an IgA protease polypeptide agent of the present disclosure may aid in the purification and / or detection of the polypeptide agent and / or may provide a means for the IgA protease polypeptide agent to form a complex with a ligand, such as, e.g., an anti-tag antibody for therapeutic purposes.

[0106] Those skilled in the art are aware of techniques by which tags can be incorporated into or otherwise attached to IgA protease polypeptides that include the tags. For example, in some embodiments, the tag can be or include a peptide, and the peptide can be chemically ligated to or incorporated into the IgA protease polypeptide (e.g., via recombinant techniques known in the art). In alternative embodiments, the tag can be or include a non-peptide moiety (e.g., biotin / avidin, etc.). Typically, such tags are associated with the IgA protease polypeptide by chemical conjugation.

[0107] In some embodiments, a tag can be removably associated with an IgA protease polypeptide agent. For example, the tag (whether a peptide tag or a non-peptide tag) can be associated via a cleavable bond (e.g., by a chemically cleavable bond and / or a protease-cleavable bond). For example, in certain embodiments, a nucleotide sequence encoding the tag can be linked (e.g., by ligation, amplification, or other means) in frame to a sequence encoding an IgA protease polypeptide, e.g., upstream of a DNA sequence encoding an IgA protease autocatalytic cleavage site, such that upon cleavage of the IgA protease precursor polypeptide, a soluble IgA protease polypeptide comprising the tag is generated (e.g., is or can be secreted from a producing cell).

[0108] In some embodiments, the tag can be or include a moiety that is specifically bound by an antibody. In some embodiments, such a tag can be or include a known moiety for which a useful antibody is readily available. Alternatively or additionally, in some embodiments, the tag can be or include a metal-binding moiety (e.g., a so-called His tag, which allows for isolation via, for example, a metal-chelating resin or bead, such as nickel-NTA beads).

[0109] In some embodiments, tags include (directly or indirectly) detectable entities, such as chromogens, enzymes (e.g., that can catalyze a reaction resulting in a color change, fluorescence, luminescence, etc.), fluorophores, luminescent materials, radioisotopes, etc. Specific detectable entities familiar to those of skill in the art include, for example, biotin / strepavidin, fluorescent dyes (e.g., fluorescein, Texas Red, rhodamine, green fluorescent protein, etc.), radioactive labels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, glucose oxidase, and others commonly used in ELISAs), and calorimetric labels such as colloidal gold. Exemplary patents that teach 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.

[0110] In some embodiments, an SD-IgAPP agent according to the present disclosure may be obtained or manufactured by producing a polypeptide having a desired amino acid sequence (e.g., as described herein) (e.g., by expressing it from a nucleic acid, such as an engineered nucleic acid), followed by, and optionally, modifying, such a polypeptide.

[0111] Nucleic acid sequences (e.g., genes encoding various IgA proteases) are known (see, e.g., Table 2), and many such sequences are deposited in the GenBank database of the National Center for Biotechnology Information. Alternatively, or additionally, nucleic acid sequences encoding desired polypeptides can be determined or designed taking into account characteristics such as, for example, codon optimization preferences of a particular expression system, sequence elements that may affect nucleic acid stability and / or expressibility, etc.

[0112] Genetic material, including genes encoding such IgA protease polypeptides, or fragments thereof, is often publicly available through gene repositories and / or through laboratories that publish gene sequence information for such IgA proteases. Such genetic material can be manipulated, for example, using molecular biology techniques, to generate nucleic acids encoding the provided IgA protease polypeptide agents (e.g., SD-IgAPP agents) described herein.

[0113] Those skilled in the art are familiar with a variety of techniques useful and suitable for producing SD-IgAPP agents. For example, in some embodiments, SD-IgAPP agents can be produced recombinantly (e.g., expressed from recombinant nucleic acids) in an expression system (e.g., an in vitro expression system or a cell system, which may utilize cells such as bacterial, insect, mammalian, and / or yeast cells). In some embodiments, the IgA protease polypeptide can be modified after expression in an expression system. In some embodiments, the IgA protease polypeptide can be modified after isolation and / or purification. For example, in some embodiments, a tag (particularly a non-peptide tag) can be linked to the polypeptide.

[0114] In some embodiments, provided SD-IgAPP agents may include a moiety fused to the N-terminus of the IgA protease sequence. In some embodiments, the moiety is an Fc fusion. In some embodiments, the SD-IgAPP agent includes an Fc region. In some embodiments, the moiety is albumin (e.g., human albumin). In some embodiments, such a moiety may extend the half-life of the SD-IgAPP agent. In some embodiments, such a moiety may stabilize the SD-IgAPP agent.

[0115] In some embodiments, a suitable stabilizing moiety is an Fc domain. In certain embodiments, the Fc domain is derived from a human immunoglobulin. In certain embodiments, the Fc domain is derived from a human IgG1 constant region. However, it is understood that the Fc domain may be derived from an immunoglobulin of another mammalian species, including, for example, a rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species. In some embodiments, the Fc domain or portion thereof may be derived from immunoglobulin classes including IgM, IgG, IgD, IgA, and IgE, and immunoglobulin isotypes including IgG1, IgG2, IgG3, and IgG4.

[0116] IgA protease activity SD-IgAPP agents provided by the present disclosure are characterized by their ability to cleave IgA (e.g., human IgA1 and / or human IgA2). In some embodiments, provided SD-IgAPP agents cleave at the hinge region of IgA. In some embodiments, SD-IgAPP agents cleave at the hinge region of IgA1. In some embodiments, SD-IgAPP agents cleave at the hinge region of IgA2. In some embodiments, SD-IgAPP agents according to the present disclosure cleave at the hinge regions of IgA1 and IgA2.

[0117] In many embodiments, provided SD-IgAPP agents, in many such embodiments, preferentially cleave IgA1 relative to IgA2. In some embodiments, provided SD-IgAPP agents exhibit proteolytic activity against IgA, such as IgA1 and / or IgA2, comparable to (e.g., to the same extent as) a reference IgA protease (see Examples 6 and 7). In some embodiments, provided SD-IgAPP agents exhibit greater proteolytic activity than a suitable reference IgA protease (e.g., when evaluated under comparable conditions, e.g., side-by-side). In some embodiments, provided SD-IgAPP agents exhibit greater preference for IgA1 (e.g., compared to IgA2) relative to a suitable reference IgA protease (e.g., when evaluated under comparable conditions, e.g., side-by-side). In some embodiments, SD-IgAPP comprising the M1-M2-M3 intermediate domain and the N-terminal domain (N), or a portion thereof, exhibits a greater preference for IgA1 (e.g., compared to IgA2) compared to an appropriate reference IgA protease. In some embodiments, provided SD-IgAPP agents comprising the intermediate domain and the N-terminal domain, or a portion thereof, exhibit greater proteolytic activity against IgA1 compared to IgA2.

[0118] In some embodiments, provided SD-IgAPP agents cleave at V222-P223 of the IgA1 and / or IgA2 hinge. In some embodiments, provided SD-IgAPP agents cleave at a site corresponding to V222-P223 (according to standard numbering) of the IgA1 hinge.

[0119] Those skilled in the art will recognize a variety of assays and techniques that may be useful for assessing IgA proteolytic activity. In some embodiments, IgA proteolytic activity is measured using the method of Plat & Bachovchin Met. Enzymol. 244:137, 1994 (entitled "IgA-specific prolyl endopeptidases: serine type," incorporated herein).

[0120] In some embodiments, the proteolytic activity of an IgA protease polypeptide (e.g., an SD-IgAPP agent and / or a reference IgA protease) may be assessed on the protease polypeptide in purified or pure form. In some embodiments, such proteolytic activity may be assessed in a complex environment, such as the system in which the protease polypeptide was produced (or an extract or other fraction thereof, e.g., a cellular (e.g., bacterial or mammalian cell, optionally an engineered cell, such as a cell engineered to express the protease polypeptide) extract or fraction thereof).

[0121] In many embodiments, proteolytic activity is assessed with respect to cleavage of human IgA (eg, human IgA1).

[0122] In some embodiments, an IgA protease polypeptide agent (e.g., an SD-IgAPP agent) is considered to have sufficient activity to be used as a therapeutic agent if it has at least one unit activity, where one unit activity is equal to 1 microgram of human IgA cleaved per minute per mg of protease at 37°C.

[0123] The examples contained herein (e.g., Example 2) document the utility of the SD-IgAPP agents described herein. For example, Example 2 demonstrates that the middle domain (MD) of Cr-IgA protease retains full proteolytic ability and cleaves the hinge region of human IgA1 to the same extent as full-length Cr-IgA protease (Figure 4).

[0124] In particular, the provided SD-IgAPP is useful in in vivo and in vitro methods of cleaving IgA, such as IgA1 and / or IgA2.

[0125] In some embodiments, a method of cleaving IgA includes 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 the IgA.

[0126] Characterization of IgA protease polypeptide agents: In many embodiments, an IgA protease polypeptide agent according to the disclosure cleaves IgA (e.g., cleaves IgA1, e.g., has IgA1 cleaving activity that is reasonably or substantially equivalent to the IgA1 cleaving activity of a reference IgA protease, e.g., a reference M64 IgA protease, e.g., the M64 IgA protease of C. ramosum of SEQ ID NO: 2).

[0127] In some embodiments, the SD-IgAPP cleavage activity is comparable to that of a reference IgA protease. In some embodiments, SD-IgAPP IgA (e.g., IgA1) cleavage activity is measured. Various methods are available for measuring IgA (e.g., IgA1) cleavage activity. IgA (e.g., IgA1) cleavage activity can be measured, for example, using a cleavage gel assay (e.g., SDS PAGE) (see, e.g., Example 3 herein) or mass spectrometry (see, e.g., Example 5 herein). In some embodiments, a method of cleaving IgA includes 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 the IgA, and then separating the generated products, for example, on an SDS-PAGE gel.

[0128] In some embodiments, provided IgA protease polypeptide agents (e.g., provided SD-IgAPP agents) having a structure that is or includes M1-M3 have IgA cleaving activity characterized by cleaving the V222 to P223 site within the IgA hinge.

[0129] As noted herein, in some embodiments, provided IgA protease polypeptide agents (e.g., provided SD-IgAPP agents) may exhibit sufficient structural and functional similarity to a known, reference M64 IgA protease (e.g., the IgA M64 protease of C. ramosum, the mature amino acid sequence of which is set forth in SEQ ID NO:2) that one of skill in the art would understand its appropriate classification as an "M64 IgA protease polypeptide" as described herein, but may nevertheless include certain amino acid sequence difference(s) and / or other modifications (e.g., the attachment of pendant groups such as glycans, PEG moieties, fusion with tags or other functional peptides)—preferably still retaining attribute(s) (e.g., smaller than full-length size) and / or advantage(s) described herein compared to such reference IgA protease.

[0130] For example, in some embodiments, a provided IgA protease polypeptide agent (e.g., an SD-IgAPP agent) may exhibit significant sequence identity to the middle domain of C. ramosum IgA M64 protease, but may have one or a few modifications relative to the middle domain of such C. ramosum IgA M64 protease, where the modification(s) may be, for example, one or more modifications of the amino acid sequence (e.g., truncations, substitutions, deletions, insertions, combinations thereof, etc.).

[0131] In some embodiments, the SD-IgAPP agents described herein, and / or compositions comprising them, can be characterized by one or more characteristics or attributes (e.g., one or more physical and / or functional parameters) that make them particularly useful in the desired context(s) (e.g., therapeutic context(s) and / or diagnostic and / or research contexts).

[0132] 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 pharmacodynamic performance, etc.

[0133] For example, in some embodiments, provided SD-IgAPP agents can be characterized in that their size makes them particularly suitable for administration. Proteins with low space-filling ratios can be delivered more easily than bulky proteins. Alternatively or additionally, shorter polypeptides may be more suitable for delivery via administration of nucleic acids, as shorter nucleic acids can be more easily incorporated into, for example, viral vectors or lipid-based delivery systems.

[0134] In some embodiments, a provided SD-IgAPP agent exhibits an acceptable level of off-target and / or other undesirable effects when delivered to a cell or organism (e.g., via administration of a composition containing or encoding it). One of skill in the art will recognize what levels may be "acceptable" in a given context and / or particular assay. In some embodiments, such levels may be lower than those observed when an appropriate reference IgA protease polypeptide is delivered.

[0135] In some embodiments, a provided SD-IgAPP agent is characterized in that it exhibits an acceptable level of toxicity (e.g., is relatively non-toxic; e.g., compared to a reference IgA polypeptide) when delivered to a cell or organism (e.g., via administration of a composition containing or encoding it).

[0136] In some embodiments, a provided SD-IgAPP agent is characterized in that it exhibits an acceptable level of immunogenicity (e.g., is relatively non-immunogenic; e.g., compared to a reference IgA polypeptide) when delivered to a cell or organism (e.g., via administration of a composition containing or encoding it).

[0137] In some embodiments, provided SD-IgAPP agents are characterized by ease of manufacture and / or stability (eg, under appropriate reference conditions).

[0138] In some embodiments, a provided SD-IgAPP agent is characterized in that it exhibits a high level of bioavailability (e.g., compared to a reference IgA polypeptide) when delivered to a cell or organism (e.g., via administration of a composition containing or encoding it).

[0139] In some embodiments, a provided SD-IgAPP agent is characterized in that it exhibits an increased immunogenic half-life when delivered to a cell or organism (e.g., via administration of a composition containing or encoding it) (e.g., compared to a reference IgA polypeptide).

[0140] Among other things, the present disclosure provides nucleic acid molecules encoding the SD-IgAPP agents described herein.

[0141] In some embodiments, the provided nucleic acid is found within or isolated from a naturally occurring cell (e.g., a source cell), hi some embodiments, the provided nucleic acid is engineered (e.g., designed and / or generated by the hand of man).

[0142] Those skilled in the art will recognize that, as described herein, certain IgA proteases are naturally expressed by microbial cells (e.g., bacterial cells). Such cells may be considered "feeder cells" for purposes of the present disclosure. These bacteria may include, but are not limited to, Clostridium ramosum, Haemophilus influenzae types 1 and 2, Neisseria meningitidis types 1 and 2, Neisseria gonorrhoeae, Neisseria lactamica, Prevotella melaninogenica, Streptococcus mitis biovar I, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus sanguis, and Ureplasma maureticum. In some embodiments, the feeder cells are C. ramosum cells.

[0143] In some embodiments, the provided nucleic acids can have nucleotide sequences that are codon-optimized for expression in a particular host cell of interest, for example.

[0144] Those skilled in the art are familiar with the preferred codons for use in a variety of different cell types of interest. Generally, 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 the genes of that host cell, while maintaining the native desired amino acid sequence.

[0145] Various species exhibit particular preferences for specific codons encoding specific amino acids. Without wishing to be bound by any particular theory, codon preference (differences in codon usage among organisms) is often correlated with mRNA translation efficiency, which is thought to depend, among other things, on the characteristics of the codon being translated and / or the availability of specific transfer RNAs (tRNAs). The dominance of particular tRNAs in a cell may generally reflect the codons most frequently used in peptide synthesis within that cell. Therefore, genes may be tailored for optimal gene expression in a given cell type (e.g., organism) based on codon optimization. Codon usage tables are readily available, for example, in the "Codon Usage Database" available at kazusa.orjp / codon / , and these tables may be adapted in numerous ways. Computer algorithms, such as GeneForge (Aptagen; Jacobus, PA), are also available to codon-optimize specific sequences for expression in a particular subject or cell.

[0146] In some embodiments, the provided nucleic acids are codon-optimized for expression in bacterial (e.g., E. coli) cells. In some embodiments, the provided nucleic acids are codon-optimized for expression in insect cells (e.g., via a baculovirus expression system). In some embodiments, the provided polynucleotides are codon-optimized for expression in mammalian cells (e.g., CHO cells or other common mammalian production cells).

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

[0148] In some embodiments, provided nucleic acids can be used as templates for, e.g., primer extension, including amplification (e.g., by polymerase chain reaction ["PCR"]), and / or for transcription (e.g., for in vitro transcription, such as that utilized to generate RNA transcripts for delivery to cells that express the RNA transcripts, and / or for in vivo transcription, e.g., by a cell into which such provided nucleic acids are introduced).

[0149] Vector construction: In some embodiments, the provided nucleic acid may be incorporated into a vector. In some embodiments, such a vector comprises one or more expression control elements (e.g., a transcriptional regulatory factor binding site such as a promoter, an enhancer or repressor site, a transcription terminator, a splice donor and / or acceptor site, a translation initiation site, a polyA tail, etc.), or their complements. Alternatively or additionally, in some embodiments, such a vector may comprise a replication site, such as an origin of replication or a primer landing site, or their complements.

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

[0151] In some embodiments, the vector may contain one or more detectable markers and / or one or more selectable markers, as known in the art, e.g., to facilitate identification of cells that have been administered the vector.

[0152] Those of skill in the art will recognize a variety of vector systems, including vector systems that are particularly useful and / or adapted for introduction into and / or expression (e.g., of heterologous nucleic acids) in particular cells (e.g., microorganisms such as bacteria or yeast cells, insect cells, mammalian cells, etc.). For example, those of skill in the art will recognize a variety of plasmid, phage, viral vectors, etc. that are useful for delivering payload nucleic acids (e.g., those encoding SD-IgAPP agents described herein) to particular cell(s) of interest in vitro and / or in vivo and / or for expressing such payload nucleic acids in such cells.

[0153] In certain embodiments, the provided nucleic acids encoding the SD-IgAPP agents described herein are incorporated into a plasmid that is useful for introduction into and / or expression by, for example, bacterial cells (e.g., E. coli cells), and / or insect cells, and / or yeast cells (e.g., S. cerevisiae cells), and / or mammalian cells (e.g., CHO cells, BHK cells, HEK293 cells, NS0 cells, etc.).

[0154] In some embodiments, provided nucleic acids encoding the SD-IgAPP agents described herein are incorporated into viral vectors, such as, for example, phage vectors, baculovirus vectors, AAV vectors, etc.

[0155] In some embodiments, provided nucleic acids encoding SD-IgAPP agents described herein are RNA, e.g., mRNA (e.g., RNA encoding SD-IgAPP without intervening intronic sequences). In some such embodiments, such provided nucleic acids are produced by in vitro transcription and / or associated with a delivery system (e.g., a lipid-based delivery system such as a lipid nanoparticle ("LNP")), as known in the art.

[0156] Pharmaceutical Composition In some embodiments, provided SD-IgAPP agents may be useful in medicine, for example, to treat a disease, disorder, or condition 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), deliver an SD-IgAPP agent described herein to such a subject, particularly when administered to a subject suffering from a disease, disorder, or condition associated with IgA deposition. Accordingly, in some embodiments, the present disclosure provides pharmaceutical compositions that comprise or deliver an SD-IgAPP agent described herein.

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

[0159] Typically, a pharmaceutical composition comprises an active agent (e.g., an SD-IgAPP agent described herein, or a nucleic acid encoding same) in combination with one or more pharmaceutically acceptable carriers or excipients, such as one or more buffers, diluents, fillers, salts, solubilizers, stabilizers, and / or other materials known in the art. Those skilled in the art will recognize various carrier components appropriate for a particular active form (e.g., polypeptide vs. nucleic acid, viral vector vs. plasmid vs. RNA, etc.) and / or route of administration (e.g., parenteral, enteral, etc.).

[0160] In some embodiments, provided pharmaceutical compositions may include or deliver an SD-IgAPP agent in a form complexed with an antibody agent (i.e., an immunoconjugate). Without wishing to be bound by any particular theory, it is proposed that such immunoconjugates may be particularly useful in treating diseases characterized by IgAl deposition in the kidney because large immunoconjugates are believed to remain in the renal glomerulus upon administration.

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

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

[0163] Alternatively or additionally, in some embodiments, provided pharmaceutical compositions may contain or deliver another active agent in addition to the SD-IgAPP agents described herein.

[0164] Use and Method In some embodiments, the provided SD-IgAPP agents may be useful as reagents (e.g., biotechnology reagents). In some embodiments, the present disclosure provides technologies (e.g., compositions, mixtures, methods, and / or systems, etc.) comprising SD-IgAPP. In particular, the present disclosure provides SD-IgAPP compositions (e.g., compositions comprising one or more SD-IgAPPs) and SD-IgAPP reaction mixtures (e.g., comprising SD-IgAPP and IgA). The compositions, mixtures, methods, and kits according to the present disclosure may be used in several applications, such as therapeutic and / or non-therapeutic applications. In some embodiments, methods using SD-IgAPP are useful in therapeutic and / or non-therapeutic applications. In some embodiments, the compositions, mixtures, methods, kits, and uses described herein may be used for non-therapeutic purposes, such as diagnostic purposes. In some embodiments, SD-IgAPP is used in an in vitro assay. In some embodiments, SD IgAPP is used in an in vitro cleavage assay.

[0165] In some embodiments, the present disclosure provides a method of cleaving IgA, the method comprising: (a) contacting a sample containing IgA with an IgA protease polypeptide agent provided herein, whereby the IgA protease polypeptide agent cleaves the IgA. In some embodiments, the IgA is IgA1. In some embodiments, the SD-IgAPP agent cleaves IgA comparably to an appropriate reference IgA protease.

[0166] host cell Among other things, the present disclosure provides host cells that express or otherwise contain (e.g., express) the SD-IgAPP agents described herein and / or nucleic acid molecules encoding such agents.

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

[0168] In some embodiments, the 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 herein being, for example, a CHO cell, a BHK cell, a HEK293 cell, an NS0 cell, etc.), a plant cell, or a yeast cell (e.g., an S. cerevisiae cell). A variety of bacteria produce IgA proteases and / or homologs of IgA proteases that may be useful in the present disclosure (e.g., as reference IgA protease polypeptides), including, but not limited to, Clostridium ramosum, Haemophilus influenzae types 1 and 2, Neisseria meningitidis types 1 and 2, Neisseria gonorrhoeae, Neisseria lactamica, Prevotella melaninogenica, Streptococcus mitis biovar I, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus sanguis, and Ureplasma maureticum.

[0169] In some embodiments, the host cell comprises a vector construct described herein (e.g., a vector construct that contains and / or expresses a nucleic acid encoding an SD-IgAPP agent described herein). Those of skill in the art will recognize a variety of techniques useful for introducing an appropriate vector construct(s) into a suitable host cell.

[0170] To give a few examples, in some embodiments, vector constructs can be introduced into suitable bacterial cells by infection with bacteriophage vector particles such as lambda or M13, or by any of several transformation methods for plasmid vectors or bacteriophage DNA. For example, standard calcium chloride-mediated bacterial transformation is still commonly used to introduce naked DNA into bacteria (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), although electroporation can also be used (Ausubel et al., Current Protocols in Molecular Biology, (1988), (John Wiley & Sons, Inc., NY, NY)). For example, methods for transforming bacterial host cells with vectors expressing SD-IgAPP according to the present disclosure, methods for inducing expression of proteins encoded by such expression vectors, and methods for isolating and purifying such proteins are known in the art. See, for example, US Pat. No. 7,407,653 and Sambrook et al. (2001), the contents of both of which are incorporated by reference in their entirety.

[0171] Use of host cells Those of skill in the art reading this disclosure will recognize various uses for the host cells provided as described herein.

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

[0173] Thus, among other things, in some embodiments, the disclosure provides a population of cells (e.g., a cell culture) in which an SD-IgAPP agent, or a nucleic acid encoding the same, is present at or above a certain desired level. In some embodiments, such a population of cells expresses or has expressed the SD-IgAPP agent. In some embodiments, such a population of cells secretes the SD-IgAPP agent. In some embodiments, the disclosure provides a cell culture, and / or medium derived therefrom, comprising an SD-IgAPP agent at or above a desired level (e.g., a desired concentration). In some embodiments, the desired level (e.g., the level of the SD-IgAPP agent and / or nucleic acid encoding the SD-IgAPP agent) is a level suitable for the manufacture of a pharmaceutical composition comprising or delivering such an SD-IgAPP agent. In some embodiments, SD-IgAPP agents and / or nucleic acids encoding same may be produced by culturing host cells containing and / or expressing such SD-IgAPP agents and / or nucleic acids encoding same. In some embodiments, a step of inducing expression of such SD-IgAPP agents is performed. Alternatively or additionally, in some embodiments, the cell(s) are separated from the culture medium, the SD-IgAPP agent is isolated from the culture medium, and / or the cell(s) are lysed, followed by isolation of the SD-IgAPP agent and / or nucleic acid encoding same. Those skilled in the art are familiar with various techniques for isolating components from cell culture medium and / or lysed cells.

[0174] Diseases, Disorders, and Conditions Those skilled in the art will understand, upon reading this disclosure, that the provided SD-IgAPP agents (and / or nucleic acid(s) encoding same and / or compositions comprising and / or delivering either) may be useful in medicine, particularly in the treatment of certain diseases, disorders, and conditions, such as one or more diseases, disorders, or conditions associated with IgA deposition.

[0175] Deposition of immunoglobulin A1 (IgA1) in human tissues and organs is a hallmark of several human diseases, including IgA nephropathy, dermatitis herpetiformis (DH), and Henoch-Schönlein purpura (HS). IgA1 deposition causes a variety of clinical symptoms, including renal failure, skin blisters, rash, arthritis, gastrointestinal bleeding, and abdominal pain.

[0176] Treatment options available to patients with abnormal IgA1 deposition include administration of corticosteroids, which have immunosuppressive and anti-inflammatory properties, dietary fish oil supplements, which reduce renal inflammation, and angiotensin-converting enzyme inhibitors, which reduce the risk of progressive renal disease and failure. Such treatments do not directly affect IgA1 deposits in tissues or organs and do not address their removal.

[0177] The present disclosure incorporates the identification of the intermediate domain of IgA proteases, such as IgA M64 protease (e.g., IgA M64 C. ramosum), in the design of novel SD-IgAPP agents with therapeutic value. In some embodiments of the present disclosure, SD-IgAPP agents, and / or nucleic acids encoding them, and / or compositions comprising and / or delivering any of the foregoing, are used to treat diseases characterized by IgA1 deposition. In some embodiments, SD-IgAPP agents, and / or nucleic acids encoding them, and / or compositions comprising and / or delivering any of the foregoing, further comprise the middle domain of an IgA protease, e.g., IgA M64 protease (e.g., IgA M64 C. ramosum), and optionally the N-terminal domain or portion thereof of an IgA protease (e.g., IgA M64 C. ramosum), and / or the C-terminal domain or portion thereof of an IgA protease (e.g., IgA M64 C. ramosum). Such embodiments may exhibit a preference for IgA1 (e.g., compared to IgA2) relative to an appropriate reference IgA protease, and thus may be particularly useful in treating IgA1 deposition diseases, such as, but not limited to, IgA nephropathy.

[0178] In some embodiments, the provided SD-IgAPP is delivered (e.g., by administration of a pharmaceutical composition described herein that contains or delivers such an agent or a nucleic acid encoding same) to a subject suffering from or susceptible to a disease, injury, or condition associated with IgA deposition described herein, or that otherwise exhibits abnormal IgA deposit(s) (e.g., in the kidney, blood vessels, skin, or elsewhere).

[0179] In some embodiments, SD-IgAPP is delivered to an individual with an IgA deposition disease. In some embodiments, the individual is a human. In some embodiments, the IgA is IgA1. In some embodiments, the IgA1 is human IgA1.

[0180] In some embodiments, the present disclosure provides a method for treating IgA nephropathy by delivering SD-IgAPP according to the present disclosure to a patient in need of such treatment. IgA nephropathy is a kidney disease. This disease is considered to be immune complex-mediated glomerulonephritis and is characterized by granular deposition of IgA1 in the glomerular mesangial region. Nephropathy results and is defined by proliferative changes in 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 present disclosure provides methods for treating dermatitis herpetiformis (DH) by delivering an SD-IgAPP according to the present disclosure to a patient in need of such treatment. Dermatitis herpetiformis is a chronic, blistering skin disease associated with IgAl deposition at the dermal-epidermal junction (Hall, RP & TJ Lawley, J. Immunol. (1985) 135(3):1760-5). DH patients have granular IgAl deposits and often have associated gluten-sensitive enteropathy (GSE).

[0182] In some embodiments, the present disclosure provides methods for treating Henoch-Schönlein purpura (HS) by delivering SD-IgAPP according to the present disclosure to a patient in need of such treatment. Henoch-Schönlein purpura is a disease of the skin, blood vessels, and kidneys. HSP is characterized by the deposition of immune complexes containing IgAl in tissues. The disease is It is diagnosed by observing evidence of IgA1 deposits in skin tissue or kidneys via immunofluorescence microscopy. Clinical symptoms typically include rash, joint pain, abdominal pain, and kidney damage.

[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., IgA1) deposition in the transplanted kidney. Without wishing to be bound by any particular theory, it is proposed that delivery of SD-IgAPP to an individual with a kidney transplant may preserve the function of the donor kidney, thereby avoiding the need for a further kidney transplant.

[0184] Administration In some embodiments of the present disclosure, provided are methods comprising delivering an SD-IgAPP agent described herein to an individual suffering from or susceptible to abnormal IgA deposition (e.g., IgA1 deposition). In some embodiments, the SD-IgAPP agent is delivered in an amount effective to reduce IgA1 deposition. In some embodiments, the individual has human IgA1 deposits.

[0185] Delivery of an SD-IgAPP agent can be achieved, for example, by oral ingestion, inhalation, topical application, or parenteral administration (e.g., cutaneous, subcutaneous, intraperitoneal, intramuscular, or intravenous injection) of a pharmaceutical composition described herein, e.g., a pharmaceutical composition comprising an SD-IgAPP agent or a nucleic acid encoding same. In many embodiments, administration is by intravenous or intramuscular injection. In some embodiments, administration can be by local administration, e.g., to the site of IgA deposition. In some embodiments, local administration can be or include topical administration (e.g., to the skin) or parenteral administration (e.g., by injection at the site of deposition, such as into the kidney).

[0186] Compositions containing therapeutic polypeptide agents of the present disclosure can be administered intravenously, such as by injection of a unit dose.

[0187] Those skilled in the art will recognize typical guidelines for formulating pharmaceutical compositions for administration by specific routes. For example, pharmaceutical compositions for parenteral injection typically include pharmaceutically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders for reconstitution into sterile injectable solutions or dispersions immediately before use. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), carboxymethylcellulose and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters such as ethyl oleate. The desired fluidity can be maintained, for example, by using coating materials such as lecithin, by maintaining the required particle size in the case of dispersions, and / or by using surfactants. In some embodiments, pharmaceutical compositions formulated for parenteral administration may contain one or more ingredients such as preservatives, wetting agents, emulsifying agents, dispersing agents, and / or one or more ingredients that reduce immunogenicity, such as protecting immunogenic determinants (e.g., epitopes) of the administered composition or the active agent (e.g., SD-IgAPP agent) it contains or delivers. In some embodiments, it may be desirable to include one or more agents, such as parabens, chlorobutanol, phenol sorbic acid, which may have antibacterial properties. Alternatively, or additionally, in some embodiments, it may be desirable to include one or more isotonic agents, such as sugars, sodium chloride, etc. In some embodiments, one or more agents that can delay absorption, such as aluminum monostearate and gelatin, may be included, particularly when extended absorption of the injectable pharmaceutical composition is desired. Alternatively, or additionally, in some embodiments, injectable depot formulations may be made by forming microencapsulating matrices of biodegradable polymers, such as polylactide-polyglycolide, poly(orthoesters), and / or poly(anhydrides). Those skilled in the art will recognize that the release rate of the active agent from such depot formulations can be adjusted by adjusting the ratio of active agent to polymer and / or the properties of the particular polymer used.Alternatively or additionally, in some embodiments, depot injectable formulations can be prepared by entrapping the active agent in liposomes or microemulsions that are compatible with body tissues. The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium immediately before use.

[0188] In some embodiments, delivery of an SD-IgAPP agent can be achieved by administration of a pharmaceutical composition described herein, such as a pharmaceutical composition comprising an SD-IgAPP agent or a nucleic acid encoding same; administration may be oral, rectal, ophthalmic (including intravitreal or intracavitary), 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 recognize typical guidelines for formulating 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 encoding same with the pharmaceutical carrier(s) or excipient(s). In some embodiments, compositions are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

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

[0190] Dosage regimen: In some embodiments, the polypeptide agent (SD-IgAPP agent) and / or nucleic acid molecule and / or pharmaceutical composition thereof is administered in a single dose in the range of 100 μg to 10 mg / kg body weight. In some embodiments, the single dose is in the range of 1 μg to 100 μg / kg body weight. This dosage may be, for example, as deemed appropriate by the treating physician.

[0191] The amount of IgA protease polypeptide agent (SD-IgAPP agent) administered in a single dose can depend on the nature and / or severity of the condition being treated and / or the nature of previous treatments the patient has undergone. In some embodiments, the attending physician determines the amount of IgA protease polypeptide agent with which to treat an individual patient. In some embodiments, the attending physician initially administers a low dose of an IgA protease polypeptide agent(s) of the present disclosure and observes the patient's response. In some embodiments, larger doses are administered until an optimal therapeutic effect is achieved for the patient, after which the dosage is not further increased.

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

[0193] In some embodiments, the pharmaceutical composition is administered via a single dose regimen. In some embodiments, the pharmaceutical composition is administered via a multiple dose regimen. In some embodiments, the pharmaceutical composition is administered via a single dose regimen in one or more cycles of administration.

[0194] In some embodiments, the SD-IgAPP agent is administered with one or more immunomodulatory agents. In some embodiments, the pharmaceutical agent is an immunostimulatory agent. In some embodiments, the pharmaceutical agent is an immunoinhibitory agent. In some embodiments, the pharmaceutical agent suppresses the adaptive immune response. In some embodiments, the SD-IgAPP agent is administered with an agent that prevents anti-drug antibodies (ADA). In some embodiments, the SD-IgAPP agent is administered with nanoparticles encapsulating rapamycin (e.g., ImmTOR™ or SVP-rapamycin). In some embodiments, the SD-IgAPP agent is administered with nanoparticles encapsulating IL-2 and rapamycin (e.g., ImmTOR-IL™). [Example]

[0195] The present disclosure will be better understood in connection with the following examples, which are intended for illustrative purposes only and do not limit 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 but not limited to, with respect to the chemical structures, substituents, derivatives, compositions, and / or methods of the present disclosure, may be made without departing from the spirit of the disclosure and the scope of the appended claims.

[0196] Example 1: Construction, production, and purification of specific peptides (e.g., exemplary IgA protease polypeptides). This example documents the polypeptide and domain structure of the Cr-IgA protease.

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

[0198] Soluble polypeptides representing each of the following domains were produced as soluble proteins: i) Full length structure (FL); ii) N-terminal domain (NTD); iii) the middle domain (MD (containing the HEKXH motif)); and iv) C-terminal domain (CTD containing the IgG-like CBDX domain).

[0199] Specifically, codon-optimized nucleotide sequences encoding each of these domains were developed. These codon-optimized sequences were cloned into a plasmid vector containing an N-terminal His6-tagged small ubiquitin-like modifier (SUMO) domain sequence, resulting in the generation of domain fusion proteins. These plasmids were transformed into BL21(DE3) E. coli cells. Saturated overnight cultures of these BL21(DE3) cells were grown and used to inoculate 1 L of ZYP-5052 autoinduction medium (50 mL). Cells were grown at 14°C with shaking at 180 rpm for 2 days. Cells were harvested by centrifugation, and the pellets were stored at -80°C until purification. The resulting cell pellets were solubilized in 25 mM HEPES (pH 7.5) + 0.5 M NaCl + 10 mM imidazole + 1 mM TCEP (Buffer A) and passed twice through a French press. The resulting solution was clarified by centrifugation, and the clarified lysate was incubated with NiNTA resin. The NiNTA resin was washed with 1 L of 25 mM HEPES (pH 7.5) + 0.1% (v / v) IgePal CA630 + 10 mM imidazole + 1 mM TCEP followed by 1 L of buffer A.

[0200] The expressed fusion protein was eluted with Buffer A supplemented with 0.3 M imidazole and cleaved overnight with SUMO protease to release the appropriate domain from the N-terminal SUMO sequence. The protein digest containing the protein of interest, SUMO fusion, and SUMO protease was dialyzed twice overnight into 25 mM HEPES (pH 7.5) + 0.5 M NaCl + 1 mM TCEP. The protein solution was incubated with NiNTA resin and loaded onto a chromatography column to remove the SUMO fusion and SUMO protease from the protein digest. The NiNTA flow-through was concentrated to less than 1 mL and injected onto an S75 gel filtration column equilibrated with 25 mM HEPES (pH 7.5) + 1 mM TCEP. The purified IgA protease was concentrated and stored at -80°C.

[0201] Figure 3 shows an SDS gel 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 polypeptide. Figure 3 illustrates that the CTD undergoes proteolysis into two components, consistent with the extended nature of its predicted structure (Figure 3D).

[0202] Example 2: Exemplary Cr-IgAPP Structures In this example, the Δ15 intermediate domain-C-terminal domain 1 329-876 The (Δ15MD+CTD1) IgA protease polypeptide structure (SEQ ID NO: 30) is recorded. This example demonstrates that the active site is located within the MD.

[0203] The crystal structure of Δ15 MD+CTD1 Cr-IgAP shows the conserved M64 fold with an additional C-terminal beta-sheet domain. The two metal-binding sites are conserved, and the 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 absent from the most active minimized Δ15 MD construct (black) (Figure 4A). The 1.60 Å structure (2Fo-Fc density at 1.3σ, fine mesh) shows a somewhat flexible active site, with the active site zinc coordinated by members of the HEKXH motif and downstream D520 (Figure 4B). The location of the metal ion was confirmed by an unusual difference peak (shown at 5.0σ, thick metal wire). A loop containing D520 and E521 bridges the active site to a nearby pocket where a second zinc-binding site is located (map contoured at the same σ value) (Figure 4C).

[0205] This example describes the structure of Δ15 MD+CTD1 IgAPP, which shows the location of the active site and coordination of the catalytic Zn ion by a HEXXH motif, with a secondary Zn ion bound near the active site and coordinated by three cysteine ​​residues and one glutamic acid.

[0206] Example 3: Evaluation of protease activity This example demonstrates that, surprisingly, the middle domain (MD) of Cr-IgA protease retained full proteolytic ability and cleaved the hinge region of IgA1 to the same extent as the reference HIN-IgA protease (Figure 5).

[0207] Materials and Methods IgA1 derived from human myeloma plasma purchased from Athens Research and Technology was incubated with 20 μg / ml of protease or its domains at 37°C for 1 hour or overnight at 37°C to cleave the heavy polypeptide chain of IgA1, and enzyme activity was measured. Products of IgA1 hydrolysis were separated on SDS / PAGE gels. Fc cleavage fragments were detected by size comparison with MW standards run on the same gel. The IgA1 control was purified human myeloma protein, and MW is a molecular weight standard.

[0208] result This example demonstrates that MD (SEQ ID NO: 22) exhibits proteolytic activity against IgA1 ( FIG. 5 ). Specifically, this example demonstrates that MD cleaves the hinge region of IgA1, thus indicating that MD is sufficient for the protease to express proteolytic activity against IgA1. In other words, the N- and C-terminal domains of Cr-IgA protease may not be essential for the protease to cleave IgA1. Note that the extent of IgA1 digestion by the reference IgAP (H. influenzae IgAP) and the MD observed in FIG. 5 is the same. Thus, this example identifies a C. ramosum IgA protease middle domain that exhibits proteolytic activity against IgAs, such as IgA1 and / or IgA2, comparable to that of the full-length C. ramosum IgA protease.

[0209] Structural studies suggest that up to 22% of the amino acids can be removed from the N-terminus of C. ramosum and up to 31% of the amino acids can be removed from the C-terminus of C. ramosum, for a total of 53% of the amino acids in the C. ramosum IgA protease. The percentage calculations are based on the length of the mature, secreted form of the polypeptide, lacking the N-terminal signal sequence. Thus, the structural architecture analysis studies described herein reveal that the C-terminal domain (approximately corresponding to amino acids 808-1234 of SEQ ID NO:1) is not essential. Furthermore, the present studies also reveal that the N-terminal domain, corresponding to amino acids 31-313 of SEQ ID NO:1, is not essential. Thus, the present disclosure surprisingly demonstrates that both the C-terminal domain (approximately corresponding to amino acids 808-1234 of SEQ ID NO:1) and the N-terminal domain (approximately corresponding to amino acids 31-313 of SEQ ID NO:1) are not essential.

[0210] Example 4: Production and purification of NTD-MD and NTD-MD-CTD1 / 2 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 half of the C-terminal domain (NTD-MD-CTD1 / 2) was generated. NTD-MD-CTD1 / 2 is resistant to proteolysis and contains a folded carbohydrate-binding domain (CBD). NTD-MD-CTD1 / 2 was generated to investigate whether the CBD domain contributes to the substrate selectivity of IgA by binding to glycans on IgA1 or IgA2. Expression and purification of both constructs (i.e., NTD-MD and NTD-MD-CTD1 / 2) were performed.

[0211] Example 5: Evaluation of IgA1 cleavage by mass spectrometry This example demonstrates that neither the full-length (FL) Cr-IgA protease nor the middle domain of the Cr-IgA protease (MD, SEQ ID NO: 22) alone cleaves the IgA hinge, as assessed by MS as described herein. However, the Cr-MD cleaves the IgA hinge in the context of the IgA1 structure (Example 3 and Figure 5).

[0212] Materials and Methods The ability of FL and MD to cleave a synthetic hinge peptide independently was tested by mass spectrometry (MS). The following synthetically produced unlabeled hinge peptide was generated as VPCPVPST. The chemical alpha-cyano-4-hydroxycinnamic acid (HCCA) was used as a matrix to ionize the sample and characterize it by MALDI-TOF MS. HCCA alone was used as a blank sample. 10 nM of FL and MD in the HCCA matrix were added to the generated synthetic hinge peptide (15 μM) and incubated at 37°C for over 12 hours.

[0213] result Figures 7A-C show the different mass spectra of the entire MS data set. Figures 7A-C show the different spectra of the HCCA matrix only (blank), the unprocessed isolated hinge peptide, the isolated hinge peptide with MD, and the isolated hinge peptide with FL. The spectra of the unprocessed isolated hinge peptide with and without MD or FL are identical, indicating that MD and FL have not been characterized to cleave the isolated synthetic IgA1 / 2 hinge peptide. Thus, neither the full-length (FL) protease nor the MD construct exhibited proteolytic activity against the hinge peptide encompassing the known IgA1 / 2 cleavage sequence (VPCP↓VPST) as assessed by mass spectrometry approaches (Figures 7A-C).

[0214] Example 6: IgA1 cleavage activity This example demonstrates that truncated IgAP peptides, including Cr-Δ15 MD IgAPP and Cr-MD IgAPP, exhibit improved IgA1 cleavage activity compared to Cr-FL IgAPP and Cr-NTD-MD IgAPP.

[0215] Gel-based cleavage assay Materials and Methods IgA2 cleavage activity was characterized using a cleavage gel assay against the following IgAPP and control IgAPs: · full length 31-1195 Cr-IgA protease (Cr-FL); N-terminal domain of Cr-IgA protease - middle 31-807 domain (Cr-NTD-MD); The middle domain of Cr-IgA protease 314-807 (Cr-MD); Δ15 intermediate domain 329-807 Cr-IgA protease (Cr-Δ15 MD); and · Haemophilus influenzae IgA protease (HIN IgAP).

[0216] 40 nM of enzyme constructs (Cr-Δ15 MD, Cr-MD, Cr-FL, and Cr-NTD-MD) were incubated with approximately 1.3 μM IgA1 (obtained from Athens Biotech, Athens, GA) in 25 mM HEPES (pH 7.5) at 37°C. Samples of the assay mixture were quenched by immersion in 6x SDS loading buffer for various time points between 15 min and 1 h. Cleavage products were visualized by standard SDS gel electrophoresis.

[0217] result These results confirm that the C. ramosum intermediate domain (Cr-MD) has proteolytic activity and can cleave IgA1, especially after contact for >15 min (Figure 8A). Quantitative comparison between IgA protease peptides shows that under these reaction conditions, IgAPP Cr-Δ15 MD and IgAPP Cr-MD exhibit high IgA cleavage rates comparable to those of Cr-FL IgAPP and Cr-NTD-MD IgAPP. Furthermore, the most truncated IgAPP (Cr-Δ15 MD) cleaves IgA faster than Cr-FL IgA protease (Figure 8A), Cr-NTD-MD (Figure 8B), and Cr-MD (Figure 8C). Cr-Δ15 MD possesses IgA1 cleavage activity comparable to that of the reference Haemophilus influenzae IgAP (Figure 8D).

[0218] Gel-based kinetic assay Materials and Methods 10 μM IgA1 was incubated with 0.5 nM C. ramosum Δ15 MD IgAP (Cr-Δ15 MD). Samples were taken at 1 h and quenched by adding SDS-PAGE loading dye and heating to 95°C. The initial steady-state rate at a single concentration of IgA1 was measured using a discontinuous gel-based assay.

[0219] result The appearance of larger cleaved heavy chain fragments (arrows) was quantified using densitometry and normalized to the intensity of the light chain (bottom band) in each lane to account for differences in loading and concentration (Figure 11A). Initial velocities were then extracted by applying linear regression to band intensities over time, which were converted to percentage / molar concentration of cleaved IgA1 using a standard curve (not shown) (Figure 11B).

[0220] The enzyme was also evaluated at longer incubation times, where the percentage of IgA1 cleaved exceeded the typical 10% of the initial rate. Even at high product concentrations, the enzyme did not deviate from linearity. The assay was also validated in human serum and shown to behave similarly to buffer (25 mM HEPES, pH 7.5).

[0221] This assay was repeated using seven different concentrations of IgA1 at a fixed enzyme concentration to obtain a Michaelis-Menten curve (Figure 11C).

[0222] The N-terminal domain of C. ramosum was combined with the intermediate domain (Cr-NTD-MD) to form the V of IgAPP, which contains the intermediate domain (Cr-MD) and the N-terminal truncated Δ15 intermediate domain (Cr-Δ15 MD). max and Km values ​​are shown in the table below. [Table 1]

[0223] These results show that the most truncated construct, Δ15 MD IgA protease (Δ15 MD329-807), has the highest activity of all truncated IgAPPs across all IgA1 concentrations.

[0224] Example 7: IgA2 cleavage activity This example demonstrates that Cr-MD can cleave IgA2 (eg, particularly after contacting Cr-MD with IgA2 for more than 15 minutes).

[0225] Materials and Methods IgA1 cleavage activity was characterized using the following cleavage gel assay for IgAPP: · full length 31-1195 Cr-IgA protease (Cr-FL); N-terminal domain-middle domain of Cr-IgA protease 31-807 (Cr-NTD-MD); and The middle domain of Cr-IgA protease 314-807 (Cr-MD).

[0226] 40 nM of enzyme constructs (Cr-MD, Cr-FL, and Cr-NTD-MD) were incubated with approximately 1.3 μM IgA2 (obtained from Athens Biotech, Athens, GA) in 25 mM HEPES (pH 7.5) at 37°C. Samples of the assay mixture were quenched by immersion in 6x SDS loading buffer for various time points between 15 min and 1 h. Cleavage products were visualized by standard SDS gel electrophoresis.

[0227] result Qualitative comparisons show that the tested C. ramosum IgAP polypeptides are capable of cleaving IgA2 to varying degrees. Similar to the IgA1 cleavage observations (Figures 8A–D), the more truncated IgAPP (Cr-MD) exhibits higher IgA2 activity, comparable to Cr-FL IgAPP (Figure 9A) and Cr-NTD-MD IgAPP (Figure 9B). Figures 8B and 9B further demonstrate that Cr-MD, when complexed with Cr-NTD, specifically cleaves IgA1 to a greater extent than IgA2, thereby demonstrating selectivity for IgA1.

[0228] Example 8: Cleavage activity of IgG1 and IgG2 In this example, it was demonstrated that none of the IgAPPs tested, including Cr-MD IgAPP, was able to cleave IgG1 and IgG2.

[0229] Materials and Methods The cleavage activity of IgG1 and IgG2 was characterized using the following cleavage gel assay for IgAPP: · full length 31-1195 Cr-IgA protease (Cr-FL); N-terminal domain of Cr-IgA protease - middle 31-807 domain (Cr-NTD-MD); and The middle domain of Cr-IgA protease314-807 (Cr-MD).

[0230] 1.3 μM IgG1 or IgG2 was incubated with 40 nM IgAPP at 37° C. Samples were taken at 1 hour, added with SDS-PAGE loading dye, and quenched by heating at 95° C.

[0231] result C. ramosum IgAPP, including full-length (Cr-FL), mid-domain (Cr-MD), and N-terminal domain combined with the mid-domain (Cr-NTD-MD), did not cleave IgG1 or IgG2, major subclasses, or antibodies upon prolonged incubation in standard assays in blood (Figure 10A-C).

[0232] Example 9: Co-crystal complex Co-crystal complexes of CR-IgAP with intact IgA1 and IgA2, as well as Fc and Fab fragments, have been generated. Co-crystallization of the MD-CR-IgAP construct with an inactive HAXXH variant may aid in the design of SD-IgAPP with proteolytic activity selective for IgA1 alone.

[0233] Example 10: Therapeutic Efficacy The therapeutic efficacy of SD-IgAPP agents for the treatment of IgA nephropathy, dermatitis herpetiformis, or Henoch-Schönlein purpura can be tested in mouse models of IgA nephropathy, dermatitis herpetiformis, or Henoch-Schönlein purpura, respectively. Useful mouse models are described, for example, in Lamm ME, Emancipator SN, Robinson Jk, Takahashi M, Takahashi 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(1):31-6. Doi:10.2353 / ajpath.2008.070131. Epub 2007 Dec 28. PMID:18165266; PMCID:PMC2189629.

[0234] Example 11: BLAST and alignment data BLAST and alignment data for the middle domain of the IgA protease of C. ramosum (SEQ ID NO: 22) were generated using the BLASTP program and the nr clustered (experimental) database. Figures 6A-H show the alignment data and BLAST results.

[0235] These data define a conserved domain within the predicted M64 IgA protease found in other bacteria. Bacteria with a conserved CR-like mid-domain structure were found to have at least 50% sequence coverage with the mid-domain of the C. ramosum IgA protease (SEQ ID NO: 4). Exemplary species with at least 50% coverage are Clostridales, Eubacterium, Caprococcus, Roseburia, Dorea, Oscillibacter, and Anaerotruncus. All belong to the Firmicutes, which have larger mid-domains. Furthermore, this analysis reveals that various other proteins, including several proteins of unknown function, share related sequences. Thus, the present disclosure teaches that these proteins can act as IgA proteases (e.g., as M64-type IgA proteases). Such proteins (and variants that conserve or otherwise include the M64 domain signature sequence defined herein) may be useful for cleaving the IgAs described herein.

[0236] In some embodiments, the present disclosure teaches that a polypeptide comprising the characteristic sequence of SEQ ID NO: 4 can be considered to be an IgA protease polypeptide described herein. Alternatively or additionally, in some embodiments, a polypeptide comprising SEQ ID NO: 7, and / or SEQ ID NO: 8, and / or SEQ ID NO: 9 can be considered to be an IgA protease polypeptide described herein.

[0237] Example 12: ELISA-based assay ELISA-based assays can be used to assess the kinetics of cleavage of IgA1 and / or IgA2 by, for example, the IgA protease polypeptide(s) described herein.

[0238] Alternatively or additionally, binding of the synthetic peptides and / or one or both of the IgA1 and Ig2 molecules to an inactive form (e.g., a HAXXH variant(s) of an exemplary IgA protease polypeptide) is tested by surface plasmon resonance.

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

[0240] Such kinetic and / or other binding studies may reveal or confirm one or more characteristics (e.g., amino acid sequence element(s)) that may be necessary and / or sufficient to characterize the useful IgA protease polypeptides described herein.

[0241] Example 13: IgAP polypeptide activity in blood This example demonstrates that Δ15 MD IgAPP cleaves IgA1 in HEPES buffer and in pooled human serum spiked with myeloma IgA1. Δ15 MD IgAPP IgA1 cleavage activity is at least maintained in human serum or improved compared to IgA1 cleavage activity in HEPES buffer.

[0242] Materials and Methods 70 μM IgA1 was incubated with 40 nM Δ15 MD IgAPP in 25 mM HEPES (pH 7.5) at 37°C or in pooled human serum collected from clots. Samples were taken at various time points, diluted (1:30 dilution) to a sample concentration of 2.33 μM IgA1, added with SDS-PAGE loading dye, and quenched by heating at 95°C. Western blots were transferred and blocked using standard procedures using PVDF membranes, incubated with mouse anti-human IgA1 Fc antibody conjugated with HRP, and developed with ECL.

[0243] result Δ15 MD IgAPP can completely cleave exogenously added myeloma IgA1 in the context of buffer (FIG. 12A) and human serum (FIG. 12B). These results suggest that Δ15 MD IgAPP is more active in serum than in buffer because it completely cleaves endogenous IgA1, as seen by the disappearance of all IgA1 heavy chains, despite the trace amounts of endogenous IgA1 present in the serum sample (FIG. 12B).

[0244] Example 14: IgA polypeptide cleavage activity of IgA1 derived from serum of IgAN patients This example demonstrates that Δ15 MD IgAP can cleave IgA1 from an IgAN patient.

[0245] IgA1 visualization Endogenous levels of IgA1 were visualized as follows: Serum from patients with IgA nephropathy (IgAN): IgAN1 to IgAN6 Pooled serum from individuals without IgAN: normal serum

[0246] Human serum was diluted 1:30 for Western blot samples. Western blots were processed as previously described (Example 13). Myeloma IgA1 and IgA2 were included as positive and negative controls, respectively. Low levels of endogenous IgA1 were observed in pooled normal serum and each IgAN patient serum (IgAN1-6) (Figure 13A), suggesting that longer exposure with mouse anti-human IgA1 Fc monoclonal antibody conjugated with HRP was sufficient to visualize cleavage of endogenous IgA1.

[0247] IgA1 from serum of an IgAN patient Materials and Methods 40nM Δ15 MD 329-807 Cr-IgAPP (Cr-Δ15 MD), full-length Cr-IgA protease (Cr-FL), or Haemophilus influenzae IgA protease, or 400 nM Δ15 MD329-807 Cr-IgAP (Cr-Δ15 MD) was incubated with IgA1 from IgAN patient serum at 37°C. Samples were taken at various time points, diluted 1:30, added with SDS-PAGE loading dye, and quenched by heating at 95°C. Western blots were processed as previously described (Example 13).

[0248] result Δ15 MD Cr-IgAPP (top three gels), Cr-FL IgAPP (middle three gels), and H. influenzae IgAP (bottom three gels) were all able to cleave endogenous IgA1 from the serum of IgAN patients (Figures 13A-C). Δ15 MD Cr-IgAPP was able to cleave endogenous IgA1 from all six IgAN patients, especially after contact for more than 1 hour. While H. influenzae IgAP was relatively inactive in the serum of IgAN patient #2, C. ramosum IgAP full-length and Δ15 MD Cr-IgAPP still retained IgA1-cleaving activity.

[0249] To verify that Δ15 MD Cr-IgAPP was able to completely cleave endogenous IgA1 from all six IgAN patient samples, especially from patients #5 and #6, which were shown to be more resistant than the others in Figure 13B, the enzyme concentration was increased 10-fold. These results indicated that all endogenous IgA1 was cleaved after 24 hours. Therefore, Δ15 MD IgAPP can indeed cleave IgA1 from IgAN patients.

[0250] Sequence Listing The amino acid sequences of particular IgA protease polypeptides (e.g., full-length Cr-IgA protease, or fragments thereof, e.g., subdomains described herein), and the nucleotide sequences encoding them, are listed in the sequence listing in Table 2. The amino acid sequence of the mature form of the IgA protease polypeptide from C. ramosum is set forth as amino acid residues 31-1234 of SEQ ID NO: 1, with residues 1-30 containing the N-terminal signal peptide that is cleaved during processing to generate SEQ ID NO: 2. That is, SEQ ID NO: 1 is the pro-form of wild-type C. ramosum IgA protease, and SEQ ID NO: 2 is the mature wild-type C. ramosum IgA protease. Table 2 lists particular CR-IgAPP candidates, such as C. ramosum IgAPP (Genbank accession AY028440). [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11]

[0251] equivalent 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 present disclosure described herein. The scope of the present disclosure is not intended to be limited to the foregoing description.

Claims

1. An IgA protease polypeptide agent having an amino acid sequence that includes elements having the following general formula: Y １ -N-M １ -M ２ -M ３ -C-Y ２ (In the formula, Y １ , N., M. １ , M ２ , M ３ , C, and Y ２ each of which comprises or consists of a contiguous sequence of amino acids, １ comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO: 7, ２ is HEX １ X ２ comprising or consisting of an H motif, １ and X ２ is an amino acid, M ３ substantially comprises or consists of an amino acid sequence having at least 70% identity to SEQ ID NO:9, and the complete amino acid sequence of the polypeptide does not comprise or consist of the amino acid sequence of SEQ ID NO:1 or 2; １ , N, C, and Y ２ is optional.

2. M ２ But HEX １ X ２ The polypeptide agent of claim 1, which consists of an H motif.

3. X １ The polypeptide agent of claim 1 or 2, wherein is an amino acid having a hydrophobic side chain.

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

5. X １ The polypeptide agent of claim 4, wherein is phenylalanine or leucine.

6. X ２ 4. The polypeptide agent of any one of the preceding claims, wherein is glycine.

7. X １ is phenylalanine, and X ２ 10. The polypeptide agent of any one of the preceding claims, which is glycine.

8. X １ is leucine, and X ２ The polypeptide agent of any one of claims 1 to 6, wherein is glycine.

9. Said M １ 10. The polypeptide agent of any one of the preceding claims, wherein the amino acid sequence is at least 80% identical to SEQ ID NO:

7.

10. Said M １ 10. The polypeptide agent of any one of the preceding claims, wherein the amino acid sequence is at least 90% identical to SEQ ID NO:

7.

11. Said M １ 10. The polypeptide agent of any one of the preceding claims, wherein the amino acid sequence is 100% identical to SEQ ID NO:

7.

12. Said M ３ 10. The polypeptide agent of any one of the preceding claims, wherein the amino acid sequence is at least 80% identical to SEQ ID NO:

9.

13. Said M ３ 10. The polypeptide agent of any one of the preceding claims, wherein the amino acid sequence is at least 90% identical to SEQ ID NO:

9.

14. Said M ３ 10. The polypeptide agent of any one of the preceding claims, wherein the amino acid sequence is 100% identical to SEQ ID NO:

9.

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

4.

16. 2. The polypeptide agent of claim 1, 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 to 300 of the amino acid sequence represented by SEQ ID NO:

5.

18. 2. The polypeptide agent of claim 1, 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 to 175 of the amino acid sequence represented by SEQ ID NO:

6.

20. 2. The polypeptide 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 to 300 of the amino acid sequence represented by SEQ ID NO:

5.

22. 2. The polypeptide 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. 17. The polypeptide agent of claim 16, wherein the N amino acid sequence is 100% identical to SEQ ID NO:

5.

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

3.

28. 23. 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. 19. The polypeptide agent of claim 18, wherein the C amino acid sequence is 100% identical to SEQ ID NO:

6.

32. The polypeptide sequence is Y １ or Y ２ 10. The polypeptide agent of any one of the preceding claims, comprising:

33. Y １ or Y ２ is a tag.

34. 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. 36. The polypeptide agent of claim 35, wherein the antibody is anti-IgA1.

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

38. 34. 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. 10. The polypeptide agent of any one of the preceding claims, wherein the polypeptide agent cleaves at the hinge region of IgA.

40. 10. The polypeptide agent of any one of the preceding claims, wherein the polypeptide agent cleaves at the hinge region of IgA1.

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

42. 10. The polypeptide agent of any one of the preceding claims, wherein the IgA protease polypeptide sequence is less than 1000 amino acids in length.

43. 43. The polypeptide agent of claim 42, wherein the polypeptide sequence is at most 750 amino acids in length.

44. 43. The polypeptide agent of claim 42, wherein the polypeptide sequence is at most 600 amino acids in length.

45. 43. The polypeptide agent of claim 42, wherein the polypeptide sequence is at most 500 amino acids in length.

46. 43. The polypeptide agent of claim 42, wherein the polypeptide sequence is at most 400 amino acids in length.

47. 43. The polypeptide agent of claim 42, wherein the polypeptide sequence is at most 350 amino acids in length.

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

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

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

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

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

53. 53. The pharmaceutical composition of claim 52, wherein the composition is admixed with an antibody.

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

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

56. 50. A host cell comprising and / or expressing an IgA protease polypeptide agent according to any one of claims 1 to 48 and / or a nucleic acid molecule according to claim 49.

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

58. 58. The method of claim 57, wherein the IgA deposits are IgA1 deposits.

59. 58. The method of claim 57, wherein the individual is suffering from a condition selected from the group consisting of IgA1 nephropathy, dermatitis herpetiformis, and Henoch-Schonlein purpura.

60. 58. The method of claim 57, wherein the individual has IgA1 nephropathy.

61. 61. The method of any one of claims 57-60, wherein the polypeptide agent is administered in an amount effective to reduce IgA deposition.

62. 61. 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. 10. A method comprising the step of administering to an individual having IgA deposits an IgA protease polypeptide agent of any one of claims 1-48, a nucleic acid molecule of claim 44, or a pharmaceutical composition of any one of claims 52-55, wherein the IgA deposits are reduced.

64. 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. 64. 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. 65. The method of claim 64, wherein the individual is administered the polypeptide agent, nucleic acid molecule, or pharmaceutical composition and the second therapy simultaneously.

67. 65. The method of claim 64, wherein the individual is not administered the polypeptide agent and the second therapy simultaneously.

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

69. 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. 70. The method of any one of claims 63 to 69, wherein the IgA deposits are IgA1 deposits.

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

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

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

74. 100. A method of producing an IgA protease polypeptide agent according to any one of claims 1 to 48, said method comprising: - providing a host cell expressing an IgA protease polypeptide agent according to any one of claims 1 to 48; - culturing said host cells under conditions that allow said host cells to produce said polypeptide agent; - optionally lysing said cells and recovering and / or isolating said polypeptide agent.

75. 1. A method for cleaving IgA, said method comprising:

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

76. 76. The method of claim 75, wherein the IgA is IgA1.

77. 77. The method of claim 75 or 76, wherein the IgA protease polypeptide agent cleaves the IgA relative to a suitable reference IgA protease.

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

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