Immunomodulatory compositions and methods

Abstract

To provide a composition for use in the treatment of an inflammatory condition.SOLUTION: Provided is a construct comprising two or more truncated T3SS bacterial effector polypeptides, wherein each truncated T3SS bacterial effector polypeptide comprises a portion of a corresponding full-length T3SS bacterial effector polypeptide, wherein the truncated T3SS bacterial effector polypeptide maintains the immunomodulatory activity of the corresponding full-length T3SS bacterial effector polypeptide, and wherein the T3SS bacterial effector polypeptide is selected from the group consisting of (a) an E3 ubiquitin ligase, (b) a RhoGTPase regulator, (c) a cysteine methyltransferase, (d) a zinc metalloprotease, (e) an acetyltransferase, (f) an O-GlcNac transferase, and (g) an LRR motif-binding and trapping polypeptide. Also provided is a pharmaceutical composition comprising the construct.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e)(1) from U.S. Provisional Patent Application No. 62 / 841,312, filed May 1, 2019, the contents of which are incorporated herein by reference. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on May 1, 2020, is named G6113-00029_SL.txt, and is 60,849 bytes in size. Technical Field The present invention relates to compositions and methods for use in the treatment of inflammatory conditions. [Background technology]

[0002] Inflammation is a physiological defense mechanism for the recognition and elimination of potentially harmful stimuli, such as pathogens, irritants, or damaged cells. Inflammation is classified as either acute or chronic. Acute inflammation refers to the body's immediate immune response, preventing further injury and promoting healing. Acute inflammation is typically self-limiting. Under some circumstances, the inflammatory process becomes continuous, leading to the development of chronic inflammation. Chronic inflammation results in chronic pain, redness, swelling, stiffness, and damage to normal tissues. Chronic inflammation is associated with a wide range of disorders, including arthritis and joint disease, cardiovascular disease, allergies, chronic obstructive pulmonary disease, diabetes, inflammatory bowel disease, and cancer, which cause significant morbidity and mortality worldwide. New and effective methods for treating inflammatory conditions are continually needed. Summary of the Invention

[0003] Constructs comprising a composition of two or more truncated T3SS bacterial effector polypeptides are described. The constructs provided herein may comprise two or more truncated T3SS bacterial effector polypeptides, including portions of the full-length bacterial effector polypeptides YopE, YopJ, YopM, NleE, NleC, NleB, OspZ, IpaH4.5, IpaH7.8, and IpaH9.8. The constructs may further comprise a protein transduction domain. The constructs may be formulated as pharmaceuticals for use in treating inflammatory conditions. These and other features and advantages of the present invention are described in, or will become apparent from, the following detailed description of preferred embodiments of the invention, which is to be considered in conjunction with the accompanying drawings, in which like numerals refer to like parts and in which: [Brief explanation of the drawings]

[0004] [Figure 1] 1 is a listing of the amino acid and nucleotide sequences of bacterial effector polypeptides. [Figure 2] FIG. 1 shows a construct comprising a YopE polypeptide and an OspZ polypeptide. [Figure 3] FIG. 1 shows constructs comprising truncated YopM and OspZ polypeptides. [Figure 4] FIG. 1 shows constructs comprising truncated YopM and NleC polypeptides. [Figure 5] FIG. 1 shows the domains of the full-length IpaH9.8 and IpaH4.5 polypeptides. DETAILED DESCRIPTION OF THE INVENTION

[0005] This description of preferred embodiments is intended to be read in conjunction with the accompanying drawings, which are considered part of the entire description of the invention. The drawings are not necessarily to scale, and certain features of the invention may be shown to scale or in some schematic form exaggerated for clarity and clarity. In the description, relative terms, such as "horizontal," "vertical," "up," "down," "top," and "bottom," and derivatives thereof (e.g., "horizontally," "downward," "upward," etc.), should be understood to refer to the orientation described or shown in the drawings under discussion. These relative terms are for convenience of description and are not generally intended to require a particular orientation. Terms, including "inward" versus "outward," and "longitudinal" versus "lateral," etc., should be interpreted as being relative to each other or to an axis of elongation or axis or center of rotation, as appropriate. Terms relating to attachment, coupling, and the like, such as "coupled" and "intercoupled," refer to both a relationship in which structures are fixed or attached to one another, directly or indirectly through intervening structures, and, unless otherwise specified, a movable or rigid attachment or relationship. The term "operably coupled" refers to such an attachment, coupling, or connection that permits the appropriate structures to operate as intended by the relationship. When referring to only a single mechanism, the term "mechanism" shall be understood to include any collection of mechanisms that individually or jointly execute a set (or sets) of instructions to perform any one or more methods discussed herein. In the claims, means-plus-function clauses, when used, are intended to encompass structures described, shown, or revealed by the description or drawings to perform the recited function, including equivalent structures as well as structural equivalents.

[0006] Disclosed herein are compositions and methods for treating inflammatory conditions. The compositions may include constructs containing two or more truncated T3SS bacterial effector polypeptides. Bacterial effector polypeptides are typically injected into host cells via the type III secretion system (T3SS) during the course of infection. By targeting host inflammatory signaling pathways, such polypeptides inhibit or neutralize the host immune response, allowing pathogens to weaken host defenses to ensure bacterial survival. The compositions and methods described herein possess immunomodulatory activity and are therefore useful for treating inflammatory conditions. Truncated T3SS bacterial effector polypeptides may offer enhanced pharmacokinetic properties and bioavailability for increased therapeutic efficacy.

[0007] The constructs provided herein may contain two or more truncated T3SS bacterial effector polypeptides, including portions of full-length T3SS bacterial effector polypeptides. The full-length T3SS bacterial effector polypeptides may have biological activity, such as E3 ubiquitin ligase activity, RhoGTPase regulatory activity, cysteine ​​methylase activity, zinc metalloprotease activity, acetyltransferase activity, or O-GIcNac transferase activity. The constructs provided herein may contain two or more truncated T3SS bacterial effector polypeptides, including portions of the full-length bacterial effector polypeptides YopE, YopJ, YopM, NleC, NleB, OspZ, IpaH4.5, IpaH7.8, and IpaH9.8. The two or more truncated T3SS bacterial effector polypeptides may be the same or different. Exemplary constructs may include truncated YopE and OspZ polypeptides, truncated YopM and OspZ polypeptides, truncated YopM and NleC polypeptides, truncated YopM and NleB polypeptides, and truncated IpaH9.8 and IpaH4.5 polypeptides. In some embodiments, the constructs provided herein may omit any one of the truncated T3SS bacterial effector polypeptides, including portions of the full-length bacterial effector polypeptides YopE, YopJ, YopM, NleC, NleB, OspZ, IpaH4.5, IpaH7.8, and IpaH9.8.

[0008] Useful bacterial effector polypeptides can have the biochemical activity, target specificity and cellular effect as shown in Table 1.

[0009] [Table 1]

[0010] composition The constructs described herein include two or more truncated T3SS bacterial effector polypeptides. The truncated bacterial effector polypeptides are continuous or contiguous portions of a reference full-length polypeptide (e.g., a 10-amino acid fragment of a polypeptide can be any 10 contiguous residues in the polypeptide). Thus, the truncated T3SS bacterial effector polypeptides can be within the reference full-length polypeptide. The truncated T3SS bacterial effector polypeptides can be at least 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acid residues shorter than the reference full-length polypeptide. In some embodiments, the amino acid sequence of the truncated T3SS bacterial effector polypeptide lacks 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid residues at the C-terminus relative to a reference full-length polypeptide. In some embodiments, the amino acid sequence of the truncated T3SS bacterial effector polypeptide lacks 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acid residues at the N-terminus relative to a reference full-length polypeptide.

[0011] In some embodiments, the truncated bacterial effector polypeptide maintains one or more activities of the reference full-length bacterial effector polypeptide. For example, the truncated E3 ubiquitin ligase may maintain all or substantially all of the E3 ubiquitin ligase activity of the reference full-length E3 ubiquitin ligase. In some embodiments, the truncated bacterial effector polypeptide lacks or substantially lacks one or more activities of the reference full-length bacterial effector polypeptide.

[0012] A reference full-length T3SS bacterial effector polypeptide can have the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:21. In some embodiments, a reference full-length T3SS bacterial effector polypeptide can have an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ ID NO:21. The construct can be a fusion protein comprising the amino acid sequence of a first truncated T3SS bacterial effector polypeptide and the amino acid sequence of a second truncated bacterial effector polypeptide. In some embodiments, the amino acid sequence of the first truncated bacterial effector polypeptide and the amino acid sequence of the second truncated bacterial effector polypeptide can be contiguous, and the amino acid sequence of the first truncated bacterial effector polypeptide and the amino acid sequence of the second truncated bacterial effector polypeptide can be linked by a peptide bond.

[0013] In some embodiments, the amino acid sequence of the first truncated bacterial effector polypeptide and the amino acid sequence of the second truncated bacterial effector polypeptide are connected by a linker. The linker can be a cleavable linker. The cleavable linker can include a pH-sensitive linker, such as a hydrazone, a phosphoramidate-based linker, a thiomaleic acid, a proteasome-specific linker, such as a Phe-Lys dipeptide linker, a Val-Cit-PABC linker, an enzyme-specific linker, such as a glucuronide-MABC linker or a β-glucuronide linker, a disulfide linker, such as a dithiocyclopeptide linker, an s Nfo-SPDB linker or an SPDB linker, a metal-assisted linker, such as a palladium linker or an iron linker, or a photocleavable linker, such as a nitrobenzyl linker or a di-6-(3-succinimidylcarbonyloxymethyl-4-nitro-phenoxy)-hexanoic acid disulfide diethanol ester (SCNE) linker.

[0014] In some embodiments, the linker may comprise at least one amino acid residue, or may be a peptide of at least or about 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 40, or 50 amino acid residues. When the linker is a single amino acid residue, it may be any naturally occurring or non-naturally occurring amino acid (e.g., Gly, Cys, Lys, Glu, or Asp) or a dipeptide containing two such residues (e.g., Gly-Lys). When the linker is a short peptide, it may be a glycine-rich peptide (which tends to be flexible), such as a peptide having the sequence [Gly-Gly-Gly-Gly-Ser]n, where n is an integer from 1 to 6, inclusive (SEQ ID NO: 25), or a serine-rich peptide linker. Serine-rich peptide linkers include those of the formula [XXXX-Gly]y, where up to two X are Thr and the remaining X are Ser, and y is an integer from 1 to 5, inclusive (SEQ ID NO: 26), (e.g., Ser-Ser-Ser-Ser-Gly (SEQ ID NO: 27), where y is greater than 1). Other linkers are rigid linkers (e.g., PAPAP (SEQ ID NO: 28) and (PT) n P, where n is 2, 3, 4, 5, 6, or 7 (SEQ ID NO: 29)) and an α-helical linker (e.g., A(EAAAK) nA, where n is 1, 2, 3, 4, or 5 (SEQ ID NO: 30). When the linker is succinic acid, one carboxyl group thereof may form an amide bond with the amino group of the amino acid residue, and the other carboxyl group may form an amide bond with, for example, the amino group of the peptide or the substituent. When the linker is Lys, Glu, or Asp, the carboxyl group thereof may form an amide bond with the amino group of the amino acid residue, and the amino group may form an amide bond with, for example, the carboxyl group of the substituent. When Lys is used as the linker, a further linker may be inserted between the ε-amino group of Lys and the substituent. The further linker may be succinic acid, which may form an amide bond with the ε-amino group of Lys and the amino group present in the substituent. In one embodiment, the further linker is Glu or Asp (e.g., which forms an amide bond with the ε-amino acid of Lys and another amide bond with the carboxyl group present in the substituent), i.e., the substituent is N ε -acrylated lysine residue.

[0015] The constructs disclosed herein may further comprise a protein transduction domain (PTD), i.e., an amino acid sequence that mediates translocation across a cell membrane. Useful protein transduction domains include the YopM protein transduction domain and the IpaH protein transduction domain. An exemplary YopM protein transduction domain may have the amino acid sequence set forth in SEQ ID NO: 17. An exemplary IpaH9.8 transduction domain may have the amino acid sequence set forth in amino acids 1-57 of SEQ ID NO: 11. A construct comprising a protein transduction domain and a first truncated T3SS bacterial effector polypeptide sequence and a second truncated T3SS bacterial effector polypeptide sequence may be a fusion protein. In some embodiments, the amino acid sequences of the protein transduction domain and the first truncated bacterial effector polypeptide may be contiguous with the amino acid sequence of the second truncated bacterial effector polypeptide, and the protein transduction domain and the first truncated bacterial effector polypeptide may be linked by a peptide bond. In some embodiments, the protein transduction domain and the amino acid sequence of the first truncated bacterial effector polypeptide and the amino acid sequence of the second truncated bacterial effector polypeptide are joined by a linker, i.e., any of the linkers described above.

[0016] Exemplary constructs and amino acid sequences of such constructs are shown in Figures 3 and 4. Figure 3 shows a fusion protein comprising a YopM protein transduction domain, a truncated YopM polypeptide, and a truncated OspZ polypeptide. The amino acid sequence of the fusion protein shown in Figure 3 is SEQ ID NO: 23. Figure 4 shows a fusion protein comprising a YopM protein transduction domain, a truncated YopM polypeptide, and a truncated NleC polypeptide. The amino acid sequence of the fusion protein shown in Figure 4 is SEQ ID NO: 24.

[0017] The polypeptides provided herein can have one or more amino acid additions, deletions, or substitutions relative to the native polypeptide amino acid sequence (also referred to herein as "variant" T3SS polypeptides), and can be made and modified as described herein. In some cases, amino acid substitutions can be made by selecting substitutions that do not significantly alter the effect on (a) the structure of the peptide backbone in the region of substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining side chain bulk. For example, naturally occurring residues can be classified into groups based on the properties of their side chains: (1) hydrophobic amino acids (norleucine, methionine, alanine, valine, leucine, and isoleucine), (2) neutral hydrophilic amino acids (cysteine, serine, and threonine), (3) acidic amino acids (aspartic acid and glutamic acid), (4) basic amino acids (asparagine, glutamine, histidine, lysine, and arginine), (5) amino acids that affect chain orientation (glycine and proline), and (6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Substitutions made within these groups can be considered conservative substitutions. Non-limiting examples of useful conservative substitutions may include, but are not limited to, substitution of valine for alanine, substitution of lysine for arginine, substitution of glutamine for asparagine, substitution of glutamic acid for aspartic acid, substitution of serine for cysteine, substitution of asparagine for glutamine, substitution of aspartic acid for glutamic acid, substitution of proline for glycine, substitution of arginine for histidine, substitution of leucine for isoleucine, substitution of isoleucine for leucine, substitution of arginine for lysine, substitution of leucine for methionine, substitution of leucine for phenylalanine, substitution of glycine for proline, substitution of threonine for serine, substitution of serine for threonine, substitution of tyrosine for tryptophan, substitution of phenylalanine for tyrosine, and / or substitution of leucine for valine.

[0018] In some embodiments, a polypeptide may contain one or more non-conservative substitutions. Non-conservative substitutions typically involve exchanging a member of one of the above-described classes for a member of another class. Such production may be desirable to provide such constructs in large quantities or alternative embodiments. Whether an amino acid change results in a functional polypeptide can be determined by evaluating the specific activity of the peptide variant.

[0019] The polypeptides provided herein can be obtained by chemical synthesis or by expression of a recombinant nucleic acid encoding the polypeptide. For example, recombinant technology using an expression vector encoding the polypeptide provided herein can be used. The resulting polypeptide can then be purified, for example, using affinity chromatography and HPLC. The degree of purification can be measured by any appropriate method, including, but not limited to, column chromatography, polyacrylamide gel electrophoresis, or high-performance liquid chromatography. The polypeptides provided herein can also be designed or engineered to contain a tag sequence that allows the polypeptide to be purified (e.g., captured on an affinity matrix). For example, tags such as c-myc, hemagglutinin, polyhistidine, or Flag™ tags (Kodak) can be used to aid polypeptide purification. Such tags can be inserted anywhere within the polypeptide, including either the carboxyl or amino terminus.

[0020] The polypeptides disclosed herein can be isolated from the inside or outside of host cells or from the medium in which the cells are cultured and purified as substantially pure and homogeneous polypeptides. A substantially pure polypeptide can be, for example, a polypeptide removed from its host cell or the medium in which it is produced, and can be at least 60%, at least 70%, at least 80%, or at least 90% pure, i.e., free or substantially free from other components, such as unrelated polypeptides, lipids, nucleic acids, or carbohydrates. Polypeptides can be isolated and purified by, for example, column chromatography, filtration, ultrafiltration, salting out, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, dialysis, and recrystallization, as appropriate. Chromatography includes, for example, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration, reversed-phase chromatography, and adsorption chromatography. Chromatography can be performed using liquid-phase chromatography, such as HPLC and FPLC.

[0021] The polypeptides provided herein can be formulated into pharmaceutical compositions by mixing with pharmaceutically acceptable non-toxic excipients or carriers. Such compositions can be administered to a subject in need thereof in an amount effective to treat inflammatory conditions. The pharmaceutical compositions can be prepared for oral or parenteral administration, such as intranasal, sublingual, buccal, intraarterial, intraarticular, intracardiac, intradermal, intramuscular, intraocular, intraosseous, intraperitoneal, intrathecal, intravenous, intravesicular, intravitreal, subcutaneous, transdermal, perivascular, intracerebral, or transmucosal administration. Intraarticular administration can be useful for treating inflammatory conditions in the joints. Compositions formulated for parenteral administration can be in the form of a liquid solution or suspension in an aqueous physiological buffer solution, particularly in the form of tablets or capsules for oral administration, or in the form of powders, nasal drops, or aerosols for intranasal administration.

[0022] Excipients or carriers may vary depending on formulation and administration route. Pharmaceutical carriers are described in Remington's Pharmaceutical Sciences (EW Martin) and USP / NF (United States Pharmacopeia and the National Formulary). Exemplary excipients may include sugars such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginic acid, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methylcellulose. In some embodiments, the formulation may include a lubricant, a wetting agent, an emulsifier, a preservative, a sweetener, or a flavoring agent.

[0023] Common excipients for parenteral administration may include sterile water or saline, polyalkylene glycols (e.g., polyethylene glycol), oils of plant origin, and hydrogenated naphthalenes. In particular, biocompatible, biodegradable lactide polymers, lactide / glycolide copolymers, or polyoxyethylene-polyoxypropylene copolymers are examples of excipients for controlling the release of polypeptides in vivo. Other suitable parenteral delivery systems include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation administration may optionally contain excipients such as lactose. Inhalation formulations may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, and deoxycholate, or oily solutions for administration in the form of nasal drops. Optionally, the compound may be formulated as a gel for intranasal application. Formulations for parenteral administration may also include glycocholate for buccal administration.

[0024] For oral administration, tablets or capsules can be prepared by conventional methods with pharmaceutically acceptable excipients, such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, or hydroxypropylmethylcellulose), fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., magnesium stearate, talc, or silica), disintegrants (e.g., potato starch or sodium starch glycolate), or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by methods known in the art. Preparations for oral administration can also be formulated to provide sustained release of the compound. Nasal formulations may be presented in liquid form or as a dry product. Atomized aqueous suspensions or solutions may contain carriers or excipients to adjust the pH and / or tonicity. In some embodiments, pharmaceutical compositions may be formulated to modulate the release of the active ingredient. Pharmaceutical compositions may also be formulated to provide rapid, sustained, or delayed release of the active ingredient after administration to a patient. The polypeptides provided herein may be formulated as sustained-release dosage forms. For example, the polypeptides may be formulated into sustained-release preparations. In some embodiments, coatings, envelopes, or protective matrices may be formulated to contain one or more of the polypeptides provided herein. In some embodiments, such coatings, envelopes, and protective matrices may be used to coat indwelling devices, such as stents, catheters, and peritoneal dialysis tubing. In some cases, the polypeptides provided herein may be incorporated into polymeric substances, liposomes, microemulsions, microparticles, nanoparticles, or waxes.

[0025] Nucleic acids encoding any of the constructs disclosed herein are also provided. An isolated nucleic acid refers to a nucleic acid that is not immediately contiguous with both of the sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it originates. For example, an isolated nucleic acid can be, but is not limited to, a recombinant DNA molecule of any length, provided that one of the nucleic acid sequences normally found immediately adjacent to the recombinant DNA molecule in the naturally occurring genome has been removed or is absent. Thus, an isolated nucleic acid includes, but is not limited to, recombinant DNA that exists as a separate molecule independent of other sequences (e.g., cDNA or genomic DNA fragments produced by PCR or restriction endonuclease treatment), as well as recombinant DNA integrated into a vector, autonomously replicating plasmid, virus (e.g., retrovirus, adenovirus, or herpesvirus), or genomic DNA of a prokaryote or eukaryote. Furthermore, an isolated nucleic acid can include a recombinant DNA molecule that is part of a hybrid or fusion nucleic acid sequence.

[0026] Isolated nucleic acids also include any nucleic acid that does not occur in nature, because a non-naturally occurring nucleic acid sequence is not found in nature and does not have a directly contiguous sequence in a naturally occurring genome. For example, a non-naturally occurring nucleic acid, such as an engineered nucleic acid, is considered an isolated nucleic acid. Engineered nucleic acids (e.g., nucleic acids encoding polypeptides comprising or consisting of the amino acid sequences set forth in SEQ ID NO:23 and SEQ ID NO:24) can be produced using molecular cloning or chemical nucleic acid synthesis techniques. Isolated non-naturally occurring nucleic acids can be independent of other sequences or can be integrated into vectors, autonomously replicating plasmids, viruses (e.g., retroviruses, adenoviruses, or herpes viruses), or genomic DNA of prokaryotes or eukaryotes. Furthermore, non-naturally occurring nucleic acids can include nucleic acid molecules that are part of hybrid or fusion nucleic acid sequences. A nucleic acid present among hundreds to millions of other nucleic acids, such as a cDNA library or genomic library, or a gel slice containing a genomic DNA restriction digest, is not considered an isolated nucleic acid.

[0027] Nucleic acids can be RNA and DNA, such as mRNA, cDNA, genomic DNA, synthetic (e.g., chemically synthesized) DNA, and nucleic acid analogs. Nucleic acids can be double-stranded or single-stranded, and the single strand can be a sense strand or an antisense strand. Furthermore, nucleic acids can be circular or linear. Nucleic acid analogs can be modified at the base moiety, sugar moiety, or phosphate backbone moiety to improve, for example, the stability, hybridization, or solubility of the nucleic acid. Modifications at the base moiety include deoxyuridine from deoxythymidine, and 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine from deoxycytidine. Modifications at the sugar moiety can include modifying the 2' hydroxyl of the ribose sugar to form a 2'-O-methyl or 2'-O-allyl sugar. The deoxyribose phosphate backbone may be modified to produce a morpholino nucleic acid, in which each base moiety is linked to a six-membered morpholino ring or peptide nucleic acid, and the deoxyphosphate backbone is replaced with a pseudopeptide backbone, maintaining the four bases. Additionally, the deoxyphosphate backbone may be replaced with, for example, a phosphorothioate or phosphorodithioate backbone, a phosphoramidate, or an alkylphosphotriester backbone.

[0028] The nucleic acids provided herein may comprise or consist of any of the nucleic acid sequences set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22. In some embodiments, the nucleic acids may comprise any truncated nucleic acid of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, or SEQ ID NO:22.

[0029] The nucleic acids encoding the first truncated T3SS bacterial effector polypeptide sequence and the second truncated T3SS bacterial effector polypeptide sequence include those that are codon-optimized. For example, the nucleic acids may be incorporated into a vector (e.g., a plasmid or viral vector), and such vectors are encompassed by the present invention. The nucleic acids may be operably linked to a regulatory region appropriate for use in either prokaryotic or eukaryotic systems. In specific embodiments, the regulatory region may be, for example, a promoter or enhancer. Useful promoters include cell-type-specific promoters, tissue-specific promoters, constitutively active promoters, and ubiquitously expressed promoters. Host cells containing vectors expressing the polypeptides of the present invention are also encompassed by the present invention, and these cells may be prokaryotic (e.g., bacterial) or eukaryotic (e.g., mammalian) cells.

[0030] Typically, the isolated nucleic acids provided herein are at least 10 nucleotides in length (e.g., 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 200, 300, 350, 400 or more nucleotides in length). Nucleic acid molecules shorter than the full length can be used, for example, as primers or probes. Isolated nucleic acid molecules can be produced by molecular cloning and chemical nucleic acid synthesis techniques. For example, polymerase chain reaction (PCR) technology can be used. Isolated nucleic acids can also be chemically synthesized as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidate technology) or as a series of oligonucleotides, which can then be ligated into a vector.

[0031] Treatment method Also provided are methods of treating a subject having or at risk of an inflammatory condition by administering a therapeutically effective amount of a pharmaceutical composition comprising any of the constructs disclosed herein. In some embodiments, the subject (e.g., a human patient) in need of treatment has been diagnosed with, is suspected of having, or is at risk for an inflammatory condition. Exemplary inflammatory conditions include, but are not limited to, arthritis and joint diseases such as rheumatoid arthritis and osteoarthritis, cardiovascular disease, allergies, asthma, chronic obstructive pulmonary disease, diabetes, gastrointestinal diseases such as inflammatory bowel disease, Crohn's disease, and ileocolitis, cancers such as kidney cancer, prostate cancer, ovarian cancer, hepatocellular carcinoma, pancreatic cancer, colon cancer, and lung cancer, and inflammatory conditions found in mesothelioma, chronic kidney disease, and Alzheimer's disease.

[0032] Generally, treatment can include one or more of inhibiting the inflammatory state (i.e., halting further progression of the pathology and / or symptomology) in an individual experiencing or exhibiting the pathology or symptomology of a disease, condition, or disorder. Treatment can also include ameliorating the inflammatory state (i.e., reversing the pathology and / or symptomology) in an individual experiencing or exhibiting the pathology or symptomology of a disease, condition, or disorder, for example, reducing the severity of the disease or reducing or alleviating one or more symptoms of the disease.

[0033] The subject can be a human or a non-human animal. Exemplary non-human species include, but are not limited to, non-human primates, livestock such as horses, pigs, cows, sheep, cats, dogs, mice, or rats. Subjects suitable for treatment can be identified by detecting symptoms commonly associated with inflammatory conditions, such as pain, fatigue, gastrointestinal symptoms such as constipation, diarrhea, and acid reflux, weight gain, and frequent infections. Subjects suitable for treatment can also be identified by laboratory tests, such as serum protein electrophoresis (SPE), high-sensitivity C-reactive protein, fibrinogen, and pro-inflammatory cytokine detection. A therapeutically effective amount can be that amount of active compound or pharmaceutical agent that elicits the biological or medical response sought in a tissue, system, animal, individual, or human by a researcher, veterinarian, physician, or other clinician.

[0034] The compositions provided herein may be administered in combination with one or more conventional therapeutic agents, including treatments for arthritis and joint diseases such as rheumatoid arthritis and osteoarthritis, cardiovascular disease, allergies, asthma, chronic obstructive pulmonary disease, diabetes, gastrointestinal diseases such as inflammatory bowel disease, Crohn's disease, and iliocolitis, cancer such as kidney cancer, prostate cancer, ovarian cancer, hepatocellular carcinoma, pancreatic cancer, colon cancer, lung cancer, and mesothelioma, chronic kidney disease, and Alzheimer's disease.

[0035] Features of the invention In general, the invention features constructs that can include two or more truncated T3SS bacterial effector polypeptides, including portions of full-length bacterial effector polypeptides. In one aspect, the construct can include a truncated YopM polypeptide linked to a truncated T3SS cysteine ​​methyltransferase polypeptide. The truncated YopM polypeptide can have an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 19. The truncated YopM polypeptide can have the amino acid sequence set forth in SEQ ID NO: 19. The truncated T3SS cysteine ​​methyltransferase polypeptide can include a portion of an OspZ polypeptide having the amino acid sequence set forth in SEQ ID NO: 3. The truncated OspZ polypeptide can have an amino acid sequence at least 90% identical to amino acids 226-446 of SEQ ID NO: 3. The truncated OspZ polypeptide can have the amino acid sequence set forth in amino acids 226-446 of SEQ ID NO: 3. The construct can further include a protein transduction domain, e.g., the YopM protein transduction domain set forth in SEQ ID NO: 17. In some embodiments, the construct comprises the amino acid sequence set forth in SEQ ID NO:23.

[0036] In other aspects, the construct may comprise a truncated YopM polypeptide linked to a truncated T3SS zinc metalloprotease polypeptide. The truncated YopM polypeptide may have an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 19. The truncated YopM polypeptide may have the amino acid sequence set forth in SEQ ID NO: 19. The truncated T3SS zinc metalloprotease polypeptide may comprise a portion of an NleC polypeptide having the amino acid sequence set forth in SEQ ID NO: 5. The truncated NleC polypeptide may have an amino acid sequence at least 90% identical to amino acids 2-187 of SEQ ID NO: 5. In some embodiments, the truncated NleC polypeptide may have the amino acid sequence set forth in amino acids 2-187 of SEQ ID NO: 5. In some embodiments, the truncated NleC polypeptide has the amino acid sequence set forth in amino acids 2-187 of SEQ ID NO: 5. The construct may further comprise a protein transduction domain, for example, the YopM protein transduction domain set forth in SEQ ID NO: 17. In some embodiments, the construct has the amino acid sequence set forth in SEQ ID NO: 24.

[0037] In one aspect, the construct may comprise a truncated YopM polypeptide linked to a truncated T3SS O-GlcNac transferase. The truncated YopM polypeptide may have an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 19. The truncated YopM polypeptide may have the amino acid sequence set forth in SEQ ID NO: 19. The truncated T3SS O-GlcNac transferase may comprise a portion of an NleB polypeptide having the amino acid sequence set forth in SEQ ID NO: 9. The truncated NleB polypeptide may have an amino acid sequence at least 90% identical to amino acids 2-226 of SEQ ID NO: 9. The truncated NleB polypeptide may have the amino acid sequence set forth in amino acids 2-226 of SEQ ID NO: 9. The construct may further comprise a protein transduction domain, for example, the YopM protein transduction domain set forth in SEQ ID NO: 17.

[0038] In one aspect, the construct can include a truncated first T3SS E3 ubiquitin ligase polypeptide linked to a truncated second T3SS E3 ubiquitin ligase. The first and second truncated T3SS E3 ubiquitin ligase polypeptides can be different. The truncated first E3 ubiquitin ligase can include a portion of an IpaH9.8 polypeptide having the amino acid sequence set forth in SEQ ID NO: 11. The truncated first IpaH9.8 polypeptide includes an amino acid sequence at least 90% identical to amino acids 56-228 of SEQ ID NO: 11. The truncated second E3 ubiquitin ligase includes a portion of an IpaH4.5 polypeptide having the amino acid sequence set forth in SEQ ID NO: 13. The truncated second IpaH4.5 polypeptide includes an amino acid sequence at least 90% identical to amino acids 62-270 of SEQ ID NO: 13. The construct may further comprise a protein transduction domain, for example the IpaH9.8 protein transduction domain shown at amino acids 1-57 of SEQ ID NO:11.

[0039] In one aspect, the construct can include a RhoGTPase modulator linked to a cysteine ​​methyltransferase, wherein the RhoGTPase modulator is linked to the cysteine ​​methyltransferase by a pH-sensitive linker. The RhoGTPase modulator can be a YopE polypeptide having the amino acid sequence set forth in SEQ ID NO: 1. The cysteine ​​methyltransferase can be an OspZ polypeptide having the amino acid sequence set forth in SEQ ID NO: 5. The pH-sensitive linker comprises a hydrazine, a phosphoramidate-based linker, or a thiomaleic acid. In one aspect, the construct may comprise a truncated YopM polypeptide linked to an acetyltransferase. The truncated YopM polypeptide may have an amino acid sequence at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 19. The truncated YopM polypeptide may have the amino acid sequence set forth in SEQ ID NO: 19. The acetyltransferase may be a YopJ polypeptide having the amino acid sequence set forth in SEQ ID NO: 9, which includes a mutation at cysteine ​​172.

[0040] In one aspect, the construct can include a truncated YopE polypeptide linked to an acetyltransferase. The truncated YopE polypeptide can include a portion of the YopE polypeptide having the amino acid sequence set forth in SEQ ID NO: 1. The construct can further include a protein transduction domain, for example, the IpaH9.8 protein transduction domain set forth at amino acids 1-57 of SEQ ID NO: 11. In one aspect, the construct may further comprise a protein transduction domain. The protein transduction domain may be a YopM protein transduction domain. The YopM protein transduction domain may have the amino acid sequence set forth in SEQ ID NO: 19. The protein transduction domain may be an IpaH9.8 protein transduction domain. The IpaH9.8 protein transduction domain may have the amino acid sequence set forth in amino acids 1 to 56 of SEQ ID NO: 23.

[0041] In one aspect, any construct may comprise a fusion protein. The first truncated T3SS bacterial effector polypeptide and the second truncated T3SS bacterial effector polypeptide may be linked by a linker. The linker may be a cleavable linker. The cleavable linker may be a pH-sensitive linker. The pH-sensitive linker may be selected from the group consisting of hydrazine, phosphoramidate-based linkers, and thiomaleic acid. In one aspect, nucleic acids encoding any of the constructs disclosed herein are also provided.

[0042] In one aspect, the construct can be formulated as a pharmaceutical composition comprising the construct and a pharmaceutically acceptable carrier. In one aspect, the present application features a method of treating a subject having or at risk of an inflammatory condition, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a construct that may include a first truncated T3SS bacterial effector polypeptide and a second truncated T3SS bacterial effector polypeptide and a pharmaceutically acceptable carrier. The method may include identifying the subject. The inflammatory condition may be a gastrointestinal disorder, a musculoskeletal disorder, an autoimmune disorder, or a skin disorder. In one aspect, the inflammatory condition may include inflammatory bowel disease, Crohn's disease, rheumatoid arthritis, osteoarthritis, cancer, allergies, cardiovascular disease, chronic obstructive pulmonary disease, and diabetes. [Example]

[0043] Example 1 We analyzed the effects of E3 ubiquitin ligases IpaH7.8 and IpaH9.8 on cytokine release from THP-1 cells. THP-1 cells were cultured in the presence or absence of increasing amounts (0.25 μg, 0.5 μg, and 1.0 μg) of recombinant IpaH7.8 or IpaH9.8. For IpaH7.8, 0.25 μg of protein is approximately 3.87 nM. For IpaH9.8, 0.25 μg of protein is approximately 4.0 nM. The cultures were then treated with lipopolysaccharide (LPS) to induce cytokine release. As shown in Table 2, recombinant IpaH7.8 inhibited the release of IL-1β, TNF-α, MCP-1, IL-6, IL-8, and IL-23. As shown in Table 3, recombinant IpaH9.8 caused dose-dependent inhibition of the release of IL-1β, TNF-α, MCP-1, IL-6, IL-8, and IL-23. These data indicated that low nanomolar concentrations of the E3 ubiquitin ligases IpaH7.8 and IpaH9.8 could efficiently downregulate cytokine levels in THP-1 cells.

[0044] [Table 2]

[0045]

Table 3

Claims

1. A construct comprising two or more truncated T3SS bacterial effector polypeptides, wherein each truncated T3SS bacterial effector polypeptide comprises a portion of a corresponding full-length T3SS bacterial effector polypeptide.

2. The construct of claim 1 , wherein the truncated T3SS bacterial effector polypeptide maintains one or more activities of the corresponding full-length T3SS bacterial effector polypeptide.

3. 2. The construct of claim 1, wherein the T3SS bacterial effector polypeptide is selected from the group consisting of an E3 ubiquitin ligase, a Rho GTPase regulator, a cysteine ​​methyltransferase, a zinc metalloprotease, an acetyltransferase, an O-GlcNac transferase, and an LRR motif-binding and capture polypeptide.

4. 4. The construct of claim 3, wherein the E3 ubiquitin ligase is IpaH7.8 or IpaH9.8, the Rho GTPase regulator is YopE, the cysteine ​​methyltransferase is OspZ or NleE, the zinc metalloprotease is NleC, the acetyltransferase is YopJ, the O-GlcNac transferase is NleB, and the LRR motif-binding and capture polypeptide is YopM.

5. 2. The construct of claim 1, comprising a truncated YopM polypeptide linked to a truncated T3SS cysteine ​​methyltransferase polypeptide.

6. The construct of claim 5 , wherein the truncated T3SS cysteine ​​methyltransferase polypeptide comprises a portion of an OspZ polypeptide having the amino acid sequence set forth in SEQ ID NO:

3.

7. 7. The construct of claim 6, wherein the truncated OspZ polypeptide comprises an amino acid sequence at least 90% identical to amino acids 226 to 446 of SEQ ID NO:

3.

8. 8. The construct of claim 7, wherein the truncated OspZ polypeptide has the amino acid sequence set forth in amino acids 226 to 446 of SEQ ID NO:

3.

9. The construct of claim 5, comprising the amino acid sequence set forth in SEQ ID NO:

23.

10. 2. The construct of claim 1, comprising a truncated YopM polypeptide linked to a truncated T3SS zinc metalloprotease polypeptide.

11. The construct of claim 10, wherein the truncated T3SS zinc metalloprotease polypeptide comprises a portion of an NleC polypeptide having the amino acid sequence set forth in SEQ ID NO:

5.

12. The construct of claim 11, wherein the truncated NleC polypeptide comprises an amino acid sequence at least 90% identical to amino acids 2 to 187 of SEQ ID NO:

5.

13. The construct of claim 12, wherein the truncated NleC polypeptide has the amino acid sequence set forth in amino acids 2 to 187 of SEQ ID NO:

5.

14. The construct of claim 10, comprising the amino acid sequence set forth in SEQ ID NO:

24.

15. 2. The construct of claim 1, comprising a truncated YopM polypeptide linked to a truncated T3SS O-GlcNac transferase.

16. 16. The construct of claim 15, wherein the truncated T3SS O-GlcNac transferase comprises a portion of an NleB polypeptide having the amino acid sequence set forth in SEQ ID NO:

9.

17. The construct of claim 16, wherein the truncated NleB polypeptide comprises an amino acid sequence that is at least 90% identical to amino acids 2 to 226 of SEQ ID NO:

9.

18. The construct of claim 17, wherein the truncated NleB polypeptide has the amino acid sequence set forth in amino acids 2 to 226 of SEQ ID NO:

9.

19. 2. The construct of claim 1, comprising a truncated first T3SS E3 ubiquitin ligase polypeptide linked to a truncated second T3SS E3 ubiquitin ligase.

20. 20. The construct of claim 19, wherein the first and second truncated T3SS E3 ubiquitin ligase polypeptides are different.

21. 20. The construct of claim 19, wherein the truncated first E3 ubiquitin ligase comprises a portion of the IpaH9.8 polypeptide having the amino acid sequence set forth in SEQ ID NO:

11.

22. 21. The construct of claim 20, wherein the truncated first IpaH9.8 polypeptide comprises an amino acid sequence at least 90% identical to amino acids 56 to 228 of SEQ ID NO:

11.

23. 23. The construct of claim 22, wherein the truncated second E3 ubiquitin ligase comprises a portion of an IpaH4.5 polypeptide having the amino acid sequence set forth in SEQ ID NO:

13.

24. 24. The construct of claim 23, wherein the truncated second IpaH4.5 polypeptide comprises an amino acid sequence at least 90% identical to amino acids 62 to 270 of SEQ ID NO:

13.

25. The construct of claim 1 , comprising a Rho GTPase modulator linked to a cysteine ​​methyltransferase.

26. The construct of claim 25 , wherein the Rho GTPase modulator is a YopE polypeptide having the amino acid sequence set forth in SEQ ID NO:

1.

27. 27. The construct of claim 26, wherein the cysteine ​​methyltransferase is an OspZ polypeptide having the amino acid sequence set forth in SEQ ID NO:

5.

28. 2. The construct of claim 1, comprising a truncated YopM polypeptide linked to a truncated acetyltransferase polypeptide.

29. 29. The construct of claim 28, wherein the truncated acetyltransferase comprises a portion of a YopJ polypeptide having the amino acid sequence set forth in SEQ ID NO:

9.

30. 29. The construct of claim 28, wherein the truncated YopJ polypeptide comprises a point mutation at cysteine ​​position 172 of SEQ ID NO:

9.

31. The construct of any one of claims 1 to 18, wherein the truncated YopM polypeptide has an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:

19.

32. 32. The construct of claim 31 , wherein the truncated YopM polypeptide has the amino acid sequence set forth in SEQ ID NO:

19.

33. 33. The construct of any one of claims 1 to 32, further comprising a protein transduction domain.

34. 28. The construct of claim 27, wherein the protein transduction domain is a YopM protein transduction domain.

35. 35. The construct of claim 34, wherein the YopM protein transduction domain has the amino acid sequence set forth in SEQ ID NO:

17.

36. 28. The construct of claim 27, wherein the protein transduction domain is an IpaH9.8 protein transduction domain.

37. 28. The construct of claim 27, wherein the IpaH9.8 protein transduction domain has the amino acid sequence set forth in amino acids 2 to 56 of SEQ ID NO:

11.

38. 38. The construct of any one of claims 1 to 37, comprising a fusion protein.

39. 39. The construct of any one of claims 1 to 38, wherein two or more truncated T3SS bacterial effector polypeptides are linked by a linker.

40. 40. The construct of claim 39, wherein the linker is a cleavable linker.

41. 41. The construct of claim 40, wherein the cleavable linker is a pH-sensitive linker.

42. 42. The construct of claim 41 , wherein the pH-sensitive linker comprises a hydrazine, a phosphoramidate-based linker, or a thiomaleic acid.

43. 40. The construct of claim 39, wherein the linker is a covalent bond.

44. A nucleic acid encoding the construct of any one of claims 1 to 43.

45. A pharmaceutical composition comprising the construct of any one of claims 1 to 44 and a pharmaceutically acceptable carrier.

46. 46. ​​A method of treating a subject having an inflammatory disorder, comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 45.

47. 47. The method of claim 46, wherein the inflammatory disorder is a skin disorder, a gastrointestinal disorder, or a musculoskeletal disorder.

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