Compositions and particles for payload delivery

Abstract

To provide compositions and particles for payload delivery.SOLUTION: The present disclosure provides complexes and compositions comprising particles, microparticles or nanoparticles, for delivery of payloads into a cell or across a polarized epithelial cell. The compositions can comprise a payload in a pill or tablet for delivery of the payload into or across a polarized epithelial cell. The present invention provides a composition comprising e.g., a carrier capable of entering a polarized epithelial cell or transcytosing across a polarized epithelial cell; and a heterologous payload, a molar ratio of the heterologous payload to the carrier being greater than 1:1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] cross reference This application is a continuation of U.S. Provisional Application No. 62 / 888,282 filed August 16, 2019, U.S. Provisional Application No. 62 / 935,615 filed November 14, 2019, U.S. Provisional Application No. 63 / 021,029 filed May 6, 2020, U.S. Provisional Application No. 63 / 033,151 filed June 1, 2020, and U.S. Provisional Application No. 63 / 033,151 filed August 16, 2019. This application claims the benefit of U.S. Provisional Patent Application No. 62 / 888,400, filed September 11, 2019, U.S. Provisional Patent Application No. 62 / 899,064, filed June 1, 2020, and PCT Patent Application No. PCT / US19 / 50708, filed September 11, 2019, which applications are incorporated herein by reference in their entireties. [Background technology]

[0002] background The inability of large macromolecules and / or protein biopharmaceuticals to be readily absorbed across epithelia, respiratory epithelia, or intestinal epithelia can be a limiting factor in developing commercially viable formulations of these agents. For example, due to this inability to absorb, unstable materials may remain in the intestinal lumen until resident enzymes degrade them, and stable materials may remain in the intestinal lumen until excreted from the body. Furthermore, challenges can exist in delivering therapeutic agents to the lungs.

[0003] Although clinical studies evaluating various biologically active agents for the treatment of diseases such as cancer, inflammatory diseases, immune diseases, growth retardation disorders, etc. have produced promising results, these agents may not reach their optimal potential.Due to inherent limitations such as short biological half-life, which may prevent the delivery of optimal therapeutically effective dosages, and / or adverse side effects and toxicity observed at therapeutically effective doses, overall efficacy may be limited or insufficient.In addition, these agents may require multiple dosing regimens, which may require continuous intravenous administration or frequent subcutaneous injections, which may be a burden to patients and caregivers. Summary of the Invention [Problem to be solved by the invention]

[0004] Thus, there is a need for new methods and compositions that can be used to administer therapeutic agents to human subjects. There is also a need for improved methods and compositions for the delivery of glucose regulating agents to subjects. [Means for solving the problem]

[0005] overview In certain embodiments, compositions are described herein that include a carrier capable of entering or transcytosing across polarized epithelial cells and a heterologous payload, wherein the heterologous payload to carrier molar ratio is greater than 1:1. In some embodiments, the composition includes a transition metal cation. In some embodiments, the transition metal cation is Fe 2+ , Mn 2+ , Zn 2+ , Co 2+ , Ni 2+ , and Cu 2+ In some embodiments, the transition metal cation is selected from the group consisting of Zn 2+ is.

[0006] In some embodiments, the composition comprises a polycation. In some embodiments, the polycation is protamine.

[0007] In some embodiments, the carrier comprises a portion of a Corix polypeptide. In some embodiments, the carrier consists of a portion of Pseudomonas exotoxin A. In some embodiments, the carrier consists of a portion of a Corix polypeptide. In some embodiments, the Corix polypeptide consists of an amino acid sequence having a C-terminus at any one of amino acid positions 206-425 of SEQ ID NO:7. In some embodiments, the Corix polypeptide consists of an amino acid sequence having a C-terminus at any one of amino acid positions 150-205 of SEQ ID NO:7. In some embodiments, the Corix polypeptide consists of an amino acid sequence having a C-terminus at any one of amino acid positions 150-195 of SEQ ID NO:7. In some embodiments, the Corix polypeptide consists of an amino acid sequence having an N-terminus at any one of amino acid positions 1-41 of SEQ ID NO:7. In some embodiments, the Corix polypeptide consists of an amino acid sequence having an N-terminus at any one of amino acid positions 35-40 of SEQ ID NO:7. In some embodiments, the Corix polypeptide consists of the amino acid sequence from amino acid 40 of the sequence set forth in SEQ ID NO:7 to any one of amino acids 150-205 of the sequence set forth in SEQ ID NO:7. In some embodiments, the Corix polypeptide has a C-terminus at any one of amino acids 150-187 of the sequence set forth in SEQ ID NO:7. In some embodiments, the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, the amino acid positions are numbered based on an alignment of the Corix polypeptide to the sequence set forth in SEQ ID NO:7, with the amino acid positions numbered from the N-terminus to the C-terminus, starting from position 1 at the N-terminus.

[0008] In some embodiments, the heterologous payload is selected from the group consisting of a macromolecule, a small molecule, a peptide, a polypeptide, a nucleic acid, an mRNA, an miRNA, an shRNA, an siRNA, an antisense molecule, an antibody, a DNA, a plasmid, a vaccine, a polymeric nanoparticle, and a catalytically active material. In some embodiments, the heterologous payload is a therapeutic payload.

[0009] In some embodiments, the heterologous payload is selected from the group consisting of dyes and radiopharmaceuticals, hormones, cytokines, anti-TNF agents, glucose-lowering agents, tumor-associated antigens, peptides, and polypeptides, hi some embodiments, the heterologous payload is a polypeptide that is a modulator of inflammation in the gastrointestinal tract.

[0010] In some embodiments, the heterologous payload is a glucagon-like peptide-2 (GLP-2) analog. In some embodiments, the GLP-2 analog is teduglutide.

[0011] In some embodiments, the heterologous payload is a cytokine. In some embodiments, the cytokine is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, and IL-30. In some embodiments, the cytokine is IL-10. In some embodiments, the cytokine is IL-22. In some embodiments, the cytokine lacks a natural secretion signal. In some embodiments, the therapeutic payload is a hormone. In some embodiments, the hormone is human growth hormone (hGH).

[0012] In some embodiments, the heterologous payload is a glucose-lowering agent, hi some embodiments, the heterologous payload is selected from the group consisting of incretin, glucagon proprotein, glucagon peptide, glucagon-like peptide 1, glucagon-like peptide 2, glicentin, glicentin-related polypeptide, gastric inhibitory polypeptide preprotein, gastric inhibitory polypeptide, dipeptidyl peptidase 4, glucose transporter member 4, preproglucagon, insulin receptor substrate 1, insulin, insulin analog, apolipoprotein A-II, solute carrier family 2, facilitative glucose transporter member 1, glycogen synthase 1, glycogen synthase 2, tyrosine-protein phosphatase non-receptor type 1, RAC-alpha serine threonine-protein kinase, peroxisome proliferator-activated receptor gamma, hexokinase 3, phosphatidylinositol-3,4,5-triphosphate 3-phosphatase, and dual specificity protein, pyruvate. Acid dehydrogenase kinase 1, calcium-binding and coiled-coil domain-containing protein 1, Max-like protein X, fructose-bisphosphate aldolase A, glucagon-like peptide 1 receptor, glucagon-like peptide 2 receptor, gastric inhibitory polypeptide receptor, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor, insulin receptor, GLP-1 agonist-exenatide, GLP-1 agonist-liraglutide, exenatide Senatide, exendin-4, exendin-3, GIPR agonist (Des-Ala2-GIP1-30), GIPR agonist truncated GIP1-30, GLP-1R agonist (amino acids 1-37 of GIP), GLP-1R agonist (amino acids 7-36 of GIP), lixisenatide (trade names Adlyxin® and Lyxumia®, Sanofi), liraglutide (trade name Victoza®, Novo Nordisk A / S), semaglutide (trade name Ozempic®, Novo Nordisk A / S), albiglutide (trade name Tanzeum®, GlaxoSmithKline, GLP-1 dimer fused to albumin), dulaglutide (trade name Trulicity®, EliLilly), glucose-dependent insulinotropic polypeptide, multispecific peptide agonists, tirzepatide (Eli Lilly), SAR425899 (Sanofi), amylin calcitonin receptor dual agonist DACRA-089, glargine / Lantus®, glulisine / Apidra®, glarine / Toujeo®, insuman®, detemir / Levemir®, lispro / Humalog® / Liprolog®, degludec / DegludecPlus, insulin aspart, insulin and analogs (e.g., LY-2605541, LY296 3016, NN1436), PEGylated insulin lispro, Humulin®, Linjeta, SuliXen®, NN1045, Insulin Plus Symlin™, PE0139, rapid- and short-acting insulins (e.g., Linjeta, PH20, NN1218, HinsBet), (APC-002) hydrogel, oral, inhaled, transdermal, and sublingual insulins (e.g., Exubera®, Nasulin®, Afrezza®, Tregopil®, TPM02, Capsulin, Oral-lyn®, Cobalamin®, oral insulin, ORMD-0801, NN1953, NN1954, NN1956, VIAtab, and Oshadi oral insulin), or exendin-4 analogs (exendin-4 analogs include desPro36-exendin-4(1-39)-Lys6NH2, H-des(Pro36,37)-exendin-4-Lys4-NH2, H-des(Pro36,37)-exendin-4-Lys5-NH2, d esPro36[Asp28]exendin-4(1-39), desPro36[IsoAsp28]exendin-4(1-39), desPro36[Met(O)14,Asp28]exendin-4(1-39), desPro36[Met(O)14,IsoAsp28]exendin-4(1-39), desPro36[Trp(O2)26,Asp28]exendin-4(1-39), or desPro36[Trp(O2)25,IsoAsp28]exendin-4(1-39), desPro36[Met(O)14 Trp(O2)25,Asp28] exendin-4(1-39), or desPro36[Met(O)14 Trp(O2)25,IsoAsp28] exendin-4(1-39).

[0013] In some embodiments, the heterologous payload is insulin or an insulin analog. In some embodiments, the heterologous payload is exenatide. In some embodiments, the heterologous payload comprises a fluorescent label. In some embodiments, the fluorescent label is fluorescein.

[0014] In some embodiments, the heterologous payload is an exenatide-fluorescein conjugate. In some embodiments, the composition is resistant to cleavage by pancreatic enzymes. In some embodiments, at least 50% of the carrier is intact at 2 hours in a pancreatin assay, the pancreatin assay comprising incubating a composition comprising 100 μg of carrier with 10 μg of pancreatin in 100 μL of phosphate-buffered saline (PBS) at 37° C. In some embodiments, the molar ratio of transition metal cation to carrier is about 100:1 to about 300,000:1. In some embodiments, the molar ratio of transition metal cation to carrier is about 1000:1 to about 30,000:1. In some embodiments, the molar ratio of transition metal cation to carrier is about 1000:1 to about 10,000:1.

[0015] In some embodiments, the molar ratio of protamine to carrier is about 10:1 to about 0.01:1. In some embodiments, the molar ratio of heterologous payload to carrier is about 2:1 to about 6000:1. In some embodiments, the molar ratio of protamine to carrier is about 1:1. In some embodiments, the molar ratio of heterologous payload to carrier is about 2:1 to about 10:1. In some embodiments, the molar ratio of heterologous payload to carrier is about 7:1. In some embodiments, the molar ratio of heterologous payload to carrier is about 7.16:1.

[0016] In some embodiments, the heterologous payload is a polypeptide comprising the sequence of SEQ ID NO: 11 or SEQ ID NO: 14. In some embodiments, the heterologous payload is a polypeptide comprising the sequence of SEQ ID NO: 18 or SEQ ID NO: 19. In some embodiments, the heterologous payload is a polypeptide comprising the sequence of SEQ ID NO: 20. In some embodiments, the heterologous payload is a polypeptide comprising the sequence of SEQ ID NO: 21. In some embodiments, the heterologous payload is a polypeptide comprising the sequence of SEQ ID NO: 22.

[0017] In some embodiments, the composition is encapsulated. In some embodiments, the encapsulated composition is configured to release a heterologous payload under a first condition but not under a second condition. In some embodiments, the encapsulated composition is configured to release a heterologous payload at a high pH but not at a low pH. In some embodiments, the encapsulated composition comprises an enteric coating. In some embodiments, the composition is a particle. In some embodiments, the composition comprises a polycation. In some embodiments, the carrier is capable of transporting a heterologous payload into or transcytosing a heterologous payload across polarized epithelial cells. In some embodiments, the carrier is linked to a polycation. In some embodiments, the polycation is protamine, poly-lysine, poly-ornithine, poly-ethyleneimine (PEI), prolamine, protamine, polyvinylpyrrolidone (PVP), polyarginine, polyvinylamine, or a combination thereof. In some embodiments, the polycation is a protamine salt. In some embodiments, the protamine salt is protamine sulfate, protamine acetate, protamine bromide, protamine chloride, protamine caproate, protamine trifluoroacetate, protamine HCO3, protamine propionate, protamine lactate, protamine formate, protamine nitrate, protamine citrate, protamine monohydrogen phosphate, protamine dihydrogen phosphate, protamine tartrate, or protamine perchlorate.In some embodiments, the protamine salt is protamine sulfate.

[0018] In some embodiments, the present specification describes a carrier that can enter polarized epithelial cells or transcytose across polarized epithelial cells, wherein at least 60% of the carrier is intact at 0.5 hours in a pancreatin assay, the pancreatin assay comprising incubating a composition comprising 100 μg of the carrier with 10 μg of pancreatin in 100 μL of phosphate-buffered saline (PBS) at 37° C.

[0019] In some embodiments, at least 90% of the carrier is intact at 2 hours in a pancreatin assay. In some embodiments, the composition further comprises a cation. In some embodiments, the cation is a metal cation or a polycation. In some embodiments, the cation is a metal cation. In some embodiments, the metal cation is a transition metal cation. In some embodiments, the carrier comprises a portion of Pseudomonas exotoxin A. In some embodiments, the carrier comprises a portion of a Corix polypeptide. In some embodiments, the Corix polypeptide comprises an amino acid sequence having a C-terminus at any one of amino acids 206-425 of SEQ ID NO:7. In some embodiments, the Corix polypeptide comprises an amino acid sequence having a C-terminus at any one of amino acids 150-205 of SEQ ID NO:7. In some embodiments, the Corix polypeptide comprises an amino acid sequence having a C-terminus at any one of amino acids 150-195 of SEQ ID NO:7. In some embodiments, the Corix polypeptide comprises an amino acid sequence having an N-terminus at any one of amino acids 1-41 of SEQ ID NO:7. In some embodiments, the Corix polypeptide consists of an amino acid sequence having an N-terminus at any one of amino acids 35-40 of SEQ ID NO:7.

[0020] In some embodiments, the Corix polypeptide consists of the amino acid sequence from amino acid 40 of the sequence set forth in SEQ ID NO:7 to any one of amino acids 150-205 of the sequence set forth in SEQ ID NO:7. In some embodiments, the Corix polypeptide has a C-terminus at any one of amino acids 150-187 of the sequence set forth in SEQ ID NO:7. In some embodiments, the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO:8. In some embodiments, the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:10. In some embodiments, the amino acid positions are numbered based on an alignment of the Corix polypeptide to the sequence set forth in SEQ ID NO:7, with the amino acid positions numbered from the N-terminus to the C-terminus, starting from position 1 at the N-terminus.

[0021] In some embodiments, the present disclosure provides a composition comprising a Corix variant ending at positions 195-347 and a heterologous payload, wherein the heterologous payload is a glucose-regulating agent. In some embodiments, the end position of the Corix variant is determined relative to SEQ ID NO: 7. In some embodiments, the carrier is capable of transcytosing the heterologous payload across polarized epithelial cells. In some embodiments, the glucose-regulating agent is a glucose-lowering agent. In some embodiments, the glucose-lowering agent is selected from the group consisting of incretin, glucagon proprotein, glucagon peptide, glucagon-like peptide 1, glucagon-like peptide 2, glicentin, glicentin-related polypeptide, gastric inhibitory polypeptide preprotein, gastric inhibitory polypeptide, dipeptidyl peptidase 4, glucose transporter member 4, preproglucagon, insulin receptor substrate 1, insulin, insulin analog, apolipoprotein A-II, solute carrier family 2, facilitative glucose transporter member 1, glycogen synthase 1, glycogen synthase 2, tyrosine-protein phosphatase non-receptor type 1, RAC-alpha serine threonine-protein kinase, peroxisome proliferator-activated receptor gamma, hexokinase 3, phosphatidylinositol-3,4,5-triphosphate 3-phosphatase, and diphosphopyridine. heavy specificity protein, pyruvate dehydrogenase kinase 1, calcium-binding and coiled-coil domain-containing protein 1, Max-like protein X, fructose-bisphosphate aldolase A, glucagon-like peptide 1 receptor, glucagon-like peptide 2 receptor, gastric inhibitory polypeptide receptor, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor, insulin receptor, GLP-1 agonist-exenatide, GLP-1 agonist-liraglutide, exenatide, exendin-4, exendin-3, GIPR agonist (Des-Ala2-GIP1-30), GIPR agonist truncated GIP1-30, GLP-1R agonist (amino acids 1-37 of GIP), GLP-1R agonist (amino acids 7-36 of GIP), lixisenatide (trade names Adlyxin® and Lyxumia®, Sanofi),Liraglutide (trade name Victoza®, Novo Nordisk A / S), semaglutide (trade name Ozempic®, Novo Nordisk A / S), albiglutide (trade name Tanzeum®, GlaxoSmithKline, GLP-1 dimer fused to albumin), dulaglutide (trade name Trulicity®, Eli Lilly), glucose-dependent insulinotropic polypeptide, a multispecific peptide agonist, tirzepatide (Eli Lilly), SAR425899 (Sanofi), amylin-calcitonin receptor dual agonist DACRA-089, glargine / Lantus®, glulisine / Apidra®, glulisine / Toujeo®, insuman®, detemir / Levemir®, lispro / Humalog® / Liprolog®, degludec / DegludecPlus, insulin aspart, insulin and analogs (e.g., LY-2605541, LY2963016 , NN1436), PEGylated insulin lispro, Humulin®, Linjeta, SuliXen®, NN1045, Insulin Plus Symlin™, PE0139, rapid- and short-acting insulins (e.g., Linjeta, PH20, NN1218, HinsBet), (APC-002) hydrogel, oral, inhaled, transdermal, and sublingual insulins (e.g., Exubera®, Nasulin®, Afrezza®, Tregopil®, TPM 02, Capsulin, Oral-lyn®, Cobalamin®, oral insulin, ORMD-0801, NN1953, NN1954, NN1956, VIAtab, and Oshadi oral insulin), or exendin-4 analogs (exendin-4 analogs include desPro36-exendin-4(1-39)-Lys6NH2, H-des(Pro36,37)-exendin-4-Lys4-NH2, H-des(Pro36,37)-exendin-4-Lys5-NH2, desPro36[Asp28]exendin-4(1-39),desPro36[IsoAsp28]exendin-4(1-39), desPro36[Met(O)14,Asp28]exendin-4(1-39), desPro36[Met(O)14,IsoAsp28]exendin-4(1-39), desPro36[Trp(O2)26,Asp28]exendin-4(1-39), or desPro36[Trp(O2)25,IsoAsp28]exendin-4(1-39), desPro36[Met(O)14 Trp(O2)25,Asp28]exendin-4(1-39), or desPro36[Met(O)14 Trp(O2)25,IsoAsp28]exendin-4(1-39).

[0022] In some embodiments, the heterologous payload comprises an incretin. In some embodiments, the heterologous payload comprises exenatide or insulin. In some embodiments, the composition comprises particles. In some embodiments, the heterologous payload is exendin-3, efpeglenatide, semaglutide, a GLP-1R agonist (amino acids 1-37 of GIP), a GLP-1R agonist (amino acids 7-36 of GIP), tirzepatide, oxyntomodulin, a GIPR agonist truncated GIP1-30, an amylin-calcitonin receptor dual agonist, preproinsulin, insulin aspart, insulin glargine, or insulin lispro.

[0023] In some embodiments, described herein are compositions comprising a carrier derived from a bacterial toxin capable of entering or transcytosing across polarized epithelial cells and a transition metal cation or polycation, wherein the polycation is a molecule or chemical complex having more than two positive charges. In some embodiments, the composition comprises a transition metal cation. In some embodiments, the transition metal cation is Fe 2+ , Mn 2+ , Zn 2+ , Co 2+ , Ni 2+ , and Cu2+ In some embodiments, the transition metal cation is selected from the group consisting of Zn 2+ In some embodiments, the composition comprises a polycation.

[0024] In some embodiments, the polycation is protamine, polylysine, polyornithine, polyethyleneimine (PEI), prolamine, protamine, polyvinylpyrrolidone (PVP), polyarginine, polyvinylamine, or a combination thereof. In some embodiments, the polycation is a protamine salt. In some embodiments, the protamine salt is protamine sulfate, protamine acetate, protamine bromide, protamine chloride, protamine caproate, protamine trifluoroacetate, protamine HCO3, protamine propionate, protamine lactate, protamine formate, protamine nitrate, protamine citrate, protamine monohydrogen phosphate, protamine dihydrogen phosphate, protamine tartrate, or protamine perchlorate. In some embodiments, the protamine salt is protamine sulfate. In some embodiments, the composition further comprises a heterologous payload. In some embodiments, the heterologous payload comprises exenatide, insulin, or human growth hormone.

[0025] In some embodiments, the carrier comprises Pseudomonas exotoxin A or a portion of Pseudomonas exotoxin A. In some embodiments, the carrier comprises the polypeptide of SEQ ID NO: 69 or a portion of SEQ ID NO: 69. In some embodiments, the carrier comprises a portion of a Corix polypeptide. In some embodiments, the Corix polypeptide comprises an amino acid sequence having a C-terminus at any one of amino acids 206-425 of SEQ ID NO: 1. In some embodiments, the Corix polypeptide comprises an amino acid sequence having a C-terminus at any one of amino acids 150-205 of SEQ ID NO: 1. In some embodiments, the Corix polypeptide comprises an amino acid sequence having a C-terminus at any one of amino acids 150-195 of SEQ ID NO: 1.

[0026] In some embodiments, the Corix polypeptide consists of an amino acid sequence having an N-terminus at any one of amino acid positions 1-41 of SEQ ID NO: 1. In some embodiments, the Corix polypeptide consists of an amino acid sequence having an N-terminus at any one of amino acid positions 35-40 of SEQ ID NO: 1. In some embodiments, the Corix polypeptide consists of an amino acid sequence from amino acid position 40 of the sequence set forth in SEQ ID NO: 7 to any one of amino acid positions 150-205 of the sequence set forth in SEQ ID NO: 7. In some embodiments, the Corix polypeptide has a C-terminus at any one of amino acid positions 150-187 of the sequence set forth in SEQ ID NO: 7. In some embodiments, the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 8. In some embodiments, the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10. In some embodiments, the amino acid positions are numbered based on an alignment of the Corix polypeptide to the sequence set forth in SEQ ID NO: 7, with the amino acid positions numbered N-terminus to C-terminus, starting from position 1 at the N-terminus. In some embodiments, the composition comprises particles.

[0027] In some embodiments, described herein are compositions comprising insulin, wherein at least 20% of the insulin is intact at 1 hour in a pancreatin assay comprising incubating the insulin-containing composition with pancreatin in PBS at 37° C. In some embodiments, described herein are compositions comprising insulin, wherein at least 20% of the insulin is intact at 1 hour in a simulated intestinal fluid assay comprising incubating insulin-containing particles at 142 μg / ml (insulin content) in simulated intestinal fluid USP (Rica Pharmaceuticals R7109000-500A, 4-fold dilution, pH 6.8) at 37° C. for 14 hours.

[0028] In some embodiments, the composition further comprises a carrier derived from a bacterial toxin, wherein the carrier is capable of transport into or transcytosis across a polarized epithelial cell. In some embodiments, the carrier comprises a portion of a Corix polypeptide. In some embodiments, the composition comprises a transition metal cation. In some embodiments, the transition metal cation is Fe 2+ , Mn 2+ , Zn 2+ , Co 2+ , Ni 2+ , and Cu 2+ In some embodiments, the transition metal cation is selected from the group consisting of Zn 2+ In some embodiments, the composition comprises a polycation. In some embodiments, the polycation is protamine. In some embodiments, the composition comprises particles. In some embodiments, the particles comprise microparticles. In some embodiments, the microparticles are formed by spray drying. In some embodiments, the particles have a diameter of about 50 nm to about 20 μm.

[0029] In some embodiments, described herein are pharmaceutical compositions comprising the compositions described herein. In some embodiments, described herein are pharmaceutical compositions comprising the compositions described herein and a preservative. In some embodiments, described herein are pharmaceutical compositions comprising the compositions described herein and a pharmaceutically acceptable excipient. In some embodiments, described herein are methods comprising administering to a subject a pharmaceutical composition described herein. In some embodiments, the subject has an inflammatory disease, an autoimmune disease, cancer, or a metabolic disorder. In some embodiments, the subject has a metabolic disorder.

[0030] In some embodiments, the metabolic disorder is selected from the group consisting of diabetes, diabetes as a result of obesity, hyperglycemia, dyslipidemia, hypertriglyceridemia, Syndrome X, insulin resistance, impaired glucose tolerance (IGT), diabetic dyslipidemia, hyperlipidemia, fatty liver disease, non-alcoholic steatohepatitis (NASH), hepatitis, obesity, vascular disease, heart disease, stroke, impaired glucose tolerance, raised fasting glucose, insulin resistance, urinary albumin secretion, central obesity, high blood pressure, elevated triglycerides, elevated LDL cholesterol and decreased HDL cholesterol, hyperglycemia, hyperinsulinemia, dyslipidemia, ketosis, hypertriglyceridemia, Syndrome X, insulin resistance, impaired fasting glucose glucose), impaired glucose tolerance (IGT), diabetic dyslipidemia, gluconeogenesis, excessive glycogenolysis, diabetic ketoacidosis, hypertriglyceridemia, hypertension, diabetic nephropathy, renal insufficiency, renal failure, hyperphagia, muscle wasting, diabetic neuropathy, diabetic retinopathy, diabetic coma, arteriosclerosis, coronary heart disease, peripheral arterial disease, or hyperlipidemia.

[0031] In some embodiments, methods are described herein that include combining a bacterially derived carrier with a heterologous payload and a cation to produce particles. In some embodiments, the heterologous payload is selected from the group consisting of a dye, a radiopharmaceutical, a hormone, a cytokine, an anti-TNF agent, a glucose-lowering agent, a tumor-associated antigen, a peptide, and a polypeptide. In some embodiments, the method further includes spray-drying the bacterially derived carrier, the heterologous payload, and the cation. In some embodiments, the method includes (a) preparing a mixture containing the isolated carrier and the payload; (b) preparing a mixture containing protamine sulfate and NaPO; and (c) combining the mixture of (a) and the mixture of (b) and allowing the combined mixture to stand overnight at room temperature. In some embodiments, the method further includes (d) increasing the ionic strength of the combined mixture obtained in step (c) to break down the particles obtained in step (c) into smaller particles. In some embodiments, the preparation in step (a) does not contain ZnCl. In some embodiments, the preparation in step (a) contains ZnCl.

[0032] One aspect of the present disclosure is a composition comprising a carrier capable of entering or transcytosing across polarized epithelial cells and a heterologous payload, wherein the molar ratio of heterologous payload to carrier is greater than 1:1.

[0033] The composition can be a particle. The particle can be a microparticle. The microparticle can be formed by spray drying. The particle or microparticle can have a diameter of about 50 nm to about 20 μm.

[0034] In some cases, the composition includes a transition metal cation. The transition metal cation is Fe 2+ , Mn 2+ , Zn 2+ , Co 2+ , Ni 2+ , and Cu 2+The transition metal cation may be selected from the group consisting of Zn 2+ It could be.

[0035] In some cases, the composition comprises a polycation. The polycation may be protamine.

[0036] The composition may include a carrier derived from a bacterial toxin. In some cases, the carrier may be capable of entering or transcytosing across polarized epithelial cells. In some cases, the carrier may be linked to a polycation.

[0037] The carrier may comprise a portion of Pseudomonas exotoxin A. In some cases, the carrier consists of a portion of Pseudomonas exotoxin A. The carrier may comprise a portion of a Corix polypeptide. In some cases, the carrier consists of a portion of a Corix polypeptide. The Corix polypeptide may consist of an amino acid sequence having a C-terminal truncation at any one of amino acid positions 206-425 of SEQ ID NO:7. The Corix polypeptide may consist of an amino acid sequence having a C-terminal truncation at any one of amino acid positions 150-205 of SEQ ID NO:7. The Corix polypeptide may consist of an amino acid sequence having a C-terminal truncation at any one of amino acid positions 150-195 of SEQ ID NO:7. The Corix polypeptide may consist of an amino acid sequence having an N-terminal truncation at any one of amino acid positions 1-41 of SEQ ID NO:7. The Corix polypeptide may consist of an amino acid sequence having an N-terminal truncation at any one of amino acid positions 35-40 of SEQ ID NO:7. The Corix polypeptide may consist of the amino acid sequence from amino acid position 40 of the sequence set forth in SEQ ID NO:7 to any one of amino acid positions 150 to 205 of the sequence set forth in SEQ ID NO:7. The Corix polypeptide may end at amino acid position 150 or amino acid position 187 of the sequence set forth in SEQ ID NO:7. The Corix polypeptide may consist of the amino acid sequence set forth in SEQ ID NO:8. The Corix polypeptide may consist of the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:10. Amino acids may be numbered based on an alignment of the Corix polypeptide to the sequence set forth in SEQ ID NO:7, and amino acid positions may be numbered from the N-terminus to the C-terminus, starting from position 1 at the N-terminus.

[0038] In some cases, the composition comprises a heterologous payload, which may be selected from the group consisting of a macromolecule, a small molecule, a peptide, a polypeptide, a nucleic acid, a messenger RNA (mRNA), a microRNA (miRNA), a small hairpin RNA (shRNA), a small interfering RNA (siRNA), a CRISPR RNA (e.g., a guide RNA, e.g., a single guide RNA (sgRNA)), an antisense molecule, a protein, an antibody, DNA, a plasmid, a vaccine, a polymeric nanoparticle, and a catalytically active material.

[0039] The heterologous payload may be a therapeutic payload. The heterologous payload may be selected from the group consisting of dyes and radiopharmaceuticals, hormones, cytokines, anti-TNF agents, glucose-lowering agents, tumor-associated antigens, peptides, and polypeptides. The heterologous payload may be a polypeptide that is a modulator of inflammation in the gastrointestinal tract. The therapeutic payload may be a glucagon-like peptide-2 (GLP-2) analog. The GLP-2 analog may be teduglutide.

[0040] The heterologous payload can be a cytokine. The cytokine can be selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, and IL-30. The cytokine can be IL-10. The cytokine can be IL-22. The cytokine can lack its natural secretory signal.

[0041] The heterologous payload can be a hormone. The hormone can be human growth hormone (hGH).

[0042] The heterologous payload can be a glucose-lowering agent, such as incretin, glucagon proprotein, glucagon peptide, glucagon-like peptide 1, glucagon-like peptide 2, glicentin, glicentin-related polypeptide, gastric inhibitory polypeptide preprotein, gastric inhibitory polypeptide, dipeptidyl peptidase 4, glucose transporter member 4, preproglucagon, insulin receptor substrate 1, insulin, insulin analog, apolipoprotein A-II, solute transporter family 2, facilitated glucose transporter member 1, glycogen synthase 1, glycogen synthase 2, tyrosine-protein phosphatase non-receptor type 1, RAC-alpha serine threonine-protein kinase, peroxisome proliferator-activated receptor gamma, hexokinase 3, phosphatidylinositol-3,4,5-triphosphate 3-phosphatase and dual specificity protein, pyruvate dehydrogenase Zeinase kinase 1, calcium-binding and coiled-coil domain-containing protein 1, Max-like protein X, fructose-bisphosphate aldolase A, glucagon-like peptide 1 receptor, glucagon-like peptide 2 receptor, gastric inhibitory polypeptide receptor, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor, insulin receptor, GLP-1 agonist-exenatide, GLP-1 agonist-liraglutide, exenatide , exendin-4, exendin-3, GIPR agonist (Des-Ala2-GIP1-30), GIPR agonist truncated GIP1-30, GLP-1R agonist (amino acids 1-37 of GIP), GLP-1R agonist (amino acids 7-36 of GIP), lixisenatide (trade names Adlyxin® and Lyxumia®, Sanofi), liraglutide (trade name Victoza®, Novo Nordisk A / S), semaglutide (trade name Ozempic®, Novo Nordisk A / S), albiglutide (trade name Tanzeum®, GlaxoSmithKline, GLP-1 dimer fused to albumin), dulaglutide (trade name Trulicity®, EliLilly), glucose-dependent insulinotropic polypeptide, multispecific peptide agonists, tirzepatide (Eli Lilly), SAR425899 (Sanofi), amylin-calcitonin receptor dual agonist DACRA-089, glargine / Lantus®, glulisine / Apidra®, glulisine / Toujeo®, Insuman®, detemir / Levemir®, lispro / Humalog® / Liprolog®, degludec / DegludecPlus, insulin aspart, insulin and analogs (e.g., LY-2605541, LY2963016 , NN1436), PEGylated insulin lispro, Humulin®, Linjeta, SuliXen®, NN1045, Insulin Plus Symlin™, PE0139, rapid- and short-acting insulins (e.g., Linjeta, PH20, NN1218, HinsBet), (APC-002) hydrogel, oral, inhaled, transdermal, and sublingual insulins (e.g., Exubera®, Nasulin®, Afrezza®, Tregopil®, TPM02, Capsulin, Oral-lyn®, Cobalamin®, oral insulin, ORMD-0801, NN1953, NN1954, NN1956, VIAtab, and Oshadi oral insulin), or exendin-4 analogs (exendin-4 analogs include desPro36-exendin-4(1-39)-Lys6NH2, H-des(Pro36,37)-exendin-4-Lys4-NH2, H-des(Pro36,37)-exendin-4-Lys5-NH2, desPro ro36[Asp28]exendin-4(1-39), desPro36[IsoAsp28]exendin-4(1-39), desPro36[Met(O)14,Asp28]exendin-4(1-39), desPro36[Met(O)14,IsoAsp28]exendin-4(1-39), desPro36[Trp(O2)26,Asp28]exendin-4(1-39), desPro36[Trp(O2)25,IsoAsp28]exendin-4(1-39), desPro36[Met(O)14 The heterologous species can be desPro36[Met(O)14 Trp(O)25,IsoAsp28]exendin-4(1-39), or desPro36[Met(O)14 Trp(O)25,IsoAsp28]exendin-4(1-39). The heterologous species can be insulin or an insulin analog. The heterologous species can be exenatide.

[0043] The heterologous payload can include a fluorescent label. The fluorescent label can be fluorescein. The heterologous payload can be an exenatide-fluorescein conjugate.

[0044] In some cases, the composition is resistant to cleavage by pancreatic enzymes. At least 50% of the carrier can remain intact after exposure to pancreatic enzymes for 2 hours. At least 50% of the carrier can remain intact after 2 hours in a pancreatin assay, which involves incubating a composition comprising 100 μg of the carrier with 10 μg of pancreatin in 100 μL of phosphate-buffered saline (PBS) at 37° C.

[0045] The molar ratio of transition metal cations to support can be from about 100:1 to about 300,000:1. The molar ratio of transition metal cations to support can be from about 1000:1 to about 30,000:1. The molar ratio of transition metal cations to support can be from about 1000:1 to about 10,000:1.

[0046] The molar ratio of protamine to carrier can be about 10:1 to about 0.01:1. The molar ratio of heterologous payload to carrier can be about 2:1 to about 6000:1. The molar ratio of protamine to carrier can be about 1:1.

[0047] The molar ratio of heterologous payload to carrier can be from about 2:1 to about 10:1. The molar ratio of heterologous payload to carrier can be about 7:1. The molar ratio of heterologous payload to carrier can be about 7.16:1.

[0048] The heterologous payload can be a polypeptide comprising the sequence of SEQ ID NO: 11 or SEQ ID NO: 14. The heterologous payload can be a polypeptide comprising the sequence of SEQ ID NO: 18 or SEQ ID NO: 19. The heterologous payload can be a polypeptide comprising the sequence of SEQ ID NO: 20. The heterologous payload can be a polypeptide comprising the sequence of SEQ ID NO: 21. The heterologous payload can be a polypeptide comprising the sequence of SEQ ID NO: 22.

[0049] The composition may be encapsulated. The encapsulated composition may be configured to release a heterologous payload under a first condition but not under a second condition. The encapsulated composition may be configured to release a heterologous payload at a high pH but not at a low pH. The encapsulated composition may include an enteric coating.

[0050] Another aspect of the present disclosure is a pharmaceutical composition comprising any of the compositions disclosed herein.

[0051] In some cases, the polycation is protamine, polylysine, polyornithine, polyethyleneimine (PEI), prolamine, protamine, polyvinylpyrrolidone (PVP), polyarginine, polyvinylamine, or a combination thereof. In some cases, the polycation is a protamine salt. In some cases, the protamine salt is protamine sulfate, protamine acetate, protamine bromide, protamine chloride, protamine caproate, protamine trifluoroacetate, protamine HCO3, protamine propionate, protamine lactate, protamine formate, protamine nitrate, protamine citrate, protamine monohydrogen phosphate, protamine dihydrogen phosphate, protamine tartrate, or protamine perchlorate. In some cases, the protamine salt is protamine sulfate.

[0052] An embodiment of the present disclosure is a composition comprising a carrier, wherein the carrier is capable of entering or transcytosing across polarized epithelial cells, and wherein at least 60% of the carrier is intact at 0.5 hours in a pancreatin assay, the pancreatin assay comprising incubating a composition comprising 100 μg of the carrier with 10 μg of pancreatin in 100 μL of phosphate-buffered saline (PBS) at 37° C. In some instances, at least 90% of the carrier is intact at 2 hours in the pancreatin assay.

[0053] In some cases, the composition further comprises a cation. In some cases, the cation is a metal cation or a polycation. In some cases, the cation is a metal cation. In some cases, the metal cation is a transition metal cation. In some cases, the carrier comprises a portion of Pseudomonas exotoxin A. In some cases, the carrier comprises a portion of a Corix polypeptide. In some cases, the Corix polypeptide comprises an amino acid sequence having a C-terminus at any one of amino acid positions 206-425 of SEQ ID NO:7. In some cases, the Corix polypeptide comprises an amino acid sequence having a C-terminus at any one of amino acid positions 150-205 of SEQ ID NO:7. In some cases, the Corix polypeptide comprises an amino acid sequence having a C-terminus at any one of amino acid positions 150-195 of SEQ ID NO:7. In some cases, the Corix polypeptide comprises an amino acid sequence having an N-terminus at any one of amino acid positions 1-41 of SEQ ID NO:7. In some cases, the Corix polypeptide comprises an amino acid sequence having an N-terminus at any one of amino acid positions 35-40 of SEQ ID NO:7. In some cases, the Corix polypeptide consists of the amino acid sequence from amino acid position 40 of the sequence set forth in SEQ ID NO:7 to any one of amino acids 150-205 of the sequence set forth in SEQ ID NO:7. In some cases, the Corix polypeptide has a C-terminus at any one of amino acids 150-187 of the sequence set forth in SEQ ID NO:7. In some cases, the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO:8. In some cases, the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:10. In some cases, the amino acid positions are numbered based on an alignment of the Corix polypeptide to the sequence set forth in SEQ ID NO:7, with the amino acid positions numbered from the N-terminus to the C-terminus, starting from position 1 at the N-terminus.

[0054] Another aspect of the present disclosure is a composition comprising a Corix variant ending at positions 195-347 and a heterologous payload, wherein the heterologous payload is a glucose regulator. In some cases, the end position of the Corix variant is determined relative to SEQ ID NO: 7. In some cases, the carrier is capable of transcytosing the heterologous payload across polarized epithelial cells. In some cases, the glucose regulator is a glucose-lowering agent. In some cases, the glucose-lowering agent is selected from the group consisting of incretin, glucagon proprotein, glucagon peptide, glucagon-like peptide 1, glucagon-like peptide 2, glicentin, glicentin-related polypeptide, gastric inhibitory polypeptide preprotein, gastric inhibitory polypeptide, dipeptidyl peptidase 4, glucose transporter member 4, preproglucagon, insulin receptor substrate 1, insulin, insulin analog, apolipoprotein A-II, solute transporter family 2, facilitated glucose transporter member 1, glycogen synthase 1, glycogen synthase 2, tyrosine-protein phosphatase non-receptor type 1, RAC-alpha serine threonine-protein kinase, peroxisome proliferator-activated receptor gamma, hexokinase 3, phosphatidylinositol-3,4,5-triphosphate 3-phosphatase and dual specificity protein, pirubicin, thiazolinone ... Phosphate dehydrogenase kinase 1, calcium-binding and coiled-coil domain-containing protein 1, Max-like protein X, fructose-bisphosphate aldolase A, glucagon-like peptide 1 receptor, glucagon-like peptide 2 receptor, gastric inhibitory polypeptide receptor, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor, insulin receptor, GLP-1 agonist - exenatide, GLP-1 agonist - liraglutide , exenatide, exendin-4, exendin-3, GIPR agonist (Des-Ala2-GIP1-30), GIPR agonist truncated GIP1-30, GLP-1R agonist (amino acids 1-37 of GIP), GLP-1R agonist (amino acids 7-36 of GIP), lixisenatide (trade names Adlyxin® and Lyxumia®, Sanofi), liraglutide (trade name Victoza®,Novo Nordisk A / S), semaglutide (trade name Ozempic®, Novo Nordisk A / S), albiglutide (trade name Tanzeum®, GlaxoSmithKline, GLP-1 dimer fused to albumin), dulaglutide (trade name Trulicity®, Eli Lilly), glucose-dependent insulinotropic polypeptide, a multispecific peptide agonist, tirzepatide (Eli Lilly), SAR425899 (Sanofi), amylin-calcitonin receptor dual agonist DACRA-089, glargine / Lantus®, glulisine / Apidra®, glulisine / Toujeo®, insuman®, detemir / Levemir®, lispro / Humalog® / Liprolog®, degludec / DegludecPlus, insulin aspart, insulin and analogs (e.g., LY-2605541, LY2963016 , NN1436), PEGylated insulin lispro, Humulin®, Linjeta, SuliXen®, NN1045, Insulin Plus Symlin™, PE0139, rapid- and short-acting insulins (e.g., Linjeta, PH20, NN1218, HinsBet), (APC-002) hydrogel, oral, inhaled, transdermal, and sublingual insulins (e.g., Exubera®, Nasulin®, Afrezza®, Tregopil®, TPM 02, Capsulin, Oral-lyn®, Cobalamin®, oral insulin, ORMD-0801, NN1953, NN1954, NN1956, VIAtab, and Oshadi oral insulin), or exendin-4 analogs (exendin-4 analogs include desPro36-exendin-4(1-39)-Lys6NH2, H-des(Pro36,37)-exendin-4-Lys4-NH2, H-des(Pro36,37)-exendin-4-Lys5-NH2, desPro36[Asp28]exendin-4(1-39), desPro36[IsoAsp28]exendin-4(1-39),desPro36[Met(O)14,Asp28]exendin-4(1-39), desPro36[Met(O)14,IsoAsp28]exendin-4(1-39), desPro36[Trp(O2)26,Asp28]exendin-4(1-39), or desPro36[Trp(O2)25,IsoAsp28]exendin-4(1-39), desPro36[Met(O)14 Trp(O2)25,Asp28]exendin-4(1-39), or desPro36[Met(O)14 Trp(O2)25,IsoAsp28]exendin-4(1-39). In some cases, the heterologous payload comprises an incretin. In some cases, the heterologous payload comprises exenatide or insulin. In some cases, the composition comprises particles. In some cases, the heterologous payload is exendin-3, efpeglenatide, semaglutide, GLP-1R agonist (amino acids 1-37 of GIP), GLP-1R agonist (amino acids 7-36 of GIP), tirzepatide, oxyntomodulin, GIPR agonist truncated GIP1-30, amylin calcitonin receptor dual agonist, preproinsulin, insulin aspart, insulin glargine, or insulin lispro.

[0055] An aspect of the present disclosure is a composition comprising a carrier derived from a bacterial toxin capable of entering or transcytosing across polarized epithelial cells and a transition metal cation or polycation, wherein the polycation is a molecule or chemical complex having more than two positive charges. In some cases, the composition comprises a transition metal cation. In some cases, the transition metal cation is Fe 2+ , Mn 2+ , Zn 2+ , Co 2+ , Ni 2+ , and Cu 2+ In some cases, the transition metal cation is selected from the group consisting of Zn 2+In some cases, the composition comprises a polycation. In some cases, the polycation is a protamine salt. In some cases, the composition further comprises a heterologous payload. In some cases, the heterologous payload comprises exenatide, insulin, or human growth hormone. In some cases, the carrier comprises Pseudomonas exotoxin A or a portion of Pseudomonas exotoxin A. In some cases, the carrier comprises a polypeptide of SEQ ID NO: 69 or a portion of SEQ ID NO: 69. In some cases, the carrier comprises a portion of a Corix polypeptide. In some cases, the Corix polypeptide comprises an amino acid sequence having a C-terminus at any one of amino acids 206-425 of SEQ ID NO: 1. In some cases, the Corix polypeptide comprises an amino acid sequence having a C-terminus at any one of amino acids 150-205 of SEQ ID NO: 1. In some cases, the Corix polypeptide comprises an amino acid sequence having a C-terminus at any one of amino acids 150-195 of SEQ ID NO: 1. In some cases, the Corix polypeptide consists of an amino acid sequence having an N-terminus at any one of amino acid positions 1-41 of SEQ ID NO: 1. In some cases, the Corix polypeptide consists of an amino acid sequence having an N-terminus at any one of amino acid positions 35-40 of SEQ ID NO: 1. In some cases, the Corix polypeptide consists of an amino acid sequence from amino acid position 40 of the sequence set forth in SEQ ID NO: 7 to any one of amino acid positions 150-205 of the sequence set forth in SEQ ID NO: 7. In some cases, the Corix polypeptide has a C-terminus at any one of amino acid positions 150-187 of the sequence set forth in SEQ ID NO: 7. In some cases, the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 8. In some cases, the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10. In some cases, the amino acid positions are numbered based on an alignment of the Corix polypeptide to the sequence set forth in SEQ ID NO: 7, with the amino acid positions numbered from N-terminus to C-terminus, starting from position 1 at the N-terminus. In some cases, the composition comprises particles.

[0056] Certain aspects of the present disclosure are compositions comprising insulin, wherein at least 20% of the insulin is intact at 1 hour in a pancreatin assay comprising incubating the insulin-containing composition with pancreatin at 37°C. In some cases, 10 μg of insulin is incubated with 10 μg of pancreatin at 37°C for 1 hour. In some cases, the composition further comprises a carrier derived from a bacterial toxin, wherein the carrier is capable of transport into or transcytosing across polarized epithelial cells. In some cases, the carrier comprises a portion of a Corix polypeptide. In some cases, the composition comprises a transition metal cation. In some cases, the transition metal cation is Fe 2+ , Mn 2+ , Zn 2+ , Co 2+ , Ni 2+ , and Cu 2+ In some cases, the transition metal cation is selected from the group consisting of Zn 2+ In some cases, the composition includes a polycation. In some cases, the polycation is protamine. In some cases, the composition includes particles. In some cases, the particles include microparticles. In some cases, the microparticles are formed by spray drying. In some cases, the particles have a diameter of about 50 nm to about 20 μm.

[0057] Another aspect of the present disclosure is a pharmaceutical composition comprising any of the compositions disclosed herein and a preservative.

[0058] Another aspect of the present disclosure is a pharmaceutical composition comprising any of the compositions disclosed herein and a pharmaceutically acceptable excipient.

[0059] In certain aspects, the present disclosure provides a method of treating a disease in a subject, the method comprising administering to the subject any of the pharmaceutical compositions disclosed herein.

[0060] The disease can be an inflammatory disease, an autoimmune disease, cancer, or a metabolic disorder.

[0061] The disease can be a metabolic disorder, including diabetes (T1D or T2D), diabetes as a result of obesity, hyperglycemia, dyslipidemia, hypertriglyceridemia, syndrome X, insulin resistance, impaired fasting glucose, impaired glucose tolerance (IGT), diabetic dyslipidemia, hyperlipidemia, fatty liver disease, nonalcoholic steatohepatitis, hepatitis, obesity, vascular disease, heart disease, stroke, impaired glucose tolerance, elevated fasting glucose, insulin resistance, urinary albumin secretion, central obesity, hypertension, elevated triglycerides, elevated LDL cholesterol, and elevated HDL cholesterol. The condition may be cholesterol lowering, hyperglycemia, hyperinsulinemia, dyslipidemia, ketosis, hypertriglyceridemia, syndrome X, insulin resistance, impaired fasting glucose, impaired glucose tolerance (IGT), diabetic dyslipidemia, gluconeogenesis, excessive glycogenolysis, diabetic ketoacidosis, hypertriglyceridemia, hypertension, diabetic nephropathy, renal insufficiency, renal failure, hyperphagia, muscle wasting, diabetic neuropathy, diabetic retinopathy, diabetic coma, arteriosclerosis, coronary heart disease, peripheral arterial disease, or hyperlipidemia.

[0062] The composition can be a particle, the composition comprises a heterologous payload, the composition comprises a polycation, and the carrier can transport the heterologous payload to or transcytose the heterologous payload across polarized epithelial cells.

[0063] The carrier may be linked to a polycation. The polycation may be protamine, polylysine, polyornithine, polyethyleneimine (PEI), prolamine, protamine, polyvinylpyrrolidone (PVP), polyarginine, polyvinylamine, or a combination thereof. The polycation may be a protamine salt. The protamine salt may be protamine sulfate, protamine acetate, protamine bromide, protamine chloride, protamine caproate, protamine trifluoroacetate, protamine bicarbonate (HCO), protamine propionate, protamine lactate, protamine formate, protamine nitrate, protamine citrate, protamine monohydrogen phosphate, protamine dihydrogen phosphate, protamine tartrate, or protamine perchlorate. The protamine salt may be protamine sulfate.

[0064] An aspect of the present disclosure is a pharmaceutical composition comprising any of the particles disclosed herein, wherein the heterologous payload is a glucose-lowering agent.

[0065] In some cases, the glucose-lowering agent is selected from the group consisting of insulin and insulin analogs. In some cases, the encapsulated particles release the payload at high pH but not at low pH.

[0066] Another aspect of the present disclosure is a pharmaceutical composition comprising any of the particles disclosed herein and a preservative. In some cases, the pharmaceutical composition further comprises a tonicity modifying agent.

[0067] In one aspect, the present disclosure provides a composition comprising a carrier derived from a bacterial toxin, wherein the carrier is capable of being transported into or transcytosed across polarized epithelial cells, and wherein at least 30% of the carriers are intact at 0.5 hours in a pancreatin assay comprising incubating a composition comprising 100 μg of the carrier with 10 μg of pancreatin in 100 μL of PBS at 37° C.

[0068] At least 40% of the carrier may be intact at 0.5 hours in a pancreatin assay comprising incubating a composition comprising 100 μg of carrier with 10 μg of pancreatin in 100 μL of PBS at 37° C. At least 50% of the carrier may be intact at 0.5 hours in a pancreatin assay comprising incubating a composition comprising 100 μg of carrier with 10 μg of pancreatin in 100 μL of PBS at 37° C. At least 60% of the carrier may be intact at 0.5 hours in a pancreatin assay comprising incubating a composition comprising 100 μg of carrier with 10 μg of pancreatin in 100 μL of PBS at 37° C. At least 90% of the carrier may be intact at 2 hours in a pancreatin assay comprising incubating a composition comprising 100 μg of carrier with 10 μg of pancreatin in 100 μL of PBS at 37° C.

[0069] The composition may further comprise a cation. The cation may be a metal cation or a polycation. The cation may be a metal cation. The metal cation may be a transition metal cation.

[0070] One aspect of the present disclosure is a composition comprising (a) a carrier derived from a bacterial toxin and (b) a transition metal cation or a polycation, wherein the carrier is capable of entering and / or transcytosing across polarized epithelial cells.

[0071] Certain aspects of the present disclosure include methods comprising combining a bacterially derived carrier with a heterologous payload and a cation to produce particles. In some cases, the heterologous payload is selected from the group consisting of a dye, a radiopharmaceutical, a hormone, a cytokine, an anti-TNF agent, a glucose-lowering agent, a tumor-associated antigen, a peptide, and a polypeptide. In some cases, the method further comprises spray-drying the bacterially derived carrier, the heterologous payload, and the cation. In some cases, the method comprises (a) preparing a mixture comprising the isolated carrier and the payload; (b) preparing a mixture comprising protamine sulfate and NaPO; and (c) combining the mixture of (a) and the mixture of (b) and allowing the combined mixture to stand overnight at room temperature. In some cases, the method further comprises (d) increasing the ionic strength of the combined mixture obtained in step (c) to break down the particles obtained in step (c) into smaller particles. In some cases, the preparation in step (a) does not include ZnCl. In some cases, the preparation in step (a) includes ZnCl.

[0072] In one aspect, provided herein is a delivery construct comprising a colix variant that does not comprise amino acids 1-348 of SEQ ID NO: 75, and is not SEQ ID NO: 76, complexed with a heterologous payload, wherein the heterologous payload is a glucose regulator, and the carrier is capable of a) transcytosing the heterologous payload across polarized epithelial cells, or b) transporting the heterologous payload into polarized epithelial cells. The payload can be an incretin or incretin mimetic. The payload can include a GLP-1 receptor agonist. The payload can include any one of SEQ ID NOs: 26-34 or 17. The payload can consist of SEQ ID NO: 15. The payload can consist of SEQ ID NO: 29. The payload can consist of SEQ ID NO: 16. The payload can consist of SEQ ID NO: 17. The payload can consist of SEQ ID NO: 33. The payload can consist of SEQ ID NO: 34. The payload can be a GIP receptor agonist. The payload may include any one of SEQ ID NOs: 33 or 36-38.

[0073] The payload can be insulin. The insulin can include SEQ ID NO: 43 and SEQ ID NO: 44, SEQ ID NO: 45 and SEQ ID NO: 46, SEQ ID NO: 47 and SEQ ID NO: 48, or SEQ ID NO: 49 and SEQ ID NO: 50. The insulin can include SEQ ID NO: 47 and SEQ ID NO: 48. The insulin can include SEQ ID NO: 49 and SEQ ID NO: 50. The payload can be any one of SEQ ID NOs: 51-63.

[0074] The colix variant may be the sequence set forth in SEQ ID NO: 81, or a fragment or sequence variant thereof. The carrier may be non-covalently attached to the heterologous payload. The carrier may be covalently attached to the heterologous payload.

[0075] The delivery construct may be a single polypeptide. The delivery construct may further comprise a cleavable linker, in which case cleavage of the linker releases the payload from the carrier. The carrier may be located at the C-terminus of the polypeptide, and the payload may be at the N-terminus of the polypeptide. The carrier may comprise SEQ ID NO:6, SEQ ID NO:2, SEQ ID NO:65, or SEQ ID NO:73. The carrier may have at least 90% amino acid identity to a C-terminal truncated variant of SEQ ID NO:1. The carrier may comprise a C-terminal truncated variant of SEQ ID NO:81. The carrier may consist of the first 195, 206, 244, 250, 266, 386, or 415 amino acid residues of SEQ ID NO:1, SEQ ID NO:12, or SEQ ID NO:81. The heterologous payload may be configured to bind to a receptor.

[0076] Also provided herein is a method of treating a metabolic disorder in a subject, comprising administering to the subject an effective amount of any one of the delivery constructs described above. The metabolic disorder can be diabetes and / or obesity.

[0077] Any aspect or case disclosed herein can be combined with any other aspect or case disclosed herein. Incorporation by Reference

[0078] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0079] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the agency upon request and payment of the necessary fee. The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized, and the accompanying drawings, in which: The present invention provides, for example, the following items. (Item 1) a carrier capable of entering or transcytosing across polarized epithelial cells; a heterologous payload, wherein the molar ratio of said heterologous payload to said carrier is greater than 1:1; A composition comprising: (Item 2) Item 1. The composition of claim 1, comprising a transition metal cation. (Item 3) The transition metal cation is Fe 2+ , Mn 2+ , Zn 2+ , Co 2+ , Ni 2+ , and Cu 2+ 3. The composition according to item 2, selected from the group consisting of: (Item 4) The transition metal cation is Zn 2+ Item 4. The composition according to item 3, wherein (Item 5) 2. The composition according to item 1, comprising a polycation. (Item 6) 6. The composition of claim 5, wherein the polycation is protamine. (Item 7) 7. The composition according to any one of items 1 to 6, wherein the carrier comprises a portion of a Corix polypeptide. (Item 8) 8. The composition of claim 7, wherein the carrier comprises a portion of Pseudomonas exotoxin A. (Item 9) 8. The composition of claim 7, wherein the carrier comprises a portion of a Corix polypeptide. (Item 10) Item 9. The composition according to Item 8, wherein the Corix polypeptide consists of an amino acid sequence having a C-terminus at any one of amino acid positions 206 to 425 of SEQ ID NO: 7. (Item 11) Item 9. The composition according to item 8, wherein the Corix polypeptide consists of an amino acid sequence having a C-terminus at any one of amino acid positions 150 to 205 of SEQ ID NO: 7. (Item 12) Item 9. The composition according to item 8, wherein the Corix polypeptide consists of an amino acid sequence having a C-terminus at any one of amino acids 150 to 195 of SEQ ID NO: 7. (Item 13) Item 9. The composition according to Item 8, wherein the Corix polypeptide consists of an amino acid sequence having an N-terminus at any one of amino acids 1 to 41 of SEQ ID NO: 7. (Item 14) 9. The composition according to item 8, wherein the Corix polypeptide consists of an amino acid sequence having an N-terminus at any one of amino acid positions 35 to 40 of SEQ ID NO: 7. (Item 15) Item 9. The composition according to Item 8, wherein the Corix polypeptide consists of an amino acid sequence from amino acid position 40 of the sequence set forth in SEQ ID NO: 7 to any one of amino acid positions 150 to 205 of the sequence set forth in SEQ ID NO: 7. (Item 16) 9. The composition according to item 8, wherein the Corix polypeptide has a C-terminus at any one of amino acid positions 150 to 187 of the sequence set forth in SEQ ID NO:7. (Item 17) 9. The composition of claim 8, wherein the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO:8. (Item 18) 9. The composition of claim 8, wherein the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10. (Item 19) 15. The composition of any one of items 10 to 14, wherein the amino acid positions are numbered based on an alignment of the Corix polypeptide to the sequence set forth in SEQ ID NO: 7, and the amino acid positions are numbered from the N-terminus to the C-terminus, starting from position 1 at the N-terminus. (Item 20) the heterologous payload is selected from the group consisting of a macromolecule, a small molecule, a peptide, a polypeptide, a nucleic acid, an mRNA, an miRNA, an shRNA, an siRNA, an antisense molecule, an antibody, a DNA, a plasmid, a vaccine, a polymeric nanoparticle, and a catalytically active material; Item 1. The composition according to item 1. (Item 21) 21. The composition of claim 20, wherein the heterologous payload is a therapeutic payload. (Item 22) 2. The composition of claim 1, wherein the heterologous payload is selected from the group consisting of dyes and radiopharmaceuticals, hormones, cytokines, anti-TNF agents, glucose-lowering agents, tumor-associated antigens, peptides, and polypeptides. (Item 23) 2. The composition of claim 1, wherein the heterologous payload is a polypeptide that is a modulator of inflammation in the gastrointestinal tract. (Item 24) 2. The composition of claim 1, wherein the heterologous payload is a glucagon-like peptide-2 (GLP-2) analog. (Item 25) 25. The composition of claim 24, wherein the GLP-2 analog is teduglutide. (Item 26) 2. The composition of claim 1, wherein the heterologous payload is a cytokine. (Item 27) The cytokine is IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, I The composition according to item 26, selected from the group consisting of L-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, and IL-30. (Item 28) 28. The composition of item 27, wherein the cytokine is IL-10. (Item 29) 29. The composition of item 28, wherein the cytokine is IL-22. (Item 30) 27. The composition of claim 26, wherein the cytokine lacks a natural secretory signal. (Item 31) 22. The composition of claim 21, wherein the therapeutic payload is a hormone. (Item 32) 32. The composition of claim 31, wherein the hormone is human growth hormone (hGH). (Item 33) 2. The composition of claim 1, wherein the heterologous payload is a glucose-lowering agent. (Item 34) the heterologous payload is selected from the group consisting of incretin, glucagon proprotein, glucagon peptide, glucagon-like peptide 1, glucagon-like peptide 2, glicentin, glicentin-related polypeptide, gastric inhibitory polypeptide preprotein, gastric inhibitory polypeptide, dipeptidyl peptidase 4, glucose transporter member 4, preproglucagon, insulin receptor substrate 1, insulin, insulin analog, apolipoprotein A-II, solute transporter family 2, facilitative glucose transporter member 1, glycogen synthase 1, glycogen synthase 2, Tyrosine-protein phosphatase non-receptor type 1, RAC-alpha serine threonine-protein kinase, peroxisome proliferator-activated receptor gamma, hexokinase 3, phosphatidylinositol-3,4,5-triphosphate 3-phosphatase and dual specificity protein, pyruvate dehydrogenase kinase 1, calcium-binding and coiled-coil domain-containing protein 1, Max-like protein X, fructose-bisphosphate aldolase A, glucagon-like peptide 1 receptor, glucagon-like peptide 2 receptor, gastric inhibitory polypeptide receptor, insulin -like growth factor 1 receptor, insulin-like growth factor 2 receptor, insulin receptor, GLP-1 agonist-exenatide, GLP-1 agonist-liraglutide, exenatide, exendin-4, exendin-3, GIPR agonist (Des-Ala2-GIP1-30), GIPR agonist truncated GIP1-30, GLP-1R agonist (amino acids 1-37 of GIP), GLP-1R agonist (amino acids 7-36 of GIP), lixisenatide (trade names Adlyxin® and Lyxumia®, Sanofi), liraglutide (trade name Victoza®, Novo Nordisk A / S), semaglutide (trade name Ozempic®, Novo Nordisk A / S), albiglutide (trade name Tanzeum®, GlaxoSmithKline, GLP-1 dimer fused to albumin), dulaglutide (trade name Trulicity®, Eli Lilly), glucose-dependent insulinotropic polypeptide, a multispecific peptide agonist, tirzepatide (Eli Lilly), SAR425899 (Sanofi), amylin-calcitonin receptor dual agonist DACRA-089, glargine / Lantus®, glulisine / Apidra®, glulisine / Toujeo®, insuman®, detemir / Levemir®, lispro / Humalog® / Liprolog®, degludec / DegludecPlus, insulin aspart, insulin and analogs (e.g., LY-2605541, LY2963016 , NN1436), PEGylated insulin lispro, Humulin®, Linjeta, SuliXen®, NN1045, Insulin Plus Symlin™, PE0139, rapid- and short-acting insulins (e.g., Linjeta, PH20, NN1218, HinsBet), (APC-002) hydrogel, oral, inhaled, transdermal, and sublingual insulins (e.g., Exubera®, Nasulin®, Afrezza®, Tregopil®, TPM 02, Capsulin, Oral-lyn®, Cobalamin®, oral insulin, ORMD-0801, NN1953, NN1954, NN1956, VIAtab, and Oshadi oral insulin), or exendin-4 analogs (exendin-4 analogs include desPro36-exendin-4(1-39)-Lys6NH2, H-des(Pro36,37)-exendin-4-Lys4-NH2, H-des(Pro36,37)-Exendin-4-Lys5-NH2, desPro36[Asp28]Exendin-4(1-39), desPro36[IsoAsp28]Exendin-4(1-39), desPro36[Met(O)14,Asp28]Exendin-4(1-39), desPro36[Met(O)14,IsoAsp28]Exendin-4(1-39), desPro36[Trp(O2)26,Asp28]Exendin-4(1-39), or desPro36[Trp(O2)25,IsoAsp28]Exendin-4(1-39), desPro36[Met(O)14 Trp(O2)25,Asp28]Exendin-4(1-39), or desPro36[Met(O)14 The composition according to item 1, wherein the exendin-4 (1-39) is a hydroxybenzoate (Trp(O2)25,IsoAsp28)). (Item 35) 2. The composition of claim 1, wherein the heterologous payload is insulin or an insulin analog. (Item 36) 2. The composition of claim 1, wherein the heterologous payload is exenatide. (Item 37) 2. The composition of claim 1, wherein the heterologous payload comprises a fluorescent label. (Item 38) 38. The composition of claim 37, wherein the fluorescent label is fluorescein. (Item 39) Item 38, wherein the heterologous payload is an exenatide-fluorescein conjugate. Composition of. (Item 40) 10. The composition of claim 1, which is resistant to cleavage by pancreatic enzymes. (Item 41) 41. The composition of claim 40, wherein at least 50% of the carrier is intact at 2 hours in a pancreatin assay, the pancreatin assay comprising incubating the composition comprising 100 μg of the carrier with 10 μg of pancreatin in 100 μL of phosphate buffered saline (PBS) at 37° C. (Item 42) 3. The composition according to item 2, wherein the molar ratio of the transition metal cation to the support is from about 100:1 to about 300,000:1. (Item 43) 3. The composition according to item 2, wherein the molar ratio of the transition metal cation to the support is from about 1000:1 to about 30,000:1. (Item 44) 3. The composition according to item 2, wherein the molar ratio of the transition metal cation to the support is from about 1000:1 to about 10,000:1. (Item 45) 7. The composition according to item 6, wherein the molar ratio of the protamine to the carrier is about 10:1 to about 0.01:1. (Item 46) 2. The composition of claim 1, wherein the molar ratio of the heterologous payload to the carrier is from about 2:1 to about 6000:1. (Item 47) 7. The composition of claim 6, wherein the molar ratio of the protamine to the carrier is about 1:1. (Item 48) 2. The composition of claim 1, wherein the molar ratio of the heterologous payload to the carrier is from about 2:1 to about 10:1. (Item 49) 2. The composition of claim 1, wherein the molar ratio of the heterologous payload to the carrier is about 7:1. (Item 50) 2. The composition of claim 1, wherein the molar ratio of the heterologous payload to the carrier is about 7.16:1. (Item 51) 2. The composition of claim 1, wherein the heterologous payload is a polypeptide comprising the sequence of SEQ ID NO: 11 or SEQ ID NO: 14. (Item 52) 2. The composition of claim 1, wherein the heterologous payload is a polypeptide comprising the sequence of SEQ ID NO: 18 or SEQ ID NO: 19. (Item 53) Item 1, wherein the heterologous payload is a polypeptide comprising the sequence of SEQ ID NO: 20. (Item 54) Item 1, wherein the heterologous payload is a polypeptide comprising the sequence of SEQ ID NO: 21. (Item 55) Item 1, wherein the heterologous payload is a polypeptide comprising the sequence of SEQ ID NO: 22. (Item 56) Item 1. The composition of item 1, which is encapsulated. (Item 57) 57. The composition of claim 56, wherein the encapsulated composition is configured to release the heterologous payload under a first condition but not under a second condition. (Item 58) 57. The composition of claim 56, wherein the encapsulated composition is configured to release the heterologous payload at high pH but not at low pH. (Item 59) 57. The composition of claim 56, wherein the encapsulated composition comprises an enteric coating. (Item 60) Item 1, wherein the composition is a particle. (Item 61) 2. The composition according to item 1, comprising a polycation. (Item 62) 2. The composition of claim 1, wherein the carrier is capable of transporting the heterologous payload into the polarized epithelial cells or transcytosing the heterologous payload across the polarized epithelial cells. (Item 63) 62. The composition of claim 61, wherein the carrier is linked to the polycation. (Item 64) 62. The composition of claim 61, wherein the polycation is protamine, poly-lysine, poly-ornithine, poly-ethylene-imine (PEI), prolamine, protamine, polyvinylpyrrolidone (PVP), polyarginine, polyvinylamine, or a combination thereof. (Item 65) 62. The composition of claim 61, wherein the polycation is a protamine salt. (Item 66) The protamine salt may be protamine sulfate, protamine acetate, protamine bromide, protamine chloride, protamine caproate, protamine trifluoroacetate, or protamine HCO 3 66. The composition of item 65, wherein the protamine monophosphate is protamine propionate, protamine lactate, protamine formate, protamine nitrate, protamine citrate, protamine monohydrogen phosphate, protamine dihydrogen phosphate, protamine tartrate, or protamine perchlorate. (Item 67) 67. The composition of claim 66, wherein the protamine salt is protamine sulfate. (Item 68) A composition comprising a carrier, wherein the carrier is capable of entering or transcytosing across polarized epithelial cells, and wherein at least 60% of the carrier is intact at 0.5 hours in a pancreatin assay, the pancreatin assay comprising incubating the composition comprising 100 μg of the carrier with 10 μg of pancreatin in 100 μL of phosphate-buffered saline (PBS) at 37°C. (Item 69) 69. The composition of claim 68, wherein at least 90% of the carrier is intact in the pancreatin assay at 2 hours. (Item 70) 69. The composition of claim 68, further comprising a cation. (Item 71) 71. The composition of claim 70, wherein the cation is a metal cation or a polycation. (Item 72) 72. The composition of claim 71, wherein the cation is a metal cation. (Item 73) 73. The composition of claim 72, wherein the metal cation is a transition metal cation. (Item 74) 69. The composition of claim 68, wherein the carrier comprises a portion of Pseudomonas exotoxin A. (Item 75) 69. The composition of claim 68, wherein the carrier comprises a portion of a Corix polypeptide. (Item 76) 75. The composition according to item 74, wherein the Corix polypeptide consists of an amino acid sequence having a C-terminus at any one of amino acid positions 206 to 425 of SEQ ID NO: 7. (Item 77) 75. The composition according to item 74, wherein the Corix polypeptide consists of an amino acid sequence having a C-terminus at any one of amino acid positions 150 to 205 of SEQ ID NO: 7. (Item 78) 75. The composition according to item 74, wherein the Corix polypeptide consists of an amino acid sequence having a C-terminus at any one of amino acids 150 to 195 of SEQ ID NO: 7. (Item 79) 75. The composition according to item 74, wherein the Corix polypeptide consists of an amino acid sequence having an N-terminus at any one of amino acids 1 to 41 of SEQ ID NO: 7. (Item 80) 75. The composition according to item 74, wherein the Corix polypeptide consists of an amino acid sequence having an N-terminus at any one of amino acids 35 to 40 of SEQ ID NO: 7. (Item 81) Item 75. The composition according to Item 74, wherein the Corix polypeptide consists of the amino acid sequence from amino acid position 40 of the sequence set forth in SEQ ID NO: 7 to any one of amino acids 150 to 205 of the sequence set forth in SEQ ID NO: 7. (Item 82) 75. The composition of claim 74, wherein the Corix polypeptide has a C-terminus at any one of amino acid positions 150 to 187 of the sequence set forth in SEQ ID NO:7. (Item 83) 75. The composition of claim 74, wherein the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO:8. (Item 84) 75. The composition of claim 74, wherein the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10. (Item 85) 85. The composition of any one of items 76 to 84, wherein the amino acid positions are numbered based on an alignment of the Corix polypeptide to the sequence set forth in SEQ ID NO: 7, and the amino acid positions are numbered from the N-terminus to the C-terminus, starting from position 1 at the N-terminus. (Item 86) A composition comprising a colix variant ending at positions 195 to 347 and a heterologous payload, wherein the heterologous payload is a glucose regulator. (Item 87) 87. The composition of claim 86, wherein the end position of the Corix variant is determined relative to SEQ ID NO: 7. (Item 88) 87. The composition of item 86, wherein the carrier is capable of transcytosing the heterologous payload across polarized epithelial cells. (Item 89) 87. The composition of claim 86, wherein the glucose regulating agent is a glucose lowering agent. (Item 90) The glucose-lowering agent is selected from the group consisting of incretin, glucagon proprotein, glucagon peptide, glucagon-like peptide 1, glucagon-like peptide 2, glicentin, glicentin-related polypeptide, gastric inhibitory polypeptide preprotein, gastric inhibitory polypeptide, dipeptidyl peptidase 4, glucose transporter member 4, preproglucagon, insulin receptor substrate 1, insulin, insulin analogs, apolipoprotein A-II, solute transporter family 2, facilitated glucose transporter member 1, glycogen synthase 1, glycogen synthase 2, tyrosine-protein phosphatase non-receptor type 1, RAC-alpha serine threonine-protein kinase, peroxisome proliferator-activated receptor gamma, hexokinase 3, phosphatidylinositol-3,4,5-triphosphate 3-phosphatase and dual specificity protein, pyruvate dehydrogenase Aldolase kinase 1, calcium-binding and coiled-coil domain-containing protein 1, Max-like protein X, fructose-bisphosphate aldolase A, glucagon-like peptide 1 receptor, glucagon-like peptide 2 receptor, gastric inhibitory polypeptide receptor, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor, insulin receptor, GLP-1 agonist-exenatide, GLP-1 agonist-liraglutide, exenatide tide, exendin-4, exendin-3, GIPR agonist (Des-Ala2-GIP1-30), GIPR agonist truncated GIP1-30, GLP-1R agonist (amino acids 1-37 of GIP), GLP-1R agonist (amino acids 7-36 of GIP), lixisenatide (trade names Adlyxin® and Lyxumia®, Sanofi), liraglutide (trade names Victoza®, Novo Nordisk A / S), semaglutide (trade name Ozempic®, Novo Nordisk A / S), albiglutide (trade name Tanzeum®, GlaxoSmithKline, GLP-1 dimer fused to albumin), dulaglutide (trade name Trulicity®, Eli Lilly), glucose-dependent insulinotropic polypeptide, a multispecific peptide agonist, tirzepatide (EliLilly), SAR425899 (Sanofi), amylin-calcitonin receptor dual agonist DACRA-089, glargine / Lantus®, glulisine / Apidra®, glulisine / Toujeo®, insuman®, detemir / Levemir®, lispro / Humalog® / Liprolog®, degludec / DegludecPlus, insulin aspart, insulin and analogs (e.g., LY-2605541, LY2963016 , NN1436), PEGylated insulin lispro, Humulin®, Linjeta, SuliXen®, NN1045, Insulin Plus Symlin™, PE0139, rapid- and short-acting insulins (e.g., Linjeta, PH20, NN1218, HinsBet), (APC-002) hydrogel, oral, inhaled, transdermal, and sublingual insulins (e.g., Exubera®, Nasulin®, Afrezza®, Tregopil®, TPM 02, Capsulin, Oral-lyn®, Cobalamin®, oral insulin, ORMD-0801, NN1953, NN1954, NN1956, VIAtab, and Oshadi oral insulin), or exendin-4 analogs (exendin-4 analogs include desPro36-exendin-4(1-39)-Lys6NH2, H-des(Pro36,37)-exendin-4-Lys4-NH2, H-des(Pro36,37)-exendin-4-Lys5-NH2, desPro36[Asp28]exendin-4(1-39), desPro36[IsoAsp28]exendin-4(1-39), desPro36[Met Item 91. The composition of Item 89, wherein the exendin-4 is desPro36[Met(O)14,IsoAsp28]exendin-4(1-39), desPro36[Trp(O2)26,Asp28]exendin-4(1-39), desPro36[Trp(O2)25,IsoAsp28]exendin-4(1-39), desPro36[Met(O)14 Trp(O2)25,Asp28]exendin-4(1-39), or desPro36[Met(O)14 Trp(O2)25,IsoAsp28]exendin-4(1-39). 87. The composition of claim 86, wherein the heterologous payload comprises an incretin. (Item 92) 87. The composition of claim 86, wherein the heterologous payload comprises exenatide or insulin. (Item 93) 87. The composition according to item 86, comprising particles. (Item 94) 87. The composition of item 86, wherein the heterologous payload is exendin-3, efpeglenatide, semaglutide, GLP-1R agonist (amino acids 1-37 of GIP), GLP-1R agonist (amino acids 7-36 of GIP), tirzepatide, oxyntomodulin, GIPR agonist truncated GIP1-30, amylin calcitonin receptor dual agonist, preproinsulin, insulin aspart, insulin glargine, or insulin lispro. (Item 95) a carrier derived from a bacterial toxin that is capable of entering or transcytosing across polarized epithelial cells; a transition metal cation or polycation, wherein the polycation is a molecule or chemical complex having more than two positive charges; A composition comprising: (Item 96) 96. The composition of claim 95, comprising a transition metal cation. (Item 97) The transition metal cation is Fe 2+ , Mn 2+ , Zn 2+ , Co 2+ , Ni 2+ , and Cu 2+ 97. The composition of claim 96, selected from the group consisting of: (Item 98) The transition metal cation is Zn 2+ Item 96. The composition according to item 95, wherein (Item 99) 96. The composition according to item 95, comprising said polycation. (Item 100) 99. The composition of claim 99, wherein the polycation is protamine, poly-lysine, poly-ornithine, poly-ethylene-imine (PEI), prolamine, protamine, polyvinylpyrrolidone (PVP), polyarginine, polyvinylamine, or a combination thereof. (Item 101) 99. The composition of claim 98, wherein the polycation is a protamine salt. (Item 102) The protamine salt may be protamine sulfate, protamine acetate, protamine bromide, protamine chloride, protamine caproate, protamine trifluoroacetate, or protamine HCO 3 , protamine propionate, protamine lactate, protamine formate, protamine nitrate, protamine citrate, protamine monohydrogen phosphate, protamine dihydrogen phosphate, protamine tartrate, or protamine perchlorate. (Item 103) 99. The composition of claim 98, wherein the protamine salt is protamine sulfate. (Item 104) 96. The composition of item 95, further comprising a heterologous payload. (Item 105) 104. The composition of claim 103, wherein the heterologous payload comprises exenatide, insulin, or human growth hormone. (Item 106) 96. The composition of claim 95, wherein the carrier consists of Pseudomonas exotoxin A or a portion of Pseudomonas exotoxin A. (Item 107) 105. The composition of claim 104, wherein the carrier consists of a polypeptide of SEQ ID NO: 69 or a portion of SEQ ID NO: 69. (Item 108) 96. The composition of claim 95, wherein the carrier comprises a portion of a Corix polypeptide. (Item 109) Item 96. The composition according to Item 95, wherein the Corix polypeptide consists of an amino acid sequence having a C-terminus at any one of amino acid positions 206 to 425 of SEQ ID NO: 1. (Item 110) Item 96. The composition according to Item 95, wherein the Corix polypeptide consists of an amino acid sequence having a C-terminus at any one of amino acid positions 150 to 205 of SEQ ID NO: 1. (Item 111) Item 96. The composition according to Item 95, wherein the Corix polypeptide consists of an amino acid sequence having a C-terminus at any one of amino acids 150 to 195 of SEQ ID NO: 1. (Item 112) Item 96. The composition according to Item 95, wherein the Corix polypeptide consists of an amino acid sequence having an N-terminus at any one of amino acids 1 to 41 of SEQ ID NO: 1. (Item 113) Item 96. The composition according to Item 95, wherein the Corix polypeptide consists of an amino acid sequence having an N-terminus at any one of amino acids 35 to 40 of SEQ ID NO: 1. (Item 114) Item 96. The composition according to Item 95, wherein the Corix polypeptide consists of the amino acid sequence from amino acid position 40 of the sequence set forth in SEQ ID NO: 7 to any one of amino acids 150 to 205 of the sequence set forth in SEQ ID NO: 7. (Item 115) 96. The composition of item 95, wherein the Corix polypeptide has a C-terminus at any one of amino acid positions 150 to 187 of the sequence set forth in SEQ ID NO:7. (Item 116) Item 96. The composition of item 95, wherein the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO:8. (Item 117) Item 96. The composition of item 95, wherein the Corix polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10. (Item 118) 116. The composition of any one of items 107 to 115, wherein the amino acid positions are numbered based on an alignment of the Corix polypeptide to the sequence set forth in SEQ ID NO: 7, and the amino acid positions are numbered from the N-terminus to the C-terminus, starting from position 1 at the N-terminus. (Item 119) 96. The composition of claim 95, comprising particles. (Item 120) A composition comprising insulin, wherein at least 20% of the insulin is intact at 1 hour in a pancreatin assay comprising incubating the composition comprising insulin with pancreatin in PBS at 37°C. (Item 121) 119. The composition of claim 118, further comprising a carrier derived from a bacterial toxin, said carrier being capable of transport into or transcytosis across polarized epithelial cells. (Item 122) 120. The composition of claim 119, wherein the carrier comprises a portion of a Corix polypeptide. (Item 123) Item 119. The composition of claim 118, comprising a transition metal cation. (Item 124) The transition metal cation is Fe 2+ , Mn 2+ , Zn 2+ , Co 2+ , Ni 2+ , and Cu 2+ Item 122. The composition according to item 121, selected from the group consisting of: (Item 125) The transition metal cation is Zn 2+ Item 122. The composition according to item 121, wherein (Item 126) 119. The composition of claim 118, comprising a polycation. (Item 127) 126. The composition of claim 125, wherein the polycation is protamine. (Item 128) 126. The composition according to any one of items 1 to 125, comprising particles. (Item 129) Item 127. The composition of item 126, wherein the particles comprise microparticles. (Item 130) Item 128. The composition of item 127, wherein the microparticles are formed by spray drying. (Item 131) Item 127. The composition according to item 126, wherein the particles have a diameter of about 50 nm to about 20 μm. (Item 132) A pharmaceutical composition comprising the composition according to any one of items 1 to 129. (Item 133) A pharmaceutical composition comprising the composition according to any one of items 1 to 129 and a preservative. (Item 134) A pharmaceutical composition comprising the composition according to any one of items 1 to 130 and a pharmaceutically acceptable excipient. (Item 135) A method comprising the step of administering to a subject the pharmaceutical composition according to any one of items 130 to 132. (Item 136) 134. The method of claim 133, wherein the subject has an inflammatory disease, an autoimmune disease, cancer, or a metabolic disorder. (Item 137) Item 135. The method of item 134, wherein the subject has a metabolic disorder. (Item 138) The metabolic disorders include diabetes, diabetes as a result of obesity, hyperglycemia, dyslipidemia, hypertriglyceridemia, syndrome X, insulin resistance, impaired glucose tolerance (IGT), diabetic dyslipidemia, hyperlipidemia, fatty liver disease, non-alcoholic steatohepatitis (NASH), hepatitis, obesity, vascular disease, heart disease, stroke, impaired glucose tolerance, elevated fasting blood glucose, insulin resistance, urinary albumin secretion, central obesity, hypertension, elevated triglycerides, elevated LDL cholesterol, and the like. Item 136. The method according to Item 135, wherein the cause is elevated cholesterol and decreased HDL cholesterol, hyperglycemia, hyperinsulinemia, dyslipidemia, ketosis, hypertriglyceridemia, syndrome X, insulin resistance, impaired fasting glucose, impaired glucose tolerance (IGT), diabetic dyslipidemia, gluconeogenesis, excessive glycogenolysis, diabetic ketoacidosis, hypertriglyceridemia, hypertension, diabetic nephropathy, renal insufficiency, renal failure, hyperphagia, muscle wasting, diabetic neuropathy, diabetic retinopathy, diabetic coma, arteriosclerosis, coronary heart disease, peripheral arterial disease, or hyperlipidemia. (Item 139) A method comprising combining a bacterially derived carrier with a heterologous payload and a cation to produce a particle. (Item 140) 138. The method of claim 137, wherein the heterologous payload is selected from the group consisting of dyes, radiopharmaceuticals, hormones, cytokines, anti-TNF agents, glucose-lowering agents, tumor-associated antigens, peptides, and polypeptides. (Item 141) Item 138. The method of item 137, further comprising spray drying the bacterial carrier, the heterologous payload, and the cation. (Item 142) (a) preparing a mixture comprising an isolated carrier and a payload; and (b) preparing a mixture comprising protamine sulfate and NaPO. 4 and (c) combining the mixture of (a) and the mixture of (b) and allowing the combined mixture to stand at room temperature overnight. (Item 143) (d) increasing the ionic strength of the combined mixture obtained by step (c) to break down the particles obtained by step (c) into smaller particles. (Item 144) The preparation of step (a) comprises ZnCl 2 Item 140. The method of claim 140, wherein the method does not include (Item 145) The preparation of step (a) comprises ZnCl 2 Item 140. The method of item 140, comprising: [Brief explanation of the drawings]

[0080] [Figure 1] Figure 1A is a digital image depicting self-assembling fusion molecule-protamine microparticles prepared as described in Example 1 suspended in a low ionic strength buffer (0.05 M). Figure 1B is a digital image depicting self-assembling fusion molecule-protamine microparticles prepared as described in Example 1 suspended in a high ionic strength buffer (>1 M). Figure 1C is a digital image depicting the microparticles of Figure 1B reconstituted in a low ionic strength buffer (0.05 M). The particles were imaged using a GE Cytel system in high magnification (10x) brightfield mode.

[0081] [Figure 2] Figure 2A is a digital image depicting self-assembling insulin-protamine microparticles prepared as described in Example 2 in bright field mode. Figure 2B is a digital image depicting self-assembling insulin-protamine microparticles prepared as described in Example 2 showing FITC fluorescence in blue field mode. Figure 2C is a digital image depicting a merged image of the bright field and blue field images shown in Figures 2A and 2B. The merged image shows the FITC fluorescence of the insulin microparticles. The particles were imaged using a GE Cytel system in high magnification (10x) bright field or blue field mode. FITC fluorescence was imaged by exciting the sample at 381 nm and recording the fluorescence emission at 435 nm. The blue arrow indicates the approximately 150 μM particle.

[0082] [Figure 3] FIG. 3A is a digital image depicting self-assembling fusogenic molecule-protamine-zinc microparticles prepared as described in Example 3 in red field mode.

[0083] Figure 3B is a digital image of the same fusion molecule-protamine-zinc microparticles in bright field mode. The particles were imaged using a GE Cytel system in high magnification (10x) bright field or red field mode. Red fluorescence was imaged by exciting the sample at 481 nm and recording the fluorescence emission at 535 nm.

[0084] [Figure 4] Figure 4A is a digital image depicting self-assembling fusion molecule-protamine (without ZnCl) microparticles prepared as described in Example 3 in bright field mode. Figure 4B is a digital image of the same particles in red field mode. The particles were imaged using a GE Cytel system in high magnification (10x) bright field or red field mode. Red fluorescence was imaged by exciting the sample at 481 nm and recording the fluorescence emission at 535 nm.

[0085] [Figure 5-1] FIG. 5A shows the dissolution profile of formulation 37-156 at pH 2 and pH 7, and after increasing the pH from pH 2 to pH 7.

[0086] FIG. 5B shows the dissolution profile of formulation 37-167 at pH 2 and pH 7, and after increasing the pH from pH 2 to pH 7.

[0087] [Figure 5-2] FIG. 5C shows the dissolution profile of the formulated nanoparticles at pH 2 and pH 7, and after increasing the pH from pH 2 to pH 7.

[0088] [Figure 6] FIG. 6 shows insulin stability after exposure of different nanoparticle formulations to pancreatin.

[0089] [Figure 7-1] Figure 7A shows a section of a rat small intestine 15 minutes after intraluminal injection of 37-49 hGH / SEQ ID NO:3 / Eudragit FS particles. hGH is seen in green and SEQ ID NO:3 is seen in red. Figure 7B shows a section of a rat small intestine 30 minutes after intraluminal injection of 37-49 hGH / SEQ ID NO:3 / Eudragit FS particles. hGH is seen in green and SEQ ID NO:3 is seen in red.

[0090] [Figure 7-2] Figure 7C shows a section of rat small intestine 45 minutes after intraluminal injection of 37-49 hGH / SEQ ID NO: 3 / Eudragit FS particles, with hGH shown in green and SEQ ID NO: 3 in red.

[0091] Figure 7D shows a section of rat small intestine 60 minutes after intraluminal injection of 37-49 hGH / SEQ ID NO: 3 / Eudragit FS particles, with hGH shown in green and SEQ ID NO: 3 in red.

[0092] [Figure 8]FIG. 8 shows serum levels of hGH in rats administered hGH-containing particles by oral gavage.

[0093] [Figure 9] FIG. 9 shows the levels of hGH transported through Caco-2 cells when delivered in different formulations.

[0094] [Figure 10-1] FIG. 10A shows the in vitro release of exenatide from five particles of Table 3 at pH 5, pH 7, pH 7.5 and pH 10 as assayed by reverse phase liquid chromatography (RPLC) at the indicated time points.

[0095] [Figure 10-2] FIG. 10B shows the in vitro release of exenatide from five additional particles of Table 3, assayed by RPLC at pH 5, pH 7, pH 7.5, and pH 10, at the indicated time points.

[0096] FIG. 10C shows the in vitro release of exenatide from five particles of Table 3 at pH 5, pH 7, pH 7.5 and pH 10 as assayed by size exclusion chromatography (SEC) at the indicated time points.

[0097] FIG. 10D shows the in vitro release of exenatide from five additional particles of Table 3, assayed by size exclusion chromatography (SEC), at pH 5, pH 7, pH 7.5, and pH 10 at the indicated time points.

[0098] [Figure 11-1]Figures 11A-11C show the pancreatin stability of SEQ ID NO:3 and / or exenatide in different formulations described herein: Figure 11A shows the pancreatin stability of SEQ ID NO:3 and / or exenatide in a zinc-containing formulation, Figure 11B shows the pancreatin stability of SEQ ID NO:3 and / or exenatide in a protamine-containing formulation, and Figure 11C shows the pancreatin stability of exenatide in a zinc and exenatide formulation. [Figure 11-2] Same as above.

[0099] [Figure 12] FIG. 12A shows a confocal microscope image of a formulation of SEQ ID NO: 3, FITC-exenatide and zinc, with FITC-exenatide visualized.

[0100] FIG. 12B shows a confocal microscopy image of a formulation of SEQ ID NO:3, FITC-exenatide and zinc, where SEQ ID NO:3 is visualized with an Alexa 647-labeled anti-SEQ ID NO:3 antibody.

[0101] FIG. 12C shows a merged image of FIGS. 12A and 12B.

[0102] [Figure 13] FIG. 13 shows the particle size distribution of the formulations in FIGS. 12A-12C.

[0103] [Figure 14] FIG. 14 shows the transport of formulations described herein through SMI-100 cells.

[0104] [Figure 15] FIG. 15 shows a schematic example of a method for producing acid-resistant particles comprising a Cholix carrier, zinc and exenatide.

[0105] [Figure 16]FIG. 16 shows how the passage of microparticles through different mucus and epithelial layers can be detected using in vitro or in vivo methods.

[0106] [Figure 17] FIG. 17A shows the pancreatin stability of SEQ ID NO: 3 and / or exenatide in different formulations described herein.

[0107] FIG. 17B shows the pancreatin stability of SEQ ID NO:3 and / or FITC-exenatide in different formulations described herein.

[0108] [Figure 18] FIG. 18 shows a reverse phase chromatogram (RPLC) showing the presence of SEQ ID NO:3 at a retention time of 6.8 minutes and exenatide at 7.5 minutes.

[0109] [Figure 19] FIG. 19 shows the in vitro release of exenatide from formulations E0, E14, E18, E0-FITC, E14-FITC and E18-FITC at pH 1, pH 5 and pH 7 at 37° C. at the indicated time points as assayed by RPLC.

[0110] [Figure 20] FIG. 20 shows the serum concentrations of exenatide in rats administered intraluminally with formulations E0, E14 and E18.

[0111] [Figure 21] FIG. 21 shows the serum concentrations of exenatide in rats administered exenatide intravenously.

[0112] [Figure 22] FIG. 22 shows the purity of SEQ ID NO: 70-exenatide (SEQ ID NO: 70 cross-linked to exenatide) run on a Coomassie blue stained SDS-PAGE gel.

[0113] [Figure 23] Figure 23 shows the in vivo transcytosis of exenatide cross-linked to carrier SEQ ID NO: 80 or SEQ ID NO: 70 across the jejunum of Sprague Dawley rats. The amount of exenatide (in pM) transported across the intestinal tissue was measured 10 and 40 minutes after treatment. The data show that both SEQ ID NO: 80-exenatide and SEQ ID NO: 70-exenatide can be transported at a faster rate than exenatide alone at 10 and 40 minutes.

[0114] [Figure 24] Figure 24 shows the time-concentration profile of blood glucose after glucose load.The effect of SEQ ID NO:70-exenatide administered by oral gavage is compared with negative control treatment (oral buffer) and the exenatide administered by intraperitoneal injection as positive control.These results demonstrate that SEQ ID NO:70-exenatide reduces the increase in blood glucose level after glucose load.

[0115] [Figure 25] FIG. 25 shows that SEQ ID NO: 11 is capable of binding to the GLP-1 receptor. DETAILED DESCRIPTION OF THE INVENTION

[0116] Detailed Description I. Overview Provided herein are methods and compositions that may include (a) a carrier derived from a bacterial toxin and (b) a transition metal cation or polycation, wherein the carrier can enter or transcytose across polarized epithelial cells. The bacterial toxin-derived carrier may be, for example, a colix polypeptide (e.g., from Vibrio cholerae) or Pseudomonas exotoxin (PE) A, for example, from Pseudomonas aeruginosa. The composition may further include a payload, for example, a heterologous payload (e.g., the payload is not a carrier, e.g., not colix or PE), for example, a therapeutic payload. The composition may be in the form of particles, for example, microparticles or nanoparticles, and can be produced, for example, by spray drying and / or freeze-drying. The methods provided herein include administering the composition to a subject. In some cases, the composition may be formulated to pass through the acidic environment of the stomach intact (e.g., the particles may be acid-resistant microparticles). Figure 16 provides a schematic diagram showing particle penetration through a mucus sample, a thin mucus layer on cultured cells, and a healthy mucus layer on epithelial cells in vivo. Particles can be soluble at pH 5-7 and can aid in binding or transport in reconstituted mucus layers assessed, for example, in transit studies using cultured endothelial tissue layers, or through intestinal tissue in either in vitro or in vivo models. In some cases, the compositions provided herein may be formulated for delivery to a subject by other routes, for example, respiratory delivery.

[0117] Also provided herein are methods and compositions comprising a carrier capable of entering or transcytosing across polarized epithelial cells and a heterologous payload, wherein the molar ratio of heterologous payload to carrier is greater than 1:1.

[0118] Provided herein are methods and compositions comprising a carrier, wherein the carrier can enter polarized epithelial cells or transcytose across polarized epithelial cells, and at least 60% of the carrier is intact at 0.5 hours in a pancreatin assay, wherein the pancreatin assay comprises incubating a composition comprising 100 μg of the carrier with 10 μg of pancreatin in 100 μL of phosphate-buffered saline (PBS) at 37° C. Methods for producing these compositions and methods for administering these compositions to a subject are provided herein.

[0119] Also provided herein are methods and compositions comprising a colix variant ending at positions 195-347 and a heterologous payload, wherein the heterologous payload is a glucose regulator. Also provided herein are compositions comprising a carrier linked to a glucose regulator.

[0120] Also provided herein are pharmaceutical compositions comprising the compositions provided herein, methods of making the compositions provided herein, and methods of administering the compositions provided herein to a subject, e.g., a subject having a deficiency in glucose regulation. II. Particles

[0121] The compositions provided herein may include one or more particles. The one or more particles may include one or more microparticles or nanoparticles. The one or more particles may include one or more carriers, one or more payloads (e.g., heterologous payloads), and / or one or more cations. The one or more cations may be one or more polycations, e.g., protamine. The one or more cations may be one or more metal cations. The one or more metal cations may be one or more transition metal cations. The one or more metal cations may be one or more divalent metal cations. The one or more transition metal cations may be Fe 2+ , Mn2+ , Zn 2+ , Co 2+ , Ni 2+ , and Cu 2+ The one or more transition metal cations can be zinc cations. The one or more divalent metal cations can be calcium (Ca 2+ ), chromium (Cr 2+ ), cobalt (Co 2+ ), iron (Fe 2+ ), magnesium (Mg 2+ ), manganese (Mn 2+ ), Nickel (Ni 2+ ), copper (Cu 2+ ), or zinc (Zn 2+ The one or more cations can be protamine, poly-lysine, poly-ornithine, poly-ethylene-imine (PEI), prolamine, polyvinylpyrrolidone (PVP), polyarginine, polyvinylamine, or a combination thereof. The one or more cations can be a protamine salt.

[0122] The one or more particles, eg, one or more microparticles or nanoparticles, can be one or more self-assembling particles, eg, stable self-assembling particles, eg, microparticles or nanoparticles.

[0123] The compositions provided herein may include one or more carriers, one or more payloads, and / or one or more cations, such as one or more polycations. The one or more carriers and one or more payloads may be directly or indirectly linked, covalently or non-covalently. The one or more carriers and one or more payloads may be in the form of a fusion molecule. In some cases, the one or more carriers and one or more payloads are not in the form of a fusion molecule. The carrier-payload fusion molecule can transport one or more payload molecules (e.g., one or more therapeutic payloads) into epithelial cells (e.g., polarized intestinal epithelial cells). The carrier may be capable of transporting the payload into or across epithelial cells using endogenous transport pathways. In contrast to using passive diffusion, utilizing endogenous transport pathways may enable the carrier to rapidly and efficiently transport the payload into or across epithelial cells without impairing the barrier function of these cells or the biological activity of the payload. The payload or payloads can be a polypeptide comprising, consisting of, or consisting essentially of a sequence set forth in any of SEQ ID NOs: 11 or 14-64 (see Table 12).

[0124] The one or more carriers and the one or more cations may be directly or indirectly, covalently or non-covalently linked. The one or more carriers and the one or more cations may be in the form of a fusion molecule. In some cases, the fusion molecule can be assembled into a microparticle or nanoparticle with one or more payloads. The carrier-cation fusion molecule can transport one or more payload molecules (e.g., one or more therapeutic payloads) into epithelial cells (e.g., polarized intestinal epithelial cells). In some cases, the one or more carriers and the one or more cations are not directly linked or covalently bound. In some cases, the one or more carriers and the one or more cations are not part of a fusion molecule.

[0125] In some cases, the one or more cations are protamine or protamine salts. Protamine may refer to a group of strongly basic proteins present in sperm cells in salt-like combinations with nucleic acids. Protamine can be obtained from salmon (salmine), rainbow trout (iridin), herring (clupeine), sturgeon (sutulin), or Spanish mackerel or tuna (chinin). The peptide composition of a particular protamine can vary depending on the family, genus, or species of fish from which it is obtained. Protamine can contain four major components, for example, a single-chain peptide containing approximately 30-32 residues, of which approximately 21-22 are arginine. The N-terminal residue can be proline for each of the four major components. Therefore, chemical modification of protamine with specific salts can be expected to be homogenous. Protamine salts can be derived from salmon. Protamine salts can be derived from herring. Protamine salts can be derived from rainbow trout. Protamine salts may be derived from tuna.

[0126] One or more particles, e.g., one or more microparticles or nanoparticles, can comprise a protamine salt selected from the group consisting of protamine sulfate, protamine acetate, protamine bromide, protamine chloride, protamine caproate, protamine trifluoroacetate, protamine bicarbonate (HCO), protamine propionate, protamine lactate, protamine formate, protamine nitrate, protamine citrate, protamine monohydrogen phosphate, protamine dihydrogen phosphate, protamine tartrate, protamine perchlorate, and a mixture of any two protamine salts. The protamine salt may be combined with other ingredients in a liquid medium prior to particle formation, e.g., by precipitation or spray drying. The protamine salt in the liquid medium prior to particle formation can have a concentration of about 0.05 mg / mL to about 0.10 mg / mL, about 0.10 mg / mL to about 0.15 mg / mL, about 0.15 mg / mL to about 0.20 mg / mL, about 0.20 mg / mL to about 0.25 mg / mL, about 0.25 mg / mL to about 0.30 mg / mL, about 0.30 mg / mL to about 0.35 mg / mL, about 0.35 mg / mL to about 0.40 mg / mL, about 0.40 mg / mL to about 0.45 mg / mL, or about 0.45 mg / mL to about 0.5 mg / mL. The protamine salt may be a mixture of two different salts, where one salt is a sulfate and the other is an acetate, propionate, lactate, formate, or nitrate salt of protamine. The molar ratio between two different salts can be from about 0.1 to about 1, from about 0.2 to about 1, from about 0.3 to about 1, from about 0.4 to about 1, from about 0.5 to about 1, from about 0.6 to about 1, from about 0.7 to about 1, from about 0.8 to about 1, and from about 0.9 to about 1.

[0127] The particles may further comprise a divalent metal ion, such as zinc, cobalt, magnesium, or calcium, or a combination of these ions. The metal ion may be zinc.

[0128] In some cases, particles can be formed by combining carriers and cations in a ratio of about 1:0.001 to about 1:2000 (carrier:cation) by weight. In some cases, carriers and cations can be combined in a ratio of about 1:0.01 to about 1:500 by weight. In some cases, carriers and cations can be combined in a ratio of about 1:0.08 to about 1:173 by weight. In some cases, carriers and cations can be combined in a ratio of about 1:0.1 to about 1:200 by weight. In some cases, carriers and cations can be combined in a ratio of about 1:0.1 to about 1:5 by weight. In some cases, carriers and cations can be combined in a ratio of about 1:0.08 to about 1:1.6 by weight. In some cases, carriers and cations can be combined in a ratio of about 1:8 to about 1:180 by weight. In other examples, the particles can be formed by combining the carrier and cation in a ratio of about 0.01:1 to about 0.2:1 by weight. In some cases, the carrier and cation can be combined in a ratio of about 1:1 by weight. In some cases, the carrier and cation can be combined in a ratio of at least about 1:0.001, 1:0.01, 1:0.08, 1:0.1, 1:0.16, 1:0.2, 1:0.3, 1:0.32, 1:0.4, 1:0.8, 1:1, 1:1.6, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:8.6, 1:9, 1:10 by weight. , 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:86, 1:90, 1:100, 1:150, 1:172, 1:200, or 1:2000 ratios.In some cases, the carrier and cation are in a ratio of about 1:0.001, 1:0.01, 1:0.08, 1:0.1, 1:0.16, 1:0.2, 1:0.3, 1:0.32, 1:0.4, 1:0.8, 1:1, 1:1.6, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:8.6, 1:9, 1:10, 1:1 They may be combined in ratios of less than 1, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:86, 1:90, 1:100, 1:150, 1:172, or 1:200, 1:2000.

[0129] In some cases, particles can be formed by combining payload fusion molecules and cations in a weight ratio of about 1:0.001 to about 1:2000 (payload fusion molecules:cations). In some cases, payload fusion molecules and cations can be combined in a weight ratio of about 1:0.01 to about 1:500. In some cases, payload fusion molecules and cations can be combined in a weight ratio of about 1:0.08 to about 1:173. In some cases, payload fusion molecules and cations can be combined in a weight ratio of about 1:0.1 to about 1:200. In some cases, payload fusion molecules and cations can be combined in a weight ratio of about 1:0.1 to about 1:5. In some cases, payload fusion molecules and cations can be combined in a weight ratio of about 1:0.08 to about 1:1.6. In some cases, the payload fusion molecule and cations may be combined in a ratio of about 1:8 to about 1:180 by weight. In other examples, particles may be formed by combining the payload fusion molecule and cations in a ratio of about 0.01:1 to about 0.2:1 by weight. In some cases, the payload fusion molecule and cations may be combined in a ratio of about 1:1 by weight. In some cases, the payload fusion molecule and cations may be combined in a ratio of at least about 1:0.001, 1:0.01, 1:0.08, 1:0.1, 1:0.16, 1:0.2, 1:0.3, 1:0.32, 1:0.4, 1:0.8, 1:1, 1:1.6, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:8.6, 1:9, They may be combined in ratios of 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:86, 1:90, 1:100, 1:150, 1:172, or 1:200, 1:2000.In some cases, the payload fusion molecule and cation are in a ratio of about 1:0.001, 1:0.01, 1:0.08, 1:0.1, 1:0.16, 1:0.2, 1:0.3, 1:0.32, 1:0.4, 1:0.8, 1:1, 1:1.6, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:8.6, 1:9, 1:10 by weight. , 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, 1:60, 1:70, 1:80, 1:86, 1:90, 1:100, 1:150, 1:172, or 1:200, 1:2000 or less.

[0130] In some examples, the particles can be formed by combining the carrier and payload in a ratio by weight of about 0.01:1 to about 1:2, about 0.0116:1 to about 1:1, or about 0.2:1 to about 1:1. In some cases, the carrier to payload ratio may be combined in a ratio of at least 0.01:1, 0.0116:1, 0.02:1, 0.0232:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.2:1, 0.232:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1 by weight. In some cases, the carrier to payload ratio may be combined in a ratio of less than 0.01:1, 0.0116:1, 0.02:1, 0.0232:1, 0.03:1, 0.04:1, 0.05:1, 0.06:1, 0.07:1, 0.08:1, 0.09:1, 0.1:1, 0.2:1, 0.232:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1 by weight.

[0131] In some cases, the particles or compositions may comprise a carrier and a cation (e.g., Zn 2+ or protamine) in a ratio of carrier to cation of about 5:1 to about 1:5 on a molar basis, about 10:1 to 1:10 on a molar basis, about 2:1 to about 1:2 on a molar basis, about 1:10 to about 1:1000 on a molar basis, about 1:30 to about 1:700 on a molar basis, about 1:100 to about 1:300,000 on a molar basis, or about 1:1000 to about 1:30,000 on a molar basis. The ratio of carrier to cation can be about 10:1, 5:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:25, 1:50, 1:100, 1:500, 1:650, 1:1000, 1:2000, 1:5000, 1:10,000, 1:20,000, 1:30,000, 1:40,000, 1:50,000, 1:75000, 1:100,000, 1:200,000, or 1:300,000 on a molar basis. In some cases, the ratio of carrier to cation is at least 1:300,000, 1:200,000, 1:100,000, 1:50,000, 1:30,000, 1:25,000, 1:20,000, 1:15,000, 1:10,000, 1:5,000, 1:3,000, 1:2800, 1:2000, 1:1500, 1:1000, 1:500, 1:100, 1:500, 1:100, 1:50, 1:25, 1:10, 1:5, 1:2, 1:1.12, 1:1, or 10:1 on a molar basis. In some cases, the ratio of carrier to cation is less than 1:300,000, 1:200,000, 1:100,000, 1:50,000, 1:30,000, 1:25,000, 1:20,000, 1:15,000, 1:10,000, 1:5,000, 1:3,000, 1:2800, 1:2000, 1:1500, 1:1000, 1:500, 1:2, 1:1.12, or 1:1 on a molar basis. In some cases, the carrier may exist in the particle as a monomer, dimer, trimer, or other complex. The ratio of carrier to cation may be calculated based on the moles of carrier monomer. When the cation is contained in a salt, the ratio can be determined using the amount or moles of the cation itself, rather than the amount or moles of the salt containing the cation.

[0132] The particle or composition may comprise carrier and payload in a ratio of carrier to payload that is from about 1:1 to about 1:10 on a molar basis, from about 10:1 to about 1:1000 on a molar basis, from about 1:1 to about 1:1000 on a molar basis, from about 1:10 to about 1:1000 on a molar basis, from about 1:10 to about 1:6000 per molar, from about 1:1 to about 1:100 on a molar basis, from about 1:5 to about 1:20 on a molar basis, from about 1:7 on a molar basis, or about 1:716 on a molar basis. In some cases, the particle or composition may comprise carrier and payload in a ratio of carrier to payload, on a molar basis, of less than about 1:1, less than about 1:2, less than about 1:5, less than about 1:7, less than about 1:10, less than about 1:20, less than about 1:30, less than about 1:60, less than about 1:70, less than about 1:80, less than about 1:90, less than about 1:100, less than about 1:125, less than about 1:150, less than about 1:200, less than about 1:300, less than about 1:400, less than about 1:500, less than about 1:600, less than about 1:750, less than about 1:1000, less than about 1:2500, less than about 1:5000, less than about 1:6000, or less than about 1:50. In some cases, the ratio of carrier to payload is at least about 1:6000, 1:5000, 1:4000, 1:3000, 1:2000, 1:1000, 1:900, 1:800, 1:700, 1:620, 1:617.06, 1:600, 1:500, 1:400, 1:310, 1:308.53, 1:200, 1:150, 1:125, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:5, or 1:1 on a molar basis. In some cases, the ratio of carrier to payload is less than about 1:6000, 1:5000, 1:4000, 1:3000, 1:2000, 1:1000, 1:900, 1:800, 1:700, 1:620, 1:617.06, 1:600, 1:500, 1:400, 1:310, 1:308.53, 1:200, 1:150, 1:125, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:5, or 1:1 on a molar basis.The ratio of carrier to payload can be about 1:6000, 1:5000, 1:4000, 1:3000, 1:2000, 1:1000, 1:900, 1:800, 1:700, 1:620, 1:617.06, 1:600, 1:500, 1:400, 1:310, 1:308.53, 1:200, 1:150, 1:125, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 5:1, or 10:1 on a molar basis. In some cases, the carrier or payload, or both, may exist as monomers, dimers, trimers, or other complexes, and the molar ratio can be calculated by comparing the carrier monomers to the payload monomers.

[0133] In some cases, the carrier is indirectly and non-covalently linked to the payload. In such cases, particles (e.g., liposomes, microparticles, nanoparticles, metal nanoparticles, polymer-based nanoparticles, etc.) can be loaded with payload molecules (e.g., IL-10, IL-22, GLP-1, etc.) (e.g., inside and / or on the surface of the particle), and carriers, such as, for example, Colicus-derived or PE-derived carrier molecules, can be linked to such nanoparticles (e.g., on the surface). In some cases, particles can be formed that include payload molecules and carrier molecules, such as, for example, Colicus-derived or PE-derived molecules.

[0134] In some cases, the ratio (e.g., molar ratio) of payload to carrier in the compositions (e.g., particles) provided herein can be at least about 15,000:1, 10,000:1, 5,000:1, 2,500:1, 1,000:1, 500:1, 250:1, 100:1, 50:1, 25:1, 10:1, 5:1, 2.5:1, or 1:1. This ratio can enable transport of such payload-containing particles (e.g., nanoparticles) into or across polarized epithelial cells (e.g., polarized intestinal epithelial cells) using a surface-bound carrier, such as a PE or colix-derived carrier. In some cases, the particles (e.g., nanoparticles) can release the payload after transcytosis or intracellular delivery. In cases where particles (e.g., nanoparticles) are transported across epithelial cells, the released payload may bind to receptors in the submucosal tissue (e.g., the lamina propria) and / or enter the systemic circulation, thus providing a specific function (e.g., a therapeutic or diagnostic function) systemically. In other cases, when particles (e.g., nanoparticles) release their payload within epithelial cells, the payload (e.g., a nucleic acid) may provide a specific intracellular function, such as production of a transgene, modulation of gene expression, etc., within those cells.

[0135] The particles, or compositions, e.g., pharmaceutical compositions, may further comprise a preservative, such as phenol, m-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, or thiomerosal, or mixtures thereof.

[0136] The composition, e.g., pharmaceutical composition, may further comprise a tonicity modifying agent, such as a sugar or sugar alcohol, an amino acid (e.g., L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), an alditol (e.g., glycerol (glycerin), 1,2-propanediol (propylene glycol), 1,3-propanediol, 1,3-butanediol), polyethylene glycol (e.g., PEG400), glycerol, mannitol, propylene glycol, dimethyl sulfone, methylsulfonylmethane, trehalose, sucrose, sorbitol, saccharose, lactose, or a mixture thereof.

[0137] The composition, e.g., pharmaceutical composition, may further comprise a buffer, such as sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)-aminomethane, or a mixture thereof.

[0138] The formulations of the present disclosure can be prepared, for example, as described in Remington's Pharmaceutical Sciences, 1985 or Remington: The Science and Practice of Pharmacy, 19th edition, 1995, where pharmaceutical techniques involve dissolving and mixing the ingredients as appropriate to achieve the desired end product.

[0139] In another aspect, provided herein is a method for preparing particles, e.g., microparticles or nanoparticles, e.g., self-assembled microparticles or nanoparticles, containing a carrier, a payload, and a cation. The carrier and payload may form a fusion molecule. The method may include: (a) preparing a mixture (e.g., as a fusion molecule) containing an isolated carrier and a payload, and optionally ZnCl2; (b) preparing a mixture containing protamine sulfate and NaPO4; and (c) combining the mixture of (a) and the mixture of (b) and allowing the combined mixture to stand overnight at room temperature. The method may further include (d) increasing the ionic strength of the combined mixture obtained by step (c) to break down the particles obtained by step (c) into smaller particles. In some cases, the preparation in step (a) does not contain ZnCl2. In some cases, the preparation in step (a) contains ZnCl2.

[0140] In some cases, the carrier and payload do not form a fusion molecule. The method may include: (a) preparing a mixture containing an isolated carrier and an isolated payload, and optionally ZnCl; (b) preparing a mixture containing protamine sulfate and NaPO; and (c) combining the mixture of (a) and the mixture of (b) and allowing the combined mixture to stand overnight at room temperature. The method may further include (d) increasing the ionic strength of the combined mixture obtained by step (c) to break down the particles obtained by step (c) into smaller particles. In some cases, the preparation of step (a) does not include ZnCl. In some cases, the preparation of step (a) includes ZnCl. In some cases, the combined mixture of (a) and (b) may be spray-dried to form microparticles or nanoparticles. In some cases, the microparticles or nanoparticles can be formed using commercially available spray-drying equipment.

[0141] In some cases, the carrier and cation may form a fusion molecule. The method may include: (a) preparing a mixture containing an isolated carrier and cation fusion molecule, an isolated payload, and optionally ZnCl; (b) preparing a mixture containing an isolated payload and NaPO; and (c) combining the mixture of (a) and the mixture of (b) and allowing the combined mixture to stand at room temperature overnight. The method may further include (d) increasing the ionic strength of the combined mixture obtained by step (c) to break down the particles obtained by step (c) into smaller particles. In some cases, the preparation of step (a) does not include ZnCl. In some cases, the preparation of step (a) includes ZnCl. In some cases, the combined mixture of (a) and (b) may be spray-dried to form microparticles or nanoparticles. In some cases, the microparticles or nanoparticles can be formed using commercially available spray-drying equipment.

[0142] The Nano Spray Dryer B-90 (Buchi, Switzerland) can produce protein particles from sub-1 micron to 2 microns in size with a narrow size distribution and controlled release of active pharmaceutical ingredients. Proteins, such as hGH and insulin, can be spray-dried in a continuous, single-step process. Counterions and polymeric excipients can be used for encapsulation and to achieve sustained release of active pharmaceutical ingredients. The system can be compatible with water-soluble excipients and aqueous emulsions. Sample feed rates can be between 1 ml / min and 3 ml / min. Gas flow can be between 100 L / min and 160 L / min. Pressure can be atmospheric.

[0143] Protein concentrations can be between about 0.02 μmole and 1 μmole. Feed rates can be between about 100 L / min and 200 L / min. Inlet temperatures can be between about 80°C and 140°C. Head temperatures can be between about 30°C and 70°C. Many different buffer solutions can be used, such as carbonate, acetate, lactate, succinate, phosphate, and Tris buffers. Examples of excipients that can be used include hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose (CMC), alginate, Eudragit, chitosan, dextran, poly(lactic-co-glycolic acid) (PLGA), Pluronic®, gum arabic, and polysorbate 20. Non-limiting examples of counterions, or molecules that contain counterions, include protamine, SEQ ID NO: 3 constructs, cationic cell-penetrating peptides, polyglutamates, and hyaluronic acid.

[0144] The particles, e.g., self-assembled or spray-dried particles, e.g., protamine particles, zinc particles, or zinc and protamine particles, can have a size of not more than about 200 nm, not more than about 300 nm, not more than about 400 nm, not more than about 500 nm, not more than about 600 nm, not more than about 700 nm, not more than about 800 nm, or not more than about 900 nm. Self-assembled microparticles, e.g., protamine microparticles, zinc particles, zinc and protamine particles, are not greater than about 10 nm, not greater than 20 nm, not greater than 30 nm, not greater than 40 nm, not greater than 50 nm, not greater than 60 nm, not greater than 70 nm, not greater than 80 nm, not greater than 90 nm, not greater than 100 nm, not greater than 110 nm, not greater than 120 nm, not greater than 130 nm. , not exceeding 140nm, not exceeding 150nm, not exceeding 160nm, not exceeding 170nm, not exceeding 180nm, not exceeding 190nm, not exceeding 200nm, not exceeding 250nm, not exceeding 300nm, not exceeding 350nm, not exceeding 400nm, not exceeding 450nm, not exceeding 500nm, not exceeding 600nm, not exceeding 700nm, not exceeding 800nm, not exceeding 900nm not more than about 1 μm, not more than about 5 μm, not more than about 10 μm, not more than about 15 μm, not more than about 20 μm, not more than about 25 μm, not more than about 30 μm, not more than about 35 μm, not more than about 40 μm, not more than about 45 μm, not more than about 50 μm, not more than about 55 μm, not more than about 60 μm, not more than about 65 μm, not more than about 70 μm, not more than about 75 μm, not more than about 80 The nanoparticles may have a size of not more than about 85 μm, not more than about 90 μm, not more than about 95 μm, not more than about 100 μm, not more than about 105 μm, not more than about 110 μm, not more than about 115 μm, not more than about 120 μm, not more than about 125 μm, not more than about 130 μm, not more than about 135 μm, not more than about 140 μm, not more than about 145 μm, and not more than about 150 μm.In some cases, the particles have a size of about 1 μm to about 20 μm. In some cases, the particles have an average size of about 5 μm ± 2 μm. In some cases, the particles have an average size of about 150 μm ± 50 μm. In some cases, the particles have an average size of about 30 nm to about 6000 nm, about 60 nm to about 6000 nm, about 30 nm to about 3000 nm, about 60 nm to about 3000 nm, about 100 nm to about 1000 nm, about 200 nm to about 800 nm, or about 300 nm to about 600 nm. In some cases, the particles have an average size of at least about 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, 650 nm, 700 nm, 750 nm, 800 nm, 850 nm, 900 nm, 950 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm, 1400 nm, 1500 nm, 2000 nm, 3000 nm, 4000 nm, 5000 nm, or 6000 nm.

[0145] Particles, such as microparticles and nanoparticles, can be encapsulated. Natural and synthetic polymer matrices can be used for drug encapsulation and controlled release. The polymer matrix used for particle encapsulation can be selected so that the particles and the drug contained therein are released at the desired pH, temperature, or time. The polymer matrix used for particle encapsulation can be selected so that the particles and the drug contained therein are protected in a low pH environment and released when the pH increases, for example, in the stomach, and then released in the intestine. The polymer matrix used for particle encapsulation can also be selected to protect the particles from the conditions in the first tissue or biological fluid, such as gastric acid. Eudragit can be used for pH- and time-controlled drug release, while chitosan, HPMC, and hyaluronic acid can be used for diffusion-controlled release. Eudragit includes a wide range of polymethacrylate-based copolymers. Examples of Eudragits that may be suitable for use in the methods and compositions of the disclosure include Eudragit RS 30 D: poly(ethyl acrylate-co-methyl methacrylate-co-trimethylammonioethyl methacrylate chloride) 1:2:0.1, Eudragit RL 30 D: poly(ethyl acrylate-co-methyl methacrylate-co-trimethylammonioethyl methacrylate chloride) 1:2:0.2, Eudragit FS 30D:poly(methyl acrylate-co-methyl methacrylate-co-methacrylic acid) 7:3:1, and Eudragit L 30 D-55:poly(methacrylic acid-co-ethyl acrylate) 1:1.

[0146] Particles can be encapsulated by any method known in the art. In some cases, particles, such as microparticles or nanoparticles, can be formed in a first step and encapsulated in a second step. For example, particles can be formed by precipitation as described above, and then encapsulated by spray drying in a second step. In another example, particles can be formed in a first spray drying step, and then encapsulated in a second spray drying step. In some cases, particles can be formed and encapsulated in a single step. For example, the encapsulating polymer matrix can be mixed with a solution containing one or more carriers, payloads, and cations before the spray drying step. In some cases, the solution containing one or more encapsulating polymer matrices, carriers, payloads, and cations can be spray dried to form nanoparticles or microparticles. III. Carriers

[0147] A carrier can be a protein or another type of molecule capable of transporting a heterologous payload across or into an epithelium (eg, the polarized intestinal epithelium of a subject).

[0148] The carrier may be derived from a polypeptide secreted by bacteria. Such a carrier may be derived from a polypeptide secreted by Vibrio cholerae or Pseudomonas aeruginosa. The polypeptide secreted by Vibrio cholerae may be a colix polypeptide. Pseudomonas The polypeptide secreted by P. aeruginosa may be Pseudomonas exotoxin (PE). Carriers derived from Corix polypeptides or PE may be naturally occurring or non-naturally occurring. Carriers derived from Corix polypeptides or PE may be truncated variants of naturally occurring Corix peptides or PE, or may be non-naturally occurring mutant variants. Mutations may include substitutions, deletions, and additions.

[0149] A. coryx

[0150] The carriers herein may have reduced (e.g., at least 50% reduced) or ablated ADP-ribosylation activity (e.g., elongation factor 2 ribosylation) compared to the carrier set forth in SEQ ID NO:1.

[0151] The carrier can be a polypeptide derived from Corix or a variant thereof further truncated at any one of positions 206-415 for transcytosis or positions 151-187 for endocytosis compared to a reference sequence, e.g., SEQ ID NO: 1, 12, or 7. Transcytotic carriers having at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of the carrier sequences shown in Table 14, or endocytotic carriers having at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of the carrier sequences shown in Table 15, are also contemplated herein. Any of the carriers herein can have a V1L substitution. In some cases, the carrier can be a Corix polypeptide having a C-terminal truncation of any one of amino acids 206-425, 150-205, or 150-195 of SEQ ID NO: 1 or 7. In some cases, the carrier can be a Corix polypeptide having an N-terminal truncation at any one of amino acids 1-40 or 35-40 of SEQ ID NO: 1 or 7. In some cases, the carrier consists of amino acid residues from position 40 at the N-terminus to any one of positions 150-205 at the C-terminus of the sequence set forth in SEQ ID NO: 7. In some cases, the carrier can be a Corix polypeptide having an N-terminal truncation at amino acid 266 of SEQ ID NO: 1, 12, or 7. Such a carrier can comprise, consist essentially of, or consist of the amino acid sequence set forth in any one of SEQ ID NOs: 65-67. In other cases, the carrier can have a C-terminus at position 150 or 187 of the sequence set forth in SEQ ID NO: 7. In some cases, the carrier consists of the amino acid sequence set forth in SEQ ID NO: 8, 9, or 10. In some cases, the Corix carrier consists of the amino acid sequence set forth in SEQ ID NO: 12 or 13. The numbering of positions can be based on an alignment of the Corix polypeptide to the sequence set forth in SEQ ID NO: 7, with positions numbered from the N-terminus to the C-terminus, starting from the N-terminus at position 1. In some cases, the carrier can have the sequence of SEQ ID NO: 3.

[0152] The carrier may be a truncated variant of a naturally occurring Corix polypeptide, or a non-naturally occurring mutant variant. Mutations may include substitutions, deletions, or additions. A carrier derived from a truncated Corix may consist of, consist essentially of, or include, for example, amino acid residues 1-415, 1-386, 1-266, or 1-206 of SEQ ID NO: 1 or 7. Thus, a carrier may be derived from a polypeptide of SEQ ID NO: 6 (Corix 1-415 Example of SEQ ID NO: 2 (Corix 1-386 Example of SEQ ID NO: 65 (Corix 1-266 ), or SEQ ID NO: 73 (Corix 1-206 The amino acid sequences may consist of, consist essentially of, or comprise the amino acid sequences set forth in Table 12 (examples of which are set forth in Table 12).

[0153] The carrier may contain one or more amino acids at its N-terminus that facilitate expression in various microorganisms (e.g., bacteria). For example, the carrier may contain an N-terminal methionine, which may be a translation initiation site. Such a carrier may contain SEQ ID NO: 65 (M+ colis). 1-386 In addition, any of the carriers herein may have a V1L substitution as set forth in SEQ ID NO: 2 (an example of a V1L colix).

[0154] The carrier may be a fragment of SEQ ID NO: 81 and may comprise, consist essentially of, or consist of no more than 386 amino acids of the amino acid sequence of SEQ ID NO: 81. The carrier may comprise, consist essentially of, or consist of amino acid residues from any one of positions 1-38 to any one of amino acid residues 195-347 of SEQ ID NO: 81. Alternatively, the carrier may comprise, consist essentially of, or consist of amino acid residues from any one of positions 1-195 to any one of amino acid residues 347 of SEQ ID NO: 81.

[0155] The carrier may comprise, consist essentially of, or consist of amino acid residues 1-195, 1-206, 1-244, 1-266, 1-386, or 1-415 of SEQ ID NO:1, SEQ ID NO:12, or SEQ ID NO:81. Similarly, the carrier may comprise, consist essentially of, or consist of amino acid residues 1-275, 1-266, 1-265, 2-265, 3-265, 4-265, 5-265, 1-250, 2-250, 3-250, 4-250, 5-250, 1-245, 2-245, 3-245, 4-245, 5-245, 1-205, 2-205, 3-205, 4-205, and 5-205 of SEQ ID NO:1, SEQ ID NO:12, or SEQ ID NO:81.

[0156] Carriers may also include variants of any of the foregoing having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of the sequences herein.

[0157] B.Pseudomonas exotoxin A

[0158] The carrier may comprise a portion of Pseudomonas exotoxin. Pseudomonas exotoxin A or "PE" can be secreted by Pseudomonas aeruginosa as a 67 kDa protein composed of three prominent globular domains (Ia, II, and III) and one small subdomain (Ib) connected to domains II and III (see, e.g., Allured et al., Proc. Natl. Acad. Sci. 83:1320 1324, 1986). Mature PE can be a 613-residue protein, the sequence of which is set forth in SEQ ID NO: 69. An example of a nucleic acid encoding mature PE used herein is set forth in SEQ ID NO: 68.

[0159] PE exotoxin domain I (e.g., SEQ ID NO: 82) can include amino acids 1-252 of SEQ ID NO: 69 and can be a receptor-binding domain that can be a ligand for a cell surface receptor and mediate binding of PE to cells. The carrier can have the amino acid sequence set forth in SEQ ID NO: 82. The carrier can include an amino acid sequence having greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90%, greater than 95%, or greater than 99% sequence homology or sequence identity to the sequence set forth in SEQ ID NO: 82. Carriers having at least about 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any of the portions of the carrier sequence of SEQ ID NO: 69 shown in Table 13 are also contemplated herein.

[0160] In some cases, the carrier comprises a polypeptide having conservative or non-conservative substitutions in the amino acid sequence of SEQ ID NO: 7. The carrier may maintain the ability to bind to cells. The carrier may be a truncated version of SEQ ID NO: 69, e.g., SEQ ID NO: 82. The carrier may comprise a receptor-binding domain polypeptide in which one or more amino acid residues of SEQ ID NO: 82 are deleted. The carrier may comprise a receptor-binding domain polypeptide in which one or more amino acid residues of SEQ ID NO: 82 are substituted with another amino acid.

[0161] Carrier PE Domain I may comprise an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID NO: 82, or at least 80% identity to a functional fragment thereof. The carrier may comprise a deletion or mutation in one or more of amino acid residues 1-252 of the amino acid sequence of SEQ ID NO: 69, e.g., the amino acid sequence of SEQ ID NO: 82. The carrier may comprise an amino acid sequence having at least 90% sequence identity to the amino acid sequence of 1-252 of SEQ ID NO: 69 (e.g., the amino acid sequence of SEQ ID NO: 82), or at least 90% sequence identity to a functional fragment thereof. The carrier may comprise an amino acid sequence having at least 95% sequence identity to the amino acid sequence of 1-252 of SEQ ID NO: 69, e.g., the amino acid sequence of SEQ ID NO: 82, or at least 95% sequence identity to a functional fragment thereof. The carrier may comprise an amino acid sequence of 1 to 252 of SEQ ID NO: 69, e.g., an amino acid sequence having at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 82, or at least 99% sequence identity to a functional fragment thereof. The carrier may comprise an amino acid sequence of 1 to 252 of SEQ ID NO: 69, e.g., an amino acid sequence having 100% sequence identity to the amino acid sequence of SEQ ID NO: 82, or 100% sequence identity to a functional fragment thereof.

[0162] The carrier may be directly or indirectly, covalently or non-covalently linked to the payload. In some cases, the carrier and the payload form a fusion protein. The carrier may be directly or indirectly, covalently or non-covalently linked to the cation. In some cases, the carrier and the cation form a fusion protein or complex. In various embodiments, the carrier, payload, carrier-payload complex, or fusion molecule of the pharmaceutical composition is encoded by a nucleic acid comprising a promoter, a regulatory element, a DNA sequence encoding the carrier or a fragment or truncated variant of such a carrier, and a DNA sequence encoding the payload or cation. The regulatory element may contain a transcription binding site for a transcription factor that activates or represses expression of the coding DNA sequence. In some cases, the regulatory element is an endogenous regulatory element of the DNA sequence encoding the carrier or a fragment or truncated variant of such a carrier. In some cases, the regulatory element is an endogenous regulatory element of the DNA sequence encoding the payload. In some cases, the regulatory element regulates both the DNA sequence encoding the carrier or a fragment or truncated variant of such a carrier and the DNA sequence encoding the payload or cation. In some cases, the carrier and payload are encoded in different nucleic acid molecules. In some cases, the carrier, payload, and cation are encoded in different nucleic acid molecules. In some cases, the carrier and payload are encoded in the same nucleic acid molecule. In some cases, the carrier, payload, and cation are encoded in the same nucleic acid molecule. IV. Payload

[0163] In addition to the carrier polypeptide, the compositions provided herein may include one or more payloads for delivery to a subject, such as one or more heterologous payloads or one or more biologically active payloads. The one or more heterologous payloads may be one or more payloads that do not have a carrier sequence, e.g., one or more payloads that do not have a colix sequence or a PE sequence. The one or more payloads, e.g., heterologous payloads, may be macromolecules, small molecules, small organic molecules, peptides, polypeptides, nucleic acids, mRNA, miRNA, shRNA, siRNA, PNA, antisense molecules, antibodies, DNA, plasmids, polysaccharides, lipids, antigens, vaccines, polymeric nanoparticles, or catalytically active materials. The one or more payloads, e.g., heterologous payloads, may be polypeptides comprising, consisting of, or consisting essentially of a sequence set forth in any of SEQ ID NOs: 11 or 14-64 (see Table 12).

[0164] The payload(s), e.g., the biologically active payload(s), may be macromolecules capable of performing a desired biological activity when introduced into a subject's bloodstream. For example, the payload(s) may have receptor binding activity, enzymatic activity, messenger activity (i.e., acting as hormones, cytokines, neurotransmitters, clotting factors, growth factors, or other signaling molecules), luminescent or other detectable activity, or regulatory activity, or any combination thereof. In various diagnostic embodiments, the payload(s) may be conjugated to a pharmaceutically acceptable gamma-emitting moiety, including, but not limited to, indium and technetium, magnetic particles, radiopaque materials such as air or barium, and fluorescent compounds (e.g., Alexa-488 or red fluorescent protein), or may itself be a gamma-emitting moiety. In some cases, the payload(s), e.g., the biologically active payload(s), do not enter the subject's bloodstream. In some cases, the payload(s) act in the lamina propria.

[0165] In various embodiments, one or more payloads are proteins that contain more than one polypeptide subunit. For example, the protein can be a dimer, trimer, or higher order multimer. In various embodiments, two or more subunits of the protein can be connected by a covalent bond, such as a disulfide bond. In other embodiments, the subunits of the protein can be held together by non-covalent interactions. Those skilled in the art can identify such proteins and determine whether the subunits are properly assembled, for example, using immunoassays.

[0166] In various embodiments, the one or more payloads, e.g., one or more therapeutic payloads, are, for example, dyes, radiopharmaceuticals, hormones, cytokines, anti-TNF agents, glucose-lowering agents, or tumor-associated antigens. In some cases, the one or more therapeutic payloads are polypeptides that are modulators of inflammation in the GI tract. In various embodiments, the one or more payloads to be delivered are glucose-lowering agents for delivery to a subject. Examples of glucose-lowering agents include incretins, glucagon, glucagon proprotein, glucagon peptide, glucagon-like peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), GLP-2 agonists, teduglutide, glicentin, glicentin-related polypeptide, gastric inhibitory polypeptide preprotein, gastric inhibitory polypeptide, dipeptidyl peptidase 4, glucose transporter member 4, preproglucagon, insulin receptor substrate 1, insulin, apolipoprotein A-II, solute transporter family 2, facilitated glucose transporter member 1, glycogen synthase 1, glycogen synthase 2, tyrosine-protein phosphatase non-receptor type 1, RAC-alpha serine threonine-protein kinase, peroxisome proliferator-activated receptor gamma, hexokinase 3, phosphatase 1, phosphatase 2 ... Atidylinositol-3,4,5-triphosphate 3-phosphatase and dual specificity protein, pyruvate dehydrogenase kinase 1, calcium-binding and coiled-coil domain-containing protein 1, Max-like protein X, fructose-bisphosphate aldolase A, glucagon-like peptide 1 receptor, glucagon-like peptide 2 receptor, gastric inhibitory polypeptide receptor, insulin-like growth factor 1 receptor, insulin-like growth factor 2 receptor, insulin receptor, GLP-1 agonist - exenatide, GLP-1 agonist - liraglutide, exendin-4, exendin-3, gastric inhibitory peptide (GIP), GIPR agonist (Des-Ala2-GIP1-30), GIPR agonist truncated GIP1-30, GLP-1R agonist (amino acids 1-37 of GIP), GLP-1R agonist (amino acids 7-36 of GIP),Lixisenatide (trade names Adlyxin® and Lyxumia®, Sanofi), liraglutide (trade name Victoza®, Novo Nordisk A / S), semaglutide (trade name Ozempic®, Novo Nordisk A / S), albiglutide (trade name Tanzeum®, GlaxoSmithKline, GLP-1 dimer fused to albumin), dulaglutide (trade name Trulicity®, Eli Lilly), glucose-dependent insulinotropic polypeptide, multispecific peptide agonist, tirzepatide (Eli Lilly), SAR425899 (Sanofi), amylin-calcitonin receptor dual agonist DACRA-089, glargine / Lantus®, glulisine / Apidra®, glulisine / Toujeo®, Insuman®, detemir / Levemir®, lispro / Humalog® / Liprolog®, degludec / degludecPlus, insulin aspart, insulin and analogs (e.g., LY-2605541, LY2963016, NN143 6), PEGylated insulin lispro (SEQ ID NOs: 40-41), Humulin®, Linjeta, SuliXen®, NN1045, Insulin Plus Symlin™, PE0139, rapid- and short-acting insulins (e.g., Linjeta, PH20, NN1218, HinsBet), (APC-002) hydrogel, oral, inhaled, transdermal, and sublingual insulins (e.g., Exubera®, Nasulin®, Afrezza®, Tregopil®, TPM 02, Capsulin, Oral-lyn®, Cobalamin®, oral insulin, ORMD-0801, NN1953, NN1954, NN1956, VIAtab, and Oshadi oral insulin), and desPro36-exendin-4(1-39)-Lys6NH2, H-des(Pro36,37)-exendin-4-Lys4-NH2, H-des(Pro36,37)-exendin-4-Lys5-NH2,desPro36[Asp28]exendin-4(1-39), desPro36[IsoAsp28]exendin-4(1-39), desPro36[Met(O)14,Asp28]exendin-4(1-39), desPro36[Met(O)14,IsoAsp28]exendin-4(1-39), desPro36[Trp(O2)26,Asp28]exendin-4(1-39), desPro36[Trp(O2)25,IsoAsp28]exendin-4(1-39), desPro36[Met(O)14 Trp(O2)25,Asp28]exendin-4(1-39), and desPro36[Met(O)14 and exendin-4 analogs selected from the group consisting of: exendin-4(1-39), exendin-4(1-39), and exendin-4(1-39). GLP-1 agonists contemplated for use in the particles or delivery constructs disclosed herein include, for example, exenatide (trade name Byetta®, Amylin / Astrazeneca, SEQ ID NO: 14, SEQ ID NO: 11), lixisenatide (trade name Adlyxin® and Lyxumia®, Sanofi, SEQ ID NO: 15), liraglutide (trade name Victoza®, Novo Nordisk A / S, SEQ ID NO: 16), semaglutide (trade name Ozempic®, Novo Nordisk A / S), albiglutide (trade name Tanzeum®, GlaxoSmithKline, GLP-1 dimer fused to albumin), and dulaglutide (trade name Trulicity®, Eli Lilly, SEQ ID NO: 17). The payload may be capable of binding to an incretin receptor, for example, an incretin receptor in gastrointestinal tissue or the hepatic portal system.

[0167] In some cases, one or more payloads, e.g., one or more therapeutic payloads, have the amino acid sequence of SEQ ID NO: 11, or any one or more of SEQ ID NOs: 14-64. Incretins

[0168] The carrier may be linked to one or more incretins. Incretins belong to a class of gastrointestinal hormones that can increase insulin release from beta cells of the islets of Langerhans after a meal, even before blood glucose levels rise. Incretins can slow the rate of nutrient absorption into the bloodstream by reducing gastric emptying, thereby reducing food intake. Incretins can inhibit the release of glucagon from alpha cells of the islets of Langerhans. Incretins can be glucagon-like peptide-1 (GLP-1) or gastric inhibitory peptide (GIP). Both GLP-1 and GIP can be rapidly inactivated by the enzyme dipeptidyl peptidase 4 (DPP-4).

[0169] The incretin or incretin mimetic peptide that can be used in the present disclosure can be a naturally occurring incretin or incretin mimetic peptide, or a modified naturally occurring incretin or incretin mimetic peptide.The peptide can be chemically synthesized using standard peptide synthesis techniques, such as solid-phase peptide synthesis, or can be prepared using recombinant DNA techniques known in the art.The peptide thus produced may or may not be identical to a naturally occurring peptide.Analogs, fragments, and conjugates of naturally occurring incretins or incretin mimetic peptides are also included as payloads in the present disclosure, as long as they retain one or more of the biological activities of naturally occurring incretins or incretin mimetic peptides.

[0170] GLP-1 may be a naturally occurring incretin hormone synthesized in intestinal L-cells by tissue-specific post-translational processing of preproglucagon. GLP-1 has been implicated in the regulation of appetite and satiety. GLP-1 may act through the GLP-1 receptor (GLP-1R), a 463-amino acid member of the G protein-coupled receptor (GPCR) superfamily (e.g., Drucker DJ et al., (See Mol Endocrinol, 17(2):161-171, 2003). Bioactive GLP-1 can exist in two molecular forms of equal potency: GLP-1(7-37) and GLP-1(7-36) amide. Bioactive GLP-1 can be rapidly cleaved by diaminopeptidyl peptidase-4 (DPP-4), resulting in the production of the largely inactive GLP-1(9-37) and GLP-1(9-36) amide molecular forms. In some cases, the majority of GLP-1 leaving the intestinal venous circulation has already been cleaved by DPP-4, which is expressed in the capillaries surrounding the intestinal L-cells. The in vivo half-life of GLP-1 has been estimated to be 1 to 2 minutes (see, e.g., Drucker DJ, Gastroenterology, 122(2):531-544, 2002).

[0171] GLP-1 can refer to glucagon-like peptide 1 from any source, including isolated, purified, and / or recombinant GLP-1 from any source or produced by chemical synthesis, for example, using solid-phase synthesis.For example, GLP-1 can have the sequence of SEQ ID NO: 26.Conservative amino acid substitutions of natural GLP-1 are also included herein.For example, conservative amino acid changes can be made, which change the primary sequence of protein or peptide, but usually do not change its function. Numerous molecular derivatives of GLP-1 have been disclosed, including several that are reported to have agonist activity and / or a longer half-life than native GLP-1; see, e.g., U.S. Pat. Nos. 6,358,924, 6,344,180, 6,284,725, 6,277,819, 6,271,241, 6,268,343, and 6,191,102, the contents of which are incorporated herein by reference.

[0172] GLP-1-related molecules, such as proteins, have also been disclosed and reported to be capable of inducing pancreatic endocrine differentiation, islet proliferation, and increased beta cell mass (Parkes et al., Metabolism 50:583, 2001). Exendin-4 (an example of which is SEQ ID NO: 14 or SEQ ID NO: 11, also known as exenatide or Byetta®) and exendin-3 (an example of which is SEQ ID NO: 28) are 39 amino acid peptides (residues 2 and 3 differ), which are approximately 53% homologous to GLP-1 and have insulinotropic activity. Numerous molecular derivatives of exendin-3 and exendin-4 have been disclosed, including several that are reported to have agonist activity, see, for example, U.S. Patent Nos. 5,424,286, 6,268,343, 6,384,016, 6,458,924, 6,858,576, 6,989,366, 7,115,569, 7,153,825, 7,223,725, 7,235,627, 7,297,761, and 7,411,562. See US Patent Nos. 9,952, 7,521,423, 7,696,161, 7,700,549, 8,097,698, 8,853,160, 8,889,619, 9,012,398, US20120283179, US20140206608, US20140206609, US20140221281, US20140213513, US20150164997, and U.S. Patent No. 9,181,305, the contents of which are incorporated herein by reference. The GLP-1 agonist may be an exendin-4 analog that potently activates GLP-1 and GIP receptors, and optionally the glucagon receptor, and may include, among other substitutions, a Tyr modification at position 1 and an Ile modification at position 12. Examples of such analogs contemplated for use include those described in, for example, US20140206608, US20140206609, 20140221281, and US20140213513, the contents of each of which are incorporated herein by reference.

[0173] The GLP-1 agonist may be, for example, exenatide (trade name Byetta®, Amylin / Astrazeneca, SEQ ID NO: 14, SEQ ID NO: 11), lixisenatide (trade name Adlyxin® and Lyxumia®, Sanofi, SEQ ID NO: 15), liraglutide (trade name Victoza®, Novo Nordisk A / S, SEQ ID NO: 16), semaglutide (trade name Ozempic®, Novo Nordisk A / S, SEQ ID NO: 30), albiglutide (trade name Tanzeum®, GlaxoSmithKline, GLP-1 dimer fused to albumin), and dulaglutide (trade name Trulicity®, Eli Lilly, SEQ ID NO: 17).

[0174] In some cases, the payload can be a glucose-dependent insulinotropic polypeptide. Glucose-dependent insulinotropic polypeptide (also known as gastric inhibitory polypeptide, GIP) can be a member of the incretin class of molecules. GIP can be derived from a 153-amino acid proprotein encoded by the GIP gene and circulates as a biologically active 42-amino acid peptide comprising the amino acid sequence set forth in SEQ ID NO: 36. GIP can be synthesized by intestinal K cells, which can be found in the mucosa of the duodenum and jejunum of the gastrointestinal tract. GIP receptors can be seven-transmembrane proteins found in beta cells in the pancreas. Various GIP antagonists can inhibit GIP-dependent insulin release in vivo and can also enhance glucose tolerance in an oral glucose tolerance test. Therefore, GIP antagonists can be used in methods for treating T2D (see, for example, US20070167363, the contents of which are incorporated herein by reference). A GIP receptor (GIPR) agonist, which is in the form of a truncated GIP analog (amino acid residues 1 to 30 of GIP) and has a substitution of D-alanine (Ala) at position 2 of SEQ ID NO: 38, is designated D-Ala2-GIP1-30 (D-GIP1-30), and includes the amino acid sequence set forth in SEQ ID NO: 37, can exert antidiabetic effects without having obesity-promoting effects (see, for example, Widenmaier et al., J. Am. Chem. Soc. 1999, 103:1111-1112, 1999). (See, e.g., PloS ONE, March 2010 | Volume 5 | Issue 3 | e9590). The glucose regulator, e.g., glucose-lowering agent, can be a GIPR agonist comprising the amino acid sequence set forth in SEQ ID NO: 60. The payload can be glucagon. Multispecific peptide agonists

[0175] In some cases, the compositions provided herein, e.g., particles or delivery constructs, can be designed to achieve dual activation of GLP-1 and GIP receptors, e.g., by combining the effects of GLP-1 and GIP in one preparation. An embodiment of a delivery construct is a composition with a carrier, or a composition with a carrier and a payload. This can result in a therapy with significantly better reduction in blood glucose levels, increased insulin secretion, and weight loss in mice with type 2 diabetes and obesity compared to the commercially available GLP-1 agonist riraglutide (e.g., VA See Gault et al., Clin Sci (Lond), 121, 107-117, 2011).

[0176] In some instances, the carriers provided herein are linked to a dual agonist, for example, tirzepatide (Eli Lilly, SEQ ID NO: 33) or SAR425899 (Sanofi).

[0177] The compositions provided herein, e.g., delivery constructs, can activate receptors for GLP-1, GIP, and glucagon. The carriers provided herein can be linked to a GGG triple agonist being investigated by Eli Lilly.

[0178] The carriers provided herein may be linked to other peptides being investigated for the treatment of diabetes and obesity, including the amylin-calcitonin receptor dual agonist DACRA-089 (SEQ ID NO: 41, also known as KBP-089, Sanofi). Insulin and insulin analogues

[0179] The payload provided herein may be insulin, or an insulin analog, or an insulin derivative. As used herein, the term "insulin analog" or "insulin derivative" may refer to a polypeptide having a molecular structure that can be formally derived from the structure of naturally occurring insulin, for example, that of human insulin (examples of which are the insulin A chain of SEQ ID NO: 43 and the insulin B chain of SEQ ID NO: 44, which may be linked by a disulfide bond), by deleting and / or replacing at least one amino acid residue present in naturally occurring insulin and / or adding at least one amino acid residue. The added and / or replaced amino acid residue may be a codable amino acid residue, or another naturally occurring residue, or a purely synthetic amino acid residue.

[0180] Insulin and insulin analogs / derivatives have been extensively described in the art (see, for example, US20150216981, US Patent No. 9,265,723, US Patent No. 8,633,156, US Patent No. 8,410,048, US Patent No. 8,048,854, US Patent No. 7,713,930, US Patent No. 7,696,162, US Patent No. 7,659,363, US Patent No. 7,291,132, US Patent No. 7,193,035, and the references cited therein, all of which are incorporated herein by reference). The insulin may have the native sequence of human insulin, in which the A chain (SEQ ID NO: 43) and the B chain (SEQ ID NO: 44) are linked by a disulfide bond.

[0181] In certain embodiments, the insulin comprises an A chain having the amino acid sequence set forth in SEQ ID NO: 43 and a B chain having the amino acid sequence set forth in SEQ ID NO: 44. Further embodiments of insulins with beneficial fast-acting or slow-acting (basal) properties include insulin aspart, having an A chain of SEQ ID NO: 45 and a B chain of SEQ ID NO: 46, insulin glargine, having an A chain of SEQ ID NO: 47 and a B chain of SEQ ID NO: 48, and insulin lispro, having an A chain of SEQ ID NO: 49 and a B chain of SEQ ID NO: 50.

[0182] The one or more payloads, e.g., heterologous payloads, can be an agent for the treatment of hemophilia, e.g., hemophilia A or hemophilia B. The agent for the treatment of hemophilia can be clotting factor VIII (e.g., clotting factor VIII concentrate), clotting factor IX (e.g., clotting factor IX concentrate), factor VIIa, Hemlibra® (ACE 910 or emicizumab), DDAVP® or Stimate® (desmopressin acetate), an antifibrinolytic agent, e.g., Amicar® (epsilon aminocaproic acid) or Lysteda® (tranexamic acid), ELOCTATE® (antihemophilic factor (recombinant), Fc fusion protein), or cryoprecipitate.

[0183] In some cases, the payload, for example, a heterologous payload, for example, a glucose regulator, for example, a glucose-lowering agent, is insulin or an insulin analogue.In some cases, the payload, for example, a heterologous payload, for example, a glucose regulator, for example, a glucose-lowering agent, is exenatide.Exenatide (SEQ ID NO: 11) can be a peptide with GLP-1-like biological activity stabilized by C-terminal amine and N-terminal H.

[0184] In some cases, the one or more payloads, e.g., one or more heterologous payloads, used herein can include an anti-neoplastic compound (e.g., a chemotherapeutic or anti-tumor agent), e.g., a nitrosourea, e.g., carmustine, lomustine, semustine, streptozotocin; a methylhydrazine, e.g., procarbazine, dacarbazine; a steroid hormone, e.g., a glucocorticoid, an estrogen, a progestin, an androgen, tetrahydrodesoxycalycosterone (tetrahydrodesoxycalycosterone); esoxycaricosterone); immunoactive compounds, such as immunosuppressants, e.g., pyrimethamine, trimethopterin, penicillamine, cyclosporine, azathioprine; and immunostimulants, e.g., levamisole, diethyldithiocarbamate, enkephalins, endorphins; antimicrobial compounds, such as antibiotics, e.g., beta-lactams, penicillins, cephalosporins, carbapenims and monobactams, beta-lactamase inhibitors, aminoglycosides, methicones ... chloride, tetracycline, spectinomycin; antimalarials, amebicides; antiprotazoals; antifungals, e.g., amphotericin beta; antivirals, e.g., acyclovir, idoxuridine, ribavirin, trifluridine, vidarbine, ganciclovir; parasiticides; antihalmintics; radiopharmaceuticals; gastrointestinal agents; hematological compounds; immunoglobulins; blood clotting proteins, e.g., antihemophilic factor, factor IX complex; anticoagulants, e.g., dicumarol, Heparin sodium; fibrolysin inhibitors, for example, tranexamic acid; cardiovascular drugs; peripheral antiadrenergics; centrally acting antihypertensives, for example, methyldopa, methyldopa HCl; antihypertensive direct vasodilators, for example, diazoxide, hydralazine HCl; drugs acting on the renin-angiotensin system; peripheral vasodilators, for example, phentolamine; antianginal drugs; cardiac glycosides; cardiac vasodilators, for example, amrinone, milrinone, enoximone, phenoximone, imazodan, sulmazole;Antidysrhythmic drugs; Calcium entry blockers; Drugs acting on blood lipids, e.g., Ranitidine, Bosentan, Rezulin; Respiratory drugs; Sypathomimetic drugs, e.g., Albuterol, Bitolterol Mesylate, Dobutamine HCl, Dopamine HCl, Ephedrine SO, Epinephrine, Fenfluramine HCl, Isoproterenol HCl, Methoxamine HCl, Norepinephrine Bitartrate, Phenylephrine HCl, Ritodrine HCl; Cholinomimetics, e.g., Acetylcholine HCl; Anticholinesterases, e.g., Edrophonium Cl; Cholinesterase reactivators; Adrenergic Blocking agents such as acebutolol HCl, atenolol, esmolol HCl, labetalol HCl, metoprolol, nadolol, phentolamine mesylate, propanolol HCl; antimuscarinics such as anisotropine methylbromide, atropine, clininium Br, glycopyrrolate, ipratropium Br, scopolamine HBr; neuromuscular blocking agents; depolarizing agents such as atracurium besylate, hexafluorenium Br, methocrine iodide, succinyl centrally acting muscle relaxants, e.g., baclofen; neurotransmitters and neurotransmitter agents, e.g., acetylcholine, adenosine, adenosine triphosphate; amino acid neurotransmitters, e.g., excitatory amino acids, GABA, glycine; biogenic amine neurotransmitters, e.g., dopamine, epinephrine, histamine, norepinephrine, octopamine, serotonin, tyramine; neuropeptides, nitric oxide, K.sup.+ Channel toxins; antiparkinsonian drugs such as amultidine HCl, benztropine mesylate, and carbidopa; diuretics such as dichlorphenamide, methazolamide, bendroflumethiazide, and polythiazide; antimigraine drugs such as carboprost tromethamine mesylate, doxorubicin, mitomycin, cisplatin, daunorubicin, bleomycin, actinomycin D, neocarzinostatin, and methysergide maleate.

[0185] In some cases, one or more payloads, e.g., one or more heterologous payloads, contemplated for use in the methods of the present disclosure include lymphokine inhibitory factors, macrophage colony-stimulating factors, platelet-derived growth factors, stem cell factors, tumor growth factor-β, tumor necrosis factors, lymphotoxins, Fas, granulocyte colony-stimulating factors, granulocyte-macrophage colony-stimulating factors, interferon-α, interferon-β, interferon-γ, growth factors and protein hormones, e.g., erythropoietin, angiogenin, hepatocyte growth factors, fibroblast growth factors, keratinocyte growth factors, nerve growth factors, tumor growth factor-α, thrombopoietin, thyroid-stimulating factors, thyroid-releasing hormones, neurotrophins, epidermal growth factors, VEGF, ciliary neurotrophic factors, LD, and the like. L, somatomedin, insulin growth factor, insulin-like growth factor I and II, chemokines such as ENA-78, ELC, GRO-α, GRO-β, GRO-γ, HRG, LEF, IP-10, MCP-1, MCP-2, MCP-3, MCP-4, MIP-1-α, MIP-1-β, MG, MDC, NT-3, NT-4, SCF, LIF, leptin, RANTES, lymphotactin , eotaxin-1, eotaxin-2, TARC, TECK, WAP-1, WAP-2, GCP-1, GCP-2; α-chemokine receptors such as CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7; and β-chemokine receptors such as CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, and CCR7. Further examples of payloads include regulatory T cells (Tregs), such as Tregs expressing CD4, CD25, and Foxp3, and inhibitors of Tregs, such as Tr1, Th3, CD8+CD28, Qa-1 restricted T cells, and IL-17 Treg cells.

[0186] In some cases, one or more payloads, e.g., one or more heterologous payloads, can be E. coli heat-labile enterotoxin (Etx).

[0187] In some cases, one or more payloads used in the methods and compositions herein, for example, one or more heterologous payloads, can be dyes or radiopharmaceuticals. One or more dyes and radiopharmaceuticals can be Alexa488, fluorescent compounds, indium, technetium, magnetic particles, radiopaque materials, and red fluorescent proteins (RFPs).

[0188] In some cases, one or more payloads used in the methods and compositions disclosed herein, e.g., one or more heterologous payloads, can be hormones.Examples of hormones include human growth hormone, NUTROPIN® (Genentech), HUMATROPE® (Lilly), GENOTROPIN® (Pfizer), NORDITROPIN® (Novo), SAIZEN® (Merck Serono), OMNITROPE® (Sandoz), SEROSTIM® (EMD Serono), ZORBITIVE® (Merck Serono), TEV-TROPIN® (Teva), pituitary hormones such as chorionic gonadotropin, cosyntropin, menotropin, somatotropin, iorticotropin, protirelin, thyroid-stimulating hormone, vasopressin, lypressin; adrenal hormones such as beclomethasone dipropionate, betamethasone, dexarnethasone, triamcinolone; pancreatic hormones such as glucagon, insulin; parathyroid hormones such as dihydrochysterol; thyroid hormones such as calcitonin etidronate disodium, levothyroxine sodium, liothyronine sodium, liotrix, thyroglobulin, teriparatide acetate; antithyroid drugs; estrogens genital hormones; progestins and antagonists; hormonal contraceptives; testicular hormones; gastrointestinal hormones, such as cholecystokinin, enteroglycan, galanin, gastric inhibitory polypeptide, epidermal growth factor urogastron, gastric inhibitory polypeptide, gastrin-releasing peptide, gastrin, pentagastrin, tetragastrin, motilin, peptide YY, secretin, vasoactive intestinal peptide, or sincalide, somatotropin, synthetic human g-hormone, partially synthetic human growth hormone, partially synthetic human growth hormone, human growth hormone 2, somatoliberin, appetite-regulating hormones, leptin, growth hormone receptor, growth hormone-releasing hormone receptor, growth hormone secretagogue receptor, growth hormone-releasing hormone receptor A form, and growth hormone receptor.

[0189] In a further example, one or more payloads, e.g., one or more heterologous payloads, can be cytokines. The one or more cytokines can be chemokines, interleukins, e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, and IL-30.

[0190] In a further example, one or more payloads, e.g., one or more heterologous payloads, can be an anti-TNF agent. Examples of anti-TNF agents that can be used include anti-TNF antibodies, infliximab (Remicade), adalimumab (Humira), etanercept (ENBREL®), tumor necrosis factor-a ("TNF-a"); NP_000585.2, lymphotoxin-a ("LT-a"), lymphotoxin-b ("LT-b"), CD30 ligand, CD40 ligand, CD70 ligand, OX40 ligand, 41BB ligand, Apo1 ligand (or FasL or CD95L), Apo2 ligand (or TRAIL, AIM-1, or AGP-1), Apo3 ligand (or TWEAK), APRIL, LIGHT, OPG ligand (or RANK ligand), BlyS (or THANK), BCMA, TACI, and the like. , TNFR1, TNFR2, lymphotoxin-bR, CD40, CD95 (or FAS or APO-1), OPG, RANK, CD30, CD27, OX40 (or CD134), 41BB, NGFR, BCMA, TAC1, EDA2R, TROY, DR6, DR5 (or TRAILR2), DR4, DR3, HVEM, LTβR, GITR, DcR3, Fn14 (or TWEAKR), BAFF, small modular immunopharmaceuticals (SMIPs), tetracyclines (e.g., tetracycline, doxycycline, lymecycline, oxytetracycline, minocycline), chemically modified tetracyclines (e.g., dedimethylamino-tetracycline), hydroxamic acid compounds, carbocyclic acids acid) and derivatives, lazaroids, pentoxifylline, naphthopyran, amrinone, pimobendan, vesnarinone, phosphodiesterase inhibitors, and small molecule inhibitors of kinases. Small molecule kinase inhibitors include, but are not limited to, small molecule inhibitors of p38MAPK, COT, MK2, P13K, IKKa, b, g, MEKK1, 2, 3, IRAK1, 4, and Akt kinases.

[0191] In some cases, one or more payloads, e.g., one or more heterologous payloads, can be tumor-associated antigens. Examples of tumor-associated antigens include Her2 / neu, Her3, Her4, EGF, EGFR, CD2, CD3, CD5, CD7, CD13, CD19, CD20, CD21, CD23, CD30, CD33, CD34, CD38, CD46, CD55, CD59, CD69, CD70, CD71, CD97, CD117, CD127, CD134, and CD1 37, CD138, CD146, CD147, CD152, CD154, CD195, CD200, CD212, CD223, CD253, CD272, CD274, CD276, CD278, CD279, CD309 (VEGFR2), DR6, PD-L1, Kv1.3, 5.00E+10, MUC1, uPA, SLAMF7 (CD319), MAGE 3, MUC 16 (CA-125), KLK3, K-ras, mesothelin, p53, survivin, G250 (renal cell carcinoma antigen), and PSMA.

[0192] In some cases, the one or more payloads, e.g., one or more heterologous payloads, can be enzymes such as hyaluronidase, streptokinase, tissue plasminogen activator, urokinase, PGE-adenosine deaminase; intravenous anesthetics such as droperidol, etomidate, fentanyl citrate / droperidol, hexobarbital, ketamine HCl, methohexital Na, thiamylal Na, thiopental Na; antiepileptic drugs such as carbamazepine, clonazepam, divalproex Na, ethosuximide, mephenytoin, paramethadione, phenyloin, primidone. In various embodiments, the biologically active cargo is an enzyme selected from hyaluronidase, streptokinase, tissue plasminogen activator, urokinase, or PGE-adenosine deaminase. V. Linking a Payload or Cation to a Carrier

[0193] In some cases, the compositions provided herein include a carrier linked to a payload, e.g., a heterologous payload. The payload, e.g., a heterologous payload, can be linked to the carrier by any method known to those skilled in the art, without limitation. The payload can be associated with the carrier by non-covalent interactions, such as ionic interactions or assembly into nanoparticles. The payload may be chemically crosslinked to the carrier through covalent interactions. In some cases, one or more payloads are fused to the carrier. In a fusion molecule, one or more payloads or one or more cations within the fusion molecule can be attached to the remainder of the fusion molecule by any method known to those skilled in the art, without limitation. The payload or cation can be introduced into any portion of the fusion molecule that does not disrupt the cell binding or transcytosis activity of the carrier. In various embodiments, the payload or cation is directly linked to the N-terminus or C-terminus of the carrier. In various embodiments, the payload or cation can be connected to the side chain of an amino acid of the carrier. The payload can be indirectly linked to the carrier via a spacer or linker. In various embodiments, the payload or cation is linked to the carrier using a cleavable linker such that cleavage of the cleavable linker separates the payload or cation from the remainder of the fusion molecule. In various embodiments, the payload or cation is a polypeptide that may also include a short leader peptide that remains attached to the polypeptide after cleavage of the cleavable linker. For example, the payload or cation may include a short leader peptide of more than 1 amino acid, more than 5 amino acids, more than 10 amino acids, more than 15 amino acids, more than 20 amino acids, more than 25 amino acids, more than 30 amino acids, more than 50 amino acids, or more than 100 amino acids. In some cases, the biologically active payload may include a short leader peptide of less than 100 amino acids, less than 50 amino acids, less than 30 amino acids, less than 25 amino acids, less than 20 amino acids, less than 15 amino acids, less than 10 amino acids, or less than 5 amino acids.In some cases, the payload or cation may include a short leader peptide of between 1 and 100 amino acids, between 5 and 10 amino acids, between 10 and 50 amino acids, or between 20 and 80 amino acids.

[0194] In embodiments in which a payload or cation is expressed as a fusion protein with another sequence, the payload or cation can be inserted into the fusion molecule by any method known to those of skill in the art, including, without limitation, for example, a nucleic acid encoding the amino acids corresponding to the payload or cation can be inserted directly into a nucleic acid encoding the other moiety or fusion molecule, with or without deletion of the native amino acid sequence.

[0195] In embodiments in which the payload or cation are not expressed together as a fusion molecule, the payload or cation can be attached by any suitable method known to those of skill in the art, without limitation. More specifically, the exemplary methods described above for attaching a receptor binding domain to the remainder of a molecule are equally applicable to attaching a payload or cation to the remainder of a molecule. VI. Generation of Nucleic Acids Encoding Carriers and / or Payloads

[0196] In various embodiments, the carriers, payloads, and / or non-naturally occurring delivery constructs, e.g., fusion molecules, of the present disclosure are prepared using techniques described, for example, in U.S. Pat. Nos. 9,090,691 and 7,713,737, each of which is incorporated herein by reference in its entirety.

[0197] In various embodiments, the carrier, payload, and / or non-naturally occurring fusion molecule is synthesized using recombinant DNA techniques. Generally, this may involve creating a DNA sequence encoding the carrier, payload, and / or fusion molecule, placing the DNA in an expression cassette under the control of a specific promoter, expressing the molecule in a host, isolating the expressed molecule, and, if necessary, folding the molecule into an active conformational form.

[0198] DNA encoding the carriers, payloads, and / or fusion molecules described herein can be prepared by, for example, cloning and restriction of appropriate sequences, or by the methods described in Narang et al. (1979) Meth. Enzymol. 68: 90-99, the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109-151, the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862, the solid support method of U.S. Pat. No. 4,458,066, or the like.

[0199] Chemical synthesis can produce single-stranded oligonucleotide.This can be converted into double-stranded DNA by hybridization with complementary sequence or by using this single strand as template and DNA polymerase polymerization.Chemical synthesis can be used to produce about 100 base DNA sequence.Longer sequences can be obtained by ligating short sequences.

[0200] Alternatively, subsequences may be cloned and the appropriate subsequences cut using appropriate restriction enzymes. The fragments can then be ligated to generate the desired DNA sequence.

[0201] In various embodiments, DNA encoding the carrier, payload, and / or fusion molecule of the present disclosure can be cloned using a DNA amplification method, such as polymerase chain reaction (PCR). Thus, for example, one or more genes of one or more payloads, e.g., one or more biologically active payloads, can be PCR amplified using, for example, a sense primer containing an NdeI restriction site and an antisense primer containing a HindIII restriction site. This can generate one or more nucleic acids encoding one or more payload sequences and having terminal restriction sites. Carriers with "complementary" restriction sites can be similarly cloned and then ligated to one or more nucleic acids encoding one or more payloads and / or linkers attached to one or more nucleic acids encoding one or more payloads. Ligation of the nucleic acid sequences and insertion into a vector generates a vector encoding one or more payloads linked to a carrier. VII. Cleavable Linkers

[0202] In various embodiments, one or more payloads to be delivered to a subject, such as heterologous payloads, are linked to a carrier using one or more cleavable linkers.The number of cleavable linkers present in a fusion molecule depends at least in part on the location of one or more payloads relative to the carrier and the nature of the biologically active payload.If one or more payloads can be separated from the rest of the fusion molecule by cleaving a single linker, the fusion molecule can contain a single cleavable linker.Furthermore, if one or more payloads are, for example, dimers or other multimers, each subunit of one or more payloads can be separated from the rest of the fusion molecule and / or other subunits of one or more payloads by cleaving a cleavable linker.

[0203] In various embodiments, the cleavable linker is cleaved by a cleaving enzyme present at or near the basolateral membrane of the epithelial cell. By selecting a cleavable linker that will be cleaved by such an enzyme, one or more payloads can be released from the epithelial cell into the basolateral membrane of the fusion molecule after transcytosis across the mucosa and released from the epithelial cell into the cellular matrix on the basolateral side of the membrane. Furthermore, a cleaving enzyme present in the epithelial cell can be used so that the cleavable linker is cleaved before the fusion molecule is released from the basolateral membrane, as long as the cleaving enzyme does not cleave the fusion molecule before it enters a transport pathway within the polarized epithelial cell that results in the release of the fusion molecule and one or more payloads from the basolateral membrane of the cell.

[0204] In various embodiments, the cleavable linker exhibits a greater propensity for cleavage than the remainder of the delivery construct. As those skilled in the art will recognize, many peptide and polypeptide sequences can be cleaved by peptidases and proteases. In various embodiments, the cleavable linker is selected so that it is preferentially cleaved relative to other amino acid sequences present in the delivery construct during administration of the delivery construct. In various embodiments, the receptor binding domain is substantially (e.g., about 99%, about 95%, about 90%, about 85%, about 80%, or about 75%) intact after delivery of the delivery construct to the subject's bloodstream. In various embodiments, the transcytosis activity is substantially (e.g., about 99%, about 95%, about 90%, about 85%, about 80%, or about 75%) intact after delivery of the delivery construct to the subject's bloodstream. In various embodiments, the macromolecule is substantially (e.g., about 99%, about 95%, about 90%, about 85%, about 80%, or about 75%) intact after delivery of the delivery construct into the subject's bloodstream. In various embodiments, the cleavable linker is substantially (e.g., about 99%, about 95%, about 90%, about 85%, about 80%, or about 75%) cleaved after delivery of the delivery construct into the subject's bloodstream.

[0205] In other embodiments, the cleavable linker can be cleaved using a cleaving enzyme found in the subject's plasma. Any cleaving enzyme known to those of skill in the art to be present in the subject's plasma can be used to cleave the cleavable linker.

[0206] In various embodiments, the cleavable linker is cleaved by a cleaving enzyme found in the subject's plasma. Any cleaving enzyme known by those skilled in the art to be present in the subject's plasma can be used to cleave the cleavable linker. In some cases, the delivery construct can be cleaved using a plasma cleaving enzyme. In other embodiments, the cleavable linker comprises a nucleic acid, such as RNA or DNA. In yet other embodiments, the cleavable linker comprises a carbohydrate, such as a disaccharide or trisaccharide.

[0207] In various embodiments, the cleavable linker can be a cleavable linker that is cleaved after a change in the environment of the fusion molecule. For example, the cleavable linker can be a pH-sensitive cleavable linker that is cleaved by the change in pH that occurs when the fusion molecule is released from the basolateral membrane of polarized epithelial cells. For example, the intestinal lumen can be strongly alkaline, while plasma can be essentially neutral. Therefore, the cleavable linker can be a moiety that is cleaved by the transition of pH from alkaline to neutral. The change in the environment of the fusion molecule that cleaves the cleavable linker can be, without limitation, any environmental change known to those skilled in the art that occurs when the fusion molecule is released from the basolateral membrane of polarized epithelial cells. VIII. Non-cleavable Linkers

[0208] In various embodiments, the carrier and one or more payloads may be separated by a linker. When a linker is used, the linker can comprise one or more amino acids. Examples of linkers contemplated herein include sequences such as S, (GS)x, (GGS)x, (GGGS)x, (GGGGS)x, or (GGGGGS)x (where x = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). In some cases, the linker does not include a terminal S residue, e.g., SEQ ID NO: 4 (GGGGSGGGGSGGGG). Generally, the linker need not have any specific biological activity other than connecting proteins or preserving a minimum distance or other spatial relationship between proteins. However, in various embodiments, the constituent amino acids of the linker can be selected to affect certain properties of the molecule, such as folding, net charge, or hydrophobicity.

[0209] In various embodiments, the linker can form a covalent bond with both the carrier and the biologically active payload. Suitable linkers include straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. In various embodiments, the linker(s) can be connected to the constituent amino acids of the carrier and / or one or more payloads via their side chain groups (e.g., via a disulfide linkage to cysteine). In various embodiments, the linker is connected to the alpha carbon amino and / or carboxyl group of the terminal amino acid of the carrier and / or biologically active payload.

[0210] Bifunctional linker can be used to form desired conjugate, which has one functional group reactive with the group on carrier and another group reactive with the group on one or more payloads.Instead, derivatization can involve chemical treatment of targeting moiety.For example, the procedure for generating free sulfhydryl group in polypeptide such as antibody or antibody fragment is known (see U.S. Patent No. 4,659,839).

[0211] Many procedures and linker molecules are known for attaching various compounds, including radionuclide metal chelates, carriers, and drugs, to proteins such as antibodies. See, for example, European Patent Application No. 188,256; U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and 4,589,071; and Borlinghaus et al. (1987) Cancer Res. 47: 4071-4075. IX. Chemical Conjugation or Complexation of Payloads or Cations to Carriers

[0212] In various embodiments, the payload to be delivered to the subject is chemically conjugated to a carrier. Means for chemically conjugating molecules are well known to those of skill in the art.

[0213] The procedures for conjugating two molecules vary according to the chemical structure of the agent. Polypeptides typically contain various functional groups, such as carboxylic acid (COOH) or free amine (--NH) groups, available for reaction with suitable functional groups on other peptides or on linkers to connect the molecules thereto.

[0214] In various embodiments of the present disclosure, isolated carriers are prepared by bacterial fermentation and purified using established methods. The purified carrier is then modified at its C-terminus to enable direct chemical coupling via a free sulfhydryl residue located near the C-terminus of the protein. The C-terminal modifications can include a cysteine-constrained loop with the consensus cleavage sequence ENLFQS for the highly selective protease from tobacco etch virus (TEV), a second cysteine, and a hexa-histidine (His6) tag (e.g., SEQ ID NO: 77 and SEQ ID NO: 78). The second Cys is included to form a disulfide bridge with the Cys ultimately used for coupling. The addition of the His6 sequence to the protein can simplify purification, and the TEV cleavage sequence provides a mechanism for selectively removing the terminal Cys residue after mild reduction. TEV cleavage and mild reduction with 0.1 mM dithiotheitol after expression and isolation of avirulent bacterial toxin constructs allows for the direct chemical coupling of glucose regulators, e.g., glucose-lowering agents, via maleimide-based reactions as a global mechanism of cargo attachment. After TEV protease cleavage, reduction, and cargo coupling via maleimide reaction with the free sulfhydryl, a second Ni 2+ Removal of the released C-terminal sequence was achieved by a column chromatography step.

[0215] In certain embodiments, the delivery construct comprises a particle covalently decorated with a carrier, and the payload is integrated into the particle. In certain embodiments, the particle can be smaller than approximately 150 nm, smaller than approximately 100 nm, or smaller than approximately 50 nm in diameter. Transcytosis test

[0216] The functionality of a carrier can be tested based on its ability to transport the carrier or a payload associated with the carrier, for example in a particle, across an epithelial membrane. Because transcytosis can first require binding to a cell, such assays can also be used to assess the functionality of cell recognition domains.

[0217] Transcytosis activity can be tested by any method known to those skilled in the art, without limitation. Transcytosis activity can be tested by assessing the ability of a composition or particle to enter non-polarized cells to which it is bound. The same properties that allow a carrier to pass through polarized epithelial cells also allow molecules carrying the carrier to enter non-polarized cells. Thus, the ability of a composition to enter a cell can be assessed, for example, by detecting the physical presence of the composition (e.g., carrier or payload) inside the cell. For example, the composition can be labeled, for example, with a fluorescent marker, and the delivery construct can be exposed to the cell. The cells can then be washed to remove any composition (e.g., carrier or payload that did not enter the cell, e.g., delivery construct), and the amount of label remaining can be determined. Detection of the label during this traction indicates that the composition has entered the cell.

[0218] The transcytosis ability of a composition can be tested by assessing the ability of a carrier or payload (e.g., a delivery construct) to pass through polarized epithelial cells. For example, the composition can be labeled with, for example, a fluorescent marker and contacted with the apical membrane of a layer of epithelial cells. Fluorescence detected at the basolateral side of the membrane formed by the epithelial cells indicates that the carrier is functioning properly. Cleavable linker cleavage test

[0219] The functionality of a cleavable linker can generally be tested in a cleavage assay. Without limitation, any suitable cleavage assay known to those skilled in the art can be used to test the cleavable linker. Both cell-based and cell-free assays can be used to test the ability of an enzyme to cleave a cleavable linker.

[0220] An exemplary cell-free assay for examining cleavage of a cleavable linker involves preparing an extract of polarized epithelial cells and exposing a labeled fusion molecule having a cleavable linker to a fraction of the extract corresponding to a membrane-associated enzyme. In such an assay, the label can be attached to either the glucose-regulating agent to be delivered, e.g., a glucose-lowering agent, or to the remainder of the fusion molecule. Among these enzymes are cleavage enzymes found near the basolateral membrane of polarized epithelial cells, as described above. For example, cleavage can be detected by, e.g., binding an antibody to the fusion molecule and washing away unbound molecules. If the label is attached to the glucose-regulating agent to be delivered, e.g., a glucose-lowering agent, little or no label should be observed in the antibody-bound molecule. Alternatively, the binding agent used in the assay can be specific for the glucose-regulating agent, e.g., a glucose-lowering agent, and can label the remainder of the construct. In either case, cleavage can be assessed.

[0221] Cleavage can also be examined using cell-based assays that examine cleavage by polarized epithelial cells assembled into membranes. For example, a labeled fusion molecule or portion of a fusion molecule containing a cleavable linker can be contacted with either the apical or basolateral side of a monolayer of suitable epithelial cells, such as Coco-2 cells, under conditions that allow for linker cleavage. Cleavage can be detected by detecting the presence or absence of the label using a reagent that specifically binds to the fusion molecule or its portion. For example, an antibody specific for the fusion molecule can be used to bind a fusion molecule containing a label distal to the cleavable linker relative to the portion of the fusion molecule bound by the antibody. Cleavage can then be assessed by detecting the presence of the label in the antibody-bound molecule. If cleavage has occurred, little or no label should be observed in the antibody-bound molecule. Such experiments can identify enzymes that cleave preferentially at the basolateral membrane rather than the apical membrane and further confirm the ability of such enzymes to cleave the cleavable linker in the fusion molecule.

[0222] Additionally, cleavage can be examined using a fluorescent reporter assay, as described in U.S. Patent No. 6,759,207. Briefly, in such an assay, a fluorescent reporter is contacted with the basolateral side of a monolayer of suitable epithelial cells under conditions that allow the cleavage enzyme to cleave the reporter. Cleavage of the reporter changes the conformation of the fluorescent reporter, converting it from a non-fluorescent to a fluorescent conformation. The amount of fluorescence observed can indicate the activity of the cleavage enzyme present in the basolateral membrane.

[0223] Additionally, cleavage can be examined using intramolecularly quenched molecular probes, such as those described in U.S. Patent No. 6,592,847. Such probes generally contain a fluorescent moiety that emits a photon when excited with light of an appropriate wavelength and a quencher moiety that absorbs the photon when in close proximity to the fluorescent moiety. Cleavage of the probe separates the quencher moiety from the fluorescent moiety, allowing fluorescence to be detected, thereby indicating that cleavage has occurred. Thus, such probes can be used to identify and assess cleavage by a specific cleavage enzyme by contacting the probe with the basolateral side of a monolayer of suitable epithelial cells under conditions that allow the cleavage enzyme to cleave the probe. The amount of fluorescence observed indicates the activity of the cleavage enzyme being examined. X.How to use

[0224] The methods and compositions, e.g., pharmaceutical compositions, of the present disclosure can be used to treat diseases or conditions, e.g., medical conditions. The methods and compositions can be suitable for oral and / or intranasal formulation and delivery. The disease or condition can be an immune disease, a metabolic disease, or a central nervous system (CNS) disease. "Metabolic disease or disorder" can refer to a combination of medical disorders that, when occurring together, increase the risk of diabetes and atherosclerotic vascular diseases, e.g., heart disease and stroke. The definition of medical parameters for metabolic syndrome includes diabetes mellitus, impaired glucose tolerance, elevated fasting blood glucose, insulin resistance, urinary albumin secretion, central obesity, hypertension, elevated triglycerides, elevated LDL cholesterol, and reduced HDL cholesterol.

[0225] The methods and compositions provided herein, e.g., pharmaceutical compositions, can be used to treat neurological conditions, immunological and endocrinological conditions, immunoneurological conditions, neuroendocrinological conditions, or immunoendocrinological conditions. The methods and compositions provided herein, e.g., pharmaceutical compositions, can be used to treat cardiovascular conditions, rare diseases, liver diseases, inflammatory bowel disorders, respiratory conditions, neurological conditions, or gastrointestinal conditions. The compositions provided herein can be vaccines. Diseases or conditions include, for example, viral diseases or infections, cancer, metabolic diseases, obesity, autoimmune diseases, inflammatory diseases, allergies, graft-versus-host disease, systemic microbial infections, anemia, cardiovascular diseases, psychiatric diseases, genetic diseases, neurodegenerative diseases, hematopoietic cell disorders, diseases of the endocrine or reproductive system, and gastrointestinal diseases. Further examples of diseases include diabetes, diabetes as a result of obesity, hyperglycemia, dyslipidemia, hypertriglyceridemia, Syndrome X, insulin resistance, impaired glucose tolerance (IGT), diabetic dyslipidemia, hyperlipidemia, fatty liver disease, non-alcoholic steatohepatitis (NASH), hepatitis, obesity, vascular disease, heart disease, stroke, impaired glucose tolerance, elevated fasting glucose, insulin resistance, urinary albumin secretion, central obesity, hypertension, elevated triglycerides, elevated LDL cholesterol and decreased HDL cholesterol, The diseases or conditions include, but are not limited to, hyperglycemia, hyperinsulinemia, dyslipidemia, ketosis, hypertriglyceridemia, syndrome X, insulin resistance, impaired fasting glucose, impaired glucose tolerance (IGT), diabetic dyslipidemia, gluconeogenesis, excessive glycogenolysis, diabetic ketoacidosis, hypertriglyceridemia, hypertension, diabetic nephropathy, renal insufficiency, renal failure, hyperphagia, muscle wasting, diabetic neuropathy, diabetic retinopathy, diabetic coma, arteriosclerosis, coronary heart disease, peripheral arterial disease, fibrosis, and hyperlipidemia. The disease or condition may be ulcerative colitis, Crohn's disease, pouchitis, psoriatic arthritis, rheumatoid arthritis, or psoriasis. The disease or condition may be a gastroenterological condition, such as short bowel syndrome (SBS). The disease or condition may be growth hormone deficiency.In some cases, the compositions provided herein include GLP-2, and the compositions are administered, for example, orally, to a subject with a gastroenterological condition, such as short bowel syndrome (SBS). In some cases, the compositions provided herein include GLP-1, and the compositions are administered, for example, orally, to a subject to treat a metabolic disease; the compositions can be formulated for oral delivery for local gastrointestinal and / or systemic exposure. In some cases, the compositions provided herein include GLP-1 or a GLP-1 analog, and the compositions are administered, for example, orally, to a subject to treat a metabolic disorder (e.g., diabetes, obesity) or non-alcoholic steatohepatitis (NASH) or a central nervous system (CNS) condition. The compositions can be formulated for oral delivery. In some cases, the compositions provided herein include human growth hormone, and the compositions are administered to a subject with growth hormone deficiency or a related disorder to treat the growth hormone deficiency or a related disorder. The compositions can be formulated for oral delivery, allowing for systemic exposure of the target location for the payload. In some cases, the payload can reach the liver. In some cases, the compositions provided herein include an incretin. In some cases, the compositions provided herein are administered to a subject to regulate glycemic function.

[0226] In many chronic diseases, the oral and / or intranasal formulations of the present disclosure can be particularly useful because they allow long-term patient care and therapy by home administration without relying on injection treatments or drug protocols.The formulations of the present disclosure can be administered orally, pulmonary, intranasally, bucally, or sublingually.Therefore, in another aspect, the present disclosure relates to the use of pharmaceutical compositions that are self-assembling particles, e.g., microparticles, containing a carrier, a payload (e.g., a heterologous payload), and / or a non-naturally occurring fusion molecule as a drug substance in a pill or tablet for oral delivery of one or more payloads in the treatment of diseases and conditions that indicate the need for the use of one or more payloads contained in such formulations.

[0227] Pharmaceutical compositions that are particles, e.g., microparticles, comprising a carrier, payload (e.g., a heterologous payload), and / or fusion molecule of the present disclosure can offer several advantages over conventional techniques for local or systemic delivery of macromolecules to a subject. Among these advantages is the ability to deliver one or more payloads to a subject without using a needle to pierce the subject's skin. Many subjects require repeated, regular doses of macromolecules. If delivery of macromolecules could be achieved without injections, avoiding the associated pain and potential complications would significantly improve the quality of life of such subjects.

[0228] In addition, coupling one or more payloads to the remainder of the fusion molecule via a linker that is cleaved by an enzyme present in the basolateral membrane of epithelial cells can allow one or more payloads to be released from the remainder of the fusion molecule immediately after transcytosis across the epithelial membrane. Such release can reduce the probability of inducing an immune response against the payload, for example, a biologically active payload. This can also allow one or more payloads to interact with their target in a form free from the remainder of the fusion molecule.

[0229] Furthermore, upon transport across the GI epithelium, the fusion molecules of the present disclosure will exhibit an extended serum half-life, i.e., the payload(s) of the fusion molecule may exhibit an extended serum half-life compared to the payload(s) in their unfused context, and oral administration of the fusion molecule may deliver a higher effective concentration of the delivered payload(s) to the liver of a subject than that observed in the subject's plasma.

[0230] The disclosed constructs can reduce the payload's sensitivity to proteolytic destruction, aid in chimera refolding, and improve chimera stability during storage. Thus, the fusion molecules can be used in the preparation of a new class of pharmaceutical compositions for oral administration of biologically active therapeutic agents.

[0231] As used herein, the terms "co-administration," "co-administered," and "in combination with," in reference to microparticles or nanoparticles of the present disclosure and one or more other therapeutic agents, include the simultaneous administration of such a combination of microparticles or nanoparticles of the present disclosure and therapeutic agent(s) to a patient in need of treatment when such components are formulated together into a single dosage form that releases the components to the patient substantially simultaneously; the substantially simultaneous administration of such a combination of microparticles or nanoparticles of the present disclosure and therapeutic agent(s) to a patient in need of treatment when such components are formulated apart from one another into separate dosage forms that are taken by the patient substantially simultaneously, thereby releasing the components to the patient substantially simultaneously; and the substantially simultaneous administration of such a combination of microparticles or nanoparticles of the present disclosure and therapeutic agent(s) to a patient in need of treatment when such components are formulated apart from one another into separate dosage forms that are taken by the patient substantially simultaneously, thereby releasing the components to the patient substantially simultaneously. and the sequential administration of such combinations of microparticles or nanoparticles of the present disclosure and therapeutic agent(s) to a patient in need of treatment when the components are formulated together in a single dosage form that releases the components in a controlled manner, whereby the components are released to the patient at the same and / or different times, in a concurrent, sequential, and / or overlapping manner, and each portion may be administered by either the same or different routes.

[0232] In various embodiments, the combination therapy comprises administering the isolated microparticle or nanoparticle composition and the second pharmaceutical composition simultaneously, either in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the isolated microparticle or nanoparticle composition and the second pharmaceutical composition are administered sequentially, i.e., the isolated microparticle or nanoparticle composition is administered either before or after administration of the second pharmaceutical composition.

[0233] In various embodiments, the administration of the particle, e.g., microparticle or nanoparticle composition and the second pharmaceutical composition is concurrent, i.e., the administration periods of the isolated particle, e.g., microparticle or nanoparticle composition and the second pharmaceutical composition overlap with each other.

[0234] In various embodiments, the administration of particles, for example, microparticle or nanoparticle compositions, and the second pharmaceutical composition is non-concurrent.For example, in various embodiments, the administration of isolated particles, for example, microparticle or nanoparticle compositions, is terminated before the administration of the second pharmaceutical composition.In various embodiments, the administration of the second pharmaceutical composition is terminated before the administration of isolated particles, for example, microparticle or nanoparticle compositions.

[0235] In various embodiments, a therapeutically effective amount of the microparticles or nanoparticles described herein will be administered in combination with one or more other therapeutic agents. Such therapeutic agents may be accepted in the art as standard treatment for the particular disease state described herein, such as metabolic disorders, fatty liver disease, inflammatory diseases, autoimmune diseases, cancer, or growth hormone (GH) deficiency growth disorders. Exemplary therapeutic agents contemplated include, but are not limited to, cytokines, growth factors, steroids, NSAIDs, DMARDs, anti-inflammatory drugs, chemotherapeutic agents, radiotherapeutic agents, or other active and ancillary agents.

[0236] In another aspect, the present disclosure provides a method for treating obesity (e.g., >30 kg / m 2 The present disclosure relates to a method of treating a subject classified as having a body mass index (BMI) of 100 or higher, the method comprising administering to the subject a therapeutically effective amount of a composition, e.g., particle, comprising a carrier and a payload (e.g., a delivery construct) of the present disclosure.

[0237] In another aspect, the present disclosure relates to a method for treating a subject diagnosed with type 1 diabetes (T1D), comprising orally administering a composition, e.g., a particle, comprising a carrier and a payload (e.g., a delivery construct) of the present disclosure in an amount sufficient to treat the disease, without insulin supplementation.

[0238] In another aspect, the present disclosure relates to a method for treating a subject diagnosed with type 1 diabetes (T1D), comprising: (a) orally administering a composition, e.g., particles, comprising a carrier and payload (e.g., a delivery construct) of the present disclosure in an amount sufficient to treat the disease; and (b) insulin supplementation. In certain embodiments, the insulin supplementation comprises administering a dose of insulin that can be between about 70%-90%, between about 50%-70%, between about 30%-50%, between about 15%-30%, between about 10%-15%, between about 5%-10%, and between zero and 5%, including 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%, of a normal daily dosage of insulin.

[0239] In another aspect, the present disclosure relates to a method for treating a subject diagnosed with type 2 diabetes (T2D), comprising orally administering a composition, e.g., a particle, comprising a carrier and a payload (e.g., a delivery construct) of the present disclosure in an amount sufficient to treat the disease.

[0240] In another aspect, the present disclosure provides a method of treating a subject having a fatty liver disease (e.g., non-alcoholic fatty liver disease (NAFLD); non-alcoholic steatohepatitis (NASH)), a gastrointestinal disease, or a neurodegenerative disease, comprising orally administering a composition, e.g., a particle, comprising a carrier and a payload (e.g., a delivery construct) of the present disclosure in an amount sufficient to treat the disease. XI. Polynucleotides Encoding Carriers, Payloads, and Fusion Molecules

[0241] In another aspect, the disclosure provides polynucleotides comprising nucleotide sequences encoding a carrier, a payload (e.g., a heterologous payload), and a non-naturally occurring fusion molecule. Such polynucleotides are useful, for example, for producing carriers, payloads (e.g., heterologous payloads), and fusion molecules. In yet another aspect, the disclosure provides expression systems comprising a recombinant polynucleotide sequence encoding a carrier, e.g., a colix carrier or PE, and a polynucleotide sequence encoding a payload, e.g., a glucose regulator, e.g., a glucose-lowering agent or cation, at a polylinker insertion site. In various embodiments, the expression system can comprise a polynucleotide sequence encoding a cleavable linker such that cleavage at the cleavable linker separates the payload, e.g., a glucose-lowering agent, encoded by the nucleic acid inserted at the polylinker insertion site from the remainder of the encoded fusion molecule. Thus, in embodiments where the polylinker insertion site is at the end of the encoded construct, the polynucleotide comprises a single nucleotide sequence encoding a cleavable linker between the polylinker insertion site and the remainder of the polynucleotide. In embodiments where the polylinker insertion sites are not at the ends of the encoded construct, the polylinker insertion sites may be flanked by nucleotide sequences that each encode a cleavable linker.

[0242] Various in vitro methods that can be used to prepare polynucleotides encoding carriers of the present disclosure, such as colix carriers or PEs, payloads, or fusion molecules, include, but are not limited to, reverse transcription, polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), self-sustained sequence replication systems (3SR), and QP replicase amplification systems (QB).

[0243] Guidance for using these cloning and in vitro amplification methodologies can be found, for example, in U.S. Pat. No. 4,683,195; Mullis et al., 1987, Cold Spring Harbor Symp. Quant. Biol. 51:263; and Erlich, ed., 1989, PCR Technology, Stockton Press, NY. Polynucleotides encoding fusion molecules or portions thereof can also be isolated by screening genomic or cDNA libraries with probes selected from the sequence of the desired polynucleotide under stringent, moderately stringent, or highly stringent hybridization conditions.

[0244] Construction of a nucleic acid encoding a carrier, payload, or fusion molecule of the present disclosure can be facilitated by introducing into the construct an insertion site for a nucleic acid encoding a glucose-lowering agent.

[0245] Additionally, the polynucleotide may encode a secretory sequence at the amino terminus of the encoded carrier, payload, or fusion molecule. Such constructs are useful for producing the carrier, payload, or fusion molecule in mammalian cells to simplify isolation of the immunogen.

[0246] Furthermore, the polynucleotide of the present disclosure also encompasses derivative versions of the polynucleotide that encodes a carrier, a payload, or a fusion molecule.For example, derivatives can be produced by site-directed mutagenesis, including the substitution, insertion, or deletion of 1, 2, 3, 5, 10, or more nucleotides of the polynucleotide that encodes a fusion molecule.Alternatively, derivatives can be produced by random mutagenesis.

[0247] Thus, in various embodiments, the present disclosure provides a polynucleotide encoding a carrier, payload, or fusion molecule. The carrier, payload, or fusion molecule can include a modified carrier and a payload to be delivered to a subject, such as a glucose regulator, e.g., a glucose-lowering agent; and, optionally, a cleavable linker. Cleavage at the cleavable linker can separate the payload, e.g., the glucose regulator, e.g., the glucose-lowering agent, from the remainder of the fusion molecule. The cleavable linker can be cleaved by an enzyme present in the basolateral membrane of a polarized epithelial cell of a subject or in the plasma of a subject.

[0248] In various embodiments, the polynucleotide hybridizes under stringent hybridization conditions to any of the polynucleotides of the present disclosure. In further embodiments, the polynucleotide hybridizes under stringent hybridization conditions to a nucleic acid encoding any of the carriers, payloads, or fusion molecules of the present disclosure.

[0249] In another aspect, the present disclosure provides an expression vector for expressing a carrier, payload, or fusion molecule. Generally, an expression vector can be a recombinant polynucleotide molecule comprising an expression control sequence operably linked to a nucleotide sequence encoding a polypeptide. Expression vectors can be easily adapted for function in prokaryotes or eukaryotes by incorporating appropriate promoters, replication sequences, selectable markers, etc., to effect stable transcription and translation of mRNA. Techniques for constructing expression vectors and expressing genes in cells containing expression vectors are well known in the art. See, for example, Sambrook et al., 2001, Molecular Cloning—A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, and Ausubel et al., eds., Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

[0250] The expression vector can contain expression and replication signals compatible with the cells in which the carrier, payload, or fusion molecule is expressed. The expression vector can be introduced into cells for expression of the carrier, payload, or fusion molecule by any method known to those skilled in the art, without limitation. The expression vector can also contain a purification moiety that simplifies isolation of the carrier, payload, or fusion molecule.

[0251] In yet another aspect, the present disclosure provides cells comprising expression vectors for expressing carriers, payloads, or fusion molecules, or portions thereof. The cells can be selected for their ability to express high concentrations of the carriers, payloads, or fusion molecules to facilitate protein purification. In various embodiments, the cells are prokaryotic cells, such as E. coli. For example, as described in the Examples, the carriers, payloads, and fusion molecules can be properly folded and contain appropriate disulfide linkages when expressed in E. coli.

[0252] In other embodiments, the cell is a eukaryotic cell. Useful eukaryotic cells include yeast and mammalian cells. Any mammalian cell known to those skilled in the art to be useful for expressing recombinant polypeptides can be used to express the carrier, payload, or fusion molecule, without limitation. For example, Chinese hamster ovary (CHO) cells can be used to express the carrier, payload, or fusion molecule.

[0253] The carriers, payloads, or fusion molecules of the present disclosure can be produced recombinantly, as described below, however, the carriers, payloads, or fusion molecules can also be produced by chemical synthesis using methods known to those skilled in the art.

[0254] Methods for expressing and purifying the carriers, payloads, and fusion molecules of the present disclosure are described extensively herein, for example, in the Examples below. Generally, these methods rely on the introduction of an expression vector encoding the carrier, payload, and / or fusion molecule into a cell capable of expressing the carrier, payload, and / or fusion molecule from the vector. The carrier, payload, and / or fusion molecule can then be purified for administration to a subject, for example, in the treatment of diseases and conditions that necessitate the use of one or more payloads contained in such formulations. XII. Use of Microparticle or Nanoparticle Pharmaceutical Compositions for Pulmonary Delivery

[0255] The particle compositions, e.g., microparticle pharmaceutical compositions disclosed herein can be used as drug substances for pulmonary delivery of payloads, e.g., biologically active payloads. Pulmonary delivery methods can include nebulization or dry powder inhalation.

[0256] Particles, for example, microparticles or nanoparticles, pharmaceutical compositions can be formulated for pulmonary delivery. Pharmaceutical compositions formulated for pulmonary administration can be easily nebulized or aerosolized. In some cases, pharmaceutical compositions formulated for pulmonary administration utilize the carrier's ability to mediate transcytosis across the pulmonary epithelium. Intranasal administration can be used for pulmonary delivery, and can include inhaling or sniffing the powder. XIII. Uses of the Compositions for Oral Delivery

[0257] The compositions, e.g., particle, e.g., microparticle or nanoparticle compositions, e.g., microparticle pharmaceutical compositions, disclosed herein can be used as drug substances in pills or tablets for oral delivery of payloads, e.g., biologically active payloads, to individuals, e.g., in the treatment of diseases and conditions indicating the use of one or more payloads contained in such formulations.

[0258] The compositions, e.g., microparticle or nanoparticle pharmaceutical compositions, can be formulated for oral delivery. Pharmaceutical compositions formulated for oral administration can be resistant to degradation in the digestive tract.

[0259] In some cases, pharmaceutical compositions formulated for oral administration utilize the ability of carriers to mediate transcytosis across gastrointestinal (GI) epithelia. Oral administration of such pharmaceutical compositions can result in absorption of the carrier and payload (e.g., as a fusion molecule) through polarized epithelial cells of the gastrointestinal mucosa, e.g., the intestinal mucosa, followed by release of the payload, e.g., one or more payloads, at the basolateral side of the mucosa. Pulmonary administration of such pharmaceutical compositions can result in absorption of the carrier and payload through polarized epithelial cells of the lungs and airways. The epithelial cells can be nasal epithelial cells, oral epithelial cells, intestinal epithelial cells, rectal epithelial cells, vaginal epithelial cells, or pulmonary epithelial cells. The pharmaceutical compositions of the present disclosure can include the addition of a transcytosis enhancer to facilitate movement of the fusion protein across the GI or pulmonary epithelia. Such enhancers are known in the art; see, e.g., Xia et al., (2000) J. Pharmacol. Experiment. Therap., 295:594-600; and Xia et al. (2001) Pharmaceutical Res., 18(2):191-195, each of which is incorporated by reference in its entirety.

[0260] Upon transport across the epithelium, compositions of the present disclosure, e.g., microparticle or nanoparticle pharmaceutical compositions, can exhibit an extended serum half-life, i.e., the payload, e.g., a biologically active payload (e.g., of a fusion molecule), can exhibit an extended serum half-life compared to the payload, e.g., a biologically active payload, in its non-fused state. Oral formulations of pharmaceutical compositions of the present disclosure can be prepared for transport to the GI epithelium and protection of the carrier, payload, or fusion molecule in the stomach. Such formulations can include carrier and dispersant components and can be in any suitable form, including aerosols (for oral or pulmonary delivery), syrups, elixirs, tablets, including chewable tablets, hard or soft capsules, troches, lozenges, aqueous or oily suspensions, emulsions, cachets or pellet granules, and dispersible powders. In various embodiments, the pharmaceutical compositions are used in solid dosage forms, e.g., tablets, capsules, etc., suitable for simple oral administration of precise dosages.

[0261] In various embodiments, the oral formulation comprises a microparticle pharmaceutical composition and one or more compounds capable of protecting the carrier, payload, or fusion molecule, or the unfused carrier and payload molecules, when present in the stomach. For example, the protective compound should be capable of preventing acid and / or enzymatic hydrolysis of the molecules. In various embodiments, the oral formulation comprises a microparticle pharmaceutical composition and one or more compounds capable of facilitating the passage of the construct(s) from the stomach to the small intestine. In various embodiments, one or more compounds capable of protecting the carrier, payload, or fusion molecule from degradation in the stomach can also facilitate the passage of the construct from the stomach to the small intestine. For example, the inclusion of sodium bicarbonate can be useful for facilitating the rapid passage of intragastrically delivered materials from the stomach to the duodenum, as described by Mrsny et al., Vaccine 17:1425-1433, 1999. Other methods for formulating a formulation so that the carrier, payload, or fusion molecule can pass through the stomach and contact the polarized epithelial membrane in the small intestine include, but are not limited to, the enteric coating techniques described in DeYoung, Int J Pancreatol, 5 Suppl:31-6, 1989, and the methods provided in U.S. Pat. Nos. 6,613,332, 6,174,529, 6,086,918, 5,922,680, and 5,807,832, each of which is incorporated herein by reference in its entirety.

[0262] In some cases, the protective compound is a cation that stabilizes acid-resistant microparticles or nanoparticles. In some cases, particles comprising a carrier, a cation, and a heterologous payload can be resistant to pancreatic enzymes without the need for an enteric coating or other stabilizing compound. For example, Figure 11 shows the results of a pancreatin digestion assay performed on a formulation containing a carrier protein (SEQ ID NO: 3) along with a cation (either zinc or protamine) and a heterologous payload (exenatide). Lanes 1-4 in Figure 11A show pancreatin degradation of a composition of SEQ ID NO: 3 protein after 0, 30, 60, and 120 minutes; after 120 minutes, the protein is completely degraded. However, lanes 5-8 show a formulation of SEQ ID NO: 3 protein and zinc salt prepared by mixing the components in a 1:1 ratio (w / w carrier:zinc salt). Lane 8 in Figure 11A shows that this formulation is only partially degraded after 120 minutes of incubation with pancreatin. Similar results were seen in formulations of SEQ ID NO:3, zinc, and exenatide mixed (missed) in a 1:1:1 or 1:2:1 ratio. Figure 11B shows similar results using protamine as the cation instead of zinc. Figure 11C shows the results of pancreatin treatment in a formulation containing exenatide and zinc without SEQ ID NO:3. In some cases, the compositions described herein may be resistant to pancreatin cleavage. In some cases, the compositions described herein may be resistant to pancreatin cleavage such that at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the heterologous payload and / or carrier protein remains intact after incubating the composition with pancreatin for 30 minutes at 37°C. In some cases, the compositions described herein may be resistant to pancreatin cleavage such that at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the heterologous payload and / or carrier protein is intact after incubating the composition with pancreatin for 60 minutes at 37°C.In some cases, the compositions described herein may be resistant to pancreatin cleavage such that at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the heterologous payload and / or carrier protein is intact after incubating the composition with pancreatin for 120 minutes at 37° C. In some cases, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the therapeutic protein is intact at 0.5, 1, or 2 hours in a pancreatin assay; the pancreatin assay comprises incubating a composition containing 100 μg of therapeutic protein with 10 μg of pancreatin in 100 μL PBS at 37° C.

[0263] Pharmaceutical compositions intended for oral use can be prepared according to any method known in the art for the manufacture of pharmaceutical compositions, and such formulations can contain one or more agents selected from the group consisting of sweeteners to provide medicament-elegant and palatable preparations.For example, to prepare orally deliverable tablets, the microparticle pharmaceutical composition is mixed with at least one pharmaceutical excipient, and the solid formulation is compressed according to known methods to form tablets for delivery to the gastrointestinal tract.Tablet compositions are typically formulated with additives, such as binders such as saccharide or cellulose carriers, starch paste or methylcellulose, fillers, disintegrants, or other additives typically commonly used in the manufacture of medical preparations.To prepare orally deliverable capsules, DHEA is mixed with at least one pharmaceutical excipient, and the solid formulation is placed in a capsule container suitable for delivery to the gastrointestinal tract.Formulations containing carriers, payloads, or fusion molecules are described in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack, 2001), which is incorporated herein by reference. Publishing Co. Easton Pa. 18042) at Chapter 89.

[0264] In various embodiments, pharmaceutical compositions are formulated as orally deliverable tablets, containing microparticle pharmaceutical compositions in a mixture with non-toxic pharmaceutically acceptable excipients suitable for tablet manufacture.Such excipients can be inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents such as corn starch, gelatin or acacia, and lubricants such as magnesium stearate, stearic acid or talc.Tablets can be coated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over a longer period of time.For example, time-delay materials such as glyceryl monostearate or glyceryl distearate can be used alone or with wax.

[0265] In various embodiments, the pharmaceutical compositions are formulated as hard gelatin capsules in which the carrier, payload, and fusion molecule are mixed with an inert solid diluent, such as calcium carbonate, calcium phosphate, or kaolin, or as soft gelatin capsules in which the carrier, payload, or fusion molecule are mixed with an aqueous or oil medium, such as peanut oil, peanut oil, liquid paraffin, or olive oil.

[0266] In various embodiments, aqueous suspensions can contain the microparticle pharmaceutical composition in a mixture with excipients suitable for the manufacture of aqueous suspensions.Such excipients are suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia; dispersing or wetting agents can be naturally occurring phosphatides, such as lecithin, or condensates of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensates of ethylene oxide with long-chain aliphatic alcohols, such as heptadecylethyloxycetanol, or condensates of ethylene oxide with partial esters derived from fatty acids and hexitols, such as polyoxyethylene sorbitol monooleate, or condensates of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, such as polyoxyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

[0267] In various embodiments, oily suspensions can be prepared by suspending microparticle pharmaceutical compositions in vegetable oils, such as peanut oil, olive oil, sesame oil or coconut oil, or in mineral oils such as liquid paraffin.Oil suspensions can contain thickeners, such as beeswax, hard paraffin or cetyl alcohol.Sweeteners such as those listed above and flavoring agents can be added to make oral preparations palatable.Such preparations can be preserved by adding antioxidants such as ascorbic acid.

[0268] In various embodiments, pharmaceutical compositions can be in the form of oil-in-water emulsion.Oil phase can be vegetable oil, for example, olive oil or peanut oil, or mineral oil, for example, gum acacia or gum tragacanth, naturally occurring phosphatides, for example, soybean lecithin, and the ester or partial ester derived from fatty acid and hexitol anhydride, for example, sorbitan monooleate, and the condensation product of the same partial ester with ethylene oxide, for example, polyoxyethylene sorbitan monooleate.Emulsion can also contain sweetener and flavoring agent.

[0269] It is possible to use encapsulated or coated tablets that release one or more payloads in a layer-by-layer manner, thereby releasing one or more payloads over a predetermined time frame while traveling along the gastrointestinal tract.In addition, tablets containing one or more payloads can be placed inside a larger tablet, thereby protecting the inner tablet from environmental and processing conditions such as temperature, chemicals (e.g., solvents), pH, and humidity.The outer tablet and coating also function to protect one or more payloads in the gastric environment.In some cases, encapsulated particles can be placed inside a larger tablet or capsule, which is encapsulated so that the larger tablet or capsule dissolves in a first environment and the encapsulated particles dissolve in a second environment.In some cases, the encapsulated particles release the payload under a first condition but not under a second condition.For example, the encapsulated particles can release the payload at a high pH but not at a low pH. In some cases, the encapsulated particles have an enteric coating.

[0270] Surface active agents or surfactants facilitate absorption of polypeptides through mucous membranes or linings. Useful surface active agents or surfactants include fatty acids and their salts, bile salts, phospholipids, or alkyl saccharides. Examples of fatty acids and their salts include the sodium, potassium, and lysine salts of caprylic acid (C8), capric acid (C10), lauric acid (C12), and myristic acid (C14). Examples of bile salts include cholic acid, chenodeoxycholic acid, glycocholic acid, taurocholic acid, glycochenodeoxycholic acid, taurochenodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, taurodeoxycholic acid, lithocholic acid, and ursodeoxycholic acid. Examples of phospholipids include single-chain phospholipids such as lysophosphatidylcholine, lysophosphatidylglycerol, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylserine; or double-chain phospholipids such as diacylphosphatidylcholine, diacylphosphatidylglycerol, diacylphosphatidylethanolamine, diacylphosphatidylinositol, and diacylphosphatidylserine. Examples of alkylsaccharides include alkylglucosides or alkylmaltosides such as decylglucoside and dodecylmaltoside.

[0271] In another aspect, the present disclosure relates to a method for orally administering the pharmaceutical composition of the present disclosure. Oral administration of a microparticle pharmaceutical composition can result in the absorption of a carrier, a payload, or a fusion molecule through polarized epithelial cells of the digestive mucosa, for example, the intestinal mucosa, followed (in some cases) by the cleavage of the fusion molecule at the basolateral side of the mucosa and the release of one or more payloads.The one or more payloads can then be directly transported to the liver via the hepatic portal vein.Therefore, if one or more payloads exert biological activity in the liver, such as the activity mediated by one or more payloads binding to their cognate receptors, the one or more payloads are considered to exert an effect greater than that expected based on the plasma concentration observed in the subject, i.e., oral administration of a carrier, a payload, or a fusion molecule can deliver a higher effective concentration of the delivered one or more payloads to the subject's liver than that observed in the subject's plasma.

[0272] In another aspect, the present disclosure relates to a method for orally administering a pharmaceutical composition of the present disclosure. Such a method can include, but is not limited to, orally administering the formulation by a patient or caregiver. Such administration can include administration at intervals such as once or twice daily, depending on the carrier, payload, or fusion molecule, the disease or patient condition, or the individual patient. Such a method can also include administration of various dosages of the individual carrier, payload, or fusion molecule. For example, an initial dosage of the pharmaceutical composition can be higher to induce a desired effect, such as a reduction in blood glucose levels. Then, once the desired effect is achieved, subsequent dosages can be reduced. Such changes or modifications to the administration protocol can be performed by an attending physician or medical professional.

[0273] Such pharmaceutical compositions can be administered to subjects at appropriate doses. The dosage regimen will be determined by the attending physician based on specific clinical factors. As is well known in the medical field, the dosage for any one patient depends on many factors, including the patient's size, body surface area, age, sex, and overall health, as well as the specific compound administered, the time and route of administration, and other drugs administered concomitantly. The therapeutically effective amount for a given situation can be easily determined by routine experimentation and is within the skill and judgment of an ordinary clinician or physician. Those skilled in the art know that the effective amount of a pharmaceutical composition administered to an individual will depend, inter alia, on the nature of the biologically active payload. The length of treatment required to observe changes and the interval after treatment for a response to occur will vary depending on the desired effect. The specific amount can be determined by conventional tests well known to those skilled in the art.

[0274] The amount of one or more payloads is the amount that is effective for achieving the purpose of a specific active agent.The amount in the composition is typically a pharmacologically, biologically, therapeutically or chemically effective amount.However, when the composition is used in a dosage unit form such as a capsule, tablet or liquid, the amount may be less than a pharmacologically, biologically, therapeutically or chemically effective amount, because dosage unit form can contain various carriers / biologically or chemically active agent formulations or can contain divided pharmacologically, biologically, therapeutically or chemically effective amounts.Then, the total effective amount can be administered in cumulative units that contain a total of pharmacologically, biologically, therapeutically or chemically active amount of biologically active payload.

[0275] In some cases, intestinal administration of a composition of the present disclosure comprising 10 μg of exenatide can result in a maximum plasma concentration of greater than about 15 ng / mL or greater than about 30 ng / mL. In some cases, intestinal administration of a composition of the present disclosure comprising 10 μg of exenatide can result in a maximum plasma concentration of about 17.3 ng / mL or about 35.7 ng / mL. In some cases, intestinal administration of a composition of the present disclosure comprising 10 μg of exenatide can result in a time to maximum plasma concentration of about 60 minutes or about 45 minutes.

[0276] Unless otherwise specified herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include plural terms and plural terms shall include the singular. Generally, the nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those commonly used and well known in the art.

[0277] The term "about" as used herein can mean plus or minus 1%, 2%, 3%, 4%, 5%, or 10% of the number to which the term refers.

[0278] As used herein, the term "percent (%) sequence identity" and related terms, in the context of amino acid sequences, refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a selected sequence after aligning the sequences and introducing gaps, if necessary, without considering any conservative substitutions as part of the sequence identity to achieve the maximum percent sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in a variety of ways within the skill of those skilled in the art, for example, using publicly available computer software such as Clustal Omega, BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment across the entire length of the sequences being compared. [Example]

[0279] Example 1 This example describes the preparation of stable self-assembling microparticles containing non-naturally occurring fusion molecules for use as drug substances in pills or tablets for oral delivery of biologically active payloads. Specifically, a non-naturally occurring fusion molecule was prepared comprising a colix carrier molecule having the amino acid sequence of SEQ ID NO:2 coupled to a human growth hormone ("hGH") molecule having the amino acid sequence of SEQ ID NO:5 using the protamine-zinc coacervate system described below.

[0280] An exemplary fusion molecule expression vector containing SEQ ID NO:2 and hGH molecules was constructed as follows: First, the polypeptide gene was amplified by PCR, incorporating restriction enzyme pairs NdeI and EcoRI, PstI and PstI, AgeI and EcoRI, or PstI and EcoRI at two ends of the PCR product. After restriction enzyme digestion, the PCR product was cloned into an appropriate plasmid for cellular expression, which was then digested with the corresponding restriction enzyme pair. The resulting construct encodes SEQ ID NO:2 and hGH, and is tagged with a 6-His motif at the N-terminus of the polypeptide to facilitate purification. The final plasmid was verified by restriction enzyme digestion and DNA sequencing.

[0281] Fusion molecules were expressed as follows: To generate fusion molecule-expressing cells, E. coli BL21(DE3)pLysS competent cells (Novagen, Madison, Wis.) were transformed using the standard heat shock method in the presence of the appropriate plasmid, selected in ampicillin-containing medium, isolated, and grown in Luria-Bertani broth (Difco; Becton Dickinson, Franklin Lakes, NJ) with antibiotics, and then induced for protein expression by the addition of 1 mM isopropyl-D-thiogalactopyranoside (IPTG) at an OD of 0.6. Two hours after IPTG induction, cells were harvested by centrifugation at 5,000 rpm for 10 minutes. Following cell lysis, inclusion bodies were isolated, and the protein was solubilized in a buffer containing 100 mM Tris-HCl (pH 8.0), 2 mM EDTA, 6 M guanidine HCl, and 65 mM dithiothreitol. The solubilized fusion molecule was refolded in the presence of 0.1 M Tris, pH 7.4, 500 mM L-arginine, 0.9 mM GSSG, and 2 mM EDTA. The refolded protein was purified by Q Sepharose ion exchange and Superdex 200 gel filtration chromatography (Amersham Biosciences, Inc., Sweden). Protein purity was assessed by SDS-PAGE and analytical HPLC (Agilent, Inc., Palo Alto, Calif.). The resulting fusion molecule was prepared at a concentration of 1 mg / mL and stored in PBS at -80°C.

[0282] Protamine-based microparticles were produced as follows: (a) fusion molecules containing SEQ ID NO:2 and SEQ ID NO:5 were added to 0.1 N HCl in a 1.5 mL microcentrifuge tube to a final concentration of 1 mg / mL fusion molecule, and then 0.02 mL ZnCl (10 mg / mL in HO) was added to the fusion molecule solution; (b) 2.0 mL protamine sulfate (0.6 mg / mL) was added to 2.0 mL 0.1 M NaPO; (c) the mixture from step (a) was combined with the mixture from step (b), and the combined mixture was allowed to stand overnight at room temperature. A precipitate formed immediately after mixing (a) and (b), as evidenced by a transition from clear to turbid.

[0283] The next morning, particles were imaged using a GE Cytel system in high magnification (10x) brightfield mode. Particles were approximately 50 μM in size and formed spontaneously at room temperature after mixing of the reagents (see Figure 1A). By increasing the ionic strength of the solution by dropwise addition of 5 M NaCl, these particles could be broken down into smaller units of approximately 5 μM (see Figure 1B). Aggregates could be reassociated by decreasing the ionic strength by dropwise addition of Milli-Q prepared water (see Figure 1C).

[0284] These data demonstrate that the protamine-zinc coacervate system can be used to prepare stable self-assembling microparticles containing carrier-derived fusion molecules, that the system will assemble into different species of particles depending on the ionic strength of the buffer, and that the association and dissociation processes are reversible for such specific molecular assemblies. Self-assembling microparticles prepared using this "tunable" system can be used as drug substances for the preparation of pills or tablets for oral delivery of biologically active payloads.

[0285] Example 2 Using the methodology of Example 1, stable insulin- and insulin-FITC-containing microparticles were prepared and evaluated as described below.

[0286] In one preparation, insulin and insulin-FITC were added to 1.0 mL of 0.1 N HCl in a 2:1 ratio to a final concentration of 5 mg / mL in a 1.5 mL microcentrifuge tube, and then 0.02 mL of ZnCl (10 mg / mL in HO) was added to the insulin solution. In a second preparation, insulin-FITC was added to 1.0 mL of 0.1 N HCl in a 2:1 ratio to a final concentration of 5 mg / mL in a 1.5 mL microcentrifuge tube, and then 0.02 mL of ZnCl (10 mg / mL in HO) was added to the insulin solution. In a separate step, 2.0 mL of protamine sulfate (1 mg / mL) was added to 2.0 mL of 0.1 M NaPO. The protamine solution was then added to the insulin-containing solution and the insulin-FITC-containing solution, and the combined mixtures were allowed to stand overnight at room temperature. A precipitate formed immediately after mixing, as evidenced by a transition from clear to cloudy.

[0287] The following morning, particles containing insulin and / or insulin-FITC were imaged using a GE Cytel system in high magnification (10x) brightfield mode. Particles were approximately 50-150 μM in size and formed spontaneously at room temperature after mixing of the reagents. Figures 2A and 2B show brightfield and blue filter images, respectively. The blue arrow indicates an approximately 150 μM particle. Figure 2C shows a merged blue and brightfield image of the microparticles. The observed blue fluorescence indicates insulin-FITC incorporation into the particles. The nature of the fluorescence emission from these microparticles is consistent with their protein-based composition.

[0288] Example 3 In this example, stable microparticles containing non-naturally occurring fusion molecules comprising a corix carrier molecule having the amino acid sequence of SEQ ID NO: 6 coupled to a red fluorescent protein ("RFP") molecule were prepared and evaluated using the protamine-zinc coacervate system described below.

[0289] A fusion molecule comprising SEQ ID NO:6 coupled to red fluorescent protein ("RFP") was prepared as follows: A plasmid construct encoding SEQ ID NO:6 was prepared as described herein. Protein expression was achieved using E. coli DH5α cells (Invitrogen, Carlsbad, CA) after transformation with the appropriate plasmid by heat shock (42°C for 1 minute); transformed cells, selected in antibiotic-containing medium, were isolated and grown in Luria-Bertani broth (Difco); protein expression was induced by the addition of 1 mM isopropyl-D-thiogalactopyranoside (IPTG); 2 hours after IPTG induction, cells were harvested by centrifugation at 5,000 x g for 10 minutes at 4°C; inclusion bodies were isolated after cell lysis, and the protein was solubilized in 6 M guanidine HCl and 2 mM EDTA (pH 8.0) plus 65 mM dithiothreitol; refolding and purification were followed by incubation at -80°C for 1 minute in Ca. 2+ and Mg 2+ Proteins were stored at approximately 5 mg / mL in PBS (pH 7.4) lacking ATP. All proteins used in these studies were determined to be >90% pure based on size exclusion chromatography.

[0290] Next, the protein shown in SEQ ID NO:6 was modified at its C-terminus to allow direct chemical coupling via a free sulfhydryl residue located near the C-terminus of the protein. The C-terminal modification included a cysteine-constrained loop with a consensus cleavage sequence for the highly selective protease from tobacco etch virus (TEV), a second cysteine, and a hexa-histidine (His6) tag. The second Cys was included to form a disulfide bridge with the Cys ultimately used for coupling. The addition of the His6 sequence to the protein simplified purification, and the TEV cleavage sequence provided a mechanism for selectively removing the terminal Cys residue after mild reduction. Following expression and isolation of the SEQ ID NO:6 construct, TEV cleavage and mild reduction with 0.1 mM dithiothreitol enabled the direct chemical coupling of one or more payloads via a maleimide-based reaction as a global mechanism for payload attachment. Following TEV protease cleavage, reduction, and RFP coupling via maleimide reaction with the free sulfhydryl, a second Ni tag was added. 2+ Removal of the released C-terminal sequence was achieved by a column chromatography step, and the fusion molecule will henceforth be referred to as FM001.

[0291] Protamine-based microparticles were produced as follows: (a) FM001 was added to 0.1 N HCl to a final concentration of 1 mg / mL FM001 in a 1.5 mL microcentrifuge tube, and then 0.02 mL ZnCl (10 mg / mL in HO) was added to the FM001 solution; in a second preparation, ZnCl was omitted; (b) 2.0 mL protamine sulfate (0.6 mg / mL) was added to 2.0 mL 0.1 M NaPO; (c) each of the two mixtures from step (a) was combined with the mixture from step (b), and the combined mixture was allowed to stand overnight at room temperature. A precipitate formed immediately after mixing (a) and (b), as evidenced by a transition from clear to turbid.

[0292] The following morning, digital images of the particles were collected using a GE Cytel system. Red fluorescence was imaged by exciting the sample at 481 nm and recording the fluorescence emission at 535 nm. At 4x magnification, particles containing protamine, zinc, and FM001 were approximately 150 μM and uniform in size (Figure 3A). When excited with 481 nm light, the particles emitted red fluorescence (Figure 3B). The observed red fluorescence indicated FM001 incorporation into the particles. The fluorescence was uniform in distribution, indicating that FM001 was homogeneously distributed and that the protein structure was not disrupted during particle formation. At 10x magnification, omission of zinc from the coacervate resulted in the formation of particles with a similar size (approximately 150 μM), but the particles were more ovoid in shape (Figure 4A). Such particles have similar fluorescent properties to zinc-containing particles (Figure 4B), indicating that zinc is not required to maintain protein structure during coacervation and that the composition of the coacervate can determine the shape of the particle.

[0293] Example 4 In this example, a variety of different conditions and formulations were tested for producing spray-dried particles with desired properties. In this example, a number of different particle formulations were produced. These particles were generally made from a carrier, drug, a polymer matrix, PEG, a surfactant, and zinc.

[0294] A detailed example of the protocol for formulations 37-49 containing hGH / SEQ ID NO: 3 / Eudragit FS30D / Polysorbate 20 / ZnCl2 is provided below. A volume of 15 μL of polysorbate was added to a Falcon tube, followed by 7.6 mL of SEQ ID NO: 3 solution (9.5 mg, 0.27 μmol). Next, 3.0 mg (0.14 μmol) of hGH powder was dissolved in 4.0 mL of DI water in a separate 20 mL glass scintillation vial to obtain a clear solution, which was then transferred to the above solution in the Falcon tube. A volume of 15 μL of ZnCl2 stock solution (5 mg / mL) (0.075 mg, 0.55 μmol) was added to the mixture, followed by 82 mg of Eudragit FS30D suspension. The suspension was shaken on a rotisserie shaker for 20 minutes and then diluted with 50 mM ammonium bicarbonate solution to a total volume of 25 mL. The solution was filtered through a 0.4 um disc filter by Pall Labs before being spray dried in a Buchi B-90 nano spray dryer.

[0295] Spray drying was performed using the following conditions: medium nozzle for atomization / atomization, inlet temperature 110°C, outlet temperature 49°C, gas flow 130 liters / min, pump speed 12%, and atomization ratio 100%. Spray drying was completed in 2 hours. The product was collected as a white, free-flowing powder. The yield was around 56% by weight. The particle size was estimated by mechanical spectroscopy to be 300-600 nm. Other particles were prepared similarly.

[0296] Particles produced by different formulation conditions were first screened by gel electrophoresis to confirm protein quality after the spray-drying process. To do this, the particles were dissolved and the released proteins were analyzed by Western blot to ensure that the proteins were intact.

[0297] Different particles were then screened for encapsulation and release efficiency at physiological pH. Figure 5A shows the dissolution assay results for particles (37-156) containing insulin, SEQ ID NO: 3, and Eudragit FS. This formulation produced particles with a slow rate of drug release at pH 2 and a gradual drug release at pH 7. Increasing the pH from pH 2 to pH 7 induced a controlled release of the previously encapsulated drug. In some cases, this may represent the desired encapsulation and release profile. Figure 5B shows the dissolution assay results for particles (37-167) containing insulin, SEQ ID NO: 3, and Eudragit L30. This formulation produced particles with fast drug release at both pH 2 and pH 7. In some cases, this may not represent the desired encapsulation and release profile. Figure 5C shows the dissolution assay results for yet another particle formulation containing hGH and Eudragit FS. This formulation resulted in low drug release at pH 2, gradual drug release at pH 7, and controlled induction of drug release with increasing pH from pH 2 to pH 7. This release profile indicates that the formulation may potentially be suitable for oral administration, for example, by oral gavage.

[0298] Next, the formulations were tested for stability in simulated intestinal fluid with pancreatin. Different particles were exposed to simulated intestinal fluid with pancreatin and the amount of drug remaining after 1 and 14 hours was assessed. Figure 6 shows the percentage of drug remaining after 1 and 14 hours for an insulin standard solution and five different formulations. Details of the formulations in Figure 6 are listed in Table 1. All five of the spray-dried particles in Figure 6 provided long-term drug protection from pancreatin. Two different formulations, 37-166 (insulin / PEG8K / SEQ ID NO: 3 / Eudragit FS / Tween®-20 / ZnCl2) and 37-224 (insulin / SEQ ID NO: 3 / 2×Eudragit L30 / ZnCl2), produced insulin concentrations at 1 hour that were higher than those seen with insulin alone. [Table 1]

[0299] Example 5 In this example, the in vivo absorption of drugs from different particle compositions is evaluated. It is hypothesized that intestinal drug retention correlates with drug absorption and is modulated by particle composition. Fluorescently labeled drugs are formulated into particles as described above. The particles are suspended in a suitable solution for administration by oral gavage. In some cases, the solution may be water. The particle suspension is administered to rats by oral gavage. Each different fluorescently labeled drug and formulation combination is administered to four rats. An additional four rats will receive a solution containing the same total amount of fluorescently labeled drug without formulation into particles. Prior to administration of the fluorescently labeled drug, and at 0.5, 1, 2, 4, 6, 8, 10, 12, and 24 hours after administration, whole-body imaging scans are performed on two of each of the four rats. The remaining two rats from each treatment group are sacrificed at 4 and 12 hours, and the intestines are collected to confirm drug concentrations.

[0300] Example 6 In this example, particles from Formulation 37-49 (hGH / SEQ ID NO: 3 / Eudragit FS) were injected into an in vivo model to evaluate in vivo transcytosis. Formulation 37-49 particles were administered to rats by intracavitary injection at a dose of 34 μg / Kg.

[0301] Male Wistar rats were housed 3-5 per cage under a 12 / 12-hour light / dark cycle and weighed 225-275 g (approximately 6-8 weeks old) at the time of study. All experiments were performed during the light phase using a non-recovery protocol with continuous isoflurane anesthesia. A 4-5 cm midline abdominal incision exposed the mid-jejunum. 3.86 × 10 -5A stock solution of 37-49 particles of M formulation was prepared, and 50 µL (per 250 g rat) was administered by intraluminal injection (ILI) using a 29-gauge needle. The injection site was marked on the mesentery with a permanent marker. At the end of the study, a 3-5 mm region capturing the marked intestinal segment was isolated and processed for microscopic evaluation.

[0302] Injected animals were sacrificed at various time points post-injection, and the intestines were harvested and sectioned. Sections of the injected area of ​​the intestine were prepared and visualized by immunofluorescence. Particle uptake was observed beginning at 15 minutes, compared to 1-5 minutes seen with the Colic construct. As can be seen in Figure 7A, at 15 minutes post-injection, hGH (green) and Colic (red) initially largely colocalized (yellow), with more free hGH observed at later time points, as can be seen in Figures 7B-7D, at 30, 45, and 60 minutes post-injection, respectively.

[0303] To further evaluate drug delivery, hGH SEQ ID NO: 3 nanoparticles were delivered to three rats (animals A, B, and C) by intrajejunal injection at a dose of 1.93 nmol / kg, resulting in an hGH dose of approximately 43 μg / kg and a total hGH dose of 11 μg per rat. Serum samples were collected at 0, 30, 45, 60, 75, 90, 105, and 120 minutes after injection. After 120 minutes, the animals were sacrificed and the intestines and livers were collected. Figure 8 shows the serum concentration of hGH in animal A. A rapid increase in serum hGH concentration was observed, peaking at 4 ng / mL around 40 minutes after administration and remaining above 3.5 ng / mL until at least 75 minutes after administration. Animals B and C did not obtain detectable levels of hGH in serum. hGH was detected in the serum and intestines of animal A and in the intestines of animal B, but not in serum. hGH was not detected in either the serum or intestine of Animal C, nor was it detected in the liver of any of the animals at the 120 minute time point.

[0304] Example 7 In this example, hGH-containing particles were applied to Caco-2 cells to evaluate their transcellular transport. Caco-2 cells are a human colon epithelial carcinoma cell line that can be used as a model for human intestinal absorption of drugs and other compounds. When cultured as a monolayer, Caco-2 cells differentiate and form tight junctions between cells. This monolayer can be used as a model for paracellular movement of compounds. Caco-2 cells express transporter proteins, efflux proteins, and phase II conjugation enzymes to model various transcellular pathways. In some cases, Caco-2 cell monolayers can be used as a mimic of human intestinal epithelium.

[0305] 1.5 × 10 cells in a transwell 5 Caco-2 cells were seeded at 100 cells / mL. Culture medium was changed every 2 days in both the apical (0.5 mL) and basolateral (1.5 mL) chambers. Cells were grown for 21 days until a functionally cohesive monolayer was formed, as assessed by transepithelial electrical resistance (TEER), before experiments were performed. On day 21, transwells were washed once with PBS. 100 μL of a suspension containing hGH particles was added to the apical chamber. 0.5 mL of PBS was added to the basolateral chamber. After 2 hours at 37°C, the solution from the basolateral chamber was collected and concentrated. Western blotting was used to assess hGH transport across the tissue. Proteins were separated by 1D gel electrophoresis on a 4-12% NuPAGE gel (BioRad, cat. #5678095). The separated proteins were transferred to a PVDF membrane (BioRad, cat. #1704157) and incubated with goat anti-hGH polyclonal antibody (1:1000, R&D AF1067) followed by AP-conjugated secondary rabbit anti-goat antibody (1:10000, Abcam ab6742). Protein bands were visualized using AP Western blotting substrate (Promega S3841). [Table 2]

[0306] Example 8 In this example, several different microparticles were produced by mixing SEQ ID NO: 3, a cation, and exenatide, as indicated in Tables 3 and 4. The particles thus produced were assayed by HPLC to determine the actual content of each component in the microparticles. [Table 3] [Table 4]

[0307] The in vitro release of exenatide from the particles produced above was evaluated by incubating the particles in solutions of different pH for up to 18 hours. The amount of released exenatide was quantified by reverse-phase liquid chromatography (RPLC, Figures 10A and 10B) or size-exclusion chromatography (SEC, Figures 10C and 10D). As can be seen in Figures 10A-10D, the compositions formed pH-stable mixtures.

[0308] Example 9 To determine the pancreatin stability of different SEQ ID NO:3 and exenatide complexes, the complexes were incubated with pancreatin enzyme for 0, 30, 60, or 120 minutes as previously described. Figures 11A-11C show the amount of remaining SEQ ID NO:3 (approximately 30,162 da), exenatide (approximately 7,000 da), and protamine (approximately 4,186 da) for the different compositions at different time points. Tables 5, 6, and 7 below show the formulations and time points for each lane in Figures 11A-11C. [Table 5] [Table 6] [Table 7]

[0309] Figures 12A-12C show confocal images of microparticles produced from Corix, FITC-labeled exenatide, and zinc. A 20 μm scale bar is shown in each image. The size distribution of approximately 100 particles is shown in Figure 13. The average size of the microparticles was approximately 5 μm ± 2 μm.

[0310] To determine the ability of particles to transcytose across human SMI-100 cells, particles were prepared as outlined in Table 8. The resulting particles were dissolved in 10 mL of PBS, and 100 μL of this solution was added to the apical side of the cells, and 500 μL of PBS was added to the basal chamber. After 1 hour at 37°C, proteins in the basal solution were concentrated and analyzed by Western blotting. Exenatide in the basal solution was quantified by HPLC. As can be seen in Figure 14 and summarized in Table 8, transcytosis was observed in the six formulations tested. E0 was a no-protein control, and EP9 was not dissolved in PBS. Higher levels of exenatide transport were observed with particles containing zinc but not protamine (E11, E13, and E14). [Table 8]

[0311] Example 10 In vivo testing Formulations were selected for in vivo testing. Such formulations were prepared on a larger scale, and samples were analyzed for content and purity as before. Formulation details and analytical test results are shown in Table 9. In a typical preparation, a solution containing the colix carrier SEQ ID NO: 3 (or substituted with water if no colix was present) was mixed with the exenatide solution for 1 minute on a stirrer plate. Zinc or protamine solution was added dropwise to the stirring solution. After 15 minutes of stirring, the entire solution / suspension was lyophilized. Three additional formulations were prepared by the same method using fluorescein-tagged exenatide; these are summarized in Table 10. Fluorescein at the side chain of lysine was added to the N-terminus of exenatide by solid-phase peptide synthesis. [Table 9] [Table 10] The purity of each powder used in preparing the formulation was as follows: exenatide % content approximately 89.0% and SEQ ID NO:3 % content approximately 71.8%, as determined by calculating the % relative peak area observed from size exclusion chromatography (SEC).

[0312] A pancreatin assay was performed to determine the stability of the different formulations; the results can be seen in Figures 17A and 17B. Samples were analyzed using 4-20% Citerion TGX stain-free precast gels (Bio-Rad, 5678094), Precision Plus unstained standards (Bio-Rad, 161-0375), and a ThermoFisher gel scanner. Formulations E14, E18, E14-FITC, and E18-FITC all showed very little degradation of SEQ ID NO:3, even after 2 hours of exposure to pancreatin enzyme. Figure 18 shows reverse-phase chromatograms (RPLC) demonstrating the presence of SEQ ID NO:3 at a retention time of 6.8 minutes and exenatide at 7.5 minutes. These formulations were also evaluated for aqueous solubility at various pH values. As can be seen in Figure 19, the FITC formulation was less soluble than the formulation without FITC. Formulation E14 showed low solubility at pH 1 and high solubility at pH 7 and higher. Formulation E0 also showed low solubility at pH 1 and high solubility at pH 5 and higher.

[0313] To evaluate in vivo pharmacokinetics and pharmacodynamics, the particles were suspended in PBS, and 100 μL of each suspension was injected into the rat intestinal lumen. Four rats were used per formulation, and the formulations were suspended to produce doses of 10 μg of exenatide for E14 and E18, and 0 μg for E0. 100 μL blood samples were collected at 15, 30, 45, 60, and 90 minutes after injection. The blood was allowed to clot and then centrifuged to prepare serum, and exenatide concentrations were measured by ELISA. As can be seen in Figure 20, exenatide was detected in the serum of both E14- and E18-injected animals. The E18 formulation produced a higher maximum serum concentration and a higher area under the curve. The Cmax of the E14 formulation was 17.3 ng / mL, and the Cmax of the E18 formulation was 35.7 ng / mL. Tmax was 60 minutes for E14 and 45 minutes for E18. No changes from baseline were observed in the blood glucose levels of either animal. For comparison, an equal amount of exenatide was intravenously injected into rats, and serum concentrations were evaluated. See Figure 21. The pharmacokinetics of enterally delivered exenatide at E14, E18, and intravenously delivered exenatide are compared in Table 11. [Table 11]

[0314] Example 11 Preparation of compositions containing exenatide Exenatide (SEQ ID NO: 11) is a peptide stabilized by a C-terminal amine and an N-terminal H, and has GLP-1-like biological activity. In this example, two non-naturally occurring isolated constructs were prepared and tested for in vivo intestinal epithelial transport: 1) a carrier having SEQ ID NO: 78 processed into a carrier having SEQ ID NO: 70 and crosslinked to SEQ ID NO: 11, and 2) a carrier having SEQ ID NO: 77 processed into a carrier having SEQ ID NO: 80 and crosslinked to SEQ ID NO: 11. Carriers having SEQ ID NO: 80 and SEQ ID NO: 70 were prepared as described herein, and exenatide (SEQ ID NO: 11) (Cat# HOR-246) was purchased from ProSpec-Tany Technogene Ltd. PO Box 6591, East Brunswick, NJ 08816. Pierce™ Controlled Protein-Protein Crosslinking Kit (Cat# 23456) containing sulfo-SMCC crosslinker was purchased from ThermoFisher.

[0315] Payload and Carrier Activation and Crosslinking: Exenatide (10 mg) was dissolved in 5 mL of HO to form a 2 mg / mL solution. Sulfo-SMCC (2 mg) was dissolved in 2 mL of PBS. Immediately, 0.088 mL (approximately a 5-fold molar excess) of sulfo-SMCC solution was added to 1.0 mL of exenatide solution and incubated at room temperature for 30 minutes. Unreacted sulfo-SMCC was removed by applying 1.0 mL of the maleimide-exenatide reaction mixture to a desalting column equilibrated with PBS and eluting with PBS, collecting 0.5 mL fractions. The absorbance of each fraction at 280 nm was measured to determine the location of the protein peak. Peak fractions containing the majority of the protein were pooled. The concentration of the pooled activated exenatide was determined by comparing its absorbance at 280 nm with that of the original protein solution.

[0316] The carriers with SEQ ID NO:77 and SEQ ID NO:78 have a C-terminal extension containing a TEV cleavage site flanked by two cysteine ​​residues that form a disulfide bond, and a C-terminal His6 tag. The carriers with SEQ ID NO:77 and SEQ ID NO:78 were purified on a HisTrap column using standard methods. 2 mg of protein (200 μL of 10 mg / ml) in PBS, pH 7.4, was activated by treatment with 2 μl of 0.1 M dithiothreitol and 5 μl of TEV protease for 2 hours at 30° C. The cleaved and reduced protein was applied to a 1-ml HisTrap column equilibrated with PBS. The C-terminal fragment bound to the column, and the activated N-terminal SEQ ID NO:80 or SEQ ID NO:70 product with a free cysteine ​​near its C-terminus was collected in the flow-through.

[0317] Maleimide-activated exenatide and carrier (sulfhydryl-SEQ ID NO: 80 protein or sulfhydryl-SEQ ID NO: 70 protein) were mixed in equal molar amounts and then incubated at room temperature for 60 minutes. The purity of the SMCC-crosslinked SEQ ID NO: 70-exenatide conjugate was assessed on a Coomassie-stained SDS gel. The conjugate was approximately the correct molecular weight and had >90% purity (Figure 22). The crosslinked delivery construct was then stored at 4°C.

[0318] Example 12 In vivo transcytosis of exenatide delivery constructs: The delivery construct of Example 11 was tested for intestinal epithelial transport as follows: Wild-type Sprague Dawley® rats (approximately 200-250 grams, approximately 6 weeks old, purchased from Charles River) were fasted overnight to clear the intestine. The following materials were prepared: microcentrifuge tubes containing 4% formaldehyde, tubes for tissue preservation, microcentrifuge tubes for blood collection, microcentrifuge tubes for serum collection, PBS, and test article. Animals were prepared for the experiment by anesthetizing with isoflorane and shaving the abdomen. Four injections were prepared per animal (two per jejunum and two per colon). The abdominal cavity was opened. Injection sites were located and marked with colors for identification. The test article was slowly injected into the lumen over 10 minutes for the colon and 40 minutes for the jejunum. Animals received 35 μg of protein per injection at a concentration of 1 μg / μL. Animals were euthanized at 50 minutes. Terminal blood was collected by cardiac puncture. The jejunum and colon were removed and placed on a plastic-lined work surface. PBS was used to flush out the contents of the jejunum and colon and discarded. A 1 cm length of intestine was excised from the injection site. The excised tissue was cut in half. One section was placed in 4% formaldehyde. The remaining tissue was then sliced ​​longitudinally and immediately placed in a microcentrifuge tube and frozen. This process was repeated for all injection sites. The liver (approximately 1 cm) 3 ) was removed and divided into two small pieces. One liver slice was placed in formaldehyde for storage and the second slice was immediately frozen. At 40 minutes post-injection, intestine, liver & serum samples were collected. Blood samples were centrifuged and the resulting serum was transferred to a container for storage. Samples were shipped on dry ice and stored at -80°C. The dosing strategy was as follows: SEQ ID NO: 70 - Exenatide 100 μL, 490 pmol / 29.4 μg (4.9 μM) SEQ ID NO: 80 - Exenatide 100 μL, 490 pmol / 30.9 μg (4.9 μM) SEQ ID NO: 11 100 μL, 490 pmol / 2 μg (4.9 μM)

[0319] Bioanalytical analysis of intestinal epithelial transport of SEQ ID NO:70-exenatide, SEQ ID NO:80-exenatide and SEQ ID NO:11 (exenatide) was performed using an Exendin-4 ELISA kit (Phoenix Pharma, Cat#EK-070-94) as follows: Brains Tissue samples were obtained online; 300 μL of assay buffer (1×) was added to each tube containing a tissue sample; the tissue was removed from the assay buffer and placed on a sterile, clean cell culture lid plate; the intestinal samples were gently scraped with a cell scraper, taking care to avoid collecting the mesentery; liver samples were treated in a similar manner with additional dissociation and homogenization; the resulting cell homogenate was returned to its original tube; the remaining tissue sample and working area were rinsed with 100 μL of buffer (2×); the cell homogenate solution was centrifuged at maximum force for 5 minutes; the supernatant was applied to an ELISA plate, which was processed according to the manufacturer's instructions; the remaining supernatant was stored at −20°C for later use.

[0320] As depicted in Figure 23, transport of both SEQ ID NO:70-exenatide and SEQ ID NO:80-exenatide across intestinal epithelial cells was observed at 10 and 40 minutes. Furthermore, both SEQ ID NO:70-exenatide and SEQ ID NO:80-exenatide were transported at a faster rate than SEQ ID NO:11 (exenatide) alone, especially at 40 minutes.

[0321] Example 13 Glucose-regulating activity of delivery constructs with exenatide payload The glucose tolerance model used to test SEQ ID NO: 70-exenatide was designed to test the ability of GLP-1-like activity to enhance the rate of recovery from glucose excursions. An IP injection of glucose was used to induce a glucose excursion event, and exenatide delivered by IP injection was used as a positive control for the timing and magnitude of the effect compared to that observed in sham IP injection controls.

[0322] Male CD1 mice used in the glucose tolerance test were 9-16 weeks old. Because plasma glucose can be responsive to handling stress, animals were acclimated to the environment, blood sampling, and dosing procedures for 1 week prior to the start of the experiment to minimize handling-induced stress. Mice were fasted for 18 hours prior to testing. Animals were weighed prior to testing. All animals had baseline blood glucose readings obtained before receiving a 2 mg / kg dose of D-glucose solution (in 50 μL of sterile PBS) via intraperitoneal (IP) injection. Animals then received either an IP injection of 10 mg of SEQ ID NO: 14 (positive control), an oral gavage of the test treatment containing 10 mg of SEQ ID NO: 14 in 200 μL of 0.2 M NaHCO3 (pH 8.5), or an oral gavage of 200 μL of 0.2 M NaHCO3 (negative control). Blood samples were obtained at t = 0, 15, 30, 45, 60 min; 2, 3, and 4 h. Blood glucose measurements were performed using 5 μL blood samples obtained from the tail using a commercially available blood glucose meter that was calibrated with glucose standards before the start of the study.

[0323] Figure 24 shows the time-concentration profiles of blood glucose levels in animals receiving the three different treatments. Correction of blood glucose excursions began as early as 15 minutes after IP injection of 10 μg of commercially available exenatide (1-40)-Gly and was complete by 120 minutes. Oral gavage of SEQ ID NO: 70-exenatide (10 mg) resulted in a similar time-concentration pattern of blood glucose. By comparison, negative control mice achieved two-fold higher blood glucose levels, which required approximately 4 hours to fully recover to baseline. These results suggest that the SEQ ID NO: 70 carrier sequence was able to facilitate epithelial cell transcytosis of biologically active exenatide, which was sufficient to achieve pharmacodynamic results in this glucose tolerance model.

[0324] Example 14 GLP-1 receptor activation by delivery constructs with an exenatide payload SEQ ID NO:71 is a fusion protein delivery construct comprising an N-terminal extendin-4 (SEQ ID NO:14) domain, a spacer (SEQ ID NO:79), and a C-terminal carrier (SEQ ID NO:73). SEQ ID NO:83 is a fusion protein delivery construct comprising an N-terminal carrier (SEQ ID NO:67), a spacer (SEQ ID NO:79), and a C-terminal extendin-4 (SEQ ID NO:14) domain. The PathHunter® β-arrestin G protein-coupled receptor (GPCR) assay (DiscoverRx) was used to assay the ability of SEQ ID NO:71, SEQ ID NO:11 (exenatide), and M+SEQ ID NO:65 to bind to the GLP-1 receptor. In the PathHunter assay, ligand binding activates the GLP-1 receptor, resulting in β-arrestin recruitment to the receptor. The receptor activation state is detected using a gain-of-signal assay based on enzyme fragment complementation. The β-galactosidase enzyme (β-gal) is split into two fragments, the enzyme donor (ED) and the enzyme acceptor (EA). These fragments have no independent activity. However, when combined through the assembly of a protein complex, they complement each other to form an active β-gal enzyme. The GLP-1 receptor was co-expressed in cells stably expressing β-arrestin tagged with the ED fragment and tagged with EA. Recruitment of EA-tagged β-arrestin by activated ED-tagged GLP-1 receptor brings together the EA and ED domains to reconstitute β-gal enzyme activity, which can be detected by the release of a luminescent product. Figure 25 illustrates that SEQ ID NO: 11 and SEQ ID NO: 71 bound to the receptor. SEQ ID NO: 83 showed reduced activity compared to SEQ ID NO: 71.

[0325] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Accordingly, those skilled in the art will recognize that numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the present disclosure described herein may be employed in practicing the present invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of such claims and their equivalents be covered thereby. [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6] [Table 12-7] [Table 12-8] [Table 12-9] [Table 12-10] [Table 12-11] [Table 12-12]

Table 12-13

Table 12-14

Table 12-15

Table 12-16

Table 12-17

Table 12-18

Table 12-19

Table 12-20

Table 12-21

Table 12-22

Table 12-23

Table 12-24

Table 12-25

Table 12-26

Table 12-27

Table 12-28

Table 12-29

Table 12-30

Table 12-31

[0326]

Table 13-1

Table 13-2

Table 14-1

Table 14-2

Table 15-1

Table 15-2

Claims

[Claim 1] The invention described in the specification.

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