Delivery constructs for transcytosis and related methods

Abstract

The present disclosure provides non-naturally occurring fusion molecules comprising therapeutic cargo moieties, such as IL-22 with a carrier. The disclosure also provides methods and compositions for the production, purification, refolding, formulation, and administration of fusion molecules. Methods and for using the purified molecules to treat and prevent diseases or disorders are also provided herein.

1 - 3 . (canceled) 
     
     
         4 . A delivery construct comprising a carrier coupled to an IL-22 payload, wherein the carrier consists of an amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 9, or an amino acid sequence having at least 90% sequence identity thereto, wherein the delivery construct is capable of transcytosing across a polarized epithelial cell. 
     
     
         5 . The delivery construct of  claim 4 , wherein the carrier has a glutamic acid at position 3 and an alanine at position 4. 
     
     
         6 . The delivery construct of  claim 4 , wherein the carrier is 266 amino acids in length. 
     
     
         7 . The delivery construct of  claim 4 , wherein the carrier consists of the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 9, or an amino acid sequence having at least 95% sequence identity thereto. 
     
     
         8 . The delivery construct of  claim 4 , wherein the carrier consists of the amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 9, or an amino acid sequence having at least 99% sequence identity thereto. 
     
     
         9 . The delivery construct of  claim 4 , wherein the carrier consists of the amino acid sequence set forth in SEQ ID NO: 7. 
     
     
         10 . The delivery construct of  claim 4 , wherein the carrier consists of the amino acid sequence set forth in SEQ ID NO: 9. 
     
     
         11 . The delivery construct of  claim 4 , wherein the IL-22 comprises an amino acid sequence set forth in SEQ ID NO: 11. 
     
     
         12 . The delivery construct of  claim 4 , wherein the carrier is coupled non-covalently to the IL-22 payload. 
     
     
         13 . The delivery construct of  claim 4 , wherein the carrier is coupled covalently to the IL-22 payload. 
     
     
         14 . The delivery construct of  claim 13 , wherein the carrier is coupled covalently to the IL-22 payload via a spacer. 
     
     
         15 . The delivery construct of  claim 14 , wherein the spacer consists of an amino acid sequence set forth in SEQ ID NO: 13. 
     
     
         16 . A pharmaceutical composition comprising the delivery construct of  claim 4 , wherein the pharmaceutical composition is formulated for oral administration. 
     
     
         17 . A method of treating an inflammatory disease in a subject, the method comprising administering to the subject an effective amount of the delivery construct of  claim 4 . 
     
     
         18 . The method of  claim 17 , wherein the inflammatory disease is hepatitis, obesity, fatty liver disease, liver inflammation, pancreatitis, Crohn's disease, ulcerative colitis, pouchitis, proctitis, multiple sclerosis, systemic lupus erythematosus, graft versus host disease, rheumatoid arthritis, or psoriasis. 
     
     
         19 . The method of  claim 18 , wherein the inflammatory disease is Crohn's disease or ulcerative colitis. 
     
     
         20 . The method of  claim 17 , wherein the delivery construct is administered orally to the subject. 
     
     
         21 . A method of obtaining a purified non-naturally occurring fusion protein, the method comprising:
 performing anion exchange chromatography on a mixture comprising the non-naturally occurring fusion protein to obtain a first fraction comprising the non-naturally occurring fusion protein;   wherein the non-naturally occurring fusion protein comprises IL-22 and a carrier that-consists of an amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 9, or an amino acid sequence having at least 90% sequence identity thereto.   
     
     
         22 . The method of  claim 21 , wherein performing anion exchange chromatography comprises binding the non-naturally occurring fusion protein to anionic exchange resin and providing an increasing salt gradient for subsequent elution of the non-naturally occurring fusion protein to obtain the first fraction. 
     
     
         23 . The method of  claim 22 , wherein performing anion exchange chromatography comprises contacting the mixture with a resin comprising amine-functionalized polymethacrylate beads. 
     
     
         24 . The method of  claim 23 , wherein the resin is an NH 2 -750F resin. 
     
     
         25 . The method of  claim 21 , further comprising refolding the non-naturally occurring fusion protein prior to performing anion exchange chromatography. 
     
     
         26 . The method of  claim 21 , further comprising subjecting a sample comprising the first fraction to a hydroxyapatite resin to obtain a second fraction comprising the non-naturally occurring fusion protein. 
     
     
         27 . The method of  claim 26 , wherein the hydroxyapatite resin is a CaPure-hydroxyapatite resin. 
     
     
         28 . The method of  claim 21 , further comprising performing cation exchange chromatography on a sample comprising the first fraction. 
     
     
         29 . The method of  claim 28 , wherein performing cation exchange chromatography comprises contacting the sample comprising the first fraction with a resin comprising sulfate-functionalized polymethacrylate beads. 
     
     
         30 . The method of  claim 29 , wherein the resin is a TOYOPEARL Sulfate-650F resin. 
     
     
         31 . A method of refolding a non-naturally occurring fusion protein comprising (1) a carrier that consists of an amino acid sequence set forth in SEQ ID NO: 7 or SEQ ID NO: 9, or an amino acid sequence having at least 90% sequence identity thereto and (2) IL-22, the method comprising:
 (i) contacting inclusion bodies comprising the non-naturally occurring fusion protein with a solubilization solution comprising a chaotropic agent to produce a soluble non-naturally occurring fusion protein;   (ii) contacting the non-naturally occurring fusion protein with a refolding solution, wherein the refolding solution comprises:
 arginine (0.75 M to 1.25 M); 
 glycerol (2% to 20% v/v); 
 cysteine (0.5 mM to 10 mm); and 
 cystamine (0.2 mM to 10 mM); 
 wherein the refolding solution has a pH of between 7.5 and 8.5. 
   
     
     
         32 . The method of  claim 31 , wherein:
 arginine is present in the refolding solution at a concentration of between 0.9 M and 1.1 M;   glycerol is present in the refolding solution at a concentration of between 7% and 13% (w/w);   cysteine is present in the refolding solution at a concentration of between 1.5 mM and 6 mM;   cystamine is present in the refolding solution at a concentration of between 0.6 mM and 3 mM; and   the refolding solution has a pH of between 7.8 and 8.2.