Cholix toxin-derived fusion molecules for oral delivery of biologically active cargo

Abstract

The present disclosure relates to pharmaceutical compositions comprising a non-naturally occurring fusion molecule and one or more pharmaceutically acceptable carriers, formulated for oral delivery to a subject, and designed to provide for improved, effective therapies for treatment of, e.g., inflammatory diseases, autoimmune diseases, cancer, metabolic disorders, and growth deficiency disorders.

1 . A pharmaceutical composition comprising a non-naturally occurring fusion molecule and one or more pharmaceutically acceptable carriers, and formulated for oral delivery to a subject, wherein the fusion molecule comprises: (i) a modified Cholix toxin consisting of at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or a fragment thereof conjugated to (ii) a biologically active cargo selected from:
 an interleukin-10 consisting of at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 82 or a fragment thereof;   an interleukin-22 consisting of at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 85 or a fragment thereof;   a human growth hormone consisting of at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 95 or a fragment thereof;   an anti-TNF-α antibody or antibody fragment consisting of at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOS: 88-91 or a combination thereof; or   a GLP-1 agonist peptide consisting of at least 90% sequence identity to the amino sequence set forth in any one of SEQ ID NOS: 93-94 or a fragment thereof.   
     
     
         2 . The pharmaceutical composition of  claim 1 , wherein the modified Cholix toxin comprises a truncation at an amino acid residue within Cholix toxin domain Ib. 
     
     
         3 . The pharmaceutical composition of  claim 1 , wherein the fusion molecule has the ability to activate the receptor for the biologically active cargo, or to enable the catalytic process of a catalytically-active material. 
     
     
         4 . The pharmaceutical composition of  claim 1 , wherein the fragment of the amino acid sequence set forth in SEQ ID NO: 1 consists of the amino acid sequence set forth in any one of SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO:48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, or SEQ ID NO: 80. 
     
     
         5 . The pharmaceutical composition of  claim 1 , wherein the fragment of the amino acid sequence set forth in SEQ ID NO: 1 consists of the amino acid sequence set forth in any one of SEQ ID NOS: 3, 42, 52, 70, or 80. 
     
     
         6 . A pharmaceutical composition of  claim 1 , wherein the modified Cholix toxin is directly coupled to the biologically active cargo. 
     
     
         7 . The pharmaceutical composition of  claim 6 , wherein the biologically active cargo is directly coupled to the C-terminus of the modified Cholix toxin. 
     
     
         8 . A pharmaceutical composition of  claim 1 , wherein the modified Cholix toxin is chemically coupled to the biologically active cargo. 
     
     
         9 . A pharmaceutical composition of  claim 1 , wherein the modified Cholix toxin is coupled to the biologically active cargo by a non-cleavable linker, wherein the modified Cholix targets said cargo to specific cells, including cells of the immune system such as macrophages, antigen-presenting cells and dendritic cells. 
     
     
         10 . The pharmaceutical composition of  claim 9 , wherein the non-cleavable linker comprises the amino acid sequence of SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98 or SEQ ID NO: 99. 
     
     
         11 . A pharmaceutical composition of  claim 1 , wherein the modified Cholix toxin is coupled to the biologically active cargo by a cleavable linker. 
     
     
         12 . The pharmaceutical composition of  claim 11 , wherein the linker is cleavable by an enzyme that is present at a basolateral membrane of a polarized epithelial cell of the subject. 
     
     
         13 . The pharmaceutical composition of  claim 11 , wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, or SEQ ID NO: 107. 
     
     
         14 . The pharmaceutical composition of  claim 11 , wherein the linker is cleavable by an enzyme that is present in the plasma of said subject. 
     
     
         15 . The pharmaceutical composition of  claim 11 , wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, or SEQ ID NO: 120. 
     
     
         16 . The pharmaceutical composition of  claim 11 , wherein the linker is cleavable by a protease of the tobacco etch virus. 
     
     
         17 . The pharmaceutical composition of  claim 11 , wherein the cleavable linker comprises the amino acid sequence of SEQ ID NO: 121. 
     
     
         18 . The pharmaceutical composition of  claim 1 , wherein the fusion molecule comprises the amino acid sequence set forth in SEQ ID NO: 114. 
     
     
         19 . The pharmaceutical composition of  claim 1 , wherein the fusion molecule comprises the amino acid sequence set forth in SEQ ID NO: 115. 
     
     
         20 . A method of treating a subject in need thereof, the method comprising orally administering to the subject a pharmaceutical composition comprising a non-naturally occurring fusion molecule and one or more pharmaceutically acceptable carriers, and formulated for oral delivery to a subject, wherein the fusion molecule comprises: (i) a modified Cholix toxin consisting of at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or a fragment thereof conjugated to (ii) a biologically active cargo selected from:
 an interleukin-10 consisting of at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 82 or a fragment thereof;   an interleukin-22 consisting of at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 85 or a fragment thereof;   a human growth hormone consisting of at least 90% sequence identity to the amino acid sequence set forth in SEQ ID NO: 95 or a fragment thereof;   an anti-TNF-α antibody or antibody fragment consisting of at least 90% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOS: 88-91 or a combination thereof; or   a GLP-1 agonist peptide consisting of at least 90% sequence identity to the amino sequence set forth in any one of SEQ ID NOS: 93-94 or a fragment thereof.   
     
     
         21 .- 29 . (canceled)