Listeria-based compositions comprising a peptide minigene expression system and methods of use thereof

Abstract

This disclosure provides compositions, including Listeria delivery vectors comprising minigene expression constructs, and methods of using the same for inducing an immune response against an antigen-expressing tumor and for treating the same, and vaccinating against the same in subjects bearing the tumors.

1 .- 118 . (canceled) 
     
     
         119 . A recombinant  Listeria  strain comprising a minigene nucleic acid construct comprising an open reading frame encoding a chimeric protein, wherein the chimeric protein comprises:
 (a) a bacterial secretion signal sequence;   (b) a ubiquitin protein; and   (c) a peptide, wherein the peptide is a minimal peptide determinant for presentation by MHC class I molecules;   wherein the recombinant  Listeria  strain is a  Listeria monocytogenes,      wherein the minigene nucleic acid construct is in an extrachromosomal plasmid in the recombinant  Listeria  strain, and   wherein the bacterial secretion signal sequence, the ubiquitin protein, and the peptide in (a) to (c) are arranged from the amino-terminus to the carboxy-terminus of the chimeric protein.   
     
     
         120 . The recombinant  Listeria  strain of  claim 119 , wherein the open reading frame encoding the chimeric protein is linked to a second open reading frame by a Shine-Dalgarno ribosome binding site nucleic acid sequence, wherein the second open reading frame encodes a second chimeric protein comprising: (a) a second bacterial secretion signal sequence; (b) a second ubiquitin protein; and (c) a second peptide, wherein the second bacterial secretion signal sequence, the second ubiquitin protein, and the second peptide in (a) to (c) are arranged from the amino terminus to the carboxy terminus of the second chimeric protein. 
     
     
         121 . The recombinant  Listeria  strain of  claim 120 , wherein each open reading frame encodes a different peptide. 
     
     
         122 . The recombinant  Listeria  strain of  claim 119 , wherein the peptide comprises one or more neoepitopes. 
     
     
         123 . The recombinant  Listeria  strain of  claim 119 , wherein the minigene nucleic acid construct further comprises a 5′ bacterial promoter driving expression of the minigene nucleic acid construct. 
     
     
         124 . The recombinant  Listeria  strain of  claim 123 , wherein the 5′ bacterial promoter is an actA promoter, an hly promoter, or a p60 promoter. 
     
     
         125 . The recombinant  Listeria  strain of  claim 119 , wherein the bacterial secretion signal sequence comprises an ActA 100  signal sequence comprising the first 100 amino acids of an ActA protein signal sequence or is a secretion signal sequence from a listeriolysin O (LLO) protein. 
     
     
         126 . The recombinant  Listeria  strain of  claim 119 , wherein the recombinant  Listeria  strain comprises genomic mutations or deletions in a D-alanine racemase (dal) gene, a D-amino acid transferase (dat) gene, and an actA gene. 
     
     
         127 . The recombinant  Listeria  strain of  claim 126 , wherein the recombinant  Listeria  strain further comprises a nucleic acid molecule comprising an open reading frame encoding a metabolic enzyme, and wherein the metabolic enzyme complements the genomic mutations or deletions in the dal gene and the dat gene. 
     
     
         128 . The recombinant  Listeria  strain of  claim 127 , wherein the nucleic acid molecule is in a plasmid in the recombinant  Listeria  strain, and wherein the plasmid is stably maintained in the recombinant  Listeria  strain in the absence of antibiotic selection. 
     
     
         129 . The recombinant  Listeria  strain of  claim 127 , wherein the metabolic enzyme is an alanine racemase enzyme or a D-amino acid transferase enzyme. 
     
     
         130 . The recombinant  Listeria  strain of  claim 119 , wherein the recombinant  Listeria  strain comprises a genomic mutation or deletion in the prfA gene. 
     
     
         131 . The recombinant  Listeria  strain of  claim 119 , wherein the bacterial secretion signal sequence is a secretion signal sequence from a listeriolysin O (LLO) protein and comprises the sequence set forth in SEQ ID NO: 4, wherein the recombinant  Listeria  strain comprises genomic mutations or deletions in a D-alanine racemase (dal) gene, a D-amino acid transferase (dat) gene, and an actA gene, wherein the recombinant  Listeria  strain further comprises a nucleic acid molecule comprising an open reading frame encoding a metabolic enzyme, and wherein the metabolic enzyme is an alanine racemase enzyme or a D-amino acid transferase enzyme. 
     
     
         132 . A pharmaceutical composition comprising the recombinant  Listeria  strain of  claim 119 , and a pharmaceutically acceptable carrier.