pHLIP® peptide-mediated epitope tethering at cell surfaces

Abstract

The invention features methods and compositions for eliciting an anti-tumor response in a subject comprising administering to the subject a pHLIP® construct comprising an antibody recruiting molecule linked to one or more pHLIP® peptides by a non-cleavable linker compound. The construct increases the amount of the antibody recruiting molecule on the surface of a diseased cell.

What is claimed is: 
     
         1 . A method of eliciting an immune response in a subject comprising administering to said subject a pHLIP® construct comprising an antibody recruiting molecule or an immune cell recruiting molecule linked to one or more pHLIP® peptides, wherein said construct increases the amount of said antibody or immune cell recruiting molecule on the surface of a diseased cell. 
     
     
         2 . The method of  claim 1 , wherein said antibody recruiting molecule or said immune cell recruiting molecule comprises an epitope. 
     
     
         3 . The composition of  claim 1 , wherein said construct comprises the formula of
   Epitope-Linker-Pept   wherein “Epitope” is an antibody or immune cell recruiting molecule;   wherein “Linker” is a non-cleavable linker compound or a membrane non-inserting end of the pHLIP® peptide further comprises an amino acid extension;   wherein “Pept” is a pHLIP® peptide comprising the sequence AXDDQNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO: ______) or AXDQDNPWRAYLDLLFPTDTLLLDLLW (SEQ ID NO: ______), where “X” is a functional group, selected from a lysine, a cysteine, or an Azido-containing amino acid;   wherein each “—” is a covalent bond.   
     
     
         4 . The method of  claim 1 , wherein said construct comprises an antibody recruiting molecule 
     
     
         5 . The method of  claim 4 , wherein 2 antibody recruiting molecules are linked to pHLIP® peptide. 
     
     
         6 . The method of  claim 1 , wherein said construct comprises the formula of
   Epitope 1-Linker-Pept-Linker-Epitope1   wherein “Epitope1” is an antibody recruiting molecule;   wherein “Linker” is a polyethylene glycol linker;   wherein “Pept” is a pHLIP® peptide comprising the sequence   
       
         
           
                 
                 
               
                     
                   (SEQ ID NO:_) 
                 
                     
                   Ac-AKQNDDQNKPWRAYLDLLFPTDTLLLDLLWA 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   (SEQ ID NO:_) 
                 
                     
                   Ac-AKQNDNDNKPWRAYLDLLFPTDTLLLDLLWA 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   (SEQ ID NO:_) 
                 
                     
                   ACQNDDQNCPWRAYLDLLFPTDTLLLDLLWA 
                 
                     
                   or 
                 
                     
                     
                 
                     
                   (SEQ ID NO:_) 
                 
                     
                   ACQNDNDNCPWRAYLDLLFPTDTLLLDLLWA 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
         wherein each “—” is a covalent bond. 
       
     
     
         7 . The method of  claim 2 , wherein said epitope comprises a peptide with a length less than 50 amino acids. 
     
     
         8 . The method of  claim 1 , wherein said diseased cell comprises a tumor cell. 
     
     
         9 . The method of  claim 1 , wherein said diseased cell comprises a cell in inflamed tissue. 
     
     
         10 . The method of  claim 1 , wherein said antibody recruiting molecule or immune cell recruiting molecule comprises an epitope. 
     
     
         11 . The method of  claim 2 , wherein said epitope comprises a peptide with a length less than 50 amino acids. 
     
     
         12 . The method of  claim 2 , wherein said epitope comprises a length of between 5 to 20 amino acids. 
     
     
         13 . The method of  claim 2 , wherein said epitope is a HA peptide. 
     
     
         14 . The method of  claim 12 , wherein said peptide comprises the amino acid sequence of YPYDVPDYA (SEQ ID NO: ______). 
     
     
         15 . The method of  claim 2 , wherein said epitope is selected from the group consisting of QVSHWVSGLAEGSFG (SEQ ID NO: ______), LSHTSGRVEGSVSLL (SEQ ID NO: ______), QMWAPQWGPD (SEQ ID NO: ______); MASMTGGQQMG (SEQ ID NO: 4); EQKLISEEDL (SEQ ID NO: 5); YTDIEMNRLGK (SEQ ID NO: 7); KETAAAKFERQHMDS (SEQ ID NO: 8); GKPIPNPLLGLDST (SEQ ID NO: 9); DYKDDDDK (SEQ ID NO: 10); GAPVPYPDPLEPR (SEQ ID NO: 11); HHHHHH (SEQ ID NO: 12); TKENPRSNQEESYDDNES (SEQ ID NO: 13); WSHPQFEK (SEQ ID NO: 14); and PDRVRAVSHWSS (SEQ ID NO: 15). 
     
     
         16 . The method of  claim 2 , wherein said epitope comprises a protein epitope with a length of 200 or less amino acids. 
     
     
         17 . The method of  claim 16 , wherein said protein epitope comprises a cytokine. 
     
     
         18 . The method of  claim 17 , wherein said cytokine comprises an interleukin (IL). 
     
     
         19 . The method of  claim 18 , wherein said interleukin comprises IL-1, IL-2, IL-6, IL-7, IL-12, and IL-17. 
     
     
         20 . The method of  claim 17 , wherein said cytokine comprises tumor necrosis factor (TNF). 
     
     
         21 . The method of  claim 17 , wherein said cytokine comprises a chemokine (CXC). 
     
     
         22 . The method of  claim 21 , wherein said chemokine is CXCL9, CXL10, or CXL11. 
     
     
         23 . The method of  claim 21 , wherein said chemokine comprises CXCL10 comprising the amino acid sequence: 
       
         
           
                 
               
                   (SEQ ID NO: ) 
                 
                   MNQTAILICCLIFLTLSGIQGVPLSRTVRCTCISISNQPVNPRSLEKLEII 
                 
                     
                 
                   PASQFCPRVEIIATMKKKGEKRCLNPESKAIKNLLKAVSKERSKRSP. 
                 
             
                
                
                
                
               
            
           
         
       
     
     
         24 . The method of  claim 2 , wherein said epitope comprises a small molecule. 
     
     
         25 . The method of  claim 24 , wherein said small molecule comprises a dinitrophenyl (DNP) or a derivative thereof. 
     
     
         26 . A composition comprising an antibody or immune cell recruiting molecule linked to one or more pHLIP® peptides by a non-cleavable linker compound. 
     
     
         27 . A composition comprising an epitope linked to one or more pHLIP® peptides, wherein the epitope is a protein epitope and is an extension of the non-inserting end of the pHLIP® peptide. 
     
     
         28 . The composition of  claim 27 , wherein said extension comprises a peptide epitope. 
     
     
         29 . A composition comprising an epitope linked to one or more pHLIP® peptides, wherein the epitope and the pHLIP® peptide are part of a single fusion construct or fusion protein. 
     
     
         30 . The method of  claim 1 , wherein said construct comprises the formula of Epitope-Linker-Peptide, wherein Peptide is a pHLIP® peptide comprising the sequence AXDDQNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______) or AXDQDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______), where “X” is a functional group, selected from a lysine, a cysteine, or an Azido-containing amino acid, wherein Linker is a linker or an extension of the pHLIP® peptide, and wherein each “—” is a covalent bond. 
     
     
         31 . A composition comprising the formula of Epitope-Linker-Peptide, wherein Peptide is a pHLIP® peptide comprising the sequence AXDDQNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______) or AXDQDNPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______), where X is a functional group, selected from a lysine, a cysteine, or an Azido-containing amino acid, and wherein Linker is a linker or an extension of the pHLIP® peptide, and wherein each — is a covalent bond. 
     
     
         32 . The composition of  claim 31 , wherein two epitopes are linked to a single pHLIP® peptide. 
     
     
         33 . The method of  claim 1 , wherein said construct comprises the formula of Epitope 2 -Linker 2 -Peptide, wherein Peptide is a pHLIP® peptide comprising the sequence AX(Z) n XPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______), wherein X is a functional group, selected from a lysine, a cysteine, an Azido-containing amino acid, or others, wherein Z comprises indicates any amino acid residue, wherein n is any integer between 1 and 10, wherein Linker is a linker or an extension of the pHLIP® peptide, and each “—” is a covalent bond. 
     
     
         34 . The composition of  claim 33 , wherein said composition comprising the formula of Epitope 2 -Linker 2 -Pept, wherein “Pept” is a pHLIP® peptide comprising the sequence AX(Z) n XPWRAYLDLLFPTDTLLLDLLWA (SEQ ID NO: ______), where X is a functional group, selected from a lysine, a cysteine, an Azido-containing amino acid, wherein Z indicates any amino acid residue, wherein n is any integer between and including 1 and 10, wherein Linker is a linker or an extension of the pHLIP® peptide, and wherein each — is a covalent bond. 
     
     
         35 . A method of inducing an immune response in a diseased tissue in a subject, comprising administering to a subject a composition comprising an epitope and a pHLIP® peptide. 
     
     
         36 . The method of  claim 35 , wherein said subject comprises a solid tumor. 
     
     
         37 . The method of  claim 35 , wherein said subject comprises an inflamed tissue. 
     
     
         38 . The method of  claim 35 , wherein said composition is injected directly into a diseased tissue tumor mass. 
     
     
         39 . The method of  claim 35 , wherein said composition is systemically administered. 
     
     
         40 . The method of  claim 35 , wherein a biological effect of said composition is at least 20% greater than that delivered in the absence of said composition. 
     
     
         41 . The method of  claim 35 , wherein said composition targets preferentially to a diseased tissue compared to a healthy tissue, thereby minimizing damage to said healthy tissue. 
     
     
         42 . A method for promoting an immune response in a subject, comprising administering to a subject the composition of  claim 1 , wherein said method comprises placement of said epitope on tumor cell or a cell in inflamed tissue of said subject.