Mhc class i epitope delivering polypeptides and cell-targeted molecules for direct cell killing and immune stimulation via mhc class i presentation and methods regarding the same

Abstract

The present invention is directed to T-cell epitope delivering polypeptides which deliver one or more CD8+ T-cell epitopes to the MHC class I presentation pathway of a cell, including toxin-derived polypeptides which comprise embedded T-cell epitopes and are de-immunized. The present invention provides cell-targeted, CD8+ T-cell epitope delivering molecules for the targeted delivery of cytotoxicity to certain cells, e.g., infected or malignant cells, for the targeted killing of specific cell types, and the treatment of a variety of diseases, disorders, and conditions, including cancers, immune disorders, and microbial infections. The present invention also provides methods of generating polypeptides capable of delivering one or more heterologous T-cell epitopes to the MHC class I presentation pathway, including polypeptides which are 1) B-cell and/or CD4+ T-cell de-immunized, 2) comprise embedded T-cell epitopes, and/or 3) comprises toxin effectors which retain toxin functions.

The invention is claimed as follows: 
     
         1 - 71 . (canceled) 
     
     
         106 . A polypeptide comprising an embedded, heterologous, CD8+ T-cell epitope, wherein the polypeptide is capable of intracellular delivery of the T-cell epitope from an early endosomal compartment to a MHC class I molecule of a cell in which the polypeptide is present; and
 wherein the embedded, heterologous CD8+ T-cell epitope replaces an equivalent number of amino acid residues in a parental polypeptide such that the polypeptide comprising the epitope has the same total number of amino acids as the parental polypeptide.   
     
     
         107 . The polypeptide of  claim 106 , comprising a proteasome delivering effector polypeptide. 
     
     
         108 . The polypeptide of  claim 106 , comprising a toxin effector polypeptide capable of exhibiting one or more toxin effector functions. 
     
     
         109 . The polypeptide of  claim 108 , wherein the toxin effector polypeptide comprises a proteasome delivering effector polypeptide. 
     
     
         110 . The polypeptide of  claim 108  or  claim 109 , wherein the heterologous, CD8+ T-cell epitope is embedded in the toxin effector polypeptide. 
     
     
         111 . The polypeptide of  claim 110 , wherein the toxin effector polypeptide is capable of exhibiting one or more toxin effector functions in addition to intracellular delivery of a CD8+ T-cell epitope from an early endosomal compartment to a MHC class I molecule of a cell in which the toxin effector polypeptide is present. 
     
     
         112 . The polypeptide of any one of  claims 108 - 111 , wherein the toxin effector polypeptide is derived from a toxin selected from the group consisting of:
 ABx toxin, ribosome inactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin, bryodin, cholix toxin, claudin, diphtheria toxin, gelonin, heat-labile enterotoxin, mitogillin, pertussis toxin, pokeweed antiviral protein, pulchellin,  Pseudomonas  exotoxin A, restrictocin, ricin, saporin, sarcin, Shiga toxin, and subtilase cytotoxin.   
     
     
         113 . The polypeptide of  claim 112 , wherein the toxin effector polypeptide is derived from a polypeptide selected from the group of polypeptides represented by:
 (i) amino acids 75 to 251 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;   (ii) amino acids 2 to 389 of SEQ ID NO:45;   (iii) amino acids 1 to 241 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3;   (iv) amino acids 1 to 251 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; and   (v) amino acids 1 to 261 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3.   
     
     
         114 . A polypeptide comprising an embedded, heterologous, CD8+ T-cell epitope disrupting an endogenous B-cell epitope and/or CD4+ T-cell epitope, wherein the embedded, heterologous CD8+ T-cell epitope replaces an equivalent number of amino acid residues in a parental polypeptide such that the polypeptide comprising the epitope has the same total number of amino acids as the parental polypeptide. 
     
     
         115 . The polypeptide of  claim 114 , wherein the polypeptide is capable of intracellular delivery of the CD8+ T-cell epitope to a MHC class I molecule from an early endosomal compartment of a cell in which the polypeptide is present. 
     
     
         116 . The polypeptide of  claim 114  or  claim 115 , comprising a toxin effector polypeptide capable of exhibiting one or more toxin effector functions. 
     
     
         117 . The polypeptide of  claim 116 , wherein the heterologous, CD8+ T-cell epitope is embedded in the toxin effector polypeptide. 
     
     
         118 . The polypeptide of  claim 117 , wherein the toxin effector polypeptide is capable of exhibiting one or more toxin effector functions in addition to intracellular delivery of a CD8+ T-cell epitope from an early endosomal compartment to a MHC class I molecule of a cell in which the toxin effector polypeptide is present. 
     
     
         119 . The polypeptide of  claim 116 , wherein the toxin effector polypeptide is derived from a toxin selected from the group consisting of:
 ABx toxin, ribosome inactivating protein toxin, abrin, anthrax toxin, Aspfl, bouganin, bryodin, cholix toxin, claudin, diphtheria toxin, gelonin, heat-labile enterotoxin, mitogillin, pertussis toxin, pokeweed antiviral protein, pulchellin,  Pseudomonas  exotoxin A, restrictocin, ricin, saporin, sarcin, Shiga toxin, and subtilase cytotoxin.   
     
     
         120 . The polypeptide of  claim 119 , wherein the toxin effector polypeptide comprises a diphtheria toxin effector polypeptide comprising amino acid sequences derived from the A and B Subunits of at least one member of the diphtheria toxin family,
 wherein the diphtheria toxin effector polypeptide comprises a disruption of at least one B-cell epitope and/or CD4+ T-cell epitope region of the amino acid sequence selected from the group of natively positioned amino acids consisting of:
 3-10 of SEQ ID NO:44, 15-31 of SEQ ID NO:44, 32-54 of SEQ ID NO:44; 33-43 of SEQ ID NO:44, 71-77 of SEQ ID NO:44, 93-113 of SEQ ID NO:44, 125-131 of SEQ ID NO:44, 138-146 of SEQ ID NO:44, 141-167 of SEQ ID NO:44, 165-175 of SEQ ID NO:44, 182-201 of SEQ ID NO:45, 185-191 of SEQ ID NO:44, and 225-238 of SEQ ID NO:45; and 
   wherein the diphtheria toxin effector polypeptide is capable of routing to a cytosol compartment of a cell in which the diphtheria toxin effector polypeptide is present.   
     
     
         121 . The polypeptide of  claim 119 , wherein the diphtheria toxin effector polypeptide is derived from the polypeptide represented by amino acids 2 to 389 of SEQ ID NO:45. 
     
     
         122 . The polypeptide of  claim 119 , wherein the toxin effector polypeptide comprises a Shiga toxin effector polypeptide comprising an amino acid sequence derived from an A Subunit of at least one member of the Shiga toxin family,
 wherein the Shiga toxin effector polypeptide comprises a disruption of at least one B-cell epitope and/or CD4+ T-cell epitope region of the Shiga toxin A Subunit amino acid sequence selected from the group of natively positioned amino acids consisting of:
 the B-cell epitope regions: 1-15 of SEQ ID NO: 1 or SEQ ID NO:2; 3-14 of SEQ ID NO:3; 26-37 of SEQ ID NO:3; 27-37 of SEQ ID NO: 1 or SEQ ID NO:2; 39-48 of SEQ ID NO: 1 or SEQ ID NO:2; 42-48 of SEQ ID NO:3; 53-66 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 94-115 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3; 141-153 of SEQ ID NO:1 or SEQ ID NO:2; 140-156 of SEQ ID NO:3; 179-190 of SEQ ID NO:1 or SEQ ID NO:2; 179-191 of SEQ ID NO:3; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; and 210-218 of SEQ ID NO:3; 240-260 of SEQ ID NO:3; 243-257 of SEQ ID NO: 1 or SEQ ID NO:2; 254-268 of SEQ ID NO: 1 or SEQ ID NO:2; 262-278 of SEQ ID NO:3; 281-297 of SEQ ID NO:3; and 285-293 of SEQ ID NO: 1 or SEQ ID NO:2, 
 and 
 the CD4+ T-cell epitope regions: 4-33 of SEQ ID NO: 1 or SEQ ID NO:2, 34-78 of SEQ ID NO: 1 or SEQ ID NO:2, 77-103 of SEQ ID NO: 1 or SEQ ID NO:2, 128-168 of SEQ ID NO: 1 or SEQ ID NO:2, 160-183 of SEQ ID NO:1 or SEQ ID NO:2, 236-258 of SEQ ID NO: 1 or SEQ ID NO:2, and 274-293 of SEQ ID NO: 1 or SEQ ID NO:2; and 
   wherein the Shiga toxin effector polypeptide is capable of routing to a cytosol compartment of a cell in which the Shiga toxin effector polypeptide is present.   
     
     
         123 . The polypeptide of  claim 119 , wherein the Shiga toxin effector polypeptide is derived from a polypeptide selected from the group of polypeptides represented by:
 (i) amino acids 75 to 251 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3;   (ii) amino acids 1 to 241 of SEQ ID NO: 1, SEQ ID NO:2, or SEQ ID NO:3;   (iii) amino acids 1 to 251 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; and   (iv) amino acids 1 to 261 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.   
     
     
         124 . A method of creating a CD8+ T-cell epitope delivery molecule capable of intracellular delivery of a T-cell epitope from an early endosomal compartment to a MHC class I molecule of a cell in which the delivery molecule is present, the method comprising the step of:
 embedding a heterologous, CD8+ T-cell epitope in a proteasome delivering effector polypeptide capable of intracellular delivery of a T-cell epitope from an early endosomal compartment to a MHC class I molecule of a cell in which the delivery molecule is present;   wherein the step of embedding involves replacing an equivalent number of amino acid residues in a parental polypeptide with the heterologous, CD8+ T-cell epitope such that the polypeptide comprising the epitope has the same total number of amino acids as the parental polypeptide.   
     
     
         125 . The method of  claim 124 , wherein the method comprises embedding the CD8+ T-cell epitope in an endogenous B-cell epitope, an endogenous CD4+ T-cell epitope, and/or a catalytic domain of the proteasome delivering effector polypeptide. 
     
     
         126 . The method of  claim 124  or  claim 125 , wherein the CD8+ T-cell epitope delivery molecule comprises a toxin effector polypeptide comprising a proteasome delivering effector polypeptide. 
     
     
         127 . The method of  claim 126 , wherein the method comprises embedding the heterologous, CD8+ T-cell epitope in the toxin effector polypeptide. 
     
     
         128 . The method of  claim 127 , wherein the embedding step results in a CD8+ T-cell epitope delivery molecule comprising a toxin effector polypeptide capable of exhibiting one or more toxin effector functions in addition to intracellular delivery of a CD8+ T-cell epitope from an early endosomal compartment to a MHC class I molecule of a cell in which the toxin effector polypeptide is present. 
     
     
         129 . A method for reducing B-cell immunogenicity of a polypeptide having a B-cell epitope, the method comprising the step of:
 disrupting the B-cell epitope in the polypeptide with one or more amino acid residue(s) of a T-cell epitope embedded in the polypeptide;   wherein the T-cell epitope is embedded in the polypeptide by replacing an equivalent number of amino acid residues in a parental polypeptide with the T-cell epitope such that the polypeptide comprising the epitope has the same total number of amino acids as the parental polypeptide.   
     
     
         130 . A method for reducing B-cell immunogenicity of a polypeptide having a B-cell epitope while simultaneously increasing CD8+ T-cell immunogenicity of the polypeptide, the method comprising the step of:
 disrupting a B-cell epitope in the polypeptide with one or more amino acid residue(s) of a heterologous, CD8+ T-cell epitope embedded in the polypeptide;   wherein the CD8+ T-cell epitope is embedded in the polypeptide by replacing an equivalent number of amino acid residues in a parental polypeptide with the heterologous, CD8+ T-cell epitope such that the polypeptide comprising the epitope has the same total number of amino acids as the parental polypeptide.   
     
     
         131 . The method of  claim 129  or  claim 130 , wherein the polypeptide has a CD4+ T-cell epitope, and wherein the B-cell epitope disrupting step comprises making one or more amino acid substitutions in the CD4+ T-cell epitope. 
     
     
         132 . A method for reducing CD4+ T-cell immunogenicity of a polypeptide having a CD4+ T-cell epitope, the method comprising the step of:
 disrupting a CD4+ T-cell epitope in the polypeptide with one or more amino acid residue(s) of a CD8+ T-cell epitope embedded in the polypeptide;   wherein the CD8+ T-cell epitope is embedded in the polypeptide by replacing an equivalent number of amino acid residues in a parental polypeptide with the CD8+ T-cell epitope such that the polypeptide comprising the epitope has the same total number of amino acids as the parental polypeptide.   
     
     
         133 . A method for reducing CD4+ T-cell immunogenicity of a polypeptide having a CD4+ T-cell epitope while simultaneously increasing CD8+ T-cell immunogenicity of the polypeptide, the method comprising the step of:
 disrupting a CD4+ T-cell epitope with one or more amino acid residue(s) of a heterologous, CD8+ T-cell epitope embedded in the polypeptide;   wherein the CD8+ T-cell epitope is embedded in the polypeptide by replacing an equivalent number of amino acid residues in a parental polypeptide with the heterologous, CD8+ T-cell epitope such that the polypeptide comprising the epitope has the same total number of amino acids as the parental polypeptide.   
     
     
         134 . The method of  claim 132  or  claim 133 , wherein the polypeptide has a B-cell epitope, and wherein the CD4+ T-cell epitope disrupting step comprises making one or more amino acid substitutions in the B-cell epitope. 
     
     
         135 . A polypeptide comprising or consisting essentially of the polypeptide shown in any one of SEQ ID NOs: 11-13, 15-19, 21-43, or 46-48.