MHC Class I Epitope Delivering Polypeptides

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.

1 - 105 . (canceled) 
     
     
         106 . A Shiga toxin A subunit effector polypeptide comprising an embedded, heterologous, CD8+ T-cell epitope;
 wherein the Shiga toxin A subunit effector polypeptide is derived from a parental Shiga toxin A subunit effector polypeptide by having the heterologous, CD8+ T-cell epitope embedded in the parental Shiga toxin A subunit effector polypeptide such that the embedded, heterologous, CD8+ T-cell epitope replaces an equivalent number of amino acid residues in the Shiga toxin A subunit effector polypeptide such that the Shiga toxin A subunit effector polypeptide comprising the embedded, heterologous, CD8+ T-cell epitope has the same total number of amino acids as the parental Shiga toxin A subunit effector polypeptide absent the embedded, heterologous, CD8+ T-cell epitope.   
     
     
         107 . The Shiga toxin A subunit effector polypeptide of  claim 106 , wherein the Shiga toxin A subunit effector polypeptide is capable of exhibiting one or more Shiga toxin effector functions selected from the group consisting of:
 cytotoxicity; enzymatic catalysis; directing subcellular routing; cellular internalization; and intracellular delivery of the embedded, heterologous, CD8+ T-cell epitope from an early endosomal compartment to a MHC class I molecule of a cell in which the Shiga toxin A subunit effector polypeptide is present.   
     
     
         108 . The Shiga toxin A subunit effector polypeptide of  claim 106 , wherein the parental Shiga toxin A subunit effector polypeptide consists of:
 (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; or   (iv) amino acids 1 to 261 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.   
     
     
         109 . The Shiga toxin A subunit effector polypeptide of  claim 108 , which comprises or consists essentially of a polypeptide represented in any one of SEQ ID NOs: 11-13, 15-19, and 21-43. 
     
     
         110 . The Shiga toxin A subunit effector polypeptide of  claim 108  which is de-immunized, wherein the embedded, heterologous, CD8+ T-cell epitope disrupts an endogenous B-cell epitope and/or CD4+ T-cell epitope of the parental Shiga toxin A subunit effector polypeptide, as evidenced by reduced B-cell antigenicity or immunogenicity and/or reduced CD4+ T-cell antigenicity or immunogenicity of the Shiga toxin A subunit effector polypeptide comprising the embedded, heterologous, CD8+ T-cell epitope, as compared to the parental Shiga toxin A subunit effector polypeptide absent the embedded, heterologous, CD8+ T-cell epitope; and
 wherein said endogenous B-cell epitope and/or CD4+ T-cell epitope is positioned at an 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; 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; and 254-268 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; and 236-258 of SEQ ID NO: 1 or SEQ ID NO: 2. 
 
 
     
     
         111 . The Shiga toxin A subunit effector polypeptide of  claim 110 , which comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 11-13, 15-19, and 21-43; or
 which comprises or consists essentially of a polypeptide represented by any one of SEQ ID NOs: 11-13 and 15-19.   
     
     
         112 . A de-immunized polypeptide derived from a parental Shiga toxin A subunit effector polypeptide, wherein the parental Shiga toxin A subunit effector polypeptide comprises an amino acid sequence having at least 70% sequence identity to:
 (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; or   (iv) amino acids 1 to 261 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3:   
       wherein the de-immunized polypeptide comprises a heterologous, CD8+ T-cell epitope embedded in said parental Shiga toxin A subunit effector polypeptide; 
       wherein the embedded, heterologous, CD8+ T-cell epitope replaces an equivalent number of amino acid residues in the parental Shiga toxin A subunit effector polypeptide, such that the de-immunized polypeptide comprising the embedded, heterologous, CD8+ T-cell epitope has the same total number of amino acids as the parental Shiga toxin A subunit effector polypeptide absent the embedded, heterologous, CD8+ T-cell epitope; 
       wherein the embedded, heterologous, CD8+ T-cell epitope disrupts an endogenous B-cell epitope and/or CD4+ T-cell epitope of the parental Shiga toxin A subunit effector polypeptide, as evidenced by reduced B-cell antigenicity or immunogenicity and/or reduced CD4+ T-cell antigenicity or immunogenicity of the Shiga toxin A subunit effector polypeptide comprising the embedded, heterologous, CD8+ T-cell epitope, as compared to the parental Shiga toxin A subunit effector polypeptide absent the embedded, heterologous, CD8+ T-cell epitope; 
       wherein said endogenous B-cell epitope is positioned at an 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; and 254-268 of SEQ ID NO: 1 or SEQ ID NO: 2; 
 and/or wherein said endogenous CD4+ T-cell epitope is positioned at an amino acid sequence selected from the group of natively positioned amino acids consisting of 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; and 236-258 of SEQ ID NO: 1 or SEQ ID NO: 2; and 
 
       wherein the de-immunized polypeptide comprising the embedded, heterologous, CD8+ T-cell epitope is capable of intracellular delivery of the embedded, heterologous, CD8+ T-cell epitope from an early endosomal compartment to a MHC class I molecule of a cell in which the polypeptide is present. 
     
     
         113 . The de-immunized polypeptide of  claim 112 , wherein the parental Shiga toxin A subunit effector polypeptide comprises or consists of:
 (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; or   (iv) amino acids 1 to 261 of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.   
     
     
         114 . The de-immunized polypeptide of  claim 113 , which comprises or consists essentially of a polypeptide represented in any one of SEQ ID NOs: 13, 17, or 18. 
     
     
         115 . A cell-targeting molecule comprising:
 a Shiga toxin A subunit effector polypeptide according to any one of  claims 106 - 111  or a de-immunized polypeptide according to any one of  claims 112 - 114 ; and   a cell-targeting moiety or agent.   
     
     
         116 . The cell-targeting molecule of  claim 115 , wherein the cell-targeting moiety comprises a binding region comprising one or more polypeptides and which is capable of specifically binding at least one extracellular target biomolecule. 
     
     
         117 . The cell-targeting molecule of  claim 116 , wherein the binding region comprises a polypeptide selected from the group consisting of:
 complementary determining region 3 fragment constrained FR3-CDR3-FR4 polypeptide, single-domain antibody fragment, nanobody, V H H heavy-chain antibody domain, variable new antigen receptor (V NAR ) fragment, single-chain variable fragment (scFv), antibody variable fragment (Fv), antigen-binding fragment (Fab), Fd fragment, immunoglobulin new antigen receptor (IgNAR) fragment, small modular immunopharmaceutical domain, fibronectin-derived 10 th  fibronectin type III domain, tenascin type III domain, ankyrin repeat motif domain, low-density-lipoprotein-receptor-derived A-domain, lipocalin, Kunitz domain, Protein-A-derived Z domain, gamma-B crystallin-derived domain, ubiquitin-derived domain, Sac7d-derived polypeptide, Fyn-derived SH2 domain, miniprotein, C-type lectin-like domain scaffold, and any genetically manipulated counterparts of any of the foregoing that retain binding functionality.   
     
     
         118 . The cell-targeting molecule of  claim 116 , whereby administration of the cell-targeting molecule to a cell physically coupled with an extracellular target biomolecule bound by the binding region, the cell-targeting molecule is capable of causing death of the cell. 
     
     
         119 . The cell-targeting molecule of  claim 118 , whereby upon administration of the cell-targeting molecule to a first populations of cells whose members are physically coupled to extracellular target biomolecules bound by the binding region, and a second population of cells whose members are not physically coupled to any extracellular target biomolecule bound by said binding region, a cytotoxic effect of the cell-targeting molecule to members of said first population of cells relative to members of said second population of cells is at least 3-fold greater. 
     
     
         120 . The cell-targeting molecule of  claim 116 , wherein the binding region is capable of binding to an extracellular target biomolecule selected from the group consisting of:
 CD20, CD22, CD40, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-specific membrane antigen, Cripto, endoglin, fibroblast activated protein, Lewis-Y, CD19, CD21, CS1/SLAMF7, CD33, CD52, EpCAM, CEA, gpA33, mucin, TAG-72, carbonic anhydrase IX, folate binding protein, ganglioside GD2, ganglioside GD3, ganglioside GM2, ganglioside Lewis-Y2, VEGFR, Alpha V beta3, Alpha5beta1, ErbB1/EGFR, Erb3, c-MET, IGFIR, EphA3, TRAIL-R1, TRAIL-R2, RANKL, FAP, tenascin, CD64, mesothelin, BRCA1, MART-1/MelanA, gp100, tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, GAGE-1/2, BAGE, RAGE, NY-ESO-1, CDK-4, beta-catenin, MUM-1, caspase-8, KIAA0205, HPVE6, SART-1, PRAME, carcinoembryonic antigen, prostate specific antigen, prostate stem cell antigen, human aspartyl (asparaginyl) beta-hydroxylase, EphA2, HER3/ErbB-3, MUC1, MART-1/MelanA, gpl00, tyrosinase associated antigen, HPV-E7, Epstein-Barr virus antigen, Bcr-Abl, alpha-fetoprotein antigen, 17-A1, bladder tumor antigen, CD38, CD15, CD23, CD53, CD88, CD129, CD183, CD191, CD193, CD244, CD294, CD305, C3AR, FceRIa, galectin-9, mrp-14, siglec-8, siglec-10, CD49d, CD13, CD44, CD54, CD63, CD69, CD123, TLR4, FceRIa, IgE, CD107a, CD203c, CD14, CD68, CD80, CD86, CD105, CD115, F4/80, ILT-3, galectin-3, CD11a-c, GITRL, MHC Class II, CD284-TLR4, CD107-Mac3, CD195-CCR5, HLA-DR, CD16/32, CD282-TLR2, CD11c, and any immunogenic fragment of any of the foregoing.   
     
     
         121 . The cell-targeting molecule of  claim 120 , which comprises or consists essentially of a polypeptide represented by any one of SEQ ID NOs: 49-54. 
     
     
         122 . The cell-targeting molecule of  claim 121 , wherein the Shiga toxin A subunit effector polypeptide further comprises a mutation relative to a naturally occurring Shiga toxin A subunit which changes the enzymatic activity of the Shiga toxin A subunit effector polypeptide. 
     
     
         123 . The cell-targeting molecule of  claim 122 , wherein the mutation is selected from at least one amino acid residue deletion, insertion, or substitution that reduces or eliminates cytotoxicity of the Shiga toxin A subunit effector polypeptide. 
     
     
         124 . A pharmaceutical composition comprising:
 a Shiga toxin A subunit effector polypeptide according to any one of  claims 106 - 111 , a de-immunized polypeptide according to any one of  claims 112 - 114 , or a cell-targeting molecule according to any one of  claims 115 - 123 ; and   at least one pharmaceutically acceptable excipient or carrier.   
     
     
         125 . A diagnostic composition comprising:
 a cell-targeting molecule according to any one of  claims 115 - 123 ; and   a detection promoting agent.   
     
     
         126 . A polynucleotide capable of encoding a Shiga toxin A subunit effector polypeptide according to any one of  claims 106 - 111 , a de-immunized polypeptide according to any one of  claims 112 - 114 , or a cell-targeting molecule according to any one of  claims 115 - 123 ; or a complement of any of the foregoing. 
     
     
         127 . An expression vector comprising the polynucleotide of  claim 126 . 
     
     
         128 . A host cell comprising a polynucleotide according to  claim 126  or an expression vector according to  claim 127 . 
     
     
         129 . A kit comprising:
 (i) a Shiga toxin A subunit effector polypeptide according to any one of  claims 106 - 111 ;   (ii) a de-immunized polypeptide according to any one of  claims 112 - 114 ;   (ii) a cell-targeting molecule according to  claim 115 - 123 ;   (iii) a pharmaceutical composition according to  claim 124 ;   (iv) a diagnostic composition according to  claim 125 ;   (v) a polynucleotide according to  claim 126 ;   (vi) an expression vector according to  claim 127 ; and/or   (vii) a host cell according to  claim 128 ; and   
       further comprising an additional reagent and/or pharmaceutical delivery device.