Cell-targeting molecules comprising de-immunized, shiga toxin a subunit effectors and cd8+ t-cell epitopes

Abstract

The present invention provides cell-targeting molecules which can deliver a CD8+ T-cell epitope cargo to the MHC class I presentation pathway of a target cell. The cell-targeting molecules of the invention can be used to deliver virtually any CD8+ T-cell epitope from an extracellular space to the MHC class I pathway of a target cell, which may be a malignant cell and/or non-immune cell. The target cell can then display on a cell-surface the delivered CD8+ T-cell epitope complexed with MHC I molecule. The cell-targeting molecules of the invention have uses which include the targeted labeling and/or killing of specific cell-types within a mixture of cell-types, including within a chordate, as well as the stimulation of beneficial immune responses. The cell-targeting molecules of the invention have uses, e.g., in the treatment of a variety of diseases, disorders, and conditions, including cancers, tumors, growth abnormalities, immune disorders, and microbial infections.

1 . A cell-targeting molecule comprising
 i) a Shiga toxin effector polypeptide having a carboxy-terminus and comprising:
 a) an embedded or inserted, heterologous, CD8+ T-cell epitope; 
 b) a disruption of at least one, endogenous, B-cell and/or CD4+ T-cell epitope region which does not overlap with the embedded or inserted, heterologous, CD8+ T-cell epitope; 
 c) Shiga toxin A1 fragment region having a carboxy-terminus; and 
 d) a disrupted furin-cleavage motif at the carboxy-terminus of the A1 fragment region; 
   ii) a binding region capable of specifically binding at least one extracellular target biomolecule, and   iii) a heterologous, CD8+ T-cell epitope cargo which is not embedded or inserted in the Shiga toxin A1 fragment region, wherein the CD8+ T-cell epitope cargo is positioned carboxy-terminal to the carboxy terminus of the Shiga toxin A1 fragment region.   
     
     
         2 . The cell-targeting molecule of  claim 1 , wherein the Shiga toxin effector polypeptide is capable of delivering the CD8+ T-cell epitope cargo from an early endosomal compartment of a cell in which the Shiga toxin effector polypeptide is present to a MHC class I molecule of the cell, and wherein 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 internalizes into the cell resulting in the presentation of the CD8+ T-cell epitope cargo complexed with a MHC class I molecule on a cellular surface of the cell. 
     
     
         3 . The cell-targeting molecule of  claim 2 , wherein the CD8+ T-cell epitope cargo is fused to the Shiga toxin effector polypeptide or the binding region. 
     
     
         4 . The cell-targeting molecule of  claim 3 , wherein the cell-targeting molecule comprises or consists of a single-chain polypeptide comprising (i) the Shiga toxin effector polypeptide, (ii) the binding region, and (iii) the CD8+ T-cell epitope cargo. 
     
     
         5 . (canceled) 
     
     
         6 . The cell-targeting molecule of  claim 1 , which comprises a molecular moiety associated with the carboxy-terminus of the Shiga toxin effector polypeptide. 
     
     
         7 . The cell-targeting molecule of  claim 6 , wherein the molecular moiety comprises the binding region. 
     
     
         8 . The cell-targeting molecule of  claim 6 , wherein the molecular moiety is cytotoxic. 
     
     
         9 . The cell-targeting molecule of  claim 7 , wherein the molecular moiety comprises an amino acid residue to which the Shiga toxin effector polypeptide is linked. 
     
     
         10 . The cell-targeting molecule of  claim 9 , wherein the molecular moiety and the Shiga toxin effector polypeptide are fused forming a continuous polypeptide. 
     
     
         11 . The cell-targeting molecule of  claim 1 , wherein the binding region comprises a:
 single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, complementary determining region 3 fragment, constrained FR3-CDR3-FR4 polypeptide, Fd fragment, antigen-binding fragment, Armadillo repeat polypeptide, fibronectin-derived 10th fibronectin type Ill 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, or a genetically manipulated counterparts of any of the foregoing which retains extracellular target biomolecule binding.   
     
     
         12 . The cell-targeting molecule of  claim 11 , wherein the Shiga toxin effector polypeptide comprises or consists of a sequence that is at least 75%, 85%, 95%, 96%, 97%, 98%/o or more identical to:
 (i) amino acids 75 to 251 of any one of SEQ ID NOs: 1-6;   (ii) amino acids 1 to 241 of any one of SEQ ID NOs: 1-18;   (iii) amino acids 1 to 251 of any one of SEQ ID NOs: 1-6; or   (iv) amino acids 1 to 261 of any one of SEQ ID NOs: 1-3.   
     
     
         13 . (canceled) 
     
     
         14 . The cell-targeting molecule of  claim 12 , wherein the disrupted furin-cleavage motif comprises a mutation, relative to a wild-type Shiga toxin A Subunit, the mutation altering at least one amino acid residue in a region natively positioned
 at 248-251 of any one of SEQ ID NOs: 1-2 and, or   at 247-250 of any one of SEQ ID NOs: 3 and 7-18.   
     
     
         15 . (canceled) 
     
     
         16 . The cell-targeting molecule of  claim 15 , wherein the amino acid residue substitution is of an arginine residue with a non-positively charged, amino acid residue selected from:
 alanine, glycine, proline, serine, threonine, aspartate, asparagine, glutamate, glutamine, cysteine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, and tyrosine.   
     
     
         17 . The cell-targeting molecule of  claim 11 , wherein the extracellular target biomolecule:
 CD20, CD22, CD40, CD74, CD79, CD25, CD30, HER2/neu/ErbB2, EGFR, EpCAM, EphB2, prostate-specific membrane antigen, Cripto, CDCP1, endoglin, fibroblast activated protein, Lewis-Y, CD19, CD21, CSI/SLAMF7, CD33, CD52, CD133, CEA, gpA33, mucin, TAG-72, tyrosine-protein kinase transmembrane receptor, 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, IGF1R, EphA3, TRAIL-R1, TRAIL-R2, RANK, 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-I, CDK-4, beta-catenin, MUM-I, 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, gp100, tyrosinase associated antigen, HPV-E7, Epstein-Barr virus antigen, Bcr-Abl, alpha-fetoprotein antigen, 17-A1, bladder tumor antigen, SAIL, CD38, CD15, CD23, CD45, CD53, CD88, CD129, CD183, CD191, CD193, CD244, CD294, CD305, C3AR, FceRIa, IL-1R, galectin-9, mrp-14, NKG2D, PD-L1, 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 I molecule, MHC class II molecule, CD284, CD107-Mac3, CD195, HLA-DR, CD16/32, CD282, CD11c, or any immunogenic fragment of any of the foregoing.   
     
     
         18 . The cell-targeting molecule of  claim 2 , wherein the Shiga toxin effector polypeptide is capable of exhibiting one or more Shiga toxin effector functions in addition to delivery of the CD8+ T-cell epitope cargo from an early endosomal compartment of a cell in which the Shiga toxin effector polypeptide is present to a MHC class I molecule of the cell. 
     
     
         19 - 20 . (canceled) 
     
     
         21 . The cell-targeting molecule of  claim 2 , whereby administration of the cell-targeting molecule to a cell physically coupled with an extracellular target biomolecule of the binding region results in death of the cell. 
     
     
         22 - 30 . (canceled) 
     
     
         31 . The cell-targeting molecule of  claim 1 , in the form of a pharmaceutically acceptable salt or solvate. 
     
     
         32 . A pharmaceutical composition comprising the cell-targeting molecule of  claim 1  and at least one pharmaceutically acceptable excipient or carrier. 
     
     
         33 . A polynucleotide capable of encoding the cell-targeting molecule of  claim 4 , or a complement thereof. 
     
     
         34 - 36 . (canceled) 
     
     
         37 . A method of delivering a CD8+ T-cell epitope to an intracellular MHC class I molecule of a cell, the method comprising the step of contacting the cell with the cell-targeting molecule  claim 1  and/or the pharmaceutical composition of  claim 32 . 
     
     
         38 - 39 . (canceled) 
     
     
         40 . A method of treating a disease, disorder, or condition in a patient, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of the cell-targeting molecule of  claim 1  or the pharmaceutical composition of  claim 32 . 
     
     
         41 - 53 . (canceled)