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.

The invention is claimed as follows: 
     
         1 . A cell-targeting molecule comprising
 i) a Shiga toxin effector polypeptide having a Shiga toxin A1 fragment region 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; 
 b); and 
 c) a disrupted furin-cleavage motif at the carboxy-terminus of the A1 fragment derived region: 
   ii) a heterologous 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.   
     
     
         2 . The cell-targeting molecule of  claim 1 , whereby administration of the cell-targeting molecule to a cell to physically coupled with an extracellular target biomolecule bound by the binding region results in internalization of the cell-targeting molecule by the cell and the cell presenting on a cellular surface the CD8+ T-cell epitope cargo complexed with a MHC class I molecule. 
     
     
         3 . The cell-targeting molecule of  claim 2 , wherein the CD8+ T-cell epitope 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 a single-chain polypeptide comprising the binding region, the Shiga toxin effector polypeptide, and the CD8+ T-cell epitope cargo. 
     
     
         5 . The cell-targeting molecule of  claim 3 , wherein the binding region comprises two or more polypeptide chains and the CD8+ T-cell epitope cargo is fused to a polypeptide comprising the Shiga toxin effector polypeptide and one of the two or more polypeptide chains. 
     
     
         6 . The cell-targeting molecule of any one of  claims 1 - 5 , wherein the CD8+ T-cell epitope cargo is positioned carboxy-terminal to the carboxy terminus of the Shiga toxin A1 fragment derived region. 
     
     
         7 . The cell-targeting molecule of any one of  claims 1 - 6 , which comprises a molecular moiety associated with the carboxy-terminus of the Shiga toxin effector polypeptide. 
     
     
         8 . The cell-targeting molecule of  claim 7 , wherein the molecular moiety comprises the binding region. 
     
     
         9 . The cell-targeting molecule of  claim 7 , wherein the molecular moiety is cytotoxic. 
     
     
         10 . The cell-targeting molecule of any one of  claims 7 - 9 , wherein the molecular moiety comprises at least one amino acid and the Shiga toxin effector polypeptide is linked to at least one amino acid residue of the molecular moiety. 
     
     
         11 . The cell-targeting molecule of  claim 10 , wherein the molecular moiety and the Shiga toxin effector polypeptide are fused forming a continuous polypeptide. 
     
     
         12 . The cell-targeting molecule of any one of  claims 1 - 11 , wherein the binding region comprises a polypeptide selected from the group consisting of:
 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 10 th  fibronectin type III domain, teascin 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 which retain binding functionality.   
     
     
         13 . The cell-targeting molecule of any one of  claims 1 - 12 , wherein the Shiga toxin effector polypeptide comprises or consists of a sequence that is at least 75%, 85%, 95%, 96%, 97%, 98% or more identical to a sequence selected from:
 (i) amino acids 75 to 251 of any one of SEQ ID NOs: 1-18;   (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: 1-18; and   (iv) amino acids 1 to 261 of any one of SEQ ID NOs: 1-18.   
     
     
         14 . The cell-targeting molecule of  claim 13 , wherein the Shiga toxin effector polypeptide comprises or consists essentially of the polypeptide sequence selected from the group consisting of:
 (i) amino acids 75 to 251 of any one of SEQ ID NOs: 1-18:   (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: 1-18; and   (iv) amino acids 1 to 261 of any one of SEQ ID NOs: 1-18.   
     
     
         15 . The cell-targeting molecule of any one of  claims 1 - 14 , wherein the disrupted furin-cleavage motif comprises one or more mutations, 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 7-18, or   at 247-250 of any one of SEQ ID NOs: 3-6.   
     
     
         16 . The cell-targeting molecule of  claim 15 , wherein the disrupted furin-cleavage motif comprises an amino acid residue substitution in the furin-cleavage motif relative to a wild-type Shiga toxin A Subunit. 
     
     
         17 . The cell-targeting molecule of  claim 16 , wherein the substitution of the amino acid residue in the furin-cleavage motif is of an arginine residue with a non-positively charged, amino acid residue selected from the group consisting of:
 alanine, glycine, proline, serine, threonine, aspartate, asparagine, glutamate, glutamine, cysteine, isoleucine, leucine, methionine, valine, phenylalanine, tryptophan, and tyrosine.   
     
     
         18 . The cell-targeting molecule of any one of  claims 1 - 17 , wherein the binding region is capable of binding to the extracellular target biomolecule selected from the group consisting of:
 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, CS1/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-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, 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, and any immunogenic fragment of any of the foregoing.   
     
     
         19 . The cell-targeting molecule of any one of  claims 1 - 18 , 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. 
     
     
         20 . The cell-targeting molecule of  claim 19 , which is capable of exhibiting a ribosome inhibition activity with a half-maximal inhibitory concentration (IC 50 ) value of 10,000 picomolar or less. 
     
     
         21 . The cell-targeting molecule of any one of  claims 1 - 20 , wherein the Shiga toxin effector polypeptide comprises
 one or more mutations, relative to a wild-type Shiga toxin A Subunit, which changes an enzymatic activity of the Shiga toxin effector polypeptide, the mutation selected from at least one amino acid residue deletion, insertion, or substitution, and   wherein the mutation(s) reduces or eliminates a cytotoxicity of the Shiga toxin effector polypeptide exhibited by the wild-type Shiga toxin A Subunit.   
     
     
         22 . The cell-targeting molecule of any one of  claims 1 - 21 , whereby administration of the cell-targeting molecule to a cell physically coupled with an extracellular target biomolecule of the binding region, the cell-targeting molecule is capable of causing death of the cell. 
     
     
         23 . The cell-targeting molecule of  claim 22 , whereby administration of the cell-targeting molecule to a first population of cells whose members are physically coupled to extracellular target biomolecules of the binding region, and a second population of cells whose members are not physically coupled to any extracellular target biomolecule of the binding region, the 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. 
     
     
         24 . The cell-targeting molecule of any one of  claims 1 - 23 , which comprises two, three, four, or more heterologous, CD8+ T-cell epitope cargos which are not embedded or inserted in the Shiga toxin A1 fragment region. 
     
     
         25 . The cell-targeting molecule of  claim 24 , wherein the two, three, four, or more heterologous, CD8+ T-cell epitope cargos are positioned carboxy-terminal to the carboxy terminus of the Shiga toxin A1 fragment region. 
     
     
         26 . The cell-targeting molecule of any one of  claims 1 - 25 , which comprises or consists essentially of the polypeptide of any one of SEQ ID NOs: 252-255 and 288-748. 
     
     
         27 . The cell-targeting molecule of any one of  claims 1 - 26 , which further comprises two or more heterologous binding regions, each of which is capable of specifically binding at least one extracellular target biomolecule. 
     
     
         28 . The cell-targeting molecule of  claim 27 , which comprises two or more components, each component comprising at least one heterologous binding region and at least one of the Shiga toxin effector polypeptide. 
     
     
         29 . The cell-targeting molecule of  claim 28 , which comprises or consists essentially of two or more protein components associated through one or more non-covalent interactions. 
     
     
         30 . The cell-targeting molecule of  claim 28 , which comprises or consists essentially of two proteins, each protein selected from any one of the proteins shown in SEQ ID NOs: 252-255 and 288-748, and which optionally comprises an amino-terminal methionine residue. 
     
     
         31 . The cell-targeting molecule of  claim 28 , which comprises or consists essentially of two or more protein components associated through one or more covalent interactions. 
     
     
         32 . The cell-targeting molecule of  claim 31 , wherein the two or more protein components are selected from any one of the polypeptides shown in SEQ ID NOs: 253, 267-278, 352, 406, 445, 498, 538, 590, 629, and 657-711;
 wherein the cell-targeting molecule comprises a cysteine disulfide bond between two or more of the protein components; and   wherein the cysteine disulfide bond involves a cysteine residue in one or both of the two protein components located, for example, at amino acid position 242, 243, 250, 251, 252, 355, 364, 386, 400, 401, 508, 510, 511, 512, 513, 517, 518, 521, or 522.   
     
     
         33 . A pharmaceutical composition comprising a cell-targeting molecule of any one of  claims 1 - 32  and at least one pharmaceutically acceptable excipient or carrier. 
     
     
         34 . A polynucleotide capable of encoding a cell-targeting molecule of any one of  claims 1 - 32 , or a protein component thereof, or a complement or fragment of any of the foregoing. 
     
     
         35 . An expression vector comprising the polynucleotide of  claim 34 . 
     
     
         36 . A host cell comprising a polynucleotide of  claim 34  and/or an expression vector of  claim 35 . 
     
     
         37 . A method of killing a cell, the method comprising the step of contacting the cell with a cell-targeting molecule of any one of  claims 1 - 32 , or a pharmaceutical composition of  claim 33 . 
     
     
         38 . A method of delivering a CD8+ T-cell epitope to an intracellular MHC class I molecule of a cell (which is optionally a tumor or cancer cell), the method comprising the step of contacting the cell with a cell-targeting molecule of any one of  claims 1 - 32  and/or the pharmaceutical composition of  claim 33 . 
     
     
         39 . The method of  claim 37  or  claim 38 , wherein the contacting occurs in vitro. 
     
     
         40 . The method of  claim 37  or  claim 38 , wherein the contacting occurs in vivo. 
     
     
         41 . 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 a cell-targeting molecule of any one of  claims 1 - 32  or a pharmaceutical composition of  claim 33 . 
     
     
         42 . The method of  claim 41 , wherein the disease, disorder, or condition is selected from the group consisting of: cancer, tumor, growth abnormality, immune disorder, and microbial infection. 
     
     
         43 . The method of  claim 42 , wherein the cancer selected from the group consisting of: bone cancer, breast cancer, central/peripheral nervous system cancer, gastrointestinal cancer, germ cell cancer, glandular cancer, head-neck cancer, hematological cancer, kidney-urinary tract cancer, liver cancer, lung/pleura cancer, prostate cancer, sarcoma, skin cancer, and uterine cancer. 
     
     
         44 . The method of  claim 42 , wherein the immune disorder associated is with a disease selected from the group consisting of:
 amyloidosis, ankylosing spondylitis, asthma, autism, cardiogenesis, Crohn's disease, diabetes, erythematosus, gastritis, graft rejection, graft-versus-host disease, Grave's disease, Hashimoto's thyroiditis, hemolytic uremic syndrome, HIV-related disease, lupus erythematosus, lymphoproliferative disorders, multiple sclerosis, myasthenia gravis, neuroinflammation, polyarteritis nodosa, polyarthritis, psoriasis, psoriatic arthritis, rheumatoid arthritis, scleroderma, septic shock, Sjögren's syndrome, systemic lupus erythematosus, ulcerative colitis, and vasculitis.   
     
     
         45 . A composition comprising a cell-targeting molecule of any one of  claims 1 - 36 , for the treatment or prevention of a cancer, tumor, growth abnormality, immune disorder, or microbial infection. 
     
     
         46 . A method of “seeding” a tissue locus within a chordate, the method comprising the step of administering to the chordate a cell-targeting molecule of any one of  claims 1 - 32  and/or a pharmaceutical composition of  claim 33 . 
     
     
         47 . The method of  claim 46 , wherein the T-cell epitope cargo of the cell-targeting molecule is selected from the group consisting of a:
 peptide not natively presented by the target cells of the cell-targeting molecule in MHC class I complexes, peptide not natively present within any protein expressed by the target cell, peptide not natively present within the transcriptome or proteome of the target cell, peptide not natively present in the extracellular microenvironment of the site to be seeded, and peptide not natively present in the tumor mass or infect tissue site to be targeted.   
     
     
         48 . The method of  claim 46 , wherein the tissue locus comprises a malignant, diseased, or inflamed tissue. 
     
     
         49 . The method of  claim 48 , wherein the tissue locus comprises the tissue selected from the group consisting of tumor mass, cancerous growth, tumor, infected tissue, or abnormal cellular mass. 
     
     
         50 . A method of treating cancer using immunotherapy, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of a cell-targeting molecule of any one of  claims 1 - 32  and/or a pharmaceutical composition of  claim 33 . 
     
     
         51 . Use of a composition of matter of any one of  claims 1 - 36  in the manufacture of a medicament for the treatment or prevention of a cancer, tumor, immune disorder, or microbial infection. 
     
     
         52 . Use of a composition of matter of any one of  claims 1 - 36  in the diagnosis, prognosis, or characterization of a disease, disorder, or condition. 
     
     
         53 . A kit comprising the composition of matter of any one of  claims 1 - 36 ; and an additional reagent and/or pharmaceutical delivery device.