De-Immunized, Shiga Toxin A Subunit Scaffolds and Cell-Targeting Molecules Comprising the Same

Abstract

The present invention relates to Shiga toxin A Subunit derived polypeptides and cell-targeting molecules comprising amino acid substitutions which equip the polypeptides with 1) de-immunization; 2) reduced, protease-cleavage sensitivity; and/or 3) a heterologous epitope cargo(s) while retaining Shiga toxin function(s), such as, e.g., potent cytotoxicity. Certain polypeptides of the invention exhibit reduced immunogenic potential in mammals and/or are capable of delivering an epitope to an MHC class molecule of a cell in which the polypeptide is present. Certain molecules comprising a polypeptide of the invention are well-tolerated by mammals while retaining one or more of the features mentioned above. The Shiga toxin polypeptides of the invention have uses as components of cell-targeting molecules for selectively killing specific cells; for selectively delivering cargos to specific cells, and as therapeutic and/or diagnostic molecules for treating and diagnosing a variety of conditions, including cancers, immune disorders, and microbial infections.

The invention is claimed as follows: 
     
         1 . Shiga toxin effector polypeptide comprising
 i) an embedded or inserted, heterologous, CD8+ T-cell epitope and   ii) 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;   
       wherein the Shiga toxin effector polypeptide is capable of exhibiting a Shiga toxin effector function selected from one or more of:
 directing intracellular routing to the cytosol of a cell in which the polypeptide is present, 
 directing intracellular routing with a cargo linked directly or indirectly to the polypeptide, 
 inhibiting a ribosome function, enzymatically inactivating a ribosome, and cytotoxicity. 
 
     
     
         2 - 4 . (canceled) 
     
     
         5 . The Shiga toxin effector polypeptide of  claim 1 , wherein the disruption comprises a mutation, relative to a wild-type Shiga toxin A Subunit, in the B-cell and/or CD4+ T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consisting of:
 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; 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; 285-293 of SEQ ID NO:1 or SEQ ID NO:2; 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:11 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:11 or SEQ ID NO:2; and 274-293 of SEQ ID NO:1 or SEQ ID NO:2.   
     
     
         6 . The Shiga toxin effector polypeptide of  claim 1 , wherein the embedded or inserted, heterologous, CD8+ T-cell epitope disrupts the endogenous, B-cell epitope and/or CD4+ T-cell epitope region selected from the group of natively positioned Shiga toxin A Subunit regions consisting of:
 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; 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; 285-293 of SEQ ID NO:1 or SEQ ID NO:2; 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.   
     
     
         7 . The Shiga toxin effector polypeptide of  claim 1 , which comprises disruptions of at least two, three, four, five, six, seven, eight, or more endogenous, B-cell and/or CD4+ T-cell epitope regions. 
     
     
         8 . The Shiga toxin effector polypeptide of  claim 1 , wherein at least one disruption comprises an amino acid residue substitution relative to a wild-type Shiga toxin A Subunit. 
     
     
         9 . The Shiga toxin effector polypeptide of  claim 8 , wherein at least one disruption comprises a plurality of amino acid residue substitutions in the epitope region relative to a wild-type Shiga toxin A Subunit. 
     
     
         10 . The Shiga toxin effector polypeptide of  claim 8  or  claim 9 , wherein at least one disruption comprises at least one, two, three, four, five, six, seven, eight, or more amino acid residue substitutions, relative to a wild-type Shiga toxin A Subunit, and optionally wherein at least one substitution occurs at the natively positioned Shiga toxin A Subunit amino acid residue selected from the group consisting of:
 1 of SEQ ID NO:11 or SEQ ID NO:2; 4 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 6 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 8 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 9 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 11 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 12 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 33 of SEQ ID NO:1 or SEQ ID NO:2; 43 of SEQ ID NO:1 or SEQ ID NO:2; 44 of SEQ ID NO:1 or SEQ ID NO:2; 45 of SEQ ID NO:1 or SEQ ID NO:2; 46 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 47 of SEQ ID NO:1 or SEQ ID NO:2; 48 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 49 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 50 of SEQ ID NO:1 or SEQ ID NO:2; 51 of SEQ ID NO:1 or SEQ ID NO:2; 53 of SEQ ID NO:1 or SEQ ID NO:2; 54 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 55 of SEQ ID NO:1 or SEQ ID NO:2; 56 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 57 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 58 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 59 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 60 of SEQ ID NO:1 or SEQ ID NO:2; 61 of SEQ ID NO:1 or SEQ ID NO:2; 62 of SEQ ID NO:1 or SEQ ID NO:2; 84 of SEQ ID NO:1 or SEQ ID NO:2; 88 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 94 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 96 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 104 of SEQ ID NO:1 or SEQ ID NO:2; 105 of SEQ ID NO:1 or SEQ ID NO:2; 107 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 108 of SEQ ID NO:1 or SEQ ID NO:2; 109 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 110 of SEQ ID NO:1 or SEQ ID NO:2; 111 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 112 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 141 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 147 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 154 of SEQ ID NO:1 or SEQ ID NO:2; 179 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 180 of SEQ ID NO:1 or SEQ ID NO:2; 181 of SEQ ID NO:1 or SEQ ID NO:2; 183 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 184 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 185 of SEQ ID NO:1 or SEQ ID NO:2; 186 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 187 of SEQ ID NO:1 or SEQ ID NO:2; 188 of SEQ ID NO:1 or SEQ ID NO:2; 189 of SEQ ID NO:1 or SEQ ID NO:2; 197 of SEQ ID NO:3; 198 of SEQ ID NO:1 or SEQ ID NO:2; 204 of SEQ ID NO:3; 205 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:1 or SEQ ID NO:2; 247 of SEQ ID NO:3; 248 of SEQ ID NO:1 or SEQ ID NO:2; 250 of SEQ ID NO:3; 251 of SEQ ID NO:1 or SEQ ID NO:2; 264 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3; 265 of SEQ ID NO:1 or SEQ ID NO:2; and 286 of SEQ ID NO:1 or SEQ ID NO:2. 
 
     
     
         11 . The Shiga toxin effector polypeptide of  claim 8 , wherein at least one of the substitutions is selected from the group consisting of:
 D to A, D to G, D to V, D to L, D to I, D to F, D to S, D to Q, D to M, D to R, E to A, E to G, E to V, E to L, E to I, E to F, E to S, E to Q, E to N, E to D, E to M, E to R, F to A, F to G, F to V, F to L, F to L G to A, G to P, H to A, H to G, H to V, H to L, H to I, H to F, H to M, I to A, I to V, I to G, I to C, K to A, K to G, K to V, K to L, K to I, K to M, K to H, L to A, L to V, L to G, L to C, N to A, N to G, N to V, N to L, N to I, N to F, P to A, P to G, P to F, R to A, R to G, R to V, R to L, R to I, R to F, R to M, R to Q, R to S, R to K, R to H, S to A, S to G, S to V, S to L, S to, S to F, S to M, T to A, T to G, T to V, T to L, T to I, T to F, T to M, T to S, V to A, V to G, Y to A, Y to G, Y to V, Y to L, Y to I, Y to F, Y to M, and Y to T.   
     
     
         12 . The Shiga toxin effector polypeptide of  claim 11 , wherein one or more substitutions is selected from the group of substitutions at native positions in a Shiga toxin A Subunit consisting of:
 K1 to A, G, V, L, I, F, M or H; T4 to A, G, V, L, I, F, M, or S; D6 to A, G, V, L, I, F, S, Q or R; S8 to A, G, V, I, L, F, or M; T9 to A, G, V, I, L, F, M, or S; S9 to A, G, V, L, I, F, or M; K1 to A, G, V, L, I, F, M or H; T12 to A, G, V, I, L, F, M, S, or K; S12 to A, G, V, I, L, F, or M; S33 to A, G, V, L, I, F, M, or C; S43 to A, G, V, L, I, F, or M; G44 to A or L; S45 to A, G, V, L, I, F, or M; T45 to A, G, V, L, I, F, or M; G46 to A or P; D47 to A, G, V, L, I, F, S, M, or Q; N48 to A, G, V, L, M or F; L49 to A, V, C, or G; Y49 to A, G, V, L, I, F, M, or T; F50 to A, G, V, L, I, or T; A51; D53 to A, G, V, L, I, F, S, or Q; V54 to A, G, I, or L; R55 to A, G, V, L, I, F, M, Q, S, K, or H; G56 to A or P; 157 to A, G, V, or M; L57 to A, V, C, G, M, or F; D58 to A, G, V, L, I, F, S, or Q; P59 to A, G, or F; E60 to A, G, V, L, I, F, S, Q, N, D, M, T, or R; E61 to A, G, V, L, I, F, S, Q, N, D, M, or R; G62 to A; R84 to A, G, V, L, I, F, M, Q, S, K, or H; V88 to A or G; 188 to A, V, C, or G; D94 to A, G, V, L, I, F, S, or Q; S96 to A, G, V, I, L, F, or M; T104 to A, G, V, L, I, F, M; or N; A105 to L; T107 to A, G, V, L, I, F, M, or P; S107 to A, G, V, L, I, F, M, or P; L108 to A, V, C, or G; S109 to A, G, V, I, L, F, or M; T109 to A, G, V, I, L, F, M, or S; G10 to A; S112 to A, G, V, L, I, F, or M; D111 to A, G, V, L, I, F, S, Q, or T; S112 to A, G, V, L, I, F, or M; D141 to A, G, V, L, I, F, S, or Q; G147 to A; V154 to A or G, R179 to A, G, V, L, I, F, M, Q, S, K, or H; T180 to A, G, V, L, I, F, M, or S; T181 to A, G, V, L, I, F, M, or S; D183 to A, G, V, L, I, F, S, or Q; D184 to A, G, V, L, I, F, S, or Q; L185 to A, G, V or C; S186 to A, G, V, I, L, F, or M; G187 to A; R188 to A, G, V, L, I, F, M, Q, S, K, or H; S189 to A, G, V, I, L, F, or M; D198 to A, G, V, L, I, F, S, or Q; R204 to A, G, V, L, I, F, M, Q, S, K, or H; R205 to A, G, V, L, I, F, M, Q, S, K or H; S247 to A, G, V, I, L, F, or M; Y247 to A, G, V, L, I, F, or M; R248 to A, G, V, L, I, F, M, Q, S, K, or H; R250 to A, G, V, L, I, F, M, Q, S, K, or H; R251 to A, G, V, L, I, F, M, Q, S, K, or H; D264 to A, G, V, L, I, F, S, or Q; G264 to A; and T286 to A, G, V, L, I, F, M, or S.   
     
     
         13 . The Shiga toxin effector polypeptide  claim 6 , wherein the Shiga toxin effector polypeptide comprises or consists essentially of the polypeptide shown in any one of SEQ ID NOs: 355-369 which further comprises a disruption of at least one, endogenous, B-cell and/or CD4+ T-cell epitope which does not overlap with an embedded or inserted, heterologous, CD8+ T-cell epitope. 
     
     
         14 . The Shiga toxin effector polypeptide of  claim 1 , wherein the Shiga toxin effector polypeptide comprises or consists essentially of the polypeptide shown in any one of SEQ ID NOs: 6-32, 340-354, and 370-438. 
     
     
         15 . (canceled) 
     
     
         16 . The Shiga toxin effector polypeptide of  claim 1 , 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. 
     
     
         17 . The Shiga toxin effector polypeptide of  claim 16 , wherein the mutation, relative to the wild-type Shiga toxin A Subunit which changes an enzymatic activity of the Shiga toxin effector polypeptide, reduces or eliminates a cytotoxicity of the Shiga toxin effector polypeptide. 
     
     
         18 . The Shiga toxin effector polypeptide of  claim 1 , wherein the Shiga toxin effector polypeptide comprises:
 a Shiga toxin A1 fragment region having a carboxy-terminus and   a disrupted furin-cleavage motif at the carboxy-terminus of the A1 fragment region.   
     
     
         19 . The Shiga toxin effector polypeptide of  claim 18 , 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 the A Subunit of Shiga-like toxin 1 (SEQ ID NO:1) or Shiga toxin (SEQ ID NO: 2), or   at 247-250 of the A Subunit of Shiga-like toxin 2 (SEQ ID NO:3).   
     
     
         20 . The Shiga toxin effector polypeptide of  claim 18  or  claim 19 , 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. 
     
     
         21 . (canceled) 
     
     
         22 . A cell-targeting molecule comprising
 i) a binding region capable of specifically binding an extracellular target biomolecule physically coupled to the cellular surface of a cell, and   ii) a Shiga toxin effector polypeptide of any one of  claim 1 ,  5 - 14 , or  16 - 20 .   
     
     
         23 - 24 . (canceled) 
     
     
         25 . The cell-targeting molecule of  claim 22 , whereby upon 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 of exhibiting a cytotoxic effect on the first population of cells that is at least 3-fold greater compared to a cytotoxic effect exhibited on the second population of cells. 
     
     
         26 . The cell-targeting molecule of  claim 25 , which comprises a molecular moiety associated with the carboxy-terminus of the Shiga toxin effector polypeptide and wherein the molecular moiety is cytotoxic. 
     
     
         27 . The cell-targeting molecule of  claim 25 , which comprise a molecular moiety associated with the carboxy-terminus of the Shiga toxin effector polypeptide and wherein the molecular moiety comprises or consists of the binding region. 
     
     
         28 - 30 . (canceled) 
     
     
         31 . The cell-targeting molecule of  claim 27 , wherein the molecular moiety and the Shiga toxin effector polypeptide are fused forming a continuous polypeptide. 
     
     
         32 . (canceled) 
     
     
         33 . The cell-targeting molecule of  claim 25 , wherein the cell-targeting molecule is capable of exhibiting a level of cytotoxicity that is at least as cytotoxic as a level of cytotoxicity observed for a reference cell-targeting molecule consisting of the cell-targeting molecule except that the Shiga toxin effector polypeptide consists of a wild-type Shiga toxin A1 polypeptide. 
     
     
         34 . The cell-targeting molecule of  claim 22 , wherein the binding region comprises or consists essentially of the polypeptide represented by any of the following:
 amino acids 1-245 of any one of SEQ ID NOs: 33, 64, and 65; 269-513 of SEQ ID NO:40 or SEQ ID NO:80; amino acids 269-520 or 269-521 of any one of SEQ ID NOs: 36, 66, and 67; amino acids 1-232, 1-233, 1-234, 1-235, 1-236, 1-242, 1-243, 1-244, 1-245, 1-246, 1-252, 1-253, 1-254, 1-255, or 1-256 of any one of SEQ ID NOs: 47-119 and 176-248; amino acids 269-498 or 269-499 of any one of SEQ ID NOs: 37-39, 68-79, and 81; or amino acids 269-499, 269-512, 269-513, or 280-510 of any one of SEQ ID NOs: 34, 35, 41-56, and 82.   
     
     
         35 . The cell-targeting molecule of  claim 34 , which comprises or consists essentially of the polypeptide shown in any one of SEQ ID NOs: 43-62, 64-82, and 439-513. 
     
     
         36 . The cell-targeting molecule of  claim 26  or  claim 27 , wherein the cell-targeting molecule is capable, upon administration of mammals, of exhibiting an in vivo tolerability associated with less mortality or moribundity as compared to an in vivo tolerability observed for a third cell-targeting molecule consisting of the cell-targeting molecule except that the Shiga toxin effector polypeptide consists of a wild-type Shiga toxin A1 polypeptide. 
     
     
         37 . (canceled) 
     
     
         38 . A pharmaceutical composition comprising
 the Shiga toxin effector polypeptide of any one of  claim 1 ,  5 - 14 , and  16 - 20  or the   cell-targeting molecule of any one of  claim 22 ,  25 - 27 ,  31 , and  33 - 36 ; and   at least one pharmaceutically acceptable excipient or carrier.   
     
     
         39 - 53 . (canceled)