Shiga toxin a subunit effector polypeptides, shiga toxin effector scaffolds, and cell-targeting molecules for site-specific conjugation

Abstract

The present invention provides Shiga toxin A Subunit derived polypeptides, scaffolds, and cell-targeting molecules comprising amino acid substitutions which equip the molecules with site-specific positions (and often unique amino acid residues in the molecule) for linking other molecules while retaining Shiga toxin function(s), such as, e.g., efficient intracellular routing and/or potent cytotoxicity. The present invention also provides cell-targeting molecules, and/or components thereof, which comprise site-specific positions for linking other molecules, such as, e.g., agents that alters a property of the cell-targeting molecule or a cargo for delivery. Certain molecules comprising a polypeptide of the present invention exhibit reduced immunogenicity and/or are well-tolerated by mammals. The cell-targeting molecules of the present invention, and compositions thereof, have uses, e.g., for the selective delivery of cargos to target-expressing cells and as diagnostic and/or therapeutic molecules for the treatment of a variety of diseases, disorders, and conditions, which include genetic disorders, genetic predispositions, infections, cancers, tumors, growth abnormalities, and/or immune disorders.

1 .- 60 . (canceled) 
     
     
         61 . A Shiga toxin effector polypeptide comprising an amino acid sequence that has at least 85% sequence identity to a wild-type Shiga toxin A Subunit sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; wherein the differences between said amino acid sequence and the wild-type Shiga toxin A Subunit sequence include one or more amino acid substitutions;
 wherein the amino acid sequence contains an amino acid residue that occurs only once within the amino acid sequence, said amino acid residue having a functional group suitable for chemical conjugation to an atom or molecule; wherein said amino acid residue is:   (i) one of the one or more amino acid substitutions as compared to the wild-type Shiga toxin A Subunit sequence, or   (ii) a lysine residue that is natively found at that position in the wild-type Shiga toxin A Subunit sequence and occurs only once within the amino acid sequence because all other lysine residues that are present within the wild-type Shiga toxin A Subunit sequence are removed by the one or more amino acid substitutions; and   wherein the Shiga toxin effector polypeptide is capable of exhibiting one or more Shiga toxin effector functions.   
     
     
         62 . The Shiga toxin effector polypeptide of  claim 61 , wherein the amino acid residue that occurs only once within the amino acid sequence is one of the amino acid substitutions as compared to the wild-type Shiga toxin A Subunit sequence. 
     
     
         63 . The Shiga toxin effector polypeptide of  claim 62 , wherein the amino acid residue that occurs only once within the amino acid sequence is selected from the group consisting of: cysteine, lysine, histidine, and a non-natural amino acid residue. 
     
     
         64 . A Shiga toxin effector polypeptide comprising an amino acid sequence selected from the group consisting of:
 (i) an amino acid sequence that is at least 95% identical to SEQ ID NO: 9, 29, or 1101, wherein the only cysteine residue in the amino acid sequence is the cysteine residue at position 33 of SEQ ID NO: 9, 29, or 1101;   (ii) an amino acid sequence that is at least 95% identical to SEQ ID NO: 11, wherein the only cysteine residue in the amino acid sequence is the cysteine residue at position 45 of SEQ ID NO: 11; and   (iii) an amino acid sequence that is at least 95% identical to SEQ ID NO: 14, wherein the only cysteine residue in the amino acid sequence is the cysteine residue at position 54 of SEQ ID NO: 14;   wherein the Shiga toxin effector polypeptide is capable of exhibiting one or more Shiga toxin effector functions.   
     
     
         65 . A Shiga toxin effector polypeptide comprising an amino acid sequence selected from the group consisting of:
 (i) an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 197, 198, and 200, wherein the only lysine residue in the amino acid sequence is the lysine residue at position 1 of SEQ ID NOs: 197, 198, and 200; and   (ii) an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 201, 202, and 205, wherein the only lysine residue in the amino acid sequence is the lysine residue at position 11 of SEQ ID NOs: 201, 202, and 205;   wherein the Shiga toxin effector polypeptide is capable of exhibiting one or more Shiga toxin effector functions.   
     
     
         66 . The Shiga toxin effector polypeptide of  claim 65 , wherein the amino acid residue that occurs only once within the amino acid sequence is covalently linked via a functional group to an atom or molecule. 
     
     
         67 . A cell-targeting molecule comprising:
 (a) a Shiga toxin effector polypeptide selected from:
 (i) a Shiga toxin effector polypeptide has at least 85% sequence identity to a wild-type Shiga toxin A Subunit sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3; wherein the differences between said amino acid sequence and the wild-type Shiga toxin A Subunit sequence include one or more amino acid substitutions; 
 wherein the amino acid sequence contains an amino acid residue that occurs only once within the amino acid sequence, said amino acid residue having a functional group suitable for chemical conjugation to an atom or molecule; wherein said amino acid residue is: 
   a. one of the one or more amino acid substitutions as compared to the wild-type Shiga toxin A Subunit sequence, or   b. a lysine residue that is natively found at that position in the wild-type Shiga toxin A Subunit sequence and occurs only once within the amino acid sequence because all other lysine residues that are present within the wild-type Shiga toxin A Subunit sequence are removed by the one or more amino acid substitutions; and   wherein the Shiga toxin effector polypeptide is capable of exhibiting one or more Shiga toxin effector functions; or
 (ii) a Shiga toxin effector polypeptide consisting of any one of SEQ ID NOs: 5-124,125-232,233-475,1101-1104, and 1109-1140; 
   
       and
 (b) a binding region capable of specifically binding at least one extracellular target biomolecule. 
 
     
     
         68 . The cell-targeting molecule of  claim 67 , which further comprises a linker peptide. 
     
     
         69 . A cell-targeting molecule comprising:
 (a) a polypeptide comprising an amino acid sequence that has at least 95% sequence identity to any one of SEQ ID NOs: 762-767, wherein the polypeptide contains an amino acid residue that occurs only once within the amino acid sequence, said amino acid residue having a functional group suitable for chemical conjugation to an atom or molecule; and wherein the polypeptide is capable of exhibiting one or more Shiga toxin effector functions; and   (b) a binding region capable of specifically binding at least one extracellular target biomolecule.   
     
     
         70 . The cell-targeting molecule of  claim 69 , wherein the amino acid residue that occurs only once within the amino acid sequence is selected from the group consisting of: cysteine, lysine, histidine, and a non-natural amino acid residue such as selenocysteine or pyrroline-carboxy-lysine. 
     
     
         71 . The cell-targeting molecule of  claim 69 , 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, 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, engineered antibody mimic, and any genetically manipulated counterparts of any of the foregoing which retain binding functionality.   
     
     
         72 . The cell-targeting molecule of  claim 69 , wherein the cell-targeting molecule is capable of inducing cellular internalization more efficiently than a reference molecule consisting of the cell-targeting molecule which does not comprise any Shiga toxin effector polypeptide. 
     
     
         73 . The cell-targeting molecule of  claim 69 , whereby upon administration of the cell-targeting molecule to a cell physically coupled with extracellular target biomolecule specifically bound by the binding region, the cell-targeting molecule is capable of causing death of the cell. 
     
     
         74 . The cell-targeting molecule of claim  33 , whereby upon administration of the cell-targeting molecule to a first population of cells whose members are physically coupled to extracellular target biomolecules specifically bound by the binding region, and a second population of cells whose members are not physically coupled to any extracellular target biomolecule specifically bound by the 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. 
     
     
         75 . The cell-targeting molecule of  claim 69 , which is capable of exhibiting a cytotoxic activity to extracellular target biomolecule positive cells with a CD 50  value of 1,000 nanomolar or less and/or a significant level of Shiga toxin cytotoxicity. 
     
     
         76 . The cell-targeting molecule of  claim 69 , wherein the Shiga toxin effector polypeptide comprises a mutation relative to a naturally occurring A Subunit of a member of the Shiga toxin family which changes the enzymatic activity of the Shiga toxin effector region, the mutation selected from at least one amino acid residue deletion or substitution. 
     
     
         77 . The cell-targeting molecule of  claim 76 , wherein the mutation reduces or eliminates cytotoxicity and wherein the Shiga toxin effector polypeptide is capable of exhibiting a Shiga toxin effector function selected from one or more of:
 stimulating cellular internalization, directing intracellular routing to the Golgi apparatus of a cell in which the polypeptide is present, directing intracellular routing to the endoplasmic reticulum of a cell in which the polypeptide is present, directing intracellular routing to the cytosol of a cell in which the polypeptide is present, and directing intracellular routing with a cargo linked directly or indirectly to the polypeptide.   
     
     
         78 . A pharmaceutical composition comprising a Shiga toxin effector polypeptide of claim  1 , and at least one pharmaceutically acceptable excipient or carrier. 
     
     
         79 . A diagnostic composition comprising a cell-targeting molecule of  claim 69 , and a detection-promoting agent. 
     
     
         80 . A polynucleotide capable of encoding a Shiga toxin effector polypeptide of claim  1  and/or a cell-targeting molecule of  claim 69 , or an expression vector thereof. 
     
     
         81 . A method of killing a cell, the method comprising contacting the cell with a composition comprising the cell-targeting molecule of  claim 69 . 
     
     
         82 . The method of  claim 81 , wherein the contacting occurs in vitro. 
     
     
         83 . A method of delivering an exogenous material into a target-expressing cell, the method comprising the step of contacting the cell with a cell-targeting molecule of claim  1 . 
     
     
         84 . The method of  claim 83 , wherein the contacting occurs in vitro. 
     
     
         85 . A method of treating a disease, disorder, or condition in a patient, the method comprising administering to a patient in need thereof a therapeutically effective amount of a composition comprising the cell-targeting molecule according to  claim 69 .