Cytotoxic proteins comprising cell-targeting binding regions and shiga toxin a subunit regions for selective killing of specific cell types

Abstract

The present invention provides cytotoxic proteins comprising immunoglobulin-type binding regions for mediating cell-type specific targeting and Shiga toxin effector regions derived from A Subunits of members of the Shiga toxin family for effectuating cytotoxicity. The cytotoxic proteins have uses for selective killing of specific cell types and as therapeutics for the treatment of a variety of diseases, including cancers, immune disorders, and microbial infections.

The invention is claimed as follows: 
     
         1 . A cytotoxic fusion protein comprising
 a) an immunoglobulin-type binding region comprising one or more polypeptides selected from the group consisting of: single-domain antibody fragment, single-chain variable fragment, antibody variable fragment, antigen-binding fragment, and Fd fragment; and   wherein the immunoglobulin-type binding region is capable of specifically binding at least one extracellular target biomolecule; and   b) a Shiga toxin A Subunit effector polypeptide;   wherein the immunoglobulin-type binding region polvpeptide is fused via a peptide bond, either directly or indirectly via one or more intervening proteinaceous linkers fused via peptide bonds, to the Shiga toxin A Subunit effector polypeptide; and   whereby administration of the cytotoxic fusion protein to a cell expressing on a cellular surface the extracellular target biomolecule bound by the immunoglobulin-type binding region, the cytotoxic fusion rotein is capable of causing death of the cell.   
     
     
         2 - 3 . (canceled) 
     
     
         4 . The cytotoxic fusion protein of  claim 1 , wereby upon administration of the cytotoxic fusion protein to a first populations of cells whose members are express an extracellular target biomolecule bound by the immunoglobulin-type binding region, and a second population of cells whose members do not express any extracellular target biomolecule of bound by the immunoglobulin-type binding region, a cytotoxic effect of the cytotoxic fusion protein to members of said first population of cells relative to members of said second population of cells is at least 3-fold, 5-fold, 10-fold, 100-fold, 500-fold, or 1000-fold greater. 
     
     
         5 . The cytotoxic fusion protein of  claim 4 , wherein the immunoglobulin-type binding region is capable of binding to an extracellular target biomolecule which is CD20, HER2/neu/ErbB2,. 
     
     
         6 . The cytotoxic fusion protein of  claim 5 , wherein the Shiga toxin effector polypeptide comprises or consists essentially of a polypeptide sequence selected from the group consisting 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, SEQ ID NO:3,   (iv) amino acids 1 to 261 of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3.   
     
     
         7 - 9 . (canceled) 
     
     
         10 . The cytotoxic fusion protein of  claim 5 , wherein the immunoglobulin-type binding region comprises or consists essentially of the polypeptide sequence of amino acids 269-508 of SEQ ID NO:4. 
     
     
         11 . The cytotoxic fusion protein of  claim 6 , whih comprises or consists essentially of the polypeptide shown in SEQ ID NO: 4. 
     
     
         12 . The cytotoxic fusion protein of  claim 5 , wherein the immunoglobulin-type binding region comprises or consists essentially of the polypeptide sequence of amino acids 1-119 of SEQ ID NO:5. 
     
     
         13 . The cytotoxic fusion protein of  claim 6 , which comprises or consists essentially of the polypeptide shown in SEQ ID NO: 5. 
     
     
         14 . The cytotoxic fusion protein of  claim 5 , 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 polypeptide, the mutation selected from at least one amino acid residuesubstitution. 
     
     
         15 . A pharmaceutical composition comprising a cytotoxic fusion protein of any one of  claims 1 ,  4 - 6 ,  10 - 14 , and  28 - 30  and at least one pharmaceutically acceptable excipient or carrier. 
     
     
         16 . A polynucleotide capable of encoding a cytotoxic fusion protein of any one of  claims 1 ,  4 - 6 .  10 - 14 , and  28 - 31 , or a complement thereof, or a fragment of any of the foregoing. 
     
     
         17 . An expression vector comprising a polynucleotide of  claim 16 . 
     
     
         18 . A transfoimed host cell comprising a polynucleotides of  claim 16  or an expression vectors of  claims 17 . 
     
     
         19 . A method of killing a cell expressing an extracellular target biomolecule, the method comprising the step of contacting the cell with a cytotoxic fusion protein of any one of  claims 1 ,  4 - 6 ,  10 - 14  and  28 - 31  or a pharmaceutical composition of  claim 15 ;
 wherein the cytotoxic fusion protein or a cytotoxic Basion protein of the pharmaceutical composition comprises an immunoglobulin-type binding region capable of specifically binding the extracel lular target biomolecule; and 
 whereby the cytotoxic fusion protein enters the cell and subsequently kills the cell. 
 
     
     
         20 . The method of  claim 19 , wherein the contacting occurs in vitro. 
     
     
         21 . The method of  claim 19 , wherein the contacting occurs in vivo. 
     
     
         22 - 27 . (canceled) 
     
     
         28 . The cytotoxic fusion protein of  claim 1 , further comprising an additional proteinaceous material which is fused via a peptide bond to the immunoglobulin-type binding region and/or the Shiga toxin A Subunit effector polypeptide. 
     
     
         29 . The cytotoxic fusion protein of  claim 28 , wherein the Shiga toxin effector polypeptide comprises or consists essentially of a polypeptide sequence selected from the group consisting 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; and   (iv) amino acids 1 to 261 of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID  - N0:3.   
     
     
         30 . The cytotoxic fusion protein of  claim 29 , wherein the extracellular target biomolecule is CD20, HER2/neu/ErbB2, or CD38. 
     
     
         31 . The cytotoxic fusion protein of  claim 28 , 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 polypeptide, the mutation selected from at least one amino acid residue substitution.