Cell-targeting molecules comprising protease-cleavage resistant, shiga toxin a subunit effector polypeptides and carboxy-terminal moieties

Abstract

The present invention provides protease-cleavage resistant molecules comprising Shiga toxin effector polypeptides capable of exhibiting potent, Shiga toxin functions (e.g. subcellular routing and cytotoxicity). The present invention also provides protease-cleavage resistant, cell-targeting molecules for targeting specific cell types, e.g., infected or malignant cells. Certain molecules of the present invention are cytotoxic, and certain cell-targeting molecules of the present invention may be used for the targeted killing of specific cell types and the treatment of a variety of diseases, disorders, and conditions, including cancers, tumors, growth abnormalities, immune disorders, and microbial infections. Certain cell-targeting molecules of the invention exhibit improved, in vivo tolerability as compared to related cell-targeting molecules comprising protease-cleavage sensitive, wild-type, Shiga toxin effector polypeptides. The cell-targeting molecules of the invention can deliver additional materials, such as, e.g., antigens, cytotoxic agents, and detection-promoting agents, into the interiors of target cells.

The invention is claimed as follows: 
     
         1 . A cell-targeting molecule comprising
 i) a heterologous, binding region capable of specifically binding an extracellular target biomolecule;   ii) a Shiga toxin effector polypeptide comprising
 (a) a Shiga toxin A1 fragment region having a carboxy terminus, wherein said Shiga toxin A1 fragment region comprises an amino acid sequence that is at least 95% identical to:
 amino acids 75 to 251 of SEQ ID NO: 1 or SEQ ID NO: 2; or 
 amino acids 1 to 251 of SEQ ID NO: 1 or SEQ ID NO: 2; 
 and 
 
 (b) a disrupted furin-cleavage motif at the carboxy terminus of the A1 fragment region comprising one or more mutations in a minimal furin-cleavage site relative to a wild-type Shiga toxin A Subunit, the one or more mutations comprising a substitution mutation of an arginine residue natively positioned at 248 or 251 of SEQ ID NO: 1 or SEQ ID NO: 2 with a non-positively charged amino acid residue; 
 wherein the Shiga toxin effector polypeptide exhibits at least one Shiga toxin effector function selected from: inducing cellular internalization, delivering an exogenous material into a cell, directing subcellular routing, and directing intracellular routing to a cytosol, and 
   iii) a molecular moiety having a mass of at least 1.5 kDa and associated with the carboxy terminus of the Shiga toxin effector polypeptide; and   wherein the cell-targeting molecule is capable of exhibiting improved stability in vitro compared to a second cell-targeting molecule, wherein said second cell-targeting molecule consists of the cell-targeting molecule except for the Shiga toxin effector polypeptide consists of a wild-type Shiga toxin A1 polypeptide without a disrupted minimal furin-cleavage site.   
     
     
         2 . The cell-targeting molecule of  claim 1 , wherein the cell-targeting molecule does not comprise a compensatory furin-cleavage site located carboxy terminal to the Shiga toxin effector polypeptide. 
     
     
         3 . The cell-targeting molecule of  claim 1 , wherein the Shiga toxin effector polypeptide further comprises at least one mutation relative to a naturally occurring A Subunit of a member of the Shiga toxin family which reduces or eliminates the cytotoxic activity of the Shiga toxin effector polypeptide. 
     
     
         4 . The cell-targeting molecule of  claim 3 , wherein the at least one mutation that reduces or eliminates the cytotoxic activity of the Shiga toxin effector polypeptide comprises a substitution at the amino acid corresponding to the asparagine residue at position 75, the tyrosine residue at position 77, the tyrosine residue at position 114, the glutamate residue at position 167, the arginine residue at position 170, the arginine residue at position 176, or the tryptophan residue at position 203 of SEQ ID NO: 1 or 2. 
     
     
         5 . The cell-targeting molecule of  claim 4 , wherein the at least one mutation that reduces or eliminates cytotoxic activity in the Shiga toxin effector polypeptide is selected from: N75A, Y77S, Y114A, E167D, R170A, R176K, or W203A of SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         6 . The cell-targeting molecule of  claim 1 , wherein the Shiga toxin effector polypeptide comprises an amino acid sequence that is at least 90% identical to residues 224 to 241 of SEQ ID NO: 1 or SEQ ID NO: 2. 
     
     
         7 . The cell-targeting molecule of  claim 1 , wherein the Shiga toxin effector polypeptide does not exhibit significant Shiga toxin A Subunit catalytic activity. 
     
     
         8 . The cell-targeting molecule of  claim 1 , wherein the Shiga toxin effector polypeptide exhibits significant Shiga toxin A Subunit cytotoxicity. 
     
     
         9 . The cell-targeting molecule of  claim 1 , which is capable of exhibiting cytotoxicity equivalent to the cytotoxicity of the second cell-targeting molecule. 
     
     
         10 . The cell-targeting molecule of  claim 1 , wherein the cell-targeting molecule is capable of exhibiting improved in vivo tolerability compared to the second cell-targeting molecule. 
     
     
         11 . The cell-targeting molecule of  claim 1 , which is linked to an additional exogenous material selected from the group consisting of: a peptide, a protein, a cytotoxic agent, a detection promoting agent, a small molecule chemotherapeutic agent, and a polynucleotide, and which delivers the additional exogenous material into a cell that expresses on a cellular surface the extracellular target biomolecule specifically bound by the heterologous, binding region. 
     
     
         12 . The cell-targeting molecule of  claim 11 , wherein the additional exogenous material is a peptide or protein comprising an antigen. 
     
     
         13 . The cell-targeting molecule of  claim 1 , wherein the molecular moiety has a mass of at least 4.5 kDa. 
     
     
         14 . The cell-targeting molecule of  claim 13 , wherein the molecular moiety comprises at least one amino acid residue and the Shiga toxin effector polypeptide is fused to at least one amino acid residue of the molecular moiety. 
     
     
         15 . The cell-targeting molecule of  claim 14 , wherein the molecular moiety comprises the heterologous binding region. 
     
     
         16 . The cell-targeting molecule of  claim 15 , wherein the molecular moiety comprises the heterologous, binding region and wherein the heterologous binding region is fused directly or indirectly to the carboxy-terminus of the Shiga toxin effector polypeptide. 
     
     
         17 . The cell-targeting molecule of  claim 1 , wherein the molecular moiety is cytotoxic. 
     
     
         18 . The cell-targeting molecule of  claim 17 , wherein the Shiga toxin effector polypeptide is not cytotoxic. 
     
     
         19 . The cell-targeting molecule of  claim 17 , wherein the cell-targeting molecule is capable of exhibiting improved in vivo tolerability compared to the second cell-targeting molecule. 
     
     
         20 . The cell-targeting molecule of  claim 1 , wherein the one or more mutations in the minimal furin-cleavage site is an arginine residue natively positioned at 248 or 251 of SEQ ID NO: 1 or SEQ ID NO: 2 with alanine residue. 
     
     
         21 . The cell-targeting molecule of  claim 1 , in the form of a homo-multimer or a hetero-multimer and/or in the form of a pharmaceutically acceptable solvate or salt. 
     
     
         22 . A pharmaceutical composition comprising the cell-targeting molecule of  claim 1 ;
 and at least one pharmaceutically acceptable excipient or carrier.   
     
     
         23 . A polynucleotide capable of encoding the cell-targeting molecule of  claim 1 , or a complement thereof. 
     
     
         24 . An expression vector comprising the polynucleotide of  claim 23 . 
     
     
         25 . A host cell comprising the polynucleotide of  claim 23  or the expression vector of  claim 24 . 
     
     
         26 . A diagnostic composition comprising: the cell-targeting molecule of  claim 1  and a detection promoting agent. 
     
     
         27 . A kit comprising the cell-targeting molecule of  claim 1 ; and an additional reagent and/or pharmaceutical delivery device. 
     
     
         28 . A method of killing a cell, the method comprising contacting the cell with the cell-targeting molecule of  claim 8 , wherein the contacting occurs in vivo. 
     
     
         29 . A method of delivering an additional exogenous material into a cell expressing the extracellular target biomolecule on a cellular surface, the method comprising the step of contacting the cell with a cell-targeting molecule according to  claim 11 . 
     
     
         30 . 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 . 
     
     
         31 . A method for improving in vitro stability of a cell-targeting molecule comprising:
 i) a Shiga toxin effector polypeptide comprising
 (a) a Shiga toxin A1 fragment region having a carboxy terminus, wherein said Shiga toxin A1 fragment region comprises an amino acid sequence that is at least 95% identical to a sequence selected from: 
 amino acids 75 to 251 of SEQ ID NO: 1 or SEQ ID NO: 2; 
 amino acids 1 to 251 of SEQ ID NO: 1 or SEQ ID NO: 2; and 
 (b) a furin-cleavage motif at the carboxy terminus of the A1 fragment region comprising a minimal furin-cleavage site, and 
   ii) a molecular moiety having a mass of at least 1.5 kDa and associated with the carboxy terminus of the Shiga toxin effector polypeptide, and   iii) a heterologous binding region capable of specifically binding an extracellular target biomolecule;   
       the method comprising the step of disrupting the furin-cleavage motif comprising the furin-cleavage site in the cell-targeting molecule. 
     
     
         32 . A cell-targeting molecule comprising
 i) a heterologous, binding region capable of specifically binding an extracellular target biomolecule;   ii) a Shiga toxin effector polypeptide comprising
 (a) a Shiga toxin A1 fragment region having a carboxy terminus,
 wherein said Shiga toxin A1 fragment region comprises an amino acid sequence that is at least 95% identical to: amino acids 1 to 251 of SEQ ID NO: 1 or SEQ ID NO: 2; and 
 
 (b) a disrupted furin-cleavage motif at the carboxy terminus of the A1 fragment region comprising one or more mutations in a minimal furin-cleavage site relative to a wild-type Shiga toxin A Subunit, the one or more mutations comprising a substitution mutation of an arginine residue natively positioned at 248 or 251 of SEQ ID NO: 1 or SEQ ID NO: 2 with a non-positively charged amino acid residue; 
 wherein the Shiga toxin effector polypeptide exhibits at least one Shiga toxin effector function selected from: inducing cellular internalization, delivering an exogenous material into a cell, directing subcellular routing, and directing intracellular routing to a cytosol, and 
   iii) a molecular moiety having a mass of at least 1.5 kDa and associated with the carboxy terminus of the Shiga toxin effector polypeptide; and   
       wherein the cell-targeting molecule is capable of exhibiting improved stability in vitro compared to a second cell-targeting molecule, wherein said second cell-targeting molecule consists of the cell-targeting molecule except for the Shiga toxin effector polypeptide consists of a wild-type Shiga toxin A1 polypeptide without a disrupted minimal furin-cleavage site.