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

A cytotoxic cell-targeting molecule comprising

• i) a heterologous, binding region capable of specifically binding an extracellular target biomolecule,
• ii) a cytotoxic, 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 241 of SEQ ID NO:
• 1 or SEQ ID NO:
• 2;
• and
• 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;
• 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;
• wherein the cytotoxic cell-targeting molecule does not comprise a compensatory furin-cleavage site located carboxy-terminal to the Shiga toxin effector polypeptide;
• and
• wherein the cytotoxic cell-targeting molecule is capable of exhibiting cytotoxicity equivalent to the cytotoxicity of a second cytotoxic cell-targeting molecule, wherein said second cytotoxic cell-targeting molecule consists of the cytotoxic 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.