Cell-targeting molecules comprising de-immunized, shiga toxin a subunit effectors and cd8+ t-cell epitopes

A cell targeting molecule comprising:

• i) a Shiga toxin effector polypeptide comprising an amino acid sequence having at least 90% identity to amino acids 1 to 251 of SEQ ID NO:
• 1;
• wherein the effector polypeptide comprises a Shiga toxin A1 fragment region with a disrupted furin-cleavage motif at the carboxy-terminus of the A1 fragment region and wherein the furin-cleavage motif has the amino acid substitutions R248A and R251A;
• wherein the effector polypeptide comprises:
• a) a heterologous, CD8+ T-cell epitope embedded at amino acid positions 53-66 and comprising amino acid substitutions V54L, R55L, I57F, P59F, E60T, and E61L;
• and
• b) amino acid substitutions S45L, G110A and R188A;
• and
• c) an amino acid substitution C242S;
• and
• wherein the effector polypeptide comprises an asparagine at the amino acid residue corresponding to position 75 of SEQ ID NO:
• 1, a tyrosine at the amino acid residue corresponding to position 77 of SEQ ID NO:
• 1, a tyrosine at the amino acid residue corresponding to position 114 of SEQ ID NO:
• 1, a glutamate at the amino acid residue corresponding to position 167 of SEQ ID NO:
• 1, an arginine at the amino acid residue corresponding to position 170 of SEQ ID NO:
• 1, an arginine at the amino acid residue corresponding to position 176 of SEQ ID NO:
• 1, and a tryptophan at the amino acid residue corresponding to position 203 of SEQ ID NO:
• 1;
• ii) a binding region capable of binding at least one extracellular target molecule wherein the binding region is positioned carboxy-terminal to the carboxy-terminus of the Shiga toxin effector polypeptide;
• and
• iii) at least one heterologous, CD8+ T-cell epitope cargo which is not embedded or inserted in the Shiga toxin effector polypeptide, wherein the at least one heterologous, CD8+ T-cell epitope cargo is positioned carboxy-terminal to the carboxy-terminus of the Shiga toxin effector polypeptide and/or the carboxy-terminus of the binding region.