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Immunotoxins (ITXs) are chimeric molecules that combine the specificity of a targeting domain, usually derived from an antibody, and the cytotoxic potency of a toxin, leading to the selective death of tumor cells. However, several issues must be addressed and optimized in order to use ITXs as therapeutic tools, such as the selection of a suitable tumor-associated antigen (TAA), high tumor penetration and retention, low kidney elimination, or low immunogenicity of foreign proteins. To this end, we produced and characterized several ITX designs, using a nanobody against EGFR (VHH 7D12) as the targeting domain. First, we generated a nanoITX, combining VHH 7D12 and the fungal ribotoxin α-sarcin (αS) as the toxic moiety (VHHEGFRαS). Then, we incorporated a trimerization domain (TIEXVIII) into the construct, obtaining a trimeric nanoITX (TriVHHEGFRαS). Finally, we designed and characterized a bispecific ITX, combining the VHH 7D12 and the scFv against GPA33 as targeting domains, and a deimmunized (DI) variant of α-sarcin (BsITXαSDI). The results confirm the therapeutic potential of α-sarcin-based nanoITXs. The incorporation of nanobodies as target domains improves their therapeutic use due to their lower molecular size and binding features. The enhanced avidity and toxic load in the trimeric nanoITX and the combination of two different target domains in the bispecific nanoITX allow for increased antitumor effectiveness.

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