Cancer is a global health problem despite the most developed therapeutic modalities. The delivery of specific therapeutic agents to a target increases the effectiveness of cancer treatment by reducing side effects and post-treatment issues. Our aim in this study was to design a recombinant protein consisting of nanobody molecules and exotoxin that targets the surface GRP78 receptor on tumor cells. Bioinformatics methods make drug design and recombinant protein evaluation much easier before the laboratory steps. Two constructs were designed from a single-variable domain on heavy chain nanobody domains and PE toxin domains II, Ib, and III. The physicochemical properties, secondary structure, and solubility of the chimeric protein were analyzed using different software. Prostate cancer DU-145 and breast cancer MDA-MB-468 cell lines were used as GRP78-positive and negative controls, respectively. Accordingly, the cytotoxicity, binding affinity, cell internalization, and apoptosis were evaluated using MTT, enzyme-linked immunosorbent assay, and western blot. The results showed that in the DU-145 cell line, the cytotoxicity of two recombinant immunotoxins is dose and time-dependent. In MDA-MB-468 and HEK-293 cells, such an event does not occur. It is possible that two constructs designed for immunotoxins can attach to GRP78-positive cancer cells and then eradicate cancer cells by internalization and apoptosis. As our in vitro results were in line with in silico data confirming the Bioinformatics predictions, it can be concluded that the designed recombinant immunotoxins may exhibit therapeutic potential against GRP78-positive tumor cells.
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