A New Combination: Anti Glypican-3 scFv and Diphtheria Toxin with the Best Flexible Linker

Abstract

Along with all cancer treatments, including chemotherapy, radiotherapy, and surgery, targeting therapy is a new treatment manner. Immunotoxins are new recombinant structures that kill cancer cells by targeting specific antigens. Immunotoxins are composed of two parts: toxin moiety, which disrupts protein synthesis process, and antigen binding moiety that bind to antigens on the surface of cancer cells. Glypican 3 (GPC3) is an oncofetal antigen on the surface of Hepatocellular carcinoma (HCC) cells. In this study, truncated Diphtheria toxin (DT389) was fused to humanized scFv YP7 by one, two and three repeats of GGGGS linkers (DT389-(GGGGS)_1-3YP7). In-silico and experimental investigation were performed to find out how many repeats of linker between toxin and scFv moieties are sufficient. Results of in-silico investigations revealed that the difference in the number of linkers does not have a significant effect on the main structures of the immunotoxin; however, the three-dimensional structure of two repeats of linker had a more appropriate structure compared to others with one and three linker replications. In addition, with enhancing the number of linkers, the probability of protein solubility has increased. Generally, the bioinformatics results of DT389-(GGGGS)₂-YP7 structure showed that expression and folding is suitable; and YP7 scFv has appropriate orientation to bind GPC3. The experimental investigations indicated that the fusion protein was expressed as near to 50% soluble. Due to the high binding affinity of YP7 scFv and the proven potency of diphtheria in inhibiting protein synthesis, the proposed DT389-(GGGGS)₂-YP7 immunotoxin is expected to function well in inhibiting HCC.