ИСТИНА

The mutant TRAIL variant DR5B has enhanced antitumor activity due to its selective interaction with the DR5 death receptor. DR5B binds to DR5 receptor as efficiently as wild type TRAIL, but has no affinity for other TRAIL receptors (DR4, DcR1, DcR2, and OPG). This makes it possible to avoid the inhibitory effect of decoy receptors and to enhance the apoptotic signaling in tumor cells. Previously, a technique was developed for the expression and purification of the recombinant DR5B protein in E. coli. In current work, we have developed a genetic construct encoding DR5B fusion protein with an iRGD peptide (CRGDKGPDC) at the Cterminus. The iRGD peptide specifically binds to integrins αvβ3 and αvβ5, overexpressed on the surface of tumor cells, with subsequent proteolytic activation and acquisition of affinity for the NRP1 receptor involved in angiogenesis. The DR5BiRGD fusion protein was expressed in a soluble form in the E. coli SHuffle B strain and purified by metal affinity and ion exchange chromatography. Affinity measurements by ELISA showed that the DR5BiRGD fusion protein binds to the DR5 receptor as efficiently as DR5B, and has a similar affinity for the integrins αvβ3 and αvβ5, as free iRGD peptide. The cytotoxic activity of the DR5BiRGD has been confirmed on various 2D tumor cell lines and 3D models of tumor spheroids in vitro. It has been shown that DR5BiRGD penetrates the 3D multicellular tumor spheroids faster and more efficiently and exhibits higher cytotoxicity towards cancer cells compared to DR5B. Bispecific targeting of various signaling pathways involved in tumor development with the DR5BiRGD fusion protein is promising for anticancer therapy. The research was funded by Russian Science Foundation grant No. 211400224, https://rscf.ru/project/211400224/.