Abstract 1707: Establishment of a targeted exosome drug delivery system containing natural bacterial toxins and antitoxins

Abstract

Abstract Background: The inactivation of p53, a tumor suppressor, and the activation of the Ras oncogene are the most frequent genetic alterations in cancer. We have shown that a unique E. coli MazF-MazE toxin-antitoxin (TA) system, controlled by Ras and p53, delivered via adenoviruses can be used to selectively and effectively eradicate Ras mutated cancer cells (Shapira et al., oncogene, 2021). Because of their low immunogenicity and known advantage as ideal cell transporters, exosomes offer great promise as cancer immunotherapy agents. CD24 is a GPI-anchored protein that highly expressed in most of human malignancies and not normal cells. Aim: Development of a drug delivery platform for effective and selective eradication of cancer cells while sparing normal cells based on the Trojan horse strategy and genetic signature. Methods: Two delivery systems have been designed and developed; targeted lentivirus and exosomes. Trex293 cells were used to establish stable scFv-CD24-expressing clones, which were adapted to suspension culture in a chemically-defined media. Exosomes were purified by polymer precipitation method and characterized by several methods including nano-particle tracking analysis, Western blot and EXO-ELISA. CD24 stable transfected HCT116-/- (P53 null) and HCT116+/+ (WTP53) clones were established, and evaluated for CD24 and P53 expression. HCT116+/+-CD24 cells were treated with exosomes loaded with GFP or MazF expressing plasmids and analyzed for GFP expression and cell viability. Results: Targeted lentiviruses carrying the TA system effiently shrinked CRC-derived xenografts tumors in mice. Then, a new genetically encoded pseudotyping platform, based on exosomes, was developed. A homogenic and pure population of exosomes was purified from 3D culture of the highest expressing clone (TREx-scFv). Stable and high expression of CD24 were shown, by Western blot and flow cytometry, in both, HCT116-/- and HCT116+/+ cells. HCT116+/+-CD24 cells treated with 5FU showed active P53. The ability of loading DNA into the purified exosomes and deliver them to the target cells were evaluated and showed high and intensive GFP expression. mazF was successfully loaded into the scFv targeted-exosomes and induced a massive cell death, in a dose-dependent manner. Conclusions: By exploiting the P53 genetic status as well as the Ras pathway, an effective and safe treatment could be developed that targets tumor cells specifically while sparing normal tissues. The effectiveness of this out-of-the-box approach holds great promise for the treatment and management of cancer. Citation Format: Shiran Shapira, Daniela Eisurovich, Dina Kazanov, Nadir Arber. Establishment of a targeted exosome drug delivery system containing natural bacterial toxins and antitoxins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1707.