Abstract 1376: Engineered exosomes for the delivery of PD-L1 siRNA for lung cancer immunotherapy
Authors: Kandimalla Raghuram , Moholkar Disha N. , Wallen Margaret , Ding Chuanlin , Gupta Ramesh C. , Aqil Farrukh 
Published: March 2024

Abstract
Cancer immunosurveillance involves a dynamic interplay between the host’s innate and adaptive immunity. The regulation of programmed death ligand-1 (PD-L1) expression on cancer cells has emerged as an attractive approach for cancer treatment. However, challenges such as the immunosuppressive tumor microenvironment, ineffective trafficking, and tumor antigen heterogeneity limit the applications of cancer immunotherapy for solid tumors. Small interfering RNA (siRNA) against PD-L1 presents a compelling alternative to antibody treatment to reduce PD-L1 presentation on cancer cells, thereby enhancing T-cell detection and preventing tumor progression. Bovine milk/colostrum serve as a biocompatible source for exosomes for delivery biologics such as siRNA. Here, we demonstrate exosomes mediated delivery of siPD-L1 for lung cancer immunotherapy. Exosomes were isolated from bovine colostrum powder using ultracentrifugation. The average particle size of exosomes was 66 ± 2.5 nm, with polydispersity index (pdi) 0.27 ± 0.01 and a zeta potential -9.2 ± 0.70 mV, as analyzed by Zetasizer and validated by atomic force microscopy (AFM) and transmission electron microscopy (TEM). Formulations of exosomes with polyethyleneimine (EPM) and siRNA (1 - 20 μg), when mixed with 5’-32P-labeled siRNA as a tracer resulted in over 90% entrapment of siRNA. Biodistribution studies of exosomes and EPM, with or without the tumor-targeting ligand folic acid (FA), revealed predominantly similar distribution patterns. Notably, FA-EPM exhibited enhanced tumor accumulation compared to EPM, attributed to the overexpression of folate receptors on tumor cells.To assess the functionality of EPM-siRNA, we screened 4 different siPD-L1 sequences in LLC-1 and A549 lung cancer cells. Following 48 h of EPM-siPD-L1 treatment, cells were harvested. Analysis of protein lysates by western blot showed a significant downregulation of the PD-L1 expression, ranging from 55% - 80% in LLC-1 cells and over 80% in A549 cells. In vivo mouse study with orthotopic Lewis lung carcinoma (LL/2-Luc2) tumors is underway to validate the effective silencing of PD-L1 and its effect on tumor inhibition and immune markers. In summary, our targeted EPM technology represents a novel nanoplatform for siRNA therapeutics delivery. As milk/colostrum are the most abundant sources of exosomes, clinical translatability of this novel approach is eminent.
Funding: This research work was supported from the Kentucky Lung Cancer Research Program (GB170558) and Immunotherapy CCII CoBRE pilot project (P20GM135004).
Keywords: Exosomes, siPD-L1, siRNA delivery, Lung cancer, Immunotherapy
Citation Format: Raghuram Kandimalla, Disha N. Moholkar, Margaret Wallen, Chuanlin Ding, Ramesh C. Gupta, Farrukh Aqil. Engineered exosomes for the delivery of PD-L1 siRNA for lung cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1376.