Abstract 585: Utilizing CAR T cell-derived exosomes for radiotheranostic applications

Abstract Chimeric antigen receptor (CAR) T cell therapy is limited by antigen escape, poor solid tumor penetration, and an off-tumor toxicity profile. CAR T cell-derived exosomes have been shown to express the parental CAR construct, allowing tumor targeting but limited inherent anti-tumor effects alone. Further engineering CAR T cells to modify their exosome cargo, such as to bind anti-tumor drugs or radionuclides, may allow for the generation of novel targeted delivery vehicles for cancer therapy. We previously engineered humanized C825 (huC825), an anti-metal-DOTA-hapten single chain variable fragment (scfv), into multiple different constructs, one of which is the 4h11 CAR against MUC16/Cancer Antigen 125, developed by Brenjens lab at Memorial Sloan Kettering Cancer Center. We showed that huC825 allows for theranostic potential based on high-affinity binding to various chelated radionuclides, such as for alpha or beta particle-based radiotherapy or diagnostic imaging. We derived exosomes from 4h11 and huC825-4h11 CAR T cells, as well as untransduced T cells, and characterized them for CAR and huC825 expression using Western blot and radio thin layer chromatography (radio-TLC), respectively, as well as with direct stochastic optical reconstruction microscopy (dSTORM). We performed binding and cytotoxicity assays in vitro after radiolabeling and purifying the exosomes with size-exclusion chromatography (SEC), or using a pretargeted approach, with various chelated radiotracers. 4h11 and huC825-4h11 CAR T cell exosomes were reliably manufactured from parental CAR T cells with high transduction rates (80-95%). Western blot using CD81 and ALIX exosomal markers illustrates that 4h11 and huC825-4h11 CAR T cell exosomes and their parental CAR T cells are positive for the 4h11 CAR. dSTORM imaging shows huC825-4h11 exosomes are positive for both 4h11 and huC825. Radio-TLC illustrates that radionuclides chelated by Y-aminobenzyl-1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid, further referred to as aminobenzyl-DOTA (ABD), such as [161Tb]Tb-ABD (therapeutic metal) or [177Lu]Lu-ABD (diagnostic and therapeutic), exhibit significant binding to huC825-4h11 CAR T cell exosomes compared to controls. We optimized a method for radiolabeling huC825-4h11 exosomes and purifying the product with SEC using various DOTA-chelated radionuclides. In vitro radiotracer binding assays using SKOV3 human ovarian cancer cells overexpressing MUC16 illustrate that only huC825-4h11 CAR T cell-derived exosomes selectively bind both MUC16-expressing tumor cells and DOTA-chelated radionuclides. Our work illustrates the potential of huC825-4h11 CAR T cell-derived exosomes as delivery vehicles for radionuclides for imaging or targeted therapy. Their 50-160 nm size may allow better solid tumor penetration in vivo than current cellular treatment modalities. Future directions include in vivo imaging and radiotherapy studies. Citation Format: Sarah Qureshy, Leah Gajecki, Darren Veach, Sang Gyu Lee, Irina Matei, Lukas Carter, David Lyden, David Scheinberg, Simone Krebs. Utilizing CAR T cell-derived exosomes for radiotheranostic applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 585.