Genetic Engineering of Solid Tumors With Enter for Fus Inducible Antigen Presentation in CAR-T Cell Therapy

Introduction: : While cell-based immunotherapy such as chimeric antigen receptor T cell (CAR-T) immunotherapy has transformed cancer treatment, especially for blood cancers, its clinical use for solid tumors presents challenges such as on-target off-tumor cytotoxicity. Moreover, targeted yet efficient delivery of gene therapies in solid tumor is also an ongoing challenge. To address this, Yu et al. developed a lentiviral-mediated cell entry system that utilizes engineered receptor-ligand interaction (ENTER) to selectively deliver gene cargo to antigen-specific T or B cells. Our goal is to re-engineer ENTER to expand its application to selectively target tumor cells and deliver focus ultrasound- (FUS) controllable synthetic antigens, such as CD19, to achieve spatiotemporal activation of SynNotch CAR-T cells, thereby kills solid tumors.

Materials and

Methods: : Various PSMA-expressing cell lines are made by lentiviral transduction of the PSMA antigen gene, followed by fluorescence-activated cell sorting (FACS) using antibody staining. PSMA ENTER pMD2.g envelope plasmid was engineered by inserting PSMA scFv into the pMD2.g envelope plasmid containing a VSV-G gene mutation. Gene-of-interest (GOI) contains a heat shock inducible truncated CD19 (tCD19) synthetic antigen with a constitutive red fluorescence protein called mCherry or a green fluorescence protein (GFP), cloned to a pHR vector. PSMA ENTER are produced by Lenti-X 293T cell transfection of PSMA ENTER pMD2.g envelope plasmid, psPAX2 packaging plasmid, and GOI using the Promega ProFection Mammalian Transfection System. Lentiviral concentration is determined by TakaraBio p24 ELISA. Heat shock of ENTER-infected cells are performed using thermal cycler at 43°C for 15 minutes. Heat shock of ENTER-infected spheroids is achieved in a 44°C water bath for 30 minutes. PSMA ENTER targeting specificity is determined by GFP or mCherry fluorescence while synthetic antigen heat inducibility is determined by APC/Cyanine7 anti-CD19 staining. Both aims are quantified via flow cytometry. LNCaP spheroids were made using an established method from Gupta et al. and Mishriki et al. Fluorescence microscopy and Incucyte live-cell imaging were conducted to monitor ENTER targetability and SynNotch T cell cytotoxicity to LNCaP spheroids. In vivo experiments were conducted in 6–8-week-old male NOD scid gamma (NSG) mice. Briefly, PSMA-expressing cell lines and its corresponding negative controls were subcutaneously inoculated on mice hindlimbs. Tumor size and ENTER targeting specificity were monitored via bioluminescence imaging (BLI) using the IVIS Lumina system.

Results, Conclusions, and Discussions:: We have verified that PSMA ENTER can achieve high targeting specificity in PSMA-expressing NALM6, K-562, PC-3, and LNCaP cell lines. FUS-responsive tCD19 is able to be expressed in ENTER-transduced cells. While PSMA ENTER does have minimal level of off-targeting in cells that do not express PSMA, this off-target effect can be rescued by the spatial-selective characteristic from FUS. Furthermore, because delivery efficiency remains a significant concern for in vivo gene therapy, we have combined our FUS-responsive gene delivery with SynNotch technology from Roybal et al. By doing so, we can initiate sufficient local SynNotch CAR T cell activation even if only a small subset of tumor cells has been transduced with FUS-inducible CD19. These activated SynNotch CAR T cells then target the tumor-associated antigen overexpressed on our target tumor cells, thereby achieving whole-tumor cytotoxicity. We further validated ENTER targeting specificity in LNCaP spheroids via fluorescence microscopy and Incucyte and observed effective cytotoxicity by SynNotch CAR T cells to LNCaP spheroids treated with a clinically relevant dosage of ENTER and heat. IVIS imaging of NSG mice with NALM6 tumors reinforces the conclusion that PSMA ENTER achieves high targeting specificity.

PSMA ENTER is able to achieve high targeting specificity in vitro, ex vivo, and in vivo. We have also demonstrated both in vitro and ex vivo that FUS-responsive gene can be robustly expressed on ENTER-transduced tumor, activates SynNotch CAR T cells, and achieves tumor elimination. The concluding step for this project is in vivo tumor elimination by SynNotch CAR T cells in NSG mice treated with ENTER and FUS.

Acknowledgements and/or References (Optional):: This research is supported by NIH and the USC Viterbi School of Engineering Graduate School Fellowship. Research is conducted under the main guidance of Peixiang He along with Longwei Liu, Yunjia (Jenny) Qu, Chi Woo (Nate) Yoon, Zhou Yuan, and my co-PIs Yingxiao (Peter) Wang and Bingfei Yu.