Nanoparticle-guided Chimeric Antigen Receptor T Cell Therapy

Introduction:: Chimeric Antigen Receptor (CAR) T cell therapy has limited efficacy against solid tumors, due to the immunosuppressive tumor microenvironment and dose-limiting on-target, off-tumor toxicity. To minimize toxicity and enhance safety, intratumoral CAR T cell delivery or drug-inducible CAR T cells coupled with local delivery of drug-inducers have been explored. However, these approaches require direct access to the tumor, and are not well-suited to treat disseminated tumors such as tumors of the peritoneum. We proposed to utilize drug-inducible Versatile ProtEase Regulatable (VIPER) CAR T cells and deliver a drug inducer, grazoprevir, by poly(glycerol carbonate) nanoparticles (PGC NPs) that readily accumulate in murine peritoneal tumors after intraperitoneal injection. We hypothesized that intraperitoneally injected grazoprevir-loaded PGC NPs will accumulate grazoprevir in the peritoneal tumor, activate tumor-infiltrating VIPER CAR T cells and enhance their tumor-specificity while minimizing on-target, off-tumor toxicity.


Materials and Methods:: PGC was synthesized by copolymerization of benzyl glycidyl ether and carbon dioxide. PGC NPs were fabricated using an emulsion-evaporation method with sodium dodecyl sulfate as surfactant, followed by 24h dialysis. The NPs were characterized by dynamic light scattering. Drug loading and encapsulation efficiency were determined by HPLC. Cytotoxicity of grazoprevir-loaded PGC NPs was studied by co-culturing human primary HER2-specific VIPER CAR or untransduced T cells with grazoprevir-loaded NPs for 24h. Cell viability was determined by flow cytometry. To determine activation of VIPER cells by NP-released grazoprevir over time, grazoprevir-loaded NPs in a mini-dialysis device (MWCO 10 kDa) were co-cultured with HER2-specific VIPER CAR Jurkat cells and HER2-expressing NALM6 cells, and VIPER CAR Jurkat cell activation was determined by flow cytometry. To evaluate NP-induced VIPER CAR T cell killing of tumor cells and cytokine secretion, HER2-specific VIPER, conventional CAR or untransduced T cells and HER2-expressing SKOV3/luciferase human ovarian cancer cells were cultured with or without 1 μM free grazoprevir or grazoprevir-loaded NPs. Tumor cell lysis was assessed by luciferase bioluminescence and cytokine secretion by ELISA. To assess grazoprevir levels in peritoneal tumors, grazoprevir-loaded NPs or free grazoprevir were injected intraperitoneally into NSG mice bearing peritoneal SKOV3 xenografts, and tumor grazoprevir concentration was quantified by LCMS. To demonstrate the NPs’ ability to spatially control VIPER CAR T cell activities, untransduced, conventional or VIPER CAR T cells with grazoprevir-loaded NPs or free grazoprevir were injected intraperitoneally into NSG mice bearing both subcutaneous and peritoneal SKOV3/luciferase tumors. Tumor growth was monitored by IVIS.

Results, Conclusions, and Discussions:: We formed monodisperse, sub-100nm grazoprevir-loaded NPs with ~65% loading efficiency. Grazoprevir-loaded NPs were minimally toxic to both human primary untransduced and VIPER CAR T cells. Grazoprevir released from the NPs through a dialysis membrane activated HER2-specific VIPER Jurkat cells as efficiently as fresh free drug over four weeks, demonstrating the NP’s ability to continuously activate VIPER cells long term. Grazoprevir-loaded NPs induced comparable SKOV3 ovarian cancer cell killing and IL-2 secretion by VIPER CAR T cells as free grazoprevir in vitro, indicating that grazoprevir bioactivity is maintained after encapsulation. NP-delivered grazoprevir was detected at significant levels ( >1 μg/g of tissue) in xenograft peritoneal tumors in mice after 5 days, whereas no free grazoprevir was detectable after 1 day. We further showed that VIPER CAR T cells with grazoprevir-loaded NPs strongly suppressed peritoneal tumors in 2/3 mice, but had minimal effect on subcutaneous tumors; in contrast, conventional CAR T cells suppressed growths of both subcutaneous and peritoneal tumors (Figure 1). Untransduced T cells did not delay subcutaneous and peritoneal tumor growths. VIPER CAR T cells with free grazoprevir suppressed peritoneal tumors only to a lesser degree compared to conventional CAR T cells. Taken together, PGC NPs demonstrate the ability to spatially control VIPER CAR T cell activities. Our NP-guided VIPER CAR T cell therapy provides a promising strategy to enhance the safety and tumor-specificity of CAR T cell therapies.

Acknowledgements (Optional): : 1) NIH U01CA265713 grant. 2) The Boston University Chemical Instrumentation Center.


References (Optional): : 1. Ekladious I, et al., Chem Sci. 2017; 8:8443-8450. 2. Li HS, et al., Cancer Cell. 2022; 40:1294-1305.e4.