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Abstract
Background Merkel cell carcinoma (MCC) is a rare skin cancer with 46% disease-associated mortality and half of patients unresponsive to immune checkpoint inhibitors.1 2 MCC and melanomas often display decreased MHC class I (MHC-I) expression on the surface of cells, which prevents antigen recognition by T cells (”signal 1”) and hampers immune activation. We therefore sought to genetically reprogram cells to express their own costimulatory molecules (”signal 2”) and immunostimulatory cytokines (”signal 3”) to increase MHC-I expression and drive a targeted immune response.
Methods We used biodegradable poly(beta-amino ester) nanoparticles (NPs) to co-deliver plasmids encoding a signal 2 molecule (4-1BBL) and two signal 3 molecules (IL-12 and IFNγ) to cancer cells. For in vitro evaluation of NPs we used two patient-derived MCC cell lines with low baseline MHC-I expression; MCC13 and UISO. Co-culture experiments were performed with human PBMCs or primary human natural killer (NK) cells. All in vitro analysis was performed 7 days following PBMC or NK cell addition. For in vivo evaluation, subcutaneous B16F10 mouse melanoma tumors were implanted in C57BL/6J mice and NPs were administered by direct injection into the tumor with and without intraperitoneal injection of αPD1. Tumors were harvested for analysis on day 16.
Results Transfection with particles delivering the three plasmids to MCC13 and UISO increased MHC-I expression (mean fluorescence intensity) 1.6- and 5.0-fold, respectively, and MHC-II expression increased 1.6- and 6.3-fold, respectively (figure 1). In co-culture with human PBMCs, signal 2/3 particles resulted in increased leukocyte proliferation (4.6- and 6.1-fold increase, respectively) and led to significantly reduced MCC viability (10.6 and 1.6% vs control particles)(figure 2). When MCC13 cells were co-cultured with primary human NK cells, NK cell expansion increased 355-fold with 4-1BBL/IL-12 particles compared to control particles and was accompanied by 2.5% MCC13 cell viability, indicating a potent innate immune response with signal 2/3 NP administration in vitro (figure 3). Following evaluation of NPs in vivo, assessment of MHC-I and MHC-II expression in the melanoma tumors found increased expression with signal 2/3 NPs compared to control NPs (figure 4). When signal 2/3 NPs were administered in combination with αPD1 treatment, 4-1BBL/IL-12 NPs with αPD1 demonstrated improved survival compared to αPD1 treatment with control NPs (p=0.0010) (figure 5).
Administration of signal 2/3 NPs to MCC13 and UISO cells led to increases in MHC-I and MHC-II expression after 7 days. MHC-I expression in transfected cells (red) and MHC-II expression in transfected cells (blue) compared to untreated control (black)
Co-culture of transfected MCC cells with human PBMCs led to increases in CD45+ cells and reduced MCC cell viability after 7 days
Co-culture of 4-1BBL/IL-12 transfected MCC13 cells with isolated CD56+ NK cells demonstrated robust NK-cell expansion and low MCC cell viability after 7 days
Direct intratumoral injection with signal 2 and 3 NPs led to increases in MHC-I and MHC-II in cancer cells in vivo.
NPs were administered intratumorally ± intraperitoneal aPD1 on day 9, 11, and 13 following B16F10 melanoma tumor implantation. 4-1BBL/IL12 particles in combination with αPD1 demonstrated a significant improvement in survival compared to control particles (Luc) with αPD1 (p=0.0010)
Conclusions Together, these results show the ability of signal 2/3 NPs to reprogram MCC and melanoma cells, leading to increased MHC-I expression in vitro and in vivo, eliciting a productive immune response against cancer cells.
References
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