Background Chimeric antigen receptor (CAR) engineered immune cells have shown unprecedented efficacy in Relapsed/Refractory hematological malignancies. However, effectiveness against solid tumors remains a challenge. The treatment of solid tumors with checkpoint blockage via PD-1/PD-L1 axis (i.e. Atezolizumab) has shown feasibility and potential benefit of targeting PD-L1 in these patients. However, lack of durable anti-tumor responses remains a major concern. Additionally, patients with low PD-L1 expression at baseline remains ineligible for the treatment.
We propose the development of a PD-L1 targeted CAR-NK cell to target PD-L1 positive or also possibly PD-L1 negative tumors. PD-L1 CAR-NK cells will be made by transducing a αPD-L1 CAR during ex vivo expansion of peripheral blood-derived NK cells, enabled by stimulation with PM21 particles that confers a highly active functional phenotype (referred as K-NK cells). We hypothesize that PD-L1 upregulation by tumor cells after exposure to IFNγ secreted by K-NK cells, upon tumor detection, can further make PD-L1low tumor cells susceptible to PD-L1 CAR K-NK cells. Additionally, these PD-L1 CAR K-NK cells are activatable by CAR and/or additional receptors that bind activating ligands on tumor cells. This allows cytotoxicity towards a range of tumors that are PD-L1+, PD-L1 inducible and also PD-L1-.
Methods PD-L1 CAR K-NK cells were generated by engineering NK cells during expansion with a PD-L1 CAR encoding vector. The expression of PD-L1 CAR and K-NK activating/inhibitory receptors was assessed using flow cytometry. PD-L1 CAR K-NK mediated cytotoxicity and cytokine secretion after co-culture with tumor cells were analyzed in vitro.
Results Stable and high PD-L1 CAR expression on K-NK cells was observed. PD-L1 CAR K-NK cells show enhanced cytotoxicity against panel of solid tumor cells, with varying PD-L1 density, compared to control WT K-NK cells. The enhancement in cytotoxic effect was particularly striking at lower E:T ratios (E:T<1). Significantly higher IFNγ secretion by PD-L1 CAR K-NK cells was observed compared to control K-NK cells, that then further enhanced induction of PD-L1 expression on cancer cell lines that normally have low base-line expression of PD-L1. Confirmatory to this mechanism of action, both enhanced cytotoxicity and enhanced IFNγ secretion by NK cells were not observed with PD-L1 knockout target cells.
Conclusions PD-L1 CAR K-NKs were able to more effectively kill tumors with varying levels of PD-L1 expression and thus have the potential to address unmet medical need for patients with PD-L1+ or also PD-L1- tumors, that currently are not optimal candidates for antibody based PD-1/PD-L1 blockade therapeutics.
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