(10d) Engineer Off-the-Shelf CAR-Neutrophils for Targeted Chemoimmunotherapy Against Glioma

Introduction: Glioblastoma (GBM), the most common type of primary brain tumor, is characterized by high mortality rate, short lifespan, and poor prognosis with a high tendency of recurrence. Functional therapeutics, including PRMT5 inhibitors, radiosensitizers, and emerging chimeric antigen receptor (CAR)-T immunotherapy, have been developed to treat GBM. However, the existence of physiological blood-brain barrier (BBB) or blood-brain-tumor barrier has impeded the efficient delivery of such promising therapeutics into the brain and limited their therapeutic efficacy. Given the native ability of neutrophils to cross BBB and penetrate the brain parenchyma [1], here we tested the therapeutic concept that neutrophils could be engineered with synthetic CARs to efficiently cross BBB and specifically target GBM for the first time. Primary neutrophils are short-lived and resistant to genetic modification. Therefore, we genetically engineered human pluripotent stem cells with different CAR constructs and differentiated them into functional CAR-neutrophils [2]. We also tested the therapeutic potential of CAR-neutrophils in delivering chemotherapeutic drugs to GBM across BBB as a dual chemoimmunotherapy.

Methods: Human pluripotent stem cell line H9 was used in this study for neutrophil and natural killer (NK) cell differentiation. CRISPR/Cas9 was used to knock different synthetic chlorotoxin (CLTX) CARs into the AAVS1 safe harbor locus of H9 for making clonal hPSC lines that express stable and homogenous CARs. Biodegradable mesoporous organic silica nanoparticles were synthesized and employed to load hypoxia-activated prodrug tirapazamine (TPZ) or clinical chemo-drug temozolomide and JNJ64619187 into hPSC-derived CAR-neutrophils. In situ xenograft mouse models of glioblastoma were constructed to evaluate antitumor activities of CAR-neutrophils and CAR-NK cells.

Results: Stable CAR-expressing hPSC lines were successfully established via CRISPR/Cas9-mediated homologous recombination (Fig. 1a). A new chemically-defined protocol was implemented to induce CAR-hPSCs into CAR-neutrophils (Fig. 1b). As compared to CAR-NK cells, systemically administered hPSC-derived CLTX CAR-neutrophils significantly reduced tumor burden in xenograft mouse models and extended their lifespan (Fig. 1c-e), suggesting the superior abilities of neutrophils in crossing BBB and penetrating GBM xenograft in mice. CAR-neutrophils could also be loaded with hypoxia-activated prodrug TPZ in silica nanoparticles with rough surfaces (R-SiO2-TPZ) and specifically delivered nanodrugs into the brain, as compared to free nanodrugs (Fig. 1f). This dual chemoimmunotherapy further inhibited tumor growth and extended lifespan in xenograft mice.

Conclusions: We demonstrated the successful generation of CAR-neutrophils from hPSCs and showed their antitumor activities against GBM in xenograft models. hPSC-derived CAR-neutrophils could also be used as an effective drug carrier, which served as a new dual chemoimmunotherapy. CAR neutrophil-mediated drug delivery may provide an effective and universal strategy for specific targeting of solid tumors.