Real-time evaluation of a hydrogel delivery vehicle for cancer immunotherapeutics within embedded spheroid cultures

Many protein immunotherapeutics are hindered by transport barriers that prevent the obtainment of minimum effective concentrations (MECs) in solid tumors. Local delivery vehicles with tunable release (infusion) rates for immunotherapeutics are being developed to achieve local and sustained release. To expedite their discovery and translation, in vitro models can identify promising delivery vehicles and immunotherapies that benefit from sustained release by evaluating cancer spheroid killing in real-time. Using displacement affinity release (DAR) within a hydrogel, we tuned the release of a CD133 targeting dual antigen T cell engager (DATE) without the need for further DATE or hydrogel modifications, yielding an injectable vehicle that acts as a tunable infusion pump. To quantify bioactivity benefits, a 3D embedded cancer spheroid model was developed for the evaluation of sustained protein release and combination therapies on T cell mediated spheroid killing. Using automated brightfield and fluorescent microscopy, the size of red fluorescent protein (iRFP670) expressing spheroids were tracked to quantify spheroid growth or killing over time as a function of controlled delivery. We demonstrate that sustained DATE release enhanced T cell mediated killing of embedded glioblastoma spheroids at longer time points, killing was further enhanced with the addition of anti-PD1 antibody (alpha PD1). The multi-cellular embedded spheroid model with automated microscopy demonstrated the benefit of extended bispecific release on T cell mediated killing, which will expedite the identification and translation of delivery vehicles such as DAR for immunotherapeutics.

Automated summary

This study demonstrates the use of displacement affinity release (DAR) to tune the release of a CD133 targeting dual antigen T cell engager (DATE) within a hydrogel. The sustained release enhances T cell mediated killing of cancer spheroids, and the addition of anti-PD1 antibody further enhances the killing effect. The development of a multi-cellular embedded spheroid model with automated microscopy facilitates the identification and translation of delivery vehicles for immunotherapeutics.