Augmenting Adoptive T Cell Therapy in Solid Malignancies via Cell Nanoengineering and Photoacoustics

Cancer immunotherapy using adoptive T cell transfer has shown remarkable success in many hematologic malignancies, however, this has not reliably translated to solid tumors. There are two main reasons that hinder T cell therapy translation in solid malignancies: 1) inability to visualize and track the infused T cells within tissues, and 2) limited delivery of T cells into deep-seated tumors that are not adjacent to vasculature. To address these two points, we introduce an approach, termed “cell nanoengineering”, based on a synergistic combination of adoptive T cells and functional nanoparticles coupled with an imaging system to track the delivery and therapy <i>in vivo</i>. Specifically, we modify T cell surfaces with light-absorbing, anticancer chemotherapeutic drug-loaded functional nanoparticles that can penetrate deeper into a 3D solid tumor, and introduce a real-time ultrasound-guided photoacoustic (US/PA) imaging system. This combined approach enables the tracking of nanoengineered adoptive T cells with excellent spatial resolution at great imaging depths to improve understanding of infused T cell trafficking, followed by selective drug release into the entire 3D tumor microenvironment for combinatorial cancer chemo-immunotherapy in solid malignancies.<br/>As a PA contrast agent and drug loading vehicle, mesoporous silica-coated, miniaturized gold nanorods (mSiO₂-AuNRs) with an aspect ratio of 6 (λ_max = 1064 nm) were synthesized. As an anticancer drug, 5-fluorouracil (5FU) or doxorubicin molecules were loaded in the mesoporous silica layer. For T cell nanoengineering, maleimide-modified mSiO₂-AuNRs were conjugated to the free thiol groups on the surface of T cells. To test the biological impact of T cell nanoengineering, T cell functions (i.e., viability, target cell killing) were tested at different nanoparticle concentrations: control cells and nanoengineered T cells at optical densities (ODs) of 1, 5, 10, and 20. Nanoengineered T cells at OD=1 and 5 showed negligible toxicity. At OD=10 and 20 significant T cell death was induced, and these nanoengineering conditions were eliminated from further analysis. Furthermore, T cell killing of matched B16-OVA cells at various ratios was maintained following cell nanoengineering, and no significant difference was observed in control vs. nanoengineered T cells. The addition of chemotherapeutic drugs (i.e., 5FU or doxorubicin) enhanced the target cell killing <i>in vitro</i> due to combinatorial chemo-immunotherapy. Furthermore, the loading of chemotherapeutic drugs selectively affected B16-OVA and not T cells at various drug amounts.<br/><i>In vivo</i> US/PA tracking, tumor regression study, and survival monitoring were carried out in ovalbumin (OVA)-expressing tumor-bearing C57BL/6 mice after intravenous injection of nanoengineered and matched OT1 murine primary T cells or the same amount of drug-loaded mSiO₂-AuNRs without T cells, followed by US/PA imaging (40 MHz, 680–970 nm and 1064 nm, Vevo LAZR, Visualsonics Inc.). Multiwavelength PA imaging successfully visualized nanoengineered T cells or nanoparticles delivered into solid tumor region. Nanoengineered T cells showed 3-fold higher PA signal (i.e., successful delivery) than the mSiO₂-AuNRs in the solid tumor, indicating the natural homing ability of matched T cells can be harnessed to improve the delivery of combination therapeutics. It followed that mice treated with nanoengineered T cells showed the best tumor regression and survival compared to other controls: T cell only or drug-loaded mSiO₂-AuNRs only.<br/>Our results indicate that nanoengineered T cells can be successfully detected within the range of pre-clinical injection doses with high sensitivity for <i>in vivo</i> US/PA tracking. Also, the ability of T cells to home to the tumor site enables better delivery of nanoparticles into solid tumors, thus achieving greater tumor regression and enhanced survival.