(10d) Nanoparticle-Augmented CAR T Cells for Combined Ultrasound and Photoacoustic Image-Guided Cancer Immunotherapy

Cancer immunotherapy using Chimeric Antigen Receptor (CAR) T cells has shown remarkable success in some hematologic malignancies, such as leukemia. In solid tumors, however, numerous obstacles must be overcome, including off-target toxicity to normal tissues, poor trafficking to the tumor, low effector cell infiltration, tumor antigen loss, an immunosuppressive tumor microenvironment, etc. To tackle some of these challenges, several approaches have been introduced. For example, CAR T cells have been equipped with “backpacks” containing synthetic antigens or chemotherapeutic drugs to enhance therapeutic efficacy. Still, clinicians and scientists are missing critical information. The ability to visualize and track the CAR T cells in the whole body or within tissues following transfer will allow evaluation of cell accumulation within the complex tumor microenvironment, assessment of local or systemic biological responses, and prediction of the therapeutic outcome. In this regard, introducing an imaging modality capable of real-time tracking of CAR T cells in vivo after the cell infusion in the context of disease characterization and monitoring will guide the selection of appropriate treatment regiments, thus resulting in significantly advanced CAR T cell therapy. Unfortunately, strategies that employ image-guidance to improve spatial targeting and treatment precision remain underdeveloped. Current image-guided CAR T cell therapy is largely based on a) labeling cells with radioactive, (super)paramagnetic, or fluorescent tracers, and b) subsequent SPECT/PET, MRI, bioluminescence, or fluorescent imaging. SPECT/PET can be used for whole body detection of CAR T cells in vivo, but there are concerns about cell viability and functionality and low spatial resolution. In MRI, the sensitivity is severely limited by the amount of contrast agent that may be loaded safely into cells. Optical imaging has low imaging depth and spatial resolution. Therefore, a widely available imaging modality capable of real-time tracking of CAR T cells in vivo with high sensitivity, specificity, depth penetration, and resolution is needed.

Combined ultrasound and photoacoustic (US/PA) imaging can inform and significantly advance current CAR T cell therapy. US/PA imaging is advantageous because US imaging can provide high resolution and deep anatomical information, while complementary PA imaging allows visualization of functional properties of the tissue. Furthermore, by utilizing optically absorbing contrast agents and leveraging the photoacoustic effect, PA imaging can be applied for tracking CAR T cells with high contrast. Herein, we present an approach based on a synergistic combination of US/PA imaging, plasmonic nanoparticles, and T cell surface engineering. This combined approach enables nanoparticle-augmented CAR T cell tracking with excellent spatial resolution at great imaging depths to improve understanding of infused CAR T cell trafficking.

As a contrast agent, miniaturized gold nanorods (AuNRs) with an aspect ratio of 6 were synthesized by a hydroquinone-based seedless method. With a strong optical absorption peak at 1064 nm, AuNRs are an excellent PA contrast agent as biological tissues have low absorption at this wavelength. The surfactant on the surface of the AuNRs was replaced with polyethylene glycol (PEG) and maleimide via surface ligand exchange and subsequent cycloaddition reaction. Maleimide-modified AuNRs (mmAuNRs) were reacted with free thiol groups on the surface of CAR T cells and purified to produce AuNR-labeled CAR T cells (Fig. A). Labeled cells were laden into a tissue-mimicking 8% gelatin phantom and imaged using a Vevo LAZR 20 MHz LZ250 transducer with a 7 ns pulsed laser operating at 1064 nm.

With the hydroquinone-based seedless synthesis method, the morphology of AuNRs appeared to be 7 nm wide and 42 nm long under transmission electron microscopy (TEM) (Fig. B). The UV-vis-NIR spectrum showed clear longitudinal surface plasmon resonance (LSPR) at 1064 nm, demonstrating successful synthesis of AuNRs for PA contrast (Fig. C). Engineering CAR T cell surface with nanoparticles is beneficial because T cells show very low endocytic behavior. To ascertain the cytocompatibility of the CAR T cell surface modification, the viability of AuNR-labeled CAR T cells was evaluated, exhibiting ~90 % cell viability following surface modification (Fig. D). This demonstrates the surface labeling of CAR T cells using AuNRs did not affect the cell function and viability. Finally, US/PA imaging of gelatin inclusions containing AuNR-labeled T cells showed a distinct PA signal, whereas unlabeled cells did not exhibit any PA signal. In addition, the co-registered US/PA images clearly showed the location of labeled cells in the context of structural features of the tissue model (Fig. E).

These results indicate that AuNR-labeled CAR T cells can be successfully detected with sensitivity within the range of pre-clinical injection doses and suggest that US/PA imaging can be used for image-guided CAR T cell therapy. In the future, we will evaluate the immunotherapeutic effect of AuNR-labeled CAR T cells in a mouse model of breast cancer under US/PA image-guidance.