Nanomaterial-mediated photoacoustic monitoring of adoptive CAR T cell therapy

Introduction: Although chimeric antigen T (CAR T) cell therapies have demonstrated considerable success in treating hematological malignancies, their therapeutic success in patients with solid tumors has been inconsistent due to tumor antigenic heterogeneity and immunosuppressive tumor microenvironment. Therefore, monitoring the spatial status of CAR T cells within solid tumors throughout the therapy duration, including CAR T cell trafficking and infiltration, can facilitate an understanding of in vivo requirements to generate therapeutic responses, thereby helping to optimize therapeutic interventions. Here we present a strategy for nanoengineering CAR T cells to non-invasively assess their spatial localization in heterogenous solid tumors by surface conjugation of photoacoustic (PA) nanomaterials. Specifically, a gold nanoassembly (GNA), composed of plasmonically-coupled gold nanospheres, is utilized for CAR T cell labeling based on the robust PA response of the GNA in the near-infrared (NIR) optical window. Surface-conjugated GNAs confer CAR T cells with the ability to generate reliable PA responses at NIR wavelengths, without compromising any key cellular functions. Consequently, ultrasound-guided PA (US/PA) imaging of GNA-conjugated CAR T cells enables real-time monitoring of their spatial distribution with high anatomical resolution over multiple imaging sessions, thus predicting therapeutic efficacy. Overall, this strategy implements nanomaterial-mediated non-invasive assessment of the antitumor activity of CAR T cells, underscoring its potential in developing an advanced nanomaterial-based theranostic platform.

Materials and

Methods: GNAs were fabricated via a template-mediated seeded growth of GNSs in an ellipsoid nanoscale space, followed by maleimide moiety functionalization. The maleimide-coupled GNAs were coupled to the surface of human CAR T cells as demonstrated by scanning electron microscopy (SEM). The optical and PA properties of GNA-conjugated CAR T cells were characterized using UV-vis-NIR spectroscopy and US/PA imaging, respectively. The influence of the GNA coupling on key cellular functions was characterized by flow cytometry. For the in vivo tumor model, NSG mice were inoculated with MDA-MB-468 cells at different HER2+ expressions (0, 10, 50, and 100%). GNA-conjugated αHER2 CAR T cells were then systemically injected to the tumor bearing mice, followed by US/PA tumor monitoring at different days post adoptive cell transfer (ACT).

Results, Conclusions, and Discussions: We fabricated GNAs by incorporating plasmonically coupled gold nanospheres via a seeded growth within an anisotropic polymer space, resulting in PA responses across the entire NIR window (Figs. 1a, b). The fabricated GNAs underwent maleimide functionalization to covalently link to the exofacial thiol groups on T cell surface proteins (Fig. 1c). The maleimide-functionalized GNAs were then incubated with CAR T cells at varying GNA doses. As the GNA dose increased, an increased surface density of GNAs on CAR T cells was observed, resulting in enhanced optical absorption within the NIR window (Figs. 1d-f). We confirmed that the GNA conjugation process preserved cell viability, proliferation, and cytotoxicity against target cancer cells, regardless of the GNA dose in the coupling process (Figs. 1g-i). Next, to understand the impact of heterogenous antigen expression on the trafficking, infiltration, and subsequent therapeutic efficacy of CAR T cells, GNA-conjugated αHER2 CAR T cells were systemically injected to mice bearing MDA-MB-468 tumors with varied HER2 compositions ranging from 0% to 100%, followed by US/PA tumor imaging. Before ACT, all tumors showed no significant difference in PA signal (Fig. 1j). However, on day 4 post ACT, tumors with 50% and 100% HER2+ cancer cells exhibited higher PA amplitude and imaging contrast than those with 0% and 10% HER2+ cells (Fig. 1j). Notably, increased PA signal was observed in 50% and 100% HER2+ tumors eight days before tumor volumes diverged among the cohorts (Fig. 1k). Together, our findings highlight the ability of US/PA monitoring of GNA-conjugated CAR T cells as a predictive tool for therapeutic efficacy in heterogenous solid tumors.

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