A laser-based nanoparticle platform to enhance T cell–Mediated immunity without direct tumor access Free

Introduction: T cell-based therapies have revolutionized cancer treatment. Chimeric antigen receptor (CAR) T cell therapy has transformed outcomes for lymphoma and multiple myeloma. Bispecific antibodies (BsAbs), which engage T cells and cancer cells, are FDA-approved for B-cell lymphoma, myeloma, small cell lung cancer, and uveal melanoma. Despite promising results, many patients do not respond or relapse, partly due to the suppressive immune environment. Improving pro-inflammatory T cell populations to boost immune activation may enhance therapy results. Photothermal therapy (PTT) elicits a strong T-cell and systemic immune response when combined with toll-like receptor (TLR) agonists, outperforming radiation therapy (RT). Unlike RT, PTT uses gold nanoparticles (NPs) to create an in situ vaccination effect via heat ablation of tumors. NPs absorb near-infrared (NIR) light, converting it into heat that triggers pro-inflammatory cytokines, such as IL-6, IL-1β, TNF-α, G-CSF, GM-CSF, and CCL2, enhancing T cell response. Despite promising pre-clinical data, PTT requires direct tumor access via a fiber optic probe, which can be costly and impractical. To address this, we describe the Nanoparticle-Focused Laser Immune Activating Method (NanoFLIAM), which mimics PTT effects without tumor access. Cancer cells with nanoparticles are irradiated with a laser to destroy them, then injected subcutaneously to elicit the same immune response.

Methods: The NanoFLIAM method: A20 murine lymphoma cells were mixed with silica gold nanoshells (NS). The cells were heat-killed by exposing the mixture to a near-infrared (NIR) laser. The mixture was then injected subcutaneously into the flank of BALB/c mice. The NIR laser irradiated the injected solution. Viability is measured by Live/Dead staining. An LDH-based assay was used to measure T cell-mediated toxicity.

Results: In a pilot study, A20 cells mixed with NS were lysed for 3 minutes at 5 watts, and PTT was conducted at 20 seconds at 4 watts after injection into the mice. Six days later, A20 tumor cells were implanted on the opposite flank. By day 23, control mice had an average tumor volume of 282 mm³, while the NanoFLIAM group averaged 39.2 mm³, indicating the platform's potential to induce anti-tumor immunity. In a treatment group, NanoFLIAM was performed twice on days 0 and 3 after tumor establishment. By day 15, the NanoFLIAM group showed a significant reduction in tumor volume compared to the control group (951 vs. 1538 mm³). However, further optimization of the NanoFLIAM platform is necessary. Initially, we aimed to determine the NS dose and laser exposure time required for complete cell lysis. We observed that ablation duration and NS concentration, measured as optical density (OD), were inversely correlated with A20 cell viability, as assessed by calcein AM fluorescence. At 10 OD NS, cell viability was reduced by 93.8% in non-ablated samples compared to those ablated for three minutes. In samples with 5 OD, viability was reduced by 78% (p<0.001). An ablation duration of 2 minutes had similar effects on cell viability as 3 minutes in both 5 OD and 10 OD samples. We observed that LDH release, correlated with cytotoxicity, was 20% greater in 1 OD and 2 OD samples that had been ablated for 2 minutes than control samples (p<0.001). There was no difference in LDH release between the 1 OD and 2 OD samples. Luminescence was 43.5% lower in the 5 OD and 72.9% lower in 10 OD samples than in the control sample that was not ablated (p<0.001). To evaluate the shorter ablation strategy, we lysed A20 cells mixed with NS at 5W for 2 minutes, injected the lysate into mice, and performed PTT at 4W for 30 seconds. The T cells were isolated and used in a cell-mediated toxicity assay. NanoFLIAM-treated mice's isolated T cells were 2.94 times more effective than those of control mice, and those that included CpG-conjugated nanoparticles were 3.39 times more effective.

Conclusion: The innovative NanoFLIAM platform provides a promising alternative to traditional photothermal therapy by effectively replicating its immune-activating effects without needing direct access to tumors. Further optimization of this approach could enhance T cell–mediated therapeutic outcomes such as CAR T therapy and bispecific antibodies.