Background Different hematological cancer types have shown promising responses to autologous chimeric antigen receptor (CAR)-T cell therapy. However, the efficacy of this treatment in solid tumors is hindered by challenges such as poor tumor infiltration, long-term retention of CAR-T cells, and systemic side effects. To address these limitations, we developed citrate-coated superparamagnetic iron oxide nanoparticles (SPIONs), which have the ability to adhere to and be taken up by T cells, thereby enabling the control of CAR-T cells through an external magnetic field (1-3). In future, magnetic guidability should help to enrich CAR-T cells in the tumor microenvironment, leading to site-specific anti-tumor responses. This study aims to investigate the influence of SPION-loading of CAR-T cells on their efficacy in anti-tumor cell responses in vitro.

Materials and Methods T cells were isolated from peripheral blood of healthy donors and received mRNA encoding a chondroitin sulfate proteoglycan 4 (CSGP4)-specific CAR via electroporation (4, 5). The cells were then incubated with SPIONs for 4h to magnetically functionalize them. Subsequently, T cells were co-incubated with melanoma tumor cells expressing CSGP4 on their surface. Afterwards, the cells were analyzed for their antigen-specific anti-tumor responses and compared to non-loaded CAR-T cells or CSGP4-negative tumor cells by flow cytometry. Additionally, tumor cell lysis was investigated via impedance-based monitoring of cell viability and microscopic analysis of the dissolution of three-dimensional tumor spheroids.

Results We observed that SPION-loading did not affect the expression of activation markers, differentiation, or proliferation of CAR-T cells. Furthermore, SPION-loaded CAR-T cells retained their capability for antigen-specific tumor cell lysis over multiple days. Additionally, these CAR-T cells demonstrated the ability to be controlled by an external magnetic field, as well as infiltrating and dissolving tumor spheroids.

Conclusions In summary, we demonstrated that SPION-loading did not compromise the functionality of CAR-T cells, as they were still able to perform the investigated effector functions with similar efficacy as the non-loaded control CAR-T cells. These findings underscore the potential of SPIONs in enhancing site-specific anti-tumor responses of CAR-T cells in the therapy of solid cancers in the future.

References

1. Mühlberger et al. J. Magn. 2019.

2. Boosz et al. Cancers. 2021.

3. Pfister et al. Front Immunol. 2023.

4. Krug et al. Cancer Immunol Immunother. 2015.

5. Harrer al. Int J Mol Sci. 2019.

References F. Pfister: None. L. Löffler: None. L.R. Carnell: None. P. Boosz: None. J. Dörrie: None. N. Schaft: None. C. Alexiou: None. C. Janko: None.