Advancing CAR-T cell therapy for solid tumors using nanobodies: Design of nanobody-based CARs and nanobody-displaying

Chimeric antigen receptor (CAR)-T cell therapy revolutionized cancer immunotherapy for hematological malignancies. So far, these successes have not been mirrored for solid tumors, while responsible for 90% of cancer-related deaths. This unfulfilled success relates to both lack of CAR-T cell efficacy and the difficulty of safely targeting tumor-associated antigens. Because solid tumors represent an important target for therapy, our goal was to advance CAR-T cell therapy for solid tumors. In addition, a general challenge of CAR-T cell therapy is the limited accessibility associated to the high cost and complex logistics inherent to manufacturing of an autologous cell product. When the translation of CAR-T cell therapies to solid tumors is intended, an increase in the eligible patient population is expected, and patient-specific therapies are even more difficult to envision. In that context, in situ CAR-T cell engineering would reduce manufacturing time and cost and improve availability by providing an off-the-shelf product.

While early research mostly focused on optimizing the intracellular signaling parts of CAR constructs, the importance of the extracellular antigen-binding domain became increasingly recognized in recent years. Reviewing this field, the potential benefit of replacing the classically used single-chain variable fragments (scFvs) with nanobodies as antigen binding domain was highlighted. This relates to their single domain nature and lack of hydrophobic interaction regions, which allows straightforward cloning in multidomain constructs, and ensures high stability and low immunogenicity. In addition, we highlighted the influence of specific antigen-binding domain parameters including affinity and epitope location, even though specific values predictive for optimal CAR function have not yet been determined.

In the context of solid tumors, there is a high clinical need to develop new therapies for glioblastoma and CAR-based approaches are ideally suited. Following the identification of B7-H3 as a promising target for glioblastoma, we developed and validated B7-H3 nanobody-based (nano)CAR-T cells in this context. We showed potent in vitro functionality of B7-H3 nanoCAR-T cells based on activation, cytokine secretion, and antigen-specific killing capacity. However, tumor growth control in vivo was associated with a limiting toxicity, which could be attributed to high sensitivity of the B7-H3 nanoCARs towards healthy tissue.

To further advance CAR therapies for solid tumors, we focused on HER2, representing a target with high expression on various solid tumor types including glioblastoma, breast cancer and melanoma. Lacking predictive values on nanobody characteristics associated with optimal CAR-T function, we performed a side-by-side evaluation of 12 HER2 nanoCARs and observed strong differences in CAR potency among constructs. Using in vitro assays evaluating activation, cytokine secretion, and target cell-specific killing, we identified a lead nanoCAR showing cytotoxicity across distinct solid tumor types, controlling tumor growth in vivo.

Finally, aiming at bringing HER2 CAR-T cells to larger patient populations, generation of T cell-targeted lentiviral vectors (LVs), for in situ CAR-T cell engineering was intended. We identified CD3, CD4, CD7 and CD8-targeting nanobodies and evaluated their capacity to generate T cell-targeted LVs. We identified CD7-targeted LVs to be most potent in generating functional HER2 CAR-T cells, showing antitumor function against glioblastoma cells in vitro and providing promising potential for further in vivo evaluation to improve therapeutic benefit.

To conclude, we developed and screened B7-H3 and HER2-targeting nanoCAR-T cells in terms of efficacy and safety, and explored the potential of using T cell-targeted nanobody-displaying LVs to enable in situ CAR-T cell engineering. Overall, this study contributes to the advancement of CAR-T cell therapies for the treatment of solid tumors, laying a foundation for the implementation of this work in follow-up projects, with the ultimate aim of making these therapies available to patients.