Switch-mediated activation and retargeting of CAR-T cells for B-cell malignancies

Abstract

Chimeric antigen receptor T (CAR-T) cell therapy has produced impressive results in clinical trials for B-cell malignancies. However, safety concerns related to the inability to control CAR-T cells once infused into the patient remain a significant challenge. Here we report the engineering of recombinant antibody-based bifunctional switches that consist of a tumor antigen-specific Fab molecule engrafted with a peptide neo-epitope, which is bound exclusively by a peptide-specific switchable CAR-T cell (sCAR-T). The switch redirects the activity of the bio-orthogonal sCAR-T cells through the selective formation of immunological synapses, in which the sCAR-T cell, switch, and target cell interact in a structurally defined and temporally controlled manner. Optimized switches specific for CD19 controlled the activity, tissue-homing, cytokine release, and phenotype of sCAR-T cells in a dose-titratable manner in a Nalm-6 xenograft rodent model of B-cell leukemia. The sCAR–T-cell dosing regimen could be tuned to provide efficacy comparable to the corresponding conventional CART-19, but with lower cytokine levels, thereby offering a method of mitigating cytokine release syndrome in clinical translation. Furthermore, we demonstrate that this methodology is readily adaptable to targeting CD20 on cancer cells using the same sCAR-T cell, suggesting that this approach may be broadly applicable to heterogeneous and resistant tumor populations, as well as other liquid and solid tumor antigens.

Versatile strategy for controlling the specificity and activity of engineered T cells

“…Indeed, in our accompanying studies using peptide neo-epitope grafted switches, antipeptide neo-epitope CAR-T-cell activity was improved when a mutation was introduced within the hinge region to enhance the dimerization of CAR via interchain disulfide bond (44,49). Notably, despite a similar FITC to antibody ratio (average DAR ∼2), the randomly conjugated anti-CD19 FITC switch demonstrated inferior in vivo antitumor activity relative to the optimized bivalent anti-CD19 AB-FITC switch, highlighting the importance of a site-specific conjugation approach to generate optimized switches.…”

Axicabtagene ciloleucel, a first-in-class CAR T cell therapy for aggressive NHL

“…Several approaches are being investigated, including CAR T cells with additional functionality, novel CAR T dosing strategies, and mechanistic studies of observed clinical correlates of safety and efficacy ( Figure 5). For example, results of preclinical studies have demonstrated the viability of various switch mechanisms for CAR T cells [51,[63][64][65]. Switch technology may give clinicians real-time, tunable control of CAR T cell activity and the ability to widen the CAR T therapeutic window.…”

CARs on the Highway: Chimeric Antigen Receptor Modified T Cells for the Adoptive Cell Therapy of Malignant Diseases

“…In an alternative approach, the CAR binds to a protein epitope, which is not encoded by the human genome and which is linked to cancer-targeting antibodies [54]. In another example, folate receptor-positive cancer cells were marked by a fluorescein isothiocyanate (FITC)-conjugated folate which binds to the cancer cells and is recognized by a FITC-specific CAR [55,56]. By using more than one FITC-labeled molecule which mark the cancer cells, the same CAR T cell can target multiple types of cells within the tumor lesion.…”