In order to create CAR T cells capable of a long-term memory response without chronic depletion of B cells, Viaud et al. developed a switchable CAR (sCAR) T cell platform in which the sCAR T cells could be reversibly turned “on” or “off” with a dosage of an antibody-based switch. In the absence of continuous antigen stimulation, this setup would allow the sCAR T cells to “rest”, which is necessary for robust memory formation, and give B cells the opportunity to replete. The researchers tracked the fate of the sCAR T cells and their impact on the B cell population over time; these results were recently published in PNAS.

Previously, the team had developed an sCAR T cell system consisting of a CD19 switch – an antigen-binding fragment of a murine CD19-targeting antibody (Fab) engrafted with a peptide neoepitope (PNE) – and a corresponding sCAR with a single-chain antibody variable fragment (scFv) that recognized the PNE. The CD19 switch coupled the CD19 on the target cell to the PNE-targeted sCAR. The rapid clearance of the CD19 switch Fab (lacking an Fc region) allowed control of timing of sCAR stimulation. Optimization efforts identified an IgG4m hinge and a wild-type CD3ζ intracellular domain as critical for persistence and expansion of the sCAR T cells. Viaud et al. then generated sCAR T cell constructs with CD28 (Ig-28z), 4-1BB (Ig-BBz), or CD28 and 4-1BB (Ig-28BBz) costimulatory domains and tested them in vivo.

The researchers utilized a syngeneic tumor model in which murine 38c13 CD19-expressing B cell lymphoma cells were subcutaneously injected into the flank of C3H mice, which have a competent immune system. After tumors were established, mice were preconditioned with cyclophosphamide and then inoculated with sCAR T cells followed by the CD19 switch. The CD19 switch was dosed every other day for two weeks, followed by 2 weeks of rest when no CD19 switch was administered. This dosing schedule was repeated for three cycles. Ig-BBz and Ig-28BBz constructs durably eliminated tumors in all mice without relapse, while Ig-28z constructs and the standard anti-CD19 CAR T cells (with a CD28 costimulatory domain) failed to control tumor growth. All treatment groups led to the depletion and eventual rebound of B cells. The Ig-BBz and Ig-28BBz sCAR T cells iteratively depleted B cells over the three switch dosing cycles, and B cells rebounded during rest phases, demonstrating the functional reversibility of activity of these sCAR T cells.

Viaud et al. then took a closer look at how timing of the switch dosing regimen affects sCAR T cell phenotype and B cell depletion. To do this, they created three different regimens: 2 week dosing, 2 weeks rest (reference); 3 weeks dosing, 1 week rest (to mimic chronic antigen stimulation); 1 week dosing, 3 weeks rest (to mimic acute stimulation). For each regimen, low and high doses of the switch were tested. The regimens were tested in C3H mice without tumors, and the Ig-28BBz sCAR T cell construct was selected because of its efficacy in the tumor model and close resemblance to a human clinical candidate that is currently in development. B cells were depleted in all dosing regimens after the first and second cycle. However, in the second cycle, the 1 week dosing regimen resulted in a dramatic expansion of sCAR T cells (the level of expansion correlated with the dosage of the switch), whereas no expansion was observed in the 3 week dosing group. This result demonstrated that the rest phase was more important to sCAR T cell expansion than the switch dose level. Phenotypically, the sCAR T cells expanded during the second cycle were mostly CD8⁺ effector/effector memory T cells (T_E/EM). The CD8⁺ T_E/EM cell population later contracted, while the central memory (T_CM) phenotype was retained – at persistently higher levels in the 1 week dosing group than in the other regimens.

Tracking the trafficking of the sCAR T cells, the team found that in tumor-bearing mice, sCAR T cells depleted B cells in the peripheral blood, spleen, tumor-draining lymph node (TDLN), and bone marrow. Phenotypically, sCAR T cells were mostly CD8⁺ T_E/EM in the blood, spleen, and bone marrow, and importantly, mostly CD8⁺ T_CM in the TDLN. During the contraction phase, the number of sCAR⁺CD8⁺ T_CM cells remained relatively constant in the tissue, whereas a dramatic decrease was observed in the blood.

The phenotypic fate of CAR T cells determines their therapeutic efficacy in the clinic, and the T_CM phenotype has been shown to effect sustained remissions in patients with acute lymphoblastic leukemia. In this study, Viaud et al. demonstrated that the switchable CAR T cell platform led to a durable antitumor response due to the development of a persistent sCAR⁺ CD8⁺ T_CM population in the secondary lymphoid tissues. The period of rest, during which the sCAR T cells were inactive, was crucial for the development of the sCAR⁺ CD8⁺ T_CM subset as well as for the repletion of B cells, which could ultimately create a favorable efficacy and safety profile in the sCAR T cell product in the clinic.

by Anna Scherer