Human CD19-specific switchable CAR T-cells are efficacious as constitutively active CAR T-cells but cause less morbidity in a mouse model of human CD19⁺malignancy

Abstract

Current Food and Drug Administration (FDA)-approved CD19-specific chimeric antigen receptor (CAR) T-cell therapies for B-cell malignancies are constitutively active and while efficacious, can cause morbidity and mortality. Their toxicities might be reduced if CAR T-cell activity was regulatable rather than constitutive. To test this, we compared the efficacies and morbidities of constitutively active (conventional) and regulatable (switchable) CAR (sCAR) T-cells specific for human CD19 (huCD19) in an immune-competent huCD19+transgenic mouse model.Conventional CAR (CAR19) and sCAR T-cells were generated by retrovirally transducing C57BL/6 (B6) congenic T-cells with constructs encoding antibody-derived single chain Fv (sFv) fragments specific for huCD19 or a peptide neoepitope (PNE), respectively. Transduced T-cells were adoptively transferred into huCD19 transgenic hemizygous (huCD19Tg/0) B6 mice; healthy B-cells in these mice expressedhuCD19Tg. Prior to transfer, recipients were treated with a lymphodepleting dose of cyclophosphamide to enhance T-cell engraftment. In tumor therapy experiments, CAR19 or sCAR T-cells were adoptively transferred intohuCD19Tg/0mice bearing a syngeneic B-cell lymphoma engineered to express huCD19. To regulate sCAR T cell function, a switch protein was generated that contained the sCAR-specific PNE genetically fused to an anti-huCD19 Fab fragment. Recipients of sCAR T-cells were injected with the switch to link sCAR effector with huCD19+target cells. Mice were monitored for survival, tumor burden (where appropriate), morbidity (as measured by weight loss and clinical scores), and peripheral blood lymphocyte frequency.CAR19 and sCAR T-cells functioned comparably regarding in vivo expansion and B-cell depletion. However, sCAR T-cells were better tolerated as evidenced by the recipients’ enhanced survival, reduced weight loss, and improved clinical scores. Discontinuing switch administration allowed healthy B-cell frequencies to return to pretreatment levels.In our mouse model, sCAR T-cells killed huCD19+healthy and malignant B-cells and were better tolerated than CAR19 cells. Our data suggest sCAR might be clinically superior to the current FDA-approved therapies for B-cell lymphomas due to the reduced acute and chronic morbidities and mortality, lower incidence and severity of side effects, and B-cell reconstitution on cessation of switch administration.

Universal CAR 2.0 to overcome current limitations in CAR therapy

“…However, multitargeted strategies and performance tuning can compensate for the reduced initial formation of the cytolytic synapse and turn the disadvantage into dominance. Additionally, adapter CAR T cell technologies facilitate a more physiological CAR recruitment and engagement compared to conventional CAR design and allow cells to recover from the massive CAR stimulation according to the administration regimen of the adapter molecules ( 237 ).…”

“…Numerous adapter CAR technologies have been developed with the aim to introduce a safety on-switch to control early onset toxicities and on-target-off-tumor toxicities as well as to establish versatile patient-individualized combinatorial immunotargeting ( 20 , 65 ). Incorporating next-generation CAR design ( 209 ) may catapult adapter CAR technologies to outcompete conventional CAR designs while retaining safety ( 237 ). The fusion of these distinct two universal CAR technologies unites key features of two concepts that might resolve our current skepticism for clinical potency and safety as well as the significant costs required for the development.…”

Antigen loss following CAR‐T cell therapy: Mechanisms, implications, and potential solutions

“…This design allows for precise control over CAR‐T cell activation, preventing off‐target effects and minimizing potential damage caused by antigen loss. Switchable CARs offer the potential to modulate CAR‐T cell activity in response to changes in antigen expression or the tumor microenvironment, thereby mitigating the impact of antigen loss 73 . sCAR‐T cells targeting CD19, along with a switch protein, were shown to produce fewer adverse effects in the mouse model.…”