convertible CAR-T cells provide a modular universal system for dose control of activity, flexible targeting, and versatile maintenance of CAR-cells

ABSTRACT Current chimeric antigen receptor (CAR) cellular therapies, while remarkably efficacious, have a number of limitations that impact their efficacy, safety and perseverance in the clinical setting, including commitment to targeting a single antigen, utilization of non-human components, and lack of dose control. We have developed a universal CAR platform to function as a flexible and controllable system to address these limitations. An inert form of the NKG2D extracellular domain (iNKG2D) was used as the surface-exposed portion of the CAR and transduced into T cells to generate convertibleCARTM-T cells. These cells were activated only when an immunological synapse was formed with a displayed antigenic target, mediated by a bispecific molecule comprised of an iNKG2D-exclusive orthogonal human ligand fused to an antigen-targeting antibody (MicAbodyTM). By altering the Fv domains of the MicAbody, cytolytic activity against a variety of antigen-expressing cell lines was demonstrated in vitro. In addition to sequential introduction of MicAbodies, arming convertibleCAR-T cells with a combination of MicAbodies resulted in simultaneous activity against different antigen-containing targets. Additionally, activation and cytolytic functions of convertibleCAR-T cells depended upon the dose of MicAbody introduced. Studies in NSG mice exploring efficacy against Raji tumors demonstrated dose-dependent control of tumor mass by rituximab-based MicAbody as well as by convertibleCAR-T cells. We have also demonstrated that the exclusive ligand-receptor partnering enabled the targeted delivery of ligand-fused payloads to convertibleCAR-T cells. Ligand fusion to CDC-enhanced Fc domains led to in vitro reduction of convertibleCAR-T cells by human complement, thus offering the ability to reduce the levels of CAR cells in a patient without necessarily completely eliminating them. Selective delivery of a mutant IL-2 drove expansion of convertibleCAR-T cells in vitro and promoted in vivo expansion and recovery. This ability to leverage the exclusive ligand-receptor interaction has profound implications for the ability to exogenously control convertibleCAR-T cell expansion, persistence, and function within the patient. This, combined with the ability to switch targets and multiplex without modifying the CAR, enables the development of a highly modular and adaptable universal system to create a single autologous cell for all targets in a single donor patient or one allogeneic cell for all patients and all targets.