A computationally designed chimeric antigen receptor provides a small-molecule safety switch for T-cell therapy

Abstract

Approaches to increase the activity of chimeric antigen receptor (CAR)-T cells against solid tumors may also increase the risk of toxicity and other side effects. To improve the safety of CART cell therapy, we computationally designed a chemically disruptable heterodimer (CDH) based on the binding of two human proteins. The CDH self-assembles, can be disrupted by a small-molecule drug and has a high-affinity protein interface with minimal amino acid deviation from wild-type human proteins. We incorporated the CDH into a synthetic heterodimeric CAR, called STOP-CAR, that has an antigen-recognition chain and a CD3ζ-and CD28-containing endodomain signaling chain. We tested STOP-CART cells specific for two antigens in vitro and in vivo and found similar antitumor activity compared to second-generation (2G) CART cells. Timed administration of the small-molecule drug dynamically inactivated the activity of STOP-CART cells. Our work highlights the potential for structure-based design to add controllable elements to synthetic cellular therapies. T cells engineered with CARs, hybrid molecules linking antigen binding to T-cell signaling endodomains, have mediated potent and durable responses against both chronic and acute B cell leukemias 1-4. CART cell therapy, however, is frequently associated with lifethreatening side effects, including cytokine release syndrome (CRS) and neurotoxicity. The clinical development of CART cells against solid tumors has proven challenging, but there is great optimism that next-generation CART cells will bring benefit to a broader range of cancer patients 5. Indeed, it is now well understood that physical and immunometabolic barriers upregulated in the solid tumor microenvironment as well as prolonged exposure to antigens can impair T-cell function and drive T-cell exhaustion 6. Innovative engineering strategies, such as the expression of cytokines, chemokines, decoy molecules or stimulatory ligands, are being developed to overcome these obstacles and have shown favorable preclinical responses 6-8. Safety, however, remains an important barrier to clinical entry because most solid tumor antigens targeted to date are also found in healthy tissues, sometimes leading to serious on-target off-tumor toxicity 9. The ability to control CART cell activity on command could greatly accelerate the clinical development of cellular immunotherapies. The above considerations have driven the development of CART cell control and safety systems 5 , such as drug-inducible suicide switches 10,11 , coinhibitory receptor (i)CARs that upon engagement with specific antigens suppress effector function 12 and split-signaling CART cells that require co-engagement of two ligands for full T-cell activation 13. SUPRA (split, universal and programmable) CARs 14 and a variety of universal CARs 15,16 have been developed that require administration of an adaptor protein to link nontumor antigen binding CART cells to tumor cells. While these approaches offer the possibility to sequentially target different tumor antigens (for ...

CAR T Cell Therapy for Solid Tumors: Bright Future or Dark Reality?

“…Several dimerizer systems have been developed; 178 of these, the inducible caspase-9 (iC9) system has been evaluated in humans with encouraging results. 179,180 Other promising strategies to increase the safety profile of CAR T cells include splitting CARs into two domains, which need to be linked with a small molecule to be active, 181,182 or taking advantage of the small molecule-assisted shut-off (SMAsh) technology. 183 The third approach relies on expressing a molecule on the cell surface of T cells that can be targeted with a clinically approved mAb (e.g., CD20 and truncated EGFR).…”

Adoptive Immunotherapy beyond CAR T-Cells

“…This emphasizes the importance of proper genetic design and comprehensive tests, and may require the introduction of a "safety switch" mechanism for regulation of immune response in the case of developed toxicity. These "safety switch" mechanisms, initially developed for CAR T-cells, include drug-inducible suicide genes, logic gate receptors (AND/OR) [29], as well as ON-switch receptors that require small molecule modulators for multimerization [87]. In addition to the suicide switches, the RNA transfection methods could be applied for initial clinical testing of new TCR and CAR-based therapeutic approaches [88].…”

A rational blueprint for the design of chemically-controlled protein switches

“…2a,b), is currently undergoing phase 1 clinical trials 16,23,31 . Leveraging the same computational design approach described for the CDH-1 18 , we utilized the structural information of the mdm2:p53 stapled peptide complex (PDB ID: 5afg) 32 and searched for proteins that could accommodate the p53 helical motif. We performed a structural search over approximately 11,000 putative protein scaffolds and after the design stage we selected for experimental characterization three designs which we refer to as LD4, LD5 and LD6 (Supp.…”