Chen Y

Adoptive T-cell therapy is a novel cancer treatment strategy in which T cells (a type of white blood cells) are isolated from a cancer patient, genetically modified to express tumor-targeting receptors, expanded in the laboratory, and then re-infused into the same patient.  

Several clinical trials have shown that T cells expressing chimeric antigen receptors (CARs) that target the B-cell marker CD19 can eradicate advanced B-cell leukemia and lymphoma for patients who had been unsuccessfully treated by conventional therapies. CD19 CAR-T cells’ remarkable clinical outcomes highlight the transformative potential of CAR–T-cell therapy for cancer. However, the CD19 CAR remains the only CAR that has consistently achieved robust clinical efficacy, and the basis of its functional superiority remains unclear. Incomplete understanding of the parameters that critically influence CAR functionality has limited the rational design of novel CARs. As a result, CAR development remains dependent on trial-and-error approaches that are costly and inefficient.  

To address this important limitation, we propose a high-throughput, performance-based method to generate robustly functional CARs. Products of the screening process will be systematically analyzed to determine whether specific molecular properties dictate CAR functionality.  

Results of these studies will enhance our ability to efficiently engineer novel CAR–T-cell therapeutics. CARs are synthetic proteins that bind to specific disease markers (antigens) via an extracellular sensing domain consisting of a single-chain variable fragment (scFv).  

Previous studies have shown that the scFv domain is not only critical to targeting specificity, but also affect the overall effectiveness of the CAR molecules and the T cells in which the CARs are expressed. Here, we describe a novel screening method by which CAR molecules with diverse scFv sequences will be expressed in human T cells and selected based on their ability to (1) prevent premature T-cell exhaustion and (2) promote T-cell proliferation upon exposure to antigens.  

The high-performance CARs generated through this process will be analyzed for specific properties—including tendency for spontaneous receptor clustering, scFv-antigen binding affinity and kinetics, and potential synergy between a given scFv sequence and particular co-stimulatory domains incorporated in the CAR. Results of these analyses will elucidate whether any of these properties can be specifically engineered to enhance CAR functionality.  

CAR–T-cell therapy has been hailed as one of the most promising breakthroughs in cancer therapy, and its full potential beyond the treatment of B-cell malignancies remains to be realized. Successful completion of the proposed research will significantly improve the efficiency by which highly functional CARs targeting new disease markers can be generated, thus facilitating the development of novel therapeutics for diseases that await better treatment options.  

Katz

Harnessing a patient’s own immune system to attack blood cancers has recently enjoyed remarkable success. In one strategy, a patient’s T cells are harvested, reprogrammed to recognize the cancer as foreign, and then returned to the body. The reprogrammed T cells produce a novel artificial protein, a chimeric antigen receptor commonly called a CAR, which brings together the ability of two other proteins: one that recognizes the tumor cells and another which can stimulate the T cell to attack.  

These so-called CAR-T cells are highly effective and specific anti-tumor agents that kill tumor cells and further stimulate other parts of the immune system. In several recent clinical trials, approximately 70% of terminally ill patients, refractory to standard treatments, were cured by CAR-T therapy. However, most protocols reprogram the T cells to express the CAR by permanently altering their DNA using retroviruses.  

We have developed an alternate system, using RNA, which transiently reprograms the T cells to attack the cancer. This approach has many benefits including increased safety, speed, control and the ability to deliver multiple beneficial proteins at the same time. The flexibility to accurately adjust therapeutic conditions and introduce different proteins into T cells at different times according to the needs of the patient makes this a useful tool for individualized medicine. Moreover, RNA reprogramming is essentially transient. After conclusion of the therapeutic treatment all of the RNAs and the proteins they make are degraded and the T cells return to their normal state, which minimizes any possible side effects of the treatment. Our technology has wide applicability for many different tumors and a high potential for further development.  

Despite the success of CAR-T cells in blood cancers, duplicating these achievements in solid cancers like breast cancer, melanoma and sarcoma remains a “holy grail” for the field. Two key barriers to success are (1) most CAR-T cells that recognize solid cancers also recognize normal tissues, and (2) solid tumors have the ability to prevent the activation of T cells. The ability of our RNA approach to make multiple helpful proteins in the patient’s T cells in one-shot has the potential to overcome both of these obstacles. In addition to the CAR, our RNA approach provides proteins that limit the T cell’s ability to attack normal tissues, and other proteins that will help the T cell survive and be active while it avoids the suppressive signals being sent to it from the solid tumor. Collectively, this approach will induce the CAR-T cells to become more sensitive, specific and effective killers of solid tumors.  
 
This Alliance for Cancer Gene Therapy Research Fellow is funded in part by Swim Across America.