Development of Intelligent Chimeric Antigen Receptor (iCAR) T-Cells for the Treatment of Hematological Malignancies
Authors: Estevam Jose , Konry Tali , Schubert Shai 
Published: December 2024

Chimeric antigen receptor T cell therapies (CAR-Ts) are regarded as one of the most promising immunotherapies for patients with hematological malignancies and continue to be a major focus of research, with almost 1,000 clinical trials actively recruiting patients. Despite the significant advancements made with CAR-T immunotherapies, not all patients benefit from this line of treatment, and many more patients eventually relapse or develop significant side effects. The reasons for this are complex, but it is becoming increasingly clear that optimal donor selection and improvements in construct design are critical factors in designing improved immunotherapies. To address this need, we have developed a proprietary function-to-omics technology platform that permits the screening of donor samples and allows us to select targeted samples for the discovery of new biomarkers and designing improved CAR-T constructs.

By applying a function-to-omics discovery workflow we can collect at the single-cell level individual T-cells that effectively lysed tumor cells from less functional or dysfunctional T-cells. By subjecting these cells to bulk RNA-sequencing, we can identify genes and biological pathways driving anti-tumor activity. By doing so, we simultaneously produce a more homogeneous population of T-cell killers and identify biomarkers that can be used to develop enhanced CAR-T constructs. To establish our technology for hematological malignancies, we use a well-characterized CD19+ cell line to demonstrate that activated T-cell killers from various donors display differential immune synapse activity, cell activation, and inhibition profiles, different use of cytolytic granules and display unique metabolic programming, despite all cells being able to target and kill tumor cells. With thousands of differentially expressed genes to select from, we noted, for example, an increase in well-characterized cytolytic genes PRF1, GZMB, and IFNG with donor-to-donor dependencies in the magnitude of gene expression as well as differences in the cytolytic genes expressed. The activation of other well-characterized genes such as ICOS, IL2 and CD28 was also detected with our approach and varied from donor to donor. Interestingly, donors displayed unique metabolic profiles which included genes from the glycolytic, oxidative phosphorylation, fatty acid metabolism and pentose phosphate pathways.

These transcriptional profiles have facilitated the design of our first iCAR cells which are currently under pre-clinical development. We have selected a novel combination of biomarkers to include in our potential best-in-class iCAR construct, which includes members of TNFSF10, FASL along with other immunomodulatory fusion proteins, to facilitate the formation of a more effective immune synapse and improve the anti-tumor activity against lymphoma cells. With the ability to produce a more homogenous cellular product using our function-to-omics approach, our iCARs are also predicted to have fewer side effects through the elimination of cells with suboptimal activity. In this way, the most potent cells can be selected for the right patient, paving the way for a more personalized approach to donor selection and generation of iCAR cells that can transform the treatment landscape for patients with hematological malignancies.