|Ph.D Student||Greenman Raanan|
|Subject||Studying the Biophysical Properties that Shape Functional|
Output of Engineered Chimeric Antigen Receptor
|Department||Department of Biology||Supervisor||Professor Yoram Reiter|
|Full Thesis text|
Adoptive cell immunotherapy with chimeric antigen receptors (CARs) has recently gained significant focus due to durable clinical responses in hematopoietic malignancies. Such CARs are composed from antigen-recognizing elements ̶ such as antibody variable domain fragments (characterized by high affinity) ̶ fused with intracellular T cell signaling moieties. We recently used antibodies that recognize peptide-MHC (TCR-like Abs) to compare a high-affinity antibody-based CAR to a native low-affinity TCR. Unexpectedly, the high-affinity CAR was less effective than the low-affinity TCR, at most antigen densities, suggesting an upper affinity threshold for effective functional outcomes of engineered T cells. We set out to further characterize this affinity threshold, in order to describe what precise combination of affinities and avidities can lead to optimized functional outcome. As affinity, avidity and the target’s antigen density dictate the binding of the T cell to its target, we generated an experimental system in which all these main parameters of CAR-T cell stimulation are controllable. We characterized a series of single chain fragment binding domains, ranging from 4nM to 434nM KD, that target the tumor-associated antigen Tyr-HLA-A2. Based on the selected binding domains we constructed antigen-specific second generation CARs with distinct affinities, and sorted each CAR-T cell population by the level of receptor expression. These cells were encountered with target cells that present the antigen at different levels. This unique system allowed understanding the interplay between CAR affinity, antigen density and CAR expression level and their effect on T cell functionality (as measured by cytokine production and T cell degranulation). We detected nonmonotonic behaviors of both affinity and antigen density upon CAR-T cell functionality. These phenomena were corroborated by measuring CAR-T cells anti-tumor activity. We further systematically analyzed the contribution of CAR affinity and expression levels to the final T cell responses. Whereas other binding biophysical parameters, such as bond stability, can affect functional avidity of CAR-T cells, affinity and avidity explained most of the response variance. Based upon these observations, we built a mathematical model of CAR-T cell activation. This model composes regulatory elements that are used to model TCR activation, such as kinetic proofreading and an inhibitory feedforward loop. However, these elements do not recapitulate the effect of CAR avidity. We therefore suggest an improved dynamical model that incorporates a slow receptor internalization process. This revised model suggests that receptor internalization provides an impetus for CAR-T cell regulation. We verified this assumption by measuring and comparing receptor downmodulation and downstream intracellular inhibitory processes. CAR downmodulation was evident at an early time-period following encounter with target cells. We observed similar kinetics of intracellular activatory and inhibitory processes, contrary to the kinetics described following TCR activation. Finally, CAR internalization inhibition, but not Shp1/Shp2 inhibition, was able to increase CAR-T cell responses. These results have a potential to improve the rational design of CAR-T cell treatments.