Enhancing chimeric antigen receptor T-cell therapy for hematological malignancies through split-signaling CAR T-cells

Given the high potential of advancing CAR T-cell therapies, this thesis specifically focuses on investigating the feasibility and application of the split-signaling CAR T-cell strategy for the treatment of MM and AML. With this specific aim, we reviewed the literature in chapter 2 and sought to find and evaluate CAR T-cell targets for MM. We also studied the different types of dual targeting strategies, to be used to improve MM CAR T-cell therapy efficacy and safety, thereby specifically focusing on the split-signaling CAR concept. We uncovered CD38 and CD138 as ideal split-signaling CAR targets. Subsequently, in chapter 3 we used this knowledge and generated CD38+CD138 targeting split-signaling CAR T-cells for MM. We tested several CAR combinations and optimized the sCAR and cCAR affinities when necessary to specifically target MM cells and spare healthy cells. In parallel to MM, we sought to apply the split-signaling CAR concept to AML. Our preliminary searches had indicated the potential unique combination of TIM3 and CLEC12A antigens on leukemic stem cells. To confirm this, we consulted the literature in more detail in chapter 4. This comprehensive literature search indeed identified TIM3 and CLEC12A, among others, as promising candidate targets for further evaluation in split-signaling CARs for AML. Using the knowledge generated in chapter 4 we first explored the use of TIM3 in chapter 5 as a mono-target for CAR T-cells in AML. TIM3 is of particular interest because of its specific expression on leukemic stem cells and its active involvement in AML relapse and progression. The other identified AML antigen, CLEC12A, was studied as a CAR target in chapter 6. First as mono-target and subsequently combined with TIM3 targeting in split-signaling CAR T-cells. Thus, this specific investigation aimed at determining the feasibility, effectivity and specificity of the CLEC12A+TIM3 targeting split-signaling CAR T-cells in AML. The methodological study described in chapter 7 was initiated to tackle the technical challenges involved in dual CAR targeting strategies. We aimed at an efficient transduction of T-cells to simultaneously express two, or even three CARs at high levels without the use of bicistronic or tricistronic vectors. Rather than relying on conventional methods, we tackled this technical problem by developing an efficient and user-friendly strategy using stable virus producer cells that can simultaneously generate up to three viral CAR vectors. We applied this method in chapter 8 to improve CAR expression of CLEC12A+TIM3 split-signaling CAR T-cells. More importantly, in this chapter we tested a recently described strategy to tune the signaling strength of the CARs. We applied this strategy in a novel way to enhance the selective killing of AML cells by CLEC12A+TIM3 split-signaling CAR T-cells. The most important findings of this thesis are summarized and discussed in chapter 9.