Evaluation of the functional performance of KLK2 chimeric antigen receptor (CAR) T cell designs

Celina Guzman

doi:10.17918/00001810

CAR T cell therapy has been making headlines with its recent success in treating refractory leukemia, lymphoma, and multiple myeloma. It represents a major advancement in immunotherapy for personalized cancer treatment. It is a type of adoptive cell therapy (ACT) that genetically reprograms a patient's own T cells to express synthetic receptors that target tumor antigen and induce cytotoxicity. This therapy, however, has many challenges in translating to an efficacious treatment for solid tumor cancers such as prostate cancer. With prostate cancer being the most common malignancy and one of the leading causes of death in men, there is a substantial lack of knowledge in treating metastatic castration-resistant prostate cancer that causes significant loss of life every day. To address this unmet clinical need, several ongoing studies have been focusing on optimizing the CAR T-cell design to be able to impart enhanced antigen-specific cytotoxicity to prostate tumor cells. On a similar note, a recent publication in Nature replaced the CD3[zeta] signaling motif of a CD19 CAR with a modified immunoreceptor tyrosine-based activation motif (ITAM) configuration (termed 1XX) to extend functional persistence of the T cell without compromising its potency. They hypothesized that the redundant signaling of a CAR incorporating all three CD3[zeta] ITAMS may foster counterproductive T cell differentiation and exhaustion, and that calibrating the ITAM activity by mutating 2 of the 3 ITAMs would reprogram T cell function and differentiation. Consequentially, they found that the CARs encoding a single functional ITAM not only outperformed the original CD28 CAR constructs in functional persistence, but balanced effector and memory programs, yielding enhanced therapeutic profiles. The studies described in this thesis aim to test whether 1XX enhanced the functional performance of KLK2 CAR T cells that target prostate cancer cells. I evaluated activation strength mediated by 1XX compared to wildtype CD3[zeta] with 3 functional ITAMs at baseline and following KLK2 stimulation using Jurkat reporter cell lines. I also measured cytotoxicity and cytokine secretion via acute killing assays using primary T cells from human donors. Although I found that the 1XX CAR configurations may reduce baseline tonic signaling; I found no conclusive evidence that this significantly impacted antigen-mediated activation signaling. Upon evaluating the functional performances of the CARs in primary T cells, I found overall comparable activities between CAR construct designs in cytotoxicity and cytokine secretion. Thus, I concluded that although the modified 1XX signaling domain may reduce hyperactivation of the CAR construct, this did not consistently significantly impact the CARs overall effector function and cytotoxicity. Thus, alternative approaches will be needed to significantly improve the functional performance of CAR designs and develop an efficacious CAR T-cell therapy for prostate cancer.

Evaluation of the functional performance of KLK2 chimeric antigen receptor (CAR) T cell designs

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Evaluation of the functional performance of KLK2 chimeric antigen receptor (CAR) T cell designs

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