Empowering CAR T Cell Therapy: Translating Resistance Mechanisms into Opportunities

Chimeric antigen receptors (CAR) T cell therapies have already demonstrated favourable clinical outcomes, however tumor resistance mechanisms remain a major obstacle. This thesis focuses on the primary function of CAR T cells in cancer therapy: the mechanisms by which they kill tumor cells. By gaining a fundamental understanding of how CAR T cells induce tumor cell death and how tumors resist this cytotoxicity, we can design new strategies to overcome resistance while avoiding systemic side effects.

The main mechanism by which CAR T cells eliminate tumor cells is through the release of cytotoxic granule contents into the target cell. The key effector protein that induces regulated cell death (apoptosis) is granzyme B. In human biology, Serpin B9 is known as a specific inhibitor of granzyme B. Chapter 2 identifies Serpin B9 as a novel intrinsic resistance mechanism to CD19- and CD20-directed CAR T cell therapy.

Previous studies have shown that preclinical inhibition of MCL-1 can effectively eliminate multiple myeloma (MM) cells. However, clinical trials have revealed severe systemic toxicity associated with MCL-1 inhibition, halting further clinical development. To make this potentially effective strategy feasible, we investigated synergistic combinations of clinically available agents. Chapter 3 demonstrates that inhibition of P70S6K1 and MCL-1 has a synergistic effect, thereby allowing treatment with lower doses of MCL-1 inhibitors and minimizing adverse effects.

In Chapter 4, we explore innovative methods to specifically target MCL-1 in MM. To this end, we developed a strategy to express the MCL-1 antagonist NOXA within the cytotoxic granules of BCMA-targeted CAR T cells. We show that NOXA can be directed to the granules by fusing it to granzyme B. Upon recognition of an MM cell, NOXA is released into the tumor cell. This arming of BCMA CAR T cells with a pro-apoptotic NOXA, enhances their antitumor activity. Besides we also reveal MCL-1 expression by MM cells as a CAR T resistance mechanism.

Chapter 5 describes an international patent filing related to fusion proteins based on the NOXA scaffold. In this approach, the effector BH3 domain of NOXA can be exchanged with BH3 domains from other pro-apoptotic BCL-2 family proteins. This design could support personalized therapies tailored to tumor-specific dependencies on survival proteins.

Cytotoxic T cells mainly induce apoptosis. This clean form of cell death does not release inflammatory mediators such as damage-associated molecular patterns (DAMPs). In Chapter 6, we take the first steps toward using our CARgo delivery method to induce immunogenic cell death in vitro. We anticipate that inducing an immunogenic form of tumor cell death could shift the balance in favor of tumor-clearing CAR T cells, especially in solid tumor settings where T cell infiltration is limited.

Collectively, this thesis provides new insights into intrinsic tumor resistance mechanisms involving Serpin B9 and MCL-1, and presents innovative strategies using CAR T cells to deliver pro-apoptotic or immunogenic cell death–inducing proteins to tumor cells. Together, these studies mark a step forward in overcoming resistance and advancing the next generation of CAR T cell therapies.