Multi-Pronged Approach to Understanding the Toxicity Associated with CAR T-Cell Therapy
Chimeric antigen receptor (CAR) T-cell therapy has revolutionised cancer immunotherapy, providing effective treatment for previously untreatable malignancies. Its success, in treat ment of haematological cancers, is now fast-advancing to solid tumours. Though, these efforts are faced with many challenges including arising therapy associated toxicities. These conditions arise from robust immune activation and cytokine release, which can necessitate intensive medical intervention and, in severe cases, be life-threatening. Understanding the mechanisms that drive toxicity, and developing strategies to mitigate it, is therefore critical for the safe and effective application of CAR T-cell therapy. This thesis takes a multi-pronged approach to address this challenge, integrating genomic, transcriptomic, and functional strategies. Virus integration is essential for CAR expression, yet the biological consequences of integration are not yet fully understood. Integration site profiling therefore provides a means to assess whether certain patterns are associated with functional differences or toxicity in CART-cell products. To achieve this, I established a pipeline to generate integration site libraries from primary CAR T-cells and developed a bioinformatic workflow for analysis. While an initial study of immunogenic capacity was underpowered to yield statistically significant correlations, the workflow provides a validated platform for integration profiling in primary material. Complementary to the integration site analysis pipeline, I optimised workflows for single cell transcriptomic analysis of CAR T-cell products. I addressed key challenges in working with sensitive patient material and established and optimised a sample processing workflow for T-cell and mononuclear cell enrichment, compatible with 10x Genomics platforms. Although the intended patient cohort study was not completed due to funding limitations, the established pipeline provides the means to study transcriptomic signatures of toxicity in future clinical material. I explored functional strategies to mitigate monocyte-driven toxicity, given the cen tral role of monocyte-derived cytokines such as IL-6 and IL-1β in toxicity. I designed fourth-generation armoured CAR T-cells capable of secreting blocking agents targeting the CCL2–CCR2 axis, the principal pathway of monocyte chemotaxis. Several approaches iv were tested, including secretion of eotaxin-3, scFvs, and a nanobody. While eotaxin-3 and anti-CCR2 scFvs showed limited efficacy, anti-CCL2 constructs were expressed and bound their target, though migration blocking was not reproducible. These studies nevertheless demonstrate proof-of-principle for activation-inducible, armoured CAR platforms that could reduce CAR T-cell associated toxicities. Collectively, this thesis provides an integrated investigation into CAR T-cell–associated toxicities, combining genomic and transcriptomic tools with novel functional CAR designs to guide the safer development of CAR T-cell therapies.
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