表达 scFv-IL-13Rα2 的嵌合抗原受体修饰 T 细胞经 (89)Zirconium oxine 放射性标记后用于 PET 成像的表征

Chimeric antigen receptor (CAR) T cell therapy is an exciting cell-based cancer immunotherapy. Unfortunately, CAR-T cell therapy is associated with serious toxicities such as cytokine release syndrome (CRS) and neurotoxicity. The mechanism of these serious adverse events (SAEs) and how homing, distribution and retention of CAR-T cells contribute to toxicities is not fully understood. Enabling in vitro methods to allow meaningful, sensitive in vivo biodistribution studies is needed to better understand CAR-T cell disposition and its relationship to both effectiveness and safety of these products.

To determine if radiolabelling of CAR-T cells could support positron emission tomography (PET)-based biodistribution studies, we labeled IL-13R 2 targeting scFv-IL-13R 2-CAR-T cells (CAR-T cells) with 89 Zirconium-oxine ( 89 Zr-oxine) and characterized and compared their product attributes with non-labeled CAR-T cells. The 89 Zr-oxine labeling conditions were optimized for incubation time, temperature, and use of serum for labeling. In addition, T cell subtype characterization and product attributes of radiolabeled CAR-T cells were studied to assess their overall quality including cell viability, proliferation, phenotype markers of T-cell activation and exhaustion, cytolytic activity and release of interferon- upon co-culture with IL-13R 2 expressing glioma cells.

We observed that radiolabeling of CAR-T cells with 89 Zr-oxine is quick, efficient, and radioactivity is retained in the cells for at least 8 days with minimal loss. Also, viability of radiolabeled CAR-T cells and subtypes such as CD4 + , CD8 + and scFV-IL-13R 2 transgene positive T cell population were characterized and found similar to that of unlabeled cells as determined by TUNEL assay, caspase 3/7 enzyme and granzyme B activity assay. Moreover, there were no significant changes in T cell activation (CD24, CD44, CD69 and IFN- ) or T cell exhaustion (PD-1, LAG-3 and TIM3) markers expression between radiolabeled and unlabeled CAR-T cells. In chemotaxis assays, migratory capability of radiolabeled CAR-T cells to IL-13R 2Fc was similar to that of non-labeled cells.

Importantly, radiolabeling has minimal impact on biological product attributes including potency of CAR-T cells towards IL-13R 2 positive tumor cells but not IL-13R 2 negative cells as measured by cytolytic activity and release of IFN- . Thus, IL-13R 2 targeting CAR-T cells radiolabeled with 89 Zr-oxine retain critical product attributes and suggest 89 Zr-oxine radiolabeling of CAR-T cells may facilitate biodistribution and tissue trafficking studies in vivo using PET.