(T1130-02-06) Biodistribution Analysis of GPC-3 CAR-T Cells in NSG Mice by a Universal ddPCR Assay to Support an IND-Enabling Toxicity Study

Purpose: The purpose of this study was to evaluate the biodistribution and tumor-targeting ability of GPC3 CAR-T cells in an IND-enabling pre-clinical toxicity study. Safety and efficacy were outside of the scope of this study.

Methods: A qualified universal droplet digital PCR (ddPCR) assay was designed to quantify the CAR transgene at the region of a conserved lentiviral gene in mouse blood and tissues. The assay platform of ddPCR was selected for its advantage of high precision, low interference, and absolute quantification without the use of a standard curve. This universal design can be applied across multiple autologous CAR-T programs. An IND-enabling toxicity study was conducted in Hep3B (GPC-3 expressing hepatocellular carcinoma) xenograft tumor-bearing female NSG mice following a single dose administration of GPC3 CAR-T cells by intravenous (IV) route in three doses (1, 3, 6 million CAR-T cells). The biodistribution patterns and levels of GPC3 CAR-T transgene in peripheral blood, normal tissues and tumor tissue were evaluated by the single plex universal ddPCR assay.

Results: For blood biodistribution, CAR transgene levels were quantified on Days 9/10 and Days 44/45 post-infusion. A dose-dependent increase was revealed on Days 9/10 (group average of 1M: 0 copies/μg DNA, 3M: 762 copies/μg DNA, 6M: 3234 copies/μg DNA), but not on Days 44/45 (group average of 1M: 40902/μg DNA, 3M: 61253/μg DNA, 6M: 52870 copies/μg DNA). Overall, a substantial expansion of the CAR-T cells was observed in the blood on Days 44/45 (up to an 80.4-fold increase) compared to Days 9/10. Tissue biodistribution results demonstrated that variable quantities of the CAR transgene were detected in all analyzed normal tissues (kidney, liver, spleen) and tumor tissue with similar observations as those in the blood. On Days 44/45, substantial increments of transgene level (up to a 208.3-fold increase), were present in tissues compared to Days 9/10. The dose-dependent effect was also observed in all tissues on Days 9/10, but only in the kidney on Days 44/45. Among all the analyzed tissues on Days 9/10, the transgene level was the highest in the tumor followed by the spleen, demonstrating the tumor-targeting profile of the GPC3 CAR-T cells.

Conclusion: The universal ddPCR assay has been successfully developed and implemented to enable determination of GPC3 CAR-T cell biodistribution and tumor-targeting ability in a hepatocarcinoma xenograft mouse model to support IND filing. Overall, a substantial in vivo expansion of the CAR-T cells in the blood and tissues was observed on Days 44/45 compared to Days 9/10, and a dose-dependent increase was detected on Days 9/10.

Acknowledgements: This study was sponsored by AstraZeneca. All the authors are employees and shareholders of AstraZeneca when the study was conducted.