(M1330-01-02) A Robust Adoptive Cell Therapy Phenotyping Panel for Assessing Cerebrospinal Fluid During and After Neurotoxicity by Flow Cytometry

Purpose: Chimeric antigen receptor T-cells (CAR-T) are an exciting area of therapeutics that are rapidly expanding into broader disease indications with the development of increasingly sophisticated and elegant CAR-T designs. Despite impressive remission rates in many disease indications, a high percentage (5-40%) of treated patients are often plagued with neurotoxicities that can be mild to life threatening in severity. The complete mechanisms are not fully understood, but a hallmark is increased permeability of the blood brain barrier, allowing immune cells including infused CAR-T to potentially accumulate in cerebrospinal fluid (CSF). Understanding which immune cells are accumulating in the CSF could help in the development of newer and safer therapies and mitigation strategies. We have developed a flow cytometry backbone assay for measuring T-Cell, B-Cell, NK cell, monocyte and neutrophil populations in CSF samples with expected low cellularity. Inclusion of an interchangeable CAR-T phenotyping channel is available for drug specific CAR-T detection as well.

Methods: 4mLs of artificial CSF samples spiked with CAR-T, isolated PBMCs, or a mix of CAR-T + PBMCs were created and then immediately transferred into the TransFix/EDTA CSF collection container (Cytomark). The samples were stained by two operators in triplicate at either 1,000 or 10,000 total events per stain tube, which included CD3, CD4, CD8, CD14, CD19, CD45, CD56 and CD66b, and acquired on two unique BD FACS Lyric (IVD) instruments. In addition, the samples were assayed at 1, 2, and 3-days post creation to mimic ex-vivo/in-transit stability. Precision and stability experiments were conducted at both 10,000 and 1,000 total staining events The sample collection was validated to be easily implementable within clinical sites, requiring minimal sample manipulation prior to shipment to testing laboratories.

Results: Our results indicate that each of the populations, (Total CD3+ T-Cells, Cytotoxic T, Helper T, B-Cells, NK-Cells, Monocytes, Granulocytes and CAR-T subsets) are robustly detected with acceptable precision at levels as low as 20-50 events, for up to 2 days post collection in the 10,000 event staining tubes, with a minimum of 786 CD45+ events and maximum of 2409 CD45+ events across donors. For the 1,000 event stain tube, there was unacceptable loss of stability in the monocytes (mean % change of 44.4%) and in the NK cells (mean % change of 50.5%) from Day 1 to Day 2, across all donors tested. Stability met acceptance for the 1,000 staining event tubes for the Cytotoxic, Helper and Total CD3+ populations, inclusive of the total CD3+ CAR-T cell, but not in the CD3+ subsets.

Conclusion: We have validated a CSF specific assay that is stable to 2 days with minimal sample manipulation by clinical sites. This customizable assay can be included in clinical trials to provide investigators a tool for understanding and potentially mitigating neurotoxicity events for future therapies.