Pb2495: quantification and immunophenotypic characterisation of car t-cells by flow cytometry and correlation with clinical outcomes for patients with large b cell lymphoma

Topic: 25. Gene therapy, cellular immunotherapy and vaccination - Clinical Background: Commercial autologous CD19 directed chimeric antigen receptor (CAR) T-cell therapy is an established therapy for relapsed/refractory large B-cell lymphomas. CAR T-cell expansion and persistence in vivo correlates with clinical response in some clinical trials. To date, measuring CAR T-cell expansion lacks consistency between labs and has been challenging to routinely perform outside of clinical trials. The value of measuring expansion with a readily available rapid assay at the centre of delivery is unclear. Aims: To evaluate and quantify by flow cytometry the in vivo CAR T-cell expansion (including CD4+ CAR T-cells and CD8+ CAR T-cells) and expression of PD1, TIGIT and CD69 on CAR T-cells in patients treated with tisagenlecleucel (tisa-cel) and axicabtagene ciloleucel (axi-cel) and correlate this with clinical outcomes. Methods: Patients with relapsed/refractory large B-cell lymphoma treated with axi-cel or tisa-cel from August 2021 to December 2022 at our institution had peripheral blood collected between D7 to D14 post CAR-T cell infusion. Samples were analysed with an eight colour flow cytometry panel containing antibodies to FMC63, CD3, CD4, CD8, CD45, CD69, PD1 and TIGIT. The anti-FMC63 antibody specifically recognises the antigen-recognition domain of FMC63 found on commercial CD19 directed CARs and allows CAR T-cells to be separated from normal T-cell populations. Data was collected via BD FACS Canto II cytometer and analysed using FlowJo (BD Life Sciences). Statistical analysis was via GraphPad PRISM. Results: 20 patients were identified as suitable for analysis. The mean absolute CAR T-cell count was 36 x106/µL of which 21.3 x106/µL were CD4+ and 16 x106/µL were CD8+ suggesting a predominance of CD4+ CAR T-cells over CD8+. CAR T-cells represented on average 4.5% of total cells, with CD4+ being 2.4% and CD8+ being 2.1%. The median CAR T count for axi-cel was 49x106/µL with tisa-cel 37.9 x106/µL suggesting this assay is suitable for both products. CAR T-cell expansion of ≥2% of total leukocytes was associated with a trend to higher progression free survival (PFS) with a sustained complete remission rate of 80%. TIGIT or CD69 expression levels on either CD4+ or CD8+ CAR T-cells were not associated with a significant difference in response rates. Higher expression of PD1 on CD8+ CAR T-cells trended towards improved PFS.Summary/Conclusion: Quantification and immunophenotypic characterisation of CAR T-cells is accessible and feasible in a treatment centre outside of a clinical trial setting. Early data suggests an improved overall response rate with increased CAR T-cell absolute number and percentage of leukocytes. There is a trend to improved PFS with higher expression of PD1 on CD8+ CAR T-cells. These findings should be explored further in a larger cohort of patients. Keywords: Immunotherapy, Diffuse large cell lymphoma, CAR-T