Background: Obe-cel is a CD19-directed autologous CAR T therapy with a fast off-rate binding domain and a 4-1BB-ζ co-stimulatory domain designed to improve persistence and minimize immunotoxicity (Roddie et al. NEJM 2024). Increasing evidence highlights the relevance of monitoring CAR T persistence in the blood for predicting clinical outcomes in pts with hematological malignancies, and this is commonly achieved using flow cytometry (FC) (Fürst et al. Blood 2022; Hamilton et al. Blood Adv 2024; Ploch et al. Cancers 2024). Anti-idiotypes are the gold standard for CAR detection, but are difficult to produce and not always commercially available. Obe-cel's unique CAT19 binder, unlike the FMC63 binder in other CAR T therapies (Smith et al. Antib Ther 2023) poses further considerations for detection. Here, we evaluated several commercially available reagents for measuring obe-cel expansion and persistence.

Methods: Five commercially available CAR binders were evaluated for their ability to detect CAR expression on obe-cel T-cells by FC: G4S linker (Cell Signaling Technology; Netherlands); CD19 CAR Detection Reagent (Miltenyi Biotech; UK); CAR Dextramer® (Immudex; Denmark); Human CD19 Protein (ACROBiosystems; DE, USA; phycoerythrin- and fluorescein isothiocyanate-labelled). Two in-development reagents from BioLegend (UK) and Becton Dickinson (NJ, USA) were also assessed. Binders were tested using CAR T products from four healthy donors, with CAR T expressing the CAT19 receptor as positive controls, compared with the CAT19 anti-idiotype antibody (Ab). Suitable binders were optimized as part of a FC panel including CD3, CD4, CD8, and viability dye FVS780, with surface staining using the specific CAR binder, and tested on samples from nine pts treated with obe-cel in ongoing clinical trials.

The G4S linker binder was also independently validated in a clinical setting at the Stanford Cancer Correlative Science Unit by titrating and staining an infused obe-cel product. The CAR detector was added to a pre-validated 25-parameter T-cell phenotypic/functional FC panel (Cytek Biosciences; CA, USA) and samples from three pts with relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL) infused with commercial obe-cel were analyzed (pre-infusion status: pt 1 with <5% bone marrow [BM] blasts; pt 2 with poor BM sample precluding blast quantification, treated as high burden given extramedullary disease; pt 3 with 95% BM blasts).

Results: Of the binders tested, only the G4S linker identified a percentage of CAR-positive cell populations that was comparable with the CAT19 anti-idiotype, across the four donors. The G4S binder was then tested in peripheral blood (PB) samples using a lyse-no-wash protocol; however, significant autofluorescence was observed, masking the positive CAR signal. To resolve this, red cells were lysed first, followed by washing, then cells were stained with the marking panel. With this optimized method, results with the G4S linker in PB were comparable with the anti-idiotype. A side-by-side analysis of the G4S linker and the CAT19 anti-idiotype Ab was performed on PB samples from nine obe-cel-treated pts. Quantification of cells/µL and percentage CAR-positivity showed strong correlation between the two reagents (p values <0.0001; r values 0.9923 and 0.9980, respectively).

All three R/R B-ALL pts treated with obe-cel in the clinical setting had detectable CARs. Pts 1 and 2 had quantifiable peaks at Day 14 (33.5 and 2,070 cells/µL, respectively). Pt 3 had peak CAR T expansion on Day 21, but too few circulating lymphocytes to accurately measure absolute expansion. In pt 2, robust expansion was primarily driven by CD4+ cells (78.3%) and an effector memory phenotype (CD45RA–/CCR7A–, 92.9% at peak). All three pts had measurable residual disease (MRD)-negative remission by NGS, following obe-cel treatment.

Conclusions:Measuring obe-cel expansion and persistence using FC is feasible with commercially available Abs that directly target regions of the CAR construct, such as the G4S linker. These reagents show high correlation with anti-idiotype Abs and provide a reliable method for tracking CAR expression in pts. Use of the G4S binder enabled tracking of CAR T expansion kinetics and phenotypic profiles in pts with different disease burdens. Reagents based on the CD19 protein, commonly used in other CAR T therapies, are unsuitable for obe-cel due to the unique features of the CAT19 binder, which limits effective detection.

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2025
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