This study included MM patients before their first anti-BCMA CAR therapy who had received their most recent dose of Teclistamab within 14 days to 1 year prior to inclusion. Peripheral blood samples were collected in EDTA tubes between April 2024 and March 2025 as part of routine care blood counts and were subsequently utilized for MFC analyses within the study. Additional clinical data, like cumulative dose of Teclistamab, body weight, and blood counts, were retrieved from electronic treatment records.
Sample preparation followed an established protocol with 2 tubes according to the fluorescence minus one (FMO) concept: Around 1 000 000 white blood cells each were transferred into two tubes labeled (1) FMO control and (2) BCMA-CAR detection. Subsequently, 1 µL of BCMA-CAR detection reagent (see Supplemental Table S1 for details) was added to tube number 2. This tube was incubated for 15 min at room temperature (RT) in the dark, and cells were washed once with phosphate-buffered saline (Gibco™ PBS, pH 7,4, ThermoFischer, Waltham, MA, USA, Cat# 10010031). The remaining antibodies (CD3, Biotin, and CD45) were then added to both tubes, and samples were incubated again for 15 min at RT in the dark. Next, 2 mL of BD Pharm Lyse™ (1:10 dilution with distilled water; BD Biosciences, San Jose, CA, USA, Cat# 555899) was added to each tube, and the samples were incubated for an additional 10 min at RT in the dark. Cells were then washed twice with PBS. All stained samples were stored at 4 °C until flow cytometric analysis, which took place on the same day as blood drawing.
All samples were measured centrally using a BD FACSCanto™ II flow cytometer and BD FACSDiva™ software (both Becton Dickinson, Franklin Lakes, NJ, USA). For analysis, Kaluza Analysis 2.1 software (Beckman Coulter, Brea, CA, USA) was used. Tube 1, as a negative control, guided the individual gates, which were then identically applied to Tube 2 (see Fig.1). After exclusion of debris and doublets by forward and sideward scatter, CD45+ Leukocytes were identified. Subsequently, all lymphocytes (CD45+SSClow), T-lymphocytes (CD45+CD3+SSClow), as well as anti-BCMA+ T-lymphocytes were identified. To ensure comparability between individual patients, the threshold between anti-BCMA-positive and -negative was set at a fixed fluorescence intensity of 3 000 following review of all negative controls. Even though no BCMA-CAR detection reagent was added, some events may still be found within the BCMA+ T-lymphocyte gate in tube 1. The percentage of BCMA+CD3+ cells among all CD3+ cells within this negative control was later subtracted from the percentage of BCMA+CD3+ cells in the second tube, giving the true percentage of BCMA+ T-cells. To ensure reliability, the population of BCMA+ T-Cells has to encompass at least 20 events.
Fig. 1: Gating strategy.
A Tube 1 is used as a fluorescence minus one control (lacking BCMAa-CARb detection reagent) and gates are set accordingly. Notably, even though no BCMA-CAR detection reagent was added, some events may still be found within the BCMA+ T-lymphocyte gate in tube 1 (upper right panel). B Gates from Tube 1 are identically applied to Tube 2, which includes the BCMA-CAR detection reagent. The example depicted belongs to a patient 27 days after the last Teclistamab injection. The majority of T-lymphocytes are still anti-BCMA+ (false positive). aBCMA: B-cell maturation antigen. bCAR: chimeric antigen receptor.
To validate the assay, we performed negative controls with samples from healthy volunteers and MM patients who had never received BCMA-directed therapy (Supplemental Fig. S3). Positive controls were performed with MM patients recently treated with a BCMA-directed CAR-therapy and who had never been exposed to Teclistamab (Supplemental Fig. S4).
