Contributions
Abstract: EP723
Type: E-Poster Presentation
Session title: Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research
Background
Two CD19-directed CAR T-cell products (Tisagenlecleucel/Tisa-cel and Axicabtagene ciloleucel/Axi-cel) have been approved for refractory/relapsed (R/R) diffuse large B cell lymphoma (DLBCL) and primary mediastinal large B cell lymphoma (PMBCL) whereas Brexucabtagene autoleucel (Brexu-cel) has been used for R/R mantle cell lymphoma (MCL). Real-world data on commercial CAR T-cell product expansion kinetics are still limited and homogeneous standardized approaches are required to in vivo quantify CAR T cells.
Aims
To monitor and compare the expansion kinetics of commercial anti-CD19 CAR T-cell products using multiparametric flow cytometry (MFC) and a novel digital PCR method (ddPCR) we have developed.
Methods
We prospectively collected samples from 42 pts, [23 Axi-cel(15 DLBCL and 8 PMBCL), 18 Tisa-cel(18 DLBCL)] and 1 MCL patient receiving Brexu-cel at our Institution between Oct 2019 and Jan 2021. CAR T cells were monitored in peripheral blood (PB) on days 0,4,7,10,14,21,28 and monthly post-infusion by MFC using the CD19 CAR Detection Reagent (Miltenyi). A unique ddPCR primer-probe assay we have developed was used to quantify CAR vectors on genomic DNA extracted from PB, infusion bag-leftovers, bone marrow (BM) samples, tissue biopsies and circulating cell-free DNA (cfDNA).
Results
By MFC, 550 time-points of the 42 pts were analyzed. Median time to maximal expansion (Tmax) was identical for both products (10 days, range 7-28 days). At the point of maximal expansion (Cmax), there was no difference between pts receiving Axi-cel and Tisa-cel, with a mean of 76 cells/ul (range 1-442) vs a mean of 186 cells/ul (range 6-591) (p, ns). At this time point, CAR+ cells were more abundant among CD3+ cells in pts receiving Axi-cel (33.06% vs 15.58% for Tisa-cel, p<0.05). Complete response (CR) at 30 days from infusion (n=18) was associated with significantly more CAR+ cells at day 10 than non-CR (mean 146 vs 18 CAR+ cells/ul, p<0.05) and the magnitude of expansion in the first 30 days was higher in CR vs non-CR pts (mean area under the curve (AUC 0-30) = 1431.2 vs 584.3; p<0.05) irrespective of the product they received. For 16 pts (10 DLBCL, 5 PMBCL and 1 MCL) CAR T copy numbers were evaluated by ddPCR in 122 PB samples already analyzed by MFC. By transgene copy evaluation, similar kinetics of expansion were observed in all pts including the MCL patient receiving Brexu-cel. Consistent with MFC data, the median Tmax was 10 days (range 7-28 days), comparable values of transgene copies per mg of DNA were detected in PB samples from Axi-cel and Tisa-cel treated patients at the Tmax (mean 24x104 copies/ug vs 13x104 copies/ug, respectively) and the mean AUC 0-30 was significantly higher in CR vs non-CR pts (mean 115.9 vs 29.9;p<0,05). Collectively, a significant correlation between all MFC and ddPCR data was observed (r=0.95,p<0,0001 by Pearson Correlation). The ddPCR assay was then used to evaluate CAR T cells in samples for which MFC analysis was not suitable such as infusion bag-leftovers (34x104-755x104 copies/ug,n=13), cryo-preserved BM samples (360-1x104 copies/ug,n=3) tumor biopsies (0-864 copies/ug,n=3) and cfDNA (0-9x104 copies/ug,n=3).
Conclusion
Both MFC and ddPCR, allow the reproducible enumeration of commercial anti-CD19 CAR T-cells with concordant results. The ddPCR assay has the advantage over MFC to allow transgene copy evaluation in samples with limited cellularity and/or cryopreserved. In our cohort Axi-cel and Tisa-cel treated pts have similar expansion kinetics and CAR peak expansion was associated with clinical responses irrespectively of the product used.
Keyword(s): CAR-T, Flow cytometry, Non-Hodgkin's lymphoma, PCR
Abstract: EP723
Type: E-Poster Presentation
Session title: Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research
Background
Two CD19-directed CAR T-cell products (Tisagenlecleucel/Tisa-cel and Axicabtagene ciloleucel/Axi-cel) have been approved for refractory/relapsed (R/R) diffuse large B cell lymphoma (DLBCL) and primary mediastinal large B cell lymphoma (PMBCL) whereas Brexucabtagene autoleucel (Brexu-cel) has been used for R/R mantle cell lymphoma (MCL). Real-world data on commercial CAR T-cell product expansion kinetics are still limited and homogeneous standardized approaches are required to in vivo quantify CAR T cells.
Aims
To monitor and compare the expansion kinetics of commercial anti-CD19 CAR T-cell products using multiparametric flow cytometry (MFC) and a novel digital PCR method (ddPCR) we have developed.
Methods
We prospectively collected samples from 42 pts, [23 Axi-cel(15 DLBCL and 8 PMBCL), 18 Tisa-cel(18 DLBCL)] and 1 MCL patient receiving Brexu-cel at our Institution between Oct 2019 and Jan 2021. CAR T cells were monitored in peripheral blood (PB) on days 0,4,7,10,14,21,28 and monthly post-infusion by MFC using the CD19 CAR Detection Reagent (Miltenyi). A unique ddPCR primer-probe assay we have developed was used to quantify CAR vectors on genomic DNA extracted from PB, infusion bag-leftovers, bone marrow (BM) samples, tissue biopsies and circulating cell-free DNA (cfDNA).
Results
By MFC, 550 time-points of the 42 pts were analyzed. Median time to maximal expansion (Tmax) was identical for both products (10 days, range 7-28 days). At the point of maximal expansion (Cmax), there was no difference between pts receiving Axi-cel and Tisa-cel, with a mean of 76 cells/ul (range 1-442) vs a mean of 186 cells/ul (range 6-591) (p, ns). At this time point, CAR+ cells were more abundant among CD3+ cells in pts receiving Axi-cel (33.06% vs 15.58% for Tisa-cel, p<0.05). Complete response (CR) at 30 days from infusion (n=18) was associated with significantly more CAR+ cells at day 10 than non-CR (mean 146 vs 18 CAR+ cells/ul, p<0.05) and the magnitude of expansion in the first 30 days was higher in CR vs non-CR pts (mean area under the curve (AUC 0-30) = 1431.2 vs 584.3; p<0.05) irrespective of the product they received. For 16 pts (10 DLBCL, 5 PMBCL and 1 MCL) CAR T copy numbers were evaluated by ddPCR in 122 PB samples already analyzed by MFC. By transgene copy evaluation, similar kinetics of expansion were observed in all pts including the MCL patient receiving Brexu-cel. Consistent with MFC data, the median Tmax was 10 days (range 7-28 days), comparable values of transgene copies per mg of DNA were detected in PB samples from Axi-cel and Tisa-cel treated patients at the Tmax (mean 24x104 copies/ug vs 13x104 copies/ug, respectively) and the mean AUC 0-30 was significantly higher in CR vs non-CR pts (mean 115.9 vs 29.9;p<0,05). Collectively, a significant correlation between all MFC and ddPCR data was observed (r=0.95,p<0,0001 by Pearson Correlation). The ddPCR assay was then used to evaluate CAR T cells in samples for which MFC analysis was not suitable such as infusion bag-leftovers (34x104-755x104 copies/ug,n=13), cryo-preserved BM samples (360-1x104 copies/ug,n=3) tumor biopsies (0-864 copies/ug,n=3) and cfDNA (0-9x104 copies/ug,n=3).
Conclusion
Both MFC and ddPCR, allow the reproducible enumeration of commercial anti-CD19 CAR T-cells with concordant results. The ddPCR assay has the advantage over MFC to allow transgene copy evaluation in samples with limited cellularity and/or cryopreserved. In our cohort Axi-cel and Tisa-cel treated pts have similar expansion kinetics and CAR peak expansion was associated with clinical responses irrespectively of the product used.
Keyword(s): CAR-T, Flow cytometry, Non-Hodgkin's lymphoma, PCR