Background: Chimeric antigen receptor (CAR) T-cells can induce durable remissions in patients with relapsed or refractory (r/r) aggressive B-cell non-Hodgkin lymphoma (B-NHL). Recent data suggest that the T-cell repertoire and the phenotype of the CAR T-cell product influence expansion and contribute to the probability of response. Samples from 12 patients (pts) treated with non-cryopreserved MB-CART2019.1 (Zamtocabtagene autoleucel) in a Phase I trial (NCT03870945) were analyzed. MB-CART2019.1 is a tandem-CAR T-cell product targeting both CD20 and CD19 antigens and contains a 4-1BB co-stimulatory domain.

Methods: MB-CART2019.1 products were manufactured on the CliniMACS Prodigy® system using a 13-days process with IL-7 and IL-15. Pts with complete response (CR) were defined by negative PET-CT by month 6. Cellular composition, viability, CD4:CD8 ratio, CAR transduction frequency, and expansion were monitored on process days 0, 5 and 12. In vivo CAR T-cell levels (Cmax, AUC0-28) and T-cell immunophenotype of leukapheresis (LP) and drug products (DP) were analyzed by comprehensive flow cytometry using common markers for differentiation (CD45RA, CD45RO, CD62L), activation (CD154, CD25, CD137), exhaustion (Lag-3, PD-1, Tim-3) and proliferative ability (CD27, CD127). Results were re-evaluated by descriptive statistics due to low pts number and correlated with expansion kinetics and/or clinical response by using the Wilcoxon test. Mean ± StDev values or ranges are shown.

Results: Five out of 12 pts achieved CR, with ongoing remission >2 years as per investigator assessment. Significantly higher mean Cmax and AUC0-28 were detected in CR vs non-CR pts (mean Cmax: CR 1092.5 cells/µL [460.1-3147.0] vs non-CR 111.0 cells/µL [3.9-458.0]; p=0.0058) (mean AUC0-28: CR 7901 d*cells/µL [2399.2; 19574.7] vs non-CR 942.0 d*cells/µL [39.3; 3471.6]; p=0.0159), equally detectable in CD4+ and CD8+ fractions (Figure 1).

LPs showed similar CD3+ cell percentages of 39.9% ± 10.9% and CD4:CD8 ratios of 1.5 ± 0.9. CR pts had higher amounts of B-cells in the LP than non-CR pts (6.0 ± 3.9 vs 0.7 ± 1.3). DPs showed a consistent T-cell expansion of 10.5 ± 3.5-fold in CD3+ cells, 9.5 ± 3.2-fold in CAR+CD4+ cells and 12.0 ± 4.5-fold in CAR+CD8+ cells, with no difference between CR and non-CR pts. Independent of the starting material, all DPs had a high CD3+ cell purity (99.3% ± 0.7%) and viability (94.0% ± 3.1%) with a higher proportion of CD4+ cells (CD4:CD8 ratio: 2.65 ± 1.9). Mean transduction frequency was 18.0% (21.0% ± 4.8% in CR vs 15.9% ± 6.7% in non-CR; p=0.19) with mean CD4:CD8 ratio of 4.5 in CAR+ cells (6.0 ± 4.5 in CR vs 3.5 ± 2.1 in non-CR; p=0.19).

LP showed different distributions of naïve (TN), central memory, (TCM), effector memory (TEM) and effector T-cells (TEFF) among CD4+ and CD8+ fractions, whereas TCM was the predominant CAR T-cell population among CD4+ (81.3% ± 20.7%) and CD8+ cells (84.6% ± 13.7%) (Figure 2). CR pts showed a slightly higher proportion of less differentiated CD4+ TN and TCM (TN + TCM: 63.9% ± 6.5% in CR vs 53.8% ± 27.5% in non-CR) in the LP and CAR+CD4+ TCM (89.8% ± 7.8% vs 75.2% ± 25.3%) and CAR+CD8+ TCM (91.3% ± 4.7% vs 79.8% ± 16.3%) in the DP. Immunophenotype of the DP showed generally a low expression of exhaustion markers Lag-3 and PD-1 in CD4+ and CD8+ CAR T-cell fractions. In vivo proliferative ability of CAR+ T-cells as assessed by CD27 and CD127 expression showed a trend for increased expression in CR pts (CAR+CD4+CD127+ in CR vs non-CR [p=0.035] and CAR+CD8+CD27+ [p=0.051]). CAR+ T cell repertoire at Cmax displayed high TCM proportions, more pronounced in CR pts at Cmax, (mean TCM content: 40.58% ± 22.37% in CR vs 26.76% ± 12.48% in non-CR) and maintained at D28 (42.48% ± 24.06% vs 27.97% ± 17.89%) in CD4+ and CD8+ subpopulations.

Conclusion: All 12 MB-CART2019.1 DPs exhibited a consistent and reproducible expansion profile, high CD4:CD8 ratio, high TCM proportion and low expression of exhaustion markers. Increased proliferative ability in the DP, higher Cmax, as well as maintaining higher levels of TCM during invivo expansion were characteristically elevated parameters of pts with CR. Other product characteristics, e.g. CD4:CD8 ratio or number of infused T-cells, were not significantly associated with in vivo performance. The high proportion of TCM is a prominent characteristic of MB-CART2019.1 DP which may be important for improved persistence and durable clinical response.

Figure 1
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Holzer:Miltenyi Biotec: Current Employment. Biedermann:Miltenyi Biotec: Current Employment. Herfort:Miltenyi Biotec: Current Employment. Friedrichs:Miltenyi Biomedicine: Current Employment. Holtkamp:Miltenyi Biomedicine: Current Employment. Hanssens:Miltenyi Biomedicine: Current Employment. Assenmacher:Miltenyi Biotec: Current Employment. Bethke:Miltenyi Biotec: Current Employment. Schneider:Lentigen Technology Inc., A Miltenyi Company: Current Employment, Patents & Royalties. Miltenyi:Miltenyi Biomedicine: Current Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Miltenyi Biotec: Current Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Lentigen Technology Inc., A Miltenyi Company: Current Employment, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Overstijns:Miltenyi Biomedicine: Current Employment, Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec: Current Employment, Membership on an entity's Board of Directors or advisory committees. Schmid:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kite: Research Funding; Abbvie: Research Funding. Ayuk:Takeda: Honoraria; Medac: Honoraria; Miltenyi Biomedicine: Honoraria; Gilead: Honoraria; Mallinckrodt/Therakos: Honoraria, Research Funding; Novartis: Honoraria; Celgene/BMS: Honoraria; Janssen: Honoraria. Scheid:Sanofi: Honoraria. Holtick:GSK: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; CLS Behring: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Novartis: Honoraria, Research Funding; Miltenyi Biotech: Honoraria; BMS/Celgene: Honoraria; Kite/Gilead: Honoraria. Gödel:Kite/Gilead: Other: Travel Grant. Borchmann:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotec: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novarts: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Bürger:Miltenyi Biotec: Current Employment.

Author notes

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Asterisk with author names denotes non-ASH members.

© 2022 by The American Society of Hematology
2022
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