Characterization and T-Cell Repertoire of MB-CART2019.1 (Zamtocabtagene autoleucel) - Data from the Phase I Trial in Patients with Relapsed or Refractory B-Cell Non-Hodgkin Lymphoma

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 (C_max, AUC_0-28) 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 C_max and AUC_0-28 were detected in CR vs non-CR pts (mean C_max: CR 1092.5 cells/µL [460.1-3147.0] vs non-CR 111.0 cells/µL [3.9-458.0]; p=0.0058) (mean AUC_0-28: 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 (T_N), central memory, (T_CM), effector memory (T_EM) and effector T-cells (T_EFF) among CD4+ and CD8+ fractions, whereas T_CM 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+ T_N and T_CM (T_N + T_CM: 63.9% ± 6.5% in CR vs 53.8% ± 27.5% in non-CR) in the LP and CAR+CD4+ T_CM (89.8% ± 7.8% vs 75.2% ± 25.3%) and CAR+CD8+ T_CM (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 C_max displayed high T_CM proportions, more pronounced in CR pts at C_max, (mean T_CM 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 T_CM proportion and low expression of exhaustion markers. Increased proliferative ability in the DP, higher C_max, as well as maintaining higher levels of T_CM 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 T_CM is a prominent characteristic of MB-CART2019.1 DP which may be important for improved persistence and durable clinical response.

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.