Third-generation anti-CD19 CAR T cells for relapsed/refractory chronic lymphocytic leukemia: a phase 1/2 study

Abstract

Third-generation chimeric antigen receptor T cells (CARTs) for relapsed or refractory (r/r) chronic lymphocytic leukemia (CLL) may improve efficacy compared to second-generation CARTs due to their enhanced CAR design. We performed the first phase 1/2 investigator-initiated trial evaluating escalating doses of third-generation CARTs (HD-CAR-1) targeting CD19 in patients with r/r CLL and B-cell lymphoma. CLL eligibility criteria were failure to two therapy lines including at least one pathway inhibitor and/or allogeneic hematopoietic cell transplantation. Nine heavily pretreated patients received HD-CAR-1 at dose levels ranging from 1 × 10⁶ to 200 × 10⁶ CART/m². In-house HD-CAR-1 manufacturing was successful for all patients. While neurotoxicity was absent, one case of grade 3 cytokine release syndrome was observed. By day 90, six patients (67%) attained a CR, five of these (83%) with undetectable MRD. With a median follow-up of 27 months, 2-year PFS and OS were 30% and 69%, respectively. HD-CAR-1 products of responders contained significantly more CD4 + T cells compared to non-responders. In non-responders, a strong enrichment of effector memory-like CD8 + T cells with high expression of CD39 and/or CD197 was observed. HD-CAR-1 demonstrated encouraging efficacy and exceptionally low treatment-specific toxicity, presenting new treatment options for patients with r/r CLL. Trial registration: #NCT03676504.

Fig. 1. Absolute B-cell and T-cell counts in the peripheral blood (PB).

A Absolute B-cell count in cells/μl. B Absolute T-cell count in cells/μl. PB of patients was assessed with flow cytometry directly before and up to 890 days after CART administration. Normal ranges of absolute values are displayed according to [39].

Fig. 2. Expansion of HD-CAR-1 CARTs in the peripheral blood (PB).

Rapid expansion of CARTs was observed in eight of nine patients. CART expansion in PB was measured by single‐copy gene duplex quantitative PCR (SCG‐DP‐PCR) as described [35].

Fig. 3. Outcome of patients after treatment with HD-CAR-1.

A Progression-free survival (PFS) of all patients achieving a complete remission (CR) after CART administration vs. non-CR patients. B Overall survival (OS) and PFS of all patients after CART treatment. C Swimmer plots of all patients after CART administration treated at dose level 5 or higher. : CART therapy; : progressive disease; : stable disease; : partial remission; : MRD-positive CR; : MRD-negative CR; : death; : 2nd allogeneic hematopoietic cell transplantation; : radiation therapy; A acalabrutinib, P pirtobrutinib, V venetoclax, Z zanubrutinib.

Fig. 4. Cellular composition of HD‐CAR‐1 CART products of responders (n = 4) vs. non-responders (n = 3).

A HD-CAR-1 CART products of seven CLL patients (#1-7) were analyzed with spectral flow cytometry using a 36-marker panel and computational analysis (see methods). B The downsampled subset of cells from all seven CART products is presented with uniform manifold approximation and projection (UMAP) visualization. Clusters are labeled depending on surface marker expression and displayed in different colors. C Frequencies of CD4+ (left) and CD8+ (right) T cells within the CART products of responders (R) vs. non-responders (NR) are displayed as bloxplots. Significance levels were determined by a two-sided Welch’s t-test. CD8+ D and CD4+ G T-cell subsets extracted from all seven CART products are presented with UMAP visualization and clusters are annotated based on surface marker expression and displayed in different colors. Differential compositions between R and NR of CD8+ E and CD4+ H T-cell subsets are displayed as density plots. Differential frequencies between R and NR of specific CD8+ F and CD4+ I T-cell subtypes are presented as boxplots of log2 fold-changes. Higher abundance in R is indicated as positive log2 fold change and negative log2 fold changes visualize higher frequencies in NR. CM central memory, EM effector memory, NK natural killer, NKT natural killer T cells, ns not significant.