Interpreting Immune Reconstitution and Clinical Outcome By Single-Cell Transcriptomic Analysis in T-ALL Patients Undergoing Donor-Derived CD7 CAR-T Immunotherapy

Background Twenty-five percent of T-ALL patients experience relapse after allo-HSCT, which indicates a dismal prognosis and represents a significant unmet clinical need. CD7 is continuously expressed on T-ALL cell surface at higher levels than that on normal T cells and is therefore considered as a viable CAR T therapeutic target. However, the impact of CD7-positive normal T and NK cell clearance on the development, function, and homeostasis of immune system remains to be addressed.

Methods We designed an open label, phase I clinical trial enrolled patients with a pathologically confirmed CD7-positive T-ALL/LBL (ChiCTR2200058969). After a 6-day manufacturing, the patients received donor-derived CD7 CAR T cells at the target dose of 1×10⁵/kg. To minimize CD7 CAR T-cell mediated fratricide, we used IntraBlock technology to prevent CD7 expression on CAR T cells surface. Single cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells (PBMCs) were adopted to profile immune statues changes before and after CD7 CAR T-cell treatment.

Results Seven eligible patients were enrolled between November 2020 and December 2021, and all were followed up until May 1, 2022. Three patients were diagnosed with R/R T-ALL and 4 with R/R T-LBL at relapse. All patients had a history of allo-HSCT prior to enrollment. Patients were heavily pretreated with a median of 4 (3-8) lines of therapies before enrollment.

The rate of CRS and ICANS was 85.7% and 14.3%, respectively. Only one patient had a grade 3 CRS and a serious grade 4 ICANS, resolved by dexamethasone and tocilizumab. Only two patients (28.6%) developed GVHD. Hematologic CAR T cells related adverse events (AE) included leukopenia, thrombocytopenia and neutropenia were observed in 6 patients (85.7%). Infections occurred in 5 patients (71.4%).

On day 28, the patients who had bone marrow involvement achieved minimal residual disease (MRD) negative complete remission (CR, n=1) or CR with incomplete hematologic recovery (CRi, n=2); The patients who had CNS leukemia achieved CR on day 28. Five patients with their EMD encountered CR at day 90. The optimal ORR and CR were 100% and 100%, respectively. The CR/CRi rate was 85.7% on month 3. The median followed-up time was 4 months (range 3-16 months). Two patients died of serious infection and one patient died of brain hemorrhage. Four patients were alive until the cut-off time.

Additionally, CD7-positive T-cells in the peripheral blood (including normal and abnormal T cells) were eliminated in seven patients on day 11 after CD7 CAR T infusion. However, CD7-negative T cells dramatically increased in all patients. In addition, the CD4/CD8 ratio decreased, driven by the increase in the proportion of CD8-positive T cells. The absolute numbers of T/B/NK cells (excluding CAR+ and leukemia T cells) showed a transient decline, and recovered by a low cells number at day 21.

PBMC samples from four patients (two alive and two died of infection) were tested by scRNA-seq. The profiling of immune cells demonstrated that the severe infection after treatment is mainly caused by the loss of monocytes. This kind of monocytes loss is related to the decline of high developmental potential subset of monocytes before treatment. Moreover, the reconstructed T cell populations may have normal or even enhanced immune functions than pre-treatment T cells in all samples, which is supported by the higher expression levels of TCRs, T cell activation pathway, and T cell function pathway in post-treatment samples. The main character of the reconstructed T cell populations is its similarity with T cells in autoimmune diseases. This includes the expansion of GZMH⁺ T cells, decline of CD4⁺ naïve T cells, and activation of autoimmune disease related pathways. We also observed consistently upregulation of S100A8 and S100A9 in leukocyte lineage as a signature of the patients with relapse lymphoma.

Conclusion Among 7 patients with r/r T-ALL enrolled in this trial, donor-derived CD7 CAR T cells exhibited efficient expansion and achieved a high complete remission rate with manageable safety profile. Data from scRNA-seq of PBMCs indicated a fulfilled T lymphocytes immune function by CD7-negativeT cells. The major challenge on prevent post-treatment infection may be the protection from loss of monocytes.

Key words CD7 CAR-T immunotherapy; Immune Reconstitution; Single-cell Transcriptomic Analysis; T-ALL

No relevant conflicts of interest to declare.