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Chimeric antigen receptor (CAR)-T cell therapy against T cell malignancies faces major challenges including fratricide between CAR-T cells and product contamination from the blasts. Allogeneic CAR-T cells, generated from healthy donor T cells, can provide ready-to-use, blast-free therapeutic products, but their application could be complicated by graft-versus-host disease (GvHD) and host rejection. Here we developed healthy donor-derived, CD7-targeting CAR-T cells (RD13-01) with genetic modifications to resist fratricide, GvHD and allogeneic rejection, as well as to potentiate antitumor function. A phase I clinical trial (NCT04538599) was conducted with twelve patients recruited (eleven with T cell leukemia/lymphoma, and one with CD7-expressing acute myeloid leukemia). All patients achieved pre-set end points and eleven proceeded to efficacy evaluation. No dose-limiting toxicity, GvHD, immune effector cell-associated neurotoxicity or severe cytokine release syndrome (grade ≥ 3) were observed. 28 days post infusion, 81.8% of patients (9/11) showed objective responses and the complete response rate was 63.6% (7/11, including the patient with AML). 3 of the responding patients were bridged to allogeneic hematopoietic stem cell transplantation. With a median follow-up of 10.5 months, 4 patients remained in complete remission. Cytomegalovirus (CMV) and/or Epstein-Barr virus (EBV) reactivation was observed in several patients, and one died from EBV-associated diffuse large B-cell lymphoma (DLBCL). Expansion of CD7-negative normal T cells was detected post infusion. In summary, we present the first report of a Phase I clinical trial using healthy donor-derived CD7-targeting allogeneic CAR-T cells to treat CD7sup+/suphematological malignancies. Our results demonstrated the encouraging safety and efficacy profiles of the RD13-01 allogeneic CAR-T cells for CD7sup+/suptumors.

Comparison of CART19 levels in different groups. A, Levels of CART19 cells in PB were shown as assessed by means of qPCR assay at serial time points before and after infusion of CART19 respectively. B, CAR DNA copies in BM and PB on day 7 to 9 after CART19 infusion were compared. CAR DNA copies in BM (Patient 2, 6, 11, 13) were higher than in PB. C, Paired measurements of CAR DNA copies in BM and PB on day 7 to 9 after CART19 infusion, percentage of leukemia cells after FC chemotherapy shows strong correlations (CAR DNA copies in BM VS in PB, Spearman r=0.956, p

Relapsed leukemia cells analysis by FACS.A, leukemia cells with bright CD19 expression obtained CR with MRD negative after CART19 therapy and relapsed with CD19 + leukemia cells (Patient 1). B, leukemia cells with dim CD19 expression obtained CR with MRD negative after CART19 therapy but relapsed with 2 subsets of CD19 + and - leukemia cells (Patient 2 and 3).

Supplementary Tables including Table 1, Table 2 and Table 3

CRS complication after CART19 therapy. A, the peak serum concentration of IL-6, IFN-γ, IL-10, CRP, D-dimer and ferritin of a representative patient (Patient 15) during CRS at the indicated time-points after CART19 infusion are showed. B, D, Peak serum levels of CRP, ferritin and D-dimer in patients who developed grade 3 CRS (n=6) compared with those without CRS or with 1 or 2 CRS (n=10). Data represented the mean{plus minus}SEM. The Mann-Whitney U test was used for statistical analysis. C, Paired measurements of peak serum IL-6, CRP, ferritin and D-dimer showed strong correlations (IL-6 VS CRP, Spearman r=0.617, p=0.014; IL-6 VS ferritin, Spearman r=0.574, p=0.028; IL-6 VS D-dimer, Spearman r=0.789, p

CART19 identification and quantification

The cumulative incidence of non-relapse mortality are shown.

Supplementary materials

A, A representative diagram of the transduction efficiency evaluation of CART19s 5 days after lentivirus infection. B, Specific target cell lysis by CART19s. T cells or CART19s were co-cultured with K562-RFP cells or CD19-RFP-K562 cells at the indicated E:T ratio for overnight co-culture (24 hours). C, IFN-γ secretion measurements in supernatants in T cells or CART19 cells co-cultured with K562-RFP cells or CD19-RFP-K562 cells at the indicated E:T ratios for 24 hours.

Patient 13 with extramedullary leukemia relapse compromising testis obtained CR after CART19 infusion. A, Ultrasound showed complete elimination of extramedullary relapsed masses involved in testis (red arrows). Images are from before treatment and 90 days after CART19 infusion. B, Pathology showed complete elimination of extramedullary relapsed masses involved in testis. Images are from before treatment and 30 days after CART19 infusion. Left: 10×10, right: 10×40.

Purpose: Patients with relapsed/refractory acute lymphocytic leukemia (R/R ALL) have a poor prognosis. Chimeric antigen receptor–modified T cells against CD19 (CART19) have displayed anti-leukemia activities. However, data from systemic trials in Chinese patients are limited.Experimental Design: T cells transduced with CD19-directed CAR lentiviral vectors were infused in patients with R/R ALL under fludarabine- and cyclophosphamide-based lymphodepletion. The postinfusion responses, toxicities, expansion, and persistence of CART19s in enrolled patients were observed and monitored.Results: We enrolled 15 patients with R/R ALL. The median transduction efficiency of CART19s was 33%. In vitro cytotoxicity assays were conducted and showed prominent antileukemia activities with CART19s. The patients received CART19s infusion at doses of 1.1 × 106/kg to 9.8 × 106/kg. Twelve patients achieved complete remission 1 month after CART19s infusion. CART19s expanded and persisted in peripheral blood and bone marrow. At 150 days, the overall survival rate and leukemia-free survival rate were 65.5% and 37.8%, respectively. The cumulative incidence of relapse and the nonrelapse mortality rate were 54.5% and 7.7%, respectively. Four patients underwent subsequent haploidentical hematopoietic stem cell transplantation. In this trial, 10 patients experienced cytokine release syndrome (CRS). Grade 3 CRS developed in 40% of patients and was associated with a higher disease burden on day −1 and a higher number of previous relapses.Conclusions: This trial demonstrated potent antileukemia activities of CART19s in Chinese patients with R/R ALL. Disease relapse remained the main obstacle. However, patients with a high risk of relapse after CART19s might benefit from subsequent haploidentical hematopoietic stem cell transplantation. Clin Cancer Res; 23(13); 3297–306. ©2016 AACR.