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Clinical Trial
. 2025 Apr 8;9(7):1644-1657.
doi: 10.1182/bloodadvances.2024015157.

Allogeneic off-the-shelf CAR T-cell therapy for relapsed or refractory B-cell malignancies

Sanam Shahid  1 , Susan E Prockop  2 , Georgia C Flynn  1 , Audrey Mauguen  3 , Charlie O White  3 , Jennifer Bieler  1 , Devin McAvoy  1 , Kinga Hosszu  1 , Maria I Cancio  1 , Ann A Jakubowski  4 , Andromachi Scaradavou  1 , Jaap Jan Boelens  1 , Craig S Sauter  5 , Miguel-Angel Perales  4 , Sergio A Giralt  4 , Clare Taylor  6 , Jagrutiben Chaudhari  6 , Xiuyan Wang  6 , Isabelle Rivière  6 , Michel Sadelain  6 , Renier J Brentjens  7 , Nancy A Kernan  1 , Richard J O'Reilly  1 , Kevin J Curran  1
Affiliations
Clinical Trial

Allogeneic off-the-shelf CAR T-cell therapy for relapsed or refractory B-cell malignancies

Sanam Shahid et al. Blood Adv. .

Abstract

Despite clinical benefit with the use of chimeric antigen receptor (CAR) T cells, the need to manufacture patient-specific products limits its clinical utility. To overcome this barrier, we developed an allogeneic "off-the-shelf" CAR T-cell product using Epstein-Barr virus (EBV)-specific T cells (EBV-VSTs) genetically modified with a CD19-specific CAR (19-28z). Patients with relapsed/refractory (R/R) B-cell malignancies were stratified into 3 treatment cohorts: cohort 1 (n = 8; disease recurrence after allogeneic or autologous hematopoietic cell transplantation [HCT]), cohort 2 (n = 6; consolidative therapy after autologous HCT), or cohort 3 (n = 2; consolidative therapy after allogeneic HCT). The primary objective of this trial was to determine the safety of multiple CAR EBV-VST infusions. Most patients (n = 12/16) received multiple doses (overall median, 2.5 [range, 1-3]) with 3 × 106 T cells per kg determined to be the optimal dose enabling multiple treatments per manufactured cell line. Severe cytokine release syndrome or neurotoxicity did not occur after infusion, and no dose-limiting toxicity was observed in the trial. Median follow-up was 48 months (range, 4-135) with 4 deaths due to disease progression. Overall survival of all patients was 81% at 12 months and 75% at 36 months. Postinfusion expansion and persistence were limited, and CAR EBV-VSTs demonstrated a unique T-cell phenotype compared with autologous 19-28z CAR T cells. Our study demonstrates the feasibility and safety of an allogeneic "off-the-shelf" CAR EBV-VST product with favorable outcomes for patients with CD19+ R/R B-cell malignancies. This trial was registered at www.ClinicalTrials.gov as #NCT01430390.

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Conflict of interest statement

Conflict-of-interest disclosure: K.J.C. has received research support from Atara Biotherapeutics, Novartis, Celgene, and Cellectis, and has consulted and participated in advisory boards for Novartis. S.E.P. has received support for the conduct of clinical trials through Boston Children’s Hospital from AlloVir, Atara Biotherapeutics, and Jasper; is the inventor of intellectual property related to development of third-party viral-specific T-cell program, with all rights assigned to Memorial Sloan Kettering Cancer Center; and reports honoraria, consulting, or participation in advisory board for Pierre Fabre, Regeneron, Cellevolve, Vor, and Ensomo, DSMB, Stanford University, and New York Blood Center. J.J.B. has consulted for Merck, Sanofi, Sobi, and SmartImmune and received compensation for serving on data monitoring committee DMC (either chair or member) for Advanced Clinical and CTI Clinical Trial Services. N.A.K. holds equity in Amgen, Johnson & Johnson, and Merck. R.B. has licensed intellectual property to and collects royalties from Bristol Myers Squibb (BMS), Caribou, and Sanofi; received research funding from BMS; is a consultant to BMS and Atara Biotherapeutics Inc; and was on the scientific advisory board of Triumvira, Cargo Tx, and CoImmune. R.J.O. received royalties, research support, and consulting from Atara Biotherapeutics. M.S. reports research support from Atara Biotherapeutics and has licensed intellectual property to Juno Therapeutics, Atara Biotherapeutics, Fate Therapeutics, Takeda Pharmaceuticals, Mnemo Therapeutics, and Minerva Biotechnologies. I.R. is a scientific cofounder of Mnemo Therapeutics and reports participation in the advisory board of Center for Commercialization of Cancer.

Figures

None
Graphical abstract
Figure 1.
Figure 1.
Process development for generating 19-28z CAR EBV-VSTs. Cryopreserved EBV-VSTs (cultured for 28-35 days before cryopreservation) are thawed and stimulated weekly with irradiated B-LCL (days 0, +7, +14). T cells are transduced with 19-28z CAR at a cell density of 1 × 106 cells per mL and 0.5 × 106 cells per mL, respectively, on days +16 and +17. Cells are then fed and evaluated on days +18, +21, and +23 and then harvested and frozen on day +24.
Figure 2.
Figure 2.
19-28z CAR EBV-VST clinical trial design. (A) Patients in cohort 1 were treated with 1 to 3 cycles of 19-28z CAR EBV-VSTs after relapse after transplant, and patients in cohorts 2 and 3 were treated with 1 to 3 cycles of 19-28z CAR EBV-VSTs as consolidation with hematopoietic cell transplant. (B) Schema showing patients in each cohort along with their diagnosis and transplant donor type. B-NHL, B-cell non-Hodgkin lymphoma; CLL, chronic lymphocytic leukemia.
Figure 3.
Figure 3.
Long-term outcomes of patients treated with 19-28z CAR EBV-VSTs. (A) OS of patients with B-ALL vs NHL. (B) OS of patients with CAR EBV-VST products from their transplant donor vs a third-party donor. (C) EFS of patients with B-ALL vs NHL. (D) EFS of patients with CAR EBV-VST products from their transplant donor vs a third-party donor. Note: patients with no initial response to treatment are included with event time of 0. NHL, non-Hodgkin lymphoma.
Figure 4.
Figure 4.
T-cell phenotypic analysis of 19-28z CAR EBV-VSTs. (A) Dot plots comparing CD3+ T-cell subsets of paired parental EBV-VSTs with 19-28z CAR EBV-VSTs. (B) Dot plots comparing CD3+ T-cell subsets of 19-28z CAR EBV-VSTs with 19-28z CAR T cells. (C) Dot plots comparing CD3+ CAR+ T-cell subsets of 19-28z CAR EBV-VSTs vs 19-28z CAR T cells. (D) Ring graphs comparing exhaustion marker (PD1, LAG3, ICOS, and CTLA4) expression of CD3+ EBV-VSTs with 19-28z CAR EBV-VSTs. (E) Ring graphs comparing exhaustion marker (PD1, LAG3, ICOS, and CTLA4) expression of CD3+ 19-28z CAR EBV-VSTs with 19-28z CAR T cells. (F) Ring graphs comparing exhaustion marker (PD1, LAG3, ICOS, and CTLA4) expression of CD3+ CAR+ 19-28z CAR EBV-VSTs with 19-28z CAR T cells. (G) Percent expression of individual exhaustion markers (PD1, LAG3, ICOS, and CTLA4) on T-cell subsets of EBV-VSTs vs 19-28z CAR EBV-VSTs. (H) Percent expression of individual exhaustion markers (PD1, LAG3, ICOS, and CTLA4) on T-cell subsets of 19-28z CAR EBV-VSTs vs 19-28z CAR T cells. P values were determined by Mann-Whitney-Wilcoxon tests.
Figure 4.
Figure 4.
T-cell phenotypic analysis of 19-28z CAR EBV-VSTs. (A) Dot plots comparing CD3+ T-cell subsets of paired parental EBV-VSTs with 19-28z CAR EBV-VSTs. (B) Dot plots comparing CD3+ T-cell subsets of 19-28z CAR EBV-VSTs with 19-28z CAR T cells. (C) Dot plots comparing CD3+ CAR+ T-cell subsets of 19-28z CAR EBV-VSTs vs 19-28z CAR T cells. (D) Ring graphs comparing exhaustion marker (PD1, LAG3, ICOS, and CTLA4) expression of CD3+ EBV-VSTs with 19-28z CAR EBV-VSTs. (E) Ring graphs comparing exhaustion marker (PD1, LAG3, ICOS, and CTLA4) expression of CD3+ 19-28z CAR EBV-VSTs with 19-28z CAR T cells. (F) Ring graphs comparing exhaustion marker (PD1, LAG3, ICOS, and CTLA4) expression of CD3+ CAR+ 19-28z CAR EBV-VSTs with 19-28z CAR T cells. (G) Percent expression of individual exhaustion markers (PD1, LAG3, ICOS, and CTLA4) on T-cell subsets of EBV-VSTs vs 19-28z CAR EBV-VSTs. (H) Percent expression of individual exhaustion markers (PD1, LAG3, ICOS, and CTLA4) on T-cell subsets of 19-28z CAR EBV-VSTs vs 19-28z CAR T cells. P values were determined by Mann-Whitney-Wilcoxon tests.
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