Off-the-Shelf CD19-Specific CAR-NKT Cells in Patients with Relapsed or Refractory B-Cell Malignancies

Autologous T cells engineered to express a CD19-specific chimeric antigen receptor (CAR) mediate high rates of complete response (CR) in patients with B-cell malignancies. However, autologous cell therapy products are time- and resource-intensive to manufacture and vary in potency and toxicity. Unlike polymorphic HLA-restricted T cells, monomorphic CD1d-restricted Vα24-invariant natural killer T cells (NKTs) are not alloreactive, and therefore therapeutic NKTs can be generated from allogeneic donors without the risk of graft-versus-host disease.We report interim results from 9 patients treated on a phase 1 dose-escalation trial of allogeneic NKTs engineered to co-express a CD19-specific CAR, IL-15, and shRNA targeting beta-2 microglobulin and CD74 for downregulation of HLA class I and class II molecules, respectively (ANCHOR, NCT00840853). NKTs for this study were isolated from the leukapheresis product of one HLA-unmatched healthy individual, transduced with the CAR construct, expanded ex vivo, and cryopreserved. Patients were enrolled on 3 dose levels (DLs): 1 × 107 (DL 1), 3 × 107 (DL 2), or 1 × 108 (DL 3) CAR-NKT cells/m2, given after lymphodepleting conditioning with cyclophosphamide and fludarabine. Seven patients with relapsed/refractory B-cell non-Hodgkin lymphoma (NHL, cohort A) were enrolled on all 3 DLs, and 2 patients with relapsed B-cell acute lymphoblastic leukemia (ALL, cohort B) were enrolled on DL 1. Adverse events were evaluated per NCI criteria. When accessible, patients underwent core biopsies of an involved site at 2-to-5 weeks post-infusion. Response to therapy was assessed at 4 weeks per Lugano Criteria (NHL) or NCCN guidelines (ALL).The most common adverse effects observed were nausea and grade 3-4 hematologic toxicities related to the lymphodepletion chemotherapy. There were no early adverse events attributable to the cellular product except grade 1 cytokine release syndrome in one patient. Of the 7 NHL patients (6 with DLBLC, 1 with FL), 3 had an initial partial response that evolved into a CR in 2 cases (Figure 1A). One ALL patient achieved a CR with incomplete hematologic recovery and showed no evidence of leukemia by next-generation sequencing at four weeks. We detected in vivo expansion of donor-derived NKT and CAR-NKT cells in the peripheral blood that peaked 1 week post-infusion in patients treated at higher DLs. While CAR-NKTs were not detected in the peripheral blood of the first 3 NHL patients (DL 1) beyond 3 hours post-infusion, they were present in biopsied tumor tissues collected from these patients (Figure 1B, C).In conclusion, initial results from our first-in-human clinical evaluation of allogeneic CAR-NKTs indicate that these cells are well tolerated and can mediate objective responses in relapsed or refractory NHL and ALL patients even at low doses. Thus, CAR-NKTs represent a promising platform for “off-the-shelf” cancer immunotherapy.