Phase I Trial of Autologous CAR T Cells Targeting NKG2D Ligands in Patients with AML/MDS and Multiple Myeloma

Abstract

NKG2D ligands are widely expressed in solid and hematologic malignancies but absent or poorly expressed on healthy tissues. We conducted a phase I dose-escalation study to evaluate the safety and feasibility of a single infusion of NKG2D-chimeric antigen receptor (CAR) T cells, without lymphodepleting conditioning in subjects with acute myeloid leukemia/myelodysplastic syndrome or relapsed/refractory multiple myeloma. Autologous T cells were transfected with a γ-retroviral vector encoding a CAR fusing human NKG2D with the CD3ζ signaling domain. Four dose levels (1 × 10⁶-3 × 10⁷ total viable T cells) were evaluated. Twelve subjects were infused [7 acute myeloid leukemia (AML) and 5 multiple myeloma]. NKG2D-CAR products demonstrated a median 75% vector-driven NKG2D expression on CD3⁺ T cells. No dose-limiting toxicities, cytokine release syndrome, or CAR T cell-related neurotoxicity was observed. No significant autoimmune reactions were noted, and none of the ≥ grade 3 adverse events were attributable to NKG2D-CAR T cells. At the single injection of low cell doses used in this trial, no objective tumor responses were observed. However, hematologic parameters transiently improved in one subject with AML at the highest dose, and cases of disease stability without further therapy or on subsequent treatments were noted. At 24 hours, the cytokine RANTES increased a median of 1.9-fold among all subjects and 5.8-fold among six AML patients. Consistent with preclinical studies, NKG2D-CAR T cell-expansion and persistence were limited. Manufactured NKG2D-CAR T cells exhibited functional activity against autologous tumor cells in vitro, but modifications to enhance CAR T-cell expansion and target density may be needed to boost clinical activity.

Figure 1:. NKG2D-CAR concept and trial design.

A) Full-length NKG2D with preserved extracellular (EC), transmembrane (TM), and cytoplasmic domain (CYTO) were fused to the cytoplasmic domain of the CD3ζ chain to create the NKG2D-CAR and cloned into the SFG vector. B) In T cells, recognition of NKG2D ligands by native NKG2D provides co-stimulation via Dap10 signaling but relies on primary T-cell activation through the TCR. In NKG2D-CAR T cells, the CAR directly activates NKG2D-CAR T cells through binding of MICA, MICB, or the UL-16 binding proteins (ULBP) 1–6 on tumor cells, independent of the TCR, and receives a costimulatory signal through the preserved transmembrane interaction with Dap10. C) Trial schema indicates a 3-week washout period from prior therapy, 9-day manufacturing process, infusion of freshly manufactured T cells according to dose level, DLT evaluation at 21 days, and response evaluation at 28 days after infusion.

Figure 2:. Patient Outcomes.

A) Swimmer’s plot indicating survival and time of subsequent therapies. B) Trajectory of peripheral blood counts in Patient 12 (AML) after NKG2D-CAR T-cell infusion.

Figure 3:. Detection and bioactivity of NKG2D-CAR T cells in peripheral blood.

A) NKG2D-CAR DNA at timepoints indicated on the horizontal axis as measured by Q-PCR. B) Absolute lymphocyte counts at time of infusion. Color scheme for patients is identical in panels A and B. C) Monitoring of absolute numbers of NKG2D⁺ CD8⁺ and CD4⁺ T cells using flow cytometry (n=12, Mean±SEM). D) RANTES detected (n=11, Mean±SEM).

Figure 4:. NKG2D-ligand expression and soluble ligands.

A) Surface NKG2D ligand detection in AML blasts (7 AML patients) and MM cells (3 MM patients) in bone marrow aspirates (BMA) at baseline measured by flow cytometry (SFI>1 indicates ligand expression exceeding isotype). AML blasts: live/CD45^dim cells expressing patient-specific AML markers including CD117, CD123, CD33, and HLA-DR. MM cells: live/CD138⁺CD38⁺ cells. B) Detection of soluble MICA and MICB in patient plasma at baseline using ELISA. Plasma of Patient 3 (MM) was separately spiked with soluble MICA and MICB as an internal positive control. ND: not detectable.

Figure 5:. Functional activity of NKG2D-CAR T cells against autologous tumor.

NKG2D-CAR T cells or mock-treated autologous T cells were cultured at a 1:1 ratio with autologous patient PBMCs containing AML blasts, patient bone marrow aspirate involved by MM, ligand-negative cell lines (P815), or healthy allogeneic donor PBMCs. IFNγ in the supernatants was measured by ELISA (**p≤0.01, ***p≤0.001, ****p≤0.0001). T cells were incubated with an NKG2D-blocking monoclonal antibody or isotype control antibody prior to addition of target cells as indicated.