CAR T Cells Administered in Combination with Lymphodepletion and PD-1 Inhibition to Patients with Neuroblastoma

Abstract

Targeting disialoganglioside (GD2) on neuroblastoma (NB) with T cells expressing a first-generation chimeric antigen receptor (CAR) was safe, but the cells had poor expansion and long-term persistence. We developed a third-generation GD2-CAR (GD2-CAR3) and hypothesized that GD2-CAR3 T cells (CARTs) would be safe and effective. This phase 1 study enrolled relapsed or refractory NB patients in three cohorts. Cohort 1 received CART alone, cohort 2 received CARTs plus cyclophosphamide and fludarabine (Cy/Flu), and cohort 3 was treated with CARTs, Cy/Flu, and a programmed death-1 (PD-1) inhibitor. Eleven patients were treated with CARTs. The infusions were safe, and no dose-limiting toxicities occurred. CARTs were detectable in cohort 1, but the lymphodepletion induced by Cy/Flu increased circulating levels of the homeostatic cytokine interleukin (IL)-15 (p = 0.003) and increased CART expansion by up to 3 logs (p = 0.03). PD-1 inhibition did not further enhance expansion or persistence. Antitumor responses at 6 weeks were modest. We observed a striking expansion of CD45/CD33/CD11b/CD163⁺ myeloid cells (change from baseline, p = 0.0126) in all patients, which may have contributed to the modest early antitumor responses; the effect of these cells merits further study. Thus, CARTs are safe, and Cy/Flu can further increase their expansion.

Keywords: GD2-CAR; immunotherapy; neuroblastoma.

Figure 1

Flow Chart of Clinical Trial NCT01822652 Black arrow indicates GD2-CAR3 T cells expanded with IL-2 and administered after a freezing step. Yellow arrows indicate conditioning with cyclophosphamide 500 mg/m²/dose on days −4, −3, and −2 and fludarabine 30 mg/m²/dose on days −4 and −3. Green arrows indicate GD2-CAR3 T cells expanded with IL-7/15 and administered without a freezing step. Blue arrows indicate PD-1 inhibitor, pembrolizumab, given at 2 mg/kg/dose on days −1 and 21. Response to therapy evaluation was completed with 3D imaging (bone marrow testing when applicable) on week 6.

Figure 2

Expansion and Persistence of GD2-CAR3 T Cells after Adoptive Transfer Peripheral blood evaluation of patients infused with GD2-CAR3 T cells at indicated time points. (A) Absolute lymphocyte count (Ly/μL). (B) Expansion of GD2-CAR3 T cells in patients in cohort 1 (black) or cohorts 2 and 3 (purple) prior to week 6 response to therapy evaluation by real-time PCR; transgene copy numbers per milliliter. Lines represent median. (C) Long-term persistence of GD2-CAR3 T cells in patients treated in cohort 1 (black) or cohorts 2 and 3 (purple). (D) Peak IL-15 levels (pg/mL) in patients detected on the day of CAR T cell infusions with (cohorts 2 and 3) and without lymphodepletion (cohort 1) measured by Luminex assay. Mean with SD. (E) Peripheral blood IL-15 levels (pg/mL) in patients in cohort 1 (black) or cohorts 2 and 3 (purple). Line represents median. (F) Association of area under the curve (AUC) of GD2-CAR3 T cells’ expansion until response to therapy evaluation at week 6 and peak IL-15 levels. (A and D) Patients in cohort 1 are represented in black, cohort 2 in green, and cohort 3 in blue; each patient per cohort is represented by a different shape. ***p < 0.001, t test.

Figure 3

Change of Peripheral Blood Myeloid Subsets after GD2-CAR3 T Cell Infusion Peripheral blood myeloid subset composition was analyzed with multiparametric flow cytometry at indicated time points. (A) Kinetics of CD45/CD33⁺, HLA-DR/CD15⁻ peripheral blood myeloid-derived suppressor cell-like (MDSC-like) subset as percentage of peripheral blood monocytes. (B) Absolute number of MDSC-like myeloid subset in peripheral blood per microliter. (C) Appearance of CD45/CD33/CD11b/CD163⁺ myeloid subset in a representative patient after GD2-CAR3 T cell infusion. (D) Kinetics of CD45/CD33/CD11b/CD163⁺ peripheral blood myeloid cell subset as percentage of peripheral blood monocytes. (E) Absolute number of CD45/CD33/CD11b/CD163⁺ peripheral blood myeloid cell subset per microliter. Patients in cohort 1 are represented by black shapes, cohort 2 by green shapes, and cohort 3 by blue shapes. Each shape and color combination represents a patient. *p < 0.05, t test.

Figure 4

Outcome of Patients Treated with GD2-CAR3 T Cells (A) Waterfall plots of the difference in Curie scores before and after therapy. (B) Kaplan-Meier survival curves of all patients. (C) Separated survival curves for patients in cohort 1 (black) and cohorts 2 and 3 (both shown in pink).