Please note that enrollment on this study terminated early due to the end of grant funding.
Following consent, subjects were enrolled onto dose-escalation cohorts. Patients will underwent leukapheresis to harvest Peripheral Blood Mononuclear Cells (PBMCs) for the generation of EGFRvIII CAR T cells prior to beginning RT and concurrent TMZ. T cells were isolated from the patient's PBMCs and transduced to express the CAR. Briefly, PBMCs were stimulated with Muromonab-cluster of differentiation 3 (CD3) (OKT3), an anti-CD3 monoclonal antibody (mAb), and transduced on RetroNectin® coated plates. Transduced cells were expanded in interleukin-2 (IL-2) for 14 days.
Patients then completed standard of care RT and concurrent TMZ. Patients who remained eligible after standard of care radiation and TMZ received up to 3 cycles of TMZ at 50-100 mg/m\^2/day for 21 days of 28 day cycles, which is the standard dose-intensified (DI) TMZ regimen. If the CAR-specific T cells did not meet release criteria, the patient was withdrawn before CAR treatment and replaced.
At least 48 hours after the last dose of DI TMZ, the total dose of EGFRvIII CAR T cells were delivered intravenously. If sufficient CAR-specific T cells could not be generated to meet the targeted assigned dose within the dose-escalation portion of the study, the patient was have been treated at a lower pre-defined dose level using available CAR-specific T cells and replaced in the assigned higher dose. The administered dose would have been the highest defined dose level for which there are sufficient CAR-specific T cells available. Within the expanded cohort, if sufficient CAR-specific T cells could not be generated to meet the MTD dose, all available T cells would be administered.
Following the infusion of EGFRvIII CARs, blood samples for immune monitoring were drawn 1, 5, and 10 days after the infusion, then 1, 3, and 6 months, then yearly until progression (or death or lost to contact). The return visits for immune monitoring at 3 months, 6 months, and yearly coincided with SOC clinic visits. Blood was also taken for Replication Competent Retrovirus (RCR) Polymerase Chain Reaction (PCR) per the Food and Drug Administration (FDA) at 3, 6, and 12 months during SOC clinic visits. Lastly, blood for evaluation of cytokine release syndrome (CRS) was drawn prior to cell infusion, 1 and 4 hours after infusion, and on days 1, 2, 5, 10, and at one month. Measurements for CRS included IL-2, IL-6, Tumor Necrosis Factor alpha (TNFα), interferon (IFN) gamma, Granulocyte-macrophage colony-stimulating factor (GM-CSF), and C-reactive protein (CRP).
Patients returned to clinic one month following the EGFRvIII CAR T cell infusion to be evaluated for cycles of SOC 5-day TMZ at 150-200 mg/m\^2/day for the first 5-day cycle, followed by 200 mg/m\^2/day for 5-days every 28 days per the treating oncologist. This resulted in a \>30 day delay between the last cycle of DI TMZ and the first cycle of 5-day TMZ.
Tumor progression was documented histologically, unless there were clinical contraindications, to exclude inflammatory responses presenting as radiographic or clinical changes, which could indicate a potentially toxic or therapeutic responses and not tumor progression. If tissue was obtained through Duke Brain Tumor Center Biorepository, it was used to confirm tumor progression histologically, and to assess immunologic cell infiltration and EGFRvIII antigen escape at the tumor site. Patients would be eligible for additional adjuvant therapy at the time of tumor progression.
A classical "3+3" study design was planned to estimate the MTD for CAR-specific T cells treatment among patients with newly-diagnosed GBM. Four dose levels were to be considered: #1: 4.5 x 10\^6/kg, #2: 1.5 x 10\^7/kg, #3: 4.5 x 10\^7/kg, and #4: 1.5 x 10\^8/kg. Starting with the lowest dose level, cohorts of 3-6 subjects would be accrued at each dose level. If a patient was lost to follow-up during the first 4 weeks after CAR treatment without experiencing a dose-limiting toxicity (DLT), then the patient was not considered evaluable for the determination of DLT and would be replaced. The MTD was considered to be the highest dose level at which ≤1 of 6 patients experienced DLT during the 4-week observation period after CAR treatment. Only 3-patient cohort was enrolled at the the first dose level due to grant funding ending.
An expanded cohort of 12 patients was planned at the MTD of EGFRvIII CAR T cells, in order to obtain a more precise estimate of the probability of unacceptable toxicity. This cohort would have the cells radiolabeled with 111In to track their distribution. Briefly, CARs would have been counted and re-suspended in phosphate buffered saline (PBS). 4-6x10\^8 cells would be labeled with a total of 500 microCi of 111In. The cells would be washed and mixed with cold CARs to achieve the desired cell dose. The labeled CARs would be infused into the patient through an intravenous catheter within the Ambulatory Bone Marrow Transplant (BMT) Unit. Distribution of 111In-labeled EGFRvIII CARs would be evaluated at 1, 2, and 3 days post-infusion using scintigraphy. There was no enrollment on the expanded cohort due to grant funding ending.
