Neural stem cell delivery of an oncolytic adenovirus in newly diagnosed malignant glioma: a first-in-human, phase 1, dose-escalation trial

Abstract

Background: Malignant glioma is the most common and lethal primary brain tumour, with dismal survival rates and no effective treatment. We examined the safety and activity of NSC-CRAd-S-pk7, an engineered oncolytic adenovirus delivered by neural stem cells (NSCs), in patients with newly diagnosed high-grade glioma.

Methods: This was a first-in-human, open-label, phase 1, dose-escalation trial done to determine the maximal tolerated dose of NSC-CRAd-S-pk7, following a 3 + 3 design. Patients with newly diagnosed, histologically confirmed, high-grade gliomas (WHO grade III or IV) were recruited. After neurosurgical resection, NSC-CRAd-S-pk7 was injected into the walls of the resection cavity. The first patient cohort received a dose starting at 6·25 × 1010 viral particles administered by 5·00 × 107 NSCs, the second cohort a dose of 1·25 × 1011 viral particles administered by 1·00 × 108 NSCs, and the third cohort a dose of 1·875 × 1011 viral particles administered by 1·50 × 108 NSCs. No further dose escalation was planned. Within 10-14 days, treatment with temozolomide and radiotherapy was initiated. Primary endpoints were safety and toxicity profile and the maximum tolerated dose for a future phase 2 trial. All analyses were done in all patients who were included in the trial and received the study treatment and were not excluded from the study. Recruitment is complete and the trial is finished. The trial is registered with ClinicalTrials.gov, NCT03072134.

Findings: Between April 24, 2017, and Nov 13, 2019, 12 patients with newly diagnosed, malignant gliomas were recruited and included in the safety analysis. Histopathological evaluation identified 11 (92%) of 12 patients with glioblastoma and one (8%) of 12 patients with anaplastic astrocytoma. The median follow-up was 18 months (IQR 14-22). One patient receiving 1·50 × 108 NSCs loading 1·875 × 1011 viral particles developed viral meningitis (grade 3) due to the inadvertent injection of NSC-CRAd-S-pk7 into the lateral ventricle. Otherwise, treatment was safe as no formal dose-limiting toxicity was reached, so 1·50 × 108 NSCs loading 1·875 × 1011 viral particles was recommended as a phase 2 trial dose. There were no treatment-related deaths. The median progression-free survival was 9·1 months (95% CI 8·5-not reached) and median overall survival was 18·4 months (15·7-not reached).

Interpretation: NSC-CRAd-S-pk7 treatment was feasible and safe. Our immunological and histopathological findings support continued investigation of NSC-CRAd-S-pk7 in a phase 2/3 clinical trial.

Funding: US National Institutes of Health.

Conflict of interest statement

Declaration of interests JP reports grants from The Ivy Foundation, during the conduct of this study. CDH reports salary payments from Southern Research, outside the submitted work. RVL reports honoraria from Novocure for advisory roles, EBSCO Publishing and Medlink Neurology for medical editing, ECRI for reviewing medical content, and the American Physician Institute for creating and presenting board review continuing medical education material, outside the submitted work. RS reports non-financial support from CarThera, and personal fees from Celularity, CranioVation, TriAct, Hemispherian, Northwest Biotherapeutics, GT Medical Technologies, Insightec, and ZaiLab, outside the submitted work. DTC, KSA, and MSL have an issued patent that is related to the study (US10238699 and US10709745). KSA was the CSO and Director of TheraBiologics (company in process of being dissolved) during the conduct of this study; she neither has assets nor receives financial benefit from the company. MSL reports grants from the National Institutes of Health, during the conduct of this study. All other authors declare no competing interests.

Copyright © 2021 Elsevier Ltd. All rights reserved.

Figures

FIGURE 1:. Survival outcomes in patients who received NSC-CRAd-S-pk7.

The median progression-free survival in all evaluable patients (n=12) is 9.05 months (95% CI 8.54, NA) (A). The median overall survival in all evaluable patients is 18.4 months (95% CI 15.7, NA) (B). In the subset of patients with high grade gliomas containing an unmethylated MGMT promoter (n=9), the median progression-free survival was 8.8 months (95% CI 6.51, NA) (C), and median overall survival was 18.0 months (95% CI 13.67, NA) (D).

FIGURE 2:. Summary of radiological responses of three patients with newly diagnosed high-grade gliomas after NSC-CRAd-S-pk7.

Magnetic resonance imaging (MRI) scans before neurosurgical procedure and at days 28 and 56 after surgery and NSC-CRAd-S-pk7 injection. Contrast enhancement and peritumoral hyperintensity on fluid-attenuated inversion recovery (FLAIR) images decreased around the resection cavity with time. Patient 1 partially responded to treatment, whereas patients 2 and 3 had stable disease.

FIGURE 3:. Immune activity in response to NSC-CRAd-S-pk7.

The neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), and total lymphocyte count were compared at different time points before surgery/product injection and throughout the treatment course. NLR and MLR spiked 3 days after surgery/product injection in all three dose levels. This peak diminished by day 14, when the number of lymphocytes increased (A). A focused comparison between days 3 and 14 post surgery/product injection showed a significant decrease in NLR (p=0.0027) and MLR (p=0.013) at dose level 2 and significant increase in absolute lymphocyte count in both dose levels 2 (p=0.011) and 3 (p=0.043) (B). Integration of lymphocyte counts into the flow cytometric analysis showed that CD8+ T cells increased in patients who were receiving the third dose level 14 days after surgery/NSC-CRAd-S-pk7 injection (C). Anti-Ad5 neutralizing antibodies were detected in low titers (1:20 dilution of plasma) 14 days after surgery and NSC-CRAd-S-pk7 treatment at the first dose level and within a week at higher dose levels (D). Analysis of circulating cytokine profiles in patients’ serum showed an initial drop in levels of IL8, IL1Ra, IL12p70, IL13, and CCL22 7 days after surgery/product injection. This was followed by an increase in levels up until day 14 for IL8, IL1Ra, IL6, IL13, and IL16; after which the levels of these cytokines plateaued or diminished. Other cytokines, such as IL12p70, CXCL10, CCL17, and CCL22, continued to increase into day 28 (E). Anti-viral and anti-tumoral immunities were evaluated through an ELISpot assay. Anti-viral immunity was determined through the detection of hexon spots, which increased as the dose of NSC-CRAd-S-pk7 increased (F). Dose level 1 (n=3), Dose level 2 (n=3), Dose level 3 (n=6). For complete blood count analysis, Dose level 3 (n=5). Student’s paired t-test was used for (C), p<0.05*, p<0.01**.

FIGURE 4:. Histopathological analysis of tumor samples before and after NSC-CRAd-S-pk7.

Samples for immunohistochemistry were collected from various regions throughout the brain for histopathological evaluation; sampled regions from the brain in the panel are detailed in Table S1, appendix p. 7. Immunohistochemical staining of collected autopsy samples did not detect E1A or hexon at the site of injection (red arrow) or in other tested samples from the resection cavity. In addition, v-myc immortalized in the injected human NSCs could not be detected via nested PCR amplification (A). Tumor samples from patients that underwent two surgical resections were collected before and after NSC-CRAd-S-pk7 injection. Tumor-specific marker staining of survivin and syndecan-1 showed a decrease in expression after treatment (B). Multiplex staining showed an increase in CD8+ T cells after NSC-CRAd-S-pk7 injection, with more cells traveling from the blood vessels to the tumoral region and sparing the peri-tumoral areas (C). These findings were seen across samples from three different patients, as more CD8+ T cells were seen in the tumor zone post injection (D). Quantitative analysis of staining results revealed an increase in CD8+ T cells and PD-1 expression and decreased numbers of CD163+ cells and SOX2+ cells that express survivin after NSC-CRAd-S-pk7 injection (E). Color key for (D) and (E): Green-CD8+, Red-CD163+, Cyan-SOX2+, Magenta-PD-1+, White-Survivin, Blue-DAPI.