Oncolytic DNX-2401 virotherapy plus pembrolizumab in recurrent glioblastoma: a phase 1/2 trial

Abstract

Immune-mediated anti-tumoral responses, elicited by oncolytic viruses and augmented with checkpoint inhibition, may be an effective treatment approach for glioblastoma. Here in this multicenter phase 1/2 study we evaluated the combination of intratumoral delivery of oncolytic virus DNX-2401 followed by intravenous anti-PD-1 antibody pembrolizumab in recurrent glioblastoma, first in a dose-escalation and then in a dose-expansion phase, in 49 patients. The primary endpoints were overall safety and objective response rate. The primary safety endpoint was met, whereas the primary efficacy endpoint was not met. There were no dose-limiting toxicities, and full dose combined treatment was well tolerated. The objective response rate was 10.4% (90% confidence interval (CI) 4.2-20.7%), which was not statistically greater than the prespecified control rate of 5%. The secondary endpoint of overall survival at 12 months was 52.7% (95% CI 40.1-69.2%), which was statistically greater than the prespecified control rate of 20%. Median overall survival was 12.5 months (10.7-13.5 months). Objective responses led to longer survival (hazard ratio 0.20, 95% CI 0.05-0.87). A total of 56.2% (95% CI 41.1-70.5%) of patients had a clinical benefit defined as stable disease or better. Three patients completed treatment with durable responses and remain alive at 45, 48 and 60 months. Exploratory mutational, gene-expression and immunophenotypic analyses revealed that the balance between immune cell infiltration and expression of checkpoint inhibitors may potentially inform on response to treatment and mechanisms of resistance. Overall, the combination of intratumoral DNX-2401 followed by pembrolizumab was safe with notable survival benefit in select patients (ClinicalTrials.gov registration: NCT02798406).

Fig. 1. Survival and response to treatment.

a, Patient flow in trial. b, Waterfall plot that displays the maximal change in tumor size for all patients who received full-dose DNX-2401 treatment (n = 42). Bars represent the maximal tumor change from baseline on the basis of contrast-enhanced MRI. Bars are colored according to responses classified according to mRANO criteria. c, Survival for each patient by DNX-2401 dose. The bar colors show the response to treatment according to the mRANO criteria. Arrows indicate that the patient remains alive. d, Overall survival for the intent to treat population. Crosses denote censored data.

Fig. 2. Complete responses to DNX-2401 and pembrolizumab.

a, Axial T1-weighted MR (top row) and FLAIR images (bottom row) obtained at baseline, 3 months, 6 months, 12 months and 38 months after infusion of DNX-2401 for one complete responder. b, The change of tumor size over time in each patient with a complete response. Dotted black line represents no change relative to baseline. Dashed red line represents the threshold for response according to the mRANO criteria. Both patients showed response to treatment at 3 months after DNX-2401 infusion, with complete response by 15–18 months.

Extended Data Fig. 1. Longitudinal volumetric changes in perilesional edema.

a, changes in perilesional FLAIR signal hyperintensity at each study MRI time point for individual patients. Patients are colored according to their mRANO responses. b, changes in perilesional FLAIR signal hyperintensity at each study MRI time point relative to baseline MRI. Data presented are mean +/− 95%CI. Patients are stratified by whether they developed clinically relevant edema requiring medical treatment. n = 9 independent patients developing edema requiring medical treatmet. * denotes statistical significance by two tailed unpaired t-test, 36 weeks P = 0.004 and 44 weeks P = 0.02.

Extended Data Fig. 2. Survival of patients.

a, Kaplan-Meier survival curve with patients stratified by response to treatment by mRANO. Crosses denote censored data. Dashes represent median overall survival for each group. b, Kaplan-Meier survival curve and associated 95%CI using landmark 6-months method with patients stratified according to objective response status. Crosses denote censored data. Dashed lines represent median overall survival for each group. Patients with objective responses (complete or partial response by mRANO) had statistically longer survival than those without objective responses (stable or progressive disease by mRANO, HR 0.20, 95%CI 0.05 to 0.87, log rank test P = 0.02).

Extended Data Fig. 3. mRNA expression prior to treatment.

a, Heatmap showing three subtypes of glioblastoma microenvironment in samples from this trial on the basis of enrichment for immune cell types using partition around medoids clustering. Scores for functional orientation markers, signature scores, and expression of immune checkpoints and biomarkers are overlayed on the heatmap. b, Distribution of expression of PD-1(PDCD-1) and PD-L1 (CD274) across different tumor size comparisons. n = 19 independent patients top row, n = 31 independent patients middle row, n = 38 independent patients bottom row. Central bars indicate medians, the box defines the upper and lower quartiles of the distribution, and whiskers define the 1.5× IQR. Statistical comparisons were performed using Welch’s two-sided t-test. c, Stacked barplot showing the rate of clinical benefit (stable disease or objective response) stratified by microenvironment subtypes in this study (left) and previously published cohort examining adjuvant PD-1 monotherapy in recurrent glioblastoma (right). Dashed box represents the proportion of objective responses by mRANO criteria in this study. d, Distribution of overall survival of patients in this trial (left) and previously published trial (right) stratified by immune microenivronment subtypes. e, Heatmap showing three subtypes of glioblastoma microenvironment on previously published cohort examining adjuvant PD-1 monotherapy in recurrent glioblastoma.

Extended Data Fig. 4. Immune cell types and signature markers.

Distribution of scores for immune cell types (top row), immune checkpoint genes and biomarkers (second row), functional orientation markers (third row) and signature scores (last row). Shown are boxplots for each TME subtype. Dots denote individual values. N = 38 independent patient samples in total. Central bars indicate medians, the box defines the upper and lower quartiles of the distribution, and whiskers define the 1.5× IQR. All comparisons have p < 0.05 unless specifically indicated with ns (p > 0.05).

Extended Data Fig. 5. Comparison of gene mRNA expression at disease progression to baseline.

a, Volcano plot showing the expression changes of individual genes after treatment compared to baseline. Genes that are differentially expressed (absolute Log₂FC greater than 1 and P-value using two-sided Welch’s t-test less than 0.05) are colored red and labelled. Blue dots indicate genes with absolute Log₂FC greater than 1. Green dots indicate genes with P value < 0.05. b, Barplot showing results of functional enrichment analysis of differentially expressed genes in (a). P-values generated using two-sided limma t-test. c, Forest plot summarizing differences in gene expression profiles and signatures for 9 patients who did not have objective response to treatment (shown in black) with expression data at both disease progression and baseline. Dot denotes average and bars represent 95%CI. Blue dot denotes one additional patient with objective partial response to treatment. FC denotes fold change.

Extended Data Fig. 6. Immunophenotyping before and after treatment.

a, Representative immunohistochemical stains for microglia (Iba1), macrophages (CD68), and lymphocytes (CD3, CD4, CD8) for patients with low, moderate, and high microenvironment subtypes in this trial. Scale bar = 100um. Experiments were completed once. b, Heatmap showing relative density (cells per mm²) of markers stratified by microenvironment subtype as determined by PAM clustering of immune cell type scores from gene expression deconvolution c, Forest plot summarizing fold differences in density of markers for 6 patients who did not have objective response to treatment (shown in black) with expression data at both disease progression and baseline. Dot denotes average and bars represent 95%CI. Blue dot denotes one additional patient with objective partial response to treatment. d, Representative immunofluorescence images of samples with different microenvironment subtypes as determined by gene expression analysis. Scale bar = 100um. Experiments were completed once. e, Immunofluoresence images of patient with objective response before (left) and after (right) treatment. Scale bar = 150um. Experiments were completed once.

Extended Data Fig. 7. Anti-adenovirus antibody levels.

a, Immunoglobulin G (IgG) levels before and after treatment across dose cohorts in the trial. b, IgG levels across dose cohorts in thet trial over time. Data shown are mean +/− SD. c-d, Shown are Kaplan-Meier survival curves with associated 95%CI. Patients stratified according to level of response in Anti-Ad5 IgG levels after treatment compared to baseline levels. + denote censored data. c, Shows survival using 4-fold threshold. d, Shows survival using 10-fold threshold.