Single intravenous administration of oncolytic adenovirus TILT-123 results in systemic tumor transduction and immune response in patients with advanced solid tumors

Abstract

Background: A limitation of approved oncolytic viruses is their requirement for intratumoral (i.t.) injection. TILT-123 (igrelimogene litadenorepvec, Ad5/3-E2F-D24-hTNFα-IRES-hIL-2) is a chimeric oncolytic adenovirus suitable for intravenous (i.v.) delivery due to its capsid modification and dual selectivity devices. It is armed with tumor necrosis alpha and interleukin-2 for promoting T-cell activation and lymphocyte trafficking to tumors, thereby enhancing the antitumor immune response. Here, we present the findings after a single i.v. administration of TILT-123 in three phase I dose escalation clinical trials.

Methods: Patients with advanced solid tumors initially received a single i.v. dose of TILT-123 ranging from 3 × 10⁹ to 4 × 10¹² viral particles (VP). Blood was collected at baseline, 1, 16, and 192 h (7 days) post-treatment for bioavailability and serum analysis. Tumor biopsies were collected prior to treatment and 7 days post-treatment for analysis of viral presence and immunological effects. Patients did not receive any other cancer therapies during this period.

Results: Across all three trials (TUNIMO, TUNINTIL, and PROTA), 52 total patients were treated with i.v. TILT-123. Overall, TILT-123 was found to be well-tolerated, with no dose-limiting toxicities observed. Post-treatment tumor biopsies showed expression of viral genes, presence of TILT-123 adenovirus proteins or DNA, and changes in immune cell infiltration from baseline. Increased virus dose did not lead to increased virus detection in tumors. Median overall survival was longer in patients with confirmed presence of TILT-123 in post-treatment biopsies (280 versus 190 days, p = 0.0405).

Conclusion: TILT-123 demonstrated safety and significant intratumoral immunomodulation following a single i.v. administration, warranting further investigation.

Trial registrations: TUNIMO-NCT04695327. Registered 4 January 2021, https://clinicaltrials.gov/study/NCT04695327 . TUNINTIL-NCT04217473. Registered 19 December 2019, https://clinicaltrials.gov/study/NCT04217473 . PROTA-NCT05271318. Registered 4 February 2022, https://clinicaltrials.gov/study/NCT05271318 .

Keywords: Adenovirus; Immunotherapy; Intravenous Delivery; Oncolytic Virus; Solid Tumors; TILT-123.

Fig. 1

Overview of sample collection analysis across trials. A Sample analysis methods for tumor biopsies and blood as well as collection timepoints. B Summary of individual trials and number of patients with samples available for analysis. C Total number of analyzed patient tumor biopsies by cancer type

Fig. 2

Detection of TILT-123 in blood and serum protein changes post-treatment. A Percentage of patients with detectable levels of TILT-123 detected by quantitative PCR (qPCR) in whole blood 1 h, 16 h, and 192 h post i.v. TILT-123 administration grouped by dose received. B Cross-trial results of circulating TILT-123 levels in blood 1-h post-injection, grouped by dose received. Data presented as mean ± SEM. Comparisons were evaluated using Kruskal–Wallis test with Dunn’s multiple comparisons test where *p < 0.05 and **p < 0.01. C Percentage of patients with detectable levels of TILT-123 in blood 16 h post-injection by cancer type. Volcano plots illustrating changes in serum proteins 16 D and 192 h E post TILT-123 administration compared to baseline, per individual trial TUNIMO, TUNINTIL and PROTA. Grey dots indicate genes which do not pass threshold values of p < 0.05. Statistical differences between groups were assessed using Mann–Whitney U tests, where non-significant results were p > 0.05. A full list of differentially expressed proteins 192 h post-injection (TUNIMO) can be found in Supplementary Fig. 1B. F Spearman correlation analysis of normalized protein expression values and TILT-123 dose (TUNIMO & TUNINTIL). Only significant results are displayed (p < 0.05)

Fig. 3

Detection of viral proteins and transcriptomic changes in biopsies. A Percentage of patients positive for viral proteins (E1A or hexon) in day 8 biopsies detected by IHC, per trial and by dose received. B IHC image of day 8 spleen biopsy from an epithelial ovarian cancer patient 30210. Red arrows highlighting speckled brown diaminobenzidine (DAB) stain indicating the presence of hexon. C Normalized gene counts of viral mRNA (hexon, fiber, and E1A) in baseline and day 8 biopsies detected using Nanostring nCounter® gene expression analysis. Nanostring analysis was performed on 14 tumor biopsies, including 9 biopsies from patients in the TUNIMO trial and 5 biopsies from patients in the PROTA trial. Data presented as mean ± SEM. D Volcano plot of differentially expressed genes analyzed by Nanostring nCounter® in day 8 biopsies compared to baseline biopsies, grouped by immunological function. Grey dots indicate genes which do not pass threshold values of –log₁₀(p-value) > 1.3 and log₂(FC) > 0.5 or < -0.5. Statistical differences between groups were assessed using either t-tests or Mann–Whitney U tests, where non-significant results were p > 0.05. E Pathway enrichment analysis of differentially expressed genes (DEGs) in day 8 biopsies

Fig. 4

Immune cell marker expression in tumor biopsies. A Percentage of cells in baseline and day 8 tumors expressing various immune cell markers, per trial. TUNIMO – CD8 + , CD4 + , CD56 + , CD20 + and PD-L1 + expressing cells in the tumor. TUNINTIL – CD8 + , CD4 + , Foxp3 + , and PD-L1 + expressing cells in the tumor. PROTA – CD8 + , CD4 + , CD56 + , and PD-L1 + expressing cells in the tumor. Data presented as mean ± SEM. Differences between groups were compared using Mann–Whitney U tests where *p < 0.05. B Multiplex immunofluorescence of baseline and day 8 liver biopsies from epithelial ovarian cancer patient 30103 indicating changes in CD8 + , CD4 + , CD56 + , and PD-L1 + expressing cells after TILT-123 treatment. C Cross-trial comparison of changes in CD8 + , CD8 + PD-1 + , CD4 + , Foxp3 + , PD-L1 + expressing cells grouped by detection of adenoviral proteins in tumors by IHC. Data presented as mean ± SEM. D CD8 + , CD4 + T cell and CD56 + expressing cells in tumor cells of post-treatment biopsies, grouped by biopsy site. Data presented as mean ± SEM

Fig. 5

Analysis of TILT-123 detection and tumor transduction in post-treatment biopsies. A Number of patients per biopsy site grouped by presence of TILT-123 in day 8 biopsies as assessed by qPCR or IHC. Only patients with results available for both assays were included. B Number of patients per biopsy site grouped by signs of tumor transduction. Signs of tumor transduction were assessed using tumor qPCR, IHC or mIF results and only patients with results available for at least two of the assays were included. C Percentage of patients showing signs of tumor transduction per individual trial and cross-trial. D Association of OS and positive TILT-123 detection (n = 8) or negative TILT-123 detection (n = 6) in day 8 biopsies as assessed by qPCR or IHC. Groups were compared using log-rank (Mantel-Cox) test where *p < 0.05