Background: Tumor-selective therapeutic viral vectors are often limited to IT delivery. NG-350A, a blood stable adenoviral vector, selectively replicates in tumors and expresses an agonist anti-CD40 payload. We assessed the impact of IV vs IT delivery during the phase 1 FORTITUDE study (NCT03852511). Methods: NG-350A was dose-escalated in separate IT or IV cohorts of patients (pts) with metastatic/advanced epithelial tumors. Pts received either 1 (Day [D] 1) or 4 IT injections (D1, 8, 15, 22) of up to 1x10¹² viral particles (vp) or 3 IV infusions completed in <9 days (doses of 1x10¹¹ to 6x10¹² vp). Serum samples were taken immediately after dosing (pharmacokinetics [PK]) and during follow-up (PD). Baseline and on-treatment (D15+) tumor biopsies were taken. Results: In total, 25 pts received NG-350A monotherapy, 9 by IT and 16 by IV dosing. Both IV and IT dosing elicited similar anti-drug (virus) antibody responses and limited viral shedding. Peak vp Cmax was variable with IT dosing but dose-related with IV dosing. Vector DNA was detected by qPCR in tumor biopsies after higher IV dose levels (including up to ~7 weeks post dosing) and IT injection. Dose-dependent, sustained and specific increases in inflammatory cytokines related to the expected effects and timing of the anti-CD40 transgene production (IL-12, IL-17A, IL-2, IFNγ) were detected in serum of pts treated at higher IV doses. By contrast, limited evidence of such PD effects was seen with IT dosing. Transgene was not detectable in core needle biopsies; however, a novel serum biomarker assay detected transgene mRNA at D8 or D15 in five pts who received higher IV dose levels. Importantly, both IV and IT dosed NG–350A was well-tolerated, with no treatment-related SAEs occurring in >1 pt and no DLTs at the highest dose levels tested. No evidence of anti-CD40 transgene-related toxicity was seen with IV or IT dosing, with few hepatic AEs and no Grade ≥3 CRS. Follow-up for imaging was limited; however, stable disease was observed in 3/6 pts at IV Dose Level 3. Conclusions: This analysis provides the first clear evidence that an IV-dosed tumor-selective viral vector can drive expression of immuno-stimulatory payloads with functional PD consequences in advanced cancers. The improved PK and PD profile of IV vs IT dosed NG-350A also highlights the benefits of systemic delivery for such vectors. Dose-escalation of NG-350A + pembrolizumab to identify a dose for efficacy assessments continues in the FORTIFY study (NCT05165433). Clinical trial information: NCT03852511.

*One pt had no follow-up beyond D1.