Background: Monotherapy with intratumoral Ad-RTS-hIL-12 (Ad), a gene therapeutic conditionally expressing IL-12 under the transcriptional control of oral veledimex (“Controlled IL-12”), was shown in a phase 1 study (NCT02026271) to elicit a new and sustained intra-tumoral infiltration of T cells with co-expression of PD-1. We report updated findings following completion of enrollment (with follow-up ongoing) for a phase 1 substudy (NCT03636477) evaluating safety and tolerability of local, Controlled IL-12 in combination with nivolumab (nivo) in adults with recurrent glioblastoma (rGBM). Methods: Multicenter, open label, dose-escalation phase 1 trial to evaluate safety and tolerability of local, Controlled IL-12 with nivo in adult subjects with rGBM. Ad was administered by single intratumoral injection (2 x 10¹¹ viral particles, Day 0 at time of resection) plus V (10 or 20 mg) PO QD x 15 with nivo (1 or 3 mg/kg) IV on Days -7, 15, then Q2W. Results: 21 subjects were treated (Cohort 1: V 10 mg, nivo 1 mg/kg, n = 3; Cohort 2: V 10 mg, nivo 3 mg/kg, n = 3; and Cohort 3: V 20 mg, nivo 3 mg/kg, n = 3 + 12 expansion). Safety data were similar to Ad+V monotherapy. Adverse reactions during follow-on nivo dosing were consistent with anti-PD-1 labeling, manageable, and generally reversible with no synergistic toxicities. Focusing on the 20mg V cohort (recommended phase 2 dose), serum IL-12 mean ± SEM (screening, 0.4 ± 0.1 pg/mL; Day 0, 0.6 ± 0.1 pg/mL), increased after Ad+V to 8.7 ± 3.3 pg/mL on Day 3. Similarly, serum IFN-g levels did not increase due to nivo alone (screening, 0 ± 0 pg/mL; Day 0, 0 ± 0 pg/mL), increasing after Ad+V to 6.2 ± 2.3 pg/mL on Day 7. Additionally, nivo alone did not significantly increase circulating T cells (CD3⁺ CD8⁺%) (paired differences comparison, Day 0 to screening) 3.1%, p =0.13, whereas Ad+V significantly increased peripheral T cells (Day 28 - Day 0) 3.6%, p =0.02. Pseudoprogression followed by a decrease in size (SPD) has been shown as evidenced by serial MRIs in a subgroup of subjects. Preliminary overall survival findings will be presented. Conclusions: Controlled IL-12 with PD-1 inhibition is a rational combination with initial data consistent with immune-mediated effects, a favorable safety profile, and early evidence of anti-tumor effects. An additional phase 2 study combining Controlled IL-12 with cemiplimab-rwlc in adults with rGBM is ongoing. Clinical trial information: NCT03636477.