Background: Interleukin-12 (IL-12) results in anti-tumor responses in preclinical models but requires tightly controlled production to achieve safety and elicit immune system activation to realize efficacy. A phase 1 “main study” (NCT02026271) enrolled subjects with Grade III or IV gliomas who at the time of resection received intratumoral administration of a replication-deficient adenovirus expressing IL-12 under control of a transcriptional switch (Ad-RTS-hIL-12, Ad) regulated by veledimex (V), referred to as “Controlled IL-12”. It was anticipated that dexamethasone (dex), a lymphocytotoxic corticosteroid used to control edema, might diminish response to immunotherapies. We report updated findings from a substudy of subjects who were dex-free during the 4 weeks prior to Ad administration. Methods: Multicenter, phase 1 substudy (NCT03679754) that assesses safety and tolerability of Controlled IL-12 by local injection (Day 0, time of resection) of Ad (2 x 10¹¹ viral particles) + V (20 mg PO QD x15 doses, Days 0-14) in subjects that were bevacizumab naïve and not receiving dex 4 weeks prior to Ad. Results: 36 subjects were treated. Of the 36, a majority received low-dose corticosteroids (≤ 20 mg dex total during V) as compared with the main study (75% vs 40%). More subjects in the substudy as compared with the main study had multifocal vs. unifocal disease (39% vs 7%). The safety profile was similar for both. Adverse reactions were mild to moderate and were manageable and reversable upon withholding V. Activation of the switch in both the main study and substudy (V 20 mg; n = 51) resulted in increased mean peak values (Day 0-28) of serum IL-12 (25.8 vs. 20.4 pg/mL) and IFN-g (57.0 vs. 39.5 pg/mL). Initial median overall survival (mOS) (unifocal, ≤ 20 mg dex cumulative, n = 20) was 16.2 (8.9, 18.5) mons (mean follow-up 12.3 mons) (Neuro Oncol 2019; 21 [suppl_6]:vi5). mOS including the impact of dex and key subject characteristics from the two studies (n = 51) will be updated and tumor response data will be provided. Conclusions: Monotherapy with Controlled IL-12 resulted in sustained increase in serum recombinant IL-12 and downstream endogenous IFN-g. There is evidence of immune-mediated anti-tumor effects which is associated with increased mOS as compared with historical controls. Follow up will investigate the adverse impact of dex, as well as the effect of additional subject characteristics (e.g., unifocal vs. multifocal disease) on mOS. Clinical trial information: NCT03679754.