Background: D2C7-IT is a recombinant immunotoxin comprised of a dual-specific antibody fragment targeting EGFRwt and EGFRvIII and a genetically engineered form of the Pseudomonas exotoxin, PE38-KDEL. We report the results of a phase I trial evaluating D2C7-IT delivered intratumorally by CED. Methods: Eligible patients were adults with recurrent supratentorial WHO grade III or IV MG; solitary tumor; ≥4 weeks after chemotherapy, bevacizumab or study drug; adequate organ function; and KPS>70%. Two patients per dose level (DL) were to enroll in the dose escalation portion (dose range: 40ng/mL to 23,354ng/mL). Results: From May 2015 to May 2018, 43 patients enrolled on study. Observed dose limiting toxicities include: grade 4 seizure (n=1) on DL3, grade 3 confusion and pyramidal tract syndrome (n=1) on DL13, and grade 4 cerebral edema (n=1) and grade 3 dysphasia (n=1) on DL17. Grade 3 or higher adverse events possibly related to D2C7-IT include: seizure (grade 4, n=2; grade 3, n=3), cerebral edema (grade 4, n=1), hydrocephalus (grade 3, n=5), headache (grade 3, n=4), hemiparesis (grade 3, n=4), dysphasia (grade 3, n=3), lymphopenia (grade 3, n=4), thromboembolic event (grade 3, n=3); and one each of grade 3 elevated ALT, urinary tract infection, fall, wound complication, generalized muscle weakness, confusion, encephalopathy, and somnolence. As of February 2020, four patients remain alive, with three patients demonstrating persistent radiographic partial response more than 54, 34 and 28 months after a single infusion of D2C7-IT. Conclusions: Dose level 13 (6,920ng/mL) was selected as the optimal phase II dose. Accrual in a dose expansion phase II trial is ongoing, and we are initiating a combination trial of D2C7-IT with checkpoint inhibitior. Clinical trial information: NCT02303678.