Background: D2C7 immunotoxin (D2C7-IT) is a dual-specific recombinant immunotoxin comprising an EGFR wild-type and mutant-specific (EGFRvIII) monoclonal antibody (Ab) fragment and a genetically engineered form of the Pseudomonas exotoxin. When injected directly into the tumor by convection enhanced delivery (CED), immunotoxins induce both direct tumor killing and secondary immune responses by activation of CD4+ and CD8+ T-cells. Tumor-associated macrophages (TAMs) are the most prominent glioma-infiltrating immune cells and constitute up to 40% of the tumor mass. Upon binding of D2C7-IT to EGFR and cellular internalization, the Pseudomonas exotoxin moiety of the D2C7-IT kills residual GBM cells, upregulates proinflammatory CD40, and induces pattern recognition receptor pathway transcriptome expression. This potentially creates a proinflammatory glioma microenvironment where TAM activation may be further stimulated by sequential CED of 2141-V11, an Fc engineered anti-human CD40 agonist antibody developed at Rockefeller University. We are conducting a first in human trial of the combination of D2C7-IT + 2141-V11 administered via CED in recurrent MG patients. Methods: Eligibility includes adult patients with recurrence of a solitary supratentorial WHO grade 3 or 4 MG; ≥ 4 weeks after chemotherapy, bevacizumab or study drug; adequate organ function; and KPS ≥ 70%. Cohorts of 3 patients are treated with increasing levels of 2141-V11 to determine the maximum tolerated dose (MTD) of the compound administered intratumorally in conjunction with D2C7-IT. Dose escalation and de-escalation are managed using a modified Bayesian optimal interval (BOIN) design to identify the MTD. Intratumoral administration of D2C7-IT via CED (4612 ng/mL over 72 hours) is followed by a 7-hour infusion of 2141-V11, both infused at 0.5 mL/hr. 2141-V11 is dose-escalated to determine the MTD when combined with D2C7-IT. Four dose levels (DL) are planned: #1: 0.70 mg; #2: 2.0 mg; #3: 7.0 mg; #4: 21.0 mg. Results: As of February 7, 2022, three patients were treated at DL1 and DL2, and two patients at DL3. No DLTs have been observed, and all eight patients remain alive and in observation on study after 7.0, 6.5, 6.0, 4.4, 2.8, 2.4, 0.9 and 0.5 months. Early signs of tumor response have been observed, with one patient at DL1 and 2 patients at DL2 without radiographic evidence of active tumor. Grade 2 or higher AEs due to D2C7-IT and/or 2141-V11 include: headache (grade 3, n = 1; grade 2, n = 2); paresthesia (grade 3, n = 1; grade 2, n = 1); dysphasia (grade 3, n = 1); pyramidal tract disorder (grade 3, n = 1; grade 2, n = 1); and depressed level of consciousness (grade 2, n = 1). Enrollment is ongoing. Conclusions: Intratumoral administration of D2C7-IT + 2141-V11 via CED is safe, and encouraging efficacy results have been observed. Clinical trial information: NCT04547777.