Background: Checkpoint blockade has become a major modality in the treatment of metastatic melanoma (MM). However, durable remission rates remain low. CD40 activation on antigen presenting cells (APCs) initiates their ability to prime and activate CD8⁺ T-cells through upregulation of co-stimulatory molecules (ie. CD80, CD86) as well as expression of effector cytokines. Furthermore, CD40 activation cause macrophages to develop a tumoricidal phenotype and increase MHC I expression on tumor cells. Direct IT immune modulation utilizes the tumor as a “vaccine site” to generate a tumor specific immune response. We hypothesize that IT injection of a CD40 Agonist (A), will “immunize” patients (Pts) against melanoma neoantigens through “licensing” of APCs for tumor specific T-cell priming and activation. In mouse models, we have shown that IT administration of the recombinant adenovirus encoding CD40L induces CD8⁺ T-cell-mediated activity against B16 melanoma and, importantly, also augmented the activity of anti-PD-1. Methods: This phase I/II trial (NCT02706353) evaluates the safety, efficacy, and immunological impact of IT administration of APX005M (CD40 A mAb) in combination with pembrolizumab in pts with MM. The phase I escalation is an accelerated 3+3 design. Pts will receive IT APX005M every 3 weeks for a total of 4 doses. Image guidance will allow for injection of visceral, nodal, and soft tissue metastases. The single-arm phase 2 expansion will evaluate the overall response rate (ORR) 12 weeks after treatment initiation. Key inclusion criteria: confirmed, measurable, metastatic cutaneous or mucosal melanoma; and at least 2 injectable lesions. Key exclusion criteria: prior immunotherapy, uveal melanoma, or active autoimmune disease. 26 pts will have 75% power to detect an improvement from a null ORR of 33% to 55%, using a one group chi-square test and assuming a one-sided α–level of 5%. Immune analysis will be performed on pre and on-treatment tumor/liquid biopsies both in injected and non-injected tumors. Dose level 3 of escalation phase has completed with 5 pts enrolled. Clinical trial information: NCT02706353