2544 Background: Improving efficacy of cancer vaccines may depend on increasing the efficiency of antigen delivery to dendritic cells and overcoming suppressive cell populations such as regulatory T cells (Treg). Alphavirus vectors can efficiently infect dendritic cells, possess a natural adjuvant effect, and express high levels of protein from heterologous genes. We tested the safety, immunogenicity, and clinical efficacy of immunizations with a novel alphavirus vector encoding the tumor-associated antigen CEA (VRP-CEA(6D), AV×701) in metastatic cancer patients with high levels of Treg. Methods: Cohorts of 3-6 patients with advanced CEA- expressing malignancies who had progressed on prior therapies were enrolled at successive dosage levels (4×107, 1×108, 4×108 IU) of VRP-CEA(6D) given as 4 IM injections every 3 weeks. Dose escalation occurred if DLT occurred in <33% of patients in a given dosage level. Following establishment of safety, an additional cohort of 14 patients received VRP-CEA(6D) at the maximum tolerated dose (MTD). Treg levels and CEA-specific T cell and antibody responses were measured before and after immunization. Results: Patients had colorectal cancer (n=23), appendiceal (1), pancreas (n=1), lung (n=2), and breast (n=1) cancer, a performance status of 80-100%, and had failed a median of 4 prior chemotherapeutic regimens. DLT was not reached and the highest dose tested (4×108 IU) was determined to be the maximal feasible dose. Immunizations were well tolerated with 6 grade 3 events, all related to tumor progression, and no grade 4 events. Patients generally possessed higher blood CD4posCD25highFoxP3pos Treg cells compared to normal donors. CEA-specific T cell and antibody responses following VRP-CEA(6D) vaccination were observed regardless of patients' Treg level. There were two patients with stable disease and one with a CR of a small liver lesion. With a median follow-up of 13 months among surviving patients, 2 of 4 patients without immune response and 9 of 11 patients with immune response are alive. Conclusions: Activation of CEA-specific T cells and functional antibodies is possible using VRP-CEA(6D) despite high Treg levels in this heavily pre-treated patient population. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Alphavax Alphavax Alphavax Alphavax