Alpha-lactalbumin (aLA) is a novel tumor antigen normally expressed only in lactating breasts but is detectable in 70% of triple-negative breast cancer (TNBC). Vaccination with aLA protected transgenic mice from developing autochthonous breast cancer and inhibited 4T1 breast tumor growth in syngeneic mice. A Phase I trial of the aLA vaccine was conducted in 3 cohorts: 1a) TNBC patients who completed standard treatment (n=26); 1b) subjects with BRCA1, BRCA2, or PALB2 mutations undergoing prophylactic mastectomies (n=4); and 1c) TNBC patients with residual cancer following pre-operative chemo-immunotherapy receiving post-operative pembrolizumab with or without capecitabine (n=5). The primary objective was to determine the maximum tolerated dose (MTD) and a secondary objective was to assess immune responses to the vaccine. Common Terminology Criteria for Adverse Events (CTCAE) Grade ≥ 2 were considered dose-limiting toxicities (DLTs). Blood was drawn prior to vaccination (Day 0) and afterwards on Days 14, 28, and 56 to assess cellular response by enzyme-linked immunosorbent spot (ELISPOT) assays and humoral response by enzyme-linked immunosorbent assay (ELISA). Injection site reactions ranged from Grade 1 (mild inflammation) to Grade 3 (ulceration). The maximum tolerated dose (MTD) is 10 mcg of aLA antigen and 10 mcg of zymosan adjuvant in Montanide vehicle. 74% of patients met protocol defined immune response by ELISPOT assay.