Purpose: HER-2/neu is overexpressed in breast cancer and is the source of immunogenic peptides. CD4+ T-helper peptides for HER-2/neu are being evaluated in vaccine trials. The addition of Ii-Key, a four-amino-acid LRMK modification, increases vaccine potency when compared with unmodified class II epitopes. We present the results of the first human phase I trial of the Ii-Key hybrid HER-2/neu peptide (AE37) vaccine in disease-free, node-negative breast cancer patients. Patients and Methods: The dose escalation trial included five dose groups, to determine safety and optimal dose of the hybrid peptide (100 μg, 500 μg, 1,000 μg) and granulocyte-macrophage colony-stimulating factor (GM-CSF; range, 0 to 250 μg). In the event of significant local toxicity, GM-CSF (or peptide in absence of GM-CSF) was reduced by 50%. Immunologic response was monitored by delayed-type hypersensitivity and [ 3H]thymidine proliferative assays for both the hybrid AE37 (LRMK-positive HER-2/neu:776-790) and AE36 (unmodified HER-2/neu:776-790). Results: All 15 patients completed the trial with no grade 3 to 5 toxicities. Dose reductions occurred in 47% of patients. In the second group (peptide, 500 μg; GM-CSF, 250 μg), all patients required dose reductions, prompting peptide-only inoculations in the third group. The vaccine induced dose-dependent immunologic responses in vitro and in vivo to AE37, as well as AE36. Conclusion: The hybrid AE37 vaccine seems safe and well tolerated with minimal toxicity if properly dosed. AE37 is capable of eliciting HER-2/neu-specific immune responses, even without the use of an adjuvant. This trial represents the first human experience with the Ii-Key modification, and to our knowledge, AE37 is the first peptide vaccine to show potency in the absence of an immunoadjuvant.