Relapse after adjuvant chemotherapy or high-dose chemotherapy with stem cell transplant for high-risk breast cancer remains high and new strategies that provide additional antitumor effects are needed. This report describes methods to generate highly effective HER2/neu-specific cytotoxic T cells by arming activated T cells with anti-CD3 x anti-HER2/neu bispecific antibody (BsAb). OKT3 and 9184 (anti-HER2) monoclonal antibodies (mAb) were conjugated and used to arm T cells that were subsequently tested in binding, cytotoxicity, and cytokine secretion assays. Armed T cells aggregated and specifically killed HER2/neu(+) breast cancer cells. Cytotoxicity emerged after 6 days of culture, was higher in armed T cells than unarmed T cells at all effector to target ratios (E/T) tested, and increased as the arming dose was increased. At an E/T of 20:1, the mean cytotoxicity of armed activated T cells (ATC) from 10 normal subjects increased by 59 +/- 11% (+/-SD) over that seen in unarmed ATC (p < 0.001) and the mean cytotoxicity of armed ATC from 6 cancer patients increased by 32 +/- 9% above that seen for unarmed ATC (p < 0.0004). After arming, the BsAb persisted on ATC up to 72 h and armed ATC continued to be cytotoxic up to 54 h. The amount of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) secreted was 1699, 922, and 3092 pg/ml/10(6) cells per 24 h, respectively, when armed T cells were exposed to a HER2/neu(+) breast carcinoma cell line. These studies show the feasibility and clinical adaptability of this approach for generating large numbers of anti-HER2-specific, cytotoxic T cells for clinical trials.

Nontoxic approaches are needed to improve overall survival (OS) and progression-free survival (PFS) for high-risk breast cancer. Combination immunotherapy (IT) consisting of activated T cells (ATC), interleukin-2 (IL-2), and CTL (GM-CSF) was given after peripheral blood stem cell transplant (PBSCT). There were no major toxicities and there appear to be improvements in OS and PFS over historical controls. In order to develop specific cytotoxic T lymphocytes (CTL), we combined ATC with the use of bispecific antibody (BiAb). By arming ATC with anti-CD3 × anti-HER2/neu BiAb (HER2BiAb), the approach converts nonspecific ATC into HER2/neu (HER2) specific CTL. ATC remain armed, kill tumor targets for days, and produce cytokines after binding to tumor. Arming ATC with BiAbs may prove to be effective for targeting a variety of tumors with and without high-dose chemotherapy.