SUMMARY Anti-HER2/neu antibody therapy is reported to mediate tumor regression by interrupting oncogenic signals and/or inducing FcR-mediated cytotoxicity. Here, we demonstrate that the mechanisms of tumor regression by this therapy also require the adaptive immune response. Activation of innate immunity and T cells, initiated by antibody treatment, was necessary. Intriguingly, the addition of chemotherapeutic drugs, while capable of enhancing the reduction of tumor burden, could abrogate antibody-initiated immunity leading to decreased resistance to re-challenge or earlier relapse. Increased influx of both innate and adaptive immune cells into the tumor microenvironment by a selected immunotherapy further enhanced subsequent antibody-induced immunity, leading to increased tumor eradication and resistance to re-challenge. Therefore, this study proposes a model and strategy for anti-HER2/neu antibody-mediated tumor clearance.

Epidermal growth factor receptor (EGFR) over-signaling leads to more aggressive tumor growth. The antitumor effect of Cetuximab, an anti-EGFR antibody, depends on oncogenic-signal blockade leading to tumor cell apoptosis and antibody dependent cell-mediated cytotoxicity (ADCC). However, whether adaptive immunity plays a role in Cetuximab-mediated tumor inhibition is unclear, as current xenograft models lack adaptive immunity and human-EGFR-dependent mouse tumor cell lines are unavailable. Using a newly developed xenograft model with reconstituted immune cells, we demonstrate that the Cetuximab effect becomes more pronounced and reduces the EGFR(+) human tumor burden when adaptive immunity is present. To further study this in a mouse tumor model, we created a novel EGFR(+) mouse tumor cell line and demonstrated that Cetuximab-induced tumor regression depends on both innate and adaptive immunity components, including CD8(+) T cells, MyD88, and FcγR. To test whether strong innate signals inside tumor tissues amplifies the Cetuximab-mediated therapeutic effect, Cetuximab was conjugated to CpG. This conjugate is more potent than Cetuximab alone for complete tumor regression and resistance to tumor rechallenge. Furthermore, Cetuximab-CpG conjugates can activate tumor-reactive T cells for tumor regression by increasing dendritic cell (DC) cross-presentation. Therefore, this study establishes new models to evaluate immune responses induced by antibody-based treatment, defines molecular mechanisms, and provides new tumor-regression strategies.

Immune tolerance to tumor-associated self-antigens poses a major challenge in the ability to mount an effective cancer vaccine response. To overcome immune tolerance to HER-2, we formulated DNA vaccines that express both human HER-2 and heterologous rat Neu sequences in separate plasmids or as single hybrid constructs that encode HER-2/Neu fusion proteins. Candidate vaccines were tested in Her-2 transgenic (Tg) mice of BALB/c (BALB), BALB/c × C57BL/6 F1 (F1), or C57BL/6 (B6) background, which exhibit decreasing immune responsiveness to HER-2. Analysis of various cocktails or hybrid vaccines defined a requirement for particular combination of HER/2/Neu sequences to effectively prime immune effector cells in HER-2 Tg mice. In B6 HER-2 Tg mice, rejection of HER-2–positive tumors protected mice from HER-2–negative tumors, providing evidence of epitope spreading. Our findings show that a strategy of combining heterologous antigen with self-antigens could produce a potent DNA vaccine that may be applicable to other tumor-associated antigens.