Depletion of human regulatory T cells.

Regulatory T cells (Treg) have become increasingly relevant in the study of human disease including cancer. Treg cells have been shown to inhibit anti-tumor immune responses, and elevated Treg levels have been associated with certain types of cancer. Similarly, depletion of Tregs by various methods can also enhance anti-tumor immune responses. We have found a prevalence of Treg in cancer patients when compared to normal volunteers. In addition, we have shown that the depletion of Treg using the IL-2 fusion protein denileukin diftitox decreased Treg function and increased antigen-specific T cell response to a cancer vaccine. These results indicate the potential for combining Treg depletion with anti-cancer vaccines to enhance tumor antigen-specific immune responses and the need to explore the dose and schedule of Treg depletion strategies in optimizing vaccine efforts.

X-linked inhibitor of apoptosis protein inhibits apoptosis in inflammatory breast cancer cells with acquired resistance to an ErbB1/2 tyrosine kinase inhibitor.

Inflammatory breast cancer (IBC) is a highly aggressive subtype of breast cancer that is often characterized by ErbB2 overexpression. ErbB2 targeting is clinically relevant using trastuzumab (anti-ErbB2 antibody) and lapatinib (small-molecule ErbB1/2 inhibitor). However, acquired resistance is a common outcome even in IBC patients who show an initial clinical response, which limits the efficacy of these agents. In the present study, using a clonal population of GW583340 (lapatinib analogue, ErbB1/2 inhibitor)-resistant IBC cells, we identified the overexpression of an antiapoptotic protein, X-linked inhibitor of apoptosis protein (XIAP), in acquired resistance to GW583340 in both ErbB2-overexpressing SUM190 and ErbB1-activated SUM149 cell lines derived from primary IBC tumors. A marked decrease in p-ErbB2, p-ErbB1, and downstream signaling was evident in the GW583340-resistant cells (rSUM190 and rSUM149) similar to parental counterparts treated with the drug, suggesting that the primary mechanism of action of GW583340 was not compromised in resistant cells. However, rSUM190 and rSUM149 cells growing in GW583340 had significant XIAP overexpression and resistance to GW583340-mediated apoptosis. Additionally, stable XIAP overexpression using a lentiviral system reversed sensitivity to GW583340 in parental cells. The observed overexpression was identified to be caused by IRES-mediated XIAP translation. XIAP downregulation in rSUM190 and rSUM149 cells using a small-molecule inhibitor (embelin), which abrogates the XIAP/procaspase-9 interaction, resulted in decreased viability, showing that XIAP is required for survival of cells with acquired resistance to GW583340. These studies establish the feasibility of development of an XIAP inhibitor that potentiates apoptosis for use in IBC patients with resistance to ErbB2-targeting agents.

An adenoviral vaccine encoding full-length inactivated human Her2 exhibits potent immunogenicty and enhanced therapeutic efficacy without oncogenicity.

PURPOSE: Overexpression of the breast cancer oncogene HER2 correlates with poor survival. Current HER2-directed therapies confer limited clinical benefits and most patients experience progressive disease. Because refractory tumors remain strongly HER2+, vaccine approaches targeting HER2 have therapeutic potential, but wild type (wt) HER2 cannot safely be delivered in immunogenic viral vectors because it is a potent oncogene. We designed and tested several HER2 vaccines devoid of oncogenic activity to develop a safe vaccine for clinical use. EXPERIMENTAL DESIGN: We created recombinant adenoviral vectors expressing the extracellular domain of HER2 (Ad-HER2-ECD), ECD plus the transmembrane domain (Ad-HER2-ECD-TM), and full-length HER2 inactivated for kinase function (Ad-HER2-ki), and determined their immunogenicity and antitumor effect in wild type (WT) and HER2-tolerant mice. To assess their safety, we compared their effect on the cellular transcriptome, cell proliferation, anchorage-dependent growth, and transformation potential in vivo. RESULTS: Ad-HER2-ki was the most immunogenic vector in WT animals, retained immunogenicity in HER2-transgenic tolerant animals, and showed strong therapeutic efficacy in treatment models. Despite being highly expressed, HER2-ki protein was not phosphorylated and did not produce an oncogenic gene signature in primary human cells. Moreover, in contrast to HER2-wt, cells overexpressing HER2-ki were less proliferative, displayed less anchorage-independent growth, and were not transformed in vivo. CONCLUSIONS: Vaccination with mutationally inactivated, nononcogenic Ad-HER2-ki results in robust polyclonal immune responses to HER2 in tolerant models, which translates into strong and effective antitumor responses in vivo. Ad-HER2-ki is thus a safe and promising vaccine for evaluation in clinical trials.

Synergism from combined immunologic and pharmacologic inhibition of HER2 in vivo.

The monoclonal antibody trastuzumab and the EGFR/HER2 tyrosine kinase inhibitor lapatinib improve the clinical outcome of patients with HER2-overexpressing breast cancer. However, the majority of metastatic cancers will eventually progress, suggesting the need for other therapies. Because HER2 overexpression persists, we hypothesized that the anti-HER2 immune response induced by cancer vaccines would be an effective strategy for treating trastuzumab- and lapatinib-refractory tumors. Furthermore, we hypothesized that the antibody response could synergize with lapatinib to enhance tumor inhibition. We developed a recombinant adenoviral vector expressing a kinase-inactive HER2 (Ad-HER2-ki) to use as a cancer vaccine. Vaccine-induced polyclonal HER2-specific antiserum was analyzed for receptor internalization and signaling effects alone and in combination with lapatinib. Ad-HER2-ki vaccine-induced potent T cell and antibody responses in mice and the vaccine-induced polyclonal HER2-specific antiserum mediated receptor internalization and degradation much more effectively than trastuzumab. Our in vitro studies demonstrated that HER2 vaccine-induced antibodies effectively caused a decrease in HER2 expression, but when combined with lapatinib caused significant inhibition of HER2 signaling, decreased pERK and pAKT levels and reduced breast tumor cell proliferation. In addition, a known mechanism of resistance to lapatinib, induction of survivin, was inhibited. The combination of Ad-HER2-ki plus lapatinib also showed superior antitumor efficacy in vivo. Based on these results, we feel clinical studies using this approach to target HER2-overexpressing breast cancer, including trastuzumab- and lapatinib-resistant tumors is warranted.