Prevention of metastases with a Mage-b DNA vaccine in a mouse breast tumor model: potential for breast cancer therapy

Abstract

Anti-tumor vaccines are a relatively non-toxic alternative to conventional chemotherapeutic strategies to control breast cancer. Immunization with tumor-associated antigens (TAAs) triggers anti-tumor cytotoxic T lymphocytes (CTL), which can limit tumor progression. Here we report on the development and effectiveness of a TAA-based DNA vaccine encoding Mage-b1/2, the mouse homologue of the human MAGE-B1/2. As model system, we used immune competent Balb/c mice with syngeneic non-metastatic (64pT) or metastatic (4TO7cg) breast tumors. First, the presence of Mage-btranscripts in the 64pT and 4TO7cg breast tumors and metastases was demonstrated by RT-PCR, Southern blotting, and DNA sequencing. A DNA-based vaccine was developed from transcripts of one of the 64pT tumors, encoding the complete Mage-b1/2 protein, and subsequently tested for its preventive efficacy in both breast tumor models. Mice were immunized two times intramuscularly with the vaccine (pcDNA3.1-Mage-b1/2-V5), the control vector (pcDNA3.1-V5), or saline. Two weeks after the last immunization, the syngeneic 4TO7cg or 64pT tumor cell lines were injected in a mammary fat pad. Mice were monitored during the next 4 weeks for tumor formation, latency and size, and subsequently sacrificed for analysis. While the Mage-b1/2 vaccine had only a minor effect on the latency and growth of primary tumors, a significant and reproducible reduction in the number of 4TO7cg metastases was observed (vaccine versus control vector, p=0.0329; vaccine versus saline, p=0.0128). The observed protective efficacy of the Mage-b DNA vaccine correlated with high levels of vaccine-induced IFNgamma in spleen and lymph nodes upon re-stimulation in vitro. These results demonstrate the potential of TAA-based DNA vaccines in controlling metastatic disease in breast cancer patients.

Mage-b vaccine delivered by recombinant Listeria monocytogenes is highly effective against breast cancer metastases

“…In previous studies, we demonstrated that DNA vaccination with mouse Mage-b in metastatic mouse breast tumour models 4TO7cg and 4T1, highly expressing Mage-b, reduced the number of metastases significantly, although not completely (Sypniewska et al, 2005;Gravekamp, 2007;Gravekamp et al, 2008). In the study presented here, we further enhanced the efficacy of Mage-b vaccine through improved delivery of the vaccine using recombinant Listeria monocytogenes (LM).…”

“…pcDNA3.1-Mage-b/V5 was developed in our laboratory (Sypniewska et al, 2005). Mouse GM-CSF plasmid (CMV1-GM-CSF) was kindly provided by Dr Stephen Johnston (the Center for Innovations in Medicine, the Biodesign Institute at Arizona State University) (Chambers and Johnston, 2003).…”

“…The first fragment located at the N-terminal site is 349 bp (position 3 -352), the second fragment adjacent to the first fragment is also 349 bp (position 311 -660), and the third fragment at the C-terminal site is 379 bp (position 610 -990). The Mage-b fragments were obtained by PCR from plasmid pcDNA3.1-Mage-b/V5, generated in a previous study (Sypniewska et al, 2005). For each construct, a restriction site Xho1 (underlined) was included in the forward primer, and a myc Tag (bold), followed by a stop codon and restriction site XmaI (underlined) in the reverse primer.…”

“…In addition, anti-IL-6 antibodies (50 mg ml À1 ) were either or not added to the wells with LNs (2 Â 10 5 ) and syngeneic 64pT tumour cells (1 Â 10 4 ; treated with mitomycin C; expressing highly Mage-b; Gravekamp et al, 2004), and secreting highly IL-6 (3000 pg ml À1 ) (Gravekamp et al, 2008), in 200 ml of RPMI containing 10% FBS. Two days later, the production of IFNg was determined by quantitative ELISA as described previously (Sypniewska et al, 2005). Anti-IL-6 antibodies and purified IL-6 were purchased from Pharmingen.…”

Vaccination with Mage-b DNA induces CD8 T-cell responses at young but not old age in mice with metastatic breast cancer

“…The mouse Mage-b DNA plasmid (pcDNA3.1-Mage-b/V5) has been described previously (Sypniewska et al, 2005). The mouse granulocyte macrophage-colony stimulating factor (GM-CSF) plasmid (CMV1-GM-CSF) was kindly provided by Dr Stephen Johnston (The Center for Innovations in Medicine, The Biodesign Institute at Arizona State University) (Chambers and Johnston, 2003).…”