*3.6. Breast Cancer*

Breast cancer is a heterogeneous disease. Triple-negative breast cancer (TNBC) is an invasive form of breast cancer, with high propensity to relapse, and characterized by the absence of epidermal growth factor receptor 2 (HER2), progesterone, estrogen. The presence of cancer stem cells (CSCs) is related to the aggressiveness of TNBC. These CSCs are resistant to conventional therapies and are conducive to tumor development and progression [100,101]. Currently, there is no successful clinical therapy for this subtype of breast cancer. TNBC resists current cytotoxic therapies due to its unique detoxification mechanism, which is greatly promoted by the overexpression of the cystine-glutamic acid transporter xCT (SLC7A11). The xCT protein is overexpressed in several human tumors but not in healthy mammary gland tissue, suggesting that the protein up-regulation occurs only upon oncogenic transformation, contributing to a decrease in patient survival [102]. xCT exports glutamate at a ratio of 1:1 in the exchange of extracellular cystine [103]. Intracellularly, cystine is reduced to cysteine, a limiting precursor in the biosynthesis of glutathione (GSH), which plays an essential role in cellular defense against oxidative stress, reducing diverse reactive oxygen species (ROS) [104]. xCT increases the intracellular concentration of GSH, reduces p38/mitogen-activated Protein Kinase activation, inhibiting ROS and preventing cell apoptosis. As a result, xCT protects CSCs from ROS, making them more resistant to conventional therapies, and promotes tumor growth [105].

Sulfasalazine (SASP) is an FDA-approved drug used to treat chronic inflammatory diseases that inhibits xCT protein function [106]; however, SASP has numerous side effects and, as a result, is not a viable therapeutic option. A vaccine using a genetic fusion of the extracellular domain 6 (ECD6) from xCT protein on the AB loop of bacteriophage MS2-VLP, named AX09-0M6, was used as a prophylactic and therapeutic vaccine in a preclinical TNBC model. The adopted vaccination regimen was able to induce high levels of IgG2a antibodies that bound to mouse and human xCT breast CSCs. Anti-AX09-0M6 antibodies reduced the number and dimension of spheres and increased ROS concentration in all breast cancer cell lines tested, demonstrating that the vaccination with AX09-0M6 prevents BCSC self-renewal. The in vivo experiments showed a significant reduction in 4T1 tumor growth and the number of spontaneous pulmonary metastases, increased natural killer (NK) cell infiltration, and CD8+ T cells, which is indicative of positive tumor microenvironment changes and antibody-dependent cellular toxicity (ADCC). No adjuvants were used in this vaccination approach, and no toxicity was detected [22,99]. Similar results were obtained with an MS2-VLP expressing the ECD3 of xCT, named AX09-0M3 (Rolih et al., data in publication).

The epidermal growth factor receptor-2 (HER2) protein is overexpressed in another aggressive form of breast cancer in humans. Nearly 30% of all invasive breast cancers overexpress HER2. Like many other tumors, HER2+ breast cancer also expresses xCT, and the AX09 VLP against xCT also demonstrated a significant activity against this type of cancer [22,99]. A VLP-based vaccine approach for HER2+ breast cancer vaccination uses Acinetobacter phage AP205 to covalently display the HER2 protein (HER2-VLP) [16]. This vaccine is able to break B cell tolerance and induces a high level of anti-HER2-neutralizing antibodies. Tumor growth was inhibited after HER2-VLP immunization in FVB mice injected with HER2+ transplantable breast cancer cells or HER2+ tumor fragments. In addition, HER2 transgenic mice that spontaneously develop aggressive HER2 mammary carcinomas were tumor free until one year of age after HER2-VLP vaccination, with high levels of anti-HER2 antibodies lasting for up to six months after vaccination [16]. The generated anti-HER2 antibodies bind to mouse and human HER2 in the same way as commercial anti-HER2 monoclonal antibodies.

Although the level of endotoxin was not shown for the AX09 VLP vaccine and not removed from the HER2-VLP vaccine, these vaccines represent an excellent example of how versatile and efficient VLP can be in overcoming B cell tolerance to tumor-associated self-antigens.
