Development of a VLP-Based Vaccine Displaying an xCT Extracellular Domain for the Treatment of Metastatic Breast Cancer.

Metastatic breast cancer (MBC) is the leading cause of cancer death in women due to recurrence and resistance to conventional therapies. Thus, MBC represents an important unmet clinical need for new treatments. In this paper we generated a virus-like particle (VLP)-based vaccine (AX09) to inhibit de novo metastasis formation and ultimately prolong the survival of patients with MBC. To this aim, we engineered the bacteriophage MS2 VLP to display an extracellular loop of xCT, a promising therapeutic target involved in tumor progression and metastasis formation. Elevated levels of this protein are observed in a high percentage of invasive mammary ductal tumors including triple negative breast cancer (TNBC) and correlate with poor overall survival. Moreover, xCT expression is restricted to only a few normal cell types. Here, we tested AX09 in several MBC mouse models and showed that it was well-tolerated and elicited a strong antibody response against xCT. This antibody-based response resulted in the inhibition of xCT's function in vitro and reduced metastasis formation in vivo. Thus, AX09 represents a promising novel approach for MBC, and it is currently advancing to clinical development.

Cancer stem cell immunology and immunotherapy: Harnessing the immune system against cancer's source.

Despite recent advances in diagnosis and therapy having improved cancer outcome, many patients still do not respond to treatments, resulting in the progression or relapse of the disease, eventually impairing survival expectations. The limited efficacy of therapy is often attributable to its inability to affect cancer stem cells (CSCs), a small population of cells resistant to current radio- and chemo-therapies. CSCs are characterized by self-renewal and tumor-initiating capabilities, and function as a reservoir for the local and distant recurrence of the disease. Therefore, new therapeutic approaches able to effectively target and deplete CSCs are urgently needed. Immunotherapy is facing a renewed interest for its potential in cancer treatment, and the possibility of harnessing the immune system to target CSCs is being addressed by a new exciting research field. In this chapter, we discuss the cancer stem cell model and illustrate CSC biological and molecular properties, critically addressing theoretical and practical issues linked with their definition and study. We then review the existing literature regarding the immunological properties of CSCs and the complex interplay occurring between CSCs and immune cells. Finally, we present up-to-date studies on CSC immunotargeting and its potential future perspective. In conclusion, understanding the interplay between CSC biology and tumor immunology will provide a deeper understanding of the mechanisms that regulate CSC immunological properties. This will contribute to the design of new CSC-directed immunotherapeutic strategies with the potential of strongly improving cancer outcomes.

Fighting breast cancer stem cells through the immune-targeting of the xCT cystine-glutamate antiporter.

Tumor relapse and metastatic spreading act as major hindrances to achieve complete cure of breast cancer. Evidence suggests that cancer stem cells (CSC) would function as a reservoir for the local and distant recurrence of the disease, due to their resistance to radio- and chemotherapy and their ability to regenerate the tumor. Therefore, the identification of appropriate molecular targets expressed by CSC may be critical in the development of more effective therapies. Our studies focused on the identification of mammary CSC antigens and on the development of CSC-targeting vaccines. We compared the transcriptional profile of CSC-enriched tumorspheres from an Her2+ breast cancer cell line with that of the more differentiated parental cells. Among the molecules strongly upregulated in tumorspheres we selected the transmembrane amino-acid antiporter xCT. In this review, we summarize the results we obtained with different xCT-targeting vaccines. We show that, despite xCT being a self-antigen, vaccination was able to induce a humoral immune response that delayed primary tumor growth and strongly impaired pulmonary metastasis formation in mice challenged with tumorsphere-derived cells. Moreover, immunotargeting of xCT was able to increase CSC chemosensitivity to doxorubicin, suggesting that it may act as an adjuvant to chemotherapy. In conclusion, our approach based on the comparison of the transcriptome of tumorspheres and parental cells allowed us to identify a novel CSC-related target and to develop preclinical therapeutic approaches able to impact on CSC biology, and therefore, hampering tumor growth and dissemination.

Bovine herpesvirus 4-based vector delivering the full length xCT DNA efficiently protects mice from mammary cancer metastases by targeting cancer stem cells.

Despite marked advancements in its treatment, breast cancer is still the second leading cause of cancer death in women, due to relapses and distal metastases. Breast cancer stem cells (CSCs), are a cellular reservoir for recurrence, metastatic evolution and disease progression, making the development of novel therapeutics that target CSCs, and thereby inhibit metastases, an urgent need. We have previously demonstrated that the cystine-glutamate antiporter xCT (SLC7A11), a protein that was shown to be overexpressed in mammary CSCs and that plays a key role in the maintenance of their redox balance, self-renewal and resistance to chemotherapy, is a potential target for mammary cancer immunotherapy. This paper reports on the development of an anti-xCT viral vaccine that is based on the bovine herpesvirus 4 (BoHV-4) vector, which we have previously showed to be a safe vaccine that can transduce cells in vivo and confer immunogenicity to tumor antigens. We show that the vaccination of BALB/c mice with BoHV-4 expressing xCT (BoHV-4-mxCT), impaired lung metastases induced by syngeneic mammary CSCs both in preventive and therapeutic settings. Vaccination induced T lymphocyte activation and the production of anti-xCT antibodies that can mediate antibody-dependent cell cytotoxicity (ADCC), and directly impair CSC phenotype, self-renewal and redox balance. Our findings pave the way for the potential future use of BoHV-4-based vector targeting xCT in metastatic breast cancer treatment.

Strengths and Weaknesses of Pre-Clinical Models for Human Melanoma Treatment: Dawn of Dogs' Revolution for Immunotherapy.

Despite several therapeutic advances, malignant melanoma still remains a fatal disease for which novel and long-term curative treatments are needed. The successful development of innovative therapies strongly depends on the availability of appropriate pre-clinical models. For this purpose, several mouse models holding the promise to provide insight into molecular biology and clinical behavior of melanoma have been generated. The most relevant ones and their contribution for the advancement of therapeutic approaches for the treatment of human melanoma patients will be here summarized. However, as models, mice do not recapitulate all the features of human melanoma, thus their strengths and weaknesses need to be carefully identified and considered for the translation of the results into the human clinics. In this panorama, the concept of comparative oncology acquires a priceless value. The revolutionary importance of spontaneous canine melanoma as a translational model for the pre-clinical investigation of melanoma progression and treatment will be here discussed, with a special consideration to the development of innovative immunotherapeutic approaches.

A Virus-Like-Particle immunotherapy targeting Epitope-Specific anti-xCT expressed on cancer stem cell inhibits the progression of metastatic cancer in vivo.

Aggressive forms of breast cancer, such as Her2+ and triple negative breast cancer (TNBC), are enriched in breast cancer stem cells (BCSC) and have limited therapeutic options. BCSC represent a key cellular reservoir for relapse, metastatic progression and therapeutic resistance. Their ability to resist common cytotoxic therapies relies on different mechanisms, including improved detoxification. The cystine-glutamate antiporter protein xCT (SLC7A11) regulates cystine intake, conversion to cysteine and subsequent glutathione synthesis, protecting cells against oxidative and chemical insults. Our previous work showed that xCT is highly expressed in tumorspheres derived from breast cancer cell lines and downregulation of xCT altered BCSC function in vitro and inhibited pulmonary metastases in vivo. We further strengthened these observations by developing a virus-like-particle (VLP; AX09-0M6) immunotherapy targeting the xCT protein. AX09-0M6 elicited a strong antibody response against xCT including high levels of IgG2a antibody. IgG isolated from AX09-0M6 treated mice bound to tumorspheres, inhibited xCT function as assessed by reactive oxygen species generation and decreased BCSC growth and self-renewal. To assess if AX09-0M6 impacts BCSC in vivo seeding, Her2+ TUBO-derived tumorspheres were injected into the tail vein of AX09-0M6 or control treated female BALB/c mice. AX09-0M6 significantly inhibited formation of pulmonary nodules. To evaluate its ability to impact metastases, AX09-0M6 was administered to mice with established subcutaneous 4T1 tumors. AX09-0M6 administration significantly hampered tumor growth and development of pulmonary metastases. These data show that a VLP-based immunization approach inhibits xCT activity, impacts BCSC biology and significantly reduces metastatic progression in preclinical models.

CSPG4: a prototype oncoantigen for translational immunotherapy studies.

Thanks to striking progress in both the understanding of anti-tumor immune response and the characterization of several tumor associated antigens (TAA), a more rational design and more sophisticated strategies for anti-tumor vaccination have been possible. However, the effectiveness of cancer vaccines in clinical trial is still partial, indicating that additional studies are needed to optimize their design and their pre-clinical testing. Indeed, anti-tumor vaccination success relies on the choice of the best TAA to be targeted and on the translational power of the pre-clinical model used to assess its efficacy. The chondroitin sulfate proteoglycan-4 (CSPG4) is a cell surface proteoglycan overexpressed in a huge range of human and canine neoplastic lesions by tumor cells, tumor microenvironment and cancer initiating cells. CSPG4 plays a central role in the oncogenic pathways required for malignant progression and metastatization. Thanks to these features and to its poor expression in adult healthy tissues, CSPG4 represents an ideal oncoantigen and thus an attractive target for anti-tumor immunotherapy. In this review we explore the potential of CSPG4 immune-targeting. Moreover, since it has been clearly demonstrated that spontaneous canine tumors mimic the progression of human malignancies better than any other pre-clinical model available so far, we reported also our results indicating that CSPG4 DNA vaccination is safe and effective in significantly increasing the survival of canine melanoma patients. Therefore, anti-CSPG4 vaccination strategy could have a substantial impact for the treatment of the wider population of spontaneous CSPG4-positive tumor affected dogs with a priceless translational value and a revolutionary implication for human oncological patients.

Protection of mice against the highly pathogenic VVIHD-J by DNA and fowlpox recombinant vaccines, administered by electroporation and intranasal routes, correlates with serum neutralizing activity.

The control of smallpox was achieved using live vaccinia virus (VV) vaccine, which successfully eradicated the disease worldwide. As the variola virus no longer exists as a natural infection agent, mass vaccination was discontinued after 1980. However, emergence of smallpox outbreaks caused by accidental or deliberate release of variola virus has stimulated new research for second-generation vaccine development based on attenuated VV strains. Considering the closely related animal poxviruses that also arise as zoonoses, and the increasing number of unvaccinated or immunocompromised people, a safer and more effective vaccine is still required. With this aim, new vectors based on avian poxviruses that cannot replicate in mammals should improve the safety of conventional vaccines, and protect from zoonotic orthopoxvirus diseases, such as cowpox and monkeypox. In this study, DNA and fowlpox (FP) recombinants that expressed the VV L1R, A27L, A33R, and B5R genes were generated (4DNAmix, 4FPmix, respectively) and tested in mice using novel administration routes. Mice were primed with 4DNAmix by electroporation, and boosted with 4FPmix applied intranasally. The lethal VVIHD-J strain was then administered by intranasal challenge. All of the mice receiving 4DNAmix followed by 4FPmix, and 20% of the mice immunized only with 4FPmix, were protected. The induction of specific humoral and cellular immune responses directly correlated with this protection. In particular, higher anti-A27 antibodies and IFNγ-producing T lymphocytes were measured in the blood and spleen of the protected mice, as compared to controls. VVIHD-J neutralizing antibodies in sera from the protected mice suggest that the prime/boost vaccination regimen with 4DNAmix plus 4FPmix may be an effective and safe mode to induce protection against smallpox and poxvirus zoonotic infections. The electroporation/intranasal administration routes contributed to effective immune responses and mouse survival.

Bovine herpesvirus 4-based vector delivering a hybrid rat/human HER-2 oncoantigen efficiently protects mice from autochthonous Her-2+ mammary cancer.

The epidermal growth factor receptor 2 (HER-2) oncogene is a major target for the immunotherapy of breast cancer. Following up to the therapeutic success achieved with Her-2-targeting monoclonal antibodies, immune-prophylactic approaches directed against Her-2 have also been investigated taking into account, and trying to overcome, Her-2 self-tolerance. Perhaps due to safety (and efficacy) concerns, the least explored anti-Her-2 active immunization strategy so far has been the one relying on viral-vectored vaccine formulations. Taking advantage of the favorable properties of bovine herpesvirus 4 (BoHV-4) in terms of safety and ease of manipulation as well as its previously documented ability to transduce and confer immunogenicity to heterologous antigens, we tested the ability of different recombinant HER-2-BoHV-4 immunogens to 8break tolerance and elicit a protective, anti-mammary tumor antibody response in HER-2 transgenic BALB-neuT mice. All the tested constructs expressed the HER-2 transgenes at high levels and elicited significant cellular immune responses in BALB/c mice upon administration via either DNA vaccination or viral infection. In BALB-neuT mice, instead, only the viral construct expressing the membrane-bound chimeric form of Her-2 protein (BoHV-4-RHuT-gD) elicited a humoral immune response that was more intense and earlier-appearing than that induced by DNA vaccination. In keeping with this observation, two administrations of BoHV-4-RHuT-gD effectively protected BALB-neuT mice from tumor formation, with 50% of vaccinated animals tumor-free after 30 weeks from immunization compared to 100% of animals exhibiting at least one palpable tumor in the case of animals vaccinated with the other BoHV-4-HER-2 constructs.