Vaccination with a nanoparticle E7 vaccine can prevent tumor recurrence following surgery in a human papillomavirus head and neck cancer model

High-risk human papillomavirus (HPV) encoding E6/E7-HPV oncogenes are responsible for a subgroup of head and neck squamous-cell carcinoma (HNSCC) and thus therapeutic E7-vaccines may be used to control HPV+HNSCC tumors. Herein we investigated the effects of an optimized nanoparticle-conjugated E7 long-peptide vaccine adjuvanted with CpG (NP-E7LP) in an orthotopic immunocompetent mouse model of HPV+HNSCC which is based on injection of HPV16 E6/E7-expressing mEERL95-cells into the submental space. In absence of surgery, vaccination performed before or after tumor-cell injection decreased tumor growth or prolonged mice survival only marginally, despite the high numbers of vaccine-induced circulating E7-specific IFN-gamma-secreting CD8(+) T-cells. This contrasts with the high-efficacy of NP-E7LP-vaccination reported in the genital and subcutaneous HPV16-E6/E7-expressing TC-1 models. Our data show that in a direct comparison, NP-E7LP-vaccination fully controlled TC-1, but not mEERL95, tumors subcutaneously growing in the flanks. Immune-cell infiltration was 10-fold higher in TC-1-tumors, than in mEERL95-tumors, suggesting that vaccine-induced CD8(+) T-cells can only poorly infiltrate mEERL95-tumors. Indeed, immunofluorescence staining of orthotopic mEERL95-tumors showed that CD3(+) T-cells are preferentially located peritumorally. However, when NP-E7LP-vaccination was performed after mEERL95-cell injection, but before resection of primary tumors, no postsurgical recurrence was observed and 100% of the mice survived until the experimental endpoint (day 70) in the NP-E7LP-vaccinated group. In contrast, we observed a 60% recurrence rate and only 35% survival in PBS-vaccinated mice. This suggests that removal of the primary tumor modified the tumor microenvironment, allowing a therapeutic effect of the vaccine-induced anti-tumor response. E7-vaccination combined with surgery may thus benefit patients with HPV+HNSCC.

Carboplatin/paclitaxel, E7-vaccination and intravaginal CpG as tri-therapy towards efficient regression of genital HPV16 tumors

Gabriele Galliverti, Douglas Hanahan

High-risk human papillomavirus (HPV) are responsible for genital and oral cancers associated with the expression of the E6/E7 HPV oncogenes. Therapeutic vaccines targeting those oncogenes can only partially control tumor progression, highlighting the necessity to investigate different treatment strategies. Using the genital orthotopic HPV16 TC-1 model, herein we sequentially investigated in progressively more stringent settings the effects of systemic administration of carboplatin/paclitaxel (C + P) chemotherapy combined with HPV16-E7 synthetic long peptide (E7LP) vaccination, followed by intravaginal immunostimulation with the synthetic toll-like-receptor-9 agonist CpG. Our data show that systemic delivery of C+ P prior to E7LP vaccination significantly increased mice survival. This survival benefit was associated with both reduced genital tumor growth at the time of vaccination, and a decreased infiltration of Ly6G myeloid cells and tumor-associated macrophages. Adding intravaginal CpG, which results in increased E7-specific CD8 T cells locally, to E7LP vaccination and the chemotherapy formed a tri-therapy, which significantly increased mice survival as compared to any of the dual treatments. When the tri-therapy was further refined by using a recently optimized nanoparticle-conjugated E7LP vaccine, even larger end-stage genital-TC-1 tumors responded, with 90% of mice showing a survival benefit as compared to 30% of mice with the tri-therapy involving the traditional E7LP 'liquid' vaccine. C + P is commonly used to treat cervical cancer patients and its combination with E7/E6 vaccination is currently being tested in a phase I/II trial (NCT02128126). Our data suggests that new vaccine formulations combined with local immunostimulation and standard-of-care chemotherapy have promise to further benefit patients with HPV-associated cancer.

BMC2019

Advancing immunotherapy for HPV-related cancers: Improving therapeutic vaccination approaches and understanding the immunosuppressive role of myeloid cells

Gabriele Galliverti

Human Papilloma Viruses (HPVs) are the most common sexually transmitted agents worldwide, and chronic infection with oncogenic HPVs can lead to the development of lesions in the cervix, the vulva, and the head and neck region. In some patients, these lesions will eventually progress to squamous cell carcinomas that have the potential to spread to distant sites. Cervical cancer is by far the most common type of HPV-related malignancy, and at the early stages can be treated quite effectively. However, for patients in the later stages of tumor progression traditional therapeutic approaches like chemotherapy are poorly effective, and the development of novel strategies based on immunotherapy is undergoing. Clinical trials using HPV E6/E7-derived synthetic long peptides (SLPs) vaccines have shown promising results. However, these vaccines are only leading to significant responses in a low percentage of patients bearing pre-malignant lesions, whereas they seem to be completely ineffective in patients with advanced tumors, suggesting the presence of immune-escape mechanisms. Here I describe an improved therapeutic vaccination approach based on conjugation of E7-derived SLPs to solid-phase ultra-small (30 nm in diameter) nanoparticles (NPs). The NP-conjugated formulation (NP-E7LP) was able to significantly enhance both the systemic and local anti-tumor immune responses, leading to higher CD8 T cell infiltrates in the tumor microenvironment (TME) resulting in a significant reduction in tumor burden and overall increased survival in mice bearing transplantable HPV+ TC1 and SC1 tumors. Next, I demonstrate that a transgenic mouse model for HPV-related cancers expressing the HPV16 early region under the keratin 14 promoter bears striking similarities to cervical cancer patients regarding the alteration of the immune system provoked by HPVs. By taking advantage of the K14HPV16 H2b model, I show that the use of a nanoparticle-conjugated vaccine is required to elicit an immune response, but that, similarly to cervical cancer patients, therapeutic vaccination alone is not sufficient to elicit efficacious anti-tumor immunity. I also illustrate that combinatorial trials involving several different therapeutic antibodies are not sufficient to significantly boost the anti-tumor immune response. Finally, I identify a substantial increase in immunosuppressive myeloid cells (MDSCs) as the mechanism likely responsible for the observed impairment of the immune response, a feature that seems to be shared by cervical cancer patients. Interestingly, 5 although the attention of most of the researchers in the field is usually focused on the TME, I provide evidence that this myeloid cell-dependent mechanism is acting upstream in the lymphoid organs where it inhibits the very first steps in the generation of the anti-tumor immune response. My data suggest that this mechanism could potentially put an early stop to the cancer-immunity cycle, thereby contributing to the ineffectiveness of immunotherapies in cervical cancer patients. In summary, this thesis provides new knowledge that will contribute to the improvement of immunotherapy-based approaches against cervical cancer and other HPV-related diseases with the potential for applicability to other types of cancer.

EPFL2018

Elucidating the role of lymphatic vessels within the tumor microenvironment of breast cancer

Ingrid Maria van Mier

Breast cancer is the most common cancer in women worldwide, with an estimated 1.7 million newly diagnosed cases in 2012. Despite the many clinical manifestations, metastatic disease is the main cause of death, and is responsible for 90% of all breast cancer-related deaths. Aggressive carcinomas of the breast preferentially spread via the lymphogenous route, and often co-opt and actively remodel the lymphatic vasculature in order to invade and metastasize to their draining lymph nodes (LNs) and, ultimately, to distant organs. Indeed, expression of vascular endothelial growth factor (VEGF)-C, the principal lymphangiogenic growth factor, by the tumor or recruited ancillary cells has been shown to correlate both with LN and distant metastasis, and shorter overall survival (OS), and LN status represents an important prognostic factor for patients diagnosed with breast cancer. Although traditionally considered as passive conduits that solely mediate tumor cell escape, more recent research aimed at discerning the interactions between the developing tumor and its associated lymphatic vessels has revealed that the lymphatic vasculature actively promotes lymphogenous metastasis of invasive tumor cells. Likewise, collaborative interactions between tumor cells and a multitude of other tissue-resident and recruited ancillary cells, which together constitute the tumor microenvironment, not only coercively contribute to tumor growth, progression and metastasis, but also suggest that interactions among primed stromal cells themselves may be important in the process of metastatic dissemination as well. Notably, expansion of tumor-associated lymphatic vessels in the tumor periphery leads to increased lymphatic drainage from the tumor to the tumor-draining LN, and interstitial flow-induced mechanical changes may alter tensional homeostasis within the tumor through stimulation of local fibroblasts that remodel and stiffen the extracellular matrix (ECM). Furthermore, VEGF-C-induced remodeling of the tumor-associated lymphatic vasculature has been shown to directly suppress the anti-tumor immune response, thereby promoting tolerance to the nascent tumor. Thus, to explore whether tumor-associated lymphatic vessels promote tumor metastasis not only via interactions with the incipient tumor, but also through cross-talk with the surrounding stromal cells, the different components of the tumor microenvironment, specifically tensional homeostasis and anti-tumor immunity, were evaluated upon modulation of lymphatic vessel growth in various experimental models of breast cancer. Accordingly, inhibition of VEGFR-3-mediated signaling upon administration of an antagonistic antibody in a genetically engineered preclinical mouse model specifically reduced the number of lymphatic endothelial cells (LECs) in the tumor, whereas conversely, orthotopic inoculation of a stable murine breast cancer cell line with enhanced expression of VEGF-C induced an expansion of the lymphatic endothelium. Although either experimental approach affected either regional or distant metastatic dissemination of the primary tumor to LN or the lungs, respectively, these effects did not seem to be mediated by interactions of the tumor-associated lymphatic vasculature with the tumor microenvironment in these particular experimental models.

EPFL2017