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

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.

New roles of the lymphatic endothelium in modulating adaptive immunity: implications for immune tolerance and cancer immunotherapy

Lambert Charles François Potin

Lymphatic vessels have traditionally been considered as passive conduits for interstitial fluid drainage and for immune cell trafficking to lymph nodes. In the last two decades, many studies have challenged this classical dogma: lymphatic endothelial cells (LECs) were shown to actively interact with immune cells by secreting chemokines and cytokines, and presenting antigens to T cells. These newly described functions pinpoint a multi-faceted role of LECs in orchestrating the immune response through both passive and active mechanisms. Yet, precise mechanisms leading to antigen-specific fine-tuning of the T cell response remain unexplored. Moreover, although lymphatics have been shown to play important roles in cancer, the contributions of LECs in regulating antitumor immunity have been largely overlooked by the biomedical community. This doctoral thesis aimed at filling these gaps. First, in order to investigate the mechanisms and implications of LEC antigen presentation to CD4+ T cells, we engineered a mouse model where LECs lacked the ability to present antigens to CD4+ T cells. In these mice, antigen-specific responses to vaccination were increased when compared to wild-type controls. Moreover, we demonstrated that LECs were poor inducers of CD4+ T cell activation in vitro, and that they could dampen CD4+ T cell activation by dendritic cells. Taken together, these findings suggest that LECs participate to the peripheral immune tolerance of CD4+ T cells. Second, we investigated the immunological consequences of the development of lymphatic vessels, called lymphangiogenesis, in mouse melanoma. Upon induction of tumor lymphangiogenesis, we observed striking tumor enrichment with immune cells, which resulted in a higher sensitivity of those tumors to immunotherapy. We subsequently demonstrated that inducing lymphangiogenesis in a mouse model of cold tumors, which lack immune infiltrates and do not respond to immunotherapies, could restore their responsiveness to immunotherapy treatments. Third, to predict which cancer types were the most likely to replicate the findings we obtained in mouse melanoma, we mined The Cancer Genome Atlas database for gene expression. We identified a set of cancers, such as colon adenocarcinoma and cholangiocarcinoma, where high expression of the lymphangiogenic growth factor was associated with a strong T cell signature. This doctoral thesis unveiled new immunomodulatory functions of LECs by which antigen-specific immune tolerance is induced. Additionally, it opened new avenues to target lymphatics therapeutically in cancer to potentiate immunotherapies. We believe this work contributes to demonstrating the immunological importance of LECs, and hope that lymphatics will be considered as an important immunomodulatory player of the tumor microenvironment when developing novel immunotherapies.

EPFL2018

Therapeutic Immunomodulation of the Lymphoid-like Tumor Environment Using Nanoparticulate Formulations

Iraklis Kourtis

Cancer is a leading cause of death worldwide. Understanding the underlying mechanisms that lead to tumor escape and evasion is pivotal for the design of new therapeutic applications. This thesis takes an immunobioengineering approach, and navigates through identifying the immunological parameters relevant in the tumor microenvironment, proposing and developing a strategy in response to address these parameters using nanosized drug and antigen carriers. It is widely accepted that changes in the immunological equilibriumof the tumor microenvironment are crucial for the progression, dissemination and metastasis of a developing tumor. Motivated by tumors' natural secretion of CCL21, a chemokine known for dendritic (DC) migration towards the lymphatics, and for attracting DC and T cells in the lymph node (LN), we perturbed the physiological secretion of CCL21 of B16-F10 melanomas, deriving both a knock-down (CCL21low) and an over-expressing (CCL21high) variant. Interestingly, in immunocompetent mice, growth in CCL21-secreting tumors had an advantage over the CCL21low sub-clones and was CCR7 dependent. CCL21low tumors harbored more antigen specific T cells, while CCL21-secreting, more T regulatory cells (Tregs), which also correlated with subsequent biochemical polarization. Moreover, CCL21-secreting tumors formed a reticular fibroblastic (FRC) network (ERTR7+gp38+CCL21+) reminiscent of the lymphoid tissue of the paracortex (T cell zone) within LNs. The lymphoidlike stroma was orchestrated by the recruitment of CCR7+ adult lymphoid tissue inducers (LTis), responsible for LN formation during embryogenesis, present only in the CCL21-secreting tumors. In mice lacking LTis (Rorc(γt)-deficient) control tumors displayed impaired growth, suggesting that tumor growth is stroma dependent. In addition, the stroma of control or CCL21high tumors was infiltrated by more myeloid suppressor cells (MDSCs) than in CCL21low variant. MDSCs are a heterogeneous population of immature myeloid precursor cells that repress T cell activation and antigen presentation in chronic inflammation and cancer, hampering the efficacy of anti-tumoral vaccinations. CCL21's ability to protect allografts was further demonstrated in non-syngeneic recipients and in ectopically CCL21 transduced βTC tumor models. By mimicking secondary lymphoid organs, tumors may hi-jack and modulate the immune response from immunogenic to tolerogenic to allow for growth and metastasis. Intrigued by the excessive stroma infiltration of MDSCs in CCL21-secreting tumors, we performed a detailed spatiotemporal biodistribution analysis of ultrasmall (sub 50 nm) nanoparticles (NPs) in naïve and tumor bearing mice. Intradermally administered NPs rapidly drained to the LNs and spleen and persistently targetedmajor phagocytic populations that play a key role in hosting an immune response in naïve and tumor bearing mice. Furthermore, tumor-induced myeloid compartments, mainlyMDSCs, were highly targeted naturally within the tumormicroenvironment and spleen (tumor macroenvironment) 12 h post administration, without the use of site specific ligand. Following the MDSCs' natural preference towards phagocytosing NPs, and taking advantage of their excessive reductive cytoplasm, we designed a macromolecular pro-drug formulation that bears a cytotoxic payload and can be released in highly reductive microenvironments. Specifically, we engineered a reducible delivery system of 6-tioguanine (TG) in various formulations. Remarkably, bothMDSC compartments were ablated, monocytic ∼20 fold (CD11b+Ly6c+), polymorphonuclear ∼100 fold (CD11b+Ly6g+), for at least 7 d after administration in tumor bearing mice. As the bioidstribution revealed that apart from MDSCs, other antigen presenting cells (APCs) have phagocytosed NPs, we observed only a ∼2 fold decrease in APCs, suggesting that the rest could be used for vaccination. Taking into account that cancer vaccines and cancer immunotherapy rely on the efficient antigen presentation and education of naïve T cells by professional APCs, we explored the ability of NPs to deliver antigens in the appropriate sub-cellular compartment for antigen presentation and activation of T cells. Using the model antigen ovalbumin, we illustrated that exogenous antigen coupled to NPs by reducible bonds were internalized by DCs in vivo and in vitro. The conjugated antigen was preferentially released from the NPs with a reducible formulation, and was successfully presented in the major histocompatibility complex (MHC) I and II, which lead to activation and proliferation of antigen specific CD8+ and CD4+ T cells in vivo, respectively. Taking together, the nanoparticle platformcould be used efficiently for both inducing cytotoxicity and invoking an immune response. From discovery of new suppressive niches to therapeutical applications, we envisioned a two prong strategy that combined I) a modulatory regime to upset the balance of theMDSC populations both within the tumor microenvoronent and systemically with II) a cancer vaccine with the potential to significantly improve anti-tumoral therapeutic applications. This thesis demonstrated that such an approach is both possible and probable.

EPFL2012

Targeting and Modulating Tumor-Associated Sites with Novel Nanoparticle-Based Immunotherapies

Laura Jeanbart

Cancer is a global disease and a leading cause of death worldwide. While surgery, radiotherapy and chemotherapy comprise the classical tools to eradicate tumors, they do not cure cancer, cannot prevent metastases, lead to side effects, and most importantly they do not instruct the patientʼs immune system to recognize and fight tumor cells. Since the discovery of the immune systemʼs implication in cancer, immunotherapies, which aim at using and educating the immune system to fight and reject cancer, have come to complement classical tumor-debulking methods. A major goal of immunotherapy is to induce or activate effector cytotoxic CD8+ T lymphocytes that can infiltrate and kill the tumor while overcoming immune suppressive mechanisms, which impede their efficacy. Moreover, cancer immunotherapies aim at inducing memory to tumor antigens to prevent relapse or metastases, which traditional therapies cannot do. Dendritic cell (DC) targeting and activation are key for inducing adaptive immunity. Our laboratory has developed synthetic nanoparticles (NPs) capable of draining through lymphatics to target skin-draining lymph nodes (LNs) and being phagocytosed by resident antigen-presenting cells (APCs) upon intradermal injection. We developed a reproducible method to conjugate tumor antigens to NPs and co-delivered them with CpG-conjugated NPs. We found that a NP vaccine composed of a single melanoma-derived peptide (TRP- 2180-188) led to dramatic tumor growth delay in melanoma and was more efficient than a NP vaccine containing several tumor antigens from whole tumor cell lysate (TL). These findings contradicted our hypothesis that immunization with TL might lead to a broader T cell response and hence improved therapeutic outcomes. Additionally, we found that the dose of CpG could modulate the magnitude of the therapeutic outcomes in both single epitope and multiple antigen based NP-vaccines. Considering the efficacy of the developed NP vaccines, we used them as a tool to ask a fundamental and extremely relevant question regarding the influence of vaccine administration site on clinical efficacy: is it therapeutically more beneficial to target a tumor- draining lymph node (tdLN), which is immune suppressed but tumor antigen-primed, or a non-tdLN, which is neither suppressed nor primed by the tumor? We found that targeting the tdLN with NP vaccines led to significantly enhanced therapeutic outcomes in two different tumor models over targeting a non-tdLN, and that efficacy relied on NP conjugation of antigen and adjuvant. This suggested that antigen drainage from the tumor might be available to APCs in the tdLN and we further hypothesized that therapies that lead to immunogenic tumor cell death might synergize with a tdLN-targeted adjuvant approach. While cytotoxic modalities, such as chemotherapy and radiotherapy, have traditionally been used for their ability to kill tumor cells, tumor cells shed antigens and debris upon cell death that drain to the tdLN. Consistent with our hypothesis, targeting an adjuvant to the tdLN enhanced therapeutic benefits of chemo- and radio- therapy, while targeting a non-tdLN did not. We hypothesized that targeting the tdLN with NP vaccines might switch the tdLN microenvironment from a suppressive to an immunogenic one. This suggested that direct targeting of active suppressive mechanisms in the tumor might further improve immunotherapy. We thus engineered a nano-sized micellar carrier loaded with 6-thioguanine (MC-6TG), a cytotoxic drug used in leukemia, and explored its use in targeting T cell- impairing myeloid-derived suppressor cells (MDSCs) in order to enhance the efficacy of therapeutic vaccines. MC-6TG entirely eradicated both Ly6c+ monocytic and Ly6g+ granulocytic MDSCs for up to 7 d in the blood, tumor and tdLN of tumor-bearing mice. Since MC-6TG also targeted certain subsets of macrophages and DCs, depleting MDSCs did not have an impact on the efficacy of a NP vaccine, which relies on APCs for efficacy. However, MC-6TG synergized with adoptively transferred CD8+ T cells, significantly delaying tumor growth and enhancing survival in a murine model of implantable melanoma. Overall, this thesis describes the design and implementation of a novel approach to immunotherapy: we engineered NP-based therapeutic vaccines, optimized their delivery route to enhance efficacy, and simultaneously tackled immune suppressive cells in the tumor microenvironment to allow optimal therapeutic outcomes. The technologies and methods developed in this work are particularly relevant to clinical oncology and have the potential to benefit cancer patients by improving cancer therapy efficacy.

EPFL2013