Nanoparticle Conjugation of Human Papillomavirus 16 E7-long Peptides Enhances Therapeutic Vaccine Efficacy against Solid Tumors in Mice

Treatment of patients bearing human papillomavirus (HPV)-related cancers with synthetic long-peptide (SLP) therapeutic vaccines has shown promising results in clinical trials against premalignant lesions, whereas responses against later stage carcinomas have remained elusive. We show that conjugation of a well-documented HPV-E7 SLP to ultra-small polymeric nanoparticles (NP) enhances the antitumor efficacy of therapeutic vaccination in different mouse models of HPV+ cancers. Immunization of TC-1 tumor-bearing mice with a single dose of NP-conjugated E7LP (NP-E7LP) generated a larger pool of E7-specific CD8(+) T cells with increased effector functions than unconjugated free E7LP. At the tumor site, NP-E7LP prompted a robust infiltration of CD8(+) T cells that was not accompanied by concomitant accumulation of regulatory T cells (Tregs), resulting in a higher CD8(+) T-cell to Treg ratio. Consequently, the amplified immune response elicited by the NP-E7LP formulation led to increased regression of large, well-established tumors, resulting in a significant percentage of complete responses that were not achievable by immunizing with the non-NP-conjugated long-peptide. The partial responses were characterized by distinct phases of regression, stable disease, and relapse to progressive growth, establishing a platform to investigate adaptive resistance mechanisms. The efficacy of NP- E7LP could be further improved by therapeutic activation of the costimulatory receptor 4-1BB. This NP-E7LP formulation illustrates a "solid-phase" antigen delivery strategy that is more effective than a conventional free-peptide ("liquid") vaccine, further highlighting the potential of using such formulations for therapeutic vaccination against solid tumors. (C) 2018 AACR.

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

Development of a nano-adjuvant cancer vaccine and its evaluation for melanoma in combination with radiotherapy

Sylvie Hauert

Cancer is the second leading cause of death in the world and melanoma is the deadliest type of skin cancer. Although surgery, radiotherapy and chemotherapy are still standard treatments, the discovery of the role played by the immune system in cancer has allowed the development of new treatments, called immunotherapies. The goal of these treatments is to help the immune system eradicate cancerous cells, for example by increasing the anti-tumor response of the patient. In this particular case, the treatment has to activate cytotoxic CD8+ and CD4+ helper T cells against the cancer cells. This can be done by using subunit vaccines composed of tumor antigens, adjuvants and a delivery system, which will be internalized by dendritic cells (DCs) and induce its activation followed by the migration to the lymph nodes where it will educate T cells against specific antigens. Our laboratory has developed two different delivery systems, nanoparticles (NPs) and polymersomes (PSs) that can enhance the vaccineâs immune response by efficiently entering the lymphatic system, due to their size, and drain to the lymph node. Once they reach that location, they will be internalized by DCs and induce an anti-tumor response. In this thesis, we used these two nanocarriers to create a vaccine composed of different nano-adjuvants capable of activating several toll-like receptors simultaneously and test their efficacy at inducing an anti-tumor response in melanoma. We have developed and characterized the different nano-adjuvants, PS-MPLA, PS-CL075 and NP-CpG-B, and demonstrated, in vitro, synergistic effects on DC activation when PS-MPLA + PS-CL075 and PS-MPLA + NP-CpG-B were combined. In vivo, we showed the enhanced immune response with the different nano-adjuvant combinations, but only PS-MPLA + NP-CpG-B was able to delay tumor growth in melanoma. We then tested this vaccine in combination with radiotherapy, mimicking a clinical situation. We first demonstrated an enhanced immune response when combining a high irradiation dose, 15 Gy, with the nano-adjuvant vaccine draining the tumor-draining lymph node. Moreover, the vaccination of NP-CpG-B + PS-MPLA and radiotherapy increased mice survival by 17 days compared to mice only receiving radiotherapy. â Overall, we have demonstrated that combining different nano-adjuvants, targeting the tumor-draining lymph node, with radiotherapy enhanced significantly the survival in a melanoma model. This approach is particularly promising since ionizing radiation is a standard treatment in cancer and our vaccine can be applied to a multitude of cancer since it does not contain any antigen and its composition can easily be modified.

EPFL2018

Antigen delivery to dendritic cells by poly(propylene sulfide) nanoparticles with disulfide conjugated peptides: Cross-presentation and T cell activation

Sachiko Hirosue, Jeffrey Alan Hubbell, Iraklis Kourtis, Melody Swartz

Vaccines aiming to activate cytotoxic T cells require cross-presentation of exogenous antigen by antigen-presenting cells (APCs). We recently developed a synthetic nanoparticle vaccine platform that targets lymph node-resident dendritic cells (DCs), capable of mounting an immune response to conjugated antigen. Here, we explore routes of processing and the efficiency of MHC I cross-presentation of OVA peptides conjugated using both reducible and non-reducible linkages, exploring the hypothesis that reduction-sensitive conjugation will lead to better antigen cross-presentation. Both clathrin and macropinocytic pathways were implicated in nanoparticle uptake by colocalization and inhibitor studies. Cross-presentation by DCs was demonstrated by direct antibody staining and in vitro stimulation of CD8(+) T cells from OT-I mice and was indeed most efficient with the reduction-sensitive conjugation. Similarly, we observed IFN-γ production by CD4(+) T cells from OT-II mice. Finally, immunization with the OVA peptide-bearing nanoparticles resulted in in vivo proliferation and IFN-γ production by adoptively transferred CD8(+) OT-I T cells and was also most efficient with reduction-sensitive linking of the peptide antigen. These results demonstrate the relevance of the poly(propylene sulfide) nanoparticle vaccine platform and antigen conjugation scheme for activating both cytotoxic and helper T cell responses.