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Etalk Session 1 eTalk Session 1 Therapeutic Vaccination & New Targets and New Leads S1-01 HPV-Associated Tumor Eradication by Vaccination with Synthetic Short Peptides and Particle-Forming Liposomes X. He1, S. Zhou1, B. Quinn1, D. Jahagirdar2, J. Ortega2, S. Abrams3, J. Lovell1 1University at bufflo, Biomedical engineering, Amherst, United States, 2McGill University, Department of Anatomy and Cell Biology, Montreal, Canada, 3Roswell Park Comprehensive Cancer Center, Department of Immunology, Buffalo, United States Text Human papilloma virus (HPV)-16 is associated with cervical cancers and induces expression of the E6 and E7 oncogenes. Using a murine cell line that expresses these, the genes were sequenced and 6 predicted major histocompatibility complex (MHC) class I (MHC-I) epitopes were identified. A liposomal vaccine adjuvant based on cobalt-porphyrin-phospholipid (CoPoP) was admixed with synthetic 9-mer epitopes appended with 3 histidine residues, resulting in rapid formation of stable peptide-liposome particles. Synthetic monophosphoryl lipid A and QS-21 were also included in the liposome bilayer. Immunization with multivalent peptides led to protection from tumor challenge. Of the peptides screened, only the previously identified E749-57 epitope was functional. The peptide-liposome particles that formed upon mixing E7HHH49-57 with CoPoP liposomes were stable in serum and avidly taken up by immune cells in vitro. Immunization with E7HHH49-57 admixed with CoPoP liposomes resulted in robust protection from tumor challenge and re- challenge. Immunization with the synthetic short peptide at a dose of 100 ng protected mice in a therapeutic tumor challenge when admixed with CoPoP liposomes, whereas 200-fold higher peptide doses were ineffective with the Poly(I:C) adjuvant. CoPoP induced strong infiltrating CD8+ T response within the tumor microenvironment with improved functional profile. Vaccine monotherapy using nanogram dosing of the E7HHH49-57 peptide admixed with CoPoP reversed the growth of large established tumors, permanently eradicating subcutaneous tumors upwards of 100 mm3, and also completely eradicated lung tumors in a metastasis model. eTalk Session 1 Therapeutic Vaccination & New Targets and New Leads S1-02 Tuning LNPs to target antigen presenting cells in spleen induces CD8 T-cell responses and tumor regression in mice S. Bevers1, 2, S. A. A. Kooijmans3, E. Van de Velde1, M. J. Evers3, S. Seghers1, J. J. Gitz-François3, L. Hassler4, K. Breckpot2, T. Bastogne4, 5, R. M. Schiffelers3, S. De Koker1 1eTheRNA Immunotherapies, Niel, Belgium, 2Laboratory of Molecular and Cellular Therapy, Vrije Universiteit Brussel, Brussels, Belgium, 3CDL Research, University Medical Center Utrecht, Utrecht, Netherlands, 4CYBERnano, Villers-lès-Nancy, France, 5CRAN, Université de Lorraine, CNRS, INRIA BIGS, Vandœuvre- lès-Nancy, France Text Intravenous (i.v.) delivery of vaccines is emerging as an appealing approach to evoke high-quality T-cell responses needed to combat tumors. Messenger RNA (mRNA) holds huge potential to use in vaccination because of its ease of production, intrinsic adjuvant activity and the high versatility in antigen design, but also requires a potent delivery vehicle to enable antigen expression and immune activation. Lipid based nanoparticles (LNP) are currently the clinically most advanced tool to protect mRNA from degradation and efficiently deliver it inside cells. Yet, i.v administered LNPs generally tend to distribute to the liver. We hypothesized LNPs can be altered to target antigen presenting cells in the spleen and hereby orchestrate specific and durable immune responses against cancer antigens. By using a design of experiment methodology we were able to screen LNPs in a cost and time effective manner. LNP compositions that were evaluated solely differed in the molar ratios of ionizable lipid, phospholipid, cholesterol and PEG-lipid and the choice of the PEG-lipid. Bayesian Regression modeling enabled us to identify LNP compositions conferring maximum immunogenicity upon i.v. administration. To link LNP compositions to T cell responses and biodistribution, we prepared the same LNPs packaging either the viral oncoprotein E7 as an antigen or Cy-5 labelled luciferase mRNA. The optimized LNP compositions showed increased localization in the spleen (of mice and non-human primates) and uptake by antigen presenting cells. Furthermore, they induced high magnitude CD8 T cell responses that conferred strong antitumor immunity and prolonged survival of TC-1 tumor bearing mice. Mechanistically, type I interferons and phagocytes were found to be essential for eliciting strong T-cell responses. Unexpectedly, we identified B cells as major mediators of the vaccine- elicited T-cell response, revealing a previously undocumented role of these cells in the immune response to mRNA LNPs. Our data highlight the potential of optimizing LNP compositions by tailoring of the molar ratio of the lipids compromising the LNPs and provide insight in the cell types involved in the mRNA LNP-induced immune response. Collectively, our results illustrate the great promise of intravenous mRNA LNP vaccination for the treatment of cancer. eTalk Session 1 Therapeutic Vaccination & New Targets and New Leads S1-03 Rapid prime-boost amplification of T cell responses using heterologous, costimulatory liposome vaccines T. Wirth1, D. Ostroumov1, B. Slütter2 1Medical School Hannover, Gastroenterology, Hepatology and Endocrinology, Hannover, Germany, 2University of Leiden, Division of Biotherapeutics, Leiden, Netherlands Text Therapeutic vaccinations for cancer therapy require rapid and reliable induction of cancer-specific T cell responses in patients. Current vaccination regimens, however, rely on repeated antigen stimulations over an extensive period of time and still fail to reach the magnitude required for therapeutic effects. Here we describe the establishment of a heterologous vaccination regimen that is able to induce robust CD4 and CD8 T cells responses in less than two weeks in mice. Following a primary immunization with liposomes (LS) containing peptide and the STING agonist cdi-GMP, the resulting immune response is boosted only seven days later with a combination of a costimulatory agonist (Co), antigen in the form of soluble peptide (A) and a TLR3 agonist (T). Using this heterologous LS-CoAT prime-boost vaccination we demonstrate robust expansion of neoantigen-specific CD8 T cells, reaching a magnitude of up to 40% of Adpgk-specific T cells in the total CD8 T cell population after 14 days. LS-CoAT could be used with both short and long peptides and was similarly efficient in inducing CD4 T cell responses or combined CD4/CD8 T cell responses. LS-CoAT therefore represents a novel heterologous prime-boost vaccination approach which induces rapid T cell responses of unprecedented magnitude, thus warranting further evaluation of this vaccination method for cancer treatment. eTalk Session 1 Therapeutic Vaccination & New Targets and New Leads S1-04 Developing a therapeutic HPV16 vaccination strategy utilizing a vaginal tumor model in MHC-humanized mice S. Zottnick1, 2, 3, S. Kruse1, 2, M. Bozza4, A. Voß1, 2, A. Klevenz1, 2, R. Yang5, F. Rösl5, R. P. Harbottle4, A. B. Riemer1, 2 1German Cancer Research Center (DKFZ), Immunotherapy and Immunoprevention, Heidelberg, Germany, 2German Center for Infection Research (DZIF), partner site Heidelberg, Molecular Vaccine Design, Heidelberg, Germany, 3Heidelberg University, Faculty of Biosciences, Heidelberg, Germany, 4German Cancer Research Center (DKFZ), DNA Vectors, Heidelberg, Germany, 5German Cancer Research Center (DKFZ), Viral Transformation Mechanisms, Heidelberg, Germany Text We recently demonstrated the therapeutic efficacy of amphiphilic peptide vaccinations in a subcutaneous HPV16 (human papillomavirus 16) tumor model in MHC-humanized A2.DR1 mice. To better mimic the situation in patients, we established the model at an orthotopic location, namely the vaginal mucosa, as the final goal is to examine tumors located in this region to mimic cervicovaginal cancers. The mucosa is more difficult to access for T cells than the subcutis, therefore the orthotopic location will allow for a more detailed understanding of the requirements for successful therapeutic HPV16 vaccination. A suitable tumor model has to express the vaccination target proteins HPV16 E6 and E7, as well as an activated oncoprotein for tumorigenicity and luciferase for eventual tracking of tumor growth. For this project, we followed two paths: first, we adapted our HPV16-positive, but E6/E7-independent, tumor model PAP-A2 for the orthotopic setting and second, we developed a new tumor model based on E6/E7-dependent lung cells from A2.DR1 mice. Both cell types were transduced with HPV16 E6 and E7, transfected with luciferase and - in the case of the lung cells - with activated H-Ras to render them tumorigenic. Western blot analysis confirmed the expression of E6, E7 and H-Ras, and luminescence measurements proved luciferase expression. After validation of tumorigenicity of subcutaneously injected cells, cells were instilled intravaginally into A2.DR1 mice. Subsequent monitoring of the genital region revealed the growth of tumors in the vaginal cavity. We also determined the minimal cell number needed for reliable tumor formation. In addition to the establishment of the vaginal tumor model in A2.DR1 mice, we examine strategies to induce the migration of HPV16-specific T cells to the mucosa. This can be either achieved by directly inducing tissue-resident
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  • Type of the Paper (Article, Review, Communication
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