Biohybrid Cloaked Nanovaccines For Cancer Immunotherapy

Fontana F., 2019. Biohybrid Cloaked Nanovaccines for Immunotherapy Dissertationes Scholae Doctoralis Ad Sanitatem Investigandam Universitatis Helsinkiensis, 47/2019, pp.78 ISBN 987-951-51-5286-2 (Paperback), ISBN 978-951-51-5287-9 (PDF, http://ethesis.helsinki.fi), ISSN 2342-3161 Immunotherapy is revolutionizing cancer treatment achieving durable and long-term responses in patients. However, only subsets of patients treated experience a positive outcome, due to immunotherapeutic resistance. Combinations of immunotherapeutics can overcome the drug resistance; the administration of a cancer vaccine or an oncolytic virus followed by immune checkpoint inhibitors is under investigation. Thereby, there is an unmet need for powerful, yet safe vaccines. Nanoparticles, in particular porous silicon nanoparticles, present ideal characteristics to formulate nanovaccines, thanks to their size-specific targeting to the lymphoid organs, to their intrinsic adjuvant effect, and to the possibility to simultaneously load adjuvants and antigens. Moreover, biohybrid cell membrane technology has been proposed as an innovative antigenic source. Thus, the aims of the current thesis were to develop a biohybrid multistage nanovaccine formulation and to evaluate its anticancer efficacy in murine tumor models. Firstly, the parameters affecting the formulation of the biohybrid nanosystems were assessed, along with the elucidation of the influence of the cell membrane coating on the colloidal stability in physiological conditions and on the biocompatibility in different cell types. Secondly, the effect of the cell membrane-wrapping on the cellular uptake was evaluated in the presence of inhibitors of selective uptake pathways, to assess the differences between naked and coated nanoparticles. Then, a multistage nanovaccine was engineered by glass capillary microfluidics, followed by the cloaking with the cell membrane. The immunological profile of the nanovaccine was investigated in vitro, assessing the expression of co-stimulatory signals and the secretion of proinflammatory cytokines. The efficacy of the biohybrid nanovaccine as a monotherapy and in combination with an immune checkpoint inhibitor was then evaluated in melanoma murine models. Finally, the adjuvant core was changed from synthetic nanoparticles to oncolytic adenoviruses to investigate the translatability of the technique, the influence of the cell membrane-coating on the viral infectivity, and the preventive and therapeutic efficacy of the vaccine in different tumor models. Overall, porous silicon and adenovirus-based biohybrid nanovaccines were developed, providing new insights on the structure and efficacy of these systems as therapeutic cancer nanovaccines.Immunoterapia on mullistamassa syöpähoitoja, koska se mahdollistaa pitkäaikaisen hoitovasteen potilaassa. Kuitenkin vain pienellä joukolla potilaista hoito onnistuu, mikä johtuu immunoterapeuttisesta resistenssistä. Resistenssi voi johtua kasvaimen immuunisolujen puutteesta tai potilaan liian heikosti toimivasta immuunijärjestelmästä; jolloin hoitovastetta ei synny. Immuuniterapeuttisen resistenssin vaikutus voidaan kumota yhdistämällä erilaisia immuunihoitoja – tällä hetkellä tutkitaan yhdistelmää, jossa annostellaan joko syöpärokote tai onkolyyttinen virus ennen tarkistuspisteen estäjää. Tehokkaille ja turvallisille yhdistelmä-syöpärokotteille on perusteltu ja kasvava tarve. Nanohiukkasilla, erityisesti huokoisilla pii-nanohiukkasilla, on ihanteellisia ominaisuuksia nanorokotteiden formuloimiseksi. Kokonsa puolesta ne kohdentuvat imusolmukkeisiin. Lisäksi ne toimivat itsessään rokotteen vaikutusta tehostavina apuaineina ja niihin voi ladata samanaikaisesti muita tehoa lisääviä materiaaleja ja antigeenejä. Uudenlaisena antigeenien lähteinä on ehdotettu biohybridisolukalvoja. Tämän väitöskirjan tavoitteena oli kehittää moniosainen biohybridi-nanorokotevalmiste ja arvioida rokotteen tehoa syöpää vastaan hiirien syöpäkasvainmalleissa. Ensin arvioitiin biohybridi-nanorokotteiden formulointiin vaikuttavia tekijöitä ja solukalvosta valmistetun päällysteen vaikutusta kolloidiseen stabiilisuuteen sekä fysiologisissa olosuhteissa että biologiseen yhteensopivuuteen eri solutyypeissä. Seuraavassa vaiheessa verrattiin päällystettyjen ja päällystämättömien nanohiukkasten eroja tutkimalla solukalvopäällysteen vaikutusta endosytoosiin kun selektiivinen soluunottomekanismi on estetty. Tämän jälkeen valmistettiin mikrofluidiikkaan perustuvalla lasikapillaarimikrokanava -menetelmällä moniosainen nanorokote, jota seurasi rokotepartikkelien kapseloiminen solukalvolla. Nanorokotteen immunologista profiilia tutkittiin in vitro-kokeissa, joissa selvitettiin kostimuloivien signaalien ilmentymistä ja proinflammatoristen sytokiinien erittymistä. Biohybridi-nanorokotteen tehoa arvioitiin sekä monoterapiana että yhdistelmäterapiana immuunitarkastuspisteiden estäjien kanssa melanoomakasvainmallilla hiirissä. Lopuksi tutkittiin tekniikan soveltuvuutta ja solukalvosta valmistetun päällysteen vaikutusta virusten infektiotehokkuuteen vaihtamalla apuaineydin synteettisistä nanopartikkeleista onkolyyttisiin adenoviruksiin. Näiden lisäksi rokotteen ehkäisevää ja terapeuttista tehoa tutkittiin eri kasvainmalleilla. Tutkimuksessa kehitettiin uusia huokoiseen piihin ja adenoviruksiin perustuvia nanorokotteita, sekä saatiin uutta tietoa näiden rakenteesta ja toimivuudesta syöpärokotteina

Bio-Nanomedicine for Cancer Therapy Preface

Non peer reviewe

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