​Protitumorski učinek avtologne tumorske vakcine v kombinaciji z obsevanjem na mišjih tumorskih modelih

Znanstveno izhodišče: Avtologne tumorske vakcine so tip terapevtskih vakcin, s katerimi želimo spodbuditi bolnikov imunski odziv za boj proti rakavemu obolenju. Sestavljene so iz gensko spremenjenih tumorskih celic, inaktiviranih z obsevanjem, ali iz tumorskega lizata. Nabor avtolognih tumorskih vakcin je majhen, med njimi pa še nobena ni bila odobrena za standardno zdravljenje. Namen doktorske raziskave je bil priprava in testiranje nove avtologne tumorske vakcine, pripravljene iz mrtvih tumorskih celic, z adjuvantnim genskim elektroprenosom plazmidne DNA z zapisom za interlevkin-12 (GEP IL-12) in vivo. Učinkovitost vakcine smo testirali v kombinaciji z lokalno radioterapijo, s katero smo želeli izpostaviti tumorske antigene, in zaviralci imunskih kontrolnih točk (ZIKT), protitelesi anti PD-1 in anti CTLA-4, s katerima smo želeli sprositi morebitno zavrtje imunskega sistema. Metode: Za raziskave smo uporabili mišje tumorske celice B16-F10 in CT26. Poskusi in vivo so potekali na miših C57Bl/6NCrl in Balb/cAnNCrl, ki so singenske izbranima tumorskima modeloma. Protitumorski učinek terapij smo spremljali z merjenjem velikosti tumorjev. Namen prvega dela raziskav je bil razvoj učinkovite tumorske vakcine B16-F10 in CT26. Pilotno vakcino smo pripravili z obsevanjem celic B16-F10 z dozo 25 Gy in jo testirali in vivo v kombinaciji z obsevanjem tumorjev. Pilotno vakcinacijo smo izvedli tako, da smo v podkožje miši, oddaljeno od tumorja, injicirali mrtve tumorske celice in izvedli GEP IL-12 okoli injiciranih mrtvih tumorskih celic (2,82 Hz, 170 V/cm, 150 ms). V nadaljevanju smo glede na rezultate histološke analize mesta pilotne vakcinacije izboljšali postopek priprave vakcine. Natančneje, izboljšano vakcino B16-F10 smo pripravili z obsevanjem 3 × 5 Gy in nato 30 Gy, izboljšano vakcino CT26 pa z obsevanjem 3 × 5 Gy in nato 2 × 30 Gy. Izboljšano vakcinacijo smo izvedli tako, da smo v podkožje miši, oddaljeno od tumorja, injicirali mešanico mrtvih tumorskih celic in plazmida IL-12 in izvedli GEP IL-12 v mešanici (5,64 Hz, 170 V/cm, 150 ms). Za določitev doprinosa vakcinacije k protitumorskemu učinku obsevanja (15 Gy) in vpliva vakcinacije na izrast tumorjev smo testirali dva odmerka vakcine (0,5 in 1 mg). Namen drugega dela raziskave je bila primerjava različnih obsevalnih in vakcinacijskih režimov. Najprej smo primerjali tri različne obsevalne režime: 1 × 5, 3 × 5 in 5 × 5 Gy v kombinaciji z vakcinacijo (1 mg) ob prvi frakciji obsevanja. Nato smo primerjali tri vakcinacijske režime: enkratno in večkratno vakcinacijo sočasno z obsevanjem ter večkratno vakcinacijo, ki smo jo začeli pred prvo dozo obsevanja. Namen tretjega dela raziskave je bilo ovrednotenje lokalnega in sistemskega imunskega odziva po kombinirani terapiji z izbranim obsevalnim in vakcinacijskim režimom pri obeh tumorskih modelih. Za imunohistokemijsko analizo smo odvzeli vzorce kože na mestu vakcinacije in tumorje, pri čemer smo označili imunske celice, pozitivne na grancim B, CD68 in FoxP3. Z analizo FluoroSpot smo analizirali prisotnost tumorsko specifičnih efektorskih imunskih celic, pozitivnih na grancim B in interferon ?, v bezgavkah. V četrtem delu raziskav smo ovrednotili doprinos ZIKT k vakcinaciji v kombinaciji z obsevanjem s protitumorsko učinkovitostjo ter histološko analizo tumorske nekroze in infiltracije imunskih celic v koži na mestu vakcinacije in v tumorjih. Rezultati: Po pilotni vakcinaciji smo na mestu vakcinacije opazili še žive tumorske celice ter večjo infiltracijo imunskih celic na mestu GEP kot na mestu injiciranih mrtvih tumorskih celic. Po izboljšanju postopka priprave vakcine na mestu izboljšane vakcinacije ni bilo več živih celic in opazili smo bolj enakomerno infiltracijo imunskih celic. Z enkratno izboljšano vakcinacijo smo doprinesli k protitumorskemu učinku obsevanja z dozo 15 Gy le pri tumorskem modelu B16-F10, ne pa tudi pri tumorskem modelu CT26. Po drugi stani pa smo z enkratno izboljšano vakcinacijo preprečili izrast do 56 % tumorjev CT26, ne pa tudi tumorjev B16-F10. Najučinkovitejši odmerek vakcine pri obeh tumorskih modelih je bil 1 mg. Najučinkovitejši obsevalni režim v kombinaciji z enkratno vakcinacijo pri obeh tumorskih modelih je bil 5 × 5 Gy. Najučinkovitejši vakcinacijski režim v kombinaciji s 5 × 5 Gy pri tumorskem modelu B16-F10 je bila enkratna vakcinacija, pri tumorskem modelu CT26 pa trikratna vakcinacija, izvedena sočasno z obsevanjem. Z vakcinacijo smo uspešno sprožili lokalni in sistemski imunski odziv, saj smo v kožo na mesto vakcine ter v tumor privabili makrofage, efektorske limfocite ter regulatorne limfocite. ZIKT niso doprinesli k protitumorskemu učinku vakcinacije v kombinaciji z obsevanjem ter niso povečali količine tumorske nekroze in infiltracije imunskih celic v tumor ali v kožo na mestu vakcinacije. Zaključki: Uspešno smo pripravili učinkovito avtologno tumorsko vakcino B16-F10 in CT26. S kombinirano terapijo z izbranimi obsevalnimi in vakcinacijskimi režimi smo pri obeh tumorskih modelih potrdili hipotezo, da vakcinacija doprinese k protitumorskemu učinku radioterapije. Prav tako smo pri obeh tumorskih modelih potrdili hipotezo, da z vakcinacijo izzovemo lokalni in sistemski imunski odziv. Zadnjo hipotezo smo ovrgli, saj smo dokazali, da ZIKT ne doprinesejo k protitumorskemu učinku vakcinacije v kombinaciji z obsevanjem. Rezultati doktorske raziskave bodo osnova za nadaljnji razvoj avtologne tumorske vakcine. Scientific Background: The goal of autologous tumor cell-based vaccines, a type of therapeutic vaccines, is to enable the patient's immune system to fight against cancer. These consist of genetically modified and with irradiation inactivated tumor cells or tumor cell lysates. The current repertoire of autologous tumor cell-based vaccines remains low. The objective of this doctoral research was to develop and test a novel autologous tumor cell-based vaccine, containing non-viable tumor cells, with adjuvant in vivo IL-12 GET. The efficacy of the vaccine was examined in combination with tumor irradiation therapy, the goal of which was to expose tumor antigens. We also examined the efficacy of vaccination in combination with tumor irradiation therapy and immune checkpoint inhibition with aniti-PD-1 and anti-CTLA-4 antibodies to release possible inhibition of the immune system. Materials and Methods: Murine B16-F10 and CT26 tumor cell lines were used during the doctoral research. In vivo experiments were performed in syngeneic C57Bl/6NCrl and Balb/cAnNCrl mice. The antitumor efficacy was determined through the measured tumor volume. In the first part of our research, we developed B16-F10 and CT26 autologous tumor cell-based vaccines. A pilot vaccine was prepared by irradiating B16-F10 cells with a dose of 25 Gy, which we then tested in vivo in combination with tumor irradiation. Pilot vaccination was performed with a subcutaneous injection of non-viable tumor cells and IL-12 GET was performed around the injection site (2.82 Hz, 170 V/cm, 150 ms). The vaccine preparation procedure was improved based on the histological analysis of the pilot vaccination site. The modified B16-F10 vaccine was prepared with 3 × 5 Gy and 30 Gy irradiation, while the modified CT26 vaccine was prepared with 3 × 5 Gy and 2 × 30 Gy irradiation. The modified vaccines were administered subcutaneously using a mixture of non-viable tumor cells and IL-12 plasmid followed by GET of IL-12 contained within the injected vaccine (5.64 Hz, 170 V/cm, 150 ms). Two vaccination doses (0.5 and 1 mg) were tested in combination with tumor irradiation (15 Gy) to determine the antitumor efficacy. The ability of vaccination (0.5 and 1 mg) to prevent tumor outgrowth was also examined. In the second part of our research, we compared different irradiation and vaccination regimens. First, we compared three irradiation regimens: 1 × 5, 3 × 5 and 5 × 5 Gy, in combination with concurrent single-dose vaccination (1 mg). Then we compared three vaccination regimens: single- and multiple-dose vaccination with concurrent irradiation as well as multiple-dose vaccination, which began before irradiation. In the third part of our research, we determined the local and systemic immune response to combination treatment with the chosen vaccination and irradiation regimens. To determine granzyme B, CD68 and FoxP3 positive immune cells, skin at the vaccination site and tumors were harvested for immunohistochemical analysis. To determine tumor specific granzyme B and interferon γ immune cells, lymph nodes were harvested for FluoroSpot analysis. In the final part of our research, we determined the antitumor efficacy of checkpoint inhibition in combination with vaccination and tumor irradiation. Histological analysis of tumor necrosis and immune cell infiltration in tumors and skin at the site of vaccination was also performed. Results: Viable tumor cells were observed at the pilot vaccination site. Additionally, greater infiltration of immune cells was observed at the site of IL-12 GET than at the site of injected non-viable tumor cells, whereas at the adjusted vaccination site, non-viable tumor cells and a more uniform distribution of immune cells was observed. Single-dose vaccination contributed to antitumor efficacy of irradiation (15 Gy) in the B16-F10 but not in the CT26 tumor model, whereas single-dose vaccination prevented outgrowth of up to 56 % CT26 tumors however, it did not prevent outgrowth of B16-F10 tumors. The most effective vaccine dose was 1 mg in both tumor models. The most effective irradiation regimen was 5 × 5 Gy with concurrent single-dose vaccination in both tumor models. The most effective vaccination regimens were single-dose vaccination and multiple-dose vaccination with concurrent irradiation (5 × 5 Gy) in the B16-F10 and the CT26 tumor models, respectively. By attracting macrophages, effector lymphocytes and regulatory lymphocytes, vaccination successfully elicited a local and systemic immune response. Finally, immune checkpoint inhibition did not contribute to antitumor efficacy of vaccination in combination with irradiation and it did not affect the amount of tumor necrosis or immune cell infiltration. Conclusions: We successfully developed effective B16-F10 and CT26 autologous tumor cell-based vaccines. By combining the chosen irradiation and vaccination regimens, we confirmed our hypothesis that vaccination contributes to irradiation in both tumor models. We also confirmed our next hypothesis that vaccination elicits a local and systemic immune response in both tumor models. We rejected the final hypothesis since immune checkpoint inhibition did not contribute to antitumor efficacy of vaccination in combination with irradiation. The results of this doctoral dissertation will be the foundation for further development of the autologous tumor cell-based vaccine.