Imunoterapija z vakcino z bakteriofagi s predstavljenimi tumorskimi peptidi in citokinom interlevkinom 12 za zdravljenje mišjega malignega melanoma

Brišar, Nuša (avtor), Cör, Andrej (mentor)

Uporaba nanotehnoloških pristopov, med njim bakteriofagov s potencialom za genski inženiring, odpira nove možnosti pri razvoju protitumorskih vakcin proti malignemu melanomu. Namen raziskave doktorske naloge je bil pripraviti bakteriofagno vakcino in preveriti njeno protitumorsko učinkovitost na mišjem modelu malignega melanoma. Ovrednotili smo želeli tudi doprinos kombinacije bakteriofagne vakcine in genskega elektroprenosa (GET) plazmida z zapisom za interlevkin 12 (IL-12) k terapevtskemu potencialu. S tehnologijo predstavitve na bakteriofagu smo pripravili gensko spremenjene bakteriofage M13, ki so na površini kapside izražali tumorske peptide MAGE-A1, gp100 ali MART-1/MELAN-A. Izražanje fuzijskih proteinov smo preverili s prenosom western ali LC-MS/MS analizo. Terapevtski potencial vakcinacije z bakteriofagi samostojno ali v kombinaciji z GET IL-12 smo preverili in vivo na miših C57BL z malignim melanomom B16F10. Odziv na terapijo smo dodatno ovrednotili tudi s histološko in imunohistološko analizo. Po potrditvi rekombinantnih fagemidnih vektorjev s Sangerjevim sekvenciranjem in namnožitvi pomožnega filamentoznega bakteriofaga VCSM13 smo uspešno optimizirali proizvodnji proces namnoževanja in čiščenja gensko spremenjenih bakteriofagov M13. Med poskusi in vivo miši niso imele negativnih stranskih učinkov. Dokazali smo, da gensko spremenjeni bakteriofagi aktivirajo celično in humoralno imunost in da bakteriofagna terapija uspešno upočasni rast tumorjev. GET IL-12 doprinese k terapevtskemu učinku gensko spremenjenih bakteriofagov in poveča zaostanek v rasti tumorjev. Terapiji z gensko spremenjeni bakteriofagi in GET IL-12 imata sinergistični učinek in omogočata popolne ozdravitve v 30% primerov. Histološka in imunohistokemična analiza je pokazala, da tako bakteriofagna monoterapija, predvsem pa v kombinaciji z GET IL-12 poveča delež nekroze ter poveča infiltracijo makrofagov, CD4+ in CD8+ limfocitov T v tumorjih. Rezultati doktorske naloge so pokazali, da imajo bakteriofagne vakcine v kombinaciji z GET IL-12 potencial, ki bi ga bilo potrebno raziskati še naprej.

• Jezik: Slovenski jezik
• Ključne besede: Bakteriofagi, tehnologija predstavitve na bakteriofagu, imunoterapija, maligni melanom, interlevkin 12
• Vrsta gradiva: Doktorsko delo/naloga
• Organizacija: MF - Medicinska fakulteta
• Leto izida: 2024
• PID: 20.500.12556/RUL-163152
• Datum objave v RUL: 03.10.2024

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Immunotherapy with vaccine with bacteriophages displaying tumor peptides and cytokine interleukin 12 for mouse melanoma treatment

Izvleček

The use of nanotechnological approaches, including genetically engineered bacteriophages, opens up new possibilities for the development of anti-tumour vaccines against malignant melanoma. The aim of the doctoral research was to prepare a bacteriophage vaccine and to test its antitumour efficacy in a mouse model of malignant melanoma. We also wanted to evaluate the contribution of the combination of a bacteriophage vaccine and a gene electrotransfer (GET) of plasmids encoding interleukin-12 (IL-12) to the therapeutic potential. Using bacteriophage display technology, we produced genetically engineered M13 bacteriophages expressing the tumour peptides MAGE-A1, gp100 or MART-1/MELAN-A on the surface of the capsid. Expression of the fusion proteins was verified by Western transfer or LC-MS/MS analysis. The therapeutic potential of bacteriophage vaccination alone or in combination with GET IL-12 was tested in vivo in C57BL mice with B16F10 malignant melanoma. Response to therapy was further evaluated by histological and immunohistological analysis. After validation of the recombinant phagemid vectors by Sanger sequencing and amplification of the filamentous helper bacteriophage VCSM13, we successfully optimised the production process for amplification and purification of genetically engineered bacteriophages M13. No negative side effects were observed during in vivo experiments in mice. We have shown that genetically engineered bacteriophages activate cellular and humoral immunity and that bacteriophage therapy successfully slows tumour growth. GET IL-12 contributes to the therapeutic effect of genetically engineered bacteriophages and increases tumour growth delay. Genetically engineered bacteriophage and GET IL-12 therapies have a synergistic effect and lead to complete responses in 30% of cases. Histological and immunohistochemical analyses have shown that both bacteriophage monotherapy and especially in combination with GET IL-12 increase the proportion of necrosis and the infiltration of macrophages, CD4+ and CD8+ T lymphocytes in tumours. The results of the doctoral thesis showed that bacteriophage vaccines in combination with GET IL-12 have a potential that should be further investigated.

• Ključne besede: Bacteriophages, bacteriophage display technology, immunotherapy, malignant melanoma, interleukin 12