Title PROTUTUMORSKA AKTIVNOST IMUNOKOMPLEKSA
αCD155/SAPORIN NA GLIOBLASTOM Title (english) ANTITUMOR EFFECT OF αCD155/SAPORIN IMMUNOCOMPLEX ON
GLIOBLASTOMA Author Hrvoje Šimić Mentor Stipan Jonjić (mentor)Mentor Bojan Polić (komentor)Committee member Astrid Krmpotić (predsjednik povjerenstva)Committee member Kamelija Žarković (član povjerenstva)Granter University of Rijeka Defense date and country 2018-12-03, Croatia Scientific / art field, BIOMEDICINE AND HEALTHCARE Scientific / art field, BIOMEDICINE AND HEALTHCARE Universal decimal classificationUDC ) 61 - Medical sciences ThesaurusMESH - Medical Subject Headings )
Glioblastoma
therapy
Antibodies, Monoclonal
therapeutic use
Immunotherapy
Abstract Cilj istraživanja:
Glioblastom multiforme predstavlja jedan od najmalignijih tumora čovjeka. Unatoč
golemom napretku, dijagnoza glioblastoma u pravilu znači kratak period preživljenja.
Standardno liječenje ne dovodi do izlječenja i zadovoljavajuće kontrole bolesti, stoga
se paralelno razvijaju napredne strategije liječenja, utemeljene na razvoju pametnih
lijekova, posebice onih utemeljenih na adaptaciji monoklonskih protutijela. Vođeni
ovim saznanjima glavni cilj nam je bio testirati koncept proizvodnje biotiniliranih
monoklonskih protutijela unutar “KAC” sustava kao modela razvoja terapijskih
protutijela, te adaptacija dobivenih protutijela u funkcionalni imunotoksin usmjeren na
receptor CD155.
Materijali i metode:
Mišji sojevi KAC C57B/6 i Balb/c korišteni su za imunizaciju. Stanična linija Sp2/O je
modificirana i testirana na sposobnost biotinilacije unutar biotinilacijskog sustava in
vivo. Proizvedena protutijela su potom testirana na specifičnost prepoznavanja
receptora CD155 u različitim tehnikama. Biotinilirano protutijelo αCD155.16
konjugirano saporinom je potom testirano citotoksičnim testom na stanicama
glioblastoma U87 MG. Nakon pozitivnog rezultata varijabilne sekvence protutijela su
izolirane metodom lančane reakcije polimeraze (PCR) te iskorištene za dizajn
imunotoksina. Imunotoksin αCD155-DT je dizajniran in silico a potom proizveden u
bakterijskom sustavu. Dobiveni imunotoksin smo testirali citotoksičnim testom na
tumorskim stanicama U87 MG u in vitro i in vivo uvjetima. Uzorci glioblastoma
pohranjeni u parafinskim kockama na Zavodu za patologiju testirani su na ekspresiju
receptora CD155 koristeći novoproizvedena protutijela.
Rezultati:
Istraživanjem je pokazano kako je sustav za proizvodnju biotiniliranih protutijela
“KAC” primjeren za navedena istraživanja. Njegovim razvojem stvorena je platforma
za razvoj monoklonskih protutijela temeljenim na ciljanoj biotinilaciji. Također,
pokazali smo kako je korištenjem navedenog sustava moguć brzi probir protutijela s
terapijskim potencijalom i njihova transformacija u “pametne lijekove” - imunotoksine.
Važnost ekspresije receptora CD155 na stanicama tumora dodatno je potvrđena imunohistokemijskom analizom banke tumora Zavoda za patologiju koja je pokazala
kako svi testirani tumori ispoljavaju receptor CD155, te kako niska ekspresija
receptora CD155 ima povoljan prognostički predznak kod oboljelih od glioblastoma.
Zaključak:
Razvili smo biotinilacijski sustav za proizvodnju monoklonskih protutijela in vivo
utemeljen na specifičnoj enzimski posredovanoj biotinilaciji. Dobiveni rezultati
proširili su spoznaje o važnosti ekspresije receptora CD155 na tumorskim
stanicama. Dodatno, rezultati su pokazali opravdanost strategije razvoja specifičnih
imunotoksina usmjerenih na receptor CD155 za specifično uništavanje tumorskih
stanica.
Abstract (english) Objectives:
Glioblastoma multiforme represent one of the most malignant tumors. Although, we
made a huge step forward in diagnostics and therapy of tumors, standard treatments
are still insufficient for disease control. In the meantime, more advanced therapy
methods are developed, most of them are based on monoclonal antibodies and their
derivatives. Following these findings, our main goal was to develop and test the
system for production of in vivo biotinylated monoclonal antibodies “KAC”. Special
interest was to adapt the system for transfer of research findings on glioblastoma
biomarkers (i.e. CD155) into effective immunotoxin. In addition, we evaluated level of
expression of receptor CD155 on glioblastoma tumor samples of patients treated on
Department of neurosurgery, Clinical Hospital Center Rijeka regarding the level of
their malignancy.
Materials and methods:
Mouse strains KAC C57BL/6 and Balb/c were used for immunization. Cell line Sp2/O
was modified to express BirA enzyme and tested for fusion. Antibodies obtained
from fusions were further tested for specificity for CD155. Cell line SP2/O was
modified to express BirA enzyme. Biotinylated antibody αCD155.16 was enriched
with saporin and tested in cytotoxic test. Variable sequences of the specific antibody
were isolated and used for design of imunotoxin αCD155-DT. Imunotoxin was further
tested on U87 MG glioblastoma cells in in vitro and in vivo experiments. Expression
of CD155 receptor on glioblastoma samples was determined by using specific
αCD155-KLH.03 antibody.
Results:
This study showed that system for biotinylated antibody production is efficient and
feasible to use. We made a platform for development of new biotinylated monoclonal
antibodies. That enable us for “quick search” for antibodies with therapeutic potential
and their transformation into “smart drugs” – immunotoxins against Furthermore, we confirmed positive correlation of CD155 expression with the
survival of patient with glioblastoma.
Conclusion:
We developed an efficient system for in vivo production of biotinylated monoclonal
antibodies with potential use in immunotherapy. Obtained results pointed on the
importance of CD155 expression on glioblastoma for the disease development and
prognosis. Finally, the data highlighted relevance of CD155 targeted therapy .
Keywords
glioblastom multiforme
receptor
CD155
imunoterapija
izražaj receptora CD155
biotinilacija protutijela
Keywords (english)
glioblastoma multiforme
CD155
receptor
immunotherapy
CD155
expression
antibody
biotinylation
Language croatian URN:NBN urn:nbn:hr:184:512275 Promotion 2020 Study programme Title: Biomedicine Postgraduate (doctoral) study programme Type of resource Text File origin Born digital Access conditions Access restricted to students and staff of home institution Terms of use Created on 2019-01-17 18:22:48
