DEVELOPMENT OF INNOVATIVE VIRUSES WITH ENHANCED IMMUNOSTIMULATORY ACTIVITIES AGAINST GLIOBLASTOMA

Glioblastoma (GBM) is a deadly tumor of the central nervous system, with a median overall survival
of 15 to 16 months after tumor diagnosis. While standard treatments such as surgical resection,
radiotherapy, and chemotherapy can help manage symptoms and prolong survival, they are not
curative. Recent advances in immunotherapy have reignited interest in utilizing immunological
approaches to fight cancer. However, current immunotherapies failed in their journey from bench
to bedside for the treatment of GBM. The challenge of developing effective immune-based
approaches is compounded by the highly immunosuppressive GBM tumor microenvironment and
by the inadequacy of current preclinical models in fully recapitulating the complex biology of GBM
tumors. There is therefore the need to develop novel immunotherapeutic approaches to enhance
anti-GBM immunity and to establish innovative preclinical models to properly address immune-
mediated responses.
This PhD project aimed to enhance anti-tumor immunity via the delivery of the Class II Major
Histocompatibility Complex Transactivator (CIITA)-encoding gene (known as CIITA or AIR-1) to the
GBM tumor cells through a replication-deficient adenovirus (AdV). CIITA is the master regulator of
major histocompatability complex class II (MHC-II) molecules, expression of which is largely limited
to professional antigen presenting cells (APCs). Upon expression and translocation to the nucleus,
CIITA acts as a non-DNA binding transcriptional coactivator by interacting with the enhanceosome
complex. This complex consists of several constitutively-expressed DNA-bound factors
preassembled at MHC-II promoters. CIITA then nucleates various transcription factors and
chromatin modifiers necessary to activate the transcription of the human leukocyte antigen (HLA)-
DM, HLA-DO and invariant chain (Ii) molecules, which are involved in intracellular antigen
processing, as well as of the cell surface “classical” HLA-DR, HLA-DP and HLA-DQ glycoproteins,
which physically present antigens to CD4+ T helper cells. Based on this, we anticipated that AdV-
mediated delivery of CIITA would convert GBM cancer cells into APC-like cells capable of priming
and activating CD4+ Th cells, which is a necessary requirement for eliciting anti-tumor effector
immune responses.
The first chapter of the thesis results covers the development and characterization of an ex vivo
preclinical model consisting of patient-derived GBM organoids co-cultured with immune cells,
defined here as immunocompetent GBM organoids. This model served the purpose of functionally
evaluating immune-mediated responses against tumors. We observed that immunocompetent
GBM organoids allow to recapitulate tumor-immune cell crosstalk ex vivo and represent an
advanced model for functional ex vivo assays in a reproducible manner.
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In the second chapter of the thesis results, we achieved successful construction and production of
a CIITA-armed AdV (Ad-CIITA), utilizing a replication-defective Ad5 backbone. AdV controls included
a promoter-only vector (Ad-null), a CIITA transgene carrying a missense point mutation (Ad-CIITA
mutant), and a green fluorescent protein (GFP)-encoding transgene (Ad-GFP). We demonstrated a
successful induction of MHC-II expression at the cell membrane mediated through CIITA in infected
cell lines and primary GBM organoids. Infection with an AdV carrying a mutant form of CIITA
resulted in the cytoplasmic accumulation of CIITA without subsequent MHC-II expression.
In the third chapter of the thesis results, we tested the functionality of the CIITA-armed viruses to
initiate adaptive immune responses in immunocompetent GBM organoids. In our ex vivo model,
tumor organoids infected with Ad-CIITA underwent significant disruption and tumor cell death, an
effect that was exclusively observed in the presence of immune cells. Of note, Ad-CIITA mutant, but
not Ad-GFP and Ad-null, induced the same immune cell-mediated killing phenotype as Ad-CIITA.
These results indicate that the effect observed follows a CIITA-induced MHC-II-independent
mechanism, challenging the initial hypothesis of MHC-II-mediated antigen presentation as the
mechanism implicated in T cell-mediated killing. We further showed that cancer cell death required
direct contact between GBM and immune cells, though none of the canonical death pathways
seemed to be activated. In conclusion, our results suggest that the delivery of CIITA transgene via
AdV vectors could be an effective approach to enhance immune-mediated responses against GBM.