| dc.description.abstract | Background Immunosuppressive therapy or T-cell
depletion in transplant patients can cause uncontrolled
growth of Epstein-Barr virus (EBV)-infected B cells
resulting in post-transplant lymphoproliferative disease
(PTLD). Current treatment options do not distinguish
between healthy and malignant B cells and are thereby
often limited by severe side effects in the already
immunocompromised patients. To specifically target EBVinfected B cells, we developed a novel peptide-selective
chimeric antigen receptor (CAR) based on the monoclonal
antibody TÜ165 which recognizes an Epstein-Barr
nuclear antigen (EBNA)−3C-derived peptide in HLA-B*35
context in a T-cell receptor (TCR)-like manner. In order
to attract additional immune cells to proximity of PTLD
cells, based on the TÜ165 CAR, we moreover generated
T cells redirected for universal cytokine-mediated killing
(TRUCKs), which induce interleukin (IL)-12 release on
target contact.
Methods TÜ165-based CAR-T cells (CAR-Ts) and TRUCKs
with inducible IL-12 expression in an all-in-one construct
were generated. Functionality of the engineered cells was
assessed in co-cultures with EBNA-3C-peptide-loaded,
HLA-B*35-expressing K562 cells and EBV-infected B
cells as PTLD model. IL-12, secreted by TRUCKs on target
contact, was further tested for its chemoattractive and
activating potential towards monocytes and natural killer
(NK) cells.
Results After co-cultivation with EBV target cells,
TÜ165 CAR-Ts and TRUCKs showed an increased
activation marker expression (CD137, CD25) and release
of proinflammatory cytokines (interferon-γ and tumor
necrosis factor-α). Moreover, TÜ165 CAR-Ts and TRUCKs
released apoptosis-inducing mediators (granzyme B and
perforin) and were capable to specifically lyse EBV-positive
target cells. Live cell imaging revealed a specific attraction
of TÜ165 CAR-Ts around EBNA-3C-peptide-loaded target
cells. Of note, TÜ165 TRUCKs with inducible IL-12 showed
highly improved effector functions and additionally led to
recruitment of monocyte and NK cell lines.
Conclusions Our results demonstrate that TÜ165 CARTs recognize EBV peptide/HLA complexes in a TCR-like
manner and thereby allow for recognizing an intracellular
EBV target. TÜ165 TRUCKs equipped with inducible
IL-12 expression responded even more effectively and
released IL-12 recruited additional immune cells which
are generally missing in proximity of lymphoproliferation in
immunocompromised PTLD patients. This suggests a new
and promising strategy to specifically target EBV-infected
cells while sparing and mobilizing healthy immune
cells and thereby ena | en_US |
