Abstract Book 2010 - CIMT Annual Meeting

016 van Nuffel | Therapeutic vaccination
T cell specificities after vaccination of melanoma patients with
mRNA loaded DC
An MT Van Nuffel 1 , Daphné Benteyn 1 , Sofie Wilgenhof 1,2 , Jurgen Corthals 1 , Carlo Heirman 1 , Bart Neyns 2 ,
Kris Thielemans 1 and Aude Bonehill 1
1 Laboratory of Molecular and Cellular Therapy, Vrije Universiteit Brussel, Brussels, Belgium
2 Department of Medical Oncology, Universitair Ziekenhuis Brussel, Brussels, Belgium
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The incidence of melanoma doubles every 10 years.
The use of full-length tumor associated antigens
(TAAs) to load dendritic cells (DCs) is proposed
to improve DC based immunotherapy because
this should allow vaccination irrespective of the
patient’s HLA-type and stimulation of a broad spectrum
of TAA-recognizing T cells.
Our objective was to investigate the in vivo stimulatory
potency of DCs loaded by electroporation with
defined, full-length TAA-encoding mRNA. Defined
mRNA was chosen as antigen carrier because it can
be modified to enhance the antigen presentation by
DCs to both CD8+ and CD4+ T cells. The exact T
cell specificities of vaccine stimulated T cells were
analyzed.
Histologically confirmed AJCC stage IV melanoma
patients included in a phase I clinical study
donated a skin biopsy one week after the last of
four biweekly DC-vaccines. Vaccination occurred
with an equal mix of TriMix-DC (i.e. DCs matured
using constitutive active TLR4, CD40L and CD70
mRNA) co-electroporated with mRNA encoding
gp100, tyrosinase, Mage-C2 or Mage-A3 fused to
the HLA-class II targeting sequence of DC-Lamp.
Skin biopsies were taken 72h after induction of the
delayed type hypersensitivity (DTH) response by
intradermal injection of a small amount of vaccine
DCs. DTH-infiltrating lymphocytes (DIL) were cultured
during 2,5 weeks in the presence of 100 IU/
ml IL-2. The DIL were re-stimulated overnight by
autologous EBV-B cells electroporated with TAA
mRNA. Activated T cells were detected by both
CD137 upregulation in flow cytometry and cytokine
secretion (IFN-γ and TNF-α) using a cytokine
bead assay. Once T cells specific for a vaccine
antigen were found, the recognized epitope was determined
using autologous EBV-B cells loaded with
overlapping peptides covering the TAAs. The HLA
restriction was assessed by allogeneic EBV-B cells
expressing relevant HLA-types and vaccine TAA.
This way, we found CD8+ and CD4+ T cells
specific for the previously unidentified epitopes
RTCQCSGNF and YPPLHEWVLREG, derived
from the tumor antigens tyrosinase and Mage-A3,
respectively. These epitopes were presented respectively
in the less common HLA-B57 and the
common HLA-DQB1*05. In vivo stimulation of the
CD8+ T cells by the TriMix-DCs was confirmed by
the analysis of blood samples obtained pre- and
postvaccination.
In conclusion, vaccination of melanoma patients
with mRNA loaded DCs leads to in vivo stimulation
of CD8+ and CD4+ T cells recognizing previously
unknown epitopes presented in both common and
less common HLA-types. This finding underscores
the broadness of the induced T cell response and
thereby the strength of using mRNA loaded DCs.
On the other hand, it shows that DC-therapy is no
longer solely suitable for a selected population with
a certain HLA-type but can be beneficial for all patients.