Abstract Book 2010 - CIMT Annual Meeting

053 Stolle | Cellular therapy
Generation of HCMV-/TAA-bispecific human T cells by
either the genetic equipment of HCMV+ T cells with
tumor-reactive TCRs or the combined retroviral transduction
of bulk human T cells with HCMV-/TAA-specific TCRs
Diana Stolle 1 , Beate Hauptrock 1 , Hakim Echchannaoui 1 , Edite Antunes 1 , Simone Thomas 1 ,
Sebastian Klobuch 1 , Wolfgang Herr 1 , Matthias Theobald 1 , Ralf-Holger Voss 1
1 Department of Hematology & Oncology, University Medical Center, Mainz, Germany
Immune suppression after allogeneic stem cell
transplantation causes reactivation of human cytomegalovirus
(HCMV) and comes along with increased
mortality in HCMV positive patients. The
aim of cellular immunotherapy is the eradication
of tumor cells and the decrease of the HCMV viral
load by adoptive transfer of HCMV-/tumor associated
antigen (TAA)-bispecific T lymphocytes.
Therefore, we performed two different strategies:
First, we generated HCMV-reactive (HCMV+) T
cells from seropositive human blood donors using
pp65(NLVPMVATV(495-503)) peptide-specific stimulation.
In this case one or two restimulations
led to the efficient accumulation of up to 80 %
HCMV-specific T cells. To obtain a pure HCMV T
cell culture we sorted the cells with CMV pp65specific
streptamers by flow cytometry. Cytotoxicity
was assessed in a 51-Chromium release assay
using either pp65- loaded K562-A2 or HCMV-infected
HLA-A*0201+ human fibroblasts as target
cells. For distinct HCMV+ donors, minimal pp65
peptide concentrations were in the range of as low
as 0.03 nM to 100 nM triggering specific lysis at
CD8+ HCMV+ to target ratios of 5:1 up to 20:1.
They were indicative of high TCR affinities for the
cognate antigen reflected by EC50-values ranging
from 0.1 nM to 1.8 nM in peptide titration. Most importantly,
HCMV T cells were efficiently capable of
lysing CMV-infected human fibroblasts. After their
antigen-specific expansion, HCMV-reactive T cells
were equipped with a p53- or gp100- tumor anti-
genspecific TCR by either RNA transfection or retroviral
transduction. RNA electroporation yielded
the generation of p53- as well as gp100- bispecific
HCMV+ T cells: Both subsets proved efficient
bifunctionality in CTL-assay and IFNg-Elisa after
endogenous (pp65)- or exogenous (p53/gp100)
peptide-specific stimulation. Preliminary data indicated
that in retroviral transduction either TCR,
the endogenous TCR CMV or the introduced TCR,
are prone to be down-regulated dependent on the
particular HCMV/TAA TCR combination.
Our second approach was the simultaneous retroviral
transduction of bulk human T cells with a
HCMV- as well as p53 tumor antigen- specific TCR.
Both retroviral TCR constructs contained drug selection
cassettes which allowed for a normalization
of TCR expression. In tetramer analysis, we
were able to detect both, single TCR+ T cells and a
substantial fraction of HCMV/TAA double TCR+ T
cells. Endogenous TCRs were still present. Bifunctionality
was corroborated in CTL-assays and in intracellular
IFNg secretion stainings for specifically
restimulated T cells.
In summary, our data demonstrate two opportunities
to generate virus (HCMV) and tumor antigen
bispecific human T cells. Most importantly, the
expression level of the HCMV- as well as tumor
antigen- specific TCR is sufficient for specific
antigen recognition.
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