Abstract Book 2010 - CIMT Annual Meeting

011 Kuhs | Therapeutic vaccination
Clinical Development of lentiviral vector-induced „SMART-DC“
for melanoma immunotherapy
Sandra Kuhs 1 , Ralf Gutzmer 2 , Bala Sai Sundarasetty 1 , Sylvia Borchers 1 , Gustavo Salguero 1 ,
Arnold Ganser 1 , Rainer Blasczyk 3 , Henk Garritsen 4 , Thomas Woelfel 5 , Farzin Farzaneh 6 and
Renata Stripecke 1
1 Dept. of Hematology, Hemostasis, Oncology and Stem Cell Transplantation
2 Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Poland
3 Dept. of Transfusion Medicine, Hannover Medical School
4 Institute for Clinical Transfusion Medicine, Städtisches Klinikum Braunschweig gGmbH
5 Hematology and Oncology, University of Mainz, Germany
6 Dept. of Hematological Medicine, Kings College, London, UK
Clinical ex vivo production of dendritic cells (DC)
is costly, time-consuming and difficult for largescale
clinical trials. In addition, ex vivo grown DCs
are not highly viable once re-infused in the body
and antigen-presentation and bio-distribution are
sub-optimal. We developed a novel technology for
production of DC consisting of lentiviral vector
(LV) transduction of growth factors and full-length
antigens into monocytes that results into induction
of “SMART-DCs” (Self-differentiated Myeloidderived
Antigen-presenting-cells Reactive against
Tumors). This concept has been extensively tested
and validated in pre-clinical melanoma mouse
models for safety and for induction of protective
immunity. Here, self-inactivating lentiviral vectors
containing interspacing 2A elements and a nonencoding
Wpre were produced in a bicistronic (coexpressing
simultaneously human GM-CSF and
IL-4) or tricistronic design (expressing in addition
the melanoma-associated antigens MART-1 or
TRP-2). Co-expression of all the transgenes was validated
in 293T cells transduced with these vectors.
Overnight transduction of fresh or cryopreserved
CD14+ monocytes resulted in persistent autocrine
production of GM-CSF (average 7 ng/ml) and IL-4
(average 0.8 ng/ml), inducing the self-differentiation
of long-lived (up to 3 weeks) human SMART-
DCs, which fully recapitulated the immunophenotype
of conventional DCs. For future GMP process
development, we evaluated monocyte transduction
in a closed GMP-grade bag system. We were able
to demonstrate that SMART-DCs were produced
more effectively in bags than in culture flasks. Monocytes
co-transduced with the bicistronic vector
plus a vector expressing full length MART-1 were
recognized by T cell clones specific for MART-1
in an HLA-restricted manner, assessed by IFN-γ-
ELISPOT-Assay. PBMCs that were primed/boosted
in vitro with autologous SMART-DCs expressing
MART-1 and assayed by IFN-γ-ELISPOT-Assay
(using peptide-pulsed T2 cells as target cells) demonstrated
the induction of MART-1-specific T cell
responses. Pre-clinical validation of SMART-DCs
engineered with tricistronic vectors for MART-1
or TRP2 expression, for the assessment of their
potency to stimulate and expand autologous T cells
obtained from melanoma patients is underway. A
pre-GMP lentiviral production for generation of
GMP compliant vector will follow, for development
of a phase I immunotherapy clinical trial for melanoma
patients.
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