Research Report 2010 2011 - Helmholtz-Zentrum für ...

106 SCIENTIFIC REPORTS | Infection and Immunity | Strategies for Prevention and Therapy
04.5 Therapeutic Cellular Vaccines
PROJECT LEADER | Dr. Werner Lindenmaier | Department of Gene Regulation and Differentiation |
wli@helmholtz-hzi.de
PROJECT MEMBERS | Dr. Kurt E. J. Dittmar | Dr. Wilhelm Meyring | Dr. Oliver Schön | Dr. Nadia Zghoul |
Claudia Preuß | Ellen Kuppe
Cell-based immunotherapies have great potential for the
treatment of tumours and persistent infections. Activation
of the cellular immune response can eliminate the cancer
cells and the cells which are persistently infected. However,
tumour cells and persistent pathogens have evolved mechanisms
to escape the normal immune response. Therefore,
specific stimulation by professional antigen presenting cells,
especially dendritic cells (DC), is necessary to overcome
these obstacles. On the other hand, a strong induction of
the immune response, especially when directed against self
tumour antigens, bears the danger of inducing pathogenic
autoimmunity. In this case control of the overwhelming
response by immunesuppressive cells like mesenchymal
stromal cells is an attractive option.
Sterile docking for cellular therapies However, the biggest
obstacle for broader application of cellular therapies is the
requirement of autologous primary cells which have to
be prepared under cGMP conditions in accordance to EU
legislation.
As a tool to overcome this hurdle we have developed completely
closed bag systems for cGMP compliant dendritic cell
generations. Using sterile docking, all steps starting from cell
isolation up to the final formulation and cryo-conservation
of adenovirally modified DC can be realized without opening
the system. In addition to the development of the cultivation
system we investigated whether the stimulatory capacity of
the DC vaccine is enhanced by infection with M. bovis BCG
using flow cytometry, expression profiling and confocal and
electron microscopy and how the balance between anti-tumour
immunity and autoimmunity after DC vaccination can
be controlled in a transgenic murine model system.
So far, the bag system only allowed the cultivation of suspension
cells. Adherently growing cells like mesenchymal stem
cells require adherence-compatible modified surfaces. This
modification can be achieved using dielectric barrier discharge
(plasma). Plasma treatment in the presence of suitable
precursors such as silanes or amino-functionalized compounds
allows selective modification of surface properties.
We have identified suitable surface coatings, which support
adherence and survival of MSC whereas cells in unmodified
bags aggregated and eventually died.
Modified bag MSC in comparison to conventional flask
MSC MSC grown on modified bag surfaces were extensively
analysed in comparison to conventional flask MSC. Analysis
of cell specific surface markers, gene transfer efficiency,
adipogenic and osteogenic differentiation and global expression
profiles did not reveal significant differences between
bag- and flask-MSC. Bag modification using dielectric barrier
discharge therefore opens up new possibilities for the cultivation
of therapeutic cells in a closed system.
Furthermore, secondary surface modification (e.g. by
coupling of cell-specific antibodies) has been used to isolate
CD14 + monocytes from peripheral blood. Leukocytes and
structured modification of the surface allowed localized
adherence and genetic modification providing a tool for the
analysis of interactions between different cell types.
Garritsen,H.S., Macke,L., Meyring,W., Hannig,H., Pägelow,U., Wörmann,B., Geffers,R.,
Dittmar,K.E., and Lindenmaier,W. (2010). Efficient generation of clinical-grade genetically
modified dendritic cells for presentation of multiple tumor-associated proteins.
Transfusion. 50, 831-842.
Adherent growth on modified surface and karyotype stability
of MSC A) Electron micrograph of MSC growing adherently on
modified bag surface (Coop.: M. Rohde, HZI).
B) Even after passage 22 a normal diploid karyotype is
maintained. (Coop.: K. Miller, MHH).
Macke,L., Garritsen,H.S., Meyring,W., Hannig,H., Pagelow,U., Wörmann,B.,
Piechaczek,C., Geffers,R., Rohde,M., Lindenmaier,W., and Dittmar,K.E. (2010). Evaluating
maturation and genetic modification of human dendritic cells in a new polyolefin
cell culture bag system. Transfusion. 50, 843-855.
Dittmar,K.E.J., Simann,M., Zghoul,N., Schön,O., Meyring,W., Hannig,H., Macke,L.,
Dirks,W., Miller,K., Garritsen,H.S.P., and Lindenmaier,W. (2010). Quality of Cell
Products: Authenticity, Identity, Genomic Stability and Status of Differentiation. Transfusion
Medicine and Hemotherapy 37, 57-64.