Abstracts (complete list) - Wissenschaft Online

Katrin Birkholz, Niels Schaft, Jan Dörrie, Michael Schwenkert, Christian Kellner, Georg
Fey, Gerold Schuler
New strategies for antigen loading of human monocytederived
DC
Although responses in dendritic cell (DC)-based vaccination of melanoma and renal cell
carcinoma patients are encouraging, further improvement of the efficiency of these
vaccines is needed. A comparison of different methods, such as electroporation of
defined tumor-antigen RNA or total tumor RNA, phagocytosis of tumor cells, direct
peptide loading, or the use of antibody-antigen constructs, is necessary to optimize DC
antigen loading. To examine this, we chose MAGE-A3, a cancer-testis antigen expressed
in malignant melanoma, multiple myeloma, and many other tumors, as a model
antigen. As a read-out for antigen-presentation efficiency, CD4 and CD8 T cells,
transfected with RNA encoding TCR recognizing MHC-presented MAGE-A3 epitopes were
used. As one possible loading strategy, we cloned antibody-antigen constructs,
consisting of a single-chain Fragment variable (scFv) directed against DEC205, an
endocytosis receptor expressed on the surface of DC, genetically linked to different
parts of MAGE-A3. After receptor-mediated endocytosis, the MAGE-A3 antigen should
be delivered into the DC, and subsequently presented on MHC class II molecules. The
expressed and purified fusion proteins were detected in western blot analysis and
displayed specific binding to mature (m)DC and DEC205-transfected CHO cells. In
another loading strategy, MAGE-A3-DCLAMP RNA was used. The DCLAMP sequence
targets the antigen to lysosomes, which leads to MHC class II presentation. Indeed,
electroporation of mDC with MAGE-A3-DCLAMP RNA resulted in HLA-DP4-restricted
presentation of the MAGE-A3 peptide KKLLTQHFVQENYLEY. Taken together, we have
established tools to optimize loading of DC with tumor antigen which might lead to
better anti-tumor DC vaccines.