Abstracts (complete list) - Wissenschaft Online

Theron Johnson, Karsten Mahnke, Dirk Nettelbeck, Volker Storn, Alexander H. Enk
Targeting of Tumor antigens to dendritic cells (DCs) using a
novel single chain fragment variable
DEC205 is a prototype receptor for antigen uptake, which is expressed specifically by
DCs and increases antigen presentation by 500-fold over other presentation pathways
such as pinocytosis. To utilize the antigen receptor to load DCs in vivo, we created novel
single chain fragment variables (ScFv) specific for DEC205, using RT-PCR with
degenerative primers on total RNA from a hybridoma cell line (HB290). The isolated
variable heavy, and variable light regions were subcloned into an expression vector,
fused to a His tag and c-myc tag, then expressed as a native protein in E. coli. The
inclusion of the two tags allows for the isolation of the protein via a nickel-NTA column
(His) and immunohistochemical detection (c-myc) via anti c-myc antibodies. In
addition, we have created a ScFv anti-murine DEC205 fused to the melanoma tumor
associated antigen gp100. In C57/Bl6 mice, initial immunohistochemical staining of
CD11c+ lymph node cells showed positive staining of ScFv comparable to the mAb anti-
DEC205. In further experiments, C57/Bl6 mice were vaccinated with the DEC205 ScFv
in the footpad or left untreated. After 8 to 16 hours, lymph nodes were isolated and
examined for DEC staining. Here, the ScFv vaccinated mice showed more c-myc
positively stained cells in comparison to the untreated controls. The staining pattern is
similar to immunohistochemical staining seen in mice injected with the mAb anti-
DEC205. Thus, we can demonstrate that murine ScFv DEC205 can target to CD11c+
DCs in vivo. Further experiments using the ScFv DEC205-gp100 will show whether this
targeting can result in induction of GP100 specific CD8+ T cells and therefore, may be a
useful tool for the induction of an anti-melanoma response.