Binding properties of the novel scBsTaFv CD8-PSCA(MB1).

<p><i>A</i>, in order to investigate binding properties of the novel single-chain bsAb, PC3-PSCA cells and isolated CD8+ T cells were stained with 10 ng/µl of recombinant Ab. Specific binding of the scBsTaFv CD8-PSCA(MB1) was detected with anti-myc/FITC. Histograms show percentage and mean fluorescence intensity (MFI) of stained antigen-positive cells (<i>black</i>) in comparison to the negative control incubated only with the secondary anti-myc/FITC mAb (white). <i>B</i>, to demonstrate simultaneous binding of the novel scBsTaFv CD8-PSCA(MB1) to PC3-PSCA cells and CD8+ T cells and, hence, to visualize a cross-linkage between the two cell types, microscopic images were taken. Therefore, PC3-PSCA cells and isolated CD8+ T cells were co-cultivated in the presence of scBsTaFv CD8-PSCA(MB1) for 22 h and fixed with 90% methanol. The scBsTaFv CD8-PSCA(MB1) was detected with anti-myc/FITC mAb (<i>green</i>) and cell nuclei were stained with DAPI (<i>blue</i>) containing sample cover medium. Microscopic image (<i>a</i>) shows DAPI-stained nuclei of one PC3-PSCA cell (<i>P</i>) surrounded by four T cells (<i>T</i>). A homogenous cell surface staining of the PC3-PSCA cell and T cells is shown in picture (<i>b</i>) after detection of the scBsTaFv CD8-PSCA(MB1) with anti-myc/FITC. Furthermore, a cross-linkage between the PC3-PSCA cell and a T cell is visible (<i>white triangle</i>). Image (<i>c</i>) is an overlay of (<i>a</i>) and (<i>b</i>).