Figure 1.
Comparison of conventional CD3-engaging bsAbs with CD8-engaging bsAbs with regard to (A) principal idea, (B) schematic structure, (C) production and purification.
A, cross-linkage of T cells and tumor cells can be mediated by conventional bsAbs via simultaneous binding to the CD3 part of the TCR/CD3 complex and a tumor-associated surface antigen (TAA) (AI). A subtype-specific cross-linkage of CD8+ T cells can theoretically be achieved by a CD8-engaging bsAb (AII). B, schematic structure of recombinant single-chain bsAbs. For construction of the novel CD8-engaging single-chain bsAb, the anti-CD3 domain of scBsTaFv CD3-PSCA(MB1) was replaced by the scFv derived from the novel anti-CD8 mAb clone MB10. Recombinant Ab constructs were further equipped N-terminally with an Igκ leader as signal peptide (SP) for Ab secretion and C-terminally with a myc- and a his-tag for protein purification and detection. C, recombinant Abs were purified via Ni-NTA affinity chromatography from cell culture supernatant. Purified bsAbs were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue G250 to estimate protein purity and concentration (CI). Purified bsAbs were further analyzed by immunoblotting (CII). After transfer onto nitrocellulose membranes recombinant Abs were detected via their C-terminal his-tag.
Figure 2.
Binding properties of the novel scBsTaFv CD8-PSCA(MB1).
A, 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 (black) in comparison to the negative control incubated only with the secondary anti-myc/FITC mAb (white). B, 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 (green) and cell nuclei were stained with DAPI (blue) containing sample cover medium. Microscopic image (a) shows DAPI-stained nuclei of one PC3-PSCA cell (P) surrounded by four T cells (T). A homogenous cell surface staining of the PC3-PSCA cell and T cells is shown in picture (b) 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 (white triangle). Image (c) is an overlay of (a) and (b).
Figure 3.
Activation and cytokine release of freshly isolated CD8+ T cells or PBMCs after cross-linkage to PC3-PSCA cells via the novel scBsTaFv CD8-PSCA(MB1).
A, freshly isolated CD8+ T cells were cultured with PC3-PSCA cells in the absence or presence of 30 pmol/ml bsAb at an e:t ratio of 10∶1 for 24 h. Up-regulation of activation markers CD69 and CD25 on T cells was measured by flow cytometry and is shown for one representative donor. Numbers in dot plots represent percentage of CD8+/CD69+ cells. B, cell culture supernatants from co-cultures of T cell activation experiments (A) were collected to determine concentrations of IFN-γ (left) or TNF (right) by ELISA. The activation experiments were additionally performed under the same conditions as already described but using freshly isolated PBMCs instead of isolated CD8+ T cells. For technical reasons isolated T cells and pre-activated PBMC samples were from independent donors. Data of one representative CD8+ T cell donor (BI) and one representative PBMC donor (BII) are shown.
Figure 4.
Tumor cell elimination mediated by the novel scBsTaFv CD8-PSCA(MB1).
To analyze killing properties of the novel CD8-engaging single-chain bsAb standard chromium release assays were performed. A, to compare the anti-tumor effect of the novel scBsTaFv CD8-PSCA(MB1) with the conventional bsAb CD3-PSCA(MB1), [51Cr]-labeled PC3-PSCA cells and either pre-activated PBMCs (left panel), isolated pre-activated CD8+ T cells (middle panel) or freshly isolated CD8+ T cells (right panel) were cultivated in the presence or absence of 30 pmol/ml of recombinant bsAbs for 22 h. Mean of specific lysis ± SD of one representative donor is shown. BI, summary of (A, left panel) seven different donors is shown. Statistical significance was determined with one-way ANOVA and Bonferroni Multiple Comparison test (***p<0.001 with respect to control: no Ab; *p<0.05 significant difference between the conventional anti-CD3 bsAb and the novel anti-CD8 bsAb). BII, In order to estimate the ratio of CD8+ to CD4+ T cells, the pre-activated PBMC preparations of all seven donors were stained for CD8+ and CD4+ T cells and analyzed by FACS.