Comparison of conventional CD3-engaging bsAbs with CD8-engaging bsAbs with regard to (A) principal idea, (B) schematic structure, (C) production and purification.

<p><i>A</i>, 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) (<i>AI</i>). A subtype-specific cross-linkage of CD8+ T cells can theoretically be achieved by a CD8-engaging bsAb (<i>AII</i>). <i>B</i>, 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. <i>C</i>, 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 (<i>CI</i>). Purified bsAbs were further analyzed by immunoblotting (<i>CII</i>). After transfer onto nitrocellulose membranes recombinant Abs were detected via their C-terminal his-tag.