Figure_1.tif (1.64 MB)

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

Download (0 kB)
posted on 21.04.2014 by Irene Michalk, Anja Feldmann, Stefanie Koristka, Claudia Arndt, Marc Cartellieri, Armin Ehninger, Gerhard Ehninger, Michael P. Bachmann

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.