Fig. 4.

Flow chart of generic pipelines for developing R-mAbs and ScFVs from existing mAbs. Top two rows show a schematic overview of the major steps involved in generation of R-mAbs from mAb-producing hybridomas. The top row shows a PCR-based cloning based approach whereby V_H and V_L region fragments are amplified using degenerate primer sets, cloned into an expression plasmid such as that shown in Fig. 2, the R-mAbs expressed and functionally characterized relative to the corresponding conventional mAb. Positive clones are then sequenced and archived. The middle row shows a high-throughput sequencing approach based approach whereby V_H and V_L region fragments are amplified using degenerate primer sets including bar codes and the amplicons from many different hybridomas pooled and sequenced. The primary V_H and V_L region fragments are synthesized and cloned into an expression plasmid such as that shown in Fig. 2, the R-mAbs expressed and functionally characterized relative to the corresponding conventional mAb. The bottom row shows a schematic of typical approach used to develop ScFVs from existing R-mAbs. One of the approaches above is used to obtain R-mAb V_H and V_L region sequences. These are used to design a synthetic gene fragment that has these sequences fused with an intervening flexible linker sequence. This gene fragment is then cloned into a mammalian or bacterial expression plasmid with suitable promoter and leader sequences and used for expression of secreted ScFVs, or expression plasmids without a leader sequence for expression as intrabodies.