Development of vectors for transient antibody expression in HEK293 cell lines. A) Illustration of the structure of scFv-Fc and scFv-Fc-RNase constructs. B) The vector pCMV-hIgG1Fc-XP based on pCMV/myc/ER backbone which contains a neomycin phosphotransferase (neo) selection marker under control of a simian virus (SV) 40 promoter (with SV40 ori) and poly A (pA) signal. The antibody gene expression cassette consists of a modified untranslated 5’ region and ribosomal binding site (modified KOZAK sequence), mouse heavy Ig chain leader comprising two exons (E1 and 2, light blue) interrupted by one intron (I) for stabilized expression, followed by a spacer sequence (grey) flanked unique NcoI/NotI sites for single step in frame cloning and fusion with following constant hinge, CH2 and CH3 regions of human IgG1 Fc gene fragment (dark blue). The gene expression cassette is driven by a CMV promoter and terminated by bovine growth hormone (bGH) poly adenylation signal (pA). C) pCMX2.5-hIgG1Fc-XP has a modified hinge without genetic upper hinge of the IgG1 heavy chain and optimized translation termination sequence at the 3’ of the Fc gene fragment. D) pCMV-neo- and pCMV-oriP1 test vectors containing CD30 specific scFv-Fc or scFv-Fc-RNase were generated from the parental pCMV vectors  by deleting the complete neo expression cassette or replacing it by a short EBV oriP variant. E) The expression vector pCSE2.5-hIgG1Fc-XP was newly generated and comprises all advantages for transient expression of the previous vectors including the optimized hinge and stop sequences of pCMX2.5, no selection marker expression cassette, minimal SV40 ori and short EBV oriP for episomal replication in SV40-large T antigen or EBNA1 positive mammalian expression cell lines.