Fig. 1.

Construction and production of anti-hDEC205-WT1 antibody fusion proteins. a Scheme of the anti-hDEC205 antibody. Heavy and light chains of the antibody were constructed by ligating the respective variable heavy and light chains to the N-terminus of the corresponding constant domains of human IgG1 isotype’s heavy chain (hIgG1–CH1–CH2–CH3) and kappa light chain. Each chain of the antibody was N-terminally FLAG-tagged. b Scheme of the scFv:hDEC205-GpL. Single chain variable fragment of anti-hDEC205 consisting of the variable heavy and light chains (anti-hDEC-VH and anti-hDEC-VL) was C-terminally fused to Gaussia princeps luciferase and tagged N-terminally by double FLAG. c Scheme of anti-hDEC205-WT1 antibody fusion proteins. The anti-hDEC205-WT1_major and -WT1_small fusion proteins were constructed by C-terminal linking of the respective WT1 fragments to the anti-hDEC205 heavy chain. Small fragments of WT1 were fused through a flexible spacer of four amino acids (little cylinders). d Production and secretion characteristics of anti-hDEC205-WT1_D_major antibody fusion proteins by SDS-PAGE and Western blot analyses. Proteins were detected with mouse anti-Flag M2 IgG (primary antibody) and goat anti-mouse IgG-IRDye 800CW (secondary antibody). S culture supernatant collected from transfected HEK293 cells, P lysate of pelleted HEK293 cells transfected with mock plasmids or encoding different anti-hDEC205-WT1_D antibody fusion proteins. e Purity and integrity of anti-hDEC205-WT1_small fusion proteins by SDS-PAGE and silver staining. PM protein marker, molecular weights in kDa, + reducing, − non-reducing condition, DTT dithiothreitol