Figure 1

Generation of bsAb CD33–CD3-releasing gene-modified hMSCs. (a) Schematic representation of the structure of the bsAb CD33–CD3 constructed as single-chain bispecific tandem fragment variable (scBsTaFv). The V_H and V_L domains of each scFv were humanized by CDR grafting and connected via a linker comprised of three repeats of four glycine and one serine residues 3*(Gly₄Ser). The N-terminus of the bsAb construct contains a signal peptide (SP) for the secretion of the bsAb into the cell culture medium, whereas its C-terminus tag harbors a myc- and his-tag used for immunochemical Ab detection and purification. (b) Flow cytometry analysis of the hMSC marker profile upon staining of the parental wild type SCP-1 line with VioBlue-, PE-, APC-, VioBlue- and PE-conjugated anti-CD90, anti-CD105, anti-CD73, anti-CD45, anti-CD33 mAbs (in black). Cells stained with matched isotype control Ab (in gray) served as negative control. Numbers represent the percentages of positive cells and the mean fluorescence intensity (MFI) of total cells. (c) Transgene expression analysis of parental and transduced hMSCs was performed by flow cytometry. The percentages of living CD45^- and EGFP⁺ cells are shown. Dead cells were excluded by propidium iodide staining. (d) Purified fractions of the bsAb CD33–CD3 secreted in the culture medium were separated on SDS-gels and thereafter stained either with Coomassie brilliant blue or analyzed by Western blotting. (e) The quantitative analysis of the released bsAb was performed by ELISA. hMSCs cells were seeded at limiting cell densities and the antibody concentration (ng/ml) in culture medium was determined after 48 h of culture. Results represent the means±s.d. of two independent experiments.