Fig. 1
Design, production, and biological function of bispecific tetravalent tandem diabody Tandab (CD3/CD19). a Schematic representation of lentiviral expression vector for Tandab (CD3/CD19). LTR, long terminal repeats; SV40, SV40 promoter; SP, signal peptide, a murine kappa light-chain leader peptide; G4S, Gly-Gly-Gly-Gly-Ser residues; His6, hexa-histidine tag. b Molecular model of Tandab (CD3/CD19). Upon expression, two polypeptide gene products dimerize in a head-to-tail fashion. c Tandab (CD3/CD19) was determined by Western blot analysis. The supernatants from 293T cells after transfection with lentiviral expression vectors were harvested and assayed for Tandab (CD3/CD19) with or without β-mercaptoethanol (BME) using anti-His tag antibodies. Lane 1, 293T cells transfected with empty vector (negative control); lane 2, 293T cells transfected with vector expressing Tandab (CD3/CD19). d Western blot analysis of the purified Tandab (CD3/CD19). e Binding specificities of Tandab (CD3/CD19) to tumor cell lines. FACS analysis with the Tandab (CD3/CD19) on different CD19-positive B cell lines (Raji, Daudi, and BJAB), on CD3-positive Jurkat cells, and on the CD3- and CD19-negative K562 cells. a, negative controls with the secondary antibody anti-His-Alexa Fluor 488 alone; b, Tandab (CD3/CD19). f Competitive binding activity with FITC-conjugated HIT19a or PE-conjugated HIT3a. Cells were firstly incubated with Tandab (CD3/CD19) for 1 h at 4 °C, then incubated with FITC-conjugated HIT19a or PE-conjugated HIT3a for 30 min at 4 °C before detection. a, Negative controls; b, FITC-conjugated HIT19a or PE-conjugated HIT3a alone; and c, Tandab (CD3/CD19) + FITC-conjugated HIT19a or PE-conjugated HIT3a. Results are representative of three independent experiments. g Specific lysis of malignant target cell lines mediated by Tandab (CD3/CD19). Cytotoxicity of IL-2 pre-activated PBMCs induced by Tandab (CD3/CD19) with the same concentration (8 pmol/mL) in different effector to target (E:T) ratios ranging from 20:1 to 1:1 against CD19-positive B cell lines (Raji, Daudi, and BJAB) was detected by LDH release assay (left panel). Specific lysis of target cells by Tandab (CD3/CD19) with different concentrations at the same E:T ratio (20:1) was also determined (right panel). K562 cells were served as negative controls. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the corresponding K562 group. Data shown are the mean ± SD of three independent experiments