Figure 2
Figure 2. Design and specific binding of the anti–DEC-205scFv-MAGE-A3-KKL and the anti–MCSPscFv-MAGE-A3-KKL fusion proteins. (A) Schematic presentation of the anti–DEC-205scFv-MAGE-A3-KKL (termed DK hereafter) and the anti–MCSPscFv-MAGE-A3-KKL (termed MK hereafter) fusion proteins. Estimated molecular weights are indicated. S indicates N-terminal Strep-tag; VL and VH, variable region light and heavy chain, respectively, of the scFv; (G4S)3 or (G4S)4, 15 or 20 amino acid linker composed of 3 or 4 repeats of the Gly4Ser unit, respectively; G4SA3, 8 amino acid linker composed of one Gly4Ser unit followed by an Ala3 unit; MAGE-A3-KKL, nucleotide sequence of MAGE-A3, presented in HLA-DP4; and H, hexahistidine-tag. (B) iDCs, mDCs, and the MCSP-positive cell line A2058 were incubated with the anti–DEC-205scFv-MAGE-A3-KKL and with the anti–MCSPscFv-MAGE-A3-KKL control proteins, and stained with murine anti–His-tag Ab and GAM-PE Ab. As a negative control, the cells were stained only with anti–His-tag Ab and GAM-PE Ab. (Bottom panels) Staining of the same cells with the FITC-labeled anti–DEC-205 parental antibody or with the anti-MCSP parental Ab and GAM-PE. As a negative control, these cells were stained with an IgG2b-FITC isotype control or GAM-PE only. (C) Preincubation with different concentrations of the parental Ab. mDCs were preincubated with the FITC-labeled parental Ab in different molar ratios to the construct. Then the mDCs were incubated with 1 μg/mL of the anti–DEC-205scFv-MAGE-A3-KKL for 30 minutes, and the construct was detected with PE-labeled anti–Strep-tag Ab. Signals were normalized to stainings with the parental Ab or the construct in absence of each other. (D) Binding of the construct to DCs in human blood. PBMCs were incubated with the anti–DEC-205scFv-MAGE-A3-KKL (DK) or with the anti–MCSPscFv-MAGE-A3-KKL (MK) constructs or stained with the parental DEC-205–specific Ab (α-DEC-205 Ab). Plasmacytoid DCs and myeloid DCs were counterstained and analyzed by multicolor flow cytometry, and lymphocytes were gated as negative control.

Design and specific binding of the anti–DEC-205scFv-MAGE-A3-KKL and the anti–MCSPscFv-MAGE-A3-KKL fusion proteins. (A) Schematic presentation of the anti–DEC-205scFv-MAGE-A3-KKL (termed DK hereafter) and the anti–MCSPscFv-MAGE-A3-KKL (termed MK hereafter) fusion proteins. Estimated molecular weights are indicated. S indicates N-terminal Strep-tag; VL and VH, variable region light and heavy chain, respectively, of the scFv; (G4S)3 or (G4S)4, 15 or 20 amino acid linker composed of 3 or 4 repeats of the Gly4Ser unit, respectively; G4SA3, 8 amino acid linker composed of one Gly4Ser unit followed by an Ala3 unit; MAGE-A3-KKL, nucleotide sequence of MAGE-A3, presented in HLA-DP4; and H, hexahistidine-tag. (B) iDCs, mDCs, and the MCSP-positive cell line A2058 were incubated with the anti–DEC-205scFv-MAGE-A3-KKL and with the anti–MCSPscFv-MAGE-A3-KKL control proteins, and stained with murine anti–His-tag Ab and GAM-PE Ab. As a negative control, the cells were stained only with anti–His-tag Ab and GAM-PE Ab. (Bottom panels) Staining of the same cells with the FITC-labeled anti–DEC-205 parental antibody or with the anti-MCSP parental Ab and GAM-PE. As a negative control, these cells were stained with an IgG2b-FITC isotype control or GAM-PE only. (C) Preincubation with different concentrations of the parental Ab. mDCs were preincubated with the FITC-labeled parental Ab in different molar ratios to the construct. Then the mDCs were incubated with 1 μg/mL of the anti–DEC-205scFv-MAGE-A3-KKL for 30 minutes, and the construct was detected with PE-labeled anti–Strep-tag Ab. Signals were normalized to stainings with the parental Ab or the construct in absence of each other. (D) Binding of the construct to DCs in human blood. PBMCs were incubated with the anti–DEC-205scFv-MAGE-A3-KKL (DK) or with the anti–MCSPscFv-MAGE-A3-KKL (MK) constructs or stained with the parental DEC-205–specific Ab (α-DEC-205 Ab). Plasmacytoid DCs and myeloid DCs were counterstained and analyzed by multicolor flow cytometry, and lymphocytes were gated as negative control.

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