Figure 1.
Expression of HER2 in different human melanoma cells.
(A) Surface expression of HER2 was evaluated by flow cytometry on seven melanoma cell lines (OCM-1, OMM-1, 92-1, Malme-3M, Mel888, SK Mel28 cell, and Mel624) and a primary melanoma (PM) cell. Shaded histograms represent cells stained with Herceptin® and open histograms represent cells stained with a non-related human IgG as control. (B) Western blot analysis showed HER2 protein band (185 kD) in cell lysates of human melanoma cells. β-actin was used as an internal control. A HER2 non-expression cell line K562 was used as a negative control.
Figure 2.
Generation of Malme-3M-luc cells.
(A) Schematic representation of the lentivirus vectors 3.7EF-1α-ER-△LNGFR-CMV-luciferase and 3.7CMV-ER-BirA. (B) Malme-3M cells were transduced with both 3.7lnluc and 3.7BirA lentiviral vectors and luc-positive cells were isolated by streptavidin beads. (C) FACS analysis of LNGFR expression in isolated Malme-3M-luc cells. Cells were labeled with anti-LNGFR monoclonal antibody CD271 followed by goat-anti-mouse-PE-CY5. Histogram indicates LNGFR expression, in which the shaded histogram represents purified Malme-3M-luc cells, and the black line represents un-transduced Malme-3M cells. (D) The bioluminescence image signal was measured with the Xenogen IVIS system, and serial dilution of Malme-3M-luc cells were plated in black 96-well plate in PBS containing D-luciferin substrate at final concentration of 0.15 mg/ml. (E) Pearson’s correlation coefficient analysis. The correlation coefficient (R) between the luciferase quantity and living cell number is 0.999.
Figure 3.
No effect of the luciferase gene on HER2 expression and the proliferation of Malme-3M cells and the binding of anti-CD3×anti-Her2 Bispecific antibody (HER2Bi-Ab) to both HER2 positive and CD3 positive cells.
(A) Proliferation of Malme-3M and Malme-3M-luc cells was assessed by CCK8 assay. The experiments were triplicated and the data are shown as the mean absorbance with SD (at OD 450 nm). Statistical analysis was conducted using Student’s t-test. (B) Surface expression of HER2 on Malme-3M-luc cells was analyzed by flow cytometry. Shaded histogram represents cells stained with Herceptin®, and open histogram represents cells stained with a control human IgG. (C) Malme-3M-luc cells were incubated with HER2Bi-Ab (1μg/ml, shaded histogram) or a combination of OKT3 and Herceptin® (1 μg/ml, open histogram) for 30 minutes at room temperature. HER2Bi-Ab binding was analyzed by flow cytometry using FITC- goat-anti-mouse IgG to detect the anti-CD3 moiety of the HER2Bi-Ab. (D) Activated T cells (ATC) were incubated with HER2Bi-Ab (1μg/ml, shaded histogram) or a combination of OKT3 and Herceptin® (1 μg/ml, open histogram) for 30 minutes at room temperature. HER2Bi-Ab binding was analyzed by flow cytometry using FITC- goat-anti-human IgG to detect the anti-HER2 moiety of the HER2Bi-Ab. (E) Malme-3M-luc cells were incubated with HER2Bi-Ab (1μg/ml, shaded histogram, OKT3 (1μg/ml, open histogram), or Herceptin® (1μg/ml, dot histogram) for 30 minutes at room temperature. HER2Bi-Ab binding was analyzed by flow cytometry using FITC- goat-anti-human IgG to detect the anti-HER2 moiety of the HER2Bi-Ab. (F) ATC were incubated with HER2Bi-Ab (1μg/ml, shaded histogram), Herceptin® (1μg/ml, open histogram), or OKT3 (1μg/ml, dot histogram) for 30 minutes at room temperature. HER2Bi-Ab binding was analyzed by flow cytometry using FITC- goat-anti-mouse IgG to detect the anti-CD3 moiety of the HER2Bi-Ab.
Figure 4.
Growth inhibition of melanoma cells by HER2Bi-armed ATC.
Malme-3M-luc cells were seeded (2 x104/well) into 96-well round bottom microplate in triplicates overnight. On the following day, the medium was removed, and fresh medium alone or containing the unconjugated mAbs OKT3 or Herceptin (1μg/ml), ATC (2 x105/well), HER2Bi-armed ATC (2 x105/well, armed with 50 ng/HER2Bi/106 ATC) or a combination of OKT3 and Herceptin® with ATC (unarmed ATC) for 18 hours. (A) Real-time photographs were taken at 200x magnification. a. Malme-3M-luc targets alone; b. Malme-3M-luc with OKT3; c. Malme-3M-luc with Herceptin; d. Malme-3M-luc with ATC; e. Malme-3M-luc with unarmed-ATC; f. Malme-3M-luc with HER2Bi-armed ATC. HER2Bi-armed ATC, but not ATC or unarmed ATC aggregated with Malme-3M-luc tumor cells in culture after 18 hours incubation. (B) The proliferation of Malme-3M-luc was assessed by CCK8 assay with the OD value measured at 450 nm. The data are mean ± SD of triplicate experiments, and a representative experiment of three was shown. Statistical analysis was conducted using Student’s t-test. *: P<0.01, HER2Bi-armed ATC compared with combination of the OKT3 and Herceptin with ATC under similar conditions.
Figure 5.
The lytic activity of HER2Bi-armed ATC against Malme-3M-luc cells
(A) Bioluminescence images of Malme-3M-luc cells after incubation with HER2Bi-armed ATC or unarmed ATC at different E/T ratio (1:1, 5:1, and 20:1) for 18 hours. (B) Bioluminescence image signal in photons per second was converted into living cell number and the cytotoxicity assay was measured at the indicated E/T. (C) The lytic activity of HER2Bi-armed ATC against B16-luc cells. (D) IFN-γ secretion by HER2Bi-armed ATC against Malme-3M–luc cells. Supernatants of co-cultures at indicated E/T were harvested at 18 hours and analyzed for IFN-γ level using IFN-γ ELISA Kit. (E) Expression of CD69 on HER2Bi-armed ATC or unarmed ATC was detected by flow cytometry after 18 hours co-culture with Malme-3M-luc cell at E/T ratio of 5:1. For B and C, the data are mean ± SD of triplicate experiments. Statistical analysis was conducted using Student’s t-test. A representative experiment of at least two was shown. *: P<0.01, HER2Bi-armed ATC compared with unarmed ATC under similar conditions.
Figure 6.
In vivo cytotoxicity of HER2Bi-armed ATC against Malme-3M-luc cells.
SCID-Beige mice were inoculated with Malme-3M-luc cells (1 x106/mouse) alone, or a mixture of Malme-3M-luc cells with un-armed ATC (250 ng Herceptin® and OKT3, combined with 1 x107ATC/mouse), or with HER2Bi-ATC (1 x107 /mouse, armed with 50 ng/HER2Bi/106 ATC) on day 0. Each group contained 5 mice. (A) Bioluminescence images of a representative mouse from each group on day 2, 7 and 14 were shown. (B) Images were analyzed using Living Image software and tumor values were represented as total flux measurements in photons/second. Mean values of tumor growth curves were shown. The significance between different groups was compared by one-way analysis of variance (ANOVA) followed by LSD for multiple comparison. **P<0.01, *P<0.05.