1. Volume 10, Issue 2, Pages 290-301 (August 2004)
Therapeutic HER2/Neu DNA Vaccine Inhibits Mouse Tumor Naturally Overexpressing Endogenous Neu 
Chi-Chen Lin, Ching-Wen Chou, Ai-Li Shiau, Cheng-Fen Tu, Tai-Ming Ko, Yi-Ling Chen, Bei-Chang Yang, Mi-Hua Tao, Ming-Derg Lai 
Molecular Therapy 
Volume 10, Issue 2, Pages (August 2004)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. Fig. 1
Characterization of DNA vaccines. (A) Schematic diagram of the N′-neu- and N′-neu–cytokine-expressing vectors. p185neu consists of an extracellular domain (black box), transmembrane domain (striped box), and intracellular kinase domain (white box). The N-terminal extracellular portion of the rat p185neu gene was fused to the mature mouse IL-2, IL-4, or GM-CSF gene. Transcription is directed by cytomegalovirus (CMV) early promoter/enhancer sequences. Four plasmids were named “N′-neu,” “N′-neu-IL-2,” “N′-neu-IL-4,” and “N′-neu-GM-CSF.” (B) Expression of N′-neu in vitro evaluated with flow cytometry. COS-1 cells were transfected with N′-neu, N′-neu-IL-2, N′-neu-IL-4, N′-neu-GM-CSF, or control plasmid DNA, and permeabilized cells were stained with monoclonal antibody against the N-terminal domain of rat p185neu, followed by FITC-conjugated goat anti-mouse secondary antibody (gray histogram). Normal mouse IgG mAb was used as the negative control (white histogram). (C) Expression of secretory cytokines in vitro. COS-1 cells were transfected with the plasmids indicated, and the amounts of IL-2, IL-4, and GM-CSF cytokines in culture supernatants were determined with ELISA. (D) Expression of N′-neu–cytokine in the muscle. Mice were inoculated intramuscularly with 100 μg of each plasmid DNA, and the cytokine content in the muscle was determined with ELISA.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. Fig. 2
Characterization of MBT-2 bladder cancer cell line. (A) MBT-2 cells overexpress p185neu. The expression of p185neu in various cell lines was analyzed by Western blotting with monoclonal antibody against p185neu. (B) Flow cytometry analysis of membrane p185neu and MHC class I molecules in MBT-2 cells. MBT-2 cells were stained with monoclonal antibody against the extracellular domain of mouse p185neu, followed by FITC-conjugated goat anti-mouse secondary antibody (gray histogram). Normal mouse IgG mAb was used as the negative control (white histogram). For MHC class I molecules, FITC-conjugated monoclonal antibody against H-2Dk MHC class I was used (gray histogram) and mouse IgG2a-FITC was the negative control (white histogram).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. Fig. 3
Therapeutic effects of neu DNA vaccines on established tumor in C3H mice. (A) Protocol for DNA vaccination. Ten days after subcutaneous tumor implantation, mice were inoculated with DNA vaccine intramuscularly three times at weekly intervals. (B) Life span of C3H mice after sc challenge of MBT-2 cells. The survival data were subjected to Kaplan–Meier analysis. The number in parentheses is the number of mice in the experiment. *Statistically significant difference compared with the control saline mice (P < 0.01). **Statistically significant difference between the N′-neu-IL-2 group of mice and the N′-neu group of mice (P < 0.05) or control mice (P < 0.001).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. Fig. 4
Anti-mouse p185neu antibody titer in the mice serum. The serum anti-p185neu antibody in mice was determined with ELISA on dishes coated with the extracellular domain of mouse p185neu. The data represent the average titer of the sera from three mice in each group.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. Fig. 5
Tumor infiltration and cytotoxicity of CD8+ T cells. Tumors were excised from mice inoculated with N′-neu, N′-neu–cytokines, or control DNA vector after the third inoculation. Analysis of (A) CD4+ and (B) CD8+ T cells in cryosections of tumors stained with primary antibody specific for CD4+ and CD8+ cells, respectively, was performed. Peroxidase-conjugated antibody was used as secondary antibody. Dark spots, peroxidase-stained cells. (C) Lysis of MBT-2 cells with splenocyte T cells derived from mice immunized with N′-neu, N′-neu-IL-2, N′-neu-IL-4, N′-neu-GM-CSF, or pRc/CMV control vector DNA vaccine. Bromodeoxyuridine-labeled MBT-2 cells were incubated with serial dilutions of splenocytes, and the lysis of MBT-2 cells was determined by release of bromodeoxyuridine-labeled DNA fragments.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. Fig. 6
The effects of CD4+ T cell depletion or CD8+ T cell depletion on the therapeutic effects of N′-neu DNA vaccine and N′-neu-IL-2 DNA vaccine. (A) Protocol for depletion of CD4+ or CD8+ T cells in vivo. Tumor-bearing mice were injected ip with 300 μg of anti-CD4 antibody or 500 μg of anti-CD8 antibody at weekly intervals starting from 2 days before the first inoculation of DNA vaccine. Life span of C3H mice after sc challenge with MBT-2 cells is depicted. (B) N′-neu DNA vaccine. The survival data were subjected to Kaplan–Meier analysis. *Statistically significant difference between the N′-neu group of mice and the N′-neu (anti-CD4) group of mice (P < 0.05). (C) N′-neu-IL-2 DNA vaccine. *Statistically significant difference compared to the control saline group of mice (P < 0.001). The number in parentheses is the number of mice in the experiment.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

8. Fig. 7
Anti-mouse p185neu antibody titer in the mice depleted of CD4+ or CD8+ T cells. The mouse serum was serially diluted and assayed by ELISA on dishes coated with N-terminal mouse neu antigen. (A) N′-neu DNA-vaccinated and T-cell-depleted mice and (B) N′-neu-IL-2 DNA-vaccinated and T-cell-depleted mice.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

9. Fig. 8
Depletion of CD4+ T cells before tumor implantation enhanced tumor growth. (A) Protocol for depletion of CD4+ T cells in vivo. Tumor-bearing mice were injected ip with 300 μg anti-CD4 antibody at weekly intervals beginning 2 days before tumor implantation. (B) Life span of C3H mice after sc challenge of MBT-2 cells. The survival data were subjected to Kaplan–Meier analysis. *Statistically significant difference between the nondepleted mice and the CD4+ T-cell-depleted mice (P < 0.05).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions