Induction of Wilms' tumor protein (WT1)-specific antitumor immunity using a truncated WT1-expressing adenovirus vaccine

Abstract

Purpose: Wilms' tumor protein (WT1) is overexpressed in most leukemias and many solid tumors and is a promising target for tumor immunotherapy. WT1 peptide-based cancer vaccines have been reported but have limited application due to HLA restriction of the peptides. We sought to vaccinate using adenoviral (Ad) vectors encoding tumor-associated antigens such as WT1 that can stimulate tumor-associated antigen-specific immunity across a broad array of HLA types and multiple class I and class II epitopes.

Experimental design: We developed a novel Ad vector encoding a truncated version of WT1 (Ad-tWT1) lacking the highly conserved COOH terminus zinc finger domains and tested its ability to stimulate WT1-specific immune responses and antitumor immunity in two murine models of WT1-expressing tumors.

Results: Despite encoding a transcription factor, we found that Ad-tWT1-transduced murine and human dendritic cells showed cytoplasmic expression of the truncated WT1 protein. In addition, vaccination of C57BL/6 mice with Ad-tWT1 generated WT1-specific cell-mediated and humoral immune responses and conferred protection against challenge with the leukemia cell line, mWT1-C1498. Moreover, in a tumor therapy model, Ad-tWT1 vaccination of TRAMP-C2 tumor-bearing mice significantly suppressed tumor growth.

Conclusions: This is the first report of a WT1-encoding Ad vector that is capable of inducing effective immunity against WT1-expressing malignancies. Based on these findings, Ad-tWT1 warrants investigation in human clinical trials to evaluate its applications as a vaccine for patients with WT1-expressing cancers.

Figure 1. WT1 protein expression by Ad-tWT1 transduced human and mouse dendritic cells

A) Seven (7) day-cultured human DC were infected with Ad-tWT1 at the indicated moi (5,000-40,000). After infection, cells were incubated for 2 days, and lysed for protein extraction. 30 μg of protein was applied for each lane, run on 12% Tris-HCl acrylamide gel, and transferred to Polyvinylidene Fluoride (PVDF) membranes. Membranes were incubated with anti-WT1 antibody or anti-actin antibody, followed by incubation with HRP-conjugated goat anti-mouse IgG antibody. An ECL kit was used for development. B) Human DC, cultured on chamber slides for 7 days, were infected with Ad-tWT1 at moi 40,000 and further incubated for 2 days. Then cells were fixed with 10% formalin and permeabilized with BD Permeabilizing Solution. Cells were stained with anti-WT1 mAb (left side of panel) or control IgG (right side of panel), followed by biotinylated anti-mouse IgG antibody. Color was developed with a Vectastain kit and DAB. Arrowheads indicate colonies of DC. C) Seven day-cultured mouse DCs were infected with Ad-tWT1 at moi 20,000, incubated for 2 days, and lysed for protein extraction. Immunoblotting with anti-WT1 mAb was performed as described in (A). D) Human DC were infected with Ad-tWT1 at the indicated moi. After infection, cells were incubated for 2 days, and stained with the indicated antibodies for analysis by flow cytometry. The mean fluorescence intensity of expression of each surface protein is indicated within each histogram.

Figure 2. Induction of WT1-specific IFN-γ production by T cells following Ad-tWT1 vaccination

C57BL/6 mice were vaccinated via footpad injection with either 40 μl of saline or 2.6 × 10¹⁰ particles of the Ad-tWT1 vector. Mice (n=4/group) were sacrificed weekly and the splenocytes were harvested. T cells producing IFN-γ in response to stimulation with WT1 peptide mix, an HIV peptide mix (negative control), media alone or PMA + ionomycin (positive control) were measured in an ELISpot assay. The mean (+/- s.d.) number of spots (T cells secreting cytokine) is depicted at each time point. * p < 0.005 for the comparison of saline control versus each time point of Ad-tWT1 vaccination. ** p < 0.01 for the comparison of the mean number of spots at day 21 versus day 7.

Figure 3. Detection of anti-WT1 antibodies in mice immunized with Ad-tWT1

C57BL/6 mice (n= 5/group) were vaccinated via footpad injection with 2.6 × 10¹⁰ particles of Ad-LacZ, or 2.6 × 10¹⁰ particles of the Ad-tWT1 vector. Mice were sacrificed 14 days post-immunization, serum samples were obtained, pooled, and the quantity of anti-WT1 antibody was measured by ELISA. *p < 0.01 for the comparison of the anti-WT1 titer for mice immunized with Ad-tWT1 compared with Ad-LacZ..

Figure 4. Prevention of mWT1-C1498 tumor growth by Ad-tWT1 vaccination

C57BL/6 mice (n= 5/group) were vaccinated via footpad injection with on days 0 and 14 with 2.6 × 10¹⁰ particles of Ad-LacZ, or 2.6 × 10¹⁰ particles of the Ad-tWT1 vector. On day 18, 5 × 10⁵ mWT1-C1498 (WT1-expressing murine leukemia line) cells were inoculated subcutaneously. Mice were euthanized 25 days after tumor cell inoculation. The mean +/-s.d. of the tumor volume is presented at each time point and a t test was performed on the day 25 data. * p < 0.05 for the comparison of the tumor size .

Figure 5. Induction of WT1-specific T cell response following Ad-tWT1 vaccinations in TRAMP-C2 tumor-bearing mice

C57BL/6 mice (n= 8/group) were inoculated with 1.0 × 10⁶ TRAMP-C2 cells subcutaneously into the flank on day 0. On days 7, 14, and 21, mice were vaccinated via footpad injection with either 40 μl of saline, 2.6 × 10¹⁰ particles of the Ad-tWT1 vector, or 2.6 × 10¹⁰ particles of the Ad-LacZ vector. Mice were sacrificed on day 35 and the splenocytes were harvested. T cells producing IFN-γ in response to stimulation with WT1 peptide mix, an HIV peptide mix (negative control), media alone or PMA + ionomycin (positive control) were measured in an ELISpot assay. The mean (+/- s.d.) number of spots (T cells secreting cytokine) is depicted for each vaccine condition. * p < 0.001 for the comparison of the mean number of spots following vaccination with Ad-tWT1 than with saline or Ad-LacZ.

Figure 6. Inhibition of tumor growth following Ad-tWT1 vaccinations in TRAMP-C2 tumor-bearing mice

C57BL/6 mice (n= 8/group) were inoculated with 1.0 × 10⁶ TRAMP-C2 cells subcutaneously into the flank on day 0. On days 7, 14, and 21, mice were vaccinated via footpad injection with either 40 μl of saline, 2.6 × 10¹⁰ particles of the Ad-tWT1 vector, or 2.6 × 10¹⁰ particles of the Ad-LacZ vector. Tumor size was serially measured and on day 56 mice were sacrificed. The mean +/-s.d. of the tumor volume is presented at each time point and a t test was performed on the day 56 data. * p < 0.05 for the comparison of the tumor volume between the Ad-tWT1 vaccinated mice and those vaccinated with Ad-LacZ or saline.