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Authors: R Zhang and LJ DeGroot Presentation: Mario E. Moreno

An Adenoviral Vector Expressing Functional Heterogeneous Proteins Herpes Simplex Viral Thymidine Kinase and Human Interleukin-2 has Enhanced in vivo Antitumor Activity Against Medullary Thyroid Carcinoma. Authors: R Zhang and LJ DeGroot Presentation: Mario E. Moreno. Introduction.

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Authors: R Zhang and LJ DeGroot Presentation: Mario E. Moreno

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  1. An Adenoviral Vector Expressing Functional Heterogeneous Proteins Herpes Simplex Viral Thymidine Kinase and Human Interleukin-2 has Enhanced in vivo Antitumor Activity Against Medullary Thyroid Carcinoma Authors: R Zhang and LJ DeGroot Presentation: Mario E. Moreno

  2. Introduction • What is an adenoviral vector? • Infect a wide range of host cells. • Can be produced at very high titers. • Can express transgenes without integration into the DNA of infected cells. • Suitable for direct in vivo gene transfer. • Suitable for gene therapy of cancer.

  3. Introduction • What is herpes simplex viral thymidine kinase (HSVtk)? • HSV enzyme: phosphorylates gancyclovir (GCV) into GCV-monophosphate (GCV-MP). • GCV-MP is phosphorylated by cellular TK to toxic GCV-triphosphate (GCV-TP). • GCV-TP is a suicide-gene/prodrug system that inhibits viral DNA polymerase.

  4. Introduction • What is human interleukin-2 (IL-2)? • Produced by CD4+ T cells and CD8+ T cells. • Stimulates proliferation of cytotoxic T cells and helper T cells, and it activates and enhances the cytolytic function of NK cells. • Stimulates synthesis of other T-cell derived cytokines and lymphotoxin.

  5. Purpose of Study • Construct adenoviral vector expressing both HSVtk and hIL-2 genes. • Analyze the functional expression of HSVtk and hIL-2 in rat medullary thyroid carcinoma (rMTC) cells. • Evaluate in vivo antitumor activity of HSVtk and hIL-2 in an rMTC animal model.

  6. Plasmid Construction: Structure of AdCMVtk and AdCMVTKhIL2 • The expression cassette for HCSVtk or HSVTKhIL2 replaces the E1 of the Ad5 genome. • Both genes are under the control of the human CMV IE promoter. • Both genes are terminated by the polyadenylation signal (PA) of simian virus 40.

  7. Direct cytotoxicity was evaluated by in vitro cell proliferation assay by measuring the incorporation of [3H] thymidine by infected cells. Inhibition was observed when cells were infected at 100 m.o.i. or more of either virus. Direct Cytotoxicity of AdCMVTKhIL2 on Infected rMTC Cells

  8. rMTC cells were infected in vitro at 100 m.o.i. The activity of hIL-2 was tested and detected in supernatants by CTLL-2 cell proliferation assay by measuring the incorporation of [3H] thymidine. More than 60,000 IU hIL-2 were produced. AdCMVTKhIL2-Directed Expression of Functional hIL-2 Infected rMTC Cells

  9. GCV sensitivity assay was used to determine the expression of HSVtk in rMTC cells transduced with AdCMVTKhIL2. The results demonstrate the expression of HSVtk and high sensitivity of infected rMTC cells to the HSVtk/GCV system. Analysis of Expression of HSVtk

  10. Tumors were injected with either AdCMVTKhIL2 or AdCMVLacZ virus. Tumors were excised and single-cell suspensions were prepared. Supernatant used to examine the presence of hIL-2 using the IL-2-dependent CTLL-2 cell proliferation assay. Results indicate the production of functional hIL-2 by the AdCMVTKhIL2 treated tumor cells. Analysis of Expression of hIL-2 After Intratumoral Administration of AdCMVTKhIL2

  11. rMTC cells were infected in vitro with different adenoviral vectors. The infected cells were injected into Wag/Rij rats. Animals were treated with either GCV or PBS. Animals were inspected every 2 days for 2 months. Tumorigenicity of rMTC Cells Infected in vitro With AdCMVTKhIL2 in Wag/Rij Rats

  12. AdCMVTKhIL2 destroyed 63% of the tumors. AdCMVIL2 destroyed 38% of the tumors. AdCMVtk destroyed 11.8% of the tumors. Superior in vivo Antitumor Activity with the Bivalent Vector

  13. Only 4 of 19 rats developed tumors. All control rats developed tumors. The results indicate that systemic and long-term antitumor immunity was established after direct intratumor injection of AdCMVTKhIL2 virus. Development of Long-Term Antitumor Immunity After Treatment with AdCMVTKhIL2

  14. Discussion and Conclusion • HSVtk/GCV treatment can kill infected tumor cells, and thus release specific tumor antigens. • IL-2 will activate and enhance the function of both non-specific and specific immune cells. • These factors will synergize and more efficiently destroy the tumor. • A combined gene strategy is the best way to obtain high therapeutic efficiency.

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