Preclinical Animal Models

 

The Thoracic Oncology Research Laboratory is focusing on the design of new treatment strategies for lung cancer and mesothelioma based on the rapidly evolving disciplines of molecular biology, immunotherapy, and gene therapy.  Multiple tumor models for mesothelioma and lung cancer are in place, including a transgenic, orthotopic model of lung cancer based on activation of the Kras oncogene.

 

These preclinical studies have allowed us to test novel agents in our animal models to determine which should be moved to clinical studies and the best way to deliver these therapies.   We have moved from “bench to bedside” at least 6 times in the last 15 years.

Suicide Gene Therapy.    Work in the 1990’s focused on the use of suicide gene therapy to treat mesothelioma using an adenoviral vector that delivered the viral enzyme herpes simplex thymidine kinase (HSVtk).  This rendered tumor cells susceptible to the normally harmless anti-viral drug ganciclovir. 

An adenovirus expressing HSVtk (Ad.HSVtk) was tested and shown to be effective in sensitizing cells in vitro to the effects of ganciclovir suggesting that a powerful bystander effect was present (Smythe et al., 1994a).  The vector was able to eradicate pre-infected tumor growing subcutaneously with a strong bystander effect and, even more importantly, was able to successfully treat established tumors growing within the peritoneal cavity of immunodeficient mice (Smythe et al., 1995b).  Formal toxicology studies in rats and primates were performed (Kucharczuk et al., 1996) and that allowed approval of a clinical trial for intrapleural administration of Ad.HSVtk in September of 1994. Our first patient was treated in November of 1994 (see below).

 

The TORL has been successful in a “bench to bedside” approach.  Preclincal data was used to inform our clinical trials program many times:

1.  Our group was the first to publish on the use of an E1/E3-deleted Ad.HSVtk vector in vitro (Smythe, 1994a) and in animal models of mesothelioma (Smythe et al., 1994b, Hwang et al, 1995, Elshami et al, 1996). Preclinical studies using this vector and toxicology studies (Kucharczuk et al. 1996) led to our original Phase 1 trial of Ad.HSVtk plus ganciclovir (GCV) in patients with mesothelioma (Sterman et al., 1998, Molnar-Kimber et al., 1998).

2. Preclinical studies suggested that steroids might augment efficacy (Elshami et al., 1995).  This was tested in a clinical trial (Sterman et al., 2000)

3.  A new “third generation” E1/E4 was developed and shown to have advantages in vitro and in vivo (Lanuti et al., 1999).  Toxicology was performed and the vector used in a clinical trial (Sterman et al., in press).

4.  Preclinical studies showed that increased doses of GCV led to increased efficacy.  A clinical trial was begun to test this hypothesis. 

 

Immunogene with Interferon-b

In 2000, clinical results from our Phase 1 Ad.HSVtk trials suggested strong, secondary immunological effects from the vector.  This prompted us to examine vectors that would intentionally generate immune responses. Preclinical studies using the Ad.IFNb vector showed remarkable activity in animal models of mesothelioma (Odaka, 2001; 2002).  Based on these studies, toxicology was performed and a new Phase 1 clinical trial was initiated and completed (see below). We have also extended these studies and showed that Ad.IFNb was very effective in models of lung cancer (Wilderman et al. 2005).

 

Augmenting Immuno- and Immuno-gene Therapy

Although preclinical and clinical studies show clear effects from immuno- and immuno-gene therapy, cures of large tumors are not often achieved.  Our recent studies have focused on ways to improve these approaches.   Our working hypothesis has been that tumors induce local and systemic immunosuppression and that these roadblocks much also be attacked.

 

We have explored a number of strategies to stimulate anti-tumor immune responses:

 

A.  Use of cyclooxygenase-2 inhibition to enhance the efficacy of immunotherapy

Given the multiple immunosuppressive effects of PGE2 and the high levels of COX-2 in mesotheliomas and lung cancers, we hypothesized that COX-2 inhibition would enhance the efficacy of immunotherapy. We have shown that  inhibition of COX-2 by oral administration of the COX-2 inhibitor, Rofecoxib, significantly augmented Ad.IFNb therapy (DeLong et al., 2003) and a lung cancer vaccine model using adenovirus to sensitive mice to a lung cancer model antigen (Hass et al., 2006). 

 

B.  Inhibiting TGF- b to Augment Host anti-Tumor Immunity

The cytokine TGF- b is made in large amounts in mesotheliomas and lung cancers.  Because TGF- b is strongly immunosuppressive, we have explored the use of TGF- b inhibitors by themselves and in combination with immunotherapy.  Our studies to date (done in collaboration with BiogenIdec) have shown strong anti-tumor activity of a TGF-b binding protein and a small molecule inhibitor of the TGF- b receptor kinase activity (called SM16) in models of mesothelioma (Suzuki et al., 2004; 2007).  In more recent studies, we have found that SM16 markedly augments the effects of Ad.IFNb and our lung cancer vaccine (papers submitted).   We have also seen marked increases in efficacy in models of adoptive T-cell transfer when we use SM16 (paper submitted).

 

C.  Depletion of B-cells to Augment Host anti-Tumor Immunity

 

 

 

D.  Efficacy and Mechanism of the Chemotherapeutic Drug Gemcitabine (Gem) in Combination with Immunotherapy (Aim 2A)        

Based on studies showing that Gem could augment an immunotherapeutic approach using ligation of CD40 with an agonist antibody, we tested the effect of Gem used in combination with Ad.IFNb in our mesothelioma model.  We studied very large tumors in which Ad.IFNb has little effect by itself.   Whereas Ad.IFNb alone and Gem alone had relatively small effects on these large tumors, the combination treatment showed a marked increase in efficacy.  The mechanism for this effect is complex, but we have data to support a number of immunologic mechanism (Suzuki – MSC paper; Suzuki- Can Biol and Rx). These studies are guiding our new combination chemotherapy/Ad.IFNb clinical trial (see below).

 

 

E.  Tumor-Associated Macrophage Activation.

DMXAA appears to activate tumor associated macrophages causing the release of cytokines (TNFa, IFN-γ) and chemokines (IP-10).  There are resultant effects on tumor endothelium causing endothelial cell necrosis, vascular thrombosis, and hemorrhagic necrosis of the tumor.  In addition, macrophage tumoricidal activity is increased, as well as NK cell activation.

 

 

 

Other Novel Therapeutics

A number of new projects are underway.   In collaboration with Novartis, we are exploring the use of histone deacetylase inhibitors for thoracic malignancies.  We are collaborating with investigators at the Mayo Clinic to evaluate a number of new replicating viral vectors for use in mesothelioma including measles, reo, and vesicular stomatitis virus.

 

 

Representative Publications

DeLong, P., Carroll, R.G., Henry, A.C., Tanaka, T., Ahmad, S., Leibowitz, M., Sterman, D.H., June, C.H., Albelda, S.M., Vonderheide, R.H.  Regulatory T cells and cytokines in malignant pleural effusions secondary to mesothelioma and carcinoma. Cancer Biology and Therapy, . 4(3); 2005.

Delong, P., Tanaka, T., Kruklitis, R. Henry, A., Kapoor, V., Kaiser, L.R., Sterman, D.H., Albelda, S.M.  Use of cycloxygenase-2 inhibition to enhance the efficacy of immunotherapy.  Cancer Research, 63:7845-7852, 2003.

Elshami, A.A., Saavedra, A., Zhang, HB., Kucharczuk, J.C., Spray, D.C., Fishman, G.I., Kaiser, L.R., Albelda, S.M.  Gap junctions play a role in the “bystander effect” of the Herpes Simplex virus thymidine kinase/ganciclovir system in vitro.  Gene Therapy, 3:85-92, 1996..

Haas, A., Sun, J., Vachani, A., Wallace, A.F., Silverberg, M., Kapoor, V., Albelda, S.M.  Cyclooxygenase-2 Inhibition augments efficacy of a cancer vaccine.  Clinical Cancer Research, 12:214-222, 2006.

Jassar, A., Suzuki, E., Kapoor, V., Sun, J., Silverberg, M., Cheung, L., Burdick^,, M., Strieter, R., Ching^, L-M., Kaiser, L.R., Albelda, S.M.  Activated Tumor-Associated Macrophages  and CD8⁺ T-cells are the Key Mediators of Anti-tumor Effects of the Vascular Disrupting Agent 5,6 Di-methylxanthenone-4-acetic Acid (DMXAA) in Murine Models of Lung Cancer and Mesothelioma”.  Cancer  Research, 65:11752-11761, 2005.

Kruklitis, R.J., Singhal, S., DeLong, P., Kapoor, V., Sterman, D.H., Kaiser, L.R., Albelda, S.M.  Immunogene therapy with interferon-b before surgical debulking delays recurrence and improves survival in a murine  model of malignant mesothelioma.  J. Thoracic and Cardiovascular Surgery, 127:123-130, 2004.

Kucharczuk, J.C., Elshami, A., Zhang, HB., Smythe, W.R., Hwang, H.C, Tomlinson, J.S., Amin, K.M., Litzky, L.A., Albelda, S.M. and Kaiser, L.R.  Pleural-based mesothelioma in immune competent rats:  A model to study adenoviral gene transfer.  Annals of Thoracic Surgery, 60:593-598, 1995.

Kucharczuk, J.C., Raper, S., Elshami, A.A., Amin, K.M., Sterman, D.H., Wheeldon, E.B., Wilson, J.M., Litzky L.A., Kaiser, L.R., Albelda, S.M.  Safety of intrapleurally administered recombinant adenovirus carrying herpes simplex thymidine kinase cDNA followed by ganciclovir therapy in non-human primates.  Hum. Gene Therapy, 7:2225-2233, 1996.

Lambright, E.S., Force, S.D., Lanuti, M., Wasfi, D.S., Amin, K., Albelda, S.M., Kaiser, L.R.  Efficacy of repeated adenoviral suicide gene therapy in a localized murine tumor model.  Annals of Thoracic Surgery, 70, 1856-1870, 2000.

Lanuti, M., Gao, G.P., Force, S.D., Chang, M.Y., el Kouri, C., Amin, K., Hughes, J.P., Wilson, J.M., Kaiser, L.R., Albelda, S.M.  Evaluation of an E1/E4-deleted adenovirus expressing the herpes simplex thymidine kinase suicide gene in cancer gene therapy.  Human Gene Therapy, 10:463-475, 1999.

Odaka, M., Sterman, D., Wiewrodt, R., Zhang, Y, Kiefer, M., Amin, K., Gao, G-P, Wilson, JM, Barsoum, J, Kaiser, L.R., Albelda,, S.M.  Eradication of intraperitoneal and distant tumor by adenovirus-mediated interferon-beta gene therapy due to induction of systemic immunity.  Cancer Research, 61:6201-6212, 2001.

Odaka, M., Wiewrodt, R., DeLong, P.A., Tanaka, T., Zhang, Y., Kaiser, L.R., Albelda, S.M.  Analysis of the immunological response generated by Ad.IFN-b during successful peritoneal tumor gene therapy.  Mol. Therapy, 6:210-218, 2002.

Smythe, W.R., Kaiser, L.R., Hwang H.C., Amin K.A., Pilewski J.M., Eck S.L., Wilson, J.M., Albelda, S.M.  Successful adenovirus-mediated gene transfer in an in vivo model of human malignant mesothelioma.  Ann Thorac Surg., 57:1395-1401, 1994.

Smythe, W.R.,, Hwang H.C., Amin K.A., Eck S.L., Davidson, B.L., Wilson, J.M., Kaiser, L.R., Albelda, S.M.  Use of recombinant adenovirus to transfer the HSV-thymidine kinase gene to thoracic neoplasms:  An effective in vitro drug delivery system.  Ca. Res., 54:2055-2059, 1994.

Suzuki E ,  Kim, S, Cheung, HK, Corbley, M,  Zhang X, Sun, L, Shan F, Singh, J., Lee, WC, Albelda,SM, Ling, LE. A Novel Small Molecule Inhibitor of TGF-beta type I Receptor Kinase (SM16) Inhibits Murine Mesothelioma Tumor Growth in vivo and Prevents the Extent of Tumor Recurrence After Surgical Resection.  Cancer Research, 67:2351-2359, 2007.

Suzuki, E., Kapoor, V., Jassar, A., Kaiser, LR., Albelda, S.M.  Gemcitabine selectively eliminates splenic Gr-1+/CD11b+ myeloid suppressor cells in tumor-bearing animals and enhances anti-tumor immune activity.  Clinical Cancer Research, 11:6713-6721, 2005

Suzuki, E., Kapoor, V., Kaiser, L..R, Albelda, S.M.  Soluble Type II Transforming Growth Factor-b Receptor Inhibits Both Small and Large Established Murine Malignant Mesothelioma Tumor Growth by Augmenting Host Anti-tumor Immunity.  Clinical Cancer Research, 10:5907-5918, 2004.

Suzuki, E., Sun, J.,  Kapoor, V.,  Jessar, A., Albelda, S.M.,  Gemcitabine Has Significant Immunomodulatory Activity in Murine Tumor Models Independent of its Cytotoxic Effects. Cancer Biology and Therapy, in press.

Tanaka, T., DeLong, P.A., Amin, K., Henry, A., Kruklitis, R., Kapoor, V., Kaiser, L.R., Albelda, S.M.  Treatment of lung cancer using clinically relevant oral doses of the cyclooxygenase-2 inhibitor, Rofecoxib:  Potential value as adjuvant therapy after surgery.  Annals of Surgery, 241:168-178, 2005.

Wallace^, A., LaRosa, D.F, Kapoor, V. Sun, J., Cheng, G, Jassar, A., Blouin, A., Ching^, L., Albelda, S.M., The Vascular Disrupting Agent, 5,6 Di-methylxanthenone-4-acetic Acid (DMXAA), Directly Activates Dendritic Cells Through a MyD88-indpendent Mechanism and Generates Anti-Tumor Cytotoxic T-Lymphocytes.  Cancer Research, 67:7011-7019, 2007

Wiewrodt, R., Amin, K., Keifer, M., Jovanovic, V., Kapoor, V., Force, S. Chang, M., Lanuti, M., Black, M., Kaiser, L., Albelda, S.  Adenovirus-mediated gene transfer of enhanced herpes simplex thymidine kinase mutants improves prodrug-mediated tumor cell killing.  Cancer Gene Therapy,  10:353-364, 2003.

Wilderman, M., Sun, J., Khan, M., Vachani, A., Suzuki, E., Kinnery, P., Steman, D., Kaiser, L., Albelda, S.M.  Intrapulmonary interferon-beta gene therapy using an adenoviral vector is highly effective in a murine orthotopic model of lung adenocarcinoma via a combination of direct toxicity, NK cell, and CD8 T-cell mediated effects, Cancer Research,  65:8379-8387, 2005.

Tumor Immunology

Thoracic Oncology Research Laboratory