We propose to test if protein transduction technology can improve the efficacy of adenoviral gene-therapy vectors for the treatment of cancer. In cell culture experiments, cancer cells can easily be eliminated with cancer targeted adenoviral gene-therapy vectors. However, in mice and humans, inefficient gene transfer remains a hurdle that has been extremely difficult to overcome. A p53-expressing adenoviral vector is in clinical use in China and has been placed on the fast track for FDA approval.
Published data indicate an extremely favorable safety profile, but overall, the therapeutic effect has been limited. Clearly, gene-transfer efficacy of adenoviral vectors has to be improved for more efficient therapy. The basic domain of HIV-1 Tat (a section of a protein made by the HIV virus, also called the protein transduction domain) can enter intact cells and has been used to deliver a wide variety of proteins to many cell types. Tat-fusion proteins, produced in bacteria, purified and then applied to cells, have been studied widely to manipulate cancer biology.
This approach is effective in cell culture experiments, but so far there is only limited evidence for efficacy in mouse tumor models. Very few studies have investigated the potential of the delivery of Tat-fusion proteins with gene therapy vectors. We hypothesize that Tat-fusion peptides expressed at high levels with an adenovirus will result in more effective treatment of solid tumors.
Furthermore, we hypothesize that smaller unfolded peptides fused to Tat will lead to more efficient distribution within tumors, compared to complexly folded large proteins. The delivery of peptides fused to a protein transduction domain with adenoviral vectors is a new approach that could find broad application.
The prevailing view suggests that lung tumors may not be good targets for vaccine therapy. The reason for this belief is the fact that in lung tumor patients we do not find any indication of immune activation including a complete absence of killer cells (cytotoxic T cells) that could recognize and kill the tumors. Lung tumors are therefore classified as non-immunogenic. We suggest that this circumstance is ideal for immunotherapy of non-small cell lung carcinoma by vaccination.
We have been developing vaccines that can generate killer cells against lung tumors (non-small cell lung carcinoma) by genetic engineering lung tumor cell lines adapted to grow in cell culture. We predict that a vaccine that can generate killer cells in lung tumor patients will be effective and that the patient tumor will be defenseless against killer cell attack because the tumor cells have never before been exposed to killer cells and have not evolved protective mechanisms.
In a phase I study we found that a lung tumor vaccine, designated B7-vaccine, was able to generate killer cells and seemed to provide clinical benefit to patients. In the study proposed here we have developed a novel vaccine approach for lung tumors based on a genetically modified, secreted form of heat shock protein gp96. The vaccine is comprised of lung tumor cells that secrete hsp-gp96 and thereby provide a very strong stimulus for the immune system to generate killer cells against lung tumors.
Our initial in vitro data and data in experimental mice indicate that the gp96-vaccine is over one million-fold more efficient in the generation of killer cells than normal proteins in the absence of gp96. These properties of tumor secreted gp96 make it an ideal vaccine. In addition, these properties allow us to study how gp96 induces immunity and how it may be able to restore immunity that is suppressed in the presence of existing tumors.
In this application we plan to pursue the clinical testing of the gp96-vaccine and to conduct further research into the underlying immune response. Understanding the underlying mechanisms will allow us to develop further reagents to improve vaccine efficiency.
Live attenuated Edmonston strain measles virus (MV) has promising anti-tumor activity against a variety of human cancers and three phase I clinical trials evaluating the safety of intraperitoneal, intratumoral or intravenous delivery of MV are in progress at Mayo Clinic Rochester.
However, most patients are immunized against measles virus and have anti-viral antibodies that can inactivate the virus. This limits our ability to use measles virus in metastatic diseases that require intravenous infusion of the virus. Since measles virus naturally spreads in white cells during natural infection, we propose to use cells as vehicles to carry the virus to the tumor sites. In this way, the virus is protected from undesirable elements in the circulation and can still effectively infect tumor cells and kill them.
In this grant, we will identify the optimal protocol to load various types of cell carriers with viruses and test them in animal models. In the end, we hope to have a feasible and effective clinical protocol based on our findings to treat relapsed multiple myeloma.
Glioblastoma multiforme (GBM) represents the most common primary malignant tumor of the adult central nervous system. The median survival after surgical intervention alone is approximately six months and the addition of radio-/chemotherapy can extend this time up to fourteen months. Consequently, efforts aimed at developing new therapies have focused on treatment strategies that target the tumor environment but spare normal and healthy surrounding brain cells.
Oncolytic adenoviral therapy is a novel modality of anti-cancer treatment. Our group has created the oncolytic vector CRAd-Survivin-pk7 (CRAd-S-pk7) for the treatment of malignant gliomas. For transcriptional targeting in gliomas, we incorporated the survivin promoter upstream from viral gene E1A. The survivin promoter is highly active in gliomas but remains silent in the surrounding brain parenchyma. To enhance viral transduction into glioma cells, the capsid of this vector was modified to bind heparan sulfate proteoglycans expressed in these tumors.
In our extensive preclinical studies, CRAd-S-pk7 exhibits potent anti-tumoral activity in mice bearing intracranial human glioma xenografts, including the highly aggressive CD133+ glioma stem cell model. In addition, we have recently shown that this virus elicits a synergistic therapeutic effect when combined with low dose radiation and with the chemotherapeutic agent temozolomide, two therapies that constitute the standard of care for patients with malignant glioma.
Since one of the major limitations of virotherapy is poor spread following injection, we have recently shown that mesenchymal stem cells (MSC) can more effectively migrate and deliver an oncolytic adenovirus to intracranial glioma than local injection of the virus alone. This form of carrier mediated delivery leads to enhanced viral replication in the tumor and a much more potent anti-tumor response than local injection of the virus alone.
Moreover, our studies further suggest that MSC suppresses the anti-adenoviral immune response, further enhancing the efficacy of oncolytic virotherapy. In order to translate our work into the clinical setting, we now propose to develop a clinical trial in which this novel virus will be delivered via MSCs.
To achieve this goal, we would like to utilize Alliance for Cancer Gene Therapy funding to complete the following aims:
- Aim 1: Validate the therapeutic efficacy of CRAd-S-pk7 loaded MSCs in vitro and in animal models of glioma.
- Aim 2: Evaluate the therapeutic efficacy and safety monitoring with CRAd-S-pk7 loaded MSCs in the presence of temozolomide-based chemotherapy and radiotherapy.
- Aim 3: Determine the migration, engraftment, and long-term fate of CRAd-S-pk7 loaded MSCs in vitro and in animal models of glioma with MRI.
- Aim 4: Perform a toxicology/biodistribution study with MSC-loaded cGMP-grade clinical lot virus.
- Aim 5: Conduct RAC and FDA meetings and assemble documents for filing an IND application for mesenchymal stem cell based.
Development and delivery of T cells directed against tumor vascular targets offers multiple theoretical advantages: it can be highly specific, efficient and sustained in time. In addition, it has the potential for significant antigen-induced amplification in vivo and is the only one that can provide long-term memory. Active immunization against tumor-derived endothelial cells has produced encouraging preclinical results but is not a practical approach. Furthermore, generation of T cells with native T cell receptor that exhibits high affinity against ‘self’ tumor endothelial antigens is not straightforward. This proposal will test the central hypothesis that T-body cell therapy is the only form of antioangiogenic gene therapy immediately translatable clinically that can deliver sustained VDA-type effect which is tumor-specific, self-amplifying in vivo and endowed with memory. Coupled with the genetic stability of tumor endothelium and the catastrophic consequences that vascular damage has on the tumor, T-body immune-gene therapy could potentially achieve tumor eradication. Presently, we are the only group in the world with the combined expertise to test this hypothesis. In this proposal we will:
(1) Generate and test in vitro human lymphocytes (T cells) engineered to recognize and attack tumor blood vessels. We will engineer lymphocytes to express several molecules that will direct them to tumor blood vessels and we will compare different molecules to identify those that yield optimal efficacy in vitro.
(2) Test engineered lymphocytes (T cells) engineered to recognize and attack tumor blood vessels in vivo. We will use specialized models of mice developed in our laboratory which can be repopulated with human tumor blood vessels. The optimal vector will be selected for clinical development from these in vivo studies. (
3) Conduct a phase I trial to test the safety and anti-tumor efficacy of lymphocytes (T cells) engineered to recognize and attack tumor blood vessels in patients with advanced, recurrent ovarian and peritoneal cancer. Taken together, a translational team of basic scientists and clinicians will provide the first comprehensive evaluation of the use of this redirected T cell concept to implement antiangiogenic immune-gene therapy in cancer patients.
Studies in recent years have documented the lytic effects of various viruses on many human cancers, and the study and re-engineering of oncolytic viruses is intensifying. Recombinant measles viruses [MV] appear to be ideal vectors for lymphoma treatment, as wild-type MV infection occasionally induces lymphoma regression in humans.
We plan to produce viruses that replicate selectively in transformed lymphocytes, viruses with modulatable cytotoxicity, and viruses with a targeted envelope. Eventually we will combine the characteristics of the most effective viruses into a ‘second generation’ virus that is armed and targeted.
