Vaccine-strain measles virus is a promising platform for the development of genetically programmable anti-cancer therapies. Measles vaccine virus has a natural preference for binding and infecting many types of tumor cells. With an impeccable safety record from decades of clinical use, the measles vaccine virus has already entered clinical trials for several cancers. 

Recently, genetic modifications to the measles vaccine virus that enhance its ability to replicate in cells have shown further improvements in tumor killing in preclinical testing. As this therapeutic virus platform is modified to be more lethal to cancer cells, however, it becomes important to be able to limit its replication to particular times and places in order retain its low toxicity to normal cells.  

We propose to develop two mechanisms for improving measles vaccine virus control and specificity. The first mechanism allows administration of an orally available nontoxic drug to rapidly shut off virus protein production. The second mechanism renders virus protein production dependent on a specific molecular abnormality commonly found in cancer cells. Both will be based on encoding in the virus genome rationally engineered protein switches to control the function of viral replication proteins. In preliminary work, we have successfully created prototypical control elements for both mechanisms.  

We propose to test the ability of these control elements to function in the context of the whole virus in order to control replication in time and by tumor type. If successful, the proposed work will establish a generalizable method by which replication of measles vaccine virus, and other RNA-based viruses, can be programmed for increased tumor specificity and safety.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia and is still considered incurable, mandating development of improved treatment strategies. While most CLL patients respond to initial chemotherapy, approximately 10-20% of newly diagnosed patients and more than 50% of relapsed patients have limited or no response to cytotoxic agents.  

These chemotherapy resistant patients frequently have CLL cells that have high-risk cytogenetic abnormalities, such as deletions in the short arm of chromosome 17 (17p-) and/or dysfunctional TP53. Patients with 17p- and/or dysfunctional TP53 respond poorly to chemoimmunotherapy and have a short median survival relative to that of patients with functional TP53. As such, the therapeutic options for patients with 17p- and/or dysfunctional TP53 are limited and usually involve high-risk treatments or allogeneic stem cell transplant, which has a high risk of mortality.  

Our proposal provides a potential solution to this problem. We discovered that adenovirus vector transduction of CLL cells with ISF35 (a recombinant CD154) could enhance in vitro and in vivo the sensitivity of leukemia cells to drugs such as fludarabine in a TP53-independent manner (Dicker F. et al, Blood 2006. Castro JE. et al; ASH-2009 meeting abstract). This results from activation of another member of the TP53 family, namely P73. We found CD40 ligation activates P73 in both transduced and bystander leukemia cells and induces the CLL cells to become efficient antigen presenting cells that are capable of inducing host anti-leukemia immune responses. Activation of this alternative pathway can circumvent the resistance of TP53-deficient CLL cells to anticancer drug therapy.  

As such, cell-gene immune therapy provides for a novel strategy to re-sensitize resistant TP53-deficient leukemia cells. Patients will receive 3 infusions of autologous CLL cells transduced with ISF35 followed by only 3 cycles of chemoimmunotherapy with FCR (fludarabine, cyclophosphamide and rituximab).  

In preliminary work we have found that infusion of Ad-ISF35 transduced autologous leukemia cells causes systemic activation of bystander leukemia cells, rendering the entire leukemia-cell population sensitive to anti-cancer treatments.  

To date, two patients with refractory TP53-deficient leukemia have completed therapy on this protocol and have achieved a complete remission. Alliance for Cancer Gene Therapy funds will be used to continue this important work and to perform correlative science studies and clinical monitoring of patients who will enroll in this clinical study. The results generated in this proposal may allow for eventual approval for such gene therapy and offer new hope for patients with intractable leukemia.  

This Alliance for Cancer Gene Therapy Research Fellow is funded in part by Swim Across America.

Efficacious cancer immunotherapies will likely require combinations of strategies that enhance tumor antigen presentation and antagonize negative immune regulatory circuits. We demonstrated that vaccination with irradiated, autologous tumor cells engineered to secrete granulocyte-macrophage colony stimulating factor (GM-CSF) followed by antibody blockade of cytotoxic T lymphocyte associated antigen-4 (CTLA-4) accomplishes clinically significant tumor destruction with minimal toxicity in a majority of stage IV metastatic melanoma patients and some advanced ovarian carcinoma patients. 

The extent of tumor necrosis in post-treatment biopsies was linearly related to the natural logarithm of the ratio of infiltrating CD8+ effector T cells to FoxP3+ Tregs, suggesting that further Treg inhibition might increase the frequency of clinical responses. Through an analysis of cytokine deficient mice, we delineated a critical role for GM-CSF in Treg homeostasis.  

GM-CSF is required for the expression of milk fat globule epidermal growth factor-8 (MFG-E8), a phosphatidylserine binding protein, in antigen presenting cells, whereby the uptake of apoptotic cells by phagocyte-derived MFG-E8 maintains peripheral Treg numbers through TGF-beta, MHC class II, and CCL22. In wild type mice, MFG-E8 restrains the potency of GM-CSF secreting tumor cell vaccines through Treg induction, while a dominant negative MFG-E8 mutant (RGE) intensifies therapeutic immunity through Treg inhibition, resulting in regressions of established tumors. 

Furthermore, an orthologous human RGE dominant negative mutant similarly manifests immunostimulatory capabilities in cultures of human peripheral blood mononuclear cells. Together, these findings suggest that combinations of GM-CSF and MFG-E8 blockade (through an RGE mutant) might improve the potency of cancer vaccines and complement the activity of CTLA-4 blockade. Here, we propose to develop a standardized platform for the cellular co-delivery of human RGE and GM-CSF for use in clinical vaccination trials for patients with diverse solid and hematologic malignancies. 

Our overall approach will be to admix irradiated K562 cells engineered by plasmid mediated gene transfer to secrete RGE or GM-CSF with irradiated autologous tumor cells. Under an investigator held IND (BB-IND 11923, Glenn Dranoff, Sponsor), we already are performing Phase I trials of vaccination with lethally irradiated, GM-CSF secreting K562 cells admixed with irradiated, autologous tumor cells in several advanced tumors. We plan to manufacture K562 cells engineered by plasmid mediated gene transfer to secrete RGE, using comparable techniques as for the GM-CSF secreting K562 cells, and then to obtain an investigator sponsored IND that will support clinical evaluation of this combinatorial vaccination strategy in advanced cancer patients.