Effectiveness of cancer vaccine to elicit immunity against tumor cells has been fundamentally proved in experimental animals or in some clinical conditions. Currently, one of the most important subjects of cancer vaccine is how to maximize its potency. In this regard, recent studies have suggested that LIGHT, a molecule belonging to tumor necrosis factor superfamily, induces potent anti-tumor immunity by a unique mechanism that facilitates both migration and activation of lymphocytes at the site of tumor.  

In this study, we will delineate the molecular-based mechanism of this phenomenon and to develop further efficient cancer vaccine using LIGHT. To this end, we will first take advantage of mutant protein of LIGHT or neutralizing monoclonal antibodies against HVEM or LTbetaR, two functional receptors of LIGHT. These experiments will elucidate the relative importance of HVEM versus LTbetaR or membrane versus soluble LIGHT in association with T cell trafficking and activation as well as anti-tumor efficacy.  

In the following experiments, we will attempt to develop innovative and more potent strategies of LIGHT cancer vaccine. By molecular engineering techniques, we will express stimulatory anti-HVEM antibody on the surface of tumor cells as cancer vaccine or generate pentameric constructs of this antibody to facilitate the signal delivery. In addition, attenuated measles virus vector that is specifically targeted to tumor cells will be utilized for tumor-selective LIGHT expression in vivo.  

Successful completion of this project will strongly support the potency of LIGHT as a target for advanced cancer immunotherapy, and thus lay the foundation of translational studies on LIGHT-based cancer gene therapy aiming at clinical applications.  

The prognosis for brain tumor patients has not improved significantly despite many innovations in surgery and radiation therapy, and the introduction of many new drugs that work well for other types of cancer. There is an urgent need for new approaches to treat brain tumors. Here, we propose investigating a new approach to brain tumor therapy based on the genetic modification of normal brain cells to create an environment that prevents tumor growth.  

Adeno-associated virus (AAV) vectors will be used as they are very efficient in introducing genes into normal brain cells. These AAV vectors will be used to introduce in normal brain cells or blood vessels a gene that makes a protein which can be released from modified normal cells and that is selectively active against tumor cells.  

In these experiments we will investigate the efficiency of this therapy principle by modifying the brain surrounding the tumor by direct injection of AAV vectors into the brain or by modifying the blood vessels in the brain by injection of these vectors into the bloodstream. These approaches may lead to the creation of widespread anti-tumor networks capable of preventing brain tumors from growing or appearing again after surgery.  

The myelodysplastic syndromes (MDS) are a group of clonal neoplastic hematologic disorders characterized by varying degrees of bone marrow failure, abnormal hematopoiesis, and proliferation of myeloid blast cells. Impaired maturation of hematopoietic progenitors is manifest clinically by peripheral cytopenias and morphologic abnormalities in the marrow (“dysplasia”). Thought to be disorders of hematopoietic stem cells, clonal cytogenetic abnormalities are frequently identified. Although the disease can evolve toward acute leukemia, morbidity and mortality most frequently result from a marrow failure syndrome.  

Evidence exists that immune activation against hematopoietic elements frequently occurs in MDS patients, based on the identification of lymphocytic infiltrates in the marrow, oligoclonal expansion of T cells, and excessive production of tumor necrosis factor alpha. Whether this represents a secondary event in response to cell injury and the generation of neo-antigens, or an initiating event inducing immunopathology, remains controversial. Nevertheless, MDS are thought to be immunologically responsive diseases, as immunomodulatory drugs can induce remissions, and allogeneic bone marrow transplantation can be curative in the small fraction of patients for whom this is an option.  

Recently, three classes of therapeutic agents have been shown to have activity in MDS; a) DNA methyltransferase inhibitors, b) histone deacetylase inhibitors (HDACi), and c) immunomodulatory derivatives (IMiDs) of thalidomide. Central to this proposal is the observation that all three classes of drugs augment discrete elements of host immunity, making their integration with therapeutic cancer vaccines ripe for exploration.  

We have developed a genetically modified tumor cell vaccine for the treatment of myeloid malignancies. The human erythroleukemia cell line K562 has been stably transfected to secrete GM-CSF. K562 cells express many of the antigens shown to be overexpressed in myeloid leukemias and MDS. In early phase clinical trials for both acute and chronic myeloid leukemias, we have observed the induction of anti-tumor immunity and associated clinical responses following K562/GM-CSF vaccination (see preliminary data). In this proposal, we seek to evaluate the integration of K562/GM-CSF vaccination with systemic therapies for MDS that alter host immunity and/or hematopoietic cellular differentiation. Specifically, we will:  

1. Examine the in vivo effect of: a) DNA methyltransferase inhibitors, b) HDACi, and c) IMiDs on the response to GM-CSF tumor vaccines in a mouse model (year 1).  

2. Conduct a clinical trial in MDS testing K562/GM-CSF vaccination integrated with the systemic agent(s) identified in aim 1 as being most active in combination with GM-CSF tumor vaccines (years 2 and 3). 

3. Evaluate immune responses specific for autologous MDS cells as well for as candidate antigens overexpressed in MDS using pre and post vaccination blood and marrow samples (years  

It is known that cancer cells are often defective in anti-viral pathways and are thus susceptible to virus infection. SV5, also known as PIV5, is not associated with symptoms or diseases in humans. This study will test the hypothesis that SV5 mutant viruses can selectively kill advanced tumors.  

SV5 viruses with mutation in SH or V proteins induce apoptosis in many cell types. Initial results have been promising with late-stage solid tumors, and in preliminary studies, mutant SV5 viruses killed human metastatic breast cancer cells, as well as Lewis lung carcinoma cells. Using cytolytic viruses as anti-tumor agent provides a viable alternative to surgery and chemotherapy. 

An ultimate goal of cancer immunotherapy is to activate tumor-specific T cells through therapeutic vaccinations to eradicate pre-established tumor. However, tumor-specific T cell tolerance remains one of the major barriers in cancer immunotherapy. Thus, to elicit effective anti-tumor immunity, it is necessary to develop vaccine strategies capable of overcoming T cell tolerance.  

In the previous application funded by Alliance for Cancer Gene Therapy, we have demonstrated an essential role of the innate immune system in shaping adaptive immune responses. In a series of 10 peer-reviewed publications, we have identified several parameters that are critical for the potency of a vaccine in overcoming T cell tolerance: 1) the ability of the vaccine to activate multiple innate immune pathways, leading to production of both type I interferons (IFNs) and pro-inflammatory cytokines; 2) the ability to activate both plasmacytoid dendritic cells (pDCs) and conventional DCs (cDCs); and 3) the ability to activate other innate immune cells such as NK cells, which further enhances adaptive immune responses.  

Based on these important parameters, we have demonstrated in a murine model of pre-established lymphoma that DC vaccines co-administered with the TLR9 ligand, CpG in vivo are effective in activating tumor-specific T cell response and treating pre-established lymphoma. This is probably related to the ability of CpG to activate both cDCs and pDCs and to produce pro-inflammatory cytokines and type I IFNs, respectively. In addition, CpG can also activate NK cells. 

 In this application, we will test the central hypothesis that DC vaccines co-administered with CpG in vivo are effective in activating tumor-specific CD8+ T cell response in patients with lymphoma through the following three specific aims: 1) To perform and analyze FDA required bio-distribution and toxicology studies in mice; 2) To obtain full regulatory approval and GMP manufacturing of DC vaccines to support the trial; and 3) To conduct a pilot phase I to study the safety and immunological efficacy of Epstein-Barr virus (EBV)-derived tumor antigen (LMP2) loaded DC vaccines in patients with EBV-associated lymphoma. We plan to administer LMP2-loaded DC vaccines twice intravenously.  

The boost vaccination will be administered 4 weeks after the first vaccination. GMP-grade CpG will be given intramuscularly with each vaccination. We will determine the safety and feasibility of the treatment by determining 1) clinical toxicology; 2) tumor antigen-specific immune responses; 3) although a secondary goal, anti-tumor effect will also be measured. In summary, this will be the first clinical trial designed to enhance anti-tumor immunity using a combined strategy of tumor. 

Natural killer (NK) cells mediate natural cytotoxicity against virus-infected or transformed cells. Alloreactive NK cells derived from haplotype mismatched hematopoietic stem cell (HSC) transplantation donors are used to successfully treat patients with high-risk acute myeloid leukemia (AML) without causing graft versus host disease (GVHD). However, equal benefit is not afforded to patients with B-cell acute lymphoblastic leukemia (B-ALL), suggesting that alloreactive NK cells fail to control B-ALL.  

This failure may be due to inhibitory signal-mediated resistance caused by B-ALL. To address this problem, mature NK cells can be genetically modified to express chimeric antigen receptors (CARs) specific for tumor antigen, thus harnessing their ability to kill B-ALL blasts. To date, adoptive transfer of ex vivo expanded mature NK cells has not shown therapeutic benefit in hematological malignancies, in part from the lack of target specificity and the short period of persistence after infusion.  

We hypothesize that CAR modified NK progenitors/precursors derived from CD34+ HSCs combined with HSC transplantation may provide enhanced anti-B-ALL-specific NK effectors with long-term persistence, thus likely increasing the efficacy of NK cell therapy for B-ALL. To test this hypothesis, two specific aims are proposed.  

Aim 1: To evaluate specific and enhanced killing of B-ALL in vitro and in immunodeficient mice by genetically modified NK progenitors/precursors. We will use the non-viral Sleeping Beauty (SB) transposon system to achieve integration and stable expression of CAR for CD19 antigen in cord blood-derived CD34+ cells. Transfected CD34+ cells will subsequently be differentiated into NK cells using the feeder free Glycostem clinical grade bioreactor system. As controls, we will use stromal cell co-culture derived NK progenitors and peripheral blood NK cells that are similarly modified by SB. Ex vivo generated NK cells will be evaluated for transgene expression, surface phenotype, cytotoxicity and cytokine production against B-ALL cells and patient blasts. NK cell expansion, persistence and anti-B-ALL activity in mice will be determined.  

Aim 2: To establish conditions for the production of genetically modified NK progenitors/precursors in a good manufacturing practice (GMP) facility. GMP grade NK cells will be evaluated for in vitro function and in vivo anti-leukemia efficacy. We are well positioned to complete these studies considering the extensive experience of our team in SB-mediated HSC gene transfer and NK cell therapeutics. Our strong preliminary data also support the likelihood of accomplishing the proposed aims.  

Our study is significant and innovative because our work can rapidly lead to a clinical trial for high-risk B-ALL using SB modified NK progenitors/precursors as universal “off-the-shelf” immunotherapy and can potentially be applied to the treatment of other hematological malignancies, solid tumors and viral infections.