Research in the Nair Research Lab involves designing and testing novel vaccines against cancer and viral infections in murine and human systems.  The major focus of the lab is generating sustained effector and memory T cell immune responses that will translate to therapeutic benefit for cancer patients.

 

Dr. Nair started working with dendritic cells during her Ph.D. training at the University of Tennessee, Knoxville in the laboratory of Dr. Barry Rouse.  Her doctoral research involved various aspects of antigen processing and presentation, in particular the role of dendritic cells as specialized antigen presenting cells.  A major focus of Dr. Nair's work was to develop dendritic cell-based vaccines against Herpes simplex virus.  This research has allowed her to work with viral systems to study dendritic cell biology, murine tumor models, murine and human dendritic cells and also work with clinical samples to demonstrate dendritic cell function in the setting of cancer.  Dr. Nair joined Dr. Eli Gilboa’s lab at Duke University Medical Center in 1993 and worked as a post-doc for 3 years prior to becoming independent at Duke.

 

The goal of this research is to induce a sustained anti-tumor response in vivo by overcoming tumor-induced immunosuppression and circumventing immune evasion.  The dendritic cell-based vaccine approach has become more feasible in humans with technology that allows us to obtain large numbers of dendritic cells from progenitor cells by culturing peripheral blood mononuclear cells in vitro.  The choice of antigen-presenting cell was based on the wealth of information demonstrating the unique ability of dendritic cells to stimulate naïve T cells and B cells.

 

Many cancers do not express a known tumor antigen and vaccination of patients with a broad repertoire of tumor antigens may have several significant advantages over vaccinating with defined tumor antigens.  Research in this lab focuses on developing a broadly applicable vaccination strategy with tumor-derived antigens that is not dependent on prior knowledge of the tumor antigens expressed in the patients, and is not limited by the availability of tumor tissue from the patient for antigen preparation.  David Boczkowski and Dr. Nair were the key inventors of a groundbreaking technology; using mRNA transfected dendritic cells as vaccines.  In a pioneering study, they demonstrated that dendritic cells pulsed with unfractionated total RNA isolated from tumor cells stimulate tumor immunity.  The researchers have shown induction of tumor immunity in murine models as well as CTL induction in vitro in human preclinical studies, using RNA-transfected dendritic cells. They have also developed protocols to amplify the mRNA content from a few tumor cells, thereby generating an unlimited supply of tumor antigen.

 

The RNA-transfected dendritic cell vaccine platform established our program at Duke and since then has been extensively investigated in many labs in the United States and Europe.  The biotech company, Argos Therapeutics Inc. in Durham, NC, is based on the use of mRNA transfected dendritic cells for cancer immunotherapy and viral infections.  In 1999, Dr. Johannes Vieweg initiated a series of phase I clinical trials at Duke University to explore the use of RNA-transfected dendritic cells in patients with renal cancer and prostate cancer with safety as the primary endpoint and induction of T cell responses and clinical responses as the secondary endpoint.  The clinical trials established the safety of the approach and also demonstrated that although we generated immunological responses in patients the clinical responses were minimal.

 

 

Vaccinating against tumor-specific antigens for cancer immunotherapy is complicated by the fact that tumor cells are genetically unstable and undergo mutations thereby giving rise to variants that can escape immune detection.  We have circumvented this limitation by targeting the antigens expressed in the tumor stroma (e.g. vascular endothelial growth factor).  It is widely recognized that tumor progression beyond a minimal size is critically dependent on normal cells known as the tumor stroma.  Moreover, since stromal cells, unlike tumor cells, are diploid, genetically stable and exhibit limited proliferative capacity, targeting the stroma could substantially reduce the incidence of immune evasion.  Stromal products also provide a source of “universal” antigens that could be targeted in every cancer patient and offer a broad spectrum of candidates from which to choose.

 

Dendritic cells used for immunotherapy are cultured ex vivo and are “matured” by the addition of various reagents that mimic infection or inflammation.  Aside from the requirement for expensive and often difficult to obtain cytokines and other chemicals, this ex vivo maturation has been shown by some studies to generate sub-optimal dendritic cells.  We developed a protocol wherein immature dendritic cells were injected into sites that were exposed to agents that induce a microenvironment conducive to functional in situ maturation of the injected dendritic cells.  The hypothesis tested in this study was that by using appropriate agents it will be possible to recapitulate the physiological conditions occurring during pathogen infection in a manner that will lead to the optimal conditions for dendritic cell maturation, migration and function.

 

Tumor-induced immune suppression is still a major obstacle in cancer immunotherapy. Our hypothesis is that preferential depletion of regulatory T (Treg) cells will enhance the potency of dendritic cell-based cancer vaccines.  The importance of thymically derived CD4+ regulatory T cells is becoming increasingly evident in many studies that demonstrate the role of Treg cells in controlling autoimmune manifestations and maintaining peripheral tolerance.  Treg cells are characterized by the expression of the IL-2R chain and the forkhead family transcription factor (Foxp3), which is critically important for the development and function of the Treg cells.  To specifically eliminate the Treg cells induced during immunization with dendritic cells transfected with tumor antigen, we co-immunize with dendritic cells transfected with Foxp3 RNA with the aim of inducing Foxp3 specific T cells that can lyse Foxp3 expressing Tregs.  Our studies indicate that co-immunization with dendritic cells transfected with tumor antigen and Foxp3 results in enhanced tumor responses.  Further analysis of the tumor demonstrated a decrease in the number of CD4+ and Foxp3+ T cells supporting our hypothesis.

 

 

Rational approach to vaccination involves the following: 1) the identification of the protective effector mechanism, 2) the choice of an antigen that can induce a response in all individuals, and 3) the use of an appropriate way to deliver the vaccine so that it will induce the desired type of response.  The effective design of vaccines therefore requires a full comprehension of each of the above parameters in vivo.

 

Dr. Nair is proposing the use of a multipronged approach to effective design of anti-viral and anti-tumor vaccines: 1] induction of antigen-specific responses plus 2] elimination of immunosuppression, specifically elimination of Treg mediated suppression along with 3] effective generation of memory T cell responses by inducing consistent CD4 T cell responses.

 

1] Local modulation of immune responses.  Systemic administration of monoclonal antibodies (mAbs) targeting immune receptors enhances the stimulation of anti-tumor immunity in mice.  However, clinical use of such mAbs is limited by toxicity associated with non-specific T-cell activation.  Targeted administration of such mAbs to sites where anti-tumor T-cells are induced would eliminate these systemic effects.  We demonstrate that when DC are transfected with RNA encoding mAbs, mAbs are secreted locally.  In preclinical studies, anti-tumor immunity was enhanced with no evidence of autoimmunity, when mice were immunized with DC transfected with RNA encoding mAbs targeting GITR or CTLA-4. 

 

The goal is to translate these promising findings into improved clinical cancer immunotherapy in patients with metastatic melanoma with our collaborator Dr. Scott Pruitt.

 

2] Inhibit the immunosuppression mediated by regulatory T cells.  Recent studies showed that CD4+ Treg cells control immune responses in cancer and viral infections.  Thus, an intervention that eliminates regulatory T cells, along with stimulation of antigen-specific immune responses could potentially be effective in enhancing the efficacy of vaccines.

We have developed a novel approach to eliminate Treg cells in vivo: inducing an immune response against Treg cells using dendritic cells transfected with Foxp3 mRNA.  The researchers are now testing another unique approach to eliminate Treg cells: immunizing against TGF-ß.  The hypothesis is that immunization with dendritic cells transfected with TGF-ß will generate TGF-ß-specific CD8+ T cells that will lyse Treg cells.  Many studies have demonstrated that membrane TGF-ß-expressing T cells interact with TGF-ß receptor-expressing CD8 T cells thereby inhibiting their ability to perform cytolytic functions.  Our studies indicate that immunological targeting of TGF-ß stimulates T cell responses that can indeed lyse Treg cells in vitro.

 

3] Generating aptamers to immune receptors.  In conjunction with Dr. Bruce Sullenger’s lab, the Nair lab is developing a novel approach to modulate the function of Treg cells and simultaneously enhancing antigen-specific T cell stimulation by the use of aptamers.  The role of glucocorticoid-induced TNFR-related gene (GITR) triggering in inducing pro- and anti-apoptotic effects, suppressing Treg function and stimulating responder T cells makes the GITR-GITR ligand interaction critical in the regulation of immunity.  To achieve this goal we would like to develop a new cell-based approach for selecting immunological aptamers and use this information to design an aptamer to GITR.   They plan to use the GITR aptamer to enhance tumor immunity by testing the following:

 

1)      Develop agonistic GITR aptamers and use them in conjunction with dendritic cell-based tumor vaccines.

2)      Use the GITR aptamer to deliver Foxp3-specific siRNA to Treg cells.

 

4] Induce antigen-specific responses.  To achieve this goal we can immunize with dendritic cells transfected with mRNA encoding viral/tumor antigens.  The drawback in the use of dendritic cells is scalability and cost for generating patient-specific vaccines.  The researchers can circumvent this problem by immunizing against tumor or viral antigens using a naked DNA or RNA vaccine.  They are currently in the process of establishing this approach for cancer vaccines using the gene gun to deliver RNA or DNA encoding tumor antigens directly in the skin.  An added benefit of using this approach is delivering biological response modifiers (like cytokines or chemokines) in the form of RNA or DNA along with the antigen.  They are also collaborating with Dr. Kam Leong to develop the next generation of drug delivery systems using polymer-based nano- and microparticles.