B. RESEARCH 5. Research Program Summary The multi-disciplinary research in my laboratory covers the entire spectrum of scientific endeavour, from basic research to industrial applications. The identification of the novel HIV co-receptor CCR5, an immune receptor for Mycobacterium TB, was published in Science (co-first author Paul A MacAry et al. Dendritic cell stimulation by mycobacterial HSP70 is mediated through CCR5 Science 2006; 314:454-8). I have extended our work on immune receptors for M.Tuberculosis in collaboration with the A*Star Genome Institute in Singapore and am now analyzing an additional Mycobacterial immune receptor (TLR-8) identified as part of genetic screens for susceptibility loci in large patient cohorts. In recent years my laboratory has discovered a talent for generating novel monoclonal antibodies and has conducted pioneering work on associated FC-receptors. We have developed antibodies with cytotoxic T-cell receptor-like specificity and target virus-infected cells in Nasopharyngeal Carcinoma and Hepatocellular Carcinoma. These are the subject of two new patent applications in 2009. We have also succeeded in making new therapeutic and diagnostic candidates for Dengue virus and Mycobacterium Tuberculosis. The TCR-like antibody that targets HBV infected Hepatocellular carcinoma cells is currently being tested for clinical applications by Roche and our anti-Dengue antibody is being assessed in pre-clinical models by Novartis. Both of these projects are covered by formal research collaboration agreements. I have a Hirsh Index of 15, a citation count of 750 (excluding self-citations), and 33 publications, with 7 in journals with an Impact Factor greater than 10. I have published 17 manuscripts in Tier 1 journals including Nature Medicine, Immunity, Blood and PNAS, USA and have 2 included in the Faculty of 1000. I have published 15 papers since my arrival at NUS in 2005, have written 3 patents and have generated over S$4.9M in peer reviewed external grant funding. I am also the co-founder and director of the British Stem Cell Registry Ltd and Co-Founder/Meetings Secretary of the Singaporean Society of Immunology (SSI). My future research objectives can be summarized as follows: To establish my laboratory as a global leader in MHC research based on unique tools and methodologies that weve established in-house and to use the same technologies to spin-out an antibody company from the National University aimed at the production of new therapeutics and diagnostics. Areas of Research Covered The MHC class-I antigen presentation pathway has been a central theme in the majority of the major investigations in which Ive taken part since the start of my PhD studies 16 years ago (Fig. 1). The MHC class-I related researches that Ive been involved in have included: projects on the molecular and cellular events associated with MHC class I presentation and cross- presentation; on the principle cell type that elicits MHC class I restricted immune responses (Dendritic cells); and on the effector immune cells that express receptors restricted to MHC class I (CD8 T cells). Recently, my focus has shifted to: molecular vehicles such as heat shock proteins that can be used to chaperone antigens to preferentially stimulate MHC class I responses; monoclonal antibody reagents that have specificity for MHC class-I/peptide complexes; and viruses such as Dengue and Influenza A that elicit MHC class I restricted responses. i). 1993-1999. PhD Thesis, Guys, Kings and St.Thomas (GKT), London-Mentor: Professor D. Michael Kemeny. Title; Investigating the role of MHC class I restricted CD8 T cells in influencing T helper 1 (Th1) versus T helper 2 (Th2) differentiation in CD4 T cells. My PhD thesis work was focused upon analyzing the role of MHC class I restricted cytotoxic T cell responses in promoting T Helper 1, CD4 T cell differentiation as a potential regulatory mechanism in a rat model of Asthma. To this end, I developed culture conditions that allowed for single cell cloning of rat ovalbumin-specific MHC-class I restricted CD8 T cells (the first rat clones of this type generated) then applied these cells in an existing model of asthma that was well-established in the Kemeny laboratory. At that time, it was postulated that a key element in the aetiology of the allergic response to inert protein antigens (allergens) was an inherent imbalance in the atopic allergen specific CD4+ T cells, resulting in the dominance of pathological, IL-4 producing, T helper 2 phenotype cells which favoured B cell IgE production. My work with the Ovalbumin-specific cytotoxic T cell clones demonstrated that a CD8 T cell response engendered at the same time as an allergic response, resulted in an immune deviation of the CD4 T helper cells towards a non-asthmatic T helper 1 phenotype. I also showed that this activity was not dependent on IFN-gamma secretion or on the cytolytic activity of the CD8 T cells. Subsequent work from the Kemeny group and others has established that CD8 T cells promote T helper 1 responses by inducing the secretion of the key regulatory cytokine IL-12p70 from Dendritic cells and that this induces the differentiation of Th1 cells from nave CD4+ precursors. This work has resulted in several manuscripts published in the Journal of.Immunology (Impact factor 6.17, Tier 1) and European Journal of Immunology (Impact Factor 4.863, Tier 1)-see published manuscripts nos: 2,7, 21 and 23-33 (section 6a). ii). Post-doctoral fellowship, Cambridge University (Mentor-Professor Paul Lehner) The immunobiology of MHC class I in human Dendritic cells 1999-2001. The Lehner group in the Cambridge Institute for Medical Research is one of the leading laboratories globally on viral and host factors that regulate MHC class I and associated CD8+ T cell responses. My initial project work was geared towards analyzing the cell biology of MHC class I molecules in an important subset of immune cells termed Dendritic cells. This required the optimization of in vitro culture systems to derive human Langerhans cells from Haematopoietic stem cells for comparison with inflammatory DCs derived from isolated human peripheral blood monocytes. Langerhans cells are a subset of dendritic cells (DCs) found in the human epidermis with unique morphological and molecular properties that enable their function as sentinels of the immune system. Given that DCs like Langerhans cells are proposed to be pivotal in the initiation and regulation of primary MHC class I restricted T lymphocyte immune responses through presentation of both endogenous and exogenous antigens on class I molecules, this represented a study with important implications for basic immunology. I showed that inflammatory stimuli induce an increase in all components of the MHC class I pathway including the Transporter for Antigen Presentation (TAP), tapasin and ERp57, and the immuno proteasome subunits LMP 2 and LMP 7. Moreover, in Langerhans cells, the rapid increase in expression of MHC class I molecules seen at the cell surface following LPS activation was due to mobilization of MHC class I molecules from HLA-DM positive endosomal compartments, a pathway not seen in monocyte-derived DCs (Fig 2). These data demonstrated the regulation of the class I pathway in concert with the maturation of the Langerhans cells and suggested potential sites for antigen loading of class I proteins. This work has resulted in two manuscripts published in PNAS USA (impact factor 10.228, tier 1) and other international journals, see published manuscripts nos: 12, 14, 18-19 and 22 (section 6a). This work also resulted in a successful Young Investigator Award application in 2005 (section 6c- Title:Dendritic Cell Generation of EBV and Human CMV specific CTL). iii) Analyzing heat shock proteins as molecular vehicles for stimulating CD8 T cells through MHC class I. Post-doctoral fellowship, Cambridge University (Mentor-Professor Paul Lehner) 2001-2004. Building upon our observations in the immunobiology of the MHC class I pathway in human Dendritic cells, I moved towards analyzing molecular vehicles that could be used to preferentially shuttle target antigens into this pathway. To this end, I developed culture systems that allowed the analysis of anti-Influenza A responses by human MHC class I restricted CD8 T cells and employed these as a read out for appropriate antigen loading. At this time, microbial heat shock proteins (HSPs) had been implicated in the induction of both the innate and adaptive arms of the immune response. I initially targeted Mycobacterial HSP70 because the structural basis of substrate antigen binding had been resolved. This allowed me to show that human dendritic cells (DC) pulsed with peptide-loaded mycobacterial HSP70 complexes generate potent antigen-specific cytotoxic lymphocyte (CTL) responses, which are dependent on an HSP70-stimulated calcium signaling cascade (Fig 3 and Fig 4). From the calculated peptide binding affinity of mycobacterial HSP70 (K(D) = 14 microM) we showed that 120 pM HSP70 bound peptide is sufficient to generate a peptide-specific CTL response that is up to four orders of magnitude more efficient than peptide alone. We also identified the functional domains of Mycobacterial HSP70 and showed that a minimal 136 amino acid, mycobacterial HSP70 peptide binding domain can generate CTL responses. Finally, we that a single amino acid mutant HSP70 designed to prevent peptide binding but retain stimulatory capacity separated antigen delivery from DC immunostimulation for the first time in HSP immunobiology. This work resulted in publications in Immunity (impact factor 20.68, tier 1) and Journal of Immunology (Impact factor 6.17, Tier 1) plus inclusion in the Faculty of 1000. See published manuscript nos: 9, 16-17 (section 6a). iv) Identification and characterization of immune receptors for Mycobacterium. 2004-present day. An interesting spin-off from our interest in Mycobacterial HSP70 was the identification of immune receptors on human monocytes and dendritic cells that allow them to respond to these mycobacterial proteins. We knew that an effective host immune response to mycobacterial infection must control pathogen dissemination without inducing immunopathology and that constitutive overexpression of mycobacterial Heat Shock Protein (myHSP70) was associated with impaired bacterial persistence, but the immune-mediated mechanisms remained unknown. The identification of the immune receptors for Mycobacterial HSP70 provided an explanation for this phenomenon. We were initially involved in the identification of CD40 as a candidate receptor for myHSP70. We then moved on to provide genetic, biochemical and functional data showing that myHSP70, in addition to enhancing antigen delivery to human dendritic cells (DC), signals through the CCR5 chemokine receptor, promoting DC aggregation, immune synapse formation between DC and T cells and the generation of effector immune responses (Fig 4). The finding that CCR5 acts as a pattern recognition receptor for myHSP70 has implications for both understanding the pathophysiology of tuberculosis and the use of myHSPs in tumour-directed immunotherapy. This work has also stimulated a general interest in my laboratory on characterizing immune receptors for Mycobacteria. In recent years we have switched focus to a poorly characterized Toll-like receptor (TLR8) that has been implicated in susceptibility to TB infection in a number of large human cohort studies. This work resulted in one publication in Science (impact factor 30.268, super Tier) and one publication in Immunity (impact factor 20.68, tier 1) plus a manuscript included in the Faculty of 1000. In recent years my work on immune receptors for TB has also resulted in two successful BMRC project grant application in 2006/7 (section 6c: Titles: Mycobacterial HSP70 Cross Presentation: Defining the roles of DC-T cell interactions and G-Protein coupled receptors; and Mycobacterial versus human HSP70-A detailed functional comparison) plus one SMART Pilot project grant in 2008 (section 6c: Title: Characterisation of a new innate immune receptor for Mycobacterial Tuberculosis). v). Assistant Professorship in the Department of Microbiology, NUS. Generation of cytotoxic T cell-receptor like (TCR-like) monoclonal antibodies. 2006-present day. On becoming an independent Principal Investigator, I was keen to establish technology platforms that would allow a more detailed analysis of the MHC class-I pathway. The advent of MHC tetramer technology has enabled the detection and analysis of rare populations of antigen-specific T lymphocytes. However, existing methodologies cannot be utilised to determine the distribution of T cell receptor (TCR) ligands on individual cells or to visualise antigen presenting cells (APC) in tissues. In our laboratory, we have made a significant technological breakthrough that allows for the development of TCR-like anti-HLA/peptide specific monoclonal antibodies using conventional hybridoma approaches with a higher degree of reliability than has been previously possible. These monoclonals represent unique immunological reagents that can be utilised to detect, visualise and quantify antigens present as MHC-class I/peptide complexes on the surface of APC, virus infected cells and tumour cells. As proof of principle, we have developed a panel of new monoclonal antibody reagents specific for several tumour virus antigens expressed in association with HLA-A201, HLA-A1101 and HLA-A2402 on the surface of Epstein-Barr virus (EBV) infected cells and Hepatitus B (HBV) infected cells. I detail our methodology for the generation of TCR-like monoclonals (Fig 5) and provide data on their specificity, affinity, isotype and function (Figures 6 and 7). These reagents will potentially revolutionize our understanding of how peptide antigens are delivered and loaded onto MHC class I molecules by allowing a level of molecular and cellular analysis that has not been possible before. They have the added advantage of targeting cells infected with tumour viruses based on complexes expressed on the cell surface. As such, they have already generated a significant degree of interest from the Pharmaceutical industry as reagents that will target tumour cells in situ. This work was initiated in 2006 and has already resulted in two US patent applications this year. Section 6b: 1)Method of Producing T-cell receptor like monoclonal antibodies and uses thereof. Authors: MacAry PA, Chan SH and Hansen B. ILO REF:09165N US. Provisional filing for US patent submitted November 2009. and 2) Hepatitis B virus specific antibody and uses thereof. Authors: MacAry PA, Bertoletti A and Chan SH, ETPL REF: SICS/P/05582/00/US. Provisional filing for US patent submitted November 2009. Our interest in antibodies and the immune receptors they utilize to mediate function has also resulted in a number of published manuscripts in Blood (impact factor 10.4, tier 1), Nature Medicine (impact factor 28.965, super tier) and BMJ (impact factor 12.87, tier 1) plus a covering article on the BBC http://news.bbc.co.uk/2/hi/health/ 6233729.stm. I also anticipate submitting 2 additional patents and 2 manuscripts in the near future on TCR-like antibodies (planned for early 2010). The generation and application of these antibodies for analyzing the MHC class I pathway plus targeting human tumors is now a major part of the research focus in my laboratory. This work is supported by two peer reviewed grant applications (section 6c: A*Star Singapore Immunology Network Title: Targeting EBV associated malignancies using anti-HLA-A2/LMP1 specific monoclonal antibodies and SMART-Pilot project: Generation of EBV-lymphoma model in humanized NOD-SCID mice). ii) Generation and characterization of anti-Dengue human monoclonal antibodies 2008-present day. The establishment of technology platforms in my laboratory to allow for the generation of novel monoclonal antibodies has led to a number of important spin-off projects. One of these involves the isolation and immortalization of human B-lymphocyte clones that secrete monoclonal antibodies with anti-viral activities. As proof of principle, we have engaged clinicians in the Infectious Disease Division at NUH and the Communicable Diseases Centre at Tan Tock Seng hospitals to recruit patients recovering from Dengue virus infection. We are now utilizing immortalized B lymphocytes from these patients as sources of cloned recombinant monoclonal antibodies that neutralize Dengue virus (Fig 8). We have already cloned and characterized a human monoclonal antibody that efficiently neutralizes Dengue serotype 1 in vitro and in vivo. This is subject to further pre-clinical testing by Novartis and we are expanding our patient cohorts to include dengue serotype 2,3 and 4. This project has direct clinical implications as Dengue is an important re-emerging disease affecting humans in more than 100 countries worldwide. Presently, over 2.5 billion people live in risk areas and 50 to 100 million people suffer from dengue fever (DF) each year. The World health Organization (WHO) estimates that currently 500,000 cases of Dengue Hemorrhagic Fever / Dengue Shock Syndrome (DHF/DSS) and more than 20,000 deaths occur per year. In the last 25 years, Dengue epidemics have had a tremendous impact on social and economical structures in countries situated in the tropics such as Singapore. At the moment, there are no effective vaccines or drugs available to prevent or treat Dengue disease. During the early stages of Dengue infection, the virus first fuses with host cell membranes through cell surface receptors. This facilitates the entry of the dengue virus into the interior of the host cell where it initiates lytic replication. Studies reported by several groups world-wide have intimated that antibodies play an important role in hindering virus entry through the blocking of virus membrane proteins. Hence, the antibodies isolated will prove useful tools for studying the immunology of Dengue virus infections and as possible therapeutic reagents to prevent virus dissemination in infected individuals. This project is part of a large 5-year STOP-Dengue TCR application (S$25M) (section 6c). As this project is still at a preliminary stage, we have not generated any IP or published manuscripts though we anticipate that characterizing the first natural human antibody monoclonal to Dengue should result in one patent plus one published manuscript in 2010. iii) Anti-Lipid Monoclonal antibodies Having established a reputation for producing rare and novel monoclonal antibodies, my laboratory was approached to participate in a research consortium aimed at producing novel reagents for lipid research. Lipids play a central role in cellular function and disease. The scope of lipid involvement in cellular function has only recently been recognized to extend well beyond its established roles in energy metabolism and membrane structure. Lipids are an extensive group of small, amphipathic molecules that are divided into a series of classes based on structure and biosynthetic origin. Detailed studies on the synthesis, trafficking and cellular location of lipids, combined with identification of their binding partners/targets have been problematic due to a scarcity of high quality molecular reagents with lipid specificities. One of the principle goals of this project is to utilize stable synthetic lipid analogues to develop a panel of monoclonal antibodies with specificity for cellular and pathogen derived lipids. A relatively small number of anti-lipid antibodies have been produced to date. This can not be ascribed to an inherent lack of antigenicity on the part of lipids (or their metabolites) but reflects a lack of application of this technology to lipid studies combined with the poor availability of synthetic lipids. In order to make anti-lipid monoclonal antibodies, my laboratory utilizes a defined phage-antibody library provided by HUMANIX. We have established methodologies for making antibodies against disease associated forms of oxidized cholesterol (15Keto Cholestane, 24S-Hydroxy Cholesterol and 7-Keto Cholesterol) and against glycolipids derived from the cellular wall of Mycobacterium Tuberculosis (Lipoarabinomannan and Mycolic Acid) (Fig 9). We are now testing the utility of these antibody reagents for studies on lipid metabolism in infection and inflammation and as diagnostic/prognostic tools for M.Tuberculosis infection, Athersclerosis and Multiple Sclerosis. This work is part of a large NRF-CRP application (S$10M) of which our allocation is S$1.13M. Our first patent application for our anti-Lipoarabinomannan antibodies will be submitted before the end of 2009 and we anticipate submitting the first methodology manuscripts in early 2010.