Novel Diagnostics and Therapeutics for Systemic Lupus Erythematosus

Paul J. Utz, M.D.

Stanford University

Funded in June, 2003: $300000 for 3 years
LAY SUMMARY . ABSTRACT . BIOGRAPHY . FINDINGS . SELECTED PUBLICATIONS .

LAY SUMMARY

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Using Autoantibody Profiling to Diagnose and Design Treatment for SLE

Autoimmune diseases affect between 3% and 5% of Americans. Connective Tissue Diseases (CTDs) such as systemic lupus erythematosus (SLE) are highly heterogeneous inflammatory disorders that target a variety of organs, including kidneys, lungs, heart, and all blood elements. A hallmark of CTDs is the production of serum proteins called "autoantibodies." These serve as critical diagnostic markers. A large proportion of these autoantibodies also play a role in the disease process. They bind to the body's own proteins (termed "autoantigens," or "self" proteins). This leads to tissue inflammation and ultimately destruction. Treatment of SLE is toxic, and largely nonspecific treatments target many blood cells, as well as unintended cells such as those that line the gut or bladder. Most importantly, most therapies, particularly those for serious manifestations such as kidney, brain, and lung disease, are not very effective.

The Stanford researchers propose to extend upon recent findings to develop a novel method to analyze thousands of different autoantibodies simultaneously. These results can then be used to design "magic bullets" that eliminate only the disease-causing blood cells, leaving the remaining healthy blood cells to fight off infections. Ultimately, this therapy could be custom designed for each patient.

The detection of serum autoantibodies remains a mainstay of clinical diagnosis in patients with SLE. Specific profiles of autoantibodies are associated with lupus disease subsets and can be used to predict future disease manifestations. The identification and characterization of unique autoantigens has been a major focus of the Utz laboratory during the last decade. Although many of the target autoantigens, or "self molecules," in SLE have been identified and extensively characterized, the initiating self molecules in this disease remain unknown, particularly in humans. The Stanford researchers have recently devised a cutting-edge method to analyze autoantibodies that bind to thousands of self molecules that are produced in test tubes and are then deposited onto the surface of glass microscope slides, where they can be analyzed. Each self molecule is printed using a robot that deposits a spot that can only be seen with a special microscope, allowing thousands of different tests to be performed simultaneously in only a few hours.

When new therapies for SLE are reviewed by the United States Food and Drug Administration (USFDA), there is no agreed-upon assay that aids in determining how effective their therapies are, a critical factor in deciding whether to approve the therapies for use by patients. The main goal of the Stanford researchers is to use their novel "protein microarray" assays to identify new biomarkers that can identify patients at risk of developing severe forms of the disease and predict which patients will respond to therapy. Customized vaccines will be developed and tested in animal models of SLE, with a goal to turn off only the destructive blood cells in diseased animals, leading to a state called "immunologic self tolerance." Arrays can then be used to monitor animals in these therapeutic trials.

 If effective, the researchers will then use the arrays to study the effects of experimental treatments on patients enrolled in clinical trials supported by other sources at Stanford's Arthritis Center. These experiments may lead to an entirely new approach to developing patient-specific therapies for SLE and perhaps other autoimmune diseases, and for monitoring the therapies' actions on the immune system.

ABSTRACT

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Novel Diagnostics and Therapeutics for Systemic Lupus Erythematosus

The broad, long-term objective of this proposal is to test the hypothesis that large-scale, parallel detection of autoantibody profiles can be used to develop customized, patient-specific tolerizing therapies, and to improve diagnosis of autoimmune diseases. We will use serum autoantibody profiling to identify autoantigens to encode directly in DNA plasmids that will be delivered as tolerizing agents, using Systemic Lupus Erythematosus (SLE) as a model disease. As an important step toward designing antigen-specific tolerizing therapeutics, we have developed large-scale autoantigen microarrays to perform autoantibody profiling. This proposal will further develop these arrays while exploring several biological questions aimed at identifying the initiating antigen(s), studying B cell epitope spreading, and employing autoantigen microarrays as diagnostic tools.

There are two aims of this proposal:
1. To test the hypothesis that autoantibody profiling can be used as a surrogate marker of tolerance in mice treated with antigen-specific, DNA plasmid-based, tolerizing vaccines;
2. To test the hypothesis that autoantibody profiling can be used to follow response to therapy in human patients enrolled in clinical trials. We will use the information derived from autoatibody profiling to rationally design antigen-specific tolerizing therapies. Therapies will first be tested in animal models of SLE.

If successful in our initial studies, in later years we hope to perform preclinical toxicology and biodistribution studies in collaboration with the NIH-sponsored Immune Tolerance Network (ITN), with the goal to enroll patients in a Phase I trial for SLE in 2007. Arrays will also be employed to analyze serum autoantibody profiles of patients enrolled in one of several trials at Stanford.

Our ultimate goal will be to identify "serologic autoantibody biosignatures" that can be used to predict response to therapy, to predict adverse events related to therapeutic interventions, and to allow better characterization of patient subsets. The results of this proposal have the potential to revolutionize how we treat autoimmune diseases, because DNA-based vaccines are easy to produce, plasmids can be constructed in a few weeks for any know autoantigen, and combination therapy is feasible through codelivery of multiple plasmids. Just as allergists deliver cocktails of allergens based on skin test profiles, we envision combining autoantibody profiling and DNA vaccination to develop customized patient-specific therapy for autoimmunity. While risky, if successful this approach will forever change the way in which autoimmune diseases are treated.

INVESTIGATOR BIOGRAPHIES

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Paul J. Utz, M.D.

Paul J. (P.J.) Utz, M.D., is an Assistant Professor of Medicine in the Division of Rheumatology and Immunology at Stanford University. While earning his M.D. degree from Stanford Medical School, he codiscovered the transcription factor Nuclear Factor of Activated T Cells (NFAT) with Jeng-Ping Shaw in Dr. Gerald Crabtree's laboratory. He completed his residency, fellowship, and post-doctoral training at Brigham and Women's Hospital before joining the Harvard Medical School Faculty in 1996.

Dr. Utz is an expert in the study of human and murine autoantibodies, and his lab is actively collaborating with Dr. Larry Steinman on the development of DNA vaccines for treatment of human autoimmune diseases. His laboratory is developing several cutting-edge proteomics technologies for immunological applications, including multiplex planar-based autoantigen microarrays, microfluidics assays, and other methodologies. Dr. Utz actively cares for patients in Stanford's Arthirits Center, and he is committed to improving the diagnosis and treatment of patients with connective tissue diseases, particularly lupus. The ultimate, long term goal of the Utz Lab is to develop patient-specific, individualized therapeutics for autoimmune diseases.

FINDINGS

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Lay Results:
We have created autoantigen microarrays for studying blood cells and antibodies in patients with systemic lupus erythematosus (SLE) and related forms of arthritis.  Arrays are printed using a robot, and slides containing thousands of microscopic spots are then probed using blood from a patient.  These arrays are then used to see how things change as patients get sicker or improve, and they are also used to design custom therapies.  This includes a novel class of drugs called “Toll-like Receptor inhibitors” that block proteins in blood cells that cause inflammatory diseases.  Arrays are now being used in many different human clinical trials of experimental drugs to monitor their efficacy.

Scientific Results:
A hallmark of autoimmune diseases is the production of high-titer, highly-specific autoantibodies that are used for diagnosis, prognostication, and patient subsetting.  We created arrays of hundreds/thousands of autoantigens and have used these to perform large-scale autoantibody profiling of immune responses in humans with SLE and other connective tissue diseases, and in mouse models of autoimmunity.  We have also used the arrays to analyze responses to specific inhibitors of TLR7 and TLR9.  We have identified a critical role for type I interferon signaling and TLR expression in the development of antibodies directed against RNA and DNA containing autoantigens.  Future studies will explore TLR inhibition and use of autoantibody profiling in humans.

SELECTED PUBLICATIONS

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Drouvalakis K.A., Bangsaruntip S., Hueber W., Kozar L.G., Utz P.J., and Dai H.   Peptide-coated nanotube-based biosensor for the detection of disease-specific autoantibodies in human serum.   Biosens Bioelectron. 2008 May 15;23(10):1413-21.

Shi X., Kachirskaia I., Walter K.L., Kuo J.H., Lake A., Davrazou F., Chan S.M., Martin D.G., Fingerman I.M., Briggs S.D., Howe L., Utz P.J., Kutateladze T.G., Lugovskoy A.A., Bedford M.T., and Gozani O.   Proteome-wide analysis in Saccharomyces cerevisiae identifies several PHD fingers as novel direct and selective binding modules of histone H3 methylated at either lysine 4 or lysine 36.   J Biol Chem. 2007 Jan 26;282(4):2450-5.

Sharp V. and Utz P.J.  Technology insight: can autoantibody profiling improve clinical practice?   Nat Clin Pract Rheumatol. 2007 Feb;3(2):96-103

Paniagua R.T., Sharpe O., Ho P.P., Chan S.M., Chang A., Higgins J.P., Tomooka B.H., Thomas F.M., Song J.J., Goodman S.B., Lee D.M., Genovese M.C., Utz P.J., Steinman L., and Robinson W.H.  Selective tyrosine kinase inhibition by imatinib mesylate for the treatment of rheumatoid arthritis. J Clin Invest. 2006 Oct;116(10):2633-42.

Kattah M.G., Alemi G.R., Thibault D.L., Balboni I., and Utz P.J.  A new two-color Fab labeling method for autoantigen protein microarrays.  Nat Methods. 2006 Sep;3(9):745-51.

Chan S.M., Olson J.A., and Utz P.J.   Single-cell analysis of siRNA-mediated gene silencing using multiparameter flow cytometry. Cytometry A. 2006 Feb;69(2):59-65.

Balboni I., Chan S.M., Kattah M., Tenenbaum J.D., Butte A.J., and Utz P.J.  Multiplexed protein array platforms for analysis of autoimmune diseases.  Annu Rev Immunol. 2006;24:391-418.

Chan S.M. and Utz P.J.   The challenge of analyzing the proteome in humans with autoimmune diseases.  Ann N Y Acad Sci. 2005 Dec;1062:61-8.

Fathman C.G., Soares L., Chan S.M., and Utz P.J.  An array of possibilities for the study of autoimmunity.  Nature. 2005 Jun 2;435(7042):605-11.

Utz P.J.  Protein arrays for studying blood cells and their secreted products.  Immunol Rev. 2005 Apr;204:264-82.