Autoantibodies in the Evolution of Systemic Lupus Erythematosus. Commercial Entities 10/25/2013

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1 // Autoantibodies in the Evolution of Systemic Lupus Erythematosus John B. Harley, MD, PhD Division of Rheumatology Center for Autoimmune Genomics & Etiology Cincinnati Children s Hospital Medical Center Cincinnati, Ohio, USA Preclinical Rheumatic Disease American College of Rheumatology :-: PM, Friday, October, San Diego Commercial Entities Erba Diagnostics, Inc. Member, Board of Directors Consultant Options Genentech, Inc. Consultant Lilly Pharmaceuticals, Inc. Consultant Regeneron Pharmaceuticals, Inc. Discussions Evidence Based Medicine Three References (required by ACR) James JA, Kaufman KM, Farris AD, Taylor-Albert E, Lehman TJ, and Harley JB. An increased prevalence of Epstein-Barr virus infection in young patients suggests a possible etiology for systemic lupus erythematosus. J Clin Invest. 997 Dec ;():9-. [PMID: 9999] Arbuckle MR, McClain MT, Rubertone MV, Scofield RH, Dennis GJ, James JA, and Harley JB. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med. Oct ;9():-. [PMID: 79] McClain MT, Heinlen LD, Dennis GJ, Roebuck J, Harley JB, and James JA. Early events in lupus humoral autoimmunity suggest initiation through molecular mimicry. Nat Med. Jan;():-9. [PMID: 9] malar rash, malaise, headache, abd pain, peripheral edema, hematuria, fever granular casts, pleural effusion, pulmonary infarction Smith & Cyr Rheum Dis Clin NA :, 9. Sequeira & Balen Br J Dermatol :7, 9 J. James

2 // ACR Classification Criteria for SLE Malar Rash Discoid Rash Photosensitivity Oral Ulcers Arthritis Serositis Pleurisy Pericarditis Kidney Involvement Neurological Disorder Seizures Psychosis Hematological Disorder Hemolytic Anemia Leukopenia Lymphopenia Thrombocytopenia Serological Disorder Anti-dsDNA Anti-Sm Antiphospholipid Antibodies IgG IgM Lupus Anticoagulant False-+VDRL Anti-Nuclear Antibody (ANA)

3 // The Army-Navy Serum Repository Patients Present to Environmental Clinicians Risk Factors Pathological Injury Contains,, Serum Samples Collected on all Active Duty Military Personnel upon entry into service and on average every two years afterwards SLE Genetic Predisposition Clinical Disease Onset of Autoimmunity & Autoantibodies GOAL: Identify SLE patients with sera stored prior to disease diagnosis J. James The ArmyNavy Serum Repository Case Serum Samples Retrieved - - Pre-clinical initial criterion in SLE patients: Comparison by ethnicity (%) (%) (%) (%) (%)..... st Clinical Criterion ANA Anti-Sm Anti-dsDNA Anti-La Time (years) Time (years) Heinlen, et al. Arthritis Rheum 7 7 (%) Post- Pre- Women # auotabs Men African-Americans European-Americans Asians Hispanics. Percent Positive Number of Samples Number of Serum Samples: Average Number of Samples per case:.9 (-) All patients had at least one sample prior to diagnosis. % of Cases also had serum samples available after diagnosis. Distribution of Samples with Respect to apl Anti-Ro Anti-nRNP Anti-Cq Yrs relative to SLE Dx Arbuckle et al. NEJM 9:. Preclinical Initial criterion in SLE patients: Comparison by gender Heinlen, et al. Arthritis Rheum 7

4 // Which autoantibodies are associated with clinical features before SLE classification criteria are met? Do autoantibodies appear before associated clinical features in the pre-clinical lupus patient? Autoantibodies Associated with SLE Clinical Features Rheumatoid factor with arthritis Anti-dsDNA with nephritis Anti-Ro with photosensitivity (EA only) Anti-Ro with leukopenia Heinlen, et al. Arthritis Rheum 7 Rheumatoid factors appear prior to arthritis Anti-Cq & anti-dsdna precede nephritis OR= p<. OR=., p<. OR=. p<. OR=.7 p<. All data are from before classification Heinlen, et al. Arthritis Rheum 7 All data are before diagnosis Heinlen, et al Arthritis Rheum 7 Rapid SLE Onset in African-American Men Nephritis No Nephritis Anti dsdna Prior to Clinical SLE 7 SLE cases (of ) Years after onset AA Males.-fold shorter Arbuckle et al. Lupus :99, Half with nephritis diagnosed at presentation AA Male Proteinuria OR=., p=. AA Male Casts OR=, p=. diagnosis (Dx) OR=.7, p=. + ādsdna with nephritis OR=., p<. Arbuckle, et al. Scand J Immunol.. Anti-dsDNA vs. Age (yrs) *AA with arthritis, nephritis, leukopenia, lymphopenia, f+vdrl, āsm, ādsdna, +ANA st post-dx (< mo) ādsdna %

5 // IFN activity level elevations are most pronounced at/after diagnosis IFN increases to SLE classification Mean IFN Activity Scores p<. N <-=9 N- to -= N - to = N>= p=. (trend) N before st=7 N at st criteria= N at diagnosis= of, + autoantibodies before detectable IFN James, Niewold, unpublished. James, Niewold, unpublished. The ArmyNavy Serum Repository Men Women African-Americans European-Americans Asians Hispanics 7 (%) Time (years) from first recorded criterion to SLE classification Hydroxychloroqine (HCQ) is associated with delayed SLE classification (Class) (%) (%) (%) (%) (%) HCQ status n Median MEAN Std. dev. Range Pre-Class Not pre-class # auotabs ANA Anti-Sm Anti-dsDNA Anti-La Time (years). apl Anti-Ro Anti-nRNP Anti-Cq * Steroids were confounded by the HCQ effect; non-steroidal anti-inflammatory agent therapies showed no significant statistical difference between those treated and those not treated. st Clinical Criterion Yrs relative to SLE Dx James, et al. Lupus :, 7 Arbuckle et al. NEJM 9:. Accrual of Select Autoabs in Individuals Receiving HCQ (A), Steroids (B), or NSAIDs (C) before SLE Classification The ArmyNavy Serum Repository Men Women African-Americans European-Americans Asians Hispanics 7 (%) (%) (%) (%) (%) (%).. # auotabs - Percent Positive Percent Positive. p-value * p= st Clinical Criterion ANA Anti-Sm Anti-dsDNA Anti-La Time (years). apl Anti-Ro Anti-nRNP Anti-Cq Yrs relative to SLE Dx. - James et al, Lupus, Arbuckle et al. NEJM 9:.

6 Number of Individuals // kd Ro and nrnp A are the most common initial targets of lupus autoimmunity Select autoantigens are more likely to be targeted early in SLE La Chrom P dsdna ds/a ds/sm ds/p ds/ chrom/ A/ScI7 /chrom ds/a A/Sm P/Sm chrom/ds nrnp A kd Ro / chrom/a /La ds/p/sm ch/ds/ a/sm/ ch/ds/sm ch/7/a P/a/Sm ch/7/sm P//La ch/a/sm //La chrom/ds/p/ chrom/p/sm/ chrom/a/sm/ a/sm//la P///La chrom/ds/a/sm chrom/7/a/sm ds/p///la ch/a/sm//la ch/p/a/sm/ ch/ds/p/a/sm ch/ds///la Heinlen, et al.. PLoS One. Initial Autoantibody Specificities P/sm///La Heinlen, et al.. PLoS One. Lupus Lessons from the DoD Serum Repository Two examples of the serial initiation of autoantibody specificities before diagnosis (Western blots) Autoantibodies commonly occur before SLE symptoms. Anti-kD Ro and anti-nrnp A are the two most common, initial autoimmune responses in pre-clinical lupus Autoantibodies precede their associated clinical findings: Anti-dsDNA before nephritis, RF before arthritis. IFN increases at clinical presentation nrnp 7K kd Ro La nrnp A Sm B nrnp 7K kd Ro La Solid Phase Peptide Assay James JA, et al. J Exp Med. : -, 99. GIMAPPPG IMAPPPGM MAPPPGMR APPPGMRP PPPGMRPP James JA, et al. J Exp Med. : -, 99. PPPGMRP(G)P - Repeated times in carboxyl terminus - Major target of all anti-sm sera - Comprises -% of anti-sm response - Target of monoclonal antibodies - Appears to be st Sm B epitope James et al. 99 JEM

7 Absorbance Absorbance Absorban ce Absorbance Platelets Creatinine WBC 7 Absorbance Absorbance Absorbance // Two Rabbits Immunized with PPPGRRP-MAP SmB/B' SmD James Ann NY Acad Sci. :, 997 PPPGMRPP of Sm Closest infectious agent sequence was PPPGRRP from Epstein-Barr Virus nuclear antigen- (EBNA-) ~% cross reaction between PPPGMRPP and PPPGRRP was demonstrated in mature anti-sm responses Immunized rabbits with PPPGRRP (as a MAP peptide) to test tolerance for anti-sm James, Ann NY Acad Sci, 997. Ro... March 9 Arbuckle. Scand J Immunol. :7, Octapeptide Number Ro Octapeptide Number aa: 9- VTKYKQRNGWSHKD..... SLE Patient # January 99 * * January * December McClain, et al.. Nat Med A kda Ro Octapeptide Number B EBNA- Octapeptide Number C..... D kda Ro ELISA EBNA- Monoclonal Purified anti-ro 9 Anti-9 inhib. w/ebna- EBV (+) SLE pt. EBV (-) Normal. EBV (+) Normal Anti-9 MBP Inhibited EBNA- Inhi bited 7 kda EBNA- -7 peptide immunized rabbits develop Anti-Ro autoimmunity, lupus-like autoimmunity and Clinical features of SLE A EBNA- -7 immunized rabbits Freund s immunized rabbits B Pre-immune kda Ro Octapeptide Number Pre-immune.. Ro 9- aa: TKYKQRNGWSHKD EBNA- aa: -7 GGSGSGPRHRDGVRR McClain, et al. Nat Med Months post-immunization.. Sm B Octapeptide Number McClain, et al. Nat Med, 7

8 Percent Negative // Seropositivity against Epstein-Barr virus in Children and Teenagers SLE is associated with EBV-VCA IgG (OR. p<.) Oklahoma City San Diego Combined # pos (total) # pos (total) # pos (total) Lupus patients 9 (9) 7 () (7) Normal controls (9) () 7 () Odds ration (OR) 9.9 9%-CI of OR 9., -.. Probability (p). Meta Analysis of EBV VCA IgG in SLE cases and controls Statistics for each study Odds ratio and 9% CI Study name Odds Lower Upper ratio limit limit Z-Value p-value Zandman-Goddard Tazi Huggins Parks James James/Kaufman Harley Tsai Origgi Favours A Favours B Meta Analysis James, et al. J Clin Invest, 997 Sestak, unpublished,. EBV DNA in PBMC from SLE cases and matched controls (James JCI 997) James, et al. J Clin Invest, 997 EBV DNA in PBMC from SLE cases & matched controls (James JCI 997) Case EBV DNA + - Control + EBV DNA - 9 OR > 7, p =. James, et al. J Clin Invest, 997 EBV serology before diagnosis in military SLE & control samples Seronegative rates of military personnel who develop SLE Positive Negative SLE Anti-VCA 9 Control Anti-VCA. Odds ratio =.7, p =.9 SLE Anti-EBNA Control Anti-EBNA. Odds ratio =.7, p =. * EBNA- CMV HSV HSV * p<. Pts Cntls James, unpublished,. James, unpublished,.

9 SLE Patient SLE Patient Anti - VCA ( - ) Normal SLE Patient SLE Patient Anti - VCA ( - ) Normal EBNA - Monoclonal Standard deviations above normal mean // Anti-EBNA- by Western Blot is Associated with SLE in Anti-EBV VCA Positive SLE Patients and Controls B9 Jijoye B9 Jijoye 7 kd Anti - VCA (+) Normal Anti - VCA (+) Normal EBNA- POS EBNA- NEG EBNA - Monoclonal SLE Anti - VCA (+) Normal c p<. =., p<. Odds Ratio Anti - VCA (+) Normal Normal McClain, et al Arthritis Rheum.. Pediatric SLE patients mount a different immune response to EBNA- compared to controls + EBNA Lupus + - c =., p<. Lupus Normal A B EBNA- Octapeptide Number CMV-IE Octapeptide Number McClain et al.. Arthritis Rheum EBV (-) Order of antibody onset Anti-EBNA- Anti-EBNA- Anti-EBNA- Any Autoantibody Any Autoantibody Anti-EBNA- EBV (-) EBV (-) Anti-EBNA- Any Autoantibody Anti-EBNA- Anti-EBNA- Anti-Ro Anti-Sm Anti-Ro Anti-Sm # Pts 7 Does Anti-ENBA- Preceed Autoimmunity Anti-ENBA- before any autoantibody N= Any autoantibody before anti-ebna- N= Odds ratio = By McNemar Χ, p x -7. McClain, et al. Nat Med The First Autoantibody Antigen presentation Autoantibody Toll-like receptors Antigen presentation through Class II pathway. Generation of specificities that would have been previously eliminated in development. Lupus Specific Differences in EBV Exposure and Responses --fold Increased EBV loads in SLE. Increased number of EBV infected B cells Increased expression of specific EBV genes in SLE Defective CD T cell responses in SLE Anti-VCA and anti-ebna- precede lupus autoimmunity. Anti-EBNA- humoral responses in pediatric SLE tend to be against the cross-reactive antigenic structures relative to controls 9

10 // The Critical Moment Properties expected of a pathogen causing autoimmunity in systemic lupus erythematosus Ig maturation Heteroimmune Antibodies Self The First Autoantibody Ig maturation Self Self Autoantibodies Harley & James. Bull NYU.. Harley, Harley, Guthridge, James. Lupus.. Ubiquitous infection Persistent immune stimulation Infection precedes autoimmunity Leads to pathogenic autoantibody Inhibits apoptosis Induces Type interferon Initiates autoimmune response Inhibits its own destruction Breaks tolerance Before Epstein-Barr Virus Systemic Lupus Erythematosus Genetic Risk (IRF, ITGAM, STAT, BLK, ) EBNA Sm Anti-EBNA- Cross-reactive EBNA/self antibodies Benign Autoimmunity Pathogenic Autoimmunity Pathologic Injury Clinical Disease Environmental Factors (EBV, IFNs, Vitamin D ) EBNA Ro Asymptomatic Life Threatening Illness James & Harley. Arthritis Rheum.. Environmental Etiology: Epstein-Barr Virus Infection At risk Normal Stages of Lupus Autoimmunity Anti- EBNA Autoimmunity Onset: Autoantigenic Bottleneck Cross-reactive anti-ebna (e.g., anti-ro) Probably important: Latent EBV infection with high viral load, INFα generation. Genetics Summary Environment Sex; Race Epstein-Barr virus > Genes Vitamin D deficiency Immune history Unified understanding of disease etiology Better diagnosis, prognosis, therapy & prevention Challenges for Preclinical Lupus Will someone do a trial in preclinical lupus with hydroxychloroquine (&?ASA): ANA positive patients with various complaints? When, where, and how do the many genes operate to increase lupus risk? What are unusual details of the Epstein- Barr virus infection in preclinical lupus that create disease risk? How do the genes interact with the environmental risks to generate lupus?

11 // ACKNOWLEDGMENTS Judith James Micah McClain Melissa Arbuckle Latisha Heinlen Brian Poole Joel Guthridge Hal Scofield Tim Niewold Morris Reichlin Mark Rubertone & DoDSR George Tsokos Michael Keith Greg Dennis & US Military Lupus Family Registry and Repository NHGI NCRR/ NIGMS Alliance for Lupus Research NIAID References (order of publication) The Center for Autoimmune Genomics and Etiology (CAGE) James JA, Gross T, Scofield RH, and Harley JB. Immunoglobulin epitope spreading and autoimmune disease after peptide immunization: Sm B/B' derived PPPGMRPP and PPPGIRGP induced spliceosome autoimmunity. J Exp Med. 99 Feb ;():-. [PMID: 77] James JA, Scofield RH and Harley JB. Lupus humoral autoimmunity after short peptide immunization. Ann NY Acad Sci. 997 Apr ;:-7. [PMID: 99] James JA, Kaufman KM, Farris AD, Taylor-Albert E, Lehman TJ, and Harley JB. An increased prevalence of Epstein-Barr virus infection in young patients suggests a possible etiology for systemic lupus erythematosus. J Clin Invest. 997 Dec ;():9-. [PMID: 9999] Arbuckle MR, Reichlin M, Harley JB, and James JA. Shared early autoantibody recognition events in the development of anti-sm B/B in human lupus. Scand J Immunol. 999 Nov;():7-. [PMID: ] Arbuckle MR, James JA, Kohlhase KF, Rubertone MV, Dennis GJ, and Harley JB. Development of anti-dsdna autoantibodies prior to clinical diagnosis of systemic lupus erythematosus. Scand J Immunol. Jul-Aug;(-):-9. [PMID: 99] Arbuckle MR, James JA, Dennis GJ, Rubertone MV, McClain MT, Kim XR, and Harley JB. Rapid clinical progression to diagnosis among African-American men with systemic lupus erythematosus. Lupus. ;():99-. [PMID: 7] Arbuckle MR, McClain MT, Rubertone MV, Scofield RH, Dennis GJ, James JA, and Harley JB. Development of autoantibodies before the clinical onset of systemic lupus erythematosus. N Engl J Med. Oct ;9():-. [PMID: 79] McClain MT, Heinlen LD, Dennis GJ, Roebuck J, Harley JB, and James JA. Early events in lupus humoral autoimmunity suggest initiation through molecular mimicry. Nat Med. Jan;():-9. [PMID: 9] Harley JB and James JA. Epstein-Barr virus infection induces lupus autoimmunity. Bull Hosp Jt Dis. ;(-):-. [PMID: 79] James JA, Kim-Howard XR, Bruner BF, Jonsson MK, McClain MT, Arbuckle MR, Walker C, Dennis GJ, Merrill JT, Harley JB. Hydroxychloroquine sulfate treatment is associated with later onset of systemic lupus erythematosus. Lupus. 7;():-9. PMID: 7 Heinlen LD, McClain MT, Merrill J, Akbarali YW, Edgeton CC, Harley JB, James JA. Clinical criteria for systemic lupus erythematosus precede diagnosis and associated autoantibodies are present before clinicl symptoms. Arthritis Rheum. 7 Jul;(7)-. [PMID:7997] Heinlen LD, McClain MT, Ritterhouse LL, Bruner BF, Edgerton CC, Keith MP, James JA, Harley JB. kd Ro and nrnp A frequently initiate human lupus autoimmunity. PLoS ONE. ; ():e999. PMID: 77; PMCID: PMC7 The End Substitute image above. Original case from: Smith & Cyr Rheum Dis Clin NA :, 9. Sequeira & BalenBr J Dermatol :7, 9 malar rash, malaise, headache, abd pain, peripheral edema, hematuria, fever granular casts, pleural effusion, pulmonary infarction