AURA June 4, 2016 Immunologic disease: Progress in wiping the slate clean

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1 AURA June 4, 2016 Immunologic disease: Progress in wiping the slate clean Gregg J. Silverman, M.D. Professor of Medicine and Pathology Laboratory of B cell Immunobiology NYU School of Medicine

2 Disclosures Dr. Silverman has been a consultant for Genentech, Roche, Pfizer, Onyx, BMS, Lilly, Crescendo, Quest Diagnostics Research support from RRF, Lupus Research Foundation, Alliance for Lupus Research and NIH-NIAID FDA approval will be mentioned when relevant 2

3 Immune system is composed of two systems: Overlay of the adaptive immune provides advantages Good Figure 1.9 The memory Immune System, Third provides Edition host advantages But Bad memory can drive autoimmunity Copyright ( 2009) from The Immune System, Third Edition by Parham. Reproduced by permission of Garland Science/Taylor & Francis Books, LLC.

4 Somatic recombination generates an effectively limitless range of antigen receptors from a limited number of germline minigenes Antigen receptor genes move during B- and T-cell differentiation

5 The adaptive immune system continuously generates lymphocyte clones with randomly generated antigen receptors. How do we maintain immune tolerance in health? Two signal hypothesis (Bretscher and Cohn) Science (3950): Signal 1 only = death/inactivation 1 Signal 1+ Signal 2 = activation 2 Signal 3 molds the immune bias of the lymphocyte Courtesy of M. Cancro, U Penn

6 Subset of MHC II genes (shared epitope) predisposes to RA autoantigen-specific T/B cell clonal expansions Log 10 P Value Chromosome The grey horizontal line indicates SNPs that are significant at a genomewide level *SNPs plotted according to chromosomal location, with the log10 P values corrected with the use of genomic control. SNPs, single nucleotide polymorphism; MHC, major histocompatibility complex; TRAF1, TNF signal transduction gene; C5, complement 5 gene. Plenge RM et al. N Engl J Med. 2007;357(12):

7 Overview of RA pathogenesis 7

8 Rheumatoid Arthritis: Hypothesis for Stages of Disease ACPA Epitope Spreading + Titer Elevation Genetics RF - HLA-DRB1 (shared epitope)* - GWAS (PTPN22, others) Cytokines Healthy Autoimmune Early Synovitis OVERT Rheumatoid Arthritis - Smoking - Hormones - Bacteria* - Diet? Pre-Clinical Phase - Arthritis - Radiographic Changes Environmental Factors Klareskog and others Courtesy of J. Scher Expanding repertoire of autoreactive lymphocytes

9 What is our interim progress in wiping the slate clean immune systems of patients with autoimmune disease? Plasma cell T cell Tabula rasa? B cell

10 Diverse approaches to eliminate B cells Stem Cell Pro/Pre B Cell Immature B cell Transitional B cell Mature B cell Antigen stimulation Memory Cell Antibody-producing plasma cells Autoreactive B cells CD19-1 (broadly reactive) SLE RA Other diseases Rituximab (anti-cd20) Belimumab/Tabalumab ( BAFF/BLyS) Atacicept (TACI-Ig) Neutralizes both BAFF/BLyS and APRIL

11 B cell subsets in the blood Switched memory Unswitched memory Plasma cells plasmablasts IgD+ IgD+CD10+ IgD- CD38++ CD27- naïve transitional/pre-naïve double negative, incl. CD27-memory CD27+ CD27++ pre-switched memory post-switched memory plasma cells plasmablasts Jacobi, et al. Arthritis Rheum. 2010;62:

12 Clinical efficacy and B-cell regeneration in RA after Rituximab (RTX) treatment 25 CD27+ Memory B Cells 20 B Cells A 5 0 baseline RTX B cell depletion regeneration RTX approved for TNF IR RA A: P < vs baseline. A A A Time (months) B cell depletion regeneration RTX Roll, et al. Arthritis Rheum. 2008;58:

13 Clinical efficacy and B cell regeneration pattern in RA after RTX treatment Percentage baseline RTX B cell depletion A regeneration A: P <.001 vs baseline; B: P <.05 vs baseline; C: P <.05 vs mos 5-6 after the first treatment. B Naive B Cells B cell depletion regeneration RTX Time (months) C Roll, et al. Arthritis Rheum. 2008;58:

14 Synovial inflammation requires trafficking in the circulation between compartments IFN γ TNF-α IL-1 IL-1 TNF-α IFNγ IL-1 Blood vessel Macrophage Neutrophil Bone marrow Synoviocytes T Cell B Cell BAFF APRIL TNF-α IFN γ B Cells Neutrophil IFN-α TNF-α Dendritic Cell B Cell T Cell TNF-α IL-1 IL-1 Dendritic Cell Silverman. Arthritis Rheum. 2006;54:

15 RTX: Accepted clinical paradigm for B cell-targeted therapy in RA Efficacy with safety Associated with reduced circulating CD27+ memory B cells and increased naïve B cells Lower activated blood B cells Lower activated blood T cells Little or no effects on protective IgG antibodies Are these lessons to judge other agents?

16 FDA approved in RA Journal of Rheumatology 2014; 41;

17 In vivo effects of anti Interleukin-6 Receptor inhibitor Tocilizumab at the B-cell level Overall memory B cells Anti-IL6R treatment causes decreases in switched and unswitched memory B cells Toculizumab approved in RA MTX IR Roll, et al. Arthritis Rheum. 2011;63(5):

18 In vivo effects of anti Interleukin-6 Receptor inhibitor Tocilizumab at the B-cell level Roll, et al. Arthritis Rheum. 2011;63(5):

19 Tociluzimab Summary Selective IL-6 blockade has focused and dramatic effects on B memory cells No significant effects on IgG levels, which is good for immune safety.

20 Anti-TNF therapy in RA lowers levels of blood Memory B lymphocytes RA patients (n=45) on MTX (n=17), anti-tnf etanercept (n=11), or MTX/anti-TNF (n =17). Normal controls (n=22). Examined Peripheral Blood B cells by flow and cells from tonsillar biopsies. Etanercept increased peripheral blood naïve and transitional B cells while decreasing memory (both switched and unswitched). * Immunohistochemistry of tonsil tissue revealed a decrease in follicular dendritic cell networks and germinal centers in etanercept treated populations Several TNFi approved for MTX IR RA Anolik, et al. J Immunol. 2008;180(2):

21 TNF Blockade Summary RA may be associated with defects in B cell tolerance Blockade of TNF mediated inflammation in RA can normalize some B cell associated defects of the cellular memory compartments TNF blockade can increase the proportion of transitional B cells and naïve B cells Can TNF blockade the frequency of anti-citrullinated self antigen memory B cells?

22 In depth characterization of the anti-citrulline circulating B cells in RA patients Manuscript in preparation 22

23 Theoretical Pathway for Breaking Tolerance in RA Anti-CCP B cells capture any citrullinated protein, giving rise to expanded T-cell responses against selfantigens More Autoreactive T Cells IgG anti-citrulline immune complexes are antigens for RF B cells RF B cell Autoreactive T Cells T Cell Anti-citrulline B Cell Anti-citrulline Plasma cell PADI RF and anti-ccp antibodies lead to immune complexes, macrophage secretion of proinflammatory cytokines Citrullinated antigens Arginine-containing antigens Adapted from: Niewold TB, et al. QJM. 2007;100(4):

24 IgG Anti-citrullinated Protein Antibodies (ACPAs) Production closely linked to SE-HLA Class II 1 ~80-90% of ACPA-positive patients express SE 2,3 Multiple autoantibodies to multiple cit*-proteins fibrinogen, α-enolase, vimentin, others Moderately sensitive and highly specific for RA: 45-80% and %, respectively 4 Rare in healthy individuals and in other conditions 4 *cit=citrullinated 1. Källberg H et al. Am J Hum Genet. 2007;80: Farragher TM et al. Arth Rheum. 2008;58(2): Irigoyen P et al. Arth Rheum. 2005; 52(12): Schellekens GA et al. Arthritis Rheum. 2000;43(1):

25 Diverse serum IgG- ACPA in RA serum CCP3+ MTX (n = 20) Citrullinated epitope IgG reactivity in CCP3-seropositive RA patients treated with TNF inhibitor + Methotrexate show similar epitope-specific ACPA patterns to patients treated with Methotrexate alone. CCP3+ TNFi/M TX (n = 20) CCP3- neg MTX (n = 10) A range of Cit-specific self antigens Cit human Fibrinogen CCP3 CitFilaggrin cfc-6 Cit FibA Cit FibB Cit FibA Cit Enolase 5-21 Cit Vimentin Cit Vimentin PC-BSA Tetanus Toxoid Anti-human IgG BSA (-)

26 Enumeration of ACPA IgG-secreting B cells in seropositive RA patient PBMC stimulated with CpG2006/IL-21/sCD40L. Healthy CCP3 RA CCP3Sero+ CCP3 Healthy CQP3 RA CCP3Sero+ CQP3 CCP3 ASC/10 6 P BMC CQP3 ASC/10 6 P BMC Tetanus Toxoid ASC/10 6 P BMC Total IgG ASC/10 6 P BMC Spontaneous IgG ASC/10 6 PBMC AVG RA CCP3sero+ (no biologic drug) (n=16) / / / (n=15) AVG RA CCP3sero+ (on biologic drug) (n=3) / / / (n=3) AVG HC & RA seroneg (n=6) 2.8 +/ / / (n=5)

27 Figure 4. Following whole PBMC stimulation, ACPA IgG-secreting B cells presence is restricted to CCP3-seropositive RA patients, while anti-tetanus Toxoid secreting B cells are present in both healthy controls and RA patients. Healthy Rheumatoid Arthritis

28 B cell sorting and stimulation strategy to enumerate ACP secreting memory B cells CD27-APC Stain PBMC on ice 30 min w/ FACS antibodies on ice, wash, perform sorting IgD-FITC CD40 L cells

29 Supplementary Figure 3a. Multiplex ACPA antigen array strategy for RA IgG autoantibody detection Cit Serum incubated with beads PE-conjugated antihuman IgG detection antibody LUMINE X 200 PE 635nm 532nm Cit Cit Cit PE Cit Cit PE Detect up to 1000 PE molecules per bead. Collect 50 beads/analyte, up to 100 analytes simultaneously.

30 Upon stimulation, memory B cells in whole PBMC cultures sorted memory ACPA IgG with reactivity patterns that reiterate ACPA patterns in serum. Serum IgG Induced memory IgG

31 High Burdens of ACPA Switched-Memory B cells after stimulation correlate levels of intertwined repertoire of serum ACPA in the same RA donor ACPA levels.

32 High Burdens of blood ACPA Switched-Memory B cells are not uniformly abolished by treatment induced clinical remission, (or low disease activity). Levels of B cell memory do not correlate significantly with DAS28 Scores.

33 Conclusion Our most commonly used therapies for RA do not eliminate the disease-associated memory B cell autoimmunity in the bloodstream 35

34 Targeting the BLyS/BAFF pathway: Atacicept and Belimumab/Tabalumab BLyS APRIL Anti-BLyS TACI-Ig BAFF-R/BR3 TACI BCMA Proteoglycan (syndecan-1) Atacicept: recomb fusion protein of the TACI* receptor and human IgG (not FDA approved) Belimumab: human monoclonal antibody which binds BLyS (approved in SLE only) *TACI: transmembrane activator and calcium modulator and cyclophilin-ligand interactor receptor Scapini, et al. Immunol Lett. 2008;116:1-6. Ramanujam & Davidson. Arth Res Therapy. 2004;6:

35 Effects on blood B cell subsets Belimumab Phase II SLE trial Total B cells Activated B cells Decreases mature and plasma B cells but spares memory B cells and B-cell progenitors Naive B cells Memory (CD27+) B cells Wallace, et al Arthritis Rheum. 2009; 61(9):

36 Belimumab in RA: no significant clinical benefits in RA Human antibody to soluble BLyS Phase II, multicenter, doubleblind, placebo- controlled, doseranging study in 283 patients with active moderate-to-severe RA 11 yr of disease DMARD IR 38% failed at least one TNF blocker significant but weak ACR20 response. Atacicept no benefit in RA Tabalumab-no benefit in RA BAFF blockade may not have depleting effects on B cell memory Cohen. J Rheumatol Suppl. 2006;77:12-7.

37 Targeting of lymphocytes

38 Abatacept in RA ABA blocks T-cell co-stimulation, competing with CD28 for binding to CD80/CD86 signal required for full T- cell activation * CD20 sublining Open trial Bx/ rebx AB in RA pts (n=16) who failed TNFi 60% DAS28 responders after 4 months treatment. 15 pts with pre and 16 wk post bx Results: Small reduction in synovial cellular content, significant reduced only for B cells (CD20+ cells) Is ABA a B cell targeted agent? approved for MTX IR RA Buch, et al. Ann Rheum Dis. 2009;68(7):1220.

39 Abatacept reduces levels of switched memory B cells, autoantibodies, and Immunoglobulins in RA. Studies of 30 RA patients before and after ABA After 6 months of treatment, significant reductions of serum IgG, IgA, and IgM, with normalization of initially abnormal values. Significant reduction of IgG- and IgA-ACPA, & IgM-, IgA-, and IgG-RF Proportion of post-switch memory B cells significantly decreased. Scarsi et al. J Rheumatol. 2014

40 Abatacept (CTLA4-Ig)- blocks T Cell co-stimulation Plasma cell B cell IL-1, IL-6, TNF-α IFN-γ T cell APC (DC or Mφ) RF, anti-ccp antibodies TNF-α IFN-γ IL-17 RANKL Immune complexes Complement fixation Induce Mφ secretion of proinflammatory cytokines MMPs Cathepsins Mφ TNF-α, IL-1, IL-6 FLS TNF-α IL-1 IL-6 MMPs Cathepsins CCP, cyclic citrullinated peptide; FLS, fibroblast-like synoviocyte; IFN, interferon; MMP, matrix metalloproteinase; RANKL, receptor activator of NF-κB ligand; RF, rheumatoid factor. Osteoclast Responsible for marginal erosions and bone loss Silverman & Boyle. Immunol Rev. 2008;223:

41 Window of Opportunity Time Pre-disease Window of opportunity Progressive disease Pathogenesis Initiation of therapy Potential pathogenetic factors are present in many individuals (anti- CCP, RF) Acceleration due to autoimmunity (further loss of tolerance) Initiation of therapy Increased production of proinflammatory cytokines Synovitis Osteoclast differentiation, development of erosions Permanent autoimmunity Initiation of therapy Lasting cytokine imbalance Synovial proliferation, chronic inflammation Structural damage Clinical trials suggest that therapy initiated during (early) window of opportunity is more likely to lead to a sustained improvement. Based on limited current data, biologicals may increase the likelihood of persistent remission. Current data suggest medication-free remission is only ~3.6 to 22%. Possible roles of disease duration, environment, and genetics should be investigated. Reliable biomarkers (or imaging technology) are needed. We need to understand the conditions that will reestablish tolerance during remission. Nagy G, van Vollenhoven RF. Arthritis Res Ther. 2015;17:181.

42 Concluding speculations Diverse biologic agents that provide clinical benefits in RA can cause shifts but not normalize the memory B cell compartment. Common approaches suppress inflammation do not substantial the autoimmune state. This explains why halting therapy often leads to flare/recurrence. Drug-free cure of RA may require normalization of adaptive immune responses, with shift in B-cell subsets deletion of pathogenic autoimmune lymphocyte clones and long-lived plasma cells. 44

43 What is in the future? Combination therapies target B cells, T cells and plasma cells? Active induction of immune tolerance? Cell based therapy (including stem cell infusions) Apoptotic cell infusions for DC bias, i.e., extracorporeal photopheresis Targeting immune checkpoints (e.g. PD-1) 45

44 taphor for effective targeted therapy 1. Rheumatoid synovitis takes on an orderly structure with coordinated activities of cells and cytokines that drive pathogenesis 2. Not every cell type and soluble inflammatory factor is essential! B cell Targeted Therapy B cells Macrophages Osteoclasts T cells IL-6 CD28 TNFa Activated FLS Complex synovitis 3. Targeted therapy may directly remove only one cell or cytokine from the process But this may cause disintegration of synovitis Revised Metaphor circa 2015 Gregg Silverman, all Jenga rights

45

46 Receptors and ligands of the CD28-B7 family

47 Fig. 3 A molecular model for Programmed Death (PD)-1-mediated in

48 Putative immune checkpoints by PD-1