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Advances in B-Cell Biology in the Treatment of Autoimmune and Inflammatory Diseases

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2 Advances in B-Cell Biology in the Treatment of Autoimmune and Inflammatory Diseases

3 Learning Objectives Discuss recent advances in understanding how B-cell biology affects autoimmune rheumatic diseases (ARDs) Review new biologic agents that target B cells and their mechanisms of action Identify how studies of B-cell–targeted therapy are changing our understanding of integrated immune responses and the pathogenesis of ARDs

4 Outline B-cell biology in autoimmune disorders
Overview of B-cell–targeting agents Recent developments in B-cell targeting in rheumatoid arthritis (RA) Recent development in B-cell targeting in other ARDs

5 B-Lineage Cells and Autoimmunity
Mature Bone Marrow Blast Activation Immature T1 T2 Plasma cell Pro B Pre B Spleen FO MZ Plasmablast Memory GC T B FDC DC Lymphoid organogenesis Multiple sclerosis Sjögren’s syndrome RA B T DC Antigen presentation and costimulation Multiple sclerosis SLE RA Inflammatory cytokines All autoimmune disorders T B TNF-a IL-6 M ? LT Immune complexes SLE RA Auto- antibodies Graves’ disease Myasthenia gravis Pemphigus vulgaris SLE (RA) DC, dendritic cell; FDC, follicular dendritic cell; FO, follicular; GC, germinal center; IL, interleukin; LT, lymphotoxin; Mφ, macrophage; MZ, marginal zone; SLE, systemic lupus erythematosus; TNF, tumor necrosis factor. Adapted from Martin F, Chan AC. Annu Rev Immunol. 2006;24: 5

6 Why Are Some Plasma Cells Short-Lived and Others Long-Lived?
Plasma cells, which arise after new immune exposures, are continuously generated in lymphoid tissues, and perhaps in pathologic ectopic lymphoid tissues (eg, rheumatoid synovium) There are different life expectancies of Ig-secreting plasmablasts (which can proliferate) and plasma cells (which cannot proliferate) Newly generated plasma cells are released into the bloodstream, then migrate to the bone marrow As long-term survival requires that they find a “niche,” they must displace “older” plasma cells from their survival niche for long-term survival Dörner T, Radbruch A. Immunity. 2007;27:

7 Complex Interactions Determine Plasma Cell Survival
Adhesion CD138 CD44, 18, 11a E-/P-selectin Chemokine systems CXCR4/CXCL12 Intracellular survival regulators ↑ Bcl-2, Blimp1, XBP1, Aiolos ↓ BACH2 and Pax5 Bone marrow GC B T Competence Plasmablasts Instruction of memory B cells by T cells CD40/154 ICOS/ICOS-L SAP This diagram explains that antigen specific B cells are selected and induced to proliferate in germinal center reactions that involve interactions with antigen specific T cells, and signals involving survival signals such as CD154 (that interacts with CD40 on the B cells) and ICOS-ligand (ICOS-L) and other potential signals. If a B cell gets all of the right signals it becomes “competent”. Some of the triggered B cells become Ig secreting cells, to secrete locally or to enter the blood stream to find a long term survival niche. In part they follow chemtactic chemical trails that encourage them to enter the bone marrow (SDF-1-CXCL12. These migrations through the vasculature and into the bone marrow are mediated through adhesion molecules such as CD138, CD44, CD18, CD11a and the selections. In the bone marrow the cell must then find the right microarchitectural site to be up against supportive cells, called nurse cells that secrete soluble survival molecules like IL-6, IL-21, BAFF and APRIL, as well as get membrane associated signals. These trigger metabolic changes in the plasma cell, which reflect its capacity for long term survival in these niches, which include high levels of Bcl-2, Blimp1 Xbp1, Aiolos as well as shut down other molecules. Niches may also be formed by activated stromal cells at sites of disease that enable the survival of plasma cells. Long-lived memory plasma cell Soluble survival factors IL-6 IL-21 (?) TNF, IL5 BAFF/APRIL Chemokine systems CXCR4/CXCL12 (?) Insoluble survival factors from nurse cells or stromal cells (within inflamed tissue) Dörner T, Radbruch A. Immunity ;27: Tarlinton D et al. Curr Opin Immunol. 2008;20:

8 Not All CD20+ B Cells Are Susceptible to Depletion
Deletion is most efficient in blood CD20+ precursors and transitional and naïve B cells appear highly susceptible to deletion CD20+ B1 cells, MZ B cells, and GC B cells are resistant (GC in GALT Peyer’s patches) Most plasma cells are not directly affected by RTX (although in the tonsil a subset may express CD20) Effect of RTX on memory B cells and plasmablasts Little is known about RTX effects on these cells in lymphoid organs IgG-producing plasma cells generally live longer than IgM producers Data are primarily from in vivo murine systems, studies of non human primates and in vitro human B cell studies. Add a references GALT, gut-associated lymphoid tissue. Gong Q et al. J Immunol. 2005;174: Silverman GJ. Arthritis Rheum. 2006;54: Withers DR et al. Blood. 2007;109: Terstappen LW et al. Blood. 1990;76: 8

9 Does Extent of Reduction in Synovial B Cells Explain Clinical Response?
CD22 staining of synovial tissue Before Treatment 4 Weeks After Treatment B-cell depletion Nondepletion Vos K et al. Arthritis Rheum. 2007;56:

10 Changes in RA Synovial Patterns After RTX
Baseline (pre-RTX) 4 Weeks 8 Weeks 16 Weeks 24 Weeks B cells* (by histochemistry or CD20 transcripts) + predictive of clinical response at 24 weeks ↓↓ in clinical responders Plasma cells (by histochemistry) (trends, NS) ND Immunoglobulins (by transcript analysis) ↓↓ predictive of clinical response at 24 weeks† T cells and lymphocyte aggregates‡ No  CD68+ Mf ↓ in lining M ↓↓ in lining and sublining M clinical responders At 12 weeks clinical nonresponders + IgM ACPA and high synovial CD79a+ CD20. *Level of immediate posttreatment synovial B-cell depletion is highly variable, even though high levels of blood-cell depletion are highly reproducible. †At 8 weeks, there was a significant decrease in Ig transcripts in patients with ACR50 or greater responses. ‡Aggregates are only found in ~1/3 of RA biopsies. ACPA, anti-citrullinated protein/peptide antibodies; ACR, American College of Rheumatology. Kavanaugh A et al. Ann Rheum Dis. 2007;66(suppl 2):291 [abstract FRI0037]; Rosengren S et al. Ann Rheum Dis. 2007;66(suppl 2):298 [abstract FRI0059]; Thurlings RM et al. Ann Rheum Dis. 2007; 66(suppl 2):123 [abstract OP0229]; Dass S et al. Ann Rheum Dis. 2007;66(suppl 2):90 [abstract OP0123]; Teng Y et al. Ann Rheum Dis. 2007;66(suppl 2):299 [abstract FRI0063]; Teng Y et al. Ann Rheum Dis. 2007; 66(suppl 2):439 [abstract SAT0034]; Teng YK et al. Arthritis Rheum. 2007;56:

11 of proinflammatory cytokines RF, anti-CCP antibodies
Model of RTX-Induced RA Synovial Changes in Clinical Responders BASELINE TNF-a, IL-1, IL-6 IL-1, IL-6, TNF-a IFN-g T cell Plasma cell Mf Immune complexes Complement fixation Induce Mf secretion of proinflammatory cytokines B cell RF, anti-CCP antibodies TNF-a IL-1 IL-6 IL-17 RANKL FLS 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. Silverman GJ, Boyle DL. Immunol Rev. 2008; 223: Responsible for marginal erosions and bone loss Osteoclast 11

12 RF, anti-CCP antibodies of proinflammatory cytokines
Model of RTX-Induced RA Synovial Changes in Clinical Responders 4 WEEKS X Early synovial B-cell depletion is necessary but not sufficient! Trends toward ↓ in plasma cells in clinical responders (at 6 months) B cell X T cell Plasma cell IL-1, IL-6, TNF-a IFN-g TNF-a IFN-g IL-17 RANKL RF, anti-CCP antibodies Immune complexes Complement fixation Induce Mf secretion of proinflammatory cytokines FLS Mf MMPs Cathepsins TNF-a IL-1 IL-6 MMPs Cathepsins TNF-a, IL-1, IL-6 Responsible for marginal erosions and bone loss Osteoclast Silverman GJ, Boyle DL. Immunol Rev. 2008; 223:

13 RF, anti-CCP antibodies of proinflammatory cytokines
Model of RTX-Induced RA Synovial Changes in Clinical Responders 8 WEEKS Early synovial B-cell depletion is necessary but not sufficient! Trends toward ↓ in plasma cells in clinical responders Significant ↓ in Ig transcripts in ACR50/70 clinical responders (at 6 months) Trends toward ↓ in lymphocytic aggregates and T cells X B cell X T cell Plasma cell IL-1, IL-6, TNF-a IFN-g TNF-a IFN-g IL-17 RANKL RF, anti-CCP antibodies Immune complexes Complement fixation Induce Mf secretion of proinflammatory cytokines FLS Mf MMPs Cathepsins TNF-a IL-1 IL-6 MMPs Cathepsins TNF-a, IL-1, IL-6 Responsible for marginal erosions and bone loss Osteoclast Silverman GJ, Boyle DL. Immunol Rev. 2008; 223:

14 of proinflammatory cytokines RF, anti-CCP antibodies
Model of RTX-Induced RA Synovial Changes in Clinical Responders 16 WEEKS IL-1, IL-6, TNF-a IFN-g T cell Mf Immune complexes Complement fixation Induce Mf secretion of proinflammatory cytokines Responsible for marginal erosions and bone loss Osteoclast TNF-a IL-1 IL-6 IL-17 RANKL FLS MMPs Cathepsins Significant synovial B-cell depletion Significant ↓ in plasma cells Significant ↓ in T cells and lymphocytic aggregates Significant ↓ in CD68+ Mf All in clinical responders at 6 months TNF-a, IL-1, IL-6 RF, anti-CCP antibodies B cell X Plasma cell ? Silverman GJ, Boyle DL. Immunol Rev. 2008; 223:

15 of proinflammatory cytokines RF, anti-CCP antibodies
Model of RTX-Induced RA Synovial Changes in Clinical Responders 24 WEEKS IL-1, IL-6, TNF-a IFN-g T cell Mf Immune complexes Complement fixation Induce Mf secretion of proinflammatory cytokines Responsible for marginal erosions and bone loss Osteoclast TNF-a IL-1 IL-6 IL-17 RANKL FLS MMPs Cathepsins Primary end point is clinical response at 24 weeks Effects on synovial cytokines/ chemokines have not been evaluated at later time points Direct studies on osteoclasts after RTX are not currently available TNF-a, IL-1, IL-6 RF, anti-CCP antibodies B cell X Plasma cell ? Silverman GJ, Boyle DL. Immunol Rev. 2008; 223:

16 Insights From RA Synovial Biopsy Studies
There are great variations in the histopathologic changes induced by RTX Based on small open synovial biopsy studies, there may be a sequence of changes in the synovia that leads to clinical response1-6 B-cell depletion at 4 weeks may predict clinical response; subsequent↓ in plasma cells and Ig transcripts may predict clinical response1,2,5 RTX-induced depletion of B-lineage cells, with loss of plasma cells and immune complex formation, may induce clinical responses by multiple pathways B-Cell Roadblock Hypothesis: As B cells are most susceptible to deletion in the blood, RTX benefits may derive from preventing reseeding of pathogenic B cells to the synovia7 Kavanaugh A et al. Ann Rheum Dis. 2007;66(suppl 2):291 [abstract FRI0037]. Rosengren S et al. Ann Rheum Dis. 2007;66(suppl 2):298 [abstract FRI0059]. 3. Thurlings RM et al. Ann Rheum Dis. 2008;67: 4. Dass S et al. Ann Rheum Dis. 2007;66(suppl 2):90 [abstract OP0123]. 5. Teng YK et al. Arthritis Rheum. 2007;56: 6. Teng Y et al. Ann Rheum Dis. 2007;66(suppl 2):439 [abstract SAT0034]. 7. Silverman GJ, Boyle DL. Immunol Rev :

17 Transcript Analysis of Synovial Biopsies May Explain How RTX Affects RA Pathogenesis
Methods: Knee synovial biopsies at baseline (T0) and 12 weeks after RTX therapy (T12) from 8 patients with RA with incomplete response to anti-TNF therapy RNA expression analyzed with GeneChip® slides Results: 206 genes ↓ and 330 genes ↑ between T0 and T12 Pathway analyses indicated down-regulated genes involved in cytokine activity and chemotaxis (eg, CCRL2, IL-2BR, CXCL1, CX3CR1) Up-regulated genes involved in bone mineralization, bone remodeling, and ossification (eg, BMP2, TWIST2, P2RX7, CD276) Conclusion: RTX induces specific changes in global gene expression in RA synovial tissue RTX may decrease overall inflammatory cytokine responses in the affected joint, but also induce increases in factors that protect and heal periosteal bone Gutierrez-Roelens I et al. Ann Rheum Dis. 2008;67(suppl 2):194 [abstract THU0182].

18 Conventional Immunosuppressive Therapy Can Deplete Peripheral Plasmablasts and Naϊve B Cells, but Not Memory B Cells Plasma cells/blasts 6.7% 2.5% 1.2% CD27 64.0% 72.1% 87.1% CD27+ memory B cells 29.2% 25.4% 11.7% CD19 07/21/03 07/28/03 09/09/03 Methylprednisolone Cyclophosphamide Odendahl M et al. J Immunol. 2000;165: Figure courtesy of T. Dörner (Berlin, Germany)

19 Are There Predictive Biomarkers of Response to RTX Treatment in RA?
Open trial of 17 patients with refractory RA who received a first cycle of rituximab 12 patients responded with good EULAR responsesa 6 patients with an early relapse (weeks 24–40) 6 patients with a late relapse (after week 40) 5 patients were classified as nonresponders 1 patient remained in remission after 1 cycle of treatment 11/17 patients received a second cycle of rituximab 2/11 were RF-negative, 3/11 were anti-CCP–negative Roll P et al. Arthritis Rheum. 2008;58: aResponse was defined as improvement in DAS28 ≥1.2. DAS28, Disease Activity Score including a 28-joint count; EULAR, European League Against Rheumatism.

20 Memory B cells are either IgD+ or IgD-
At Baseline, Higher CD27+ Memory B Cell Counts Correlated With Early Relapse in Clinical Responders After RTX Treatment CD27+ B cells/µL Before Therapy memory 60.0 CD27 50.0 40.0 30.0 naive 20.0 10.0 P = .045 IgD 0.0 Memory B cells are either IgD+ or IgD- Early Relapse Late Relapse (Week 24–40) (After Week 40) At Time of B-cell Regeneration, Clinical Nonresponders Had Higher Levels of IgD+ CD27+ Memory B Cells – Lack of Persistent Memory B Cell Reductions CD27+/IgD+ B cellsa 2.0 P = .019 In RA, clinical responders to B cell depletion therapy had higher disease activity at baseline and reduced CD27+/IgD+ memory B cells during repopulation. Early relapse after the first RTX cycle was associated with enhanced CD27+ memory B cells prior to therapy and higher IgD+/CD27+ and IgD-/CD27+ B-cells after first and second treatment cycle, compared to late relapsing patients. After a subsequent cycle of B cell depletion, reduction of circulating plasma cells and enhanced naïve B-cells were identified. Yet even for non responders, RTX retreatment can still be effective (AB 0377) Clinical response to anti-CD20 treatment may be primarily dependent on the capacity to reduce blood memory B-cells, and this may be linked to a reduction of plasmablasts. (if don‘t kill memory, unlikely to have a clinical benefit) CD19+/CD27+ B Cells/ml 0.0 Responder Nonresponder (n = 12) (n = 5) aFirst time-point of regeneration Roll P et al. Arthritis Rheum. 2008;58: 20

21 1. Fekete et al. J Autoimmun. 2007;29:154-163.
Summary In RA, levels of CD27+ memory B cells increase with disease duration1 After RTX treatment, at time of B-cell regeneration, clinical nonresponders had higher levels of IgD+ CD27+ memory B cells Lack of lasting memory B cell reductions Early relapse after the first RTX cycle was associated with higher CD27+ memory B cell counts prior to therapy and higher IgD+/CD27+ and IgD-/CD27+ B cell counts after first and second treatment cycle, compared with late-relapsing patients Yet even for nonresponders after the first cycle, RTX retreatment can still be effective Clinical response to anti-CD20 treatment may be primarily dependent on the reduction of blood memory B cells, and this may be linked to a reduction of plasmablasts 1. Fekete et al. J Autoimmun. 2007;29:

22 Epratuzumab: A Humanized Anti-CD22 Therapeutic Antibody
CD22 first appears on B cells at the stage of antigen responsiveness CD22 acts as a coreceptor with the antigen receptor that can down-regulate antigen responses Interacts with inhibitory 2,6-linked sialic acid–associated glycoproteins Epratuzumab induces rapid CD22 internalization and phosphorylation In clinical trials for ARD and NHL Open phase I trial in 12 patients with SLE showed safety and some efficacy based on BILAG scores All patients fulfilled ACR criteria Majority of patients had low disease activity (median SLEDAI = 2) More modest effects than rituximab Different moa than RTX How does epratuzumab work? What are the effects on the human immune system? BILAG, British Isles Lupus Assessment Group; ACR, American College of Rheumatology; NHL, non-Hodgkin‘s lymphoma; SLEDAI, systemic lupus erythematosus disease activity index. Dörner T et al. Arthritis Res Ther. 2006;8:R74. doi: /ar1942. Jacobi AM et al. Ann Rheum Dis. 2008;67:

23 Epratuzumab Preferentially Reduced Peripheral Blood Naϊve B Cells; Plasmablasts and CD27+ Memory B Cells Are Less Affected B cells absolute cell number CD27- naive B cells/ml CD27+ memory B cells/ml CD27++ plasmablasts/ml Jacobi AM et al. Ann Rheum Dis. 2008;67:

24 Epratuzumab Modulates the Activation and Proliferation of B Cells in Patients With SLE
Epratuzumab was shown to be safe in a phase I open trial Surface levels of CD22 were rapidly down-modulated Average reduction of 30% of total blood B-cell levels, that lasted for 12 weeks after last infusion Reductions primarily in naïve B cells, trend to increase in plasmablasts. No change in memory B cells Laboratory studies show epratuzumab causes impaired B-cell proliferative responses to in vitro experimental stimulation of T-cell–independent pathway CD22-targeted therapy may provide mechanistic advantages in patients with certain autoimmune diseases More studies are warranted Jacobi AM et al. Ann Rheum Dis. 2008;67:

25 Conclusions Studies with RTX have demonstrated an attractive safety/efficacy profile in RA and encouraged the development of newer agents A diversity of additional strategies for targeting B cells, as well as design of biologic agents, are currently under investigation Different B-cell subsets, including naïve B cells, plasma cells/blasts, and memory B cells, appear to make different contributions to pathogenesis The capacity to inhibit or delete each B-cell subset varies greatly based on the mechanism of action of the therapeutic agent Investigations into B-cell–targeted therapy are providing new therapeutic options as well as helping to elucidate previously unknown immunologic mechanisms of pathogenesis.

26 B-Cell–Directed Therapy for RA
RA, rheumatoid arthritis.

27 Learning Objectives State the rationale of B-cell depletion for the management of RA and list at least 3 agents that are being studied in clinical trials Summarize the efficacy of B-cell–targeted therapy following DMARD and anti-TNF therapy failure Describe the safety of long-term and repeated courses of B-cell–targeted therapy and the nuances of fixed or on-demand retreatment Summarize the radiographic changes noted following B-cell–targeted therapy for RA DMARD, disease-modifying antirheumatic drug; TNF, tumor necrosis factor.

28 CD20: A Target on B cells 297-AA membrane-associated phosphoprotein (33–37 kDa) Not shed: No known membrane/secreted molecular analogues Selective expression: Not expressed on stem cells, pro-B cells, plasma cells, dendritic cells Anti-CD20 binding: Does not rapidly modulate expression Does not cause substantial internalization Differences in epitope binding are of unclear clinical significance with regard to safety or efficacy Ofatumumab Ocrelizumab TRU-015 CD20 AA, amino acid. Kehrl JH et al. Immunol Today. 1994;15: ; Golay J et al. Blood. 2000;95:

29 RTX in RA Timeline 2002 Data from studies featuring RTX in the treatment of RA are presented at the American College of Rheumatology (ACR) annual meeting 2004 The New England Journal of Medicine publishes the results of a phase IIa study of RTX in patients with moderate to severe RA DANCER, a phase IIb study of RTX in RA, meets its primary end point 2005 REFLEX, a phase III clinical study of RTX in RA, meets its primary end point 2006 RTX is approved in combination with MTX to reduce signs and symptoms in adult patients with moderately to severely active RA who have had an inadequate response to one or more TNF-antagonist therapies The REFLEX trial, a 2-year multicenter, randomized, double-blind, placebo-controlled phase III study of RTX therapy, is published, showing that a single course of RTX with concomitant MTX therapy provides significant and clinically meaningful improvements in disease activity in patients with active, long-standing RA who had an inadequate response to 1 or more anti-TNF therapies 2007 Results of an open-label extension analysis published: patients treated with repeated courses of RTX have sustained clinical responses with no new adverse events DANCER, Dose-Ranging Assessment International Clinical Evaluation of Rituximab in RA; MTX, methotrexate; REFLEX, Randomized Evaluation of Long-Term Efficacy of Rituximab in RA; RTX, rituximab; TNF, tumor necrosis factor.

30 RTX (REFLEX): EULAR Responses Based on RF/Anti-CCP
RF/Anti-CCP+ (Either or Both)a While significantly more patients receiving RTX demonstrated ACR20 and EULAR responses compared with placebo recipients, seronegative patients (for RF and anti-CCP) did not respond as well RF/Anti-CCP−b aP > vs placebo, seropositive. bP = .05 vs placebo, seronegative. Anti-CCP, anti–cyclic citrullinated peptide; EULAR, European League Against Rheumatism; RF, rheumatoid factor. Tak PP et al. Ann Rheum Dis. 2007;66(suppl 2):338 [abstract FRI0192].

31 Repeated Treatment With RTX Produces Sustained Efficacy in Patients With RA With an Inadequate Response to DMARDs Week 24 (n = 57) Patients (%) DMARD, disease-modifying antirheumatic drug; DAS28, Disease Activity Score including a 28-joint count. Emery P et al. Arthritis Rheum. 2007;56(9 suppl):S151-S152 [abstract 266].

32 Patients (%) Achieving MCID
Patient-Reported Outcomes During RTX Treatment of Anti–TNF-a Refractory RA in Addition to ACR Responses a a a a Patients (%) a Patients (%) Achieving MCID a RTX produced rapid, clinically meaningful, and statistically significant improvements in: Patient-reported pain Fatigue, functional disability Health-related quality of life Disease activity MCID definitions: FACIT-F: Decreased at least Last observation carried forward. HAQ DI: Decreased at least Last observation carried forward. PCS: Increased at least 3. MCS: Increased at least 3. aP < vs placebo + MTX. MCID, minimum clinically important difference; FACIT-F, Functional Assessment of Chronic Illness Therapy–Fatigue; HAQ-DI, Health Assessment Questionnaire Disability Index; PCS, physical component score; MCS, mental component score. Cohen SB, et al. Arthritis Rheum 2006;54: Keystone E et al. Arthritis Rheum. 2008;59:

33 Repeated Treatment With RTX Produces Sustained Efficacy in Patients With an Inadequate Response or Intolerance to TNF Inhibitors Week 24 (n = 96) Patients (%) Emery P et al. Arthritis Rheum. 2007;56(9 suppl):S151-S152 [abstract 266].

34 REFLEX: Change in Radiographic Outcomes at Week 56
Mean Change JSN, joint space narrowing; TGS, total Genant-modified Sharp score. Keystone E et al. Ann Rheum Dis. 2007;66(suppl 2): [abstract SAT0011].

35 REFLEX: Change in Radiographic End Points at Week 56
Change in Sharp–Genant Total Score at Week 56 by Anti-CCP Status at Baseline1 ACR20 Nonresponders at Week 242 Mean Change in X-ray Score Mean Change (n = 11) (n = 78) Missing (n = 21) (n = 33) Negative (n = 85) (n = 129) Positive The effect of RTX in inhibiting joint damage was consistent for all subgroups examined (baseline: total Sharp score, DAS28, disease duration, CRP, HAQ, SJC, and TJC). The study was not powered to detect treatment differences within each subgroup; however, numerically similar and consistent results were observed for nearly all subgroups analyzed. CRP, C-reactive protein; SJC, swollen joint count; TJC, tender joint count. 1. Cohen S et al. Ann Rheum Dis. 2007;66(suppl 2):428 [abstract SAT0002]. 2. Keystone E et al. Ann Rheum Dis. 2007;66(suppl 2): [abstract SAT0012].

36 REFLEX: RTX Radiographic Findings in RA at 2 years
X-rays at weeks 0, 24, 56, 104 Total Genant-modified Sharp score used Linear extrapolation used for missing week 104 x-rays (30% of patients) Effects on JSN and erosions were similar 87% of RTX patients who did not progress in year 1, did not progress in year 2 The data are confounded by multiple therapeutic changes 4 RTX (n = 187) Placebo (n = 281) 3 2.81 TGS 2 1.78 1.14 1 0.66 Year 1 Year 2 80 RTX Placebo 68 60 60 54 46 No  TGS (%) 40 20 Baseline to 1 Year 1 Year to 2 Years Cohen S et al. Ann Rheum Dis. 2008;67(suppl 2):189 [abstract THU0167].

37 RTX Fixed Retreatment vs On-Demand Retreatment of RA
Fixed and on-demand retreatment with RTX showed equal efficacy and safety Fixed retreatment was more effective in moderate responders and nonresponders to the first course Nonresponders improved significantly only after fixed retreatment 0% 10% 20% 30% 40% 50% 60% 70% 64% 53% 28% 18% 4% 6% ACR 20 ACR 50 ACR 70 Fixed Retreatment Patients (%) On-Demand Retreatment Teng Y et al. Ann Rheum Dis. 2008;67(suppl 2):339 [abstract FRI0167].

38 Type of Prior Anti–TNF-α Failure
Switching Strategies When an Anti–TNF-α Fails: RTX or Alternate Anti–TNF-α Type of Prior Anti–TNF-α Failure Overall (%) Not effective ≥AE 65 35 Switch to alternate anti-TNF-α (%) Adalimumab Etanercept Infliximab 56 25 19 Significance Overall evolution of DAS 28 more favorable in RTX vs alternate anti–TNF-α Concomitant DMARD use Type of anti–TNF-α agent switch Type of prior anti–TNF-α failure Efficacy AEs P = .01 NS (Graph) Signif Not Effective AEs Significant Not significant I M P R O V E M E N T -0.5 -0.77 DAS 28 Δ From Baseline -1 -0.86 -1.03 Switching Strategies When an Anti–TNF-α Fails: RTX or Alternate Anti–TNF-α This observational study suggests that RTX is more effective than switching to an alternative anti-TNF in RA patients who have persistent active disease despite anti-TNF treatment. These results were not significantly modified by DMARD co-therapy or by the type of anti-TNF agent. However, when the motive for interrupting anti-TNF treatment was other than ineffectiveness, unsurprisingly both RTX and alternative anti-TNF agents appear to offer similar levels of effectiveness. Reference Finckh A, Ciurea A, Brulhart L, et al, SCQM. Which subgroup of rheumatoid arthritis patients benefit most from switching to rituximab versus alternative anti-TNF agents after previous failure to anti-TNF agent?  Presented at the 9th Annual EULAR Meeting. Paris, France; June 11-14, Abstract OP0249. -1.5 RTX (n = 101) Switch anti–TNF-α agent (n = 199) -1.55 -2 N = 300. Finckh A et al. Ann Rheum Dis. 2008;67(suppl 2):127 [abstract OP-0249].

39 Subsequent RTX Treatment Courses: Incidence of Acute Infusion Reactions by Treatment Course
Patients (%) SIEs occurred in <1% of patients during course 1 and 2 No SIEs were observed during course 3 or 4 All-exposure population by treatment course. SIE = serious infusion-associated event. van Vollenhoven RF et al. Arthritis Rheum. 2007;56(9 suppl):S147 [abstract 257].

40 Design of B-Cell–Targeting Agents
Chimeric antibody (Synthetic) fully human antibody Mouse (red) Human variable regions Human Human IgG constant region Ocrelizumab (CD20) Ofatumumab (CD20) Belimumab (BAFF) Epratuzumab (CD22) Rituximab (CD20) SMIP Mouse (red) Synthetic peptide Decoy receptors Human Atacicept (BAFF and APRIL) (TACI-Ig) Human (green) TRU-015 (CD20) BR3-FC (BAFF) Baminercept-alpha (LT-) AMG623 (BAFF)

41 Therapeutic Approaches to B-Cell Depletion
Memory Cell Immature B cell Transitional B cell Mature B cell Antigen stimulation Pro- B Cell Antibody-producing Plasma cells Stem Cell Pre- B Cell Epratuzumab (Anti-CD22) CD19 Modulates B cell function With limited depletion CD22 CD20 SLE Autoreactive B cells RTX (anti-CD20) Binds and eliminates CD20-positive cells RA Other diseases Belimumab (antisoluble BAFF) Acts later in B-cell development Potently depletes Ig-secreting cells Atacicept (TACI-Ig) Neutralizes both BAFF and APRIL Acts broadly in B-cell development Potently depletes Ig-secreting cells SLE, systemic lupus erythematosus.

42 Therapeutic Approaches to B-Cell Depletion: Clinical Trial Status
Description Indications Approved Rituximab Chimeric anti-CD20 RA Phase 3a Ocrelizumab Humanized anti-CD20 antibody based on RTX Belimumab Human antibody that binds/sequesters BAFF/BLyS, B-cell survival factor SLE Phase 2a Epratuzumab Humanized anti-CD22 antibody Baminercept Alfa Soluble LTß receptor IgG1 fusion protein TRU-015 Anti-CD20 SMIP (small modular immunopharmaceutical) Ofatumumab Human IgG antibody from humanized transgenic mice aStatus as of July 17, 2008.

43 Ocrelizumab (OCR): Humanized Anti-CD20 mAb in RA
Phase I/II dose-escalation RCT with OCR + MTX 175 patients in whom 1–6 DMARDs failed ≥8 TJC and SJC plus CRP ≥1 mg/dL or ESR ≥28 mm/h Single infusion of OCR (400, 1000, 1500, or 2000 mg) or placebo No peri-infusional corticosteroids 1° end point: ACR20 at week 24 Complete peripheral B-cell depletion Patients with incomplete response require 100 mg IV corticosteroids ACR Results at Week 24 70 Placebo 60 58 58 OCR 400 50 OCR 1000 OCR 1500 40 38 34 33 OCR 2000 Patients (%) 32 30 27 25 23 19 20 14 13 9 10 3 ACR20 ACR50 ACR70 ESR, erythrocyte sedimentation rate; IV, intravenous; OCR, ocrelizumab; mAb, monoclonal antibody; RCT, randomized controlled trial. Tak PP et al. Ann Rheum Dis. 2008;67(suppl 2):127 [abstract OP-0250].

44 CD20 – Ofatumumab: Human Anti-CD20 IgG1 mAb
Phase II trial in 225 DMARD/TNF incomplete responders Ofatumumab IV day 1/15 or placebo; premedication: 100 mg corticosteroid; antihistamines; acetaminophen Primary end point: ACR20 week 24 No significant change in IgA, IgM, or IgG; all patients were human anti-human antibody–negative at 24 weeks 51% of all AEs occurred on infusion days Majority were mild Common Terminology Criteria (CTC) grade 3 None in placebo group 1 serious AE (SAE) in 700 group 3 SAEs in 1000 group No CTC grade 4 or 5 events Østergaard M et al. Arthritis Rheum. 2007;56(9 suppl):S793-S794 [abstract 2086].

45 CD20 – Ofatumumab: Human Anti-CD20 IgG1 mAb – Phase II Study
ACR20 ACR50 ACR70 60 Placebo 300 700 1000 AE (%) 57 81 84 83 SAE (%) 4 7 9 11 SIE (%) 2 49 50 46 41 40 Patients (%) b 30 26 26 a 19 20 15 Conclusions No apparent dose response No limiting safety signals as yet 9 10 5 6 4 Placebo 300 700 1000 n = 55 n = 58 n = 57 n = 54 aP < .05; bP <.01 Østergaard M et al. Arthritis Rheum. 2007;56(9 suppl):S793-S794 [abstract 2086].

46 TRU-015 in Patients With RA: Phase II Dose-Ranging Study
24-week double-blind RCT in patients with incomplete response to MTX 227 patients in United States ≥6 tender joints and swollen joints; RF+ Either increased ESR/CRP or morning stiffness >45 min 1 infusion, TRU-015 or placebo 200, 400, 800, or 1600 mg Premedicated 1 grade 3 AE on infusion day (400-mg group) Conclusion: 800 and 1600 mg of TRU-015 IV once statistically superior to placebo at week 24 Pbo n = 53 200 n = 56 400 n = 57 800 1600 SAE (%) 2 5 4 DAS28 −1.2 −1.6 −2.1a −2a ACR20% 33 49 37 65b 61c ACR50% 9 19 16 26a 13 ACR70% 6 ∆ HAQ −0.37 −0.5 −0.64 −0.7 %↓ CRP 28 38 42 47 48 aP < .05; bP = .003; cP = .008. Burge D et al. Abstract presented at: ACR/ARHP Annual Scientific Meeting; November 6–8, 2007; Boston, Massachusetts; abstract L7.

47 Repeated Administration of TRU-015 in Patients With RA
AEs >10% SMIP protein against CD20 Dose-dependent B-cell depletion Retreatment in 36/54 patients from phase I/IIa RCT IV infusion: 5 or 15 mg/kg × 1 INF every 6 months if B-cell recovery Up to 6 courses given 4 AEs on day of infusion 3 mild (facial flushing, facial erythema, pruritus) 1 moderate (Baker’s cyst) Conclusions: Retreatment after B-cell recovery well tolerated AEs (%) Initial (n = 54) R1 (n = 36) R2 (n = 29) Headache 31 8 7 URI 24 14 Fatigue 20 3 Edema 19 11 Bronchitis 17 6 Insomnia 15 Sinusitis 13 10 Rash Pruritus R1, first retreatment; R2, second retreatment; URI, upper respiratory tract infection. Burge D et al. Ann Rheum Dis. 2008;67(suppl 2):128 [abstract OP-0252].

48 LTβR-Ig (Baminercept)
Human lymphotoxin-b receptor (LTb) extracellular domain fused to human IgG1 Fc receptor acts as a decoy receptor for LTb Effective in murine models of arthritis: Blocks induction of arthritis and decreases severity of arthritis in established disease Activated T cells Dendritic cells B cells, T cells, NK cells Lymphotoxin a/b Pathway is implicated in: Liver, spleen, and lymph nodes Gut mucosa and Peyer’s patches LIGHT Pathway is implicated in: Mucosal and hepatic inflammation LIGHT LT Baminercept alfa HVEM LTR T Cells B cells NK cells Control of Lymphoid Microenvironments Costimulation of T-cell activity Affects T-cell responsiveness (proposed) and enhanced survival HVEM, herpes virus entry mediator. Baldassare A et al. Ann Rheum Dis. 2008;67(suppl 2):86 [abstract OP-0122].

49 LTβR-Ig (Baminercept) in RA
Placebo dose-finding Phase IIa RCT: 47 pts – IR ≥1 DMARD; +MTX SC weekly injections x4 and observation to day 77: 6 dose cohorts: 0.01, 0.05, 0.1, 0.3, 1 and 3 mg/kg BAM versus PBO AEs >5%: PBO 55% and LTβR-Ig 67% Headache: Placebo 9% and LTβR-Ig 19% Flu-like symptoms in 25% within 24 h of first injection; ↓ with subsequent injections to 6–9% Continued clinical responses after week 4 despite discontinuation of treatment Phase IIb planned: 5, 50, 200 mg weekly Sample size: 10 6 4 5 mg/kg 30 50 40 67 25 33 20 17 60 70 80 PBO BAM 0.01 BAM 0.05 BAM 0.1 BAM 0.3 BAM 1.0 BAM 3.0 Patients (%) ACR20 ACR50 ACR70 OP-0122   PRELIMINARY SAFETY AND EFFICACY OF BAMINERCEPT ALFA (BG9924, LTBR-IG ) IN THE TREATMENT OF RHEUMATOID ARTHRITIS (RA) A. Baldassare*1, J. Fiechtner2, A. Filipowicz-Sosnowska3, S. Jeka4, J. O'Gorman5, M. Weaver5, E. Beckman5 1Rheumatology, Arthritis Consultants, St. Louis, 2Rheumatology, Michigan State University, Lansing, United States, 3Rheumatology, Institute of Rheumatology, Warsaw, 4Rheumatology, NZOZ, Torun, Poland, 5Immunology, Biogen Idec, Cambridge, United States Objectives: To determine safety and explore efficacy of lymphotoxin (LT) beta receptor IgG1 (baminercept alfa, LTBR-Ig, BG9924) with MTX in patients with moderate/severe RA. Baminercept alfa consists of the human LTBR extracellular domain fused to the human IgG1 Fc domain. It binds to LT alpha1/beta2 and LIGHT ligands on activated lymphocytes and natural killer cells, blocking interactions with their receptors on peripheral antigen-presenting and stromal cells. Methods: This was a blinded, randomized, placebo-controlled, multicenter, Phase 2a trial. Patients with moderate/severe RA and inadequate response to >=1 DMARDs continued their stable MTX dose and other allowed concomitant RA therapy (low-dose prednisone, NSAIDs, hydroxychloroquine sulfate), and received subcutaneous injections of baminercept alfa (0.01, 0.05, 0.1, 0.3, 1.0, or 3.0 mg/kg) or placebo once weekly for 4 weeks followed by 8 weeks of observation. Safety and efficacy were monitored throughout. Exploratory efficacy endpoints were DAS28, EULAR response and ACR core set measures (swollen joint count [SJC], tender joint count [TJC], investigator and patient global assessments [IGA, PGA], Health Assessment Questionnaire [HAQ], pain visual analog scale [VAS], C-reactive protein [CRP], erythrocyte sedimentation rate [ESR]). Results: (FINAL data) 49 patients were randomized (per cohort: baminercept alfa n=6; placebo n=2); 47 patients received >1 dose. All groups had similar baseline (BL) demographics and DAS28, SJC, TJC, HAQ, IGA, PGA, pain VAS, RF, and anti-CCP. Incidence of all adverse events (AEs) was 55% in placebo vs 67% in baminercept alfa patients (Table); none was severe. No drug-related serious infections or serious AEs were reported. The most common AE in baminercept alfa patients was headache (7 [19%] patients vs 1 [9%] placebo patient). At Day 77, the mean change in DAS28 was from BL in baminercept alfa patients vs in placebo patients, with the 1.0 and 3.0 mg/kg dose group showing the greatest mean change from baseline with and respectively (Table). 33% patient in the 3.0 mg/kg dose group achieved remission compared to 0% placebo patients. Similar patterns were observed in EULAR response and ACR core set measures. Table: Final Safety & Efficacy Data  Safety Placebo Total Active # of Pts Any AE (% pts) 6 (55) 3 (50) 4 (67) 5 (83) 3 (50) 5 (83) 4 (67) 24 (67) Efficacy at Day 77 # of Ptsa Baseline DAS Mean DAS Change Mean% DAS change Low Dis. Act. (%)b 1 (9) (50) 1 (20) 1 (17) 3 (50) Remission (% Pts)c (50) 1 (20) 1 (17) 2 (33) a: Pts w/ efficacy at day 77; b: Defined as DAS28 <=3.2; c: Defined as DAS28 <2.6 Conclusion: Baminercept alfa was well tolerated. DAS28, EULAR responses and other efficacy measurements improved among patients who received baminercept alfa compared with placebo. Baminercept alfa is a promising novel therapeutic option for RA and warrants further study. Baldassare A et al. Ann Rheum Dis. 2008;67(suppl 2):86 [abstract OP-0122].

50 Summary B-cell–directed therapy has demonstrated clinical and laboratory efficacy in RA Multiple other diseases potentially may be treated with B-cell–directed therapy Safety issues concerning long-term B-cell depletion are of concern, but data thus far are cautiously reassuring New strategies of B-cell targeting may provide more potent therapeutics in the future 50

51 Assessing Immunocompetence in Patients Treated With B-cell–Directed Therapies

52 Learning Objectives Define immunocompetence and review the impact of B-cell–directed therapies on humoral immunity Summarize the safety of B-cell–directed therapies with regard to the following clinical scenarios: repeated use, safety of switching to DMARD or TNF inhibitor after RTX failure, effects on serum immunoglobulins, and serious infection rates State clinical considerations for immunizing patients who are candidates for B-cell–directed therapy DMARD, disease-modifying antirheumatic drug; RTX, rituximab; TNF, tumor necrosis factor.

53 Outline Immunocompetence and the effects of B-cell–directed therapies
Safety of B-cell–directed therapies Long-term safety of repeated use Effects on serum immunoglobulins Serious infection rates Safety of switching to DMARD or TNF inhibitor after RTX failure Immunization and B-cell–directed therapy Considerations for immunocompetence of newer B-cell– targeting agents BAFF, APRIL Progressive multifocal leukoencephalopathy (PML) in rheumatic diseases 53

54 Defining Immunocompetence: Promises and Problems With Biologic Therapies
Immunocompetence is broadly defined as the capacity of the integrated immune response to defend against infections and malignancies An increased rate of infections is the gold standard for detecting compromised immune function, but: Clinical trials are generally underpowered for rare events Data collected across clinical trials and databases of such events are not uniform

55 Immunocompetence and B-Cell– Directed Therapy
Humoral immunity is vital in protecting the host from bacterial infections Patients deficient in Ab are prone to polysaccharide encapsulated bacterial infections Experience in oncology fails to show that RTX adds risk of SIEs to traditional chemotherapy (except in HIV infections) Pro-B Pre-B Immature Transitional Naïve Memory CD20 RTX Plasmablast (short-lived) Plasma Cell (long-lived) Protective vs pathogenic antibodies Protective vs pathogenic antibodies Antimicrobial Anti-dsDNA RF and anti-CCP antibodies Antimicrobial Anti-dsDNA Anti-RBP antibodies (Ro, La, Sm/RNP) Survive weeks after RTX Survive months to years after RTX Figure 1 from Looney RJ, Srinivasan R, Calabrese LH. The effects of rituximab on immunocompetency in patients with autoimmune disease. P 6. Arthritis Rheum Jan;58(1):5-14. RTX spares long-lived plasma cells, which are the primary source of antimicrobial Abs Ab, antibody; CCP, cyclic citrullinated peptide; dsDNA, double-stranded DNA; HIV, human immunodeficiency virus; RF, rheumatoid factor; SIE, severe infectious event. Looney RJ, et al. Arthritis Rheum. 2008;58:5-14. 55

56 Immunoglobulin Levels Following RTX Therapy
Total immunoglobulin (Ig), IgM, IgG Every 8–12 weeks after each course Always normal IgG & IgM: 761 (72%) >1 Ig level <LLN (at any point) >1 low IgM: 261 (25%) >1 low IgG: 67 (6%) Decreases are nonprogressive in most cases Patients undergoing repeated courses of RTX have been reported with severely depressed IgM levels, but background therapy has been nonstandardized and has included CTX Percent of Patients IgM/IgG <LLN 24 Weeks After RTX C1 C2 C3 C4 IgM 10.1 20.4 20.9 31.4 IgG 1.4 3.5 4.0 4.3 SIEs per 100 pt-yr 5.4 4.6 6.3 CTX, cyclophosphamide; LLN, lower limit of normal. Genovese M et al. Arthritis Rheum. 2007;56(9 suppl):S149-S150 [abstract 261]. Popa C et al. Rheumatology (Oxford). 2007;46:

57 Long-Term Safety of RTX With Repeated Use
1053 patients (2438 patient-years) from 3 double-blind trials 1014 >6 months 957 >1 year 701 >2 years 120 >3 years Up to 7 treatments (1000 mg  2 IV, 2 weeks; ≥4 months from last infusion) 684 ≥2 courses; 400 ≥3 courses; 142 ≥4 courses Acute infusion reactions decreased with subsequent courses 1st infusion: C1, 23%; C4, 11% 2nd infusion: C1, 7%; C2, 2% 702 (67%) had ≥1 infection URI: 32%; UTI: 11% No tuberculosis, opportunistic infections, or viral reactivation 36 malignancies in 32 patients (3%): 4 fatal; no lymphomas Extended Follow-up of the Long-term Safety of Rituximab in Rheumatoid Arthritis Category:18. RA treatment: small molecules Author(s): Ronald F. van Vollenhoven1, Paul Emery2, Clifton O. Bingham III3, Edward Keystone4, Maria Greenwald5, Larry W. Moreland6, Marianne Sweetser7, Karen Rowe8, Bridget Wagner9, Fabio Magrini8. 1Karolinska University Hospital, Stockholm, Sweden; 2Leeds General Infirmary, Leeds, United Kingdom; 3Johns Hopkins University, Baltimore, MD; 4University of Toronto, Toronto, ON, Canada; 5Desert Medical Advances, Palm Desert, CA; 6University of Alabama School of Medicine, Birmingham, AL; 7Biogen Idec, Cambridge, MA; 8Roche Products Ltd, Welwyn Garden City, United Kingdom; 9Genentech, Inc, South San Francisco, CA Purpose: To provide an update on the long-term safety evaluation of rituximab (RTX) in patients (pts) with active rheumatoid Arthritis (RA). Methods: Safety analyses were performed on 1053 RA pts exposed to RTX as of September 15, 2006 in the clinical trial program. Data on pts receiving up to 4 treatment courses are reported. Results: Data represent 2438 pt-years [yrs] of exposure; pts received up to 7 treatment courses. A total of 684, 400 and 142 pts received ≥2, ≥3 and ≥4 courses of RTX, respectively. Incidence of adverse events (AEs) decreased from 88% (931/1053) of RTX pts after Course 1 (C1) to 81%, 72%, and 65% after C2, C3, and C4, respectively (Table). Serious AEs (SAEs) followed a similar pattern (Table). Acute infusion reactions (first infusion, each course) decreased from 23% (C1) to 11% (C4) and from 7% (C1) to 2% (C2) (second infusion, each course). A total of 702 pts (67%) experienced ≥1 infection. The most common infections were upper respiratory tract infections (32%) and urinary tract infections (11%). The proportion of pts with IgM and IgG levels below the lower limit of normal increased with further treatment courses. No opportunistic infections, viral reactivations or tuberculosis were reported. A total of 36 malignancies (18 in C1, 13 in C2, 4 in C3, and 1 in C4) occurred in 32 pts (3%). No lymphoproliferative malignancies and no increased risk of malignancy with additional courses of treatment were observed. Conclusions: This further update on the long-term follow-up (2438 pt-yrs) of RA pts receiving RTX showed a safety profile consistent with that reported previously. After 4 courses, a slight upward trend was observed in the rate of infections; however, the rate of serious infections remained stable with repeated treatment. C1 (n=1053)C2 (n=684)C3 (n=400)C4 (n=142)Any AE n (%)931 (88)553 (81)228 (72)93 (65)SAE n (%)187 (18)105 (15)39 (10)4 (3)No. of infections Infections/ 100 pt-yrs (CI)79.46 ( )84.76 ( )96.97 ( )101.4 ( )No. of serious infections Serious infections/ 100 pt-yrs (CI)5.36 ( )4.6 ( )6.34 ( )5.41 ( )CI = confidence interval C, course; URI, upper respiratory tract infection; UTI, urinary tract infection. van Vollenhoven RH et al. Arthritis Rheum. 2007;56(9 suppl):S [abstract 257]. 57 57

58 Long-Term Safety of RTX With Repeated Use
Safety of Additional Courses of RTX in Patients With Active RA: Open-Label Extension Analysis C1 C2 C3 C4 AEs 4570 1358 339 48 SAEs 222 73 18 3 Infections 954 347 71 11 Infections per 100 pt-yr 83 80 88 SIEs 59 19 5 1 SIE per 100 pt-yr 5.1 4.6 5.6 8.0 Keystone E, Fleischmann R, Emery P, et al. Safety and efficacy of additional courses of rituximab in patients with active rheumatoid arthritis: an open-label extension analysis. Arthritis Rheum. 2007;56: Multiple occurrences of the same event in a single individual are counted multiple times. Events with a start date prior to the date of the last visit are included. Includes acute infusion-related events. Adverse events (AEs) plausibly related to infusion included pruritus, urticaria/rash, angioedema, fever, chills, rigors, sneezing, throat irritation/tightness, cough, and bronchospasm, with or without hypotension or hypertension. Reported as serious and/or treated with intravenous antibiotics. A total of 1,039 patients received >1 course of rituximab. Of these, 570 received 2 courses, 191 received 3 courses, and 40 received 4 courses, for a total of 1,669 patient-years. AE, adverse event; RA, rheumatoid arthritis; SAE, serious adverse event. Keystone E et al. Arthritis Rheum. 2007;56: 58 58

59 Serious Infection Rates by Immunoglobulin Levels With Repeated Use of RTX
All-Exposure Population (n = 1053) Patients With Normal IgG and IgM (n = 761) Patients with Low IgM at Any Time (n = 261) Patients With Low IgG at Any Time (n = 67) Percentage of patients, % 100 72.2 24.7 6.3 Serious infections per 100 pt-yr (95% CI) 5.4 (4.3–6.38) 4.9 (3.93–6.06) 6.4 (4.74–8.68) 6.8 (4.03–11.49) Genovese M et al. Arthritis Rheum. 2007;56(9 suppl):S149-S150 [abstract 261]. 59

60 Long-Term Safety of RTX With Repeated Use
Conclusion A progressive increase in the number of patients who have ≥1 Ig level below LLN is seen at some point with repeated infusions of RTX The rate of infections has been stable RTX should be used with caution in patients previously treated with biologics, and long-term studies are needed A more complete safety profile of RTX will emerge as more patients are treated, followed up for longer periods of time, and given more courses of RTX van Vollenhoven RH et al. Arthritis Rheum. 2007;56(9 suppl):S [abstract 257].

61 Rate of Serious Infections With Rituximab and Other RA Therapies
Incidence of Serious Infections per 100 Patient-Years Rituxan-exposed patients (n=1053) Biologic-treated patients (n=9868) (BSR Biologics Register) Biologic-treated patients (n=928) (German Biologics Register) DMARD-treated patients (n=1352) (BSR Biologics Register) DMARD-treated patients (n=601) (German Biologics Register) Key Points In the BSR Biologics Register, biologic-treated patients received anti-TNF therapy. In the German Biologics Register, biologic-treated patients received anti-TNF therapy or anti-IL-1 therapy. BSR, British Society for Rheumatology. van Vollenhoven RH et al. Arthritis Rheum. 2007;56(9 suppl):S [abstract 257]. Listing J et al. Arthritis Rheum. 2005;52: Dixon WG et al. Arthritis Rheum. 2006;54: van Vollenhoven RF, Emery P, Bingham C, et al. Extended follow-up of the long-term safety of rituximab in rheumatoid arthritis. American College of Rheumatology Annual Meeting; Boston, MA; November 6-11, Poster 257. Listing J, Strangfeld A, Kary S, et al. Infections in patients with rheumatoid arthritis treated with biologic agents. Arthritis Rheum. 2005:52; Dixon WG, Watson K, Lunt M, et al. Rates of serious infection, including site-specific and bacterial intracellular infection, in rheumatoid arthritis patients receiving anti-tumor necrosis factor therapy. Arthritis Rheum. 2006:54; 61

62 Serious Infection Rates During Treatment With DMARDS or Anti-TNF Agents Following RTX Therapy
All Patients Receiving Any DMARD or Biologic (n = 153) Patients Receiving DMARD/ Non–TNF Inhibitor or Biologic (n = 46) Patients Receiving a TNF Inhibitor (n = 46) Before After Total exposures, pt-yr 143.22 147.55 50.13 42.02 100.83 97.79 SIEs, no. 8 12 1 2 9 Serious infections per 100 pt-yr (95% CI) 4.19 (1.88–9.33) 8.13 (4.82–14.3) 1.99 (0.28–14.1) 4.78 (1.19–19.0) 5.95 (2.67–13.3) 9.20 (4.79–17.7) All-exposure population. Genovese M et al. Arthritis Rheum. 2007;56(9 suppl):S150 [abstract 262].

63 Safety of Switching to DMARD or TNF Inhibitor After RTX Failure
All Patients DMARDs Added TNF Inhibitor Added Before After Total exposures, pt-yr 143 148 50 42 101 98 SAEs/100 pt-yr (n = 75) 28 (40) 24 (35) NR SIEs, no. 6 12 1 2 9 SIE/100 pt-yr 4 8 5 95% CI 1.9–9.4 4.6–14.9 0.3–14.2 1.2–19 2.9–19.3 4.8–17.7 153 patients (of 1053) withdrew from RTX extension studies Entered safety follow-up for up to 48 weeks for SAEs Could be treated with DMARD(s) or TNF inhibitor 107 (70%) treated with TNF inhibitor and 46 (30%) DMARD(s) Majority had CD19 counts <LLN 18 SAEs were infectious – typical clinical course – resolved with treatment Conclusion: No increased SIEs demonstrated with switching to DMARD/TNF inhibitor “Further investigations in larger numbers over time is warranted” Keystone E, Fleischmann R, Emery P, et al. Safety and efficacy of additional courses of rituximab in patients with active rheumatoid arthritis: an open-label extension analysis. Arthritis Rheum. 2007;56: Genovese M et al. Arthritis Rheum. 2007;56(9 suppl):S150 [abstract 262]. 63

64 Can Patients Given Biologics, Including B-Cell–Depleting Therapy, Be Effectively Immunized?
64

65 Immunization and Immune Response
Responses to immunization depend on intact immune response T-cell–dependent antigens (peptide) T-cell–independent (carbohydrate) Response to neoantigens Response to recall (booster) antigens How do newer targeted immunomodulatory therapies affect immunization responses? 65

66 Biologics and Immunization
TNF antagonists may slightly decrease immune response to influenza immunization compared with MTX1 B-cell depletion in RTX-treated patients with SLE significantly decreased response to pneumococcal vaccine and tetanus immunization in almost all patients, although partial responses were observed on return of peripheral B cells2 Abatacept decreased response to neoantigens (fX174 and KLH) in patients with psoriasis3 KLH, keyhole limpet hemocyanin; MTX, methotrexate; SLE, systemic lupus erythematosus. Kapetanovic MC et al. Rheumatology (Oxford). 2007;46: Albert DA et al. Arthritis Rheum. 2006;54(9 suppl):S550 [abstract 1323]. Abrams JR et al. J Clin Invest. 1999;103: 66

67 Response to Influenza Vaccine in RA Treated With Abatacept
Proportion of Patients Responding* to Different Numbers of Influenza Serotypes – 1 Month Post-immunization Previous data suggest that abatacept may blunt response to the T-cell–independent Ag pneumococcus in healthy controls and patients with RA Influenza is a T-cell–dependent Ag, with healthy individuals demonstrating seroconversion (4-fold) to 33%–44% of the time ARRIVE trial studied abatacept in patients in whom TNF inhibitors failed Substudy of 21 patients given influenza vaccine 7 days prior to abatacept in patients receiving long-term therapy 75% of patients mounted an adequate response to at least 1 strain 1 month post-vaccination; 50% to 2 strains; 15% to all 3 MTX, consistent with previous studies, blunts response Abatacept does not appear to meaningfully impair response to influenza *Positive response defined as post-immunization antibody titer at least 4 times above baseline value. Ag, antigen; ARRIVE, Abatacept Researched in RA Patients With an Inadequate Anti-TNF Response to Validate Effectiveness. Schiff M et al. Arthritis Rheum. 2007;56(9 suppl):S392 [abstract 943]. 67

68 RTX and Vaccination Following RTX administration, post-vaccination protection rates are decreased in response to Pneumococcal vaccine1 Tetanus toxoid1 Trivalent influenza vaccine2,3 1. Albert DA et al. Arthritis Rheum. 2006;54(9 suppl):S550 [abstract 1323]. 2. Gelinck L et al. Ann Rheum Dis. 2007;66(suppl 2):160 [abstract THU0116]. 3. Oren S et al. Ann Rheum Dis. 2007;66(suppl 2):363 [abstract FRI0275]. 68

69 Immunization Responses With RTX in Patients With RA
4 patients treated with RTX (1000 mg  2) Immunization with trivalent influenza A/B at 84 days during B-cell depletion 19 patients receiving TNF antagonists 20 healthy controls Evaluation of preimmunization and postimmunization titers to antigens Gelinck LB et al. Ann Rheum Dis. 2007;66: 69

70 RTX Impairs Ability to Respond to Influenza Vaccine in Patients With RA
A/H3N2 A/H1N1 Influenza B 1024 HC RA - TNF RA - RTX 1024 1024 512 512 512 256 256 256 128 128 128 64 64 GMT Pre-vaccination Post-vaccination 64 32 32 32 16 16 8 8 16 4 4 8 2 2 4 1 1 Pre Post Pre Post Pre Post Vaccination Vaccination Vaccination Response Defined as GMT > 40 units RA-RTX N = 4 RA-TNF N = 19 HC N = 20 A/H3N2 (%) 50 84 100 A/H1N1 (%) 23 88 Influenza B (%) 24 96 Gelinck LB, Teng YK, Rimmelzwaan GF, et al. Poor serological responses upon influenza vaccination in patients with rheumatoid arthritis treated with rituximab. Ann Rheum Dis. 2007;66:1402-3 Decrease with TNF is consistent with prior observations. This is based on only 4 patients in RTX though. Background: Annual influenza vaccination is recommended for immunocompromised patients at the beginning of each winter season, under the assumption that it will provide protection against the circulating viral strains during the influenza season. Objectives: We examined the impact of RTX on influenza vaccination outcomes in rheumatoid Arthritis (RA) patients. Methods: Four RA patients treated with Rituximab, 25 RA patients treated with tumor necrosis factor alpha (TNF)-blocking agents, and 20 healthy controls, were vaccinated with a trivalent influenza vaccine. (figure 1). For B-cell counts blood samples were obtained at baseline and at days 28, 84 and 168 after starting RTX treatment. Absolute B-lymphocytes were detected by flow cytometry using TruCOUNT tubes with a mixture of monoclonal antibodies against CD45, CD3 and CD19. Hemagglutination inhibition (HI) titers were measured (in duplo) just before vaccination and 28 days later in order to determine the response upon vaccination. Results: The influenza vaccine was administered between 87 – 140 days after the first RTX infusion. B-cells were completely depleted (< 1x106 cells/L) in all four patients at day 84 after the first RTX infusion. Significantly lower postvaccination titers were found in the RA patients treated with RTX, compared to both control groups. Pre- and postvaccination serum geometric mean titers (GMT with standard deviation) against influenza A/H3N2, A/H1N1 and Influenza B for a group of RA patients treated with RTX (RA - RTX), compared to RA patients treated with anti-TNF (RA - TNF) and healthy controls (HC). (respectively p=0.02 and p < 0.001). Postvaccination protection rates were also significantly lower in the RTX group (25-50%) compared to both control groups (84% for all three antigens in the RA group and % in healthy controls, p < 0.05). No major side effects were observed after vaccination nor any effect on disease activity. Conclusion: Rituximab impairs the ability to respond adequately to influenza vaccination. This is the first study to demonstrate significantly lower postvaccination titers and protection rates, for all three influenza vaccine strains, in RA patients treated with RTX, compared to a group of RA patients treated with anti-TNF and a group of healthy controls. In conclusion, our findings suggest that influenza vaccination, although not completely ineffective, will most likely not sufficiently protect RTX treated RA patients from influenza infection during the whole winter season. GMT, geometric mean titers; HC, healthy controls. Gelinck LB et al. Ann Rheum Dis. 2007;66: 70

71 Immunization: Summary
Consider immunization responses when treating patients with RA and when introducing new targeted therapies Live attenuated virus vaccines should be avoided in all patients receiving immunosuppressants (based on absence of data) Some immunomodulatory therapies may significantly attenuate the efficacy of other immunizations Timing of immunizations may be clinically important Evaluating larger numbers of patients treated with these agents alone and in combination with other DMARDS and concomitantly evaluating appropriate controls using standardized definitions of response are very important in guiding clinical care Appropriate immunization should be performed prior to initiation of RTX therapy Gelinck LB et al. Ann Rheum Dis. 2007;66: . 71

72 Considerations for Immunocompetence of Newer B-Cell–Targeting Agents: BAFF, APRIL

73 Considerations for Immunocompetence of Newer B-Cell–Targeting Agents: Baminercept (LTßR-Ig)
LTß-Ig is a fusion protein binding to LTα1ß2 and LIGHT (members of the TNF family) on T, B, and NK cells Blocks interaction with antigen-presenting and stromal cells The LT pathway regulates organization of the lymphoid microarchitecture and lymphoid trafficking LTß-deficient mice have absent secondary lymphoid tissue and profound immunodeficiency Partially deficient LTß mice are susceptible to a wide variety of pathogens: Listeria, Mycobacterium tuberculosis, viruses, and parasites1,2 Watch for opportunistic, viral infections 1. Xu G et al. J Immunol. 2007;179: 2. Lin X et al. Int Immunol. 2003;15: 73

74 PML in Rheumatic Diseases
PML is a neurologic disease caused by infection with the JC polyomavirus that occurs in susceptible patient populations Recently reported in 0.1% of patients receiving natalizumab, a promising drug for multiple sclerosis (also in clinical trials for RA and Crohn’s disease) Mechanism of action is unknown 70-year-old woman with SLE and HA previously treated with CTX, AZA, and long-term GC; developed vertigo and ataxia after 4 infusions of RTX MRI had multiple brain lesions and biopsy showed PML The patient died 1 year later 45-year-old woman with SLE since 1982, previously treated with CTX, IV methylprednisolone, and daily GC. CD4 count was low (<200). The patient received 3 courses of RTX with daily GC in 2003–2006 In April 2006, she developed neurologic symptoms and signs and had multiple brain lesions by MRI, and was found to have JC virus in the CSF The patient died in July 2006 AZA, azathioprine; CSF, cerebrospinal fluid; GC, glucocorticoids; HA, hemolytic anemia; MRI, magnetic resonance imaging. US Food and Drug Administration. FDA Alert: rituximab (marketed as Rituxan). December Available at: Accessed December 18, 2007. 74

75 PML and RTX PML has not been diagnosed in any patient to date in clinical trials Systematic reviews of PML and of rheumatic disease and PML have suggested that patients with SLE may have a unique susceptibility to PML1 Rheumatologists should familiarize themselves with PML and consider it in the differential diagnosis of immunosuppressed patients with unexplained neurologic disease Calabrese LH, Molloy ES, Huang D, Ransohoff RM. Progressive multifocal leukoencephalopathy in rheumatic diseases: evolving clinical and pathologic patterns of disease. Arthritis Rheum Jul;56(7): Calabrese LH, et al. Arthritis Rheum. 2007;56: 1. Calabrese LH et al. Arthritis Rheum. 2007;56: 75

76 Immunocompetence and Biologic Therapies: Promises and Problems
Efforts to improve our assessment of compromised immune responses in patients with rheumatic disease treated with biologics include: Uniform assessment of infections (bacterial, opportunistic, and viral) across trials and databases Uniform assessment of vaccine response from early clinical trials Detailed assessment of in vitro immunity, including cellular humoral and innate responses

77 The Use of B-cell– Directed Therapies in SLE, Vasculitis, and Other Autoimmune Diseases
SLE, systemic lupus erythematosus.

78 Learning Objectives Describe the rationale of B-cell depletion for the management of SLE, vasculitis, and other autoimmune diseases Summarize recent clinical trial data on the use of rituximab, belimumab, and epratuzumab in the treatment of SLE Summarize recent clinical trial data on the use of B-cell targeting in the treatment of Sjögren’s syndrome Summarize recent clinical trial data on the use of B-cell targeting in other vasculitides 78

79 B-Cell–Depletion Therapy in SLE
B lymphocytes may play a central role in the pathogenesis of SLE As precursors of antibody-secreting cells, B cells are the source of pathogenic autoantibodies B cells are not merely the passive producers of immunoglobulins, but also play a central role in autoimmunity B-cell depletion has recently emerged as a promising therapeutic approach to the treatment of autoimmune diseases, including SLE

80 RTX B-Cell–Depletion Therapy in Patients With SLE: Long-Term Follow-Up and Predictors of Response
Observations of nonrandomized clinical experience (since June 2000) in 41 patients with refractory SLE at University College, London1-3 Mean duration of follow-up 37 months (2 patients lost to follow-up) Patients must have failed to respond to CTX or methotrexate RTX 1 g IV  2 (14 days apart) + CTX RTX therapy showed efficacy Mean duration of B-cell depletion was 4 months (range 2–15) Serum immunoglobulins decreased but not below lower limit of normal Anti-dsDNA antibody levels significantly reduced 6 months after B-cell–depletion therapy Protein/creatinine ratio fell (but change did not reach statistical significance) 13 patients re-treated Background: Open uncontrolled studies have suggested that B-cell depletion therapy (BCDT) will be a promising therapy for the treatment of patients with refractory SLE. However, the factors which predict outcome and the long term safety of this treatment are unknown. Objectives: We describe the clinical outcome and safety profile of BCDT in patients with SLE during long term follow up. Autoantibody profile was also examined as a possible predictor of flare of disease. Methods: Since 2000, 41 patients with refractory SLE have been treated with BCDT (based on RTX) using a combination protocol with cyclophosphamide and steroid cover, of whom 35 have had a minimum of 6 months follow up. Patients were assessed clinically with the BILAG activity index at 1 to 3 monthly intervals. Baseline autoantibody profiles were measured by ELISA (Shield Diagnostics, Dundee) at the time of diagnosis. Adverse events and duration of B-cell depletion (CD19 < x 109/L) were noted. Flare was defined as a new BILAG A or two new subsequent Bs in any organ system. Results: The mean duration of follow up was 37 months (range 6 -79) and 28 patients had follow up for more than a year. Two patients were lost to follow up and were excluded from the analysis. Of the 33 patients (minimum follow up duration 6 months), 12 patients have remained well. The mean time to flare (TTF) was 10 months post-BCDT. Of the 20 patients who flared, 11 flared between 6 to 12 months post-BCDT. The median duration of B-cell depletion was 4 months (range 2 -15). Two patients remain depleted at the time of analysis (73 and 8 months). One patient did not deplete at all. Patients with anti-ENA antibodies (20/32) were more likely to flare at any time after BCDT with an odds ratio (OR) of 6 (p = 0.03). Patients with anti-Sm (9/32) or anti-La (5/32) were more likely to flare (OR 9, p = and OR 10, p = 0.04 respectively). Patients with low serum C3 at baseline (27/32) had a shorter time to flare post-BCDT (median TTF 12 months). The median TTF for normal C3 patients has not been reached (p = ). B-cell depletion was beneficial clinically with a decrease of median global BILAG scores from 13 to 5 at 5 to 8 months (p < ). There was improvement of serological parameters with median dsDNA titers decreasing from 203 to 74 IU/mL at 6 months (p = 0.002) and increase of median C3 from 0.73 to 0.9 gm/L at 6 months (p = ). Thirteen patients have been retreated with at least another cycle of RTX. Serious adverse events observed were pneumococcal sepsis, severe serum sickness reaction and seizure related to hyponatriaemia. Two patients have died. One developed varicella pneumonitis shortly after the second cycle of RTX and the other had pancarditis related to active lupus after B cell repopulation. Conclusion: One third of patients remain well after BCDT without needing further standard immunosuppressive agents. Most flares occurred between 6 to 12 months post-BCDT. Autoantibody profiling may help identify which patients will have a more sustained response. Patients with low baseline serum C3 or the presence of anti-ENA antibodies were more likely to flare at any time post-BCDT. Although the long term safety profile of B-cell depletion therapy is so far favorable, ongoing vigilance is recommended. CTX, cyclophosphamide; dsDNA, double-stranded DNA; IV, intravenous; RTX, rituximab. 1. Edwards JC et al. N Engl J Med. 2004;350: 2. Ng KP et al. Ann Rheum Dis. 2007;66(suppl 2):56-57 [abstract OP0020]. 3. Ng KP et al. Ann Rheum Dis. 2006;65:

81 RTX B-Cell–Depletion Therapy in Patients With SLE: Long-Term Follow-Up and Predictors of Response
11 (55%) of 20 patients who flared did so 6–12 months after treatment Predictors of lack of response Patients with anti-ENA antibodies (anti-Sm or anti-La) were more likely to flare Lower serum C3 at baseline was associated with shorter time to flare following B-cell–depletion therapy 2 (5%) of 39 patients in the cohort have died (varicella pneumonitis and SLE pancarditis) AEs: single cases of hematuria, self-limiting neutropenia, pneumococcal sepsis/pneumonia, serum sickness–like reaction, active pancreatitis, seizure Background: Open uncontrolled studies have suggested that B-cell depletion therapy (BCDT) will be a promising therapy for the treatment of patients with refractory SLE. However, the factors which predict outcome and the long term safety of this treatment are unknown. Objectives: We describe the clinical outcome and safety profile of BCDT in patients with SLE during long term follow up. Autoantibody profile was also examined as a possible predictor of flare of disease. Methods: Since 2000, 41 patients with refractory SLE have been treated with BCDT (based on RTX) using a combination protocol with cyclophosphamide and steroid cover, of whom 35 have had a minimum of 6 months follow up. Patients were assessed clinically with the BILAG activity index at 1 to 3 monthly intervals. Baseline autoantibody profiles were measured by ELISA (Shield Diagnostics, Dundee) at the time of diagnosis. Adverse events and duration of B-cell depletion (CD19 < x 109/L) were noted. Flare was defined as a new BILAG A or two new subsequent Bs in any organ system. Results: The mean duration of follow up was 37 months (range 6 -79) and 28 patients had follow up for more than a year. Two patients were lost to follow up and were excluded from the analysis. Of the 33 patients (minimum follow up duration 6 months), 12 patients have remained well. The mean time to flare (TTF) was 10 months post-BCDT. Of the 20 patients who flared, 11 flared between 6 to 12 months post-BCDT. The median duration of B-cell depletion was 4 months (range 2 -15). Two patients remain depleted at the time of analysis (73 and 8 months). One patient did not deplete at all. Patients with anti-ENA antibodies (20/32) were more likely to flare at any time after BCDT with an odds ratio (OR) of 6 (p = 0.03). Patients with anti-Sm (9/32) or anti-La (5/32) were more likely to flare (OR 9, p = and OR 10, p = 0.04 respectively). Patients with low serum C3 at baseline (27/32) had a shorter time to flare post-BCDT (median TTF 12 months). The median TTF for normal C3 patients has not been reached (p = ). B-cell depletion was beneficial clinically with a decrease of median global BILAG scores from 13 to 5 at 5 to 8 months (p < ). There was improvement of serological parameters with median dsDNA titers decreasing from 203 to 74 IU/mL at 6 months (p = 0.002) and increase of median C3 from 0.73 to 0.9 gm/L at 6 months (p = ). Thirteen patients have been retreated with at least another cycle of RTX. Serious adverse events observed were pneumococcal sepsis, severe serum sickness reaction and seizure related to hyponatriaemia. Two patients have died. One developed varicella pneumonitis shortly after the second cycle of RTX and the other had pancarditis related to active lupus after B cell repopulation. Conclusion: One third of patients remain well after BCDT without needing further standard immunosuppressive agents. Most flares occurred between 6 to 12 months post-BCDT. Autoantibody profiling may help identify which patients will have a more sustained response. Patients with low baseline serum C3 or the presence of anti-ENA antibodies were more likely to flare at any time post-BCDT. Although the long term safety profile of B-cell depletion therapy is so far favorable, ongoing vigilance is recommended. AE, adverse event; ENA, extractable nuclear antigen. 1. Ng KP et al. Ann Rheum Dis. 2007;66(suppl 2):56-57 [abstract OP0020]. 2. Ng KP et al. Ann Rheum Dis. 2006;65:

82 2-Year Extended Follow-Up of Open-Label Phase I/II Trial of RTX for Active, Refractory SLE
RTX (500 mg IV weekly  4 in 5 patients and 1000 mg IV  2 on days 0 and 14 in 10) was administered to patients with active, refractory SLE (mean BILAG score 12.5, range 8–17) Peripheral B cells rapidly decreased by day 14 in all patients and remained low until 6 months post-treatment, except for 1 patient whose peripheral B cells remained low for 2 years No significant change in Ig levels  autoantibody levels and  complement levels in a majority of patients RTX resulted in sustained improvement of disease activity in 5 of 14 patients at 2 years Mean BILAG scores improved from 12.5 to 4.6 Mean background corticosteroid dose decreased from 25.2 to 7.7 mg 6 patients required repeated courses of RTX for persistent disease activity Severe AEs occurred in 7 patients, all with antiphospholipid antibodies (DVT [2], multiple infarctions [2], pulmonary infarction, MI, cerebral hemorrhage, transverse myelitis) 2 patients died (cerebral hemorrhage and catastrophic APS) Objectives: We reported safety and efficacy of rituximab for SLE which was conducted as an open-label, multi-center study of 15 patients with active and refractory SLE. Accordingly, a 2 year-extended follow-up study of patients with refractory SLE who were enrolled in the trial were performed and long-term safety, efficacy and problems were estimated. Methods: Rituximab (4 infusions of 500 mg/body every week in 5 patients and 2 infusions of 1000 mg every other week in 10) was administered to patients with active and refractory SLE (mean BILAG score was 12.5 and ranged from 8 to 17). Assessment of safety, adverse effects and efficacy was estimated by the 2-year follow-up. Results: One patient withdrew at 2 months because of the use of immunosuppressant. B cells rapidly reduced in all patients and remained low until 3-9 months post-treatment, except for 1 patient whose peripheral B cells remained low for 2 years. At week 28, 2 patients satisfied major clinical response (MCR) and 7 satisfied partial CR (PCR) criteria defined by BILAG scores, but 5 were grouped as non-CR (NCR). At year 2, 5 cases satisfied MCR (3 were scored 0), 6 did PCR and 1 did NCR and means of BILAG scores improved from 12.5 to 4.6 at the 2-year. The dosage of corticosteroid was decreased from 25.2 mg to 7.7 mg by the 2-year follow-up. However, 6 patients required additional courses of rituximab after the more than 24-week intervals because of the less efficacy of the treatment. During 2-year follow-up, severe adverse events were observed in 7 cases and among them 1 patient died of homophagocytic syndrome and cerebral hemorrhage at 7 months after the rituximab therapy and another died of catastrophic antiphospholipid syndrome (APS) at 7 months after the second course of the therapy. Also, a patient suffered from transverse myelitis. One week after the third course therapy, paraplegia of legs with pain was developed, and MRI confirmed the diagnosis, but severe infusion reaction was observed with high level of HACA at the second course of rituximab therapy. It is noteworthy that the 3 patients were complicated with APS. Conclusion: This 2 year-extended follow-up study provides that rituximab offers sustained improvement of the disease activity in 5 of 14 patients. However 6 patients required repeated uses of rituximab and severe adverse events occurred in 7 cases and 2 patients died, indicating that special attention should be paid in SLE patients with APS or those who develop severe infusion reactions. APS, antiphospholipid syndrome; BILAG, British Isles Lupus Assessment Group; DVT, deep venous thrombosis; Ig, immunoglobulin; MI, myocardial infarction. Tanaka Y et al. Ann Rheum Dis. 2008;67(suppl 2):54 [abstract OP-0020].

83 SLE: Case Series of RTX (anti-CD20) Treatment of Patients With SLE Refractory to Conventional Therapy Among 22 patients, RTX treatment1: Resolved anemia, thrombocytopenia, and/or cryoglobulinemia in all 18 patients with severe hematologic manifestations Decreased daily proteinuria by ≥50% at 6 months in 4 (66%) of 6 patients with lupus nephritis Among 19 patients, RTX treatment2: Improved thrombocytopenia in 7 (64%) of 11 patients within 3 months, resolved anemia in 3 (75%) of 4 patients, and improved cutaneous manifestations of lupus in 4 of 4 patients Decreased levels of antiplatelet and anti-RBC antibodies, but not of anti-dsDNA antibodies Among 16 patients, RTX treatment3: Improved disease activity ( BILAG score) in 9 (56%) of 16 patients Did not prevent progression to stage 5 CKD in 5 patients with severe proliferative, crescentic lupus nephritis Although some manifestations of refractory SLE may improve, some forms of lupus nephritis may progress despite RTX therapy Title: Efficacy of Rituximab in Systemic Lupus Erythematosus: A Series of 22 Cases Category: 25. SLE: clinical aspects Author (s): Zahir Amoura1, Karin Mazodier2, Marc Michel3, Jean-François Viallard4, Du Le Thi Huong1, Nathalie Chalumeau1, Patrice Cacoub1, Jean-Robert Harlé2, Jean-Luc Pellegrin4, Gilles Kaplanski2, Bertrand Godeau3, Jean-Charles Piette1. 1Service de Médecine Interne, Hopital Pitié Salpétrière, Paris, France; 2Service de Médecine Interne, Hopital de La Conception, Marseille, France; 3Service de Médecine Interne, Hopital Henri Mondor, Créteil, France; 4Service de Médecine Interne, Hopital Haut-Lévêque, Bordeaux, France Background Rituximab is a chimeric monoclonal antibody which is growingly used for the treatment of Systemic Lupus Erythematosus. Objective To describe a series of 22 SLE patients treated with Rituximab Results Eighteen women and 4 men, mean age: 35 ± 11.4 years, received Rituximab for: lupus nephritis refractory to conventional immunosuppressants (n = 6; 4 class IV and 2 class III), refractory auto-immune hemolytic anemia (n = 4), severe thrombocytopenia refractory to splenectomy (n = 1) or high dose steroids (n = 2), thrombotic thrombocytopenic purpura resistant to plasmapheresis (n = 2), systemic cryoglobulinemia (n = 1), hemophagocytic syndrome (n = 1), severe polyarthritis associated to cutaneous lupus refractory to conventional treatment (n = 3), anti-phospholipid antibody syndrome with repeated thrombosis despite of an accurate anticoagulation treatment (n = 1). Doses of Rituximab were 375 mg/m2/week X 4 (n = 16), 750 mg at day 1 and day 15, or 1000 mg on day 1 and day 15. Rituximab was used without immunosuppressant and/or immunomodulating drugs (n = 9), with IV Cyclophosphamide (n = 2), with Mycophenolate (n = 5), with plasmapheresis (n = 3), or with IVIg (n = 2). In 18 cases (81%), outcome was considered as favorable by the physician in charge of the patient. All the hematological manifestations improved: platelet counts and haemoglobin levels returned to normal range, and Willebrand protease inhibitor and cryoglobulinemia disappeared. Four nephritis (66%) improved with a ≥ 50% decrease of the daily proteinuria at 6 months. One patient evolved to end stage renal disease. Lymphocyte B depletion was assessed in 12 cases (54%) and was complete (< 1%) in all the cases. Anti-DNA antibody levels did not significantly decrease. Two patients exhibited hypogammaglobulinemia without clinical complications. Conclusion Rituximab is effective in severe hematological manifestations of SLE refractory to conventional therapy. For lupus nephritis, results are interesting but must be confirmed by larger studies. Sangle – see hard copy. CKD, chronic kidney disease; RBC, red blood cell. 1. Amoura Z et al. Arthritis Rheum. 2007;56(9 suppl):S458 [abstract 1124]. 2. Lindholm C et al. Ann Rheum Dis. 2008;67(suppl 2):344 [abstract FRI0184]. 3. Sangle SR et al. Arthritis Rheum. 2007;56(9 suppl):S215 [abstract: 441]. 83

84 RTX + IV CTX Induction for Patients With Lupus Nephritis
RTX + IV CTX may serve as an alternative induction regimen for patients with severe lupus nephritis1 18 patients with biopsy-proven lupus nephritis (10 proliferative GN, 7 membranous GN, 1 unknown) RTX 375 mg/m2 IV weekly  4 + IV CTX (at weeks 1 and 4) After 6 months, 17 (94%) of 18 patients had clinical improvement; 1 had early relapse Histologic examination showed significant improvement in most patients at 6 months At 2 years (n = 17), 15 (88%) of 17 patients had persistent clinical improvement; 2 had relapsed MMF may serve as a maintenance regimen for patients with severe lupus nephritis, following RTX + IV CTX induction regimen2 35 patients with SLE and biopsy-confirmed active nephritis (ISN/RPS class IV [70%] or V [30%]), refractory to standard treatment Treated with RTX 750 mg IV + methylprednisolone 500 mg IV + CTX IV on days 1 and 15 Followed by maintenance MMF 1–2 g/d for 2 years At end of 2 years, significant improvement in C3, C4, anti-dsDNA antibodies, and proteinuria in all patients; serum creatinine remained stable No SAE; 6 patients had mild viral infections No comparison with induction regimens using methylprednisolone+ CTX alone or RTX alone Objectives: To evaluate the long-term renal outcome of B-cell depleting therapy in patients with biopsy-verified Lupus Nephritis. Methods: Eighteen female patients with biopsy-verified lupus nephritis (LN) were treated with rituximab (RTX) in a combination protocol with cyclophosphamide. No immunosuppressive drugs were allowed before B-cell repopulation. In all patients but one, nephritis had been confirmed by renal biopsy before onset of therapy. Patients were evaluated with renal BILAG at baseline, 6 months and after 2 years of follow-up. Relapse was defined as a new BILAG A. Repeated renal biopsy was performed in 16 out of 17 patients after 6-12 months and at renal relapse in 2 out of 3 patients. Results: All patient had a renal BILAG A before treatment. Ten patients had proliferative nephritis at baseline (WHO III or IV) and 7 had membranous nephritis (WHO V), one unknown. After 6 months, 8 patients had a BILAG B, 7 a C, 2 a D and one patient had an early relapse (BILAG A). During follow-up, two additional patients relapsed after 10 and 18 months. Of the remaining 15 patients, 3 had a renal BILAG B, 4 a C and 8 a BILAG D at 2 years of follow-up. On repeat renal biopsy after 6 months, the histology showed considerable improvements in most patients, with significant improved activity indices and stabilized chronicity indices. Electronmicroscopic evaluation showed striking resolution of electron-dense subepithelial deposits in the patients with membranous disease. In 8/18 patients, maintenance therapy with immunosuppressants were started after B-cell repopulation. The remaining ten patients were on low dose prednisolone only. The three patients that relapsed belonged to the group treated with only low dose prednisolone. Conclusion: In these patients with biopsy-verified lupus nephritis, B-cell depleting therapy with rituximab provided long-term renal improvement in 67% of the patients. Repeat renal biopsies were useful in order better to characterize the therapeutic results. ISN, International Society of Nephrology; MMF, mycophenolate mofetil; RPS, Renal Pathology Society; SAE, serious adverse event. 1. Jónsdóttir T et al. Ann Rheum Dis. 2008;67(suppl 2):54 [abstract OP-0021]. 2. Guzman RA et al. Ann Rheum Dis. 2008;67(suppl 2):219 [abstract THU0260].

85 EXPLORER: Efficacy and Safety of RTX in Patients With Moderate to Severe SLE
Phase II/III randomized, double-blind, placebo-controlled study to evaluate efficacy and safety of RTX in patients (n = 257) with moderate to severe SLE receiving background prednisone therapy Primary end point: Proportion of patients who achieved either a major clinical response or partial clinical response measured by the BILAG index at 52 weeks Secondary end points: Time-adjusted AUC of BILAG disease activity, improvement in BILAG disease activity, time to flare, QOL, and proportion taking <10 mg prednisone daily Study did not meet its primary end point or any of its six secondary endpoints Reasons for failure unclear Clinical trial in lupus nephritis ongoing Slide Notes: Study excluded patients with lupus nephritis (LN). 257 patients randomized 2:1 to receive: RTX IV on days 1 & 15 + prednisone or Placebo IV on days 1 & 15 + prednisone Each group was retreated 6 months later with the same regimen. Study of RTX in patients with ISN/RPS Class III or IV lupus nephritis (LUNAR) is ongoing. AUC, area under the curve; EXPLORER, A Study to Evaluate the Efficacy and Safety of Rituximab in Patients With Moderate to Severe Systemic Lupus Erythematosus; QOL, quality of life.

86 RTX B-Cell–Depletion Therapy in Patients With SLE: Summary
Observations of nonrandomized clinical experience in patients with refractory SLE at 7 institutions suggests therapeutic efficacy of RTX when used in addition to standard (anchor) therapy1-8 Serum immunoglobulin and anti-dsDNA antibody levels decreased for at least 6 months after treatment1,2 Presence of anti-Sm or anti-La antibodies or low C3 at baseline may identify patients who will flare after treatment1,2 However, a prospective randomized, double-blind, placebo-controlled study of RTX in patients with moderate to severe SLE receiving background prednisone therapy failed to demonstrate efficacy9 23 (64%) of 36 reported cases of PML in patients with rheumatic diseases have occurred in patients with SLE, most of whom had not received RTX10 Background: Open uncontrolled studies have suggested that B-cell depletion therapy (BCDT) will be a promising therapy for the treatment of patients with refractory SLE. However, the factors which predict outcome and the long term safety of this treatment are unknown. Objectives: We describe the clinical outcome and safety profile of BCDT in patients with SLE during long term follow up. Autoantibody profile was also examined as a possible predictor of flare of disease. Methods: Since 2000, 41 patients with refractory SLE have been treated with BCDT (based on RTX) using a combination protocol with cyclophosphamide and steroid cover, of whom 35 have had a minimum of 6 months follow up. Patients were assessed clinically with the BILAG activity index at 1 to 3 monthly intervals. Baseline autoantibody profiles were measured by ELISA (Shield Diagnostics, Dundee) at the time of diagnosis. Adverse events and duration of B-cell depletion (CD19 < x 109/L) were noted. Flare was defined as a new BILAG A or two new subsequent Bs in any organ system. Results: The mean duration of follow up was 37 months (range 6 -79) and 28 patients had follow up for more than a year. Two patients were lost to follow up and were excluded from the analysis. Of the 33 patients (minimum follow up duration 6 months), 12 patients have remained well. The mean time to flare (TTF) was 10 months post-BCDT. Of the 20 patients who flared, 11 flared between 6 to 12 months post-BCDT. The median duration of B-cell depletion was 4 months (range 2 -15). Two patients remain depleted at the time of analysis (73 and 8 months). One patient did not deplete at all. Patients with anti-ENA antibodies (20/32) were more likely to flare at any time after BCDT with an odds ratio (OR) of 6 (p = 0.03). Patients with anti-Sm (9/32) or anti-La (5/32) were more likely to flare (OR 9, p = and OR 10, p = 0.04 respectively). Patients with low serum C3 at baseline (27/32) had a shorter time to flare post-BCDT (median TTF 12 months). The median TTF for normal C3 patients has not been reached (p = ). B-cell depletion was beneficial clinically with a decrease of median global BILAG scores from 13 to 5 at 5 to 8 months (p < ). There was improvement of serological parameters with median dsDNA titers decreasing from 203 to 74 IU/mL at 6 months (p = 0.002) and increase of median C3 from 0.73 to 0.9 gm/L at 6 months (p = ). Thirteen patients have been retreated with at least another cycle of RTX. Serious adverse events observed were pneumococcal sepsis, severe serum sickness reaction and seizure related to hyponatriaemia. Two patients have died. One developed varicella pneumonitis shortly after the second cycle of RTX and the other had pancarditis related to active lupus after B cell repopulation. Conclusion: One third of patients remain well after BCDT without needing further standard immunosuppressive agents. Most flares occurred between 6 to 12 months post-BCDT. Autoantibody profiling may help identify which patients will have a more sustained response. Patients with low baseline serum C3 or the presence of anti-ENA antibodies were more likely to flare at any time post-BCDT. Although the long term safety profile of B-cell depletion therapy is so far favorable, ongoing vigilance is recommended. See hard copy for Amoura and Sangle abstracts. PML, progressive multifocal leukoencephalopathy. 1. Ng KP et al. Ann Rheum Dis. 2007;66(suppl 2):56-57 [abstract OP0020]; 2. Ng KP et al. Ann Rheum Dis. 2006;65: Tanaka Y et al. Ann Rheum Dis. 2008;67(suppl 2):54 [abstract OP-0020]; 4. Amoura Z et al. Arthritis Rheum. 2007;56(9 suppl):S458 [abstract 1124]; 5. Lindholm C, et al. Ann Rheum Dis 2008;67(suppl 2):344; 6. Sangle SR et al. Arthritis Rheum. 2007;56(9 suppl):S215 [abstract: 441]; 7. Jónsdóttir T et al. Ann Rheum Dis. 2008;67(suppl 2):54 [abstract OP-0021]; 8. Guzman RA, et al. Ann Rheum Dis. 2008;67(suppl 2):219 [abstract THU0260]; Calabrese LH et al. Arthritis Rheum. 2007;56: 86

87 Phase II RCT of Belimumab in SLE: Combined Response Rate Is Significantly Higher for Belimumab-Treated Patients 1, 4, and 10 mg/kg belimumab dosed on days 0, 14, 28, and then every week through week 52 Changes in immunosuppressants, glucocorticoids permitted 46% combined response rate for patients with serologically active disease receiving belimumab versus 29% for placebo at week 521 No dose response observed2 56% combined response rate for patients receiving belimumab at week 762 In subjects with serologically active disease, significant improvements in SLE disease activity shown by SELENA-SLEDAI, Physician’s Global Assessment and QOL (SF-36 PCS), and decreased BILAG 1A or 2B flares RCT, randomized controlled trial; SELENA, Safety of Estrogens in Lupus Erythematosus National Assessment; SLEDAI, SLE Disease Activity Index; SF-36 PCS, physical component score of theSF-36 health survey. 1. Ginzler E et al. Ann Rheum Dis. 2007;66(suppl 2):56 [abstract OP0018]. 2. Furie R et al. Ann Rheum Dis. 2008;67(suppl 2):53 [abstract OP-0017].

88 B-Cell Depletion With Belimumab in Patients With SLE
Efficacy1-3 Although the primary study end point (a reduction in the SELENA-SLEDAI score) was not achieved, there were indications of efficacy over 3 years: Combined response rate of 46% at week 52 (n = 235); increased to 65% with belimumab treatment continued to week 160 (n = 170) Rate of SLE flares decreased progressively with belimumab treatment to 7% after 3 years Belimumab treatment allowed reduction of prednisone dose to 7.5 mg/d from >7.5 mg/d at baseline in up to 44% of patients after 1 year and in up to 62% of patients by 3 years Sustained response to belimumab is independent of type of autoantibody present at baseline Safety4 Belimumab is well tolerated in combination with antimalarials and immunosuppressive drugs Similar incidence rates of AEs, SAEs, serious infections, and malignancies between belimumab- and placebo-treated patients during the first year of treatment Incidence rate of AEs did not increase over time, during more than 3 years of belimumab exposure See hard copy abstracts. 1. Petri M et al. Arthritis Rheum. 2007;56(9 suppl):S527 [abstract 1316]. 2. Furie R et al. Ann Rheum Dis. 2008;67(suppl 2):53 [abstract OP-0017]. 3. Ginzler E et al. Ann Rheum Dis. 2008;67(suppl 2):217 [abstract THU0253] Merrill JT et al. Ann Rheum Dis 2008;67 (suppl 2):217 [abstract THU0254].

89 B-Cell Depletion With Belimumab in Patients With SLE
Biomarkers Changes correlated with clinical improvement observed over 2.5 years of belimumab treatment:  in C3 and C4 levels  in anti-dsDNA antibody levels  in IgG, IgM, and IgE levels (more in subjects with elevated levels at baseline) Reversion of anti-Sm and anti-RNP serologic findings from positive to negative in some belimumab-treated subjects See hard copy abstracts. Stohl W et al. Arthritis Rheum. 2007;56(9 suppl):S210 [abstract 426]. 89

90 B-Cell Depletion With Epratuzumab in SLE: RCTs
90 patients with SLE and moderate to severe flares randomized in 2 phase II RCTs, each of which was terminated prematurely because of interruptions in medication supply Up to 4 treatment cycles over a 48-week period Placebo Epratuzumab 360 mg/m2 Epratuzumab 720 mg/m2 Primary end point: Reduction of all BILAG A to B, BILAG B to C, no worsening in other systems, no addition or increase in immunosuppressives/antimalarials or corticosteroids above tapering levels Petri M et al. Ann Rheum Dis. 2008;67(suppl 2):53 [abstract OP-0016].

91 B-Cell Depletion With Epratuzumab in Patients With SLE
Patients in both epratuzumab groups had greater reductions in total BILAG scores from weeks 4 through 48, compared with placebo1 Physician and patient global assessment scores were also significantly improved in the epratuzumab-treated groups1 Overall efficacy was most consistent in the epratuzumab 360-mg/m2 group1 Epratuzumab-treated patients used less corticosteroid than placebo-treated patients over 24 weeks2 AE incidence was similar among epratuzumab- and placebo-treated patients2 Low incidence of immunogenicity (HAHA)2 Placebo (n = 30) Epratuzumab 360 mg/m2 (n = 34) Epratuzumab 720 mg/m2 (n = 10) HAHA, human antihuman antibody; ITT, intention to treat. 1. Petri M et al. Ann Rheum Dis. 2008;67(suppl 2):53 [abstract OP-0016]. 2. Wallace D et al. Ann Rheum Dis. 2008;67(suppl 2):212 [abstract THU0238].

92 B-Cell–Depletion Therapy in Patients With SLE: Summary
To date, no B-cell–directed therapy is approved for the treatment of SLE Most clinical trials of both B-cell depletion and anti-BAFF therapy, thus far, have been of either inadequate design (open-label and uncontrolled) or flawed by interruptions in medication supply, making it difficult to determine the functionality of these therapies in this setting Safety signals in controlled clinical trials to date are modest, but caution clearly is indicated until better safety and efficacy data are available Background: Open uncontrolled studies have suggested that B-cell depletion therapy (BCDT) will be a promising therapy for the treatment of patients with refractory SLE. However, the factors which predict outcome and the long term safety of this treatment are unknown. Objectives: We describe the clinical outcome and safety profile of BCDT in patients with SLE during long term follow up. Autoantibody profile was also examined as a possible predictor of flare of disease. Methods: Since 2000, 41 patients with refractory SLE have been treated with BCDT (based on RTX) using a combination protocol with cyclophosphamide and steroid cover, of whom 35 have had a minimum of 6 months follow up. Patients were assessed clinically with the BILAG activity index at 1 to 3 monthly intervals. Baseline autoantibody profiles were measured by ELISA (Shield Diagnostics, Dundee) at the time of diagnosis. Adverse events and duration of B-cell depletion (CD19 < x 109/L) were noted. Flare was defined as a new BILAG A or two new subsequent Bs in any organ system. Results: The mean duration of follow up was 37 months (range 6 -79) and 28 patients had follow up for more than a year. Two patients were lost to follow up and were excluded from the analysis. Of the 33 patients (minimum follow up duration 6 months), 12 patients have remained well. The mean time to flare (TTF) was 10 months post-BCDT. Of the 20 patients who flared, 11 flared between 6 to 12 months post-BCDT. The median duration of B-cell depletion was 4 months (range 2 -15). Two patients remain depleted at the time of analysis (73 and 8 months). One patient did not deplete at all. Patients with anti-ENA antibodies (20/32) were more likely to flare at any time after BCDT with an odds ratio (OR) of 6 (p = 0.03). Patients with anti-Sm (9/32) or anti-La (5/32) were more likely to flare (OR 9, p = and OR 10, p = 0.04 respectively). Patients with low serum C3 at baseline (27/32) had a shorter time to flare post-BCDT (median TTF 12 months). The median TTF for normal C3 patients has not been reached (p = ). B-cell depletion was beneficial clinically with a decrease of median global BILAG scores from 13 to 5 at 5 to 8 months (p < ). There was improvement of serological parameters with median dsDNA titers decreasing from 203 to 74 IU/mL at 6 months (p = 0.002) and increase of median C3 from 0.73 to 0.9 gm/L at 6 months (p = ). Thirteen patients have been retreated with at least another cycle of RTX. Serious adverse events observed were pneumococcal sepsis, severe serum sickness reaction and seizure related to hyponatriaemia. Two patients have died. One developed varicella pneumonitis shortly after the second cycle of RTX and the other had pancarditis related to active lupus after B cell repopulation. Conclusion: One third of patients remain well after BCDT without needing further standard immunosuppressive agents. Most flares occurred between 6 to 12 months post-BCDT. Autoantibody profiling may help identify which patients will have a more sustained response. Patients with low baseline serum C3 or the presence of anti-ENA antibodies were more likely to flare at any time post-BCDT. Although the long term safety profile of B-cell depletion therapy is so far favorable, ongoing vigilance is recommended.

93 B-Cell Targeting in Sjögren’s Syndrome (SS)
Epithelial inflammatory disease driven by cellular and humoral factors No evidence of effective remittive therapy Strong evidence of B-cell hyperactivity Polyclonal B-cell activation Hypergammaglobulinemia Multiple autoantibodies: SSA/SSB, antifodrin, others Elevated levels of BAFF Micrograph courtesy of Leonard Calabrese, DO Ramos-Casals M et al. Ann Rheum Dis. 2005;64:

94 SS: Studies of B-Cell–Directed Therapy (anti-CD20 and anti-CD22)
Uncontrolled studies have demonstrated symptomatic improvement (sicca) especially in early disease Extraglandular disease (vasculitis, GN, neuropathy) has been reasonably responsive QOL improved Non-Hodgkin’s lymphoma and SS often respond Serum sickness-like reactions have been reported GN, glomerulonephritis. Looney RJ. Arthritis Rheum. 2007;56:

95 RTX for Primary SS Open trial in 12 patients with SS and 1 or more severe disease manifestations (fatigue, parotid enlargement, neuropathy, interstitial pulmonary disease [IPD], purpura) treated with 1 g RTX, 100 mg methylprednisolone  2 10/10 fatigue; 3 parotid, 4 neuropathy, 1 IPD, 1 purpura Significant decrease in physician and patient global assessment, fatigue, and joint pain; no significant change for sicca assessments; no serum sickness or SAE related to drug All patients became B-lymphopenic; reconstitution was similar to that seen in studies of RTX in RA and SLE, with transitional and plasmablastic phenotype and a paucity of memory (CD27) B cells Larger placebo-controlled trials are needed See hard copy for abstracts. 1. St. Clair EW et al. Arthritis Rheum. 2007;56: (suppl 9):S [abstract 1102]. 2. Levesque MC et al. Arthritis Rheum. 2007;56: (suppl 9):S445-S446 [abstract 1091].

96 Safety and Efficacy of RTX in SS: First Randomized Placebo-Controlled Trial
Primary SS causes significant symptoms but has no effective therapy Subjects: Primary SS by EU-USA criteria, antibodies to Ro and La, and fatigue >50% on VAS Treatment: RTX 1 g  2 with prednisone (tapering 60→30→0 over 14 days) or placebo Primary end point: ≥20% improvement in fatigue Title: Safety and Efficacy of Rituximab in Sjogren's Syndrome: Results of a Randomised, Placebo Controlled Pilot StudyCategory: 23. Sjogren's syndrome Author(s): Shouvik Dass1, Simon J. Bowman2, Edward M. Vital1, Kei Ikeda1, Colin T. Pease1, Paul Emery1. 1University of Leeds, Leeds, United Kingdom; 2Department of Rheumatology, Selly Oak Hospital, Birmingham, United Kingdom Purpose: Primary Sjögren’s Syndrome (pSS) causes significant systemic symptoms but has no effective therapy. From open label studies there is preliminary evidence of efficacy of rituximab (RTX) in pSS. We undertook a double-blind, randomized, placebo controlled pilot study of RTX in the treatment of systemic symptoms of pSS. Methods: Patients were included if they had a diagnosis of pSS (by modified EU-USA criteria), anti-Ro/La antibodies and a fatigue visual analogue scale (VAS) score of >50mm. Patients were randomized to infusions of either 1g RTX or placebo on days 1 and 15 with 100mg methylprednisolone prior to each infusion. Patients also received oral prednisolone 60mg daily on days 2-7 and 30mg daily on days The primary endpoint was 20% reduction in fatigue VAS at 6 months. Secondary outcomes included: 20% improvement in somatic fatigue, mental fatigue and sicca mean scores of the PROFAD and FACIT-F questionnaires; SF36 mean vitality domain scores; and reduction in immunoglobulins and antibodies (all at 6 months). Results: 9 patients were randomized to each group. 1 patient received only the first infusion because of a subsequent serum-sickness like reaction. Groups were well matched at baseline for mean fatigue VAS score (placebo 69.2, RTX 76.6), disease duration, serum IgG and antibody status. There was a trend to greater improvement in the primary outcome measure (fatigue) in the RTX group, with mean improvement of 48.4% (range %) for those receiving RTX against 20.0% (range -110%-98.7%) for those receiving placebo. However, there was no significant difference in the primary endpoint between the two groups: 8/9 receiving RTX and 5/9 receiving placebo achieved 20% improvement in fatigue VAS score (p=0.294). Patients receiving RTX demonstrated improvements across all domains of SF36, especially the social functioning (p=0.01) and the mental health domain scores (p=0.06). No significant differences were noted in PROFAD outcomes. Patients receiving RTX had significant reduction in RF (p=0.05); no significant changes were seen in IgG levels or anti-Ro/La. 3 patients had infusion reactions, all mild and with first infusions. 3 serious adverse events occurred in the RTX group (1 serum sickness, 1 appendicitis, 1 palpitations (benign)). B cells were depleted below x 109/L in all patients and had returned to a mean of 59% of baseline by 6 months. No correlation was observed between B cell levels and clinical response at this timepoint. Conclusions: This is the first RCT of RTX in pSS. Results suggest that RTX may improve fatigue and systemic symptoms without influencing IgG levels. The steroids/placebo effect was quite marked with considerable variability in responses in both groups. Based on these results, a study with 37 patients per arm would be adequately powered to detect a significant difference in the primary endpoint. EU, European Union; USA, United States of America; VAS, visual analog scale. Dass S et al. Arthritis Rheum 2007;56:(suppl):S446-S447 [abstract 1094]. 96

97 Dass S et al. Arthritis Rheum. 2007;56:(9 suppl):S446 [abstract 1094].
Safety and Efficacy of RTX in SS: Results of the First Double-blind Randomized Placebo-Controlled Trial 18 available for analysis Failure to reach primary end point for fatigue (VAS) VAS fatigue improved 48% RTX vs 20% placebo (NS); 8/9 versus 5/9 achieved ≥20% improvement in fatigue VAS (NS); significant improvement in SF-36 RTX patients had significant reductions in rheumatoid factor (RF) but not Ro, La, or IgG 3 SAEs with 1 serum sickness Marked variability of clinical responses Title: Safety and Efficacy of Rituximab in Sjogren's Syndrome: Results of a Randomised, Placebo Controlled Pilot StudyCategory: 23. Sjogren's syndrome Author(s): Shouvik Dass1, Simon J. Bowman2, Edward M. Vital1, Kei Ikeda1, Colin T. Pease1, Paul Emery1. 1University of Leeds, Leeds, United Kingdom; 2Department of Rheumatology, Selly Oak Hospital, Birmingham, United Kingdom Purpose: Primary Sjögren’s Syndrome (pSS) causes significant systemic symptoms but has no effective therapy. From open label studies there is preliminary evidence of efficacy of rituximab (RTX) in pSS. We undertook a double-blind, randomized, placebo controlled pilot study of RTX in the treatment of systemic symptoms of pSS. Methods: Patients were included if they had a diagnosis of pSS (by modified EU-USA criteria), anti-Ro/La antibodies and a fatigue visual analogue scale (VAS) score of >50mm. Patients were randomized to infusions of either 1g RTX or placebo on days 1 and 15 with 100mg methylprednisolone prior to each infusion. Patients also received oral prednisolone 60mg daily on days 2-7 and 30mg daily on days The primary endpoint was 20% reduction in fatigue VAS at 6 months. Secondary outcomes included: 20% improvement in somatic fatigue, mental fatigue and sicca mean scores of the PROFAD and FACIT-F questionnaires; SF36 mean vitality domain scores; and reduction in immunoglobulins and antibodies (all at 6 months). Results: 9 patients were randomized to each group. 1 patient received only the first infusion because of a subsequent serum-sickness like reaction. Groups were well matched at baseline for mean fatigue VAS score (placebo 69.2, RTX 76.6), disease duration, serum IgG and antibody status. There was a trend to greater improvement in the primary outcome measure (fatigue) in the RTX group, with mean improvement of 48.4% (range %) for those receiving RTX against 20.0% (range -110%-98.7%) for those receiving placebo. However, there was no significant difference in the primary endpoint between the two groups: 8/9 receiving RTX and 5/9 receiving placebo achieved 20% improvement in fatigue VAS score (p=0.294). Patients receiving RTX demonstrated improvements across all domains of SF36, especially the social functioning (p=0.01) and the mental health domain scores (p=0.06). No significant differences were noted in PROFAD outcomes. Patients receiving RTX had significant reduction in RF (p=0.05); no significant changes were seen in IgG levels or anti-Ro/La. 3 patients had infusion reactions, all mild and with first infusions. 3 serious adverse events occurred in the RTX group (1 serum sickness, 1 appendicitis, 1 palpitations (benign)). B cells were depleted below x 109/L in all patients and had returned to a mean of 59% of baseline by 6 months. No correlation was observed between B cell levels and clinical response at this timepoint. Conclusions: This is the first RCT of RTX in pSS. Results suggest that RTX may improve fatigue and systemic symptoms without influencing IgG levels. The steroids/placebo effect was quite marked with considerable variability in responses in both groups. Based on these results, a study with 37 patients per arm would be adequately powered to detect a significant difference in the primary endpoint. Dass S et al. Arthritis Rheum. 2007;56:(9 suppl):S446 [abstract 1094].

98 Vasculitis and B-Cell Targeting: Rationale
Cryoglobulinemia-Mediated Vasculitis ANCA-Associated Vasculitis Sansonno D et al. Blood. 2003;101: Zaja F et al. Blood. 2003;101: Multiple case reports and small series: Lamprecht P et al. Ann Rheum Dis. 2003;62: ; Cai FZ. J Rheumatol. 2006;33: ; Basse G et al. Transplantation. 2005;80: ; Quartuccio L et al. Rheumatology (Oxford). 2006;45: Kay J et al. N Engl J Med. 2005;353: Keogh KA et al. Am J Respir Crit Care Med. 2006;173: Smith KG et al. Arthritis Rheum. 2006;54: Golbin JM et al. Arthritis Rheum. 2006;54(9 suppl):S527 [abstract 1265]. Aries PM et al. Ann Rheum Dis. 2006;65: ; Flossmann O et al. Ann Rheum Dis. 2006;65: Golbin JM et al. Clin Exp Rheumatol. 2007;25(1 suppl 44): S74-S76. Brihaye B et al. Clin Exp Rheumatol. 2007;25 (1 suppl 44):S23-S27. ANCA, antineutrophil cytoplasmic antibody. 1. Ruddy S et al, eds. Kelley’s Textbook of Rheumatology. 6th ed. Philadelphia, Pa: W.B. Saunders; Vassilopoulos D et al. AIDS. 2005;19(suppl 3):S123-S127.

99 Repeated B-Cell Depletion in ANCA-Associated Vasculitis
2006: Golbin et al suggested repeated treatment with RTX and profound B-cell depletion are well tolerated in relapsing WG; B-cell depletion appears effective for maintaining remission and relapse was not seen in the absence of B cells or ANCA 2007: Molloy et al retrospectively reported 4 patients with refractory ANCA disease each responding initially to RTX therapy; all patients eventually relapsed despite ongoing B-cell depletion Until controlled studies are completed, the decision to use RTX therapy in ANCA-associated disease should be based on assessment of risk/benefit to the individual patient. Title: Relapses in Rituximab-Treated Wegener’s Granulomatosis Patients Despite Peripheral B Cell Depletion Category: 30. Vasculitis Author(s): Eamonn S. Molloy, Curry L. Koening, Jose Hernandez-Rodriguez, Tiffany M. Clark, Carol A. Langford, Gary S. Hoffman. Cleveland Clinic, Cleveland, OH Purpose: Wegener’s granulomatosis (WG) is a chronic relapsing disorder, with significant disease- and treatment-related morbidity, emphasizing the need for safer, more effective therapies. Rituximab (RIT), an anti-CD20 monoclonal antibody that leads to B cell depletion, has demonstrated encouraging safety and efficacy in uncontrolled series of WG patients. A multicenter, randomized, controlled trial is currently underway investigating the efficacy and safety of RIT for the treatment of WG. We describe the occurrence of relapse in WG patients treated with open-label RIT. Methods: Patients were included if they had a WG relapse despite depletion of circulating B cells following RIT treatment. Four such patients were identified by retrospective chart review. All 4 fulfilled the ACR classification criteria for WG. Disease activity was assessed by BVAS/WG. Results: Median age was 36.5 years (range 23-57); three patients were female; all were Caucasian. Mean disease duration was 8 years (range 4-15); patients had previously been treated with a median of 3 immunosuppressive drugs in addition to prednisone (range 3-4). All were cANCA-PR3 positive at diagnosis; one was positive at the time of RIT treatment. Three patients were treated with 4 weekly RIT doses of 375mg/m2; and one with 2 doses of 1g, 2 weeks apart. All received concomitant intravenous methylprednisolone (≤500mg) with each RIT infusion, as well as oral prednisone. None received concomitant cyclophosphamide. One received RIT for immune-mediated thrombocytopenia concurrent with WG; this resolved with RIT treatment. The other 3 patients were treated with RIT for active WG, all of whom had a good initial response to RIT; median BVAS/WG prior to RIT was 4 (range 3-7); this had reduced to a median of 1 (range 1-4) 1 month after RIT. However, all 4 patients had a WG relapse at a median interval of 2 months (range months) following initiation of RIT treatment. All 4 had peripheral B cell depletion at the time of relapse. Median BVAS/WG at the time of relapse was 4 (range 3-6). The items scored for each patient included 1. sinusitis, arthralgias and enlarging pulmonary nodules; 2. sinusitis, arthritis, conjunctivitis/lacrimal duct inflammation and new pulmonary nodules; 3. sinusitis, arthritis, new sensory peripheral neuropathy; and 4. sinusitis, epistaxis, fatigue/weight loss and enlarging pulmonary nodules. Conclusions: In this small cohort of WG patients, clinical improvement and B cell depletion occurred in all patients after treatment with RIT. However, all 4 relapsed despite B cell depletion. These cases demonstrate that absence of detectable peripheral blood B cells does not preclude the possibility of WG relapse. Whether such relapses were related to the influence of B cells in lymphoid organs or bone marrow mediating injury or yet other pathways of immune-mediated disease is unknown. WG, Wegener’s granulomatosis. 1. Golbin JM et al. Arthritis Rheum. 2006;54(suppl 9):S527 [abstract 1265]. 2. Molloy E et al. Arthritis Rheum. 2007;56(suppl 9):S769 [abstract 2020]. 99

100 Rituximab: Updates in Vasculitis
Wegener’s granulomatosis Efficacy of RTX for refractory WG with ear, nose, and throat manifestations (N = 34) 30 (88%) of 34 patients with refractory WG responded well to RTX; therapeutic responses maintained during follow-up No long-term adverse effects documented; therapy failed in 3 patients Patients with systemic WG refractory to, or intolerant of, other therapies randomly assigned to infliximab (3 mg/kg/mo for 1 month then 5 mg/kg/mo) or RTX (375 mg/m2/wk for 4 weeks, then once every 4 months) for 12 months (21 evaluable patients) Infliximab and RTX achieved comparable outcomes, and improved clinical disease course in 6/11 and 7/10, respectively Martinez Del Pero MA et al. Ann Rheum Dis. 2008;67(suppl 2):68 [abstract OP-0060]. Cohen P et al. Ann Rheum Dis. 2008;67(suppl 2):223 [abstract THU0273].

101 RTX in ANCA-Associated Vasculitis: Summary to Date
In ANCA-associated vasculitis, most but not all preliminary (noncontrolled) studies of B-cell depletion suggest that this strategy is a safe and effective mechanism-based approach for remission induction and maintenance Repeated courses are equally effective and thus far associated with few infectious complications Limited trials in cryoglobulinemia with and without HCV infection have demonstrated efficacy and reasonable safety of anti-CD20 based therapy A large controlled trial of RTX for ANCA-associated vasculitis (RAVE) is fully recruited and analysis is pending (www.clinicaltrials.gov) HCV, hepatitis C virus.


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