B cell elimination in MS, Predictors of good Response and Changes in CSF
Anne H. Cross Washington University School of Medicine St. Louis, Missouri
Disclosures
Anne Cross has served as a consultant for: Abbvie, Biogen, EMD Serono, Genentech, Genzyme, Mallinckrodt, Novartis, Roche, Teva And has research grants from Biogen and Genentech Outline
1. Biomarkers predicting response to anti-CD20 monoclonal antibodies 2. Animal models 3. B cell-targeted therapy that failed in MS 4. Is there a more focused B cell target?
Reduction in ARR vs IFNB1a in RRMS: OPERA I and II 12 and 24 week Confirmed disability worsening reduced 40% in OPERA I & II
>90% reduction in Gad+ lesions in RRMS: OPERA I and II Surface markers during B cells Development
Immature B cell Naïve Memory Stem Cell mature B B cell Plasmablast Plasma cell
CD20 CD19 CD138 CD27 (memory)
Major histocompatibility complex-Class II
Based on Mei HE, et al. Rationale of anti-CD19 immunotherapy: an option to target auto-reactive plasma cells in autoimmunity Arthritis Research & Therapy 2012, 14(Suppl 5):S1.
No agent that specifically targets B-cells has yet been approved for clinical use in MS
• Two monoclonal antibodies (mAbs) that eliminate B cells (ocrelizumab, ofatumumab) by targeting CD20 are in MS pipeline. • It is expected that one or more agents will receive agency approval for MS within the coming 1-2 years Using animal studies to unravel the roles of B cells
How might B cells work with T cells to induce CNS demyelination?
Murine experimental autoimmune encephalomyelitis model
Animal model studies B cells essential when long protein (not peptide) used as antigen
Lyons & Cross, 1999; Weber, Zamvil 2010 Goverman Lab Th1 antigen-specific T cells reactivated in CNS by B cells • Most MHC II+ cells in naïve mouse brain are B cells (CD19+) • Passive transfer MOG-specific T cells normal vs. B cell deficient mice. Similar numbers of T cells in CNS at day 4 (pre-clinical), but by day 7 the encephalitogenic T cells had ↑d in numbers in normal mice and ↓d in B-cell deficient mice. • Not due to ↑d in situ proliferation, • Increased T cells were due to ↑d recruitment from the periphery
Pierson ER, Stromnes IM, Goverman JM. B cells promote induction of EAE by facilitating reactivation of T cells in the central nervous system. J. Immunol 2014
Greg Wu Lab: MHCII expressed only by B cells Resistant to passive EAE
4.0 WT WT CD19Cre- 3.0 BMHCII
2.0
Clinical Score Score Clinical 1.0
0.0 0 10 20 30 Day post-transfer
Archambault, et al. J Immunol. 2013; 191:545-550. B cells abundantly express BCR targeting MOG susceptible to passive EAE
BMHCII x BCRMOG 4.0 WT WT CD19Cre-BMHCII 3.0 BMHCII MOG-IgH/CreBMHCII x BCR + MO MOG BCR Mouse 2.0
Clinical Score Score Clinical 1.0
0.0 0 10 20 30 Day post-transfer
Parker Harp et al, J Immunol. 2015;194:5077-84.
Conclusions
•Animal studies: antigen-specific B cells are key for activation of autoimmune T cells in response to a protein antigen
•B cells in the CNS recruit T cells into CNS, as well as reactivate them Evidence-based treatment selection
•14 MS disease-modifying treatment options now FDA approved.
•Choice of treatment remains largely empirical.
•Methods are needed to predict who will respond to specific treatments: How do we choose the best patients for anti-CD20 therapy?
Objective: Determine characteristics of patients who responded best to anti-CD20 B cell depletion
•Phase II study of the B cell depleting anti-CD20 mAb, rituximab, as an add-on agent in 30 relapsing MS patients who were sub-optimally treated with an injectable.
• Some patients responded with “no evident disease activity”.
•Clinical data along with MRI metrics were collected as part of this study. Additionally, 24 of the 30 patients had CSF banked pre- and post-treatment.
•We sought predictors of optimal response to rituximab, a chimeric anti-CD20 mAb Phase II Trial of Rituximab as “add-on” to BIFN or GA
• Dosing was oncology dosing – 375mg/m2 x 4, 1 week apart. • 30 patients enrolled in study, 24 of whom had CSF collected pre and post treatment.
Naismith et al, Neurology, 2010
Phase II Trial of Rituximab: 88% reduction in Gad+ lesions
Pre- mean =2.81
Post – mean = 0.33
Pre
1. Cross AH, et al.. J. Neuroimmunol. 2006; 180:63-70. 2. Naismith RT et al. Neurology 2010; 74: 1860-1867 Definitions: NEDA vs non-NEDA
• Patients were categorized into:
• Ideal responders (NEDA) had no Gad-enhancing lesions, no relapses and stable or improved clinical exams after treatment. N=9
• Non-responders had a relapse or clinical deterioration, or no reduction in number of enhancing-lesions after B cell depletion. N=21
Clinical & statistical methods
• Expanded Disability Status Scale (EDSS) determined by single examiner (AHC)
• MSFC done by coordinator blinded to assay
• MRI results- blinded to order in time and to assay results Demographics of NEDA responders and non-NEDA subjects
Alvarez E, et al. MSJ-ETC ,2016.
Baseline Predictors of NEDA: Clinical and Imaging
Optimal responders had faster (A) 9-hole peg test times (dominant and non- dominant), (B) 25 FTW times, (C) lower Expanded Disability Status Scale, (D) higher numbers of contrast enhancing lesions Alvarez E, et al. MSJ-ETC ,2016. Blood and Spinal Fluid Assays
• CSF Levels of CXCL13, CCL19, B cell activating factor (BAFF), MBP, Neurofilament Light and Heavy chain, antibodies to recombinant hMOG
• ELISAs performed blinded to response status
• Index values for CXCL13, BAFF and IgG index: Index= (CSF level/serum level) ÷ (CSF albumin/serum albumin)
Baseline CSF IgG Index is predictive of response
Median baseline CSF IgG Index 1.17 vs 0.67, p=0.04. 60% of the ideal responders had Index>1.0, vs only 20% of those in the non-ideal group Results: Baseline Predictors
Optimal responders had higher Intrathecally synthesized CXCL13 calculated by “CXCL13 Index” (p=0.028). However, much overlap. ROC curve : 0.8.
Combined CSF IgG Index and CXCL13 Index ROC Curve Sensitivity as a function of specificity – 1.0 is ideal predictor BAFF Index, NF levels & MBP levels did not predict NEDA
BAFF/Blys (B cell activating factor / B-Lymphocyte Stimulator) enhances B-cell maturation, function and survival. BAFF upregulated in MS patient brains; astrocytes are a source as well as T cells & dendritic cells. B cells express the receptors (TACI and BCMA) for BAFF.
CSF Neurofilament levels and CSF MBP levels were not predictive of response to rituximab either
Limitations
• Low number of patients • Exploratory in nature • Retrospective • Clinical assessments not blinded Biomarker responses supported NEDA: MBP, NFL and NFH levels were stable or ↓ in all NEDA responders
CSF (A) myelin basic protein, (B) neurofilament light, (C) neurofilament heavy, and (D) CXCL13 levels pre- and post-rituximab. Optimal responders meeting NEDA are in solid lines; non-NEDA responders in dotted lines.
CSF from Rituximab Ph2 study: B cells greatly reduced as well as T cells reduced
CSF T Cells Pre- vs Post-Rituximab CSF B Cells Pre- vs Post-Rituximab 5000 300 4000 p=0.0001 Wilcoxon matched pairs test p=0.0001, 250 3000 Wilcoxon matched pairs 2000 200 test 1800
40 1200
20 600 #CD3+ cells/ml CSF # CD19+ cells/ml CSF 0 0 Week 0 Week 24-30 Pre- vs Post-Rituximab Week 0 Week 24 Rituximab Ph2 study: Decline in CSF T cells correlated with decline in CSF CXCL13
(r=0.45, p=0.03, Spearman rank correlation, n=23 pairs)
R= 0.45 P = 0.03 0.25
0.00
-0.25
-0.50
-0.75 CXCL13 % change % CXCL13
-1.00 0 5 5 0 .0 .7 .2 .5 -1 -0 -0.50 -0.25 0.00 0 0 0.75 1.00
CD3+ cells/ml % change
Conclusions
•Patients who achieve NEDA in response to B cell depletion: Higher IgG indices and higher CXCL13 indices •Contrast enhancing lesions and better baseline neurologic status increase chances of NEDA. • Anti-CD20 rituximab treatment reduced CSF biomarkers of tissue destruction (MBP, neurofilament) and inflammation, especially in the ideal responders. •CXCL13 greatly decreased by the depletion of B cells About CXCL13
• CXCL13 is an essential chemokine in establishing and maintaining germinal centers in lymphoid tissues. • Receptor is CXCR5 • Chemotactic for B cells, activated T cells, some DCs • CXCL13 is critical to ectopic lymphoid follicles, found in progressive forms of MS, assoc’d with worse outcomes
B220 CD3 CXCL13 B CELLS T CELLS
CXCL13 and MS
• CSF CXCL13 Levels higher in MS patients than controls
Khademi et al, Mult Scler 2011 Higher CXCL13 not MS-specific: ↑ in inflammatory CNS diseases
• MS, NMO and other inflammatory CNS control patients have higher CXCL13 levels in CSF than non-inflammatory controls. • CSF CXCL13 had moderate correlation with baseline EDSS, also correlated
in our study with WBC #, oligoclonal band #, NFH and IgG Index
Alvarez E et al. Mult Scler. 2013;19:1204-8.
CSF CXCL13 correlates with total WBCs and B cell numbers in CSF in RRMS patients
Sellebjerg et al, Neurology 2009 CSF CXCL13 levels associated with MS activity
Khademi et al, 2010
Do Approved Disease Modifying therapies affect B cells?
We reviewed published research articles and pivotal clinical trial data
Longbrake and Cross JAMA Neurol. 2016 Most Approved Disease Modifying therapies affect B cells
Drug Mechanism of Action related to B-cells Effect on Circulating B-cells
Increased BAFF Increased total numbers Decreased expression of costimulatory molecules Rela ve ↑ transi onal Interferon-Beta Impaired antigen presentation Rela ve ↓ class-switched memory Inhibits pro-inflammatory cytokines (e.g. IL-1β, IL-23) Increased anti-inflammatory cytokines (IL-10) Decreased BAFF Decreased total numbers Glatiramer Impaired antigen presentation Rela ve ↑ naïve acetate ↓ pro-inflammatory cytokines (e.g. IL-17, IL-6, TNFα, LT) Rela ve ↓ plasmablast, memory Increased anti-inflammatory cytokines (IL-4, IL-10) Sequesters B-cells in lymphoid tissue; Decreased total numbers ↓ expression of costimulatory molecules and pro-inflammatory Rela ve ↑ naïve Fingolimod cytokines (e.g. TNF-α) Rela ve ↓ newly produced B-cells, memory Increased anti-inflammatory cytokines (IL-10) Dimethyl ↑ B1 B-cells (secrete IL-10) Slight ↓ in total numbers fumarate ↑ CD24highCD38high T2-MZP (secrete IL-10) ↓s pyrimidine synthesis enzyme required for T & B cell Unknown Teriflunomide proliferation ↓s multiple transcription factors and enzymes (COX-2, iNOS) Impairs transmigration into CNS, as well as other tissues Increased total circulating numbers ↑ precursors, regulatory, marginal-zone like, and Natalizumab memory cells Rela ve ↓ naïve B-cells Increased BAFF ↑ newly produced cells (soon after infusions); Alemtuzumab Temporary depletion of B-cells with subsequent reconstitution; mature naïve cells long term depletion of T-cells Rela ve ↓ memory B cells Mitoxantrone Cytotoxic for rapidly dividing cells Decreased total numbers after infusions
Failed Human MS trial targeting B cells: Atacicept
• BAFF and APRIL are factors that normally enhance B-cell maturation, function and survival.
• Atacicept -- human recombinant fusion protein with receptor binding site for BAFF and APRIL. Acts on mature B cells and blocks plasma cells and late stages of B-cell development, but somewhat spares memory B cells.1 • Increased disease activity in trial in relapsing MS and optic neuritis development program in MS has been halted.
• In a Phase 1 trial, CD27+CD19+ B cells were increased1
1. Hartung HP and Kieseier BC. 2010 Memory B cells
• Most MS DMTs induced a relative decrease in circulating memory B-cells with concomitant expansion of circulating B-cell precursors and/or naïve B-cells. • Failed atacicept trial; memory B cells relatively spared • Animal studies suggest B cells as CNS APC and antigen-specific B cells are critical
MEMORY B CELLS
B-Cell Receptor Maturation –all CD20+
Naïve B Cell Activated Germinal Memory B cell B Cell Center B cell Efficient Antigen uptake APC to T cell Antigen Cytokines (LTα) Abs in small & chemokines scale •IgD-negative, CD27+ Long-lived •Can be IgG+ or IgM+ CD20-neg •Found in blood, and in germinal centers, along with naïve B Plasma Cell cells •Upon Antigen exposure, produce first wave of Abs and many differentiate to Plasma cells. •A small fraction persist as memory B cells, having developed in mutative GC reaction & survive long-term. . Dendritic Cell CD20+ Memory B cells: a more precise target for future therapies?
Immature B cell Naïve Memory Stem Cell mature B B cell Plasmablast Plasma cell
CD20 CD19 CD138 CD27 (memory)
Major histocompatibility complex-Class II
Acknowledgements • Washington University: – Enrique Alvarez MD PhD – Laura Piccio, MD/PhD – Eric Klawiter, MD (MGH) – Becky Parks, MD – Rob Naismith, MD – Samantha Lancia • Our Patients • National MS Society - Sylvia Lawry Fellowship(EA) - Harry Weaver Scholar Award (LP) • Funding: – National Center for Research Resources UL1 TR000448 – National MS Society USA – Barnes-Jewish Hospital Foundation.