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

Gregg J. Silverman, M.D. Professor of Medicine and Pathology Laboratory of B cell Immunobiology NYU School of Medicine

Disclosures

• Dr. Silverman has been a consultant for Genentech, Roche, Pfizer, Onyx, BMS, Lilly, Crescendo, Quest Diagnostics

• Research support from RRF, Lupus Research Foundation, Alliance for Lupus Research and NIH-NIAID

• FDA approval will be mentioned when relevant

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Copyright (©2009) from The Immune System, Third Edition by Parham. Reproduced by permission of Garland Science/Taylor & Francis Books, LLC.

Figure 1.9 The Immune System, Third Edition

Immune system is composed of two systems: Overlay of the adaptive immune provides advantages

Good memory provides host advantages But Bad memory can drive autoimmunity

Antigen receptor genes move during B- and T-cell differentiation

Somatic recombination generates an effectively limitless range of antigen receptors from a limited number of germline minigenes

Signal 1+ Signal 2 = activation

1

2

1

Signal 1 only = death/inactivation

Courtesy of M. Cancro, U Penn Signal 3 – molds the immune bias of the lymphocyte

The adaptive immune system continuously generates lymphocyte clones with randomly generated antigen receptors. How do we maintain immune tolerance in health?

Two signal hypothesis (Bretscher and Cohn) Science 1970 169(3950):1042-9.

Subset of MHC II genes (shared epitope) predisposes to RA autoantigen-specific T/B cell clonal expansions

Plenge RM et al. N Engl J Med. 2007;357(12):1199-1209.

Chromosome The grey horizontal line indicates SNPs that are significant at a genomewide level *SNPs plotted according to chromosomal location, with the log10 P values corrected with the use of genomic control.

SNPs, single nucleotide polymorphism; MHC, major histocompatibility complex; TRAF1, TNF signal transduction gene; C5, complement 5 gene.

6

Log 1

0 P V

alue

Overview of RA pathogenesis

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Rheumatoid Arthritis: Hypothesis for Stages of Disease

Healthy

Pre-Clinical Phase

Autoimmune Early Synovitis

- Arthritis - Radiographic Changes

OVERT Rheumatoid Arthritis

- HLA-DRB1 (shared epitope)* - GWAS (PTPN22, others)

Genetics

- Smoking - Hormones - Bacteria* - Diet ?

Environmental Factors

Epitope Spreading + Titer Elevation

ACPA RF

Cytokines

Klareskog and others Courtesy of J. Scher Expanding repertoire of autoreactive lymphocytes

What is our interim progress in wiping the slate clean immune systems of patients with autoimmune disease?

Tabula rasa ? T cell

Plasma cell

B cell

Diverse approaches to eliminate B cells

Atacicept (TACI-Ig)

Rituximab (anti-CD20)

Belimumab/Tabalumab ( BAFF/BLyS)

Neutralizes both BAFF/BLyS and APRIL

CD19-1 (broadly reactive)

Autoreactive B cells

SLE RA

Other diseases

Stem Cell

Mature B cell

Transitional B cell

Pro/Pre B Cell

Immature B cell

Antigen stimulation

Memory Cell Antibody-producing plasma cells

B cell subsets in the blood

Jacobi, et al. Arthritis Rheum. 2010;62:201-10.

Unswitched memory Plasma cells plasmablasts Switched

memory

IgD+ IgD+CD10+ IgD- CD38++

CD27- naïve transitional/pre-naïve double negative, incl. CD27-memory

CD27+ pre-switched memory post-switched

memory

CD27++ plasma cells plasmablasts

CD27+ Memory B Cells

B C

ells

Time (months) RTX RTX

B cell depletion

B cell depletion

baseline regen- eration

1-2 3-4 5-6 regen- eration

1-2 3-4 5-6 0

25

20

15

10

5 A A A

A

Roll, et al. Arthritis Rheum. 2008;58:1566-75.

Clinical efficacy and B-cell regeneration in RA after Rituximab (RTX) treatment

A: P < 0.001 vs baseline. RTX approved for TNF IR RA

Naive B Cells

Perc

enta

ge

Time (months) RTX RTX

0 10 20 30 40 50 60 70 80 90

B cell depletion

B cell depletion

A

C

baseline regen- eration

1-2 3-4 5-6 regen- eration

1-2 3-4 5-6

B

A: P <.001 vs baseline; B: P <.05 vs baseline; C: P <.05 vs mos 5-6 after the first treatment.

Roll, et al. Arthritis Rheum. 2008;58:1566-75.

Clinical efficacy and B cell regeneration pattern in RA after RTX treatment

Synovial inflammation requires trafficking in the circulation between compartments

Silverman. Arthritis Rheum. 2006;54:2356-67.

TNF-α

Macrophage

Dendritic Cell

IFN-α

BAFF APRIL

TNF-α

Dendritic Cell

B Cells

B Cell T Cell TNF-α

IL-1

IFNγ

IL-1 TNF-α

IFNγ

IL-1 TNF-α IFNγ IL-1

Neutrophil

IL-1

B Cell

T Cell

Neutrophil

Synoviocytes

Bone marrow

Blood vessel

RTX: Accepted clinical paradigm for B cell-targeted therapy in RA

•Efficacy with safety •Associated with reduced circulating CD27+ memory B cells and increased naïve B cells

•Lower activated blood B cells •Lower activated blood T cells •Little or no effects on protective IgG antibodies

•Are these lessons to judge other agents?

16 Journal of Rheumatology 2014; 41; 824-828 FDA approved in RA

Anti-IL6R treatment causes decreases in switched and unswitched memory B cells

Roll, et al. Arthritis Rheum. 2011;63(5):1255-64.

In vivo effects of anti–Interleukin-6 Receptor inhibitor Tocilizumab at the B-cell level

Toculizumab approved in RA MTX IR

Overall memory B cells

Roll, et al. Arthritis Rheum. 2011;63(5):1255-64.

In vivo effects of anti–Interleukin-6 Receptor inhibitor Tocilizumab at the B-cell level

•Selective IL-6 blockade has focused and dramatic effects on B memory cells

•No significant effects on IgG levels, which is good for immune safety.

Tociluzimab Summary

Anti-TNF therapy in RA lowers levels of blood Memory B lymphocytes

• RA patients (n=45) on MTX (n=17), anti-TNF etanercept (n=11), or MTX/anti-TNF (n =17). Normal controls (n=22).

• Examined Peripheral Blood B cells by flow and cells from tonsillar biopsies.

• Etanercept increased peripheral blood naïve and transitional B cells while decreasing memory (both switched and unswitched).

• Immunohistochemistry of tonsil tissue revealed a decrease in follicular dendritic cell networks and germinal centers in etanercept treated populations

Anolik, et al. J Immunol. 2008;180(2):688-92.

*

Several TNFi approved for MTX IR RA

•RA may be associated with defects in B cell tolerance •Blockade of TNF mediated inflammation in RA can normalize some B cell associated defects of the cellular memory compartments

•TNF blockade can increase the proportion of transitional B cells and naïve B cells

•Can TNF blockade the frequency of anti-citrullinated self antigen memory B cells?

TNF Blockade Summary

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

• Manuscript in preparation

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Theoretical Pathway for Breaking Tolerance in RA

RF B cell Autoreactive

T Cells More

Autoreactive T Cells

Anti-citrulline B Cell

Citrullinated antigens

RF and anti-CCP antibodies lead to immune complexes, macrophage secretion of proinflammatory cytokines

Arginine-containing antigens

Anti-citrulline Plasma cell

PADI

Anti-CCP B cells capture any citrullinated protein, giving rise to expanded T-cell responses against self-antigens

T Cell

IgG anti-citrulline immune complexes are antigens for RF B cells

Adapted from: Niewold TB, et al. QJM. 2007;100(4):193-201.

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IgG Anti-citrullinated Protein Antibodies (ACPAs)

•Production closely linked to SE-HLA Class II1

•~80-90% of ACPA-positive patients express SE2,3 •Multiple autoantibodies to multiple cit*-proteins

•fibrinogen, α-enolase, vimentin, others

•Moderately sensitive and highly specific for RA:

•45-80% and 96-100%, respectively4

•Rare in healthy individuals and in other conditions4

1. Källberg H et al. Am J Hum Genet. 2007;80:867–875. 2. Farragher TM et al. Arth Rheum. 2008;58(2):359-369. 3. Irigoyen P et al. Arth Rheum. 2005; 52(12):3813-3818. 4. Schellekens GA et al. Arthritis Rheum. 2000;43(1):155–163.

*cit=citrullinated

Citrullinated epitope IgG reactivity in CCP3-seropositive RA patients treated with TNF inhibitor + Methotrexate show similar epitope-specific ACPA patterns to patients treated with Methotrexate alone.

CCP3+ MTX

(n = 20)

CCP3+ TNFi/M

TX (n = 20)

CCP3-neg MTX

(n = 10)

Cit

hum

an

Fibr

inog

en

CC

P3

CitF

ilagg

rin c

fc-6

Cit

FibA

616

-35

Cit

FibB

36-

52

Cit

FibA

36-

50

Cit

Enol

ase

5-21

Cit

Vim

entin

59-

78

Cit

Vim

entin

419

-445

PC-B

SA

Teta

nus

Toxo

id

Anti-

hum

an Ig

G

BSA

(-)

Diverse serum IgG-ACPA in RA serum

A range of Cit-specific self antigens

Enumeration of ACPA IgG-secreting B cells in seropositive RA patient PBMC stimulated with CpG2006/IL-21/sCD40L.

RA CCP3Sero+ CCP3

RA CCP3Sero+ CQP3

Healthy CCP3

Healthy CQP3

CCP3 ASC/106P

BMC

CQP3 ASC/106P

BMC

Tetanus Toxoid

ASC/106PBMC

Total IgG ASC/106P

BMC

Spontaneous IgG

ASC/106PBMC

AVG RA CCP3sero+ (no biologic drug) (n=16) 41.3 +/- 0.5 1.9 +/- 0.6 71.1 +/- 2.6 30808.3 200.0 (n=15)

AVG RA CCP3sero+ (on biologic drug) (n=3) 12.5 +/- 0.8 0.8 +/- 1.2 52.5 +/-1.2 49166.7 320.8 (n=3)

AVG HC & RA seroneg (n=6) 2.8 +/- 3.0 3.2 +/- 2.7 65.4 +/- 1.2 42856.4 206.0 (n=5)

Healthy Rheumatoid Arthritis

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

B cell sorting and stimulation strategy to enumerate ACP secreting memory B cells

CD

27-A

PC

IgD-FITC

Stain PBMC on ice 30 min w/ FACS antibodies on ice, wash, perform sorting

CD40L cells

Supplementary Figure 3a. Multiplex ACPA antigen array strategy for RA IgG autoantibody detection

LUMINEX

200 Cit

Cit

Cit

Cit Cit Cit

PE

PE

PE

Serum incubated with

beads

PE-conjugated anti-human IgG detection

antibody

635nm 532nm

Detect up to 1000 PE

molecules per bead.

Collect 50 beads/analyte,

up to 100 analytes

simultaneously.

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

Serum IgG Induced memory IgG

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

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

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

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Conclusion

Targeting the BLyS/BAFF pathway: Atacicept and Belimumab/Tabalumab

BLyS APRIL

BAFF-R/BR3 TACI BCMA

Anti-BLyS TACI-Ig

• Atacicept: recomb fusion protein of the TACI* receptor and human IgG (not FDA approved)

• Belimumab: human monoclonal antibody which binds BLyS (approved in SLE only)

• *TACI: transmembrane activator and calcium modulator and cyclophilin-ligand interactor receptor Scapini, et al. Immunol Lett. 2008;116:1-6.

Ramanujam & Davidson. Arth Res Therapy. 2004;6:197-202.

Proteoglycan (syndecan-1)

Effects on blood B cell subsets Belimumab Phase II SLE trial

Wallace, et al Arthritis Rheum. 2009; 61(9): 1168–78.

Total B cells

Naive B cells

Activated B cells

Memory (CD27+) B cells

Decreases mature and plasma B cells but spares memory B cells and B-cell progenitors

Belimumab in RA: no significant clinical benefits in RA

•Human antibody to soluble BLyS

•Phase II, multicenter, double-blind, placebo- controlled, dose-ranging study in 283 patients with active moderate-to-severe RA 11 yr of disease DMARD IR 38% failed at least one TNF blocker significant but weak ACR20 response.

Cohen. J Rheumatol Suppl. 2006;77:12-7.

Atacicept – no benefit in RA Tabalumab-no benefit in RA

BAFF blockade may not have depleting effects on B cell memory

Targeting of lymphocytes

Abatacept in RA • ABA blocks T-cell co-stimulation, competing with CD28 for binding to CD80/CD86 signal required for full T-cell activation

• Open trial Bx/ reBx AB in RA pts (n=16) who failed TNFi

• 60% DAS28 responders after 4 months treatment.

• 15 pts with pre and 16 wk post bx

• Results: Small reduction in synovial cellular content, significant reduced only for B cells (CD20+ cells)

• Is ABA a B cell targeted agent?

Buch, et al. Ann Rheum Dis. 2009;68(7):1220.

approved for MTX IR RA

* CD20 sublining

Abatacept reduces levels of switched memory B cells, autoantibodies, and Immunoglobulins in RA.

Studies of 30 RA patients before and after ABA After 6 months of treatment, significant reductions of serum IgG, IgA, and IgM, with normalization of initially abnormal values. Significant reduction of IgG- and IgA-ACPA, & IgM-, IgA-, and IgG-RF Proportion of post-switch memory B cells significantly decreased.

Scarsi et al. J Rheumatol. 2014

CCP, cyclic citrullinated peptide; FLS, fibroblast-like synoviocyte; IFN, interferon; MMP, matrix metalloproteinase; RANKL, receptor activator of NF-κB ligand; RF, rheumatoid factor.

Osteoclast

TNF-α, IL-1, IL-6

T cell Plasma cell

Mφ Immune complexes Complement fixation Induce Mφ secretion of proinflammatory cytokines

B cell

RF, anti-CCP antibodies

TNF-α IL-1 IL-6

TNF-α IFN-γ IL-17 RANKL

FLS

MMPs Cathepsins

MMPs Cathepsins

Responsible for marginal erosions and bone loss

IL-1, IL-6, TNF-α IFN-γ

Abatacept (CTLA4-Ig)- blocks T Cell co-stimulation

APC (DC or Mφ)

Silverman & Boyle. Immunol Rev. 2008;223:175-85.

Window of Opportunity

• Clinical trials suggest that therapy initiated during (early) window of opportunity is more likely to lead to a sustained improvement.

• Based on limited current data, biologicals may increase the likelihood of persistent remission.

• Current data suggest medication-free remission is only ~3.6 to 22%. • Possible roles of disease duration, environment, and genetics should be investigated. • Reliable biomarkers (or imaging technology) are needed.

Nagy G, van Vollenhoven RF. Arthritis Res Ther. 2015;17:181.

Pre-disease Window of opportunity Progressive disease Time

Path

ogen

esis

Initiation of therapy

• Potential pathogenetic factors are present in many individuals (anti-CCP, RF)

Initiation of therapy

• Increased production of proinflammatory cytokines

• Synovitis • Osteoclast differentiation,

development of erosions Acce

lera

tion

due

to a

utoi

mm

unity

(fu

rther

loss

of t

oler

ance

) Initiation of therapy

• Lasting cytokine imbalance

• Synovial proliferation, chronic inflammation

• Structural damage Perm

anen

t au

toim

mun

ity

We need to understand the conditions that will re-establish tolerance during remission.

Concluding speculations •Diverse biologic agents that provide clinical benefits in RA can cause shifts but not normalize the memory B cell compartment.

•Common approaches suppress inflammation do not substantial the autoimmune state.

•This explains why halting therapy often leads to flare/recurrence.

•Drug-free “cure” of RA may require normalization of adaptive immune responses, with shift in B-cell subsets deletion of pathogenic autoimmune lymphocyte clones and long-lived plasma cells .

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What is in the future?

•Combination therapies target B cells, T cells and plasma cells?

•Active induction of immune tolerance ? Cell based therapy (including stem cell infusions)

Apoptotic cell infusions for DC bias, i.e., extracorporeal photopheresis

Targeting immune checkpoints (e.g. PD-1)

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etaphor for effective targeted therapy

1. Rheumatoid synovitis takes on an orderly structure with coordinated activities of cells and cytokines that drive pathogenesis

2. Not every cell type and soluble inflammatory factor is essential !

3. Targeted therapy may directly remove only one cell or cytokine from the process But this may cause disintegration of synovitis

Revised Metaphor circa 2015

B cells B cell Targeted Therapy

T cells

IL-6

TNFa

CD28

Osteoclasts Macrophages

Activated FLS

Com

plex

syn

oviti

s

Gregg Silverman, all Jenga rights reserved

Receptors and ligands of the CD28-B7 family

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

Putative “immune checkpoints” by PD-1