Evidence-based Medicine

Inducing antigen-specific tolerance in rheumatic

diseases

Ranjeny Thomas

Arthritis Qld Professor of Rheumatology

Diamantina Institute

University of Queensland

Disclosure

• Director of Dendright

• Dendright Pty Ltd in R&D collaboration with

Janssen Biotech, Inc to develop and

commercialise tolerizing immunotherapy for the

treatment of rheumatoid arthritis

Evidence-based Medicine

Thomas R. Dendritic cells and the promise of

antigen-specific therapy. Arthritis Res Ther 2013, 15:204

Josefowicz S, Rudensky A. Control of regulatory T

cell lineage commitment and maintenance.

Immunity 2009, 30:616

McGaha TL et al. Amino acid catabolism: a pivotal

regulator of innate and adaptive immunity. Immunol

Rev 2012, 249:135-57

•Unmet needs

•Long-term drug-free remission (compliance)

•Incomplete response and adverse events current drugs

•poor stratification using biomarkers

•Lower cost of treatment

•Avoid suppression of immunity to infection or cancer

•Prevention in at-risk subjects

•However, unlike cytokine or global suppression, antigen-specific

tolerance is difficult to achieve in human autoimmune diseases

•

Understanding disease pathogenesis as well as immune tolerance

is the key to designing therapies with greater specificity

Why we need antigen-specific therapy

•Finding appropriate disease-specific protein or peptide T cell epitopes

to use

•Good progress in RA, type 1 diabetes, multiple sclerosis

•SLE, Sjogren’s, Scleroderma need work!

•Spondyloarthropathies need to address microbial pathogenesis

•TCR to model antigens in mice have high affinity and expression

•In human autoimmune disease low affinity, T cell responses are

difficult to measure:

•need better surrogate markers

•There are multiple mechanisms of tolerance:

•dendritic cell biology

•Translation: mouse models induced and may not reflect human

•Mouse mucosal immunity doesn’t always = human, role of the

microbiome may be underappreciated

•need delivery systems that promote tolerance to delivered antigen e.g. DC,

nanoparticles, targeting vectors or mAb

Some of the problems, gaps

_{Disease context: what causes RA?}

Genes

Environment

Essential disease

pathway

The problem of self-tolerance is the balance

between infection control and self-control

Genes

Environment

Essential disease

pathway

Most important genetic

association is with HLA:

identifies self.

Contributes at least 50%

heritability of RA, other

autoimmune diseases

E.COLI

Peripheral self tolerance: dendritic cells educate T cells

to respond to antigens

 Dendritic cells ingest and bring

antigens from tissues to lymphoid

organs, educating T cells to respond

to these or to self antigens

 Microbial antigens

 Self: apoptotic bodies, ubiquitous

 Altered self – neo-self Ag e.g

cit-peptides

 Dendritic cell type and activation state

affects the trigger supplied to T cells,

hence the outcome of the encounter

 Polymorphic HLA affects epitope

binding, presentation, and T cell

signal

Dendritic cells

Antigen presentation also occurs in the thymus for

central tolerance

Adapted from Germain (2002) Nature Reviews Immunology 2:309-322

joint antigens DC transport tissue antigens AIRE-depdt antigens mTEC e.g. collagen II

Regulatory T cells are produced in the thymus and gut

Adapted from Josefowicz and Rudensky (2009) Immunity:30:616

Markers: FoxP3, CD25 (IL-2Ra)

Specialized migratory DC expressing CD103, making retinoic acid (ALDH+)

Natural (n)Treg Induced (i)Treg

Tissue-derived apoptotic bodies

Markers: FoxP3, Helios

Regulation of inflammation

Dioxin, drugs, endogenous and microbial ligands

DC maturation, T cell memory Endotoxin tolerance (epigenetic regulation)

IDO Foxp3 Treg activation IL-22 Mucosal tolerance

Myeloid and monocyte-derived DC expressing CD11b, CD1c

IL-10+ IL-27+

Tr1 cells: IL-10+ (IFNg+) Foxp3- LAG3+ CD49d+ Exert bystander suppression when activated by their antigen

Peripheral tolerance: regulation of inflammation,

avoidance of self reactivity

E.COLI

CTL and CD4 effectors

Finding antigens in RA: HLA-DR genetic susceptibility connects with autoantigenic

peptide presentation or selection

DRb chain a-helix N-terminus of peptide C-terminus of peptide DRa chain a-helix P4Cit

T cell

cytokine

response

Gregersen et al, Bell et al, Snir et al, Law et al

H N O NH NH2 - - - - - - O L-citrulline residue (U) H N O NH NH2 - - - - - - O L-citrulline residue (U)

Five amino acids in three HLA proteins explain most of the

association between MHC and seropositive RA

Raychaudri et al. Nat Genetics 2012. 3-D ribbon models of structure



AA positions 11, 13, 71, 74 in HLA-DRB1, 9 in HLA-B, 9 in HLA-DRB1

explain MHC association. All in peptide binding grooves



Very compelling evidence for pathogenic role of specific peptide

interactions in RA SE alleles for seropositive RA

Data Collection at the Australian Synchrotron

Australian Synchrotron

Diffraction DR0401VimCit7166-78

Purification and crystallisation

Scally S et al. J Exp Med 2013 In press

Crystal structure: citrullinated self-antigens bind to HLA-DR SE

alleles: native peptides do not bind

Scally S et al. J Exp Med 2013 In press

Citrullinated and naive autopeptides bind to resistance allele

HLA-DRB1*0402

Scally S et al. J Exp Med 2013 In press

Stain PBMC with tetramer and cell surface markers.

Surrogate markers: Direct detection of antigen-specific

T cells in HLA-DRB1*0401+ RA patients

Purified biotinylated MHC class II molecule MHC class II CD4 Antigen-specific CD4+ T cell

The PB repertoire of HLA-DRB1*0401+ individuals contains T cells specific

for citrullinated self antigens

Scally S et al. J Exp Med 2013 In press

Citrullinated self-specific T cells are deficient in resting and activated

Treg in HLA-DRB1*0401+ RA patients

Resting Treg Activated Treg

Naive Effector-memory

Scally S et al. J Exp Med 2013 In press

RA HC

HLA-DRB1*04 binding motifs and protease sensitivity of citrullinated

epitopes

Vimentin 59-71

Endogenous peptides from T2 cell lines Cathepsin L digestion patterns of native and citrullinated vimentin by LC-MS/MS



Disease association with exclusivity to bind citrulline



SE alleles restrict the repertoire of peptides that can be presented

where R would be present at P4 – this would include infectious

antigens



Citrullination confers resistance to epitope degradation in endosome



Capacity to respond to cit-peptides present in SE and non-SE alleles



Tetramer biomarkers identify autoreactive T cells, which are likely

expanding with disease activity and exposure to cit-peptides, and a

Treg deficiency in SE+ RA patients



Applications of tetramer biomarkers:



Understand when the regulatory defect occurs (prior to or at RA

onset?)



Use to monitor response to antigen-specific therapies: expand

functional Treg?

Summary and implications

How are cit-peptides presented?

Peripheral lymphoid organ and thymic

DC constitutively present cit-peptide:

transgenic mouse model

Ireland and Unanue, J Exp Med 2011

2006: HEL Ag immunization – T cells recognize native and cit-HEL epitopes i.e. citrullination constitutive, extends range of epitopes

Membrane-HEL TG mice: all APC express HEL Hybridomas recognising HEL48-62, HEL 48-62cit 61 A-F Purified APC, incubated with each hybridoma G, H Primed non-TG mice with HEL protein

Demonstrate also that DC constitutively citrullinate during autophagy, B cells citrullinate under stress when autophagy induced

If also true in human, selection of cit-autoantigen-specific T cells and development of Treg could occur in the thymus

This would be the only way these T cells could be selected as no native peptide binding. Implications for cit-peptides derived from micro-organisms

Arthritogenic antigen

DC

Skin

Joint

Lymph node

_{Antigen-specific}

Regulatory T cells

DC

Martin E/Capini C et al: Arthritis Rheum 2007.

Delivery systems for tolerance: Generating regulatory T cells through

dendritic cells to suppress RA

• RelB-deficient DC, or DC generated in presence of NF-

k

B

inhibitor Bay11-7082 exposed to antigen suppress primed

immune responses.

• Induce CD4+ antigen-specific regulatory cells, IL-10

dependent. Martin E et al: Immunity 2003.

• POC antigen-induced arthritis

• Liposome containing curcumin and antigen

• Targets APC, including DC in vivo

– NF-

k

B inhibitor promotes Treg

– Cit-peptides provide specificity

Phospholipid bilayer entraps curcumin

Aqueous core entraps antigen

Schematic of product

Lipophilic phenolic natural product from Curcuma longa (turmeric)

Other antigen-specific strategies targeting dendritic cells for tolerance

Capini et al: J Immunol 2009.

DC targeted with curcusomes-Ag suppress arthritis, promote Ag-specific regulatory T cells

• Chimeric mAb-antigen

• Anti-DEC-205-OVA deleted CD8

• Mouse: DEC-205 not specific in

human

• Peptide immunotherapy

• Intact protein stimulates mast

cells, induces AB: peptide safer

• Much evidence for clinical

benefit using allergen extracts

• Fel-d1 cat allergen peptide s.c.

induced tolerance (phase 2)

• MBP peptide i.n. induces IL-10+

Treg

HLA-citrullinated peptide

250ml peripheral blood

Density gradient separation PBMCs Elutriation in a closed system Monocytes 48h culture in the CLINICell®25 with IL-4, GM-CSF and BAY11-7082 Tolerogenic autologous DCs

3h exposure to citrullinated peptides: cit-vimentin 447-455, cit-fibrinogen beta chain 433-441, cit-fibrinogen alpha chain 717-725, cit-collagen type II 1237-1249

Tolerogenic autologous DC specific for RA auto-antigens:

Rheumavax

Adapted from Berzofsky et al., 2001

Proof of concept phase I clinical trial of tolerising DC and

cit-peptides

i.d. injection upper thigh

Proof of concept phase I clinical trial Rheumavax: study design

Aims

Demonstrate safety of Rheumavax

Obtain evidence of mechanism

Phase I clinical data to describe effects in man

Major inclusions

HLA-SE+ ACPA+ RA any duration Treated by a rheumatologist Max prednisone 10mg

Major exclusions

Malignancy

Allergy (history and RAST test) Serious infection last 28 days

Primary outcomes

Safety Tolerance

Secondary outcomes

Efficacy

Swollen joints, tender joints, CRP and DAS

Groups

Rheumavax 1 million x 1 Rheumavax 5 million x 1. Control (no placebo)

dose

Screening, disease activity, blood, radiology

d2 1 mth

AE, disease activity, blood

2 mth 3 mth d6

Clinical, lab AE

AE, disease activity, blood

AE, disease activity, blood

6 mth

AE, disease activity, blood , radiology

Changes in joint counts and PB correlates month 1



Proof-of-concept: tolerising immuno-therapy using cit-peptides

and autologous dendritic cells in RA



Safe



Immune tolerance biomarkers



Efficacy signal

Summary

Environmental risk factors interact with genes e.g. smoking, stress, infection

Damage or inflammation subclinical Clinical onset Joint inflammation Treatment Control is major determinant of outcome Mortality Age (years) Birth Atherosclerotic disease Infection Lifestyle modification Innate immune dysregulation

Adaptive immune dysregulation

UA Autoantibodies

RF, ACPA Other biomarker

1 yr

Acknowledgements

Helen Pahau

Claire Hyde Shayna Street

Christelle Capini

Brendan O’Sullivan Emily Duggan

Mechanisms and liposomes

Suman Yekollu Karen Herd Hanno Nel

Roland Ruscher

Soi Law Srinivas Mutalik

Peptides and T cell responses Pt recruitment, cell production, database, stats Sanjoy Paul Tetramers and binding

studies: University of Melbourne, Monash University Stephen Scally Hugh Reid Jamie Rossjohn Kim Lau Anthony Purcell Nadine Dudek Rene Toes: LUMC

Manipal University

Marion Brunck Jennifer Ng Nigel Davies Uniquest commercial team:

Crossing the valley of death

Lisa Bidwell Don Kakuda Craig Belcher Dean Moss Victor Argeat

Peptide-specific ACPA: LUMC

Ellen van der Voort Leendert Troew Rene Toes