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DEVELOPMENT*OF*GLIADIN*CONTAINING*TOLEROGENIC*IMMUNE*MODIFYING*NANOPARTICES*FOR*CELIAC*DISEASE*
Daniel$R.$GeQs,$Charles$B.$Smarr,$Derrick$McCarthy,$Marcel$$Messing,$$Leif$C.$Andersson,$Nicholas$J.C.$King,$Stephen$D.$Miller,$Lonnie$D.$Shea$,$Seppo$Meri,$Tobias$L.$Freitag$$
1th$Interna-onal$Celiac$Disease$Symposium,$Prague$2015$
1.$Abstract$
3.3$COUR1NP1GLI$successfully$inhibits$DTH$in$an$an-gen$specific$fashion$
In$ celiac$ disease$ (CD),$ tolerance$ to$ gluten$ proteins$ from$ cereals$ is$ lost.$ Toleragenic$ Immune$ Modifying$
nanoPar-cles$ (TIMP)$ are$ poly(lac-de1co1glycolide)$ that$ contain$ autoreac-ve$ protein$ or$ pep-de$ epitopes.$ These$
nanopar-cles$ have$ been$ shown$ to$ induce$ immune$ tolerance$ in$ numerous$ autoimmune$ condi-ons.$ The$
iden-fica-on$of$gliadins$as$the$primary$epitopes$in$celiac$disease$suggest$that$TIMP$containing$gliadin$may$serve$as$
a$tool$to$induce$tolerance$to$gluten$and$poten-ally$cure$CD.$Here$we$developed$gliadin$containing$TIMP,$referred$
to$ as$ COUR1NP1GLI,$ and$ examined$ its$ safety$ and$ ability$ to$ induce$ immune$ tolerance$ in$ a$ delayed$ type$
hypersensi-vity$(DTH)$$and$CD$animal$model$sekng.$
A$
• 
Presently,$there$is$no$cure$for$CD$
BLOOD
BLOOD
SPLEEN/LIVER
Gliadin$specifIc$T$cells$are$the$primary$cause$
TIMPs bind to monocytes
Antigen Presenting Cells (APC)
Control of Autoreactive
Peripheral
– divert to spleen
take up & process TIMP
T cells
T cell
of$ pathology$ in$ CD$ pa-ent1$ immune* Proprietary
surface
regulation
modification:
ζ = < 50 mV
Disease
tolerance*is*the*ideal*treatment*for*CD*
SPLEEN
LIVER
specific
autoreactive
The$ induc-on$ of$ tolerance$ using$ TIMP$
antigen
requires$ that$ the$ Gliadin1loaded$ par-cles$
- -Deletion
- 500 nm
must$be$delivered$intravenously.$
The$ par-cles$ are$ then$ taken$ up$ via$
MARCO
TIMP
Anergy
scavenger$ receptor$ mediated$ processes,$
1
MHC TCR
with$ MARCO$ shown$ to$ be$ a$ primary$
APC
receptor$media-ng$uptake$and$down$stream$
PD1
tolerance$induc-on.$$
Monocyte
PD-L1
TFG$β%%
IL$10%
MARCO$ is$ expressed$ on$ circula-ng$
2
3 CD28
inflammatory$ monocytes,$ as$ well$ as$
Apoptosis
Antigen Presentation
Immune Tolerance
•  Apoptosis of TIMPmarginal$zone$macrophages.$Based$on$TIMP$
1 Autoreactive antigen is:
2 T cell deletion or anergy from:
monocytes in the spleen/
•  processed by APCs
•  negative co-stimulation
liver
size$(500nm)$phagocytosis$is$likely$to$be$the$
•  presented to T cells
•  lack of positive stimulation
•  TIMP and apoptotic
T cells get:
debris picked up by
primary$ uptake$ mechanism.$ Upon$ uptake,$
•  negative co-stimulation – PD-L1
3 IL-10 and TGF-β induce Tregs
antigen presenting cells
•  anti-inflammatory milieu (IL-10 /
•  maintain tolerance
(APCs) in spleen/liver
TGF-β)
these$macrophage$popula-ons$produce$IL10$
Figure*1.*TIMP*Tolerance*InducFon$
and$TGF.$
When$ integrated$ these$ APC$ responses$ co1ordinate$ the$ regula-on$ of$ auto1reac-ve$ T$ cells$ via$ three$ predominate$
pathways:$
1.  Naïve$T$cell$anergy$and$dele-on$results$from$signal$1$in$the$absence$of$signal$2$
2.  Immune$regula-on$of$ac-vated$T$cells$is$the$result$of$nega-ve$co1s-mula-on,$T$cell$anergy$and$dele-on$
3.  Conversion$and$expansion$of$effector$T$cells$and$naïve$T$cells$to$Treg$results$in$tolerance$maintenance$
Naïve
T Cell
Naïve
T Cell
Naïve
T Cell
Autoreactive
T cell
CPM
**
40
*
5000
20
0
3.4$COUR1NP1GLI$induce$robust$tolerance$in$rodent$model$of$celiac$disease$
Model$of$gluten$induced$enteropathy$
U-lized$to$examine$the$pathogenesis$of$CD$
First$-me$used$to$examine$an-gen$specific$tolerance$
COUR1NP1GLI$ability$to$induce$tolerance$was$examined$afer$two$
infusions$(day$7$and$day$14$post$T$cell$transfer)$
•  Body$weight,$histology$and$cytokines$were$examined$as$efficacy$
measures$
• 
• 
• 
• 
Figure*5.*Mouse*model*of*glutenG
s e n s i F v e * e n t e r o p a t h y .$$
CD4+CD45RBlow$ CD252$ T$ cells$
from$ gliadin1immunised$ donors,$
were$transferred$into$$Rag1/1$mice.$
R e c i p i e n t s$ w e r e$ e i t h e r$
maintained$on$GFD$(gliadin/GFD),$
or$ challenged$ with$ gluten$
(gliadin/gluten,$ ovalbumin/
gluten).$$
3.5$COUR1NP1GLI$showed$an-gen$specific$disease$modifica-on$on$all$end$point$measures$
A$
Ameliora-on$of$weight$loss$
B$
Reduc-on$in$histopathology$
C$
Reduced$IFN1γ$
D$
Reduced$IL17$
iTreg
Naïve
T Cell
Naïve
T cell
later$ears$were$challenged$with$10ug$gliadin$an$ear$swelling$(A)$
and$ ex, vivo$ T$ cell$ prolifera-on$ was$ examined$ (B).$ Animals$
receiving$ TIMP1OVA$ control$ par-cles$ prior$ to$ immunisa-on$
with$gliadin$developed$robust$ear$swelling.$In$contrast,$animals$
treated$with$COUR1NP1GLI$showed$a$significant$reduc-on$in$ear$
swelling$ (*p<0.05).$ Treatment$ also$ resulted$ in$ a$ reduc-on$ of$
splenic$ T$ cell$ prolifera-on$ in$ response$ to$ gliadin$ res-mula-on$
ex$vivo$(*p<0.05).$$
Naïv
T Ce e
ll
iTreg
• 
Naive
either$with$NP1COUR1GL,$TIMP1OVA$(irrelevant$an-gen$specific$
OVA TIMP (n=5) control)$or$nothing,$as$described$in$Fig$3.$TIMP1OVA$and$COUR1
NP1GLI$ were$ subsequently$ immunized$ with$ gliadin.$ 10$ days$
COUR-NP-GLI
10000
60
0
Naïve
T Cell
• 
80
Figure* 4.* COURGNPGGLI* inhibit* DTH* response* and* T* cell*
proliferaFon.$ Three$ groupd$ of$ 5$ Balb/c$ mice$ were$ treated$
Spleen Restimulation
Naive
15000
OVA TIMP (n=5)
COUR-NP-GLI
-4
Ear Swelling (x10 inches)
2.$Introduc-on$
2.1$Toleragenic$Immune$Modifying$Nanopar-cles$
• 
• 
B$
Mean Ear Swelling
100
iTreg
Naïve
T Cell
Naïv
T Ce e
ll
iTreg
iTreg
• 
$
2.2$Development$of$the$COUR1NP1GLI$Tolerizing$Nanopar-cle$
A$ COUR1NP1GLI$
B$
Gliadins(
The$Par-cle$
C$ PLGA$with$proprietary$surface$modifica-ons$
The$An-gen$
Iden+fica+on(of(gliadin1binding(pep+des(by(phage(display,(Tingsu(et#al#
Proteomic(anlysis(of(wheat(Gliadins(Dziuba,(et#al(
((
(
10
Figure* 2.* COURGNPGGLI* nanoparFcle* development.* (A)$ A$
schema-c$ of$ the$ gliadin$ containing$ nanopar-cle.$ (B)$ SDS1
Page$ and$ western$ blot$ of$ giadins.$ (C)* COUR1NP1GLI$ is$
derived$from$PLGA,$a$regulaotry$body$approved$biodegrable$
inert$polymer.$**$$
3.$Results$
•  Synthesized$ using$ double$ emulsion$
technique$
•  Incorpora-on$ of$ PEMA$ to$ enhance$
surface$charge$
•  An-gen$load$~2.9110ug/mg$of$PLGA$
•  An-gen$fully$encapsulated$for$safety$
•  Lyophilized$vials$
•  An-gen$ composi-on$ determined$ by$
mass1spectrometry$
•  All$ immunodominate$ epitopes$ described$
in$the$literature$detected$
•  O v a l b u m i n$ a n d$ L y s o z y m e$ T I M P$
generated$as$controls$
$
Figure*6.*COURGNPGGLI*treatment*effect*vs.*TIMPGLYS*treatment*control*in*a*CD*mouse*model,*and*vs.*GFD*or*GLUTEN*dietary*controls.*$(A)$body$weight$development$and$(B)$histological$duodeni-s$
score.(C*&*D)*T$cell$cytokine$secre-on$in$response$to$gliadin$res-mula-on$of$spleen$cells$ex,vivo.$$***p<0.001,$*p<0.05).$$
4.$Conclusions$
•  The$ results$ support$ the$ therapeu-c$ concept$ of$ re1establishing$ T$ cell$ tolerance$ to$ gluten$ in$ CD$ pa-ents.$
Importantly,, gluten, tolerance, induc3on, carries, cura3ve, poten3al,, in, contrast, to, most, other, novel, treatments,
for,CD,that,are,designed,as,adjunc3ve,therapies,,
1. 
2. 
3. 
4. 
COUR1NP1GLI$are$safe$and$effec-vely$induced$immune$tolerance$in$models$of$delayed$hypersensi-vity$and$celiac$disease.$$
Treatment$restored$tolerance$to$gluten$and$allowed$for$a$complete$gluten$containing$diet$to$be$consumed$
An-gen$presen-ng$cells$capable$of$libera-ng$epitopes$from$Gliadin,$meaning$that$exquisite$epitope$iden-fica-on$is$not$necessary$
COUR1NP1GLI$contain$no$other$immune$suppression$agents$(eg$Rapamycin)$and$do$not$depend$on$red$cell$apoptosis.$!
5.$Clinical$transla-on$
3.1$Physiochemical$characteris-cs$of$COUR1NP1GLI$
Protein*encapsulated*
(ug/g*PLGA)*
ζ*average*(nm)*
PDL*
Peak*(nm)*
Ζ*potenFal*(mv)*
Gliadin$
3$
579.6$
0.238$
568.6$
149.1$
Lysozyme$
9$
392.8$
0.213$
474.1$
165$
Ovalbumin$
25$
378.2$
0.246$
411.8$
162.4$
AnFgen*
3.2$Tes-ng$in$delayed$hypersensi-vity$model$(DTH)$$
NP1COUR1GLI$
Immuniza-on$
Re1challenge$
Figure*7.*Phase*2*COURGNPGGLI*clinical*trial*schemaFc$
6.$References$
Day$17$
Day$%7$tolerance$
COUR1NP1GLI$
Gliadin'Par*cles'made'with'TFA'
Day$11$
Day$0$
Day$%1$tolerance$
Day$0$Immuniza4on$
COUR1NP1GLI$
Immuniza-on$
Gliadin'Par*cles'made'with'TFA' Immuniza*on'with'Gliadin'
dissolved'in'TFA'
Average'zeta'poten*al=;48.6'
Average'size'605.6nm'
Figure*3*.*TesFng*of*NPGCOURGGLI*in*delayed*type*hypersensiFvity*model
$
dissolved'gliadin''
dissolved'gliadin''
Average'zeta'poten*al=;48.6'
Average'size'605.6nm'
Day$10$
Day$10$DTH$
Rechallenge$
Challenged'with'gliadin'
dissolved'in'50mm'ace*c'acid'
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4.  Freitag,,et,al.,$Gut,$2009$
5.  GeQs,$et,al.,,Journal$Experimental$Medicine,$2008$
6.  GeQs,$et,al.,,Nature$Biotechnology,$2012$