Blake Rust1, Veronique Bajzik1, Kelly Aldridge1, Kavitha Gilroy1, Brian Vickery2, Erik Wambre1

INTRODUCTION

1 Translational Immunology Division, Benaroya Research Institute at Virginia Mason, Seattle, Washington, USA2 Aimmune Therapeutics, Brisbane, California, USA

METHODS

CONCLUSIONS

Funding: Aimmune grant, ITN grant, NIH/NIAID grant R01 AI108839 and Food Allergy Research & Education grant 0728101

Dual assessment of peanut-specific effector and regulatory T cells

in patients undergoing oral immunotherapy: A preliminary review of ARC003

Blinded samples to the operator are provided during a randomized 3:1, double blind, placebo

controlled Phase 3 ongoing trial (ARC003) of the efficacy and safety of AR101 in a characterized

desensitization (CODIT™) approach in patients with peanut allergy (Figure 1). Eligible subjects, 4-55

years of age, reacted to ≤ 100 mg peanut protein during a screening double blind Placebo controlled

food challenge (DBPCFC). To measure basophil allergen threshold sensitivity, basophils are

stimulated in vitro with increased dilution of peanut crude extract until threshold sensitivity was

reached based on detection of the activation marker CD203c. The magnitude and quality of peanut

specific effector and regulatory T cell responses are determined ex vivo using both CD154 and an

adapted CD137 upregulation assay respectively, following short restimulation of PBMCs with a pool of

peanut peptides library derived from Ara h 1, h 2, h 3, h 6 and Ara h 8. Following bead-enrichment, the

CD154+ and CD137+ enriched fraction are separately stained with various combinations of surface

and intra-nuclear antibodies for phenotyping using 16 color flow cytometry platform.

Peanut allergy is one of the most serious hypersensitivity reactions to foods in terms of persistence and severity. Currently, there is no approved treatment to address the growing prevalence of severe peanut allergies, leaving these

patients at risk of life-threatening anaphylaxis upon accidental exposure to peanuts. To address this need, food oral immunotherapy (OIT) has been investigated as a treatment and potentially disease modifying approach. OIT is the

process of gradual increases in antigen ingestion until relevant quantities of foods that correspond to accidental exposure can be consumed without life threatening symptoms. While peanut oral immunotherapy has demonstrated

encouraging safety and efficacy as a desensitization therapy for peanut allergy, little is known about corresponding immune responses. It is generally accepted that enumeration and characterization of antigen-specific T cells provide

essential information about the potency of the immune response and can serve as useful biomarker. The clinical application of food immunotherapy would greatly benefit from the development of reliable immune-monitoring assays

that could address the complexity and functional heterogeneity of food allergy. The aim of this study is to evaluate the frequency and phenotypic heterogeneity of peanut specific effector and regulatory CD4+ T cells at baseline in a

subset of peanut allergic patients undergoing characterized oral desensitization immunotherapies (CODIT) with AR101, an experimental orally administered biological drug containing the antigenic profile found in peanuts. This dual

assessment of peanut-specific effector (Teff) and regulatory T cells (Treg) as a potential to provide essential information determining which patients are most likely to tolerate up-dosing and which patients are at risk for developing GI

symptoms associated with early discontinuation from AR101 therapy thereby helping to reveal the best course of treatment for the individual patient.

Initial Escalation

0.5 mg

3mg

Day 1

12 mg20 mg

240 mg

200 mg

160 mg120 mg

80 mg40 mg

6 mg

Highest

Eliciting Dose:

Secondary Endpoint:

Primary Endpoint:Draw 1

(n=55)

Draw 2

(n=28)

Draw 4 Draw 5Draw 3

300mg

Figure 1. Study design of Phase 3 trial (ARC003). Characterized oral

desensitization IT (CODIT TM) is Aimmune's approach to provide a

systematic, validated and optimized OIT protocol. This therapeutic

strategy involves the gradual introduction of precisely controlled doses of

antigenic protein at regulated intervals. During this ongoing phase 3 trial

with AR101, patients receive 3 mg of protein on day 1, and subsequently

spend two weeks at each double dose level before eventually reaching a

maintenance dose of 300 mg daily. A blind placebo control food challenge

(DBPCFC) with peanut flour is used pre and post therapy to determine

which subjects have been successfully desensitized. During this

randomized, double-blinded, placebo-controlled trial, coded samples from

subjects providing additional consent for these studies are provided at

baseline and at the end of the maintenance both pre- and post- DBPCFC,

and at the end of the up-dosing dose.

Here we have used methods for parallel ex vivo assessment of peanut-specific effector and regulatory CD4+ T cells to provide meticulous immune-monitoring of patients receiving patients receiving AR101 or placebo through the

CODIT approach, in order to evaluate induced immune responses and allow for the optimization of the treatment strategy. At baseline, peanut allergic patients (positive during DBPCFC) had significantly higher sensitivity values to

peanut extract compared with those patients who did not react to a 100 mg DBPCFC (Figure 2b). The absence of a clinical response in DBPCFC non-reactors patient was accompanied by a significantly lower number of peanut–

reactive effector CD4+ T cells (Figure 2c). These cells also displayed a predominantly CD27+ CRTH2- memory phenotype consistent with protective immune responses observed in non-allergic individuals (Figure 2d). Surprisingly,

subjects who did not react to the entry DBPCFC also exhibit a significantly lower frequencies of peanut-specific regulatory T cells as compared to their counterparts who reacted to the oral challenge (Figure. 3). In all peanut

allergic subject enrolled in ARC003 trial, the DBPCFC protocol at baseline led to significant increased expression of the cell-surface activation marker CD38 within peanut-reactive T cells (Figure 2e), confirming their role in food

allergic pathogenesis. All together, the data suggest a model wherein a pathogenic subpopulation of peanut-specific CD4+ T cells possessing a distinct feature in effector function are generated in vivo in atopic individuals and are

crucial for allergic pathogenesis, regardless of the balance of T helper cell subsets. Our data at baseline of the ARC003 trial also emphasize the heterogeneity of allergen-reactive effector T cell responses in peanut allergic

subjects, with two mutually exclusive immunotypes associated with food allergy (Figure 4). This raises important questions regarding the pathophysiological role of each peanut specific CD4+ T cell subset in food allergy and

suggests that cellular phenotypes may indicate or predict clinical treatment outcomes following CODIT in subjects with peanut allergy. These immunotypes are likely the result of different immunologic mechanisms and therefore

may require different immunotherapeutic approaches to bring about resolution. These biomarkers are currently being examined prospectively during the Aimmune Phase III (ARC003) trial to determine if it can be used to help

predict which patients are most likely to benefit from this investigational therapy, and which immune mechanisms are involved in the response and screen out those candidates in whom OIT may lead to unnecessary risks. .

RESULTS

DBPCFC

non-reactors

10-2

10-4

10-6

10-8

Ba

so

ph

il s

en

sit

ivit

y t

o p

ea

nu

t

ex

tra

ct

dilu

tio

n

*b) c)

Pe

an

ut

sp

ec

ific

eff

ec

tor

CD

4+

T

ce

lls

pe

r 1

06

CD

4+

me

mo

ry T

ce

lls e)d)

CD27 CRTH2 CCR4 CCR6

% p

ea

nu

t e

ffe

cto

r C

D4

+ T

ce

lls

Figure 4: Two distinct immunotypes are

defined by mutually exclusive CRTH2 and

CCR6 expression within peanut-reactive

effector T cells in DBPCFC reactors peanut

allergic patients. a) Representative dot plot

examples showing expression of CCR4, CRTH2,

CD27 and CCR6 in ex vivo enriched peanut-

reactive effector CD4+ T cells between a

DBPCFC reactor patient with a TH2A high

immunotype and a DBPCFC reactor patient with

TH2A low immunotype. Immunotypes were

defined according to proportion of peanut-reactive

effector T cells expressing CRTH2 or CCR6 within

an individual. b) Percentage of peanut-reactive

effector T cells expressing CRTH2 or CCR6 from

DBPCFC reactors peanut allergic individual are

plotted by scatterplot. Th2A-low and Th2A-high

patterns are inversely correlated. Each data point

represent a single ARC003 patient. Clustering

CRTH2 and CCR6 percentage was utilized to

classify subjects as CRTH2+ dominant (Th2A-

high, red) and CCR6+ dominant (Th2A-low, blue).

c) Representative CCR6 and CRTH2 staining and

profile of peanut reactive effector T cells in an

DBPCFC reactors peanut allergic patient. Integrin

β7 is used as a gut-homing marker.

b)

% p

ea

nu

t e

ffe

cto

r C

D4

+ T

ce

lls

Pre-OFC Post-OFC

CD38

a) DBPCFC reactor peanut allergic : TH2A-high Immunotype

DBPCFC reactor peanut allergic : TH2A-low Immunotype

CCR4

CD

15

4

CRTH2 CD27 CCR6

CCR4

CD

15

4

CRTH2 CD27 CCR6

Figure 3: Peanut allergic individuals exhibit

low frequencies of peanut-specific regulatory

T cells at baseline but higher than DBPCFC

non-reactors patients. Representative dot plot

examples showing expression of CD127, CD25,

Foxp3, Helios, CTLA-4 and CD27 on ex vivo

enriched peanut-reactive effector T cells (green)

and peanut-reactive Tregs cells (red) using dual

CD154 and adapted CD137 assay during

ARC003 mechanistic study. b) Peanut-reactive

regulatory CD4+ T cell frequencies between

groups at baseline. Differences between groups

were analyzed by using the Mann-Whitney U test.

**P < .005.Pe

an

ut

sp

ec

ific

re

gu

lato

ry C

D4

+

T c

ells

pe

r 1

06

CD

4+

me

mo

ry T

ce

llsb)

TH2A-low

Immunotype

TH2A-high

Immunotype

Figure 2: Differentiation between DBPCFC reactors patients with peanut allergy (PA) and those with peanut sensitization who did not react to the 100 mg DBPCFC. a) Representative flow cytometric analysis of the ex vivo enriched peanut-reactive effector CD4+ T cells during ARC003 trial. b)

basophil allergen threshold sensitivity test to peanut crude extract between groups at baseline. c) Peanut-reactive effector CD4+ T cell frequencies between groups at baseline. d) Statistical summary showing the percentage of CD27, CRTH2, CCR4 and CCR6 on ex vivo enriched peanut-reactive effector

CD4+ T cells between DBPCFC non-reactors (n=12) and DBPCFC reactors patient (n=16) at baseline. e) Percentage of peanut-reactive CD4+ T cells expressing the cell surface marker of activation CD38 between groups. b, c and d) Whiskers in box plots indicate maximum and minimum values

measured. Line indicates the median. Differences between groups were analyzed by using the Mann-Whitney U test. **P < 0.005. e) Differences between groups were analyzed by using the Wilcoxon matched-pairs test *P < 0.005.

DBPCFC reactors*

CCR4

CD

15

4

CRTH2 CD27 CD161 CCR6 CD38 Integrin β7 CD49b LAG3CD45RACD4

a) 5%44%26%41%18%60%38%31%84%6%99%

Peanut-specific Treg

CD

15

4

CD127 CD25 Foxp3 Helios CTLA-4 CD27Pea

nu

t-s

pec

ific

Tre

gC

D1

54

Within CD4+ CD45RA- T cells

a) 68% 54% 1% 70% 87% 6%

6% 100% 92% 90% 92% 95%

86% 75% 20% 2%

65% 5% 69% 56%

DBPCFC

non-reactors

DBPCFC

non-reactors

DBPCFC

reactors

DBPCFC

reactorsDBPCFC

reactors

DBPCFC non-reactors