selective induction of activated regulatory t-cells in ... · • nktr-358 is also being evaluated...
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Selective Induction of Activated Regulatory T-Cells in Healthy Volunteers by NKTR-358, a Novel IL-2 Conjugate Treg Stimulator, in Development for the Treatment of Autoimmune DiseasesC. Fanton, N. Dixit, S. Siddhanti, L. Lu, B. Kotzin, J. ZalevskyNektar Therapeutics, San Francisco, CA
Background Methods Results
Conclusions• Safe and well tolerated in this �rst in human study
• Marked dose-dependent expansion of numbers and proliferating CD25bright Treg cells, as demonstrated by �ow cytometric and epigenetic analyses
• The induction of Tregs is selective, with no measurable changes in numbers and percentages of CD4+ and CD8+ Tcons at all doses tested and low-level increases of NK cell numbers at highest doses tested
• Tregs induced by NKTR-358 are activated, as measured by �ow cytometry and RNA expression analyses
• Data provide strong support for studying NKTR-358 in autoimmune and in�ammatory diseases
• NKTR-358 is also being evaluated in a multiple ascending dose MAD study in patients with systemic lupus erythematosus (NCT03556007) and two additional Phase 1b studies in adults with psoriasis (NCT04119557) and atopic dermatitis (NCT04081350)
References1. Langowski J, et al. Arthritis Rheumatol. 2017;69 (suppl 10).2. Baron U, Eur J Immunol. 2007;37:2378.
*All authors are employees of Nektar Therapeutics and have ownership interest (including stock, patents, etc.) in the same. Study funded by Nektar Therapeutics.
IL-2 is Critical for Treg Expansion, Function and Control of Immune Responses by Regulatory T-cells (Tregs)
Many autoimmune disorders, including systemic lupus erythematosis (SLE), are associated with:• Reduced Treg numbers• Impaired Treg function• Reduced systemic IL-2
NKTR-358: PEG-conjugated rhIL-2 Selectively Induces Tregs and Their Suppressive Activity
Compared with IL-2, PEG-conjugation in NKTR-358: • Alters binding pro�le of NKTR-358 with lower binding af�nity to
IL-2Rβ and different binding bias for IL-2Rα & IL-2Rβ• Imparts selectivity for effect on Tregs over conventional T-cells
(Tcon)• Increases half-life
NKTR-358 has shown activity in animal models of SLE and cutaneous hypersensitivity1
Assay Methodology• Immunophenotyping by multicolor �ow cytometry to quantify multiple immune cell
subsets, using whole blood collected at multiple time points pre- and post-NKTR-358 administration
– Total Tregs: CD4+FoxP3+CD25+ total Tregs, evaluated as absolute numbers and percentage of CD4+ or CD3+ T cells
– CD25bright Tregs: Treg subpopulation with highest CD25 expression; expected to have highest suppressive capacity
– Ki67: marker of proliferation – ICOS, CTLA4, Helios: markers of Treg activation• Evaluation of epigenetic modi�cations conducted with Epiontis ID qPCR-based
assay, to monitor methylation status of Treg-speci�c demethylation region (TSDR) of FoxP3 gene, using whole blood collected at multiple time points from pre-dose through Day 50 post-dose
• Gene expression measured with NanoString platform, with whole blood collected at multiple time points from pre-dose through Day 15 post-dose
• Pharmacodynamic studies conducted to measure selective induction of Tregs and to further characterize their activity following NKTR-358 administration
• Assess the effects of subcutaneous administration of single-ascending doses of NKTR-358 in healthy volunteers on:
NKTR-358 was Safe and Well Tolerated in Healthy Volunteers• No dose-limiting toxicities, deaths, or AEs leading to study discontinuation• No clinically signi�cant vital sign, ECG, or physical examination abnormalities• Adverse events primarily limited to mild or moderate (Grade 1 or 2) injection site reactions • 4 subjects experienced Grade 1 mild events of headache • 1 subject at the highest dose tested (28.0 µg/kg) experienced mild (Grade 1) signs and symptoms of vomiting, diarrhea, anorexia,
tachycardia, and myalgia attributed to elevated cytokine levels• No anti-drug antibodies detected• NKTR-358 Cmax and AUC values demonstrated dose proportional increase, with maximal concentrations reached in 5-7 days, and
with an estimated half-life of 8-11 days
NKTR-358 Leads to Sustained, Dose-dependent Increases in Numbers and Proliferation (%Ki67+) of Total and CD25bright Tregs
Identification of NKTR-358-induced Tregs Supported by Correlation with Demethylation Status of FoxP3 Gene
• At 28 µg/kg NKTR-358: – 3.5-fold mean peak increase (above predose levels) in numbers of total Tregs and 17-fold mean peak increase in numbers of
CD25bright Tregs, suggesting a large increase in most suppressive Treg population – Treg levels peak at Days 10-12 and do not return to baseline until Days 20-25 following administration – 6-fold mean increase in Ki67+ CD25bright Tregs above predose value
• Published studies have demonstrated that an epigenetically active (demethylated) FoxP3 gene is observed solely in Tregs and not in activated, conventional (non-Treg) CD4+ T cells2
– FoxP3 expressed transiently in activated conventional T cells – Constitutive FoxP3 expression in Tregs regulated at
epigenetic level by demethylation of TSDR in FoxP3 gene
• After treatment with 28 µg/kg NKTR-358, signi�cant correlation observed between NKTR-358-induced Tregs identi�ed by �ow cytometry (CD4+CD25+FoxP3+ cells) and Tregs identi�ed by epigenetic analysis (% demethylation of FoxP3 TSDR)
• Increase in number and magnitude of differentially expressed genes in response to NKTR-358 administration• 84 genes identi�ed that show consistent trend of dose-dependent changes in expression in response to NKTR-358 administration• NKTR-358-dependent differential expression of 13 genes signi�cantly correlated with induction of Tregs as measured by �ow
cytometry
• Sustained increase in percentage of Treg activation markers CTLA4+ and ICOS+ at 20 and 28 µg/kg NKTR-358• Increase in Helios expression (MESF) on Total Tregs at highest dose of NKTR-358• Preliminary results indicate suppressive activity observed in ex vivo Treg suppression assay performed with whole blood collected
from a limited number of subjects at 8-11 days post-NKTR-358 administration (data not shown)
IL-2RβIL-2Rγ
IL-2RαNKTR-358
CTLs
CD8+ T-Cellsand NK Cells
NKTR-358
Stimulates ImmuneResponse to Kill
Tumor Cells
Tregs
Down-Regulates Proliferation of
Conventional T-Cells
Stimulated Tregs with Potential for
Long Term Control ofImmune Response
βγ αβγ
Selectively ExpandsTreg Cells
CD4+ Regulatory T-Cells
Study Design: Randomized Double-blind Study of Subcutaneous Single-Ascending Doses (SAD) of NKTR-358 in Healthy Volunteers
Study Subjects Follow-up Completed
HealthyVolunteers
(N=100)Age: 18 – 54 yrs
Males: 60%Females: 40%
Each cohort followedfor 50 days
Coho
rt 7
Coho
rt 2
Coho
rt 3
Coho
rt 4
Coho
rt 5
Coho
rt 6
Coho
rt 1
Coho
rt 8
NKTR-358 0.3 µg/kg (n=9)Placebo (n=3)
NKTR-358 1.0 µg/kg (n=9)Placebo (n=3)
NKTR-358 3.0 µg/kg (n=9)Placebo (n=3)
NKTR-358 6.0 µg/kg (n=9)Placebo (n=3)
NKTR-358 9.0 µg/kg (n=9)Placebo (n=3)
NKTR-358 13.5 µg/kg (n=9)Placebo (n=3)
NKTR-358 28.0 µg/kg (n=9)Placebo (n=3)
NKTR-358 20.0 µg/kg (n=13)Placebo (n=3)
Study Objectives
• Safety and tolerability in subjects as evaluated by:
– Adverse events – Vital signs – Clinical safety lab results – Cytokine levels
• Time course and extent of changes in the numbers and activity of Tregs, Tcons, T cell subsets, and NK cells
• Pharmacokinetics (PK) of NKTR-358
• Other immunological effects: cytokine levels, peripheral blood cell populations,serum proteins and gene expression
Primary Secondary
0 10 20 300
10
20
30
40
50
Day
CD
25br
ight T
regs
, cel
ls/µ
l
0 10 20 300
20
40
60
80
100
Day
Tota
l Tre
gs, c
ells
/µl
0 10 20 300
10
20
30
40
Day
% o
f CD
25br
ight
Tre
gs(m
ean
+/- S
EM)
Absolute Numbers ofCD25bright Tregs
Proliferation (%Ki67+) of CD25bright Tregs
Helios MESF on Total Tregs
Absolute Numbers ofTotal Tregs
Correlation of % Total Tregs and % Demethylated FoxP3 Gene at 28 µg/kg
Pattern of Differential Expressionof 13 Correlated Genes at 28 µg/kg Dose-dependent Changes in 2 Genes Correlated with Treg Induction
IDO-1 CD38
NKTR-358 Increases Expression of Treg Activation Markers
Genes Associated with Treg Regulation Show Correlation with Treg Induction
%ICOS+ Total Tregs %CTLA4+ Total Tregs
0 10 20 300
2
4
6
8
10
12
Day%
ICO
S+ T
regs
fold
cha
nge
from
bas
elin
e
0 10 20 300.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
Day
Hel
ios
MES
F (T
regs
)fo
ld c
hang
e fr
om b
asel
ine
0 10 20 300
1
2
3
4
Day
%C
TLA
4+ T
regs
fold
cha
nge
from
bas
elin
e
0 5 10 15 200
5
10
15
20
%CD4+FoxP3+CD25+ of CD3+ T cells(flow cytometry)
%Fo
xP3
TSD
Rof
CD
3+T
cells
(Epi
gene
ticas
say)
Spearman r = 0.647 (n=76)p-value <0.0001
Each data point represents an individual blood sample from 9 NKTR-358-treated subjects where data was available for both flow cytometry and demethylation assays
Transiently induced
Sustained induction
Repressed
0 5 10 150
2
46
8
10
12
Day
Fold
cha
nge
from
bas
elin
e
L
No Changes in Numbers of Tcon Cells and Low-level Increases in Numbers of CD56+ NK Cells in Response to NKTR-358
Mean Fold Change of CD4+ Cells
0 10 20 300
1
2
3
4
5
Day
CD
4+T
cells
(cel
ls/u
l)m
ean
fold
cha
nge
from
pre
dose
Poster #0098
Mean Fold Change of CD8+ Cells
0 10 20 300
1
2
3
4
5
Day
CD
8+T
cells
(cel
ls/u
l)m
ean
fold
cha
nge
from
pre
dose
Mean Fold Change of CD56+ NK Cells
0 10 20 300
2
4
6
8
10
Day
CD
56+
NK
cel
ls (c
ells
/ul)
mea
n fo
ld c
hang
efr
om p
redo
se
TIGIT
Day
Day of administration6 µg/kg3 µg/kg1 µg/kgPlacebo 13.5 µg/kg 20 µg/kg 28 µg/kg
Day of administration6 µg/kg3 µg/kg1 µg/kgPlacebo 13.5 µg/kg 20 µg/kg 28 µg/kg
Day of administration6 µg/kg3 µg/kg1 µg/kgPlacebo 13.5 µg/kg 20 µg/kg 28 µg/kg
Day of administration6 µg/kg3 µg/kg1 µg/kgPlacebo 13.5 µg/kg 20 µg/kg 28 µg/kg
0 5 10 15
1
2
3
Day
Fold
chan
gefr
omba
selin
e