selective induction of activated regulatory t-cells in ... · • nktr-358 is also being evaluated...

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)








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




0 5 10 15

1

2

3

Day

Fold

chan

gefr

omba

selin

e

selective induction of activated regulatory t-cells in ... · • nktr-358 is also being evaluated...

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