NKTR-255: an IL-15-based therapeutic with optimized biological activity
and anti-tumor efficacy
Peter Kirk1, Murali Addepalli1, Thomas Chang1, Ping Zhang1, Marina Konakova1, Katsunobu Hagihara2, Steven Pai2, Laurie VanderVeen1, Palakshi Obalapur1, Peiwen Kuo1, Phi Quach1,
Mekhala Maiti1, Christie Fanton1, Takahiro Miyazaki1, Poorna Chandra1, Arunasree Lanka1, Ravi Nutakki1, Lawrence Fong2, Deborah Charych1, Jonathan Zalevsky1
Nektar Therapeutics, San Francisco, California; 2Helen Diller Family Comprehensive Cancer Center, UCSF, San Francisco, California
Results
Mice received a single i.v. dose of 0.3 mg/kg NKTR-255 (left) or IL-15 (right), then STAT5
phosphorylation in lymphocyte subpopulations from whole blood was assessed by flow cytometry.
0
1.49 x 10-4
0.26
NKTR-255
8.29x104
1.62 x 10-4
1.96
• Retention of binding to IL-15Rα is an important determinant of the biological activity of
NKTR-255
• Surface Plasmon Resonance was used to determine the binding kinetics and affinity
of NKTR-255 for IL-15Rα
Results
80
IL-15
Test Article
NKTR-255
60
40
20
0
-12
-11
-10
-9
-8
-7
log Conc (g/mL)
-6
EC50 (ng/ml)
EC50 (pM)
IL-15
0.27
21
NKTR-255
5.6
430
CTLL-2 cells were treated with test article
for 10 minutes, then cells lysed and STAT5
phosphorylation assessed using a
MesoScaleDevice immunoassay.
10000
1000
100
10
1
0.1
IL-15 Conjugate 1, IV, Plasma_conc
IL-15 Conjugate 2, IV, Plasma_conc
IL-15, IP, Plasma_conc
0
10000
1000
16 32 48 64 80 96
Time (hr)
IL-15 Conjugate 1, IV, Plasma_conc
IL-15 Conjugate 1, IV, Tumor_conc
IL-15 Conjugate 2, IV, Plasma_conc
IL-15 Conjugate 2, IV, Tumor_conc
IL-15, IP, Plasma_conc
IL-15, IP, Tumor_conc
1
0
16 32 48 64 80 96
Time (hr)
• NKTR-255 achieves >100x greater tumor exposure for the same dose
Vehicle
NKTR-255
40
20
0
0
100
Hours
200
Vehicle
Ki-67
300
Vehicle
NKTR-255
1500
NK cells per µL
60
CD8:Treg Ratio – Spleen
8
0
100
Hours
200
1500
1000
500
0
20
40
60
80
Days post-implantation
n
4
2
0
0.06
Vehicle
CD8:Treg Ratio – Tumor
30
20
10
0
NKTR-255
**
0.02
NKTR-255
Vehicle
NKTR-255
CD8+IFNγ+TNFα+ – Lymph Node
0.020
CD8+TNFα+IFNγ+ – Spleen
0.04
*
0.015
0.010
0.005
0.000
Vehicle
NKTR-255
NKTR-255 reduces tumor burden in CT-26 and B16F10 lung
metastasis models
CT-26 Lung Metastasis
in Balb/c
100
***
50
Vehicle
**
100
***
Vehicle
****
IL-15 NKTR-255
CT-26 Lung Metastasis
in Balb/c
60
40
***
20
0
IL-15 NKTR-255
150 B16F10 Lung Metastasis
in C57BL/6 Mice
50
80
100
80
Vehicle
IL-15
**
NKTR-255
B16F10 Lung Metastasis
in C57BL/6 Mice
60
40
***
*
IL-15
NKTR-255
20
0
Vehicle
300
Discussion
NKTR-255
CD122
2000
Tumors injected i.v. on day 0. NKTR-255 dosed i.v. at 0.3 mg/kg on d1, 5, 10; IL-15 dosed at 0.3 mg/kg
i.p. on d1,2,3,4,5,8,9,10,11,12. Metastases counted on d13
500
0
*
6
0
1000
80
NKTR-255 treatment results in reduced tumor growth rate, an increase in the CD8:Treg ratio in
spleen and tumor, and an increase in the frequency of IFNγ+/TNF+ CD8 T-cells
0
A single i.v. dose of NKTR-255 induces expression of proliferation marker Ki-67 on NK cells
peaking at 72hr, with a corresponding increase in frequency of NK cells in blood. Expression of
NK activation marker CD122 was also increased – histograms shows 72 hour timepoint.
60
Vehicle
NKTR-255
-500
NKTR-255 induces NK cell proliferation and activation
NK % Ki-67+
NKTR-255 binds to IL-15Rα, and induces STAT5
phosphorylation in CTLL-2 cells with subnanomolar
EC50. Following intravenous administration, NKTR-255
demonstrates a greatly reduced clearance rate
compared to IL-15, with plasma t1/2 of 22-26h versus
<1h for IL-15. Tumor exposure of NKTR-255 was 50-fold
greater than IL-15 in B16F10-bearing C57/Bl6 mice and
110-fold greater in CT-26-bearing Balb/c mice.
Immunophenotyping studies in normal mice showed an
induction of Ki-67 and CD122 expression in NK cells,
indicating proliferation and activation. In tumor-bearing
mice, NKTR-255 treatment resulted in an increased
CD8:CD4 and CD8:Treg ratio in tumor and spleen, and
an increased frequency of CD8+TNF+IFNγ+ T-cells.
Tumor growth inhibition was observed in both CT-26
and TRAMP-C2 models.
NKTR-255 (n=10)
150
• Unmodified IL-15 is cleared rapidly, so frequent administration of a high dose is required
to achieve therapeutically relevant exposure
Day 47
Immune monitoring
(Spleen, LN, Tumor)
Vehicle (n=6)
Vehicle
10
0.1
Day 7 Day 12 Day 17
Day 0
0.00
100
-20
40
Hours
2500
Pharmacokinetic data are shown for IL-15 and two PEG-IL-15 conjugates with linkers tuned to
provide different PEG release kinetics. All were administered i.v. at 0.5 mg/kg.
B16F10 Tumor-bearing Mice
20
Immunophenotypic changes induced by NKTR-255 in
tumor-bearing mice
Compared to IL-15, NKTR-255 has greatly improved plasma
and tumor exposure
Naïve Mice
0
• Signaling induced by IL-15 is gone by 6 hours
• NKTR-255 sustains signal in NK and CD8 cells for more than 3 days
TRAMP-C2
(subcutaneous)
NKTR-255 is bioactive, with sub-nanomolar EC50
20
Tumor volume (mm3)
5.78x105
40
CD8:T-reg ratio
IL-15
80
60
Frequency
KD (nM)
60
80
Total Lesion
Area (mm)
koff (s-1)
40
Hours
%pSTAT5 (CD4)
%pSTAT5 (CD8)
%pSTAT5 (NK)
Total Lesion
Area (mm)
kon (M-1 s-1)
Mean Plasma Conc.
(ng/mL)
Binding kinetics and affinity of NKTR-255 for IL-15Rα were
measured by surface plasmon resonance using
immobilized IL-15Rα. Cell-based potency was determined
by treating CTLL-2 cells with NKTR-255 at a range of
concentrations and measuring phosphorylation of STAT5
in cell lysate by immunoassay. Pharmacokinetic analysis
was performed following single-dose intravenous
administration of IL-15 and NKTR-255 in normal mice and
in mice bearing subcutaneous B16F10 and CT-26 tumors,
with analytes quantified in tumor and plasma by ELISA.
Immunophenotyping studies were performed by flow
cytometry on lymphocytes from peripheral blood of
NKTR-255-treated and IL-15-treated normal mice and
from spleen, tumor, and draining lymph node of treated
mice carrying subcutaneous TRAMP-C2 tumors. Efficacy
was determined by measuring tumor volume of
subcutaneous TRAMP-C2 and CT-26, with q5dx3
treatment with NKTR-255.
20
-20
100
Methods
20
CD8:T-reg ratio
!"
40
Frequency
$%
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60
IL-15
100
%pSTAT5
80
Total Lesions
Per Mouse
!"
%pSTAT5 (CD4)
%pSTAT5 (CD8)
%pSTAT5 (NK)
Total Lesions
Per Mouse
"!#
NKTR-255
100
Analyte
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NKTR-255 induces sustained signaling in lymphocytes in vivo
NKTR-255 binds to IL-15Rα
%pSTAT5
Interleukin-15 has been identified as a promising
candidate for use as an immuno-oncology
therapeutic, but the native cytokine has poor
drug-like properties. NKTR-255 is a novel
immunotherapeutic
agent
consisting
of
polymer-engineered IL-15 designed to optimally
engage the IL-15 receptor complex and provide
durable pathway activation in vivo. Here we show
that NKTR-255 has greatly improved plasma and
tumor exposure relative to IL-15, induces NK and
CD8 T-cell activation and proliferation, and has
single-agent efficacy in syngeneic tumor models.
Analyte Conc. (ng/mL)
Introduction
% Phospho-STAT5
1
Ki-67
CD122
NKTR-255 treatment results in sustained IL-15 activity, which
induces CD8 T-cell and NK cell activation and proliferation, and
produces long-lived immunophenotypic changes in tumor-bearing
mice. The design of NKTR-255 enables a potential drug-like
therapeutic strategy for accessing IL-15-based immunotherapy.
Poster #342 | Presented at SITC2016, National Harbor, Maryland