Time (h) % CD3 + pSTAT5 + (Mean ±SEM) NKTR-214 IL-2 0 5 72 96 120 144 0 5 15 10 20 10 days post-dose 20 15 24 48 10 Anti-tumor activity of NKTR-214; a CD122-biased agonist that promotes immune cell activation in the tumor microenvironment and lymphoid tissues John Langowski, Murali Addepalli, Yolanda Kirksey, Ravi Nutakki, Shalini Kolarkar, Rhoneil Pena, Ernesto Iacucci, Ute Hoch, Jonathan Zalevsky, Stephen K. Doberstein, Deborah H. Charych Nektar Therapeutics, San Francisco CA Introduction Results Conclusions Results • IL-2 has pleiotropic immune stimulatory effects that may limit its anti-tumor activity – Binding to the low-affinity heterodimeric receptor IL-2Rβγ leads to expansion of tumor-killing CD8 memory effector T cells – Binding of the high-affinity IL-2Rαβγ on Treg leads to expansion of Treg numbers, antagonizing antitumor immunity • NKTR-214 delivers a controlled, sustained, and biased signal through the IL-2 receptor pathway 1 – NKTR-214 is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol – Sustained signaling through the heterodimeric IL-2 receptor pathway preferentially activates and expands effector CD8+ T and NK cells over Tregs • NKTR-214 delivers a controlled, sustained, and biased signal through the IL-2 receptor, stimulating CD8 and NK cells and substantially increasing the CD8/Treg ratio preferentially in tumors • A 10-fold lower dose of NKTR-214 with reduced administration frequency relative to aldesleukin delivers superior efficacy and superior immune cell activation • NKTR-214 administration leads to significant upregulation of genes associated with activation and chemotaxis; increased T cell infiltration is a significant component of NKTR-214’s anti-tumor efficacy • A Phase 1/2 study as a single-agent in multiple tumor types was initiated in late 2015 and is currently ongoing • In collaborative clinical studies, NKTR-214 will also be evaluated in combination with nivolumab in multiple indications NKTR-214 : Prodrug design, proposed metabolic scheme, and comparative tumor pharmacokinetics NKTR-214 provides superior single-agent efficacy compared to aldesleukin with 10-fold lower dose in the murine B16F10 melanoma tumor model NKTR-214 treatment increases expression of genes associated with lymphocyte activity and chemotaxis Lymphocyte recruitment to tumor required for optimal efficacy with NKTR-214 A single NKTR-214 administration leads to increased NK and CD8 T cells and amplifies the CD8/Treg ratio relative to five aldesleukin administrations: effects are preferential in tumor over spleen In vivo, NKTR-214 and IL-2 provide contrasting IL-2R engagement as measured by pSTAT5 in blood lymphocytes Cell type CD25 (IL-2Rα) CD132 (IL-2Rβ) CD132 (IL-2Rγ) Naïve T cell - -/+ + Effector T cell +++ ++ + Memory T cell - +/++ + NK cell - ++ + Treg cell +++ + + Mean Fluorescence Intensity (MFI) of pSTAT5+ cells at indicated time point C57BL/6 mice were treated with either one dose of NKTR-214 or aldesleukin and pSTAT5 in peripheral blood CD3+ T cells was assessed using flow cytometry. Histograms on right depict pSTAT5 MFI for IL-2 and NKTR-214. Mice bearing subcutaneous B16F10 tumors were treated with NKTR-214 (2 mg/kg, single-dose) or aldesleukin (3 mg/kg daily for 5 doses). (A) Tumor-infiltrating lymphocytes and (B) splenocytes were isolated and immune cell populations assessed by flow cytometry. (*, p<0.05 with bars indicating comparisons) NKTR-214 (2 mg/kg, i.v., q9d x3; three doses) provides superior single-agent efficacy to aldesleukin (3 mg/kg, i.p. bid x5, two cycles; 20 doses). *, p<0.05 relative to vehicle; ‡, p<0.05 relative to aldesleukin B16F10 tumors were harvested 1, 3, 5 and 7 days after a single dose of NKTR-214 (0.8 mg/kg i.v.) or after dosing initiation with aldesleukin (3 mg/kg i.p. qd days 0-5). RNA-Seq was performed on tumor samples to assess gene expression changes, and data were normalized (Deseq2, R) using a general linearized model followed by ANOVA analysis. (A) Venn diagram of differentially expressed genes (DEGs) in tumor samples showing NKTR-214 treatment induced far greater gene expression. (B) Heatmap representing genes in the NKTR-214 treated group, belonging to chemotaxis family as defined by Gene Ontology. Significant increases are evident at days 1 and 3 following NKTR-214 administration. (C and D) DEGs from the NKTR-214 treated group, annotated by function (DAVID, NIH) to identify biological pathways with significant gene induction, including cytokine-cytokine receptor interaction and leukocyte transendothelial migration (Kyoto Encyclopedia of Genes and Genomes). Stars indicate NKTR-214 induced DEG genes. B16F10-bearing mice were treated with either NKTR-214 (0.8 mg/kg, i.v., q9d), fingolimod (FTY720, 5μg p.o. qd), or the two in combination. Fingolimod reduced peripheral lymphocyte numbers and significantly abrogated the anti-tumor activity of NKTR-214, suggesting tumor lymphocyte infiltration contributes to the mechanism of action. A) B16F10 tumor-infiltrating lymphocytes B) Spleen, tumor-bearing animals Poster #343 | Presented at SITC 2016, National Harbor, Maryland 0 2 4 6 8 10 0 20 40 60 80 100 120 140 160 180 Days Mean Tumor Volume (mm 3 ±SEM) * * * 0 2 4 6 8 10 Blood Lymphocytes Cell Count (x 10 3 / μl blood) Vehicle NKTR-214 Fingolimod NKTR-214 + Fingolimod * Vehicle NKTR-214 Fingolimod NKTR-214 + Fingolimod * * * * 0 8 16 24 32 * * * * * * * * 0 1 2 3 4 5 * * * * * 0 10 20 30 * 0 4 8 12 16 0 10 20 30 40 Activated NK CD8 T Cells Memory CD8 T Cells (NKp46 + DX5 + CD122 + ) CD4 Treg CD8 / Treg (CD25 + FOXP3 + ) (CD122 + CD44 hi ) Day 5 Day 7 Day 10 Day 5 Day 7 Day 10 Day 5 Day 7 Day 10 Vehicle Aldeleukin NKTR-214 Day 5 Day 7 Day 10 Day 5 Day 7 Day 10 % of Lymphocytes % of Lymphocytes Spleen Ratio % of Lymphocytes % of Lymphocytes * * * * * * * * * * 0 20 40 60 Activated NK CD8 T Cells Memory CD8 T Cells (NKp46 + DX5 + CD122 + ) CD4 Treg CD8 / Treg (CD25 + FOXP3 + ) (CD122 + CD44 hi ) * * * Day 5 Day 7 Day 10 0 1 2 3 4 % of TIL TIL Ratio * * * * * * 0 100 200 500 1500 2500 Vehicle Aldeleukin NKTR-214 * * Day 5 Day 7 Day 10 Day 5 Day 7 Day 10 Day 5 Day 7 Day 10 % of TIL Day 5 Day 7 Day 10 0 20 40 60 % of TIL 0 20 40 60 % of TIL 0 2 4 6 8 10 12 14 16 0 50 100 150 * ‡ 0 5 10 15 20 25 30 0 25 50 75 100 Days Days Tumors Below 4x Initial Volume (%) Mean Tumor Volume (mm 3 ±SEM) Vehicle Aldeleukin NKTR-214 * * , ‡ Treatment Duration 80 60 Vehicle IL-2 40 20 0 10 2 10 3 10 4 10 5 80 100 60 Vehicle NKTR-214 40 20 0 10 2 10 3 10 4 10 5 Count Count pSTAT5 intensity CLONAL EXPANSION Stimulates Immune Response to Kill Tumor Cells LEGEND: NKTR-214 – Inactive 2-PEG – Active Cytokine 1-PEG – Active Cytokine NKTR-214 (6-PEG) Irreversible Release 2-PEG Active Cytokine 1-PEG Active Cytokine Irreversible Release IL-2Rαβγ α β γ β γ IL-2Rβγ Immunosuppressive cells limit anti-tumor response NK NK CD8 + CD8 + CD8 + CD4 + Helper CD4 + Helper CD4 + T reg NK NK NK, CD4 + , and CD8 + T cells CD4 + Helper CD8 + CD4 + Helper CD4 + Helper CD8 + NK CD4 + Helper NK CD8 + CD4 + Helper CD4 + Helper CD8 + CD8 + NK NK NK CD8 + CD4 + Helper CD4 + Helper NK CD8 + NKTR-214 Aldesleukin Vehicle 4469 (74.7%) 363 (6.1%) 307 (5.1%) 87 (1.5%) 172 (2.9%) 246 (4.1%) 342 (5.7%) A) B) C) D) Adapted from Boyman and Sprent, Nat Rev Immunol 12(3):180-90 (2012) Reference: 1) Charych DH, et al. Clin Cancer Res. 2016;22(3):680-90. Cytokine-Cytokine Receptor Interaction Leukocyte Transendothelial Migration
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Time (h)
% C
D3+
pS
TA
T5+
(M
ean
±S
EM
)
NKTR-214
IL-2
0 5 72 96 120 1440
5
15
10
2010 days post-dose
2015 24 4810
Anti-tumor activity of NKTR-214; a CD122-biased agonist that promotes immune cell activation in the tumor microenvironment and lymphoid tissues John Langowski, Murali Addepalli, Yolanda Kirksey, Ravi Nutakki, Shalini Kolarkar, Rhoneil Pena, Ernesto Iacucci, Ute Hoch, Jonathan Zalevsky, Stephen K. Doberstein, Deborah H. Charych
Nektar Therapeutics, San Francisco CA
Introduction
Results
Conclusions
Results
• IL-2 has pleiotropic immune stimulatory effects that may limit its anti-tumor activity– Binding to the low-affinity heterodimeric
receptor IL-2Rβγ leads to expansion of tumor-killing CD8 memory effector T cells
– Binding of the high-affinity IL-2Rαβγ on Treg leads to expansion of Treg numbers, antagonizing antitumor immunity
• NKTR-214 delivers a controlled, sustained, and biased signal through the IL-2 receptor pathway1
– NKTR-214 is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol
– Sustained signaling through the heterodimeric IL-2 receptor pathway preferentially activates and expands effector CD8+ T and NK cells over Tregs
• NKTR-214 delivers a controlled, sustained, and biased signal through the IL-2 receptor, stimulating CD8 and NK cells and substantially increasing the CD8/Treg ratio preferentially in tumors
• A 10-fold lower dose of NKTR-214 with reduced administration frequency relative to aldesleukin delivers superior efficacy and superior immune cell activation
• NKTR-214 administration leads to significant upregulation of genes associated with activation and chemotaxis; increased T cell infiltration is a significant component of NKTR-214’s anti-tumor efficacy
• A Phase 1/2 study as a single-agent in multiple tumor types was initiated in late 2015 and is currently ongoing
• In collaborative clinical studies, NKTR-214 will also be evaluated in combination with nivolumab in multiple indications
NKTR-214 : Prodrug design, proposed metabolic scheme, and comparative tumor pharmacokinetics
NKTR-214 provides superior single-agent efficacy compared to aldesleukin with 10-fold lower dose in the murine B16F10 melanoma tumor model
NKTR-214 treatment increases expression of genes associated with lymphocyte activity and chemotaxis
Lymphocyte recruitment to tumor required for optimal efficacy with NKTR-214
A single NKTR-214 administration leads to increased NK and CD8 T cells and amplifies the CD8/Treg ratio relative to five aldesleukin administrations: effects are preferential in tumor over spleen
In vivo, NKTR-214 and IL-2 provide contrasting IL-2R engagement as measured by pSTAT5 in blood lymphocytes
Cell typeCD25
(IL-2Rα)CD132
(IL-2Rβ)CD132(IL-2Rγ)
Naïve T cell - -/+ +
Effector T cell +++ ++ +
Memory T cell - +/++ +
NK cell - ++ +
Treg cell +++ + +
Mean Fluorescence Intensity (MFI) ofpSTAT5+ cells at indicated time point
C57BL/6 mice were treated with either one dose of NKTR-214 or aldesleukin and pSTAT5 in peripheral blood CD3+ T cells was assessed using flow cytometry. Histograms on right depict pSTAT5 MFI for IL-2 and NKTR-214.
Mice bearing subcutaneous B16F10 tumors were treated with NKTR-214 (2 mg/kg, single-dose) or aldesleukin (3 mg/kg daily for 5 doses). (A) Tumor-infiltrating lymphocytes and (B) splenocytes were isolated and immune cell populations assessed by flow cytometry. (*, p<0.05 with bars indicating comparisons)
NKTR-214 (2 mg/kg, i.v., q9d x3; three doses) provides superior single-agent efficacy to aldesleukin (3 mg/kg, i.p. bid x5, two cycles; 20 doses). *, p<0.05 relative to vehicle; ‡, p<0.05 relative to aldesleukin
B16F10 tumors were harvested 1, 3, 5 and 7 days after a single dose of NKTR-214 (0.8 mg/kg i.v.) or after dosing initiation with aldesleukin (3 mg/kg i.p. qd days 0-5). RNA-Seq was performed on tumor samples to assess gene expression changes, and data were normalized (Deseq2, R) using a general linearized model followed by ANOVA analysis. (A) Venn diagram of differentially expressed genes (DEGs) in tumor samples showing NKTR-214 treatment induced far greater gene expression. (B) Heatmap representing genes in the NKTR-214 treated group, belonging to chemotaxis family as defined by Gene Ontology. Significant increases are evident at days 1 and 3 following NKTR-214 administration. (C and D) DEGs from the NKTR-214 treated group, annotated by function (DAVID, NIH) to identify biological pathways with significant gene induction, including cytokine-cytokine receptor interaction and leukocyte transendothelial migration (Kyoto Encyclopedia of Genes and Genomes). Stars indicate NKTR-214 induced DEG genes.
B16F10-bearing mice were treated with either NKTR-214 (0.8 mg/kg, i.v., q9d), fingolimod (FTY720, 5μg p.o. qd), or the two in combination. Fingolimod reduced peripheral lymphocyte numbers and significantly abrogated the anti-tumor activity of NKTR-214, suggesting tumor lymphocyte infiltration contributes to the mechanism of action.
A) B16F10 tumor-infiltrating lymphocytes
B) Spleen, tumor-bearing animals
Poster #343 | Presented at SITC 2016, National Harbor, Maryland
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20
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*
**
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Blood Lymphocytes
Cel
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(x 1
03 /
µl b
loo
d)
VehicleNKTR-214Fingolimod
NKTR-214+ Fingolimod
*
Vehicle
NKTR-214
Fingolimod
NKTR-214+ Fingolimod
**
**
0
8
16
24
32
**
**
**
**
0
1
2
3
4
5 *
***
*0
10
20
30*
0
4
8
12
16
0
10
20
30
40
Activated NK
CD8 T Cells Memory CD8 T Cells
(NKp46+DX5+CD122+)CD4 Treg
CD8 / Treg
(CD25+FOXP3+)
(CD122+CD44hi)
Day 5 Day 7 Day 10
Day 5 Day 7 Day 10
Day 5 Day 7 Day 10
Vehicle
Aldeleukin
NKTR-214
Day 5 Day 7 Day 10 Day 5 Day 7 Day 10
% o
f Ly
mp
hocy
tes
% o
f Ly
mp
hocy
tes
Sp
leen
Rat
io
% o
f Ly
mp
hocy
tes
% o
f Ly
mp
hocy
tes
**
**
**
*
**
*
0
20
40
60
Activated NK
CD8 T Cells Memory CD8 T Cells
(NKp46+DX5+CD122+)CD4 Treg
CD8 / Treg
(CD25+FOXP3+)
(CD122+CD44hi)
*
**
Day 5 Day 7 Day 10
0
1
2
3
4
% o
f T
IL
TIL
Rat
io
**
**
*
*
0
100
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500
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Vehicle
Aldeleukin
NKTR-214
**
Day 5 Day 7 Day 10
Day 5 Day 7 Day 10 Day 5 Day 7 Day 10
% o
f T
IL
Day 5 Day 7 Day 10
0
20
40
60
% o
f T
IL
0
20
40
60
% o
f T
IL
0 2 4 6 8 10 12 14 160
50
100
150
*
‡
0 5 10 15 20 25 300
25
50
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DaysDays
Tum
ors
Bel
ow
4x
Init
ial V
olu
me
(%)
Mea
n T
umo
r V
olu
me
(mm
3 ±
SE
M) Vehicle
Aldeleukin
NKTR-214
* * ,‡
Treatment Duration
80
60
Vehicle IL-2
40
20
0
102 103 104 105
80
100
60Vehicle
NKTR-21440
20
0
102 103 104 105
Co
unt
Co
unt
pSTAT5 intensity
CLONAL EXPANSION
Stimulates Immune Response to Kill Tumor Cells
LEGEND:NKTR-214 – Inactive2-PEG – Active Cytokine1-PEG – Active Cytokine
NKTR-214 (6-PEG)
IrreversibleRelease
2-PEGActive Cytokine
1-PEG Active Cytokine
IrreversibleRelease
IL-2Rαβγ
α
β γβ γ
IL-2Rβγ
Immunosuppressive cells limit anti-tumor response
NKNK
CD8+
CD8+
CD8+
CD4+
Helper
CD4+
Helper
CD4+
T reg
NK NK
NK, CD4+, and CD8+ T cells
CD4+
HelperCD8+
CD4+
Helper
CD4+
HelperCD8+
NK CD4+
Helper
NK
CD8+
CD4+
HelperCD4+
HelperCD8+
CD8+
NKNK
NK
CD8+
CD4+
Helper
CD4+
Helper
NKCD8+
NKTR-214
Aldesleukin
Vehicle
4469(74.7%)
363(6.1%)
307(5.1%)
87(1.5%)
172(2.9%)
246(4.1%)
342(5.7%)
A)
B)
C)
D)
Adapted from Boyman and Sprent, Nat Rev Immunol 12(3):180-90 (2012)
Reference:1) Charych DH, et al. Clin Cancer Res. 2016;22(3):680-90.
Cytokine-Cytokine Receptor Interaction
Leukocyte Transendothelial Migration
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