The CD122-biased immunostimulatory cytokine NKTR-214 combined with checkpoint blockade leads to
mobilization of anti-tumor immunity and synergistic activity
John L. Langowski, Murali Addepalli, Laurie VanderVeen, Rhoneil Pena, Ravi Nutakki, Yolanda Kirksey, Ute Hoch, Jonathan Zalevsky, Stephen K. Doberstein, Deborah H. Charych | Nektar Therapeutics, San Francisco CA
RESULTS
• Recombinant IL-2 (aldesleukin) is an effective immunotherapy for metastatic
melanoma and renal cell carcinoma with durable responses in up to 10% of
patients, but side-effects are dose-limiting
NKTR-214 administration increases immune activation biomarkers and inhibits tumor growth; synergy with checkpoint blockade is
concomitant with increases in T cell infiltration and T cell clonality
+
• Binding of the high-affinity
IL-2Rα on Treg leads to
expansion which antagonizes
antitumor immunity
Treg cell
-
NK cell
Endothelial cell
++
+
+++
+
+
+
+
+
Adapted from Boyman and Sprent, Nat Rev Immunol 12(3):180-90 (2012)
• NKTR-214 is a prodrug consisting of recombinant human IL-2 chemically
conjugated with 6 releasable chains of polyethylene glycol (PEG)‡
• Slow release of PEG chains generates active PEG-conjugated IL-2 metabolites
150
600
100
0
1
2
3
4
5
6
Time post dose (days)
• Active NKTR-214 metabolites possess biased IL-2R activation, favoring
IL-2Rβγ binding, with improved pharmacokinetics and tolerability compared
to aldesleukin
Mean Tumor Volume (mm3 ± SEM)
Charych DH, et al. Clin Cancer Res, 22(3):680-90, 2016
NKTR-214 : Prodrug design, proposed metabolic scheme
and comparative tumor pharmacokinetics
IL-2Rα
NKTR-214
Prodrug
(Inactive)
0
1
2
3
4
5
6
Time post dose (days)
7
1-PEG-IL-2
(EC50 = 0.052 nM)
20 h
Free IL-2
(EC50 = 0.026 nM)
Rapidly cleared in vivo
EC50, pSTAT5 CTLL-2
Science 310(5751):1159-63 (2005)
NKTR-214 Prodrug
NKTR-214 Active cytokine
Aldesleukin
A single 2 mg/kg
administration of
NKTR-214 delivers
50-times higher active
cytokine than five 3
mg/kg administrations
of aldesleukin
Abstract 311 | Presented at the 2016 CRI-CIMT-EATI-AACR Cancer Immunotherapy Conference
(Fraction; Mean ± SEM)
T cellnfiltration
i
20 h
γc
500
0
0
500
0
10 20 30 40 50 60
Days
2000
1000
0
1000
500
*
dosing duration
0
2
4
6
Days
8
10
1500
1000
500
0
12
500
0
Days
2000
0
NKTR-214 + Anti-PD-1
Days
Vehicle
aCTLA-4
aPD-1
0.10
2000
90%
tumor-free
500
10 20 30 40 50 60
0
500
0
10 20 30 40 50 60
Days
10%
tumor-free
0
NKTR-214 + aPD-1
0.3
0.4
T cell clonality (Mean ± SEM)
0.5
IL-6
IFNg
0.4
IL-17A
GranzymeB
TNFa
0.2
0.0
TNFa
0.2
GranzymeB
IL-4
0.0
Days
sVEGF
NKTR-214 + Anti-CTLA-4
uPA
-0.2
67%
tumor-free
-0.2
Dosing duration
1000
500
0
10
20
Days
30
NKTR-214 + aCTLA4
(45)
aCTLA-4 + aPD1
(38)
7
15
19
13
3
6
17
0.00
NKTR + Keytruda
NKTR
Keytruda
The DiscoverX human BioMAP system was utilized to assess biomarker changes in a human in vitro stromal/tumor/immune microenvironment model.
The StroHT29 system combines the HT-29 colon adenocarcinoma cell line, human PBMC and primary human fibroblasts, while the VascNSCLC system
combines the human H1299 NSCLC cell line, human PBMC and primary human endothelial cells. The most active species of the NKTR-214 prodrug (stable
1-PEG-IL2, 110ng/ml), Keytruda (1000ng/ml) or the two in combination were tested. Following 48 hours in culture, quantitative protein readouts were
measured by ELISA, with data represented as the log transformed average of the test agent divided by the average of the control. The combination of both
agents led to increases in lymphocyte markers of cytotoxicity and activation, and decreases in the angiogenesis markers VEGF and uPA.
D) Unique and shared T cell clones between treatment groups
aCTLA-4 + aPD-1
0.05
IL-6
IL-17A
CONCLUSIONS
•
NKTR-214 is synergistic with anti-CTLA-4 and anti-PD-1 checkpoint blockade, producing durable anti-tumor
responses
•
The combination of NKTR-214 with checkpoint blockade leads to increased T cell clonality and TIL infiltration
•
Activation of immune markers provide mechanistic rationale for increased lymphocytes in the tumor
microenvironment after treatment with NKTR-214
•
The combination of NKTR-214 and anti-PD-1 potentiates the increase of multiple biomarkers in a
tumor-immune co-culture system, suggesting these findings may be translatable from mouse to human
•
NKTR-214 is currently in a Phase 1 clinical trial to evaluate the pharmacokinetics, pharmacodynamics and
activity in an outpatient setting
NKTR-214
NKTR-214 + aCTLA-4
IFNg
10 20 30 40 50 60
1500
0
NKTR + Keytruda
NKTR
Keytruda
0.4
1000
Days
1000
0
1500
10 20 30 40 50 60
1500
60%
tumor-free
0
1000
10 20 30 40 50 60
Anti-CTLA-4 + Anti-PD-1
2000
1500
0.15
IL-2
IL-2Rβ
1000
Vehicle
C) T cell clonality and TIL infiltration (Adaptive ImmunoSEQ)
2-PEG-IL-2
(EC50 = 1.0 nM)
1500
2000
1500
NKTR-214
0.8mg/kg q9d
0
Region
of PEG
attachment
0
7
1500
• NKTR-214 delivers a controlled, sustained and biased signal through the
IL-2 receptor pathway
‡
400
200
50
0
Vehicle
NKTR-214
2000
0.6
VascNSCLC : NCI-H1299 + Endothelial cells + PMBC
M
C
VC P-1
A
M
-1
C
D
40
C
D
69
uP
A
R
IP
PB
-1
M P 0
C A
G cy I-1
ra to
nz to
ym x
e
B
sI
FN
sI γ
L1
sI 0
LsI 13
L17
A
sI
L4
sI
LsM 6
D
C
SR
sT B
N
Fα
+/++
-
Memory T cell
Vehicle
NKTR-214
2000
0.6
Log Ratio
+
800
Vehicle
StroHT29 : HT-29 + Fibroblasts +PBMC
10
6/
C
EA C VC
C D8 A M
A 7/ M u 1
5 PA
/C R
C D
ol 66
C la g e
C olla en
XC g
L 1 en I
0
K /IP III
er -1
at 0
in
M 20
M
sG
Pra P 9
nz A
ym I-I
e
sI B
FN
sI g
sT sIL L-6
N -17
F- A
al
ph
sI a
L10
SR
sV B
EG
TI F
M
P2
tP
A
uP
A
++
200
MCP-1
NKTR-214
0.8 mg/kg q9dx3
C
D
+++
Effector T cell
IFNγ
Tumor Volume (mm3)
+
Anti-PD-1
200 µg twice-weekly
Tumor Volume (mm3)
-/+
Tumor Volume (mm3)
-
Anti-CTLA-4
100 µg twice-weekly
Tumor Volume (mm3)
Naïve T cell
Tumor Volume (mm )
• Binding to the low-affinity
heterodimeric receptor IL-2Rβγ
leads to expansion of tumorkilling CD8 memory effector
T cells
3
CD132
(IL-2Rγ)
Tumor Volume (mm3)
CD122
(IL-2Rβ)
B) Single and combination efficacy in CT26 colon carcinoma model
Tumor Volume (mm3)
CD25
(IL-2Rα)
pg/ml ± SEM
Cell type
A) NKTR-214 activation of serum biomarkers, single-agent efficacy
pg/ml ± SEM
• IL-2 has pleiotropic immune
stimulatory effects
Human biomarker translation : NKTR-214 combined with Keytruda increases
immune-related and decreases angiogenesis-related soluble mediators in a human
in vitro co-culture system
Log Ratio
INTRODUCTION
NKTR-214 + aPD1
(39)
A) Top, a single administration of NKTR-214 to CT26 tumor-bearing mice (n=5/time point) increases serum markers of T cell activation (IFN-γ) and chemotaxis (MCP-1). Bottom, repeat administration leads to 58% tumor growth inhibition (*p<0.05).
B) While the CT26 mouse colon carcinoma model is refractory to single-agent treatment, the combination of NKTR-214 with checkpoint inhibition generates a significant proportion of tumor-free mice (n=10-12/group). NKTR-214 combined with
anti-PD-1 generates more complete responses than when both checkpoint antibodies are combined. C) TCR Vβ and Jβ gene usage was determined utilizing the ImmunoSEQ platform (Adaptive Biotechnologies), and T cell clonality and TIL infiltration
were assessed in CT26 tumors 7 days after treatment initiation (n=4/group). Treatment with single agent anti-CTLA-4 or NKTR-214 led to increases in T cell clonality and slight increases in T cell infiltration. The combination of NKTR-214 with either mode
of checkpoint inhibition led to superior increases in both clonality and tumor infiltration relative to the combination of anti-CTLA-4 and anti-PD-1. The most significant effects were achieved when NKTR-214 was combined with anti-PD-1. D) T cell clones
with ≥1% frequency (in parenthesis) were compared between combination treatment groups. While 13 clones expanded by therapy are shared by all three, each combination modality also elicited a proportion of clones which were discreet.