Download NKTR-214, an Engineered Cytokine with Biased IL2 Receptor

Clinical
Cancer
Research
Cancer Therapy: Preclinical
NKTR-214, an Engineered Cytokine with Biased
IL2 Receptor Binding, Increased Tumor Exposure,
and Marked Efficacy in Mouse Tumor Models
Deborah H. Charych, Ute Hoch, John L. Langowski, Steve R. Lee, Murali K. Addepalli,
Peter B. Kirk, Dawei Sheng, Xiaofeng Liu, Paul W. Sims, Laurie A. VanderVeen,
Cherie F. Ali, Thomas K. Chang, Marina Konakova, Rhoneil L. Pena, Rupesh S. Kanhere,
Yolanda M. Kirksey, Chunmei Ji, Yujun Wang, Jicai Huang, Theresa D. Sweeney,
Seema S. Kantak, and Stephen K. Doberstein
Abstract
Purpose: Aldesleukin, recombinant human IL2, is an effective
immunotherapy for metastatic melanoma and renal cancer, with
durable responses in approximately 10% of patients; however,
severe side effects limit maximal dosing and thus the number of
patients able to receive treatment and potential cure. NKTR-214
is a prodrug of conjugated IL2, retaining the same amino acid
sequence as aldesleukin. The IL2 core is conjugated to 6 releasable
polyethylene glycol (PEG) chains. In vivo, the PEG chains slowly
release to generate active IL2 conjugates.
Experimental Design: We evaluated the bioactivity and receptor binding of NKTR-214 and its active IL2 conjugates in vitro; the
tumor immunology, tumor pharmacokinetics, and efficacy of
NKTR-214 as a single agent and in combination with anti–
CTLA-4 antibody in murine tumor models. Tolerability was
evaluated in non-human primates.
Results: In a murine melanoma tumor model, the ratio of
tumor-killing CD8þ T cells to Foxp3þ regulatory T cells was greater
than 400 for NKTR-214 compared with 18 for aldesleukin,
supporting preferential activation of the IL2 receptor beta over
IL2 receptor alpha, due to the location of PEG molecules. NKTR214 provides a 500-fold greater exposure of the tumor to conjugated IL2 compared with aldesleukin. NKTR-214 showed efficacy
as a single agent and provided durable immunity that was
resistant to tumor rechallenge in combination with anti–CTLA4 antibody. NKTR-214 was well tolerated in non-human
primates.
Conclusions: These data support further evaluation of NKTR214 in humans for a variety of tumor types, adding to the
repertoire of potent and potentially curative cancer immunotherapies. Clin Cancer Res; 22(3); 680–90. 2016 AACR.
Introduction
IL2 stimulates immune cell proliferation and activation
through a receptor-signaling complex containing alpha (IL2Ra,
CD25), beta (IL2Rb, CD122), and common gamma chain
receptors (g c, CD132; refs. 7, 8), but the effects are pleiotropic
and contextual. At high doses, IL2 binds to heterodimeric
IL2Rbg receptor leading to desired expansion of tumor killing
CD8þ memory effector T (CD8 T) cells (9). IL2 also binds to its
heterotrimeric receptor IL2Rabg with greater affinity, which
expands immunosuppressive CD4þ CD25þ regulatory T cells
(Tregs) expressing high constitutive levels of IL2Ra. Expansion
of Tregs represents an undesirable effect of IL2 for cancer
immunotherapy (7–13).
We hypothesized IL2 conjugation with polyethylene glycol
(PEG) could be used to mask the region of IL2 that interacts with
the IL2Ra subunit responsible for activating immunosuppressive
Tregs, biasing activity towards tumor killing CD8þ T cells. PEGylation could alter the immunostimulatory profile of aldesleukin
without mutation of its amino acid sequence while creating an
inactive prodrug, mitigating rapid systemic immune activation,
and improving tolerability (14).
We report the development of, NKTR-214, a biologic prodrug
consisting of IL2 bound by 6 releasable polyethylene glycol (PEG)
chains. We evaluated receptor bias, bioactivity, tumor immunology, pharmacokinetics, pharmacodynamics, and tolerability
of NKTR-214. Antitumor effects of NKTR-214 were compared
with aldesleukin as a single agent and in combination with an
Immunotherapy offers potential for durable responses in a
growing list of tumor types (1). Aldesleukin, recombinant human
IL2, was the first cancer immunotherapy, and one of the first
recombinant proteins, approved by the FDA in 1992. High-dose
IL2 (HD-IL2) administration results in severe hypotension and
vascular leak syndrome, which has relegated its use to specialized
care centers. With use of appropriate supportive care, aldesleukin
has demonstrated complete cancer regression in about 10% of
patients treated for metastatic melanoma and renal cancer (2–4).
Approximately 70% of patients with complete responses have
been cured, maintaining complete regression for more than 25
years after initial treatment (2–6). Unfortunately, the side-effect
profile restricts optimal IL2 dosing, limiting the number of
patients who might achieve durable responses.
Nektar Therapeutics, San Francisco, California.
Note: Supplementary data for this article are available at Clinical Cancer
Research Online (http://clincancerres.aacrjournals.org/).
Corresponding Author: Deborah H. Charych, Nektar Therapeutics, 455 Mission
Bay Blvd South, San Francisco, CA 94158. Phone: 415-482-5558; Fax: 415-3395300; E-mail: [email protected]
doi: 10.1158/1078-0432.CCR-15-1631
2016 American Association for Cancer Research.
680 Clin Cancer Res; 22(3) February 1, 2016
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2016 American Association for Cancer Research.
NKTR-214, an Engineered Cytokine for Cancer Immunotherapy
Translational Relevance
Recent attention in immunotherapy for treatment of cancer
has focused on the successes of checkpoint inhibitor antibodies. However, administration of the recombinant cytokine
IL2 aldesleukin produces durable responses lasting up to two
decades in approximately 10% of patients treated for metastatic melanoma and renal cancer. IL2 is an activator and
suppressor of the immune system and is associated with severe
side effects such as hypotension and vascular leak syndrome,
limiting widespread use. Recent studies have shown that
durable responses may increase with combined immunotherapies. We evaluated NKTR-214, a polymer conjugate
of IL2 that tips the balance in favor of immune activation over
suppression in the tumor, with improved efficacy and tolerability compared with aldesleukin. Our data provide the
rationale for further evaluation of NKTR-214 in clinical studies
as both a single agent and in combination with other immunotherapies including checkpoint inhibitor antibodies.
anti-CTLA-4 antibody, using murine tumor models. The results
support the future development of NKTR-214 for treatment of
cancer.
Materials and Methods
Recombinant protein and antibodies
Aldesleukin was purchased from Myoderm and diluted in the
recommended product formulation buffer for in vivo studies.
NKTR-214 was manufactured by Nektar Therapeutics. ELISA
study materials are described in Supplementary Materials and
Methods.
For in vivo murine studies, aldesleukin and anti–CTLA-4 antibody were administered intraperitoneally; NKTR-214 was administered intravenously by tail-vein injection. For each animal, the
concentration was adjusted to achieve the specified dose at an
injection volume of 0.2 mL for aldesleukin, 0.1 mL for anti–CTLA4 antibody, and 8 to 10 mL/kg for NKTR-214. NKTR-214 dosing
was based on the IL2 equivalent content of the conjugate.
Animals
Animal studies were conducted under accreditation by the
Association for Assessment and Accreditation of Laboratory Animal Care or the Canadian Council on Animal Care. All studies met
the ethical and humane criteria for transportation, housing, and
care established by United States NIH guidelines or Canadian
animal care regulators. Female C57BL/6 NCrl mice aged 6 to 8
weeks weighing 15 to 20 g and female Balb/C NCrl mice aged 6 to
8 weeks weighing 16 to 20 g were purchased from Charles River
Laboratories. Male and female cynomolgus monkeys, aged 2 to 3
years weighing 2.3 to 2.8 kg at treatment initiation, were obtained
from Primus Bio-Resources Inc. Male na€ve adult cynomolgus
monkeys, aged 3 to 4 years weighing 3.7 to 4.3 kg at treatment
initiation, were obtained from Worldwide Primates Inc.
Cell lines
IL2-dependent CTLL-2 mouse T-lymphocyte cell line, B16F10
melanoma, EMT6 mammary, and CT26 colon carcinoma cell
lines were obtained from ATCC. All cells were verified pathogen
www.aacrjournals.org
and mycoplasma (IDEXX BioResearch) free and authenticated by
ATCC using Cytochrome C subunit I PCR assay.
Production and purification of anti–CTLA-4
UC10-4F10-11 hybridoma (15), which secretes hamster antimouse CTLA-4 antibody with mouse IgG2a isotype, was purchased from ATCC (HB-304). PFHM-II medium (Invitrogen) was
used to culture the cell line under 5% CO2. The cell line was
maintained between 1 105 and 1 106 cells/mL and anti–
CTLA-4 was expressed and purified (16).
Degree of PEGylation for NKTR-214
A releasable conjugate of glycine was used as a reference
standard. Reference standard or NKTR-214 was incubated at room
temperature for 45 minutes with 37.5 mmol/L NaOH to release
PEG from IL2 or glycine. The reaction was neutralized with an
equal volume of 100 mmol/L HCl. Samples were analyzed by
reverse phase high-performance liquid chromatography (HPLC)
to quantify released protein, glycine, and PEG. PEG moles
released from NKTR-214 were calculated using the standard curve
from releasable PEG-glycine standard. Degree of PEGylation was
calculated as ratio of PEG moles to protein moles, or ratio of area
under the curve (AUC) of released PEG to AUC of IL2. Procedures
for mapping PEGylation sites are included in the Supplementary
Materials and Methods.
In vitro hydrolysis kinetics of NKTR-214
Hydrolysis of NKTR-214 was monitored in vitro in PBS at pH 7.4
at 37 C. At indicated time points, levels of released IL2 conjugates
were measured using reverse phase HPLC with corresponding IL2
conjugate reference standards. Data were fit to a kinetic model
using the Berkeley-Madonna software. The study was performed
long enough to obtain the half-life of release.
Identification of the active released IL2 conjugates
Activity of NKTR-214 and its released IL2 conjugates was
measured by activation of pSTAT5 in CTLL-2 cells using Sector
Imager 2400 plate reader and assay reagents from Meso Scale
Discovery, LLC as described in Supplementary Materials and
Methods. Total and phosphorylation of STAT5 (pSTAT5) levels
were detected using the MSD Phospho(Tyr694)/Total STAT5a,b
Whole Cell Lysate Kit. Methods for biophysical characterization
and affinity of NKTR-214 binding to IL2 receptor subunits are
described in Supplementary Materials and Methods.
Tumor exposure of NKTR-214 compared with aldesleukin
C57BL/6 mice were implanted subcutaneously into the right
flank with B16F10 melanoma cells (1 106 per animal). Seven
days after implantation, when tumors measured 200 mm3, animals were administered NKTR-214 (2 mg/kg 1) or aldesleukin
(3 mg/kg daily 5). Tumors were harvested (n ¼ 4 per observation time), homogenized in ice-cold PBS containing protease
inhibitor (Roche) and 0.25% acetic acid, and centrifuged to
obtain supernatant. To quantify NKTR-214 levels in tumor tissue,
PEG was released from IL2 by incubating the supernatant in a pH
9 buffer at 37 C overnight. IL2 was measured by sandwich ELISA
specific for human IL2. To calculate AUC, data were fit with
Pheonix WinNonLin using a noncompartmental model. AUC
after aldesleukin was estimated on the basis of day 1 AUC
multiplied by 5.
Clin Cancer Res; 22(3) February 1, 2016
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2016 American Association for Cancer Research.
681
Charych et al.
Immune cell phenotyping
Mice bearing B16F10 melanoma tumors (as described
above) were administered NKTR-214 (2 mg/kg 1), aldesleukin
(3 mg/kg daily 5), or vehicle (daily 5). Tumors were collected
5, 7, and 10 days following initial dose and were within one SD of
mean tumor volume for each group. Following mechanical and
enzymatic digestion, phenotypic assessment of immune cell
subtypes in tumor microenvironment was determined by flow
cytometry. Immunohistochemical analysis of T-cell density was
performed on frozen tumor sections 7 days after administration
(Supplemental Materials and Methods).
Activity of NKTR-214 in murine tumor models
All studies were conducted in therapeutic mode with dosing
initiated in the presence of established tumors with volume in
excess of 100 mm3. Tumor measurements and body weights were
recorded 3 times per week. For all studies, mean tumor volume for
each group was graphically represented until >20% of the original
cohort was removed due to tumor volume constraints. For the
single-agent study, mice bearing B16F10 melanoma tumors were
randomized 7 days after implantation and administered NKTR214 2 mg/kg every 9 days for 3 doses (n ¼ 9 mice), aldesleukin 3
mg/kg twice daily for 5 days (2 cycles, n ¼ 9 mice), or vehicle for 5
doses (n ¼ 12 mice). For EMT6 mammary (n ¼ 10 per group) and
CT26 colon carcinoma (n ¼ 12 per group) combination efficacy
studies, 2 106 cells were implanted into the right flank of BALB/c
mice. Seven days after implantation, mice were randomized and
administered NKTR-214 0.8 mg/kg every 9 days for 3 doses, anti–
CTLA-4 100 mg twice weekly through day 18, vehicle control, or
anti–CTLA-4 and NKTR-214 given in combination. Two groups
implanted with EMT6 were administered either aldesleukin 0.5
mg/kg daily for 5 days (2 cycles) or aldesleukin combination with
anti–CTLA-4.
For the tumor rechallenge study, Balb/c mice were implanted
subcutaneously in the right flank with 2 106 EMT6 mammary
carcinoma cells. Seven days after inoculation, mice were randomized and administered antibody control (n ¼ 10 mice) or a
combination of anti–CTLA-4 and NKTR-214 (n ¼ 80 mice).
Forty-nine days after dosing initiation, tumor-free animals exhibiting complete response to combined immunotherapy were
enrolled into two groups receiving either rechallenge of 2 106 EMT6 or unrelated CT26 colon carcinoma cells. An additional
cohort of age-appropriate na€ve animals was challenged with
EMT6 cells (n ¼ 10 mice). After 109 days, animals that rejected
the EMT6 rechallenge were inoculated with 2 106 EMT6 cells
(n ¼ 14 mice). Age-appropriate na€ve animals (n ¼ 5 mice) were
similarly challenged; all groups were followed with no additional
treatment.
Evaluation of immune markers and potential hypotension and
vascular leak syndrome in non-human primates
For evaluation of immune markers, cynomolgus monkeys
(n ¼ 3/sex/dose) received a single intravenous bolus dose into
the saphenous vein of NKTR-214 0.01, 0.03, and 0.1 mg/kg. Blood
samples were collected to evaluate effects of NKTR-214 on lymphocyte and soluble CD25 counts as described in Supplementary
Materials and Methods.
Arterial blood pressure was used to monitor potential hypotension and vascular leak syndrome. Male monkeys (n ¼ 4) were
surgically implanted with telemetry transmitters (TL11M2-D70PCT; Data Sciences International). Each received NKTR-214 as a
682 Clin Cancer Res; 22(3) February 1, 2016
slow intravenous bolus every 14 days for 4 doses (0, 0.01, 0.03,
and 0.1 mg/kg) according to Latin-square study design. Continuous telemetry recordings were collected from each conscious
unrestricted monkey for 0 to 24 hours (time for peak NKTR-214
concentration), 30 to 54 hours (time for peak active 2-PEG-IL2
and 1-PEG-IL2 concentration), and 80 to 104 hours (peak immunological and clinical signs observed) after each dose. Arterial
blood pressure was collected every 15 minutes over each telemetry-recording period.
Inflammatory cytokines TNFa and IFNg were analyzed in
monkeys at 1, 3, 6, 12, 24, 48, 72, and 168 hours after a single
0.1 mg/kg dose of NKTR-214 by flow cytometry using a Cytometric Bead Array for Non-Human Primate (NHP) Th1/Th2 kit
(BD Biosciences). One milliliter of blood was collected via saphenous vein and resulting serum samples were diluted 1 in 10 in
tubes containing a mixture of beads coated with capture antibodies specific to IFNg and TNFa. Bound cytokines were detected
using phycoerythrin-conjugated detection antibodies. The IFNg
and TNFa concentrations were interpolated using a 5-parameter
logistic nonlinear regression equation, established from the
human Th1/Th2 cytokine standard curves ranging from 20 to
5,000 pg/mL for IFNg and of 10 to 5,000 pg/mL for TNFa.
Statistical analyses
For identification of active released IL2 conjugates, EC50 values
for each compound were determined from sigmoidal dose–
response curves using GraphPad Prism 5.01. In mouse efficacy
studies, results are shown as mean SEM. Tumor growth inhibition was calculated as percent reduction in volume for treated
groups relative to vehicle. The significance between experimental
and vehicle groups was compared using ANOVA, with the Tukey
post test. Differences between NKTR-214 and aldesleukin were
analyzed using unpaired Student t test. P values < 0.05 were considered significant. Kaplan–Meier survival curves represent percent
of tumors in each group with a volume less than their 4-fold of
initial day 0 value (tumor volume quadrupling time, TVQT). Statistical significance was calculated utilizing GraphPad Prism v6.04.
Results
Design and characterization of NKTR-214
NKTR-214 is a prodrug consisting of IL2 conjugated to 6
releasable PEG chains. The amino acid sequence of the IL2 is the
same as aldesleukin. The PEG reagent and conjugation reaction
were optimized to facilitate localization on IL2 at lysine residues
clustered at the IL2/IL2Ra interface (Fig. 1). Enzymatic digestion
of NKTR-214 followed by hydrolytic removal of PEG on the
peptides confirmed on average 6 PEG chains conjugated and
distributed at K31, K34, K42, K47, K48, and K75 (cleavage
peptides 20–51 and 68–94). Amino acids within these cleavage
peptides form the hydrophobic center of the IL2/IL2Ra interface,
surrounded by a polar periphery containing key ion-pairing
residues (17). These key hydrophobic and charged IL2/IL2Ra
interaction sites are at or near sites of PEGylation. When NKTR214 is hydrolyzed to its most active 1-PEG-IL2 state (described
below), the last remaining PEG is distributed at the IL2/IL2Ra
interface.
Because of the high degree of PEGylation initially, NKTR-214
is an inactive prodrug and serves as "reservoir" revealing active
conjugated IL2 after slow release of PEG chains in vivo (Fig. 2A).
The prodrug effect is designed to mitigate rapid systemic immune
Clinical Cancer Research
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2016 American Association for Cancer Research.
NKTR-214, an Engineered Cytokine for Cancer Immunotherapy
Biophysical affinity characterization of NKTR-214 binding to IL2
receptor subunits indicates PEG chains impact affinity to IL2Ra to
a greater extent than IL2Rb compared with IL2 (Supplementary
Table S1; Supplementary Fig. S2).
Figure 1.
Model demonstrating region of PEGylation sites at IL2 interface with IL2Ra.
NKTR-214 is IL2 at its core (green) bound by lysines to 6 PEG chains per
molecule as determined by reverse phase HPLC quantification of completely
released PEG and protein (8 replicates for 4 lots of NKTR-214). Sites of
PEGylation are depicted by red circles. The g c receptor is depicted in pink,
IL2Rb in aqua blue, and IL2Ra in dark blue. Only peptide 68–94
75
(VLNLAQSK NFHLRPRDLISNINVIVLE) and peptide 20–51
31
34
42
47 48
(LQMILNGINNYK NPK LTRMLTFK FYMPK K ATE) were bound by PEG.
The pegylation results represent 6 separate experiments, each with 2
replicates, using 3 batches of NKTR-214.
activation such as what might occur with HD-IL2 regimen, while
allowing sustained concentrations of active conjugated IL2 in the
tumor. In vitro hydrolysis studies demonstrate PEG release occurs
sequentially, with a half-life of 20 hours for each step in the
release, supporting the lack of a "burst" IL2 release effect (Supplementary Fig. S1). Unconjugated IL2 is cleared rapidly in vivo
with a half-life of 13 minutes in mice and 1.4 hours in humans
(18, 19). Therefore, slow release of PEG relative to fast clearance of
IL2 provides minimal exposure to free IL2 in vivo.
Identification of the active IL2 conjugates released from
NKTR-214
To assess bioactivity of NKTR-214 and identify active conjugated IL2 species formed after PEG release, we evaluated levels of
pSTAT5 in murine T cells (Fig. 2B) after treatment in culture. As
anticipated, NKTR-214 was not active with an EC50 > 4,000 ng/
mL, consistent with the prodrug design. Unconjugated aldesleukin had an EC50 of 0.4 ng/mL, similar to reported values (18). In
vitro, PEG chains were released hydrolytically from NKTR-214,
forming a mixture of conjugated IL2 species ranging from 5-PEGIL2 to 1-PEG-IL2, which were each purified. 1-PEG-IL2 and 2PEG-IL2 species were identified as the most active IL2 conjugates
with EC50 values of 2 and 20 ng/mL, respectively. Positional
isomer analysis of synthesized 1-PEG-IL2 indicated two positional isomers (of the six potential K sites described above), which
were identically bioactive in the pSTAT assay. The 3-PEG-IL2 was
found to be inactive with EC50 > 1,300 ng/mL. Therefore, species
containing 3-PEGs are inactive; those with < 3-PEGs are active.
www.aacrjournals.org
NKTR-214 provides 500-fold increased tumor exposure relative
to aldesleukin
To assess tumor exposure, NKTR-214 or aldesleukin levels were
measured in mice bearing B16F10 melanoma tumors. As shown
in Fig. 3, tumor aldesleukin levels rapidly reached Cmax and then
rapidly declined, leading to <4 ng/g concentrations 24 hours after
each dose and a daily AUC of 0.09 0.02 mg/hour/g. In contrast,
NKTR-214 was detectable in tumors for up to 8 days after a single
dose achieving an AUC of 30 6.9 mg/hour/g. On the basis of
AUC, a single dose of NKTR-214 led to a 67-fold higher exposure
compared with 5 daily doses of aldesleukin, even though 7.5
fewer IL2 equivalents were dosed using NKTR-214 (3 mg/kg daily
5 ¼ 15 mg/kg vs. 2 mg/kg). Thus, normalizing exposure on the
basis of IL2 equivalents, NKTR-214 achieved a 500-fold increased
exposure relative to aldesleukin. The active conjugated IL2 form of
NKTR-214 (2-PEG-IL2 and 1-PEG-IL2 together) was also quantified and remained detectable in tumor for up to 5 days yielding
an AUC of 23 4.4 mg/g. Hence, exposure to active conjugated IL2
was 50-times higher compared with aldesleukin, translating to
380 times increased exposure relative to an equivalent dose of
aldesleukin. The tumor exposure of NKTR-214 allows dosing once
every 9 days in mice compared with twice daily for two 5-day
cycles for aldesleukin.
Immune cell phenotyping: NKTR-214 favors proliferation and
infiltration of memory CD8 T cells over Tregs in the tumor
The in vitro binding and activation profiles described above
suggest PEGylation interfered with the interaction between IL2
and IL2Ra relative to aldesleukin. We therefore tested whether
these effects bias the profile of immune cell subtypes in vivo. The
number of CD8 T and Treg cells in the tumor following administration of either NKTR-214 or IL2 is a key measure of whether
pleiotropic effects of IL2 have been shifted due to conjugation of
IL2 to PEG at the IL2/IL2Ra interface. To address the question,
mice bearing subcutaneous B16F10 mouse melanoma tumors
were treated with a single dose of NKTR-214 or 5 doses of
aldesleukin, and immune cells in the tumor microenvironment
were quantified by flow cytometry. In tumors of aldesleukintreated mice, total and memory CD8 cells were increased as a
percentage of tumor-infiltrating lymphocytes; however, these
effects were transient, reaching significance relative to vehicle on
day 5 (Fig. 4A). In contrast, significant (P < 0.05) and sustained
total and memory CD8 T-cell stimulation was achieved following
a single NKTR-214 administration, with superior percentages of
memory CD8 (day 7) and total CD8 (days 7 and 10) relative to
aldesleukin. Both NKTR-214 and aldesleukin treatment resulted
in increased activated natural killer (NK) cells 5 and 7 days after
treatment initiation, though this effect was diminished by day 10.
CD4 cell percentages of tumor-infiltrating lymphocytes were
diminished following NKTR-214 treatment relative to vehicle on
day 5. On day 10, NKTR-214 resulted in fewer CD4 cell percentages compared with vehicle and aldesleukin. The CD4 cell
population was further analyzed for the FoxP3þ subset, which
defines the Treg population. NKTR-214 administration reduced
percentage of Tregs at every time point, consistent with reduced
access to the IL2Ra subunit arising from the PEG chains. In
Clin Cancer Res; 22(3) February 1, 2016
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2016 American Association for Cancer Research.
683
Charych et al.
Figure 2.
A, schematic of the inactive NKTR-214 prodrug releasing to IL2 conjugates bound by fewer PEG chains with increasing bioactivity. Red depicts the IL2 cytokine core,
and orange depicts the polymer chains located at the IL2/IL2Ra interface. B, identification of the most active IL2 conjugates created by hydrolysis of
NKTR-214 as measured by phosphorylation of STAT5 in murine CTLL-2 T cells. Purified forms of the released conjugated IL2 forms were assayed. On the basis of the
EC50, the 2-PEG-IL2 and 1-PEG-IL2 derived from NKTR-214 are identified as the most active conjugates released from NKTR-214. EC50 values for STAT5
phosphorylation were determined from two or more independent experiments, each performed in triplicate. The IL2 control is a purified reference standard.
contrast, Treg reduction with aldesleukin was modest achieving
significance on day 5. The increase of CD8 T cells and reduction of
Tregs led to a marked elevation of the CD8/Treg ratio in the tumor
by day 7. The ratio of CD8/Treg for NKTR-214, aldesleukin, and
vehicle was 449, 18, and 4, respectively.
While these results provide quantification of immune cells in
the tumor microenvironment, the spatial relationship between T
cells and tumor cells cannot be visualized. Therefore, immunohistochemical staining was performed (Fig. 4B), with representative images showing CD8 T cells are not only increased in
number but are interspersed with tumor cells.
These results indicate NKTR-214 is capable of inducing more
robust in vivo memory effector CD8 T-cell response, than seen with
aldesleukin, without a commensurate stimulation of Tregs in
tumor, consistent with in vitro IL2Rb-biased binding profile of
active IL2 conjugates derived from NKTR-214. These in vivo effects
arise although 7.5-fold fewer IL2 equivalents were administered
for NKTR-214 compared with aldesleukin.
NKTR-214 provides superior single-agent efficacy compared
with aldesleukin in an aggressive mouse melanoma model
Given the markedly increased ratio of CD8 T cells/Tregs in
the tumor microenvironment following NKTR-214 treatment, we
compared the efficacy of NKTR-214 to aldesleukin in the B16F10
684 Clin Cancer Res; 22(3) February 1, 2016
murine melanoma model. NKTR-214 and aldesleukin led to
significant tumor growth inhibition at day 9 compared with
vehicle, 80% and 91%, respectively (Fig. 5A). In animals who
received vehicle, the tumors rapidly grew to the endpoint of 1,500
mm3 8 days after treatment, leading to median tumor growth
delay, defined as time to reach 4 times the initial volume or TVQT
of 5 days. Aldesleukin significantly increased the TVQT to 10 days
compared with vehicle. For animals treated with NKTR-214, the
TVQT was increased 16.7 days providing significant tumor growth
inhibition compared with aldesleukin, despite 10-fold fewer IL2
equivalents and reduced dosing frequency (Fig. 5B). Although no
significant body weight loss was recorded, mice in the aldesleukin
group became hypothermic and exhibited shivering behavior,
unlike the NKTR-214–treated group where mild weakness was
observed.
NKTR-214 is synergistic with an anti–CTLA-4 antibody
Checkpoint inhibitor antibody anti–CTLA-4 antagonizes the
inhibitory interaction between CTLA-4 and its ligand B7 on
antigen-presenting cells (20). To test the concept of T-cell activation using dual mechanisms of checkpoint blockade and direct
cytokine stimulation, NKTR-214 and murine anti–CTLA-4 were
evaluated in two syngeneic tumor models. No significant effect on
tumor growth was seen when the EMT6 murine breast tumor
Clinical Cancer Research
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2016 American Association for Cancer Research.
NKTR-214, an Engineered Cytokine for Cancer Immunotherapy
Figure 3.
Tumor pharmacokinetics of NKTR-214 and its released active conjugatedIL2 forms. B16F10 mouse melanoma tumor tissue was analyzed after a
single dose of NKTR-214 or 5 daily doses of aldesleukin. NKTR-214 (all
conjugated IL2 species, black squares) and active conjugated IL2 from
NKTR-214 (1-PEG-IL2 and 2-PEG-IL2 blue circles) are shown. Aldesleukin is
indicated by red triangles. When adjusted for dose equivalency, tumor
exposure for NKTR-214 was approximately 500 times higher as 7.5-fold
fewer total IL2 equivalents were administered for NKTR-214. Active
conjugated IL2 forms from NKTR-214 are 380 times higher than
aldesleukin. For aldesleukin, solid red lines are measured; dotted red lines
represent extrapolated values based on measured values after day 1 and 5.
Graph shows mean SD, n ¼ 4 mice per time point.
model was treated with single-agent NKTR-214, aldesleukin, or
anti–CTLA-4, Fig. 6A. When aldesleukin was combined with anti–
CTLA-4, tumor growth inhibition of 83% was achieved on day 18;
however, the combination of NKTR-214 and CTLA-4 proved more
efficacious leading to tumor regression of 3.8%. After treatment
ended on day 21, both groups were followed for long-term tumor
growth delay. At the end of study (day 100), the aldesleukin
combination group maintained 40% tumor-free animals, with a
mean TVQT of 32.5 days, which was significant compared with
vehicle (TVQT 10.8 days). On the other hand, the TVQT of the
NKTR-214 and anti–CTLA-4 group could not be determined as
70% of the animals were tumor-free at day 100; 78 days after the
last dose of NKTR-214 was given. Although the CT26 murine
colon tumor model also exhibits aggressive growth, single-agent
NKTR-214 and anti–CTLA-4 each produced significant, if moderate, tumor growth inhibition of 51% and 60%, respectively, at
day 11 compared with vehicle (Fig. 6B). Neither single agent led to
significant mean TVQT changes, with 10% of animals remaining
tumor free at day 100. However, the combination of these agents
proved synergistic, with 89% tumor growth inhibition at day 11
and 67% tumor-free animals at day 100. For all treatment groups,
the combinations were well tolerated with no body weight loss
and no other obvious clinical signs. These data indicate synergy
between NKTR-214 and checkpoint blockade, with each using a
complementary mechanism of immune activation. Data demonstrating the antitumor immunity stimulated by this combination
requires innate and adaptive immune responses are shown in
Supplementary Fig. S3.
NKTR-214 combined with anti–CTLA-4 induces durable and
specific antitumor immunity
The combination studies described above suggest durability
of response; animals remain tumor-free for an extended period
after their last therapeutic dose. To more fully explore the
durability and specificity of antitumor activity induced by
www.aacrjournals.org
NKTR-214 combined with anti–CTLA-4 in the EMT6 model,
mice with complete responses to therapy (n ¼ 30) were rechallenged with a second EMT6 tumor implant or nonrelated CT26
implant (n ¼ 10) and monitored without additional therapy
(Fig. 6C and D). Tumor-free animals proved resistant to EMT6
rechallenge with 70% of the mice remaining tumor-free, while
CT26 tumor implants proceeded to grow. The tumor-free mice
from the EMT6 tumor rechallenge were challenged a third time
with EMT6, and followed without additional treatment. After
60 days, 100% of these animals remained tumor-free. The
resistance to tumor growth strongly suggests antitumor immunity induced by this combination therapy is tumor-specific and
long-lasting. Antibody depletion experiments (Supplementary
Materials and Methods and Supplementary Fig. S3) demonstrated CD8 T and NK cells were required for protection from
rechallenge.
NKTR-214 does not produce hypotension or vascular leak
syndrome in non-human primates at the maximum
tolerated dose
To proceed with clinical testing of NKTR-214 in humans,
safety was evaluated in cynomolgus monkeys. To establish the
cynomolgus monkey as a suitable toxicology species, human
and monkey peripheral blood mononuclear cells from three
donors each were exposed to human IL2 and active 1-PEG-IL2
from NKTR-214. The resulting IL2R activation measured by
pSTAT5 signaling was similar between monkey and human
primary T cells (Supplementary Table S2). The maximum
tolerated dose (MTD) was 0.1 mg/kg. Soluble CD25 and total
lymphocyte counts were used as pharmacodynamic markers of
immune stimulation (21). Both biomarkers increased in a
dose-responsive manner above baseline producing 24-fold and
4-fold increases in soluble CD25 and lymphocytes respectively
at MTD (Supplementary Fig. S4A). As vascular leak syndrome is
a known toxicity of HD-IL2 that is detected through hypotension, cardiovascular safety (ECG and blood pressure) was
monitored in cynomolgus monkeys using telemetry. No hypotension or ECG abnormalities were observed at MTD for 104
hours after dose administration (Supplementary Fig. S4B). Lack
of pulmonary edema was further confirmed by histopathology
(data not shown) with no evidence of lung infiltrates. Studies of
inflammatory cytokines TNFa and IFNg showed no detectable
levels, suggesting lack of toxicity-associated cytokine release,
despite robust immune activation (22). Together with the
murine antitumor efficacy, these results support the improved
safety and tolerability of NKTR-214.
Discussion
This report describes the development of NKTR-214 for
cancer therapy. To our knowledge, this is the first use of a
prodrug design for biologic therapy using PEG, releasable PEG
linkers, and site-directed PEG to alter the immunologic, pharmacokinetic, and biodistribution profile of the molecule (Figs.
3 and 4). The agent shows marked efficacy in aggressive murine
tumors as a single agent and in combination with anti–CTLA-4
(Figs. 5 and 6). NKTR-214 is a highly "combinable cytokine"
dosed more like an antibody than a cytokine; anticipated
dosing schedule in humans is once every 21 days. Yet NKTR214 provides a mechanism of direct immune stimulation
Clin Cancer Res; 22(3) February 1, 2016
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2016 American Association for Cancer Research.
685
Charych et al.
Figure 4.
Immune cell alterations in B16F10 mouse melanoma models following treatment with a single dose of NKTR-214 or 5 daily doses of aldesleukin. Tumorinfiltrating lymphocytes were isolated from animals at the time points indicated and immune cell populations were assessed by flow cytometry. Each data
point represents an individual mouse tumor and the line represents the mean. Data are combined from 2 to 4 independent studies with 3 to 4 replicates at
each time point. A, in the tumor, NKTR-214 administration led to marked increases in total and memory CD8 T cells, greater in magnitude and duration
than with aldesleukin. Both treatments led to increased NK cells. NKTR-214 also led to significant decreases in Treg percentages, leading to higher
CD8/Treg ratios of >400 compared with aldesleukin (ratio of 18) and vehicle (ratio of 4). B, immunohistochemical staining of B16F10 tumors showing
CD8 T cells (brown) in close proximity to tumor cells (purple). Representative fields from 30 to 60 images obtained per group are shown, n ¼ 3 mice per group.
Data shown are for day 7 after dose administration.
characteristic of cytokines. The combination of NKTR-214 with
murine anti–CTLA-4 results in tumor-free animals remaining
tumor-free for up to 100 days and resistant to tumor rechallenge (Fig. 6). NKTR-214 is well tolerated in non-human
primates, with no evidence of hypotension and vascular leak
syndrome at its MTD (Supplementary Fig. S4B).
PEGylation technology has existed for decades leading to
availability of multiple protein conjugates across diverse therapeutic areas including oncology (pegfilgrastim), infectious
disease (peginterferon alpha-2b) and autoimmune disease
(certolizumab pegol; ref. 23). In the past, the primary function
of PEG was to improve half-life using standard stable conjugation chemistry (24–26). In this report, PEG is used to bias
686 Clin Cancer Res; 22(3) February 1, 2016
receptor selectivity and alter pharmacokinetics. No mutations
were introduced into the molecule; therefore, the core of NKTR214 is identical to the well-understood aldesleukin amino acid
sequence. The presence of multiple PEG chains creates an
inactive prodrug, which prevents rapid systemic immune activation upon administration. Use of releasable linkers allows
PEG chains to slowly hydrolyze, continuously forming active
conjugated IL2 bound by 2-PEGs or 1-PEG. PEGylation dramatically alters the pharmacokinetics of NKTR-214 compared
with IL2, providing a 500-fold increase in AUC in the tumor
compared with an IL2 equivalent dose. The location of PEG
chains at the IL2/IL2Ra interface interferes with binding to
high-affinity IL2Ra, while leaving binding to low-affinity IL2Rb
Clinical Cancer Research
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2016 American Association for Cancer Research.
NKTR-214, an Engineered Cytokine for Cancer Immunotherapy
Figure 5.
Single-agent efficacy of NKTR-214 compared with aldesleukin in wellestablished subcutaneous B16F10 mouse melanoma tumors. Mice were
treated with NKTR-214, aldesleukin, or vehicle. Changes in mean tumor
volume over time (A) and the percentage of tumors below 4 times the initial
volume over time (B) are shown. Symbols are mean SEM. , statistical
significance (P < 0.05) compared with vehicle (ANOVA, Tukey post test);
z, P < 0.05 compared with aldesleukin (unpaired Student t test). The
experiment was repeated a total of 7 times with similar results.
unperturbed (Supplementary Fig. S2). The bias observed in vitro
changes the immune cell profile in vivo in the tumor microenvironment, leading to a maximal CD8/Tregs ratio of >400 for
NKTR-214 compared with 18 for aldesleukin treatment (Fig.
4A). Overcoming immune tolerance in the tumor microenvironment may require tipping the balance in favor of CD8 T over
Tregs in the tumor (10–13). Furthermore, CD8 T cells are
extensively interspersed with tumor cells (Fig. 4B). Infiltration
of CD8 T cells is relevant because previous immunotherapies
such as tumor antigen–specific vaccines may have failed due to
poor trafficking of these cells into the tumor (11). NK cell
responses are also mobilized by NKTR-214 treatment; both
innate and adaptive immune responses appear to play a role in
antitumor immunity. Together, these results suggest NKTR-214
favors immune activation over suppression in the tumor, with a
www.aacrjournals.org
mechanism different from the original cytokine and complementary to checkpoint blockade antibodies.
Numerous approaches to improve the safety profile of
aldesleukin have been reported; however, these were challenged by the absence of a defined mechanism for IL2 toxicity.
One hypothesis implicated NK cells as the mechanism of
toxicity; a mutein biased towards IL2Ra and against activation
of NK cells was developed. However, this proved ineffective in
the clinic with a lower MTD than aldesleukin (27, 28). Subsequently, other reports described modified forms of IL2 that
have attempted to favor ILRbg binding, prolong exposure, or
mitigate toxicity. These include mutation of the IL2 amino acid
sequence, fusion to a targeting antibody, or use of IL2/anti-IL2
antibody complexes (29–31). These alterations, like every new
agent, need to be evaluated clinically. Notably, NKTR-214
provides increased tumor exposure without assuming tumor
target selectivity and without the complications of fusing an
antibody to a cytokine. Unnatural protein fusion junctions
pose greater risk for immunogenicity. Receptor bias shown
with NKTR-214 is achieved using the clinically validated aldesleukin amino acid sequence and PEG, which is regarded as
safe and nonimmunogenic.
To assess the impact of receptor bias, prodrug design, and high
tumor concentrations on in vivo efficacy, we chose the subcutaneous B16F10 mouse melanoma model, a tumor known to be
aggressive and difficult to treat (32). NKTR-214 showed significantly improved efficacy in this model as a single agent compared
with aldesleukin. The improvement was achieved despite using
10-fold fewer IL2 equivalents. While 20 total doses of aldesleukin
were needed to achieve modest efficacy, only 3 doses of NKTR-214
were administered (Fig. 5). Interestingly anti–CTLA-4 was not
active in the B16F10 melanoma model as a single agent (32, 33).
The combination of NKTR-214 with anti–CTLA-4 proved synergistic in mouse efficacy models of breast and colon tumors, with
83% and 67% of animals remaining tumor-free up to 100 days,
respectively (Fig. 6A and B). Tumor-free animals could be challenged twice with the same tumors, and up to 100% of these
animals resisted tumor regrowth without further therapy (Fig.
6C). Recent clinical and preclinical studies combining immunotherapies such anti–CTLA-4 and anti–PD-1 indicate more
patients may benefit when multiple mechanisms of immune
activation are targeted (34–37). Our results suggest NKTR-214 is
positioned to provide a complementary mechanism of immune
activation in combination, using dosing schedules concurrent
with antibody therapy.
NKTR-214 is administered in vivo as an inactive prodrug,
potentially mitigating toxicities associated with rapid off-target systemic immune activation, which would occur upon
administration of HD-IL2 or other biologically active cytokines. Ability to dose fewer IL2 equivalents at a significantly
reduced frequency may also contribute to the improved tolerability observed with NKTR-214. In cynomolgus monkeys,
NKTR-214 could be dosed at MTD without cardiovascular
adverse effects, reflected in the absence of hypotension and
vascular leak syndrome. Total lymphocyte and soluble CD25
levels were used as pharmacodynamic markers and indicate
clear dose-dependent immune stimulation in monkeys, with
no evidence of cytokine release syndrome. These results suggest unlike HD-IL2, NKTR-214 may be dosed safely in
humans, potentially increasing the number of patients achieving durable responses.
Clin Cancer Res; 22(3) February 1, 2016
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2016 American Association for Cancer Research.
687
Charych et al.
Figure 6.
Combination of NKTR-214 with
checkpoint inhibition and tumor
rechallenge. A, EMT6 breast model:
aldesleukin or NKTR-214 in
combination with anti–CTLA-4
generated significant tumor growth
inhibition (left; , day 18) compared
with vehicle. A higher percentage of
tumor-free animals was achieved
when NKTR-214 was combined with
anti–CTLA-4 (right; z, 70% day 100);
n ¼ 12 mice per group. B, CT-26 colon
model: anti–CTLA-4 and NKTR-214 as
single agents generated significant
growth inhibition ( ), but the
combination of both agents was
synergistic (left; z, day 11) and lead to
significant percentage of tumor-free
animals (right; , 67% day 100);
n ¼ 12 mice per group. Tumor
rechallenge data: mean tumor
volumes (C) and individual animal
measurements (D). Combination
therapy induced complete responses
in 44% of treated animals. Tumor-free
responders were resistant to first
EMT6 rechallenge, with 70%
remaining tumor-free, but were
susceptible to CT26 tumor growth.
Complete responders from the first
rechallenge group were rechallenged
a second time with EMT6 implants, and
100% remained tumor-free without
additional treatment. Symbols
represent mean SEM. , P < 0.05
compared with vehicle (ANOVA,
Tukey post test); z, P < 0.05 compared
with aldesleukin (unpaired
Student t test).
Although it is difficult to directly translate efficacy in mouse
tumor models to human cancers, it is encouraging that the IL2
pathway is already a validated cancer immunotherapy target with
a history of durable benefit for some patients. The efficacy of
combination therapy using checkpoint blockade antibodies
appears to translate between mice and humans, increasing the
probability that NKTR-214 will produce robust efficacy in patients with cancer (34, 37). Although much of the current focus
688 Clin Cancer Res; 22(3) February 1, 2016
in cancer immunotherapy is on immune checkpoint blockade
using antibodies, cytokines will play an important role in providing complementary mechanisms of action, as single agents
and in combination. The data presented in this report indicate
the immunologic, pharmacokinetic, and pharmacologic properties of the endogenous cytokine IL2 were significantly altered
by strategic polymer conjugation. As such, NKTR-214 is currently
being evaluated in a Phase 1 clinical study in solid tumors.
Clinical Cancer Research
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2016 American Association for Cancer Research.
NKTR-214, an Engineered Cytokine for Cancer Immunotherapy
Disclosure of Potential Conflicts of Interest
D. Sheng, T.K. Chang, and J. Huang have ownership interest (including
patents) in Nektar Therapeutics. No potential conflicts of interest were disclosed
by the other authors.
Authors' Contributions
Conception and design: D.H. Charych, U. Hoch, J.L. Langowski, S.R. Lee,
P.B. Kirk, X. Liu, C.F. Ali, J. Huang, S.S. Kantak, S.K. Doberstein
Development of methodology: D.H. Charych, U. Hoch, J.L. Langowski,
S.R. Lee, M.K. Addepalli, P.B. Kirk, D. Sheng, X. Liu, L.A. VanderVeen, C.F. Ali,
T.K. Chang, M. Konakova, R.S. Kanhere, Y. Wang, J. Huang, S.S. Kantak,
S.K. Doberstein
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): D.H. Charych, S.R. Lee, M.K. Addepalli, P.B. Kirk,
X. Liu, P.W. Sims, L.A. VanderVeen, C.F. Ali, T.K. Chang, Y.M. Kirksey, C. Ji,
Y. Wang, T.D. Sweeney, S.S. Kantak
Analysis and interpretation of data (e.g., statistical analysis, biostatistics,
computational analysis): D.H. Charych, U. Hoch, J.L. Langowski, S.R. Lee,
M.K. Addepalli, P.B. Kirk, X. Liu, L.A. VanderVeen, C.F. Ali, T.K. Chang, R.L. Pena,
R.S. Kanhere, Y.M. Kirksey, C. Ji, Y. Wang, T.D. Sweeney, S.S. Kantak
Writing, review, and/or revision of the manuscript: D.H. Charych, J.L. Langowski, S.R. Lee, P.B. Kirk, D. Sheng, P.W. Sims, L.A. VanderVeen, C.F. Ali, T.K.
Chang, M. Konakova, C. Ji, J. Huang, T.D. Sweeney, S.S. Kantak, S.K. Doberstein
Administrative, technical, or material support (i.e., reporting or organizing
data, constructing databases): J.L. Langowski, S.R. Lee, M.K. Addepalli,
Y.M. Kirksey, C. Ji, S.S. Kantak
Study supervision: D.H. Charych, U. Hoch, J.L. Langowski, S.R. Lee,
M.K. Addepalli, R.L. Pena, C. Ji, S.S. Kantak, S.K. Doberstein
Other (initiated and developed the concept of the program and molecule
with help from the team): D.H. Charych
Acknowledgments
The authors thank Dr. Mario Sznol, Yale University, for his careful reading
and critique of this manuscript. The authors also thank Pai Charaweeyawong,
Nektar Therapeutics, for his technical contributions and Phillips Gilmore
Oncology Communications, Inc. for technical support with manuscript
preparation.
Grant Support
This study was financially supported by Nektar Therapeutics.
The costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received July 14, 2015; revised October 21, 2015; accepted October 23, 2015;
published online February 1, 2016.
References
1. Pennock G, Chow L. The evolving role of immune checkpointe inhibitors
in cancer treatment. Oncologist 2015;20:1–11.
2. Rosenberg SA. Raising the bar: the curative potential of human cancer
immunotherapy. Sci Transl Med 2012;4:127ps8.
3. Smith FO, Downey SG, Klapper JA, Yang JC, Sherry RM, Royal RE, et al.
Treatment of metastatic melanoma using interleukin-2 alone or in conjunction with vaccines. Clin Cancer Res 2008;14:5610–8.
4. Klapper JA, Downey SG, Smith FO, Yang JC, Hughes MS, Kammula US,
et al. High-dose interleukin-2 for the treatment of metastatic renal cell
carcinoma: a retrospective analysis of response and survival in patients
treated in the Surgery Branch at the National Cancer Institute between 1986
and 2006. Cancer 2008;113:293–301.
5. Atkins MB, Lotze MT, Dutcher JP, Fisher RI, Weiss G, Margolin K, et al. Highdose recombinant interleukin 2 therapy for patients with metastatic melanoma: analysis of 270 patients treated between 1985 and 1993. J Clin
Oncol 1999;17:2105–16.
6. Rosenberg SA, Yang JC, White DE, Steinberg SM. Durability of complete
responses in patients with metastatic cancer treated with high-dose interleukin-2 - Identification of the antigens mediating response. Ann Surg
1998;228:307–19.
7. Wang X, Rickert M, Garcia KC. Structure of the quaternary complex of
interleukin-2 with its alpha, beta, and gammac receptors. Science 2005;
310:1159–63.
8. Margolin K. Cytokine therapy in cancer. Expert Opin Biol Ther 2008;
8:1495–505.
9. Boyman O, Sprent J. The role of interleukin-2 during homeostasis and
activation of the immune system. Nat Rev Immunol 2012;12:180–90.
10. Curran MA, Montalvo W, Yagita H, Allison JP. PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and
myeloid cells within B16 melanoma tumors. Proc Natl Acad Sci U S A
2010;107:4275–80.
11. Gajewski TF, Woo SR, Zha Y, Spaapen R, Zheng Y, Corrales L, et al. Cancer
immunotherapy strategies based on overcoming barriers within the tumor
microenvironment. Curr Opin Immunol 2013;25:268–76.
12. Zhou P, Zheng X, Zhang H, Liu Y, Zheng P. B7 blockade alters the balance
between regulatory T cells and tumor-reactive T cells for immunotherapy of
cancer. Clin Cancer Res 2009;15:960–70.
13. Wang HY, Lee DA, Peng G, Guo Z, Li Y, Kiniwa Y, et al. Tumor-specific
human CD4þ regulatory T cells and their ligands: implications for immunotherapy. Immunity 2004;20:107–18.
14. Rickert M, Wang X, Boulanger MJ, Goriatcheva N, Garcia KC. The structure
of interleukin-2 complexed with its alpha receptor. Science 2005;308:
1477–80.
www.aacrjournals.org
15. Walunas TL, Lenschow DJ, Bakker CY, Linsley PS, Freeman GJ, Green JM,
et al. CTLA-4 can function as a negative regulator of T cell activation.
Immunity 1994;1:405–13.
16. UC10-4F10-11 (ATCC HB-304) culture method; 2015 [accessed
2015 Jun 29]. Available from http://www.atcc.org/Products/All/HB-304.
aspx#culturemethod.
17. Stauber DJ, Debler EW, Horton PA, Smith KA, Wilson IA. Crystal structure
of the IL-2 signaling complex: paradigm for a heterotrimeric cytokine
receptor. Proc Natl Acad Sci U S A 2006;103:2788–93.
18. Gibbons JA, Luo ZP, Hannon ER, Braeckman RA, Young JD. Quantitation
of the renal clearance of interleukin-2 using nephrectomized and ureterligated rats. J Pharmacol Exp Ther 1995;272:119–25.
19. Konrad MW, Hemstreet G, Hersh EM, Mansell PW, Mertelsmann R, Kolitz
JE, et al. Pharmacokinetics of recombinant interleukin 2 in humans. Cancer
Res 1990;50:2009–17.
20. Leach DR, Krummel MF, Allison JP. Enhancement of antitumor immunity
by CTLA-4 blockade. Science 1996;271:1734–6.
21. Thompson JA, Curti BD, Redman BG, Bhatia S, Weber JS, Agarwala SS, et al.
Phase I study of recombinant interleukin-21 in patients with metastatic
melanoma and renal cell carcinoma. J Clin Oncol 2008;26:2034–9.
22. Anderson JA, Lentsch AB, Hadjiminas DJ, Miller FN, Martin AW, Nakagawa
K, et al. The role of cytokines, adhesion molecules, and chemokines in
interleukin-2-induced lymphocytic infiltration in C57BL/6 mice. J Clin
Invest 1996;97:1952–9.
23. Patel JN, Walko CM. Sylatron: a pegylated interferon for use in melanoma.
Ann Pharmacother 2012;46:830–8.
24. Parveen S, Sahoo SK. Nanomedicine: clinical applications of polyethylene
glycol conjugated proteins and drugs. Clin Pharmacokinet 2006;45:
965–88.
25. Fishburn CS. The pharmacology of PEGylation: balancing PD with PK to
generate novel therapeutics. J Pharm Sci 2008;97:4167–83.
26. Kunugi K, Yamaoka T. Polymers in nanomedicine. Springer-Verlag, Heidelberg; New York, NY; 2012. p.xi, 281p.
27. Shanafelt AB, Lin Y, Shanafelt MC, Forte CP, Dubois-Stringfellow N,
Carter C, et al. A T-cell-selective interleukin 2 mutein exhibits potent
antitumor activity and is well tolerated in vivo. Nat Biotechnol
2000;18:1197–202.
28. Margolin K, Atkins MB, Dutcher JP, Ernstoff MS, Smith JW, Clark JI, et al.
Phase I trial of BAY 50-4798, an interleukin-2-specific agonist in advanced
melanoma and renal cancer. Clin Cancer Res 2007;13:3312–9.
29. Krieg C, Letourneau S, Pantaleo G, Boyman O. Improved IL-2 immunotherapy by selective stimulation of IL-2 receptors on lymphocytes and
endothelial cells. Proc Natl Acad Sci U S A 2010;107:11906–11.
Clin Cancer Res; 22(3) February 1, 2016
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2016 American Association for Cancer Research.
689
Charych et al.
30. Gillies SD, Lan Y, Hettmann T, Brunkhorst B, Sun Y, Mueller SO, et al. A
low-toxicity IL-2-based immunocytokine retains antitumor activity
despite its high degree of IL-2 receptor selectivity. Clin Cancer Res
2011;17:3673–85.
31. Levin AM, Bates DL, Ring AM, Krieg C, Lin JT, Su L, et al. Exploiting a natural
conformational switch to engineer an interleukin-2 `superkine'. Nature
2012;484:529–33.
32. Sundstedt A, Celander M, Eriksson E, Torngren M, Hedlund G. Monotherapeutically nonactive CTLA-4 blockade results in greatly enhanced
antitumor effects when combined with tumor-targeted superantigens in a
B16 melanoma model. J Immunother 2012;35:344–53.
33. Quezada SA, Peggs KS, Curran MA, Allison JP. CTLA4 blockade and GMCSF combination immunotherapy alters the intratumor balance of effector
and regulatory T cells. J Clin Invest 2006;116:1935–45.
690 Clin Cancer Res; 22(3) February 1, 2016
34. Ott PA, Hodi FS, Robert C. CTLA-4 and PD-1/PD-L1 blockade: new
immunotherapeutic modalities with durable clinical benefit in melanoma
patients. Clin Cancer Res 2013;19:5300–9.
35. Wolchok JD, Kluger H, Callahan MK, Postow MA, Rizvi NA, Lesokhin AM,
et al. Nivolumab plus ipilimumab in advanced melanoma. N Engl J Med
2013;369:122–33.
36. Hafalla JC, Claser C, Couper KN, Grau GE, Renia L, de Souza JB, et al. The
CTLA-4 and PD-1/PD-L1 inhibitory pathways independently regulate host
resistance to Plasmodium-induced acute immune pathology. PLoS Patholog 2012;8:e1002504.
37. Duraiswamy J, Kaluza KM, Freeman GJ, Coukos G. Dual blockade
of PD-1 and CTLA-4 combined with tumor vaccine effectively
restores T-cell rejection function in tumors. Cancer Res 2013;73:
3591–603.
Clinical Cancer Research
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2016 American Association for Cancer Research.
NKTR-214, an Engineered Cytokine with Biased IL2 Receptor
Binding, Increased Tumor Exposure, and Marked Efficacy in Mouse
Tumor Models
Deborah H. Charych, Ute Hoch, John L. Langowski, et al.
Clin Cancer Res 2016;22:680-690.
Updated version
Supplementary
Material
Cited articles
Citing articles
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://clincancerres.aacrjournals.org/content/22/3/680
Access the most recent supplemental material at:
http://clincancerres.aacrjournals.org/content/suppl/2016/01/29/22.3.680.DC1
This article cites 35 articles, 18 of which you can access for free at:
http://clincancerres.aacrjournals.org/content/22/3/680.full#ref-list-1
This article has been cited by 1 HighWire-hosted articles. Access the articles at:
http://clincancerres.aacrjournals.org/content/22/3/680.full#related-urls
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at
[email protected].
To request permission to re-use all or part of this article, contact the AACR Publications Department at
[email protected].
Downloaded from clincancerres.aacrjournals.org on June 15, 2017. © 2016 American Association for Cancer Research.