Target Journal: Cancer Discover Research Briefs...NKTR-214 EXCEL Manuscript Page 1 of 21 Target...

NKTR-214 EXCEL Manuscript

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Target Journal: Cancer Discover – Research Briefs

Title: A First-in-Human Study and Biomarker Analysis of NKTR-214, a Novel IL-2-Receptor Beta/Gamma

(βγ)-Biased Cytokine, in Patients With Advanced or Metastatic Solid Tumors

Authors: Salah-Eddine Bentebibel1*

, Michael E. Hurwitz2*

, Chantale Bernatchez1*

, Cara Haymaker1, Courtney

W. Hudgens1, Harriet M. Kluger

2, Michael T. Tetzlaff

1, Mary A. Tagliaferri

3, Jonathan Zalevsky

3, Ute Hoch

3,

Christie Fanton3, Sandra Aung

3, Patrick Hwu

1, Brendan D. Curti

4, Nizar M. Tannir

1, Mario Sznol

2, Adi Diab

1

Affiliations:

1. The University of Texas MD Anderson Cancer Center, Houston, TX

2. Yale School of Medicine, New Haven, CT

3. Nektar Therapeutics, San Francisco, CA

4. Providence Cancer Institute and Earle A. Chiles Research Institute, Portland, OR

*These authors contributed equally

Running Title: NKTR-214 in Patients With Advanced Solid Tumors

Key Words: NKTR-214, IL-2R, tumor microenvironment, lymphocytes, immune activation

Financial Support: Nektar Therapeutics, San Francisco, CA

Corresponding Author:

Adi Diab, MD

The University of Texas MD Anderson Cancer Center

1515 Holcombe Blvd

Houston, TX 77030

Phone Number: (713)792-2921

Fax Number: (713) 745-1046

Email: [email protected]

Research. on June 20, 2020. © 2019 American Association for Cancercancerdiscovery.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on April 15, 2019; DOI: 10.1158/2159-8290.CD-18-1495

mailto:[email protected]

http://cancerdiscovery.aacrjournals.org/


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Conflict of Interest Disclosure Statement:

Salah-Eddine Bentebibel: No Relationships to Disclose

Michael Hurwitz: Consulting or Advisory Role Nektar

Chantale Bernatchez: Employment - Lexicon (Family member); Stock and Other Ownership Interests -

Lexicon (Family member); Consulting or Advisory Role - Lion Biotechnologies; Research Funding - Idera;

Nektar; Patents, Royalties, Other Intellectual Property - Patent pending on BTLA as a marker for better CD8 T

cells for adoptive immunotherapy

Cara Haymaker: Research Funding - Idera; Nektar

Courtney W. Hudgens: No Relationships to Disclose

Harriet Kluger: Consulting or Advisory Role - Alexion Pharmaceuticals; Biodesix; Corvus Pharmaceuticals;

Genentech/Roche; Nektar; Pfizer; Research Funding - Bristol-Myers Squibb (Inst); Merck (Inst); Travel,

Accommodations, Expenses - Bristol-Myers Squibb

Michael Tetzlaff: Consulting or Advisory Role - Myriad Genetics; Novartis

Mary Tagliaferri: Employment – Nektar; Leadership – Nektar; Stock and Other Ownership Interests - Nektar;

Patents, Royalties, Other Intellectual Property – Nektar

Jonathan Zalevsky: Employment – Nektar; Leadership – Nektar; Stock and Other Ownership Interests - Nektar;

Patents, Royalties, Other Intellectual Property - Nektar

Ute Hoch: Employment – Nektar; Stock and Other Ownership Interests - Nektar; Patents, Royalties, Other

Intellectual Property - Nektar

Christie Fanton: Employment – Nektar; Stock and Other Ownership Interests - Nektar; Novartis

Sandra Aung: Employment – Nektar; Leadership – Nektar; Stock and Other Ownership Interests - Nektar;

Travel, Accommodations, Expenses - Nektar

Patrick Hwu: Stock and Other Ownership Interests - immatics; Lion Biotechnologies; Consulting or Advisory

Role - Lion Biotechnologies; Research Funding - Genentech (Inst)

Brendan Curti: Consulting or Advisory Role - Alligator Bioscience; Eisai; Speakers' Bureau - Bristol-Myers

Squibb; Prometheus Laboratories; Research Funding - Bristol-Myers Squibb (Inst); Galectin Therapeutics





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(Inst); MedImmune (Inst); Prometheus Laboratories (Inst); Viralytics (Inst); Patents, Royalties, Other

Intellectual Property - Biomarkers for OX40 response (Inst); Travel, Accommodations, Expenses - Bristol-

Myers Squibb; MedImmune; Prometheus Laboratories

Nizar M. Tannir: Honoraria - Argos Therapeutics; Bristol-Myers Squibb; Calithera Biosciences; Exelixis;

Nektar; Novartis; Pfizer; Consulting or Advisory Role - Argos Therapeutics; Bristol-Myers Squibb; Exelixis;

Nektar; Novartis; Pfizer; Research Funding - Bristol-Myers Squibb; Epizyme; Exelixis; Mirati Therapeutics;

Novartis; Travel, Accommodations, Expenses - Argos Therapeutics; Argos Therapeutics; Bristol-Myers Squibb;

Calithera Biosciences; Exelixis; Nektar; Novartis; Pfizer

Mario Sznol: Stock and Other Ownership Interests - Adaptive Biotechnologies; Amphivena; Intensity

Therapeutics; Consulting or Advisory Role - Adaptimmune; Agonox; Alexion Pharmaceuticals;

AstraZeneca/MedImmune; Baxalta/Shire; Biodesix; Bristol-Myers Squibb; Genentech/Roche; Ignyta; Immune

Design; Inovio Pharmaceuticals; Janssen Oncology; Kyowa Hakko Kirin; Lilly; Lion Biotechnologies; Lycera;

Merck Sharp & Dohme; Modulate; Molecular Partners; Nektar; Newlink Genetics; Novartis; Omniox; pfizer;

Pierre Fabre; Seattle Genetics; Symphony Evolution; Theravance; Vaccinex; Other Relationship -

AcademicCME; Clinical Care Options; DAVAOncology; Haymarket Media; Imedex; Physician Education

Resource; Prime Oncology; Research to Practice; TRM Oncology; Vindico

Adi Diab: Consulting or Advisory Role with Celgene; CureVac; Nektar, Research Funding Celgene (Inst); Idera

(Inst); Nektar (Inst); Pfizer (Inst); and Travel, Accommodations, Expenses Nektar





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Abstract

NKTR-214 (bempegaldesleukin) is a novel IL-2 pathway agonist, designed to provide sustained signaling

through heterodimeric IL-2-Receptor Beta/Gamma to drive increased proliferation and activation of CD8+ T

and NK cells without unwanted expansion of T regulatory cells (Tregs) in the tumor microenvironment. In this

first-in-human multicenter phase 1 study, NKTR-214 administered as an outpatient regimen was well tolerated

and showed clinical activity including tumor shrinkage and durable disease stabilization in heavily treated

patients. Immune activation and increased numbers of immune cells were observed in the periphery across all

doses and cycles with no loss of NKTR-214 activity with repeated administration. On-treatment tumor biopsies

demonstrated that NKTR-214 promoted immune cell increase with limited increase of Tregs. Transcriptional

analysis of tumor biopsies showed that NKTR-214 engaged the IL-2 receptor pathway and significantly

increased genes associated with an effector phenotype. Based on safety and pharmacodynamic markers, the

recommended phase 2 dose was determined to be 0.006 mg/kg q3w.

Statement of Significance

We believe that IL-2 and IL-2 pathway targeted agents such as NKTR-214 are key components to an optimal

immunotherapy treatment algorithm. Based on its biological activity and tolerability, NKTR-214 is being

studied with approved I-O agents including checkpoint inhibitors.





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INTRODUCTION

In the treatment of patients with cancer, low levels of tumor-infiltrating lymphocytes (TILs) prior to the

introduction of checkpoints inhibitors is predictive of a poor response.1-3

Stimulation of the interleukin-2

receptor (IL-2R) pathway is a potent means of expanding and activating immune cells, specifically lymphocytes

including cytotoxic CD8+ T cells.

4 FDA-approved high-dose (HD) IL-2 (aldesleukin) treatment mediates

durable clinical benefit in a limited number of patients with melanoma and renal cell carcinoma (RCC),

presumably through CD8+

T cell expansion.5 However, the frequent and rapid induction of the IL-2 cytokine

signaling cascade is associated with an increased risk for serious adverse events (SAEs).6-10

The ability of IL-2

to broadly expand many lymphocyte cell populations is a critical feature of its mechanism of action as an

immunotherapy and a non-redundant role of IL-2 biology.

NKTR-214 (bempegaldesleukin) is a human recombinant IL-2 attached to an average of six releasable

polyethylene glycol (PEGs) chains.11,12

In the 1980s, polyethylene-glycol modified interleukin-2 (PEG-IL-2)

was developed in an effort to improve the solubility and decrease the plasma clearance of IL-2.13

Results of a

phase I study describing the use of polyethylene-glycol modified interleukin-2 (PEG-IL-2) in combination with

high dose IL-2 to treat patients with metastatic renal cell carcinoma and melanoma were reported in 1995.14

Results from the phase I study concluded that the toxicities and tumor response rates to combination treatment

with high-dose IL-2 followed by administration of PEG-IL-2 were similar to those of high dose unconjugated

IL-2 alone. Further development of PEG-IL-2 was discontinued. In the case of NKTR-214, attachment of PEG

chains can also be creatively utilized to not only alter a molecule’s PK but also to alter its receptor binding.

NKTR-214 is engineered to bias receptor selectivity and alter pharmacokinetics of IL-211,12

, while leaving the

well-understood amino acid sequence of IL-2 (aldesleukin) unaltered. When fully PEGylated, NKTR-214 is a

pro-drug that has essentially no biological activity. Upon intravenous administration, the PEG chains slowly

release to generate active cytokine species with limited binding to the IL-2Rα subunit, thereby biasing signaling

to favor the dimeric IL-2Rβγ (CD122). Consequently, NKTR-214 increases the proliferation, activation, and

effector function of CD8+ T cells and natural killer (NK) cells within the tumor microenvironment (TME)

without expanding the undesirable intra-tumoral T regulatory cells (T regs).11

In murine tumor models, NKTR-

214 generated a ratio of CD8+ cytotoxic T cells to Tregs of > 400:1 in tumor, and when combined with a

checkpoint inhibitor antibody, either anti-cytotoxic T lymphocyte antigen 4 (anti-CTLA-4) or anti-programmed

cell death protein 1 (anti-PD-1), there was a strong synergistic effect.11

Based on supportive preclinical efficacy,

immunobiology data, and pharmacology and toxicology studies performed in rats and monkeys, a first-in-

human study was initiated using NKTR-214 in patients with advanced solid tumors. We report here on the





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safety, pharmacodynamics, immunological and antitumor activity of NKTR-214 monotherapy in patients

treated with multiple prior lines of therapy.

RESULTS

Patient characteristics and disposition

Twenty-eight patients with advanced or locally advanced solid tumor malignancies were enrolled across three

centers from December 2015 to April 2017. Demographics and disease characteristics are summarized in

Supplementary Table S1. The majority of patients had a diagnosis of metastatic RCC (n=15; 53.6%) or

melanoma (n=7; 25.0%). All patients had received prior anti-cancer treatment: 16 (57.1%) had received targeted

therapy; 16 (57.1%) had received an immune checkpoint inhibitor; and, 6 (21.4%) had received an immune

checkpoint inhibitor in addition to other immunotherapy. The median number of prior therapies was 2 (range: 1-

12) (Supplementary Fig. S1A). The most common reason for study drug discontinuation was progressive

disease by RECIST (n=15; 53.6%) or physician decision (n=6; 21.4%), primarily for patients who did not

respond rapidly and had other therapeutic options. One patient discontinued treatment due to an infusion-related

reaction. Patients received a median of 3 infusions of NKTR-214 (range: 1–25 infusions), with a median

duration of exposure of 69.5 days (range: 15–533 days).

Safety and tolerability

All 28 patients enrolled in the study had treatment-emergent adverse events (TEAEs), with twenty-six patients

having TEAEs that were considered related to study drug. TEAEs and Treatment-related adverse events

(TRAEs) occurring in ≥3 patients across all doses are listed in Table 1 and in Supplementary Table S2,

respectively. Six of 28 (21.4%) patients reported grade 3 TRAEs; there were no grade 4 or 5 TRAEs. At the

highest NKTR-214 dose tested (0.012 mg/kg), one patient experienced two DLTs (grade 3 hypotension and

grade 3 syncope), which resolved within 24 hours following intravenous fluid treatment and one dose of

tocilizumab. This patient was discharged from the hospital two days after admission for the events and

continued on study receiving two additional doses of NKTR-214 0.006 mg/kg that were well tolerated. No other

patient was dosed at 0.012 mg/kg. At a NKTR-214 dose of 0.009 mg/kg, one patient discontinued the study due

to an SAE of grade 3 infusion-related reaction. The reaction occurred following the first dose of NKTR-214 and

resolved with treatment. This patient had a history of an infusion-related reaction to a previous immuno-

oncology agent. There was one reported irAE of hypothyroidism associated with NKTR-214, which was treated

with replacement therapy. The MTD was defined as the highest dose of NKTR-214 that was administered





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without causing any unacceptable side effects or AEs. DLTs and AEs were considered in the determination of

the MTD, which was deemed to be 0.009 mg/kg q3w.

The most common TRAEs included fatigue (71%), flu-like symptoms (68%), pruritus (64%), hypotension

(57%), rash (50%), decreased appetite (46%), and arthralgia and cough (each 32%). Flu-like symptoms were

managed with acetaminophen and nonsteroidal anti-inflammatory drugs. The median duration of flu-like

symptoms, including fever, was 72 hours and became less pronounced after cycle 2. Rash and pruritus were

either self-limited or treated with antihistamines. Hypotension usually occurred around 48–72 hours following

the first NKTR-214 infusion. Declines in blood pressure following administration of NKTR-214 were generally

clinically asymptomatic.

All grade 3 TEAEs of hypotension (5 of 28 [18%] patients) were rapidly reversed with intravenous fluid

administration, and no patients discontinued treatment due to hypotension. Risk minimization guidelines, which

included oral and IV fluids during the first week of dosing, were implemented and successfully reduced the

incidence of grade 3 hypotension. The frequency of drug administration, q2w or q3w at a NKTR-214 dose of

0.006 mg/kg, did not appear to impact the safety profile. Based on safety and pharmacodynamics markers, the

RP2D was determined to be 0.006 mg/kg q3w.

Pharmacokinetic and pharmacodynamics

NKTR-214-related cytokine (RC) represents a mixture of compounds containing IL-2 independent of the PEG-

conjugation status, and NKTR-214-active cytokine (AC) represents a mixture of 2-PEG-IL-2 and 1-PEG-IL-2,

and potentially unconjugated IL-2, although this species is often undetectable from patient samples; the mean

observed plasma concentration-time profiles are shown in Supplementary Fig. S2A. NKTR-214-RC maximal

concentrations were achieved shortly after the end of the infusion and declined monoexponentially thereafter.

NKTR-214-RC concentrations remained detectable for 8 to 11 days post-dose. NKTR-214-AC concentrations

increased gradually after dosing with Tmax between 24 and 48 hours. NKTR-214-RC and NKTR-214-AC

exposure were similar across patients within a dose cohort and cycles, without indication of accumulation with

either the q3w or q2w administration schedules (data not shown). Unconjugated IL-2 levels were detected in 76

of 425 samples with reportable results. Unconjugated IL-2 concentrations in samples with detectable levels

ranged between 0.509 and 4.44 ng/mL with one concentration at 19.4 ng/mL. Out of 425 samples analyzed, 378

samples (89%) had concentrations of unconjugated IL-2 that were below the limit of quantitation or less than 1





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ng/mL (the lower limit of quantitation was 0.5 ng/mL) (data not shown). Soluble CD25 (sCD25) and

lymphocyte levels were measured as pharmacodynamic indicators of immune system activation

(Supplementary Fig. S2B and S2C). Changes in absolute lymphocyte counts were observed with each q3w

treatment cycle and included lymphopenia on day 3 followed by lymphocytosis, which peaked between days 8

to 11, and returned to near baseline levels prior to the next cycle. Similarly, sCD25 levels increased within each

treatment cycle, peaked around day 8 and returned to near baseline levels prior to each qw3cycle. PD kinetics

were observed at all dose levels and at the q2w dosing schedule (data not shown). Anti-NKTR-214 or anti-IL-2

anti-drug antibodies were not present in any samples tested for this study (data not shown).

Antitumor activity

Twenty-eight patients were enrolled in the study, and 26 were evaluable for efficacy. Treatment exposure is

shown in Supplementary Fig. S1B. While no objective responses were observed using RECIST criteria, 9 out

of 26 patients (35%) experienced maximum tumor reductions ranging from 2%-30%. One patient with RCC had

a 40% reduction on the right adrenal gland at the first on-treatment scan (the overall response was stable

disease) and experienced a 30% reduction of the sum of 2 target lesions at the second post-treatment scan; a CT

scan of a lung lesion is shown Supplementary Fig. S1C. Best overall response included SD in 14 patients

(53.8%) (Supplementary Table S3). One patient with metastatic melanoma, previously treated with

ipilimumab and a BRAF inhibitor, received 25 cycles of NKTR-214 and had durable, stable disease (SD) for 15

months. A second patient with metastatic RCC, who had progressed on high-dose IL-2 and was refractory to

single-agent OX40 and nivolumab, was treated with 19 cycles of NKTR-214 and had durable SD for 13

months. Of note, there were 3 immunotherapy-naïve patients with RCC that showed immunological changes in

the blood and tumor, who received standard-of-care nivolumab within 1 month immediately following end of

treatment with NKTR-214, all three patients experienced a partial response at their 1st post-treatment scan after

follow-on therapy with nivolumab.

Treatment-related changes in peripheral blood

To determine if NKTR-214 could induce functional immune cells in the blood, a multiparametric flow

cytometry analysis was performed on peripheral blood mononuclear cells (PBMCs) collected during the first 2

cycles, before treatment on day 1, and after treatment on day 8. NKTR-214 treatment significantly increased

proliferation of CD4+

T cells, CD8+

T cells, and NK cells at day 8 after each cycle (Fig. 1A). Proliferating (Ki-

67+) cells expressed higher levels of inducible costimulator (ICOS) and PD-1 activation markers, compared

with non-proliferating (Ki-67-) cells, consistent with an activated phenotype (Fig. 1B). Analysis of the





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frequencies of CD4+

T cells, CD8+ T cells, and NK cells within the CD45

+ immune cell population and absolute

number of cells is shown in Supplementary Fig. S3A-C. While there was no increase in the frequencies of

peripheral CD4+

and CD8+

T cells within CD45+ immune cells (Supplementary Fig. S3A), treatment with

NKTR-214 resulted in a significant increase in the absolute numbers of CD4+ and CD8

+ T cells

(Supplementary Figure S3B). There were also increases in the frequencies of CD4+, CD8

+ T and NK cells

expressing ICOS and PD-1, increases were observed regardless of dose level or regimen (Supplementary Fig.

S4A-C). Expression of co-stimulatory molecule OX-40, and inhibitory molecule CTLA-4, was significantly

increased in proliferating NK and T cells, and this appeared to be more pronounced in CD4+ T cells

(Supplementary Fig. S3D). An increase in the frequency and absolute number of NK cells after treatment was

also noted (Supplementary Fig. S3C). The gating strategy for flow cytometry analysis is shown in

Supplementary Fig. S5A and S5B.

Treatment-related changes in the tumor

To demonstrate NKTR-214 activated genes associated with the IL-2 pathway, targeted analysis of

immune networks using NanoString gene analysis to quantify transcript levels was performed on 12 matched

baseline and on-treatment tumor biopsies. The Human PanCancer Immune Profiling panel was used to analyze

genes associated with immune networks making it applicable to assess the TME from tumors with diverse

pathology. Among the 770 genes assayed, 62 genes were significantly increased in the tumor biopsies on-

treatment compared to baseline biopsies (Fig. 2A and Supplementary Table S4), and there were no genes of

the 770 genes that were significantly decreased. The top 29 most statistically significant differentially expressed

genes with P values < 0.01 are listed in Supplementary Table S5. The capacity of NKTR-214 to induce

immune activation, cytotoxic and effector gene signatures in the tumor was assessed. Our analysis revealed an

increased expression of genes associated with T-cell infiltration and signaling (CD3G, CD3D, CD3E, CD247

and ZAP70, P≤0.05), T-cell activation and co-inhibitory molecules (ICOS, TNFRSF9, PDCD1, CTLA-4,

TIGIT, and LAG3, P≤0.05), and of cytotoxic effector genes (PRF1, GZMB, GZMA, and GZMK, P≤0.05).

Genes encoding for Programmed death-ligand 1 (PD-L1) and PD-L2 (CD274, PDCD1LG2, P≤0.05) (Fig. 2 B-

E), SOCS-1 and IDO1 were also significantly increased, while HAVCR2 (TIM-3), and MHC-class 1 molecules

had no change (Supplementary Fig. S6A). Notably, CD8 and CD4+ Th1 associated genes were increased after

treatment (CD8A, CD8B, EOMES, TBX21, and IFNG, P≤0.05), with no significant change noted in Th2, Th17

or inhibitory cytokine-associated genes (IL4, GATA3, IL17A, RORC, TGFB1, TGFB2, and IL10) (Fig. 2F and

2G). Several immune NK cell genes (KLRC2, KLRD1, KLRK1 and KLRC1, P≤0.05) were increased (Fig.

2H). CXCR3, a chemokine receptor expressed by NK cells, CD4+

Th1, and CD8+

cytotoxic T lymphocytes, is





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essential for their localization to the tumor and was also increased after treatment (Fig. 2F). Consistently, the

gene signatures associated with the presence and activity of T cells including CD4+ Th1 cells, CD8

+ T cells, NK

cells, and cytotoxic cells were all significantly increased (Supplementary Fig. S6B). Taken together, our

results show that NKTR-214 induced a cytotoxic and effector gene signature in the TME consistent with its

mechanism of action.

To determine if NKTR-214 promoted an increase in tumor infiltrating lymphocytes (TIL), 14 matched pre-

treatment and on-treatment tumor biopsies were analyzed by flow cytometry and showed a marked increase in

the frequency of CD45+ live cells in 8 out of 14 patients after treatment (Fig. 3A). Similarly, the frequency of

NK and CD8+ T cells within live CD45

+ immune cells increased after treatment in 11 out of 14 and 9 out of 14

patients, respectively (Fig. 3B). To further assess the increase in immune cells within the tumor, the density of

infiltrating CD8+ T-cell infiltrate was measured by IHC, the majority of patients, 6 out of 10, showed an

increase in the number of CD8+ staining per area (mm

2) after treatment compared to baseline, data from one

patient are shown (Fig. 3C). Since tumor tissue was limited in several patients, not all samples could be

analyzed for flow cytometry, gene expression, and IHC. For example, some patients had samples available for

flow cytometry and NanoString analysis but not IHC. However, there were 6 patients for which both IHC and

NanoString data were available (Supplementary Fig. S7A), there was a good correlation between CD8+ T cells

changes measured by these two different approaches (Supplementary Fig. S7B). In pre- and on-treatment

tumor biopsies, sequencing of the TCRβ repertoire showed an increase in T cell infiltrates in 10 out of 12

patients and changes in clonality with a trend towards increased frequency of specific clones post-treatment in 8

out of 12 patients (Fig. 3D). One patient with melanoma who had received 12 prior lines of therapy was

analyzed for changes in productive clonality and clonal frequency, NKTR-214 appeared to induce an increase in

both existing and new clones at week 3 (Supplementary Fig. S8A). Furthermore, an increase in clonality was

associated with increased T cell infiltration in 7 out of 12 patients reflecting the induction of a more oligoclonal

T cell repertoire (Fig. 3D and Supplementary Fig. S8B).

Despite the robust proliferation and increase in the absolute number of CD4+CD25

hiFoxp3

+ Tregs in the

peripheral blood after treatment (Fig. 3E and Supplementary Fig. S9A and S9B) and upregulation of markers

associated with enhanced suppressive function (ICOS and CTLA-4) (Supplementary Fig. S9C),4,15

NKTR-214

administration did not appear to have the same effect on CD4+CD25

hiFoxp3

+ Tregs in the TME as in the

peripheral blood. Flow cytometry analysis showed that the percentages of CD4+CD25

hiFoxp3

+ Tregs were

slightly increased in 4 patients and either reduced or did not change in 10 patients (Fig. 3F). From the same





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tumor biopsies analyzed, FoxP3 staining by IHC showed little to no increase in FoxP3 expression after NKTR-

214 administration compared to baseline, Foxp3+ cells were increased in 2 patients or did not change in 8

patients (Fig. 3G). The ratio of CD8+ T cells over CD4

+CD25

hiFoxp3

+ Tregs, as measured by flow cytometry is

shown in Supplementary Fig. S8C. The gating strategy for TIL flow cytometry analysis is shown

Supplementary Fig. S8D.

DISCUSSION

This is a first-in-human report of NKTR-214. Treatment with NKTR-214 was well tolerated and feasible as an

outpatient regimen. Grade 3 TRAEs were experienced by 21.4% of patients; there were no grade 4 TRAEs or

any treatment-related deaths. The most common TRAEs were of grade 1 and 2 severity and included flu-like

symptoms, rash, and pruritus, the majority of which coincided with the peak plasma concentrations of the active

cytokine and resolved spontaneously or were mitigated by non-prescription oral or topical treatments. NKTR-

214-related hypotension was predictable, manageable, and reversible. The incidence of grade 3 hypotension was

reduced once hypotension risk mitigation strategies were implemented. Notably, 3 of the 5 patients who

experienced grade 3 hypotension had preexisting adrenal insufficiency, were managed conservatively, and

continued to be dosed on the study. The Safety Review Committee (SRC) decided to halt enrollment into the

0.012 mg/kg cohort after dosing one patient. This decision was based on the composite of hypotension,

syncope, and cytokine release syndrome that necessitated a hospital admission of this patient, and the goal to

identify a NKTR-214 dose that could be delivered as a safe, outpatient treatment. Based on the tolerability,

convenient dosing schedule, and pharmacodynamic changes at all NKTR-214 doses tested, a NKTR-214 dose

of 0.006 mg/kg q3w was selected as the RP2D.

NKTR-214 engaged the IL-2 receptor pathway and provided robust systemic immune activation with repeated

administration, as demonstrated by dynamic changes in absolute lymphocyte counts and sCD25. Most

importantly, these pharmacodynamic changes did not wane after repeated cycles. NKTR-214 induced robust

proliferation of CD4+

and CD8+ T and NK cells in the blood, and the cells were activated as indicated by high

levels of ICOS, PD-1, CTLA-4, and OX40 expression. Importantly, treatment with NKTR-214 resulted in a

significant increase in the absolute numbers of CD4+ T cells, CD8

+ T cells, and NK cells. The induction of

CD4+ T cells, CD8

+ T cells, and NK cell proliferation in the peripheral blood was also observed in patients

treated with human recombinant IL-15 (rhIL-15), these changes were most evident in NK and CD8+ T cells and

to a lesser degree in CD4+ T cells.

16 IL-15 shares the IL-2/IL-15Rβ (CD122) chain with IL-2 and is known to

stimulate proliferation of T cells, induce generation of cytotoxic lymphocytes (CTLs), and stimulate prolonged





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expansion of NK cells. In contrast to IL-2, IL-15 has no marked effects on the stimulation of Tregs and does not

cause substantial toxicity necessitating intense supportive care. However, the use of rhIL-15 as a therapeutic

agent may not be optimal due to the low expression of IL-15Rα and the need for high doses to achieve

biological responses in vivo.17

The administration of rhIL-15 as a daily intravenous bolus infusion for 12

consecutive days to 5 patients with metastatic malignant melanoma or metastatic renal cell cancer proved to be

difficult due to clinical toxicities produced by intense cytokine secretion that occurred in the first 2 hours after

treatment.16

Importantly, treatment with NKTR-214 induced meaningful immunological changes within the TME. At the

RP2D, freshly isolated TIL showed that there was a 15.1, 3.5, and 0.7-fold change increase in CD8+ T cells, NK

cells, and Treg cells, respectively (Supplementary Fig. S10). Transcriptional analysis of the on-treatment

tumor biopsies highlighted differentially expressed genes that were significantly increased on-treatment, which

support a fundamental reshaping of the TME towards immune cell activation and effector functions. NKTR-214

also triggered immune regulatory pathways as seen in the increased gene expression of PD-L1/PD-L2, TIGIT,

LAG3, SOC1, and IDO1. Interestingly, NKTR-214 induced a gene signature associated with the presence and

activity of T cells including Th1 cells and CD8+ T cells. The increase of this Th1/CD8 gene signature was not

associated with an increase in the gene signature representing Th2 or Th17 cells that are known to be mediators

of inflammation associated with anti-PD-1 and anti-CTLA4 therapy-induced irAEs.18,19

A study of melanoma

patients treated with anti-CTLA-4 combined with high-dose IL-2 demonstrated a trend towards a decreased

incidence of grade 3/4 irAEs compared to patients receiving anti-CTLA-4 without IL-2 (17% vs 29% and

32%).20,21

Prieto et al proposed an elegant theory that the Tregs induced by high-dose IL-2 may decrease the

activity of autoreactive T cells to reduce the incidence of irAEs.21

It is well established that IL-2 signals control

the polarization of CD4+

T cell subsets leading to enhancement of Th1 and Tregs while limiting the

differentiation into Th17.22,23

Several mechanisms have been proposed to explain the negative effect of IL-2 on

Th17, including lowering the expression of the IL-6Rβ on CD4+ T cells, thereby reducing the IL-6 mediated

STAT3 phosphorylation that is essential for the induction of Th17.24

Other studies showed that the IL-2

signalizing through STAT5, which competes with STAT3 for binding the IL-17 gene locus, constrains the

generation of Th17.25,26

Hence, the immune effect of NKTR-214 on the tumor appeared to be Th1 dominant

with no induction of Th2 or Th17 gene signatures. Despite the increases in Tregs in the periphery with NKTR-

214 administration, expansion of these cells was limited in the tumor. Preclinical evidence from mouse tumor

models indicates that NKTR-214 limits intratumoral Treg proliferation and survival by way of promoting

apoptosis, thereby polarizing the TME toward CD8+ T cells and consequently driving a very high CD8

+/Treg





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ratio.12,27

Exploratory studies are in progress to further probe the mechanisms by which peripheral T regs are

preserved while intratumoral T regs are not.

A key immunological observation was the increase in the percentage of proliferating CD8+ T cells also

expressing increased levels of PD-1 in the TME (Fig. 3H). Studies have demonstrated that PD-1 identifies

CD8+ tumor-reactive T cells in infiltrating tumors.

2,28-31 The increase in the percentage of CD8

+ PD-1

+ T cells in

patients’ tumors and the tumor shrinkage observed in 35% of patients, including one unconfirmed partial

response, is promising. Durable, stable disease greater than one year was seen in two patients, with one patient

having failed 3 prior immunotherapies. It is possible that compensatory mechanisms, namely PD-L1/PD-L2

(Fig. 2E), may have dampened the potential of these CD8+

PD-1+ T cells to convert patients into responders.

31-33

Intriguingly, 3 patients with RCC, who were anti-PD-1 treatment-naïve experienced TIL elevation with tumor

reductions of 1%, 10% and 20%; all 3 patients ended NKTR-214 with stable disease and within 1 month started

anti-PD-1 treatment and experienced rapid tumor reductions at first scan on-treatment resulting in partial

responses.34

These anecdotal observations supported the hypothesis that NKTR-214 treatment may have

conditioned the TME by expanding activated TILs, thereby potentially providing synergy with therapies that

block inhibitory signals, such as those of the PD-1/PD-L1 axis.

In summary, every patient evaluated showed evidence for activation of the immune response, at all four

doses tested, and these effects were reproduced with repeated administration. Given the small number of

patients evaluable at q2w (n=4), no conclusions can be made on the impact of immune activation with a shorter

frequency of dosing. Based on the biological activity and tolerability of NKTR-214 as well as the non-

overlapping toxicities with approved checkpoint inhibitors, NKTR-214 is being combined with approved anti-

PD-1/PD-L1/CTLA-4 agents in on-going clinical trials. NKTR-214, as a T cell and NK cell growth factor, may

serve as a central mechanism for boosting tumor-specific T cell proliferation and activation in the TME and has

the potential to serve as a backbone therapy for combination therapy with a variety of agents.

MATERIALS AND METHODS

Patients and study design

This was a multicenter, open-label dose-escalation study using a 3+3 design. Eligible patients were

treatment-refractory to ≥1 prior therapy for advanced or locally-advanced disease. Twenty-eight patients with a

histologically confirmed diagnosis of RCC, melanoma, urothelial carcinoma, breast cancer, leiomyosarcoma,

chondrosarcoma, or colorectal carcinoma not amenable to curative therapy were enrolled. All patients provided

written informed consent before any protocol-specified procedures. The primary objectives were to assess the





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safety, maximum tolerated dose (MTD), recommended phase 2 dose (RP2D), and anti-tumor clinical activity.

Secondary objectives were to characterize pharmacokinetics, pharmacodynamics, and immunological changes

in blood and tumor. NKTR-214 was administered at 0.003, 0.006, 0.009, and 0.012 mg/kg as a 15-minute

intravenous infusion every 3 weeks (q3w) or 0.006 mg/kg every 2 weeks (q2w) until disease progression or

unacceptable toxicities occurred. Patients were held for 24 hours in an infusion center after the first dose of

NKTR-214; however, subsequent cycles were administered on an outpatient basis. If no dose-limiting toxicities

(DLTs) were observed in a cohort after 21 days, the dose was escalated for the following cohort after review by

the SRC, which consisted of the sponsor and at least one site investigator. Additional patients were added to a

lower dose cohort to further assess the safety and tolerability. All patients who received study treatment and one

post-treatment scan were evaluable for antitumor activity. Investigator-assessed anti-tumor activity was

evaluated using Response Evaluation Criteria in Solid Tumors, version 1.1 (RECIST 1.1). Baseline tumor

imaging was completed within 4 weeks before starting therapy and every 8 weeks thereafter. Tumor responses

were confirmed with a repeat scan at least 4 weeks later.

Safety

All patients who received any study drug were included in the safety analyses. Adverse events (AEs),

SAEs, and laboratory abnormalities were graded per the National Cancer Institute Common Terminology

Criteria for Adverse Events (CTCAE), Version 4.03 and monitored throughout the study until 30 days after the

last dose of study drug. Treatment-related AEs (TRAEs) and treatment-emergent adverse events (TEAEs) were

reported throughout the observation period and recorded once per patient at the highest grade. Dose-limiting

toxicity (DLT) was defined as any non-transient grade 3, 4, or 5 hematologic or non-hematologic toxicity that

occurred within the first 21 days after dosing and was deemed related or possibly related to the administration

of NKTR-214. The MTD was defined as the highest dose of NKTR-214 that was administered without causing

any unacceptable side effects or AEs. Immune-related adverse events (irAEs) were considered AEs of special

interest due to their potential clinical significance.

Biomarker analysis

Serial blood and tumor tissue samples were collected to perform in-depth analysis of immune cells using

flow cytometry, nCounter® gene expression assay (NanoString, Seattle WA), immunohistochemistry (IHC),

and TCR sequencing; details are provided in the supplement.

Statistical methods





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Summary statistics were used for any continuous variables; numbers and percentage were used for

categorical variables. For all analyses, baseline values were defined as the last value before the first dose of

NKTR-214. All statistical calculations were performed using Statistical Analysis System (SAS) version 9.4

(SAS Institute, Cary, NC). Statistical analyses for immune profiling by flow cytometry were performed with

GraphPad Prism 7.0 (GraphPad Software, La Jolla, CA). The significance of the difference between groups in

the experiments was evaluated by a two-tailed paired t-test. A value of P<0.05 was considered statistically

significant.

Authors’ Contributions:

Conception and design: Michael Hurwitz, Harriet Kluger, Mary Tagliaferri, Patrick Hwu, Brendan Curti,

Nizar M. Tannir, Mario Sznol, Adi Diab

Development of methodology: Salah-Eddine Bentebibel, Michael Hurwitz, Chantale Bernatchez, Cara

Haymaker, Harriet Kluger, Mary Tagliaferri, Jonathan Zalevsky, Ute Hoch, Christie Fanton, Sandra Aung,

Brendan Curti, Nizar M. Tannir, Mario Sznol, Adi Diab

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): Salah-

Eddine Bentebibel, Michael Hurwitz, Chantale Bernatchez, Cara Haymaker, Courtney W. Hudgens, Harriet

Kluger, Patrick Hwu, Brendan Curti, Nizar M. Tannir, Mario Sznol, Adi Diab

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): Salah-

Eddine Bentebibel, Michael Hurwitz, Chantale Bernatchez, Cara Haymaker, Courtney W. Hudgens, Harriet

Kluger, Michael Tetzlaff, Mary Tagliaferri, Jonathan Zalevsky, Ute Hoch, Christie Fanton, Sandra Aung,

Patrick Hwu, Brendan Curti, Nizar M. Tannir, Mario Sznol, Adi Diab

Writing, review, and/or revision of the manuscript: Salah-Eddine Bentebibel, Michael Hurwitz, Chantale

Bernatchez, Cara Haymaker, Courtney W. Hudgens, Harriet Kluger, Michael Tetzlaff, Mary Tagliaferri,

Jonathan Zalevsky, Ute Hoch, Christie Fanton, Sandra Aung, Patrick Hwu, Brendan Curti, Nizar M. Tannir,

Mario Sznol, Adi Diab

Administrative, technical, or material support (ie, reporting or organizing data, constructing

databases): Salah-Eddine Bentebibel, Chantale Bernatchez, Cara Haymaker, Courtney W. Hudgens, Michael

Tetzlaff, Ute Hoch, Christie Fanton, Sandra Aung, Adi Diab

Study supervision: Michael Hurwitz, Harriet Kluger, Michael Tetzlaff, Mary Tagliaferri, Patrick Hwu,

Brendan Curti, Nizar M. Tannir, Mario Sznol, Adi Diab





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Acknowledgments

This project was supported in part, by the Translational Molecular Pathology-Immunoprofiling lab (TMP-IL) at

the Department Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center.

Funding for this study was provided by Nektar Therapeutics. We thank the patients, their caregivers and

families, and the investigators who participated in this study. We also thank Phillips Gilmore Oncology

Communications (Philadelphia, USA) for providing editorial support, funded by Nektar Therapeutics

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Figure 1: Increase in proliferation and activation of immune cells in peripheral blood after NKTR-214

treatment. PBMC samples were obtained from 22 patients before treatment on day 1 and after treatment on day

8. (A) Percentage of Ki-67+ cells within CD4

+ T cells (top), CD8

+ T cells (center) and CD3

-CD56

+ NK cells

(bottom) were analyzed by flow cytometry. Representative dot plots are shown on the left (B) ICOS (top) and

PD-1 (bottom) expression by proliferating (Ki-67+) and non-proliferating (Ki-67

-) cells at day 8 after treatment

with NKTR-214. Paired T-test, P-value ****<0.0001, *** <0.001, ** < 0.01, * <0.05.

Figure 2: Differential expression of mRNA in the tumor microenvironment after NKTR-214 treatment.

NanoString gene analysis using the nCounter PanCancer Immune Profiling Panel to quantify transcript levels of

770 genes was performed on 12 matched baseline, and on-treatment tumor biopsies (A) volcano plot displaying

each gene’s −log 10 (P-value) and log 2-fold change with the selected covariate. Highly statistically significant

genes fall at the top of the plot, and highly differentially expressed genes fall to either side. Horizontal line

indicates thresholds for P = 0.05. P-values were corrected using Benjamin-Yekutieli adjusted false discovery

rate. (B-H) Transcriptional analysis of 12 matched tumor biopsies at baseline (Blue) and after NKTR-214 after

treatment (Orange), p values were obtained using paired T-test, P-value ** < 0.01, * <0.05. (B) Selected genes

related to T cell infiltration and signaling (C) Genes reflecting T cell activation and co-inhibitory receptors (D)

Genes associated with cytotoxic effector functions (E) Gene expression for PD-L1 (CD274) and PD-L2

(PDCD1LG2) (F) CD8+ T and Th1 associated genes (G) Th2/Th17 cells and inhibitory cytokines associated

genes (H) Markers found on infiltrating tumor-associated NK cells..

Figure 3: Intratumoral changes in critical lymphocyte populations and numbers after NKTR-214

treatment. Tumor biopsies were obtained at baseline (Blue) and after NKTR-214 treatment (Orange). Immune

infiltrates, including CD45+ live cells, NK and CD8

+ T cells were analyzed by flow cytometry on freshly

disaggregated tumor tissue. Patients #13 and #14 received a NKTR-214 dose of 0.006 mg/kg q2w (A)

Percentage of live CD45+ cells within total cells (B) Percentage of NK cells (top panel) and CD8

+ T cells

(bottom panel) within CD45+ live cells. (C) A portion of the tumor biopsy from the chest wall of an RCC

patient (#13) dosed q2w was further analyzed for CD8 staining by IHC (left), 10 patients had pretreatment and

on-treatment IHC data (right), each circle represents one individual tumor sample with red circles indicating

increased and black circles indicating similar or reduced number of CD8+ T cells on-treatment compared to

pretreatment (D) TCR sequencing examining the percentage of T-cell infiltration (the fraction productive of

cells mass estimate values obtained through the immuSeq Analyzer software) (top panel), and clonality (bottom





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panel) in tumor biopsy samples, n=12 (E) Percentage of CD4+CD25

hiFoxp3

+ Tregs within total CD4

+ T cells in

the blood before and 8 days after treatment with NKTR-214. A representative result is shown on the left. Paired

T-test, P-value **< 0.01. (F) Percentage of CD4+CD25

hiFoxp3

+ Tregs within CD3

+ T cells in tumor biopsies

samples were analyzed by flow cytometry, n=14 (G) Quantification of the number of Foxp3+ cells by IHC,

n=10, each circle represents one individual tumor sample with red circles indicating increased and black circles

indicating unchanged or reduced Foxp3+cells on-treatment compared to pretreatment (H) PD-1 expression

within CD4+ (top panel) and CD8

+ T cells (bottom panel) in tumor biopsies samples were analyzed by flow

cytometry, n=14.




Table 1. Treatment emergent adverse events occurring in 3 or more patients across all doses

Grade 1-2 Grade 3-5 Preferred Term1 0.003 mg/kg

(q3w) (n=4)

0.006 mg/kg (q3w) (n=11)

0.006 mg/kg (q2w) (n=6)

0.009 mg/kg (q3w) (n=6)

0.012 mg/kg (q3w) (n=1)

0.003 mg/kg (q3w) (n=4)

0.006 mg/kg (q3w) (n=11)

0.006 mg/kg (q2w) (n=6)

0.009 mg/kg (q3w) (n=6)

0.012 mg/kg (q3w) (n=1)

Total (%)

15/28 (54%) Patients Reported Grade ≥ 3 TEAE Hypotension 2 (50) 8 (73) 2 (33) 1 (17) 1 (9) 2 (33) 1 (17) 1 (100) 18 (64) Syncope 1 (9) 1 (17) 1 (100) 3 (11) Headache 3 (75) 2 (18) 1 (17) 2 (33) 1 (9) 1 (17) 10 (36) Anemia 2 (50) 1 (9) 1 (17) 1 (9) 1 (17) 6 (21) Metastases to central nervous system 2 (18) 1 (17) 3 (11) Constipation 2 (50) 3 (27) 2 (33) 1 (17) 1 (25) 9 (32) Edema peripheral 4 (36) 1 (17) 1 (17) 1 (17) 7 (25) Abdominal pain

1 (25) 1 (9) 1 (17) 2 (33) 5 (18)

Pleural effusion

1 (25) 2 (18) 1 (17) 1 (25) 5 (18)

Confusional state 2 (33) 1 (9) 3 (11) Infusion related reaction 1 (9) 1 (17) 2 (7) Presyncope 1 (9) 1 (9) 2 (7) Brain edema 1 (9) 1 (4) Disease progression 1 (17) 1 (4) Gastroduodenal hemorrhage 1 (9) 1 (4) Hemiparesis 1 (9) 1 (4) Hypovolemic shock 1 (17) 1 (4) Lipase increased 1 (25) 1 (4) Liver function test abnormal 1 (17) 1 (4) Lower gastrointestinal hemorrhage 1 (17) 1 (4) Metastases to meninges 1 (9) 1 (4) Sepsis 1 (17) 1 (4) Fatigue 3 (75) 9 (82) 5 (83) 5 (83) 1 (100) 23 (82) Flu-like symptoms2

3 (75) 8 (73) 4 (67) 5 (83) 20 (71)

Pruritus 2 (50) 7 (64) 5 (83) 4 (67) 1 (100) 19 (68) Decreased appetite

1 (25) 6 (55) 4 (67) 4 (67) 15 (54)

Rash3 3 (75) 6 (55) 4 (67) 2 (33) 15 (54) Arthralgia 1 (25) 3 (27) 5 (83) 2 (33) 1 (100) 12 (43) Cough 1 (25) 5 (45) 2 (33) 3 (50) 1 (100) 12 (43) Nausea 3 (75) 3 (27) 3 (50) 2 (33) 11 (39) Diarrhea 3 (75) 2 (18) 1 (17) 3 (50) 9 (32) Dyspnea 3 (75) 4 (36) 1 (17) 8 (29) Nasal congestion

1 (25) 2 (18) 2 (33) 3 (50) 8 (29)

Dizziness 2 (50) 3 (27) 1 (17) 1 (17) 7 (25) Myalgia 3 (27) 1 (17) 2 (33) 6 (21) Vomiting 1 (25) 2 (18) 2 (33) 1 (17) 6 (21) Weight decreased

2 (50) 1 (9) 2 (33) 5 (18)

Flushing 1 (9) 2 (33) 1 (17) 4 (14) Anxiety 1 (25) 1 (9) 1 (17) 3 (11) Blood creatinine increased

1 (9) 1 (17) 1 (17) 3 (11)

Cytokine release syndrome

2 (33) 1 (100) 3 (11)

Dry skin 1 (25) 2 (18) 3 (11) Dyspepsia 1 (17) 1 (17) 1 (100) 3 (11) Generalized edema 1 (9) 1 (17) 1 (100) 3 (11) Muscular weakness

1 (25) 1 (9) 1 (17) 3 (11)

Oropharyngeal pain

1 (25) 2 (18) 3 (11)

Pain in extremity 1 (9) 1 (17) 1 (17) 3 (11) 1Does not include events that occurred in <3 patients at grade 1 and 2 severity 2Flu-like symptoms include the following preferred terms: ‘Influenza-like illness,’ ‘Influenza,’ ‘Pyrexia,’ ‘Chills’ 3Rash includes the following preferred terms: ‘Erythema,’ ‘Rash,’ ‘Rash erythematous, ‘Rash maculo-papular,’ Rash pruritic,’ ‘Rash pustular,’ ‘Rash vesicular,’ ‘Rash

generalized,’ ‘Rash macular’

Patients reporting more than one AE within the same preferred term are counted once at the highest severity reported

Abbreviation: AE, adverse event; TEAE, treatment emergent adverse event




Gated on CD4+ T cells

CD4+ T

CD4+ T

A

Fig1C

D4

Ki-67

Ki-67

Ki-67

Cycle1 D1 Cycle1 D8

Gated on CD8+ T cells

Gated on NK cells

Cycle1 D1 Cycle1 D8

CD

8

Cycle1 D1 Cycle1 D8

CD

56

8.3 79.5

****

% K

i-6

7

****

Cycle 1 Cycle 2

D1 D8 D1 D8

NK

NK

****

% K

i-6

7

****

Cycle 1 Cycle 2

D1 D8 D1 D8

CD8+ T

CD8+ T

****

% K

i-6

7

****

Cycle 1 Cycle 2

D1 D8 D1 D8

0

20

40

60

80

0

20

40

60

80

0

25

50

75

100

B

D8 after treatment with NKTR-214

% I

CO

S

**** ****

Ki-67–

Ki-67+

% P

D1

*** ***

% I

CO

S

**** ****

% P

D1

*** **

% I

CO

S

** *

CD4+ T NKCD8+ T

% P

D1

* *

Cycle 1 Cycle 2

Cycle 1 Cycle 2

Cycle 1 Cycle 2 Cycle 1 Cycle 2

Cycle 1 Cycle 2 Cycle 1 Cycle 2

6.2 44.1

6.7 65.3

0

5

10

15

20

0

25

50

0

5

10

15

0

25

50

0

20

40

60

0

5

10

15




Pretreatment On-treatmentB

CD3G CD3D CD3E CD247 ZAP70

*

**

* **

**

RN

A C

ou

nts

(L

og

2)

G

RN

A C

ou

nts

(L

og

2)

IL17A RORC IL4 GATA3 TGFB1 TGFB2 IL10

C

ICOS TNFR-SF9

PDCD1 CTLA4 TIGIT LAG3

RN

A C

ou

nts

(L

og

2)

***

**** *

*

D

RN

A C

ou

nts

(L

og

2)

GZMK

* **

* *E

RN

A C

ou

nts

(L

og

2)

CD274 PDCD-1LG2

**

HF

A

Fig2

Adjusted P = 0.05

−L

og10

(P

-va

lue)

CD247

CD8ATIGIT

KLRD1

CD3D

TNFRSF9IL32 GZMA

PSMB9CTLA4

IFNG

GZMBCXCR3

CD27

Differential Expression On-treatment/Pre-treatment

-0.5 0.0 0.5 1.0 1.5 2.0

0

1

2

3

4

5

6

7

Significantly highly expressed mRNA

Log 2 (Fold Change in Gene Expression)

4

8

12

4

8

12

5

10

15

5.0

7.5

10.0

RN

A C

ou

nts

(L

og

2)

KLRC1 KLRC2 KLRD1 KLRK1

*** *

*

0

4

8

12

RN

A C

ou

nts

(L

og

2)

CXCR3CD8A CD8B EOMES TBX21 IFNG

**

***

* **

3

6

9

12

0

5

10

15

PRF1 GZMB GZMA




A

Fig3

C

E

% N

K in

CD

45

% C

D8

+ T

in

CD

45

1 2 3 4 5 6 7 8 9 10 11121314

1 2 3 4 5 6 7 8 9 10 11121314

Pretreatment On-treatmentB

0

10

20

40

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Published OnlineFirst April 15, 2019.Cancer Discov Salah-Eddine Bentebibel, Michael E Hurwitz, Chantale Bernatchez, et al. Patients With Advanced or Metastatic Solid Tumors

)-Biased Cytokine, inγβNovel IL-2-Receptor Beta/Gamma (A First-in-Human Study and Biomarker Analysis of NKTR-214, a

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