A First-In-Human Study and Biomarker Analysis of NKTR-214, a Novel Il2raf- Biased Cytokine, in Patients with Advanced Or Metastatic Solid Tumors

Published OnlineFirst April 15, 2019; DOI: 10.1158/2159-8290.CD-18-1495

RESEARCH BRIEF

A First-in-Human Study and Biomarker Analysis of NKTR-214, a Novel IL2Raf- Biased Cytokine, in Patients with Advanced or Metastatic Solid Tumors

Salah-Eddine Bentebibel1, Michael E. Hurwitz2, Chantale Bernatchez1, Cara Haymaker1, Courtney W. Hudgens1, Harriet M. Kluger2, Michael T. Tetzlaff1, Mary A. Tagliaferri3, Jonathan Zalevsky3, Ute Hoch3, Christie Fanton3, Sandra Aung3, Patrick Hwu1, Brendan D. Curti4, Nizar M. Tannir1, Mario Sznol2, and Adi Diab1

ABSTRACT NKTR-214 (bempegaldesleukin) is a novel IL2 pathway agonist, designed to provide sustained signaling through heterodimeric IL2 receptor βγ to drive increased proliferation and activation of CD8+ T and natural killer cells without unwanted expansion of T regulatory cells (Treg) in the tumor microenvironment. In this first-in-human multicenter phase I study, NKTR-214 administered as an outpatient regimen was well tolerated and showed clinical activity including tumor shrinkage and durable disease stabilization in heavily pretreated 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 IL2 receptor pathway and significantly increased genes associated with an effector phenotype. Based on safety and pharmacodynamic markers, the recommended phase II dose was determined to be 0.006 mg/kg every three weeks.

SIGNIFICANCE: We believe that IL2- and IL2 pathway–targeted agents such as NKTR-214 are key com- ponents to an optimal immunotherapy treatment algorithm. Based on its biological activity and tolerability, NKTR-214 is being studied with approved immuno-oncology agents including checkpoint inhibitors.

INTRODUCTION potent means of expanding and activating immune cells, specifically lymphocytes including cytotoxic CD8+ T cells In the treatment of patients with cancer, low levels of (4). FDA-approved high-dose (HD) IL2 (aldesleukin) treat- tumor-infiltrating lymphocytes (TIL) prior to the introduc- ment mediates durable clinical benefit in a limited number tion of checkpoint inhibitors is predictive of a poor response of patients with melanoma and renal cell carcinoma (RCC), (1–3). Stimulation of the IL2 receptor (IL2R) pathway is a presumably through CD8+ T-cell expansion (5). However, the

1The University of Texas MD Anderson Cancer Center, Houston, Texas. Corresponding Author: Adi Diab, The University of Texas MD Anderson 2Yale School of Medicine, New Haven, Connecticut. 3Nektar Therapeutics, Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: San Francisco, California. 4Providence Cancer Institute and Earle A. Chiles 713-745-7336; Fax: 713-745-1046; E-mail: [email protected] Research Institute, Portland, Oregon. doi: 10.1158/2159-8290.CD-18-1495 Note: Supplementary data for this article are available at Cancer Discovery Cancer Discov 2019;9:1–11 Online (http://cancerdiscovery.aacrjournals.org/). ©2019 American Association for Cancer Research. S.-E. Bentebibel, M.E. Hurwitz, and C. Bernatchez contributed equally to this article.

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RESEARCH BRIEF Bentebibel et al. frequent and rapid induction of the IL2 cytokine signaling therapy. The median number of prior therapies was 2 (range, cascade is associated with an increased risk for serious adverse 1–12; Supplementary Fig. S1A). The most common reason events (SAE; refs. 6–10). The ability of IL2 to broadly expand for study drug discontinuation was progressive disease by many lymphocyte cell populations is a critical feature of its RECIST (n = 15; 53.6%) or physician decision (n = 6; 21.4%), mechanism of action as an immunotherapy and a nonredun- primarily for patients who did not respond rapidly and had dant role of IL2 biology. other therapeutic options. One patient discontinued treat- NKTR-214 (bempegaldesleukin) is a human recombinant ment due to an infusion-related reaction. Patients received a IL2 attached to an average of six releasable polyethylene median of 3 infusions of NKTR-214 (range, 1–25 infusions), glycol (PEG) chains (11, 12). In the 1980s, PEG-modified with a median duration of exposure of 69.5 days (range, IL2 (PEG-IL2) was developed in an effort to improve the 15–533 days). solubility and decrease the plasma clearance of IL2 (13). Results of a phase I study describing the use of PEG-IL2 in Safety and Tolerability combination with HD IL2 to treat patients with metastatic All 28 patients enrolled in the study had treatment-emergent RCC and melanoma were reported in 1995 (14). Results adverse events (TEAE), with 26 patients having TEAEs that from the phase I study concluded that the toxicities and were considered related to the study drug. TEAEs and treat- tumor response rates to combination treatment with HD ment-related adverse events (TRAE) occurring in ≥3 patients IL2 followed by administration of PEG-IL2 were similar to across all doses are listed in Table 1 and in Supplementary those of HD unconjugated IL2 alone. Further development Table S2, respectively. Six of 28 (21.4%) patients reported of PEG-IL2 was discontinued. In the case of NKTR-214, grade 3 TRAEs; there were no grade 4 or 5 TRAEs. At the attachment of PEG chains can also be creatively utilized to highest NKTR-214 dose tested (0.012 mg/kg), 1 patient expe- not only alter a molecule’s pharmacokinetics but also alter rienced two dose-limiting toxicities (DLT; grade 3 hypoten- its receptor binding. NKTR-214 is engineered to bias recep- sion and grade 3 syncope), which resolved within 24 hours tor selectivity and alter pharmacokinetics of IL2 (11, 12), following i.v. fluid treatment and one dose of tocilizumab. while leaving the well-understood amino acid sequence of This patient was discharged from the hospital 2 days after IL2 (aldesleukin) unaltered. When fully PEGylated, NKTR- admission for the events and continued on study, receiving 214 is a prodrug that has essentially no biological activity. two additional doses of NKTR-214 0.006 mg/kg that were Upon i.v. administration, the PEG chains slowly release well tolerated. No other patients were dosed at 0.012 mg/kg. to generate active cytokine species with limited binding At a NKTR-214 dose of 0.009 mg/kg, 1 patient discontinued to the IL2Rα subunit, thereby biasing signaling to favor the study due to an SAE of grade 3 infusion-related reaction. the dimeric IL2Rβγ (CD122). Consequently, NKTR-214 The reaction occurred following the first dose of NKTR-214 increases the proliferation, activation, and effector func- and resolved with treatment. This patient had a history of tion of CD8+ T cells and natural killer (NK) cells within an infusion-related reaction to a previous immuno-oncology the tumor microenvironment (TME) without expanding agent. There was one reported immune-related adverse event the undesirable intratumoral T regulatory cells (Treg; ref. (irAE) of hypothyroidism associated with NKTR-214, which 11). In murine tumor models, NKTR-214 generated a ratio was treated with replacement therapy. The MTD was defined of CD8+ cytotoxic T cells to Tregs of >400:1 in tumor, and as the highest dose of NKTR-214 that was administered with- when combined with an anti-CTLA4 checkpoint inhibitor out causing any unacceptable side effects or AEs. DLTs and antibody, there was a strong synergistic effect (11). Based AEs were considered in the determination of the MTD, which on supportive preclinical efficacy, immunobiology data, was deemed to be 0.009 mg/kg every three weeks (q3w). and pharmacology and toxicology studies performed in The most common TRAEs included fatigue (71%), flu- rats and monkeys, a first-in-human study was initiated like symptoms (68%), pruritus (64%), hypotension (57%), using NKTR-214 in patients with advanced solid tumors. rash (50%), decreased appetite (46%), and arthralgia and We report here on the safety, pharmacodynamics, and cough (each 32%). Flu-like symptoms were managed with immunologic and antitumor activity of NKTR-214 mono- acetaminophen and NSAIDs. The median duration of flu- therapy in patients treated with multiple prior lines of like symptoms, including fever, was 72 hours and became therapy. less pronounced after cycle 2. Rash and pruritus were either self-limited or treated with antihistamines. Hypotension RESULTS usually occurred around 48 to 72 hours following the first NKTR-214 infusion. Declines in blood pressure follow- Patient Characteristics and Disposition ing administration of NKTR-214 were generally clinically Twenty-eight patients with advanced or locally advanced asymptomatic. solid tumor malignancies were enrolled across three cent- All grade 3 TEAEs of hypotension [5 of 28 (18%) patients] ers from December 2015 to April 2017. Demographics and were rapidly reversed with i.v. fluid administration, and no disease characteristics are summarized in Supplementary patients discontinued treatment due to hypotension. Risk Table S1. The majority of patients had a diagnosis of meta- minimization guidelines, which included oral and i.v. fluids static RCC (n = 15; 53.6%) or melanoma (n = 7; 25.0%). All during the first week of dosing, were implemented and success- patients had received prior anticancer treatment: 16 (57.1%) fully reduced the incidence of grade 3 hypotension. The fre- had received targeted therapy; 16 (57.1%) had received an quency of drug administration, every two weeks (q2w) or q3w immune checkpoint inhibitor; and 6 (21.4%) had received an at a NKTR-214 dose of 0.006 mg/kg, did not appear to affect immune checkpoint inhibitor in addition to other immuno- the safety profile. Based on safety and pharmacodynamic

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NKTR-214 in Patients with Advanced Solid Tumors RESEARCH BRIEF

Table 1. TEAEs occurring in 3 or more patients across all doses

Grades 1–2 Grades 3–5

0.003 0.006 0.006 0.009 0.012 0.003 0.006 0.006 0.009 0.012 mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg (q3w) (q3w) (q2w) (q3w) (q3w) (q3w) (q3w) (q2w) (q3w) (q3w) Preferred terma (n = 4) (n = 11) (n = 6) (n = 6) (n = 1) (n = 4) (n = 11) (n = 6) (n = 6) (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 symptomsb 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) Rashc 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)

(Continued)

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Table 1. TEAEs occurring in 3 or more patients across all doses (Continued)

Grades 1–2 Grades 3–5

NOTE: Patients reporting more than one AE within the same preferred term are counted once at the highest severity reported. aDoes not include events that occurred in <3 patients at grade 1 and 2 severity. bFlu-like symptoms include the following preferred terms: “Influenza-like illness,” “Influenza,” “Pyrexia,” and “Chills.” cRash includes the following preferred terms: “Erythema,” “Rash,” “Rash erythematous,” “Rash maculo-papular,” “Rash pruritic,” “Rash pustular,” “Rash vesicular,” “Rash generalized,” and “Rash macular.”

markers, the recommended phase II dose (RP2D) was deter- tosis, which peaked between days 8 and 11, and returned to mined to be 0.006 mg/kg q3w. near-baseline levels prior to the next cycle. Similarly, sCD25 levels increased within each treatment cycle, peaked around Pharmacokinetics and Pharmacodynamics day 8, and returned to near-baseline levels prior to each q3w NKTR-214–related cytokine (NKTR-214-RC) represents cycle. PD kinetics were observed at all dose levels and at the a mixture of compounds containing IL2 independent of q2w dosing schedule (data not shown). Anti–NKTR-214 or the PEG-conjugation status, and NKTR-214–active cytokine anti-IL2 antidrug antibodies were not present in any samples (NKTR-214-AC) represents a mixture of 2-PEG-IL2 and tested for this study (data not shown). 1-PEG-IL2, and potentially unconjugated IL2, although this species is often undetectable in patient samples; the mean Antitumor Activity observed plasma concentration–time profiles are shown in Twenty-eight patients were enrolled in the study, and 26 Supplementary Fig. S2A. NKTR-214-RC maximal concentra- were evaluable for efficacy. Treatment exposure is shown in tions were achieved shortly after the end of the infusion and Supplementary Fig. S1B. Although no objective responses declined monoexponentially thereafter. NKTR-214-RC con- were observed using RECIST criteria, 9 of 26 patients (35%) centrations remained detectable for 8 to 11 days post-dose. experienced maximum tumor reductions ranging from 2% NKTR-214-AC concentrations increased gradually after dos- to 30%. One patient with RCC had a 40% reduction on ing with Tmax between 24 and 48 hours. NKTR-214-RC and the right adrenal gland at the first on-treatment scan [the NKTR-214-AC exposures were similar across patients within overall response was stable disease (SD)] and experienced a a dose cohort and cycles, without indication of accumula- 30% reduction of the sum of 2 target lesions at the second tion with either the q3w or q2w administration schedules post-treatment scan; a CT scan of a lung lesion is shown (data not shown). Unconjugated IL2 levels were detected in Supplementary Fig. S1C. Best overall response included in 76 of 425 samples with reportable results. Unconjugated SD in 14 patients (53.8%; Supplementary Table S3). One IL2 concentrations in samples with detectable levels ranged patient with metastatic melanoma, previously treated with between 0.509 and 4.44 ng/mL with one concentration at ipilimumab and a BRAF inhibitor, received 25 cycles of 19.4 ng/mL. Out of 425 samples analyzed, 378 samples (89%) NKTR-214 and had durable SD for 15 months. A second had concentrations of unconjugated IL2 that were below the patient with metastatic RCC, who had progressed on HD IL2 limit of quantitation or less than 1 ng/mL (the lower limit and was refractory to single-agent anti-OX40 and nivolumab, of quantitation was 0.5 ng/mL; data not shown). Soluble was treated with 19 cycles of NKTR-214 and had durable SD CD25 (sCD25) and lymphocyte levels were measured as for 13 months. Of note, there were 3 immunotherapy-naïve pharmacodynamic indicators of immune system activation patients with RCC that showed immunologic changes in the (Supplementary Fig. S2B and S2C). Changes in absolute lym- blood and tumor, who received standard-of-care nivolumab phocyte counts were observed with each q3w treatment cycle within 1 month immediately following end of treatment with and included lymphopenia on day 3 followed by lymphocy- NKTR-214, and all 3 patients experienced a partial response

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NKTR-214 in Patients with Advanced Solid Tumors RESEARCH BRIEF

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Figure 1. Increase in proliferation and activation of immune cells in peripheral blood after NKTR-214 treatment. Peripheral blood mononuclear cell 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) was 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 nonproliferating (Ki-67−) cells at day 8 after treatment with NKTR-214. Paired t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. at their first post-treatment scan after follow-on therapy with less of dose level or regimen (Supplementary Fig. S4A–S4C). nivolumab. Expression of costimulatory molecule OX-40, and inhibitory molecule CTLA4, was significantly increased in proliferating Treatment-Related Changes in Peripheral Blood NK and T cells, and this appeared to be more pronounced in To determine if NKTR-214 could induce functional CD4+ T cells (Supplementary Fig. S3D). An increase in the immune cells in the blood, a multiparametric flow cytom- frequency and absolute number of NK cells after treatment etry analysis was performed on peripheral blood mononu- was also noted (Supplementary Fig. S3C). The gating strat- clear cells (PBMC) collected during the first 2 cycles, before egy for flow cytometry analysis is shown in Supplementary treatment on day 1 and after treatment on day 8. NKTR- Fig. S5A and S5B. 214 treatment significantly increased proliferation of CD4+ T cells, CD8+ T cells, and NK cells at day 8 after each cycle Treatment-Related Changes in the Tumor (Fig. 1A). Proliferating (Ki-67+) cells expressed higher levels To demonstrate NKTR-214 activated genes associated of inducible costimulator (ICOS) and PD-1 activation mark- with the IL2 pathway, targeted analysis of immune networks ers, compared with nonproliferating (Ki-67−) cells, consistent using NanoString gene analysis to quantify transcript levels with an activated phenotype (Fig. 1B). Analysis of the fre- was performed on 12 matched baseline and on-treatment quencies of CD4+ T cells, CD8+ T cells, and NK cells within tumor biopsies. The Human PanCancer Immune Profiling the CD45+ immune cell population and absolute number panel was used to analyze genes associated with immune net- of cells is shown in Supplementary Fig. S3A–S3C. Although works, making it applicable to assess the TME from tumors there was no increase in the frequencies of peripheral CD4+ with diverse pathology. Among the 770 genes assayed, 62 and CD8+ T cells within CD45+ immune cells (Supplemen- genes were significantly increased in the tumor biopsies on- tary Fig. S3A), treatment with NKTR-214 resulted in a sig- treatment compared with baseline biopsies (Fig. 2A; Supple- nificant increase in the absolute numbers of CD4+ and CD8+ mentary Table S4), and there were no genes of the 770 genes T cells (Supplementary Fig. S3B). There were also increases that were significantly decreased. The top 29 most statisti- in the frequencies of CD4+ T cells, CD8+ T cells, and NK cells cally significant differentially expressed genes withP values expressing ICOS and PD-1; increases were observed regard- <0.01 are listed in Supplementary Table S5. The capacity

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Figure 2. Differential expression of mRNA in the TME 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 −log10 (P value) and log2 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 FDR. 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 < 0.05; **, P < 0.01. B, Selected genes related to T-cell infiltration and signaling. C, Genes reflecting T-cell activation and coinhibitory 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 cytokine associated genes. H, Markers found on infiltrating tumor- associated NK cells. of NKTR-214 to induce immune activation, cytotoxic and ment (Fig. 2F). Consistently, the gene signatures associated effector gene signatures in the tumor was assessed. Our with the presence and activity of T cells including CD4+ analysis revealed an increased expression of genes associated Th1 cells, CD8+ T cells, NK cells, and cytotoxic cells were with T-cell infiltration and signaling CD3G,( CD3D, CD3E, all significantly increased (Supplementary Fig. S6B). Taken CD247, and ZAP70; P ≤ 0.05), T-cell activation and coinhibi- together, our results show that NKTR-214 induced a cyto- tory molecules (ICOS, TNFRSF9, PDCD1, CTLA4, TIGIT, and toxic and effector gene signature in the TME consistent with LAG3; P ≤ 0.05), and of cytotoxic effector genes (PRF1, GZMB, its mechanism of action. GZMA, and GZMK; P ≤ 0.05). Genes encoding for PD-L1 and To determine if NKTR-214 promoted an increase in TILs, PD-L2 (CD274, PDCD1LG2; P ≤ 0.05; Fig. 2B–E), SOCS1, and 14 matched pretreatment and on-treatment tumor biop- IDO1 were also significantly increased, whereas HAVCR2 sies were analyzed by flow cytometry and showed a marked (TIM3) and MHC class 1 molecules had no change (Supple- increase in the frequency of CD45+ live cells in 8 of 14 patients mentary Fig. S6A). Notably, CD8 and CD4+ Th1-associated after treatment (Fig. 3A). Similarly, the frequency of NK and genes were increased after treatment (CD8A, CD8B, EOMES, CD8+ T cells within live CD45+ immune cells increased after TBX21, and IFNG; P ≤ 0.05), with no significant change noted treatment in 11 of 14 and 9 of 14 patients, respectively (Fig. in Th2, Th17, or inhibitory cytokine-associated genes (IL4, 3B). To further assess the increase in immune cells within the GATA3, IL17A, RORC, TGFB1, TGFB2, and IL10; Fig. 2F and tumor, the density of infiltrating CD8+ T-cell infiltrate was G). Several immune NK-cell genes (KLRC2, KLRD1, KLRK1, measured by IHC; the majority of patients, 6 of 10, showed and KLRC1; P ≤ 0.05) were increased (Fig. 2H). CXCR3, an increase in the number of CD8+ staining per area (mm2) encoding a chemokine receptor expressed by NK cells, CD4+ after treatment compared with baseline, and data from 1 Th1, and CD8+ cytotoxic T lymphocytes, is essential for their patient are shown (Fig. 3C). Because tumor tissue was lim- localization to the tumor and was also increased after treat- ited in several patients, not all samples could be analyzed

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NKTR-214 in Patients with Advanced Solid Tumors RESEARCH BRIEF

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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 cells, 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 plot) and CD8+ T cells (bottom plot) within CD45+ live cells. C, A portion of the tumor biopsy from the chest wall of a patient with RCC (#13) dosed q2w was further analyzed for CD8 staining by IHC (left), 10 patients had pretreatment and on-treatment IHC data (right), and 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 with 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 plot) and clonality (bottom plot) in tumor biopsy samples, n = 12. E, Percentage of CD4+CD25hiFOXP3+ 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 < 0.01. F, Percentage of CD4+CD25hiFOXP3+ Tregs within CD3+ T cells in tumor biopsies samples was analyzed by flow cytometry,n = 14. G, Quantification of the number of FOXP3+ cells by IHC, n = 10, and each circle represents one individual tumor sample with red circles indicating increased and black circles indicating unchanged or reduced FOXP3+ cells on-treatment compared with pretreatment. H, PD-1 expression within CD4+ (top plot) and CD8+ T cells (bottom plot) in tumor biopsy samples was analyzed by flow cytometry n( = 14). for flow cytometry, gene expression, and IHC. For example, Despite the robust proliferation and increase in the some patients had samples available for flow cytometry absolute number of CD4+CD25hiFOXP3+ Tregs in the and NanoString analysis but not IHC. However, there were peripheral blood after treatment (Fig. 3E; Supplementary 6 patients for whom both IHC and NanoString data were Fig. S9A and S9B) and upregulation of markers asso- available (Supplementary Fig. S7A), and there was a good ciated with enhanced suppressive function (ICOS and correlation between CD8+ T-cell changes measured by these CTLA4; Supplementary Fig. S9C; refs. 4, 15), NKTR-214 two different approaches (Supplementary Fig. S7B). In pre- administration did not appear to have the same effect on treatment and on-treatment tumor biopsies, sequencing CD4+CD25hiFOXP3+ Tregs in the TME as in the peripheral of the T-cell receptor β (TCRβ) repertoire showed an increase blood. Flow cytometry analysis showed that the percent- in T-cell infiltrates in 10 of 12 patients and changes in clonal- ages of CD4+CD25hiFOXP3+ Tregs were slightly increased ity with a trend toward increased frequency of specific clones in 4 patients and either reduced or did not change in 10 after treatment in 8 of 12 patients (Fig. 3D). One patient patients (Fig. 3F). From the same tumor biopsies analyzed, with melanoma who had received 12 prior lines of therapy FOXP3 staining by IHC showed little to no increase in was analyzed for changes in productive clonality and clonal FOXP3 expression after NKTR-214 administration com- frequency, and NKTR-214 appeared to induce an increase in pared with baseline, and FOXP3+ cells were increased in 2 both existing and new clones at week 3 (Supplementary Fig. patients or did not change in 8 patients (Fig. 3G). The ratio S8A). Furthermore, an increase in clonality was associated of CD8+ T cells over CD4+CD25hiFOXP3+ Tregs, as meas- with increased T-cell infiltration in 7 of 12 patients, reflect- ured by flow cytometry, is shown in Supplementary Fig. ing the induction of a more oligoclonal T-cell repertoire (Fig. S8C. The gating strategy for TIL flow cytometry analysis is 3D; Supplementary Fig. S8B). shown in Supplementary Fig. S8D.

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RESEARCH BRIEF Bentebibel et al.

DISCUSSION and 0.7-fold increase in CD8+ T cells, NK cells, and Treg cells, respectively (Supplementary Fig. S10). Transcriptional This is a first-in-human report of NKTR-214. Treatment analysis of the on-treatment tumor biopsies highlighted dif- with NKTR-214 was well tolerated and feasible as an out- ferentially expressed genes that were significantly increased patient regimen. Grade 3 TRAEs were experienced by 21.4% on-treatment, which support a fundamental reshaping of the of patients; there were no grade 4 TRAEs or any treatment- TME toward immune cell activation and effector functions. related deaths. The most common TRAEs were of grade 1 NKTR-214 also triggered immune regulatory pathways as and 2 severity and included flu-like symptoms, rash, and seen in the increased gene expression of PD-L1/PD-L2, TIGIT, pruritus, the majority of which coincided with the peak LAG3, SOC1, and IDO1. Interestingly, NKTR-214 induced a plasma concentrations of the active cytokine and resolved gene signature associated with the presence and activity of spontaneously or were mitigated by nonprescription oral or T cells including Th1 cells and CD8+ T cells. The increase topical treatments. NKTR-214–related hypotension was pre- of this Th1/CD8 gene signature was not associated with an dictable, manageable, and reversible. The incidence of grade increase in the gene signature representing Th2 or Th17 cells 3 hypotension was reduced once hypotension risk mitigation that are known to be mediators of inflammation associated strategies were implemented. Notably, 3 of the 5 patients with anti–PD-1 and anti-CTLA4 therapy–induced irAEs (18, who experienced grade 3 hypotension had preexisting adrenal 19). A study of patients with melanoma treated with anti- insufficiency, were managed conservatively, and continued to CTLA4 combined with HD IL2 demonstrated a trend toward be dosed on the study. The Safety Review Committee (SRC) a decreased incidence of grade 3/4 irAEs compared with decided to halt enrollment into the 0.012 mg/kg cohort after patients receiving anti-CTLA4 without IL2 (17% vs. 29% and dosing 1 patient. This decision was based on the composite 32%; refs. 20, 21). Prieto and colleagues proposed an elegant of hypotension, syncope, and cytokine release syndrome that theory that the Tregs induced by HD IL2 may decrease the necessitated a hospital admission of this patient and the goal activity of autoreactive T cells to reduce the incidence of to identify a NKTR-214 dose that could be delivered as a irAEs (21). It is well established that IL2 signals control the safe, outpatient treatment. Based on the tolerability, conveni- polarization of CD4+ T-cell subsets leading to enhancement ent dosing schedule, and pharmacodynamic changes at all of Th1 and Tregs while limiting the differentiation into Th17 NKTR-214 doses tested, a NKTR-214 dose of 0.006 mg/kg (22, 23). Several mechanisms have been proposed to explain q3w was selected as the RP2D. the negative effect of IL2 on Th17, including lowering the NKTR-214 engaged the IL2R pathway and provided expression of the IL6Rβ on CD4+ T cells, thereby reducing robust systemic immune activation with repeated admin- the IL6-mediated STAT3 phosphorylation that is essential istration, as demonstrated by dynamic changes in absolute for the induction of Th17 (24). Other studies showed that the lymphocyte counts and sCD25. Most importantly, these IL2 signaling through STAT5, which competes with STAT3 pharmacodynamic changes did not wane after repeated for binding the IL17 gene locus, constrains the generation of cycles. NKTR-214 induced robust proliferation of CD4+ Th17 (25, 26). Hence, the immune effect of NKTR-214 on the T cells, CD8+ T cells, and NK cells in the blood, and the cells tumor appeared to be Th1-dominant with no induction of were activated as indicated by high levels of ICOS, PD-1, Th2 or Th17 gene signatures. Despite the increases in Tregs CTLA4, and OX40 expression. Importantly, treatment with in the periphery with NKTR-214 administration, expansion NKTR-214 resulted in a significant increase in the absolute of these cells was limited in the tumor. Preclinical evidence numbers of CD4+ T cells, CD8+ T cells, and NK cells. The from mouse tumor models indicates that NKTR-214 limits induction of CD4+ T-cell, CD8+ T-cell, and NK-cell prolif- intratumoral Treg proliferation and survival by way of pro- eration in the peripheral blood was also observed in patients moting apoptosis, thereby polarizing the TME toward CD8+ treated with human recombinant IL15 (rhIL15), and these T cells and consequently driving a very high CD8+/Treg ratio changes were most evident in NK and CD8+ T cells and to a (12, 27). Exploratory studies are in progress to further probe lesser degree in CD4+ T cells (16). IL15 shares the IL2/IL15Rβ the mechanisms by which peripheral Tregs are preserved (CD122) chain with IL2 and is known to stimulate prolifera- while intratumoral Tregs are not. tion of T cells, induce generation of cytotoxic lymphocytes, A key immunologic observation was the increase in the and stimulate prolonged expansion of NK cells. In contrast to percentage of proliferating CD8+ T cells also expressing IL2, IL15 has no marked effects on the stimulation of Tregs increased levels of PD-1 in the TME (Fig. 3H). Studies have and does not cause substantial toxicity necessitating intense demonstrated that PD-1 identifies CD8+ tumor-reactive T supportive care. However, the use of rhIL15 as a therapeu- cells in infiltrating tumors (2, 28–31). The increase in the tic agent may not be optimal due to the low expression of percentage of CD8+PD-1+ T cells in patients’ tumors and IL15Rα and the need for high doses to achieve biological the tumor shrinkage observed in 35% of patients, includ- responses in vivo (17). The administration of rhIL15 as a daily ing one unconfirmed partial response, is promising. Durable i.v. bolus infusion for 12 consecutive days to 5 patients with SD greater than 1 year was seen in 2 patients, with 1 patient metastatic malignant melanoma or metastatic RCC proved having failed 3 prior immunotherapies. It is possible that to be difficult due to clinical toxicities produced by intense compensatory mechanisms, namely PD-L1/PD-L2 (Fig. 2E), cytokine secretion that occurred in the first 2 hours after may have dampened the potential of these CD8+PD-1+ treatment (16). T cells to convert patients into responders (31–33). Intriguingly, Importantly, treatment with NKTR-214 induced mean- 3 patients with RCC who were anti–PD-1 treatment-naïve ingful immunologic changes within the TME. At the RP2D, experienced TIL elevation with tumor reductions of 1%, 10%, freshly isolated TILs showed that there was a 15.1-, 3.5-, and 20%; all 3 patients ended NKTR-214 with SD and within

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NKTR-214 in Patients with Advanced Solid Tumors RESEARCH BRIEF

1 month started anti–PD-1 treatment and experienced rapid as the highest dose of NKTR-214 that was administered without caus- tumor reductions at first scan on-treatment, resulting in ing any unacceptable side effects or AEs. irAEs were considered AEs of partial responses (34). These anecdotal observations sup- special interest due to their potential clinical significance. ported the hypothesis that NKTR-214 treatment may have conditioned the TME by expanding activated TILs, thereby Biomarker Analysis potentially providing synergy with therapies that block inhibi- Serial blood and tumor tissue samples were collected to perform tory signals, such as those of the PD-1/PD-L1 axis. in-depth analysis of immune cells using flow cytometry, nCounter In summary, every patient evaluated showed evidence for gene expression assay (NanoString), IHC, and TCR sequencing; details are provided in the Supplementary Information. activation of the immune response, at all four doses tested, and these effects were reproduced with repeated administration. Statistical Methods Given the small number of patients evaluable at q2w (n = 4), Summary statistics were used for any continuous variables; num- no conclusions can be made on the impact of immune activa- bers and percentage were used for categorical variables. For all tion with a shorter frequency of dosing. Based on the biological analyses, baseline values were defined as the last value before the first activity and tolerability of NKTR-214 as well as the nonoverlap- dose of NKTR-214. All statistical calculations were performed using ping toxicities with approved checkpoint inhibitors, NKTR-214 Statistical Analysis System (SAS) version 9.4 (SAS Institute). Statisti- is being combined with approved anti–PD-1/PD-L1/CTLA4 cal analyses for immune profiling by flow cytometry were performed agents in ongoing clinical trials. NKTR-214, as a T-cell and with GraphPad Prism 7.0 (GraphPad Software). The significance of NK-cell growth factor, may serve as a central mechanism for the difference between groups in the experiments was evaluated by a boosting tumor-specific T-cell proliferation and activation in two-tailed paired t test. A value of P <0.05 was considered statistically the TME and has the potential to serve as a backbone therapy significant. for combination therapy with a variety of agents. Disclosure of Potential Conflicts of Interest M.E. Hurwitz reports receiving commercial research grants from METHODS Apexigen, Astellas, Innocrin, Iovance, Merck, Nektar Therapeutics, Novartis, Pfizer, Progenics, Sanofi Aventis, Seattle Genetics, Astra- Patients and Study Design Zeneca, Bayer, Bristol-Myers Squibb, Corvus, Eli Lilly, Endocyte, This was a multicenter, open-label dose-escalation study using Genentech, and Genmab; is a consultant/advisory board mem- a 3+3 design. Eligible patients were treatment-refractory to ≥1 ber for Nektar Therapeutics, Janssen, and CRISPR Therapeutics; prior therapy for advanced or locally advanced disease. Twenty- and reports the receipt of remuneration from Gamida Cell. H.M. eight patients with a histologically confirmed diagnosis of RCC, Kluger reports receiving commercial research grants from Bris- melanoma, urothelial carcinoma, breast cancer, leiomyosarcoma, tol-Myers Squibb, Merck, and Apexigen; is a consultant/advisory chondrosarcoma, or colorectal carcinoma not amenable to curative board member for Nektar, Iovance, Merck, Genentech, Corvus, therapy were enrolled. All patients provided written informed con- Immunocore, Biodesix, Apexigen, and Alexion; and reports receiv- sent before any protocol-specified procedures. The primary objec- ing other remuneration from Pfizer and Nektar. M.T. Tetzlaff is tives were to assess the safety, MTD, RP2D, and antitumor clinical a consultant/advisory board member for Novartis LLC, Myriad activity. Secondary objectives were to characterize pharmacokinetics, Genetics, and Seattle Genetics. M.A. Tagliaferri is CMO at Nektar pharmacodynamics, and immunologic changes in blood and tumor. and has ownership interest (including stock, patents, etc.) in the NKTR-214 was administered at 0.003, 0.006, 0.009, and 0.012 mg/ same. J. Zalevsky is Chief Scientific Officer at Nektar Therapeutics kg as a 15-minute i.v. infusion q3w or 0.006 mg/kg q2w until disease and has ownership interest (including stock, patents, etc.) in the progression or unacceptable toxicities occurred. Patients were held same. U. Hoch has ownership interest (including stock, patents, for 24 hours in an infusion center after the first dose of NKTR-214; etc.) in Nektar. C. Fanton has ownership interest (including stock, however, subsequent cycles were administered on an outpatient patents, etc.) in Nektar Therapeutics. S. Aung has ownership inter- basis. If no DLTs were observed in a cohort after 21 days, the dose est (including stock, patents, etc.) in Nektar Therapeutics. P. Hwu was escalated for the following cohort after review by the SRC, which is a consultant/advisory board member for Dragonfly, Glaxo consisted of the sponsor and at least one site investigator. Additional SmithKline, Immatics, and Sanofi. N.M. annirT reports receiving patients were added to a lower-dose cohort to further assess the a commercial research grant from Nektar Therapeutics and is a safety and tolerability. All patients who received study treatment consultant/advisory board member for the same. M. Sznol is a and one post-treatment scan were evaluable for antitumor activity. consultant/advisory board member for Genentech-Roche, Bristol- Investigator-assessed antitumor activity was evaluated using RECIST Myers, Merck US, Theravance, Biodesix, Vaccinex, Janssen/Johnson 1.1. Baseline tumor imaging was completed within 4 weeks before & Johnson, Modulate Therapeutics, Baxalta-Shire, Incyte, Newlink starting therapy and every 8 weeks thereafter. Tumor responses were Genetics, Lion Biotechnologies, AstraZeneca/Medimmune, Ago- confirmed with a repeat scan at least 4 weeks later. nox, Arbutus, Celldex, Inovio, Gritstone, Molecular Partners, Innate Pharma, AbbVie, Immunocore, Genmab, Pfizer, Almac, Hinge, Alla- Safety kos, Anaeropharma, Array, Symphogen-Scientific (advisory board), All patients who received any study drug were included in the Adaptimmune-Scientific (advisory board), Omniox-Scientific (advi- safety analyses. AEs, SAEs, and laboratory abnormalities were graded sory board), Lycera (formerly on scientific advisory board), Pieris per the National Cancer Institute Common Terminology Criteria for (scientific advisory board), Novartis, Amphivena (stock options Adverse Events, Version 4.03, and monitored throughout the study only), Adaptive Biotechnologies (stock options only), Intensity until 30 days after the last dose of study drug. TRAEs and TEAEs (stock options only), Axtym (stock options only), Torque, Torque were reported throughout the observation period and recorded once (stock options), Kyowa-Kirin, Seattle Genetics, Nektar, Pierre-Fabre, per patient at the highest grade. DLT was defined as any nontransient and Lilly. A. Diab reports receiving a commercial research grant grade 3, 4, or 5 hematologic or nonhematologic toxicity that occurred from Nektar Therapeutics and is a consultant/advisory board within the first 21 days after dosing and was deemed related or possi- member for the same. No potential conflicts of interest of were bly related to the administration of NKTR-214. The MTD was defined disclosed by the other authors.

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Authors’ Contributions 9. Kruit WH, Punt KJ, Goey SH, de Mulder PH, van Hoogenhuyze DC, Henzen-Logmans SC, et al. Cardiotoxicity as a dose-limiting factor in a M.E. Hurwitz, C. Haymaker, H.M. Kluger, Conception and design: schedule of high dose bolus therapy with interleukin-2 and -interferon. M.A. Tagliaferri, J. Zalevsky, U. Hoch, S. Aung, P. Hwu, B.D. Curti, An unexpectedly frequent complication. Cancer 1994;74:2850–6. N.M. Tannir, A. Diab 10. Lotze MT, Matory YL, Ettinghausen SE, Rayner AA, Sharrow SO, Development of methodology: S.-E. Bentebibel, M.E. Hurwitz, Seipp CA, et al. In vivo administration of purified human interleukin M.T. Tetzlaff, M.A. Tagliaferri, U. Hoch, C. Fanton, S. Aung, A. Diab 2. II. Half-life, immunologic effects, and expansion of peripheral lym- Acquisition of data (provided animals, acquired and managed phoid cells in vivo with recombinant IL 2. J Immunol 1985;135:2865– patients, provided facilities, etc.): S.-E. Bentebibel, M.E. Hurwitz, 75. H.M. Kluger, M.T. Tetzlaff, S. Aung, B.D. Curti, N.M. Tannir, M. Sznol, 11. Charych DH, Hoch U, Langowski JL, Lee SR, Addepalli MK, Kirk PB, A. Diab et al. NKTR-214, an engineered cytokine with biased IL2 receptor Analysis and interpretation of data (e.g., statistical analysis, bio- binding, increased tumor exposure, and marked efficacy in mouse statistics, computational analysis): S.-E. Bentebibel, M.E. Hurwitz, tumor models. Clin Cancer Res 2016;22:680–90. C. Bernatchez, C. Haymaker, C.W. Hudgens, M.T. Tetzlaff, M.A. 12. Charych D, Khalili S, Dixit V, Kirk P, Chang T, Langowski J, et al. Tagliaferri, J. Zalevsky, U. Hoch, C. Fanton, S. Aung, P. Hwu, N.M. Modeling the receptor pharmacology, pharmacokinetics, and phar- Tannir, M. Sznol, A. Diab macodynamics of NKTR-214, a kinetically-controlled interleu- Writing, review, and/or revision of the manuscript: S.-E. Bentebibel, kin-2 (IL2) receptor agonist for cancer immunotherapy. PLoS One M.E. Hurwitz, C. Bernatchez, C. Haymaker, H.M. Kluger, M.T. Tetzlaff, 2017;12:e0179431. M.A. Tagliaferri, J. Zalevsky, U. Hoch, C. Fanton, S. Aung, P. Hwu, 13. Katre N, Knauf MJ, Laird WJ. Chemical modification of recombinant interleukin 2 by polyethylene glycol increases its potency in B.D. Curti, N.M. Tannir, M. Sznol, A. Diab the murine Meth A sarcoma model. Proc Natl Acad Sci U S A 1987; Administrative, technical, or material support (i.e., reporting 1487–91. or organizing data, constructing databases): S.-E. Bentebibel, 14. Yang JC, Topalian SL, Schwartzentruber DJ, Parkinson DR, Marincola C. Fanton, S. Aung FM, Weber JS, et al. The use of polyethylene glycol-modified interleu- Study supervision: C. Bernatchez, M.A. Tagliaferri, S. Aung, A. Diab kin-2 (PEG-IL-2) in the treatment of patients with metastatic renal cell carcinoma and melanoma. 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A First-in-Human Study and Biomarker Analysis of NKTR-214, a Novel IL2R γβ -Biased Cytokine, in Patients with Advanced or Metastatic Solid Tumors

Salah-Eddine Bentebibel, Michael E. Hurwitz, Chantale Bernatchez, et al.

Cancer Discov Published OnlineFirst April 15, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/2159-8290.CD-18-1495

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