The Novel IL-2 Cytokine Immune Agonist NKTR-214 Harnesses the Adaptive and Innate Immune System for the Treatment of Solid Cancers

Salah Eddine Bentebibel1, Chantale Bernatchez1, Cara Haymaker1, Michael Hurwitz3, Patrick Hwu1, Mario Sznol3, Nizar Tannir1, Anthony Conley1, Harriet Kluger3, Sandra Aung2, Michael Imperiale2, Mary Tagliaferri2, Christie Fanton2, Ernesto Iacucci2, Jonathan Zalevsky2, Ute Hoch2, Adi Diab1 1The University of Texas MD Anderson Cancer Center, Houston, TX, United States of America, 2Nektar Therapeutics, San Francisco, CA, United States of America, 3Yale School of Medicine, New Haven, CT, United States of America

BACKGROUND RESULTS

NKTR-214 Figure 1. NKTR-214 Mechanism of Action Figure 5. NKTR-214 induces activating markers and co-inhibitory Figure 10. Increase in T cell clonality in tumors after treatment • NKTR-214 is a CD122-biased cytokine agonist + NKTR-214 2-PEG 1-PEG receptors in proliferating CD4 T cells in peripheral blood with NKTR-214 conjugated with multiple releasable chains of (6-PEG) Active Cytokine Active Cytokine PD-1 CTLA-4 OX40 ICOS Irreversible Irreversible 15 40 20 80 5 polyethylene glycol (PEG) designed to provide Release Release • An increase in overall T cell n=11 n=15 n=9 n=15 4 (%CD4)

sustained signaling through the heterodimeric + 30 60 3

(%CD4) 15 (%CD4) + (%CD4)

+ clonality observed in 8/12 + IL-2 receptor pathway (IL-2R) to preferentially 10 + 2 20 10 40 1 patients after NKTR-214 PD-1 ICOS OX40

+ + 10 + CTLA-4 LEGEND: + + 0 activate and expand effector CD8 T and NK 5 α Ki-67 treatment 1 NKTR-214 – Inactive 5 20 -1 Melanoma 5 Ki67 Ki-67 + Ki-67

cells over Tregs (Figure 1). 2-PEG – Active Cytokine + + Ki-67 -2 + Renal Cell Carcinoma 1-PEG – Active Cytokine γ γ β β In ltrate (Log2) • Increase in clonality associated CD4 -3 CD4 0 0 IL-2Rβγ IL-2Rαβγ 0 0 CD4 Chondrosarcoma C1D1 C1D8 CD4 C1D1 C1D8 C1D1 C1D8 C1D1 C1D8 Day Day Day Day -4 Each line represents a different patient with increased T cell infiltrate in NKTR-214 MONOTHERAPY STUDY NK, CD4+, and CD8+ T cells 15 40 20 80 -6 -5 -4 0 1 2 3 4 n=8 n=15 7/12 patients • A phase 1, multicenter, open-label, dose- n=10 n=16 Clonality (Log2)

(%CD4) 30 (%CD4) + 15

− 60 (%CD4) (%CD4) + + + escalation study (EXCEL) was conducted CLONAL EXPANSION 10 CD4+ Treg 20 10 40 ICOS PD-1

10 OX40 to assess the safety, preliminary efficacy, − − − NK + CTLA-4 NK CD4 − Figure 11. Case study: Patient with RCC stage IV, age 59, male, NK + + Ki-67 5 CD8 CD4 Helper Immunosuppressive pharmacokinetics, and pharmacodynamics CD8+ Helper 5 5 20 Ki-67 cells limit anti-tumor Ki-67 Ki-67 + + progressed on prior TKI + + Ki-67 NK + of NKTR-214 in 28 patients with locally NK CD8+ CD4 response + Helper CD8+ CD4

+ CD4 CD4 0 0 0 0 2-4 CD8 Helper CD4 Immunological Changes on advanced or metastatic solid tumors. CD4+ C1D1 C1D8 CD4 C1D1 C1D8 C1D1 C1D8 C1D1 C1D8 Duration of Time Interval to NK Helper Day Day Day Day NK CD4+ NKTR-214 Therapy Treatment NK + + Helper Treatment Next Treatment CD8 CD8+ CD4 CD4+ Activating and co-inhibitory marker gates set on C1D1 sample Legend is same as for Figure 6 Helper Helper • Outpatient regimen with a convenient NK CD4+ 1027 mean 675 mean Prior Therapy Sunitinib ~ 67 mos (PD) ~ 2 mos + Helper CD8+ CD4 Baseline 2 2 NK + Helper CD8 CD8+ cells/mm cells/mm IV dosing regimen every 2 or 3 weeks. Therapies NKTR-214 ~ 5 mos (SD) ~ 1 mo Figure 6. NKTR-214 induces activating markers and co-inhibitory Administered • NKTR-214 has a favorable safety and Stimulates Immune Response to Kill Tumor Cells receptors in proliferating CD8+ T cells in peripheral blood Nivolumab > 9 mos Ongoing tolerability profile.4 PD-1 CTLA-4 ICOS NKTR-214 (8 cycles) † Nivolumab – No evidence of immune-mediated AEs or organ 25 8 10 PD-L1 status = neg ~1 mo Blood and Tumor Biopsy Collection and Analysis n=10 n=11 n=10 related inflammation (eg, colitis, pneumonitis, 20 (%CD8) 8 (%CD8) + 6 Melanoma (%CD8) NKTR-214 NKTR-214 NKTR-214 + + 4 PR on 1st scan Conrmed PR + 2017 mean 1620 mean dermatitis, hepatitis, endocrinopathies). Q2W or Q3W 15 Renal Cell Carcinoma Week 3 2 2 4 cells/mm cells/mm ICOS Chondrosarcoma PD-1 +

– Grade 3 hypotension occurred in 14% of + = Blood Sample 10 CTLA-4 Cycle (C)/Day (D): C1D1 C1D8 C2D1 C2D8 C3D1 + 2 Breast Cancer patients and was rapidly reversible with IV fluids. Ki-67 2

Ki-67 Bladder Cancer Ki-67

5 + + Ki-67 – No patients experienced capillary leak syndrome. + Each line represents a

0 CD8 0 CD8 0 different patient

EOT C1D1 C1D8 CD8 C1D1 C1D8 C1D1 C1D8 – No Grade 4 TRAEs or treatment-related deaths Day Day Day 25 8 10 Pre NKTR-214 EOT NKTR-214 POST Nivolumab • Maximum-administered dose (MAD) was n=8 n=12 n=8 Left Lung Nodule Tumor 20 (%CD8) 8 Baseline Week 3 Week 8 EOT + H&E CD3 CD8 (%CD8) 6 (%CD8) 0.012 mg/kg q3w. − + (Max tumor response -10% per RECIST 1.1) Biopsies: + 4 Tumor Analysis 15 Blood Analysis 4 †PD-L1 status was obtained using the Cell Signaling antibody (Cell Signaling #13684, PD-L1 PD-1 ICOS

• Sustained exposure and robust PD changes − −  Fresh TIL analysis by ow cytometry 10 CTLA-4 m  Flow cytometry − (E1L3N)) at a 1:100 dilution on the Leica BOND RX 3,5 Ki-67 2 after a single dose of NKTR-214 (Figure 3).  IHC  Cytokines 2 Ki-67

5 Ki-67 + + Ki-67 +  T cell receptor gene sequencing  PK + Robust immune-stimulatory response

CD8 0 • NKTR-214 substantially increased CD8 T cells 0 0 CD8

 Gene expression analysis  PD (sCD25, lymphocytes) C1D1 C1D8 CD8 C1D1 C1D8 C1D1 C1D8 + 5 Day Day Day Increase in T cell receptor >2-fold induction of Increase in proliferation that were newly proliferative (Ki-67 ) (Figure 4). EOT, end of treatment. + Activating and co-inhibitory marker gates set on C1D1 sample clonality at wk3 immune checkpoint genes and and PD-1 expression 1.00 CD8-related genes at wk3 following NKTR-214 40 50 Baseline LAG3 CD4+ Ki67+ Table 1. NKTR-214 induces gene expression changes in tumors Week 3 ICOS CD8+ Ki67+ CD8 0.75 IDO1 40 30 + RESULTS Ki-67 associated with innate and adaptive immune pathways CTLA-4

(%CD4) 30 Pathway name (database source) pSize Genes in pathway differentially expressed post-treatment TIGIT +

20 +

PD-1 (%CD8) 0.50 ICOS; ITK; LCK; MAPK11; ZAP70; CD3E; CD247; CD8A; PD-L1 20 Figure 2. Time on Study and Best Overall Response T cell receptor signaling pathway (KEGG) 37 Ki-67 CD8B; CD40LG; CTLA-4; PDCD1 PD-L2 + 10 Melanoma SD TCR signaling in naïve CD8+ T cells (NCI) 20 CD8A; CD3D; LCK; CD3E; CD3G; CD8B; CD247; ZAP70; PRF1 Tim3 10 Melanoma SD 0.25 OX40 CD4 Bladder Ca SD IL-12-mediated signaling events (NCI) 43 SOCS1; IFNG; IL-2RA; CD8A; CD3D; CD3E; CD3G; CD8B; IL-2RB; TP53 0 0 Melanoma SD CD247; IL-2RG; STAT4; IL-12RB2; EOMES; LCK; GZMB; TBX21 CD19 C1D1 C1D8 SD - Best Overall Response is Stable Disease Day RCC SD(uPR) of reads Cumulative percentage PD - Best Overall Response is Progressive Disease IL-2-mediated signaling events (NCI) 21 SOCS1; IL-2RA; LCK; IL-2RB; IL-2RG; MAPK11; IFNG 0.00 0 2 4 6 8 10 CD8 Case Study RCC SD Fold change (wk3/baseline) 6 15 0.003 mg/kg q3w GZMB; IFNG; KLRC1; KLRC2; KLRD1; LCK; SH2D1A; CD244; 0.00 0.25 0.50 0.75 1.00 CD4+ Ki67+ PD-1+

RCC SD Nature killer cell mediated cytotoxicity (KEGG) 48 + + + + 0.006 mg/kg q3w Cumulative percentage of Ki-67 RCC PD ZAP70; CD247; KLRK1; NCR1; BID; SH2D1B; TNFRSF10C IFNG CD8 Ki67 PD-1 0.012 mg/kg q3w unique sequences RCC SD pSize: Total # of genes in pathway covered by NanoString assay (%CD4)

PRF1 + 0.009 mg/kg q3w 4 10

RCC PD +

• Increased T cell clonality and GZMB PD-1 Colorectal PD 0.006 mg/kg q2w • NanoString analysis includes 10 patients with matched baseline and wk3 samples and 5 additional patients with induction of immune-related genes TBX21 Discontinued treatment due to RECIST PD PD-1

RCC PD either baseline or wk3 sample + in the tumor, along with an increase CD8A Melanoma PD Discontinued treatment due to AE + − RCC (8), melanoma (4), chondrosarcoma (1), leiomyosarcoma (1), and triple negative breast cancer (1) 2 5 (%CD8) Melanoma PD Discontinued treatment due to other reasons in the proliferative index of immune CD8B

• 89 genes differentially expressed between pre- and post-treatment when all patients used in the analysis Ki-67 Triple Neg BC PD cells in the blood, indicate the IL-6 + RCC PD ability of NKTR-214 to induce SMAD3 RCC SD robust immune changes in patients CD4 0 0 RCC SD Figure 7. NKTR-214 induces gene expression changes in the 0 1 2 3 4 5 6 7 C1D1 C1D8 Melanoma SD Fold change (wk3/baseline) Day RCC SD tumor associated with T cell activation RCC SD Melanoma 12.5 NE Gene Gene name W3/C1 W15/C5 W30/C10 W45/C15 W60/C20 W75/C25 W90/C30 symbol RCC PD Leiomyosarcoma SD 10.0 CD8A CD8 alpha chain isoform Chondrosarcoma PD CONCLUSIONS RCC SD CD8B CD8 beta chain isoform RCC PD Th1 cell-specific transcription Data cut-off: October 4, 2017 TBX21 • Immunological activity in peripheral blood and tumor tissue consistent with Breast Ca NE 7.5 factor 0 W8/C4 W16/C8 W24/C12 W32/C16 W40/C20 W48/C24 W56/C28 W64/C32 W72/C36 W80/C40 W88/C44 W96/C48 W104/C52 IFNG Interferon gamma NKTR-214’s biological mechanism of biased IL-2 pathway activation Time on Study (Weeks/Cycles) 5.0 GZMB Granzyme B

Log−counts • NKTR-214 has a robust PK-PD profile PRF1 Perforin-1 Figure 3. Sustained exposure and robust PD changes after a single dose of NKTR-214 • Robust immune-stimulatory response in the tumor and blood 2.5 Baseline n=14 Mean Week 3 n=11 Median – NKTR-214 induced change in proliferative index of immune cells and Pharmacokinetics Pharmacodynamics p-value ≤0.05 for all genes, costimulatory/checkpoint markers only on newly proliferating immune cells (0.006 mg/kg, n=15) (0.006 mg/kg, n=15) 0.0 comparing baseline vs wk3 means 1000 Lymphocytes sCD25 CD8A CD8B TBX21 IFNG GZMB PRF1 3 6 – RNA expression changes consistent with increased immune cell infiltrate and NKTR-214-RC activation of effector mechanisms 100 NKTR-214-AC Figure 8. NKTR-214 induces immune checkpoint and 2 4 – Increased frequency of specific T cell clones and associated increase in T cell 10 co-inhibitory genes in the tumor

(Mean+SE) infiltrate suggests efficient remodeling of the T cell repertoire 12.0 Gene 1 2 Gene name

1 /L (Mean+SE)

9 symbol • The ability of NKTR-214 to increase TILs and increase PD-1 expression on ng/mL 10 PDCD1 PD-1 0.1 ng/mL (Mean+SE) 0 10.0 immune cells provides a sound biological basis for combination with 0 Cytotoxic T lymphocyte CTLA-4 1 8 15 22 1 8 15 22 1 8 15 22 associated protein 4 anti-PD1 checkpoint inhibitors Time (days) Time (Days) Time (Days) 8.0 LAG3 Lymphocyte activating 3 • Clinical trials currently enrolling patients with NKTR-214 in combination • Sustained exposure with half-life of ~20 hours • Transient decrease followed by increase in lymphocytes from Day 3 to Day 9 ICOS Inducible T cell costimulator 6.0 • Gradual increase in active cytokine species, • Transient increase in soluble IL-2 receptor alpha (sCD25) from Day 1 to Day 8, IDO1 Indoleamine 2,3-dioxygenase 1 with anti-PD1 checkpoint inhibitors: shed from activated T cells

reaching C 1-2 days post dose Log−counts max T cell immunoreceptor 4.0 TIGIT – PIVOT-02 clinical trial combining NKTR-214 + nivolumab in patients with • Exposure increases in proportion to dose • sCD25 levels return to baseline by Day 15 with Ig and ITIM domains • Free IL-2 is not detectable • Similar PD effects observed across dose levels metastatic melanoma, renal cell carcinoma, non-small cell lung cancer, 2.0 Baseline n=14 Mean Week 3 n=11 Median urothelial cancer and triple-negative breast cancers (NCT02983045) RC: Related Cytokine, total protein detection assay measures 6-PEG NKTR-214 and all derived species; p-value ≤0.05 for all genes, – PROPEL clinical trial combining NKTR-214 with atezolizumab or AC: Active Cytokine, detection assay for 2-PEG and lower active species 0.0 comparing baseline vs wk3 means PDCD1 CTLA-4 LAG3 ICOS IDO1 TIGIT pembrolizumab in patients with metastatic melanoma, urothelial and non-small cell lung cancers (NCT03138889) Figure 4. NKTR-214 promotes proliferation of CD4+ T cells, CD8+ T cells, and NK cells in Figure 9. NKTR-214 does not alter expression of Th17- or – A study of NKTR-214 + nivolumab in patients with metastatic and/or locally peripheral blood Treg-associated genes in the tumor advanced sarcoma (NCT03282344) 15.0 + - + Gene CD4+ T cells CD8 T cells CD3 CD56 NK cells Gene name symbol 70 100 12.5 70 n=16 n=13 Melanoma RORC ROR gamma; transcription factor ACKNOWLEDGEMENTS n=16 60 60 Renal Cell Carcinoma 80 Chondrosarcoma TGFB1 TGF beta 1 50 10.0 • The authors would like to acknowledge the contribution of patients and their families in

50 (%CD56) Breast Cancer

+ TGFB2 TGF beta 2 Bladder Cancer participation of this clinical trial.

(%CD8) 60 (%CD4)

+ 40 + 40 Each line represents a 7.5 IL-10 Interleukin-10 Ki-67 30 30 + 40 different patient Ki-67 Ki-67 + Log−counts + REFERENCES 20 20

CD56 5.0 - 20 Baseline n=14 Mean CD8 CD4 10 10 Week 3 n=11 Median 1) Charych DH, Hoch U, Langowski JL, et al. Clin Cancer Res. 2016;22(3):680–90.

CD3 2) Bernatchez C, Haymaker C, Tannir NM, et al. Presented at SITC 2016, National Harbor, MD. Poster #387. 0 0 0 2.5 C1D1 C1D8 C1D1 C1D8 3) Hurwitz ME, Diab A, Bernatchez C, et al. Presented at ASCO GU 2017, Orlando, FL. Poster #D17. C1D1 C1D8 Day Day Day p-value ≥ 0.1 for all genes, 4) Bernatchez C, Bentebibel SE, Hurwitz ME, et al. Presented at ASCO 2017, Chicago, IL. 0.0 comparing baseline vs wk3 means Ki-67+ set on baseline sample at ~5% 5) Nektar Therapeutics Analyst & Investor Event at ASCO 2017 Annual Meeting. [online] RORC TGFB1 TGFB2 IL-10 Available at: http://ir.nektar.com/events.cfm

Poster #P77: Society for Immunotherapy of Cancer 2017 Annual Meeting