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