967224 480 120 144

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967224 480 120 144time (hr)

0

5

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25 IL2-Rβγ

time (hr)

0

1

IL2-RαβγNKTR-214-ACIL21-PEG-IL22-PEG-IL2

Modeling of PEG chain release in vivo indicate that conjugated IL-2 derived from NKTR-214 occupies the IL-2Rβγ with an AUC that is 90-fold greater than IL-2

Mobilization of lymphocytes from the periphery into the tumor is an inherent property of NKTR-214

The combination of NKTR-214 and anti-CTLA4 delivers durable anti-tumor activity and vigorous immune memory recall when rechallenged over six months later

The mechanistic model was developed using in vitro release kinetics, in vitro receptor binding kinetics at IL-2Rαβγ and IL-2Rβγ and in vivo pharmacokinetic data. The model provided an estimate of receptor subunit occupancy of the indicated daughter species that evolve from NKTR-214.

Combining Complementary Mechanisms of Immune Activation: NKTR-214, a Biased IL-2 Pathway Agonist and Immune Checkpoint AntagonistsDeborah Charych, Peiwen Kuo, Murali Addepalli, Vidula Dixit, Rhoneil Pena, Werner Rubas, Samira Khalili, Ute Hoch, Peter Kirk, John Langowski, Stephen K. Doberstein, Jonathan Zalevsky | Nektar Therapeutics, 455 Mission Bay Blvd South, San Francisco, CA 94158

CONCLUSIONS

• NKTR-214 activates the IL-2 pathway by slowly releasing the PEG chains in vivo, evolving into an array of IL-2 conjugates that occupy the IL-2Rβγ with greater AUC than unconjugated IL-2 • In vivo, the NKTR-214 mechanism of action delivers a controlled, sustained and biased signal to the IL-2 pathway for several immune cell subtypes, In contrast, IL-2 results in a rapid burst and decline of signaling. The controlled activation profile from NKTR-214 may mitigate systemic toxicities • Mobilization of T cells from the periphery into the tumor is an inherent property of NKTR-214 • NKTR-214 synergizes with checkpoint blockade providing marked anti-tumor activity and durable survival • Mice treated with NKTR-214 and checkpoint blockade resist new tumors, even when tumor re-challenge occurs > 6 months after their treatment has ended • NKTR-214 is being evaluated in an ongoing outpatient Phase 1/2 clinical trial for the treatment of solid tumors as a single agent and in combination with anti-PD1

Abstract 3048, ESMO ANNUAL MEETING, October 9, 2016, Copenhagen, Denmark

INTRODUCTION

• Recombinant human IL-2 (aldesleukin) is an effective immunotherapy for metastatic melanoma and renal cell carcinoma with durable responses in ~ 10% of patients, but side effects limit its use

• IL-2 has pleiotropic immune modulatory effects[1] which may limit its anti-tumor activity • Binding to the heterodimeric receptor IL-2Rβγ leads to expansion of tumor-killing CD8+ memory effector T cells and NK cells • Binding to the heterotrimeric IL-2Rαβγ leads to expansion of suppressive Treg which antagonizes anti-tumor immunity

• NKTR-214 was engineered for biased immune-stimulatory effects and for providing a sustained signal through the IL-2 receptor pathway • The prodrug design of NKTR-214 comprises recombinant human IL-2 chemically conjugated with multiple releasable chains of polyethylene glycol (PEG) • Slow release of PEG chains over time generates active PEG-conjugated IL-2 metabolites of increasing bioactivity, improving pharmacokinetics and tolerability compared to aldesleukin • Active NKTR-214 metabolites bias IL-2R activation towards CD8 T cells over Treg[2]

REFERENCES[1] Boyman et al, Nature Reviews, 2012[2] Charych et al, Clinical Cancer Research, 2016[3] Spranger et al, J. Immunoth.. Cancer, 2014

In mice, a single dose of NKTR-214 gradually builds and sustains pSTAT5 levels in several immune cell subtypes. In contrast, IL-2 produces a rapid burst and decline of pSTAT5 levels

Mice were treated with either one dose of NKTR-214 (blue) or aldesleukin (red); at 0.8 mg/kg. Peripheral blood samples were collected at various time points post-dose. Levels of intracellular pSTAT5, (indicative of activation) and immune cell surface markers were assessed by flow cytometry. NK cells exhibited the greatest proportion of pSTAT5 signaling after a single dose of either NKTR-214 or IL-2. However, NKTR-214 induced a strikingly different kinetic profile that gradually increased to ~80% pSTAT5+ over several days and lasted to at least 72 hours. In contrast, NK activation after IL-2 peaked within a few minutes and receded to baseline < 6 later. Inset: Full time course of pSTAT5 signaling as a percentage of total CD3+ cells isolated from mouse peripheral blood. Signaling lasts ~1 week after one dose of NKTR-214. NKTR-214 (blue), IL-2 (red).

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NKTR-214 Vehicle

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NKTR-214 %pSTAT5 (CD3)IL-2 % pSTAT5 (CD3)

RESULTS, CONTINUED…

A single 2 mg/kg administration of NKTR-214 delivers 50-times higher active cytokine than five 3 mg/kg administrations of aldesleukin

IL-2 (green) with sites of PEGylation denoted as red dots

2-PEG-IL-2 (EC50 = 1.0 nM)

EC50, pSTAT5 CTLL-2

1-PEG-IL-2 (EC50 = 0.052 nM)

Free IL-2(EC50 = 0.026 nM)

Rapidly cleared in vivo

NKTR-214 Prodrug(Inactive)

20 h

20 h

NKTR-214 : Molecular design, prodrug and comparative tumor pharmacokinetics[2] NKTR-214 releases to an array of ‘daughter’ conjugated IL-2 species

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RESULTS

NKTR-214 was engineered to release PEG at physiological pH with predictable kinetics that follow the Arrhenius Law

The kinetics of PEG release was evaluated in vitro by quantifying free PEG over time using HPLC. The release of PEG from IL-2 followed first order kinetics. A. Log concentration-time profiles showed linearity and identical slopes at different initial concentrations. B. Release rate versus temperature followed the Arrhenius law and provided an activation energy for PEG release of 130 kJ/mole.

1/Temp

Ln(K

)

0.0032 0.0033 0.0034 0.0035 0.0036

-15

-10

Slope: -16989 ± 509.2

AUC of active species derived from NKTR-214*Receptor Free IL-2 from

NKTR-214± 1-PEG-IL2 2-PEG-IL2

1 90 114

IL-2Rαβγ

IL-2Rβγ

1 0.48 0.004

Efficacy of NKTR-214 in combination with checkpoint blockade. Balb/c mice bearing established subcutaneous CT26 colon tumors were dosed with NKTR-214, 0.8 mg/kg i.v. q9dx3 or checkpoint inhibitors, 200 ug/mouse 2x/week. (*, p<0.05 relative to vehicle)

T cell infiltration after treatment with checkpoint antibodies, NKTR-214, or combinations. Infiltration of T cells into mouse CT26 colon tumors was determined by TIL DNA fraction 7 days after the indicated treatments. Adaptive Biotechnologies, n=4 per group

Lymphocyte migration into tumors after NKTR-214. NKTR-214 was co-administered with Fingolimod (FTY720), an agent that blocks lymphocyte trafficking.[3] Fingolimod was dosed 5 ug/animal, qd p.o. Lymphocyte counts in blood were significantly reduced for study duration. Tumor growth inhibition (TGI) is shown at study endpoint. The addition of Fingolimod reduced the efficacy of NKTR-214. (One-way ANOVA, Dunnets multiple comparison test ***=p<0.001, ****=p<0.0001 vs. vehicle; #=p<0.05 vs. NKTR-214)

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Mice bearing EMT6 tumors became tumor-free from NKTR-214+anti-CTLA4 therapy. These were rechallenged > 6 months later with either EMT6, CT26 or Sham buffer. No further treatment was given. The mice rechallenged with the same tumor type immediately rejected the implanted cells shown by lack of tumor outgrowth. Immune cells in spleen, lymph and blood were enumerated by flow cytometry, n=4/group

Tumor free mice challenged with EMT6 tumor cells mounted a vigorous proliferative (Ki67+) effector CD8 T cell response. The response was greatest for mice previously treated with NKTR-214 and anti-CTLA4 rechallenged with the same tumor type (blue). In contrast, there was no response when mice were implanted with a different tumor (red) or mice who were never treated (brown, gray). Treated mice had not received therapy for over 6 months when rechallenged. Top row shows total CD8+ cells, bottom row shows effector memory CD8+ (CD44hi) in 3 tissues. Immunophenotyping was evaluated 7 days after tumor rechallenge

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30.45mg/ml0.225mg/ml0.1125mg/ml0.05626mg/ml

* The AUC of 1-PEG and 2-PEG are shown as fold-change over the AUC of IL-2 at each receptor complex. The AUC values for IL-2 at IL-2βγ and IL-2Rαβγ are 5.3 and 40.3 %-hr, respectively± not detected in plasma after administration of NKTR-214

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eEMT6 rechallengSham rechallenge

CT26 rechallengeTx naive EMT6

Tx naive Sham

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EMT6 rechallengeNaive (CT26)Naive (EMT6)

51/70 (73%)tumor-free tumor-free

dosing duration

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