NKTR-214, an engineered cytokine, synergizes and improves efficacy of anti-cancer vaccination in the treatment of established murine melanoma tumor Meenu Sharma 1 , Faisal Fa’ak 1 , Louise Janssen 1 , Hiep Khong 1,2 , Zhilan Xiao 1 , Yared Hailemichael 1 , Manisha Singh 1 , Christina Vianden 1 , Adi Diab 1 , Jonathan Zalevsky 3 , Ute Hoch 3 , Willem W. Overwijk 1,2 1 Department of Melanoma Medical Oncology, University of Texas MD Anderson Cancer Center, Houston, TX 77030 2 University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX 77030 3 Nektar Therapeutics, 455 Mission, Bay Blvd South, San Francisco, CA 94158 Pmel-1 CD8+ T cells CD25+ Foxp3+ Tregs Pmel-1/Tregs Ratio • NKTR-214 efficiently synergized with vaccination, potently suppressing tumor growth and improving survival of mice compared to vaccination with IL-2. • NKTR-214 significantly enhanced pmel-1 CD8+ T cell numbers in tumor and spleen. • NKTR-214 specifically decreased numbers of immune-suppressive Tregs in the tumor while maintaining their numbers in spleen. • Despite the induction of very strong CD8+ T cell responses and anti-tumor activity, no gross toxicity was observed. • Accumulating evidence suggests that low baseline tumor infiltrating lymphocytes (TILs) are predictive for poor response to checkpoint inhibitor immunotherapies, 1,2 thus agents designed to specifically activate and expand TILs may improve the overall success and utility of checkpoint inhibitor therapies in patients with low TILs • Interleukin-2 (IL-2) is a cytokine that activates and expands tumor killing lymphocytes, but also potently activates suppressive T regulatory cells (Tregs) by binding to the heterotrimeric IL-2Rαβγ • NKTR-214 is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol and designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand effector CD8+ T and NK cells over Tregs 3 In this pre-clinical study, we investigated whether NKTR-214 can promote expansion and function of vaccination-induced, tumor specific effector CD8+ T cells using the murine B16 melanoma model. We also studied how NKTR-214 impacts the localization of effector CD8+ T cells and Tregs to tumor and spleen. 1. No treatment 2. Aldesleukin (100,000 IU X 5 doses - D0,D1,D2)_every 8 days 3. NKTR-214 (0.2 mg/kg) _every 8 days 4. Vaccine 5. Vaccine + aldesleukin (100,000 IU X 5 doses D0,D1,D2)_every 8 days 6. Vaccine+ NKTR-214 (0.2 mg/kg)_every 8 days Mice: C57BL/6 D 0 Gp100 peptide TLR-7 agonist Anti- CD40 IL-2 or NKTR-214 D 1 IL-2 D 2 IL-2 NKTR-214 Or IL-2 D 8 NKTR-214 Or IL-2 D 16 D -6 Tumor Induction 300,000 B16 wild type cells NKTR-214 Or IL-2 D 24 Treatment repeated till experimental end point o Gp100 peptide (50 ug/mouse)-L-tyr (adjuvant) o anti –CD40 (50 ug/mouse) o TLR-7 agonist (Imiquimod- 5 mice/pack) Vaccine formulation 0 20 40 60 80 100 0 20 40 60 80 100 No treatment NKTR-214 Aldesleukin Vaccine + Aldesleukin Vaccine + NKTR-214 Vaccine ** p= 0.0031 Days a=er vaccina?on Percent survival Pmel-1 T cell response Tregs Survival 0 20 40 60 80 0 50 100 150 Untreated NKTR3214 Aldesleukin Vaccine Vaccine + Aldesleukin p=0.039 Vaccine + NKTR3214 * p=0.018 * Days a@er vaccinaBon Tumor size (mm ² ) 0 50 100 150 0 10 20 30 40 50 60 70 80 No treatment 0 50 100 150 0 10 20 30 40 50 60 70 80 Vaccine Days a9er vaccina;on 0 50 100 150 0 10 20 30 40 50 60 70 80 NKTR?214 0 50 100 150 0 10 20 30 40 50 60 70 80 Aldesleukin 0 50 100 150 0 10 20 30 40 50 60 70 80 Vaccine + Aldesleukin 0 50 100 150 0 10 20 30 40 50 60 70 80 Vaccine + NKTR?214 Tumor size (mm 2 ) Tumor size No treatment Vaccine + Aldesleukin Vaccine + NKTR7214 Vaccine 45±8.8 % 12±5.2 % 6±3.3 % 1.2±0.8 % No treatment Vaccine Vaccine + Aldesleukin Vaccine + NKTRB214 Foxp 3 CD4 Introduction Objectives NKTR-214 improves anti-tumor efficacy of peptide vaccines NKTR-214 impacts the localization of Pmel-1 cells and Tregs between tumor and spleen Tumor Spleen Conclusions References Pmel-1 CD8+ T cells CD25+ Foxp3+ Tregs Pmel-1/Tregs Ratio Pmel%1/Treg ra-o (tumor) 0 1000 2000 3000 4000 ** 0 200000 400000 600000 * ** Number of Pmel,1 CD8+ T cells/ gram of tumor 10.5±2.3% 11.4±1.2 % 12.2±3.3 % 11±2.4 % No treatment Vaccine Vaccine + Aldesleukin Vaccine + NKTR@214 Foxp 3 CD4 0 500000 1000000 1500000 2000000 *** Number of Pmel,1/ Spleen 0 20000 40000 60000 80000 100000 * ns Number of Tregs / Spleen 0 50 100 150 * ** Pmel%1/Treg ra-o (Spleen) Tregs (% of CD4+ T cells) Pmel-1 (% of CD8+ T cells) 3.4±2.2% 72.3±5.2 % 91.1±3.5 % 95.2±3.5 % No treatment Vaccine Vaccine + Aldesleukin Vaccine + NKTRA214 Thy1.1 CD8 Thy1.1 0.1±0.8% 34.3±4.2 % 65.8±3.5 % 75.2±3.5 % No treatment Vaccine Vaccine + Aldesleukin Vaccine + NKTRE214 CD8 transfer of TCR transgenic pmel-1 CD8+ T cells D 0 Gp100 pep(de TLR-7 agonist An(- CD40 Pmel-1 NKTR-214 T cell response : Tumor/spleen D -6 Tumor Induc(on 300,000 B16 wild type cells D 7 1) Daud AI, Wolchok JD, Robert C, et al. J Clin Oncol. [Epub ahead of print] DOI:10.1200/JCO.2016.67.2477 2) Daud AI, Loo K, Pauli ML, et al. J Clin Invest. 2016;126(9):3447-52 3) Charych DH, Hoch U, Langowski JL, et al. Clin Cancer Res. 2016;22(3):680-90. 0 10 20 30 40 50 60 70 0 20 40 60 80 100 Untreated NKTR,214 Aldesleukin Vaccine Vaccine + Aldesleukin Vaccine + NKTR,214 *** *** < 0.0001 < 0.0001 Days a/er vaccina6on Thy1.1 + Pmel-1 T cells (% of CD8+ T cells) 0 10 20 30 40 50 60 70 0 20 40 60 80 100 Untreated NKTR,214 Aldesleukin Vaccine Vaccine + Aldesleukin Vaccine + NKTR,214 * * p=0.03 p=0.01 Days a/er vaccina6on CD25+ Foxp3+ Tregs (% of CD4+ T cells) 0 1000 2000 3000 4000 * ** * Number of Tregs/ gram of tumor