Combination of NKTR-214 and Radiotherapy (RT) to reverse anergy and expand specific CD8 T cells Joshua M. Walker , Michael J. McNamara , Melissa J. Kasiewicz , Ian Hilgart-Martiszus , Ute Hoch , Annah S. Rolig , Deborah H. Charych and William L. Redmond Oregon Health & Science University, Portland, OR Earle A. Chiles Research Institute, Providence Portland Medical Ctr., Portland, OR; Nektar Therapeutics, San Francisco, CA INTRODUCTION TUMOR-SPECIFIC T CELL RESPONSES TRANSCRIPTOME PROFILING We investigated therapeutic and mechanistic synergy between single-dose radiotherapy (RT) A and systemic administration of NKTR-214. NKTR-214 is a CD122-biased cytokine agonist CD4 T Cells: Percent Nur77-GFP+ CD8 T Cells: Percent Nur77-GFP+ 40 6 conjugated with releasable chains of polyethylene glycol. NKTR-214 provides sustained p<0.05 signaling though the IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand effector CD8 T and NK cells over regulatory T cells. Preclinical models demonstrated 30 79 t t 4 n NKTR-214 preferentially expands effector CD8 and NK cells in the tumor resulting in marked n tumor growth suppression as a single agent and in combination with checkpoint inhibitors. RT 20 erc e erc e P can induce antigen-release and epitope spreading, while NKTR-214 activates and expands P 2349 genes differentially 2 1251 genes DE antigen-specific effector populations. We hypothesized the combination of systemic 10 expressed (DE) NKTR-214/RT vs RT NKTR-214 and RT would generate better therapeutic responses than either treatment alone. NKTR-214/RT vs RT abscopal tumor A phase I/II trial is in progress to evaluate NKTR-214 safety and efficacy in an outpatient 0 0 setting as mono-therapy and in combination with nivolumab. irradiated tumor Blood BlooLymphd :Node T CellT umorGzmA Blood Lymph Node Tumor NKTR-214 synergizesMETHODS with radiotherapy to drive tumor regression CD8 T Cells: Percent AH1-Tetramer+ 50 40 Tumor models: CT26 (Colon Carcinoma, BALB/C, MCA-205 (Soft Tissue Sarcome, C57BL/6). 40 Control Tumor implant: Dual flank, 1 million cells per implant on day 0. t Treatments: Control (Vehicle), NKTR-214 (0.8 mg/kg, iv), Radiation (20gy x 1, n 30 30 administered to right flank target tumor only), Combination NKTR-214 plus t NKTR-214 B erc e radiation. All treatments administered on day 10. n − P 20 10 5 0 5 10 Radiotherapy and CT imaging Platform: Xtrahl Small Animal Radiation Change in gene expression from RT Research Platform (SARRP) RT (20 Gy x 1) 10 20 (Log2) Experimental timepoints: Day 18 (8 days after treatemtn) for all assays. erc e

Replicates: Tumor growth/ survival and peripheral activation (MCA-205 n=7, CT26 P Irr. Abs. Irr. Abs. Irr. Abs. n=21-22), Tumor specificity assays (Nur-77-GFP: CT26 n=3, AH1 tetrament: CT26 0 NKTR-214+RT CXCR6 CXCL12 Model of NKTR-214 GZMD CXCR6 CCL19 n=27, TIL (CT26 n=6), tumor transcriptome (CT26 n=3) Blood Lymph Node Tumor LTB4R1 CCL5 PEGylation sites at IL2 10 ITGAX RARRES2 interface with IL2Rα GZMD CCR2 XCL1 . Figure 2: CT26 tumors were implanted into a single flank of Nur77-GFP BALB/c mice on day 0. CCR5 TNFa NKTR-214 (green) with GZMG CCR5 CCL8 Animals received treatment (NKTR-214 +/- RT) on day 10 after implantation and blood, tumor, CSF2Ra CXCL10 sites of PEGylation (red CCR2 CCL4 and lymph nodes were0 harvested on day 18. GFP expression in CD4 and CD8 T cell populations in- GZME CCR2 RARRES2 circles). IL2γ chain CSF2Rb CCL27 dicates T cell receptor ligation. Frequency of AH1 tumor-specific positive CD8 T cells were mea- GZMF ITGA9 CCL27 receptor (pink), IL2Rβ ITGAV CXCL16 CSF2Rb RARRES2 (aqua blue), IL2Rα (dark sured using AH1 specific MHC tetramers. Combination radioimmunotherapy resulted in greater GZME CCR5 CCRL2 CXCL11 blue). CD4 & CD8 T cell activation and increased tumor-specific CD8 T cell frequency. CXCR3 CXCL4 GZMC SELP ITGA1 SELP ITGA1 SELP CD4 T CellsCD8 T Cells PRF1 ITGA9 CMKLR1 CXCL11 CSF1R CXCL16 FASL CCR1 CCL25 TUMOR GROWTH AND SURVIVAL PERIPHERAL ACTIVATION ITGB5 CXCL12 CCL25 FASL ITGA9 ITGB5 CCL12 CCR1 CCL1 Survival: MCA-205 Survival: CT26 GZMK CSF1R CXCL9 CXCL9 40 40 CSF1R T cell recruitment Control Cytotoxic effectors CMKLR1 SELE Ki-67 Granzyme A GZMA CCL28 100 100 CSF2Ra p=0.0004 RT (20 Gy x 1) ITGB5 CCL20 l l GZMN ITGAV CCL28 a a 30 30 ITGAV CCL11 v v NKTR-214 t t i i ITGB5 CXCL12 v v n n GZMB r r ITGBL1 CCL17

u NKTR-214+RT u ITGAV CCL2 s s

ITGB8 CCL21 t t KLRK1 20 20 ymphocyte homing to periphery n 50 p=0.04 n 50 CX3CR1 CCL7 erc e erc e L e e ITGAV CCL27 P p<0.0001 P GZMM CX3CR1 CCL22 er c er c P P 10 10 Figure 4. 8 d post-trreatment, tumors harvested from CD26 bearing mice were processed for 0 0 RNA isolation for gene expression analysis with Affymetrix Mouse 430.2 arrays. N=3 biological 0 50 100 0 20 40 60 0 0 replicates per group. Not false discovery rate corrected prior to statistical analysis. (A) Number Days Post Implant Days Post Implant CD4 T Cells CD8 T Cells CD4 T Cells CD8 T Cells of genes differentially expressed (DE) in the NKTR-214+RT group relative to RT in the IgG (Left Flank) IgG (Right Flank) IgG (Left Flank) IgG (Right Flank) 200 200 200 200 irradiated (red) and abscopal tumor (blue). Of those, 79 genes were differentially expressed in 40 60 150 150 150 150 CD25+ (IL-2Ra) CD122+ (IL-2Rb) both the irradiated and abscopal tumor. (B) Change in gene expression relative to RT treated 100 100 100 100 mice from the irradiated and abscopal tumors. Transcriptome profiling reveals increased CD8

50 50 50 50 30 activity and lymphocyte homing/recruitment with NKTR-214+RT treatment. t t n

n 40 0 0 0 0 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 20Gy RT (Abscopal side; L) 20Gy RT (Irradiated side; R) 20Gy RT (Abscopal side; L) 20Gy RT (Irradiated side; R) 20 erc e 200 200 200 200 erc e P P 150 150 150 150 20 Conclusions & Future Directions 10 100 100 100 100

50 50 50 50 Conclusions 0 0 0 0 0 0 -The addition of NKTR-214 to localized, single fraction RT controls tumor growth in both 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 CD4 T Cells Nektar-214 (Left side) Nektar-214 (Right side) Nektar-214 (Left side) Nektar-214 (Right side) CD8 T Cells CD4 T Cells CD8 T Cells irradiated and abscopal tumors and improves survival. 200 200 200 200 -NKTR-214 plus RT increases the frequency of tumor-reactive T cells in the target 150 150 150 150 20 2.5 tumors. 100 100 100 100 PD-1 TIM-3 -NKTR-214 induces robust T cell activation and differentiation in the peripheral blood, 50 50 50 50 2.0 15

t while the addition of RT did not have a significant impact on T cell phenotypes in the 0 0 0 0 t 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 n n ) ) e 2 RT+NKTR-214 (Absc.; L) RT+NKTR-214 (Irr.; R) 2 RT+NKTR-214 (Absc.; L) RT+NKTR-214 (Irr.; R) 1.5 PBL. 200 200 200 200 10

er c Ongoing Studies erc e 150 150 1.0 150 150 P P ea (mm ea (mm -Multispectral imaging (IHC) analysis of target and abscopal tumors. 100 100 100 100 5 0.5 -Evaluate the ability of NKTR-214/RT to resuce anergic tumor-reactive CD8 T cells. 50 50 50 50 umor a r umor a r T 0 0 T 0 0 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 10 20 30 40 50 0 0.0 Days post implant Days post implant CD4 T Cells CD8 T Cells CD4 T Cells CD8 T Cells Acknowledgements Figure 1: CT26 (BALB/c mice) or MCA-205 (C57BL/6) tumors were implanted into bilateral Figure 3: CT26 tumors were implanted and treated as in Figure 2. At the time of tumor harvest pe- flanks on d0. Established tumors (40-60 mm2) were treated (NKTR-214+/-RT) d10 post-implant. ripheral blood levels of proliferation, activation, and exhaustion markers on CD4 and CD8 T cells were -Nektar Therapeutics Tumor area and animals survival were determined following therapy. NKTR-214 synergizes measured. Significant increases in peripheral T cell activation, proliferation, and exhaustion appear -EACRI Flow Core and Mouse Husbandry Facility (CRAD) with RT to increase survival and tumor control in both irradiated and abscopal tumors. to be primarily driven by NKTR-214 treatment. -OHSU Gene Microarray Shared Resource