IL2 Variant Circumvents ICOS Regulatory T-Cell Expansion And

Published OnlineFirst October 3, 2016; DOI: 10.1158/2326-6066.CIR-15-0195

Research Article Cancer Immunology Research IL2 Variant Circumvents ICOSþ Regulatory T-cell Expansion and Promotes NK Cell Activation Geok Choo Sim1,2, Chengwen Liu1, Ena Wang3, Hui Liu4, Caitlin Creasy1, Zhimin Dai1, Willem W.Overwijk1, Jason Roszik1, Francesco Marincola3, Patrick Hwu1, Elizabeth Grimm1, and Laszlo Radvanyi1,2

Abstract

Clinical responses to high-dose IL2 therapy are limited due to Bcl-2 expression. Stimulation of PBMCs with F42K induced þ þ þ selective expansion of CD4 CD25 Foxp3 T-regulatory cells expression of more NK cell activation molecules, such as NKp30, þ (Treg), especially ICOS Tregs, rather than natural killer (NK) NKp44, DNAM-1, NKG2D, 4-1BB/CD137, and Tim-3, than WT þ cells and effector T cells. These ICOS Tregs are highly suppressive IL2. F42K induced greater upregulation of TRAIL, and NK- and constitutively express high levels of IL2Ra (CD25) and CD39. mediated cytolytic activity was increased against both autolo- Here, we characterized the effect of a mutant form of IL2 (F42K), gous and HLA-mismatched melanoma cells compared with WT which preferentially binds to the lower affinity IL2Rbg with IL2. Gene expression analysis revealed distinct gene expression reduced binding to CD25, on Tregs, effector NK cells, and T-cell profiles stimulated by F42K, WT IL2, and IL15. F42K therapy subsets. Unlike wild-type (WT) IL2, F42K did not efficiently in vivo also induced a dramatic reduction in the expansion of þ þ induce the expansion of highly suppressive ICOS Tregs in ICOS Tregs, promoted NK cell expansion, and inhibited mel- peripheral blood mononuclear cells (PBMC) from healthy con- anoma tumor growth more efficiently than WT IL2 and more trols and melanoma patients. Instead, it promoted the expan- effectively than anti–CTLA-4. Our findings suggest that F42K þ þ sion of CD16 CD56 NK cells and CD56hiCD16 NK cell could be a potential substitute for WT IL2 as a cytokine therapy subsets in both short- and long-term cultures, with enhanced for cancer. Cancer Immunol Res; 4(11); 983–94. 2016 AACR.

þ þ high level by CD4 Foxp3 regulatory T cells (Treg), especially Introduction þ þ highly suppressive ICOS Tregs, and some activated CD8 T and IL2 is one of the most well studied cytokines since its initial NK cells, whereas the intermediate IL2Rbg complex is constitu- discovery as "T cell growth factor" and later for its immunosti- þ þ tively expressed on effector T cells and CD16 CD56 NK cells. mulatory effects on natural killer (NK) cells and NK T cells. IL2 The discovery that IL2 regulates the survival, proliferation, and belongs to the g cytokine family and binds to the IL2 receptor, c differentiation of activated T and NK cells has led to the clinical which is composed of the three subunits IL2Ra (CD25), IL2Rb development of IL2 therapy and was one of the first FDA- (CD122), and IL2Rg (CD132). IL2 binds with different affinities, approved immunotherapies for metastatic melanoma and renal depending on the subunit composition, with binding to the cell cancer (5–7). It is also the first successful cancer immuno- IL2Rabg trimeric complex of greater affinity than binding to therapy to demonstrate that modulating the self-immune system either a single IL2 receptor subunit or to the IL2Rbg heterodimer with cytokine therapy could completely eradicate tumor cells (1–4). The IL2Rabg trimeric complex is expressed constitutively at under certain conditions. This success has also led to extensive clinical studies for treatment of metastatic melanoma that com- bine IL2 with adoptive T-cell transfer (8–10), peptide vaccines 1Department of Melanoma Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, Texas. 2Department of Immunology, H. Lee (11), and chemotherapeutic agents (12). Moffitt Cancer Center and Research Institute, Tampa, Florida. 3Division of Although IL2 therapy can induce long-lasting complete remis- Translational Medicine, Sidra Medical and Research Center, Doha, Qatar. 4Cell sions in metastatic melanoma and renal cell carcinoma patients, Processing Section, Department of Transfusion Medicine, Clinical Center, these effects are observed only in about 5% to 6% of treated National Institutes of Health, Bethesda, Maryland. patients. For many years, the use of high-dose (HD) IL2 therapy Note: Supplementary data for this article are available at Cancer Immunology (Proleukin, 720,000 IU/kg) has remained relatively limited due to Research Online (http://cancerimmunolres.aacrjournals.org/). its severe toxicities (13–16), such as the release of large amounts of Current address for Zhimin Dai: EMD Serono, 45A Middlesex Turnpike, Billerica, proinflammatory cytokines, including IL1b, TNFa, IL6, and IFNg þ MA 01821; current address for Geok Choo Sim: MerckKGaA, Frankfurter Str. (15, 16), and the direct binding of IL2 to CD25 endothelial cells 250, 64293 Darmstadt, Germany. that induce acute vasodilation effects (14). Another key issue with Corresponding Authors: Laszlo G. Radvanyi, EMD Serono, 45A Middlesex IL2 therapy is the dual functions of IL2 that acts not only as a driver Turnpike, Rockland, MA 02370. Phone: 978-944-7277; Fax; 978-944-7277; of effector lymphocyte responses, but at the same time it para- þ E-mail: [email protected]; and Geok Choo Sim, Department of doxically drives the expansion and suppressive function of CD4 Immunology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, þ Foxp3 regulatory T cells (Treg). Evidence from many clinical 33612. Phone: 813-745-3420; E-mail: [email protected] studies suggests that both high- and low-dose IL2 therapy pref- þ þ þ doi: 10.1158/2326-6066.CIR-15-0195 erentially expand CD4 CD25 Foxp3 Tregs (17–21), which 2016 American Association for Cancer Research. remain elevated after each cycle of HD IL2 therapy (19). This is

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a major factor that compromises the therapeutic efficacy of IL2 informed consent, and the collection and use of patient blood and thereby limits its clinical application. Our previous data show samples for laboratory analysis were approved by the Institu- that HD IL2 treatment induces expansion of a more activated and tional Review Board at The University of Texas MD Anderson suppressive subset of Tregs that are CD45RA /low and express Cancer Center (Protocol# LAB06-0762). higher levels of the inducible T-cell costimulator (ICOS) molecule (17, 22) and CD39 than ICOS Treg subset. Microarray gene profiling analysis Novel IL2-like cytokines are needed that do not induce PBMCs (2 106 cell/mL) from HC or melanoma patients cytokine storms and can circumvent the expansion as well as were stimulated with WT IL2, F42K, or IL15 at 0.4 nmol/L in effect on Tregs while differentially activating NK and T effector 24-well plate for 24 hours. Cells were harvested and total RNA cells. Attempts at improving IL2 have been developed by were isolated using AllPrep DNA/RNA/miRNA Universal Kit a generating IL2 variants with altered IL2R binding domains, (Qiagen) according to the manufacturer's instruction. The gene – such as F42K and R38A (23 25), which greatly decreases their profiling process and analysis were described in Supplementary fi a fi af nity for IL2R , while having a similar af nity as native Information. IL2 for binding to the IL2Rbg complex. However, the differential effects of how these IL2 variants on specific Treg subsets, effector T cells, and NK cells remain largely undefined. Based on Caspase-3 cleavage tumor target killing assay the findings by Heaton and colleagues (23–25), we sought in WT IL2- or F42K-treated PBMCs from melanoma patients for this study to compare the effects of F42K and WT IL2 on the 6 days were assayed for cytotoxicity activity by measuring active expansion of Tregs, NK cells, and T-cell subsets from healthy caspase-3 in tumor target cells (26). Autologous or HLA-mis- controls and patients with metastatic cancer. We also present matched melanoma cell lines and PBMCs derived from patients preclinical findings on the therapeutic potential of F42K and on were used for the cytotoxicity assay and the details were described the differential gene expression profiles elicited by F42K, IL2, or in Supplementary Information. IL15 in immune cells. Multiplex cytokine assays Materials and Methods PBMCs (2 106 cells) from HC and melanoma patients were PBMC expansion and immunophenotyping analysis plated and stimulated with equal concentration (0.4 nmol/L) of Freshly isolated peripheral blood mononuclear cells (PBMC) WT IL2 or F42K for 48 hours. Supernatants were collected and from normal healthy controls (HC) and melanoma patients cytokines measured using the Luminex multiplex cytokine detec- were seeded at 1 106 cell/mL in 24-well plate and were tion platform (Bio-Rad Laboratories). stimulated with WT IL2 (Proleukin; Prometheus Therapeutics and Diagnostics), F42K (provided by Dr. Elizabeth Grimm, PhD, Mouse tumor model studies The University of Texas MD Anderson Cancer Center) or IL15 Eight- to 12-week-old female C57BL/6 mice were purchased (R&D Systems) at 0.4 nmol/L or otherwise indicated for 6 days. from the National Cancer Institute, Bethesda, MD. All mice were Cells were harvested, counted, and stained for Treg, T, and NK cell maintained in a pathogen-free barrier facility at The University of markers. The expansion of immune cell subsets was determined Texas MD Anderson Cancer Center. All experiments and mice by combination of total cell count measurement and flow cyto- handling were performed according to the protocols approved by metry staining and was calculated by dividing the cell number the Institutional Animal Care and Use Committee. obtained on day 6 by the cell number obtained on day 0. Cells Mice were injected subcutaneously (s.c.) with 1 105 MCA205 were subjected to acquisition on a FACSCanto II flow cytometer fibrosarcoma cells or intravenously (i.v.) with 3 105 B16F10 (Becton Dickinson) and were analyzed using FlowJo software murine melanoma cells on day 0. Mice were randomized and (Treestar). treated i.v. with 2 mL of WT IL2, F42K, or control vector plasmid in saline on day 7 and 14 using hydrodynamic gene transfer (HGT) Cloning of IL2 technique (27, 28). For the MCA205 tumor model, tumor size was The full-length original IL2 gene amplified from human PBMCs measured 2 to 3 times per week using digital calipers and was was cloned into the commercial vector pcDNA3.1/V5-His-TOPO calculated by multiplying the length and width of each tumor. For (Life Technologies) and IL2 mutant with single-site mutation of lung metastasis model, mice were treated with plasmid DNA on phenylalanine to lysine at position 42 in the mature pre-protein day 3 with or without second dose on day 5. In some experiments, (F42K) was generated using the QuickChange II Site Directed 100 mg of anti–CTLA-4 (clone 9H10) was injected intraperitone- mutagenesis kit (Agilent Technologies) according to manufac- ally alone or with WT IL2, F42K, or control plasmids. Blood was turer's instructions. Endotoxin-free plasmids encoding WT IL2 or collected and stained for immune cells. Mice were sacrificed, and F42K and the control empty pcDNA3.1/V5-His/lacZ plasmid were lungs were removed and fixed in Fekete's solution (70% ethanol, prepared and purified using the EndoFree Plasmid Mega kit 3.7% paraformaldehyde, 0.75 mol/Lglacial acetic acid) on day 17. (Qiagen). Lung tumor nodules were counted under a binocular microscope.

Blood sampling and PMBC isolation Statistical analysis PBMCs from age-matched healthy controls (HC) and mel- Two-tailed Mann–Whitney U test, two-tailed Wilcoxon anoma patients (Pt) were either freshly isolated or cryopre- matched paired test, or one-way ANOVA was used to deter- served as described in a previous study (17). PBMCs from mine statistical significance (95% confidence interval), as patientswerecollected2to3daysafterthelastdoseofHD indicated. A P value of < 0.05 was considered statistically IL2 treatment. All patients enrolled in this study provided significant.

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þ þ Results CD8 T and CD4 Foxp3 cells as efficient as WT IL2 (Fig. 1B and C). By measuring the ratio of different effector cell subsets to F42K is less potent than WT IL2 at inducing Treg expansion, but þ ICOS Tregs, based on the absolute numbers obtained on day 6, supported NK cells expansion F42K stimulation also skewed the PBMC populations toward a F42K has been shown to bind to IL2Rbg and possessed reduced higher T effector:Treg ratio than WT IL2 (Fig. 1D). These biological affinity for IL2Ra chain (23–25). In this study, we further inves- effects of F42K suggest that it can efficiently drive NK cell expan- tigate how F42K affects the expansion of Tregs, especially þ sion while circumventing the expansion of immunosuppressive the highly suppressive ICOS Treg subset that correlates with þ ICOS Tregs better than WT IL2. clinical outcome during WT IL2 therapy (17). Freshly isolated PBMCs from HC were stimulated either with WT IL2 or F42K at 0.4 nmol/L (based on the initial dose response assays) for 6 days F42K supports expansion and long-term culture of NK cells and (Supplementary Fig. S1). Because accumulation of suppressive induces Bcl-2 expression cells (e.g., Tregs) is typically found in patients with metastatic Next, we evaluated the potential of F42K to support longer term cancers, we performed our analysis also on primary immune cells culture of Tregs and NK cell subsets. After 14 days of culture with from patients with advanced cancer to provide insight on how F42K, we observed similar differences with a preferential expan- þ F42K modulates the immune compartment of these patients. We sion of both NK cell subsets but not ICOS and ICOS Tregs (Fig. þ found that WT IL2 facilitated the expansion of highly suppressive 2A and B). The ICOS Tregs in long-term culture consistently þ þ CD39 CD127loICOS Tregs, but not effector T cells (Fig. 1A–C; expressed low levels of CD127 when stimulated with WT IL2 or Supplementary Figs. S2 and S3), more effectively than F42K in F42K, suggesting the cells were Tregs rather than T effector cells PBMCs from HC and melanoma patients. In contrast, F42K (Supplementary Fig. S3). The capacity of F42K to sustain long- þ þ preferentially promoted more expansion of CD16 CD56 cyto- term expansion of NK cell subsets was associated with increased lytic NK and CD56hiCD16 cytokine-secreting NK cell subsets cell survival and/or proliferation, with a significantly higher levels than WT IL2 (Fig. 1A–C). F42K stimulation induced expansion of (P < 0.05) of Bcl-2 (a prosurvival marker) expression and higher

Figure 1. F42K is less potent than WT IL2 in supporting the expansion of Tregs but supports the expansion of NK cells. PBMCs (2 106) from healthy controls (HC) and patients with stage IV melanoma patients were treated with 0.4 nmol/L of WT IL2 or F42K. Cells were harvested and stained for Treg, T-cell, and NK cell markers on day 0 and 6 after stimulation. Dot and scatter plots depict the gating strategy (A) for the identiﬁcation of immune cell subsets in response to F42K and WT IL2 from HC (B)and melanoma patients (C). D, graphs depict the ratio of effector to Treg cells upon stimulation. Horizontal bars represent median values, and nine independent experiments were performed. Pt, melanoma patients.

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Figure 2. F42K support long-term survival and expansion of NK cell subsets. PBMCs (2 106) from healthy controls (HC) and melanoma patients (Pt) were treated with 0.4 nmol/L WT IL2 or F42K. Cells were harvested and stained for Treg, T-cell, and NK cell markers on day 0 and 14 after stimulation. The fold expansion of immune cells subsets is indicated for PBMCs from HC (A) and melanoma patients (B). C (left), histograms from one representative melanoma patients and HC and scatter plots (right) show the Bcl-2 expression levels and median ﬂuorescence intensity (MFI) for NK cell subsets after 18 days of WT IL2 or F42K stimulation, and six independent experiments were performed. Pt, melanoma patients.

þ þ þ þ percentage of Ki67 CD16 CD56 and Ki67 CD56hiCD16 NK cells. After 3 days of stimulation, F42K induced a higher propor- cells than WT IL2 (Fig. 2C and Supplementary Fig. S4). Both NK tion of NK cells that expressed NKp30, NKp44, and CD137 cell subsets in bulk PBMCs showed more expansion when treated than WT IL2, at different concentrations (Supplementary Fig. þ þ with F42K than with WT IL2, whereas purified NK cells treated S6). Similarly, a greater percentage of CD16 CD56 and with either cytokine showed similar fold expansions. The fold CD56hiCD16 NK cell subsets in PBMC treated with F42K þ expansion of ICOS Tregs was lower in both bulk PBMCs and expressed significantly more (P < 0.05) NKp30, NKp44, and þ sorted CD4 T cells with F42K stimulation than with WT IL2 DNAM-1 than WT IL2 in HC and melanoma patients after 6 days (Supplementary Fig. S5). Thus, the biological effect of F42K likely of culture (Fig. 3A and B and Supplementary Fig. S7). The acts indirectly on NK cells by preventing expansion of Tregs. expression of NK cell–inhibitory KIR molecules CD158a and CD158b on NK cell subsets from HC and melanoma patients F42K induced a more activated NK cell phenotype than WT IL2 was induced to a similar extent (Supplementary Fig. S8). Like Next, we evaluated the efficiency of F42K at upregulating NK T cells, NK cells also express costimulatory molecules such as Tim- cell activation markers and costimulatory molecules that are 3 and 4-1BB (CD137) that regulate NK cell function in response to critical in mediating tumor cell recognition and killing by NK IL2 and IL15. Both F42K-treated PMBCs from HC and melanoma

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Figure 3. Phenotype and activation status of NK cells upon F42K treatment. PBMCs (2 106) from healthy controls (HC) and melanoma patients (Pt) were treated with 0.4 nmol/L WT IL2 or F42K. Cells were harvested and phenotyped for NK cell-activation markers and costimulatory molecules on day 0 and 6 after stimulation. The MFI of Nkp30, Nkp44, NKG2D, and DNAM-1 of NK cell subsets are depicted in the scatter plots from HC (A) and melanoma patients (B). The MFI of CD137 (C)and TIM-3–expressing NK cell subsets (D) are shown in scatter plots. Horizontal bars represent median values, and 10 independent experiments were performed. Pt, melanoma patients.

þ þ patients also showed a higher expression and percentage of Tim-3 and perforin expression by CD16 CD56 and CD56hiCD16 þ þ and 41BB-expressing CD16 CD56 and CD56hiCD16 NK cells NK cells, F42K-treated PBMCs from melanoma patients had than WT IL2 (Fig. 3C and D and Supplementary Fig. S7). Taken increased cytolytic activity against both autologous and HLA- together, F42K induced a more activated and mature phenotype mismatched melanoma cell lines than WT IL2 (Fig. 4C). By of both NK cell subsets compared with WT IL2. blocking HLA class I molecule, F42K stimulation also induced higher caspase-3 cleavage in the tumor cells than WT IL2 (Fig. Higher TRAIL expression and enhanced NK cell-mediated 4C). Thus, F42K induced significantly higher (P < 0.05) expres- killing with F42K sion of cytolytic molecules and promoted more NK-mediated The higher expression of NK cell activation molecules cytolytic function, than WT IL2. prompted us to further ask how F42K modulates the expression of cytolytic molecules and NK cell cytotoxic activity in Cytokine profiles induced by F42K and WT IL2 PBMCs. F42K induced higher percentage of TRAIL-expressing One of the major side effects of IL2 therapy is induction of þ þ CD16 CD56 and CD56hiCD16 NK cells and sustained cytokine storms by promoting the secretion of large amounts of higher proportion of granulysin expression than WT IL2 (Fig. proinflammatory cytokines, such as IL6, IL1b, IFNg, and TNFa 4A). TRAIL and perforin expression in NK cell subsets was also (13). F42K stimulation has been shown to induce a lower amount significantly higher (P < 0.05) upon F42K stimulation than WT of proinflammatory cytokines (24). We examined the cytokine IL2 (Fig. 4B and Supplementary Fig. S9). The granzyme B secretion profile of PBMCs from HC and melanoma patients þ þ expression level was higher in F42K-stimulated CD16 CD56 upon F42K or WT IL2 stimulation using a more extended panel and CD56hiCD16 NK cell subsets from melanoma patients but of inflammatory and immunosuppressive cytokines not studied not from HC (Supplementary Fig. S9). before with IL2 variants. PBMCs from HC and melanoma patients The higher proportion and expression of cytolytic mediators treated with F42K tended to produce less proinflammatory IFNg per cell seen in F42K- stimulated NK cells led us to further and TNFa than WT IL2 (Fig. 5). In addition, the secretion examine the cytolytic function of F42K-stimulated PBMCs from of immunosuppressive cytokines, including IL4, IL5, IL10, and melanoma patients against autologous or HLA-mismatched IL13, was significantly lower (P < 0.05) in PBMCs treated with melanoma tumor targets. Consistent with the higher TRAIL F42K than WT IL2 in melanoma patients samples (Fig. 5). No

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Figure 4. Functional properties of NK cells upon F42K stimulation. PBMCs (2 106) from healthy controls (HC) and melanoma patients (Pt) were treated with 0.4 nmol/L WT IL2 or F42K and were harvested and then stained for cytolytic markers on day 0 and 6 after stimulation. A, the percentage of CD56þCD16þ and CD56hiCD16NK cells that expressed cytolytic molecules is shown in the scatter plots. B, the MFI of TRAIL by NK cell subsets in response to WT IL2 or F42K is depicted in (left) histograms from one representative patient and (right) in scatter plots. After 6 days of WT IL2 or F42K stimulation, PBMCs from melanoma patients were cocultured with autologous, HLA-matched, or mismatched tumor cell lines in the presence or absence of W6/32 blocking at an effector-to-target ratio of 25:1 for 2 hours. Ten independent experiments were performed. C, the percentages of caspase-3 expressing tumor cells are shown in the graphs. Horizontal bars represent median values, and three independent experiments were performed. Pt, melanoma patients.

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Figure 5. Cytokine production induced by F42K. Cytokine secretion by PBMCs (2 106) stimulated with WT IL2 or F42K (0.4 nmol/L) from healthy controls (HC) and melanoma patients was measured using the Luminex multiplex cytokine assay. Cell cultured supernatant was harvested after 48 hours of WT IL2 or F42K stimulation. One independent experiment was performed. Pt, melanoma patients.

substantial IL4, IL5, IL10 and IL13 were detected in PBMCs from in inhibiting MCA205 tumor growth and in controlling B16F10 HC upon F42K or WT IL2 stimulation. melanoma lung metastases with significant (P < 0.05) reduction of lung tumor nodules in mice treated with F42K (Fig. 6A and B). Greater in vivo antitumor effects with F42K than WT IL2 Because F42K did not induce Treg expansion (especially highly þ To investigate the in vivo effect of F42K versus WT IL2 against suppressive ICOS Tregs), we also compared its effects against tumors, we generated F42K- and WT IL2-encoding plasmids that melanoma to anti–CTLA-4, which has been demonstrated to could be delivered in vivo using an HGT approach (27, 28). Upon deplete Tregs in melanoma mouse models (29). F42K alone is plasmid injection, we detected in vivo expression of both soluble superior to anti–CTLA-4 monotherapy in inhibiting lung metas- WT IL2 and F42K in serum of mice as early as 1 day after treatment tases. Furthermore, F42K and anti–CTLA-4 combination did (Supplementary Fig. S10). The F42K plasmid (up to 10 mg) treat- not further enhance the initial superior efficacy of F42K alone ment was well tolerated and did not induce significant toxicity, no over WT IL2 in controlling metastasis. The combination of anti– lung edema and other motor function or observable defects in mice CTLA-4 with WT IL2 induced moderate increased in controlling (data not shown). Single dose of WT IL2 but not F42K treatment metastasis compared with control IgG or anti–CTLA-4 monother- þ þ þ þ þ þ þ induced a dramatic increase of CD4 CD25 Foxp3 ICOS Tregs, apy (Fig. 6C; ref. 17). The percentage of bulk CD4 CD25 Foxp3 þ þ þ þ whereas there was a trend of higher levels of NK cells in F42K Tregs, highly activated CD4 CD25 Foxp3 ICOS Tregs, and þ þ þ þ treatment than WT IL2 on days 3, 6, and 9 (Supplementary GITR Lag3 ICOS OX40 Treg subsets was markedly lower in Fig. S11). F42K was more effective than WT IL2 and control vector blood after two doses of F42K treatment alone or together with

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Figure 6. F42K exhibited greater antitumor effect than WT IL2. A, C57BL/6 mice were injected s.c. with 1 105 murine MCA205 fibrosarcoma cells on day 0 and were treated with 2 doses of 10 mg WT IL2 or F42K encoding plasmid or control plasmid on days 7 and 14 using HGT. Tumor sizes were shown in the graph. B and C, mice received tail-vein i.v. injection with 3 105 B16F10 murine melanoma cells on day 0 and were treated with a single dose of WT IL2, F42K-encoding plasmid, or control plasmid at 10 mg. C, mice also received control IgG, anti–CTLA-4 at 3- to 4-day interval in combination with two doses of WT IL2, F42K, or control plasmid on days 3 and 5 after tumor injection. Mice were sacrificed on day 17, and lung tumor nodules were counted under binocular microscope. Blood was collected 3 or 9 days after first treatment and stained for Treg (D), T-cell (E), and NK cell markers. Horizontal bars represent median values, and three independent experiments were performed.

anti–CTLA-4, than in mice that received WT IL2 alone or with anti– complex and not IL2Ra/CD25. Principal component analysis CTLA-4 (Fig. 6D). F42K also preferentially promoted the expansion (PCA) showed a clear segregation of each treatment group and þ of NK cells and CD8 T cells with a higher frequency of a gene cluster with F42K treatment was found in between WT IL2 þ þ þ þ CD3 NK1.1 DX5 NK cells and CD3 CD8 T cells found in the and IL15 gene clusters with close proximity to WT IL2 cluster blood than WT IL2 or control vector treatment (Fig. 6E). The (Fig. 7A). IL15-treated PBMCs showed a markedly larger number biological effect of F42K found in vivo is consistent with our in of differentially expressed genes (4,449 genes, P < 0.005) than WT vitro data, suggesting that F42K has the potential to generate a more IL2, underlying the distinctness of the two cytokines (Fig. 7B). effective therapeutic antitumor effector response by circumventing F42K treatment rendered 374 significant (P < 0.05) differentially the expansion of highly activated and suppressive Treg subsets. expressed genes compared with WT IL2 of which 288 genes overlapped to genes modulated by IL15. A total of 2,206 distinct Gene expression profiling of PBMCs treated with WT IL2, F42K, genes were found upregulated or downregulated when comparing or IL15 F42K versus IL15 treatment (Fig. 7B). Supervised hierarchical We evaluated how F42K altered the gene expression profile of clustering of the 374 differentially regulated genes (P < 0.005) PBMCs in comparison with WT IL2 to provide more mechanistic between WT IL2 and F42K treatment revealed distinct upregulated insight into these differential effects of F42K. We included IL15 in or downregulated gene clusters that were modulated by F42K (Fig. these experiments, as it also preferentially binds to the IL2Rbg 7C). Cluster visualization showed that the F42K-modulated gene

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Figure 7. Gene expression profile induced by WT IL2, F42K, or IL15. PBMCs (2 106) from healthy controls (HC) were treated with 0.4 nmol/L of WT IL2, F42K, or IL15 for 24 hours. Cells were harvested, and total RNA were isolated for Affymetrix ST 1.0 microarray gene profiling analysis. A, PCA graph of global gene expression data shows that each sample is representative of a single color-coded sphere and each color code corresponds to WT IL2 (green), F42K (red), and IL15 (blue) stimulation. B, Venn diagram shows the number of overlapping and complementary genes that were differentially expressed with P < 0.005 in the comparison between F42K, WT IL2, and IL15 stimulation. C, heat map with hierarchical clustering comparing the log2 fold changes for genes significantly differed in WT IL2-, F42K-, and IL15-treated PBMCs. D, forest plot analysis shows upregulated or downregulated genes induced by F42K in comparison with WT IL2 stimulation based on their gene ontology enrichment. E, heat map shows immune response genes that were differentially regulated in response to F42K as compared with WT IL2 at P < 0.005. One independent experiment was performed.

profile had a similar upregulation or downregulation pattern as Discussion IL15, with a majority of the genes upregulated rather than down- WT IL2 therapy is a relatively potent immunotherapy. How- regulated (Fig. 7C). Gene ontology enrichment analysis based on ever, induction of Treg expansion caused by the preferential the 374 differentially expressed genes induced by F42K in com- binding of IL2 to the high-affinity IL2R, and toxicities induced parison with WT IL2 revealed a predominant upregulated by high proinflammatory cytokine secretion, have limited the immune gene signature related to regulation of immune response, broad application of IL2 in the clinic. In this study, we charac- immune effector processes, and antigen processing and presen- terized the effects of F42K, an IL2 mutant that has been shown by tation (Fig. 7D). Among those immune function-related genes, fi IL2Ra, HMGB1, IL1R1, IL13, TNFRSF21, NRG1, F2RL1, THBS1, others to have reduced af nity to IL2Ra (23, 25), on different subsets of immune cells and assessed its therapeutic potential in MERTK, and CD93 were significantly (P < 0.05) downregulated in generating antitumor responses and controlling tumor growth F42K-treated PBMCs relative to IL2 treatment (Fig. 7E). In addi- and metastasis in comparison with WT IL2. Although F42K has tion, NKp30 (NCR3) and perforin (PRF1) genes were among the been studied previously for its effect on LAK cells (23–25), it is not immune response genes significantly (P < 0.05) upregulated in known how it affects different subpopulations of Tregs, NK, and F42K-treated PBMCs compared with WT IL2 (Fig. 7E and Sup- effector cells and how it regulates antitumor responses. Several IL2 plementary Table S1).

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variants have similar reduced binding affinity to CD25 and and may induce activation rather than an inhibitory function facilitate antitumor responses in vivo (23–25, 30–32). However, in NK cells. these studies have not addressed how those IL2 mutants affect the Although F42K binds similarly to IL15 to the IL2Rbg hetero- balance between specific Treg and NK cell subsets in human dimer complex and shares some unique gene expression pat- PBMCs, especially in patients with metastatic cancer. We charac- terns, F42K and IL15 induce a distinct overall gene expression þ terized the effect of F42K on Treg subsets, including ICOS and profile in PBMCs of HC and patients. These differences may be ICOS Tregs, NK cell subsets, and T cells, not only from HC but attributed due to IL15 being trans-presented by IL15Ra also those from advanced cancer patients. We also compared the expressed on DC and monocytes/macrophages, whereas F42K effects of IL2 variant to IL15 and WT IL2 on gene expression. As far is not trans-presented to the bg receptor complex. The trans- as we know, no gene expression data has compared the effects of presentation of IL15 to IL15Ra induces different dynamics of IL2 variants to IL15 and WT IL2 have been presented so far. receptor occupancy and activation leading to stronger and Our results have revealed the biological effects of F42K, which longer-lasting downstream signaling events compared with supports short- and long-term expansion and survival of both soluble cytokine binding (46). Although it might be advanta- þ þ human CD16 CD56 and CD56hiCD16 NK cell subsets, while geous that IL15 can be trans-presented in the in vivo setting, one circumventing immunosuppression. The balance in the immune potential caveat is the availability of DC and macrophages for compartment shifts toward high effector to Treg ratios in both HC IL15 trans-presentation as cancer patients frequently have and patients. The biological effects of F42K on Tregs imply that it reduced DC numbers and dysregulated DC and macrophage could also circumvent the activation of IL2Ra-expressing endo- functions. Also, both IL15Ra and IL2Ra are shed via proteolytic thelial cells that mediate WT IL2 toxicity (14). Additionally, F42K cleavage by matrix metalloproteinases and can act as endoge- induced significantly less (P < 0.05) proinflammatory and immu- nous antagonists inhibiting IL15 and IL2 activity, respectively nosuppressive cytokines than WT IL2 in vitro and was nontoxic at (47, 48). Therefore, F42K may have some distinct advantages in the doses tested in vivo using HGT. Our findings add to our cancer immunotherapy through its interaction with IL2Rbg in a knowledge on the effects of F42K, in comparison with WT IL2 non-complexed fashion without the need of trans-presentation on Tregs and NK cell subsets from both HC and melanoma and the requirements of accessory DC, while also bypassing the patients. Although HD IL2 therapy activates and expands effector potential adverse effects posed by soluble IL15Ra and IL2Ra T and NK cells, IL2 increases the numbers of circulating Tregs even mentioned above. more, which potently suppress activation and proliferation of In addition, F42K has therapeutic potential for cytokine ther- þ þ effector CD4 , CD8 T, and NK cells, rather than inducing apy for cancer with an increased capacity of F42K over WT IL2 to antitumor responses (17, 33, 34). Indeed, failure of HD IL2 prevent in vivo melanoma lung metastasis development, a key role therapy in large numbers of patients is attributed to expansion of NK cells in tumor immunosurveillance. Similar F42K-mediated þ þ þ of CD4 CD25 Foxp3 Tregs, particularly the highly suppressive effects in the human system were recapitulated in our in vivo þ ICOS Treg subset that we have shown to correlate with IL2 mouse tumor models where F42K circumvented the expansion of þ therapy outcome (17, 18). F42K, in contrast, limited the expan- activated Treg subsets, particularly ICOS Tregs, without deplet- þ sion of ICOS Treg subset, the major Treg subset in the tumor ing the endogenous Treg pool, and was associated with activation þ microenvironment of melanoma (35, 36). ICOS Tregs are acti- and expansion of NK and T cells. HGT is an efficient way to induce vated and more suppressive than ICOS Tregs, with memory production of cytokines by hepatocytes and to allow assessment phenotype and coexpressing low level of CD127 but with high of in vivo activity of cytokines. One caveat with the HGT approach levels of CD25, Foxp3, PD-1, CD39, TGF-b, and IL10 compared is its limitation on frequent, repeated, multiple tail-vein injections þ with the bulk Treg population. ICOS Tregs may use a wider of plasmid to induce higher systemic cytokine levels to assess variety of suppressive mechanisms, including ATP depletion and toxicity that is achievable using recombinant protein injection. adenosine generation through the high expression of the CD39/ However, HGT allowed us to investigate efficiently the biological CD73 axis (37). Thus, the ability of F42K to circumvent the effect of F42K in vivo when a large numbers of purified recombi- þ expansion of these highly suppressive ICOS Tregs is a previously nant cytokines were not available due to the expense and difficulty unknown feature of this IL2 variant that can have important in manufacturing. Although F42K concentrations in the serum impact for improving IL2 therapy. were slightly higher than in WT IL2, the difference was not þ Increasing evidence demonstrates a significant role of NK statistically significant (P < 0.05), and the percentage of ICOS cells in tumor immunosurveillance and in preventing metas- Tregs induced by F42K remained at least 3-fold lower than WT tases in melanoma and other cancer types (38, 39). NKp30 and IL2. The slightly lower concentrations of WT IL2 in the serum NKp44 have critical roles in facilitating melanoma recognition could be due to several factors, including longer half-life of by NK cells, indicating that a higher expression of these F42K than WT IL2 and/or cytokine sink effect due to WT IL2 molecules induced by F42K is advantageous (39). A higher consumptions by CD25-expressing Tregs. Although our in vivo gene and protein expression of NKp30 induced by F42K may studies revealed no synergistic effect of the combination further enhance NK–dendritic cell (DC) interaction and DC of F42K with anti–CTLA-4, our findings indicated that F42K maturation via the NKp30/NKp30L axis (40, 41). It could be alone was more efficient than WT IL2 and was more efficient advantageous that F42K maintained a larger proportion of than anti–CTLA-4 in controlling lung metastasis. This is a both NK cell subsets that expressed higher TIM-3 and 4-1BB critical point considering the toxicity of anti–CTLA-4 therapy expression than IL2. The expression of 4-1BB by NK cells is key and the demonstrated mechanism of action of anti–CTLA-4 in in enhancing antibody-dependent cytolytic function of NK depleting Treg in patients and mouse models (29). F42K has a cells in the presence of agonistic 41BB antibody (42–44). similar mechanism of action by leaving Tregs "un-touched" TIM-3 expression is known to be an "exhaustion" marker on while expanding effector NK cells and would significantly T cells, but it is associated with a more mature NK phenotype increase (P < 0.05) the NK cell-to-Treg ratio. Moreover, unlike

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Effect of IL2 Variant on Tregs and NK Cells

anti–CTLA-4, F42K would not cause an indiscriminant deple- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, tion of natural (unactivated) Tregs, which are also needed in computational analysis): G.C. Sim, E. Wang, Z. Dai, J. Roszik, F. Marincola, preventing autoimmune reactions in patients. L. Radvanyi Writing, review, and/or revision of the manuscript: G.C. Sim, E. Wang, Taken together, the data presented here suggest that F42K can W.W. Overwijk, P. Hwu, L. Radvanyi circumvent the expansion and negative immunoregulatory effects Administrative, technical, or material support (i.e., reporting or organizing þ of highly suppressive ICOS Tregs, while promoting NK cell data, constructing databases): G.C. Sim, C. Liu, H. Liu, P. Hwu expansion and function. F42K also induces a unique gene expres- Study supervision: E. Grimm, L. Radvanyi sion profile and does not activate many IL2-induced genes, although it has the capacity to activate NK cells and NK cell– Acknowledgments associated activation genes, costimulatory molecules, and NK- We thank Dr. Karen Dwyer and her team from the Flow Cytometry Core mediated cytolytic function. As such, it may serve as a way of Facility and Immunomonitoring Core Lab of The University of Texas MD activating and maintaining NK cell function in cancer patients to Anderson Cancer Center for their support. We are also grateful for the hard work in acquiring patient samples by the research nurses at the Melanoma prevent metastasis and as immunosurveillance enhancer in a Medical Oncology Department, in particular Edwina Washington and Amber nontoxic way, avoiding Treg expansion during long-term therapy. Richardson. We also thank the support from Dr. Nallaparaju Kalyan of the Lion F42K may serve as a unique niche to overcome defects in NK cell Biotechnologies and Dr. Richard Wu Cheng-Han of The University of Texas MD function in advanced cancer patients through its selectivity for NK Anderson Cancer Center. cell activation. Grant Support fl Disclosure of Potential Con icts of Interest This work was supported by The University of Texas MD Anderson Cancer W.W. Overwijk is a consultant at GLG Consulting; reports receiving a Center SPORE in MelanomaP50 CA093459funded from the NCI (to E. Grimm commercial research grant from Nektar, Inc., 7 Hills Pharmaceuticals, and and G.C. Sim), the Dr. Miriam and Sheldon G. Adelson Medical Research Immatics, Inc.; and is a consultant/advisory board member for Immatics, Inc. Foundation (to L. Radvanyi), and The University of Texas MD Anderson Cancer No potential conflicts of interest were disclosed by the other authors. CenterP30 CA016627CCSG grant (to UTMDACC Flow Cytometry Core Facility and Immunomonitoring Core Facility). Authors' Contributions The costs of publication of this article were defrayed in part by the payment of advertisement Conception and design: G.C. Sim, P. Hwu, E. Grimm, L. Radvanyi page charges. This article must therefore be hereby marked in Development of methodology: G.C. Sim, C. Liu, E. Grimm, L. Radvanyi accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): G.C. Sim, C. Liu, E. Wang, C. Creasy, Z. Dai, Received August 9, 2015; revised August 22, 2016; accepted August 23, 2016; W.W. Overwijk, E. Grimm published OnlineFirst October 3, 2016.

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IL2 Variant Circumvents ICOS+ Regulatory T-cell Expansion and Promotes NK Cell Activation

Geok Choo Sim, Chengwen Liu, Ena Wang, et al.

Cancer Immunol Res 2016;4:983-994. Published OnlineFirst October 3, 2016.

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