Deborah H. Charych

NKTR-214, an Engineered Cytokine with Biased IL2 Receptor Binding, Increased Tumor Exposure, and Marked Efficacy in Mouse Tumor Models

Purpose: Aldesleukin, recombinant human IL2, is an effective immunotherapy for metastatic melanoma and renal cancer, with durable responses in approximately 10% of patients; however, severe side effects limit maximal dosing and thus the number of patients able to receive treatment and potential cure. NKTR-214 is a prodrug of conjugated IL2, retaining the same amino acid sequence as aldesleukin. The IL2 core is conjugated to 6 releasable polyethylene glycol (PEG) chains. In vivo, the PEG chains slowly release to generate active IL2 conjugates. Experimental Design: We evaluated the bioactivity and receptor binding of NKTR-214 and its active IL2 conjugates in vitro; the tumor immunology, tumor pharmacokinetics, and efficacy of NKTR-214 as a single agent and in combination with anti–CTLA-4 antibody in murine tumor models. Tolerability was evaluated in non-human primates. Results: In a murine melanoma tumor model, the ratio of tumor-killing CD8+ T cells to Foxp3+ regulatory T cells was greater than 400 for NKTR-214 compared with 18 for aldesleukin, supporting preferential activation of the IL2 receptor beta over IL2 receptor alpha, due to the location of PEG molecules. NKTR-214 provides a 500-fold greater exposure of the tumor to conjugated IL2 compared with aldesleukin. NKTR-214 showed efficacy as a single agent and provided durable immunity that was resistant to tumor rechallenge in combination with anti–CTLA-4 antibody. NKTR-214 was well tolerated in non-human primates. Conclusions: These data support further evaluation of NKTR-214 in humans for a variety of tumor types, adding to the repertoire of potent and potentially curative cancer immunotherapies. Clin Cancer Res; 22(3); 680–90. ©2016 AACR.

Modeling the receptor pharmacology, pharmacokinetics, and pharmacodynamics of NKTR-214, a kinetically-controlled interleukin-2 (IL2) receptor agonist for cancer immunotherapy

Cytokines are potent immune modulating agents but are not ideal medicines in their natural form due to their short half-life and pleiotropic systemic effects. NKTR-214 is a clinical-stage biologic that comprises interleukin-2 (IL2) protein bound by multiple releasable polyethylene glycol (PEG) chains. In this highly PEG-bound form, the IL2 is inactive; therefore, NKTR-214 is a biologic prodrug. When administered in vivo, the PEG chains slowly release, creating a cascade of increasingly active IL2 protein conjugates bound by fewer PEG chains. The 1-PEG-IL2 and 2-PEG-IL2 species derived from NKTR-214 are the most active conjugated-IL2 species. Free-IL2 protein is undetectable in vivo as it is eliminated faster than formed. The PEG chains on NKTR-214 are located at the region of IL2 that contacts the alpha (α) subunit of the heterotrimeric IL2 receptor complex, IL2Rαβγ, reducing its ability to bind and activate the heterotrimer. The IL2Rαβγ complex is constitutively expressed on regulatory T cells (Tregs). Therefore, without the use of mutations, PEGylation reduces the affinity for IL2Rαβγ to a greater extent than for IL2Rβγ, the receptor complex predominant on CD8 T cells. NKTR-214 treatment in vivo favors activation of CD8 T cells over Tregs in the tumor microenvironment to provide anti-tumor efficacy in multiple syngeneic models. Mechanistic modeling based on in vitro and in vivo kinetic data provides insight into the mechanism of NKTR-214 pharmacology. The model reveals that conjugated-IL2 protein derived from NKTR-214 occupy IL-2Rβγ to a greater extent compared to free-IL2 protein. The model accurately describes the sustained in vivo signaling observed after a single dose of NKTR-214 and explains how the properties of NKTR-214 impart a unique kinetically-controlled immunological mechanism of action.

Synergistic antitumor activity of the CD122-biased immunostimulatory cytokine NKTR-214 when combined with anti-PD-1 in murine tumor models.

Meeting abstracts NKTR-214 is an immunotherapeutic drug that exerts its biological activity by binding and activating the interleukin-2 (IL-2) receptor beta subunit (IL-2Rb), thereby causing expansion of memory effector T cells in the tumor. NKTR-214 consists of 4-6 releasable polyethylene glycol (

Abstract 1604: NKTR-214 synergizes with radiotherapy to drive tumor regression

The purpose of this study was to investigate therapeutic and mechanistic synergies between single-dose radiotherapy and systemic administration of NKTR-214. NKTR-214 is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol. NKTR-214 is 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 Regulatory CD4 T cells. Preclinical models demonstrated NKTR-214 preferentially expands effector CD8+ T cells and NK cells within the tumor resulting in marked tumor growth suppression as a single-agent and in combination with checkpoint inhibitors. A phase I/II trial is in progress to evaluate NKTR-214 safety and efficacy in an outpatient setting. Radiation therapy can induce antigen-release and epitope spreading, while NKTR-214 can active and expand antigen-specific effector populations. We hypothesized that the combination of systemic NKTR-214 and local radiotherapy would generate better therapeutic responses than either treatment alone. In this study, we evaluated the combination of systemic NKTR-214 treatment with single fraction high-dose radiotherapy (20 Gy) in multiple murine models. We used flow cytometry, multi-spectral histology, and whole tumor mRNA profiling to investigate local, systemic and potential abscopal immune responses. We used Nur77-GFP reporter mice to enable detection of T cell receptor ligation in vivo and to evaluate the effects of NKTR-214+RT on tumor-reactive T cells. The results from these studies indicate that the combination of NKTR-214 and radiotherapy is synergistic, providing significantly better anti-tumor responses than either monotherapy. Consistent with previous observations, NKTR-214 alone induces expression of a wide range of activation markers expressed by CD4 and CD8 T cells as well as NK cells in the blood, lymph nodes and tumor. The combination of radiotherapy and NKTR-214 was found to have several unique effects including a significant increase (>75%) in the absolute numbers of lymphocytes in the peripheral blood, increased expression of activation markers (CD25, PD-1) by CD8 T cells in the blood and tumor, and increased density of tumor-infiltrating NK cells. Evaluation of tumor infiltrating lymphocytes (TIL) in Nur77-GFP reporter mice revealed that the combination of NKTR-214+RT resulted in a higher frequency of recently activated (Nur77-GFP+) CD8 T cells in treated (irradiated) and abscopal (non-irradiated) tumors. Whole tumor mRNA profiling and multi-spectral histology of these tumors is being assessed to identify key differences in the tumor-microenvironment that may help to define the underlying mechanism of action. Taken together, these data provide evidence of synergy between localized radiotherapy and systemic NKTR-214 treatment via an expansion of activated, tumor-specific CD8 T cells. Citation Format: Michael J. Mcnamara, Melissa Kasiewicz, Ian Hilgart-Martiszus, Ute Hoch, Deborah H. Charych, William L. Redmond. NKTR-214 synergizes with radiotherapy to drive tumor regression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1604. doi:10.1158/1538-7445.AM2017-1604

Abstract 2671: Antitumor activity of NKTR-214 in combination with pmel-1 ACT in an aggressive murine melanoma model

The adoptive cell transfer (ACT) of genetically engineered T cells expressing cancer-specific T-cell receptors (TCR) has been shown to induce effective anti-tumor response. However, tumors frequently relapse after an initial response. Another strategy towards stimulating the immune system is the use of high-dose interleukin-2 (IL-2) to target the IL-2 receptor (IL2R), leading to immune cell expansion. However, clinically approved IL-2 expands both tumor-killing CD8+ effector T cells (CD8T) as well as regulatory T cells (Tregs) through binding the IL-2Rβγ and IL-2Rαβγ complexes, respectively. Tregs in the tumor lead to immune suppression, which hampers the antitumoral response. 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. We used the pmel-1 ACT/B16 melanoma tumor model to test the anti-tumor activity of NKTR-214 and evaluate its effects on tumor-specific TCR transgenic T cells. On Day 0 (D0) C57BL/6 mice were implanted with B16-F10 mouse melanoma cells and lymphodepleted with 500 cGy on D6. On D7, mice were treated with either the combination of ACT (T lymphocytes activated in vitro with 1 μg/ml gp100) plus NKTR-214 (0.8 mg/kg, q9dx3, i.v.) or with C57/B6 T cells plus PBS (vehicle control). The tumors of the vehicle control mice (n=12) rapidly grew to the 1500mm3 endpoint in 12 days post-treatment, versus 35 days for the NKTR-214 group (n=12) with only 1 out of 12 mice reaching endpoint. Bioluminescence imaging was used to visualize the in vivo distribution and tumor-homing of antigen-specific T cells. Interestingly, the reporter T cells were retained in the spleen until D7 and could be seen migrating to the tumor at D9 reaching peak of bioluminescence at day 12, a delayed time point compared to the 5 days usually observed in mice treated with standard IL-2. The signal persisted in the NKTR-214 + ACT group until D20 versus D7 in the vehicle-control animals. These data suggest that NKTR-214 + ACT is well tolerated and provides a robust anti-tumor response in the aggressive B16F10 model. Treatment with NKTR-214 + ACT robustly mobilizes T cells into the tumor where they durably persist. The robust and long-lasting effect of NKTR214 supports its potential use in combination with cell-based therapeutics. Citation Format: Giulia Parisi, Justin Saco, Siwen Hu-Lieskovan, Ruixue Zhang, Paige Krystofinski, Cristina Puig Saus, Deborah H. Charych, Antoni Ribas. Antitumor activity of NKTR-214 in combination with pmel-1 ACT in an aggressive murine melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2671. doi:10.1158/1538-7445.AM2017-2671

Abstract 1617: Mechanistic modeling of a new kinetically-controlled CD122 agonist for cancer immunotherapy: NKTR-214 pharmacokinetics, pharmacodynamics, and receptor pharmacology

Introduction: NKTR-214 is a biologic prodrug currently in a Phase 1 / 2 clinical trial in patients with solid tumors, as a single agent and in combination with anti-PD1. It 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. Here we describe a mechanistic mathematical model that quantifies conjugated aldesleukin (IL-2) species that are generated from NKTR-214, their sustained exposure, and biased receptor pharmacology in rodent models. Methods: A mechanistic mathematical model, using ordinary differential equations (ODE), was developed using Matlab® programming language to describe the dynamics of NKTR-214 PEG release, clearance, distribution, and receptor occupancy in vivo. The model was conditioned using experimental data for in vitro binding of unconjugated IL-2 and active conjugated IL-2 derived from NKTR-214 to IL-2 receptors using surface plasmon resonance, in vitro PEG release data, and mouse pharmacokinetics data. Simulations were performed to 1) quantify the concentration-time profiles of the various conjugated IL-2 species derived from NKTR-214 and contribution of each of them to the NKTR-214 mechanism of action, and 2) compare occupancy at IL-2Rβγ and IL-2Rαβγ receptors after administration of therapeutic doses of NKTR-214 and aldesleukin. Results: After NKTR-214 administration, PEG chains are released slowly from the prodrug and active conjugated IL-2 species gradually increase and reach maximum concentrations about one day post-dose, followed by sustained exposure for up to a week. Compared to an equivalent dose of aldesleukin, the active conjugated IL-2 species derived from NKTR-214 achieve a 26-fold higher area under the curve (AUC) of IL-2Rβγ occupancy, and a 0.34-fold lower AUC of IL-2Rαβγ occupancy. The significant difference is due to the combined effects of slow release of active conjugated IL-2 species from NKTR-214 and their favorable binding kinetics towards IL-2Rβγ. Aldesleukin, even when simulated at repeated daily doses or constant infusion, is incapable of increasing the receptor occupancy at IL-2Rβγ without simultaneously increasing the receptor occupancy at IL-2Rαβγ in this model. Conclusions: The mechanistic model demonstrated how NKTR-214’s intrinsic design enables both biased receptor pharmacology and sustained exposure in vivo. Such biased receptor binding could not be achieved by a sustained delivery of aldesleukin. Citation Format: Samira Khalili, Aleksandrs Odinecs, Deborah H. Charych, Vidula Dixit, Peter Kirk, Thomas Chang, John Langowski, Werner Rubas, Steve Doberstein, Jonathan Zalevsky, Michael A. Eldon, Ute Hoch. Mechanistic modeling of a new kinetically-controlled CD122 agonist for cancer immunotherapy: NKTR-214 pharmacokinetics, pharmacodynamics, and receptor pharmacology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1617. doi:10.1158/1538-7445.AM2017-1617

Abstract 1598: Single agent NKTR-214, a biased IL2 pathway agonist, increases immune cell infiltrates in brain tumors and prolongs survival in rodent (rattus) glioblastoma (GBM)

Background: Immunotherapy is an attractive option for brain tumor therapy if a robust infiltrative T cell response can be elicited in the tumor. 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. To assess the potential activity of single agent NKTR-214 in GBM, we used an orthotopic rat glioblastoma survival model. Methods:NKTR-214 was administered at 0.1 or 0.3 mg/kg q2w iv to Sprague-Dawley rats starting 2 or 7 days (D2, D7) post (p)-implantation of 106 C6 glioma cells into the right striatum. The model requires euthanasia by ~D14 due to tumor burden. Brain tumors were characterized by magnetic resonance (MR) and immunohistochemistry (IHC) for infiltration of CD4+ and CD8+ T cells and for retention of PEG polymer in brain tumor. Results: Compared to rats receiving vehicle (n = 15), survival was significantly prolonged after NKTR-214 treatment (n = 43, mean 17.2 vs. 10.0 days (P Conclusions: NKTR-214 is well tolerated, prolongs survival and induces immunological activity in the brain when administered to rats harboring orthotopic GBM. While there was no significant dose dependence, a marked increase in survival was observed when larger D7 tumors were treated with NKTR-214 compared to microscopic D2 tumors, associated with increased intratumoral CD8+ T cells. Levels of CD4+ were unchanged, consistent with the mechanism of CD-122 biased activation of the IL2 pathway. While requiring further study, it is intriguing that the increased sensitivity of larger tumors also corresponds to onset of angiogenesis and rapid tumor growth in this model. NKTR-214 is currently being evaluated in an outpatient Phase 1 / 2 clinical trial for the treatment of solid tumors. The results presented suggest a potential role for NKTR-214 in the treatment of patients afflicted with GBM. Citation Format: Lawrence Recht, Seema Nagpal, Taichang Jang, Milton Merchant, Irene Choi, Ute Hoch, Deborah Charych. Single agent NKTR-214, a biased IL2 pathway agonist, increases immune cell infiltrates in brain tumors and prolongs survival in rodent (rattus) glioblastoma (GBM) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1598. doi:10.1158/1538-7445.AM2017-1598

Immune memory in nonclinical models after treatment with NKTR-214, an engineered cytokine biased towards expansion of CD8+ T cells in tumor.

11545Background: NKTR-214 is an immune-stimulatory cytokine designed to provide a controlled, sustained, and CD122-biased receptor signal through the heterodimeric IL2 receptor pathway (IL2Rbg) res...

Abstract B50: Antineoplastic activity of Top I inhibitor etirinotecan pegol (NKTR-102) and PARP inhibitor rucaparib (CO-388) in platinum-resistant high-grade serous BRCA WT ovarian cancer PDX models.

Background: At the time of ovarian cancer platinum resistance, standard therapies have limited activity. New anti-cancer drug combinations may lead to improved clinical outcomes. Activity of topoisomerase I (Top I) inhibitors or poly(ADP-ribose) polymerase (PARP) inhibitors have been reported in ovarian cancer and have demonstrated potentiation in other models. Objective: To investigate the efficacy of the Top I inhibitor etirinotecan pegol (NKTR-102) and the PARP inhibitor rucaparib (CO-338) as single agents and in combination in patient-derived xenograft (PDX) models of ovarian cancer. Methods: Five ovarian cancer PDX models in SCID mice were treated for 28 days with NKTR-102 (10 mg/kg/q7d IV), rucaparib (150 mg/kg/qd PO), or combination NKTR-102 plus rucaparib at the same doses and schedules. Of the five PDX models, four were platinum resistant and one was a platinum-sensitive positive control. Each treatment arm had a total of 10 mice and animals were observed daily for body weight and general condition. Tumor size was assessed weekly by ultrasound. The primary endpoint was change in tumor cross-sectional area (cm2) from pretreatment baseline to final tumor burden. Western blot was performed to investigate potential biomarkers of response. Results: Compared to vehicle-treated controls, statistically significant improvement (p Conclusion: NKTR-102 and rucaparib are well tolerated in mice and demonstrate activity in platinum-resistant and platinum-sensitive ovarian cancer PDX models. Combination therapy demonstrated a trend towards decreased tumor size when compared to single agent activity during treatment. Current efforts are focused on extending the time on study for the PDX models evaluated to determine if statistical superiority of the NKTR-102/rucaparib combination can be established. Citation Format: Caroline C. Nitschmann, Rachel M. Hurley, Marc A. Becker, Xiaonan Hou, Ute Hoch, Thomas C. Harding, Deborah H. Charych, Scott H. Kaufmann, Paul Haluska, Saravut J. Weroha. Antineoplastic activity of Top I inhibitor etirinotecan pegol (NKTR-102) and PARP inhibitor rucaparib (CO-388) in platinum-resistant high-grade serous BRCA WT ovarian cancer PDX models. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B50.

Abstract B015: Antitumor activity of the CD122-biased immunostimulatory cytokine combined with immune checkpoint blockade requires innate and adaptive immunity

Background: While immune checkpoint blockade is a promising therapeutic approach, combination with agents that modulate complementary pathways may improve responses. Interleukin-2 (IL-2) immunotherapy leads to long-term responses in a small percentage of cancer patients, but systemic toxicity limits its use. In addition, IL-2 expands T regulatory cells, antagonizing antitumor immunity and resulting in a poorer clinical outcome. NKTR-214 is a novel CD122-biased immunostimulatory cytokine which combines biased activation of the IL-2R beta receptor subunit, greatly favoring activation of effector over regulatory T cells, with improved pharmacokinetics and tolerability compared to Proleukin in non-human models. Here we examine the efficacy and mechanism of NKTR-214 combined with anti-CTLA-4 in murine tumor models. Methods: Mice bearing subcutaneous EMT6 mammary tumors were treated with NKTR-214 q9d, murine anti-CTLA-4 or anti-PD-1 twice-weekly, or both in combination. Immune cell profiling was assessed by flow cytometry following treatment. CD8 or NK cells were depleted in vivo by serial anti-CD8 or anti-asialo-GM1 antibody injections, respectively. Antitumor memory and specificity was assessed in complete responders by challenging with EMT6 or CT26 colon carcinoma implants with no additional treatment. Results: While NKTR-214 and anti-CTLA-4 were not as efficacious individually, their combination was synergistic and well-tolerated with 83% of test animals tumor-free. Combination treatment increased NK and memory effector CD8 cells in both tumor and spleen. Antitumor immunity by the combination was durable and specific as 70% and 100% of mice remained tumor-free after challenge with a second and third EMT6 implant, but not after a subsequent CT26 implant. NKTR-214 combined with anti-PD-1 also proved synergistic with 40% of animals remaining tumor free following treatment. In vivo depletion of either CD8 effector or NK cells abrogated efficacy suggesting both contribute to the response. Conclusions: The mechanism of NKTR-214 antitumor immunity is complementary to checkpoint inhibition. Favorable pharmacokinetics of NKTR-214 allows sustained tumor exposure and dosing schedules commensurate with other therapies. This new therapeutic combination of NKTR-214 and checkpoint inhibition may similarly enable durable responses in humans. Citation Format: John L. Langowski, Seema S. Kantak, Rhoneil Pena, Yolanda Kirksey, Murali Addepalli, Steve Lee, Ute Hoch, Deborah H. Charych, Stephen K. Doberstein. Antitumor activity of the CD122-biased immunostimulatory cytokine combined with immune checkpoint blockade requires innate and adaptive immunity. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B015.