Interleukin-2 Pathway Research Articles

Cellular Immunobiology and Molecular Mechanisms in Alloimmunity-Pathways of Immunosuppression.

Current maintenance immunosuppression commonly comprises a synergistic combination of tacrolimus as calcineurin inhibitor (CNI), mycophenolic acid, and glucocorticoids. Therapy is often individualized by steroid withdrawal or addition of belatacept or inhibitors of the mechanistic target of rapamycin. This review provides a comprehensive overview of their mode of action, focusing on the cellular immune system. The main pharmacological action of CNIs is suppression of the interleukin-2 pathway that leads to inhibition of T cell activation. Mycophenolic acid inhibits the purine pathway and subsequently diminishes T and B cell proliferation but also exerts a variety of effects on almost all immune cells, including inhibition of plasma cell activity. Glucocorticoids exert complex regulation via genomic and nongenomic mechanisms, acting mainly by downregulating proinflammatory cytokine signatures and cell signaling. Belatacept is potent in inhibiting B/T cell interaction, preventing formation of antibodies; however, it lacks the potency of CNIs in preventing T cell-mediated rejections. Mechanistic target of rapamycin inhibitors have strong antiproliferative activity on all cell types interfering with multiple metabolic pathways, partly explaining poor tolerability, whereas their superior effector T cell function might explain their benefits in the case of viral infections. Over the past decades, clinical and experimental studies provided a good overview on the underlying mechanisms of immunosuppressants. However, more data are needed to delineate the interaction between innate and adaptive immunity to better achieve tolerance and control of rejection. A better and more comprehensive understanding of the mechanistic reasons for failure of immunosuppressants, including individual risk/benefit assessments, may permit improved patient stratification.

JAK1 Pseudokinase V666G Mutant Dominantly Impairs JAK3 Phosphorylation and IL-2 Signaling

Overactive Janus kinases (JAKs) are known to drive leukemia, making them well-suited targets for treatment. We sought to identify new JAK-activating mutations and instead found a JAK1-inactivating pseudokinase mutation, V666G. In contrast to other pseudokinase mutations that canonically lead to an active kinase, the JAK1 V666G mutation led to under-activation seen by reduced phosphorylation. To understand the functional role of JAK1 V666G in modifying kinase activity we investigated its influence on other JAK kinases and within the Interleukin-2 pathway. JAK1 V666G not only inhibited its own activity, but its presence could inhibit other JAK kinases. These findings provide new insights into the potential of JAK1 pseudokinase to modulate its own activity, as well as of other JAK kinases. Thus, the features of the JAK1 V666 region in modifying JAK kinases can be exploited to allosterically inhibit overactive JAKs.

Understanding the spatial and functional link between the IL-2R and TCR at the immunological synapse.

The immunological synapse is the molecular platform for signal integration that controls T cell activation in response to antigen presentation. Interleukin 2 (IL-2) is an essential signal for effective T cell activation and proliferation after T cell receptor (TCR) engagement. However, the molecular mechanisms that link IL-2 signaling with TCR activation are not fully understood. Here, we investigate the spatial and temporal relationship between the IL-2 receptor (IL-2R) and the TCR function by visualizing their localization and activity during the formation of a synaptic contact. We use a supported lipid bilayer (SLB) system to mimic the surface of a virus-infected cell and we visualize antigen-specific CD8+ T cells forming a contact with it. The SLB system allows us to manipulate the two-dimensional accessibility of IL-2R and TCR ligands at the contact. For this purpose, we use the novel biologic therapeutics Immuno-STATs™. The full Immuno-STATs™ framework consists of a Fc-formatted peptide-HLA complex and a modified IL-2 with reduced affinity. Using structural variants of these compounds, we can manipulate the spatial link between IL-2 and TCR signaling pathways and evaluate its relevance for an efficient synapse formation. Our data sheds light into the mechanisms of early IL-2 action and IL-2R assembly during the immunological synapse.

Pilot study of bempegaldesleukin in combination with nivolumab in patients with metastatic sarcoma

PD-1 blockade (nivolumab) efficacy remains modest for metastatic sarcoma. In this paper, we present an open-label, non-randomized, non-comparative pilot study of bempegaldesleukin, a CD122-preferential interleukin-2 pathway agonist, with nivolumab in refractory sarcoma at Memorial Sloan Kettering/MD Anderson Cancer Centers (NCT03282344). We report on the primary outcome of objective response rate (ORR) and secondary endpoints of toxicity, clinical benefit, progression-free survival, overall survival, and durations of response/treatment. In 84 patients in 9 histotype cohorts, all patients experienced ≥1 adverse event and treatment-related adverse event; 1 death was possibly treatment-related. ORR was highest in angiosarcoma (3/8) and undifferentiated pleomorphic sarcoma (2/10), meeting predefined endpoints. Results of our exploratory investigation of predictive biomarkers show: CD8 + T cell infiltrates and PD-1 expression correlate with improved ORR; upregulation of immune-related pathways correlate with improved efficacy; Hedgehog pathway expression correlate with resistance. Exploration of this combination in selected sarcomas, and of Hedgehog signaling as a predictive biomarker, warrants further study in larger cohorts.

A Phase 2/3, Randomized, Open-Label Study of Bempegaldesleukin Plus Pembrolizumab vs. Pembrolizumab Alone in First-Line Treatment of Patients with Metastatic or Recurrent Head and Neck Squamous Cell Carcinoma with PD-L1-Expressing Tumors (PROPEL-36)

<h3>Purpose/Objective(s)</h3> Head and neck squamous cell carcinoma (HNSCC) represents the 8th most common cancer worldwide.¹ However, survival rates remain low at ≈50%, despite standard first-line treatment with the immune checkpoint inhibitor (ICI) pembrolizumab (PEMBRO) ± chemotherapy.² Recombinant interleukin-2 (IL-2) is associated with improved overall survival (OS) in HNSCC patients.³ Research has shown that high levels of tumor-infiltrating lymphocytes are prognostic of disease-free survival in this patient population. Bempegaldesleukin (BEMPEG; NKTR-214), a first-in-class immunostimulatory IL-2 cytokine prodrug, is engineered to deliver a controlled, sustained, and preferential signal to the clinically validated IL-2 pathway to selectively stimulate antitumor responses. Earlier studies of BEMPEG in combination with ICIs evaluated in patients with immune-sensitive cancers have shown the potential to increase and deepen treatment responses vs historical rates for ICIs alone.⁴ Here, we present the design of the PROPEL-36 study (NCT04969861) that evaluates BEMPEG + PEMBRO vs PEMBRO alone in patients with previously untreated metastatic or recurrent programmed death ligand-1 (PD-L1)-positive HNSCC (combined positive score ≥1). <h3>Materials/Methods</h3> This phase 2/3, multicenter study is enrolling ≈500 patients with cancer of the oropharynx, oral cavity, hypopharynx, or larynx (excluding nasopharyngeal tumors) and an Eastern Cooperative Oncology Group performance status of 0 or 1. Randomization will be stratified by disease status, PD-L1 expression, and human papillomavirus status. Patients are treated with BEMPEG and/or PEMBRO every 3 weeks for up to 35 cycles (≈2 years). The primary endpoints are OS and objective response rate. Secondary endpoints include progression-free survival, time to deterioration in global health status/quality of life, pain, and swallowing, change in quality of life, and safety. This study is now enrolling. <h3>Results</h3> TBD <h3>Conclusion</h3> TBD

PIVOT IO 011: A phase 1/2 study of bempegaldesleukin (BEMPEG; NKTR-214) plus nivolumab (NIVO) and tyrosine kinase inhibitor (TKI) versus NIVO and TKI alone in patients (pts) with previously untreated advanced or metastatic renal cell carcinoma (mRCC).

TPS403 Background: The current standard of care for advanced RCC (aRCC) utilizes immune checkpoint inhibitors (ICIs) and targeted agents. Novel combinations of these agents, such as NIVO + TKIs, have demonstrated clinical benefit over TKI monotherapy. In CheckMate 9ER, NIVO + cabozantinib (CABO) in aRCC demonstrated clinically meaningful efficacy results and a favorable safety profile, and was FDA approved (01/2021). High-dose interleukin-2 (IL-2) was used historically for durable responses in mRCC but had unfavorable toxicity with complex inpatient treatment regimens. BEMPEG, an immunostimulatory IL-2 cytokine prodrug, is engineered to deliver a controlled, sustained, and preferential signal to the clinically validated IL-2 pathway. Preferential binding of BEMPEG to the IL-2 heterodimeric receptor (IL-2Rβγ) activates and expands CD8+ T cells and NK cells over immunosuppressive Tregs. In the phase 1/2 PIVOT-02 study, BEMPEG + NIVO displayed encouraging antitumor activity in pts with mRCC, with a tolerable safety profile. The clinical activity and the manageable, non-overlapping toxicity profiles of the individual agents in this combination warrants exploration of triplet regimens in previously untreated aRCC. Methods: PIVOT IO 011 (NCT04540705) is a 2-part, phase 1/2, randomized, open-label study assessing the safety and efficacy of BEMPEG + NIVO + TKI triplet in pts with untreated aRCC or mRCC. In Part 1, pts will receive BEMPEG + NIVO + TKI (Part 1A: axitinib [AXI] or Part 1B: CABO; n≈6–24 pts in each TKI arm). While BEMPEG + NIVO dosing will remain consistent across phases, each TKI will have 2 doses evaluated in Part 1, and results will determine the recommended phase 2 dose for AXI/CABO in Part 2. In Part 2, pts will be randomized 1:1 to receive either BEMPEG + NIVO + CABO or NIVO + CABO (N≈250), stratified by IMDC prognostic score and prior nephrectomy status. If the CABO triplet has an unacceptable toxicity profile, Part 2 may be amended to use the AXI triplet. Key inclusion criteria: aRCC or mRCC with clear cell component; no prior systemic therapy for RCC, except 1 prior adjuvant/neoadjuvant therapy for completely resectable RCC (must have included an anti–VEGF agent with recurrence ≥6 months after last dose); and Karnofsky PS ≥70%. Key exclusion criteria: active CNS brain/leptomeningeal metastases; active, known, or suspected autoimmune disease; and inadequately treated adrenal insufficiency. Primary endpoints in Part 1: dose-limiting toxicities and safety; and in Part 2: overall response rate per RECIST v1.1 by investigator. Secondary endpoints in Part 2: progression-free survival, overall survival, safety. Duration of BEMPEG + NIVO will be ≤2 years, and treatment with TKI will continue until disease progression. The study is currently recruiting. Clinical trial information: NCT04540705.

An IL-2 mutein engineered to promote expansion of regulatory T cells arrests ongoing autoimmunity in mice.

Interleukin-2 (IL-2) controls the homeostasis and function of regulatory T (Treg) cells, and defects in the IL-2 pathway contribute to multiple autoimmune diseases. Although recombinant IL-2 therapy has been efficacious in certain inflammatory conditions, the capacity for IL-2 to also activate inflammatory effector responses highlights the need for IL-2-based therapeutics with improved Treg cell specificity. From a panel of rationally designed murine IL-2 variants, we identified IL-2 muteins with reduced potency and enhanced Treg cell selectivity due to increased dependence on the IL-2 receptor component CD25. As an Fc-fused homodimer, the optimal Fc.IL-2 mutein induced selective Treg cell enrichment and reduced agonism of effector cells across a wide dose range. Furthermore, despite being a weaker agonist, overall Treg cell growth was greater and more sustained due to reduced receptor-mediated clearance of the Fc.IL-2 mutein compared with Fc-fused wild-type IL-2. Preferential Treg cell enrichment was also observed in the presence of activated pathogenic T cells in the pancreas of nonobese diabetic (NOD) mice, despite a loss of Treg cell selectivity in an IL-2R proximal response. These properties facilitated potent and extended resolution of NOD diabetes with infrequent dosing schedules.

Abstract 903: In vivo efficacy of bempegaldesleukin, immune checkpoint inhibition, and targeted radionuclide therapy in immunocompetent murine model of head and neck cancer

Abstract In preclinical studies, we have demonstrated that delivering low dose radiation therapy to all sites of metastatic cancer using targeted radionuclide therapy (TRT) can improve the response to immune checkpoint blockade. NM600 is a tumor-targeting alkylphosphocholine radiolabeled with 90Y. Following intravenous administration, NM600 is selectively taken up and retained in most murine and human cancer cells. Bempegaldesleukin (NKTR-214) is a first in class, CD122-preferential interleukin-2 (IL2) pathway agonist being studied for its ability to influence the IL2 pathway and selectively stimulate an immune response. The primary objective of this study was to test the hypothesis that NKTR-214 and 90Y-NM600 would increase the response to immune checkpoint blockade in the immunologically “cold” MOC2 syngeneic mouse model of head and neck squamous cell carcinoma (HNSCC). C57BL/6 female mice were engrafted with MOC2, a murine HNSCC cell line, in the right flank. When mean tumor volume reached ~100mm3, mice were randomized into eight treatment groups using a 2 × 2 × 2 study design for combinations of NKTR-214, 90Y-NM600, and anti-CTLA4. 100 µCi 90Y-NM600 was administered intravenously (IV, treatment day 1). Prior in vivo dosimetry performed using the Monte Carlo based RAPID platform following serial 86Y-NM600 PET/CT imaging demonstrated that this activity delivered ~8Gy to the MOC2 tumor. 200 µg anti-CTLA4 was delivered by intraperitoneal injection on days 4, 7, and 10. 16 µg NKTR-214 was given IV on days 6, 15, and 24. Tumor growth was monitored. In a parallel study, cohorts of mice were treated with PBS (control), NKTR-214, 90Y-NM600, or NKTR-214 + 90Y-NM600, and tumors were collected at day 14 for flow cytometry analysis. In the spontaneously metastatic, immunologically “cold” MOC2 HNSCC tumor model, 62.5% of mice treated with the combination of 90Y-NM600, NKTR-214, and anti-CTLA4 experienced complete tumor response, and these mice showed no observable primary or metastatic disease 60 days after treatment initiation. No mice receiving single or dual therapy combinations exhibited complete tumor response (p = &amp;lt;0.01). Treatment with NKTR-214 + 90Y-NM600 resulted in increased tumor infiltration by CD8+ T cells. Intriguingly, 90Y-NM600 triggered increased expression of the IL-2β receptor, CD122, on the surface of tumor infiltrating CD8+ T cells, suggesting a novel mechanism of synergy with NKTR-214. Combination therapy with NKTR-214, 90Y-NM600, and anti-CTLA4 resulted in complete and durable tumor response in a difficult to treat murine model of HNSCC. Therapeutic interaction between TRT and NKTR-214 may be a result from low dose radiation increasing the expression of CD122 on tumor infiltrating lymphocytes. Further preclinical and early phase clinical investigations are warranted to evaluate the therapeutic potential of such combinations. Citation Format: Gustavo A. Sosa, Amber M. Bates, Ravi Patel, Reinier Hernandez, Joseph J. Grudzinski, Ian Marsh, Bryan Bednarz, Alexander Pieper, Erin Nystuen, Sarah Emma, Elizabeth G. Sumiec, Jamey P. Weichert, Zachary S. Morris. In vivo efficacy of bempegaldesleukin, immune checkpoint inhibition, and targeted radionuclide therapy in immunocompetent murine model of head and neck cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 903.

Low-dose Interleukin-2: Biology and therapeutic prospects in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic aggressive arthritis that is characterized with systemic inflammation response, the production of abnormal antibodies, and persistent synovitis. One of the key mechanisms underlying the pathogenesis of RA is the imbalance of CD4 + T lymphocyte subsets, from T helper (Th) 17 cells and regulatory T (Treg) cells to T follicular helper (Tfh) cells and T follicular regulatory (Tfr) cells, which can mediate autoimmune inflammatory response to promote the overproduction of cytokines and abnormal antibodies. Although the treatment of RA has greatly changed due to the discovery of biological agents such as anti-TNF, the remission of it is still not satisfactory, thus, it is urgently required new treatment to realize the sustained remission of RA via restoring the immune tolerance. Interleukin-2 (IL-2) has been discovered to be a pleiotropic cytokine to promote inflammatory response and maintain immune tolerance. Low-dose IL-2 therapy is a driver of the imbalance between autoimmunity and immune tolerance towards immune tolerance, which has been tried to treat various autoimmune diseases. Recent researches show that low-dose IL-2 is a promising treatment for RA. In this review, we summarize the advances understandings in the biology of IL-2 and highlight the impact of the IL-2 pathway on the balance of Th17/Treg and Tfh/Tfr aiming to investigate the role of IL-2-mediated immune tolerance in RA and discuss the application and the therapeutic prospect of low-dose IL-2 in the treatment of RA.

Abstract 4508: Modulating the release rate of polyethylene glycol (PEG) polymers from interleukin-2 (IL-2) PEG conjugates to optimize anti-tumor efficacy

Abstract The cytokine interleukin-2 (IL-2) is an endogenous agonist of the IL-2 pathway that activates and expands tumor killing lymphocytes. High doses of IL-2 are needed to activate CD8 T cells and NK cells, through binding on low-affinity IL-2 receptor beta gamma subunits (IL-2Rβγ). Severe toxic side effects associated with high doses, as well as the poor PK profile of IL-2, have limited its clinical usage. In addition of the toxicities, IL-2 also stimulates proliferation and activation of regulatory T cells (Tregs) through binding on the high-affinity heterotrimeric IL-2 receptor alpha beta gamma subunits (IL-2Rαβγ), which may antagonize the intended anti-tumor immunity. We report on an IL-2 PEGylation strategy to improve the pharmacokinetic properties of IL-2. Utilizing our proprietary PEGylation technology, IL-2 was covalently connected with multiple polyethylene glycol (PEG) polymers through releasable linkers. Our technology enabled the conjugation of IL-2 with average of 5-6 polyethylene glycol (PEG) polymers in high efficiency and 80-90% isolated yield. Anti-tumor efficacy of IL-2 was optimized by modulating the hydrolysis rate of the releasable linkers connecting IL-2 and polymers. The releasable linkers used in the study had the hydrolysis rate in the range of 15 to 120 hours to release one PEG polymer from the conjugates. The conjugates with hydrolysis rate of 20-60 h showed the optimal anti-tumor efficacy in B16F10 animal model. These results suggest that our novel approach of IL-2 PEGylation may optimize IL-2 biological activity and anti-tumor efficacy. Modulating the releasable rate of the PEG polymer would optimize the pharmacokinetic properties of the released active IL-2 species and enable the optimized anti-tumor efficacy. We are currently exploring the usage of our technology platform to further engineer IL-2 as a non-IL2Rα binding conjugate for improved toxicity profile. Citation Format: Hui Li, Yuntao Song, Haiping Zhou, Chuan Liao, Meng Xu, Chuang Li. Modulating the release rate of polyethylene glycol (PEG) polymers from interleukin-2 (IL-2) PEG conjugates to optimize anti-tumor efficacy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4508.

Bempegaldesleukin selectively depletes intratumoral Tregs and potentiates T cell-mediated cancer therapy

High dose interleukin-2 (IL-2) is active against metastatic melanoma and renal cell carcinoma, but treatment-associated toxicity and expansion of suppressive regulatory T cells (Tregs) limit its use in patients with cancer. Bempegaldesleukin (NKTR-214) is an engineered IL-2 cytokine prodrug that provides sustained activation of the IL-2 pathway with a bias to the IL-2 receptor CD122 (IL-2Rβ). Here we assess the therapeutic impact and mechanism of action of NKTR-214 in combination with anti-PD-1 and anti-CTLA-4 checkpoint blockade therapy or peptide-based vaccination in mice. NKTR-214 shows superior anti-tumor activity over native IL-2 and systemically expands anti-tumor CD8+ T cells while inducing Treg depletion in tumor tissue but not in the periphery. Similar trends of intratumoral Treg dynamics are observed in a small cohort of patients treated with NKTR-214. Mechanistically, intratumoral Treg depletion is mediated by CD8+ Teff-associated cytokines IFN-γ and TNF-α. These findings demonstrate that NKTR-214 synergizes with T cell-mediated anti-cancer therapies.

Superior Treg-Expanding Properties of a Novel Dual-Acting Cytokine Fusion Protein.

Autoimmune diseases are caused by uncontrolled endogenous immune responses against healthy cells. They may develop due to an impaired function of regulatory T cells (Tregs), which normally suppress self-specific effector immune cells. Interleukin 2 (IL-2) and tumor necrosis factor (TNF) have been identified as key players that promote expansion, function, and stability of Tregs. In vivo, both low-dose IL-2 therapy and TNF receptor 2 (TNFR2) agonism were shown to expand Tregs and alleviate autoimmunity. We here designed a novel dimeric dual-acting fusion cytokine, where mouse IL-2 is genetically linked to a TNFR2-selective single-chain TNF mutein (IL2-EHD2-sc-mTNFR2). IL2-EHD2-sc-mTNFR2 showed high affinity to TNFR2 and efficiently activated IL-2 and TNFR2-selective signaling pathways. Further, IL2-EHD2-sc-mTNFR2 promoted superior Treg expansion, with both the IL-2 and the TNFR2 agonist (sc-mTNFR2) component necessary for this biological response. Ultimately, we propose that IL2-EHD2-sc-mTNFR2 is a dual-acting cytokine that efficiently promotes Treg expansion and might have a superior therapeutic window than conventional IL-2-based drugs.

Restoring the balance in autoimmunity: the promise of IL-2 mutein

Abstract The development and function of regulatory T cells (Treg) relies on interleukin-2 (IL-2), and Treg efficiently compete with other cells for IL-2 by expressing the highest levels of the IL-2 receptor alpha-chain (CD25). Accordingly, defects in Treg and the IL-2 pathway have been reported in several autoimmune diseases including type 1 diabetes (T1D). Low-dose IL-2 therapy has shown promising results in autoimmunity; however, autoreactive immune cells can also be activated by IL-2, and the pleiotropic effects of IL-2 raise concerns regarding its widespread utility. Hence, there is a need to develop novel IL-2 based therapeutics with enhanced Treg-specificity. Hypothesizing that reduced IL-2 affinity for IL-2Rβ should increase dependency on high CD25 expression and enhance Treg-selectivity, we generated and screened twenty-eight Fc-fused murine IL-2 muteins (Fc.IL-2 mutein), and identified an optimized mutein that showed reduced signaling but prolonged association with CD25. In vivo, this optimal mutein led to greater enrichment and Ki-67 induction in Treg compared to wild-type (WT) IL-2, and reduced agonism of effector cells, resulting in an improved Treg:effector ratio. In the non-obese diabetic mouse model of T1D, we observed significant protection from disease in mice treated with Fc.IL-2 mutein compared to WT IL-2. The enhanced efficacy of the mutein was most pronounced at high doses, revealing preferential Treg stimulation across a wide dose range. Thus, Fc.IL-2 mutein treatment represents a safer and more efficient alternative for the WT IL-2 therapy, allowing for selective Treg enrichment and a targeted control of the autoimmune response.

Effect of microRNA-186 on oxidative stress injury of neuron by targeting interleukin 2 through the janus kinase-signal transducer and activator of transcription pathway in a rat model of Alzheimer's disease.

Recent studies have proposed that microRNAs (miR) function as novel diagnostic and prognostic biomarkers and therapeutic targets in Alzheimer's disease (AD), a common disease among the elderly. In the current study, we aim to explore the effect of miR-186 on oxidative stress injury of neuron in rat models ofAD with the involvement of the interleukin-2 (IL2) and the Janus kinase/signal transducers and activators of transcription (JAK-STAT) pathways. AD rat models were established, and dual-luciferase reporter assay and online software were used to confirm the targeting relationship between miR-186 and IL2. Immunohistochemistry was usedevaluating the positive rate of IL2. Afterward, to define the role of miR-186 in AD, miR-186, IL2, and JAK-STAT related protein (JAK2, STAT3) expressions were quantified. Cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide, and cell apoptosis was detected by flow cytometry. We observed downregulated miR-186 and IL2 and upregulated JAK-STAT signaling pathway related genes in AD. The overexpression of miR-186 was shown to significantly promote cell proliferation while suppressing cell apoptosis along with the expression of the IL2 and JAK-STAT signaling pathway related protein. Collectively, the key findings obtained from the current study define the potential role of miR-186 as an inhibitor of AD development by downregulation of IL2 through suppression of the JAK-STAT signaling pathway.

Selectively Agonizing Treg in Type 1 Diabetes with IL-2 Muteins

Regulatory T cells (Treg) play a pivotal role in maintaining self-tolerance. For their development and function, Treg rely on interleukin-2 (IL-2) and efficiently compete with other immune cells for IL-2 by expressing the highest levels of the IL-2 receptor alpha-chain (IL-2Ra or CD25). Treg deficiencies and alterations of the IL-2 pathway have been reported in several autoimmune diseases including type 1 diabetes (T1D) where the loss of self-tolerance leads to the destruction of insulin-producing beta cells in the pancreas. Low-dose IL-2 therapy has been shown to be successful in several autoimmune and inflammatory conditions; however, the potential for IL-2 to activate pro-inflammatory immune cells raises concerns regarding its widespread utility, particularly in type 1 diabetes (T1D) where pathogenic autoreactive T cells are activated by IL-2. Hence, there is a need to develop novel IL-2 based therapeutics with enhanced Treg-specificity. We have generated a series of Fc-fused murine IL-2 muteins (Fc.IL-2 mutein) to optimize Treg-selectivity, tissue distribution and PK. Twenty-eight Fc.IL-2 muteins were screened for their signaling potency and surface retention on Treg. Eight of the mutein candidates, showing a reduced signaling in Treg but prolonged association with CD25, were further characterized in vitro. Two of the weakest muteins were tested in vivo and showed a greater enrichment and Ki-67 induction in Treg compared to wild type (WT) IL-2, and less effect on CD4+ T effector cells, CD8+ cells and NK cells. Finally, in the non-obese diabetic mouse model of T1D, we observed a significantly improved protection in mice treated infrequently with Fc.IL-2 mutein compared to WT IL-2, and, unexpectedly, differences in efficacy between WT and mutein were more pronounced at a very high dose level. Altogether, our data suggest that Fc.IL-2 mutein treatment represent a safer and more efficient alternative for the WT IL-2 therapy, allowing a selective Treg enrichment and a targeted control of the autoimmune response. Disclosure L. Khoryati: None. S. Kumari: None. T. Pham: None. M. Bogdani: None. M. Gavin: None.

Abstract 558: Durable antitumor activity of the CD122-biased immunostimulatory cytokine NKTR-214 combined with immune checkpoint blockade

Abstract 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. NKTR-214 is a novel CD122-biased immunostimulatory cytokine which combines biased activation of the IL-2R beta receptor, greatly favoring activation of effector over regulatory T cells in the tumor microenvironment, with improved pharmacokinetics and tolerability compared to Proleukin® (aldesleukin) in non-clinical models. Here we examine the efficacy and mechanism of NKTR-214 combined with checkpoint blocking antibodies in murine tumor models. Methods: Mice bearing subcutaneous B16, LLC, CT26 or EMT6 tumors were treated with single agent NKTR-214 (q9d), aldesleukin (bid or qd), murine anti-CTLA-4 or anti-PD-1 (twice-weekly), or combinations of these agents. Tumor volume was measured before, during and after treatment. Immune cells were profiled by flow cytometry; CD8 or NK requirements for efficacy were assessed by in vivo depletion through 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 followed by observation with no additional test article treatments. Results: In the B16 model, superior single-agent efficacy was achieved by NKTR-214 with a 10-fold lower dose than aldesleukin, yet significantly increased CD8/Treg ratio in the tumor (&amp;gt;400). In the EMT6 model, while both aldesleukin and NKTR-214 synergized with anti-CTLA-4, a greater percentage of complete responders were consistently observed with NKTR-214 (73% versus 44%). Antitumor immunity induced by the combination required both NK and CD8 T cell activity, and was durable and specific with mice remaining tumor-free after re-challenge with an EMT6 but not CT26 implant. Finally, NKTR-214 combined with anti-PD-1 provided superior activity in the CT26 model compared with the combination of anti-CTLA-4 and anti-PD-1 (90% versus 60% tumor-free, respectively). Conclusions: NKTR-214 enables access to the potent IL-2 pathway, providing a mechanism of action complementary to checkpoint inhibition. Favorable pharmacokinetics of NKTR-214 allows sustained tumor exposure and dosing schedules commensurate with antibody therapies. The nonclinical results support the ongoing Phase 1 trial of NKTR-214 in patients with solid tumors. Citation Format: John L. Langowski, Rhoneil Pena, Yolanda M. Kirksey, Murali K. Addepalli, Ute Hoch, Jonathan Zalevsky, Stephen K. Doberstein, Deborah H. Charych. Durable antitumor activity of the CD122-biased immunostimulatory cytokine NKTR-214 combined with immune checkpoint blockade. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 558.

Anti-CD25 monoclonal antibody Fc variants differentially impact regulatory T cells and immune homeostasis.

Interleukin-2 (IL-2) is a critical regulator of immune homeostasis through its non-redundant role in regulatory T (Treg) cell biology. There is major interest in therapeutic modulation of the IL-2 pathway to promote immune activation in the context of tumour immunotherapy or to enhance immune suppression in the context of transplantation, autoimmunity and inflammatory diseases. Antibody-mediated targeting of the high-affinity IL-2 receptor α chain (IL-2Rα or CD25) offers a direct mechanism to target IL-2 biology and is being actively explored in the clinic. In mouse models, the rat anti-mouse CD25 clone PC61 has been used extensively to investigate the biology of IL-2 and Treg cells; however, there has been controversy and conflicting data on the exact in vivo mechanistic function of PC61. Engineering antibodies to alter Fc/Fc receptor interactions can significantly alter their in vivo function. In this study, we re-engineered the heavy chain constant region of an anti-CD25 monoclonal antibody to generate variants with highly divergent Fc effector function. Using these anti-CD25 Fc variants in multiple mouse models, we investigated the in vivo impact of CD25 blockade versus depletion of CD25(+) Treg cells on immune homeostasis. We report that immune homeostasis can be maintained during CD25 blockade but aberrant T-cell activation prevails when CD25(+) Treg cells are actively depleted. These results clarify the impact of PC61 on Treg cell biology and reveal an important distinction between CD25 blockade and depletion of CD25(+) Treg cells. These findings should inform therapeutic manipulation of the IL-2 pathway by targeting the high-affinity IL-2R.

Reduced interleukin-2 responsiveness impairs the ability of Treg cells to compete for IL-2 in nonobese diabetic mice.

Enhancement of regulatory T cell (Treg cell) frequency and function is the goal of many therapeutic strategies aimed at treating type 1 diabetes (T1D). The interleukin-2 (IL-2) pathway, which has been strongly implicated in T1D susceptibility in both humans and mice, is a master regulator of Treg cell homeostasis and function. We investigated how IL-2 pathway defects impact Treg cells in T1D-susceptible nonobese diabetic (NOD) mice in comparison with protected C57BL/6 and NOD congenic mice. NOD Treg cells were reduced in frequency specifically in the lymph nodes and expressed lower levels of CD25 and CD39/CD73 immunosuppressive molecules. In the spleen and blood, Treg cell frequency was preserved through expansion of CD25(low), effector phenotype Treg cells. Reduced CD25 expression led to decreased IL-2 signaling in NOD Treg cells. In vivo, treatment with IL-2-anti-IL-2 antibody complexes led to effective upregulation of suppressive molecules on NOD Treg cells in the spleen and blood, but had reduced efficacy on lymph node Treg cells. In contrast, NOD CD8(+) and CD4(+) effector T cells were not impaired in their response to IL-2 therapy. We conclude that NOD Treg cells have an impaired responsiveness to IL-2 that reduces their ability to compete for a limited supply of IL-2.

Copy number variations of HLA-I and activation of NKp30 pathway determine the sensitivity of gastric cancer cells to the cytotoxicity of natural killer cells.

Nude mice are important in vivo model for characterization of cell malignancy behavior; however, many cancer cells fail to form tumors in it. Understanding this defective mechanism may provide novel insights into tumorigenesis and how tumor cells escape innate immunity. Whole-genome sequencing was conducted on two gastric cancer (GC) cells, BGC823 and AGS, which do and do not form tumors in nude mice, to identify their genomic differences relevant to natural killer (NK) cells. We found that the tumorigenic capacity of human GC cell lines was dependent on the recruitment and activation of NK cells in xenograft tumors. We used whole-genome sequence (WGS) on GC cell lines to identify potential genes controlling susceptibility to NK-mediated killing. The tumorigenic cell line BGC823 expressed high levels of HLA-I because of copy gain and was resistant to NK cell killing. In contrast, another cell line AGS expressing low levels of HLA-I with activated NKp30/MAPK/IL-12 (interleukin-12) or IL-2 (interleukin-2) pathway was susceptible to NK lysis. Treatment of tumor bearing mice with systemic administration of IL-12 in combination with intratumor injection of anti-HLA-I antibody significantly increased NK cell recruitment into xenograft tumors, which became sensitive to NK killing, resulting in reduced tumor progression. In human GC specimens, decreased HLA-I expression and increased NK cells surrounding tumor cells were correlated with decreased metastasis potential and better prognosis of patients. Our results provide a mechanistic basis for GC cells to escape NK lysis and a promising prospect of NK immunotherapy for GC cells.

SILAC-based quantification of changes in protein tyrosine phosphorylation induced by Interleukin-2 (IL-2) and IL-15 in T-lymphocytes.

This data article presents the first large-scale quantitative phosphoproteomics dataset generated to decipher the signaling networks initiated by IL-2 and IL-15 in T-lymphocytes. Data was collected by combining immunoprecipitation of tyrosine phosphorylated proteins and TiO2-based phosphopeptide enrichment with SILAC-based quantitative mass spectrometry. We report all the proteins and phosphotyrosine-containing peptides identified and quantified in IL-2- and IL-15-stimulated T-lymphocytes. The gene ontology analysis of IL-2 and IL-15 effector proteins detected in the present work is also included. The data supplied in this article is related to the research work entitled “Simultaneous dissection and comparison of IL-2 and IL-15 signaling pathways by global quantitative phosphoproteomics” [1]. All mass spectrometry data have been deposited in the ProteomeXchange with the identifier PXD001129.