Native IL-15 Research Articles

Abstract 4057: Beyond IL-2: The promise of anti PD-1-attenuated IL-2 fusion protein in cancer immunotherapy

Abstract PD1+ CD8+ tumor-infiltrating T-cells have been at the forefront of cancer immunotherapies due to their antigen-specificity, cytotoxicity, proliferative capacity, and stem-like/memory properties. IL-2 is essential for T cell growth and proliferation, however, its systemic administration at a high dose was associated with intolerable toxicities, while low-dose treatment contradicted the intended therapeutic effect by activating regulatory T cells.To overcome the limitations of IL-2, we fused modified IL-2 sequences with reduced affinity for the murine IL-2 receptor to a PD1 antibody designed to target the PD1+ T cells found in tumors. The resulting murine PD1 antibody-attenuated-IL-2 (mAnti-PD1-IL2Att) demonstrated similar IL-2 potency on PD1+ cells as native IL-2, but diminished potency by orders of magnitude on PD1- cells. The agent was furthermore designed to bind PD1 non-competitively with known PD1-blocking antibodies, so that it could be used in combination with them to achieve effective anti-tumor response. We demonstrated that mAnti-PD1-IL2Att exhibits potent anti-tumor activity, resulting in complete tumor regression in a syngeneic MC38 colorectal cancer model, and significant anti-tumor activity in other models of different tumor types. The mechanism was evaluated by immunophenotyping and single-cell RNAseq. Shortly after the first administration of mAnti-PD1-IL2Att, CD8+ T cells, rather than Tregs, emerged as the primary responding cells; these CD8+ T cells exhibited increased proliferation and activation with reduced expression of exhaustion markers (PD1+Tim3+). Following subsequent doses, there was a notable increase in the number of effector cells, followed by a prolonged generation of effector and memory cells at a later stage. Myeloid cells exhibited increased transcriptional programs related to chemotaxis, co-stimulation, and co-inhibition, including the upregulation of the PD1-PDL1 pathway. By contrast, blockade of PD1 with a naked PD1 antibody led to distinct cellular responses and less pronounced anti-tumor activity, and a combination of the blocking antibody with PD1-attIL-2 was superior to either agent alone. Together, our findings suggest that mAnti-PD1-IL2Att potent anti-tumor activity was driven by its direct targeting of PD1+ on tumor-infiltrating CD8+ T-cells, shedding light on the specific changes in their dynamic differentiation trajectory and their impact on the tumor microenvironment. Citation Format: Sarah Amar, Yulia Liubomirski, Eilam Yeini, Michael Monsonego, Tomer Weiss, Galia Tiram, Anshika Katyal, Opal Avramoff, Anne Krinsky, Keren Reshef, Shai Dulberg, Ayelet Kaminitz, Dana Bar On, Inbal Ben Eliezer, Ortal Iancu, Tetsuya Taura, David Wilson, Paul Ayton, Ronit Satchi-Fainaro, Asaf Madi. Beyond IL-2: The promise of anti PD-1-attenuated IL-2 fusion protein in cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4057.

PEGylation Strategy for Improving the Pharmacokinetic and Antitumoral Activity of the IL-2 No-alpha Mutein.

In a previous work, an IL-2Rβγ biased mutant derived from human IL-2 and called IL-2noα, was designed and developed. Greater antitumor effects and lower toxicity were observed compared to native IL-2. Nevertheless, mutein has some disadvantages, such as a very short half-life of about 9-12 min, propensity for aggregation, and solubility problems. In this study, PEGylation was employed to improve the pharmacokinetic and antitumoral properties of the novel protein. Pegylated IL-2noα was characterized by polyacrylamide gel electrophoresis, size exclusion chromatography, in vitro cell proliferation and in vivo cell expansion bioassays, and pharmacokinetic and antitumor studies. IL-2noα-conjugates with polyethylene glycol (PEG) of 1.2 kDa, 20 kDa, and 40 kDa were obtained by classical acylation. No significant changes in the secondary and tertiary structures of the modified protein were detected. A decrease in biological activity in vitro and a significant improvement in half-life were observed, especially for IL-2noα-PEG20K. PEGylation of IL-2noα with PEG20K did not affect the capacity of the mutant to induce preferential expansion of T effector cells over Treg cells. This pegylated IL-2noα exhibited a higher antimetastatic effect compared to unmodified IL-2noα in the B16F0 experimental metastases model, even when administered at lower doses and less frequently. PEG20K was selected as the best modification strategy, to improve the blood circulation time of the IL-2noα with a superior antimetastatic effect achieved with lower doses.

Enhancing CAR T-cell Therapy Using Fab-Based Constitutively Heterodimeric Cytokine Receptors.

Adoptive T-cell therapy aims to achieve lasting tumor clearance, requiring enhanced engraftment and survival of the immune cells. Cytokines are paramount modulators of T-cell survival and proliferation. Cytokine receptors signal via ligand-induced dimerization, and this principle has been hijacked utilizing nonnative dimerization domains. A major limitation of current technologies resides in the absence of a module that recapitulates the natural cytokine receptor heterodimeric pairing. To circumvent this, we created a new engineered cytokine receptor able to constitutively recreate receptor-heterodimer utilizing the heterodimerization domain derived from the IgG1 antibody (dFab_CCR). We found that the signal delivered by the dFab_CCR-IL2 proficiently mimicked the cytokine receptor heterodimerization, with transcriptomic signatures like those obtained by activation of the native IL2 receptor. Moreover, we found that this dimerization structure was agnostic, efficiently activating signaling through four cytokine receptor families. Using a combination of in vivo and in vitro screening approaches, we characterized a library of 18 dFab_CCRs coexpressed with a clinically relevant solid tumor-specific GD2-specific chimeric antigen receptor (CAR). Based on this characterization, we suggest that the coexpression of either the common β-chain GMCSF or the IL18 dFab_CCRs is optimal to improve CAR T-cell expansion, engraftment, and efficacy. Our results demonstrate how Fab dimerization is efficient and versatile in recapitulating a cytokine receptor heterodimerization signal. This module could be applied for the enhancement of adoptive T-cell therapies, as well as therapies based on other immune cell types. Furthermore, these results provide a choice of cytokine signal to incorporate with adoptive T-cell therapies.

NEO-TRA1: A CD25-Targeted De Novo non-Alpha Agonist of the IL-2 Receptor Selectively Expands Regulatory T Cells

Introduction: Regulatory T cells (Tregs) are crucial for maintaining immune tolerance and their dysfunction contributes to inflammatory and autoimmune conditions, such as graft versus host disease (GVHD). IL-2 is a pleiotropic cytokine that activates both immunosuppressive Tregs and inflammatory cells, including NK cells, cytotoxic T cells, and helper T cells. Although low dose IL-2 therapy effectively expands Tregs, this intervention is limited by coincident stimulation of conventional T cells and natural killer cells. Investigational therapeutics derived from directed mutagenesis of native IL-2 sequences have resulted in some improvements in Treg selectivity. To optimize the magnitude and selectivity of Treg expansion, we utilized de novo protein design technology to fine-tune the individual receptor interfaces of IL-2 mimetic proteins targeted to Tregs with CD25 antibodies. We found decoupling receptor agonism and targeting is an effective means of improving Treg stimulation and corresponding therapeutic candidates elicit robust expansion of Treg populations in vivo without detectable stimulation of inflammatory immune cells. Methods: We designed a series of de novo IL-2Ra independent agonists of the IL-2 receptor that demonstrate reduced binding affinity to CD122 and CD132, the beta and gamma chains of the IL-2 receptor complex, by up to 5 logs when compared to IL-2. These non-alpha IL-2 mimetic variants were fused to a humanized anti-CD25 mAb that lacks effector function for cis-targeting to Tregs. Candidate molecules were screened on primary human T cells to assess pSTAT5 signaling. Treg-specific molecules were administered to humanized NSG mice weekly (four times) and peripheral blood was collected for immune cell profiling via flow cytometry. Results: Screening of TRA candidates in primary human T cells identified several highly selective Treg agonists, including NEO-TRA1 (Fig1). At all concentrations, CD8+ and CD4+ non-Tregs showed low or negligible levels of pSTAT5 signaling upon treatment with NEO-TRA1. Nevertheless, CD4+CD25highCD127low Tregs show increasing pSTAT5 signaling at a wide range of concentrations starting at 20pM, and demonstrate preferential signaling in Tregs by NEO-TRA1. To establish the ability of our TRA to expand Treg in vivo, NSG mice engrafted with human hematopoietic stem cells to create a humanized immune system received weekly injections of NEO-TRA1, vehicle control, or a previously described Treg selective IL-2 mutein (IL-2v-Fc). Characterization of Day 13 CD4+CD25+Foxp3+ Treg percentages in peripheral blood mononuclear cells show that the Treg population expanded to ~30% of total CD4+ cells in mice receiving NEO-TRA1, while Treg percentages reached ~22% in mice receiving IL-2v-Fc (Fig2). Despite a slight decrease at Day 20, the Treg percentages remained higher in NEO-TRA1-treated cohorts than in control groups throughout the study. Conclusions: NEO-TRA1 exhibits potent expansion of Treg populations within primary human samples and in humanized mouse models. The observed profile of STAT5 phosphorylation demonstrates selective activation of Tregs over other lymphocyte populations. NEO-TRA1 is the first Treg agonist based on de novo protein design. NEO-TRA1 is both specific and potent and has high therapeutic potential for the treatment of GVHD and other autoimmune or inflammation diseases. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Nemvaleukin alfa combination therapy for gastrointestinal (GI) cancers: Preclinical evidence and clinical data from the ARTISTRY-1 trial.

659 Background: Nemvaleukin alfa (nemvaleukin, ALKS 4230) is a novel engineered cytokine that selectively binds to the intermediate-affinity IL-2 receptor, preferentially activating and expanding antitumor CD8+ T and NK cells, with minimal expansion of regulatory T cells. Inherently active, nemvaleukin neither requires metabolic/proteolytic conversion nor degrades into native IL-2. Preclinical characterization confirmed receptor selectivity and antitumor activity of nemvaleukin alone and with checkpoint inhibitors (CPIs) and informed dose selection for the first-in-human study ARTISTRY-1; other combination regimens are ongoing evaluation. In ARTISTRY-1, responses with nemvaleukin in combination with pembrolizumab were observed in a variety of tumors, including GI. In addition to the clinical outcomes of these patients (pts), we describe preclinical evaluation of nemvaleukin with tyrosine kinase inhibitors (TKIs)—a drug class indicated for some GI cancers. Methods: Pts with GI cancers who had progressed on prior therapy were enrolled into cohorts of mixed tumor types in ARTISTRY-1 (NCT02799095) and received IV nemvaleukin (3 µg/kg) and pembrolizumab (200 mg). Outcomes presented include antitumor activity and safety as of August 2021. Combinations of nemvaleukin with TKIs were evaluated in mouse tumor models, including colorectal adenocarcinoma (MC38). Results: Clinically, 26 pts with GI cancer (colon/colorectal, n = 14; esophageal, n = 5; hepatocellular, n = 3; pancreatic, n = 3; gastric, n = 1) received nemvaleukin + pembrolizumab (median 3 cycles [range: 1-24]). Median age was 55 y (range: 26-82), ECOG performance status was 0 (n = 6) or 1 (n = 20), and median prior lines of therapy was 3 (range: 1-6). Four pts had a partial response, 2 with esophageal, 1 with MSI-H colorectal, and 1 with pancreatic cancer, with target lesion decreases of 37% to 63%. Six pts had stable disease. Two responders remain on treatment (> 36 and > 80 wks for colorectal and esophageal cancer, respectively). Frequent (> 25%) nemvaleukin-related adverse events (AEs) among all pts receiving nemvaleukin combination (n = 156) were chills (58%), pyrexia (53%), nausea (29%), and fatigue (29%). Grade ≥3 nemvaleukin-related AEs (≥8%) were reported in 48%, including anemia (12%), neutrophil count decreased (10%), and neutropenia (8%). In mice, the combination of the mouse ortholog of nemvaleukin with VEGF TKIs showed improved antitumor activity and survival, along with increased immune activation and angiogenesis blockade in the tumor microenvironment compared with any compound alone. Conclusions: Emerging clinical data show responses in GI tumors may be achieved with nemvaleukin/pembrolizumab with manageable safety. Preclinical evidence of antitumor activity of nemvaleukin with CPIs or TKIs warrants further exploration of nemvaleukin in new combinations for pts with GI cancers. Clinical trial information: NCT02799095.

Immunogenicity profile in African green monkeys of a vaccine candidate based on a mutated form of human Interleukin-15

BackgroundInterleukin (IL)-15 is a proinflammatory T-cell growth factor overexpressed in several autoimmune diseases such as rheumatoid arthritis. Our initial strategy to neutralize the increased levels of IL-15 consisted in a vaccine candidate based on the recombinant modified human IL-15 (mhIL-15) mixed with the alum adjuvant. A previous study in non-human primates Macaca fascicularis has shown that vaccination induces neutralizing antibodies against native IL-15, without affecting animal behavior, clinical status, or the percentage of IL-15-dependent cell populations. However, the mhIL-15 used as an antigen was active in the IL-2-dependent cytotoxic T-cell line CTLL-2, which could hinder its therapeutic application. The current article evaluated the immunogenicity in African green monkeys of a vaccine candidate based on IL-15 mutant D8SQ108S, an inactive form of human IL-15.ResultsIL-15 D8SQ108S was inactive in the CTLL-2 bioassay but was able to competitively inhibit the biological activity of human IL-15. Immunization with 200 µg of IL-15 mutant combined with alum elicited anti-IL-15 IgG antibodies after the second and third immunizations. The median values of anti-IL-15 antibody titers were slightly higher than those generated in animals immunized with 200 µg of mhIL-15. The highest antibody titers were induced after the third immunization in monkeys vaccinated with 350 µg of IL-15 D8SQ108S. In addition, sera from immunized animals inhibited the biological activity of human IL-15 in CTLL-2 cells. The maximum neutralizing effect was observed after the third immunization in sera of monkeys vaccinated with the highest dose of the IL-15 mutant. These sera also inhibited the proliferative activity of simian IL-15 in the CTLL-2 bioassay and did not affect the IL-2-induced proliferation of the aforementioned T-cell line. Finally, it was observed that vaccination neither affects the animal behavior nor the general clinical parameters of immunized monkeys.ConclusionImmunization with inactive IL-15 D8SQ108S mixed with alum generated neutralizing antibodies specific for human IL-15 in African green monkeys. Based on this fact, the current vaccine candidate could be more effective than the one based on biologically active mhIL-15 for treating autoimmune disorders involving an uncontrolled overproduction of IL-15.

Abstract 1512: OrthoCARs: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance CAR T cell antitumor efficacy by driving T cell expansion and fitness

Abstract CAR T cell therapy (CAR) has demonstrated remarkable clinical efficacy in hematological malignancies. However, barriers such as poor T cell effector function, lack of proliferation, and limited persistence prevent CARs from reaching their full curative potential. IL-2 is a potent stimulator of T cell proliferation, survival, and cytotoxic function, making it an attractive cytokine to support CARs. However, therapeutic use of IL-2 is limited by systemic toxicity due its promiscuous activation of undesired immune cell populations. To facilitate selective expansion and activation of CARs we have developed a human orthogonal ligand/receptor system consisting of a pegylated IL-2 mutein (STK-009) that does not significantly activate the wild type IL-2 receptor and a mutated IL-2 Receptor Beta (hoRb) that is fully activated by STK-009, but does not respond to the native IL-2 ligand. This system enables in vivo IL-2 signaling in CARs engineered to express hoRb while avoiding signaling bystander T cells and NK cells. Here, we demonstrate the ability of the STK-009/hoRb receptor pair to selectively enhance the anti-tumor efficacy of hoRb expressing CD19 CARs (SYNCAR-001) in preclinical lymphoma mouse models at will. We also demonstrate, in non-human primates (NHP), that STK-009 is selective for hoRb expressing cells. SYNCAR-001 + STK-009 can lead to complete responses in subcutaneous Raji mouse models, even with SYNCAR-001 dosed at sub-efficacious levels (400,000 CAR-Ts/mouse). Subcutaneous dosing of STK-009 expands SYNCAR-001 systemically and drives infiltration of SYNCAR-001 into tumors. UMAP analysis of STK-009 treated SYNCAR-001 reveal a small PD1+LAG3+ subpopulation during tumor rejection which disappeared once tumors are controlled. The majority of T cells showed no exhaustion markers. Further, an IL-7R+ population arises and remains after tumor control, indicating long term memory development. Nanostring analysis confirms upregulation of IL-7R and other critical genes involved in cytotoxic activity and persistence in SYNCAR-001 when treated with STK-009. These data demonstrate that STK-009 treatment expands SYNCAR-001 and delivers a gene signature indicative of enhanced T cell fitness and activity. Subcutaneous administration of STK-009 in NHP shows no evidence of toxicity or physiological IL-2 mediated activity on immune cells, including Teffs, Tregs, and NK cells. Pharmacokinetic analysis of STK-009 shows stable exposure with minimal clearance, demonstrating the selectivity of STK-009. These findings validate an orthogonal platform that selectively drives potent T cell effector functions of engineered cells without the toxicities mediated by NK cells or non-tumor specific T cells associated with high dose IL-2 therapy. These results demonstrate the ability of this orthogonal platform to improve the efficacy and durability of CARs. Citation Format: Paul-Joseph P. Aspuria, Sandro Vivona, Michele Bauer, Marie Semana, Navneet Ratti, Scott McCauley, Romina Riener, Rene de Waal Malefyt, Deepti Rokkam, Jan Emmerich, Patrick J. Lupardus, Rob A. Kastelein, Martin Oft. OrthoCARs: Engineered human IL-2/IL-2Rb orthogonal pairs selectively enhance CAR T cell antitumor efficacy by driving T cell expansion and fitness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1512.

Selective expansion of regulatory T cells using an orthogonal IL-2/IL-2 receptor system facilitates transplantation tolerance.

Adoptive transfer of Tregs has been shown to improve alloengraftment in animal models. However, it is technically challenging to expand Tregs ex vivo for the purpose of infusing large numbers of cells in the clinic. We demonstrate an innovative approach to engineering an orthogonal IL-2/IL-2 receptor (IL-2R) pair, the parts of which selectively interact with each other, transmitting native IL-2 signals, but do not interact with the natural IL-2 or IL-2R counterparts, thereby enabling selective stimulation of target cells in vivo. Here, we introduced this orthogonal IL-2R into Tregs. Upon adoptive transfer in a murine mixed hematopoietic chimerism model, orthogonal IL-2 injection significantly promoted orthogonal IL-2R+Foxp3GFP+CD4+ cell proliferation without increasing other T cell subsets and facilitated donor hematopoietic cell engraftment followed by acceptance of heart allografts. Our data indicate that selective target cell stimulation enabled by the engineered orthogonal cytokine receptor improves Treg potential for the induction of organ transplantation tolerance.

Dual Modulators of Selected Plant Secondary Metabolites Targeting COVID-19 Main Protease and Interleukin-2: An In-Silico Approach based Novel Hypothesis

Background: Owing to the recent scenario on this ongoing Coronavirus pandemic outbreak around the world, the present study has been undertaken. Aim: In this study, we adopted two strategies, i.e., via computational method, a search for the novel plant secondary metabolites from the Indian Traditional Medicine to target and combat the enduring novel 2019 CoVs main protease that causes pneumonia, followed by the effect of these selected secondary metabolites on the host’s immune system for their immunomodulatory potential on Interleukin-2. Methods: A detailed literature review has been done to identify the assorted plant secondary metabolites from the natural sources, which have been extensively used traditionally for their immunomodulatory potential. Next, the resulting compounds have processed for the molecular docking study to predict whether the compounds have the potency to fight against 2019- CoVs protein or it could have the tendency to battle the cytokines, which are responsible for the immune response of the host, thereby preventing the CoVs caused infection in humans. Furthermore, to explore molecular mechanics, the insilico docking study with COVID-19 Mpro and Interleukin-2 has been performed. Results & Discussion: Among the six secondary metabolites selected, five compounds showed its possible promising potency with COVID-19 and IL-2 proteins, which are compared with the standard drug Remdesivir, one of the anti-viral drugs for treating and managing the present coronavirus condition and an IL-2 inhibitor, which is the native IL-2 ligand protein (i.e., from PDB Id- 1PW6) itself. Besides, based on the docking scores, the Curcumin (from Curcuma longa) showed the highest score towards these two targets taken for this study. The identified compounds have a promising binding affinity with the Mpro receptors, in the narrow range of binding energy for the protein PDB Id: 6LU7 and the score range between -10.9102 to -19.8790 kcal/mol: when compared to the standard -21.8600 kcal/mol. Whereas, the binding affinity with the Interleukin -2 receptor, for the protein PDB Id: 1PW6 the range between -11.3899 to -17.1366 kcal/mol: when compared to that of standard -16.9554 kcal/mol. Conclusion: Our result findings demonstrate that the integrated Indian traditional herbal treatment might be hopefully used for the viral respiratory infection due to either it may have acted directly on the viral protein or through regulating the immune response, which could lead to the rapid drug discovery of the drug leads with clinical potency towards the novel infectious disease, where there is no drug or vaccines are available.

THU0054 NKTR-358, A NOVEL IL-2 CONJUGATE, STIMULATES HIGH LEVELS OF REGULATORY T CELLS IN PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS

Background:Impaired IL-2 production and dysfunction of regulatory T cells (Tregs) have been identified as key immunological defects leading to the breakdown of immune self-tolerance in SLE. Low-dose IL-2 can expand Tregs, but the effect is limited by a narrow therapeutic window for Treg selectivity. Furthermore, the short half-life of IL-2 necessitates frequent administration. NKTR-358 is a polyethylene glycol (PEG) conjugate of recombinant human IL-2 (aldesleukin sequence) and is differentiated from native IL-2 by its altered binding to the IL-2 receptor and prolonged biological activity. NKTR-358 resulted in marked and selective stimulation of Tregs when administered as a single SC injection to healthy volunteers.Objectives:This multiple ascending dose study assessed the safety, tolerability, pharmacokinetics (PK), and immune effects of NKTR-358 in patients with SLE after repeated administration of SC doses. The time course and extent of changes in numbers and percentages of Tregs, conventional CD4+ and CD8+ T cells, NK cells, and cytokine levels in peripheral blood were investigated.Methods:In this double-blind, multiple ascending dose study, patients with mild to moderate SLE received 3 SC doses q2w in 4 cohorts ranging from 3.0 to 24.0 µg/kg (9 active:3 placebo per cohort); patients were followed for a total of 79 days.Results:There were no dose-limiting toxicities, deaths, or clinically significant abnormalities in either vital signs or electrocardiograms. Adverse events attributed to NKTR-358 were primarily limited to mild (grade 1) injection site reactions. At the highest dose, one subject had transient and mild (grade 1) symptoms of a flu-like syndrome after administration, without associated elevated cytokine levels, and another subject had dosing stopped due to elevated eosinophil levels. No other individual at any dose level had systemic signs or symptoms known to be associated with IL-2 therapy. No anti-drug antibodies were detected. NKTR-358 demonstrated dose-proportional PK with repeated dosing; plasma levels peaked 3-6 days post-dose and declined with a terminal half-life of ~10-13 days.The primary and consistent effect of NKTR-358 was seen on Tregs. In the four dose cohorts, dose-dependent and sustained increases in absolute numbers and percentages of circulating CD4+FoxP3+CD25brightTregs were observed. Treg levels remained elevated throughout the dosing period, peaking at Day 10 after the first administration of NKTR-358 and returning to baseline ~ 20-30 days following last administration. At 24.0 µg/kg, the mean peak increase in numbers of CD25brightTregs was 11-fold above baseline. In addition, there was an increase in Treg activation markers at doses ≥12.0 µg/kg. In contrast to effects on Tregs, no changes in percentages or numbers of conventional CD4+ or CD8+ T cells were observed at any dose tested. At the highest dose, there were low-level increases in the percentages and numbers of NK cells. Overall, NKTR-358 selectively induced Tregs, evidenced by a 12-fold increase in the mean peak Treg:CD8 ratio over baseline in the 24.0 µg/kg group.Conclusion:NKTR-358, an IL-2 conjugate Treg stimulator, was well tolerated when repeatedly administered (q2w) at doses up to 24 µg/kg. Its administration led to marked, selective, prolonged, and dose-dependent increases in circulating CD25brightTregs. This clinical study in SLE patients extends the previous results in healthy volunteers and provides strong support for continued testing of NKTR-358 as a new therapeutic in SLE and other inflammatory diseases.Disclosure of Interests:Suresh Siddhanti Shareholder of: Nektar Therapeutics, Employee of: Nektar Therapeutics, Christie Fanton Shareholder of: Nektar Therapeutics, Employee of: Nektar Therapeutics, Neha Dixit Shareholder of: Nektar Therapeutics, Employee of: Nektar Therapeutics, Lin Lu Shareholder of: Nektar Therapeutics, Employee of: Nektar Therapeutics, Vishala Chindalore Grant/research support from: Nektar Therapeutics for conducted studies, Speakers bureau: > 5 years ago, Robert Levin Grant/research support from: Payments for clinical research for industry-sponsored trials, Consultant of: Gilead, Exagen, Myriad Rheumatology, Speakers bureau: Sanofi/Genzyme, Regeneron, Bristol-Myers Squibb, AbbVie, Isam Diab: None declared, Richard Furie Grant/research support from: Nektar Therapeutics to Northwell Rheumatology to conduct this study, Consultant of: Nektar Therapeutics, Jonathan Zalevsky Shareholder of: Nektar Therapeutics, Employee of: Nektar Therapeutics, Brian Kotzin Shareholder of: Nektar Therapeutics, Employee of: Nektar Therapeutics

Engineered IL-2 Cytokine-Cytokine Receptor Complex Enables Selective Expansion of Regulatory T Cells and Facilitates Establishment of Organ Transplantation Tolerance

Mixed hematopoietic cell chimerism induction is a promising approach to establish transplantation tolerance, though achievement of donor cell engraftment in patients given non-myeloablative conditioning remains challenging. Adoptive transfer of regulatory T cells (Tregs) has been shown to improve alloengraftment in animal models. However, ex vivo Treg expansion for the purpose of infusing large numbers is technically challenging. An innovative approach is to engineer an orthogonal IL-2 (oIL-2) cytokine-receptor pair that selectively interacts with each other and transmits native IL-2 signals, but does not interact with the natural cytokine or receptor counterparts, thereby enabling selective stimulation of target cells in vivo. Here, we introduced this ortho cytokine-receptor combination into Treg with the goal of improving donor hematopoietic cell engraftment upon adoptive transfer in a murine mixed chimerism model to induce tolerance to a transplanted organ allograft. CD4⁺CD25^hiFoxp3^GFP+cells were isolated from Foxp3^GFP-BALB/c mice. Purified Tregs were preactivated and transduced with the construct for murine oIL-2 receptor β chain (oIL-2Rβ) through a retroviral vector. Transduction efficiency was approximately 40%. Transduced oIL-2Rβ⁺Tregs (oTreg) were injected at 1 × 10⁶ /mouse together with 15 × 10⁶ bone marrow cells (BMCs) that were obtained from luciferase transgenic C57BL/6 donor mice into WT BALB/c recipient mice that received 3.3-Gy total body irradiation and anti-CD40L mAb (0.3 mg, ip on d0) as preconditioning. Recipients were treated with PBS, WT IL-2, or oIL-2 (both 25K IU/day) for 14 days. Without oTreg transfer, donor cells were rejected by d10 as measured by bioluminescence. Improvement of engraftment by oTreg transfer was not statistically significant without IL-2 injection (P = 0.13 compared to BMC + PBS group on d30). WT IL-2 injection increased CD8⁺T cells and resulted in rapid graft rejection. On the other hand, oIL-2 injection significantly increased %Foxp3^GFP+ in CD4⁺T cells (PBS; 4.3±1.6%, oIL-2; 9.3±4.7%, each n=10, P=0.007) without increasing CD8⁺T cells. Consequently, donor cell engraftment was significantly improved in this group (P<0.0001 compared to BMC + PBS group on d30). Those recipients accepted heart allografts that were transplanted from C57BL/6 donor mice after chimerism induction, indicating the establishment of donor specific tolerance. Because ortho IL-2 did not have an impact when Tregs were transferred without oIL-2Rβ transduction, we concluded that specific interactions between oIL-2 and oIL-2Rβ resulted in improved acceptance of the heart allografts. Our data indicates that selective IL-2 stimulation improves Treg potential for facilitation of engraftment and for the induction of transplantation tolerance. Cytokine receptor engineering could be a powerful option to manipulate target cells such as Treg in vivo.

NKTR-255, a Polymer-Conjugated IL-15 Enhances Antibody-Dependent Cellular Cytotoxicity Mediated By NK Cells in a B Cell Lymphoma Model

Background IL-15 is a cytokine that activates and provides survival benefit to T and NK cells and has great potential as an immunotherapeutic agent for the treatment of cancer. Exploiting the therapeutic value of native IL-15 has been challenging due to its unfavorable pharmacokinetic properties and poor tolerability. NKTR-255 is a polymer-conjugated IL-15 designed to retain binding affinity to the IL-15 receptor alpha chain and have decreased clearance to provide a sustained pharmacodynamics response. NKTR-255 has an enhanced immunotherapeutic effect when combined with certain targeted monoclonal antibodies that mediate tumor killing by antibody dependent cellular cytotoxicity (ADCC). Here we further characterized the effect of NKTR-255 on NK cell-mediated ADCC in a B cell lymphoma model. Methods For in vitro ADCC, NKTR-255 pre-treated human NK cells (effector cells) were co-cultured with RPMI-8226 (target cells) and with daratumumab for 3 hours. The ability of NK cells to lyse target cells was evaluated by detecting 7-AAD stained RPMI-8226. In the Daudi lymphoma model, SCID or SCID beige mice were inoculated IV with 1x107 Daudi cells on Day 0. NKTR-255 (0.3 mg/kg IV) was administered on Days 14, 21 and 28 and a single dose of antibody treatment was given on Day 14 (daratumumab 0.5 mg/kg IP). Survival rate was determined by onset of hind limb paralysis or moribundity as surrogate parameters. Concurrent vs staggered regimen assessment was conducted with NKTR-255 dosed on day 14 and daratumumab on Day 14, 17, or 21. For dose examination, high (0.5 mg/kg) and low (0.05 mg/kg) doses of daratumumab were combined with low (0.03 mg/kg) and high (0.3 mg/kg) doses of NKTR-255 in a concurrent manner. Daudi cells in the bone marrow were assessed 10 days after the NKTR-255 dose by flow cytometry. Results In vitro, NKTR-255 pre-treatment of human purified NK cells enhanced daratumumab-mediated ADCC against RPMI-8226. The ability of NKTR-255 to enhance in vitro ADCC was translated into an enhanced therapeutic efficacy of daratumumab in the Daudi lymphoma model with SCID mice. NKTR-255 combined with daratumumab synergistically provided long-term survival benefit in a NKTR-255 dose-dependent manner. To confirm the contribution of NK cells as effector cells for ADCC in vivo, the therapeutic effect of the combination treatment was assessed in the tumor model in SCID beige mice, which have a selective impairment of NK cell cytotoxic function. The long-term survival benefit by the combination treatment, which was observed in SCID mice (median survival of 27 days in control and 59.5 days in treatment), was attenuated in SCID beige mice (median survival of 23 days in control and 30 days in treatment). Concurrent vs staggered dose regimens and dose ranging for NKTR-255 and daratumumab were evaluated in the Daudi model with SCID mice to understand optimal therapeutic use of NKTR-255 in future clinical studies. Tumor depletion in bone marrow was most effective in concurrent treatment of NKTR-255 and daratumumab and three days-staggered treatment (NKTR-255 first and the antibody 3 days later). In contrast, a 7-day stagger showed no benefit of the combination in tumor depletion. Combination of low dose daratumumab and high dose NKTR-255 was equally effective in tumor depletion as a combination of high dose daratumumab and low dose NKTR-255. Conclusions NKTR-255 was shown to enhance in vivo therapeutic efficacy of daratumumab, a monoclonal antibody that mediates tumor killing by ADCC. The effect was mainly mediated by NK cells. The treatment scheduling experiments demonstrated that antibody should be present during NKTR-255-driven NK cell activation/expansion phase to efficiently deplete Daudi tumor cells in bone marrow. In addition, even low dose of daratumumab treatment showed effective tumor depletion when combined with high dose of NKTR-255. This suggests that NKTR-255 has a potential to expand the therapeutic window of existing tumor-targeted antibodies to patients with, for example, low antibody concentration in target tumor site or low expression of tumor antigen. Disclosures Miyazaki: Nektar Therapeutics: Employment, Equity Ownership. Kivimäe:Nektar Therapeutics: Employment, Equity Ownership. Hennessy:Nektar Therapeutics: Employment, Equity Ownership. Pena:Nektar Therapeutics: Employment, Equity Ownership. Quach:Nektar Therapeutics: Employment, Equity Ownership. Moffet:Nektar Therapeutics: Employment, Equity Ownership. Nieves:Nektar Therapeutics: Employment, Equity Ownership. Zhang:Nektar Therapeutics: Employment, Equity Ownership. Marcondes:Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Zalevsky:Nektar Therapeutics: Employment, Equity Ownership.

A Phase 1, Open-Label, Multi-Center, Dose Escalation and Dose Expansion Study of NKTR-255 As a Single Agent in Relapsed or Refractory Hematologic Malignancies and in Combination with Daratumumab As a Salvage Regimen for Multiple Myeloma

A Phase 1, Open-Label, Multi-Center, Dose Escalation and Dose Expansion Study of NKTR-255 As a Single Agent in Relapsed or Refractory Hematologic Malignancies and in Combination with Daratumumab As a Salvage Regimen for Multiple Myeloma

Abstract 3265: NKTR-255, a polymer-conjugated IL-15 enhances anti-tumor NK cell responses and synergizes with monoclonal antibodies to provide long-term survival in human lymphoma model

Abstract Background: IL-15 is a cytokine that activates and provides survival benefit to NK cells. Exploiting the therapeutic value of native IL-15 has been challenging due to its unfavorable pharmacokinetic properties and tolerability. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. NKTR-255 has potential for providing an enhanced immunotherapeutic effect when combined with monoclonal antibodies that mediate tumor killing by antibody dependent cellular cytotoxicity (ADCC). Here we investigate the pharmacological properties of NKTR-255 on NK cells and the therapeutic effect of NKTR-255 when combined with tumor-directed monoclonal antibodies in a B cell lymphoma model. Methods: KHYG-1 cells (human NK cell line) were used to measure phosphorylated STAT5 (pSTAT5) and cell proliferation. Human PBMCs were stimulated with NKTR-255 and/or daratumumab for in vitro NK cell characterization. In the cytotoxic assay, mice received single IV doses of 0.3 mg/kg of NKTR-255 and splenic NK cells were co-cultured with YAC-1 cells (mouse T lymphoma cell line) to measure cytotoxic function. In the Daudi lymphoma model, 1x107 Daudi cells were inoculated IV on Day 0. NKTR-255 (0.03, 0.1 or 0.3 mg/kg SC) was administered on Days 14, 21 and 28 and antibody treatment was administered on Day 14 (daratumumab 0.5 mg/kg IP) or on Days 14 and 17 (rituximab 40 mg/kg IP). Survival rate was determined by onset of hindlimb paralysis as a surrogate parameter. Results: NKTR-255 dose-dependently induced pSTAT5 and proliferation in KHYG-1 cells (EC50 values for pSTAT5: 0.2 ng/ml, proliferation: 5 ng/ml). NKTR-255 also resulted in enhanced pSTAT5 and NKG2D surface expression on human primary NK cells. In addition NKTR-255 increased NK cell degranulation in co-culture experiments with U266 cells (myeloma cell line) with/without daratumumab (anti-human CD38 antibody), as determined by enhanced CD107a surface expression. In vivo pretreatment with NKTR-255 resulted in sustained cytotoxic function of NK cells in both ex vivo and in vivo studies. Finally, NKTR-255 synergistically provided long-term survival benefit in a dose-dependent manner when administered with daratumumab or rituximab (anti-human CD20 antibody) in the Daudi B cell lymphoma model. Conclusions: NKTR-255 is an immune stimulator of NK cells that provides a dose-dependent effect in the proliferation and activation of NK cells. This property of NKTR-255, when administered with daratumumab or rituximab, translates into enhanced therapeutic efficacies of the antibodies in a B cell lymphoma model. These results indicate that combining NKTR-255 with a tumor-directed antibody having an ADCC mechanism can provide a synergistic effect for treating cancers. Citation Format: Takahiro Miyazaki, Saul Kivimäe, Rhoneil Pena, Peiwen Kuo, Marlene Hennessy, Murali Addepalli, Neha Dixit, Wildaliz Nieves, Sara Sheibani, Mekhala Maiti, Laurie VanderVeen, Joanna Wilczek, Loui Madakamutil, Jonathan Zalevsky. NKTR-255, a polymer-conjugated IL-15 enhances anti-tumor NK cell responses and synergizes with monoclonal antibodies to provide long-term survival in human lymphoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3265.

De Novo Cytokine Mimics Have Improved Therapeutic Properties.

A de novo IL2/IL15 mimic has improved antitumor activity and reduced toxicity compared with native IL2.

Pharmacokinetic and Pharmacodynamic Study of NKTR-255, a Polymer-Conjugated Human IL-15, in Cynomolgus Monkey

IntroductionIL-15 is a common gamma chain cytokine that activates and provides a survival benefit to T-cells and NK cells and has long been recognized as having potential as an immunotherapeutic agent for the treatment of cancer. Therapeutic use of native IL-15 has been challenging due to, for example, its unfavorable pharmacokinetic and safety properties. NKTR-255 is a polymer-conjugated human IL-15 that retains binding affinity to the alpha subunit of IL-15 receptor and exhibits reduced clearance to thereby provide a sustained pharmacodynamics response. Here we investigate the biological effects of NKTR-255 in naïve cynomolgus monkey.MethodsIn vitro monkey whole blood was treated with NKTR255 and the percentage of pSTAT5 positive populations in each NK, CD4 T and CD8 T cells was determined by flow cytometry. In an PK/PD study, monkeys received single IV doses of 0.001, 0.003, 0.01, 0.03, or 0.1 mg/kg NKTR-255. Blood samples were collected to determine the plasma concentrations of NKTR-255 and to assess the effects of NKTR-255 on NK and CD8 T cells at multiple time points; flow cytometry was used to measure STAT5 phosphorylation, Ki-67 expression and frequency of cell populations. Granzyme B expression was assessed in NK and CD8 T cells by flow cytometry.ResultsNKTR-255 induced dose-dependent phosphorylation of STAT5 in monkey whole blood (EC50 values NK cells: 6.9 ng/ml, CD8 T cells: 39 ng/ml, CD4 T cells: 53 ng/ml). The half-life and clearance of NKTR-255 were 26x longer and 38x lower, respectively, than IL-15. NKTR-255 engaged the IL-15 signaling pathway, in vivo, demonstrating both robust and sustained STAT5 phosphorylation in lymphocytes. NKTR-255 drove the proliferation of total CD8 T cells and NK cells in a dose-dependent manner, with dramatic and durable increases observed in Ki67 positive population and absolute cell numbers (NK cells: 6.1 fold; CD8 T cells: 7.8 fold from baseline on day 5 at 0.1 mg/kg). These effects were strongly biased towards CD8 T cells and NK cells, with substantially less induction of CD4 T cells. The Ki67 response analyses of the T cell subpopulation revealed a higher response of memory populations than for naive T cells. Among memory T cells, effector memory T cells showed the highest response over stem cell memory T cells and central memory T cells. Finally, NKTR-255 also increased the expression of Granzyme B in both NK and CD8 T cells, concomitant with an enhancement in target cell lysis.ConclusionsNektar has generated a novel and potent molecule in NKTR-255 that not only preserves the relevant biology of IL-15, but additionally provides enhanced PK and PD properties relative to the native IL-15 cytokine. NKTR-255 is being developed as an immune-stimulatory agent to target NK and CD8 T cell biology for the treatment of cancer. DisclosuresMiyazaki:Nektar Therapeutics: Employment, Equity Ownership. Kuo:Nektar Therapeutics: Employment, Equity Ownership. Maiti:Nektar Therapeutics: Employment, Equity Ownership. Obalapur:Nektar Therapeutics: Employment, Equity Ownership. Addepalli:Nektar Therapeutics: Employment, Equity Ownership. Rubas:Nektar Therapeutics: Employment, Equity Ownership. Sims:Nektar Therapeutics: Employment, Equity Ownership. Zhang:Nektar Therapeutics: Employment, Equity Ownership. Madakamutil:Nektar Therapeutics: Employment, Equity Ownership. Zalevsky:Nektar Therapeutics: Employment, Equity Ownership.

Targeted IL-15-based Protein Fusion Complexes as Cancer Immunotherapy Approaches

This mini review provides an overview and rationale for creating IL-15-based fusion protein complexes to be used as targeted immunotherapeutic agents. IL-15 stimulates proliferation and activation of CD8+ T and natural killer cells which result in augmentation of their anti-tumor activities. We have created ALT-803, an IL-15 superagonist complex which exhibits longer serum half-life, longer retention in lymphoid tissues, and better immunostimulatory and anti-tumor activities compared to native IL-15. When used alone or in combination with other immunotherapeutic molecules in various mouse tumor models, ALT-803 effectively reduces tumor burden and prolongs survival by stimulating the innate and adaptive arms of the immune system. To evaluate whether ALT-803 could be used as a protein scaffold to create IL-15-based tumor cell-specific molecules, we genetically fused it with a single chain anti-CD20 antibody derived from the variable regions of rituximab. This novel fusion protein exhibits enhanced anti-tumor activity compared to rituximab while maintaining IL-15 immunostimulating properties. Thus, ALT-803 may be exploited as a versatile scaffold to produce multivalent targeted fusion proteins to improve the anti-tumor efficacy of other therapeutic agents in the clinic.

Active immunization with human interleukin-15 induces neutralizing antibodies in non-human primates.

BackgroundInterleukin-15 is an immunostimulatory cytokine overexpressed in several autoimmune and inflammatory diseases such as Rheumatoid Arthritis, psoriasis and ulcerative colitis; thus, inhibition of IL-15-induced signaling could be clinically beneficial in these disorders. Our approach to neutralize IL-15 consisted in active immunization with structurally modified human IL-15 (mhIL-15) with the aim to induce neutralizing antibodies against native IL-15. In the present study, we characterized the antibody response in Macaca fascicularis, non-human primates that were immunized with a vaccine candidate containing mhIL-15 in Aluminum hydroxide (Alum), Montanide and Incomplete Freund’s Adjuvant.ResultsImmunization with mhIL-15 elicited a specific antibodies response that neutralized native IL-15-dependent biologic activity in a CTLL-2 cell proliferation assay. The highest neutralizing response was obtained in macaques immunized with mhIL-15 adjuvanted in Alum. This response, which was shown to be transient, also inhibited the activity of simian IL-15 and did not affect the human IL-2-induced proliferation of CTLL-2 cells. Also, in a pool of synovial fluid cells from two Rheumatoid Arthritis patients, the immune sera slightly inhibited TNF-α secretion. Finally, it was observed that this vaccine candidate neither affect animal behavior, clinical status, blood biochemistry nor the percentage of IL-15-dependent cell populations, specifically CD56+ NK and CD8+ T cells.ConclusionOur results indicate that vaccination with mhIL-15 induced neutralizing antibodies to native IL-15 in non-human primates. Based on this fact, we propose that this vaccine candidate could be potentially beneficial for treatment of diseases where IL-15 overexpression is associated with their pathogenesis.

IL15N72D superagonist/IL15Rα-Fc fusion complex (ALT-803) exhibits anti-metastatic activity in murine breast tumor model

Interleukin (IL)-15N72D-superagonist-complexed with IL15Rα-Fc fusion protein (IL15-SA/IL15Rα-Fc, also known as ALT-803) has been reported to have greater anti-myeloma activity, due to its increased in vivo half-life and unique tissue biodistribution, than native IL-15 alone in murine models. In order to test the anti-tumor efficacy of IL15-SA/IL15Rα-Fc in a non-hematologic murine cancer model, we examined the monotherapy effect of IL15-SA/IL15Rα-Fc in non-tumor bearing mice and in the 4T1-breast tumor model. In non-tumor bearing Balb/c mice, IL15-SA/IL15Rα-Fc (1 µg i.p.), a 10-fold increase occurred in NK cells followed by CD8+ T cells (3-fold), both peaking on Day 3 post treatment. In examining NK cell population subsets, the greatest significant change was in high effector (CD11b+, CD27hi) NKs as compared with terminal effector (CD11b+, CD27lo) NKs on Day 3 post IL15-SA/IL15Rα-Fc-treatment, leading to increased NK function on a per-cell basis. CD8 subset analysis determined that IL15-SA/IL15Rα-Fc significantly increased IL15-responding, memory (CD122+, CD44+) CD8+ T cells, in particular those having the innate (NKG2D+, PD1-) phenotype. In 4T1 tumor bearing mice, IL15-SA/IL15Rα-Fc induced significant anti-metastatic activity, and thus consequently resulted in a longer median survival of IL15-SA/IL15Rα-Fc-treated mice following surgical resection of the primary tumor. Finally, T cell depletion revealed that the anti-metastatic property of IL15-SA/IL15Rα-Fc was dependent on CD8+ T cells and not CD4+ T cells. Altogether, these studies showed for the first time that IL15-SA/IL15Rα-Fc a) promoted the development of high effector NKs, b) enhanced per-cell function of NKs, and c) played a vital role in reducing tumor metastasis and ultimately survival.

Combination therapy of an IL-15 superagonist complex, ALT-803, and a tumor targeting monoclonal antibody promotes direct antitumor activity and protective vaccinal effect in a syngenic mouse melanoma model

Cytokine-based and antibody-targeted immunotherapies have both been important approaches in the treatment of malignant cancers. However, combinational therapies of cytokines and tumor-targeting antibodies remain to be further explored, especially in advanced solid tumors. In this study, C57BL/6 mice bearing established subcutaneous B16F10 melanoma were treated with mouse melanoma targeting anti-gp75 monoclonal antibody (mAb), TA99, combined with interleukin (IL)-15 based superagonist ALT-803. This soluble protein complex consists of an IL-15 superagonist mutant (IL-15N72D) associated with an IL-15 receptor α Sushi domain - human IgG1 Fc fusion protein. Compared to native IL-15, ALT-803 possesses superior in vivo biologic activity for stimulating NK and CD8+ memory T cells. The combined ALT-803+TA99 therapy significantly exceeded either monotherapy in inhibiting melanoma tumor growth (p < 0.001) and prolonging survival (p < 0.01) of B16F10 tumor-bearing mice. Through immune cell depletion studies and immunophenotyping of peripheral cells as well as tumor-infiltrating leukocyte subsets, we found that ALT-803 enhances TA99-mediated antitumor immunity through activation of NK cells and expansion of the CD8+CD44high memory T cell arm. In contrast, CD4+ T cells were shown to play more of a suppressive role in the therapeutic effect of ALT-803+TA99, possibly through involvement of regulatory T cells or ALT-803-mediated induction of PD-L1 on CD4+ T cells in the periphery and tumor microenvironment. Addition of anti-PD-L1 mAb to ALT-803+TA99 therapy resulted in a further increase in antitumor activity against subcutaneous B16F10 tumors. Furthermore, tumor-bearing mice that survived due to ALT-803+TA99 combination therapy exhibited long term antitumor memory against B16F10 tumor cell rechallenge. Immune-depletion studies revealed that this antitumor memory was associated with CD4+ T cells, CD8+ T cells and NK cells. Our findings suggest a therapeutic opportunity for ALT-803 in combination with tumor-targeting antibodies to simultaneously augment targeted antitumor activities of therapeutic antibodies and induce a long-term vaccinal effect which will provide durable responses in the treated host.