Engineering IL-2 to Give New Life to T Cell Immunotherapy.

Interleukin-2 (IL-2) is integral to immune system regulation. Its opposing immunostimulatory and immunosuppressive actions make it an attractive therapeutic target for cancer and autoimmune diseases. A challenge in developing IL-2-directed anticancer therapies has been how to stimulate effector T cells (Teffs) without inducing regulatory T cells (Tregs) in the tumor microenvironment; conversely, IL-2 therapy for autoimmune diseases requires Treg induction without further stimulation of Teffs. High-dose IL-2 is approved for melanoma and renal cell carcinoma, but its therapeutic value is limited by a need for frequent dosing at specialist centers, its short half-life, severe toxicity, and a lack of efficacy in most patients. Re-engineered IL-2 therapeutics are designed to have longer in vivo half-lives, target specific IL-2 receptor conformations to stimulate specific T cell subsets, or localize to target tissues to optimize efficacy and reduce toxicity. We discuss recent studies that elucidate the potential of newly engineered IL-2-based therapeutics for cancer and autoimmune diseases.

Bempegaldesleukin (BEMPEG; NKTR-214) efficacy as a single agent and in combination with checkpoint-inhibitor therapy in mouse models of osteosarcoma.

Survival of patients with relapsed/refractory osteosarcoma has not improved in the last 30 years. Several immunotherapeutic approaches have shown benefit in murine osteosarcoma models, including the anti-programmed death-1 (anti-PD-1) and anti-cytotoxic T-lymphocyte antigen-4 (anti-CTLA-4) immune checkpoint inhibitors. Treatment with the T-cell growth factor interleukin-2 (IL-2) has shown some clinical benefit but has limitations due to poor tolerability. Therefore, we evaluated the efficacy of bempegaldesleukin (BEMPEG; NKTR-214), a first-in-class CD122-preferential IL-2 pathway agonist, alone and in combination with anti-PD-1 or anti-CTLA-4 immune checkpoint inhibitors in metastatic and orthotopic murine models of osteosarcoma. Treatment with BEMPEG delayed tumor growth and increased overall survival of mice with K7M2-WT osteosarcoma pulmonary metastases. BEMPEG also inhibited primary tumor growth and metastatic relapse in lungs and bone in the K7M3 orthotopic osteosarcoma mouse model. In addition, it enhanced therapeutic activity of anti-CTLA-4 and anti-PD-1 checkpoint blockade in the DLM8 subcutaneous murine osteosarcoma model. Finally, BEMPEG strongly increased accumulation of intratumoral effector T cells and natural killer cells, but not T-regulatory cells, resulting in improved effector:inhibitory cell ratios. Collectively, these data in multiple murine models of osteosarcoma provide a path toward clinical evaluation of BEMPEG-based regimens in human osteosarcoma.

Preclinical Assessment of the Analgesic Pharmacology of NKTR-181 in Rodents.

OBJECTIVE: Pharmacological evaluation of the mu-opioid receptor (MOR) agonist properties of NKTR-181 in rodent models. METHODS: Graded noxious stimulus intensities were used in rats to establish the antinociceptive potency and efficacy of NKTR-181 relative to morphine, fentanyl, and oxycodone. Characteristics of MOR agonist actions, as measured by antinociceptive tolerance and cross-tolerance, as well as opioid-induced hyperalgesia (OIH) and naloxone-precipitated withdrawal in NKTR-181- and morphine-dependent in mice, were compared. RESULTS: NKTR-181 showed dose- and time-related antinociception with similar maximal effects to morphine in the rat and mouse hot-water tail-flick test. No sex or species differences were observed in NKTR-181 or morphine antinociception. Rats treated with NKTR-181 and morphine exhibited decreases in both potency and maximal efficacy as nociceptive stimulus intensity was increased from a water temperature of 50 °C to 54 °C. Evaluation of antinociception at a high stimulus intensity revealed that oxycodone and fentanyl exhibited greater efficacy than either NKTR-181 or morphine. The relative potency difference between NKTR-181 and morphine across all tail-flick studies was determined to be 7.6-fold (90% confidence interval, 2.6, 21.5). The peak antinociceptive effect of NKTR-181 was delayed compared to that of the other opioids and cumulative drug effects were not observed. Repeated treatment with escalating, approximately equi-analgesic doses of NKTR-181 or morphine, produced antinociceptive tolerance and cross-tolerance. Under these pharmacological conditions, OIH and naloxone-precipitated physical dependence were similar for NKTR-181 and morphine. CONCLUSIONS: NKTR-181 had a slower onset, but similar efficacy, to morphine in the models studied supporting reduced abuse potential while maintaining analgesic effect in comparison with current opioids.

Phase I study protocol: NKTR-255 as monotherapy or combined with daratumumab or rituximab in hematologic malignancies.

NKTR-255 is an investigational polyethylene glycol-modified recombinant human IL-15 (rhIL-15) receptor agonist, designed to improve the immunotherapeutic and anti-cancer benefit observed with rhIL-15 while circumventing the toxicities associated with this therapy. In preclinical studies, NKTR-255 has demonstrated enhanced proliferation and function of CD8+ T cells and natural killer cells, as well as enhanced anti-tumor activity and survival both as monotherapy and in combination with monoclonal antibodies in multiple cancer models. Here, we describe the rationale and design of the first-in-human Phase I, dose-escalation and dose-expansion study of NKTR-255 alone and in combination with daratumumab or rituximab in adults with relapsed/refractory multiple myeloma or non-Hodgkin's lymphoma that will determine the maximum tolerated dose and recommended Phase II dose for NKTR-255.

Lay abstract Interleukin-15 (IL-15) is a protein that helps the body's natural immune system to defend itself against infections and diseases like cancer. This article discusses a clinical trial in patients with multiple myeloma or non-Hodgkin's lymphoma that evaluates a new investigational medicine, NKTR-255, a polymer-modified form of IL-15 that has been engineered to improve its ability to provide a sustained anti-tumor immune response. The trial will explore different doses of NKTR-255 to determine patient side effects and to find the highest acceptable dose that patients can tolerate. Based on this, a dose will be chosen that offers an optimal balance between having a positive anti-cancer effect and minimizing side effects. This dose will be tested further in patients who have had different treatments in the past. If the side effects are acceptable, this dose will be tested in a new trial in a large number of patients. Clinical Trial Registration: NCT04136756 (ClinicalTrials.gov).

Human Abuse Potential of Oral NKTR-181 in Recreational Opioid Users: A Randomized, Double-Blind, Crossover Study.

OBJECTIVE: To evaluate the human abuse potential, pharmacokinetics, pharmacodynamics, and safety of oral NKTR-181 (oxycodegol), a novel full mu-opioid receptor agonist, relative to oral oxycodone. DESIGN: This double-blind, randomized, single-dose, crossover human abuse potential study was conducted in healthy, adult, non-physically dependent recreational opioid users. SETTING: Inpatient clinical research site. SUBJECTS: Seventy-one subjects randomized (95.7% male, 65.2% African American, mean age = 31.7 years). METHODS: The primary objective was to compare two therapeutic doses of NKTR-181 (400 and 600 mg) with 40 and 60 mg of oxycodone and a supratherapeutic dose (1200 mg) of NKTR-181 with 60 mg of oxycodone using visual analog scale (VAS) ratings for Drug Liking "at this moment" (Drug Liking). Secondary objectives included VAS ratings for other subjective measures, and central nervous system (CNS) mu-opioid effects were assessed using pupillometry. Each subject received single oral doses of five treatments and matching placebo. RESULTS: Compared with 40 and 60 mg of oxycodone, the maximum mean Drug Liking score at 400 and 600 mg NKTR-181 was significantly lower, and the rate of onset and extent of Drug Liking for all NKTR-181 doses in the first two hours postdose were also significantly lower. Delayed attenuated Drug Liking and pupillary miosis response following administration of NKTR-181 vs oxycodone were consistent with slower NKTR-181 CNS entry kinetics and mu-opioid receptor binding. No adverse events were rated as severe, and somnolence and dizziness occurred more frequently when subjects received oxycodone. CONCLUSIONS: NKTR-181 at oral doses of 400 and 600 mg showed significantly fewer and less severe subjective effects accepted as representative of opioid abuse potential, such as lower peak Drug Liking in recreational opioid users, than 40 and 60 mg of oxycodone.

A Reduction in B, T, and Natural Killer Cells Expressing CD38 by TAK-079 Inhibits the Induction and Progression of Collagen-Induced Arthritis in Cynomolgus Monkeys.

Ectoenzyme CD38 is increased on lymphocytes in response to an antigenic challenge and it is hypothesized that targeting these activated lymphocytes could ameliorate pathologic activities in autoimmune diseases. The cynomolgus monkey is an appropriate model for assessing potential effects of targeting CD38 in humans because these species exhibit similar expression profiles. TAK-079 is a human monoclonal antibody (IgG1 λ ) that binds to CD38 and lyses bound cells by complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity. TAK-079 binds to monkey CD38 with an affinity at EC50 4.5 nM, and the potential activity of TAK-079 was investigated in a monkey collagen-induced arthritis model of autoimmune disease. Prophylactic administration of TAK-079 (3 mg/kg i.v. weekly) was well tolerated and prevented arthritis development compared with vehicle-treated control animals, which exhibited progressive disease with radiographic damage and worsening clinical scores over the study course. Therapeutic treatment of arthritic monkeys with TAK-079 (3 mg/kg i.v. weekly) was also well tolerated and reduced disease progression and symptoms. Arthritis scores and joint swelling were significantly lower than the vehicle control, accompanied by decreases in blood levels of C-reactive protein, alkaline phosphatase, and natural killer, B, and T cells. Histopathology, morphometry, and radiology revealed significantly less joint damage in animals exposed prophylactically to TAK-079 treatment compared with vehicle-treated animals and significantly less damage in animals treated therapeutically with TAK-079 or dexamethasone (0.1 mg/kg oral gavage daily), illustrating potential disease-modifying activity. In conclusion, these data indicate that depletion of CD38-expressing cells could be a therapeutic mechanism for treating autoimmune diseases. SIGNIFICANCE STATEMENT: This study demonstrates that targeting CD38-expressing leukocytes with a cytolytic antibody can ameliorate autoimmune disease in cynomolgus monkeys. The study gives a unique perspective into this therapeutic strategy because the three other anti-CD38 cytolytic antibodies in clinical development (daratumumab, isatuximab, and MOR202) cannot be tested in similar models because they do not crossreact with CD38 expressed by new world primates.

NKTR-181: A Novel Mu-Opioid Analgesic with Inherently Low Abuse Potential.

The increasing availability of prescription opioid analgesics for the treatment of pain has been paralleled by an epidemic of opioid misuse, diversion, and overdose. The development of abuse-deterrent formulations (ADFs) of conventional opioids such as oxycodone and morphine represents an advance in the field and has had a positive but insufficient impact, as most opioids are still prescribed in highly abusable, non-ADF forms, and abusers can tamper with ADF medications to liberate the abusable opioid within. The abuse liability of mu-opioid agonists appears to be dependent on their rapid rate of entry into the central nervous system (CNS), whereas analgesic activity appears to be a function of CNS exposure alone, suggesting that a new opioid agonist with an inherently low rate of influx across the blood-brain barrier could mediate analgesia with low abuse liability, regardless of formulation or route of administration. NKTR-181 is a novel, long-acting, selective mu-opioid agonist with structural properties that reduce its rate of entry across the blood-brain barrier compared with traditional mu-opioid agonists. NKTR-181 demonstrated maximum analgesic activity comparable to that of oxycodone in hot-plate latency and acetic-acid writhing models. NKTR-181 was distinguishable from oxycodone by its reduced abuse potential in self-administration and progressive-ratio break point models, with behavioral effects similar to those of saline, as well as reduced CNS side effects as measured by the modified Irwin test. The in vitro and in vivo studies presented here demonstrate that NKTR-181 is the first selective mu-opioid agonist to combine analgesic efficacy and reduced abuse liability through the alteration of brain-entry kinetics.

Discovery of TAK-659 an orally available investigational inhibitor of Spleen Tyrosine Kinase (SYK).

Spleen Tyrosine Kinase (SYK) is a non-receptor cytoplasmic tyrosine kinase that is primarily expressed in hematopoietic cells. SYK is a key mediator for a variety of inflammatory cells, including B cells, mast cells, macrophages and neutrophils and therefore, an attractive approach for treatment of both inflammatory diseases and oncology indications. Using in house co-crystal structure information, and structure-based drug design, we designed and optimized a novel series of heteroaromatic pyrrolidinone SYK inhibitors resulting in the selection of the development candidate TAK-659. TAK-659 is currently undergoing Phase I clinical trials for advanced solid tumor and lymphoma malignancies, a Phase Ib study in advanced solid tumors in combination with nivolumab, and PhIb/II trials for relapsed/refractory AML.

Bempegaldesleukin Plus Nivolumab Versus Sunitinib or Cabozantinib in Previously Untreated Advanced Clear Cell Renal Cell Carcinoma: A Phase III Randomized Study (PIVOT-09).

PURPOSE: Bempegaldesleukin (BEMPEG) is a pegylated interleukin (IL)-2 cytokine prodrug engineered to provide controlled and sustained activation of the clinically validated IL-2 pathway, with the goal of preferentially activating and expanding effector CD8+ T cells and natural killer cells over immunosuppressive regulator T cells in the tumor microenvironment. The open-label, phase III randomized controlled PIVOT-09 trial investigated the efficacy and safety of BEMPEG plus nivolumab (NIVO) as first-line treatment for advanced/metastatic clear cell renal cell carcinoma (ccRCC) with intermediate-/poor-risk disease. METHODS: Patients with previously untreated advanced/metastatic ccRCC were randomly assigned (1:1) to BEMPEG plus NIVO, or investigator's choice of tyrosine kinase inhibitor (TKI; sunitinib or cabozantinib). Coprimary end points were objective response rate (ORR) by blinded independent central review and overall survival (OS) in patients with International Metastatic RCC Database Consortium (IMDC) intermediate-/poor-risk disease. RESULTS: Overall, 623 patients were randomly assigned to BEMPEG plus NIVO (n = 311) or TKI (n = 312; sunitinib n = 225, cabozantinib n = 87), of whom 514 (82.5%) had IMDC intermediate-/poor-risk disease. In patients with IMDC intermediate-/poor-risk disease, ORR with BEMPEG plus NIVO versus TKI was 23.0% (95% CI, 18.0 to 28.7) versus 30.6% (95% CI, 25.1 to 36.6; difference, -7.7 [95% CI, -15.2 to -0.2]; P = .0489), and median OS was 29.0 months versus not estimable (hazard ratio, 0.82 [95% CI, 0.61 to 1.10]; P = .192), respectively. More frequent all-grade treatment-related adverse events (TRAEs) with BEMPEG plus NIVO versus TKI included pyrexia (32.6% v 2.0%) and pruritus (31.3% v 8.8%). Grade 3/4 TRAEs were less frequent with BEMPEG plus NIVO (25.8%) versus TKI (56.5%). CONCLUSION: First-line BEMPEG plus NIVO for advanced/metastatic ccRCC did not improve efficacy in patients with intermediate-/poor-risk disease but led to fewer grade 3/4 TRAEs versus TKI.

Antibody-mediated coengagement of FcγRIIb and B cell receptor complex suppresses humoral immunity in systemic lupus erythematosus.

Engagement of the low-affinity Ab receptor FcγRIIb downregulates B cell activation, and its dysfunction is associated with autoimmunity in mice and humans. We engineered the Fc domain of an anti-human CD19 Ab to bind FcγRIIb with high affinity, promoting the coengagement of FcγRIIb with the BCR complex. This Ab (XmAb5871) stimulated phosphorylation of the ITIM of FcγRIIb and suppressed BCR-induced calcium mobilization, proliferation, and costimulatory molecule expression of human B cells from healthy volunteers and systemic lupus erythematosus (SLE) patients, as well as B cell proliferation induced by LPS, IL-4, or BAFF. XmAb5871 suppressed humoral immunity against tetanus toxoid and reduced serum IgM, IgG, and IgE levels in SCID mice engrafted with SLE or healthy human PBMC. XmAb5871 treatment also increased survival of mice engrafted with PBMC from a unique SLE patient. Unlike anti-CD20 Ab, coengagement of FcγRIIb and BCR complex did not promote B cell depletion in human PBMC cultures or in mice. Thus, amplification of the FcγRIIb inhibitory pathway in activated B cells may represent a novel B cell-targeted immunosuppressive therapeutic approach for SLE and other autoimmune diseases that should avoid the complications associated with B cell depletion.

Selective expansion of regulatory T cells by NKTR-358 in healthy volunteers and patients with systemic lupus erythematosus.

Objective: To evaluate NKTR-358, a polyethylene glycol-interleukin-2 conjugate composition designed to selectively induce regulatory T cells (Tregs), in first-in-human studies. Methods: Healthy volunteers and patients with systemic lupus erythematosus (SLE) received single- or multiple-dose (biweekly) NKTR-358 or placebo in two sequential, randomized, phase 1 studies (single ascending dose [SAD; NCT04133116] and multiple ascending dose [MAD; NCT03556007]). Primary objectives were safety and tolerability; secondary objectives included pharmacokinetics (PK) and immune effects of NKTR-358; exploratory objectives included effects on SLE disease activity. Results: There were eight ascending dose cohorts in the SAD study (0.3-28.0 µg/kg: n = 76; placebo: n = 24) and four in the MAD study (3-24.0 µg/kg: n = 36; placebo: n = 12). Most adverse events (AEs) were grade 1-2 injection-site reactions, with no treatment-related serious or severe AEs, or deaths. PK data showed dose proportionality and prolonged exposure (mean half-life: 7.4-12.9 days). Dose-dependent, selective, and sustained increases in percentages and absolute numbers of total CD4+ Tregs and CD25bright Tregs were observed, with no significant changes in conventional CD4+ and CD8+ T cells, and low-level increases in natural killer cells. At the highest doses tested, administration of NKTR-358 resulted in a 12-17-fold increase in CD25bright Tregs over baseline that was sustained for 20-30 days. Conclusion: NKTR-358 was well tolerated, had a suitable PK profile for biweekly dosing, and led to marked and selective dose-dependent increases in CD25bright Tregs, with no significant changes in conventional T cells. These results provide strong support for further testing in SLE and other inflammatory diseases.

Bempegaldesleukin plus Nivolumab in First-line Metastatic Urothelial Carcinoma: Results from PIVOT-02.

BACKGROUND: Despite recent changes in the treatment landscape, there remains an unmet need for effective, tolerable, chemotherapy-free treatments for patients with advanced/metastatic urothelial carcinoma (mUC), especially cisplatin-ineligible patients. OBJECTIVE: To evaluate the immunostimulatory interleukin-2 cytokine prodrug bempegaldesleukin (BEMPEG) plus nivolumab in patients with advanced/mUC from the phase 2 multicenter PIVOT-02 study. DESIGN, SETTING, AND PARTICIPANTS: This open-label, multicohort phase 1/2 study enrolled patients with previously untreated locally advanced/surgically unresectable or mUC (N = 41). INTERVENTION: Patients received BEMPEG 0.006 mg/kg plus nivolumab 360 mg intravenously every 3 wk. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary objectives were safety and the objective response rate (ORR) in patients with measurable disease at baseline and at least one postbaseline tumor response assessment (response-evaluable). Secondary objectives were overall survival (OS) and progression-free survival (PFS). Exploratory biomarker analyses via univariate logistic regression were performed to test the association between potential biomarkers (CD8+ tumor-infiltrating lymphocytes, tumor mutational burden, and IFN-γ gene expression profile) and response. RESULTS AND LIMITATIONS: The ORR was 35% (13/37 evaluable patients) and the complete response rate was 19% (7/37 patients); the median duration of response was not reached. Median PFS was 4.1 mo (95% confidence interval [CI] 2.1-8.7) and median OS was 23.7 mo (95% CI 15.8-not reached). Overall, 40/41 patients (98%) experienced at least one treatment-related adverse event (TRAE); grade 3/4 TRAEs occurred in 11 patients (27%), most commonly pyrexia (4.9%; 2 patients). Exploratory biomarker analyses showed no association between biomarkers and response. Limitations include the small sample size and single-arm design. CONCLUSIONS: BEMPEG plus nivolumab was well tolerated and showed antitumor activity as first-line treatment in patients with locally advanced/mUC. PATIENT SUMMARY: We investigated an immune-stimulating prodrug called bempegaldesleukin plus the antibody nivolumab as the first therapy for patients with advanced or metastatic cancer of the urinary tract. This combination had manageable treatment-related side effects and was effective in a subset of patients. This trial is registered at ClinicalTrials.gov as NCT02983045.

Bempegaldesleukin plus nivolumab in first-line renal cell carcinoma: results from the PIVOT-02 study.

BACKGROUND: Immune checkpoint inhibitor-based combinations have expanded the treatment options for patients with renal cell carcinoma (RCC); however, tolerability remains challenging. The aim of this study was to evaluate the safety and efficacy of the immunostimulatory interleukin-2 cytokine prodrug bempegaldesleukin (BEMPEG) plus nivolumab (NIVO) as first-line therapy in patients with advanced clear-cell RCC. METHODS: This was an open-label multicohort, multicenter, single-arm phase 1/2 study; here, we report results from the phase 1/2 first-line RCC cohort (N=49). Patients received BEMPEG 0.006 mg/kg plus NIVO 360 mg intravenously every 3 weeks. The primary objectives were safety and objective response rate (ORR; patients with measurable disease at baseline and at least one postbaseline tumor response assessment). Secondary objectives included overall survival (OS) and progression-free survival (PFS). Exploratory biomarker analyses: association between baseline biomarkers and ORR. RESULTS: At a median follow-up of 32.7 months, the ORR was 34.7% (17/49 patients); 3/49 patients (6.1%) had a complete response. Of the 17 patients with response, 14 remained in response for >6 months, and 6 remained in response for >24 months. Median PFS was 7.7 months (95% CI 3.8 to 13.9), and median OS was not reached (95% CI 37.3 to not reached). Ninety-eight per cent (48/49) of patients experienced ≥1 treatment-related adverse event (TRAE) and 38.8% (19/49) had grade 3/4 TRAEs, most commonly syncope (8.2%; 4/49) and increased lipase (6.1%; 3/49). No association between exploratory biomarkers and ORR was observed. Limitations include the small sample size and single-arm design. CONCLUSIONS: BEMPEG plus NIVO showed preliminary antitumor activity as first-line therapy in patients with advanced clear-cell RCC and was well tolerated. These findings warrant further investigation.

The impact of Fc engineering on an anti-CD19 antibody: increased Fcgamma receptor affinity enhances B-cell clearing in nonhuman primates.

CD19, a B cell-restricted receptor critical for B-cell development, is expressed in most B-cell malignancies. The Fc-engineered anti-CD19 antibody, XmAb5574, has enhanced Fcgamma receptor (FcgammaR) binding affinity, leading to improved FcgammaR-dependent effector cell functions and antitumor activity in murine xenografts compared with the non-Fc-engineered anti-CD19 IgG1 analog. Here, we use XmAb5574 and anti-CD19 IgG1 to further dissect effector cell functions in an immune system closely homologous to that of humans, the cynomolgus monkey. XmAb5574 infusion caused an immediate and dose-related B-cell depletion in the blood (to <10% of baseline levels) concomitant with a sustained reduction of natural killer (NK) cells. NK cells had fully recovered by day 15, whereas B-cell recovery was underway by day 57. B cells in secondary lymphoid tissues were depleted (to 34%-61% of vehicle), with involuted germinal centers apparent in the spleen. Anti-CD19 IgG1 had comparable serum exposure to XmAb5574 but demonstrated no B-cell depletion and no sustained NK-cell reduction. Thus, increasing FcgammaR binding affinity dramatically increased B-cell clearing. We propose that effector cell functions, possibly those involving NK cells, mediate XmAb5574 potency in cynomolgus monkeys, and that enhancing these mechanisms should advance the treatment of B-cell malignancies in humans.

NKTR-255 is a polymer-conjugated IL-15 with unique mechanisms of action on T and natural killer cells.

NKTR-255 is a PEG conjugate of recombinant human IL-15 (rhIL-15) being examined as a potential cancer immunotherapeutic. Since IL-15 responses can be mediated by trans or cis presentation via IL-15Rα or soluble IL-15/IL-15Rα complexes, we investigated the role of IL-15Rα in driving NKTR-255 responses using defined naive and memory OVA-specific CD8+ T cells (OT-I) and NK cells in mice. NKTR-255 induced a 2.5- and 2.0-fold expansion of CD8+ T and NK cells, respectively, in WT mice. In adoptive transfer studies, proliferation of naive and memory WT OT-I T cells in response to NKTR-255 was not impaired in IL-15Rα-/- mice, suggesting trans presentation was not utilized by NKTR-255. Interestingly, naive IL-15Rα-/- OT-I cells had deficient responses to NKTR-255, while memory IL-15Rα-/- OT-I cell responses were partially impaired, suggesting that naive CD8+ T cells are more dependent on cis presentation of NKTR-255 than memory CD8+ T cells. In bone marrow chimera studies, IL-15Rα-/- and WT NK cells present in WT recipients had similar responses to NKTR-255, suggesting that cis presentation is not utilized by NK cells. NKTR-255 could form soluble complexes with IL-15Rα; binding to murine IL-15Rα generated superagonists that preferentially stimulated NK cells, showing that conversion to IL-15Rß agonist biases the response toward NK cells. These findings highlight the ability of NKTR-255 to utilize IL-15Rα for cis presentation and act as an IL-15Rαß agonist on CD8+ T cells.

In vivo and in vitro Characterization of a Partial Mu Opioid Receptor Agonist, NKTR-181, Supports Future Therapeutic Development.

Mu opioid receptor (MOPr) agonists are well-known and frequently used clinical analgesics but are also rewarding due to their highly addictive and often abusive properties. This may lead to opioid use disorder (OUD) a disorder that effects millions of people worldwide. Therefore, novel compounds are urgently needed to treat OUD. As opioids are effective analgesics and OUD often occurs in conjunction with chronic pain, these novel compounds may be opioids, but they must have a low abuse liability. This could be mediated by diminishing or slowing blood-brain barrier transport, slowing target receptor binding kinetics, and showing a long half-life. NKTR-181 is a PEGylated oxycodol and a MOPr agonist that has slowed blood-brain barrier transport, a long half-life, and diminished likeability in clinical trials. In this study, we examined the signaling and behavioral profile of NKTR-181 in comparison with oxycodone to determine whether further therapeutic development of this compound may be warranted. For this preclinical study, we used a number of in vitro and in vivo assays. The signaling profile of NKTR-181 was determined by the electrophysiological assessment of MOPr-Ca2+ channel inhibition in the nociceptive neurons of rodent dorsal root ganglia. Heterologous cell-based assays were used to assess biased agonism and receptor trafficking. Different rodent behavioral models were used to define the NKTR-181-induced relief of effective and reflexive nociception and drug-seeking behavior as assessed by an intravenous self-administration (IVSA) of NKTR-181. We found that NKTR-181 and oxycodone are partial agonists in G-protein signaling and Ca2+ channel inhibition assays and promote limited MOPr desensitization. However, NKTR-181 inhibits Ca2+ channels by a different mechanism than oxycodone and induces a different pattern of arrestin recruitment. In addition, NKTR-181 has a slower receptor on-rate and a slower rate of Ca2+ channel coupling than oxycodone. This signaling profile is coupled with a slower onset of antinociception and limited drug-seeking behavior in comparison with oxycodone. Together with its known long half-life and slow blood-brain barrier transport, these data suggest that NKTR-181 could be further studied as a pharmacotherapeutic treatment modality for OUD.

Maximizing cancer therapy via complementary mechanisms of immune activation: PD-1 blockade, neoantigen vaccination, and Tregs depletion.

BACKGROUND: A number of different immune pathways are involved in the effective killing of cancer cells, collectively named as the 'Cancer Immunity Cycle'. Anti-PD-1 checkpoint blockade (CPB) therapy is active on one of these pathways and reinvigorates anticancer T cell immunity, leading to long-term responses in a limited fraction of patients with cancer. We have previously shown that neoantigens-based adenovirus vectored vaccine in combination with anti-PD-1 further expands pre-existing anticancer immunity and elicits novel neoantigen-specific T cells thereby increasing efficacy to 50% of tumor clearance in mice. Here we added a third component to the CPB plus vaccine combination, which is able to modify the suppressive tumor microenvironment by reducing the number of tumor-infiltrating regulatory T cells (Tregs), as strategy for improving the therapeutic efficacy and overcoming resistance. METHODS: The antitumor efficacy of anti-PD-1, neoantigen vaccine and Treg modulating agents, either Bempegaldesleukin (BEMPEG: NKTR-214) or an anti-CTLA-4 mAb with Treg-depleting activity, was investigated in murine tumor models. We evaluated tumor growth in treated animals, neoantigen-specific T cells in tumors, tumor-infiltrating lymphocytes (TILs) and intratumoral Tregs. RESULTS: The addition of BEMPEG or anti-CTLA-4 to the combination of vaccine and anti-PD-1 led to complete eradication of large tumors in nearby 100% of treated animals, in association with expansion and activation of cancer neoantigen-specific T cells and reduction of tumor-infiltrating Tregs. CONCLUSION: These data support the notion that the integrated regulation of three steps of the cancer immunity cycle, including expansion of neoantigen-specific T cells, reversal of the exhausted T cell phenotype together with the reduction of intratumoral Tregs may represent a novel rationally designed drug combination approach to achieve higher cure rates.

NKTR-255, a novel polymer-conjugated rhIL-15 with potent antitumor efficacy.

BACKGROUND: NKTR-255 is a novel polyethylene glycol-conjugate of recombinant human interleukin-15 (rhIL-15), which was designed to retain all known receptor binding interactions of the IL-15 molecule. We explored the biologic and pharmacologic differences between endogenous IL-15 receptor α (IL-15Rα)-dependent (NKTR-255 and rhIL-15) and IL-15Rα-independent (precomplexed rhIL-15/IL-15Rα) cytokines. METHODS: In vitro pharmacological properties of rhIL-15, NKTR-255 and precomplex cytokines (rhIL-15/IL-15Rα and rhIL-15 N72D/IL-15Rα Fc) were investigated in receptor binding, signaling and cell function. In vivo pharmacokinetic (PK) and pharmacodynamic profile of the cytokines were evaluated in normal mice. Finally, immunomodulatory effect and antitumor activity were assessed in a Daudi lymphoma model. RESULTS: NKTR-255 and rhIL-15 exhibited similar in vitro properties in receptor affinity, signaling and leukocyte degranulation, which collectively differed from precomplexed cytokines. Notably, NKTR-255 and rhIL-15 stimulated greater granzyme B secretion in human peripheral blood mononuclear cells versus precomplexed cytokines. In vivo, NKTR-255 exhibited a PK profile with reduced clearance and a longer half-life relative to rhIL-15 and demonstrated prolonged IL-15R engagement in lymphocytes compared with only transient engagement observed for rhIL-15 and precomplexed rhIL-15 N72D/IL-15Rα Fc. As a consequent, NKTR-255 provided a durable and sustained proliferation and activation of natural killer (NK) and CD8+ T cells. Importantly, NKTR-255 is more effective than the precomplexed cytokine at inducing functionally competent, cytotoxic NK cells in the tumor microenvironment and the properties of NKTR-255 translated into superior antitumor activity in a B-cell lymphoma model versus the precomplexed cytokine. CONCLUSIONS: Our results show that the novel immunotherapeutic, NKTR-255, retains the full spectrum of IL-15 biology, but with improved PK properties, over rhIL-15. These findings support the ongoing phase 1 first-in-human trial (NCT04136756) of NKTR-255 in participants with relapsed or refractory hematologic malignancies, potentially advancing rhIL-15-based immunotherapies for the treatment of cancer.

Bempegaldesleukin Plus Nivolumab in First-Line Metastatic Melanoma.

PURPOSE: Therapies that produce deep and durable responses in patients with metastatic melanoma are needed. This phase II cohort from the international, single-arm PIVOT-02 study evaluated the CD122-preferential interleukin-2 pathway agonist bempegaldesleukin (BEMPEG) plus nivolumab (NIVO) in first-line metastatic melanoma. METHODS: A total of 41 previously untreated patients with stage III/IV melanoma received BEMPEG 0.006 mg/kg plus NIVO 360 mg once every 3 weeks for ≤ 2 years; 38 were efficacy-evaluable (≥ 1 postbaseline scan). Primary end points were safety and objective response rate (blinded independent central review); other end points included progression-free survival, overall survival (OS), and exploratory biomarkers. RESULTS: At 29.0 months' median follow-up, the objective response rate was 52.6% (20 of 38 patients), and the complete response rate was 34.2% (13 of 38 patients). Median change in size of target lesions from baseline was -78.5% (response-evaluable population); 47.4% (18 of 38 patients) experienced complete clearance of target lesions. Median progression-free survival was 30.9 months (95% CI, 5.3 to not estimable). Median OS was not reached; the 24-month OS rate was 77.0% (95% CI, 60.4 to 87.3). Grade 3 and 4 treatment-related and immune-mediated adverse events occurred in 17.1% (7 of 41) and 4.9% (2 of 41) of patients, respectively. Increased polyfunctional responses in CD8+ and CD4+ T cells were seen in blood after treatment, driven by cytokines with effector functions. Early on-treatment blood biomarkers (CD8+ polyfunctional strength difference and eosinophils) correlated with treatment response. CONCLUSION: BEMPEG in combination with NIVO was tolerated, with relatively low rates of grade 3 and 4 treatment-related and immune-mediated adverse events. The combination had encouraging antitumor activity in first-line metastatic melanoma, including an extended median progression-free survival. Exploratory analyses associated noninvasive, on-treatment biomarkers with response, before radiologic evidence was observed.

NKTR-358: A novel regulatory T-cell stimulator that selectively stimulates expansion and suppressive function of regulatory T cells for the treatment of autoimmune and inflammatory diseases.

Impaired interleukin-2 (IL-2) production and regulatory T-cell dysfunction have been implicated as immunological mechanisms central to the pathogenesis of multiple autoimmune and inflammatory diseases. NKTR-358, a novel regulatory T-cell stimulator, is an investigational therapeutic that selectively restores regulatory T-cell homeostasis in these diseases. We investigated NKTR-358's selectivity for regulatory T-cells, receptor-binding properties, ex vivo and in vivo pharmacodynamics, ability to suppress conventional T-cell proliferation in mice and non-human primates, and functional activity in a murine model of systemic lupus erythematosus. In vitro, NKTR-358 demonstrated decreased affinity for IL-2Rα, IL-2Rß, and IL-2Rαß compared with recombinant human IL-2 (rhIL-2). A single dose of NKTR-358 in cynomolgus monkeys produced a greater than 15-fold increase in regulatory T-cells, and the increase lasted until day 14, while daily rhIL-2 administration for 5 days only elicited a 3-fold increase, which lasted until day 7. Repeated dosing of NKTR-358 over 6 months in cynomolgus monkeys elicited cyclical, robust increases in regulatory T-cells with no loss in drug activity over the course of treatment. Regulatory T-cells isolated from NKTR-358-treated mice displayed a sustained, higher suppression of conventional T-cell proliferation than regulatory T-cells isolated from vehicle-treated mice. NKTR-358 treatment in a mouse model (MRL/MpJ-Faslpr) of systemic lupus erythematosus for 12 weeks maintained elevated regulatory T-cells for the treatment duration and ameliorated disease progression. Together, these results suggest that NKTR-358 has the ability to elicit sustained and preferential proliferation and activation of regulatory T-cells without corresponding effects on conventional T-cells, with improved pharmacokinetics compared with rhIL-2.