Phase I study of single agent NIZ985, a recombinant heterodimeric IL-15 agonist, in adult patients with metastatic or unresectable solid tumors

Background NIZ985 is a recombinant heterodimer of physiologically active interleukin (IL-)15 and IL-15 receptor alpha. In preclinical models, NIZ985 promotes cytotoxic lymphocyte proliferation, killing function, and organ/tumor infiltration, with resultant anticancer effects. In this first-in-human study, we assessed the safety, pharmacokinetics, and immune effects of NIZ985 in patients with metastatic or unresectable solid tumors. Methods Single agent NIZ985 dose escalation data are reported from a phase I dose escalation/expansion study of NIZ985 as monotherapy. Adult patients (N=14) received 0.25, 0.5, 1, 2 or 4 µg/kg subcutaneous NIZ985 three times weekly (TIW) for the first 2 weeks of each 28-day cycle, in an accelerated 3+3 dose escalation trial design. IL-15 and endogenous cytokines were monitored by ELISA and multiplexed electrochemiluminescent assays. Multiparameter flow cytometry assessed the frequency, phenotype and proliferation of peripheral blood mononuclear cells. Preliminary antitumor activity was assessed by overall response rate (Response Evaluation Criteria in Solid Tumors V.1.1). Results As of March 2, 2020, median treatment duration was 7.5 weeks (range 1.1–77.1). Thirteen patients had discontinued and one (uveal melanoma) remains on treatment with stable disease. Best clinical response was stable disease (3 of 14 patients; 21%). The most frequent adverse events (AEs) were circular erythematous injection site reactions (100%), chills (71%), fatigue (57%), and fever (50%). Treatment-related grade 3/4 AEs occurred in six participants (43%); treatment-related serious AEs (SAEs) in three (21%). The per-protocol maximum tolerated dose was not reached. Pharmacokinetic accumulation of serum IL-15 in the first week was followed by significantly lower levels in week 2, likely due to more rapid cytokine consumption by an expanding lymphocyte pool. NIZ985 treatment was associated with increases in several cytokines, including interferon (IFN)-γ, IL-18, C-X-C motif chemokine ligand 10, and tumor necrosis factor-β, plus significant induction of cytotoxic lymphocyte proliferation (including natural killer and CD8+ T cells), increased CD16+ monocytes, and increased CD163+ macrophages at injection sites. Conclusions Subcutaneous NIZ985 TIW was generally well tolerated in patients with advanced cancer and produced immune activation paralleling preclinical observations, with induction of IFN-γ and proliferation of cytotoxic lymphocytes. Due to delayed SAEs at the two highest dose levels, administration is being changed to once-weekly in a revised protocol, as monotherapy and combined with checkpoint inhibitor spartalizumab. These alterations are expected to maximize the potential of NIZ985 as a novel immunotherapy. Trial registration number NCT02452268.


Methodology
• Briefly, up to 4x10 6 cryopreserved PBMCs were thawed quickly in a 37ºC water bath and washed by centrifugation with pre-warmed RPMI medium containing 10% FBS. After washing, cells were stained with fixable viability dye eFluor™506 (ThermoFisher, San Diego, CA) at a dilution of 1:100 in wash buffer (phosphate-buffered saline containing 0.1% sodium azide and 2% fetal bovine serum) followed by staining with fluorochrome-conjugated surface antibodies for 30 minutes at room temperature in the dark. Previously prepared and qualified antibody cocktails consisting of 12 surface antibodies were utilized.

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After incubation, cells were washed once by centrifugation in wash buffer and fixed with 1x Fixation/Permeabilization Buffer from the Foxp3/Transcription Factor Staining Buffer Set (ThermoFisher, San Diego, CA) for 30 minutes at room temperature in the dark. After fixation, cells were washed once by centrifugation with 1x Permeabilization Buffer from the Foxp3/Transcription Factor Staining Buffer Set and resuspended in 1x Permeabilization Buffer. Fluorochrome-conjugated intracellular antibody (Ki-67) was added and cells were incubated for 30 minutes at room temperature in the dark. Cells were then washed twice by centrifugation with 1x Permeabilization Buffer and resuspended in 0.5% Formalin solution.
• Cells were then acquired on a BD LSRFortessa X-20 equipped with 5 lasers (BD Biosciences, San Jose, CA) and data was analyzed using FlowJo software (FlowJo LLC, Ashland, OR). For data analysis of the T-cell-Proliferation assay, after exclusion of debris, doublets and dead cells, T cells were further gated for CD4+ and CD8+ subsets. Both CD4+ and CD8+ T cells were then subsequently analyzed for the expression of various biomarkers to define proliferating (Ki-67), checkpoint inhibitor-expressing (PD-1, LAG-3, TIM-3), activated (CD38, HLA-DR) and memory (CCR7, CD45RA) T cell subsets. For data analysis of the NK-cell-Proliferation assay, after exclusion of debris, doublets and dead cells, NK cells were further gated for NK subsets (CD56, CD16), as well as inhibition (CD159a) and activation (CD25, CD335, NKG2D) biomarkers. Monocytes were also assessed for CD14, CD16 and HLA-DR expression.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance  Table S2. NCI flow cytometry panel details.
The following monoclonal antibodies were used for the flow analyses in the first nine patients treated in the study: from BD Biosciences: CD11c-BV650 (clone 3.9) and CD86-BV711 (clone IT2.2). From ThermoFisher: Vd1-FITC (clone TS8.2). From Beckman Coulter: NKG2a-PECy7 (clone Z199). The CD11b antibody (clone ICRF44) was conjugated in-house with FITC. The viability dye (LIVE/DEAD fixable Blue dead cell stain) was from Invitrogen. Two different staining cocktails were used (T cell panel, NK and myeloid panel) as indicated below.

Detector
Marker Clone Fluor Source    BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)  Grade 3 hyponatremia in one patient; grade 3 and/or 4 events of lymphocyte count decreased (2 events), productive cough, dysphonia, dysphagia, stridor, bronchial obstruction, tracheal stenosis, esophageal stenosis, esophageal pain, decreased appetite, dehydration, hypotension, embolism (2 events), atrial fibrillation, fatigue, peripheral edema, purpura, acute kidney injury, oliguria and vasculitis (2 events) in one patient.
ISR, injection site reaction.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance  Figure S1. Duration of treatment IO, immuno-oncology; PD, progressive disease; SD, stable disease. BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Figure S3: Best percentage change from baseline in sum of diameters of target lesions.
IO, immuno-oncology treatment; PD, progressive disease; SD, stable disease; UNK, unknown. One patient (1.0 µg/kg) had no recorded data and is excluded from the plot. BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) Figure S4. Individual proportions of Ki67 + CD4 + cells, CD8 + cells,  T cells and NK cells over the first 2-3 treatment cycles among the last five patients treated in the study. (see table S1 for methodology).
NK, natural killer. BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)