Phase I Trial of N-803, an IL15 Receptor Agonist, with Rituximab in Patients with Indolent Non-Hodgkin Lymphoma

Abstract

Purpose: N-803 is an IL15 receptor superagonist complex, designed to optimize in vivo persistence and trans-presentation, thereby activating and expanding natural killer (NK) cells and CD8+ T cells. Monoclonal antibodies (mAbs) direct Fc receptor-bearing immune cells, including NK cells, to recognize and eliminate cancer targets. The ability of IL15R agonists to enhance tumor-targeting mAbs in patients has not been reported previously.

Patients and methods: Relapsed/refractory patients with indolent non-Hodgkin lymphoma were treated with rituximab and intravenous or subcutaneous N-803 on an open-label, dose-escalation phase I study using a 3+3 design (NCT02384954). Primary endpoint was maximum tolerated dose. Immune correlates were performed using multidimensional analysis via mass cytometry and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) which simultaneously measures protein and single-cell RNA expression.

Results: This immunotherapy combination was safe and well tolerated and resulted in durable clinical responses including in rituximab-refractory patients. Subcutaneous N-803 plus rituximab induced sustained proliferation, expansion, and activation of peripheral blood NK cells and CD8 T cells, with increased NK cell and T cells present 8 weeks following last N-803 treatment. CITE-seq revealed a therapy-altered NK cell molecular program, including enhancement of AP-1 transcription factor. Furthermore, the monocyte transcriptional program was remodeled with enhanced MHC expression and antigen-presentation genes.

Conclusions: N-803 combines with mAbs to enhance tumor targeting in patients, and warrants further investigation in combination with immunotherapies.

Conflict of interest statement

Conflict of Interest Statement: This clinical trial was supported by ImmunityBio (previously Altor BioScience). ADR, JL, and PS-S are employees or have financial interest in ImmunityBio. TAF has consulted for Nektar, Wugen, Indapta, Kiadis, Orca Bio, and has received research funding from ImmunityBio, Affimed, and Compass Therapeutics. BK has received research funding and consulting fees from Roche, Genentech, and Celgene. NMS has received research funding from Verastem Pharmaceuticals, Corvus Pharmaceuticals, Innate Pharmaceuticals, Bristol Myers Squibb, Genetech/Roche, Celgene and consulted for Kiowa Hakka Kirin, C4 Therapeutics, Karyopharma Therapeutics. NE has received funding from Verastem-Speaker’s Bureau and Pharmacyclics-Honoraria. JAF has pending patents (WO 2019/152387, US 63/018,108) unrelated to the present work that are licensed to Kiadis, and a monoclonal antibody unrelated to the present work licensed to EMD Millipore. MBH is a co-inventor on a patent (US2010/0159594A1). MMBE is a consultant and has equity interest in Wugen, and may receive royalty income based on a technology developed by TAF and MMBE and licensed by Washington University to Wugen. MMBE has received travel funds from Fluidigm. All other authors have no conflicts of interest to report.

©2021 American Association for Cancer Research.

Figures

Figure 1.. N-803 and Rituximab induce clinical responses and immune modulation in a Phase 1 trial.

A) Schematic of the phase 1 clinical trial and dosing regimens. B) Waterfall plot depicting the percent maximal change in the sum of the products of the greatest diameter (SPD) of the lymphoma tumor burden and the best clinical response for all patients in the IV and SQ cohort by color. n = 21 C) Swimmer plot for SQ N-803 patients depicting best clinical responses across follow-up by dose. n = 12. Red X denotes PD. * denotes rituximab-refractory patient. D) Representative tSNE visualization of major immune cell lineages in high-dimensional mass cytometry data. E) Percentage of NK, CD8+ T cells, and CD4+ T cells in patient PBMC pre-treatment (D1) and during N-803 and rituximab treatment (D8-22). Mean ± SEM depicted. n = 5, 3 independent experiments. 2-way ANOVA with Dunnett’s multiple comparison’s test. F) Representative tSNE density plot of PBMC lineages across time. Numbers in orange denoting mean of NK cell percentages across time. * = p < 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

Figure 2.. N-803 and rituximab induce activation, expansion, and proliferation of PB NK cells.

A) Representative Flow Cytometry plots of NK cells at each time points. Black box denotes NK cell gate, and numbers denote percent of NK cells in lymphocytes at each time point. B) Line graph and C) dot plot depicting the percentage of NK cells by time. D) Line graph and E) dot plot of absolute number of NK cells per μl of blood. F) Representative flow histograms of Ki-67 expression in NK cells at each time point. Numbers denote percent of Ki-67+ NK cells. G) Line graph and H-I) Dot plot of Ki-67+ NK cells at each time point during induction and consolidation. J) Dot Plot depicting percent of granzyme B+ NK cells by patient. Linear mixed model for all of the above. K) tSNE visualization and density plots gated on NK cells assessed by mass cytometry. L) Representative mass cytometry viSNE plots of Eomes, CD38, NKp30, and NKp44 expression in NK cells; blue- low expression, red- high expression. Line graphs depicting M) percent of Eomes+, CD38 and NKp30 Median Expression, and NKp44+ NK cells. n = 5-6, ≥ 3 independent experiments. Friedman’s test for NKp44 and Dunn’s multiple comparisons, RM one-way ANOVA and Dunnett’s multiple comparisons for all others. Mean ± SEM for all line and dot plots. Line graphs depict Mean ± SEM of values for all patients. Individual dots represent individual patients. Significance for mass cytometry comparisons p < 0.05, all others p < 0.01. * = p < 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

Figure 3.. N-803 and rituximab expand and activate a subset of CD8+ T cells.

A) Dot Plot of the percent and B) absolute number of CD8 + T cells. C) Line graph and D) dot plots of percent of Ki-67+ CD8+ T cells by time point. Linear mixed model for all. E) Representative flow histograms of Ki-67 expression in CD8+ T cells at each time point. Numbers denote percent of Ki-67+ CD8+ T cells. F) tSNE density plot gated on CD8+ T cells and visualization of metaclusters from mass cytometry as determined by FlowSOM analysis, and bar graph of percent of each metacluster at each time point. 2-way ANOVA with Tukey’s multiple comparisons. G) Relative expression of select CD8+ T cell markers in each metacluster. Expression is scaled by row. Friedman’s test with Dunn’s multiple comparisons. H) Representative plots of CD38 and HLA-DR expression in CD8+ T cells across time and I) percent of CD38 and HLA-DR double-positive T cells. RM one-way ANOVA with Dunnett’s multiple comparisons test. Mean ± SEM for all line and dot plots. Line graphs depict Mean ± SEM of values for all patients. n = 5-6, ≥ 3 independent experiments. Significance for mass cytometry comparisons p < 0.05, all others p < 0.01. * = p < 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001.

Figure 4.. CITE-seq reveals activation of NK cells following N-803 plus rituximab.

A) tSNE visualization of PBMC transcriptomes colored by immune cell type in patient 027-015 (15) before (D1) and at D15 of N-803 plus rituximab. Dashed lines depict major immune cell lineages: NK (navy), T cells (red), Monocytes (grey). B-C) Volcano plots depicting summary data of the DEGs of all 3 patients in red in CD56bright and CD56dim NK cells with N-803 plus rituximab with an absolute log2 FC cut-off of ≥ 0.5. D) UMAP visualization of NK cell clusters in patients 027-016 (16) & 027-017 (17) clustered using CiteFuse with protein and RNA data. E) UMAP colored by patient. F) Feature Plots depicting protein (ADT) and RNA expression of key NK maturation markers. Min. cutoff = quantile 2 (q02), Max.cutoff = q98. G) Split violin plots depicting expression of select genes differentially expressed between D1 and D15 in each NK cell cluster. * denotes a significant change. White= Day 1, Blue = Day 15. H) Heatmap depicting select differentially expressed genes in γδ T cells between pre-treatment (D1) and Day 15. I) Line graphs depicting the proportion of CD16 Monocytes in each patient at each timepoint. Two-sided fisher’s Exact test with Holm’s multiple comparisons p-value adjustment. * = p < 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, **** = p ≤ 0.0001. J) Select enriched GO BP in CD16+ Monocytes between D15 and D1. Colored boxes depict average log2 FC of the genes in the BP gene set. White boxes correspond to genes not included in the GO BP gene set. Wilcoxon Rank-Sum test for all DEGs with an adjusted p-value of < 0.05 and fold change of ≥ 0.5 absolute log2 fold change. Enriched GO terms p < 0.05 and q-value threshold of 0.05. n=2-3 patients for all. 2 independent experiments.