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

This article is featured in Highlights of This Issue, p. 3267

Immunotherapy is a rising modality in cancer therapeutics, harnessing concepts from immunology to enhance, trigger, or rescue immune responses against malignant targets. Established immunotherapies include engineered therapeutic monoclonal antibodies (mAbs), such as the anti-CD20 mAb rituximab, which targets endogenous Fc-receptor bearing immune cells, including natural killer (NK) cells, to B-cell malignancies (1). More recent successes in a variety of cancers have been the translation of anti–PD-1/PD-L1 and anti–CTLA-4 immune checkpoint blockade, which release induced brakes on tumor-reactive T cells (2). Recently, complementary immunotherapy approaches utilizing cytokine receptor agonists are being tested as single agents and in combination with immunotherapies to potentiate immune cell survival and function against cancer cells (3). Currently, the use of this class of drugs is limited by adverse side effects including fevers and chills, hypotension, and expansion of suppressive regulatory T cells (Treg; refs. 4–6). To address this unmet need in cancer immunotherapy, a new class of immune therapy biologics seeks to overcome these hurdles via innovative protein engineering, led by IL15 receptor (IL15R) agonists.

IL15 is a key cytokine for the development, survival, and function of NK cells (7). Recent in vitro and in vivo work demonstrated the ability of IL15 to prime noncytotoxic CD56^bright NK cells to become antitumor effectors, a function previously believed to be largely restricted to the CD56^dim NK cell subset (8). IL15 also plays a key role in supporting memory CD8⁺ T cells, thus augmenting two types of antitumor effector lymphocytes (9). In vivo, IL15 is trans-presented by IL15Rα from accessory cells such as monocytes/macrophages and dendritic cells to ligate the IL2/15Rγβ heterodimer expressed on NK and T cells, resulting in activation of multiple signaling pathways, and hence multiple antitumor functions (9). Although IL15 and IL2 share signaling components, including the beta (CD122) and gamma chain (CD132), they have divergent effects, with IL2 promoting Treg expansion (10.) Initial pioneering studies tested E. coli–derived recombinant human (rh)-IL15, which demonstrated immune modulation in patients. However, rhIL15 has a short half-life and dose levels that modulated immune cells were limited by unacceptable adverse events (AE; ref. 4). N-803 (formerly ALT-803) is an IL15R superagonist complex with a prolonged in vivo half-life, physiologic trans-presentation, and accumulation in secondary lymphoid tissues (11). The initial study of N-803 in patients with hematologic malignancies who relapsed after hematopoietic cell transplantation (HCT) demonstrated safety and modulation of immunity by N-803 but modest single-agent clinical activity (12).

We hypothesized that N-803 synergizes with the anti-CD20 lymphoma-targeting mAb rituximab to promote antilymphoma immune responses, which was supported by preclinical models (13). To test this hypothesis, we designed a clinical trial combining N-803 with rituximab for patients with relapsed/refractory (rel/ref) indolent NHL (iNHL). Here, we report the safety, pharmacokinetics, antilymphoma activity, and immunologic effects in the phase I study cohort.

Patients were treated on an open-label, multicenter [Washington University (St. Louis, MO); University of Minnesota (Minneapolis, MN; Medical University of South Carolina (Charleston, SC)], dose-escalation phase I study registered on clinicaltrials.gov (NCT02384954) using a 3+3 design. Patients were enrolled beginning April 17, 2015 and ending July 23, 2017. Clinical data were analyzed by individual site investigators (T.A. Fehniger, B.T. Hess, V. Bachanova, N. Epperla) and verified and summarized by ImmunityBio; all authors had access to primary clinical trial data. Progression-free survival (PFS), overall survival, and duration of response of all treated patients was be assessed at least every 3 months during years 1 and 2, every 4 months during year 3, and then every 6 months (± 2 months) during years 4 and 5 from the start of study treatment, or through the point designated as the end of the study follow-up (5 years). Information about disease response assessments and other therapies (chemotherapy, surgery, radiotherapy, other investigational treatment) received after discontinuation or completion of study treatment will be collected as available during the follow-up period. Patients with iNHL (follicular lymphoma, marginal zone lymphoma, small lymphocytic lymphoma, lymphoplasmacytic lymphoma) that were rel/ref after ≥2 prior lines of therapy were eligible. Patients were considered anti-CD20 mAb refractory if they progressed on anti-CD20 mAb therapy or within 6 months of their last dose of anti-CD20 mAb. Treatment consisted of intravenous rituximab 375 mg/m² and intravenous or subcutaneous N-803 (in increasing dosing cohorts of 1, 3, 6, 10, 15, 20 μg/kg) on days 1, 8, 15, and 22 of cycle 1, followed by a rest period, and then consolidation with four additional treatments on days 78, 134, 190, and 246 (total of eight treatments). For the initial three intravenous cohorts, rituximab was administered on day 1, and N-803 on day 2. Responses were assessed using the 2007 International Harmonization Project criteria with assessment modifications to incorporate indeterminate response criteria (14, 15). The primary endpoints for the phase I cohort was determination of the maximum tolerated dose or tested dose of N-803 and the recommended phase II dose (RP2D). AEs were monitored and graded using the NCI Common Terminology Criteria for Adverse Events v4.0. Samples were collected for immune cell number and phenotyping pre-therapy [cycle 1 day 1(C1D1)] and days 2, 5, 8, 15, 22, 23, and 26 of cycle 1, and days 78, 79, and 82 of the first consolidation cycle. Serum for cytokine and pharmacokinetic analysis was collected pre-therapy (0 minutes), and after N-803 at 30 minutes, 2 hours, 6 hours (C1D1) and days 2, 5, and 8.

This clinical trial was approved at participating institutions Institutional Review Boards and was conducted in accordance with recognized ethical guidelines (Declaration of Helsinki), and all patients provided written informed consent prior to study evaluation and treatment.

Immunogenicity, N-803, IL6, and IFNγ serum measurements were conducted as described previously (12).

Patient peripheral blood mononuclear cells (PBMCs) were stained as described previously (Supplementary Materials and Methods; ref. 16). Absolute cell count of flow cytometry measured cell populations were only performed when absolute lymphocyte counts were available. Mass cytometry was performed (Supplementary Table S1) on thawed patient PBMCs as described previously (12). Data were collected on a Helios mass cytometer (Fluidigm) and analyzed using Cytobank (17, 18). Briefly, for each individual donor, cell subsets were identified using viSNE (v1, Supplementary Table S1). These individually viSNE-gated subsets were then assessed. CD8 and CD4 T cells were assessed with an additional viSNE (v2), and subsets therein gated using unbiased FlowSOM analysis (Supplementary Table S1, using all events, 36 clusters, 6 Metaclusters, 10 iterations, random seed, RRID:SCR_016899; ref. 19).

PBMCs were thawed in batch (batch 1: 15, batch 2: 16 and 17) and mixed with 5% murine spleen cells where indicated (batch 2), and stained with oligonucleotide-tagged antibodies [antibody-derived tags (ADTs); Supplementary Table S2; Supplementary Materials and Methods]. Following sequencing, FASTQ files were aligned to a custom genome consisting of the GRCh38 and mm10 reference and all patients and timepoints were aggregated using CellRanger (default settings, v.3.0.1; RRID:SCR_017344), and the resulting matrix was imported into the R-based package, Seurat (v.3.1/3.2; RRID:SCR_016341; refs. 20–22) for normalization, batch-correction, clustering, visualization, and differential expression (23). NK cells from patients 027-016 and 027-017 were imported into CiteFuse (RRID:SCR_019321) for clustering and UMAP projection using all ADTs and variable genes were selected for clustering using default settings (24). For gene ontology analysis, we utilized clusterprofiler (RRID:SCR_016884) (25).

Differential expression analysis was conducted in either R v 3.5/6 (RRID:SCR_001905), GraphPad Prism (v7/8; RRID:SCR_002798), or SAS (v 9.4, SAS Institutes; RRID:SCR_008567) with the appropriate parametric or nonparametric test as listed in the figure legend. For specific immune correlates as described in the figure legends, statistical analyses were performed comparing pretreatment (D1) to the peak value for each variable within the time frames as indicated in the figures. For these analyses, the over-time changes were assessed using linear mixed models and post hoc multiple comparisons for the differences between specific time-points of interest were only performed on those biomarkers that were significant in the overall test for improved control of the familywise error rate. A logarithm transformation was applied to the absolute cell counts to better satisfy the normality and homoscedasticity assumptions.

Raw sequencing files will be available in dbGaP under accession phs001229 to qualified investigators at the time of publication. Clinical data will be deposited at clinicaltrials.gov in accordance with guidelines. For all other data requests, contact tfehnige@wustl.edu.

Patients with rel/ref iNHL were enrolled in two sequential N-803 cohorts: intravenous (N = 9 total; N = 3 at each of 1, 3, and 6 μg/kg) and subcutaneous (N = 12 total; N = 3 at each of 6, 10, 15, and 20 μg/kg) for a total of 21 patients (Fig. 1A; Table 1). Most patients had follicular lymphoma (76%), with a median of 2 (range, 1–7) prior treatments, a median time from last prior anti-CD20 therapy to study therapy of 25 (range, 1–134) months, and five patients were refractory to their last anti-CD20 mAb containing regimen. For the patients with follicular lymphoma, 94% of patients had intermediate or high-risk Follicular Lymphoma International Prognostic Index (FLIPI) category. For study therapy, a standard dose and schedule of rituximab (375 mg/m²) was chosen (26), to allow comparison with historical clinical trials of single-agent rituximab, comprised of four weekly doses, followed by consolidation with four additional doses every 2 months (Fig. 1A). N-803 was administered on the same schedule as rituximab at patient-specific doses. No patients discontinued therapy due to a treatment-related AE.

Patients in the N-803 intravenous cohort encountered grade 1 to 2 AEs, including a high frequency of fever and chills, typically occurring within 4 hours of the intravenous injection that responded to supportive care measures (Table 2). Correlative laboratory analysis of the pharmacokinetics of N-803 revealed that the intravenous cohort patients had an early high C_max with near complete clearance within 24 hours (Supplementary Fig. S1A). This coincided with elevations in both serum IFNγ and IL6, suggesting that intravenous N-803 was triggering acute cytokine release, responsible for the fever and rigors. Although these were not dose limiting, they were concerning for prolonged clinical dosing schedules, and a second cohort was initiated via the subcutaneous route. Patients who received subcutaneous N-803 uniformly experienced an injection site reaction consisting of nonpainful erythema, warmth, and edema, which peaked at 7 to 10 days postinjection, and resolved with minimal supportive measures by 2 weeks. Patients also experienced fevers and chills, but these were transient and occurred 2 to 3 days after the subcutaneous injection, resolving with supportive measures. Consistent with this change in constitutional symptoms based on N-803 route of administration, pharmacokinetic analysis revealed a lower C_max and prolonged serum concentration of N-803 following subcutaneous administration, with minimal elevations in IFNγ and IL6 (Supplementary Fig. S1B). Grade 3 nonhematologic toxicities in the subcutaneous cohort were transient and included fever (N = 1), hypertension (N = 2), and hyperglycemia (N = 1). Overall, the combination of rituximab and N-803 was well tolerated, without treatment-related grade 4 or 5 AEs (Table 2). Immunogenicity testing was also performed on days 1, 22, and week 11 and demonstrated no evidence of anti–N-803 antibodies developing in these patients at all timepoints (Supplementary Table S3). On the basis of the dose escalation and pharmacokinetics, the RP2D was 15 or 20 μg/kg via the subcutaneous route.

Overall response rate (ORR) for the combined intravenous and subcutaneous cohorts across all doses and CD20 mAb refractory status was 57% (12/21), with 44% (4/9) in the IV and 67% (8/12) in the subcutaneous cohort. The majority of patients experienced reductions in the size of their lymph nodes (Fig. 1B). For patients with anti-CD20 mAb-sensitive disease, the ORR in the intravenous cohort was 43% (3/7) and in the subcutaneous cohort was 78% (7/9). In the subcutaneous cohort, seven of seven (100%) responses were complete remissions (CR). Moreover, patients in the subcutaneous cohort had prolonged stable disease (SD) and conversion of SD and/or partial response (PR) to CR with a prolonged duration without progression (eight of 12 patients without progression at 18 to 24 months; Fig. 1C). In the subcutaneous cohort, six of seven CRs occurred at either the first (11 weeks) or second evaluation (40 weeks). In the intravenous cohort, CR responses occurred following the second evaluation and as late as 18 months follow-up. For the five patients with anti-CD20 mAb refractory disease in both intravenous and subcutaneous cohorts, the ORR was two of five (40%) with one CR, one PR, one SD, and two PD (Fig. 1C; Supplementary Fig. S1C). The PR and SD are ongoing in the subcutaneous patients at > 18 months (Fig. 1C). The subcutaneous cohort patient with PD was highly refractory, having received five prior lines of therapy.

Here, we focused our correlative immunology on patients treated with 15 and 20 μg/kg subcutaneous of N-803 because they had a similar pharmacokinetic profile (Supplementary Fig S1B) and relevant as the RP2D. Weekly subcutaneous N-803 combined with rituximab resulted in a marked expansion in the frequency of total PB NK cells as assessed using multidimensional mass cytometry (Fig. 1D–F), with no significant change observed in the overall frequency of CD4⁺ or CD8⁺ T cells. The substantial increase in PB NK cells was confirmed using flow cytometry (Fig. 2A–E). This increase occurred early (days 5–15 of therapy) and was maintained throughout the 4 weeks of induction (days 22–27). Absolute NK cell counts remained elevated at D78, 8 weeks after the last N-803 dose (Fig. 2E). NK cell proliferation was increased, with ≥95% of NK cells expressing the cell-cycle transit marker Ki-67 after the first dose of N-803 (Fig. 2F–H). This proliferative effect was maintained throughout induction as well, with Ki-67⁺ NK cells >50% when assessed at days 15 and 21 (prior to N-803 injections). The response of NK cells to N-803 was examined 8 weeks after induction at the time of the first consolidation therapy. Here, N-803 induced increases in Ki-67⁺ NK cells, comparable to the first dose of N-803, suggesting a prolonged administration schedule was feasible (Fig. 2I). As an indicator of functionality, significant upregulation of the cytotoxic protein granzyme B was observed following N-803 plus rituximab therapy (Fig. 2J).

To provide a multidimensional assessment of NK cells, the mass cytometry data were analyzed gating on the NK cell viSNE islands. NK cells demonstrated a marked change in their multidimensional phenotype at days 8 and 22 (Fig. 2K), which was driven by the activation marker CD38, and activating receptors NKp30 and NKp44 (Fig. 2L and M), and EOMES, a T-box transcription factor associated with NK cell functionality (27–29). Thus, N-803 in the presence of a therapeutic mAb resulted in a sustained expansion and alteration of the NK cell activation state following therapy.

We hypothesized that N-803 would also result in changes in the CD8⁺ T-cell compartment. A modest increase in absolute CD8⁺ T-cell numbers were observed at the later timepoints of N-803 induction and maintained 8 weeks after induction therapy at D78, while no change was observed in CD8⁺ T-cell percentage (Fig. 3A and B). In addition, subsets of CD8⁺ T cells expressed Ki-67, at both early and later timepoints of N-803 induction, although at a lower peak frequency (∼50%) than in NK cells (Fig. 3C–E; Fig. 2G and H). Using mass cytometry and viSNE to gate on major T-cell subsets within PB lymphocytes, no changes in the overall frequency of naïve (T_N), effector (T_E), and central memory (T_CM) CD8⁺ T cells were observed as defined using FlowSOM clustering, however a subset of effector memory (T_EM) (MC2) was increased after N-803 administration (Fig. 3F). This metacluster was defined by high Ki67, CD38, HLA-DR, and TIM-3 expression along with intermediate expression of PD-1 (P < 0.05) compared with the other T_EM subsets (Fig. 3G,–I). However, by day 22, the CD8⁺ T-cell profile had returned to a pretherapy phenotype.

Next, we examined the impact of N-803 plus rituximab therapy on CD4⁺ T cells. The frequency of CD4⁺ T cells did not increase over the course of N-803 induction therapy; however, there was a minimal but significant increase in overall CD4⁺ T-cell numbers which persisted at D78 (Supplementary Fig. S2A–S2D). This was accompanied by a minor increase in the percentage of Ki67⁺ CD4⁺ T cells with a mean peak of 22% at D2–15 (Supplementary Fig. S2E and S2F). Clustering analysis on the CD4⁺ T cells in mass cytometry, revealed no significant changes in the FlowSOM identified metaclusters, indicating minimal phenotypic changes with N-803 and rituximab (Supplementary Fig. S2G). There was a small increase in the frequency of CD4⁺ Tregs during the early phase of induction and immediately following the first dose of N-803 in consolidation, and a modest increase in Ki-67⁺ Tregs during the early phase of induction, but no change in absolute Treg numbers (Supplementary Fig. S2H–S2L). Thus, while modest and of unclear biological significance, activation of a small fraction of CD4⁺ T cells was observed with N-803, consistent with established IL15R biology.

To better understand the impact of N-803 plus rituximab, we performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq; ref. 20) using a custom antibody panel designed for NK cells, which measures protein using barcode-tagged antibodies (ADTs) while simultaneously performing single-cell RNA sequencing (scRNA-seq) on PBMC samples at day 1 (pre-N-803, n = 3), day 8 (n = 2), and day 15 (n = 3; Fig. 4A; Supplementary Fig. S3A–S3C). Distinct and shared impact of N-803 plus rituximab on NK-cell subsets was evident (Fig. 4B–C; Supplementary Fig. S3D). In CD56^bright NK cells, a significant enrichment in Gene Ontology (GO) Biological Processes (BP) related to NFκB and MAPK signaling, cytotoxicity, cell adhesion, and proliferation was observed, while CD56^dim NK cells were enriched for leukocyte activation and both NK subsets were enriched for cytokine-related BP (Supplementary Fig. S3D). These changes in BP corresponded to increased expression of granzymes (GZM) A, B, K, and H, and perforin (PRF1), and the T-box transcription factor, T-BET (TBX21) in CD56^bright NK cells, providing evidence of in vivo priming (Fig. 4B). CD56^dim NK cells had increased expression of chemotaxis genes (CCL3, CXCR4), FOS and CEBPB transcription factors, and IFN-inducible genes (IFITM3, IFI6; Fig. 4C). In agreement with our cytometry data, we observed increased CD38 RNA (Fig. 4B and C) and protein (Supplementary Fig. S3E). NK cell molecular changes were evident by D8 with no further differentially expressed genes in CD56^bright and only two differentially expressed genes in CD56^dim (CCL4L2, AREG) between day 8 and day 15, suggesting that N-803 alters the NK transcriptome after the first dose.

Identification of KIR⁺ and terminally mature (CD57⁺) CD56^dim NK-cell subsets with scRNA-seq has been hindered by high transcript drop-out rate of current technologies, resulting in poor enrichment for CD57 (B3GAT1) and KIRs. To address this, we performed similarity network fusion implemented in CiteFuse (24, 30), reclustering NK cells using both protein and RNA (Fig. 4D and E). Unsupervised clustering revealed increased heterogeneity of CD56^dim NK, but only one cluster of CD56^bright NK, accurately recapitulating known biology of NK cell maturation (Fig. 4D and 4F; Supplementary Figs. S3F and S3G and S4A and S4B; ref. 31). In addition, CiteFuse uncovered a conserved decrease in CD57⁺ NK cells, as well as patient-specific changes in CD56^dim NK cell subsets, which may represent a repair of a dysfunctional CD56^dim compartment in patient 027-016 (Supplementary Fig. S4A). Transcriptional changes were enriched in subsets of CD56^dim NK cells, including upregulation of AP-1 subunits JUN and FOS, and CD69 (Fig. 4G; Supplementary S4C and S4D), suggesting that N-803 and rituximab may preferentially activate specific CD56^dim NK cell subsets in vivo. In addition, IL15 pathway gene expression changes were driven by only one patient indicating that N-803 may have both shared and individual responses (Supplementary Fig. S4D).

Evidence of augmented HLA-DR and CD38 was found in CD8⁺ T_EM, consistent with mass cytometry findings (Supplementary Fig. S4E and S4F). In γδ T cells, a significant increase in the expression of cytotoxicity genes (GNLY, GZMB, GZMH, NKG7), transcription factors (TBX21, JUN), secreted proteins (FGFBP2, CCL5), and MHC Class II (HLA-DRB1, HLA-DPA1, CD74), were observed at D15 (Fig. 4H). In the monocyte compartment, striking shifts in the tSNE space of CD14 monocytes was accompanied by a significant decrease in the proportion of CD14 monocytes (Supplementary Fig. S4G). Similarly, a significant decrease in CD16⁺ monocytes was evident (Fig. 4I). Gene expression changes driving this marked shift included increased type 1 IFN and IFNγ-inducible genes (Supplementary Fig. S4H; Fig. 4J), humoral immune responses, complement activation (C1QA, C1QB, C1QC), antigen processing and presentation (HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, CD74), and antibody-responses (FCGR1A; Fig. 4J).

Here we report the first-in-human clinical trial of an IL15R agonist in combination with a tumor-targeting mAb. Administration of subcutaneous N-803 with rituximab had an excellent safety profile and demonstrated prolonged PFS and a 78% CR rate in rituximab-sensitive patients. Correlative immunology in the PB revealed distinct activation signatures of CD56^bright and CD56^dim NK cells, including priming of CD56^bright NK cells, along with increased NK proliferation which contrasts with previous study following single-agent rituximab (32). Within the T-cell compartment, CD8⁺ T_EM exhibited marked but transient activation and expansion characterized by high expression of CD38 and HLA-DR. Using scRNA-seq, we observed γδ T-cell activation along with a profound change in monocytes and enhanced antigen presentation and type I IFN genes. These studies support the mechanism of action of N-803 as an activator of NK and CD8⁺ T cells, and provide insights into the potential for broad immune modulation with this novel combination therapy.

As intravenous rhIL15 promotes NK and CD8⁺ T-cell proliferation, but results in unacceptable AE, several IL15R agonist drugs have since been developed and translated into the clinic to mitigate these barriers (6). N-803 has been the pioneer in this class of drugs, and was proven to have a prolonged in vivo half-life, while maintaining the ability to stimulate NK and CD8⁺ T cells (11, 33, 34), and is currently being investigated in several cancer types including NSCLC where it has demonstrated efficacy in difficult to treat patient populations and an excellent safety profile (35). Here we report that subcutaneous administration of N-803 is well tolerated, and identify 15 to 20 μg/kg N-803 as the RP2D doses for future study in this clinical setting (12, 35, 36). Clinical responses were observed in 78% of subcutaneous rituximab-sensitive patients warranting further clinical study. In comparison, single-agent rituximab has an ORR of 40% to 53% and a CR rate of 11% to 18% (26, 37, 38). We observed responses in two of five rituximab-refractory patients, further supporting the importance of N-803 in the observed responses. It is intriguing to postulate that N-803 may restore iNHL responses when used in conjunction with a tumor targeting mAb, which should be addressed in larger phase II trial cohorts.

N-803 is currently being investigated in several cancer clinical trials as a combination therapy including with gemcitabine and in combination with adoptive NK cell therapy. On the basis of this study, broad testing of N-803 in concert with cancer-targeting mAbs is warranted. Thus, this class of IL15R agonists has the potential to transform immunotherapy for multiple cancer types.

Distinct from the focused monitoring of previously reported N-803 trials (12, 35), we applied scRNA-seq to comprehensively define N-803 and rituximab mediated alterations of immune cell molecular programs revealing unexpected immune modulation. For example, we demonstrate activation of antigen-presentation genes in CD16 monocytes and type 1 IFN and IFNγ family genes, suggesting that immune cross-talk between NK cells, T cells, and monocytes occur. Furthermore, the decrease in blood monocytes following therapy coupled with the increased expression of FCGR1A, may be indicative of trafficking to the site of the lymphoma where the monocytes mediate antibody-directed effects and T-cell activation. The enhanced antigen presentation program and MHC class II expression suggests potential utility as an adjunct to vaccines. These findings suggest that N-803 may simultaneously enhance innate and adaptive immune responses to lymphoma. Future studies correlating the magnitude of immune changes within monocytes, T cells, and NK cells with clinical response will be informative to understanding the mechanisms behind N-803 and rituximab-mediated clinical outcomes.

Consistent with previous work on single-agent N-803 post-HCT (12), NK cells exited the circulation immediately following N-803 treatment, and then rapidly expanded with sustained Ki67 expression. Previous work on N-803 has demonstrated increased NK-cell localization in the lymph node shortly after N-803 administration, providing a potential explanation for the transient decrease in NK cells observed in this study (39). Future studies will be of interest to identify the prevalence and phenotypic changes of immune cells within the lymph nodes following N-803 and anti-CD20 mAbs, particularly as this relates to lymphoma immune surveillance. Our findings add to prior work on N-803 demonstrating here that NK and CD8⁺ T-cell total counts remain elevated 8 weeks after N-803 treatment, suggesting a prolonged effect of N-803 on the immune milieu in patients with lymphoma. This finding may represent restoration of blood lymphocyte counts by N-803, as lymphopenia is frequently present in patients with lymphoma and is adversely correlated with poor clinical outcome (40).

Using scRNA-seq profiling, we find cytotoxic effector molecules increased (GZMA, GZMB, GZMK, GZMH, PRF1, GNLY) in CD56^bright NK cells consistent with previous in vitro reports on IL15 priming of CD56^bright NK cells. These findings stand in contrast to previous work in single-agent rituximab-treated patients with lymphoma, where rituximab-treated patients had decreased NK cell counts; however, future in depth studies comparing the effect of single-agent anti-CD20 mAbs and N-803 are required to better elucidate the contribution of each therapy and are being accrued as part of the larger ongoing phase II trial (32).

Furthermore, CITE-seq revealed a significant change in the transcription factor profile of N-803 expanded NK cells, with AP-1 member, JUN, and CEBPB significantly increased across most NK clusters. These data lead to important, novel molecular characterization of NK cells in vivo, and generate hypotheses related to key molecular changes that occur following IL15 administration. The addition of cell-surface protein to scRNA-seq data in CITE-seq overcomes many of the current technical limitations of scRNA-seq for NK cells, facilitating the identification of CD57⁺ NK cells as a subset significantly decreased following treatment, potentially due to N-803–induced proliferation of other NK cell subsets, as CD57⁺ NK cells are reported to have low proliferative potential (41). Furthermore, CiteFuse revealed increased heterogeneity in the N-803 and rituximab changes in CD56^dim NK cell populations but not CD56^bright, indicating that CD56^dim NK cell heterogeneity is not captured by scRNA-seq alone. In addition, we observed increased differences in the proportion of CD56^dim clusters across patients, likely driven by known individual differences in KIR repertoires (42, 43). The CITE-seq approach will be critical for comprehensively evaluating NK cells at the single-cell level in response to immunotherapy.

Here we report preferential activation of CD8⁺ T_EM subsets consistent with prior publications in humans and macaques (12, 44, 45). This effect may be due to the increased sensitivity of human CD8⁺ T_EM to IL15, consistent with increased IL15 receptor expression on T_EM. (45) In contrast, in mice, IL15 activates and expands T_CM to a greater degree than T_EM, likely due to increased expression of CD122 on murine T_CM. (46, 47) Together, these studies suggest a gradient of IL15 responsiveness across T-cell subsets driving IL15 activation and proliferation responses.

Here, we demonstrate that a new combination immunotherapy of the IL15R agonist N-803, and the tumor-targeting antibody, rituximab, is safe and shows preliminary clinical activity in a phase I trial of rel/ref patients with iNHL. Using multi-dimensional mass cytometry and CITE-seq, we define protein and transcriptional changes, demonstrating remodeling of iNHL patient immune landscapes by promotion of an activation signature in nearly all main immune cell lineages including NK cells, CD8⁺ T_EM, γδ T cells, and CD14 and CD16 monocytes. These findings warrant additional investigations on the efficacy of N-803 and anti-CD20 mAbs in ongoing trials for iNHL (NCT02384954) and the testing of N-803 in combination with other therapeutic antibodies in additional cancers. Extended studies on the long-term effects of N-803 and anti-CD20 mAbs on the NHL immune environment are currently underway as part of the phase II trial.

J.A. Foltz reports grants from the American Association of Immunologists during the conduct of the study; grants from NIH T32HL007088 outside the submitted work; in addition, J.A. Foltz has a patent for USPTO 16/966,367 and WO 2019/152387 A1 pending, licensed, and with royalties paid from Kiadis and a patent for US 63/018,108 pending, licensed, and with royalties paid from Kiadis; and canine antibody licensed to EMD Millipore. B.T. Hess reports personal fees from Bristol Meyers Squibb, ADC Therapeutics, and AstraZeneca outside the submitted work. V. Bachanova reports grants from ALTOR Bioscience during the conduct of the study; grants and other from Gamida Cell; grants from Incyte; other from Karyopharma and Kite outside the submitted work. N.L. Bartlett reports other from ADC Therapeutics, Roche/Genentech, Seattle Genetics, BTG, and Acerta; grants from Affimed, Gilead, Immune Design, and Pfizer outside the submitted work. M.M. Berrien-Elliott reports personal fees from WUGEN outside the submitted work. B. Kahl reports grants and personal fees from Genentech and Roche during the conduct of the study. N. Mehta-Shah reports personal fees from Kyowa Hakka Kirin, Ono Pharmaceuticals, Karyopharm, Secura Bio, and Daiichi Sankyo outside the submitted work; and also receives institutional research funding from Celgene, Bristol Myers-Squibb, Genentech/Roche, Innate Pharmaceuticals, Corvus Pharmaceuticals, and Verastem/Secura Bio. N. Epperla reports other from Verastem, Beigene, Karyopharm, and Genzyme outside the submitted work. A.D. Rock reports other from ImmunityBio during the conduct of the study. J. Lee reports other from immunitybio during the conduct of the study; other from immunitybio outside the submitted work; and during the time of the work for this publication J. Lee was employed by immunitybio as the person responsible for collaboration. J. Lee helps work in study design, review and editing of manuscript. P. Soon-Shiong reports other from ImmunityBio during the conduct of the study; in addition, P. Soon-Shiong has a patent for ImmunityBio pending, issued, and licensed to ImmunityBio. T.A. Fehniger reports grants and other from ImmunityBio and Altor BioScience during the conduct of the study; personal fees from Nektar, Wugen, personal fees from Kiadis; other from Indpata, Orca Bio; grants from Affimed, and other from Compass Therapeutics outside the submitted work; and not directly relevant to this work, T.A. Fehniger has equity interest, consulting, and royalty interest in Wugen. This includes intellectual property with T.A. Fehniger as a co-inventor, licensed to Wugen from Washington University not related to this work. No disclosures were reported by the other authors.

J.A. Foltz: Data curation, formal analysis, funding acquisition, validation, investigation, visualization, methodology, writing–original draft, writing–review and editing. B.T. Hess: Data curation, investigation, writing–review and editing. V. Bachanova: Data curation, investigation, writing–review and editing. N.L. Bartlett: Data curation, investigation, writing–review and editing. M.M. Berrien-Elliott: Conceptualization, formal analysis, investigation, visualization, methodology, writing–review and editing. E. McClain: Data curation, investigation, writing–review and editing. M. Becker-Hapak: Data curation, formal analysis, investigation, writing–review and editing. M. Foster: Data curation, investigation, writing–review and editing. T. Schappe: Data curation, investigation, writing–review and editing. B. Kahl: Data curation, investigation, writing–review and editing. N. Mehta-Shah: Data curation, investigation, writing–review and editing. A.F. Cashen: Data curation, investigation, writing–review and editing. N.D. Marin: Formal analysis, writing–review and editing. K. McDaniels: Data curation, investigation, writing–review and editing. C. Moreno: Data curation, investigation, writing–review and editing. M. Mosior: Resources, data curation, methodology, writing–review and editing. F. Gao: Formal analysis, validation, investigation, methodology, writing–review and editing. O.L. Griffith: Supervision, methodology, writing–review and editing. M. Griffith: Supervision, methodology, writing–review and editing. J.A. Wagner: Data curation, investigation, writing–review and editing. N. Epperla: Data curation, investigation, writing–review and editing. A.D. Rock: Conceptualization, supervision, methodology, writing–review and editing. J. Lee: Conceptualization, supervision, methodology, writing–review and editing. A.A. Petti: Formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing–review and editing. P. Soon-Shiong: Supervision, writing–review and editing. T.A. Fehniger: Conceptualization, supervision, funding acquisition, methodology, writing–original draft, writing–review and editing.

We would like to acknowledge the patients and clinical lymphoma teams for making this study possible. We would like to acknowledge the Siteman Flow Cytometry (Bill Eades), Immune Monitoring Lab (Stephen Oh), and McDonnell Genome Institute (Catrina Fronick, Bob Fulton, Alex Paul).

The clinical trial was supported by R44 CA195812 and ImmunityBio. NIH T32 HL007088 (to J.A. Wagner, J.A. Foltz), SPORE in Leukemia P50CA171963 (to M.M. Berrien-Elliott, A.F. Cashen, T.A. Fehniger), K12CA167540 (to M.M. Berrien-Elliott), R01CA205239 (to T.A. Fehniger), American Association of Immunologists Intersect Fellowship Program for Computational Scientists and Immunologists (to J.A. Foltz, A.A. Petti, T.A. Fehniger); MGI Pilot Grant (to T.A. Fehniger), Jamie Erin Follicular Lymphoma Research Fund (to T.A. Fehniger). This work was also supported by the NCI CCSG P30 C091842 (Siteman Cancer Center).

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