<div>AbstractPurpose:<p>Natural killer (NK)-cell recognition and function against NK-resistant cancers remain substantial barriers to the broad application of NK-cell immunotherapy. Potential solutions include bispecific engagers that target NK-cell activity via an NK-activating receptor when simultaneously targeting a tumor-specific antigen, as well as enhancing functionality using IL12/15/18 cytokine pre-activation.</p>Experimental Design:<p>We assessed single-cell NK-cell responses stimulated by the tetravalent bispecific antibody AFM13 that binds CD30 on leukemia/lymphoma targets and CD16A on various types of NK cells using mass cytometry and cytotoxicity assays. The combination of AFM13 and IL12/15/18 pre-activation of blood and cord blood–derived NK cells was investigated <i>in vitro</i> and <i>in vivo</i>.</p>Results:<p>We found heterogeneity within AFM13-directed conventional blood NK cell (cNK) responses, as well as consistent AFM13-directed polyfunctional activation of mature NK cells across donors. NK-cell source also impacted the AFM13 response, with cNK cells from healthy donors exhibiting superior responses to those from patients with Hodgkin lymphoma. IL12/15/18-induced memory-like NK cells from peripheral blood exhibited enhanced killing of CD30<sup>+</sup> lymphoma targets directed by AFM13, compared with cNK cells. Cord-blood NK cells preactivated with IL12/15/18 and <i>ex vivo</i> expanded with K562-based feeders also exhibited enhanced killing with AFM13 stimulation via upregulation of signaling pathways related to NK-cell effector function. AFM13–NK complex cells exhibited enhanced responses to CD30<sup>+</sup> lymphomas <i>in vitro</i> and <i>in vivo</i>.</p>Conclusions:<p>We identify AFM13 as a promising combination with cytokine-activated adult blood or cord-blood NK cells to treat CD30<sup>+</sup> hematologic malignancies, warranting clinical trials with these novel combinations.</p></div>
<div>AbstractPurpose:<p>N-803 is an IL15 receptor superagonist complex, designed to optimize <i>in vivo</i> persistence and trans-presentation, thereby activating and expanding natural killer (NK) cells and CD8<sup>+</sup> 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.</p>Patients and Methods:<p>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.</p>Results:<p>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.</p>Conclusions:<p>N-803 combines with mAbs to enhance tumor targeting in patients, and warrants further investigation in combination with immunotherapies.</p></div>
Establishing peripheral CD8+ T cell tolerance is vital to avoid immune mediated destruction of healthy self-tissues. However, it also poses a major impediment to tumor immunity since tumors are derived from self-tissue and often induce T cell tolerance and dysfunction. Thus, understanding the mechanisms that regulate T cell tolerance versus immunity has important implications for human health. Signals received from the tissue environment largely dictate whether responding T cells become activated or tolerant. For example, induced expression and subsequent ligation of negative regulatory receptors on the surface of self-reactive CD8+ T cells are integral in the induction of tolerance. We utilized a murine model of T cell tolerance to more completely define the molecules involved in this process. We discovered that, in addition to other known regulatory receptors, tolerant self-reactive CD8+ T cells distinctly expressed the surface receptor neuropilin-1 (Nrp1). Nrp1 was highly induced in response to self-antigen, but only modestly when the same antigen was encountered under immune conditions, suggesting a possible mechanistic link to T cell tolerance. We also observed a similar Nrp1 expression profile on human tumor infiltrating CD4+ and CD8+ T cells. Despite high expression on tolerant CD8+ T cells, our studies revealed that Nrp1 had no detectable role in the tolerant phenotype. Specifically, Nrp1-deficient T cells displayed the same functional defects as wild-type self-reactive T cells, lacking in vivo cytolytic potential, IFNγ production, and antitumor responses. While reporting mostly negative data, our findings have therapeutic implications, as Nrp1 is now being targeted for human cancer therapy in clinical trials, but the precise molecular pathways and immune cells being engaged during treatment remain incompletely defined.
Abstract Natural killer (NK) cells are innate lymphoid cells critical for host defense against viral infection and malignant transformation. NK cells exhibit innate immunologic memory in response to specific haptens, viruses, or combined cytokine pre-activation. Human cytokine-induced memory-like NK cells, generated by overnight pre-activation with IL-12, IL-15, and IL-18, respond more robustly to numerous stimuli (including leukemia target cells) for weeks to months following the initial activation. The mechanisms responsible for the enhanced effector function of human memory-like NK cells are poorly understood. We hypothesized that memory-like NK cell differentiation alters the balance of signal integration downstream of activating and inhibitory receptors. NK cells achieve functional competence through self-MHC class I interactions (licensing) during maturation; if this does not occur, NK cells remain anergic. We tested this hypothesis using normal donors with licensed and unlicensed NK cell populations. We observed that cytokine pre-activation increases IFN-γ production by both licensed and unlicensed NK cells in response to tumor targets, activating receptor (FcγRIIIa) ligation, or cytokine stimulation. In fact, in response to cytokine stimulation, memory-like NK cells produce significantly more IFN-γ than controls with no impact of licensing. We observed no difference in the expression level of several signaling adaptor molecules or in phospho-signaling downstream of FcγRIIIa ligation in memory-like vs. control NK cells. We are currently investigating epigenetic modification of the IFN-γ gene locus in memory-like NK cells as a potential mechanism for their enhanced function; updated results will be presented.
Abstract Purpose: Treatment of advanced melanoma is a clinical challenge. Natural killer (NK) cells are a promising cellular therapy for T cell–refractory cancers, but are frequently deficient or dysfunctional in patients with melanoma. Thus, new strategies are needed to enhance NK-cell antitumor responses. Cytokine-induced memory-like (ML) differentiation overcomes many barriers in the NK-cell therapeutics field, resulting in potent cytotoxicity and enhanced cytokine production against blood cancer targets. However, the preclinical activity of ML NK against solid tumors remains largely undefined. Experimental Design: Phenotypic and functional alterations of blood and advanced melanoma infiltrating NK cells were evaluated using mass cytometry. ML NK cells from healthy donors (HD) and patients with advanced melanoma were evaluated for their ability to produce IFNγ and kill melanoma targets in vitro and in vivo using a xenograft model. Results: NK cells in advanced melanoma exhibited a decreased cytotoxic potential compared with blood NK cells. ML NK cells differentiated from HD and patients with advanced melanoma displayed enhanced IFNγ production and cytotoxicity against melanoma targets. This included ML differentiation enhancing melanoma patients' NK-cell responses against autologous targets. The ML NK-cell response against melanoma was partially dependent on the NKG2D- and NKp46-activating receptors. Furthermore, in xenograft NSG mouse models, human ML NK cells demonstrated superior control of melanoma, compared with conventional NK cells. Conclusions: Blood NK cells from allogeneic HD or patients with advanced melanoma can be differentiated into ML NK cells for use as a novel immunotherapeutic treatment for advanced melanoma, which warrants testing in early-phase clinical trials.
Abstract Follicular lymphoma (FL) is the most common indolent non-Hodgkin lymphoma and although often responsive to treatment remains incurable with conventional therapies. Approximately 30% of patients will undergo transformation to a more aggressive, clonally related malignancy (t-FL). Our understanding of the genomic landscape of FL is limited when compared to other hematologic malignancies such as acute myeloid leukemia or diffuse large B-cell lymphoma. To better understand the FL genomic landscape and identify novel recurrent mutations, we performed exome sequencing on fresh frozen samples from a discovery cohort of 24 patients with FL prior to treatment (12), FL relapsed after prior therapy (6) or t-FL (6) with paired normal skin samples. From this analysis, we identified 898 genes harboring mutations. We combined these results with a list of 819 recurrently mutated genes from 10 sequencing studies of other B-cell malignancies to develop a custom capture reagent targeting the coding regions and UTRs of 1717 genes. We applied this reagent to the initial discovery samples and an additional 81 FFPE tumor samples from patients with FL (80) or t-FL (1), achieving >20x coverage for >75% of the targeted region. Through this approach, we confirmed previously described mutations in chromatin/epigenetic modifiers (MLL2 [60%], CREBBP [55%], EP300 [19%], EZH2 [17%], MEF2B [7.6%]), histones linkers (HIST1H1C/E [21%]), transcription factors (IRF8 [13%], STAT6 [12%]), and the BCR signaling pathway (CD79B [5.7%], CARD11 [11.4%], BCL10 [2.8%], TNFAIP3 [2.8%]). Additionally, we identified mutations in novel genes within the previously implicated BCR pathway (CD22 [3.8%], BTK [8.6%], HVCN1 [7.6%]) and multiple hits in complexes not previously described in association with FL such as the Swi/Snf nucleosome remodeling complex (ARID1A/B [8.6%], SMARCA4 [4.8%], SMARCB1 [1.9%], BCL7A [21%], BCL11A [1.9%], PBRM1 [1.9%], DPF1 [1%], ACTB [1%]), hotspot mutations in vacuolar ATPases (ATP6V1B2 [8.6%], VMA21 [4.8%]) and others (EGR1/2 [6.7%], POU2AF1 [6.7%]). Though many of these mutations have been independently reported in patients, those studies lacked sufficient size to evaluate their statistical significance. In contrast, the strength of our approach is illustrated in our finding of 54 genes significantly mutated above background mutation rates (FDR<0.15). Clinical outcomes and treatment regimen data were available for 100 patients, and as expected, the FLIPI score was predictive of PFS. Notably, improved PFS was observed in treated patients harboring novel HVCN1 mutations (p<0.05). In contrast, CREBBP mutations were associated with reduced PFS (p<0.05). Although larger cohort sizes are required to more robustly associate clinical outcomes and mutation profiles, we have identified novel recurrent mutations and pathways in FL. Such discoveries are key to understanding the etiology of and the development of novel therapeutic approaches for FL. Citation Format: Kilannin Krysiak, Felicia Gomez, Brian S. White, Matthew Matlock, Chris A. Miller, Robert S. Fulton, Friederike Kreisel, Amanda F. Cashen, Kenneth R. Carson, Melissa M. Berrien-Elliott, Nancy L. Bartlett, Richard K. Wilson, Elaine R. Mardis, Malachi Griffith, Obi L. Griffith, Todd A. Fehniger. Identification of novel recurrent mutations in follicular lymphoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-326.
Natural killer (NK) cells are innate lymphoid cells specialized to eliminate malignant cells via direct cytotoxicity and immunoregulatory cytokine production. As such, NK cells are ideal as cellular therapy for cancer patients, and several studies have provided proof of principle that adoptively transferred NK cells can induce remissions in patients with leukemia. A clear understanding of the mechanisms underlying NK cell antitumor responses, including target cell recognition, activation status, and negative regulatory signals will improve NK cellular therapy for cancer patients.Clinical studies have demonstrated the safety and preliminary efficacy of NK cell adoptive transfer, especially in hematologic malignancies. Various NK cell sources, isolation techniques, activation approaches, and ex-vivo expansion strategies are under investigation. New approaches have been developed and are being tested to optimize NK cell therapy, including ways to better target NK cells to malignant cells, increase their functional competence, facilitate expansion in patients, and limit inhibitory signals or cells.NK cells represent a promising cellular immunotherapy for the treatment of cancer. In addition to adoptive cellular therapy, adjunct treatments that optimize NK cell targeting and function will enhance their potency and broaden their potential use to many cancer types.
