Natural killer cell therapies.

Natural killer (NK) cells are lymphocytes of the innate immune system. A key feature of NK cells is their ability to recognize a wide range of cells in distress, particularly tumour cells and cells infected with viruses. They combine both direct effector functions against their cellular targets and participate in the generation, shaping and maintenance of a multicellular immune response. As our understanding has deepened, several therapeutic strategies focused on NK cells have been conceived and are currently in various stages of development, from preclinical investigations to clinical trials. Here we explore in detail the complexity of NK cell biology in humans and highlight the role of these cells in cancer immunity. We also analyse the harnessing of NK cell immunity through immune checkpoint inhibitors, NK cell engagers, and infusions of preactivated or genetically modified, autologous or allogeneic NK cell products.

On blood and tissue-resident natural killer cells.

Conventional natural killer (cNK) cells patrol the organism via circulation and invade tissues in response to infection or inflammation. In this issue of Immunity, Torcellan et al. report that circulating cNK cells are recruited into infected skin and differentiate into long-lived tissue-resident NK cells capable of mediating an accelerated response upon reinfection.

New immune cell engagers for cancer immunotherapy.

There have been major advances in the immunotherapy of cancer in recent years, including the development of T cell engagers - antibodies engineered to redirect T cells to recognize and kill cancer cells - for the treatment of haematological malignancies. However, the field still faces several challenges to develop agents that are consistently effective in a majority of patients and cancer types, such as optimizing drug dose, overcoming treatment resistance and improving efficacy in solid tumours. A new generation of T cell-targeted molecules was developed to tackle these issues that are potentially more effective and safer. In addition, agents designed to engage the antitumour activities of other immune cells, including natural killer cells and myeloid cells, are showing promise and have the potential to treat a broader range of cancers.

Germinal center-dependent and -independent immune responses of tumor-infiltrating B cells in human cancers.

B cells play essential roles in immunity, mainly through the production of high affinity plasma cells (PCs) and memory B (Bmem) cells. The affinity maturation and differentiation of B cells rely on the integration of B-cell receptor (BCR) intrinsic and extrinsic signals provided by antigen binding and the microenvironment, respectively. In recent years, tumor infiltrating B (TIL-B) cells and PCs (TIL-PCs) have been revealed as important players in antitumor responses in human cancers, but their interplay and dynamics remain largely unknown. In lymphoid organs, B-cell responses involve both germinal center (GC)-dependent and GC-independent pathways for Bmem cell and PC production. Affinity maturation of BCR repertoires occurs in GC reactions with specific spatiotemporal dynamics of signal integration by B cells. In general, the reactivation of high-affinity Bmem cells by antigens triggers GC-independent production of large numbers of PC without BCR rediversification. Understanding B-cell dynamics in immune responses requires the integration of multiple tools and readouts such as single-cell phenotyping and RNA-seq, in situ analyses, BCR repertoire analysis, BCR specificity and affinity assays, and functional tests. Here, we review how those tools have recently been applied to study TIL-B cells and TIL-PC in different types of solid tumors. We assessed the published evidence for different models of TIL-B-cell dynamics involving GC-dependent or GC-independent local responses and the resulting production of antigen-specific PCs. Altogether, we highlight the need for more integrative B-cell immunology studies to rationally investigate TIL-B cells as a leverage for antitumor therapies.

Myc controls NK cell development, IL-15-driven expansion, and translational machinery.

MYC is a pleiotropic transcription factor involved in cancer, cell proliferation, and metabolism. Its regulation and function in NK cells, which are innate cytotoxic lymphocytes important to control viral infections and cancer, remain poorly defined. Here, we show that mice deficient for Myc in NK cells presented a severe reduction in these lymphocytes. Myc was required for NK cell development and expansion in response to the key cytokine IL-15, which induced Myc through transcriptional and posttranslational mechanisms. Mechanistically, Myc ablation in vivo largely impacted NK cells' ribosomagenesis, reducing their translation and expansion capacities. Similar results were obtained by inhibiting MYC in human NK cells. Impairing translation by pharmacological intervention phenocopied the consequences of deleting or blocking MYC in vitro. Notably, mice lacking Myc in NK cells exhibited defective anticancer immunity, which reflected their decreased numbers of mature NK cells exerting suboptimal cytotoxic functions. These results indicate that MYC is a central node in NK cells, connecting IL-15 to translational fitness, expansion, and anticancer immunity.

NKG2A blocks the anti-metastatic functions of natural killer cells.

Pancreatic ductal adenocarcinoma (PDAC)-derived liver metastasis represents a major unmet medical need. Liu et al. show that circulating tumor cells (CTCs) from the hepatic portal vein (HPV), and not from primary or metastatic sites, are protected from natural killer (NK) cells through the NKG2A/HLA-E axis. Interfering with this pathway unleashes NK cells and prevents PDAC metastasis.

Natural killer cells and type 1 innate lymphoid cells in cancer.

Innate lymphoid cells (ILCs) are a group of innate lymphocytes that do not express RAG-dependent rearranged antigen-specific cell surface receptors. ILCs are classified into five groups according to their developmental trajectory and cytokine production profile. They encompass NK cells, which are cytotoxic, helper-like ILCs 1-3, which functionally mirror CD4+ T helper (Th) type 1, Th2 and Th17 cells respectively, and lymphoid tissue inducer (LTi) cells. NK cell development depends on Eomes (eomesodermin), whereas the ILC1 program is regulated principally by the transcription factor T-bet (T-box transcription factor Tbx21), that of ILC2 is regulated by GATA3 (GATA-binding protein 3) and that of ILC3 is regulated by RORγt (RAR-related orphan receptor γ). NK cells were discovered close to fifty years ago, but ILC1s were first described only about fifteen years ago. Within the ILC family, NK and ILC1s share many similarities, as witnessed by their cell surface phenotype which largely overlap. NK cells and ILC1s have been reported to respond to tissue inflammation and intracellular pathogens. Several studies have reported an antitumorigenic role for NK cells in both humans and mice, but data for ILC1s are both scarce and contradictory. In this review, we will first describe the different NK cell and ILC1 subsets, their effector functions and development. We will then discuss their role in cancer and the effects of the tumor microenvironment on their metabolism.

Control of acute myeloid leukemia by a trifunctional NKp46-CD16a-NK cell engager targeting CD123.

CD123, the alpha chain of the IL-3 receptor, is an attractive target for acute myeloid leukemia (AML) treatment. However, cytotoxic antibodies or T cell engagers targeting CD123 had insufficient efficacy or safety in clinical trials. We show that expression of CD64, the high-affinity receptor for human IgG, on AML blasts confers resistance to anti-CD123 antibody-dependent cell cytotoxicity (ADCC) in vitro. We engineer a trifunctional natural killer cell engager (NKCE) that targets CD123 on AML blasts and NKp46 and CD16a on NK cells (CD123-NKCE). CD123-NKCE has potent antitumor activity against primary AML blasts regardless of CD64 expression and induces NK cell activation and cytokine secretion only in the presence of AML cells. Its antitumor activity in a mouse CD123+ tumor model exceeds that of the benchmark ADCC-enhanced antibody. In nonhuman primates, it had prolonged pharmacodynamic effects, depleting CD123+ cells for more than 10 days with no signs of toxicity and very low inflammatory cytokine induction over a large dose range. These results support clinical development of CD123-NKCE.

Canonical IRE1 function needed to sustain vigorous natural killer cell proliferation during viral infection.

The unfolded protein response (UPR) aims to restore ER homeostasis under conditions of high protein folding load, a function primarily serving secretory cells. Additional, non-canonical UPR functions have recently been unraveled in immune cells. We addressed the function of the inositol-requiring enzyme 1 (IRE1) signaling branch of the UPR in NK cells in homeostasis and microbial challenge. Cell-intrinsic compound deficiency of IRE1 and its downstream transcription factor XBP1 in NKp46+ NK cells, did not affect basal NK cell homeostasis, or overall outcome of viral MCMV infection. However, mixed bone marrow chimeras revealed a competitive advantage in the proliferation of IRE1-sufficient Ly49H+ NK cells after viral infection. CITE-Seq analysis confirmed strong induction of IRE1 early upon infection, concomitant with the activation of a canonical UPR signature. Therefore, we conclude that IRE1/XBP1 activation is required during vigorous NK cell proliferation early upon viral infection, as part of a canonical UPR response.

Tissue-specific transcriptional profiles and heterogeneity of natural killer cells and group 1 innate lymphoid cells.

Natural killer (NK) cells and type 1 innate lymphoid cells (ILC1s) are populations of non-T, non-B lymphocytes in peripheral tissues. Although NK and ILC1 subsets have been described, their identification and characteristics remain unclear. We performed single-cell RNA sequencing and CITE-seq to explore NK and ILC1 heterogeneity between tissues. We observed that although NK1 and NK2 subsets are conserved in spleen and liver, ILC1s are heterogeneous across tissues. We identified sets of genes expressed by related subsets or characterizing unique ILC1 populations in each organ. The syndecan-4 appeared as a marker discriminating murine ILC1 from NK cells across organs. Finally, we revealed that the expressions of EOMES, GZMA, IRF8, JAK1, NKG7, PLEK, PRF1, and ZEB2 define NK cells and that IL7R, LTB, and RGS1 differentiate ILC1s from NK cells in mice and humans. Our data constitute an important resource to improve our understanding of NK-ILC1 origin, phenotype, and biology.

Avdoralimab (Anti-C5aR1 mAb) Versus Placebo in Patients With Severe COVID-19: Results From a Randomized Controlled Trial (FOR COVID Elimination [FORCE]).

OBJECTIVES: Severe COVID-19 is associated with exaggerated complement activation. We assessed the efficacy and safety of avdoralimab (an anti-C5aR1 mAb) in severe COVID-19. DESIGN: FOR COVID Elimination (FORCE) was a double-blind, placebo-controlled study. SETTING: Twelve clinical sites in France (ICU and general hospitals). PATIENTS: Patients receiving greater than or equal to 5 L oxygen/min to maintain Sp o2 greater than 93% (World Health Organization scale ≥ 5). Patients received conventional oxygen therapy or high-flow oxygen (HFO)/noninvasive ventilation (NIV) in cohort 1; HFO, NIV, or invasive mechanical ventilation (IMV) in cohort 2; and IMV in cohort 3. INTERVENTIONS: Patients were randomly assigned, in a 1:1 ratio, to receive avdoralimab or placebo. The primary outcome was clinical status on the World Health Organization ordinal scale at days 14 and 28 for cohorts 1 and 3, and the number of ventilator-free days at day 28 (VFD28) for cohort 2. MEASUREMENTS AND MAIN RESULTS: We randomized 207 patients: 99 in cohort 1, 49 in cohort 2, and 59 in cohort 3. During hospitalization, 95% of patients received glucocorticoids. Avdoralimab did not improve World Health Organization clinical scale score on days 14 and 28 (between-group difference on day 28 of -0.26 (95% CI, -1.2 to 0.7; p = 0.7) in cohort 1 and -0.28 (95% CI, -1.8 to 1.2; p = 0.6) in cohort 3). Avdoralimab did not improve VFD28 in cohort 2 (between-group difference of -6.3 (95% CI, -13.2 to 0.7; p = 0.96) or secondary outcomes in any cohort. No subgroup of interest was identified. CONCLUSIONS: In this randomized trial in hospitalized patients with severe COVID-19 pneumonia, avdoralimab did not significantly improve clinical status at days 14 and 28 (funded by Innate Pharma, ClinicalTrials.gov number, NCT04371367).

Antitumor immunity induced by antibody-based natural killer cell engager therapeutics armed with not-alpha IL-2 variant.

Harnessing innate immunity is emerging as a promising therapeutic approach in cancer. We report here the design of tetraspecific molecules engaging natural killer (NK) cell-activating receptors NKp46 and CD16a, the ß-chain of the interleukin-2 receptor (IL-2R), and a tumor-associated antigen (TAA). In vitro, these tetraspecific antibody-based natural killer cell engager therapeutics (ANKETs) induce a preferential activation and proliferation of NK cells, and the binding to the targeted TAA triggers NK cell cytotoxicity and cytokine and chemokine production. In vivo, tetraspecific ANKETs induce NK cell proliferation and their accumulation at the tumor bed, as well as the control of local and disseminated tumors. Treatment of non-human primates with CD20-directed tetraspecific ANKET leads to CD20+ circulating B cell depletion, with minimal systemic cytokine release and no sign of toxicity. Tetraspecific ANKETs, thus, constitute a technological platform for harnessing NK cells as next-generation cancer immunotherapies.

Bringing natural killer cells to the clinic.

Cancer is a leading cause of mortality worldwide, with around 10 million deaths every year. Despite huge advances due to immunotherapy, the majority of cancer patients present primary or secondary resistance to these treatments. In this Found in Translation, we focus on the approaches developed to harness the anti-tumor function of NK cells, suggesting promising strategies to complete the therapeutic arsenal of cancer immunotherapies.

Role of the ITAM-Bearing Receptors Expressed by Natural Killer Cells in Cancer.

Natural Killer (NK) cells are innate lymphoid cells (ILCs) capable of recognizing and directly killing tumor cells. They also secrete cytokines and chemokines, which participate in the shaping of the adaptive response. NK cells identify tumor cells and are activated through a net positive signal from inhibitory and activating receptors. Several activating NK cell receptors are coupled to adaptor molecules containing an immunoreceptor tyrosine-based activation motif (ITAM). These receptors include CD16 and the natural cytotoxic receptors NKp46, NKp44, NKp30 in humans. The powerful antitumor NK cell response triggered by these activating receptors has made them attractive targets for exploitation in immunotherapy. In this review, we will discuss the different activating receptors associated with ITAM-bearing cell surface receptors expressed on NK cells, their modulations in the tumor context and the various therapeutic tools developed to boost NK cell responses in cancer patients.

Targeting CISH enhances natural cytotoxicity receptor signaling and reduces NK cell exhaustion to improve solid tumor immunity.

BACKGROUND: The success and limitations of current immunotherapies have pushed research toward the development of alternative approaches and the possibility to manipulate other cytotoxic immune cells such as natural killer (NK) cells. Here, we targeted an intracellular inhibiting protein 'cytokine inducible SH2-containing protein' (CISH) in NK cells to evaluate the impact on their functions and antitumor properties. METHODS: To further understand CISH functions in NK cells, we developed a conditional Cish-deficient mouse model in NK cells (Cishfl/flNcr1Ki/+ ). NK cells cytokine expression, signaling and cytotoxicity has been evaluated in vitro. Using intravenous injection of B16F10 melanoma cell line and EO711 triple negative breast cancer cell line, metastasis evaluation was performed. Then, orthotopic implantation of breast tumors was performed and tumor growth was followed using bioluminescence. Infiltration and phenotype of NK cells in the tumor was evaluated. Finally, we targeted CISH in human NK-92 or primary NK cells, using a technology combining the CRISPR(i)-dCas9 tool with a new lentiviral pseudotype. We then tested human NK cells functions. RESULTS: In Cishfl/flNcr1Ki/+ mice, we detected no developmental or homeostatic difference in NK cells. Global gene expression of Cishfl/flNcr1Ki/+ NK cells compared with Cish+/+Ncr1Ki/+ NK cells revealed upregulation of pathways and genes associated with NK cell cycling and activation. We show that CISH does not only regulate interleukin-15 (IL-15) signaling pathways but also natural cytotoxicity receptors (NCR) pathways, triggering CISH protein expression. Primed Cishfl/flNcr1Ki/+ NK cells display increased activation upon NCR stimulation. Cishfl/flNcr1Ki/+ NK cells display lower activation thresholds and Cishfl/flNcr1Ki/+ mice are more resistant to tumor metastasis and to primary breast cancer growth. CISH deletion favors NK cell accumulation to the primary tumor, optimizes NK cell killing properties and decreases TIGIT immune checkpoint receptor expression, limiting NK cell exhaustion. Finally, using CRISPRi, we then targeted CISH in human NK-92 or primary NK cells. In human NK cells, CISH deletion also favors NCR signaling and antitumor functions. CONCLUSION: This study represents a crucial step in the mechanistic understanding and safety of Cish targeting to unleash NK cell antitumor function in solid tumors. Our results validate CISH as an emerging therapeutic target to enhance NK cell immunotherapy.

Advancing natural killer therapies against cancer.

Natural killer (NK)-based therapies against cancer are emerging, but the understanding of NK cell functions needs to be completed to optimize these treatments. In this issue, Pan et al. (2022) show that pro-apoptotic molecules, such as BH3-mimetics, synergize with NK cells to induce mitochondria-driven apoptosis in tumor cells, thereby enhancing the efficacy of NK cell therapies.

Innate lymphoid cells and cancer.

The innate lymphoid cell (ILC) family is composed of natural killer (NK) cells, ILC1, ILC2 and ILC3, which participate in immune responses to virus, bacteria, parasites and transformed cells. ILC1, ILC2 and ILC3 subsets are mostly tissue-resident, and are profoundly imprinted by their organ of residence. They exhibit pleiotropic effects, driving seemingly paradoxical responses such as tissue repair and, alternatively, immunopathology toward allergens and promotion of tumorigenesis. Despite this, a trickle of studies now suggests that non-NK ILCs may not be overwhelmingly tumorigenic and could potentially be harnessed to drive anti-tumor responses. Here, we examine the pleiotropic behavior of ILCs in cancer and begin to unravel the gap in our knowledge that exposes a new horizon for thinking about modifying ILCs and targeting them for immunotherapy.