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Abstract Natural killer (NK) cells are a subset of cytotoxic lymphocytes within the innate immune system. While they are naturally cytotoxic, genetic modifications can enhance their tumor-targeting capability, cytotoxicity, persistence, tumor infiltration, and prevent exhaustion. These improvements hold the potential to make NK-cell-based immunotherapies more effective in clinical applications. Currently, several viral and non-viral technologies are used to genetically modify NK cells. For nucleic acid delivery, non-viral methods such as electroporation, lipid nanoparticles, lipofection, and DNA transposons have gained popularity in recent years. On the other hand, viral methods including lentivirus, gamma retrovirus, and adeno-associated virus, remain widely used for gene delivery. Furthermore, gene editing techniques such as clustered regularly interspaced short-palindromic repeats-based, zinc finger nucleases, and transcription activator-like effector nucleases are the pivotal methodologies in this field. This review aims to provide a comprehensive overview of chimeric antigen receptor (CAR) arming strategies and discuss key gene editing techniques. These approaches collectively aim to enhance NK cell/NK cell CAR-based immunotherapies for clinical translation. Graphical Abstract Graphical Abstract

Natural killer cells are an important part of the innate immune system mediating robust responses to virus-infected and malignant cells without needing prior antigen priming. NK cells have always been thought to be short-lived and with no antigen specificity; however, recent data support the presence of NK cell memory including in the hapten-specific contact hypersensitivity model and in certain viral infections. The memory-like features can also be generated by short-term activation of both murine and human NK cells with cytokine combination of IL-12, IL-15 and IL-18, imparting increased longevity and enhanced anticancer functionality. Preclinical studies and very early clinical trials demonstrate safety and very promising clinical activity of these c ytokine- i nduced memory-like (CIML) NK cells, making them an attractive cell type for developing novel adoptive cellular immunotherapy strategies. Furthermore, efforts are on to arm them with novel gene constructs for enhanced tumor targeting and function.

Natural killer cells constitute a part of the innate immune system that mediates an effective immune response towards virus-infected and malignant cells. In recent years, research has focused on exploring and advancing NK cells as an active immunotherapy platform. Despite major advances, there are several key challenges that need to be addressed for the effective translation of NK cell research to clinical applications. This review highlights some of these challenges and the innovative strategies being developed to overcome them, including in vitro expansion, in vivo persistence, infiltration to the tumor site, and prevention of exhaustion.

BackgroundResponses to conventional donor lymphocyte infusion for postallogeneic hematopoietic cell transplantation (HCT) relapse are typically poor. Natural killer (NK) cell-based therapy is a promising modality to treat post-HCT relapse.MethodsWe initiated this ongoing phase I trial of adoptively transferred cytokine-induced memory-like (CIML) NK cells in patients with myeloid malignancies who relapsed after haploidentical HCT. All patients received a donor-derived NK cell dose of 5 to 10 million cells/kg after lymphodepleting chemotherapy, followed by systemic IL-2 for 7 doses. High-resolution profiling with mass cytometry and single-cell RNA sequencing characterized the expanding and persistent NK cell subpopulations in a longitudinal manner after infusion.ResultsIn the first 6 enrolled patients on the trial, infusion of CIML NK cells led to a rapid 10- to 50-fold in vivo expansion that was sustained over months. The infusion was well tolerated, with fever and pancytopenia as the most common adverse events. Expansion of NK cells was distinct from IL-2 effects on endogenous post-HCT NK cells, and not dependent on CMV viremia. Immunophenotypic and transcriptional profiling revealed a dynamic evolution of the activated CIML NK cell phenotype, superimposed on the natural variation in donor NK cell repertoires.ConclusionGiven their rapid expansion and long-term persistence in an immune-compatible environment, CIML NK cells serve as a promising platform for the treatment of posttransplant relapse of myeloid disease. Further characterization of their unique in vivo biology and interaction with both T cells and tumor targets will lead to improvements in cell-based immunotherapies.Trial RegistrationClinicalTrials.gov NCT04024761.FundingDunkin' Donuts, NIH/National Cancer Institute, and the Leukemia and Lymphoma Society.

Allogeneic cellular immunotherapy exhibits promising efficacy for cancer treatment, but donor cell rejection remains a major barrier. Here, we systematically evaluate human leukocyte antigens (HLA) and immune checkpoints PD-L1, HLA-E, and CD47 in the rejection of allogeneic NK cells and identify CD8 + T cells as the dominant cell type mediating allorejection. We demonstrate that a single gene construct that combines an shRNA that selectively interferes with HLA class I but not HLA-E expression, a chimeric antigen receptor (CAR), and PD-L1 or single-chain HLA-E (SCE) enables the one-step construction of allogeneic CAR-NK cells that evade host-mediated rejection both in vitro and in a xenograft mouse model. Furthermore, CAR-NK cells overexpressing PD-L1 or SCE effectively kill tumor cells through the upregulation of cytotoxic genes and reduced exhaustion and exhibit a favorable safety profile due to the decreased production of inflammatory cytokines involved in cytokine release syndrome. Thus, our approach represents a promising strategy in enabling “off-the-shelf” allogeneic cellular immunotherapies. The use of donor-derived CAR-NK cells is limited by CD8 T cell-mediated allorejection. Here, the authors describe a one-step approach, based on selective HLA knockdown and overexpression of PD-L1, that allows allogeneic modified CAR-NK cells to escape rejection by the host immune system while exhibiting enhanced anti-tumor activity and safety in preclinical mouse models.

Background C ytokine i nduced m emory- l ike natural killer (CIML NK) cells combined with an IL-15 super-agonist (N-803) are a novel modality to treat relapsed/refractory head and neck cancer. Methods We report data from a phase I trial of haploidentical CIML NK cells combined with N-803 with or without ipilimumab (IPI) in relapsed/refractory head and neck cancer patients after a median of 6 prior lines of therapy. The trial adhered to a 3 + 3 dose de-escalation design, with primary endpoint being safety. High-resolution immunophenotypic and transcriptional profiling characterized the NK cells and their interacting partners in vivo. Results The primary safety endpoint was established, with dose-limiting toxicity in 1/10 patients. A transient disease control rate correlated with donor NK cell expansion, the latter occurring irrespective of IPI. The combination of CIML NK cells with N-803 and IPI was associated with increased early NK cell proliferation, contraction of Treg: Tcon, rapid recovery of recipient CD8 + T cells, and subsequent accelerated rejection of donor NK cells. Conclusions CIML NK cells combined with N-803 and ipilimumab to treat head and neck cancer is safe, and associated with a more proliferative NK cell phenotype. However, the combination leads to reduced HLA mismatched NK cell persistence, resulting in an important limitation affecting NK cell combination therapies in clinical trials. These results inform evaluation of CIML NK therapy for advanced malignancies, with considerations for combination with IPI. Trial Registration NCT04290546.

Innate immune cells, including natural killer cells, macrophages and γδ T cells, are gaining prominence as promising candidates for cancer immunotherapy. Unlike conventional T cells, these cells possess attributes such as inherent antitumor activity, rapid immune responses, favorable safety profiles and the ability to target diverse malignancies without requiring prior antigen sensitization. In this Review, we examine the engineering strategies used to enhance their anticancer potential. We discuss challenges associated with each cell type and summarize insights from preclinical and clinical work. We propose strategies to address existing barriers, providing a perspective on the advancement of innate immune engineering as a powerful modality in anticancer treatment. The unique attributes of innate immune cells position them as attractive candidates for cancer immunotherapy, with engineering strategies to enhance their antitumor potential, offering notable therapeutic promise.

Acute myeloid leukemia (AML) remains a therapeutic challenge, and a paucity of tumor-specific targets has significantly hampered the development of effective immunebased therapies. Recent paradigm-changing studies have shown that natural killer (NK) cells exhibit innate memory upon brief activation with IL-12 and IL-18, leading to cytokine-induced memory-like (CIML) NK cell differentiation. CIML NK cells have enhanced antitumor activity and have shown promising results in early phase clinical trials in patients with relapsed/refractory AML. Here, we show that arming CIML NK cells with a neoepitope-specific chimeric antigen receptor (CAR) significantly enhances their antitumor responses to nucleophosphmin-1 (NPM1)-mutated AML while avoiding off-target toxicity. CIML NK cells differentiated from peripheral blood NK cells were efficiently transduced to express a TCR-like CAR that specifically recognizes a neoepitope derived from the cytosolic oncogenic NPM1-mutated protein presented by HLA-A2. These CAR CIML NK cells displayed enhanced activity against NPM1-mutated AML cell lines and patient-derived leukemic blast cells. CAR CIML NK cells persisted in vivo and significantly improved AML outcomes in xenograft models. Single-cell RNA sequencing and mass cytometry analyses identified up-regulation of cell proliferation, protein folding, immune responses, and major metabolic pathways in CAR-transduced CIML NK cells, resulting in tumor-specific, CAR-dependent activation and function in response to AML target cells. Thus, efficient arming of CIML NK cells with an NPM1-mutation-specific TCR-like CAR substantially improves their innate antitumor responses against an otherwise intracellular mutant protein. These preclinical findings justify evaluating this approach in clinical trials in HLA-A2⁺ AML patients with NPM1c mutations.

Abstract Background Despite recent advances, metastatic clear cell renal cell carcinoma (ccRCC) remains largely incurable in most patients, and novel immunotherapeutic strategies to address checkpoint inhibitor refractory renal cell carcinoma are needed. Natural killer (NK) cells are immune effector lymphocytes that are specialized in the cytotoxic elimination of cancer cells. Chimeric antigen receptor (CAR) NK cells have also shown promising effects in clinical trials. IL-12 is a potent cytokine that mediates type 1 immunity and antitumor responses but with systemic toxicity. We hypothesize that IL-12 secreted by CD70-CAR NK cells will allow “tumor-directed” IL-12 delivery and with the incorporation of a novel motif, collagen-binding domain A3, we will further minimize toxicity. Methods We engineered lentivectors to express CD70-CAR, CD70-CAR+IL-12, and CD70-CAR+IL-12/A3, and evaluated their transduction efficiencies in human NK cells. The phenotypes of IL-12-secreting CD70-CAR NK cells were analyzed using flow cytometry and bulk RNA sequencing. Untransduced (UTD) and modified NK cells were cocultured with ccRCC tumor cell lines (A498 and ACHN) at various effector to target (E: T) ratios to assess target cell lysis. NK cell activation was determined by measuring degranulation (CD107a) and IFNγ expression. IFNγ secretion was quantified from coculture supernatants. The killing of ccRCC PDXs was similarly evaluated. In vivo, the therapeutic efficacy of CD70-CAR+IL-12/A3 NK cells was tested using ccRCC xenograft models using A498 cells injected subcutaneously into NSG mice. The mice were divided into five groups: no treatment (PBS), NK cell, CD70-CAR NK cell, CD70-CAR+IL-12 NK cell, and CD70-CAR+IL-12/A3 NK cell. When tumors reached ∼100 mm³, 1x10^6 NK cells were administered intravenously. Tumor volumes and mouse survival were monitored. For safety evaluation, the animals were closely monitored for their weight and wellbeing and plasma collected on day 7 post CAR NK cell injection to evaluate the production of IL-12 and other key inflammatory cytokines including IL-1β, IL-2, IL-6, IFNγ, IL-18 and GM-CSF that are associated with cytokine release syndrome (CRS). Results High transduction efficiency was achieved in the NK cells with all three CAR constructs (50–85%). IL-12-engineered CD70-CAR NK cells exhibited significantly increased surface expression of CD25, LFA1, CXCR4, and sLex compared to regular CD70-CAR NK cells. Gene expression analysis revealed upregulation of IL12A, IL12B, IFNG, GZMB, IRF1, SOCS3, and ABCA1, alongside downregulation of CISH. These cells also displayed enhanced metabolic fitness, with an increased oxygen consumption rate (OCAR). CD70-CAR NK cells that secrete IL-12 and IL-12/A3 exhibited significantly increased cytotoxicity in vitro against ccRCC tumor cell lines A498 and ACHN, as well as CD70+ ccRCC patient-derived xenografts (PDXs). The presence of IL-12 and IL-12/A3 effectively stimulated NK cell degranulation and IFNγ production following coculture with ccRCC tumor cells. In vivo studies showed that CD70-CAR NK cells secreting IL-12 and IL-12/A3 achieved notably better control of ccRCC tumors compared to CD70-CAR NK cells alone. Mice treated with CD70-CAR+IL-12/A3 NK cells demonstrated better survival rates than those treated with CD70-CAR+IL-12 NK cells or other constructs (Fig. 1). Additionally, CD70-CAR+IL-12/A3 NK cells resulted in significantly lower systemic levels of IL-12 7 days after CAR NK cell infusion compared to CD70-CAR+IL-12 NK cells, suggesting that fusing IL-12 to a collagen binding domain can reduce systemic IL-12 exposure. Conclusions Incorporating IL-12/A3 into CAR NK cells is feasible, safe, and significantly enhances their cytotoxicity against ccRCC tumor cells in vitro and in vivo. These findings provide strong rationale for further clinical evaluation of CD70-CAR+IL-12/A3 NK cells in patients with advanced ccRCC.