Natural killer (NK) cells have gained attention as a promising adoptive cell therapy platform for their potential to improve cancer treatments. NK cells offer distinct advantages over T-cells, including major histocompatibility complex class I (MHC-I)-independent tumor recognition and low risk of toxicity, even in an allogeneic setting. Despite this tremendous potential, challenges persist, such as limited in vivo persistence, reduced tumor infiltration, and low absolute NK cell numbers. This review outlines several strategies aiming to overcome these challenges. The developed strategies include optimizing NK cell expansion methods and improving NK cell antitumor responses by cytokine stimulation and genetic manipulations. Using K562 cells expressing membrane IL-15 or IL-21 with or without additional activating ligands like 4-1BBL allows “massive” NK cell expansion and makes multiple cell dosing and “off-the-shelf” efforts feasible. Further improvements in NK cell function can be reached by inducing memory-like NK cells, developing chimeric antigen receptor (CAR)-NK cells, or isolating NK-cell-based tumor-infiltrating lymphocytes (TILs). Memory-like NK cells demonstrate higher in vivo persistence and cytotoxicity, with early clinical trials demonstrating safety and promising efficacy. Recent trials using CAR-NK cells have also demonstrated a lack of any major toxicity, including cytokine release syndrome, and, yet, promising clinical activity. Recent data support that the presence of TIL-NK cells is associated with improved overall patient survival in different types of solid tumors such as head and neck, colorectal, breast, and gastric carcinomas, among the most significant. In conclusion, this review presents insights into the diverse strategies available for NK cell expansion, including the roles played by various cytokines, feeder cells, and culture material in influencing the activation phenotype, telomere length, and cytotoxic potential of expanded NK cells. Notably, genetically modified K562 cells have demonstrated significant efficacy in promoting NK cell expansion. Furthermore, culturing NK cells with IL-2 and IL-15 has been shown to improve expansion rates, while the presence of IL-12 and IL-21 has been linked to enhanced cytotoxic function. Overall, this review provides an overview of NK cell expansion methodologies, highlighting the current landscape of clinical trials and the key advancements to enhance NK-cell-based adoptive cell therapy.

Epithelial ovarian cancer (EOC) remains one of the most lethal gynecological cancers. Cytokine-induced memory–like (CIML) natural killer (NK) cells have shown promising results in preclinical and early-phase clinical trials. In the current study, CIML NK cells demonstrated superior antitumor responses against a panel of EOC cell lines, increased expression of activation receptors, and up-regulation of genes involved in cell cycle/proliferation and down-regulation of inhibitory/suppressive genes. CIML NK cells transduced with a chimeric antigen receptor (CAR) targeting the membrane-proximal domain of mesothelin (MSLN) further improved the antitumor responses against MSLN-expressing EOC cells and patient-derived xenograft tumor cells. CAR arming of the CIML NK cells subtanstially reduced their dysfunction in patient-derived ascites fluid with transcriptomic changes related to altered metabolism and tonic signaling as potential mechanisms. Lastly, the adoptive transfer of MSLN-CAR CIML NK cells demonstrated remarkable inhibition of tumor growth and prevented metastatic spread in xenograft mice, supporting their potential as an effective therapeutic strategy in EOC.

Memory formation, guided by microbial ligands, has been reported for innate immune cells. Epigenetic imprinting plays an important role herein, involving histone modification after pathogen-/danger-associated molecular patterns (PAMPs/DAMPs) recognition by pattern recognition receptors (PRRs). Such “trained immunity” affects not only the nominal target pathogen, yet also non-related targets that may be encountered later in life. The concept of trained innate immunity warrants further exploration in cancer and how these insights can be implemented in immunotherapeutic approaches. In this review, we discuss our current understanding of innate immune memory and we reference new findings in this field, highlighting the observations of trained immunity in monocytic and natural killer cells. We also provide a brief overview of trained immunity in non-immune cells, such as stromal cells and fibroblasts. Finally, we present possible strategies based on trained innate immunity that may help to devise host-directed immunotherapies focusing on cancer, with possible extension to infectious diseases.

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.

Tumor immune escape is a major barrier to durable cancer immunotherapy, as advanced malignancies create a tumor microenvironment (TME) that preferentially exhausts and disables T cell responses. While most approved cell therapies are T cell‐based, this limitation motivates the exploration of an alternative effector cell platform. Natural killer (NK) cells, innate cytotoxic lymphocytes capable of antigen‐independent recognition and killing, offer a compelling foundation for next‐generation therapies with an improved safety profile. In this review, we first outline the cellular, molecular, and metabolic features of the immunosuppressive TME that restrict cytotoxic lymphocyte function, emphasizing mechanisms that limit immune cell‐mediated responses. We then summarize key aspects of NK cell biology that can circumvent these barriers and critically evaluate current NK‐based strategies, including engineered chimeric antigen receptor (CAR)‐NK products and metabolic and trafficking interventions. Finally, we highlight emerging in vivo viral and mRNA/lipid nanoparticle platforms for CAR‐NK generation and their potential to enhance scalability and therapeutic durability.

Introduction Bladder Cancer (BC) is the ninth most common cancer worldwide. Non-Muscle Invasive Bladder Cancer (NMIBC) is a heterogeneous disease that includes tumours with low and high risk of progression to muscle invasion. Nowadays, clinico-pathological features alone are not sufficient to effectively classify these patients and the discovery of new biomarkers is essential to increase quality of their life, who should only be treated with a radical high-morbidity approach, whenever needed, depending on the type of NMIBC tumour developed. ▵Np63 has previously been reported by our group as a protective factor of High Grade (HG) NMIBC progression: From the patients whose tumours showed nuclear ▵Np63 expression, none suffered disease progression after a median follow-up of 62,1 months. Furthermore, when ▵Np63 was knocked down (ΔNKO), cells showed higher proliferation and invasive capacities in vitro and higher tumour and metastasis formation in vivo. Interestingly, ΔNKO cells displayed AGR2 gene up-regulation and protein overexpression. Material and methods Two commercially available HG-NMIBC cell lines (RT112 and BF-TC905) were used to study the role of AGR2 in tumour progression. A stable knockdown of AGR2 (using shRNA technology) was performed on ΔNKO cells. In vitro functional studies, such as cell cycle, proliferation and invasion assays were performed comparing parental cells, ΔNKO cells and cells with knockdown of both ▵Np63 and AGR2 (AKO). The orthotopic mouse model is being used to study in vivotumour initiation and metastatic capacities. Immunofluorescence analyses of AGR2 in human HG-NMIBC tissue specimens will be performed to see if AGR2 expression is a prognostic and predictive biomarker for HG-NMIBC. Results and discussions After knocking down AGR2 in RT112 and BF-TC905 ▵NKO cells, we observed a reversal of the aggressive phenotype of ΔNKO cells, with lower cell proliferation (cell cycle analysis and proliferation assay) and invasion, thus confirming the functional importance of AGR2. If in vivo results corroborate our hypothesis, we will start a collaboration with Dr. Li’s lab to test the efficiency of AGR2 antibody (Agtuzumab) intravesical therapy in NMIBC. Conclusion This work uncovered AGR2 as a progression biomarker in HG-NMIBC, allowing a better understanding of the mechanisms of BC progression, and opening the possibility of novel therapies for patients affected by this disease, ultimately improving patients overall survival and quality of life.