Allogeneic natural killer (NK) cell-based therapies with an outstanding safety profile, are a compelling alternative to autologous T cell-based approaches for cancer immunotherapy, offering innate tumor-killing ability that can be further augmented via introduction of tumor antigen-specific chimeric antigen receptors (CARs). In this study, we genetically engineered primary human hematopoietic stem cells using an optimized lentiviral backbone carrying CD19 CAR cassettes with varied hinge, transmembrane, and signaling domains to evaluate their role in CAR-NK development and function. Our platform integrates early genetic modification with our unique expansion/differentiation system, enabling high CAR expression with low vector copy numbers. Notably, CARs incorporating CD28 transmembrane and signaling domains with CD3ζ, promoted enhanced tonic signaling, accelerated NK differentiation, enhanced antigen-specific kinome activation, and improved cytotoxicity and persistence both in vitro and in vivo. These findings offer a robust strategy for development of stem cell-based CAR-NK immunotherapies, combining potent innate, and antigen-specific antitumor responses.

Immune checkpoints are critical in modulating immune responses and maintaining self-tolerance. Cancer cells can exploit these mechanisms to evade immune detection, making immune checkpoints attractive targets for cancer therapy. The introduction of immune checkpoint inhibitors (ICIs) has transformed cancer treatment, with monoclonal antibodies targeting CTLA-4, PD-1, and PD-L1 demonstrating clinical success. However, challenges such as immune-related adverse events, primary and acquired resistance, and high treatment costs persist. To address these challenges, it is essential to explore alternative strategies, including small-molecule and peptide-based inhibitors, aptamers, RNA-based therapies, gene-editing technologies, bispecific and multispecific agents, and cell-based therapies. Additionally, innovative approaches such as lysosome-targeting chimeras, proteolysis-targeting chimeras, and N-(2-hydroxypropyl) methacrylamide copolymers are emerging as promising options for enhancing treatment effectiveness. This review highlights significant advancements in the field, focusing on their clinical implications and successes.

Immunotherapy has revolutionized the oncology treatment paradigm, and CAR-T cell therapy in particular represents a significant milestone in treating hematological malignancies. Nevertheless, tumor resistance due to target heterogeneity or mutation remains a Gordian knot for immunotherapy. This review elucidates molecular mechanisms and therapeutic potential of next-generation immunotherapeutic tools spanning genetically engineered immune cells, multi-specific antibodies, and cell engagers, emphasizing multi-targeting strategies to enhance personalized immunotherapy efficacy. Development of logic gate modulation-based circuits, adapter-mediated CARs, multi-specific antibodies, and cell engagers could minimize adverse effects while recognizing tumor signals. Ultimately, we highlight gene delivery, gene editing, and other technologies facilitating tailored immunotherapy, and discuss the promising prospects of artificial intelligence in gene-edited immune cells.

Cardiac fibrosis (CF) is a common pathophysiological process in the development of various cardiovascular diseases, during which many cardiac fibroblasts undergo myofibroblast transdifferentiation. Fibroblast activation protein (FAP) can serve as a specific target for myofibroblasts, and chimeric antigen receptor (CAR)-based therapy is a promising immunotherapy strategy. In this study, we attempted to construct CAR natural killer (NK) cells that target FAP and explored their potential therapeutic role in CF. Our results suggested FAP CAR-NK-92 cells can specifically recognize and kill FAP+ cells in vitro. In addition, compared with parental NK-92 cells, FAP CAR-NK cells cocultured with FAP HEK-293 T cells presented increased cytotoxicity, cytokine secretion, and degranulation, indicating an effect-to-target ratio dependence. Coculturing FAP CAR-NK cells with mouse cardiac fibroblast lines (MCFs) eliminated the activated fibroblasts, reduced fibrosis-related protein secretion, and significantly reversed the contractile phenotype of myofibroblasts, which is characterized by alpha-smooth muscle actin (α-SMA) and stress fiber formation. Intravenous injection of FAP CAR-NK cells in mice 7 days after Ang II/PE-induced injury significantly improved cardiac function and reduced fibrosis. In terms of the killing mechanism, the early apoptosis rate of target cells was significantly increased, the antiapoptotic protein Bcl-2 was significantly decreased, and the proapoptotic proteins Bax and Caspase 3 were markedly increased. Our findings demonstrate that FAP CAR-NK-92 cells can specifically recognize FAP+ target cells and exert potent anti-fibrotic effects both in vitro and in vivo. Therefore, FAP CAR-NK-92 cells could be considered an effective therapeutic option for CF patients.

NK cells are a class of innate lymphocytes capable of nonspecifically killing tumor cells without MHC restriction or prior sensitization. Recent advancements in biotechnology, particularly the development of chimeric antigen receptors (CAR) and related technologies, have enabled targeted tumor cell elimination. CAR endows NK cells with enhanced functionality, with the extracellular domains typically consisting of single-chain variable fragments (scFv) for targeting specific antigens. CAR-NK cells have shown excellent results in several preclinical studies and clinical trials for hematologic malignancies. However, their clinical application in the treatment of solid tumors is still insufficient. Current treatments for gynecological cancers primarily involve surgery, chemotherapy, and radiotherapy, all of which often present substantial side effects and variable efficacy. While CAR-T cell therapy has shown effectiveness in certain gynecological tumors, its clinical application is hindered by severe side effects, such as Cytokine Release Syndrome (CRS) and Graft-Versus-Host Disease (GVHD). CAR-NK cell therapy offers improved safety profiles in clinical applications.

This review aims to systematically evaluate recent methodological innovations in CAR-NK engineering and their translational potential in tumor-targeted treatment, providing valuable insights for clinical trials and studies.

Electronic databases, including PubMed and Web of Science were searched for relevant literature. Keywords are as follows: CAR-NK cell; Chimeric antigen receptor; Solid tumor; cell therapy; gynecological cancers.

CAR-NK engineering has innovations such as multi-targeted CAR design, gene editing for enhanced persistence, and "off-the-shelf" CAR-NK cells compared to CAR-T cells.

CAR-NK cell therapy combines safety and anti-tumor efficacy, particularly for gynecological cancers.

A substantial proportion of patients diagnosed with rheumatologic and musculoskeletal diseases (RMDs) exhibit resistance to conventional therapies or experience recurrent symptoms. These diseases, which include autoimmune disorders such as multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus, are marked by the presence of autoreactive B cells that play a critical role in their pathogenesis. The persistence of these autoreactive B cells within lymphatic organs and inflamed tissues impairs the effectiveness of B-cell-depleting monoclonal antibodies like rituximab. A promising therapeutic approach involves using T cells genetically engineered to express chimeric antigen receptors (CARs) that target specific antigens. This strategy has demonstrated efficacy in treating B-cell malignancies by achieving long-term depletion of malignant and normal B cells. Preliminary data from patients with RMDs, particularly those with lupus erythematosus and dermatomyositis, suggest that CAR T-cells targeting CD19 can induce rapid and sustained depletion of circulating B cells, leading to complete clinical and serological responses in cases that were previously unresponsive to conventional therapies. This review will provide an overview of the current state of preclinical and clinical studies on the use of CAR T-cells and other cellular therapies for RMDs. Additionally, it will explore potential future applications of these innovative treatment modalities for managing patients with refractory and recurrent manifestations of these diseases.

In vitro hematopoiesis systems can be used to define mechanisms for blood cell formation and function, produce cell therapeutics, and model blood cell contributions to systemic disease. Hematopoietic progenitor cell (HPC) production remains inefficient, precluded by knowledge gaps related to specification and morphogenesis of specialized hemogenic endothelial cells, which undergo an endothelial-to-hematopoietic transition (EHT) to form HPCs. We elected to define changes in gene expression and chromatin organization during HPC formation to reveal regulatory mechanisms. Using paired single cell RNA/ATAC sequencing together with Hi-C, we profiled cells before and after EHT. Pathway analysis and pseudotime inferences confirmed a continuum of stromal and endothelial cells undergoing development into HE cells and lineage-based HPCs in vitro. In these cell types, we characterize cis-regulatory elements and transcriptional regulatory activities that facilitate EHT and HPC homeostasis, including for SNAI1, SOX17, TGFβ, STAT4, as well as for GFI1b and KLF1 in megakaryocyte- and erythroid-biased progenitors, respectively. We then leveraged our insights into chromatin organization among in vitro-derived cells to assess enrichments corresponding to human trait variation reported in human genome wide association studies. HPCs revealed locus enrichment for quantitative blood traits and autoimmune disease predisposition, which were particularly enriched in myeloid- and lymphoid-biased populations. Stromal and endothelial cells from our in vitro cultures were specifically enriched for accessible chromatin at blood pressure loci. Our findings reveal genes and mechanisms governing in vitro hematopoietic development and blood cell-related disease pathology.

Chimeric antigen receptor (CAR)-engineered immune cell therapy represents an important advance in cancer treatments. However, the complex ex vivo cell manufacturing process and stringent patient selection criteria curtail its widespread use. In vivo CAR engineering is emerging as a promising off-the-shelf therapy, providing advantages such as streamlined production, elimination of patient-specific manufacturing, reduced costs and simplified logistics. A large set of preclinical findings has inspired further investigation into treatments for hard-to-treat diseases such as solid tumours and has facilitated the development of advanced products to enhance in vivo CAR engineering efficacy, the persistence of the cellular therapeutic and safety. In this Review, we summarize current in vivo CAR engineering strategies, including nanoparticle-based and viral delivery systems as well as bioinstructive implantable scaffolds, and discuss their advantages and disadvantages. Additionally, we provide a systematic comparison between in vivo and conventional ex vivo CAR engineering methods and address the challenges and future prospects of in vivo CAR engineering.

Resveratrol (RSV) is a dietary polyphenolic compound with anticancer property. However, its clinical translation as an anticancer chemotherapeutic agent is hindered by multiple challenges. Chimeric antigen receptor (CAR) natural killer (NK) cell therapy has emerged as a promising immunotherapeutic strategy against refractory malignancies. The role of RSV in CAR NK cell therapy remains unexplored. This study pioneers the investigation of RSV's role in CAR NK immunotherapy. Specially, RSV preconditioning improved CAR NK cells' resistance to oxidative stress, and augmented energetic metabolism in CAR NK cells. More importantly, the enhanced cytotoxicity against cancer cells of non-Hodgkin lymphoma (NHL) and stronger cytokine secretions were observed in RSV-treated anti-CD19 CAR NK cells. In vivo tracking revealed RSV extended CAR NK tissue residency and enhanced therapeutic efficacy in NHL xenografts. RSV-adjuvanted anti-CD19 CAR NK therapy achieved an obvious reduction in intraperitoneal tumor burden and improvement in the mice survival. Our mechanistic investigation revealed that nuclear factor erythroid 2-related factor 2 (NRF2) serves as a critical mediator in RSV-induced functional augmentation of CAR NK cells. These findings established RSV as a potential polyphenol adjuvant capable of improving CAR NK therapy, providing a translatable strategy for polyphenols to overcome current limitations in cancer therapy.

High-grade serous ovarian cancer (HGSOC) remains an urgent unmet clinical need, with more than 70% of patients presenting with metastatic disease. Many patients develop large volumes of ascites, which promotes metastasis and is associated with poor therapeutic response and survival. Immunotherapy trials have shown limited success, highlighting the need to better understand HGSOC immunology. Here, we analyzed cytotoxic lymphocytes [natural killer (NK), T, and innate T cells] from patients with HGSOC and observed widespread dysfunction across primary and metastatic sites. Although nutrient rich, ascites was immunosuppressive for all lymphocyte subsets. NK cell dysfunction was driven by uptake of polar lipids, with associated dysregulation in lipid storage. Phosphatidylcholine was a key immunosuppressive metabolite, disrupting NK cell membrane order and cytotoxicity. Blocking lipid uptake through SR-B1 protected NK cell antitumor functions in ascites. These findings offer insights into immune suppression in HGSOC and have important implications for the design of future immunotherapies.

There has been significant progress in the clinical development of allogeneic off-the-shelf chimeric antigen receptor (CAR)-engineered cell therapies for the treatment of cancer and autoimmune diseases. Unlike autologous CAR cell therapies, allogeneic approaches overcome challenges such as high costs, labor-intensive manufacturing, and stringent patient selection. This makes allogeneic therapies a more universally applicable option for a diverse patient population. In this review, we examine recent clinical advancements in allogeneic CAR cell therapies, including CAR-T cell therapy derived from healthy donor peripheral blood mononuclear cells, as well as CAR-NK cell therapy from cord blood or induced pluripotent stem cells. We provide an overview of their genetic engineering strategies, clinical designs, and outcomes, highlighting their promising efficacy and safety. Additionally, we summarize key preclinical developments, address key challenges, and explore future directions to provide insights into emerging trends in the field.

The clinical potential of current chimeric antigen receptor-engineered T (CAR-T) cell therapy is hampered by its autologous nature that poses considerable challenges in manufacturing, costs and patient selection. This spurs demand for off-the-shelf therapies. Here we introduce an ex vivo feeder-free culture method to differentiate gene-engineered hematopoietic stem and progenitor (HSP) cells into allogeneic invariant natural killer T (AlloNKT) cells and their CAR-armed derivatives (AlloCAR-NKT cells). We include detailed information on lentivirus generation and titration, as well as the five stages of ex vivo culture required to generate AlloCAR-NKT cells, including HSP cell engineering, HSP cell expansion, NKT cell differentiation, NKT cell deep differentiation and NKT cell expansion. In addition, we describe procedures for evaluating the pharmacology, antitumor efficacy and mechanism of action of AlloCAR-NKT cells. It takes ~2 weeks to generate and titrate lentiviruses and ~6 weeks to generate mature AlloCAR-NKT cells. Competence with human stem cell and T cell culture, gene engineering and flow cytometry is required for optimal results.

In recent years, immunotherapy has become a novel antitumor strategy in addition to traditional surgery, radiotherapy, and chemotherapy and has exhibited promising results in clinical applications. Despite significant breakthroughs in immunotherapy, such as immune checkpoint blockade and CAR-T cell therapy, it remains necessary to develop more efficacious, safer, and cheaper immunotherapeutic drugs due to factors including small reaction populations, acquired resistance, adverse side effects, and high costs. Natural killer (NK) cells are preeminent cytotoxic lymphocytes of the innate immune system that act as the first line of defense against tumors and synergistically enhance the adaptive immune response of T lymphocytes. Therefore, boosting the antitumor function of NK cells is an important direction in the development of immunotherapy. For decades, various immunotherapies such as adoptive cell therapy, antibody drugs, cytokines supplement, and chemical immunomodulators have been developing rapidly to improve the function of NK cells. Compared to biological immunotherapy, immunomodulators derived from natural products have outstanding advantages of low immunogenicity, multi-targeting, and cost-effectiveness. Currently, increasing attention is being focused on discovering NK cell-stimulating agents from natural products, such as polysaccharides, alkaloids, terpenoids, saponins, phenolics, and quinones. This review aims to categorize and summarize the comprehensive research progress on these natural products, discuss their potential molecular mechanisms in regulating NK cells, and explore their clinical applications as standalone treatments or in combination with conventional chemotherapy regimens.

Natural killer (NK) cells show significant potential in targeting hard to treat cancers, but these cells need effective preservation methods to maintain viability and efficacy after cryopreservation. Traditional methods of preserving NK cells result in low post-thaw recovery and function. Dimethyl sulfoxide (DMSO) is a very common cryoprotectant for preserving NK cells, but its infusion into patients post-thaw can cause dose-dependent adverse effects, including nausea, discomfort and cardiac arrest. The aim of this work was to evaluate low DMSO- and osmolyte-based cryopreservation solutions across multiple steps in the cryopreservation process for NK cells.

This study investigated NK cell membrane responses to cryoprotectants, dependence of cell survival on cooling rate and nucleation temperature and influence of osmotic shock and pH changes with regard to freezing NK cells using the NK cell line NK-92 as a model system.

Exposure to cryoprotectants reduced the membrane fluidity and NK cell-induced cytotoxicity of the cells before freezing, but combinations of osmolytes mitigated this loss. The introduction of cryoprotectants did not reduce perforin or granzyme content, and slow or rapid dilution after thawing did not reduce viability, recovery or proliferation. Controlled rate freezing and Raman cryomicroscopy studies revealed that NK cells tolerated fast cooling rates, and the optimal cooling rate for NK cells was 4-5°C/min. Raman cryomicroscopy mapped the distribution of cryoprotectants and ice of frozen NK cells at -50°C, showing a reduction in cytotoxic granule signal.

The large, osmotically inactive volume of NK cells demonstrates cell sensitivity to cryoprotectants and freezing. Exposure to cryoprotectants can reduce NK cell-induced cytotoxicity and membrane fluidity. We hypothesize that cell dehydration and freezing disrupt cytolytic granules, causing NK intracellular damage. These data emphasize the importance of developing robust techniques to enhance the cryopreservation of NK cells and indicate the points where cell damage occurs.

iPSC-derived natural killer cells (iNKs) have emerged as a promising cellular therapy, especially for the refractory or relapsed acute myeloid leukemia (R/R AML) patients, but limited research focused on the chemotaxis of iNKs. Here we demonstrate that C-X-C chemokine receptor type 4 (CXCR4) is significantly reduced in iNKs, resulting in impaired bone marrow (BM) infiltration, which cannot be rescued by constitutively expressed CXCR4 in iPSC due to CXCR4-induced differentiation failure. To address this, we developed a strategy to allow specific expression of CXCR4 during the iNK maturation stage without compromising the final iNK yield and function. The engineered iNKs exhibited enhanced BM infiltration, resulting in improved therapeutic effects in AML murine models. This, brought attention to iNK chemotaxis, provided a meaningful strategy by incorporating well-designed gene editing with stem cells for cell product development, and obtained improved effective NK cells for AML therapy.

Tumor therapy is still a tough clinical challenge, and cancer immunotherapy has drawn increasing attention. T cells and natural killer (NK) cells play crucial roles in the immune response. Induced pluripotent stem cell (iPSC) technology opens up a new way to produce functionally improved universal iPSC-derived chimeric antigen receptor (CAR) T (CAR-iT) and iPSC-derived CAR-NK (CAR-iNK) cells. This study aims to comprehensively review the generation and clinical applications of iPSC-derived universal CAR-iT and CAR-iNK cells to explore their potential and future directions in cancer immunotherapy.

We searched EBSCO, PubMed, and Web of Science databases for relevant literature from 1975 to 2024 on the transformation of iPSCs into universal immune cells.

iPSC technology enables the generation of enhanced CAR-iNK cells. Genetic modifications can boost the antitumor activity of iPSC-derived immune cells. CAR-iT cells have cytotoxicity issues. In contrast, CAR-iNK cells have advantages as they can be sourced from different origins and enhanced via genetic engineering.

This review outlines iPSC technology's application in oncology, iNK cells' properties, and the pros and cons of CAR cells in cancer treatment. It also focuses on the current clinical status and modification strategies of CAR-iT and CAR-iNK therapies, facilitating the development of future effective off-the-shelf blood cell therapies.

Natural killer (NK) cells are emerging as a promising tool for cancer immunotherapy due to their innate ability to selectively recognize and eliminate cancer cells. Over the past 3 decades, strategies to harness NK cells have included cytokines, small molecules, antibodies, and the adoptive transfer of autologous or allogeneic NK cells, both unmodified and genetically engineered. Despite favorable safety profiles in clinical trials, challenges such as limited in vivo persistence, exhaustion, and the suppressive tumor microenvironment continue to hinder their efficacy and durability. This review categorizes NK cell-based therapies into 3 major approaches: (i) cellular therapies, including unmodified and chimeric antigen receptor-engineered NK cells; (ii) cytokine-based strategies such as interleukin-2 and interleukin-15 derivatives; and (iii) antibody-based therapies, including immune checkpoint inhibitors and NK cell engagers. We highlight these advancements, discuss current limitations, and propose strategies to optimize NK cell-based therapies for improved cancer treatment outcomes.

Chimeric antigen receptor natural Killer (CAR-NK) cells therapy represents a next-generation immunotherapeutic approach following CAR-T cells therapy, offering inherent "off-the-shelf" compatibility and mitigated off-tumor toxicity. Despite these advantages, clinical translation remains constrained by poor in vivo persistence and functional exhaustion in immunosuppressive tumor microenvironments (TME). This review examines recent advancements in synthetic biology aimed at enhancing the physiological characteristics of CAR-NK cells. By delineating the synergy between NK cells and synthetic biology toolkits, this work provides a roadmap for developing next-generation CAR-NK therapies capable of addressing solid tumor challenges while maintaining favorable safety profiles.

Invariant natural killer T (iNKT) cells are a distinct subset of T lymphocytes that possess unique properties making them highly suitable for addressing the challenges of solid tumor immunotherapy. Unlike conventional T cells, which are restricted by polymorphic major histocompatibility complex (MHC) molecules and recognize peptide antigens, iNKT cells are restricted by the non-polymorphic CD1d molecule and respond to lipid antigens. Chimeric antigen receptor (CAR)-redirected iNKT (CAR-iNKT) cells represent a significant advancement in cancer immunotherapy. However, optimizing sustained activation and long-term persistence of CAR-iNKT cells remains a critical need for effective solid tumor treatment. To address these limitations, we develop the iNKT cell-targeted microparticle recruitment and activation system (iMRAS), a biomimetic platform designed to enhance iNKT cell functionality through localized immunostimulation in vivo. This biomimetic platform is designed to function as an in vivo "charging station" containing chemotactic and activation signals for the recruitment, activation, and expansion of CAR-iNKT cells, leading to more effective tumor killing and longer persistence of CAR-iNKT cells, as demonstrated in the therapy of lymphoma and melanoma. Through its biomimetic design and localized immunostimulatory effects, iMRAS helps overcome the limitations of current therapies for solid tumors, establishing a robust platform for enhancing systemic CAR-iNKT cell-mediated immunotherapy.

Chimeric antigen receptor (CAR) T and natural killer (NK) cell therapies represent a promising strategy for the treatment of cancers and other chronic diseases. Engineered CAR constructs endow immune cells with the ability to target desired antigens with high specificity, allowing for directed responses to antigen-expressing cells. CAR T and NK cells have shown marked success in the treatment of hematologic malignancies, although there remains a large population of patients with disease that fails to respond to CAR therapies, and their efficacy in solid tumors is still limited. In this review, we provide a broad overview of the development, design, and implementation of CAR therapies from bench to bedside. We discuss the building blocks of CAR constructs and how these can be manipulated to optimize CAR functionality, review the possible sources of T and NK cells for CAR therapies, and examine the limitations of both CAR T and CAR NK cells. Finally, we discuss recent breakthroughs in the CAR field and consider how these advances may affect the success of CAR therapies in the years to come.

Chimeric antigen receptor (CAR)-T-cell therapy has been highly successful in the treatment of B-cell hematological malignancies. CARs are modular synthetic molecules that can redirect immune cells towards target cells with antibody-like specificity. Despite their modularity, CARs used in the clinic are currently composed of a limited set of domains, mostly derived from IgG, CD8α, 4-1BB, CD28 and CD3ζ. The current low throughput CAR screening workflows are labor-intensive and time-consuming, and lie at the basis of the limited toolbox of CAR building blocks available. High throughput screening methods facilitate simultaneous investigation of hundreds of thousands of CAR domain combinations, allowing discovery of novel domains and increasing our understanding of how they behave in the context of a CAR. Here we review the growing body of reports that employ these high throughput screening and computational methods to advance CAR design. We summarize and highlight the important differences between the different studies and discuss their limitations and future considerations for further improvements. In conclusion, while still in its infancy, high throughput screening of CARs has the capacity to vastly expand the CAR domain toolbox and improve our understanding of CAR design. This knowledge could be foundational for translating CAR therapy beyond hematological malignancies and push the frontiers in personalized medicine.

High expression of externally injected in vitro transcribed (IVT) mRNA in natural killer (NK) cells is a prerequisite for NK cell-mediated cell therapy. To enhance the transfection efficacy of IVT mRNA-loaded polyplexes, we exposed NK cells to a hypertonic condition during transfection, which facilitated endo/exocytosis to maintain the isotonic state of the cells. The transfection efficacy of IVT mRNA was significantly enhanced after 24 h, which was mainly due to the facilitated cellular uptake and endosomal escape of the polyplexes. Interestingly, osmotic alterations in NK cells significantly affect the expression levels of endosome-escape-related genes in ion channels. Treatment with a mild hypertonic condition exhibited negligible toxicity to NK cells, without disturbing the integrity of the cellular membranes or the innate cytotoxic abilities of NK cells against cancer cells. These results demonstrate that the hypertonic treatment of NK cells enhances the transfection efficacy of IVT mRNA to produce genetically engineered NK cells.

Over the past few years, cellular immunotherapy has emerged as a promising treatment for certain hematologic cancers, with various CAR-T therapies now widely used in clinical settings. However, challenges related to the production of autologous cell products and the management of CAR-T cell toxicity highlight the need for new cell therapy options that are universal, safe, and effective. Natural killer (NK) cells, which are part of the innate immune system, offer unique advantages, including the potential for off-the-shelf therapy. A recent first-in-human trial of CD19-CAR-NK infusion in patients with relapsed/refractory lymphoid malignancies demonstrated safety and promising clinical activity. Building on these positive clinical outcomes, current research focuses on enhancing CAR-NK cell potency by increasing their in vivo persistence and addressing functional exhaustion. There is also growing interest in applying the successes seen in hematologic malignancies to solid tumors. This review discusses current trends and emerging concepts in the engineering of next-generation CAR- NK therapies. It will cover the process of constructing CAR-NK cells, potential targets for their manufacturing, and their role in various solid tumors. Additionally, it will examine the mechanisms of action and the research status of CAR-NK therapies in the treatment of solid tumors, along with their advantages, limitations, and future challenges. The insights provided may guide future investigations aimed at optimizing CAR-NK therapy for a broader range of malignancies.

Glioblastoma (GBM) is the most prevalent and aggressive primary brain malignancy in adults. Nevertheless, the cellular heterogeneity and complexity within the GBM microenvironment (TME) are still not fully understood, posing a significant obstacle in the advancement of more efficient immunotherapies for GBM. In this study, we conducted an integrated analysis of 48 tumor fragments from 24 GBM patients at the single-cell level, uncovering substantial molecular diversity within immune infiltrates. We characterized molecular signatures for five distinct tumor-associated macrophages (TAMs) subtypes. Notably, the TAM_MRC1 subtype displayed a pronounced M2 polarization signature. Additionally, we identified a subtype of natural killer (NK) cells, designated CD56dim_DNAJB1. This subtype is characterized by an exhausted phenotype, evidenced by an elevated stress signature and enrichment in the PD-L1/PD-1 checkpoint pathway. Our findings also highlight significant cell-cell interactions among malignant glioma cells, TAM, and NK cells within the TME. Overall, this research sheds light on the functional heterogeneity of glioma and immune cells in the TME, providing potential targets for therapeutic intervention in this immunologically cold cancer.

Ovarian cancer (OC) presents significant challenges due to late diagnosis and high recurrence rates, necessitating a deeper understanding of the molecular and cellular characteristics of OC and the exploration of novel therapeutic approaches. Here, we provide a protocol to characterize primary OC patient samples, including the tumor and the tumor microenvironment (TME), using flow cytometry. Additionally, we detail the design and evaluation of various cell-based immunotherapies aimed at targeting primary OC tumor and the TME through in vitro killing assays. For complete details on the use and execution of this protocol, please refer to Li et al.1.

Cellular senescence, a state of stable cell cycle arrest, acts as a double-edged sword in cancer biology. In young organisms, it acts as a barrier against tumorigenesis, but in the aging population, it may facilitate tumor growth and metastasis through the senescence-associated secretory phenotype (SASP). Natural killer (NK) cells play a critical role in the immune system, particularly in the surveillance, targeting, and elimination of malignant and senescent cells. However, age-related immunosenescence is characterized by declining NK cell function resulting in diminished ability to fight infection, eliminate senescent cells and suppress tumor development. This implies that preserving or augmenting NK cell function may be central to defense against age-related degenerative and malignant diseases. This review explores the underlying mechanisms behind these interactions, focusing on how aging influences the battle between the immune system and cancer, the implications of senescent NK cells in disease progression, and the potential of adoptive NK cell therapy as a countermeasure to these age-related immunological challenges.

Myeloid malignancies include various types of cancers that arise from the abnormal development or proliferation of myeloid cells within the bone marrow. Chimeric antigen receptor (CAR) T cell treatments, which show great potential for B cell and plasma cell cancers, face major challenges when used for myeloid malignancies. CAR natural killer (NK) cell-based immunotherapy encounters several challenges in treating myeloid cancers, including (i) poor gene transfer efficiency and expansion platforms in vitro, (ii) limited proliferation and persistence in vivo, (iii) antigenic heterogeneity, and (iv) an immunosuppressive tumor microenvironment. Despite these hurdles, "off-the-shelf" CAR-NK treatments showed encouraging results, marked by enhanced proliferation, prolonged persistence, enhanced tumor infiltration, and improved adaptability. This review offers a summary of the biological traits and cellular sources of NK cells along with a discussion of contemporary CAR designs. Furthermore, it addresses the challenges observed in preclinical research and clinical trials related to CAR-NK cell therapy for myeloid cancers, suggesting enhancement strategies.

The recent advancements in cancer immunotherapy have spotlighted the potential of natural killer (NK) cells, particularly chimeric antigen receptor (CAR)-transduced NK cells. These cells, pivotal in innate immunity, offer a rapid and potent response against cancer cells and pathogens without the need for prior sensitization or recognition of peptide antigens. Although NK cell genetic modification is evolving, the viral transduction method continues to be inefficient and fraught with risks, often resulting in cytotoxic outcomes and the possibility of insertional mutagenesis. Consequently, there has been a surge in the development of non-viral transfection technologies to overcome these challenges in NK cell engineering. Non-viral approaches for CAR-NK cell generation are becoming increasingly essential. Cutting-edge techniques such as trogocytosis, electroporation, lipid nanoparticle (LNP) delivery, clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR-Cas9) gene editing and transposons not only enhance the efficiency and safety of CAR-NK cell engineering but also open new avenues for novel therapeutic possibilities. Additionally, the infusion of technologies already successful in CAR T-cell therapy into the CAR-NK paradigm holds immense potential for further advancements. In this review, we present an overview of the potential of NK cells in cancer immunotherapies, as well as non-viral transfection technologies for engineering NK cells.

In recent times, the application of CAR-T cell treatment has significantly progressed, showing auspicious treatment outcomes in hematologic malignancies. However, along with these advances, certain limitations and challenges hurdle the widespread utilization of this technology. Recently, CAR-NK cells have gained attention in cancer treatment, as this approach has an important advantage over CART therapy (i.e. no need for HLA matching) for targeting foreign cells. This review aims to explore the benefits of CAR NK cell therapy, and generation strategies, as well as the challenges and limitations hindering the application of CAR NK cells in experimental studies and trials on hematologic malignancies.

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.

This study aims to explore the potential correlation between circulating inflammatory proteins and carcinoid syndrome (CS). Using summary data from genome-wide association studies (GWAS), we conducted a Mendelian randomization (MR) analysis with two samples, treating 91 circulating inflammatory proteins as exposure factors and CS as the outcome. Based on genetic loci closely associated with circulating inflammatory proteins selected as instrumental variables, we primarily employed the inverse-variance weighted (IVW) method for analysis, combined with the weighted median method (WM), simple median method (SM), weighted mode estimation (WME), and MR-Egger regression for comprehensive analysis. Initial IVW results revealed significant causal effects of six circulating inflammatory proteins on CS. Specifically, Interleukin-17C levels were negatively correlated with CS risk, indicating a protective effect; whereas beta-nerve growth factor, C-C motif chemokine 20, Natural killer cell receptor 2B4, C-X-C motif chemokine 5, and Leukemia inhibitory factor levels were positively correlated with CS risk, suggesting detrimental effects. In heterogeneity tests, the selected single-nucleotide polymorphisms (SNPs) did not show heterogeneity, and analysis using Egger intercept and MR-PRESSO test did not detect pleiotropy of SNPs, thus validating the reliability of the study. Furthermore, sensitivity analysis using the leave-one-out method further confirmed the robustness of the results. In summary, this study identified significant causal relationships between six inflammatory proteins-Interleukin-17C, beta-nerve growth factor, C-C motif chemokine 20, Natural killer cell receptor 2B4, C-X-C motif chemokine 5, and Leukemia inhibitory factor-and CS risk through MR analysis. This finding not only emphasizes the important role of inflammation in the pathogenesis of CS but also suggests the potential value of inflammatory proteins as targets for early diagnosis and therapeutic interventions.

Viral infections persist as a significant cause of morbidity and mortality worldwide. Conventional therapeutic approaches often fall short in fully eliminating viral infections, primarily due to the emergence of drug resistance. Natural killer (NK) cells, one of the important members of the innate immune system, possess potent immunosurveillance and cytotoxic functions, thereby playing a crucial role in the host's defense against viral infections. Chimeric antigen receptor (CAR)-NK cell therapy has been developed to redirect the cytotoxic function of NK cells specifically towards virus-infected cells, further enhancing their cytotoxic efficacy. In this manuscript, we review the role of NK cells in antiviral infections and explore the mechanisms by which viruses evade immune detection. Subsequently, we focus on the optimization strategies for CAR-NK cell therapy to address existing limitations. Furthermore, we discuss significant advancements in CAR-NK cell therapy targeting viral infections, including those caused by severe acute respiratory syndrome coronavirus 2, human immunodeficiency virus, hepatitis B virus, human cytomegalovirus, and Epstein-Barr virus.

Therapeutic strategies that can target multilevel immunoregulatory pathways in inflammatory bowel disease (IBD) and efficiently target the site of inflammation are expected to greatly enhance the therapeutic efficacy. Here, we have developed a DNA nanorobot-armed bidirectional resistant mesenchymal stem cell (MSC) for IBD treatment, which blocks lymphocyte infiltration at the site of inflammation by bidirectional inhibition of integrin-ligand inter-recognition via resistant aptamer-hands. And this strategy can induce MSC homing for immunomodulation and tissue repair. Herein, in this nanorobot, tetrahedral DNA (TDN) serves as a communication bridge, Integrin α4 and VCAM 1 aptamers are equipped to two vertices of TDN, and the other two cholesterol vertices of TDN are used for immobilization on MSC. In murine colitis models, tail vein-injected resistant MSC preferentially and rapidly accumulated in the inflamed colon and have been more effective in reducing colonic inflammation than pure MSC or aptamers bidirectional inhibitors. The therapeutic strategy proposed in this work has minimal systemic side effects and holds therapeutic promise for a subgroup of IBD patients who do not respond to single anti-inflammatory therapies.

Chimeric antigen receptors (CARs) are synthetic receptors that reprogram the target specificity and functions of CAR-expressing effector cells. The design of CAR constructs typically includes an extracellular antigen-binding moiety, hinge (H), transmembrane (TM), and intracellular signaling domains. Conventional CAR constructs are primarily designed for T cells but have been directly adopted for other effector cells, including natural killer (NK) cells, without tailored optimization. Given the benefits of CAR-NK cells over CAR-T cells in terms of safety, off-the-shelf utility, and antigen escape, there is an increasing emphasis on tailoring them to NK cell activation mechanisms.

We first have taken a stepwise approach to modifying CAR components such as the combination and order of the H, TM, and signaling domains to achieve such tailoring in NK cells. Functionality of NK-tailored CARs were evaluated in vitro and in vivo in a model of CD19-expressing lymphoma, along with their expression and signaling properties in NK cells.

We found that NK-CAR driven by the synergistic combination of NK receptors NKG2D and 2B4 rather than DNAM-1 and 2B4 induces potent activation in NK cells. Further, more effective CAR-mediated cytotoxicity was observed following the sequential combination of DAP10, but not NKG2D TM, with 2B4 signaling domain despite the capacity of NKG2D TM to recruit endogenous DAP10 for signaling. Accordingly, an NK-CAR incorporating DAP10, 2B4, and CD3ζ signaling domains coupled to CD8α H and CD28 TM domains was identified as the most promising candidate to improve CAR-mediated cytotoxicity. This NK-tailored CAR provided more potent antitumor activity than a conventional T-CAR when delivered to NK cells both in vitro and in vivo.

Hence, NK receptor-based domains hold great promise for the future of NK-CAR design with potentially significant therapeutic benefits.

MDS is a heterogeneous group of myeloid neoplasms originating from hematopoietic stem cells, with a high risk of transformation into acute myeloid leukemia (AML). Natural Killer (NK) cells, crucial for their role in immune surveillance and efficient tumor cell lysis, experience functional impairments due to the complex microenvironment and cytokine dynamics in MDS. This article focuses on the mechanisms of NK cell dysfunction in MDS and the latest strategies to enhance NK cell activity to restore their anti-MDS efficacy, highlighting their key role and potential in MDS therapy.

Chimeric antigen receptor (CAR) engineered NK cells (CAR-NK) are a novel approach to the immunotherapy of hematologic malignancies which seeks to overcome some of the challenges faced by CAR-T cells (CAR-T). With few published clinical studies, preclinical studies can identify strategies to accelerate clinical translation. We conducted a systematic review on the preclinical in vivo use of CAR-NK for the treatment of hematologic malignancies to assess these therapies in a holistic and unbiased manner.

Our protocol was registered with PROSPERO (ID: CRD42023438375). We performed a search of OVID MEDLINE, OVID Embase, and Embase for animal studies employing human CAR-NK cells in the treatment of hematologic malignancies. Screening of studies for eligibility criteria was performed in duplicate. Our primary outcomes were survival and reduction in tumor volume. Data extraction from individual experiments was performed by one reviewer using DigitizeitTM software and verified by a second reviewer. Meta-analysis and subgroup analyses were performed using Comprehensive Meta-AnalysisTM software. Information for descriptive outcomes was extracted in duplicate by two independent reviewers. Risk of bias was assessed using the SYRCLE Risk of Bias Tool for Animal Studies.

A total of 34 papers met eligibility criteria. Overall, CD19 was the most common antigen targeted however there was substantial diversity in antigenic targets, source material for generating CAR-NK cells, and NK cell modifications. Mice treated with CAR-NK therapy survived significantly longer than untreated mice (median survival ratio of 1.18, 95% CI: 1.10-1.27, P < 0.001), and mice treated with nonengineered NK cells (median survival ratio 1.13, 95% CI: 1.03-1.23, P < 0.001). Similarly, treatment with CAR-NK significantly reduced the tumor burden when compared to untreated mice (ratio of mean tumor volume 0.23, 95% CI: 0.17-0.32, P < 0.001) or mice treated with nonengineered NK cells (ratio of mean tumor volume 0.37, 95% CI: 0.28-0.51, P < 0.001). Subgroup analysis showed that cotreatment with IL-15 reduced tumor volume but did not increase survival. In general, CAR-NK cell persistence was short but was increased by IL-15.

CAR-NK shows promise for the treatment of hematologic malignancies in preclinical models.

Over the past few decades, immunotherapy has emerged as a powerful strategy to overcome the limitations of conventional cancer treatments. The use of extracellular vesicles, particularly exosomes, which carry cargoes capable of modulating the immune response, has been extensively explored as a potential therapeutic approach in cancer immunotherapy. Exosomes can deliver their cargo to target cells, thereby influencing their phenotype and immunomodulatory functions. They exhibit either immunosuppressive or immune-activating characteristics, depending on their internal contents. These exosomes originate from diverse cell sources, and their internal contents can vary, suggesting that there may be a delicate balance between immune suppression and stimulation when utilizing them for immunotherapy. Therefore, a thorough understanding of the molecular mechanisms underlying the role of exosomes in cancer progression is essential. This review focuses on the molecular mechanisms driving exosome function and their impact on the tumor microenvironment (TME), highlighting the intricate balance between immune suppression and activation that must be navigated in exosome-based therapies. Additionally, it underscores the challenges and ongoing efforts to optimize exosome-based immunotherapies, thereby making a significant contribution to the advancement of cancer immunotherapy research.

Chimeric antigen receptor (CAR) immune cell therapies have revolutionized oncology, particularly in hematological malignancies, yet their efficacy against solid tumors remains limited due to challenges such as dense stromal barriers and immunosuppressive microenvironments. With advancements in nanobiotechnology, researchers have developed various strategies and methods to enhance the CAR cell efficacy in solid tumor treatment. In this Review, we first outline the structure and mechanism of CAR-T (T, T cell), CAR-NK (NK, natural killer), and CAR-M (M, macrophage) cell therapies and deeply analyze the potential of these cells in the treatment of solid tumors and the challenges they face. Next, we explore how biomaterials can optimize these treatments by improving the tumor microenvironment, controlling CAR cell release, promoting cell infiltration, and enhancing efficacy. Finally, we summarize the current challenges and potential solutions, emphasize the effective combination of biomaterials and CAR cell therapy, and look forward to its future clinical application and treatment strategies. This Review provides important theoretical perspectives and practical guidance for the future development of more effective solid tumor treatment strategies.

Chimeric antigen receptor (CAR) T cell therapy is a promising immunotherapy against cancer. Although there is a growing interest in other cell types, a comparison of CAR immune effector cells in challenging solid tumor contexts is lacking. Here, we compare mouse and human NKG2D-CAR-expressing T cells, natural killer (NK) cells, and macrophages against glioblastoma, the most aggressive primary brain tumor. In vitro we show that T cell cancer killing is CAR dependent, whereas intrinsic cytotoxicity overrules CAR dependence for NK cells, and CAR macrophages reduce glioma cells in co-culture assays. In orthotopic immunocompetent glioma mouse models, systemically administered CAR T cells demonstrate superior accumulation in the tumor, and each immune cell type induces distinct changes in the tumor microenvironment. An otherwise low therapeutic efficacy is significantly enhanced by co-expression of pro-inflammatory cytokines in all CAR immune effector cells, underscoring the necessity for multifaceted cell engineering strategies to overcome the immunosuppressive solid tumor microenvironment.