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.

Cell-based immunotherapies, including CAR-T, CAR-NK, and TCR-T therapies, represent a transformative approach to cancer treatment by offering precise targeting of tumor cells. Despite their success in hematologic malignancies, these therapies encounter significant challenges in treating solid tumors, such as antigen heterogeneity, immunosuppressive tumor microenvironments, limited cellular infiltration, off-target toxicity, and difficulties in manufacturing scalability. CAR-T cells have demonstrated exceptional efficacy in blood cancers but face obstacles in solid tumors, whereas CAR-NK cells offer reduced graft-versus-host disease but encounter similar barriers. TCR-T cells expand the range of treatable cancers by targeting intracellular antigens but require meticulous antigen selection to prevent off-target effects. Alternative therapies like TIL, NK, and CIK cells show promise but require further optimization to enhance persistence and overcome immunosuppressive barriers. Manufacturing complexity, high costs, and ensuring safety and efficacy remain critical challenges. Future advancements in gene editing, multi-antigen targeting, synthetic biology, off-the-shelf products, and personalized medicine hold the potential to address these issues and expand the use of cell-based therapies. Continued research and innovation are essential to improving safety, efficacy, and scalability, ultimately leading to better patient outcomes.

Chimeric antigen receptor (CAR)-modified NK (CAR-NK) cells are candidates for next-generation cancer immunotherapies. Here we generated CD19-specific CAR-NK cells with 4-1BB and CD3ζ signaling endo-domains (CD19-BBz CAR-NK) by transduction of cord blood-derived NK cells using baboon envelope pseudotyped lentiviral vectors and demonstrated their antitumor activity in preclinical B cell lymphoma models in female mice. We next conducted a phase 1 dose-escalation trial involving repetitive administration of CAR-NK cells in 8 patients with relapsed/refractory large B cell lymphoma (NCT05472558). Primary end points were safety, maximum tolerated dose, and overall response rate. Secondary end points included duration of response, overall survival, and progression-free survival. No dose-limiting toxicities occurred, and the maximum tolerated dose was not reached. No cases of cytokine release syndrome, neurotoxicity, or graft-versus-host disease were observed. Results showed an overall response rate of 62.5% at day 30, with 4 patients (50%) achieving complete response. The median progression-free survival was 9.5 months, and the median overall survival was not reached. A post hoc exploratory single-cell RNA sequencing analysis revealed molecular features of CAR-NK cells associated with therapeutic efficacy and efficacy-related immune cell interaction networks. This study met the pre-specified end points. In conclusion, CD19-BBz CAR-NK cells were feasible and therapeutically safe, capable of inducing durable response in patients with B cell lymphoma.

Gastric cancer remains a significant global health burden, characterized by regional variations in incidence and poor survival prospects in advanced stages. Natural killer (NK) cells play a crucial role in the body's anti-cancer defense, and chimeric antigen receptor (CAR)-NK cell therapy is gaining attention as a cutting-edge and promising treatment method. This study aims to tackle the challenge of TGF-β-mediated tumor immune evasion within the immunosuppressive tumor microenvironment by designing a novel chimeric cytokine receptor TRII/21 R, which consists of extracellular domains of TGF-β receptor II (TRII) and transmembrane and intracellular domains of IL-21 receptor (21 R) and can convert the immunosuppressive signal from TGF-β in the tumor microenvironment (TME) into an NK cell activation signal through the IL-21R-STAT3 pathway. We successfully constructed NKG2D-CAR-NK cells expressing TRII/21 R and demonstrated strong anti-tumor activity against cancer cells both in vitro and in vivo. The co-expression of TRII/21 R in CAR-NK cells enhanced the cytotoxicity, promoted proliferation and survival capabilities, and reduced the expression of exhaustion markers. In the xenograft mouse model, TRII/21R-CAR-NK cells significantly inhibited tumor growth and improved the survival rate of tumor-bearing mice compared to the mice receiving control CAR-NK cells. Additionally, TRII/21 R co-expression enhanced NK cells' infiltration, activation, and persistence within the tumor, indicating a robust anti-tumor response mediated by the JAK-STAT3 signaling pathway. This study underscores the therapeutic potential of TRII/21R-modified CAR-NK cells as a breakthrough strategy for combating cancer.

ErbB3 is markedly overexpressed in breast cancer cells and is associated with resistance and metastasis. Additionally, ErbB3 expression levels are positively correlated with low densities of tumor-infiltrating lymphocytes, a marker of poor prognosis. Consequently, ErbB3 is a promising therapeutic target for cancer immunotherapy. Here, we report the generation of ErbB3-targeted chimeric antigen receptor (CAR)-modified natural killer (NK) cells by transducing cord blood-derived primary NK cells using vsv-g envelope-pseudotyped lentiviral vectors. Transduced cells displayed stable CAR-expressing activity and increased cytotoxicity against ErbB3-positive breast cancer cell lines. Furthermore, anti-ErbB3 (aErbB3) CAR-NK cells strongly reduced the tumor burden in the SK-BR-3 xenograft mouse model without observable side effects. These findings underscore the potential of aErbB3 CAR-NK cells as targeted immunotherapy for ErbB3-positive breast cancer, suggesting a promising alternative to conventional treatments.

CAR-T cell therapy, a cutting-edge cellular immunotherapy with demonstrated efficacy in treating hematologic malignancies, also exhibits significant promise for addressing autoimmune diseases. This innovative therapeutic approach holds promise for achieving long-term remission in autoimmune diseases, potentially offering significant benefits to affected patients. Current targets under investigation for the treatment of these conditions include CD19, CD20, and BCMA, among others. However, CAR-T therapy faces difficulties such as time-consuming cell manufacturing, complex and expensive process, and the possibility of severe adverse reactions complicating the treatment, etc. This article examines CAR-T therapy across various rheumatic autoimmune diseases, including systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), Sjögren's syndrome (SS), systemic sclerosis (SSc), antisynthetase syndrome (ASS), and ANCA-associated vasculitis (AAV), highlighting both therapeutic advancements and ongoing challenges.

Immune cell effector therapies, including chimeric antigen receptor (CAR)-T cells, T-cell receptor (TCR) T cells, natural killer (NK) cells, and macrophage-based therapies, represent a transformative approach to cancer treatment, harnessing the immune system to target and eradicate malignant cells. CAR-T cell therapy, the most established among these, involves engineering T cells to express CARs specific to cancer cell antigens, showing remarkable efficacy in hematologic malignancies like leukemias, B-cell lymphomas, and multiple myeloma. Similarly, TCR-modified therapies, which reprogram T cells to recognize intracellular tumor antigens presented by major histocompatibility complex (MHC) molecules, offer promise for a range of solid tumors. NK-cell therapies leverage NK cells' innate cytotoxicity, providing an allogeneic approach that avoids some of the immune-related complications associated with T-cell-based therapies. Macrophage-based therapies, still in early stages of the development, focus on reprogramming macrophages to stimulate an immune response against cancer cells in the tumor microenvironment. Despite their promise, socioeconomic and regulatory challenges hinder the accessibility and scalability of immune cell effector therapies. These treatments are costly, with CAR-T therapies currently exceeding $400,000 per patient, creating significant disparities in access based on socioeconomic status and geographic location. The high manufacturing costs stem from the personalized, labor-intensive processes of harvesting, modifying, and expanding patients' cells. Moreover, complex logistics for manufacturing and delivering these therapies limit their reach, particularly in low-resource settings. Regulatory pathways further complicate the landscape. In the United States., the Food and Drug Administrations' (FDA) accelerated approval processes for cell-based therapies facilitate innovation but do not address cost-related barriers. In Europe, the European Medicines Agency (EMA) offers adaptive pathways, yet decentralized reimbursement systems create uneven access across member states. Additionally, differing regulatory standards for manufacturing and quality control worldwide pose hurdles for global harmonization and access. To expand the reach of immune effector cell therapies, a multipronged approach is needed-streamlined regulatory frameworks, policies to reduce treatment costs, and international collaborations to standardize manufacturing. Addressing these socioeconomic and regulatory obstacles is essential to make these life-saving therapies accessible to a broader patient population worldwide. We present a literature review on the current landscape of immune effector cell therapies and barriers of access to currently approved standard of care therapy at various levels.

Cancer immunotherapy is gaining increasing attention. However, immune checkpoints are exploited by cancer cells to evade anti-tumor immunotherapy. Here, we knocked out NKG2A, an immune checkpoint expressed on natural killer (NK) cells, in human pluripotent stem cells (hPSCs) and differentiated these hPSCs into NK (PSC-NK) cells. We show that NKG2A knockout (KO) enhances the anti-tumor and anti-viral capabilities of PSC-NK cells. NKG2A KO endows PSC-NK cells with higher cytotoxicity against HLA-E-expressing glioblastoma (GBM) cells, leukemia cells, and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected cells in vitro. The NKG2A KO PSC-NK cells also exerted potent anti-tumor activity in vivo, leading to substantially suppressed tumor progression and prolonged survival of tumor-bearing mice in a xenograft GBM mouse model. These findings underscore the potential of PSC-NK cells with immune checkpoint KO as a promising cell-based immunotherapy. The unlimited supply and ease of genetic engineering of hPSCs makes genetically engineered PSC-NK an attractive option for easily accessible "off-the-shelf" cancer immunotherapy.

Gastrointestinal (GI) cancers represent a significant health burden worldwide. Their incidence continues to increase, and their management remains a clinical challenge. Chimeric antigen receptor (CAR) natural killer (NK) cells have emerged as a promising alternative to CAR-T cells for immunotherapy of GI cancers. Notably, CAR-NK cells offer several advantages, including reduced risk of graft-versus-host disease, lower cytokine release syndrome, and the ability to target cancer cells through both CAR-dependent and natural cytotoxic mechanisms.

This review comprehensively discusses the development and applications of CAR-NK cells in the treatment of GI cancers. We explored various sources of NK cells, CAR design strategies, and the current state of CAR-NK cell therapy for GI cancers, highlighting recent preclinical and clinical trials. Additionally, we addressed existing challenges and propose potential strategies to enhance the efficacy and safety of CAR-NK cell therapy.

Our findings highlight the potential of CAR-NK cells to revolutionize GI cancer treatment and pave the way for future clinical applications.

Autologous chimeric antigen receptor (CAR)-modified T (CAR-T) cell therapy has displayed high efficacy in the treatment of hematological malignancies. Up to now, 11 autologous CAR-T cell products have been approved for the management of malignancies globally. However, the application of autologous CAR-T cell therapy has many individual limitations, long time-consuming, highly cost, and the risk of manufacturing failure. Indeed, some patients would not benefit from autologous CAR-T cell products because of rapid disease progression. Allogeneic CAR-T cells especially universal CAR-T (U-CAR-T) cell therapy are superior to these challenges of autologous CAR-T cells. In this review, we describe basic study and clinical trials of U-CAR-T cell therapeutic methods for malignancies. In addition, we summarize the problems encountered and potential solutions.

In the past decade, chimeric antigen receptor (CAR)-T cell therapy has emerged as a promising immunotherapeutic approach for combating cancers, demonstrating remarkable efficacy in relapsed/refractory hematological malignancies in both pediatric and adult patients. CAR-natural killer (CAR-NK) cell complements CAR-T cell therapy by offering several distinct advantages. CAR-NK cells do not require HLA compatibility and exhibit low safety concerns. Moreover, CAR-NK cells are conducive to "off-the-shelf" therapeutics, providing significant logistic advantages over CAR-T cells. Both CAR-T and CAR-NK cells have shown consistent and promising results in hematological malignancies. However, their efficacy against solid tumors remains limited due to various obstacles including limited tumor trafficking and infiltration, as well as an immuno-suppressive tumor microenvironment. In this review, we discuss the recent advances and current challenges of CAR-T and CAR-NK cell immunotherapies, with a specific focus on the obstacles to their application in solid tumors. We also analyze in depth the advantages and drawbacks of CAR-NK cells compared to CAR-T cells and highlight CAR-NK CAR optimization. Finally, we explore future perspectives of these adoptive immunotherapies, highlighting the increasing contribution of cutting-edge biotechnological tools in shaping the next generation of cellular immunotherapy.

Cell surface engineering with exogeneous receptors holds great promise for various applications. However, current biological methods face problems with safety, antigen escape, and receptor stoichiometry. The purpose of this study is to develop a biochemical method for displaying polyvalent antibodies (PAbs) on the cell surface. The PAbs are synthesized through the self-assembly of DNA-Ab conjugates under physiological conditions without the involvement of any factors harsh to cells. The data show that PAb-functionalized cells can recognize target cells much more effectively than monovalent controls. Moreover, dual Ab incorporation into the same PAb with a defined stoichiometric ratio leads to the formation of a polyvalent hybrid Ab (DPAb). DPAb-functionalized cells can effectively recognize target cell models with antigen escape, which cannot be achieved by PAbs with one type of Ab. Therefore, this work presents a novel biochemical method for Ab display on the cell surface for enhanced cell recognition.

We have developed and validated a highly specific, versatile antibody to the extracellular domain of human LGR5 (α-LGR5). α-LGR5 detects LGR5 overexpression in >90% of colorectal cancer (CRC), hepatocellular carcinoma (HCC) and pre-B-ALL tumour cells and was used to generate an Antibody-Drug Conjugate (α-LGR5-ADC), Bispecific T-cell Engager (α-LGR5-BiTE) and Chimeric Antigen Receptor (α-LGR5-CAR). α-LGR5-ADC was the most effective modality for targeting LGR5+ cancer cells in vitro and demonstrated potent anti-tumour efficacy in a murine model of human NALM6 pre-B-ALL driving tumour attrition to less than 1% of control treatment. α-LGR5-BiTE treatment was less effective in the pre-B-ALL cancer model yet promoted a twofold reduction in tumour burden. α-LGR5-CAR-T cells also showed specific and potent LGR5+ cancer cell killing in vitro and effective tumour targeting with a fourfold decrease in pre-B-ALL tumour burden relative to controls. Taken together, we show that α-LGR5 can not only be used as a research tool and a biomarker but also provides a versatile building block for a highly effective immune therapeutic portfolio targeting a range of LGR5-expressing cancer cells.

Natural Killer (NK) cells play a crucial role as effector cells within the tumor immune microenvironment, capable of identifying and eliminating tumor cells through the expression of diverse activating and inhibitory receptors that recognize tumor-related ligands. Therefore, harnessing NK cells for therapeutic purposes represents a significant adjunct to T cell-based tumor immunotherapy strategies. Presently, NK cell-based tumor immunotherapy strategies encompass various approaches, including adoptive NK cell therapy, cytokine therapy, antibody-based NK cell therapy (enhancing ADCC mediated by NK cells, NK cell engagers, immune checkpoint blockade therapy) and the utilization of nanoparticles and small molecules to modulate NK cell anti-tumor functionality. This article presents a comprehensive overview of the latest advances in NK cell-based anti-tumor immunotherapy, with the aim of offering insights and methodologies for the clinical treatment of cancer patients.

Ovarian cancer is a lethal gynaecological malignancy with unsatisfactory 5 year survival rates of 30-50 %. Cell immunotherapy is a promising new cancer treatment where immune cells, such as Natural Killer (NK) cells, are administered to enable the patient to fight cancer through direct cytotoxicity. NK cells orchestrate an adaptive immune response by enabling the release of tumour antigens. NK cell cytotoxicity and effector responses are largely driven by TRAIL engagement. In this study we investigated the cytotoxic potential of a human NK cell line that were modified to express a potent DR5 specific TRAIL variant. We hypothesised that this modification would enhance NK cell cytotoxicity against TRAIL sensitive and resistant ovarian cancer cell lines in vitro.

KHYG-1 human NK cells were modified with a TRAIL variant targeting DR5 (TRAILv-KHYG-1). Human ovarian cancer cell lines, OVCAR-3 and SKOV-3, were cultured with modified or non-modified NK cells at different effector:target (E:T) ratios for 4 or 16 h. Apoptosis was assessed by Annexin-APC and 7-AAD and measured using flow cytometry. Apoptotic cells were defined as annexin V 7-AAD double positive. Cytokine expression was measured by multiplex ELISA, and analysed by flow cytometry.

Modified and non-modified NK cells significantly reduced OVCAR-3 cell viability as compared to OVCAR-3 cells that were cultured alone after 4 and 16 h treatment. OVCAR-3 cell viability was reduced after treatment with 1:1 E:T ratio with TRAILv-KHYG-1 cells after 16 h. On the contrary, neither NK cell line had any effect of SKOV-3 cell viability despite SKOV-3 cells having more DR5 surface expression compared to OVCAR-3 cells.

TRAILv-KHYG-1 cells significantly reduced OVCAR-3 cell viability as compared to non-modified NK cells. However, no significant reduction in viability was observed when SKOV-3 cell were cultured with either NK cells, despite having more DR5 surface expression compared to OVCAR-3 cells. These data indicate that mechanisms other than DR5 expression drive TRAIL resistance in ovarian cancer.

Using natural killer (NK) cells to treat hematopoietic and solid tumors has great promise. Despite their availability from peripheral blood and cord blood, stem cell-derived NK cells provide an "off-the-shelf" solution.

In this study, we developed two CAR-NK cells targeting PD-L1 derived from lentiviral transduction of human umbilical cord blood (UCB)-CD34+ cells and UCB-CD34+-derived NK cells. The transduction efficiencies and in vitro cytotoxic functions including degranulation, cytokine production, and cancer cell necrosis of both resultants PD-L1 CAR-NK cells were tested in vitro on two different PD-L1 low and high-expressing solid tumor cell lines.

Differentiated CAR‑modified UCB-CD34+ cells exhibited enhanced transduction efficiency. The expression of anti-PD-L1 CAR significantly (P < 0.05) enhanced the cytotoxicity of differentiated CAR‑modified UCB-CD34+ cells and CAR-modified UCB-CD34+-derived NK cells against PD-L1 high-expressing tumor cell line. In addition, CAR-modified UCB-CD34+-derived NK cells significantly (P < 0.05) restored the tumor-killing ability of exhausted PD-1 high T cells.

Considering the more efficient transduction in stem cells and the possibility of producing CAR-NK cell products with higher yields, this approach is recommended for studies in the field of CAR-NK cells. Also, a pre-clinical study is now necessary to evaluate the safety and efficacy of these two CAR-NK cells individually and in combination with other therapeutic approaches.

Natural killer (NK) cells are crucial components of innate immunity, known for their potent tumor surveillance abilities. Chimeric antigen receptors (CARs) have shown promise in cancer targeting, but optimizing CAR designs for NK cell functionality remains challenging. CAR-NK cells have gained attention for their potential to reduce side effects and enable scalable production in cancer immunotherapy. This study aimed to enhance NK cell anti-tumor activity by incorporating PD1-synthetic Notch (synNotch) receptors. A chimeric receptor was designed using UniProt database sequences, and 3D structure models were generated for optimization. Lentiviral transduction was used to introduce PD1-Syn receptors into NK cells. The expression of PD1-Syn receptors on NK cell surfaces was assessed. Engineered NK cells were co-cultured with PDL1+ breast cancer cells to evaluate their cytotoxic activity and ability to produce interleukin-12 (IL-12) and interferon-gamma (IFNγ) upon interaction with the target cells. This study successfully expressed the PD1-Syn receptors on NK cells. CAR-NK cells secreted IL-12 and exhibited target-dependent IFNγ production when engaging PDL1+ cells. Their cytotoxic activity was significantly enhanced in a target-dependent manner. This study demonstrates the potential of synNotch receptor-engineered NK cells in enhancing anti-tumor responses, especially in breast cancer cases with high PDL1 expression.

Chimeric antigen receptor natural killer (CAR-NK) cells have been found to be successful in treating hematologic malignancies and present potential for usage in solid tumors.

In this study, we created CD276-targeted CAR-expressing NK cells from pluripotent stem cells (iPSC CD276-targeted CAR-NK cells) and evaluated their cytotoxicity against esophageal squamous cell carcinoma (ESCC) using patient-specific organoid (PSO) models comprising of both CD276-positive and CD276-negative adjacent epithelium PSO models (normal control PSO, NC PSO) as well as primary culture of ESCC cell models. In addition, in vitro and in vivo models such as KYSE-150 were also examined. iPSC NK cells and NK-free media were used as the CAR-free and NK-free controls, respectively.

The positive CD276 staining was specifically detected on the ESCC membrane in 51.43% (54/105) of the patients of all stages, and in 51.35% (38/74) of stages III and IV. The iPS CD276-targeted CAR-NK cells, comparing with the iPS NK cells and the NK-free medium, exhibited specific and significant cytotoxic activity against CD276-positive ESCC PSO rather than CD276-negative NC PSO, and exhibited significant cytotoxicity against CD276-expressing cultured ESCC cells, as well as against CD276-expressing KYSE-150 in vitro and in BNDG mouse xenograft.

The efficacy of the iPSC CD276-targeted CAR-NK cells demonstrated by their successful treatment of CD276-expressing ESCC in a multitude of pre-clinical models implied that they hold tremendous therapeutic potential for treating patients with CD276-expressing ESCC.

The clinical efficacy of CAR-NK cells against CD19-expressing blood cancers has been demonstrated, and they have shown potential for treating solid tumors as well. However, the efficacy of CAR-NK cells for treating human oral tongue squamous cell carcinoma (OTSCC) has not been examined.

We assessed MUC1 expression in human OTSCC tissue and a cell line using immunohistochemistry and immunofluorescence. We constructed NK cells that express CAR targeted to MUC1 from pluripotent stem cells (iPSC-derived MUC1-targeted CAR-NK cells) and evaluated their effectiveness against OTSCC in vitro using the xCELLigence Real-Time Cell Analysis system and CCK8 assay, and in vivo by measuring xenograft growth daily in BNDG mice treated with MUC1-targeted CAR-NK cells. As controls, we used iPSC-derived NK cells and NK-free media, which were CAR-free and blank, respectively.

MUC1 expression was detected in 79.5% (66/83) of all OTSCC patients and 72.7% (24/33) of stage III and IV. In stage III and IV MUC1 positive OTSCC, 63.6% (21/33) and 48.5% (16/33) patients had a MUC1-positive cancer cell rate of more than 50% and 80%, respectively. The iPSC-derived MUC1-targeted CAR-NK cells exhibited significant cytotoxicity against MUC1-expressing OTSCC cells in vitro, in a time- and dose-dependent manner, and showed a significant inhibitory effect on xenograft growth compared to both the iPSC-derived NK cells and the blank controls. We observed no weight loss, severe hematological toxicity or NK cell-mediated death in the BNDG mice.

The MUC1-targeted CAR-NK cells had significant efficacy against human OTSCC, and their promising therapeutic response warrants further clinical trials.

Establishing reliable and reproducible animal models for disease modelling, drug screening and the understanding of disease susceptibility and pathogenesis is critical. However, traditional animal models differ significantly from humans in terms of physiology, immune response, and pathogenesis. As a result, it is difficult to translate laboratory findings into biomedical applications. Although several animal models with human chimeric genes, organs or systems have been developed in the past, their limited engraftment rate and physiological functions are a major obstacle to realize convincing models of humans. The lack of human transplantation resources and insufficient immune tolerance of recipient animals are the main challenges that need to be overcome to generate fully humanized animals. Recent advances in gene editing and pluripotent stem cell-based xenotransplantation technologies offer opportunities to create more accessible human-like models for biomedical research. In this article, we have combined our laboratory expertise to summarize humanized animal models, with a focus on hematopoietic/immune system and liver. We discuss their generation strategies and the potential donor cell sources, with particular attention given to human pluripotent stem cells. In particular, we discuss the advantages, limitations and emerging trends in their clinical and pharmaceutical applications. By providing insights into the current state of humanized animal models and their potential for biomedical applications, this article aims to advance the development of more accurate and reliable animal models for disease modeling and drug screening.

Treatment for renal cell carcinoma (RCC) has improved dramatically over the last decade, shifting from high-dose cytokine therapy in combination with surgical resection of tumors to targeted therapy, immunotherapy, and combination therapies. However, curative treatment, particularly for advanced-stage disease, remains rare. Cell therapy as a "living drug" has achieved hematological malignancy cures with a high response rate, and significant research efforts have been made to facilitate its translation to solid tumors. Herein, we overview the cellular therapies for RCC focusing on allogeneic hematopoietic stem cell transplantation, T cell receptor gene-modified T cells, chimeric antigen receptor (CAR) T cells, CAR natural killer (NK) cells, lymphokine-activated killer (LAK) cells, γδ T cells, and dendritic cell vaccination. We have also included perspectives for using other recent approaches, such as CAR macrophages, dendritic cell-cytokine induced killer cells and regulatory CAR-T cells to shed light on preclinical development of cell therapy and advancing cell therapy into clinic to achieve cures for RCC.

Chimeric antigen receptor T-cell (CAR-T) therapy has emerged as a highly efficacious treatment modality for refractory and relapsed hematopoietic malignancies in recent years. Furthermore, CAR technologies for cancer immunotherapy have expanded from CAR-T to CAR-natural killer cell (CAR-NK), CAR-cytokine-induced killer cell (CAR-CIK), and CAR-macrophage (CAR-MΦ) therapy. Nevertheless, the high cost and complex manufacturing processes of ex vivo generation of autologous CAR products have hampered broader application. There is an urgent need to develop an efficient and economical paradigm shift for exploring new sourcing strategies and engineering approaches toward generating CAR-engineered immune cells to benefit cancer patients. Currently, researchers are actively investigating various strategies to optimize the preparation and sourcing of these potent immunotherapeutic agents. In this work, the latest research progress is summarized. Perspectives on the future of CAR-engineered immune cell manufacturing are provided, and the engineering approaches, and diverse sources used for their development are focused upon.

The last decade has witnessed great progress in the field of adoptive cell therapies, with the authorization of Kymriah (tisagenlecleucel) in 2017 by the Food and Drug Administration (FDA) as a crucial stepstone. Since then, five more CAR-T therapies have been approved for the treatment of hematological malignancies. While this is a great step forward to treating several types of blood cancers, CAR-T cell therapies are still associated with severe side-effects such as Graft-versus-Host Disease (GvHD), cytokine release syndrome (CRS) and neurotoxicity. Because of this, there has been continued interest in Natural Killer cells which avoid these side-effects while offering the possibility to generate allogeneic cell therapies. Similar to T-cells, NK cells can be genetically modified to improve their therapeutic efficacy in a variety of ways. In contrast to T cells, viral transduction of NK cells remains inefficient and induces cytotoxic effects. Viral vectors also require a lengthy and expensive product development process and are accompanied by certain risks such as insertional mutagenesis. Therefore, non-viral transfection technologies are avidly being developed aimed at addressing these shortcomings of viral vectors. In this review we will present an overview of the potential of NK cells in cancer immunotherapies and the non-viral transfection technologies that have been explored to engineer them.

Numerous studies are exploring the use of cell adoptive therapies to treat hematological malignancies as well as solid tumors. However, there are numerous factors that dampen the immune response, including viruses like human immunodeficiency virus. In this study, we leverage human-derived microphysiological models to reverse-engineer the HIV-immune system interaction and evaluate the potential of memory-like natural killer cells for HIV+ head and neck cancer, one of the most common tumors in patients living with human immunodeficiency virus. Here, we evaluate multiple aspects of the memory-like natural killer cell response in human-derived bioengineered environments, including immune cell extravasation, tumor penetration, tumor killing, T cell dependence, virus suppression, and compatibility with retroviral medication. Overall, these results suggest that memory-like natural killer cells are capable of operating without T cell assistance and could simultaneously destroy head and neck cancer cells as well as reduce viral latency.

Chimeric antigen receptor natural killer cells (CAR-NK) promote off-the-shelf cellular therapy for solid tumors and malignancy.However,, the development of CAR-NK is due to their immune surveillance uncertainty and cytotoxicity challenge was restricted. Natural killer cell-derived exosome (NK-Exo) combine crucial targeted cellular therapies of NK cell therapies with unique non-toxic Exo as a self-origin shuttle against cancer immunotherapy. This review study covers cytokines, adoptive (autologous and allogenic) NK immunotherapy, stimulatory and regulatory functions, and cell-free derivatives from NK cells. The future path of NK-Exo cytotoxicity and anti-tumor activity with considering non-caspase-independent/dependent apoptosis and Fas/FasL pathway in cancer immunotherapy. Finally, the significance and implication of NK-Exo therapeutics through combination therapy and the development of emerging approaches for the purification and delivery NK-Exo to severe immune and tumor cells and tissues were discussed in detail.

The reprogramming of terminally differentiated cells over the past few years has become important for induced pluripotent stem cells (iPSCs) in the field of regenerative medicine and disease drug modeling. At the same time, iPSCs have also played an important role in human cancer research. iPSCs derived from cancer patients can be used to simulate the early progression of cancer, for drug testing, and to study the molecular mechanism of cancer occurrence. In recent years, with the application of cellular immunotherapy in cancer therapy, patient-derived iPSC-induced immune cells (T, natural killer, and macrophage cells) solve the problem of immune rejection and have higher immunogenicity, which greatly improves the therapeutic efficiency of immune cell therapy. With the continuous progress of cancer differentiation therapy, iPSC technology can reprogram cancer cells to a more primitive pluripotent undifferentiated state, and successfully reverse cancer cells to a benign phenotype by changing the epigenetic inheritance of cancer cells. This article reviews the recent progress of cell reprogramming technology in human cancer research, focuses on the application of reprogramming technology in cancer immunotherapy and the problems solved, and summarizes the malignant phenotype changes of cancer cells in the process of reprogramming and subsequent differentiation.

Acute myeloid leukemia (AML) is a devastating disease. Intensive chemotherapy is the mainstay of treatment but results in debilitating toxicities. Moreover, many treated patients will eventually require hematopoietic stem cell transplantation (HSCT) for disease control, which is the only potentially curative but challenging option. Ultimately, a subset of patients will relapse or have refractory disease, posing a huge challenge to further therapeutic decisions. Targeted immunotherapies hold promise for relapsed/refractory (r/r) malignancies by directing the immune system against cancer. Chimeric antigen receptors (CARs) are important components of targeted immunotherapy. Indeed, CAR-T cells have achieved unprecedented success against r/r CD19+ malignancies. However, CAR-T cells have only achieved modest outcomes in clinical studies on r/r AML. Natural killer (NK) cells have innate anti-AML functionality and can be engineered with CARs to improve their antitumor response. CAR-NKs are associated with lower toxicities than CAR-T cells; however, their clinical efficacy against AML has not been extensively investigated. In this review, we cite the results from clinical studies of CAR-T cells in AML and describe their limitations and safety concerns. Moreover, we depict the clinical and preclinical landscape of CAR used in alternative immune cell platforms with a specific focus on CAR-NKs, providing insight into the future optimization of AML.

The current high prevalence of malignant tumors has attracted considerable attention, and treating advanced malignancies is becoming increasingly difficult. Although immunotherapy is a hopeful alternative, it is effective in only a few people. Thus, development of preclinical animal models is needed. Humanized xenotransplantation mouse models can help with selecting treatment protocols, evaluating curative effects and assessing prognosis. This review discusses the establishment of humanized mouse models and their application prospects in cancer immunotherapy to identify tailored therapies for individual patients.

Metastatic tumors cause the most deaths in cancer patients. Treating metastasis remains the primary goal of current cancer research. Although the immune system prevents and kills the tumor cells, the function of the immune system in metastatic cancer has been unappreciated for decades because tumors are able to develop complex signaling pathways to suppress immune responses, leading them to escape detection and elimination. Studies showed NK cell-based therapies have many advantages and promise for fighting metastatic cancers. We here review the function of the immune system in tumor progression, specifically focusing on the ability of NK cells in antimetastasis, how metastatic tumors escape the NK cell attack, as well as the recent development of effective antimetastatic immunotherapies.

Adoptive cell therapy with chimeric antigen receptor (CAR)-expressing natural killer (NK) cells is an emerging approach that holds promise in multiple myeloma (MM). However, the generation of CAR-NK cells targeting CD38 is met with obstacles due to the expression of CD38 on NK cells. Knock-out of CD38 is currently explored as a strategy, although the consequences of the lack of CD38 expression with regards to engraftment and activity in the bone marrow microenvironment are not fully elucidated. Here, we present an alternative approach by harnessing the CD38^dim phenotype occurring during long-term cytokine stimulation of primary NK cells.

Primary NK cells were expanded from peripheral blood mononuclear cells by long-term IL-2 stimulation. During expansion, the CD38 expression was monitored in order to identify a time point when introduction of a novel affinity-optimized αCD38-CAR confered optimal viability, i.e. prevented fratricide. CD38^dim NK cells were trasduced with retroviral vectors encoding for the CAR trasngene and their functionality was assessed in in vitro activation and cytotoxicity assays.

We verified the functionality of the αCD38-CAR-NK cells against CD38⁺ cell lines and primary MM cells. Importantly, we demonstrated that αCD38-CAR-NK cells derived from patients with MM have increased activity against autologous MM samples ex vivo.

Overall, our results highlight that incorporation of a functional αCD38-CAR construct into a suitable NK-cell expansion and activation protocol results in a potent and feasible immunotherapeutic strategy for the treatment of patients with MM.

A new therapy strategy for relapsing patients who have received trastuzumab treatment urgently needs to be explored. HER2-specific chimeric antigen receptor (CAR)-expressing NK cells are being rapidly developed for solid tumor therapy, as they have many advantages over HER2-CAR-T cells. Endogenous soluble PD-1 (sPD-1) from the PD-1 extracellular domain blocks PD-1/PD-L1 interaction to promote cancer immunology. Herein, we engineered a new HER2-CAR-NK cell that co-expresses sPD-1 (designed as sPD-1-CAR-NK cells) and assessed its cytotoxic activities toward various cancer cells, activation of immunity and sPD-1 release in vitro and in mouse models bearing breast cancer cells with high HER2 expression, with or without trastuzumab resistance. We demonstrated that sPD-1-CAR-NK cells were able to release bioactive sPD-1, thereby enhancing the cytolytic activities of HER2-CAR-NK cells against HER2 and PD-L1 highly expressing target cells accompanied by increases in the secretion of perforin, granzyme B and IFN-γ. In vivo, sPD-1-CAR-NK cells had superior immunological anticancer efficacy compared to HER2-CAR-NK cells, and they had advantages over HER2-CAR-NK cells in the intraperitoneal injection of sPD-1. Moreover, the infiltration and activation of NK and T cells into tumor tissue were increased in mice with sPD-1-CAR-NK cells. There was no significant change in the body temperature, organ tissue and body weight in all groups except for the group with the PD-1 injection. Together, these data indicate that HER2-specific sPD-1-CAR-NK cells can transport sPD-1 into cancer tissues with high HER2 expression, further improving the efficacy of HER-CAR-NK cells without obvious side effects. sPD-1-CAR-NK is a promising cytotherapeutic agent for patients bearing HER2-positive breast cancer, including those with trastuzumab resistance.

Rapid advancements have taken place in gene therapy technology. However, effective methods for treating aging- or age-related chronic diseases, which are often closely related to genes or even multiple genes, are still lacking. The path to developing cures is winding, while gene therapy that targets genes related to aging represents an exciting research direction with tremendous potential. Among aging-related genes, some candidates have been studied at different levels, from cell to organismal levels (e.g., mammalian models) with different methods, from overexpression to gene editing. The TERT and APOE have even entered the stage of clinical trials. Even those displaying only a preliminary association with diseases have potential applications. This article discusses the foundations and recent breakthroughs in the field of gene therapy, providing a summary of current mainstream strategies and gene therapy products with clinical and preclinical applications. Finally, we review representative target genes and their potential for treating aging or age-related diseases.

The incidence and mortality rates of lung cancer are among the highest in the world. Traditional treatment methods include surgery, chemotherapy, and radiotherapy. Although rapid progress has been achieved in the past decade, treatment limitations remain. It is therefore imperative to identify safer and more effective therapeutic methods, and research is currently being conducted to identify more efficient and less harmful drugs. In recent years, the discovery of antitumor drugs based on the essential trace element selenium (Se) has provided good prospects for lung cancer treatments. In particular, compared to inorganic Se (Inorg-Se) and organic Se (Org-Se), Se nanomedicine (Se nanoparticles; SeNPs) shows much higher bioavailability and antioxidant activity and lower toxicity. SeNPs can also be used as a drug delivery carrier to better regulate protein and DNA biosynthesis and protein kinase C activity, thus playing a role in inhibiting cancer cell proliferation. SeNPs can also effectively activate antigen-presenting cells to stimulate cell immunity, exert regulatory effects on innate and regulatory immunity, and enhance lung cancer immunotherapy. This review summarizes the application of Se-based species and materials in lung cancer diagnosis, including fluorescence, MR, CT, photoacoustic imaging and other diagnostic methods, as well as treatments, including direct killing, radiosensitization, chemotherapeutic sensitization, photothermodynamics, and enhanced immunotherapy. In addition, the application prospects and challenges of Se-based drugs in lung cancer are examined, as well as their forecasted future clinical applications and sustainable development.

Immunotherapy has remained at the vanguard of promising cancer therapeutic regimens due to its exceptionally high specificity for tumor cells and potential for significantly improved treatment-associated quality of life compared to other therapeutic approaches such as surgery and chemoradiation. This is especially true in the digestive system, where high rates of mutation give rise to a host of targetable tumor-specific antigens. Many patients, however, do not exhibit measurable improvements under immunotherapy due to intrinsic or acquired resistance, making predictive biomarkers necessary to determine which patients will benefit from this line of treatment. Many of these biomarkers are assessed empirically by pathologists according to nuanced scoring criteria and algorithms. This review serves to inform clinicians and pathologists of extant and promising upcoming biomarkers predictive of immunotherapeutic efficacy among digestive system malignancies and the ancillary testing required for interpretation by pathologists according to tumor site of origin.

Myelodysplastic syndrome (MDS) treatment remains a big challenge due to the heterogeneous nature of the disease and its ability to progress to acute myeloid leukemia (AML). The only curative option is allogeneic hematopoietic stem cell transplantation (HSCT), but most patients are unfit for this procedure and are left with only palliative treatment options, causing a big unmet need in the context of this disease. Natural killer (NK) cells are attractive candidates for MDS immunotherapy due to their ability to target myeloid leukemic cells without prior sensitization, and in recent years we have seen an arising number of clinical trials in AML and, recently, MDS. NK cells are reported to be highly dysfunctional in MDS patients, which can be overcome by adoptive NK cell immunotherapy or activation of endogenous NK cells. Here, we review the role of NK cells in MDS, the contribution of the tumor microenvironment (TME) to NK cell impairment, and the most recent data from NK cell-based clinical trials in MDS.

Natural killer (NK) cell therapy is emerging as a cancer treatment. NK cells are innate cytotoxic lymphocytes that act as first-line responders to kill target cells without prior encounters. NK cells recognize cancer cells, virus-infected cells, and other types of stressed cell through a reservoir of germline-encoded receptors. NK cells are safe for allogeneic applications. Therefore, they are the ideal off-the-shelf cell, which overcome the low efficiency issue caused by the patient-by-patient nature of autologous cell therapy. Unlike T cells, NK cells cannot form a strong immune memory; therefore, they suffer from short in vivo persistence. However, different from T cells, NK cells have a reservoir of innate immune receptors targeting a variety of malignant cells. In addition, they can utilize antibody guidance in target recognition. With suitable engineering, NK cells can function as universal anticancer drugs that are not restricted to HLA and cancer types, which will benefit the large cohort of patients with rare cancer types and patients with no convenient drug targets for precision and personalized medicine. Here, we summarize and discuss the designs of current anticancer NK cell therapies.

Chimeric antigen receptor (CAR) engineering of natural killer (NK) cells is an attractive research field in tumor immunotherapy. While CAR is genetically engineered to express certain molecules, it retains the intrinsic ability to recognize tumor cells through its own receptors. Additionally, NK cells do not depend on T cell receptors for cytotoxic killing. CAR-NK cells exhibit some differences to CAR-T cells in terms of more precise killing, numerous cell sources, and increased effectiveness in solid tumors. However, some problems still exist with CAR-NK cell therapy, such as cytotoxicity, low transfection efficiency, and storage issues. Immune checkpoints inhibit immune cells from performing their normal killing function, and the clinical application of immune checkpoint inhibitors for cancer treatment has become a key therapeutic strategy. The application of CAR-T cells and immune checkpoint inhibitors is being evaluated in numerous ongoing basic research and clinical studies. Immune checkpoints may affect the function of CAR-NK cell therapy. In this review, we describe the combination of existing CAR-NK cell technology with immune checkpoint therapy and discuss the research of CAR-NK cell technology and future clinical treatments. We also summarize the progress of clinical trials of CAR-NK cells and immune checkpoint therapy.