Building a Better Defense: Expanding and Improving Natural Killer Cells for Adoptive Cell Therapy

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


What Are Natural Killer Cells?
Natural killer (NK) cells are large granular lymphocytes representing 5-10% of circulating lymphocytes in healthy adults, playing a crucial role in recognizing and eliminating transformed and infected cells [1].Distinguished by the absence of CD3 and CD19 and the presence of CD56, NKp46, and NKp80 expression, NK cells consist of two principal subsets: the immature or regulatory CD56 bright CD16 +/− cells and the mature or cytotoxic CD56 dim CD16 + cells (Figure 1A) [1][2][3].The CD56 bright subset, more prevalent in the lymph nodes, secretes cytokines and chemokines in an inflammatory milieu, recruiting and modulating immune cells such as neutrophils, macrophages, T-cells, B-cells, and dendritic cells (Figure 1B) [4][5][6].However, CD56 bright cells can be rapidly primed to acquire potent cytotoxic function upon cytokine activation [7].In contrast, the CD56 dim subset exhibits higher direct cell cytotoxicity against tumor targets, mediating antibody-dependent cell cytotoxicity (ADCC) through the CD16 expression, which binds to the Fc region of IgG1 antibodies (Figure 1C) [8][9][10].Furthermore, both NK cell subsets induce tumor cell apoptosis by expressing death ligands like FasL and TRAIL (Figure 1C) [11].

What Are Natural Killer Cells?
Natural killer (NK) cells are large granular lymphocytes representing 5-10% of circulating lymphocytes in healthy adults, playing a crucial role in recognizing and eliminating transformed and infected cells [1].Distinguished by the absence of CD3 and CD19 and the presence of CD56, NKp46, and NKp80 expression, NK cells consist of two principal subsets: the immature or regulatory CD56 bright CD16 +/− cells and the mature or cytotoxic CD56 dim CD16 + cells (Figure 1A) [1][2][3].The CD56 bright subset, more prevalent in the lymph nodes, secretes cytokines and chemokines in an inflammatory milieu, recruiting and modulating immune cells such as neutrophils, macrophages, T-cells, B-cells, and dendritic cells (Figure 1B) [4][5][6].However, CD56 bright cells can be rapidly primed to acquire potent cytotoxic function upon cytokine activation [7].In contrast, the CD56 dim subset exhibits higher direct cell cytotoxicity against tumor targets, mediating antibody-dependent cell cytotoxicity (ADCC) through the CD16 expression, which binds to the Fc region of IgG1 antibodies (Figure 1C) [8][9][10].Furthermore, both NK cell subsets induce tumor cell apoptosis by expressing death ligands like FasL and TRAIL (Figure 1C) [11].
NK cell activation is mediated by a balance between activating and inhibitory signals (Figure 2).The "missing-self" hypothesis proposes that loss or downregulation of MHC-I molecules on tumor cells leads to decreased inhibitory signals, thus favoring NK cell activation [22].In contrast, "stress-induced" activation occurs when stress ligands such as MIC-A/B are upregulated on infected or malignant cells, engaging activating receptors like NKG2D on NK cells [12,23].In addition, NK cell function can be restrained by immune checkpoints, including NKG2A, TIM-3, TIGIT, and CD96 [24].Although not highly prevalent, programmed death-1 (PD-1) expression has been observed in NK cells, thereby inducing dysfunction of the NK cells within the tumors with increased PD-L1 expression [25,26].
NK cell activation is mediated by a balance between activating and inhibitory signals (Figure 2).The "missing-self" hypothesis proposes that loss or downregulation of MHC-I molecules on tumor cells leads to decreased inhibitory signals, thus favoring NK cell activation [22].In contrast, "stress-induced" activation occurs when stress ligands such as MIC-A/B are upregulated on infected or malignant cells, engaging activating receptors like NKG2D on NK cells [12,23].In addition, NK cell function can be restrained by immune checkpoints, including NKG2A, TIM-3, TIGIT, and CD96 [24].Although not highly prevalent, programmed death-1 (PD-1) expression has been observed in NK cells, thereby inducing dysfunction of the NK cells within the tumors with increased PD-L1 expression [25,26].

What Are the Major Advantages of NK Cells and Their Respective Sources for Adoptive Cell Therapy?
Chimeric antigen receptor (CAR)-T cells have significantly advanced cellular immunotherapy for cancer treatment [27][28][29][30].Other T-cell-based approaches, including tumor-infiltrating lymphocytes (TIL) and T-cell receptor (TCR) T-cells, have also demonstrated promising efficacy, leading to the approval of several products by the FDA [30][31][32][33][34][35].Nevertheless, T-cell-based therapies are associated with a relatively high risk of developing cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and hemophagocytic lymphohistiocytosis (HLH), resulting in prolonged cytopenia and risk of graph-versus-host disease (GVHD) in an allogeneic setting [36][37][38][39].In contrast, NK/CAR-NK-cell-based products are not associated with these side effects, making them an attractive alternative [40].In addition, the versatility of NK cell sources, including peripheral blood (PB), cord blood (CB), hematopoietic stem cells (HSC), and induced pluripotent stem cells (iPSC), increases their accessibility, supporting NK-cell-based products as a viable alternative to T-cells [41,42].The major advantages and disadvantages of NK cell sources are described below (Figure 3).

Peripheral Blood (PB)
NK cells constitute approximately 5 to 10% of total lymphocytes in PB, exhibiting a mature phenotype with high cytotoxicity against tumors (Figure 3A) [1].Within the PB-NK cells, the CD56 bright subset accounts for approximately 10%, while the remainder is mostly CD56 dim NK cells [43].NK cells are commonly isolated from peripheral blood mononuclear cells (PBMC), obtained through leukapheresis, followed by a bead-based selection process [44].Despite being readily available, using PB as a source for NK cells has several limitations, including relatively low cell numbers and donor-dependent variability [44].

Cord Blood (CB)
NK cells constitute around 23% of CB cells, with similar proportions of the CD56 bright and CD56 dim subsets found in PB (Figure 3B) [45].CB-NK cells exhibit lower levels of adhesion molecules (CD2, CD11a, CD18, and CD54) and maturation receptors (KIR and CD57) while maintaining similar expression of the key cytotoxic molecules like granzyme B and perforin compared to PB-NK cells [46][47][48].Although CB presents a limited number of NK cells per CB unit, recent efficient expansion strategies (described in

What Are the Major Advantages of NK Cells and Their Respective Sources for Adoptive Cell Therapy?
Chimeric antigen receptor (CAR)-T cells have significantly advanced cellular immunotherapy for cancer treatment [27][28][29][30].Other T-cell-based approaches, including tumor-infiltrating lymphocytes (TIL) and T-cell receptor (TCR) T-cells, have also demonstrated promising efficacy, leading to the approval of several products by the FDA [30][31][32][33][34][35].Nevertheless, T-cell-based therapies are associated with a relatively high risk of developing cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and hemophagocytic lymphohistiocytosis (HLH), resulting in prolonged cytopenia and risk of graph-versus-host disease (GVHD) in an allogeneic setting [36][37][38][39].In contrast, NK/CAR-NK-cell-based products are not associated with these side effects, making them an attractive alternative [40].In addition, the versatility of NK cell sources, including peripheral blood (PB), cord blood (CB), hematopoietic stem cells (HSC), and induced pluripotent stem cells (iPSC), increases their accessibility, supporting NK-cell-based products as a viable alternative to T-cells [41,42].The major advantages and disadvantages of NK cell sources are described below (Figure 3).

Peripheral Blood (PB)
NK cells constitute approximately 5 to 10% of total lymphocytes in PB, exhibiting a mature phenotype with high cytotoxicity against tumors (Figure 3A) [1].Within the PB-NK cells, the CD56 bright subset accounts for approximately 10%, while the remainder is mostly CD56 dim NK cells [43].NK cells are commonly isolated from peripheral blood mononuclear cells (PBMC), obtained through leukapheresis, followed by a bead-based selection process [44].Despite being readily available, using PB as a source for NK cells has several limitations, including relatively low cell numbers and donor-dependent variability [44].

Cord Blood (CB)
NK cells constitute around 23% of CB cells, with similar proportions of the CD56 bright and CD56 dim subsets found in PB (Figure 3B) [45].CB-NK cells exhibit lower levels of adhesion molecules (CD2, CD11a, CD18, and CD54) and maturation receptors (KIR and CD57) while maintaining similar expression of the key cytotoxic molecules like granzyme B and perforin compared to PB-NK cells [46][47][48].Although CB presents a limited number of NK cells per CB unit, recent efficient expansion strategies (described in the following section) have allowed the generation of several infusion products [49][50][51].Alternatively, NK cells can be differentiated from the CD34 + HSC that are highly enriched in the CB and display many similarities to PB-NK cells, though with a lower inhibitory receptor expression [52][53][54][55].
the following section) have allowed the generation of several infusion products [49][50][51].Alternatively, NK cells can be differentiated from the CD34 + HSC that are highly enriched in the CB and display many similarities to PB-NK cells, though with a lower inhibitory receptor expression [52][53][54][55].

Feeder-Cell-Based Expansion
Feeder-cell-based expansion systems, typically involving immortalized or tumor cell lines in combination with cytokines, have become a common approach for expanding and activating NK/CAR-NK cells (Table 2) [76][77][78]96].Using Epstein-Barr Virus-Immortalized Lymphoblastoid cell line (EBV-LCL) as feeder cells resulted in a 53-fold expansion of NK cells after one week with IL-21 stimulation and superior NK cell cytotoxicity [76].
In addition, CB-NK cells cultured with EBV-LCL cells showed an up to 6092-fold expansion after 35 days [77].Furthermore, NK cells from patients with advanced cancer were cultured with NK-cell-depleted-PBMC from diseased and healthy donors [87].A higher NK cell expansion rate was observed when using healthy donors' feeder cells (300-fold) compared to diseased feeder cells (164.9-fold)[87].

Culture Materials Used for NK Cell Expansion
Selecting appropriate culture materials, including flasks, bags, or bioreactors, is crucial in ensuring efficient NK cell expansion.Effector cells, particularly CD4 + , CD8 + , CD8 + CD56 + T-cells, and CD56 + NK cells cultured in bags and flasks, exhibited no significant differences in expansion, phenotype, or function over a 7-day period [100].In contrast, NK cells reached a 530-fold expansion in bags compared to 1100-fold using flasks [101].Culture in flasks carries the risk of exposure and contamination, which can be minimized in a GMP laboratory [101].On the other hand, culturing in bags requires maintaining a certain cell concentration, and the gas exchange is restricted by the media's volume, limiting the supply of nutrients and impacting NK cell proliferation [102].Bioreactors are considered the most practical method despite being more expensive due to minimal hands-on time.However, bioreactors typically require higher starting cell numbers, and the expansion rate might be lower than other methods [101].

How Do Cell Culture Strategies Improve NK Cell Activity?
Although naturally cytotoxic, NK cells' activity can be further improved during cell culture.The previous section described several expansion methods, while the current section outlines the changes in phenotype, persistence, and cytotoxicity that NK cells undergo during expansion.

Telomere Length
Telomere shortening is a limiting factor for NK cell expansion [104].Exposure to K562-mb15-41BBL cells limited proliferation due to telomere shortening [104].To overcome this, NK cells overexpressing human telomerase reverse transcriptase (TERT) demonstrated an extended lifespan, maintaining a high percentage of cells in the S/G2 phase [105].TERT-NK cells cultured with K562-mb15-41BBL cells showed prolonged proliferation for over a year, maintaining a normal karyotype and genotype [106].NK cells in culture with K562 cells and IL-2 stimulation showed higher phosphorylation of STAT3, an activator of human TERT [73,107].In addition, NK cells expanded with K562-mbIL21 cells, demonstrated increased telomere length, upregulation of STAT3, and reduced senescence [82].
Expansion strategies influence NK cell phenotype, function, and cytotoxicity, determining the efficacy of NK-cell-based therapies.

Which Other Strategies Improve NK Cell Antitumor Response?
Despite improvements in NK cell expansion and cytotoxicity during cell culture, their clinical application is hampered by major challenges, including short half-life, limited tumor infiltration, and low in vivo persistence [110].Key strategies to further enhance NKcell-based immunotherapeutic approaches include stimulation of memory-like NK cells, genetic manipulation including CAR-NK cells, and, more recently, TIL-NK cells [111][112][113].

Chimeric Antigen Receptor (CAR) Technology
CAR therapy involves modifying immune cells to express a synthetic receptor binding to a tumor-associated antigen like CD19, expressed by B-cells (both normal and B-cell lymphomas), and BCMA, expressed by normal and malignant plasma cells [29,125,126].While T-cells are the most widely used immune cell for CAR therapy, they present several limitations, such as the alloreactivity and GVHD risks, making autologous T-cells a preferential choice [38,127].In contrast, CAR-NK cells, derived from allogeneic sources, provide an "off-the-shelf" product without inducing GVHD, avoiding the massive release of cytokines and neurotoxicity [51,128].

Tumor-Infiltrating Lymphocytes (TILs), including TIL-NK Cells
TILs primarily comprise T-cells that have migrated into a tumor [139].TIL therapy has shown significant clinical results, particularly in melanoma patients [140][141][142].Furthermore, ongoing studies in breast and colorectal cancers highlight the potential of TILs as a source of antigen-specific immune cells [113,143,144].Recent studies have emphasized that TILs include NK cells (TIL-NK cells), associated with improved prognosis in multiple malignancies [145][146][147][148][149][150].In lung cancer, TIL-NK cells are predominantly CD56 bright perforin low , exhibiting lower cytotoxicity but with similar cytokine production compared to the PB-NK cells [151].In soft tissue sarcoma, NK cells represent around 20% of the TIL population [152].In comparison, less than 0.5% of TIL-NK cells are found in pancreatic ductal adenocarcinoma (PDAC), attributed to the low expression of the chemokine receptor CXCR2 [153].Transgenic expression of CXCR2 facilitates NK cell infiltration, although proliferation is limited in the hypoxic TME [154].CCR7 expression, involved in lymphocyte migration to lymph nodes, remains unchanged, while CXCR3, mediating NK cell recruitment to tumor sites, is enhanced in expanded NK cells [79].Additionally, CXCL9 and CXCL10 expression in the TME and IL-15 stimulation promote the recruitment of NK cells and cytotoxic CD8 + T-cells via CXCR3 into the tumors [155].
While until now, no method has been explicitly published for the expansion of TIL-NK cells, it is crucial to consider the promising prospects of TIL-NK-cell-based therapies in cancer [149,156].Despite the limited cell numbers of TIL-NK cells, their superior tumorinfiltration capacities and association with improved overall survival of cancer patients underscore significant potential [149,156].Additionally, the recent FDA approval of Amtagvi, an autologous TIL therapy for advanced melanoma in adults, emphasizes the promising potential of TIL therapy as a treatment option for cancer [157,158].

Which Are Currently the Major NK-Cell-Based Clinical Trials?
The current clinical application of NK cells includes autologous and allogeneic NKcell-based approaches [159].So far, the NK cell clinical trials have predominantly focused on patients with hematological diseases, though promising data from recent preclinical studies strongly support their evaluation in solid tumors [160][161][162].The following section summarizes key clinical trials, highlighting the advantages of expanded NK cells.

Autologous NK Cells
In the autologous setting, NK cells have been safely infused and expanded in vivo with IL-2 administration; however, their efficacy has been limited [78,163,164].In a phase I trial, autologous NK cells expanded with K562-mbIL15-41BBL cells reported stable disease combined with trastuzumab in 6 of 19 patients with HER2-positive malignancies [163].Infusion of activated autologous NK cells into MM patients post-autologous HCT also supported the broader feasibility of this therapy [165].Despite the prolonged survival of ex vivo IL-2-activated autologous NK cells in preclinical studies, no clinical responses were observed in patients with metastatic melanoma or renal cell carcinoma [166].Their minimal clinical activity can be due to a lack of KIR/ligand mismatch in the autologous tumor cells and/or due to their limited in vivo persistence after adoptive transfer [166].These challenges represent a significant hurdle for autologous NK-cell-based therapy [166].Several ongoing clinical trials are evaluating autologous NK cell therapy for hepatocellular carcinoma (NCT06044506) and MM in combination with low IL-2 (NCT04634435).

Allogeneic NK Cells
The use of allogeneic NK cells has been associated with inducing remission and preventing relapse in AML and MM patients [167].Using KIR-mismatched donor NK cells after haploidentical HCT demonstrated a significantly reduced risk of relapse in high-risk AML [168,169].Subsequently, allogeneic NK cells from unrelated healthy donors were assessed in advanced lymphoma and solid tumors [160].Among 17 patients, 47.1% showed stable disease, highlighting the safety and the potential efficacy of administrating randomdonor allogeneic NK cells and thus expanding cell donor options [160].In another study, the use of haploidentical PBMC (CD3 + T-cell-depleted and NK-cell-enriched) was safe and induced complete response (CR) in 5 of 19 poor-prognosis AML patients [170].Similarly, IL-2-activated allogeneic NK cells combined with anti-CD20 mAb yielded responses in 14 of 15 relapsed/refractory CD20 + lymphoma patients in a phase II clinical trial [171].
While haploidentical NK cell infusion induced remissions, the presence of Tregs may have contributed to their diminished efficacy [172].Depletion of host Tregs with IL-2diphtheria fusion protein improved the efficacy of haploidentical NK cell therapy, resulting in higher donor NK cell expansion in relapsed-refractory AML patients [172].Substituting IL-2 with IL-15 showed promising results, with 36% of patients exhibiting robust in vivo NK cell expansion and 32% achieving CR, avoiding Treg stimulation [173].In a phase II trial, IL-15 administered subcutaneously (SC) resulted in NK cell expansion in 27% of the patients, and 40% achieved remission [173].However, while IL-15 improved in vivo NK cell expansion and remission rates, it was also associated with previously unreported CRS after SC administration [173].Moreover, IL-15 superagonist complex ALT-803 was well tolerated, stimulating NK and CD8 + T-cells without increasing Tregs [174].
Considering the high risk of relapse after allogeneic HCT, donor-derived CIML NK cells are attractive in myeloid malignancies that have relapsed after haploidentical HCT [175].A first-in-human phase I trial with donor CIML NK cells in relapsed or refractory AML reported four of nine patients achieving CR and one achieving morphologic leukemia-free state (MLFS), resulting in an overall response rate of 55% and a CR rate of 45% [122].Additionally, CIML NK cell infusion led to rapid 10-to 50-fold expansion in vivo, sustained over months, supporting CIML NK cells as a platform for post-transplant relapse myeloid disease treatment [175].These findings highlight the importance of expanding and stimulating NK cells before infusion, whether as CIML or conventional NK cells, to improve immunotherapy efficacy.

Allogeneic CB-NK Cells
In a first-in-human trial, CB-NK cells, expanded with IL-2 and K562-mbIL21 cells, were infused in MM patients receiving high-dose chemotherapy and autologous HCT [176].This trial demonstrated safety and efficacy, with 10 patients achieving at least a good partial response, including 8 near CR [176].Additionally, in recurrent ovarian carcinoma, CB-NK cells exhibited safety and in vivo expansion capacity [177].The "off-shelf" CB-NK cell product, oNKord, has obtained approval for AML patients [178].The phase I trial demonstrated safety and efficacy in elderly AML patients, ineligible for allogeneic HCT, while an ongoing phase II trial is evaluating oNKord in patients with minimal residue disease (MRD) (NCT04632316) [178].Recently, a phase I/II trial of CB-NK cells expressing CD19-CAR and IL-15 was evaluated in patients with CD19 + B-cell malignancies [40,51].The 1-year overall survival (OR) and progression-free survival were 68% and 32%, respectively, with patients achieving OR correlated with higher levels and longer persistence of CAR-NK cells [40].No notable toxicities were observed, including CRS and GvHD [40].

What Is the Future of NK-Cell-Based Therapy?
While NK cells hold great promise, challenges remain, including low in vivo persistence and proliferation capacity, which can compromise their effectiveness in cancer therapy [179].This review aims to elucidate several strategies used to improve NK cell proliferation and antitumor function, either through expansion methodologies, cytokine stimulation, or genetic modifications.Diverse culture methods are explored using various cytokines (such as IL-2, IL-12, IL-15, IL-18, and IL-21) and feeder cells (including genetically modified or nongenetically modified cell lines, as well as autologous or allogeneic cells), with emphasis on their effects on NK cell expansion, phenotype, telomere length, and cytotoxic activity.Notably, stimulation with IL-2 and IL-15 has been shown to promote NK cell expansion, while IL-12 and IL-21 have been associated with enhancing cell cytotoxicity [71][72][73][74].In addition, using genetically engineered feeder cells, like K562-mbIL21 cells, has demonstrated remarkable ex vivo NK cell expansion capacities [82,180].
Furthermore, the generation of memory-like NK cells, characterized by superior IFN-γ production and cytotoxicity, represents another strategy to improve in vivo persistence, providing a sustained and potent functional alternative [122].On the other hand, genetic manipulation of NK cells to develop CAR-NK cells has shown promising results, enhancing NK cell targeting without inducing severe side effects [40,51,128,129,136,137].Moreover, exploring TIL therapy, particularly TIL-NK cells, presents a compelling alternative to improve tumor treatment, given their association with improved overall survival in cancer patients [149,156].Recently, FDA approval of an autologous TIL therapy for advanced melanoma has underscored the promising antitumor potential of TILs [157,158].
Despite the promising outcomes in cancer, especially in hematological malignancies, translating NK-cell-based therapies in solid tumors faces significant challenges, with poor tumor trafficking and highly immunosuppressive TME as major barriers for both NKand non-NK-cell-based cellular therapy approaches [110].The TME can induce NK cell dysfunction and exhaustion through various mechanisms, including suppressive immune or nonimmune cells (e.g., Tregs and myeloid-derived suppressor cells (MDSC)), cytokines like TGF-β, overexpression of inhibitory ligands (e.g., HLA-E), and downregulation of activating ligands [181].Several strategies are being evaluated to overcome these challenges, including exploring TIL's superior tumor infiltration capacity, and developing novel CAR-engineered cells [182][183][184].Engineered NK cells with synthetic receptors, sustained cytokine production, safety mechanisms like drug-inducible suicide genes, or "on/off switches" through small molecule administrations promise great potential [185].Targeting cancer and other cells in the TME that help tumor growth, like cancer-associated fibroblasts (CAFs), can potentially improve NK-cell-based approaches.
Furthermore, the most effective NK-cell-based immunotherapy may involve combining them with other immune cells, such as CAR-T cells, TIL, or CAR-macrophages, taking advantage of the unique strengths of each approach to enhance tumor control significantly.Additionally, combining NK cell therapies with immune checkpoint inhibitors might be a great strategy, especially considering the dysfunction and exhaustion markers expressed by NK and T-cells within the TME, such as CD161, TIGIT, and CD96 [152].Lastly, there is a huge interest in developing the in vivo "arming" of NK and other immune cells to mitigate labor-intensive and expensive adoptive cell therapies [186].Recent progress in RNA-based approaches combined with nanoparticle-based technology makes in vivo modulation of the immune cells feasible.However, challenges like low transfection efficacy, short RNA half-life, and limited cell specificity remain the major barriers to these efforts [187].
In summary, this review offers insights into strategies aimed at enhancing NK cell function through expansion methodologies or genetic modifications, elucidating their impact on NK cell phenotype, proliferation, and cytotoxicity.In addition, this review highlights the current clinical trial landscape and key advancements aimed at further enhancing NK-cell-based adoptive cell therapy.

Figure 1 .
Figure 1.Representation of the NK cell subsets and NK-cell-mediated immunomodulation and cytotoxicity mechanisms.(A) NK cells are classified by CD56 and CD16 surface markers, including regulatory or immature NK cells (CD56 bright CD16 +/− ), primarily releasing IFN-γ molecules, and cytotoxic or mature NK cells (CD56 dim CD16 + ), mainly releasing perforin and granzyme B molecules.(B) Activated NK cells, in particular regulatory NK cells, release immunomodulatory mediators such as CCL5, XCL1, IL-8, IFN-γ, TNF-α, and GM-CSF within the TME, recruiting and activating other immune cells, including neutrophils, macrophages, dendritic cells (DC), T-cells, and B-cells.(C) Upon activation, NK cells exert cytotoxic effects through FasL and TRAIL-induced apoptosis, degranulation of perforin and granzyme molecules, and through antibody-dependent cell cytotoxicity (ADCC) via CD16, particularly cytotoxic NK cells, binding to antibodies on tumor cells, inducing apoptosis.

Figure 1 .
Figure 1.Representation of the NK cell subsets and NK-cell-mediated immunomodulation and cytotoxicity mechanisms.(A) NK cells are classified by CD56 and CD16 surface markers, including regulatory or immature NK cells (CD56 bright CD16 +/− ), primarily releasing IFN-γ molecules, and cytotoxic or mature NK cells (CD56 dim CD16 + ), mainly releasing perforin and granzyme B molecules.(B) Activated NK cells, in particular regulatory NK cells, release immunomodulatory mediators such as CCL5, XCL1, IL-8, IFN-γ, TNF-α, and GM-CSF within the TME, recruiting and activating other immune cells, including neutrophils, macrophages, dendritic cells (DC), T-cells, and B-cells.(C) Upon activation, NK cells exert cytotoxic effects through FasL and TRAIL-induced apoptosis, degranulation of perforin and granzyme molecules, and through antibody-dependent cell cytotoxicity (ADCC) via CD16, particularly cytotoxic NK cells, binding to antibodies on tumor cells, inducing apoptosis.

Figure 2 .
Figure 2. NK cell activation mechanisms.A balance of inhibitory and activating signals regulates NK cells.This balance is maintained when NK cells encounter normal cells, resulting in NK cell inactivity.However, certain conditions can activate NK cells, such as recognizing "missing self" when MHC-I molecules, serving as negative signals, are absent.Additionally, NK cells can be activated through "stress-induced" mechanisms when stress ligands, acting as positive signals, are overexpressed in stressed cells, including tumor-or viral-infected cells.

Figure 2 .
Figure 2. NK cell activation mechanisms.A balance of inhibitory and activating signals regulates NK cells.This balance is maintained when NK cells encounter normal cells, resulting in NK cell inactivity.However, certain conditions can activate NK cells, such as recognizing "missing self" when MHC-I molecules, serving as negative signals, are absent.Additionally, NK cells can be activated through "stress-induced" mechanisms when stress ligands, acting as positive signals, are overexpressed in stressed cells, including tumor-or viral-infected cells.

Figure 3 .
Figure 3. Sources of NK cells.(A) In PB-NK cells represent 5-10% of total lymphocytes, with approximately 90% being CD56 dim CD16 + NK cells and the remaining 10% comprising CD56 bright CD16 +/− NK cells.(B) CB-NK cells can be obtained from CB or by differentiating CD34 + cells through exposure to specific cytokines.NK cells comprise 23% of the total cells in CB, and the proportions of CB-NK cell subsets are similar to PBMC-NK cells.(C) Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can generate NK cells with functional and phenotype similarities to PB-NK cells, although presenting a lower CD16 expression.

Figure 3 .
Figure 3. Sources of NK cells.(A) In PB-NK cells represent 5-10% of total lymphocytes, with approximately 90% being CD56 dim CD16 + NK cells and the remaining 10% comprising CD56 bright CD16 +/− NK cells.(B) CB-NK cells can be obtained from CB or by differentiating CD34 + cells through exposure to specific cytokines.NK cells comprise 23% of the total cells in CB, and the proportions of CB-NK cell subsets are similar to PBMC-NK cells.(C) Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can generate NK cells with functional and phenotype similarities to PB-NK cells, although presenting a lower CD16 expression.

Table 1 .
Analysis of NK cell expansion rates across diverse cytokine-based expansion methods, varying in source, media, duration, and culture material.

Table 2 .
Analysis of NK cell expansion rates across diverse feeder-cell-based expansion methods, varying in source, media, feeder cells, duration, and culture material.