TriKEs are a combination of 3 distinct domains that are constructed from antigen-specific antibodies or part of antibodies (Figure 1). Formerly, TriKEs or other multi-specific immune cell engager were constructed by only scFv which consist of heavy chain and light chain variable domain which are connected with short amino acid sequences, called flanking [7, 13]. Lately, there have been a lot of formats to be used to construct. We can divide it into 2 groups of formats. One of them is immunoglobulin G (IgG)-like formats which include fragment crystallizable (Fc) region. Another is non-IgG-like formats which consists of antibody-derived building blocks without Fc region. So, the domain of non-IgG-like formats can be antigen-binding fragment (Fab) portion, scFv, or sdAb which have relatively smaller sizes than the IgG-like one with Fc portion [22, 23]. Each domain is also linked together by flanking [7].

The construction of TriKEs is composed of several combinations of antigen-specific antibodies or ligands of specific receptors. The most popular domain is composed of anti-CD16 and IL-15 which take roles in the activation of NK cells and enhancement of NK cell function and survival respectively. This review focuses on anti-CD16/IL-15 TriKE which is the most popular and effective combination of TriKE. While another part is cancer-specific and varies depending on the targeted cancer [7, 13–15].

In the early stages of TriKE development, the first generation of TriKEs employed anti-human CD16 scFv as a crucial component together with N72D modified human IL-15 superagonist where the asparagine at position 72 has been changed to aspartic acid [14, 24]. However, researchers encountered limitations in this configuration, primarily related to steric hindrance caused by the relatively large size of the two scFvs that directly affects to IL-15 accessibility, demonstrated with a function 25 times lower than that of recombinant human IL-15 [7, 25]. Recognizing the need for a more compact design, they embarked on a journey to streamline TriKE composition [20, 25]. To overcome this challenge, the research team innovatively introduced a humanized anti-CD16 heavy chain camelid sdAb, with a relatively smaller size, replacing the anti-human CD16 scFv. This strategic modification aimed to minimize steric hindrance and prevent undesirable pairing between multiple scFvs within the construct. The success of this innovation was remarkable, demonstrating prominently improved results. Notably, the newly incorporated anti-CD16 sdAb showcased superior efficacy in enhancing the impact of the IL-15 moiety. This achievement ensured that the use of mutated IL-15 within the construct was no longer necessary [22, 23, 25]. Building upon these notable findings, the second generation of TriKEs embraced the incorporation of anti-CD16 sdAbs, along with the critical presence of wild type IL-15 (wtIL-15). This strategic evolution marked a vital achievement in TriKE development, enhancing their effectiveness in targeting cancer cells and optimizing NK cell activation. For all previous studies of TriKE are listed in Table 1, specifying their format, specificity, tumor type, and target model.

Moreover, there have been questioned that, instead of using TriKE including IL-15, induction of BiKE separately to IL-15 will not be better [14]. The reports from previously experiments confirmed that IL-15 in the TriKE construct has higher specificity to NK cell due to anti-CD16 which also improves preferable to NK cells instead of T cells. Demonstrated by higher in both lytic degranulation and interferon-γ (IFN-γ) secretion which are important signatures illustrated NK cell function. Besides, IL-15 specified to NK cell attenuates toxicity caused by undesired binding of IL-15 also [7, 14].

NK cells are immune cell in innate immunity which take important roles in immunosurveillance and spontaneous cytolytic activity. NK cell function is driven by the balance of activating and inhibiting signal on the NK cell surface [21, 28, 29]. The signals are composed of various cytokines or antigens on the target cell surface those are normally different between host cells and malignant cells or foreign cells. The activating signals of NK cells can be stimulated through NK cell group 2 isoform C (NKG2C), NKG2D, DNAX accessory molecule-1 (DNAM-1), and NKp80, etc., by their ligands, such as IL-2, IL-15, IL-18, tumor necrosis factor-α (TNF-α), C-X-C motif chemokine ligand 10 (CXCL10) and IFN-γ. While the inhibiting receptors are composed of NKG2A, programmed cell death protein-1 (PD-1), killing Ig-like receptors (KIRs), and T cell immunoreceptor with Ig and ITIM domains (TIGIT), etc., stimulated through ligands, such as human leukocyte antigen (HLA) class I, IL-10, prostaglandin E2 (PGE2), transforming growth factor-β (TGF-β), and indoleamine 2,3-dioxygenase (IDO) [21, 29, 30].

In malignant cells, the mutation alters the cell surface protein to express more activating ligands while losing some inhibiting ligands, resulting in NK cell activation and degranulation to kill those malicious cells [21, 30]. Once NK cells are activated, there are two main cascades to act against the abnormal cells. On one hand, NK cells exhibit CD107a-dependent functions which relate to the degranulation of NK cells. NK cells will secrete perforin and granzyme to induce apoptosis in target cells and inflammatory cytokines and chemokines to stimulate inflammation and recruit further immune cells, both innate and adaptive, to the site [31]. On the other hand, NK cells also perform CD107a-independent functions through the death receptor pathway, like the TNF-related apoptosis-inducing ligand (TRAIL) or fatty acid synthase ligand (FASL). Those receptors take a role in binding to their respective receptors or ligands on the target cells, ultimately inducing apoptosis [32, 33]. However, in some types of cancer, these cells express more inhibitory ligands, such as HLA, and/or exhibit reduced expression of activating ligands. Furthermore, certain tumor microenvironments (TMEs) can alter the expression of surface receptors on NK cells and secrete some factors, like IL-6, IL-10, PGE2, TGF-β [30], acids [34] and reactive oxygen species (ROS) [35] to prevent NK cell activation through both direct and indirect pathway [36, 37]. For example, TGF-β decrease the expression of activating receptors, NKp30 and NKG2D, while promoting regulatory T cells (Tregs) function. These effects result in the attenuation of NK cell function in TMEs. TriKEs can address these issues by efficiently activating NK cells through a highly effective pathway, called antibody-dependent cell cytotoxicity (ADCC) achieved through CD16 activation which is naturally stimulated by the Fc portion of IgG (Figure 2) [30, 36, 38, 39].

Additionally, those issues from TMEs can also diminish NK cell proliferation. To cope with this, previously, IL-2 had been used to enhance NK cell function in tumor suppression [42, 43]. But the researchers found that IL-2 also stimulate Tregs causing NK cell exhaustion, and potentially inducing vascular leak syndrome in mice in vivo studies [44]. Later, IL-15 was discovered to be a promise representative of IL-2 in NK cell immunotherapies with their similar function and processing through the same pathway. Importantly, IL-15 does not stimulate Treg but cytotoxic T cell and is not capable to induce vascular leak syndrome [8, 44–46]. In conclusion, TriKE incorporates IL-15 can deal with the immunosuppressive function of TMEs by promoting NK cell proliferation and persistence within TMEs excluding negative results in both NK cells and healthy cells. The last domain of TriKE is antigen-specific which takes a role in specifically target the cancer cells to complete their bridge function which connects enhanced NK cells to the targeted cancer cells. Previously, various types of specificity were designed, such as anti-CD33 AML [7, 25], anti-EpCAM against carcinomas [13], anti-CD19 against B-CLL [15] anti-TEM8 and anti-mesothelin against NSCLC [10, 27], anti-CLEC12A against AML [9], and anti-HER2 against ovarian cancer [26]. However, there are some interesting target antigens those have been used as potential target immunotherapy in other platforms, like monoclonal antibody (mAb), CAR-T cell, and other immune cell-engager: T cell engager and macrophage engager which can be applied to TriKEs construction [47].

Until now, there have been a lot of TriKE constructs that are progressing on in vitro studies (Table 1) [9–11, 13–16, 20, 25–27], but there is only one reached clinical studies. GTB-3550 TriKE which is a primal construct with CD16 scFv and targets on CD33⁺ AML and myelodysplastic syndromes (MDS) is the first TriKE in phase I clinical trials (NCT03214666) [7, 12]. Patients received treatment at doses of 5 µg/kg per day and 10 µg/kg per day, with two patients assigned to each dose group. The compromised treatment was observed with the dose-dependent manner of CD33⁺ blast clearance and NK cell proliferation and activation in AML and MDS patients. Furthermore, the short half-life was observed, illustrated the rapid clearance of TriKE without advance stimulation. Nevertheless, there were no observed instances of severe side effects [12]. Afterward, GTB-3550 was substituted by second-generation TriKE, GTB-3650, with camelids anti-CD16 sdAb which proved their superior affinity in binding to CD16 and stimulation of ADCC in preclinical studies [25]. Although the results in the preclinical stage are promising and have not shown any negative outcomes, further preclinical studies are still needed to validate the novel findings.

Chimeric antigen receptor (CAR) represents a groundbreaking genetic modification of immune cells, primarily T and NK cells, to express a CAR that is specific to tumor-associated antigens (TAAs) [48, 49]. In general, this recombinant protein consists of four different domains: the antigen recognition domain, the hinge region, a transmembrane domain, and an intracellular signaling domain. This specific feature of CAR offers the advantage of overriding cancer immune escape via major histocompatibility complex (MHC) downregulation. The recognition of CAR is MHC-independent, achieved by using the antigen recognition domain (i.e., single chain variable fragment and peptide) to directly target the tumor antigen on the cancer cell surface. The binding of antigen and CAR triggers the signaling domain to activate the anti-tumor activity, resulting in the cancer elimination. Consequently, it can redirect the specificity and improve the function of T and NK cells. Notably, adoptive cell transfer of genetically engineered cytotoxic immune cells armed with CAR holds greater promise for cancer treatment. This innovation has demonstrated remarkable potential as a cancer treatment, both in vitro and in vivo, and several types of CAR-T platform have obtained Food and Drug Administration (FDA) approval following successful clinical studies [50–54]. Currently, there are six FDA-approved CAR-T cells for blood cancer treatment: (1) CD19 specific CAR-T cells; tisagenlecleucel (Kymriah) for the treatment of pediatric and young adult acute lymphoblastic leukemia (ALL) in addition to axicabtagene ciloleucel (Yescarta), brexucabtagene autoleucel (Tecartus), and lisocabtagene maraleucel (Breyanzi) for the treatment of different B cell malignancies. (2) BCMA specific CAR-T cells; idecabtagene vicleucel (Abecma) and ciltacabtagene autoleucel (Carvykti) for the treatment of MM [55].

However, as CAR-T cell therapies have gained prominence, concerns have arisen regarding their adverse effects, prompting a closer examination of their specificity and potential toxicity [49]. One of the critical aspects of CAR-T cell therapy is its specificity. CARs are engineered to target particular cancer cells bearing associated antigens. However, this specificity extends not only to target cancer cells but also to non-target, normal cells that may express the same antigen, even at lower levels. This phenomenon can lead to off-tumor effects, where CAR-T cells inadvertently attack healthy tissues that share the targeted antigen with cancer cells. This off-tumor effect can have severe and harmful consequences [49, 56, 57]. Moreover, CAR-T cell therapy’s mechanism of action relies on the hyperactivation of T cells upon encountering the antigen, regardless of MHC receptor activation [48, 57]. While this can effectively target tumor cells, it can also result in excessive cytokine release and massive tumor necrosis. These processes can give rise to two notable complications. The first one is cytokine release syndrome (CRS) which can cause systemic inflammation, impacting various organs and leading to symptoms such as fever, fatigue, and even organ dysfunction [53, 54, 58–61]. Another one is tumor lysis syndrome (TLS) leading to electrolyte imbalances [56, 62, 63]. The underlying factor in these adverse outcomes is the hyperactivity of T cells.

In response to these challenges, there has been a growing interest in the development of NK cell-related immunotherapy approaches to mitigate these undesired effects. Unlike the CAR-T cells, CAR-NK cells have demonstrated promising outcomes with fewer side effects. NK cells employ an MHC-independent mechanism to recognize the cancer through the imbalance of activating and inhibitory molecules. This balance makes NK cells highly effective in distinguishing between normal and cancer cells [21, 28, 29] and subsequently activate the cytotoxicity by releasing lytic granule or inducing death receptor-mediated apoptosis through fatty acid synthase (FAS) or TRAIL cascade. In the CAR-NK platform, the CAR molecule, providing specificity and a distinct anti-tumor signaling cascade, greatly improves the cytotoxicity and specificity of NK cells. The MHC-independent mechanism of CAR-NK confers several advantages over CAR-T cells, including a low risk of graft-versus-host disease, CRS, and neurotoxicity, particularly, when compared to allogenic CAR-T cells.

However, the CAR platform utilizes engineered technology to embed the CAR molecule, which necessitates high-cost technology and is time consuming. Alternatively, TriKEs have emerged as promising strategies to redirect NK cells and limit complications related to T cell hyperactivity [8, 10, 64]. TriKEs with anti-CD16/IL-15/anti-TAA target NK cells as their effectors and can effectively modulate NK specificity and enhance NK cell function. As mentioned NK cell property earlier, NK cell activation via TriKEs presents a potentially safer pathway in immunotherapy with minimized cost and time production. Recombinant TriKEs provide as off-the-shelf products without the requirement of engineering process and autologous cells from patients resulting in more safety and highly promising. By harnessing the inherent specificity of NK cells and their ability to discern between healthy and cancerous cells, these approaches aim to reduce the risk of off-tumor effects and the complications associated with the hyperactivation of T cells. As the field of immunotherapy continues to evolve, the development and refinement of NK cell-based strategies hold great promise in improving the safety and efficacy of cancer treatments.

A therapeutic vaccine is a form of active immunization designed to manipulate anti-tumor activity. One of the most well-known therapeutic vaccines for cancer treatment is the dendritic cell (DC) vaccine. DCs function as professional antigen presenting cells (APCs), capable of processing antigens and presenting them via MHC I and MHC II to CD8⁺ and CD4⁺ T cells, respectively. DCs play a crucial role as a link between innate and adaptive immunity, activating both arms of the immune system, innate and adaptive immunity. DCs loaded with tumor antigen serve as an immunized vaccine, inducing antigen-specific cytotoxic T cells to recognize and eliminate cancer cells. Clinical trials in melanoma, prostate cancer, malignant glioma, and renal cell carcinoma have demonstrated safety without serious side effects, even in patients with advanced disease [65]. Unfortunately, while vaccine immunization has shown the induction of the specific anti-tumor immune response, the clinical response has been suboptimal and unsatisfactory. The objective tumor response rate has rarely exceeded 15% [65].

Due to the DC vaccine is a personalized approach that modulates the upstream of anti-tumor stimulation, its failure could be influenced by several factors. These may include the immunosuppressive environment, the absence of specific T cell clones, the presence of immune checkpoint proteins, or individual immune status. Furthermore, the production of a DC vaccine involves multiple steps based on the source of DC and faces limitations in mass production due to the difficulty of DC proliferation. In contrast, TriKEs technology offers a straightforward method to redirect the function of the NK population to specifically target and eliminate cancer cells. The ex vivo expansion of NK and their allogenic use overcome limitations in both the quality and quantity aspects of immune cell preparation compared to DC vaccines. Moreover, as off-the-shelf technology, TriKEs are more robust and can serve as a frontier immunotherapy. Given that TriKEs and DC vaccines promote different arms of immunity, their combination as therapy is plausible to enhance anti-tumor activity and provide the highest potential to combat cancer growth.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer immunotherapy by enhancing the activation of immune cells, particularly T cells, to improve their ability to target and kill cancer cells by blocking the regulatory checkpoint [66]. The main targets of ICIs composed of cytotoxic T-lymphocyte antigen-4 (CTLA-4) [67, 68] and PD-1/programmed cell death ligand-1 (PD-L1) [66]. Until now, there have been various agents based on ICI that were approved by the FDA and in clinical trials [66]. However, the clinical use of ICIs is associated with certain adverse effects, broadly called immune-related adverse events (irAEs), that warrant careful consideration [69, 70]. The irAEs can initially be characterized by two primary short-term adverse effects, which may eventually accumulate into more impactful long-term adverse effects or life-threatening [70, 71]. The first one is hyperactivation of T cells leading to on-target off-tumor effects. Several ICIs overstimulate T cells through blocking of main regulation pathway, resulting in damage to both malignant and non-malignant cells which can prolong to be hypophysitis, thyroiditis, pneumonitis, and autoimmune diabetes [70, 71]. Another one is tissue pathway inhibition. Some tissues in the body rely on immune checkpoint pathways for stability and regulation. When ICIs disrupt these pathways, it can lead to instability in these tissues, compromising their normal function. This disruption can give rise to autoimmune-like diseases, as ICIs inadvertently interfere with the tissue’s immune-regulatory mechanisms [70].

While ICIs have shown remarkable efficacy in cancer treatment, these adverse effects are remarkable concerns that need to be addressed. Here comes the role of TriKEs. TriKEs represent a promising advancement in immunotherapy that can address these adverse effects. Unlike ICIs, which primarily target immune checkpoints, TriKEs can be designed to target specific TAAs, bypassing the immune checkpoint altogether. The key advantage of TriKEs is their mechanism of action through NK cells [7]. TriKEs activate NK cells with high precision, inducing a targeted immune response against cancer cells without the same adverse effects associated with T cell hyperactivation seen in ICIs [8, 64]. NK cells are known for their lower propensity to cause cytokine storms and off-tumor effects, making them an attractive alternative for cancer immunotherapy [64]. In summary, while ICIs have notably improved cancer treatment, their adverse effects remain a challenge. TriKEs offer a promising solution by targeting specific TAAs through NK cell activation, mitigating the adverse effects seen with ICIs. Future research and clinical trials will further elucidate the potential of TriKEs in cancer therapy, offering hope for improved cancer treatments with fewer complications.

Cytokines serve as central regulators of the immune system, acting as messengers between immune cells and exerting control over various cellular functions, including the induction of inflammation and apoptosis in malignant cells [47]. Over the years, several cytokines have received approval for use in human therapies, offering promising avenues for cancer treatment. These include IFN-α, IFN-γ, IL-2, IL-12, IL-15, IL-21, and granulocyte-macrophage colony-stimulating factor (GM-CSF), among others [42, 43, 47]. Nonetheless, it’s important to acknowledge that cytokine therapies come with their set of difficulties. Even though these therapies have proven effective in enhancing immune responses against cancer, they have also been linked to various side effects, including some that can be severe and potentially life-threatening [43, 46, 71, 72]. For instance, IFN-α, employed in melanoma patients [47], has been linked to flu-like symptoms, hematological toxicity, elevated liver enzyme levels, nausea, fatigue, and psychiatric sequelae. These side effects often correlate with the dose administered, making high-dose treatments particularly challenging [73]. Similarly, IL-2, used in metastatic renal cancer and melanoma [47], can lead to a life-threatening condition known as vascular-leak syndrome [46]. Given the limitations associated with high-dose cytokine therapy due to its potential for severe adverse effects, it is increasingly recognized as a complementary treatment to be combined with other primary therapies [47, 72]. The question arises as to whether low-dose cytokine therapy possesses sufficient potential to combat cancer effectively [47]. TriKEs offer a promising solution that bridges the gap between cytokine therapy and targeted immunotherapy [7]. These innovative molecules incorporate one IL-15 domain, a cytokine known for its role in stimulating NK cell proliferation and prolonging their function. In addition to their cytokine activity, TriKEs activate NK cells through CD16 engagement and target cancer cells through a third TAA-specific domain [7–11]. This unique combination of features positions TriKEs as a versatile tool in cancer immunotherapy. By harnessing the benefits of cytokine stimulation while precisely directing immune cell activity, TriKEs aim to provide a more focused and effective approach to combating cancer [8, 23, 64, 74]. Moreover, their ability to engage NK cells in a highly specific manner reduces the risk of off-target effects and severe adverse reactions, offering a more balanced and safer immunotherapeutic option [64]. In summary, while cytokine therapy has made prominent contributions to cancer treatment, it comes with challenges and limitations, necessitating careful consideration in clinical practice. TriKEs represent an innovative approach that combines the benefits of cytokine stimulation with targeted immunotherapy, potentially offering a more effective and safer solution for cancer patients.

mAbs are precision-engineered immunotherapeutic agents used widely in modern medicine. They are designed to target specific antigens, making them valuable in various fields, including cancer treatment [75, 76]. The mAbs have a dual structure, comprising the Fab region for antigen recognition and the constant Fc region for interacting with the immune system [76]. The Fc region plays a crucial role in stimulating immune cell responses [77]. While mAbs show remarkable therapeutic potential, they have factors to consider, including their size, persistence, and potential for unintended immune cell activation, leading to off-target effects and complications [76].

TriKEs were initially based on the function of antibodies, including mAbs [2]. However, their construct has been influentially altered to enhance their effectiveness beyond traditional antibodies [41]. TriKEs, with their compact size due to the absence of the Fc portion [7, 25], facilitate efficient biodistribution, allowing them to navigate through the circulatory system and reach target tissues, including solid tumors [41]. This advantage becomes particularly pronounced in the challenging microenvironments of solid tumors, where TriKEs can penetrate barriers effectively [20]. In contrast, the larger size of mAbs can hinder their distribution within solid tumors, potentially limiting their therapeutic reach [78]. However, the smaller size of TriKEs also means a shorter half-life, requiring careful dosing strategies [41].

In summary, mAbs and TriKEs have distinct characteristics, with TriKEs offering advantages in biodistribution due to their smaller size and altered construct. This property enhances their potential as a valuable tool in cancer immunotherapy, striking a balance between size-related advantages and therapeutic effectiveness. Some functions and adverse effects of those immunotherapies are summarized in Table 2.