Principles and current clinical landscape of NK cell engaging bispecific antibody against cancer

ABSTRACT

Monoclonal antibody-based targeted therapies have greatly improved treatment options for patients by binding to the innate immune system. However, the long-term efficacy of such antibodies is limited by mechanisms of drug resistance. Over the last 50 years, with advances in protein engineering technology, more and more bispecific antibody (bsAb) platforms have been engineered to meet diverse clinical needs. Bispecific NK cell engagers (BiKEs) or tri-specific NK cell engagers (TriKEs) allow for direct targeting of immune cells to tumors, and therefore resistance and serious adverse effects are greatly reduced. Many preclinical and clinical trials are currently underway, depicting the promise of antibody-based natural killer cell engager therapeutics. In this review, we compile worldwide efforts to explore the involvement of NK cells in bispecific antibodies. With a particular emphasis on lessons learned, we focus on preclinical and clinical studies in malignancies and discuss the reasons for the limited success of NK-cell engagers against solid tumors, offering plausible new ideas for curing some advanced cancers shortly.

NK cell engaging bispecific antibody as an emerging therapy

Immunotherapy is the preferred agent for systemic cancer treatment, and antibody therapy in particular has become the preferred treatment choice for cancer because of its ability to specifically target molecules and low toxicity.¹ However, in complex disease pathogenesis, a wide range of mediators help to stimulate separate signaling pathways or promote duplicated signaling cascades, which limit the effectiveness of therapies targeting individual molecules.² Tumor types are considered to express diverse receptors that interact to activate such tumor cells to proliferate indefinitely, metastasize, and inhibit apoptosis. For this reason, in 1961, Nisonoff et al. made a large class of molecules capable of recognizing two different epitopes or antigens, known as bispecific antibodies, by chemically cross-linking the Fab’ fragments of two polyclonal antibodies that bind different antigens.³ With the rapid development of genetic engineering techniques and new insights into the mechanisms by which tumors evade immune control, the next generation of bispecific or multi-specific antibodies is being developed to eliminate cancer by redirecting immune cells (e.g., T cells, NK cells) to the tumor. Various forms of bispecific antibodies (bsAbs) have been generated, which in clinical practice may show better clinical efficacy than monoclonal antibodies or other conventional anti-tumor therapies.^4,5

The redirection of immune cells is not necessarily restricted to T cells. Natural killer (NK) cells are cytotoxic lymphocytes of the innate immune system that can kill tumor or virus-infected cells with effector functions.⁶ Significantly, a higher percentage of NK cell infiltration in solid tumors is associated with better clinical therapeutic efficacy, as has been demonstrated in breast cancer,⁷ head and neck cancer,⁸ and clear cell renal cancer.⁹ The composition and release of NK lysis granules filled with perforin and granzyme are comparable to that of cytotoxic T cell subpopulations but without strong cytokine release.¹⁰ Targeting NK cells by immunotherapy is, therefore, an attractive anti-tumor strategy.

In addition, NK cells could express various activating and inhibitory receptors to respond to the tolerance of or activation signaling of cytotoxicity against the target cells.¹¹ Where the total level of inhibitory receptor signaling surpasses the total level of activating receptor signaling, NK cell activation is thwarted, resulting in the induction of cellular tolerance.¹² For instance, NK cells normally upregulate stimulatory ligands for NK cell activation receptors such as natural-killer group 2 (NKG2D) in response to viral infection or transformation. The resulting interaction induces activation of signaling levels that exceed those of receptors at the level of intrinsic signaling via inhibitory signaling (e.g., killer immunoglobulin-like receptors (KIR) and NKG2A), activating the release of NK cell cytokines including products of cytotoxicity to target cells.¹³

NK cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is another key mechanism for the killing of cancer cells. Several studies have confirmed that the Fc receptor CD16 (CD16A, FcγRIII) is the most effective activating receptor expressed in NK cells, resulting in ADCC-triggered tumor cell lysis when IgG-coated target cells bind to it and occur independently of other co-activating receptors.^14,15 Indeed, several therapeutic monoclonal antibodies (mAbs), such as rituximab (Rituxan®), cetuximab (Erbitux®), and trastuzumab (Herceptin®) based on this mechanism are in wide use and have shown the significant therapeutic potential of NK cells^16–19(Table 1).

Table 1.

Overview of pre-clinical or clinical trials based on NK cells and MAbs.

Treatment	Condition or disease	Target	Reference or clinical trial
Rituximab-RLIb	B-cell malignancies	CD20	Vincent et al.20
c.60C3-RLIc	Neuroblastoma cells.	GD2	Vincent et al.21
Hul4.18-IL2	Neuroblastoma cells	GD2	Shusterman et al.22 Delgado et al.23
KM2812	Prostate cancer cells	PSMA	Sugimoto et al.24
293C3-SDIE	Acute myeloid leukemia cells	CD133	Koerner et al.25 Riegg et al.26
7C6 mAb	Cholangiocarcinoma	NKG2D receptor	Oliviero et al.27
Rituximab	Non-Hodgkin’s lymphoma	CD20	Cartron et al.28
Obinutuzumab	Chronic lymphocytic leukemia	CD20	Goede et al.29 Capuano et al.30
Dinituximab	High-risk neuroblastoma	GD2	Yu et al.31
Trastuzumab	Breast cancer	HER2	Junttila et al.32
Cetuximab	Metastatic colorectal cancer and head and neck cancer	EGFR	Juliá et al.33
Hu3F8	GD2+ tumors	GD2	NCT01419834 NCT01662804 NCT01757626

A recently revisited approach is to redirect NK cells to tumor cells by targeting one tumor antigen (e.g., CD19) and one CD16A molecule simultaneously, similar to the BiTE® format which consists of a target on the surface of cancer cell and CD3 on the surface of T-cells. Some groups have named these bispecific antibodies “bispecific killer cell engagement (BiKEs).”¹⁰ These BiKEs work by bringing NK cells and target cells into similar proximity, forming cytolytic synapses that lead to tumor cell death. Tri-specific killer engagers (TriKEs) are trivalent molecules that redirect the killing of NK cells against different tumor antigens and enhance NK cell functions through the addition of a third single-chain variable fragment (scFv) targeting another cancer target (e.g., CD22) or immune cell-stimulating cytokines, like interleukin (IL)-15.³⁴

Although these molecules vary in structure, they all have small molecular weights and structural domains that bind to target-associated antigens (TAAs) and NK cell-activated receptors, including CD16A, NKG2D, NKp30, and NKp46.³⁵ In this review, we discuss the emergence of Bi- and trispecific NK cell engagers as a new generation of immunotherapy, as well as the advantages, obstacles, and development prospects for future clinical applications.

Bi-specific and tri-specific NK cell engagers mechanism of action

As previously mentioned, NK cells are cytotoxic lymphocytes of the innate immune system and play an important role in tumor immune surveillance. One strategy to re-direct NK cells to cancer cells is based on tandem scFvs, like bi- or tri-specific NK cell engagers,³⁶ which can target both scFv binding CD16A on NK cells and one or two TAA on various solid tumor types (e.g., CD19, CD133, EpCAM, or B7-H3) (Figure 1).^37,38 Alternatively, two scFvs can be linked by a modified human IL-15 cross-linker to produce TriKEs that promote the persistence and activation of NK cells in vivo. For example, AFM13, a bispecific antibody targeting CD30 and CD16A, mediates in vivo ADCC and NK cell retention activity, with CD30 being a prominent marker for relapsed/refractory (R/R) Hodgkin lymphoma (HL), increased cytotoxicity.^39,40 In addition, revisiting the CD133-expressing cancer stem cells (CSCs), a TriKE expressing IL-15, named 1,615,133, showed in vitro the activity of NK cell efficient cytotoxicity and proliferation.⁴¹

Figure 1.

Design and function of BiKEs and TriKEs (a) BiKEs are consisting of individual heavy (VH) and light (VL) chains of the variable region of each different antibody with the VH and VL structural domains linked via short flexible peptide junctions to prevent dissociation. These scFv are specific for CD16 on NK cells and tumor antigens on tumor cells. (b) TriKEs of the same design including the cytokine, IL-15, are included as a linker between the two scFv components. (c) shown here is a mechanism diagram. BiKEs and TriKEs redirect NK cells to target tumor cells through a tumor-specific target on the one hand and to CD16A-positive NK cells on the other hand, forming lytic immune synapses with cytokines, granzymes, and perforins release, ultimately leading to tumor cell death.

BsAbs can be divided into IgG-like and non-IgG-like based on their structure for the presence of the Fc region of the IgG-like BsAbs with Fc-mediated antibody effect function, such as ADCC. In addition, the retention of the constant region is conducive to the purification of the antibody, improves the solubility and stability of the antibody drug, and in vivo has a higher half-life.⁴² Although NK cells can also be recruited, what needs to be solved in the process of practical project development is the problem of antibody chain mismatch, which often leads to low efficiency of double antibody industrialization and high impurity proteins. In contrast, non-IgG-like BsAbs like BiKEs or TriKEs are less immunogenic and relatively safer because of their relatively small molecular weight (55-60 kDa).⁴³ Although it has a short half-life, BiKEs have high tumor tissue permeability. In addition, NK cells are safer than T cells compared to BiTEs, and NK cell infusion does not induce graft versus host disease; therefore, adverse events with BiKEs and TriKEs are usually less severe.⁴⁴ In summary, these BiKEs or TriKEs showed promising translational potential.

Examples of bi-specific and tri-specific NK cell engagers

Hematological malignancies

BiKE has demonstrated promising treatment by targeting specific hematopoietic cell lines and their corresponding malignant counterpart antigens, like CD19, considered a key component of the B-cell receptor complex, as well as CD33 highly expressed by myeloid cells and acute myeloid leukemia (AML) cells.⁴⁵ For example, CD16 × CD19 BiKEs showed the enhancing effect of in vitro NK cell-mediated cytotoxicity.⁴⁶ In a study of AML and biphenotypic, ALL blasts from pediatric patients with primary CD33⁺ blasts, CD16 × CD33 BiKEs elicited the inhibitory effect of KIR signaling and enhanced NK cell-mediated lysis of AML blasts.⁴⁷ Moreover, CD16 × CD33 BiKEs were also demonstrated to redirect NK cells to myelodysplastic syndrome (MDS), expressing CD33 in pre-clinical studies. Gleason MK et al. published the results about 67 patients with MDS who could be assessed for a response by a pre-clinical study against high-risk MDS to express CD33.⁴⁸ In this treatment, BiKEs played an essential role in targeting MDS and other myeloid malignancies and significantly enhanced degranulation and cytokine production.

TriKEs serve as an alternative and innovative version, and 161533 TriKEs (GTB-3550), including an anti-CD16 and an anti-CD33 scFv with IL-15 presented as a linker, have anti-leukemic activity in patients with relapsed and/or refractory AML or MDS, according to results from pre-clinical studies and a phase I/II clinical trial (NCT03214666).^49,50 CD19 as an antigen target for Trike technology was confirmed in studies assessing its expression in patients with primary chronic lymphocytic leukemia (CLL), and the cytotoxicity of the 161,519 TriKE (CD16 × CD19 × IL-15 TriKEs) showed better results in the lysis of CLL cells in vitro, when compared with that of rituximab or those treated with 1619 BiKE (CD16 × CD19 BiKE).⁵¹ Additionally, multiple clinical trials are currently underway for BiKEs or TriKEs treatment of R/R HL patients (e.g., NCT02321592, NCT01221571, NCT03192202, and NCT04074746),^39,40,52 or in combination with pembrolizumab (NCT02665650), or for BCMA⁺ relapsed or refractory multiple myeloma (known as RO7297089, NCT04434469) (Table 2)^53–55 are currently underway.

Table 2.

Overview of clinical current trials of bi- and tri-specific NK cell engager.

Drug	Condition or disease	Targeted antigen	ClinicalTrials.gov Identifier	Phase status
AFM13 (anti-CD30/CD16A BiKE)	Lymphoma, T-Cell, Cutaneous	CD30	NCT03192202	Phase 1/2 Completed
	Hodgkin Lymphoma	CD30	NCT01221571	Phase 1 Completed
	R/R CD30-positive T-cell Lymphoma, Transformed Mycosis Fungoides	CD30	NCT04101331	Phase 2 Active, not recruiting
	R/R Hodgkin lymphoma	CD30	NCT02321592	Phase 2 Completed
The combination therapy of AFM13 and pembrolizumab (MK-3475)	R/R Hodgkin lymphoma	CD30	NCT02665650	Phase 1 Completed
The combination therapy of AFM13 and UCB- derived NK cell therapy	R/R Hodgkin or Non-Hodgkin Lymphomas	CD30	NCT04074746	Phase 1/2 Recruiting
AFM24 (anti-EGFR/CD16A BiKE)	Advanced Solid Tumor	EGFR	NCT04259450	Phase 1/2 Recruiting
The combination therapy of AFM24 and SNK01	Advanced or metastatic EGFR-expressing cancers	EGFR	NCT05099549	Phase 1/2 Recruiting
The combination therapy of AFM24 and atezolizumab	Advanced Solid Tumor	EGFR	NCT05109442	Phase 1/2 Recruiting
AFM26 (anti-BCMA/CD16A BiKE)	R/R Multiple Myeloma	BCMA	NCT04434469	Phase 1 Completed
GTB-3550 (anti-CD16/IL-15/CD33 TriKETM)	High-risk MDS and R/R AML	CD33	NCT03214666	Phase 1/2 Recruiting
The combination therapy of DF1001 (anti-HER2 TriNKETM) and Nivolumab	HER2+ solid tumors	HER2	NCT04143711	Phase 1/2 Recruiting

*All listed clinical studies are sourced from ClinicalTrials.gov. BCMA B cell maturation antigen, R/R relapsed or refractory, UCB umbilical cord blood, EGFR epithelial growth factor receptor, MDS myelodysplastic syndrome, AML acute myeloid leukemia, HER2 human epidermal growth factor receptor 2.

Solid tumor

One of the most significant issues in the area of NK cell-engaging therapies is whether these treatments are effective outside of hematological malignancies. AFM24 is another novel bispecific, IgG1-scFv fusion antibody developed by Affimed, based in Heidelberg, Germany, that mediates tumor cell depletion by targeting EGFR on tumor cells and CD16A on, NK cells, i.e., innate immune cells and macrophages.⁵⁶ A pre-clinical investigation has emphasized robustly enhanced killing of tumor cells expressing varying levels of EGFR, regardless of KRAS/BRAF mutational status both in vitro and in vivo with AFM24.⁵⁷ Importantly, there was the absence of systemic immune activation or any other toxicity found in the cynomolgus monkey model in vivo; thus, the promising potential clinic trial (NCT04259450) of AFM24 is currently enrolling patients (Table 2).

The epithelial cell-adhesion molecule (EpCAM) is a mutated TAA that is expressed in various types of carcinomas like prostate cancers, colon, breast, and ovary. In 2013, Vallera DA and colleagues showed strong preclinical evidence that CD16 × EpCAM BiKEs were previously presented in vitro with significantly higher NK cell cytotoxicity at low effect target ratios.^35,38 A sufficient NK cell proliferative capacity seems to be a further prerequisite for NK cell cytolytic activity and correlates with the level of inflammatory cytokine production. Thus, to enhance the efficacy of specific engagers, an EpCAM TriKE that incorporates an IL-15-crosslinker into a BiKE construct relative to CD16 × EpCAM BiKEs is preferable.⁵⁸ These authors proposed that the CD16 × EpCAM × IL-15 TriKEs markedly enhance its therapeutic potential and NK cell proliferation in a manner like exogenous IL-15 although its intramolecular conformation. In line with the TriKEs platform, B7-H3 expressed by prostate cancer, ovarian cancer, and lung cancer,⁵⁹ and tumor endothelial marker 8 (TEM8) expressed by lung cancer is also currently being tested preclinically.⁶⁰ These data all suggest that TriKEs may be a valuable NK cell-based immunotherapy for patients with various solid tumors.

Taken together, advances in BiKEs and TriKEs technology could substantially improve the treatment of hematologic and solid tumors.

Limitations of bi-specific and tri-specific NK cell engagers

NK cell engagers are considered less immunogenic and relatively safer than Fc-mediated ADCC compared to conventional IgG because of their relatively small molecular size.^61,62 In contrast to antibody-drug conjugates (ADCs), which utilize NK cell redirection for cytotoxic effects and are more dependent on the host’s immune system, they have a better safety profile.^63,64

A deeper understanding of the mechanisms of structural selection, tumor escape, resistance, and toxicity confronted in Bi-specific and Tri-specific NK cell engagers may identify opportunities to enhance the effectiveness of NK cell-based therapies and the long-term outcomes of cancer patients. Notably, to compensate for these shortcomings, researchers are currently performing several different modifications to improve their efficacy (Figure 2).

Figure 2.

Different formats of NK cell engagers. A BiKE molecule consisting of two single chain fragment variables (scFvs), including one targeting a target-associated antigen (TAA) and (a) CD16A, (b) NKG2D, (c) NKp30 on NK cells; a TriKE adding (d) an interleukin (IL)-15-crosslinker or (e) another TAA against tumor cells; (f) a Tetraspecific killer engager (TetraKE) comprising three scFvs and an IL-15-crosslinker; (g) BiKEs and the PD-1/PD-L1 blockade combination therapy; (h) in addition, oncolytic viruses (OVs) have been modified for NK cell engagers, forming OVs-armed BiKEs.

Selection of structure

It is generally known that bsAbs can simultaneously engage two different antigens together. This increased functionality may effectively lead to fewer side effects and fewer injections compared to conventional mAbs, which can only bind a single epitope.^65,66 It is important to understand how to design and stabilize bsAbs in vivo for developing effective NK cell-mediated anti-cancer therapeutics. The design of these structures is a complex process and with the rapid development of protein engineering technology in recent years, researchers have become increasingly interested in nanobodies.

Nanobodies are known to be univariate immunoglobulin structural domains originating from naturally occurring heavy chain antibodies (hcAbs) in camels.^67,68 It has been shown that nanobody-based BiKEs (nano-BiKEs, approx. 30 kDa) are smaller in size and more soluble than scFv-based BiKEs (approx. 50 kDa), resulting in superior stability and greater penetration of tissues in vivo.⁶⁹ Antibodies binding selectively to CD16A have an essential role in improving treatment efficacy and decreasing off-target toxicity. Nikkhoi et al. designed a functional bispecific killer cell engager (BiKE) with high affinity and specificity/selectivity for the CD16A receptor using anti-CD16A and anti-HER2 nanobodies.⁷⁰ This BiKE induced higher ADCC on ovarian and breast cancer cells compared to trastuzumab. Besides, Van Faassen H and his colleagues constructed bi-specific sdAb-sdAb and sdAb-scFv BiKEs bound by fusion with tumor-specific sdAb or scFv (anti-CD19, anti-HER2, or anti-EGFR) by isolating camel anti-CD16 monoclonal antibodies. The experimental results suggest that the smaller size of sdAbs may confer better tumor penetration to sdAb-based BiKEs and TriKEs compared to full-length IgGs, Fabs, or scFv; also, the fact that the nanobodies have only heavy chains without the need for light-heavy chain pairing makes them suitable for modular assemblies, allowing for the easy construction of multispecific and multivalent molecules.⁶⁸

Despite the advantages of hcAbs over full-length IgG, Fabs, or scFvs in the design of NK cell engagers, their smaller size which is below the kidney filtration barrier leads to a shorter serum half-life in vivo. To prolong in vivo half-life, several research groups have designed nano-BiKE with an extended half-life (HLE nano-BiKE), that is, nano-based BiKE is fused into albumin-specific nanobody.^71,72 Recently, Hambach J et al. reported that CD38-specific HLE-nano-BiKEs recognizing three distinct and non-overlapping epitopes of CD38-induced NK92 hCD16-mediated cell lysis more efficiently than the conventional CD38-specific antibody daratumumab (ADCC) and induced in vitro CD38-positive myeloma cell line cytotoxicity.⁷³ These nanobody-trimers bind CD38 on myeloma cells (N-terminal nanobody), bind and activate NK cells via targeting CD16 with central nanobodies, and bind to albumin via a C-terminal nanobody. Thus, the whole trimer not only targets the antigen and activates NK cells but also extends the half-life of the construct.

In general, different NK cell engagers have their structural advantages and shortcomings, and the selection and development of next-generation NK cell engagers are still the centers of future research.

Tumor drug resistance

Antigen escape

The first step in generating BiKEs is the identification of suitable tumor cell-specific antigens. Ideally, the target antigen needs to be clearly and predominantly expressed in tumor cells, rather than in normal cells to prevent nonspecific toxicity, and they are closely associated with malignant phenotypes or signaling pathways to prevent immune tolerance caused by antigen mutations. However, most cancer targets do not meet these criteria.¹⁰ CD19, the most representative TAA, is restricted to specific immune cell lineages and is a key antigenic target for BiKEs-based immunotherapy. It has been reported that anti-CD19 × CD3 BiTEs in B-ALL therapy result in loss of extracellular structural domains due to mutations in CD19, conformational changes, and ultimately therapeutic failure.^74,75 For this reason, some researchers have come up with the idea of TriKEs. It can simultaneously recognize multiple antigens on the surface of cancer target cells and is expected to improve tumor targeting specificity and exert better anti-tumor efficacy through synergistic signal inhibition and accelerated tumor cell degradation. For example, generating a tri-specific antibody, which recognizes both CD33 and CD123 on AML cancer cells.⁷⁶ Similarly, Schmohl et al. recently developed a tetraspecific killer engager (TetraKE), named 1615EpCAM133, which targets an anti-CD16 scFv on NK cells, an anti-EpCAM scFv on carcinoma cells and an anti-CD133 scFv on cancer stem cells to promote dual-antigen ADCC.⁷⁷ Importantly, although an IL-15-crosslinker enhanced NK cell-related superior efficacy and cytokine production, there is no evidence of excessive cytokine release.

Furthermore, the search for tumor-specific antigens (TSAs) and the identification of the associated intrinsic mechanisms is challenging but imminent, paving the way for improved anti-tumor strategies.⁷⁸ Although most immunotherapies target TAAs as they are usually overexpressed in a group of tumor types, numerous clinical reports of TAA-based therapies have shown their potential damage to normal tissues and are accompanied by unsatisfactory clinical outcomes.⁷⁸ However, most TSAs are derived from non-synonymous somatic mutations that are completely absent in normal cells by contrast. It can be selectively expressed in tumor cells, providing an opportunity for personalized TSA-dependent immunotherapy due to differences between individuals.^10,79

The suppression of NK cells is another explanation for tumor escape. Thus, another attractive option is the addition of scFvs that block checkpoint receptors such as PD-1, KIR, NKG2A, or TIGIT through structural improvements to TriKEs to bypass checkpoint blockade and further drive NK-mediated anti-tumor responses.⁸⁰ More recently, one study has shown that the use of a PD-1 blocking antibody (CT-011) enhances target cell killing and cytokine production by NK cells in response to PD-L1 expressing primary multiple myeloma cells or myeloma cell lines in vitro.⁸¹

Immunosuppressive microenvironment

The ability of NK cells to kill tumor cells is greatly hampered in the face of sensitivity to the multiple immunosuppressive mechanisms active within TME although their activity in tumor growth control. It could limit the persistence of NK cells and inhibit their cytotoxic functions, by producing immune-suppressive cells and secreting factors, IL-6, IL-10, prostaglandin E2 (PGE2), and indoleamine 2,3-dioxygenase (IDO).^82,83 To benefit patients, several potential strategies are a focus of future research.

ADAM17 (a disintegrin and metalloprotease 17), an enzyme that cleaves the outer membrane of L-selectin, promotes the decay of NK cell function by cleaving CD16 from the cell surface and is another mechanism by which MDSC suppresses antitumor immunity.^84,85 Wiernik et al. found that combining CD16 × 33 BiKEs with an ADAM17 inhibitor increased both NK cell toxicity and cytokine secretion, preventing the failure of NK cell therapy due to the immunosuppressive effects of the TME.³⁷

Generally, while the current generation of TriKEs triggers NK responses through CD16 ligation and cytokine signaling, other components can be added to enhance the NK cell anti-tumor response. For example, another mechanism by which NK cells evade tumors is by shedding soluble NKG2D ligands.⁸⁶ Therefore, ligand expression on the tumor surface is reduced, decreasing the susceptibility to NK-mediated tumor cell killing. Tregs have been reported to downregulate NKG2D and NCR NKp30 expression on NK cells through cell–cell contacts, membrane-bound TGF-β, and soluble TGF-β.⁸⁷ In the future, including an scFv blockade of TGF-β to reduce negative signaling in the tumor microenvironment might enhance NK cell-mediated tumor killing based on a bispecific antibody platform.

Pharmacokinetics and toxicity

Although a variety of NK cell engagers have demonstrated good in vitro targeting capabilities, their safety including preclinical studies to clinical trials and finally to the formal approval of the product for the treatment of tumors is a key point that must be considered and evaluated. BiKEs or BiTEs have a short half-life in serum, thus one of the drawbacks of BiKEs or BiTEs is the need for continuous intravenous infusion. The development of arming oncolytic viruses (OVs) with a therapeutic transgene encoding BiTE is an attractive platform for bsAbs delivery.⁸⁸ OVs enter cells by interacting with specific surface receptors that are highly expressed in cancer cells. Viral replication is achieved, targeting tumor cells while leaving normal cells undamaged. For example, Fajardo et al. engineered the adenovirus ICOVIR-15K to express antibodies against BiTE (cBiTE) targeting EGFR, and ICOVIR-15K-cBiTE induced the persistence and accumulation of tumor infiltration in vivo.⁸⁹ In another similar construct, Freedman et al. modified an anti-EpCAM × CD3 BiTE-armed oncolytic group B adenovirus EnAdenotucirev (EnAd) that could enhance infiltration and activation of CD4⁺ and CD8⁺ T cells, thereby enhancing T cell-mediated tumor killing in clinical tissue biopsies.⁹⁰ Although no relevant engineered BiKE-producing OVs are currently undergoing preclinical evaluation, future applications are extremely valuable.

In addition, TriKEs have also been reported to potentially activate systemic immune responses by triggering cytokine cascades, leading to a risk of systemic toxicity.⁹¹ Thus, ongoing development of treatments is imperative, both in terms of alternative and potentially safer ways of detecting cytokine pathway activation.

Conclusions and future directions

The utilization of immunotherapy to target malignant cells has emerged as a novel approach for the treatment of various cancers. Especially, NK cells serve as a unique set of anti-tumor response agents with non-MHC-restricted cytotoxicity, and cytokine production. The success of preclinical studies by multiple groups and the results of ongoing clinical studies with BiKEs have demonstrated specific killing activity. However, in the long term, a large proportion of patients will not benefit from immunotherapy based on bi-specific and tri-specific NK cell engagers.

The major challenge is the treatment of solid tumors involving immunosuppressive TME. Since inhibitory receptors, downregulation of activated receptors, molecular checkpoints, and inhibitors of the tumor environment all lead to immune escape of NK cells, combinations of immune checkpoint inhibitors targeting the synergistic response of NK cells are being attempted. The observation that the antitumor activity of a CD47 × CD19 bispecific antibody (NI-1701) was improved in the presence of NK cells by CD47/SIRPα blockade in vivo,⁹² and the evidence that CD47 antibody treatment could thereby promote activation of systemic and tumor-associated NK cells.⁹³

Another important aspect of NK cell therapy is to increase the lifespan of NK cells, and TriKEs provide another choice for NK cell persistence in vivo regardless of genetic modification. However, insight is needed into the biological effects of TriKEs on NK cells, including their activation, maturation, and depletion; their activity after cessation of administration; and their tendency to develop into a long-life memory cell population.

For example, a novel multifunctional molecule （ ANKET4 ）based on the versatile NK cell engager platform is available in preclinical development that binds an Interleukin-2 (IL-2) variant that enhances NK cell activation and proliferation through cis-binding of NKp46, CD16a, and IL-2 Rβ (CD122) without binding to IL-2 Rα (CD25) in Tregs or endothelial cells.⁹⁴ In testing with non-human primates, targeting CD20 to ANKET4 showed that ANKET4 not only has no clinical signs of toxicity but also severe depletion of circulating B cells with a favorable safety profile. During clinical trials, as more bsAbs and cellular therapies are investigated, understanding of the optimal design and/or construct for targeting specific tumor types is increasing. We expect that as more NK cell-based immunotherapy is applied in clinical treatment in the future, more and more patients will benefit from painful diseases and endless treatment processes.

Funding Statement

This work was supported by the Health Research Project of Huai’an City in 2020, General Program, Jiangsu, China. Grant number [33].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Tian Huan and Bugao Guan collected the data and wrote the manuscript. Hongbo Li, Xiu Tu, and Chi Zhang provided substantial contributions to revise the paper critically for important intellectual content. Bugao Guan and Bin Tang contributed to the scientific discussion and critical review of the manuscript. All authors contributed to the article and approved the submitted version.