As NK cells are often impaired in cancer patients, the use of adoptive NK cellular immunotherapy aims to restore NK cell functionality through supplementation or complete replacement of the NK cell populations with functionally active NK cells. As a result, tumor load, and the immunosuppressive TIME could be reduced. Earliest attempts of adoptive NK cell transfer failed to show meaningful clinical responses using ex vivo purified and unstimulated NK cells (122).

Therefore, combination of an NK cellular product with cetuximab could enhance the functionality of these NK cells and achieve overall responses through the induction of ADCC. A phase I trial in CRC administered ex vivo expanded patient-derived NK cells following cetuximab treatment (123). Noteworthy, the majority of expanded NK cells showed high expression of NKG2D and CD16, and high lymphocyte-activation gene 3 (LAG-3) and TIGIT expression. Cytotoxic effects toward the tumor remained elevated up to 4 weeks following NK cell administration, indicating a favor towards NK activation rather than inhibition. Addition of expanded NK cells following cetuximab treatment displayed an increased cytotoxic activity against tumor cell lines and reduced overall tumor size of heavily pretreated cetuximab-resistant patients. Lastly, patients treated with expanded NK cells following cetuximab showed enriched levels of circulating interferon gamma (IFNγ) and reduced Treg frequencies, suggesting an induction of a Th1‐type adaptive immune response (123).

With the increased understanding of self-regulation in NK cells, a possible alternative for the limited number of patient-derived NK cells has been the use of allogeneic NK cells. This approach may hold several benefits including the ability to obtain NK cells from healthy donors which may retain greater antitumor activity and the development of off-the-shelf application due to easier and greater availability of NK cells (124). Furthermore, several models to predict alloreactivity of NK cells (graft-versus-host disease) have been described (125), the ‘Receptor–ligand mismatch’ model remains one the most established predictive models. Briefly, donor NK cells bearing inhibitory KIR for which the corresponding HLA ligands are missing in the recipient become uninhibited. The presence of (non-HLA-restricted) activating signals can then induce alloreactivity (126, 127).

Sources for alloreactive NK cells include (i) acquiring umbilical cord blood (128),; (ii) partially KIR/HLA matched peripheral blood (126); or (iii) engineered NK cell lines (129). Investigations using the former primary NK cells yielded increased expression of activation markers CD69 and CD16 and strong ADCC responses towards NSCLC and B cell lymphoma in vitro and in mice (128). Adoptive transfer of the modified NK-92 cell line (haNK) cells with cetuximab harbored the capacity to efficiently kill HNSCC tumor cells in a dose-dependent manner and enhanced ADCC response (130, 131). In a clinical trial in NSCLC, ex vivo stimulated KIR/HLA matched healthy donor NK cells were administered together with cetuximab. This combination led to a significantly improved PFS and OS compared to cetuximab alone (132). A phase I trial in gastrointestinal carcinoma used allogeneic IL-2 stimulated NK cells in combination with cetuximab and obtained beneficial clinical responses and a tolerable safety profile (133). Interestingly, while addition of adoptive NK cells increased the number of circulating lymphocytes (CD8⁺, CD4⁺, B and NK cells), cetuximab alone, albeit to a lesser degree, was also able to significantly increase lymphocyte levels. This suggests that part of the increased levels may be related to improvement of cellular immunity and prevention of apoptosis of T cells. Indeed, levels of IFNγ and pro-inflammatory cytokines were significantly more present through combination of cetuximab with adoptive NK cell transfer, indicating an enhanced Th1-response (132). These first and promising results of cetuximab stimulating adoptive NK cell therapy in solid tumors are encouraging, since to date clinical effectiveness of adoptive NK cell therapy is only observed in hematological malignancies. Therefore, more research on cetuximab unlocking the potential of adoptive NK cell therapy for solid tumors is warranted.

A more recent and promising approach for adoptive NK cell therapy is the use of chimeric antigen receptor-engineered NK (CAR-NK) cells. These can be developed either through lenti-/retroviral transduction of primary adult NK cells or immortalized NK-92 cells to recognize a specific tumor antigen (134). CAR-NK cells have several advantages over CAR-T cells. First, they are more robust as they still maintain their intrinsic target cell recognition. Therefore, a reduction of the target CAR is less likely to be an effective tumor escape mechanism (135). Second, cytokines released by activated NK cells are less associated with the induction of a cytokine release syndrome (136, 137). Third, as NK cells do not clonally expand, the cytokine levels they release is found to be less sufficient to induce a cytokine release syndrome (138, 139). Fourth, NK cells are known to suppress graft-versus-host reactions which are induced by T cells due to strict HLA-matching (135, 136, 138).

While CAR-NK therapy research is developing at a rapid pace, combination treatments using CAR-NK together with already established treatments are still limited. Recently, combined treatment of a CRC mouse model with epithelial cell adhesion molecule (EpCAM)-CAR-NK-92 and regorafenib (a sorafenib-related multikinase inhibitor) achieved a synergistic tumor suppression than either treatment alone (140). The basis for this investigation was the observation that regorafenib could modulate the TIME through alteration of Fas and PD-L1 expression in CRC cell lines (140). Similarly, efficacy of cetuximab in HNSCC is also linked to its immunostimulatory activities which include downregulation of PD-L1 expression. Therefore, although not validated yet, this suggests that cetuximab combined with CAR-NK cells against a specific tumor antigen could alter the TIME towards tumor cell killing as a potentially promising treatment strategy. As a proof of concept, CAR-T cells transduced with CD32A or CD16 in combination with cetuximab, achieved a greater cytotoxic response and improved survival of a CRC mouse model bearing EGFR mutations compared to either treatment alone (141, 142). Taken together, although definitive evidence for this regimen is still missing, these early results support the potential strength of cetuximab-based dual-targeting CAR-NK therapy as an adoptive therapy.

A last consideration is that adoptive transfer of (un)modified NK cells in solid tumors is inferior compared to responses observed in hematological malignancies. The most evident cause for this discrepancy is the poor migration of infused NK cells inside the tumor. This may be caused by altered chemokine receptors following ex vivo activation. For example, CXCR2/3/4 are important chemokine receptors on immune cells that facilitate migration towards CXCL9/10/12-expressing tumor cells (143–146). Loss of CXCR2/3 following ex vivo activation prevented NK cells from migrating towards B16 melanoma tumors (147). Therefore, more recent expansion protocols such as the one described by Somanchi et al. (148) consider the chemokine repertoire in order to achieve efficient expansions of specific NK cell phenotypes that may provide a better invasion in the tumor.

The PD-1/PD-L1 axis has become one of the most studied pathways in cancer immunotherapy, with promising results guiding the approval of several inhibitors (150, 151). Interestingly, early investigations of PD-1 expression on NK cells found 25% of healthy individuals to have PD-1⁺ NK cells which correlated well with prior human cytomegalovirus infections (152). This prompted the idea that PD-1 expression on NK cells is a result of activation rather than exhaustion, which is the case for T cells following chronic stimulation (153). In cancer patients, peripheral blood NK cells are often found to be PD-1 positive (154–156) and intratumoral NK cells often express high levels of PD-1 (40, 156).

Interestingly, PD-1⁺ NK cells were found to have downregulated CD16 expression and induce PD-L1 expression on tumor cells via IFNγ secretion, thus possibly inhibiting ADCC induction (157). However, inhibition of EGFR-signaling via cetuximab is known to interrupt INF-γ signaling and prevent PD-L1 upregulation on tumor cells (117). Thus, combining cetuximab with a PD-1 inhibitor could be viewed as a valuable strategy to prevent CD16 downregulation and PD-1/PD-L1-axis mediated silencing of ADCC. A study in HNSCC found an increased number of PD-1⁺ NK cells in patients, which correlated with a diminished NK cell activity, as observed by a downregulated expression of CD16, CD107a and GZMB. In addition, PD-L1 expression correlated with a lack of response to cetuximab alone. Administration of cetuximab in combination with the anti-PD-1 mAb nivolumab successfully reversed NK cell diminishment and enhanced cetuximab-mediated ADCC in vitro (157). Early results from a phase I trial in HNSCC patients also reported an increased objective response rate compared to either treatment alone (158). Currently, several trials investigating this combination are ongoing, with preliminary results indicating potentially synergistic effects in advanced solid tumors (159, 160).

Recent years have seen a growing interest in the TIGIT signaling pathway due to its complex immunomodulatory role. Similar to the B7/CD28/CTLA-4 pathway, the TIGIT axis consists of a network of inhibitory receptors (TIGIT, CD96 and CD112R) that compete with the activating receptor (DNAM-1/CD226) for their shared ligands (CD111/NECTIN1, CD112/NECTIN2, CD113/NECTIN3, CD155/PVR) (161, 162). In contrast to DNAM-1, only marginal TIGIT expression is observed on resting NK and T cells while stimulation and tumor infiltration showed upregulated TIGIT expression (161). As a stimulatory receptor, DNAM-1 signaling induces pro-inflammatory cytokine secretion and enhances cytotoxic activity of NK cells. Meanwhile, TIGIT induces an anti-inflammatory, non-proliferative and non-cytotoxic profile in NK cells (163).

Targeting of TIGIT is still in early development but positive early (pre)clinical investigations have enabled further clinical investigations. Interestingly, in vitro co-culture and in vivo transgenic HNSCC mice models were able to restore the cytotoxic effects of T and NK cells following anti-TIGIT treatment (164). Initial clinical studies in solid tumors demonstrated strong antitumor activity as a single agent (163, 165), that could be further improved when combined with anti-PD1 mAb (NCT03119428, NCT02794571). Furthermore, disruption of the TIGIT/CD155 interaction can also beneficially impact the TIME, in particular by incapacitating myeloid-derived suppressor cells and depleting Tregs. Although not investigated yet, this observation suggests the possible combination of anti-TIGIT-mAbs with cetuximab, thereby reducing the suppressive action of Tregs and targeting specific tumor antigens.

Alternatively to TIGIT, CD155 (PVR), has been suggested as a potential target due to its greater affinity towards TIGIT compared to DNAM-1 and its frequent overexpression in solid tumors (166, 167). However, clinical trials of CD155 are still scarce and preclinical investigations of CD155 in combination with cetuximab are limited as well. However, one study in CRC cell lines reported an improvement of cetuximab-mediated ADCC following effective signaling of DNAM-1/CD155. Blocking this interaction abrogated this effect entirely (168). The same effect was observed by blocking NKG2D/MICA/B signaling. A possible reason for the limited progress in CD155 targeting might be that CD155 inhibition disrupts both TIGIT and DNAM-1 signaling, therefore potentially robbing NK cells from activating signals. However, this concern is not completely warranted, as CD155 under normal circumstances has a greater affinity towards inhibitory receptors, thus preferentially signaling via TIGIT even in the presence of DNAM-1 (169). Lastly, administration of anti-CD155 also showed upregulation of DNAM-1 on peripheral blood lymphocytes. As CD155 is not the only ligand capable of binding DNAM-1, this interaction could potentially shift the balance towards increased antitumor immunity (170).

Altogether, this suggests that strategies targeting the TIGIT-axis could reverse immune inhibition through reduced inhibitory signaling and that combinations with cetuximab could enhance ADCC, resulting in an enhanced antitumor response (167).

Another ITIM-containing signaling pathway expressed on NK and T cells is the NKG2A-HLA-E interaction. Although NKG2A is expressed on a low number of peripheral NK cells, both antigen and cytokine stimulation upregulate its expression (171, 172). While binding of NKG2A to HLA-E is known to inhibit NK cell responses, ovarian cancer cell lines that were treated with the anti-NKG2A mAb monalizumab showed profound antitumor responses and significantly improved cetuximab-mediated ADCC (173, 174). Moreover, monalizumab combined with cetuximab was tested in a phase II trial with recurrent and metastatic HNSCC patients showing promising improvements with an easily manageable safety profile similar to either treatment alone (173). Another trial, where monalizumab was combined with durvalumab (anti-PD-1 mAb) in CRC showed encouraging activity as well (175). Meanwhile, a phase III randomized trial in HNSCC has been announced for this combination (176). Therefore, an anti-NKG2A mAb could be a promising checkpoint inhibitor to enhance antitumor immunity of both T and NK cells.

KIRs play a major role in regulating NK cell activity through various inhibitory and activating receptors and are most frequently found on intratumoral CD56^dim NK cells (29, 171). Similar to IFNγ, the inhibition of EGFR can increase HLA-C expression through STAT-1 signaling (26, 177). Thus, this could potentially limit NK cell responses through an increased interaction of KIRs with HLA-C. The use of mAbs, such as lirilumab (IPH2102), targeting KIR2DL-1/-2/-3, can mimic the mismatch of KIR with HLA-C and prevent inhibitory signaling. Indeed, various (pre-)clinical reports have described an improved NK cell cytotoxicity following lirilumab treatment (178–180). Furthermore, combination of lirilumab with an anti-CD20 mAb enhanced ADCC against lymphoma cells in vitro and in vivo (180). Similarly, lirilumab in combination with cetuximab induced a significantly higher cytotoxic response against HNSCC cell lines in co-culture experiments (149). Hence, despite the lack of extensive literature, investigations of lirilumab in combination with cetuximab suggest that could generate clinical benefit and therefore warrant further investigation. Importantly however, long-term treatment with lirilumab may also hold some drawbacks. To fully develop into functionally mature cells, NK cells undergo a process of ‘education’ whereby their level of exposure and interaction to ‘self’ antigens with inhibitory receptors will determine their responsiveness in cases where these antigens are missing (181). Therefore, it is thought that persistent inhibition of KIRs could, besides stimulating the activity of mature NK cells, impede the development of new, functionally competent NK cells (178). In this regard, future clinical trials will have to resolve the optimal scheduling of blockade of inhibitory receptors.

Similar to KIRs although far less understood, leukocyte immunoglobulin‐like receptors (LILRs)can regulate immune activity through ligation with MHC class I molecules. However, in contrast to the extensive KIR repertoire being expressed, NK cells predominantly express LILRB1 (182, 183). Interestingly, LILRB1 expression negatively correlated with cetuximab-induced ADCC against breast cancer patients (184). Furthermore, blocking LILRB1 increased both natural cytotoxicity as well as cetuximab-mediated ADCC, especially when both NK cells and cancer cells expressed LILRB1. Interestingly, LILRB1 expression and cetuximab-mediated ADCC were positively correlated in this context, indicating a greater inhibition at higher LILRB1 expression levels. However, LILRB1 research is still limited and factors impacting the regulation of LILRB1 expression should be the focus of future research to assess the potential for clinical implementation of this combination.

Of interest for the context of this review is the molecule CD137 (4-1BB), expressed on various immune cells following pro-inflammatory stimuli (187). Signaling through CD137 delivers an enhanced tumor-selective cytotoxicity and IFNγ secretion (188). Interestingly, CD137 agonistic mAbs are classified as either strong or weak agonistic Abs. The difference is that strong agonistic Abs (Urelumab) can activate 4-1BB without FcγR-mediated crosslinking, while the weak agonistic Abs (Utomilumab) require FcγR-mediated crosslinking to activate 4-1BB. However, the effects of both classes can still be enhanced through separate FcγR-crosslinking (189). In this regard, although urelumab alone in a breast cancer xenograft model had no effect on tumor size, combined treatment with trastuzumab enhanced trastuzumab-mediated killing significantly (190). Furthermore, urelumab together with cetuximab greatly improved survival of HNSCC patients and elevated DC maturation and T cell cross-presentation together with an increased cytokine secretion (185, 186). Interestingly, TINK but not peripheral blood NK cells substantially increased CD137 expression following treatment with cetuximab. Both urelumab and cetuximab alone also upregulated anti-apoptotic proteins (Bcl-xL and Bcl-2) in NK cells, suggesting an improved survival of activated NK cells, that was further increased following combination treatment (186). These results suggest that urelumab could indeed be combined with cetuximab to enhance immune activity. However, the early clinical observations remain to be investigated in larger cohorts and various tumor types to develop a stronger support for this notion.

A critical role in pathogen recognition is carried out by toll-like receptors (TLRs). As part of the innate immunity, TLRs play a vital role in activating immune responses as well. This is achieved through recognition of pathogen- or damage-associated molecular patterns (PAMPs and DAMPs) expressed by microorganisms or released from damaged or dying cells (191). While a total of 11 TLRs have been identified, TLR7/8 are of particular interest in cancer research due to their direct immune stimulatory effect and simultaneous ablation of Treg function (192, 193). Therefore, stimulation of TLR7/8 could be an interesting treatment in tumors that are highly infiltrated with effector and suppressive immune cells. Stimulation through TLR7/8 could potentially polarize the TIME towards tumor killing by producing Th1-polarizing cytokines such as TNF-α, IFNγ and IL-12 (192). In this regard, the use of the TLR8 agonist motolimod, increased peripheral blood mononuclear cell cytotoxicity against HNSCC cell lines, together with a higher production of inflammatory cytokines and chemokines by DCs, monocytes and NK cells (194). Additionally, ADCC was enhanced through combination with cetuximab as well (194, 195), showing a possible way to effectively activate innate and adaptive anticancer immune responses. A phase I trial in HNSCC reported encouraging antitumor activity without dose limiting toxicities when motolimod was combined with cetuximab. Furthermore, increases in plasma cytokine levels and in frequency and activation of circulating NK cells were observed as well (196). Currently, this combination is being further investigated in a phase II randomized trial (NCT01836029) of chemotherapy plus cetuximab in combination with motolimod in patients with recurrent or metastatic HNSCC.

As part of the PRR family, the stimulator of interferon genes (STING) DNA sensing pathway forms an important part of the innate immunity, as it recognizes cytoplasmic DNA through Cyclic GMP-AMP synthase (cGAS), gamma-interferon-inducible protein 16 (IFI16) and probable ATP-dependent RNA helicase (DDX41) (197). Therefore, STING also recognizes tumor-DNA and induces downstream signaling of NF-κB and interferon regulatory factor 3 (IRF-3). This results in the induction IFNs and inflammatory cytokines such as TNF-α, IL-1β and IL-6 (198). However, STING can also induce mitochondrial apoptosis through Bcl-2-associated X protein (Bax) induction (199). Therefore, the use of STING agonists to induce an inflammatory microenvironment and induce direct tumor apoptosis may be a valuable treatment. However, some reports suggest that STING may play a dual role in cancer, potentially promoting tumor growth in tumors with low antigenicity (200). Therefore, combined treatment of STING agonists with other treatments may achieve a good clinical outcome. Interestingly, EGFR was found to affect IRF-3 phosphorylation, suggesting a possibility for cetuximab to be combined with a STING agonist to enhance IRF-3 signaling and thereby lead to an enhanced antitumor response (201). Indeed, STING activation enhanced cetuximab-mediated ADCC of NK cells against HNSCC cell lines and promoted NK : DC crosstalk, suggesting an important role of STING in effective antitumor immunity (202). A phase I trial of the STING agonist dimethylxanthone acetic acid (DMXAA) (murine STING agonist) plus carboplatin, paclitaxel and cetuximab only demonstrated limited activity due to limited binding to human STING (NCT01031212). However, other clinical trials using human counterparts of STING agonists have provided clinical evidence for its therapeutic effectiveness. However, as no phase III trials have been registered yet, it remains to be seen what the exact clinical benefit of this combination will be. Regardless, the accumulated data so far point towards integration of immune-stimulatory molecules into standardized treatment regimens to induce clinically exploitable systemic responses.

Characterization of immunosuppressive factors and their involvement in tumor immune escape mechanisms has prompted researchers to reverse these impaired cytotoxic interactions through implementation of immunostimulatory cytokines. A study in HSNCC patients displayed elevated plasma levels TGF-β1 and soluble MHC I chain-related peptide A (sMICA) to diminish NKG2D expression, TNF-α and IFNγ release by NK cells, suppressing their antitumor responses (204). Interestingly, although NKG2D was downregulated due to high sMICA/TGF-β1 levels, CD16 expression and cetuximab-induced ADCC remained unaltered (204). Furthermore, IL-2 stimulation improved ADCC of sMICA inhibited NK cells resulting in a restored TNF-α and IFNγ secretion (204). Similarly, several other investigations in solid tumors have reported a significantly enhanced antitumor activity with tolerable safety profiles and improved ADCC following combined treatment with IL-2 and cetuximab (133, 205, 206). However, IL-2 administration in patients also causes expansion of FoxP3⁺ Tregs, which highly express the IL-2α receptor (207). Tumor types with relatively low intratumoral Tregs could potentially still benefit from this combination, as shown by the studies above (133, 208, 209). In contrast, tumors such as HNSCC and melanoma have been characterized as the most Treg infiltrated tumor types, making the use of IL-2 in combination with cetuximab less attractive (29, 210). Therefore, the makeup of the TIME is an important consideration that must be evaluated on a tumor type basis for this combination to be of value.

One of the first alternatives to IL-2 was IL-12, a cytokine produced by DCs and macrophages. IL-12 has anti-bacterial and anti-angiogenic effects and enhances the immune response to Ab-coated tumor cells (211). Stimulation of NK cells with IL-12 leads to secretion of IFNγ and TNF-α, as well as increased levels of chemokines such as MIP-1α, IL-8 and RANTES, further stimulating the infiltration of CD8⁺ T cells into the tumor. Additionally, IL-12 increases IL-2α expression by NK cells, further enhancing NK cell activity in response to endogenous IL-2 (212). A phase I/II trial of heavily pretreated HNSCC patients investigated the combination of IL-12 with cetuximab and achieved stable disease in 69% of patients, with prolonged PFS. Additionally, ADCC responses were increased together with higher levels of IFNγ, CXCL10 and TNF-α secretion (213). IL-12 was also able to suppress Treg function through downregulation of FoxP3 (207, 214). Thus, in addition to stimulating NK cells, IL-12 administration may also reverse immune tolerance and creates a less suppressive TIME, enhancing antitumor immunity.

IL-15 is a cytokine produced primarily by monocytes and macrophages and stimulates various NK and T cell functions (215). Similar to IL-2, stimulation with IL-15 is able to enhance the antitumor effects of NK cells against various tumor types and significantly increases cytokine and chemokine secretions (216, 217). Interestingly, besides upregulation of CD16, NKG2D and IFNγ, levels of NKp30 and NKp46 on NK cells of CRC patients were restored following IL-15 stimulation (216). However, IL-15 based therapies face some limitations as well, including a short serum half-life, narrowing down the therapeutic window, and the requirement for IL-15 receptor α-chain (IL-15Rα)-binding prior to activating effector cells, which limits the therapeutic application (218, 219). More recently, the genetically modified IL-15 compound ALT-803, consisting of IL-15 plus the IL-15Rα fused to the Fc portion of IgG1, has been developed in order to address the limitations of IL-15-based therapies. As a result, ALT-803 has higher biological activity and a longer serum half-life compared with free IL-15. Consistently, ALT-803 was able to enhance the ADCC response following cetuximab treatment in HNSCC cell lines to a level similar to or better than IL-15. In mice, while single-agent treatment partially reduced tumor growth, co-administration of cetuximab with ALT-803 showed complete tumor regression and increased secretion of IFNγ, RANTES and IL-8 (218). Early clinical trials with ALT-803 alone have reported promising efficacy and activity, showing an increased expansion of NK and CD8⁺ T cells (220). Interestingly, combination of ALT-803 with rituximab, another ADCC inducing mAb, gave similar results as ALT-803 plus cetuximab, thus supporting the exploration of ALT-803 to enhance cetuximab therapy (221).

IL-21 belongs to the IL-2 family of cytokines, based on the shared cytokine receptor γ chain (γ_c). In comparison to IL-2 and IL-15, single-agent treatment with IL-21 was shown to be the most potent antitumor cytokine with longer lasting responses and clearing mice from tumors in settings where both IL-2 and IL-15 only showed limited effect (222). Additionally, IL-21 stimulation was also shown to increase levels of IL-2α in addition to IFNγ, perforin and GZMB (223). Interestingly, the combination of IL-21 with cetuximab was also able to enhance the ability of NK cells to recognize and eliminate cetuximab-coated tumor cells (223–225). Clinical trials using IL-21 in combination with cetuximab confirm preclinical findings, reporting increased cytokine secretion, enhanced ADCC and achieving stable disease in patients with different tumor types (225, 226).

Although we have discussed the drawback involved in IL-2 treatment regarding Treg expansion, cytokines also have faced criticism as a potential immunotherapeutic approach, due to additional limitations. These include the relatively short serum half-life, requiring careful exploration of clinical doses that could otherwise lead to severe toxic responses (227). Furthermore, IL-2 and IL-12 induce vascular leaking due to alterations in vascular permeability, which is only minimally present with IL-15 and IL-21 treatment (228, 229). These limitations lie at the basis of the functional properties of cytokines. However, they have not stopped researchers from investigating ways to enhance the effectiveness of cytokines through, for example, genetic engineering. The works of Skrombolas et al. and Berraondo et al. provide a detailed and comprehensive review regarding these strategies (203, 208). Taken together, the combined use of cytokines with cetuximab as an ADCC inducing agent has the ability to restore/enhance cytolytic activity of NK cells. Future research likely will include genetically cytokine engineering or consider the use of cytokine cocktails. These could help provide optimal enhancement of NK cells and prevent the limitations involved with single cytokine administration.

Cancer cells rely on DNA damage repair mechanisms to maintain their survival, making these repair pathways ideal targets for cancer treatment, e.g. poly Adenosine diphosphate (ADP)-ribose polymerase (PARP) (230). PARP enzymes act as DNA damage sensors when single-strand DNA breaks occur. Thus, PARP inhibition can severely inhibit cell survival, trigger cell cycle arrest and apoptosis through accumulation of DNA damage. Interestingly, PARP inhibition also activates the STING DNA sensing pathway, subsequently leading to production of type I IFN and pro-inflammatory cytokines, thus priming an antitumor immune response (231, 232). Therefore, the possibility to combine PARP inhibition with immunotherapy seems highly interesting.

EGFR inhibition with cetuximab diminishes DNA synthesis and double-strand break repair and therefore can increase tumor susceptibility to PARP inhibitors (233, 234). Indeed, combining cetuximab with PARP inhibitors significantly increased ADCC in both Breast cancer susceptibility protein (BRCA)-WT and -mutant cell lines (235). Clinically, a phase I study in locally advanced HNSCC patients demonstrated promising responses and tolerable toxicities (236), although results were confounded by continued smoking during treatment of non-responders (237). Thus, this combination warrants further study in a phase II setting to further investigate its effectiveness. The biggest risk involved with PARP inhibition is the potential to develop secondary myelodysplastic syndrome/acute myeloid leukemia due to impaired DNA damage repair. This was limited to patients that additionally received chemotherapy and had germline DNA repair deficiencies, further inducing DNA damage (238).

Despite the severe side effects observed with thalidomide in the 1960s, its mechanisms of action have revealed immunomodulatory and anti-angiogenic activity. Analogues such as lenalidomide and pomalidomide are more potent immunomodulators and have fewer side effects. Lenalidomide has been approved for treatment of multiple hematological malignancies, as it is known to activate cytokine production, regulate T cell co-stimulation and augment NK cell cytotoxicity (239, 240). Lenalidomide is believed to enhance NK cell functionality in an indirect manner, mainly related to the release of IL-2 by other immune cells (240). Lenalidomide also enhanced ADCC following combination with several IgG1 mAbs, including cetuximab (168, 241). So far, the suggested mechanisms report that this enhancement is likely the result of an increased CD16 expression (168) and partly attributable to an increased presence of IL-2 and/or IL-12 cytokines secreted by T cells or other immune cells (242). On the other hand, lenalidomide-enhanced ADCC was abrogated through blocking of either DNAM-1/CD155 interactions or NKG2D with its ligands, indicating that optimal enhancement of ADCC requires interactions of DNAM-1 and NKG2D (168). Clinical trials investigating the combination of lenalidomide with cetuximab are currently in phase I/II and report a well-tolerated treatment with promising clinical activity in patients with CRC and HNSCC. Moreover, a dose-dependent increase in NK cytotoxic activity was demonstrated, with increasing doses of lenalidomide. This was associated with a significantly increased ADCC activity and an increased number of CD8⁺ T cells and circulating NK cells (243, 244).

Thus, immunomodulating agents such as PARP inhibitors or lenalidomide combined with EGFR-directed therapies show promising preclinical and early clinical results but remain to be investigated in more detail.