Among the 21 studies on radiolabelled imaging, the radionuclide-based VHHs were directed against tumor targets and proteins important in immune response including: epidermal growth factor receptor (EGFR) (19, 20), macrophage mannose receptor (MMR) (21, 22), human epidermal growth factor receptor type 2 (HER2) (23–30), carcinoembryonic antigen (CEA) (31), signal regulatory protein alpha (SIRPα) (32), Class II major histocompatibility complex antigens (MHC-II) (33), CD8 (34, 35), carbonic anhydrase IX (CAIX) (36), spliced EIIIB (EDB) domain of fibronectin (FN) (37), multiple myeloma M-protein (38) and prostate-specific membrane antigen (PSMA) (39).

VHHs conjugated with radionuclides have been successfully exploited in research settings with the single-positron emission tomography (SPECT) combined with micro-computed tomography (micro-CT) for imaging in cancer (19, 22, 25, 26, 31, 32, 36, 38, 39). In these studies, ^99mTc, ¹¹¹In, ²²⁵Ac and ¹³¹I, ¹⁷⁷Lu are radionuclides that were used for imaging. These studies show that radiolabelled VHHs exerts efficient tumor targeting, low non-target binding and excellent imaging results. VHHs show high specificity to detect primary tumors and metastatic sites in multiple models of melanoma, breast cancer, ovarian cancer, early pancreatic lesions such as pancreatic intraepithelial neoplasia and advanced pancreatic ductal adenocarcinoma with excellent clarity and signal-to-noise ratios through PET/CT imaging (30, 37). One study showed that humanized anti-CEA ^99mTc-VHHs display high targeting specificity with low signals in non-targeted organs, high heat-stability and rapid renal clearance using SPECT/micro-CT in a colon carcinoma mice model (31). High tissue penetration and high tumor targeting allow VHHs to easily reach brain targets by overcoming the BBB. For example, ^99mTc-labeled VHHs could successfully target SIRPα in glioblastoma (32). Puttemans et al. also found that VHHs labelled with either [¹³¹I] or [²²⁵Ac] display a high and specific tumor uptake in HER2 positive brain metastasis lesions, whereas their counterparts mAbs Trastuzumab-[¹³¹I] and Trastuzumab-[²²⁵Ac] are unable to accumulate in intracranial tumors (26). The study showed that ¹¹¹In-labeled anti-HER2 VHHs display high specific uptake in HER2 positive brain tumor from 1 h up to three days post injection, whereas ¹¹¹In-labeled mAb Trastuzumab causes high non-specific uptake in highly vascularized organs including heart, spleen and liver (26).

VHHs can also be used for detection of subset of immune cells. For instance, ^99mTc-labeled anti-MMR VHHs accumulate in hypoxic regions by targeting a specific subset of tumor-associated macrophages (TAMs), which are associated with strong angiogenic properties. This has been visualized through SPECT/micro-CT imaging (22). There are also multiple examples of the use of VHHs to detect biomarkers for response to immunotherapy. Real-time PET imaging of ⁸⁹Zr-labeled anti-CD8⁺ and CD11b⁺ VHHs show the potential to assess the dynamic distribution of CD8⁺ and CD11b⁺ cells as predictive biomarkers for response to anti-PD-1 treatment (35). Furthermore, assessing the distribution of intratumoral CD8⁺ T cells by PET imaging after application of ⁸⁹Zr-labeled anti-CD8⁺ VHHs is associated with the response to CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) therapy (34). One study demonstrated that ^99mTc-VHHs and SPECT/micro-CT successfully real-time monitor the expression change of M-protein in a mice model with multiple myeloma, with high uptake in M-protein expressing malignant plasma cells in the blood and no uptake in healthy mice (38). ¹⁸F-labelled anti-MHC-II VHHs successfully detected inflammation through PET/CT imaging at melanoma tumor growth sites before tumors were visible or detectable by palpation (33). ¹⁸F-labelled anti-MHC-II VHHs and anti-CD11b VHHs, which bind to targets expressed by multiple immune cells, detected primary melanoma, as an alternative to melanoma-specific markers in a melanoma mice model (33).

As a result of their small size, VHHs are able to rapidly shift from blood to targeted tissues and blood clearance and elimination is also relatively fast. This makes the time between administration and imaging relatively short, allowing a high image quality at early time points following injection. ¹⁸F-labelled anti-MHC-II VHHs were rapidly cleared from circulation 20 min post injection in a melanoma mice model (33). In another study, tumor imaging was possible as early as 3 h following the administration of radiolabeled anti-EGFR VHHs (20), compared to 16 h for radiolabeled anti-EGFR mAbs. Moreover, imaging by the latter was accompanied with relatively high background signals from the liver and the gastrointestinal tract (40). One study reported that fast blood clearance limits the absolute uptake of the VHH and thereby limits imaging of low-abundant targets in therapeutic applications (36). To prolong the blood residence time of VHHs and increase tumor uptake, one feasible option is the introduction of an albumin-binding domain to the VHHs while preserving the inherent high affinity and specificity of the molecules (36).

In a comparative study of in vivo tumor uptake of VHHs- and mAbs-coated nanoparticles in CT molecular imaging, VHHs with longer blood residence time (20 h for VHH- and 11 h for mAbs-coated nanoparticles) failed to make up for its lower binding affinity, leading to significantly lower in vivo tumor accumulation of VHHs than mAbs (41). This data suggests that VHHs with a long circulation time but low target affinity perform less with regards to tumor accumulation and imaging. The binding affinity can have more impact on tumor accumulation than blood residence time in certain conditions, but studies to confirm this observation are warranted.

There are 11 studies focusing on the application of VHHs in optical imaging (8, 11, 41, 62–64, 66–70). Optical imaging uses light to obtain detailed images of molecules ( Figure 2 ). As observed in the study of molecular optical imaging with IRDye 800CW (IR)-labeled anti-HER2 VHHs, VHHs-based optical imaging exhibits two attractive characteristics: rapid accumulation in the tumor and high T/B ratio. T/B ratio is determined by two factors: specific target binding capability and rapid clearance of unbound antibodies in the body. VHHs generally exhibit higher T/B ratios than conventional mAbs when used for molecular imaging in vivo. VHHs have better tissue penetration and fast clearance from the circulation, whereas mAbs display slow clearance. These pharmacokinetics are of importance for optimizing dose and time points of administration and imaging in clinical practice.

Specific target binding capability has been investigated in multiple studies. VHHs-IR were highly specific with accumulation only in HER2-positive tumors, as opposed to the mAb Trastuzumab-IR, which accumulated in both HER2-positive and HER2-negative tumors (62). In a glioblastoma multiforme (GBM) mice model and in ex vivo human GBM tissue, IRDyeCy5.5-labelled anti-IGFBP7 VHHs did bind to GBM vessels at 10 min to 24 h after injection, but did not to normal brain vessels (67). Alexa Fluor 647 (a NIR dye)-labelled anti-MHC-II VHHs exert high specificity, high cytotoxicity and fast cellular internalization (within 1 h) in localized and metastatic tumor cells in B-cell lymphoma models, with signals from 30 min to 96 h after injection (11). Imaging of anti-EGFR VHHs showed highest fluorescence at 2 h post injection of anti-EGFR VHHs, compared to 24 h for anti-EGFR mAbs Cetuximab (70).

Moreover, excellent T/B ratio and good contrast between tumor and background enable surgical resection guided by fluorescence derived probes specifically accumulated in the tumor. NIRF-dye AF680-labelled VHHs displayed a 6-fold faster tumor accumulation and 2-fold better T/B ratio than mAbs at the same dosage in a lymphoma mice model (8). One study showed that the anti-HER2 VHHs-IR significantly outperform Trastuzumab-IR on overall T/B ratio and serves as a useful imaging-guide in a HER2-positive xenograft (62). Anti-HER2 VHHs-IR have a ∼20 times faster tumor accumulation than the anti-HER2 mAbs Trastuzumab-IR (62). This was associated with intravenous administration of VHHs-IR a few hours before surgery, compared to 3 days for the mAb Trastuzumab-IR. There is evidence that anti-CAIX VHHs-IR can provide pre-, intra-, and postoperative optical imaging of (pre-invasive) breast cancer in a xenograft breast cancer mouse model (64).

Expression of tumor markers may alter in the primary tumor and metastatic lesions during cancer progression. Employing one unique probe might be insufficient to adequately image all tumor lesions. This was addressed in a recent study, which investigated whether a combination of two optical VHH probes, which specifically recognize two independent breast cancer markers, could improve tumor imaging by increasing the T/B ratios. As expected, application of two optical VHH probes effectively increased the T/B ratio compared to that of a single VHH probe, with successful detection of small metastases (69). In this way, the expression status of different tumor markers can be observed simultaneously within the same tumor, enabling a more complete tumor characterization. Another benefit is that it provides a rapid, non-invasive assessment of tumor marker expression in a pathological setting. Moreover, combining multiple optically labelled VHHs to target neighboring molecules can be performed without steric hindrance compared to mAbs. Imaging by using the combination of different probes has also been reported in other studies (71–73).

Moreover, bimodal VHHs can be developed to enable application of one agent for multiple imaging and/or treatment modalities. In addition to conventional optical imaging, a bimodal VHH that constituted by site-specific dual-labeling of ¹¹¹In and IRDye700DX was investigated and developed for SPECT imaging and targeted photodynamic therapy (63). The bimodal ¹¹¹In-labeled DTPA-IRDye700DX VHH was found to specifically accumulate at the EGFR overexpressed tumor sites in mice bearing A431 xenografts. This enabled successful visualization with both near infrared fluorescence and SPECT imaging. This novel bimodal VHH displayed a high internalization ratio and retained high specificity and affinity. Furthermore, application of a bimodal VHH can circumvent the limitations of a single imaging modality. This bimodal VHH could potentially be applied in oncologic surgery for EGFR overexpressing tumors, such as lung cancer, head-and-neck cancer, brain tumors and bladder cancer (63).

Another study underscored the potential of VHHs for monitoring the efficacy of mAb Daratumumab therapy, since they can still detect their target CD38 after binding of Daratumumab (66). This is because anti-CD38 VHHs bind to different epitopes with the ones that Daratumumab does. Therefore, VHHs hold promise as novel tools, which can be applied collaboratively with Daratumumab for diagnosis and treatment of CD38-expressing malignancies.

A limitation of most of the imaging studies was neglecting dose optimization (62, 70). The excess antibodies may have contributed to non-specific accumulation in non-target organs (74). Similar as for the application of VHHs in radiolabeled imaging, the VHHs’ short circulation half-life might limit clinical application in optical imaging. Efforts to prolong the half-life of VHH in circulation is performed by engineering strategies, such as pegylation, multimerization and fusion (75). In a comparative study, the synthesis and pharmacokinetic properties of bivalent VHH EG2-hFc (80 kDa) and pentavalent V2C-EG2 (128 kDa) constructs based on monovalent EG2 were described (68). As expected, the half-life of EG2-hFc and V2C-EG2 in circulation was improved and EG2-hFc showed a significant increase in affinity for its target antigen compared to the monovalent VHH. The EG2-hFc construct effectively targeted intracranial brain tumors in in vivo molecular imaging with a good balance between affinity, serum half-life, molecular size and tumor penetration. Such a balance with effective in vivo molecular imaging and therapy was achieved only for the bivalent VHH constructs but not in higher-valent constructs (68),. In this way, excessive repeat dosing can be avoided, which reduces the burden for patients during molecular imaging.

Conjugation of a fluorophore to a VHH for optical imaging might affect its binding affinity. To resolve this problem, protein site-directed conjugation has been developed to replace conventional random conjugation and to avoid affinity loss (62). Conjugating fluorophores specifically to the C-terminal region of a VHH is a good option, because the C-terminus is situated opposite to its epitope-binding region. The VHHs applied for recognition of two independent breast cancer markers as described above, were site-specifically conjugated to their respective fluorophores using a cysteine that was introduced in the C-terminal region (69), avoiding the effect of fluorophore conjugation on the binding affinity.

There are five studies on photodynamic therapy in this review (79–83). Photodynamic therapy involves a light source of a particular wavelength, a photosensitizer (PS) and oxygen to destroy cancer cells (84). Most PSs used in the clinic are hydrophobic, which makes them easier to bind to cells, but also leads to non-specific binding. In several studies hydrophilic PSs were created by conjugating the PS to a VHH. The VHH-PS conjugates proved to be able to more specifically target antigens on tumor cells (79, 80, 82, 83).

Four research groups have confirmed the efficacy of VHH-PS in preclinical studies (79, 80, 82, 83). All studies used IRDye700DX as PS to form VHH-PS conjugates with VHHs. For example, anti-Met VHH-PS specifically killed targeted tumor cells upon illumination. The binding affinity of the target cells was very high (79). Another study showed that a conjugate of anti-EGFR VHH and hydrophilic PS IRDye700DX strongly binds to high EGFR expressing tumor cells and hardly bind to low EGFR expressing tumor cells, whereas PS IRDye700DX alone doesn’t bind to any cells (82). Anti-EGFR VHH-PS monovalent conjugates resulted in tumor necrosis in approximately 90% of tumor cells, which was significantly better than the mAbs Cetuximab-PS in an orthotropic mouse tumor model of head and neck cancer (82). Notably, the anti-EGFR VHH-PS conjugates were observed to homogenously distribute throughout the solid tumor, which is beneficial for the treatment efficacy. Another study confirmed that anti-EGFR VHHs are more homogeneously distributed throughout the tumor, whereas the anti-EGFR mAbs Cetuximab are confined to the core of the tumor (70).

Rapid penetration of VHHs was also displayed in the application of PDT. Effective illumination could already be performed 1 h post administration of VHH-PS conjugates, whereas illumination effects could only be seen 24 h post-administration with mAbs Cetuximab-PS conjugates (82). Nevertheless, several strategies to enhance the efficacy of VHH-PS were investigated. Cell-penetrating peptides (CPPs) are short peptides which facilitate CPP/drug complexes to translocate across the cell membrane (85). CPP increased the internalization of VHH-PS conjugates but slightly decreased their affinity, leading to reduced PDT efficacy (83). Biparatopic antibodies bind to two distinct epitopes of the same antigen (86). The potential of biparatopic antibodies in PDT has been explored. The biparatopic VHH-PS conjugates exert more toxic effect to tumor cells because they could deliver more PSs and enhance internalization (82). One study reported that internalized biparatopic VHH-PS significantly increased phototoxicity compared to monovalent VHH-PS conjugates located on the outer of cells (80). The mixture of Met targeted VHH-PS and EGFR targeted VHH-PS could further enhance therapeutic efficacy of VHH-PS and decrease potential therapy resistance (79). In addition, the degree of conjugation of the VHH-PS impacts treatment efficacy and photocytoxicity (82). Approximately only 50% of monovalent VHHs form VHH-PS with PSs, whereas for an equal quantity of biparatopic VHHs, all VHHs were conjugated with PSs. The competition between conjugated monovalent VHH-PS and unconjugated VHHs for target antigen EGFR leads to lower phototoxicity and T/B ratio compared to biparatopic VHHs (82).

It is speculated that although the advantage of PDT lies in restricting light dose specifically to tumor cells, normal cells near the tumor to some extent may encounter illumination. To eliminate unwanted effects on normal cells near the tumor from low T/B ratio, it is important to set an appropriate threshold dose for PDT. Heukers and colleagues performed co-culturing of high- and low- EGFR expressing tumor cells and adjusted the illumination and found that VHH-PS could be 100% specific to high EGFR expressing cells and safe for low EGFR expressing cells (80), which is in agreement with other studies (87, 88).

Moreover, as an alternative to PSs, branched gold nanoparticles can be used to form VHHs-branched gold nanoparticles conjugates. These have been applied for PDT and showed effective therapeutic efficacy for cancer treatment in a SKOV3 (HER2⁺) cell model (81).

CAR T-cell therapy is a special type of immunotherapy in which T-cells isolated from patients are genetically modified to express specific receptors directed against antigens of cancer cells, leading to T-cell attacks to destroy cancer cells (89). We found five studies on VHH-based immunotherapy (90–94), among which four studies on VHH based CAR T-cell therapy (91–94). Optimization of CAR T therapy has been investigated through various approaches. One study transduced the human natural killer cell line NK-92 to stably express VHH-based chimeric antigen receptors (VHH-CARs) specific for CD38 and as such developed anti-CD38 VHH-CAR NK-92 cells which exhibited promising efficacy for killing multiple myeloma cells in primary human bone marrow samples (91) ( Figure 3 ). NK-92 cells are considered as a ready-to-use reagent compared to regular CAR T therapy which applies T cells that need to be isolated from patients. Phase-I clinical trials have confirmed that NK-92 cells (without VHH-CARs) have a high safety profile when used for patients (95, 96). Unfortunately, there are no clinical trials on anti-CD38 VHH-CAR NK-92 yet.

B-cell maturation antigen (BCMA) of multiple myeloma is one of most studied tumor antigens among CAR T therapy, which showed promising efficacy and clinical manageable side effects in different clinical trials on multiple myeloma (97–99). Single VHH targeting one epitope of BCMA in CAR-T therapy has also been explored (92). Anti-BCMA CAR-T cells produced by using a humanized VHH sequence were able to kill lymphoblastic B cells with high expression of BCMA. Interestingly, increased numbers of BCMA tumor cells stimulated anti-BCMA CAR-T cell proliferation in a preclinical model. These anti-BCMA CAR-T cells were also tested in human subjects in a phase I trial, and anti-BCMA CAR-T was successful in all the patients and achieved an overall response rate of 88.2% and sustained tumor remissions up to 12.5 months after therapy. Except for a slight cytokine release syndrome as the most common effect, no other toxicity was observed in the clinical treatment of multiple myeloma.

The one problem of CAR T is its suboptimal efficacy for solid tumors because of lack of specific tumor antigens (100, 101). Development of novel VHH-based CAR T cells against antigens in the tumor microenvironment can circumvent this hurdle. For example, targeting PD-L1, which is widely expressed in the tumor microenvironment, by VHHs-based anti-PD-L1 CAR T cells significantly enhanced immune infiltration and inhibited growth of melanoma and colon adenocarcinoma and improved the survival in mice (94). Another study showed that anti-PD-L1 VHH fused with two cytokines IL2 and IFNγ overcame the delivery barrier caused by an immunosuppressive tumor microenvironment and dense stroma surrounding tumors in an orthotropic pancreatic tumor model (90). Furthermore, the anti-PD-L1 VHH displayed a ~100-fold higher binding affinity than the commercial mAbs 10F.9G2 (90). Therefore, delivery of cytokines by VHHs could be a promising therapy. Another study showed that secretion of anti-CD47 VHHs by CAR T cells can modify the intratumoral immune landscape in immunocompetent animal models (93). CD47 is a tumor antigen that can block phagocytosis of tumor cells by macrophages. Secretion of anti-CD47 VHHs by CAR T cells effectively improved antitumor efficacy in an animal model with an immunocompetent immune system. Further engagement of VHHs in CAR T therapy may support and accelerate personalized, multi-modal immunotherapy based on individual tumor characteristics.

There are five studies included on targeted radionuclide therapy with VHHs (102–106). The radiolabelled VHHs are designed to maximize retention of radioactivity in tumor tissues with overexpression of target antigens and minimize retention of radioactivity in normal tissues. Treatment of VHHs conjugated with radionuclides in in vivo studies resulted in high and specific tumor uptake in multiple myeloma (38), HER2⁺ breast cancer (103, 105), ovarian cancer (103), TS/A mammary carcinoma (102), multiple myeloma (106)and non-Hodgkin lymphoma (104).

One study shows that ¹³¹I-labelled anti-HER2 VHHs bind to the epitopes that are distinct to the ones recognized by mAbs (Trastuzumab and Pertuzumab) in HER2⁺ breast cancer (103). The VHHs displayed a 4-fold binding efficiency to Trastuzumab-resistant tumor cells compared to Trastuzumab and significantly improve survival of mice, without detected toxicity in an ovarian cancer mice model (103). Another study shows that ¹⁷⁷Lu-labelled anti-CD20 VHHs is stable in human serum with >91% intact complexes at 144 h post injection (104). The highest tumor uptake of ¹⁷⁷Lu-labelled anti-CD20 VHHs was at 1.5 h post injection, compared to 120 h for ¹⁷⁷Lu-labelled anti-CD20 mAbs Rituximab. Furthermore, ¹⁷⁷Lu-labelled anti-CD20 VHHs significantly improved survival in a lymphoma mice model compared to controls (104). The important drawback of Rituximab is that radioactivity in the circulation is higher than in the tumor at all time points after injection. Also, radioactivity in non-target organs was much higher with Rituximab than with the VHHs.

The heterogeneity of biomarker expression in tumor cells compromises the effectivity of targeted therapy. To develop an efficient targeted radionuclide therapy for a variety of cancers, Bolli et al. proposed to target TAMs, a type of tumor stromal cells, presented in the majority of tumor types that stably express particular targets instead of cancer cells. The radiolabelled monovalent VHHs directed against MMR expressed by TAMs, so called ¹⁷⁷Lu-labeled anti-α-MMR VHHs, were developed to target stromal cells in a preclinical study (ex vivo and in vivo) (102). This study first optimized the route of administration to avoid undesirable non-tumoral targeting. The co-injection of ¹⁷⁷Lu-labeled anti-α-MMR VHHs and 100-fold unlabelled bivalent anti-α-MMR VHHs most efficiently reduced non-tumoral uptake of VHHs, while high tumor uptake was maintained. In a terminal animal experiment, 83.3% of mice carrying mammary adenocarcinoma died within 24 days under administration of the monovalent anti-MMR VHHs alone, while all mice survived for longer than 35 days after co-injection with 100-fold unlabelled bivalent α-MMR VHHs. Therefore, blocking non-tumoral binding to decrease therapy-induced toxicity seems to be of high importance in targeted radionuclide therapy. Of note, this therapy targeting MMR in stromal cells pronouncedly outcompeted other common treatments including chemotherapy (doxorubicin, paclitaxel), anti-angiogenic therapy (α-VEGFR2) and immune checkpoint blockade (α-PD1), while the therapeutic efficacy was not improved upon the combination with any of these treatments in the mice model (102). Moreover, ¹⁷⁷Lu-labeled VHHs successfully targeted stromal cells and delivered a high radiation dose to the stromal cell regions and significantly impaired the progression of tumors that were resistant to conventional therapies (102).

In multiple myeloma, patients are often staged with minimal residual disease (MRD) before cancer relapses. Paraprotein, as an idiotype protein, is expressed on the cell membrane of malignant plasma cells in the MRD stage of multiple myeloma (107). The idiotype involves the presence of a specific set of antigen-binding sites which characterizes an antibody produced by a particular clone (108). The paraprotein sequence is unique to an individual, which makes paraprotein valuable tumor antigens to develop personalized targeted radionuclides. Starting from serum-isolated paraprotein for immunization of llamas, patient-specific anti-idiotype VHHs were successfully generated. These anti-idiotype VHHs (anti-id VHHs) were generated and radiolabelled with ¹⁷⁷Lu and ²²⁵Ac for preclinical experiments in a mice model mimicking MRD of multiple myeloma (106). Mice treated with anti-id VHHs had significantly prolonged survival and delayed damage to end-organs compared to the control group. Concluding from this research, radiolabeled anti-id VHHs generate strong therapeutic efficacy against multiple myeloma in the MRD stage, which suggests that radiolabeled anti-id VHHs are valuable therapeutic agents to prevent cancer relapse in multiple myeloma patients.

To overcome limitations due to degradation of radionuclides-labeled VHHs by lysosomes and sustain cellular internalization rate, a recent study radioiodinated anti-HER2 antibodies with ¹³¹I with the use of a specific radioiodination prosthetic agent (IB-Mal-D-GEEEK containing a D-peptide core) (105). This radioiodinated prosthetic agent was added to facilitate trapping of the radioactivity in the cell after proteolysis of the labeled protein upon internalization in cells (109). The specific tumor-cellular retention of [¹³¹I]IB-Mal-D-GEEEK-VHHs is lower at 1 hour after administration, but much higher at 24 hours after administration compared to the directly labeled [¹²⁵I]-VHHs (56.9 ± 4.1% at 1h, 58.3 ± 4.6% at 24h versus 61.4 ± 4.3% at 1h and 32.4 ± 2.0% at 24h). Importantly, tumor targeting properties and T/B ratio were also significantly enhanced both in vitro and in vivo for treatment with [¹³¹I]IB-Mal-D-GEEEK-VHHs compared with directly labelled VHHs ([¹²⁵I]-VHHs). This radiolabeling strategy needs to be evaluated in clinical studies for the treatment of HER2 positive cancers.

Several potentially therapeutic VHHs have been developed to target tumor-specific targets. For instance, in the 26 included studies on molecular targeted therapy (110–135), the targets include the chemokine receptor CXCR7, actin cytoskeleton CapG, receptor tyrosine kinases HER2, EGFR, MET, cluster of differentiation CD7, CD16, CD38, HPV viral-like protein E6, tumor signaling associated proteins BMP2/4, PD-L1, and CD8 T cells.

Earlier studies only focused on the impact of VHHs on tumor cell proliferation, migration and invasion, regardless of specificity, affinity, internalization and biodistribution of the VHHs (110). Anti-EGFR VHHs are the most frequently reported treatment in cancer using VHHs. Anti-EGFR VHHs effectively inhibit proliferation of tumor cells in vitro and outgrowth of solid tumors in vivo by blocking EGF-mediated signaling (122). Other studies often focus on comparative preclinical experiments between novel VHH candidates and conventional mAbs, inhibitors or antagonists. Anti-EGFR VHHs linked to a CPP (non-arginine) resulted in 1000 fold higher cytotoxic activity in tumor cells compared to the small molecular EGFR tyrosine kinase inhibitors in preclinical experiments (126). Viability, mitosis and colony formation of tumor cells were damaged using HER2 targeting VHHs in in vitro experiments (132).

VHH targets are often distinct to epitopes recognized by larger biological molecules, for instance, mAbs, resulting in distinct therapeutic mechanisms. Indeed, Zhai et al. found that VHH enhanced agonistic activity of tumor-necrosis superfamily member 4-1BB by binding a novel epitope of 4-1BB as determined by X-ray crystallography (134). This has the advantage that the VHHs do not affect natural tumor cytotoxicity generated through interaction between 4-1BB and its natural ligand. The most variable portion of immunoglobulin molecules is the third complementarity determining region (CDR3) of the heavy chain (136). In another study, VHHs interacted with EGFR tyrosine kinase residues in the vicinity of their catalytic area (the part to which the natural ligand binds) by using CDR3 and thereby hindered the function of EGFR, impeding tumor cell proliferation and migration (126). A recent study confirmed that VHHs elicit distinct pathways compared to other anticancer drugs, for instance, VHHs blocked the phosphorylation of MET and promoted its degradation via the endo-lysosomal pathway, whereas anti-MET mAbs promoted MET activation (125).

Extended studies further optimized the potential of VHHs for therapeutic efficacy in cancer treatment. Increasing the valence of VHHs from monovalent to multivalent VHHs, for example, trivalent, tetravalent and pentavalent, has been shown to enhance the antitumor effect in a death receptor 5-targeting preclinical study (116). Producing tetravalent or pentavalent VHHs highly increased the binding affinity compared to monovalent VHHs. In addition, tetravalent and pentavalent VHHs both maximally induced activation of caspase-8 and caspase-3/7, resulting in the formation of death-inducing signaling complexes, leading to significant enhancement in anticancer response in vitro and in vivo. In general, the valence of VHHs is one of the key factors that determines the anticancer capability of VHHs as anticancer drugs. Studies suggested that the binding affinity and targeting potency of VHHs vary with their valence (68, 121). However, one study found that there is no significant difference between monovalent and bivalent VHHs in respect to their tumor targeting potential (112). The discrepancy between the different studies is possibly due to a limited amount of epitopes being expressed on cancer cells, which limits the measurable effect on affinity by increasing agent concentrations. Otherwise, the latter study supports the existence of an affinity threshold value beyond which no further improvement of tumor binding can be achieved. Furthermore, the study also found that higher affinity does not necessarily enhance tumor penetration. This is probably due to the presence of a binding site barrier effect, resulting from VHHs binding to the first epitopes encountered at the tumor cells in the superficial tumor layer, which impedes deeper penetration of the VHHs to reach the core of the tumor (137). One study reported that the mixture of four VHHs targeting four distinct epitopes of HER2 improved target binding potential and therapeutic efficacy in an in vitro model (114). This suggests that targeting different epitopes of the same antigen simultaneously might be a potential strategy to improve efficacy.

Our group developed two VHHs C4C4 and C8C8 directed against BMP4 and BMP2/4, respectively. We demonstrated that specificity, effectiveness and affinity of C4C4 and C8C8 are higher than conventional anti-BMP2 and/or BMP4 antibodies (138, 139). We also found that bivalent VHHs C4C4 and C8C8 lead to higher affinity than monovalent C4 and C8, respectively (139). Furthermore, in vitro experiments confirmed that C4C4 and C8C8 inhibit cell migration and chemo-resistance to cisplatin in primary esophageal adenocarcinoma (EAC) cell line. In a patient-derived xenograft model, IRDye800cw-labelled C4C4 and C8C8 effectively target BMP4 and BMP2/4, respectively, and inhibit tumor growth (119). Overall, our study demonstrated that VHHs inhibited tumor growth and aggressiveness of EAC in vitro and in vivo through inhibiting BMP4 and BMP2/4 (119). Furthermore, C4C4 and C8C8 also were able to inhibit the growth of esophageal adenocarcinoma premalignant lesions, known as Barrett`s esophagus in an in vivo organoid model (111).

Although VHHs contain no Fc region, a study generated anti-CD38 VHH-Fc by genetically fusing VHH with the Fc-domain of human IgG1 (123). The VHH-Fc with a high specificity and affinity binds to a different epitope than the conventional mAb Daratumumab. The VHH-Fc induced potent antibody-dependent cellular cytotoxicity in a myeloma cell line and primary multiple myeloma cells. Furthermore, the VHH-Fc inhibited tumor growth and prolong survival time compared to a control and Daratumumab group in a mouse xenograft model.

VHHs that simultaneously target immune cells, such as NK cells in the tumor microenvironment, next to tumor cells, result in a more aggressive antitumor therapy than treatment of targeting tumor cells only. Indeed, innovative studies have shown that bispecific and even tri-specific VHHs which interact with NK cells elicited robust cytotoxicity and cytolysis of tumor cells due to stronger activity of the induced NK cell responses (128, 129). The tri-specific VHHs trigger significant NK cell expansion and further enhance immunity response of NK cells on tumor cells as shown in in vivo experiments. The advantage of these multi-specific VHHs lies in the combination of molecular targeted therapy on tumor cells and immunotherapy focused on NK cells and the removal of non-specific killing by NK cells. In another application in immune therapy, anti-PD-L1 VHH in combination with an anti-OX40 antibody were used to construct the bispecific antibody PD-L1/OX40, that had better antitumor effects than the anti-PD-L1 VHH alone, anti-OX40 antibody alone, or a combination therapy of anti-PD-L1 VHH plus anti-OX40 in colon cancer and lung cancer mice models (118).

To address tumor cell resistance in conventional therapies, a VHH was designed as an immunotoxin by fusion of the VHH with peptide toxins (de-immunized pseudomonas exotoxin) with distinctive anticancer mechanisms (115, 127). The novel VHH-based immunotoxin displayed superior antigen-restricted cytotoxicity against tumor cells and evident survival benefit in a mice model (127). Further optimization of the immunotoxin was performed by addition of a streptococcal albumin binding domain and the removal of T-cell epitopes, leading to longer serum half-life and lower immunogenicity (115). It is important to note that adding albumin to immunotoxins may lower the effective dose, as shown in mouse liver cancer xenografts (115). Another study demonstrated the targeting specificity of humanized anti-CD7 immunotoxin in vitro and in vivo (133). The anti-CD7 immunotoxin significantly inhibits the proliferation of T-cell acute lymphoblastic leukemia cells and extends the survival time of mice in a tumor transplant model and patient tumor-derived xenograft model by exerting a cytotoxicity function via endocytosis into the cytoplasm of CD7-positive cells. Moreover, study reported that anti-MHC-II VHH-drug conjugates are superior than commercial mAbs on pharmacokinetics, such as internalization and circulatory clearance and display efficient anticancer outcomes for an aggressive murine B-cell lymphoma (11).

VHHs are also used for targeting intracellular protein, besides proteins on the cell-membrane. In a breast cancer study, breast cancer cells expressing anti-CapG VHHs lost the capability of migration and invasion (130). Tumor-bearing mice inoculated with breast cancer cells expressing anti-CapG VHHs showed significant slower tumor growth and prevention of lung metastasis compared to the control group. Other studies were performed to enhance tumor cell penetration and to increase the intracellular concentrations of VHHs. Solid tumors are characterized by high interstitial fluid pressure, hypoxia and homeostatic imbalance, contributing to the difficult penetration of anticancer drugs within tumor tissues. The anticancer activity of anti-EGFR VHHs was further enhanced by introduction of a CPP called iRGD in gastric cancer therapy in ex vivo and in vivo models (124). Anti-EGFR VHHs fused with iRGD significantly improved penetration and were able to reach the core area of the tumor mass in established multicellular spheroids. The favorable penetration ability of anti-EGFR-iRGD VHHs was reflected by the change of tumor volume in a mice model. The tumor volume shrunk by about 63.7% for anti-EGFR-iRGD VHHs, outcompeting the anti-EGFR VHHs group. Combining other anti-cancer drugs including DOX (20 µg/mL), liposomes (1.5 µg/mL) and mAbs Bevacizumab (1 mg/mL) with anti-EGFR-iRGD VHHs significantly enhanced their penetration capability and tumor inhibiting activity in mice models (124). Moreover, considering that leaky vasculature exists in most solid tumors, a delivery carrier so called PEGylated liposomes were investigated in the therapy of HER2 overexpressed cancer in vitro (114). This approach displayed great therapeutic potential by combining the drug delivery advantages of liposomes and tumor-specific binding capabilities of the VHHs.

One recent study has extended the utilization of VHHs in cancer treatment. VHHs as delivery carriers have been used for development of cancer vaccines against melanoma. These therapies significantly decreased tumor growth in a mice model (113). Woodham and colleagues established a therapeutic vaccine platform based on VHHs for treatment of HPV induced cancer (131). The novel vaccine, VHH_CD11b-E7, is composed of a HPV tumor antigen peptide E7 and a VHH directed against CD11b as expressed on antigen-presenting cells by site-specific conjugation. The VHH-conjugated vaccine could elicit potent cellular immunity to attack tumor cells in established HPV positive tumors, as opposed to regular prophylactic vaccinations against HPV. The VHH_CD11b-E7 recognized an epitope, which is different from the epitope recognized by the conventional anti-CD11b mAbs. This could have contributed to the high affinity of VHH_CD11b-E7 for CD11b expressing cells. The extent of CD8⁺ T-cell response determines the efficacy of the therapeutic vaccine against HPV induced cancers (140). An enhanced cellular immune response could be attributed to VHH_CD11b-E7, as suggested by a two-fold increase in the number of CD8⁺ T cells compared to E7 alone. VHH_CD11b-E7 also significantly outperformed E7 alone in terms of tumor regression. Although VHH_CD11b-E7 is developed as a therapeutic vaccine, prophylactic vaccination with VHH_CD11b-E7 also significantly lowered the tumor burden and improved overall survival compared to E7 alone in mice. Overall, the application of tumor antigens conjugated with VHHs, which are of importance to acquire an immune response, such as VHH_CD11b-E7, should be considered in the clinic as a potential therapeutic vaccine for HPV induced cancers.

In addition to their use in vaccines in HPV induced cancers, VHHs can also be designed to directly target oncoproteins such as oncoproteins E6 and E7 which play key roles in HPV16 induced cancer (120, 135). This showed effective inhibition of tumor growth, which suggests that targeting E6/E7 in therapy of HPV16 induced cancer holds great promise.

Therapeutic stem cells can be designed to secrete VHHs in the treatment of Basal-like breast cancer (BLBC). BLBC is associated with poor prognosis and survival with a high incidence of brain metastasis and poor delivery of drugs through the BBB. In the past decade, intrathecal stem cell therapy has been investigated and applied in patients with neurodegenerative diseases and trauma (141–143). Building on these experiences, scientists developed human mesenchymal stem cells (hMSCs) secreting VHHs (E_VDR_L) directed against EGFR and DR_L simultaneously (117). hMSCs-E_VDR_L treatment significantly lowered tumor volumes and prolonged survival in mice BLBC models of brain metastasis. In a mice model, histopathology showed hMSCs-E_VDR_L being widely distributed at residual tumor cells along with the brain vasculature after surgical resection of the primary tumor, suggesting a perivascular niche of micro-metastasis. Treatment with hMSCs-E_VDR_L significantly increased macro-metastasis-free survival and overall survival of mice. In a mice model of BLBC leptomeningeal metastasis, hMSCs-E_VDR_L survived in cerebrospinal fluid (CSF) space longer than 2 weeks after intrathecal injection. This suggests that hMSCs-E_VDR_L can overcome the therapeutic difficulty of fast clearing drugs by CSF. Treatment with hMSCs-E_VDR_L exhibits powerful therapeutic efficacy by targeting residual invasive tumor cells after surgical resection in the perivascular niche and leptomeningeal metastasis of BLBC. Therefore, the delivery of stem cell secreting VHHs could be an effective therapeutic strategy for BLBC treatment.