and is caused by (i) *Trypanosoma brucei gambiense* (Western and central Africa) which is an anthroponotic disease with a minor role for animal reservoirs accounting for 98% of the reported HAT cases, and causing a chronic, gradually progressing disease with limited symptoms, whereby the late meningoencephalitic stage is reached after months/years of infection (7–9), and (ii) *Trypanosoma brucei rhodesiense* (Eastern/southern Africa) which is a zoonotic disease affecting mainly animals (livestock and wildlife), with humans being only occasionally infected, and representing 2% of the reported HAT cases, whereby the infections are more acute and virulent/lethal with a rapid progression (within weeks) to the late meningoencephalitic stage (3, 9–12). Hence, the zoonotic nature of *T. b. rhodesiense* infections makes them more difficult to control compared to *T. b. gambiense* infections (8, 13, 14). Animal African trypanosomiasis (AAT) or Nagana is the second form of AT affecting sub-Saharan Africa, which is mainly caused by *Trypanosoma congolense*, *Trypanosoma vivax*, and to a lesser extent *Trypanosoma brucei brucei*, whereas Surra and Dourine are also forms of AAT caused by *Trypanosoma evansi* and *Trypanosoma equiperdum*, respectively (15–17). Overall, *T. congolense* is a major constraint for livestock production in sub-Saharan Africa, whereby cattle succumb to infection primarily due to parasite-induced anemia or complications resulting from secondary, opportunistic infections (18). In addition, the estimated annual losses associated with AAT are about US\$5 billion (1, 19–21), which is mainly due to a combined result of political, sociocultural, environmental, entomological, and livestock management factors (22–24). So far, chemotherapy is the only strategy available to treat the disease, whereby unique organelles of trypanosomes (glycosomes or kinetoplast) that are absent in the mammalian host or trypanosome metabolic pathways that differ from their host counterparts (carbohydrate metabolism, protein and lipid modifications, and programmed cell death) are targeted (25–27). Given that chemotherapy is associated with high drug toxicity, there is an urgent need to optimize trypanocide usage and delivery in order to decrease the risk of toxicity and/or resistance development (28–30). Control of AT is also hampered due to inefficient diagnosis of the infection especially for AAT and *T. b. rhodesiense* HAT where microscopical parasite detection (cheap but with low sensitivity), detection of the parasite's DNA (expensive but with high sensitivity), or anti-parasite antibodies remain the only available tools for diagnosis. Yet, these techniques require specialized equipment and personnel and hence are not suitable for direct use in the field. Only for *T. b. gambiense*, monitoring tools are available for both detection and staging of the disease (4, 31–33). Existing field applicable antibody-based diagnostic tests still suffer from a lack of positive predictive value and cannot differentiate between active or cured infections (32, 34, 35). Direct diagnosis aimed at parasite antigen detection is often hampered by sequestration of parasite antigens by the host's antibodies or by concealing of epitopes from the diagnostic monoclonal antibodies (mAbs) (36). Although immunodiagnostics based on antigen detection would be preferable, they are currently not available for trypanosomiasis in the field (32).

In contrast to conventional antibodies, nanobodies [Nbs or VHHs, i.e., camelid-derived single-domain antibody fragments (~15 kDa) that are selected through phage display technology and panning methodologies] (37, 38) could be used to overcome certain challenges faced by mAb-based tests (see above). Hereby, Nbs exhibit characteristic features such as (i) a nanomolar affinity for their target (39), (ii) a unique epitope recognition spectrum different from conventional antibodies, thereby allowing detection of both free antigens and those bound by host antibodies (40), (iii) high solubility (40), (iv) easy tailorability (multimerization or tagging) for molecular imaging and drug-delivery applications (41–44), and (v) small size that circumvents problems of tissue or blood–brain barrier (BBB) penetrability (44, 45). Due to these unique biochemical and biophysical properties, they are considered as promising next-generation therapeutics with great potential in pharmaceutical and industrial applications (46, 47). Indeed, Nbs are increasingly exploited in protein structure/function studies and in the development of alternative or new medical diagnostic and therapeutic applications (48, 49). Nbs also possess a relatively high thermostability (50, 51) and are consequently attractive for the development of immunodiagnostic tests that could be applicable in hot climatic conditions (i.e., sub-Saharan Africa).

In the next sections, we will give an overview of how the Nb technology can be implemented in the fight against AT both at the level of diagnosis and treatment and finally how acquired knowledge on Nbs in AT might lead to new insights in the function of conventional antibodies in the immune system.

### LIFE CYCLE OF AFRICAN TRYPANOSOMES

In order to point out at which stages Nbs might be applicable to fight AT, it is appropriate to overview briefly their life cycle. African trypanosomes exhibit a digenetic life cycle, alternating between the blood/tissues of the mammalian host and alimentary tract of the tsetse fly vector, whereby they exist as procyclic or trypomastigote forms (52), respectively (see **Figure 1**). The lifecycle within the mammalian host is initiated upon the bite of a trypanosome-infected tsetse fly when taking a blood meal (see **Figure 1**). Hereby, metacyclic parasites are inoculated in the host dermis in concert with tsetse saliva components that play a key role in the modulation of the host early immune response and in sculpturing an immune privileged microenvironment for infection initiation (53–55). From the dermal infection site, parasites reach the blood circulation through the lymphatics (55). Subsequently, these metacyclic parasites expressing a heterogeneous metacyclic variable surface glycoprotein (VSG) coat will differentiate into dividing long slender (LS) bloodstream dividing forms (BF), which express a unique VSG coat and are adapted to survive in the glucose-rich and highly oxygenated blood of the mammalian host. Next, these BFs rapidly multiply, giving rise to a first parasitemia peak. At the peak of parasitemia, most likely *via* a quorum sensing mechanism (56, 57), the LS parasites differentiate into non-dividing short stumpy (SS) forms pre-adapted for survival in the tsetse fly vector. Within the tsetse fly vector, these SS forms differentiate within the midgut into procyclic forms (PF) that express a procyclin coat,

which are adapted to survive in the proline-rich (carbon source) and low-oxygenated environment. Within the tsetse fly, these parasites undergo several differentiation stages in the different parts of the alimentary tract, mouthparts, and salivary glands (58, 59). In order to adapt to the growth conditions imposed by the different environments of their hosts, trypanosomes undergo essential morphological and metabolic changes (52), consisting of fine-tuning their energy metabolism, a dedicated iron and nutrient uptake, organelle reorganization, and biochemical and ultrastructural remodeling (60–65). Hence, tools to interfere with the various stages in the parasite life cycle might be an attractive strategy to combat AT.

### Nbs AS VERSATILE TOOLS FOR AT

The control over AT would benefit from more efficient diagnosis and treatment intervention strategies. Hereby, since their serendipitous discovery 30 years ago, Nbs attracted a progressively growing interest from fundamental research on antibody structure and ontogeny to diagnostical and therapeutical applications (66–68). With respect to fundamental research, the Nb technology was found to provide a novel tool in structural biology, whereby they can be used as crystallization-aid, or as a tool to design novel drugs based on co-crystallization of Nbs with their cognate antigen (69, 70). Although such applications in the field of AT are not yet documented, this will most likely become an emerging field of study in the near future. Moreover, given the tremendous efforts to identify novel targets for AT through proteomic approaches (71), the merging of the Nb technology with the current technologies might pave the way to develop additional tools/targets to fight AT. In this section, we will give an overview of the applications of the Nb technology in the field of AT.

### Nbs As Tools for Diagnosis

To date, several obstacles hamper an efficient and reliable diagnosis of AT, whereby (i) inefficient antibody-based detection due to interferences caused by the host's antibody response (i.e., antibodies remaining in circulation for long periods of time) impairs discrimination between active and cured infections and (ii) antigen-based trypanosome detection methods that might allow circumventing the problems encountered in the antibodybased detection system are not yet available. In this context, Nbs are emerging as promising tools to overcome the limitations of antibody-based diagnosis and to improve antigen-based detection of African trypanosomes (see **Figure 2** and **Table 1**). For instance, though the binding interference caused by the host's antibody response upon infection might hamper sensitive detection, this response might also be exploited using the Nb technology. Indeed, Caljon et al. (72) showed that following immunization of an alpaca with the sialome of the savannah tsetse fly vector (*Glossina morsitans morsitans*), Nbs could be generated against an abundant highly immunogenic tsetse salivary gland (Tsal) endonuclease protein and could subsequently be used to monitor tsetse fly bite exposure (73). Monitoring this bite exposure level in a target host population is important as the probability of a trypanosome transmission event to a new host is directly linked to the exposure level of the host to tsetse bites in that area (=risk factor). Indeed, only a very limited number of flies in a natural tsetse population carries the final infective parasite stage, so the more frequent a host is bitten by tsetse the higher the risk is that it will trap a trypanosome infection through the bite of a rarely occurring infected fly in that area. The assay principle relies on the detection of specific

Figure 2 | Overview of strategies used to generate nanobodies (Nbs) and their applications. Nbs can be obtained from llama's that are either immunized with factors derived from (i) tsetse flies (i.e., sialome or procyclin coat isolated form procyclic forms) or (ii) purified blood stream parasites originally grown in the mammalian host [i.e., secretome, soluble lysate, or variable surface glycoprotein (VSG)] or (iii) from infected (naturally or experimentally) with trypanosomes. After immunization, lymphocytes are isolated from the blood and *via* the PCR, phage display, and different selection procedures, individual Nbs can be obtained and purified. These Nbs can find application in diagnosis, therapy, or be used for fundamental research aiming at developing novel strategies to fight African trypanosomiasis.

*In addition, the Nb specificity and applicability (diagnosis/treatment) is mentioned.*

*PF, procyclic forms; BP, bloodstream form; Tb, Trypanosoma brucei; Tc, Trypanosoma congolense; Te, Trypanosoma evansi; Tv, Trypanosoma vivax.*

*Trypanosome species in bold refers to the species against which the target was originally generated.*

*"?" stands for not tested or unknown.*

Stijlemans et al. Nbs As Tools Against AT

anti-Tsal1 antibodies in an assay that measures the competitive binding onto the immunogenic Tsal1protein of diagnostic Nbs (Tsal1Nb-5 and -11) and host antibodies that are typically induced by bite exposure. This Nb-based competition assay allows specific detection of exposure to a range of important tsetse fly species in the context of sero-epidemiological surveys based on salivary proteins. This could not only allow monitoring/ estimating the intensity of the host exposure to tsetse fly bites but also reveal the efficacy of applied and/or ongoing tsetse fly control activities. This approach might be further extended by generating anti-proteome and/or anti-infectome Nb libraries in order to identify diagnostic Nbs (74–76). Indeed, the work by Obishakin et al. (77) showed that upon immunization of a llama with *T. evansi* lysate, Nbs against the paraflagellar rod (PFR) protein of trypanosomes could be generated and used in a solid phase antigen-ELISA to detect this protein in different *T. evansi* strains. Although the assay was not sensitive enough to detect *T. congolense* and *T. vivax* lysates in ELISA, one of the anti-PFR Nbs (Nb392) was found to cross-react with multiple parasite species such as *T. brucei*, *T. congolense*, and *T. vivax* as well as PF. Detection was achieved using fixed and permeabilized parasites and *via* flow cytometry and immunofluorescence microscopy, inferring that this Nb could be used to develop a broad spectrum diagnostic reagent. Moreover, this Nb could also be exploited as a PFR marker and/or as a useful research tool to isolate PFR proteins. More recently, Odongo et al. (78) identified following immunization of a llama with the soluble proteome of bloodstream form (BF) *T. congolense* a Nb (Nb474) recognizing glycosomal aldolase (*Tco*ALD) that could be used in a Nb-based sandwich ELISA to specifically detect active *T. congolense* infections in experimentally and naturally infected cattle. In experimental *T. congolense* infection models, parasitemia and detected antigenemia followed the same trend and the assay was suggested to be suitable as a test of cure. Although no formal detection limit was determined, the Nb474-based test was able to detect *T. congolense* infections in two field collected cattle blood samples that underwent the traditional parasitological diagnosis using the buffy coat technique followed by 18S-PCR-RFLP-based parasite species identification. Furthermore, it was suggested that the robustness of this Nb474-ELISA to specifically monitor *T. congolense* infections in the field might be improved once the structural and biophysical determinants of the specific Nb474–*Tco*ALD interaction can be determined. Of note, also in other parasitic diseases such as malaria, aldolase was reported as a proficient biomarker for the detection of *Plasmodium vivax* (79). In addition, in regions of sub-Saharan Africa where animals are infected with human and animal infective trypanosomes, this selective test for *T. congolense* using aldolase as a biomarker would allow discriminating between the two parasite groups, hence enabling assessment of the potential risk for human infection (80).

Besides recognizing low abundant proteins, Nbs have also been generated against the highly abundant VSG coat of the *T. congolense* parasite, yet due to the system of antigenic variation their diagnostic value is rather limited (unpublished data). However, the work by Stijlemans et al. (81), using the *T. brucei* model parasites, showed that the reduced size of a Nb allows to target conserved, less-immunogenic, cryptic VSG epitopes, that are inaccessible to conventional antibodies. Hereby, the fluorescently labeled anti-VSG Nb-33 was found to be very proficient to detect different isoforms of the *T. brucei* family and to specifically stain trypanosomes in infected blood. This suggests that even Nbs directed against specific conserved regions of the VSG molecule can be used as diagnostic tools.

Collectively, these data suggest that a Nb-based strategy could be a unique approach for diagnosis development and might bring us a step closer toward obtaining an antigen-detection test that can be used for rapid and reliable detection of vector exposure as well as the presence of pathogen infections in reservoir hosts (see **Figure 2** and **Table 1**). In addition, Nbs could also be used for target discovery given that following immunizations with complex protein mixtures and *via* different purification techniques, their cognate antigens with diagnostic potential can be identified.

#### Nbs As Therapeutic Devices Nbs As Tools for Drug/Toxin Delivery

Although the currently used chemotherapeutics are proficient in killing trypanosomes, their *in vivo* application suffers from systemic drug toxicity and occurrence of drug resistance (82). Therefore, delivering the chemotherapeutic (or toxin) directly to the parasite could be a more efficient way for treating AT. The possibility of using Nbs as targeting entity was investigated using the Nb-33 as model Nb (**Figure 2**; **Table 1**). With respect to using Nb-33 as a *toxin-delivery system* to African trypanosomes, apolipoprotein L-1 (ApoL-1) was selected as a trypanolytic component. ApoL-1 is a component of normal human serum (NHS) that exerts a direct trypanolytic effect on all AAT species, except resistant forms such as *T. brucei rhodesiense* (83). Indeed, *T. b. rhodesiense* expresses the apoL-I-neutralizing serum resistance-associated (SRA) protein, endowing this parasite with the ability to infect humans and cause HAT (83, 84). Hence, Nb-33, recognizing a conserved/cryptic region within the VSG coat, was coupled to a truncated form of Apo-L1 (i.e., Tr-apoL-1), which is engineered by deleting the SRA-interacting domain. This engineered immunotoxin was shown to function curatively and to alleviate effects on acute and chronic infections in mice infected with both NHS-resistant and sensitive parasites (85, 86). The Nb-33 was also used as a *delivery system for therapeutics* using *T. b. gambiense* parasites as a model. Hereby, nanoparticles loaded with pentamidine [i.e., drug used for treating the early disease stage, before central nervous system involvement (87), as the second-line option to suramin] were coupled to Nb-33. This targeted drug-bullet allowed decreasing the halfinhibitory concentration (IC50) 7-fold compared to free drug *in vitro* and cured all mice at a 10-fold lower dose than the minimal full curative dose of free pentamidine (88–90). Moreover, recently an improved version of this nanocarrier allowed reducing the curative dose 100-fold and circumvented drug resistance that is due to mutations in aquaglyceroporin 2 (i.e., the surface channel protein that mediates pentamidine uptake in *T. brucei*) (29, 91, 92). Overall, targeting the trypanosome surface using Nb-coated drug-loaded nanoparticles can be an elegant way to deliver drugs *via* endocytosis and bypass the usual drug delivery route altogether. Recently, using the Nb-33, it was shown that Nbs are able to pass the BBB during the late stage of *T. brucei* infection using murine and rat models, which suggests they might be valuable tools to target toxins even at the levels of the BBB. Yet, they were incapable of accumulating in the brain at therapeutically relevant concentrations (93), which is most likely due to the systemic pharmacokinetics of monovalent Nbs (42, 94). Hence, efforts should be undertaken to tailor these Nbs *via* for instance CDR grafting to yield improved brain penetrating properties without losing their beneficial size (95, 96). In addition, to quantitatively study the penetration of Nbs into the CNS *in vivo*, intracerebral microdialysis represents a powerful and sensitive technique. Given that much effort is put into developing new drugs against African trypanosomes, the Nb-based drug/toxin-delivery approach might allow increasing their efficacy, even against drug resistant parasites, and be applicable for many other diseases (97).

#### Nbs As Direct Trypanolytic Entities by Blocking Endocytosis

By serendipity, besides a drug-targeting potential, Nbs were found to exert direct trypanolytic activities. Indeed, it was shown that some Nbs specific for the variable part of VSG of *T. b. brucei* parasites were able to induce parasite lysis *in vitro*. The lytic process consists of a rapid immobilization of the parasites, followed by massive enlargement of the flagellar pocket and a major blockage of endocytosis (98). Given that endocytosis is essential for trypanosome survival, playing a key role in nutrient uptake and in regulating intracellular ATP-levels as well as in maintaining the mitochondrial membrane potential, it is a prime candidate target for therapeutic interventions (99). Moreover, the endocytosis is confined to the flagellar pocket, which can be considered as the gateway to and from the cell surface that regulates the host–parasite interface as well as significantly contributes toward interactions with therapeutics. Hence, this might be the trypanosomal Achilles' heel and offer perspectives for directed drug delivery focusing on proteins essential in endocytosis (100). Also, targeting specific receptors essential for nutrient uptake could be considered as therapeutic targets. For instance, given that iron is essential for the survival of both the trypanosome and the mammalian host and that their receptor for iron/transferrin uptake differs (i.e., a heterodimer and homodimer, respectively) (101), specific targeting of the parasite transferrin receptor using Nbs might be an opportunity to selectively deprive trypanosomes from this essential nutrient or alternatively be used as drug delivery or diagnostic tool. Also, the fact that low-molecular weight VSG-specific trypanolytic Nbs can impede endocytosis suggests that Nbs can be used as tools to further unravel the fascinating endocytosis mechanism used by trypanosomes. This in turn may offer new opportunities for developing novel trypanosomiasis therapeutics aimed at affecting endocytosis.

One aspect that could compromise the therapeutic applications of Nbs, in for instance HAT patients, is the potential immunogenicity of Nbs, especially in treatments that require repeated injections. Currently, the immunogenicity of Nb-based therapeutic applications is controversial. For instance, a clinical trial study conducted by GSK revealed the occurrence of anti-TNFR1 Nb autoantibodies and in another study with a tetravalent anti-DR5 receptor Nb hepatotoxicity in patients with such pre-existing antibodies has been described (102, 103). By contrast, no anti-HER2 Nb autoantibodies could not be detected in patients who received a non-humanized Nb (104), nor in patients receiving a Nb against von Willebrand factor (105). Also in the murine model, so far, multiple injections of Nbs in different disease settings did not result in immunogenicity, neither at the level of specific antibodies against Nbs nor at the level of cell proliferation and cytokine levels (68, 106–108). Hence, it seems that the occurrence of immunogenicity might depend on the target and disease situation (46). Yet, to reduce/minimize the risk of an immune response within the mammalian host, there are strategies currently implemented, such as humanization of the Nbs, whereby the camelid-specific amino acid sequences are mutated to their human heavy chain variable domain equivalent. In this context, a universal humanized Nb scaffold has been generated that allows grafting the antigen-binding loops from other Nbs, thereby transferring the antigen specificity and affinity (109).

#### Paratransgenesis As Tool to Deliver Nbs within the Tsetse Fly Vector

The applications of Nbs against AT may be not restricted to the BF of the parasites within the mammalian host, but could be applied/extrapolated to the vector. In this context, the possibility to exploit the tsetse fly bacterial symbiont *Sodalis glossinidius* as a paratransgenic platform organism for the expression and delivery of trypanosome-interfering proteins (i.e., Nbs) within the tsetse fly vector was evaluated. To this end, both the non-lytic Nb-33 and the trypanolytic Nbs were shown to be successfully expressed without affecting *S. glossinidius* fitness/viability (110). Moreover, using the trypanolytic Nb as proof of concept, recombinant *S. glossinidius* could settle in different tsetse fly tissues at high densities. Furthermore, significant levels of functional anti-trypanosome Nbs were released in several tissues including the midgut where important developmental stages of the parasite reside (111). Here, the level of Nb expression was estimated to be in the low nanogram (<10 ng), which was calculated to be sufficient to lyse the expected low number of transforming blood stream trypanosomes (around 103 parasites) in the tsetse midgut during the early developmental period after ingestion by the fly (111). Accordingly, this paratransgenic approach using *Sodalis* to deliver Nbs that target the parasite or the trypanosome–tsetse fly cross talk could open new avenues to unravel the molecular determinants of this specific parasite–vector interplay and to ultimately render tsetse flies trypanosome resistant [reviewed by Caljon et al. (112)]. Given that the trypanosome is not exposed to an adaptive immune system in the tsetse vector, this parasite stage is not undergoing antigenic variation with the major surface antigen being encoded by a limited set of procyclin genes. In this context, the potential of Nbs delivered using the *Sodalis* endosymbiont targeting the major developmental stages in the tsetse fly, such as the procyclic trypanosomes that need to overcome the midgut barrier in order to achieve colonization of the tsetse fly vector, is currently being investigated. Stable integration of Nb expression cassettes in the *Sodalis* genome (e.g., by using recently established procedures) and efficient vertical transfer of the transgenic *Sodalis* have been achieved (113). Important issues, such as the identification of highly potent infectionblocking Nbs and the increased proteolytic stability, are still to be addressed (114). The latter feature will be highly beneficial to maintain potent effector levels in the strong proteolytic digestive environment of the insect midgut. Yet, such strategies could potentially culminate in a drug-targeting strategy to eliminate trypanosomes within the tsetse fly vector. An important achievement in the context of the *Sodalis*-based paratransgenesis is the efficient transfer of genetically modified *Sodalis* from the mother tsetse fly to its offspring through intrauterine nourishment (113). This implies that a large-scale tsetse fly colony can be established of flies harboring a Nb-expressing *Sodalis*. The paratransgenic trypanosome-resistant male flies from these colonies can then be released (after sterilization through irradiation) at a massive scale in the context of the Sterile Insect Technique (SIT). SIT was already successfully used to eradicate the tsetse fly in Zanzibar and is currently an important pillar in the tsetse fly control campaigns in Ethiopia and Senegal (http://www.fao. org/in-action/senegal-celebrates-first-victory-against-tsetse-flyeradication/en/).

### LYTIC Nbs: RELEVANCE *IN VIVO*?

The observation that the antigen-binding domain (i.e., Nb) on itself, in the absence of the Fc part can exert a significant Fc-independent killing of African trypanosomes *in vitro* and *in vivo* is remarkable (98). Moreover, it was found that both the size and affinity were of crucial importance for this trypanolytic activity (98, 115). Indeed, whereas polyclonal antibodies (including heavy-chain antibodies) specific for the VSG of African trypanosomes are completely harmless to trypanosomes in the absence of complement or any other bystander effector (116, 117), polyclonal Fabs or Nbs derived from the serum antibody pools and monoclonal/polyclonal Fabs or Nbs that are deprived of all effector functions (i.e., Fc) could exhibit an intrinsic trypanolytic activity *in vitro* (98). It is surprising that removal of the Fc part from antibodies unveils a novel but deadly situation, because trypanosomes and other extracellular pathogens mostly coped during evolution with intact immunoglobulins and thus developed multiple ways to avoid the destructive action of such large molecules (118, 119). In case of African trypanosomes such escape mechanisms include (i) antigenic variation of the VSGs, (ii) dense packing of VSG molecules on the parasite's coat prohibiting the recognition of conserved and/or physiologically important epitopes by intact antibodies, (iii) clearing of VSGbound antibodies by endocytosis of the VSG–antibody complex [reviewed in Ref. (120–123)]. In contrast to intact Abs, the small-sized Fabs or Nbs may penetrate the dense VSG coat and trigger new processes or avoid removal of VSG–antibody complexes, which is dictated by the bivalency of Abs (possibly due to cross-linking of the VSGs) and/or antibody size, e.g., whereby the presence of the Fc part leads to steric occlusion (123, 124).

The concept that the Fc part within an antibody is masking the intrinsic destructive capacity of the antigen-binding fragment is intriguing. Consequently, other polyclonal or mAbs may share similar features and harbor hidden activities that remain occluded within intact Abs and might manifest themselves upon generation of monovalent Fabs or Nbs (see **Figure 3**). Evidence for a possible intrinsic anti-pathogen activity of *in vivo* generated Fabs or Nbs within the *bona fide* antibody independent of the Fc part is difficult to provide as there is so far no simple assay to demonstrate such an event. Nevertheless, such mechanisms might exist as Nbs with competitive enzyme-inhibiting activity or Fabs with catalytic activity (termed Abzymes) have been identified (117–120). Under certain conditions, antibodies were documented to exert bactericidal activities in the absence of complement or phagocytes (125). For instance, antibodies catalyze the generation of hydrogen peroxide (H2O2) from singlet molecular oxygen and water, thereby producing an additional molecular species with a chemical signature similar to that of ozone (126). Interestingly, this singlet molecular oxygen is only present when the host is under assault, thereby making it an "event-triggered" substrate and consequently suggests that the additional function of an antibody might only be apparent under "inflammatory" conditions (127). In addition, it was shown that high H2O2 in concert with transition metal ions (FeCl2) (i.e., factors that are typically produced/released *via* apoptotic neutrophils during inflammation) can generate hydroxyl radicals (*via* a Fenton-like reaction) that induce hinge fragmentation of IgG1 mAb [(128) consisting of (i) a Fab domain and the upper hinge of one of the Fc domains and (ii) another Fab domain linked to the Fc domain (see proposed model in **Figure 3**)]. This indicates that under certain *in vivo* inflammatory conditions hidden biological activities could become unmasked from *bona fide* antibodies. To the best of our knowledge, the observation that an antibodyderived fragment on itself can exert a biological function is a new conceptual insight which might broaden the potentiality of Ab applications in different fields.

# CONCLUSION AND FUTURE PERSPECTIVES

Over the years, Nbs have been found to be valuable tools in the field of AT both at the level of diagnosis and treatment. They were shown to have potential to circumvent problems encountered with antibody-based detection systems and in addition allow the development of antigen-based approaches that were so far lacking (32). With the era of proteomics (71), allowing additional biomarkers to be discovered, the application of a Nb-based immunoproteomic approach might allow developing more efficient tools to improve trypanosomiasis control (diagnosis/treatment) in the near future. With respect to treatment against AT, it seems that Nbs are a very proficient tool to deliver drugs/toxins to parasites, thereby reducing the side effects due to drug toxicity and possibly the probability to develop drug resistance. Their small size, low immunogenicity, and tailorability furthermore favor their application in AT with respect to BBB drug delivery, routine/systemic administration, and generation of half-life extended formats. Also for research purposes, their advantages are increasingly appreciated and

they will most likely become a useful tool with respect to crystallization and imaging. Finally, the concept that a Nb or a Fab fragment derived from an intact IgG might exert a biological function by itself in the absence of the Fc-bystander warrants further investigation. Moreover, this suggests that intact antibodies harbor hidden functions that only become apparent upon fragmentation into a Fab, a process that might occur under certain conditions, and this might have a broad range of implications and applications in other diseases.

# AUTHOR CONTRIBUTIONS

BS, PB, GC, JA, JG, and SM collectively wrote the manuscript.

### REFERENCES

# FUNDING

We acknowledge the financial support of the Interuniversity Attraction Pole Program (PAI-IAP N. P7/41, http://www.belspo. be/belspo/iap/index\_en.stm) and grants from the FWO (FWO G015016N and G.0.028.10.N.10) and the Bill and Melinda Gates Foundation grant 641K760. BS is a research fellow supported by the Strategic Research Program (SRP3, VUB): targeting inflammation linked to infectious diseases and cancer (Nanobodies for Health). GC is funded by a research fund of the University of Antwerp (TT-ZAPBOF 33049). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

*Copyright © 2017 Stijlemans, De Baetselier, Caljon, Van Den Abbeele, Van Ginderachter and Magez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Isolation of Single-Domain Antibody Fragments That Preferentially Detect Intact (146S) Particles of Foot-and-Mouth Disease Virus for Use in Vaccine Quality Control

*Michiel M. Harmsen1 \*, Julian Seago2 , Eva Perez <sup>2</sup> , Bryan Charleston2 , Phaedra L. Eblé1 and Aldo Dekker <sup>1</sup>*

*1Wageningen Bioveterinary Research, Lelystad, Netherlands, 2 The Pirbright Institute, Woking, United Kingdom*

#### *Edited by:*

*Kevin A. Henry, National Research Council Canada, Canada*

#### *Reviewed by:*

*Ming Yang, Canadian Food Inspection Agency, Canada Mehdi Arbabi Ghahroudi, National Research Council Canada (NRC-CNRC), Canada*

> *\*Correspondence: Michiel M. Harmsen<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology*

*Received: 25 April 2017 Accepted: 26 July 2017 Published: 17 August 2017*

#### *Citation:*

*Harmsen MM, Seago J, Perez E, Charleston B, Eblé PL and Dekker A (2017) Isolation of Single-Domain Antibody Fragments That Preferentially Detect Intact (146S) Particles of Foot-and-Mouth Disease Virus for Use in Vaccine Quality Control. Front. Immunol. 8:960. doi: 10.3389/fimmu.2017.00960*

Intact (146S) foot-and-mouth disease virus (FMDVs) can dissociate into specific (12S) viral capsid degradation products. FMD vaccines normally consist of inactivated virions. Vaccine quality is dependent on 146S virus particles rather than 12S particles. We earlier isolated two llama single-domain antibody fragments (VHHs) that specifically recognize 146S particles of FMDV strain O1 Manisa and shown their potential use in quality control of FMD vaccines during manufacturing. These 146S-specific VHHs were specific for particular O serotype strains and did not bind strains from other FMDV serotypes. Here, we describe the isolation of 146S-specific VHHs against FMDV SAT2 and Asia 1 strains by phage display selection from llama immune libraries. VHHs that bind both 12S and 146S particles were readily isolated but VHHs that bind specifically to 146S particles could only be isolated by phage display selection using prior depletion for 12S particles. We obtained one 146S-specific VHH—M332F—that binds to strain Asia 1 Shamir and several VHHs that preferentially bind 146S particles of SAT2 strain SAU/2/00, from which we selected VHH M379F for further characterization. Both M332F and M379F did not bind FMDV strains from other serotypes. In a sandwich enzyme-linked immunosorbent assay (ELISA) employing unlabeled and biotinylated versions of the same VHH M332F showed high specificity for 146S particles but M379F showed lower 146S-specificity with some cross-reaction with 12S particles. These ELISAs could detect 146S particle concentrations as low as 2.3–4.6 µg/l. They can be used for FMD vaccine quality control and research and development, for example, to identify virion stabilizing excipients.

Keywords: enzyme-linked immunosorbent assay, single-domain antibody, foot-and-mouth disease, virion stability, foot-and-mouth disease virion, vaccine quality control

### INTRODUCTION

Foot-and-mouth disease (FMD) is an animal disease that is caused by a picornavirus, FMD virus (FMDV), which encompasses seven serotypes: A, O, C, Asia1, SAT1, SAT2, and SAT3. Infection with any one serotype does not produce significant humoral immunity against other serotypes. In FMD endemic areas vaccination is used as a preventive method (1). Due to differences in serotype prevalence in the field most vaccines are used for serotypes O and A. Further vaccines generally are specific for Asia1 or SAT2 serotypes. Conventional FMD vaccines (2) are based on chemically inactivated FMDVs that are formulated with an adjuvant. FMD virions consist of an RNA molecule and a capsid composed of 60 copies each of VP1, VP2, VP3, and VP4 proteins (3). Intact virions sediment at 146S in sucrose gradients. Some FMDV strains also produce empty capsids that lack the RNA molecule and sediment at 75S. Mild heating or incubation at pH below 6.5 leads to irreversible dissociation of 146S or 75S particles into stable 12S particles that contain five copies each of VP1, VP2, and VP3. Dissociation into 12S particles results in a strongly reduced immunogenicity (4–7).

Several methods have been developed to measure the concentration of 146S particles of the crude FMDV antigen preparation used for vaccine preparation. This is traditionally measured by sucrose density gradient (SDG) centrifugation (8). Novel methods that are more easy to automate are based on size-exclusion high-performance liquid chromatography (9, 10) or lateral flow immunoassay (11). All these methods have the advantage of being suitable for all FMDV strains, but the disadvantage of low sensitivity, limited sample throughput, and inability to discriminate different vaccine strains in multivalent vaccines. Double antibody sandwich (DAS) enzyme-linked immunosorbent assays (ELISAs) using monoclonal antibodies (mAbs) were also developed for FMDV antigen quantification (7, 12–16). They are more sensitive, but often not specific for intact 146S particles. Only two DAS ELISAs were specific for 146S particles due to use of a mAb showing such specificity. They were suitable for detection of A or O serotype strains (15, 16). We have recently developed two DAS ELISAs using two recombinant llama singledomain antibody fragments (VHHs) that bind specifically to either 146S particles or 12S particles of strain O1 Manisa (17, 18). In each ELISA, the same VHH was used for coating as well as for detection of captured antigen using biotinylated VHH. The DAS ELISA employing 146S-specific VHH M170 was specific for particular O serotype strains, including strain O1 Manisa. The DAS ELISA employing 12S-specific VHH M3 could detect FMDV antigen of several A, O, and Asia 1 strains but not SAT2 strain. We were also able to measure 146S particles of A and Asia 1 serotype strains employing the M3 DAS ELISA of heated and untreated samples (17). However, this latter ELISA approach is not suitable for detecting 146S particles in the presence of higher concentrations of 12S particles. An ELISA approach employing a 146S-specific VHH is therefore preferred. We here describe the isolation of two VHHs for specific detection of 146S particles of strains Asia 1 Shamir and SAT2 SAU/2/00.

Many conventional mAbs against FMDV have been isolated in the past. Most of those mAbs bind to both 12S and 146S particles (7, 19–22). However, mAbs that specifically detect 146S particles are rarely isolated (15, 16). VHHs against FMDV have recently been isolated (23, 24) without reporting their particle specificity. M170 is the only 146S-specific VHH that is currently described (17). It was isolated by screening a panel of 24 VHHs against strain O1 Manisa that were earlier isolated from llama immune libraries (25). It is not surprising that 146S-specific VHHs or mAbs are rare since 12S and 146S particles share many epitopes.

VHHs are normally isolated by phage display selection from immunized llamas or dromedaries (26). One of the advantages of using VHHs for this purpose is the high functional diversity of immune libraries derived from heavy chain antibodies since random shuffling of antibody heavy and light chains does not occur. Phage display VHH immune libraries therefore contain VHHs with many different specificities. The isolation of rare specificities requires dedicated selection procedures. The removal from libraries of clones with unwanted cross-reaction to particular antigens can be accomplished by prior depletion of libraries on these antigens before performing selection of VHHs on the target antigen (27), although such depletion often is inefficient (28, 29). We here describe the selection of VHHs specific for 146S particles by depletion on 12S particles. We focus on VHHs specific for Asia 1 and SAT2 FMDV since 146S-specific VHHs or mAbs are not yet available for these serotypes.

# MATERIALS AND METHODS

#### mAbs, VHHs, and FMDV Antigens

Monoclonal antibody 13A6 was raised against SAT1 Zimbabwe/89 that does not neutralize FMDV *in vitro*, cross-reacts to SAT2 Zimbabwe/86 and binds in a trypsin-sensitive manner in ELISA (30). VHHs were produced in baker's yeast as a fusion to the natural llama heavy-chain antibody long hinge region and a hexahistidine tag using vector pRL188 (25) and purified from culture supernatant using immobilized-metal affinity chromatography as described earlier. VHHs produced in this manner are indicated by the suffix "F." At least 20% of the VHH amount produced in this manner is dimerized through the single cysteine present at the C-terminus of the VHH, immediately preceding the his6 tag. Both such monomeric and dimeric VHHs are useful for functional immobilization of VHHs to polystyrene surfaces by passive adsorption (31). Yeast-produced VHHs were biotinylated at a weight ratio of protein to biotin of 10 using amine-reactive sulfo-*N*-hydroxysuccinimide-LC-biotin (Pierce, Rockford, IL, USA). Buffer was exchanged to PBS and free biotin removed by at least three consecutive 10-fold dilutions and concentration using Amicon Ultra 3-kDa molecular weight cutoff concentration devices (Millipore, Billerica, MA, USA). The VHH2 M3ggsVI4Q6E specific for 12S particles has been previously described (32).

The FMD vaccine strains Asia 1 Shamir/Israel/89, SAT2 SAU/2/00, O1 Manisa/Turkey/69, O1 British Field Strain 1860 (BFS)/67, A Turkey/98, and A24 Cruzeiro/Brazil/55 were used for antigen production. FMDV antigen originated from the virus production facilities in Lelystad. FMDV was cultured using BHK-21 cells grown in suspension in industrial size bioreactors. FMDV present in the clarified culture was inactivated with 0.01 M binary ethylenimine and concentrated using two consecutive polyethylene glycol-6000 precipitations, resulting in crude antigen. Infectious FMDV was prepared at laboratory scale by growing them on monolayers of BHK-21 cells and harvesting the culture supernatant without further treatment. Work with infectious FMDV was done in a high containment unit at Wageningen Bioveterinary Research using appropriate measures for virus handling and waste disposal to prevent FMDV introduction into the environment. Wageningen Bioveterinary Research is authorized to work with live FMDV according to EU directive 2003/85/EC.

Foot-and-mouth disease virus antigens were fractionated using 10–40% SDGs that were centrifuged for 2.5 h at 10°C and 200,000 × *g*. The gradients were fractionated into 20 0.61 ml aliquots and the absorbance at 254 nm was determined to identify the 146S peak. The 146S concentration in milligrams per liter was then calculated by multiplying the absorbance at 254 nm with 126.7. 12S particles were prepared from 146S preparations by acidification (12SA) or heat treatment for 1 h at 56°C (12SH) as previously described (33).

# Llama Immunization and Phage Library Construction

Llama immunizations were performed after ethical review by Wageningen Bioveterinary Research and in accordance with Dutch national guidelines on animal use. The immunization of llama 6058 with crude antigen of FMDV strain Asia 1 Shamir has been described earlier (25). The immunization of llamas 3049 and 3050 with influenza antigens and subsequent phage library construction was earlier described (34). Simultaneously with these influenza antigens llamas 3049 and 3050 were immunized with FMDV 146S particles purified from SDG. For each of the three immunizations llama 3049 received 40 µg Asia 1 Shamir 146S particles and 45 µg A24 Cruzeiro 146S particles, whereas llama 3050 received 50 µg SAT2 SAU/2/00 146S particles. Phage display immune libraries in phagemid vector pRL144 (35) of at least 107 independent clones were generated as previously described (25, 34).

## Phage Display Selection of Antigen Binding VHHs

Phage display selections were performed by two consecutive rounds of biopanning (36) in polystyrene 96-well plates (Greiner, Solingen, Germany, Cat. No. 655092), using 100 µl/well for each incubation. Many variations on the procedure were used (**Table 1**). In general, antigen was immobilized to 96-well plates at three serial 10-fold dilutions and a control without antigen was included. VHHs were normally coated at concentrations of 1, 0.1, and 0.01 mg/l in 0.05 M carbonate/bicarbonate buffer, pH 9.6 (coating buffer), overnight at 4°C. Purified 146S particles (1 mg/l) were separately incubated in PBS buffer containing 1% milk and 0.05% Tween-20 (PBSTM) for 1 h at room temperature (RT). Controls included wells coated with VHH without subsequent incubation with FMDV antigen and uncoated wells incubated with 1 mg/l FMDV antigen. Plates were then incubated with 1 × 109 transducing phage units per well. In initial experiments, phages were preincubated in PBSTM containing 10 mg/l 12SA particles and then added to wells with captured 146S particles to compete for binding to 12SA. In later experiments, phages binding to 12SA particles were depleted prior to biopanning by incubation on plates containing immobilized 12SA particles. For this purpose, separate plates were coated with coating buffer containing 1 mg/l M3ggsVI4Q6E or M311F specific for SAT2 SAU/2/00 antigen and subsequently incubated with 5 mg/l 12SA particles of strains Asia 1 Shamir or SAT2 SAU/2/00, respectively. Phages were then incubated on these plates with captured 12SA particles after which the unbound phages were transferred

*FMDV, foot-and-mouth disease virus.*

to biopanning plates containing captured 146S particles. To ensure binding of most 12SA reactive phages we used 20-fold lower amounts of phage (5 × 107 transducing phage units per well) when employing depletion. Bound phages were finally eluted by incubation with 1 mg/ml trypsin in PBS for 30 min at 37°C and immediately transduced to *Escherichia coli* TG1 [(F′ *traD36 proAB lacIqZ* Δ*M15*) *supE thi-1* Δ*(lac-proAB)* Δ*(mcrB-hsdSM)5(rK<sup>−</sup> mK<sup>−</sup>)*] cells. In each selection round, we performed a phage ELISA simultaneous with the phage display selection for evaluation of the phage display selection. For this purpose, a duplex plate containing similar concentrations and types of antigen and phage was incubated with a peroxidaseconjugated mAb against M13 phage instead of incubation with trypsin. The amount of bound antigen-specific phage was then measured by phage ELISA.

#### Production of Soluble VHH in *E. coli*

After the second round of panning phages was transduced to *E. coli* TG1 cells, individual colonies were picked and the VHH genes were induced with 1 mM isopropyl β-d-thiogalactopyranoside. Recombinant VHHs, extracted from the periplasm, were tested for binding to FMDV antigens at 10-fold dilution as described below in Section "Enzyme-Linked Immunosorbent Assays."

#### Sequence Analysis

Sequence analysis of the VHH encoding region was performed, as previously described (37). The deduced VHH amino acid sequences were aligned according to the IMGT system (38) for alignment, numbering, and complementarity-determining region definition of immunoglobulins. VHHs were classified into subfamilies as earlier defined (37). Potential N-glycosylation sites were defined as Asn-X-Ser/Thr, where X represents any amino acid, except Pro.

#### Enzyme-Linked Immunosorbent Assays

Three different ELISA procedures were used. We first describe the basic ELISA procedure that was commonly used in these three procedures. ELISAs were performed by coating high-binding polystyrene 96-well plates (Greiner) with 0.1–1 mg/l of unlabeled VHH, VHH2, or mAb in 50 mM carbonate/bicarbonate buffer, pH 9.6 (coating buffer), overnight at 4°C. Coating and subsequent incubations were performed using 100 µl per well. After washing, the coated plates were incubated with FMDV antigens in ELISAbuffer (1% skimmed milk; 0.05% Tween-20; 0.5 M NaCl; 2.7 mM KCl; 2.8 mM KH2PO4; 8.1 mM Na2HPO4; pH 7.4) or PBSTM for 1 h at RT. The 146S concentration of the FMDV antigens was determined by measuring the absorbance at 254 nm as described above. The concentration of the 12S antigens was derived from the 146S concentration of the sample from which it was prepared assuming complete conversion of 146S into 12S particles. Plates were next incubated with either phage-displayed VHH, *E. coli*produced VHH, or yeast-produced biotinylated VHH, and subsequently with a suitable specific peroxidase conjugate, using the same buffer as used for incubation with FMDV antigen (ELISAbuffer or PBSTM). The peroxidase conjugate was subsequently detected by staining with 3,3′,5,5′-tetramethylbenzidine. After stopping the reaction by addition of 0.5 M H2SO4 (50 µl per well) the absorbance at 450 nm was measured using a Multiskan Ascent spectrophotometer (Thermo Labsystems, Helsinki, Finland).

For phage ELISA plates were preferably coated with low concentrations of VHH (0.1 mg/l) and FMDV antigen (0.5 mg/l) as described in **Table 1**. Many variations on the procedure were used during phage display selection as described above. Bound phage displayed VHH was detected by incubation with peroxidaseconjugated mAb against the M13 p8 coat protein (GE Healthcare, Little Chalfont, UK) in PBSTM.

For ELISA using *E. coli*-produced soluble VHH plates were preferably coated with 1 mg/l VHH and then subsequently incubated with 1 mg/l FMDV antigen in ELISA buffer. Bound FMDV antigens were detected by incubation with 10-fold diluted *E. coli*-produced soluble VHH, which contains a myc-tag, and a peroxidase-conjugated mAb against the c-myc tag (clone 9E10; Roche Applied Science).

For DAS ELISA to measure the FMDV antigen concentration plates were coated with 0.5 mg/l of unlabeled VHH or VHH2. These plates were then incubated with serial 2-fold dilution series of FMDV antigen preparations in ELISA buffer. Normally, standards of untreated FMDV antigen were included in the ELISAs for quantification of 146S particles. However, 12SA preparations of FMDV antigen were used as standards in the ELISA employing M3ggsVI-4Q6E. Standards were serial twofold dilutions of 1 mg/l start concentration. Plates were next incubated with 0.25 mg/l of biotinylated VHH(2). Bound biotinylated VHH(2) was detected with 1 mg/l PO-conjugated streptavidin (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA). Absorbance data were evaluated using an Excel® spreadsheet template (Microsoft Corporation, Redmond, WA, USA). A fourparameter logistic curve was fitted to absorbance and FMDV antigen concentrations of standards by non-linear least squares using the Excel® solver tool. The FMDV antigen concentration in unknown samples was then determined by interpolation. For determination of the quality of binding of VHHs to particular FMDV antigens these antigens were titrated in twofold dilution series. The effective antigen concentration required to reach a particular absorbance value was then interpolated after four-parameter logistic curve fitting of absorbance and antigen concentrations.

The limit of detection (LOD) of ELISAs was measured by titrating FMDV antigens in triplicate and interpolating the VHH concentration required to reach the average and three times the SD of nine blank measurements without antigen.

### RESULTS

#### Selection of 146S-Specific VHHs

We earlier immunized llama 6058 with crude antigen of FMDV strains O1 Manisa, A Turkey, A22 Iraq, and Asia 1 Shamir. We isolated many FMDV O1 Manisa binding VHHs from this llama by phage display employing biotinylated FMDV antigen captured with streptavidin-coated magnetic beads (25). The VHH M98 was similarly selected from llama 6058 using biotinylated FMDV Asia 1 Shamir antigen. Yeast-produced M98F VHH neutralized Asia 1 Shamir FMDV *in vitro*, confirming M98F binds FMDV virions rather than a host protein present in the crude FMDV antigen used to perform the selection. As further confirmed below M98F was found to bind both 12S and 146S particles. This was not unexpected since 146S-specific VHHs were also rarely retrieved during phage display selection of O1 Manisa binding VHHs (25).

To improve the selection process of 146S-specific VHHs we purified 146S particles by SDGs. The purified 146S particles were then used for both novel llama immunization and phage display selection of VHHs. One llama (3049) was immunized with FMDV Asia 1 Shamir 146S and another llama (3050) with SAT2 SAU/2/00 146S. As observed with many other proteins (39) FMDV loses much of its antigenicity upon direct passive adsorption to polystyrene (40). We, therefore, used M98F VHH that binds Asia 1 Shamir and mAb 13A6 that binds SAT2 SAU/2/00 to capture purified 146S particles in ELISA. We initially tried to select VHHs that bind specifically to 146S particles by competition during phage display selection with a surplus of 12SA particles. This approach was not successful as all VHHs isolated bound both 146S and 12SA particles when tested as soluble VHH produced from *E. coli* TG1 cells (results not shown). After sequence analysis (**Table 1**), we identified five unique clones (M311-M317) binding SAT2 SAU/2/00 and eight unique clones (M301–M308) binding Asia 1 Shamir 146S. Their cross-reaction with 12S was later confirmed using yeast-produced VHH (**Table 2**). To increase enrichment of 146S-specific VHHs we improved phage display selections in two ways. Since mAb 13A6 preferentially binds 12SA particles (see below), it was replaced by a novel isolated VHH that was highly produced in yeast, M311F, for capture of SAT2 SAU/2/00 146S particles. Furthermore, novel selections were performed using depletion with immobilized 12SA particles instead of competition with soluble 12SA particles. Using these novel selection procedures we retrieved seven unique SAT2 SAU/2/00 binding VHHs comprising three novel CDR3 groups (**Table 1**) that are different from earlier isolated SAT2 SAU/2/00 binding VHHs (**Table 2**) and seven unique Asia 1 Shamir binding VHHs comprising six CDR3 groups (**Table 1**) that are mostly different from earlier isolated VHHs (**Table 2**).

#### 146S Specificity of VHHs

Seventeen VHHs with different CDR3 sequences (**Table 2**) were produced by yeast expression. They all lacked potential *N-*glycosylation sites to prevent compromised antigen binding due to fortuitous *N*-glycosylation. The purified VHHs were biotinylated for use in DAS ELISA in combination with the same unlabeled VHH for coating. To assess the specificity of the biotinylated VHHs for the different FMDV particles we performed DAS-ELISAs with a titration series of untreated antigen, 146S particles and 12SH. Similar to earlier methods (17), the FMDV antigen concentration required to reach an absorbance value of 0.4 was calculated for each FMDV antigen. The absorbance value of 0.4 was chosen because for all different ELISAs used it is sufficiently above background absorbance values to be indicative of antigen binding and sufficiently below the maximal absorbance value. The ratio between these antigen concentration values for 12SH and 146S was taken as a measure for particle

*a As defined by reference (37).*

*bNA, Not applicable.*

*c* >*1,000 indicates absorbance does not exceed 0.4 at the highest VHH concentration analyzed (1 mg/l).*

*DAS, double antibody sandwich; ELISAs, enzyme-linked immunosorbent assays; mAb, monoclonal antibody.*

specificity (**Table 2**). A ratio > 1 corresponds to 146S specificity and a ratio < 1 corresponds to 12S specificity.

In the case of SAT2 SAU/2/00 antigen mAb 13A6 shows 12S specificity with a ratio below 0.16, the four VHHs selected by competition with 12SA show no or limited 146S specificity with ratios of 0.85–4.7, whereas the three VHHs selected by depletion with 12SA (M377F, M379F and M380F) show higher 146S specificity with ratios of 10–28 (**Table 2**). M379F exhibited the highest 146S specificity and recognized relatively low antigen and 146S concentrations as compared to other SAT2 SAU/2/00 binding VHHs. Among the Asia 1 Shamir binding VHHs, M332F that was selected by depletion on 12SA particles showed highest 146S specificity with a ratio of 57. Furthermore, M332F recognized lower concentrations of untreated antigen and 146S as compared to most of the other nine VHHs. Only M311F recognized slightly lower antigen concentrations, although with far lower 146S specificity (**Table 2**). In conclusion, M332F exhibited high 146S specificity for strain Asia 1 Shamir, whereas M379F showed some 146S specificity for strain SAT2 SAU/2/00.

We next titrated FMDV antigens and 12SH of strains SAT2 SAU/2/00 and Asia 1 Shamir in triplicate in the M3ggsVI4Q6E, M311F, M379F, M98F, and M332F ELISAs using the same VHH as used for detection also for coating. The LOD was calculated for each ELISA (**Table 3**). The M332F ELISA had an LOD of 4.6 µg/l and the M379F ELISA of 2.3 µg/l for antigen. We also titrated the antigens in these latter two ELISAs in the presence of a constant amount of 12SH particles to determine if antigen quantification is affected by the presence of 12SH particles. Asia 1 Shamir antigen quantification in the M332F ELISA was not affected by the presence of 4 mg/l 12SH (**Figure 1B**) but SAT2 SAU/2/00 quantification in the M379F ELISA was affected already by the lowest 12SH concentration analyzed (0.15 mg/l; **Figure 1E**). This is consistent with the earlier observed higher specificity of M332F for 146S particles than M379F.

#### FMDV Strain Specificity

To further characterize the 17 VHHs, their respective ability to bind to strains of other FMDV serotypes was investigated. As a control we included mAb 13A6 and M8ggsVI4Q6E, to demonstrate successful immobilization of the various antigens (**Table 4**). All seven SAT2 SAU/2/00 binding VHHs did not cross-react to strains Asia 1 Shamir, A Turkey, A24 Cruzeiro, O1 BFS, or O1

*a* >*2,000 indicates LOD not reached at the highest VHH concentration used (2 mg/l). DAS, double antibody sandwich; ELISAs, enzyme-linked immunosorbent assays; LOD, limit of detection.*

Manisa. Similarly, all 10 Asia 1 Shamir binding VHHs did not cross-react to strains SAT2 SAU/2/00, A Turkey, A24 Cruzeiro, O1 BFS, or O1 Manisa (**Table 4**).

### Binding to 12S, 75S, and 146S Particles in SDGs

Next, we analyzed the specificity of M332F and M379F, as well as the earlier isolated M170F, for different FMDV particles (12S, 75S, and 146S) present in SDG fractions. As controls, we also analyzed each fraction using VHHs that detect both 12S and 146S particles of Asia 1 Shamir, SAT2 SAU/2/00, or O1 Manisa.

When FMDV Asia 1 Shamir, SAT2 SAU/2/00, or O1 Manisa purified 146S particles were again separated on SDG a peak was seen in fractions 15–18 that corresponds to 146S particles (**Figures 2A–C**). The absence of further peaks indicated virions had not dissociated into smaller particles during virus handling. The peak in fraction 20 of Asia 1 Shamir FMDV (**Figure 2A**) probably represents aggregated 146S particles. When 146S particles purified from SDG were acidified and again subjected to SDG only fractions 2–5 contained FMDV antigen (**Figures 2D–F**), indicating full conversion into 12SA particles. When crude antigen was separated on SDG some antigenic material was detected in fractions 6–14 by ELISA (**Figures 2G–I**). This could represent 75S particles that sediment between 12SA and 146S particles, although 75S particles would be expected to form a sharper peak. The amount of putative 75S particles as compared to 146S particles is especially high for strain Asia 1 Shamir (**Figure 2G**), somewhat lower but still considerable for strain SAT2 SAU/2/00 (**Figure 2H**) and quite low, but clearly detectable for strain O1 Manisa (**Figure 2I**). To visualize the low amount of putative 75S particles of strain O1 Manisa the material in fractions 1–15 was plotted on a scale of 0–0.5 mg/l on the right axis (**Figure 2I**). When crude antigen was acidified prior to SDG fractionation a 12S peak was readily observed for Asia 1 Shamir (**Figure 2J**) and O1 Manisa (**Figure 2L**), but was less evident for SAT2 SAU/2/00 (**Figure 2K**). This could have been due to a combination of reduced 12SA particle reactivity in the M311F ELISA and the use of untreated antigen that predominantly consisted of 146S particles as a standard. Acidified O1 Manisa antigen consisted only of 12S particles, indicating full dissociation of 146S and 75S particles into 12S (**Figure 2L**). However, acidified SAT2 SAU/2/00 antigen also contained a broad peak in the fractions corresponding to 75S particles (**Figure 2K**), whereas Asia 1 Shamir antigen contained both a broad peak corresponding to putative 75S particles as well as a 146S peak, although both peaks were considerably lower as compared to untreated antigen (**Figure 2J**). This indicates that, unlike SDG purified 146S particles, 75S and 146S particles present in Asia 1 Shamir crude antigen, and 75S particles present in SAT2 SAU/2/00 antigen are only partially converted into 12S by acidification. Such a difference in acid liability between SDG purified and crude antigen could be due to matrix effects due to different buffer composition, as was also reported earlier (41).

The M170F DAS ELISA detects only O1 Manisa 146S particles (**Figures 2C,F,I,L**). The M332F DAS ELISA detects both Asia 1 Shamir 146S and the putative 75S particles, although the latter are detected with reduced efficiency as compared to the M98F DAS

constant amount of 12SH particles was added. In the legend the concentration of the constant amount of 12SH particle is indicated. ELISAs were performed in

ELISA (**Figures 2A,D,G,J**). The M379F DAS ELISA detects both SAT2 SAU/2/00 146S and putative 75S particles with an efficiency that is equal to the M311F DAS ELISA, whereas SAT2 SAU/2/00 12SA particles are detected with reduced efficiency as compared to the M311F ELISA (**Figures 2B,E,H**). The binding of M379F to 12SA particles, although with reduced efficiency compared to 146S particles, is consistent with the relatively low 146S specificity observed for M379F.

To elucidate the nature of the putative 75S particles we fractionated infectious FMDV on SDG and determined the presence of infectious virus in each fraction by plaque assay. In addition, we determined the concentration of total FMDV antigen present in each fraction by M3 ELISA after acidification. The M3 ELISA facilitates reliable quantification of 12S, 75S, and 146S particles against a 12S standard. This analysis was only done for strains Asia 1 Shamir and O1 Manisa since strain SAT2 SAU/2/00 is not detected by M3 ELISA (**Figures 3A,B**). Notably, FMDV O1 Manisa infectious virus contained a considerable putative 75S peak (**Figure 3B**). Infectious virus was only found in fractions 16–19, consistent with the notion that this represents 146S particles and that FMDV antigen in fractions 9–11 represent 75S particles.

# DISCUSSION

We aimed to isolate VHHs that specifically bind to FMDV 146S particles of strains SAT2 SAU/2/00 and Asia 1 Shamir. For this purpose, llamas 3049 and 3050 were immunized with SDG purified 146S particles of these strains. We preferentially used a 12S depletion step in phage display selections to enrich for 146S-specific VHHs. In this manner, we isolated three novel SAT2 SAU/2/00 binding VHHs showing reasonable 146S specificity and one novel Asia 1 Shamir binding VHH showing high 146S specificity. However, when using a depletion step there is some risk of detachment of captured 12S particles and carryover of this material into the wells containing 146S

triplicate. Mean and SD are indicated.

#### Table 4 | Binding of biotinylated VHHs to different FMDV strains.

*a 1 mg/l FMDV antigen captured with 1 mg/l M311F.*

*b1 mg/l FMDV antigen captured with 1 mg/l M98F.*

*c 1 mg/l FMDV antigen captured with 1 mg/l M8F.*

*d1 mg/l FMDV antigen captured with 1 mg/l M170F.*

*e* >*1,000, absorbance does not exceed 0.4 at the highest VHH concentration used (1 mg/l).*

*BFS, British Field Strain 1860; FMDV, foot-and-mouth disease virus; mAb, monoclonal antibody.*

particles for panning, resulting in binding to capturing VHH and unwanted enrichment for 12S binding phage clones. Since 12S binding VHHs are generally not serotype specific this can be prevented by using 12S particles from another strain for depletion in combination with a serotype-specific VHH for capturing 146S particles. We currently favor this approach for isolation of 146S-specific VHHs. Selection of VHHs specifically recognizing *Taenia solium* antigen without cross-reacting with other *Taenia* species was earlier described (27). Furthermore, VHHs recognizing a recombinant immunotoxin without binding an undesired deamidated derivative of this immunotoxin were isolated recently (42). Together with our study these are further examples where the high functional diversity of camelid heavy-chain antibody immune libraries in combination with careful phage display selection schemes allows isolation of rare VHHs that can discriminate closely related antigens.

We developed two DAS ELISAs that utilize the same yeastproduced VHH for coating, as well as detection when in a biotinylated form, for quantification of FMDV 146S particles. The ELISA with M379F VHH for quantification of SAT2 SAU/2/00 FMDV required 28-fold higher 12S concentrations than 146S concentrations to reach similar absorbance values. Thus, it shows reasonable specificity for 146S particles. However, it cannot be used for quantification of 146S particles in the presence of high concentrations of 12S particles due to cross-reaction with 12S particles. Since the 12S content of vaccines is generally less than 20% of total FMDV antigen (18), such specificity is sufficient to measure the 146S content of SAT2 vaccines with only an about 1% error. Similar analyses performed with M332F VHH to quantify Asia 1 Shamir exhibited a 51-fold increase in specificity for 146S particles, revealing this VHH could be used for 146S quantification in the presence of high concentrations of 12S particles. Both ELISAs showed an LOD of 2.3–4.6 µg/l 146S. Assuming that the antigen concentration in FMDV vaccines is about 10 mg/l and that extraction of antigens from vaccines for use in VHH-ELISA involves a 10-fold dilution of vaccines (18), such sensitivity is amply sufficient for quantification of FMDV antigens in vaccines as well as stability studies of vaccines, which requires measurement of FMDV antigen concentrations that decrease to even lower levels. However, when needed for other applications the LOD could possibly be further increased by genetic fusion of VHH domains to increase affinity as was earlier demonstrated for an antitumor necrosis factor alpha VHH (43) and many further VHHs. In addition, both ELISAs did not detect strains belonging to other serotypes. The M332F ELISA also does not recognize strain Asia 1 Bahrain (results not shown). Both 146S-specific VHHs bind in a serotype-specific manner, and M332F binding is also strain specific. Such coincidence of strain and serotype specificity and 146S specificity was observed before with the 146S-specific O1 Manisa binding VHH M170F (25) and two 146S-specific A serotype strain binding mAbs (15, 16). This suggests that the 146S-specific antigenic sites that we now detect on strains of O, SAT2, and Asia 1 serotypes are located on the same region of the FMDV capsid. This notion is consistent with the conservation of three of the four neutralizing antigenic sites between O, A, C, and Asia 1 serotype FMDV strains (44). The 146S-specific antigenic site most likely overlaps with the interface of two 12S pentamers.

Figure 2 | Specificity of double antibody sandwich (DAS) enzyme-linked immunosorbent assays (ELISAs) for inactivated foot-and-mouth disease virus (FMDV) antigen fractionated by sucrose density gradient (SDG). FMDV strains Asia 1 Shamir (A,D,G,J), SAT2 SAU/2/00 (B,E,H,K), and O1 Manisa (C,F,I,L) were used. FMDV 146S particles purified by SDG were again layered on SDG without further treatment (A–C) or after prior acidification for conversion into 12SA particles (D–F). Crude FMDV antigen was similarly fractionated on SDG without further treatment (G–I) or after prior acidification (J–L). Twenty fractions of each SDG were analyzed by ELISA using either a VHH that is specific for 146S particles (open diamonds) or that binds both 12S and 146S particles (closed squares). The different 146S-specific VHHs used were M332F (A,D,G,J), M379F (B,E,H,K), and M170F (C,F,I,L). The different 12S- and 146S-recognizing VHHs used were M98F (A,D,G,J), M311F (B,E,H,K), and M8F (C,F,I,L). In panel (I), the low amount of FMDV antigen detected in fractions 1–15 by M8 ELISA is visualized by plotting on a different scale (right axis; gray circles). FMDV antigen concentrations in fractions were calculated from titration series in ELISA against a standard of untreated FMDV antigen with known 146S content. Fraction 1 corresponds to top of gradient.

Double antibody sandwich enzyme-linked immunosorbent assay analysis of SDG fractionated inactivated FMDV antigen of strains SAT2 SAU/2/00, Asia 1 Shamir, and O1 Manisa reveals that some immunoreactive material fractionates between the 12S and 146S peaks (**Figure 2**). This material could represent 75S empty capsids, although the peak is shallower compared to the 12S and 146S peaks, especially for strain Asia 1 Shamir. Consistent with this notion SDG fractionation of infectious Asia 1 Shamir and O1 Manisa FMDV confirms that this material is not infectious (**Figure 3**). These putative 75S particles derived from inactivated antigen are not recognized by M170F, recognized with reduced efficiency by M332F, and recognized well by M379F (**Figure 2**). Acidification of crude inactivated antigen did not fully convert the putative 75S particles of strains SAT2 SAU/2/00 and Asia 1 Shamir and the 146S particles of strain Asia 1 Shamir into 12S particles. We, therefore, recommend that preparation of a 12S standard for use in the 12S-specific M3 ELISA is preferably done by SDG purification of 146S particles and subsequent heating. The preparation of 12S standards by conversion of 146S present in crude antigen into 12S as done

earlier by us (17) is inaccurate as it ignores the 12S particles already present in the sample and the 12S particles derived from 75S particles.

The ELISAs developed here for quantification of 146S particles of strains Asia 1 Shamir and SAT2 SAU/2/00 can be used for quality control of FMD vaccines during and after manufacturing. There is a need for such quality control as many locally produced FMD vaccines show poor quality (45, 46). Several other ELISAs have been described recently for this purpose (7, 12–17). There are only three reports of ELISAs specific for 146S particles (15–17). They were only suitable for O and A serotype strains. Our VHH-based ELISAs that detect 146S particles of SAT2 and Asia 1 serotype strains thus complement the currently available 146S-specific ELISAs. The serotype and strain specificity of the VHHs are advantageous for independent quantification of different FMD strains in multivalent vaccines but has the disadvantage that their use is limited to the FMD strains recognized by these VHHs. The ELISAs developed here can also be used for analysis of FMDV stability in oil-adjuvanted vaccines as described earlier for O1 Manisa FMDV using the M170F ELISA (17, 18). As compared to the recently described thermofluor assay for measuring

#### REFERENCES

FMDV stability (47) our ELISAs have the advantage of increased sensitivity but disadvantage of strain specificity.

#### ETHICS STATEMENT

Llama immunizations were performed after ethical review by Wageningen Bioveterinary Research and in accordance with Dutch national guidelines on animal use.

#### AUTHOR CONTRIBUTIONS

MH and EP performed experiments. MH, JS, PE, BC, and AD conceived and designed experiments and analyzed data. MH, JS, and AD wrote the manuscript. All the authors read and critically reviewed the manuscript.

#### ACKNOWLEDGMENTS

The authors want to thank Froukje van Hemert-Kluitenberg at Wageningen Bioveterinary Research for performing many experiments.

O, A and Asia 1 using a generic RapidAssay device. *Virol J* (2013) 10:125. doi:10.1186/1743-422X-10-125

gap analysis: 3 – vaccines. *Transbound Emerg Dis* (2016) 63(S1):30–41. doi:10.1111/tbed.12518

47. Kotecha A, Zhang F, Juleff N, Jackson T, Perez E, Stuart D, et al. Application of the thermofluor PaSTRy technique for improving foot-and-mouth disease virus vaccine formulation. *J Gen Virol* (2016) 97(7):1557–65. doi:10.1099/ jgv.0.000462

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer, MG, and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.

*Copyright © 2017 Harmsen, Seago, Perez, Charleston, Eblé and Dekker. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

# Nanobody-Based Delivery Systems for Diagnosis and Targeted Tumor Therapy

#### *Yaozhong Hu1,2,3, Changxiao Liu1,3 and Serge Muyldermans2 \**

*1Department of Pharmaceutical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin, China, 2 Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Brussels, Belgium, 3State Key Laboratory of Drug Delivery Technology and Pharmacokinetics, Tianjin Institute of Pharmaceutical Research, Tianjin, China*

The development of innovative targeted therapeutic approaches are expected to surpass the efficacy of current forms of treatments and cause less damage to healthy cells surrounding the tumor site. Since the first development of targeting agents from hybridoma's, monoclonal antibodies (mAbs) have been employed to inhibit tumor growth and proliferation directly or to deliver effector molecules to tumor cells. However, the full potential of such a delivery strategy is hampered by the size of mAbs, which will obstruct the targeted delivery system to access the tumor tissue. By serendipity, a new kind of functional homodimeric antibody format was discovered in camelidae, known as heavy-chain antibodies (HCAbs). The cloning of the variable domain of HCAbs produces an attractive minimal-sized alternative for mAbs, referred to as VHH or nanobodies (Nbs). Apart from their dimensions in the single digit nanometer range, the unique characteristics of Nbs combine a high stability and solubility, low immunogenicity and excellent affinity and specificity against all possible targets including tumor markers. This stimulated the development of tumor-targeted therapeutic strategies. Some autonomous Nbs have been shown to act as antagonistic drugs, but more importantly, the targeting capacity of Nbs has been exploited to create drug delivery systems. Obviously, Nb-based targeted cancer therapy is mainly focused toward extracellular tumor markers, since the membrane barrier prevents antibodies to reach the most promising intracellular tumor markers. Potential strategies, such as lentiviral vectors and bacterial type 3 secretion system, are proposed to deliver target-specific Nbs into tumor cells and to block tumor markers intracellularly. Simultaneously, Nbs have also been employed for *in vivo* molecular imaging to diagnose diseased tissues and to monitor the treatment effects. Here, we review the state of the art and focus on recent developments with Nbs as targeting moieties for drug delivery systems in cancer therapy and cancer imaging.

Keywords: nanobody, targeted cancer therapy, drug delivery, intracellular targeting, type III secretion system, molecular imaging

# INTRODUCTION

To date, the development of effective strategies for cancer therapy remains a huge challenge. The conventional chemotherapy and radiotherapy appear to have a potent effect to kill tumor cells, but they also eliminate healthy cells. Therefore, massive attention went to the development of more effective curable options by targeted cancer therapy (1). Over the years, antibodies have

#### *Edited by:*

*Colin Roger MacKenzie, National Research Council Canada (NRC-CNRC), Canada*

#### *Reviewed by:*

*Oscar R. Burrone, International Centre for Genetic Engineering and Biotechnology, Italy Guy Caljon, University of Antwerp, Belgium*

> *\*Correspondence: Serge Muyldermans<EMAIL_ADDRESS>
#### *Specialty section:*

*This article was submitted to Vaccines and Molecular Therapeutics, a section of the journal Frontiers in Immunology*

*Received: 23 July 2017 Accepted: 17 October 2017 Published: 02 November 2017*

#### *Citation:*

*Hu Y, Liu C and Muyldermans S (2017) Nanobody-Based Delivery Systems for Diagnosis and Targeted Tumor Therapy. Front. Immunol. 8:1442. doi: 10.3389/fimmu.2017.01442*

been employed, first as antivenom therapeutic and later as a valued research and clinical diagnostic tool. The first injection of monoclonal antibodies (mAbs) into patients dates back some 30 years ago (2, 3). But murine mAbs elicit immunogenicity problems in patients. Nevertheless, mAb-based cancer therapy has obtained remarkable successes, emphasizing the attention to evolve therapeutic treatments into a personalized curable proposal. To date, the Food and Drug Administration has approved over 30 mAbs for clinical application. Among these mAbs, seven blockbusters are combatting tumors, including Rituximab (anti-CD20), Trastuzumab (directed to HER2), Bevacizumab (anti-VEGF), Alemtuzumab (anti-CD52), Cetuximab, Panitumumab, and Matuzumab (all targeting EGFR) (4, 5). These mAbs were selected for their capacity to disturb the normal function of their targets in tumor cells. The intact mAbs, containing a fully functional Fc domain, evoke antibody-dependent cell-mediated cytotoxicity (ADCC). In addition, mAbs showed significant potential for tumor diagnosis through molecular imaging (6, 7). The mAbs have also been engineered to carry various toxic loads to produce immunotoxins, antibody drug conjugates (ADCs), nanomedicines, or nanoparticles (NPs) encapsulating cytotoxic agents, that work as drug delivery systems (8). For example, mAbs have been directly conjugated to cytotoxic drugs [e.g., auristatin, maytansine, calicheamicin, or doxorubicin (DOX)] and several of these ADCs reached the clinical trials (9). Nevertheless, the large size of mAb (MW 150,000; dimensions: 14.2 nm × 8.5 nm × 3.8 nm), which might be further increased after conjugation with NPs, constitutes a manifest drawback. An enlarged size will lead to a suboptimal biodistribution and a limited tumor penetration (10). Considerable efforts have been put into the development of smaller antibody formats (11, 12), such as the naturally derived or recombinant antigen-binding fragment (Fab; ~50 kDa), variable fragment (Fv; ~28 kDa), or single-chain variable fragment (scFv; ~30 kDa) (**Figure 1**) (13).

Figure 1 | Schematic representation of antibodies and their derivatives from conventional and heavy chain-only antibodies. Schematic structure of a monoclonal antibody (central top part) and its derivatives: Fab (right, top), Fv, and scFv (left, top part); and of a HCAb (central, lower part), together with its antigen-binding fragment, known as VHH or nanobody (Nb) (right, lower part). Besides the monovalent format, Nbs have been engineered into bivalent monospecific constructs (lower part, right). Two different Nbs can be fused into (i) a biparatopic construct where each Nb recognizes a different epitope on the same molecule or (ii) a bispecific construct targeting two independent molecules (lower, left part). The fusion of the Nb-based construct with a large molecule (star-like shaped) or with an Nb with specificity for albumin are standard strategies to prolong the half-life of the construct in the bloodstream. The molecular weight of each Ab format is also given.

To reinstall the bivalency and concomitant avidity effects, the minibody (an engineered antibody fragment made by fusing the scFv binding domain to human CH3) was introduced as a better candidate (14). Important successes were obtained with these size-reduced Abs of which some reached clinical trials, however, detailed immunogenicity studies are underrepresented and research is still ongoing (15–17).

By serendipity, a new type of antibody naturally devoid of light chains and their first constant domain (CH1) in the heavy chain was discovered in the early 90s in camelids (18). These heavy chain-only antibodies (HCAbs) exhibit a similar affinity to their cognate antigen compared to conventional antibodies, despite only one single variable domain (VHH) is involved in antigen recognition (19). It was demonstrated that the autonomous VHH retains its full antigen-binding potential, and it was considered to be the smallest natural intact antigen-binding fragment (20, 21). With a size of below 15 kDa and dimensions in the nanometer range (~2.5 nm in diameter and ~4 nm in height), the VHH molecule was also named nanobody (Nb, **Figure 1**). The immense efforts in the Nb research field underscoring the remarkable prospects of these molecules formed eventually the basis for the foundation of spin-offs such as Ablynx, Chromotek, Agrosavfe, QvQ, Camel-IDS, Hybrigenics, Confo-Therapeutics, and many other companies offering the technology of generating and selecting Nbs (20, 22). The focus of all these companies ranges from service providers to developing therapeutic Nbs, currently tested in clinical trials (23) with ~9 candidates in advanced stage and more than 15 at the discovery and preclinical stage.

In this report, the beneficial characteristics of Nbs will be reviewed and different Nb conjugation systems for tumor targeting and drug delivery will be discussed, as well as strategies to target intracellular tumor markers. The latter will not only facilitate the exploration of new potential therapeutic approaches but also expand our understanding of particular signaling cascades. Finally, the *in vivo* molecular imaging using Nbs will be summarized.

# CHARACTERISTICS OF Nbs

The ontogeny and emergence of dedicated genes to produce HCAbs in camelids, including VHH domains generated after gene rearrangement events have been comprehensively covered (22, 24–27).

# Nbs Are Easily Selected by Phage Display

The VHH repertoire from peripheral blood cells of the immunized camelid is cloned and phage displayed to retrieve Nbs with highest affinity and specificity for the target (28). The procedure has been adapted to construct large non-immune (naive) or synthetic Nb libraries, from which to select binders. Naive libraries use the VHH repertoire of non-immunized animals. For synthetic libraries, the codons of the antigen-binding loop regions of a robust VHH scaffold are randomized. In all cases, selected Nbs can be produced easily in microorganisms, mammalian cells, or plants (29–32).

# The Smaller Size of Nbs Assists in Reaching and Recognizing Unique Epitopes

The Nb holds great promises (33), mainly due to a unique paratope architecture, monomeric, and robust behavior (34–36) and favorable solubility (21). Due to their small size, a rapid extravasation of intravenously administered Nbs and diffusion into tissues is obtained to deliver interesting reagents to the target. Many Nbs possess a long complementarity determining region 3 (CDR3), forming a finger-like structure that penetrates into cavities on the antigen surface (36). For those VHHs that do not have a long CDR3, the prolate shape of the Nb creates a convex paratope that interacts deeply into antigen concave surfaces. Consequently, Nbs are directed against unique antigen epitopes that are low or not antigenic for classical antibodies (37–39).

# The Smaller Size of Nbs Is Beneficial for Engineering

The small size and monomeric single-domain nature forms the basis for the flexible engineering of Nbs. Engineering of Nbs facilitates the conjugation of additional proteins, reporter molecules, or drugs. Most methods, employed for the chemical conjugation, depend on presence of lysines. However, the occurrence of multiple lysines (on average 3–4 per Nb) and their random conjugation creates a mixture of conjugates whereby a fraction might have lost its antigen-binding capacity when lysines within the antigen-binding region reacted. The introduction of an extra cysteine at a distant location from the paratope and preferably at the C-terminal end of the domain remediates these issues (40, 41). Alternatively, the C-terminal end of the Nbs have been equipped with short peptide tags, such as the Sortag that undergoes the Sortase A-mediated protein ligation reaction to attach any probe (42, 43).

# Inconveniences of Nbs and How to Remediate

The minimal size of an Nb is often considered as an advantage; however, it might also be a handicap. For example, all molecules with a size below 50,000 Da are rapidly cleared from the bloodstream through kidney glomerular filtration. Although a fast blood clearance of Nbs is certainly beneficial for non-invasive *in vivo* imaging (33, 44, 45), for optimal tumor therapy, a longer blood residence time would permit lower injected doses, longer time intervals between two consecutive administrations and still yield a higher load of Nb-based drug at the target. To increase the blood residence time, Tijink et al. (46) generated a tandem fusion of a bivalent Nb against EGFR with an Nb cross-reacting with mouse and human serum albumin (α-EGFR-αEGFR-αAlb, **Figure 1**) (47). Since human serum albumin has a half-life of around 19 days (48), the half-life of the bispecific trivalent α-EGFR-αEGFR-αAlb was prolonged to around 2–3 days in mice. Furthermore, compared to the monovalent Nb, the longer circulation of the trivalent Nb in blood increased its tumor uptake as well. Similar levels of tumor loading were noted with the trivalent Nb and Cetuximab, while a faster and deeper tumor penetration was obtained with the former (47).

For conventional antibodies, it is well established that upon antigen binding, the ADCC and complement-dependent cytotoxicity are triggered by the Fc region (47, 49, 50). These two mechanisms are known to be important in the process of tumor eradication, as they both contribute to activation of cell lysis, and hence apoptosis cascades (47). For this reason, it was proposed to extent Nbs with an Fc region, although the advantages of a small size (extravasation, tumor penetration) will be lost (50).

# High Stability of Nbs Admit Their Application under Stringent Conditions

Nanobodies seem to be extraordinarily resistant when exposed to various stress conditions. The Nbs have a long shelf-life and tolerate storage for several months at 4°C, and even longer at −20°C, while maintaining full antigen-binding capacity. Incubating Nbs at 37°C for several weeks seems to be well tolerated as well (28). Although some reports indicate that Nbs might resist temperatures above 90°C (35), this will be more an exception than the rule and Nbs are certainly not always refolding quantitatively upon heat denaturation. Also exposure to elevated pressure does not seem to harm the Nbs. Altogether, most Nbs exhibit a high stability against elevated temperature, high pressure, or chemical denaturants as demonstrated by thermo fluorescence or circular dichroism measurements (34, 51, 52).

#### Low Immunogenicity of Nbs

The detailed Nb sequence information (21) revealed that VHHs share a high degree of sequence identity with human VHs (of family 3). This feature is considered to contribute to the low immunogenicity, thus allowing prolonged and repeated administrations of Nbs in patients (53). Furthermore, strategies have been developed to humanize Nbs to minimize the possible immune reaction of patients (54, 55). Data from Phase I clinical trials performed by Ablynx (Belgium) also support the notion that Nbs are endowed with low immunogenicity (56, 57).

As it is difficult for Nbs, as well as for other proteins, to migrate across cell membranes, most current investigations had a focus on extracellular targets, such as receptor ligands or transmembrane proteins. However, possible applications of Nbs directed against intracellular tumor markers have been proposed. For example, scFv or Nbs might be transcribed and translated inside the tumor cell. Such intracellular antibodies (known as intrabodies), when folded properly might immediately target the intracellular antigen protein. Groot et al. (58, 59) produced intrabodies against HIF-1α and evaluated its targeting efficacy. Obviously, explorative experiments with Nb-based tools either expressed intracellularly (intrabodies) or introduced *via* viral vectors are underway (60–62).

# Nb-CONJUGATED PARTICLES FOR THERAPY AND DIAGNOSIS

From the very beginning, the potential of Nbs as cancer therapeutic agent has been evaluated, whereby the Nb targets the ectodomain or cell surface exposed loops of receptors or biomarkers, aiming at the inactivation of the transcriptional pathways or signaling cascades. In the following section, the therapeutic agents and the different formats of drug delivery systems based on Nbs will be described.

#### Nbs with an Intrinsic Therapeutic Activity

To date, the most investigated extracellular targets for Nbs include EGFR1 or EGFR2 (HER1 and HER2, respectively), vascular endothelial growth factor receptor-2 (VEGFR2), c-Met and CXCR7, or hepatocyte growth factor (HCG), which all play a crucial role in making a link with the signaling cascades. Binding of Nbs to these tumor markers can potentially block the signaling pathways to halt the growth and proliferation of tumor cells. As such, Nbs against EGFR and c-Met have been evaluated (47, 63). Both Nbs showed potent antagonistic effects *in vitro*, as well as an inhibition of the tumor growth *in vivo* in case of a trivalent biparatopic anti-EGFR Nb 7D12-9G8-Alb (47).

Furthermore, Nbs have been developed to combat different infections and diseases, such as thrombotic thrombocytopenic purpura (64, 65), respiratory syncytial virus (66), and rheumatoid arthritis (67–69). These Nbs reached various stages of preclinical or clinical testings. Some particular Nbs are being developed as allosteric inhibitors that are able to modulate the enzymatic activity of their target protein, such as carbonic anhydrase (CAIX) (70), which plays a significant role for hypoxic tumor cells so that the enzymatic CAIX neutralization with Nbs could reduce malignancy and survival of tumor cells. All these results will expand the research focus and stimulate applications of Nbs for cancer diagnosis and therapy. Theoretically, potent and intrinsic effective Nbs that can completely inhibit tumor growth and lead to cell death should be employed, rather than Nbs that are just inhibiting tumor cell proliferation.

#### Nb-Toxin Conjugation

However, most Nbs do not exhibit an inherent therapeutic activity, but need to be conjugated with a toxic load or any other effector function. In these applications, the conjugated Nbs are employed for drug delivery, irrespectively whether the conjugate is a single effector domain or a nanocargo containing antitumor drugs (71–73).

The conjugation of Nbs with an enzyme or toxin molecule increases the Nb circulation time in blood due to its enlarged size. Therefore, the constructs become more effective to transfer their load to tumors or diseased tissues. Two strategies can be applied for conjugation, either by chemical conjugation or by gene fusion of the Nb and a toxic protein and cloning in an expression vector (55, 74). For chemical conjugation, the conjugation of the effector moiety to the Nb—mostly to lysine residues—might be heterogeneous as several lysines are present in the Nb and if a Lys in the CDR reacted then the reactant might shield the CDRs from access to antigen, thus resulting in a decrease or loss of antigen recognition by Nbs. In another strategy, a single cysteine—inserted at the C-terminal end of the Nb—allows for a unique site-directed conjugation of a toxic load distant to the paratope so that the disturbance of antigen binding will be minimal (41). Recently, the Sortase A-mediated modification of the C-terminal end of Nbs has been explored to attach chelators and nuclides (42, 43). Anyway, the antigen-binding properties must be confirmed after conjugation. Hence, it is necessary to ensure the accumulation and retention of specific Nbs at the tumor tissue, rather than normal healthy tissue.

#### Anti-Carcinoembryonic Nbs with *Enterobacter cloacae* β-Lactamase

In 2004, Cortez-Retamozo et al. (75) introduced the genetic conjugation of anti-carcinoembryonic Nbs to *E. cloacae* β-lactamase, which is an enzyme with excellent catalytic properties for converting a non- or low-toxic prodrug into a potent cytotoxic agent. *In vivo* biodistribution and therapeutic activity of the conjugation was evaluated in nude mice bearing LS174T xenografts. Effective accumulation of the Nb conjugate at the tumor xenograft was noticed and no, or very low, accumulation in kidneys. Regression of the grafted tumor was observed in mice and even complete remission was obtained in this antibodydirected enzyme prodrug therapy model. Although the bacterial origin of β-lactamase will make the immuno-enzyme highly immunogenic and therefore less practical, the study provides the proof of concept to generate highly cytotoxic compounds in the vicinity of the tumor and shows its potential as a promising approach for cancer therapy.

#### Anti-EGFR Nbs with Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL)

In 2012, a unique conjugation based on anti-EGFR Nbs was introduced for malignant glioblastoma multiforme (GBM). As demonstrated, both neural stem cells (NSCs) and mesenchymal stem cells can migrate toward brain tumors. In this study, bivalent and bispecific Nbs against EGFR were conjugated to TRAIL and packaged into lentivirus (LV) virions to transduce NCS. The secretion of specific Nbs and Nb-TRAIL from engineered NSCs was confirmed and NSCs retained the ability to differentiate. Furthermore, these Nb constructs secreted from NSCs were designed to target GBM tumor tissues, which show an enhanced EGFR expression (76). The therapeutic effect of anti-EGFR Nbs and their variants was evaluated both *in vitro* and in xenografted models. NSC released anti-EGFR Nbs can inhibit EGFR signaling dramatically *in vitro* and reduced the tumor growth in mice bearing GBM. By taking advantage of tropism of the NSCs that could provide on-site delivery of therapeutic Nbs, significant inhibitory effects on GBM were noticed. Direct comparison of the inhibition activity between the bivalent anti-EGFR Nb and the Nb-TRAIL conjugate further revealed that the combined therapeutic approaches were more potent (76). The NSC-delivered Nbs inhibit the proliferation and migration, whereas their conjugation with cytotoxic molecules enhances the therapeutic efficacy, significantly.

#### Anti-VEGFR2 Nbs with Recombinant *Pseudomonas* Exotoxin A

The conjugation of anti-VEGFR2 Nbs with recombinant *Pseudomonas* exotoxin A (PE38) was proposed to inhibit growth of tumors, highly expressing VEGFR2 (77). PE38 was designed to enter the cell in an endocytic vesicle and to bind to the ADPribosylating elongation factor II to kill subsequently tumor cells. As demonstrated by an *in vitro* proliferation assay, this conjugation system could efficiently recognize VEGFR2 expressed on the surface of 293KDR cells and inhibit their proliferation *in vitro* (77). Thus, this anti-VEGFR2–PE38 conjugate act as a potent immuno-cytotoxic effector; however, data of an *in vivo* evaluation have not been reported.

In conclusion, the conjugation of Nbs and toxins combines the advantages of the tumor-specific targeting Nbs and the tumor killing toxins within one molecule. In this case, an effective cell penetration was also achieved, which will help to enhance the therapeutic efficacy *via* this particular effector domain. Several Nb conjugates are in the pipeline for research purposes or clinical evaluation.

# Nbs As Targeting Modules for Drug Delivery Systems

Drug delivery systems also involve nano-sized drug carriers or NPs with a diameter below 200 nm. The design of nanoscale vehicles for drug delivery has been one of the most exiting strategies in medicine and pharmaceutical technology. Different drug delivery systems based on NPs have been developed, including inorganic, magnetic, and polymeric NPs (**Figure 2**) (78). These systems can protect drugs against oxido-reduction and enzymatic reactions, increase their bioavailability by reducing the effective dose and they will diminish the potential immunogenicity of the drug. The packaged, administered toxic compounds can avoid damage and negative side effects to normal tissues, solubilize hydrophobic drugs in lipidic bilayers (e.g., liposomes) or hydrophobic cores (micelles). The NPs allow administering larger amounts of drugs in one single dose, and the slow but prolonged drug release will result in a reduced frequency of the administration (79).

The conjugation of drug cargo's to targeting moieties, especially those against receptors that mediate cellular internalization, was introduced to facilitate the transport of drugs or functional agents in target cells and tissues (**Figure 3**) (8, 80, 81). Poly-ethylene glycol (PEG) molecules or surface-charge-shielded NPs have been conjugated on the surface of NPs to extend the circulation in the bloodstream leading to more significant accumulation at tumor sites and reduced liver uptake (82–84). The PEG-ylation of NPs also provides chemical reactive moieties to attach biofunctional molecules for specific cell or organ targeting (85, 86). The damaged vasculature around tumor cells will encourage the enhanced permeability and retention (EPR) effect and enhance the accumulation of NPs in the tumor vasculature (**Figure 3**) (87). After extravasation of NPs into the tumor microenvironment, the interaction between NPs and tumor cells can be enhanced by targeting moieties. Nbs have been employed to serve as targeting molecules, and the delivery systems based on Nbs will be reviewed in the following sections.

#### Nbs Conjugated to Liposomes

Since the discovery of liposome by Bangham in the 1960s, liposomes have been considered as a valuable drug-carrier system, with a morphology and characteristics very similar to those of cellular membranes (88, 89). Liposomes can be constructed in a broad size range from 100 to 400 nm, which might be useful

(10–1,000 nm). The blue parts of the polymer NP represent the solid hydrophobic polymer matrix with optionally an aqueous core. The nanosphere is composed of a solid polymer matrix, able to encapsulate hydrophobic drugs. The nanocapsule is composed of a spherical polymeric matrix with an aqueous or oily core (light blue part in lower right panel). The poly-ethylene glycol-ylation prolongs the circulation of NPs in the bloodstream; antigen-specific Nbs are conjugated to the surface of NPs for targeting purposes.

in view of the importance of size in tumor targeting. Significant progress was made over the years and several candidates are at the stage of preclinical evaluation or clinical application (90). The external chemical differences of liposomes can facilitate the construction of targeted systems with Nbs or any other protein and will ultimately result in the accumulation of encapsulated liposomes to tumor tissues (89, 91). In conclusion, Nb–liposome systems play a suitable role for a combined therapeutic strategy and have the potential to enhance the antitumor effect.

#### *Anti-EGFR Nbs Conjugated to Empty Liposomes*

Oliveira et al. (72) introduced a multivalent Nb–liposome platform without incorporated drugs to target tumors expressing epidermal growth factor receptor (EGFR). Nb EGa1 directed against EGFR was conjugated to the surface of liposomes *via* PEG-ylation. It was shown that the EGa1 C-end modification did not compromise the antigen recognition capacity or change the effective epitope targeting. However, a lower affinity was observed, probably caused by steric hindrance from the attached liposome particles. Nevertheless, a remarkable reduction of more than 90% of cell surface EGFR was observed. Increase of EGa1 on liposomes from 0.4 to 0.8 nmol can even lower the EGFR levels and further enhances the inhibition of tumor growth. The total EGFR protein level in tumors was checked at the end of the study and revealed a remarkable drop in the treated group. Supposedly, the combination of EGa1 to liposomes will retard the clearance from circulation, and the presence of EGa1 ensures specific target interactions, resulting in a measurable increase of accumulated particles in tumor tissues. The poor inhibition of tumor growth *in vivo* is attributed to "blank" liposomes without any drugs incorporated (72).

#### *Anti-EGFR Nbs Conjugated to Liposome with Anti-Insulin-Like Growth Factor 1 Receptor (IGF-1R) Kinase Inhibitor (AG538) Encapsulated*

In 2012, an improved version of the EGa1-liposome system was introduced by loading the liposomes with an anti-IGF-1R kinase inhibitor (AG538) (**Figure 2**) (73). IGF-1R plays a crucial role during the progression of particular tumors. It was demonstrated

that EGFR inhibition will raise the IGF-1R levels in tumors. Hence, a combined therapeutic strategy against EGFR and IGF-1R was chosen as starting point for this targeted system.  Compared to empty EGa1 liposome, a stronger growth inhibition is observed from EGa1- AG538-liposome on 14C cells and empty EGa1-liposomes could decrease the total number of cells by about 40%. This percentage can be increased to nearly 80 by exposure to EGa1-liposome (2 mM TL) with a high dose of free AG538 (80–160 µM) (73). However, the EGa1-AG538-liposomes seemed to be a more robust approach as it reached almost the same inhibition efficacy with just 0.25 mM TL, corresponding to 20 µM AG538. The A431 cell line responded similarly as the 14C cell line. These results strongly support the effective delivery of AG538 by this Nb–liposome system. Next, the *in vivo* antitumor efficacy of EGa1-AG538-liposomes was evaluated in a xenograft model of 14C and MB-468. A strong inhibitory response was observed in the group treated with EGa1-AG538-liposomes, confirming the result of the *in vitro* tests (92). Overall, this kind of platform will encourage the study on different combinations of antitumor drug encapsulated systems and specific antibodies.

#### *Anti-EGFR Nbs Conjugated to Extracellular Vesicles (EVs)*

Recently, Kooijmans et al. (93) introduced a potent delivery system based on EVs. In this strategy, anti-EGFR Nbs were expressed on the surface of EV fused by glycosylphosphatidylinositol (GPI) anchor signal peptides derived from the decay-accelerating factor. It is demonstrated that the GPI-linked Nbs were successfully displayed and accumulated strongly on the surface of EV. As a result, the targeting efficacy of EVs was dramatically improved *via* their anti-EGFR Nbs, under static conditions. The cancer cell recognition and association was also demonstrated under flow conditions, highlighting the potential of the GPI-anchoring approach and GPI-anchoring drug delivery systems.

#### *Nbs Conjugated to Microbubbles*

The development of Nb-microbubble (μB) conjugates as a novel molecular tracer has been reported (94). The biotinylated antieGFP (cAbGFP4) and anti-VCAM-1 (cAbVCAM1-5) Nbs were site specifically coupled to lipid μBs containing streptavidin. The specific binding of eGFP to μB-cAbGFP4 was confirmed by fluorescent microscopy, as well as the ability of μB-cAbVCAM1-5 to bind VCAM-1 in fast flow. The application of VCAM-1 conjugated μBs as novel molecular ultrasound contrast agent was demonstrated both *in vitro* and *in vivo* (94). It was further proposed that the encapsulation of specific agents in μBs might be used to control a slow release at the tumor site.

#### Nb Conjugation to Micelles

A micelle is an aggregate of amphiphilic block molecules dispersed in aqueous solution with the hydrophilic head regions in contact with the surrounding solvent, sequestering the hydrophobic single-tail regions in the micelle center, ranging from 10 to 100 nm based on the composition and concentration. Micelles are commonly used as platform to deliver hydrophobic drugs, which are difficult to carry through the bloodstream. Micelles remain stable upon dilution and assist in the solubility of these hydrophobic drugs. Their nanoscale dimensions permit an efficient accumulation in tumor tissues *via* the EPR effect (95). For an optimal EPR effect, a long circulation time of drugs or particles is necessary, which might be obtained by coating the small drugs with PEG or to attach drugs to the surface of carriers. Thus, according to the last strategy, the coupling of a targeting moiety (e.g., antibody, scFv, or Nb) to the surface of micelles will increase the accumulation of carriers in target tissue and promote uptake of the specific drugs. Coupling of specific Nbs to the micelle surface will generate a targeted drug delivery system promoting the internalization of carried drugs (71).

#### *Anti-EGFR Nbs Conjugated to Micelles*

In 2011, a new kind of Nb-micelle drug delivery system was introduced (71). The actively targeted polymeric micelle comprised 80% mPEG-b-p (HPMAm-Lacn) and 20% PDP–PEG-b-p (HPMAm-Lacn) block copolymers. This micelle was decorated with the EGFR antagonist Nb, EGa1, captured to the micellar surface through a disulfide linker (96). The A431 and 14C cell lines and a low endogenous EGFR expressing NIH 3T3 cell line were selected to assess the binding characteristics and uptake of Nb-conjugated micelles. The particles could target effectively to EGFR-positive cells, and no binding was observed to EGFRnegative cells. The results demonstrated that the coupling of EGa1 to the surface of micelles enhanced the recognition of, and uptake by, EGFR-positive target cells.

#### *Anti-EGFR Nbs Conjugated to Micelles with Encapsulated DOX*

In a follow-up study, an upgraded version (EGa1-DOX-micelle) of this delivery system with encapsulated DOX was proposed (97). Polymeric micelles without Nbs were developed with covalently entrapped DOX through a pH-sensitive linker (**Figure 2**) (98). Such DOX-micelles showed an increased cytostatic activity against ovarian carcinoma and B16F10 melanoma cells compared to pure DOX. Likewise, in comparison to the free drug, the DOX-micelles exhibited an increased therapeutic efficacy in B16F10 melanoma bearing nude mice. More importantly, mice treated with DOXmicelles showed a prolonged survival compared to the group that received free DOX. The inhibition efficacy to tumor growth dramatically improved by coupling anti-EGFR EGa1 Nbs to the surface of these micelles. EGa1-DOX-micelles were more toxic than the untargeted polymeric micelles for cell lines and xenografted tumors. Early 2010, another biological cargo system was introduced, with EGFR-positive Nbs (EGa1) conjugated to PEG-ylated micelles (98). The subsequent investigation with this delivery system highlighted the importance of the post-insertion strategy, which should target microvesicles to cell lines of interest.

#### Nbs Conjugated to Polymer NPs

Polymer NPs have attracted the interest and have been exploited in different fields over the past decade. This trend originates from their versatile capacities to meet the demands in various applications and marketing requirements. Several types of NPs, including dendrimers, nanospheres, and nanocapsules, have been exploited for enhanced cancer therapy (99–101).

#### *Dendrimers*

Dendrimers are monodisperse, branched structures, with a size ranging from 3 to 20 nm (102). The surface of dendrimers can be functionalized by coupling targeting moieties. Functional agents can be encapsulated in the dendrimer's multifunctional core to facilitate drug delivery. Drug molecules, such as paclitaxel, can also be attached to the exterior of the dendrimer for special purposes. Recently, DOX was conjugated to carboxyl-terminated poly(amidoamine) dendrimers (PAMAM) and assessed against lung metastases for improved pharmacokinetics and biodistribution (103). A dramatic increase in efficacy of DOX treatment was observed, upon pulmonary administration, in a lung metastasis mouse model bearing the B16-F10 melanoma. A decreased tumor weight and increased survival rates of the animals (C57BL/6) were noted. Compared to free DOX, this conjugate was demonstrated to further increase the therapeutic efficacy as indicated by the fewer number of nodules observed in lungs. The results demonstrated that pulmonary administration of DOX conjugated to PAMAM dendrimer is a useful strategy to enhance the therapeutic efficacy and decrease systemic toxicity of DOX. The conjugation of specific Nbs to the surface of dendrimer is expected to further facilitate tumor targeting (**Figure 2**).

#### *Nanospheres*

A nanosphere is a delivery vehicle composed of a spherical polymeric matrix ranging from 1 to 100 nm, where the drug can be encapsulated inside the aqueous or oily core from where it is released slowly during the circulation in the bloodstream. The surface of the nanosphere can also be PEG-ylated to increase the half-life and to facilitate the binding of Nbs for targeted therapy (**Figure 2**) (104, 105).

#### *Nanocapsules*

Nanocapsules are nanoscale shells of 10–1,000 nm with drugs encapsulated inside their core and separated from the environment by a polymeric membrane (106, 107). Nanocapsules are used in a myriad of fields, including medical applications for drug delivery, food enhancement, nutraceuticals, and self-healing materials. The most attractive current application is the targeted delivery of agents to particular tissues. Monomer, bivalent, or even trivalent Nbs can be attached to the surface of this delivery system to obtain a specific targeting (**Figure 2**) (108). Although there is no publication yet where such multimeric Nbs are conjugated on nanocapsules, it remains a very attractive material of high potential for future research.

#### *Nbs Conjugated to Albumin NPs*

Another type of a highly potent delivery module comprises albumin NPs. Albumin is the most abundant plasma protein in the bloodstream, participating in several important regulations. Moreover, albumin is biocompatible and bio-safe, and albumin NPs as a drug delivery system was proposed by Muller et al. (109). The work inspired several researchers to develop such albumin NPs into a safe drug delivery system (110, 111). In one of these publications, a novel albumin nanoparticle drug carrier system (NANAPs) was loaded with the multi-kinase inhibitor 17864.  PEG-ylated NPs without EGa1 on their surface showed lower targeting and internalization efficacy compared to PEG-NP-EGa1. After binding to the cancer cells, the clathrin-mediated endocytosis leads eventually to the lysosomal degradation of the NPs releasing the multi-kinase inhibitor 17864 inside cells and provoking a notable anti-proliferative effect on tumor cells. The importance of a targeted effect from the EGa1 Nb on NANAPs was demonstrated *in vitro*, whereas the cell proliferation inhibition was not observed by treating cancer cells with non-targeted NPs encapsulated with 17864.

### Targeted Therapy with Nb-Decorated Viral Vectors

Gene therapy with the assistance of viral vectors has become a very important technology in basic life sciences and applied medicine. To date, viruses, such as adenovirus, adeno-associated virus (AAV), and herpes simplex virus, are favored for this task (112, 113). LV and AAV are well-established vectors (114, 115), and these viruses can be employed to transfer genes, including those encoding Nbs into the host cells to produce intracellular Nbs (i.e., intrabodies). The LV is the most studied model for gene delivery and immunotherapy. Unfortunately, it remains challenging to deliver the genes of interest within the lentiviral particles after an *in vivo* administration to relevant target cells such as tumor cells or antigen-presenting cells (APCs) (116). The administration of wild-type AAV and LV vectors usually results in virion accumulation in liver and spleen.

Breckpot et al. (116) developed an interesting approach, whereby a modified LV vector was assembled with a bindingdefective, but fusion competent, envelope glycoprotein derived from VSV-G decorated with Nb DC2.1 (**Figure 4A**). This Nb targets dendritic cells (DCs) that together with macrophages are imperative for activation of antigen-specific T cells. Such APCs are often targeted in immunotherapeutic strategies for the treatment of cancer and infectious diseases (117, 118). The modified LVs contain genes for tumor-associated antigens (TAAs) (119). Upon transduction of the APC, the TAA will be processed and presented to oncolytic effector cells that will subsequently eradicate cancer cells, both primary and metastasized.

The transducing specificity of Nb DC2.1 displaying LVs was evaluated on both, mouse and human APCs. It was demonstrated that DC2.1 Nb-displaying LVs showed an Nb-dependent and APCspecific transduction on murine cell lines and *in vitro* generated DCs (116). *In vivo* transduction with the DC2.1 Nb-displaying LVs was demonstrated after intranodal injection by bioluminescence imaging, and the transduction results were confirmed by nested PCR (116). Phenotypic characterization of the *in situ* transduced lymph node (LN)-derived DCs demonstrated that the entry of DC2.1 Nb-displaying LVs showed a tendency to accumulate at macrophages, conventional DCs (cDCs), and plasmacytoid DCs. Importantly, myeloid DCs are supposed to mediate immune responses upon LV transduction, and the transduction was enhanced with Nb-displaying LVs.

The use of Nbs on LVs (such as R3\_13 LVs) can target specifically to human LN-resident myeloid DCs (120). Later on, an extended study was performed to check the targeting of LVs to cDCs, which are assumed to play a central role in the induction of a TH1-mediated antitumor immune response (117). In this study, the *in vivo* transduction profile and immune stimulatory potential of broad tropism LVs was compared with non-targeting BCII10 Nbs and APC-targeting Nb DC1.8 or DC2.1 displaying LVs. It was demonstrated that the DC1.8-LVs can exclusively transduce cDCs, while also macrophages and pDCs can be transduced with DC2.1-LVs. The transduction of these different cell types opens the potential to stimulate both, the antigen-specific CD8+ and CD4+ T cells (121). Intranodal immunization with ovalbumin encoding LVs induces the proliferation of antigen-specific CD4+ T cells. It has been demonstrated that all targeted LVs were able to stimulate cytotoxic T lymphocytes, and the APC-targeted LVs were as potent in therapy as broad tropism LVs and as such meet their expectations as safer and efficacious LV-based vaccines (117).

# INTRACELLULAR TARGETING OF TUMORS WITH Nbs

To date, the majority of the research focuses on extracellular tumor markers, including cytokines, signaling receptors, and extracellular domains of cell surface proteins (122). However, most of the signaling regulation linked to the growth and proliferation of tumor cells occurs intracellularly, and thus intracellular markers should be promising therapeutic targets as well (123). The barrier of the lipid membrane halts the transfer of Nbs to the intracellular compartment, but many researchers try to develop potent delivery strategies to transport intact or at least functional Nbs into cells, and several strategies have been explored for their intracellular transportation. The LVs can be engineered to target to tumor cells *via* decoration with tumor surface marker-specific Nbs, while encoded Nbs within the LVs might produce intracellular Nbs that could associate with intracellular tumor markers to inhibit growth and proliferation (**Figure 4A**).

Alternatively, bacteria have developed sophisticated systems, such as the type III secretion system (T3SS), to translocate

Figure 4 | Strategies for intracellular tumor targeting. (A) Gene delivery of specific nanoboides (Nbs) against intracellular tumor targets based on lentiviral vectors. The lentivirus displays Nbs directed toward antigen-presenting cells (APCs) such as dendritic cells (DCs). The initial attachment of the virus to the cellular receptor on the surface of APC allows internalization of the viral contents. The viral nucleoprotein core containing the genomic RNA is released into the cytoplasm after entry. Reverse transcription and synthesis of full-length chimeric viral DNA produces an integration-competent nucleoprotein complex that mediates integration of viral DNA into the host cell genome. Integrated chimeric viral DNA serves as a transcription template for the synthesis of tumor-associated antigens that after proteolysis will be presented on MHC to stimulate oncolytic T cells inducing tumor cell death (red arrows). In an alternative approach, the LVs could contain genes encoding Nbs against intracellular tumor markers. The targeting of such LVs to tumor cells could then produce intrabodies (Nbs) that will associate with the intracellular tumor marker to inhibit tumor growth and proliferation (black arrows). (B) Transport of specific Nbs into tumor cells *via* bacterial type III secretion system (T3S) for intracellular tumor targeting. Gram-negative bacteria use a specialized secretion apparatus known as the T3S system to inject proteins directly into the eukaryotic cells, such as *Y. enterocolitica* T3S, *S. typhimurium* T3S, and *E. coli* T3S. Bacterial proteins that are delivered by a T3S are injected through the eukaryotic cell membrane *via* a proteinaceous transmembrane channel known as the type III translocon. The schematic components of the T3S nanosyringe are shown and Gram-negative bacteria were engineered to produce antigen-specific Nbs against intracellular tumor markers. The attachment of T3S and tumor cells will facilitate the export of Nb proteins inside tumor cells, such as HeLa cells. These internalized Nbs will block and inhibit the signaling cascades or processes of tumor metastasis, leading to targeted cancer therapy.

exogenous proteins into eukaryotic host cells (124). This needlelike system serves as a sensory probe to detect eukaryotic organisms and to inject bacterial proteins directly in the host cell to prepare these cells and to assist the pathogen to survive and to escape the host immune system.

# *E. coli* Bacteria T3SS

An old observation, that bacteria are homing at the hypoxic environment of solid cancers, inspired researchers to harness bacteria with Nbs that would be translocated *via* the T3SS inside the cancer cells to cause damage (**Figure 4B**). In a first step to explore this strategy, it was demonstrated that the non-invasive *E. coli* bacteria carrying a T3SS could translocate successfully the Nbs into mammalian cells (125). Several constructs of Nb fused to EspF20 T3 signal (T3s) were cloned into non-pathogenic *E. coli* cells and shown by western blot to get inside HeLa cells. The immunoprecipitation further demonstrated the intact antigen-binding activity of the injected Nbs (125). Higher levels of injected T3s-Nbs remained inside the HeLa host when they express the cognate antigen. Thus, the non-invasive bacterial T3SS is a promising strategy to deliver Nbs into mammalian cells to target intracellular cell components and signaling pathway molecules.

# The *Y. enterocolitica* T3SS

Likewise, Ittig et al. (126) reported a protein delivery system based on the type III secretion of *Y. enterocolitica*. In their research, YopE, a *Y. enterocolitica* effector with Rho GTPase-activating protein activity, was utilized for the expression of protein–YopE fusions in *Y. enterocolitica* and translocation of proteins into mammalian cells. The secretion of multiple proteins was evaluated, including cellcycle proteins (i.e., Mad2, CDK1, CDKN2A/INK4A, CDKN2B/ INK4B, and CDKN2C/INK4C), apoptosis-related proteins(Bad, FADD, caspase-3 [CASP] p17 and p12, zebrafish BID, and zebrafish t-BID), and signaling proteins (TRAF6, TIFA, and the GPCR Gα subunit GNA12). Furthermore, an anti-GFP Nb (VHH GFP4) and a VHH GFP4 fusion construct for targeted protein degradation was also tested (126). The delivery of these functional fusion Nb proteins of different size and structure was demonstrated after infection of HeLa cells. The Nb against GFP was employed to assess the translocation of functional Nbs from *Y. enterocolitica* into HeLa cells. Interestingly, the translocation of YopE1-Nb fusions occurs first in the cytoplasm, whereas translocated YopE1-Nb fusions against GFP were exclusively detected in the nucleus of cells expressing histone 2B-GFP, illustrating the mobility of the fusion inside the cell and its organelles and the interaction between the Nbs and their target antigen (126). The results indicated that YopE fusion are effectively secreted and delivered into eukaryotic cells and that the *Y. enterocolitica*-based delivery is fast, homogenous, and controllable. However, the unspecific targeting of bacteria to normal and tumor cells indicates that further engineering of the bacteria will be required to obtain an exclusive tumor-specific Nb translocation. This might be achieved by anchoring tumor-specific Nbs to the surface of the engineered bacteria.

# *Clostridium*-Directed Antibody Therapy

A non-toxic bacteriolytic strain of *Clostridium* has been engineered for the production of tumor therapeutic proteins (127). This obligatory anaerobic *Clostridium* specifically colonizes hypoxic and necrotic regions present in solid tumors but normally absent in other parts of the body. The efficacy of *Clostridium*directed tumor therapy (CDAT) has been demonstrated in experimental models as a vehicle for tumor-specific delivery of prodrug converting enzymes (128, 129) and to enhance radiotherapy and chemotherapy (130–132).

Nanobodies targeting HIF-1 were cloned in *Clostridium novyi-NT* and *C. sporogenes* strains. The expression of HIF-specific intrabodies in an oncolytic *C. novyi* strain opened the path for developing a *Clostridium*-directed antibody therapy (133).

# MOLECULAR IMAGING WITH Nbs FOR EARLY STAGE DIAGNOSIS OF TUMORS

Early diagnosis is essential to increase chances on a successful treatment of tumors. Recently, Nbs supported by their small size, high stability, and high target specificity and affinity have been engineered into Nb-detective constructs for non-invasive *in vivo* molecular imaging (33). These molecules reach rapidly a maximal contrast between signal in the pathological tissues and that in healthy tissues, which is crucial for optimal *in vivo* molecular imaging. The short half-life of Nbs in the bloodstream due to rapid clearance of excess of non-targeting Nbs *via* kidney and bladder guarantees a high tumor to background ratio at early time points after administering the Nb probe. To date, several imaging techniques have been developed and applied for clinical application, such as positron emission tomography (PET), single photon emission computed tomography (SPECT), computed tomography (CT), magnetic resonance imaging (MRI), optical, ultrasound, and photo-acoustic imaging (134–136). In following sections, radionuclide imaging (by SPECT and PET) will be described as it passed a phase I study (136) and probes based on Nbs developed during the past decade are listed in **Table 1**.

# Nbs in Nuclear Imaging