Nanobodies are extremely promising imaging reagents in different clinical applications such as fluorescence-guided surgery (44), positron emission tomography (PET) and ingle-photon emission computed tomography (SPECT) (45, 46).

In cancer in vivo imaging, nanobodies have been successfully used to specifically deliver radionucleotides to tumor cells, to biomarkers of the tumor microenvironment and to monitor immune infiltration in animal models and clinical trials (16). The major drawback of the conventional PET protocol that exploits the preferential accumulation of glucose in tumors to deliver radionucleotide-labeled glucose analogues to the cancerous site is that organs such brain or heart, which have high glucose consumption, are difficult to imagine because of the elevated background signal. Antibodies complexed to radionuclides and specific for tumor surface biomarkers represent a rational alternative for selective targeting but the large dimension of IgGs impairs the fast clearance of the unbound fraction. IgGs persist for a couple of days in the vessels determining a diffuse background that prevents high resolution imaging in the first 24-48 hour after administration. However, waiting for days between radionucleotide injection and imaging is impracticable in the clinical organization. Therefore, the optimal solution appears using antibody fragments such nanobodies that are cleared in 15 min by kidney filtration but still provide excellent target accumulation, effective tumor penetration and improved tumor-to-background signal with respect to labeled glucose (47, 48). This approach brings a further advantage: since the imaging can be performed within one hour after the reagent injection, it is possible to use the short half-life positron-emitting nuclides (18F, 68Ga or 89Zr) for PET and the γ-emitting nuclide (99mTc) for SPECT to diminish unnecessary patient irradiation (17). Given the simple engineering of nanobodies, they can be simply expressed as bivalent molecules that still preserve low mass but have increased avidity. For instance, with respect to the monovalent α-EGFR construct, its corresponding bivalent format resulted in higher accumulation at the tumor site and improved PET imaging (24).

B cell lymphoma and leukemia are the most common subtypes of malignant lymphomas and represent 80-85% of non-Hodgkin lymphomas (NHLs). About 20%-40% of B-cell lymphoma/leukemia patients die due to relapse after Rimximab treatment (49). CAR-T cell therapy has shown potential efficacy in the treatment of B cell leukemias and lymphomas and the contemporary targeting of multiple antigen epitopes has been envisaged to overcome the emergence of single antigen-resistant leukemic cells and avoid immune escape (50, 51) ( Figure 1 ). Nanobodies targeting CD19, CD20, CD30 and CD22 (52) have been successfully used for CAR applications.

De Munter et al. (22) reported the generation of a bispecific CAR comprising two nanobodies specific for CD20 and HER2, respectively. T cells expressing the bispecific nanoCAR were able to kill tumor cells over-expressing CD20, HER2, or both antigens. Since the individualized manufacturing process of (nano)CARs is costly, the attention moved to donor-derived γδT cells to use as a CAR backbone because γδT cells lack allogenicity and are known to mediate natural anti-tumor responses (54). De Bruin et al. constructed a bispecific nanobody-based structure that targets Vγ9Vδ2-T cells and EGFR for cancer immunotherapeutic therapy (23). Biotech companies such as PersonGen BioTherapeutics developed nanobody-based CAR-γδT cells to treat B-cell leukemia. A clinical trial is currently under way to test the safety and effectiveness of donor γδT cell infusion to prevent relapsed/refractory leukemia rescue relapse after allogeneic hematopoietic stem cell transplantation. Moreover, Vγ9Vδ2-T cells have become a novel potential immunotherapeutic for Chronic Lymphocytic Leukemia (CLL) due to their capacity to be triggered by phosphoantigens which are overproduced by CLL. A nanobody-based CD1d-specific Vγ9Vδ2-T cell engager was generated to induce robust activation and degranulation of Vg9Vd2-T cells and consequent lysis of autologous leukemic cells (29).

Acute myeloid leukemia (AML) includes all acute non-lymphocytic leukemia. It is related to the insurgency of pluripotent stem cells or slightly differentiated progenitor cell nuclear type mutations. A Sequentially Tumor-Selected Antibody and Antigen Retrieval (STAR) system (31) was developed for screening multiple nanobodies that specifically target AML cells. Nanobodies were used to enhance the binding efficacy of CAR-T cells to AML cells. To this aim, the anti-CD13 nanobody Nb157 was isolated and used to target CD13+ AML cells. Further, bispecific CAR-T cells targeting CD13 and TIM3 were designed to eradicate patient-derived AML and to promote decreased toxicity to human bone marrow stem cells and peripheral myeloid cells in mouse models ( Figure 1 ).

CD47 is overexpressed in gastric, ovarian and colon cancer, as well as in AML (55) and an anti-CD47 nanobody (HuNb1-IgG4) with high affinity and specificity (32) effectively empowered macrophage-mediated phagocytosis of tumor cells in vitro. In vivo it showed potent anti-ovarian cancer and anti-lymphoma activity and its efficacy was further increased when it was combined with rituximab to build a bispecific antibody for the simultaneous targeting of CD47 and CD20.

Acute lymphoblastic leukemia is a highly invasive type of blood cancer. Blinatumomab, which was approved by FDA in late 2014 for the treatment of Fischer-negative precursor ALL ( Figure 2 ), is a bispecific antibody based on the BiTE technology and employing two scFvs. One targets the CD19 antigen on the surface of tumor cells, whereas the other targets the CD3 receptor on the surface of cytotoxic T lymphocytes (57). Nanobodies were used in alternative configurations to contrast ALL. CD7 is a convenient ALL biomarker of T-cells (59) because it is rapidly endocytosed once complexed by antibodies and therefore can be exploited for biotherapeutic uptake. A set of humanized anti-CD7 nanobodies was characterized. With respect to the monovalent version, bivalent formats fused to a truncated derivative of Pseudomonas exotoxin A showed significant higher cytotoxicity (60, 61). The fusion dhuVHH6-PE38 was used in vivo in NOD-Prkdc^em26IL2rg^em26Nju (NGG) mouse model and significantly extended the survival of the animals. The same nanobody was successfully used to create CAR constructs which significantly inhibited disease progression in xenograft mouse models of T-ALL primary tumor cells (21).

Multiple myeloma is a neoplastic plasma-cell disorder that arises from an asymptomatic premalignant proliferation of monoclonal plasma cells derived from post–germinal-center B cells. CD38 is considered as a biomarker overexpressed in multiple myeloma and the anti-CD38 monoclonal antibody daratumumab demonstrated its high therapeutic efficacy (62).

Schütze et al. isolated a set of nanobodies that recognized three different non-overlapping epitopes of CD38 extracellular domain and then prepared biparatopic constructs by fusing two nanobodies, specific for alternative epitopes, to human IgG1 Fc-domain (26). These constructs outscored both the monoclonal daratumumab and bivalent constructs sharing the same geometry by displaying two identical nanobodies when compared for their capacity of mediating complement-dependent cytotoxicity toward CD38-expressing myeloma cells.

B cell maturation antigen is expressed extensively in malignant plasma cells, seems to be involved in their proliferation and survival and is regarded as a target for CAR-T therapy, despite the possible side effects (63, 64). Nanobody-based CAR-T molecules targeting such receptor are under development in biotech companies but no scientific publication confirms their efficacy.

Tumors require active angiogenesis for securing the energy necessary for their growth and the factors regulating this process become potential drug targets. Vascular endothelial growth factor (VEGF) plays a critical role in the angiogenesis (65) and the high-affinity anti-VEGFR2 nanobody (3VGR19) (28) was tested for its capacity in inhibiting proliferation, tube formation, and migration of human endothelial cells. When built into a bivalent format using the hinge region of llama IgG2c, its half-life in vivo in a C57BL/6 mice model was almost doubled and its inhibitory activity was significantly higher than those obtained using the monovalent nanobody.

Placental growth factor (PlGF) is a structurally related member belonging to the same superfamily of VEGF (66) and might be involved in pathogenic angiogenesis, probably by recruiting myeloid progenitors (67). Nikooharf et al. (34) developed a bivalent anti-PLGF nanobody to test as suppressor of the angiogenesis progression and observed that it could inhibit cell proliferation, capillary-like structure formation and motility.

Glioblastoma is the most common primary brain tumor in adults and usually leads to rapid death. A therapeutic option considers targeting the death receptor (DR) to activate tumor cell death pathway but the variable response degree in tumor cells to DR agonist-mediated apoptosis represents a major limitation of this approach.

A bispecific construct (ENb-TRAIL) composed by an anti-EGFR nanobody and the DR ligand TRAIL demonstrated therapeutic efficacy in tumor cells that do not respond to either EGFR antagonist or DR agonist monotherapies ( Figure 1 ). ENb-TRAIL induced a triple anti-tumor effect by inducing DR5 aggregation in the plasma membrane, by initiating caspase-mediated apoptosis of tumor cells and by blocking the EGFR signaling pathway. In vivo assays proved that ENb-TRAIL treatment significantly alleviated tumor burden and increased survival (20, 68). A tetravalent anti-DR5 agonistic nanobody construct (TAS266) showed more potency than the ENb-TRAIL compound in pre-clinical studies but did not passed Phase I due to its strong hepatotoxicity (69).

Brain is also the site of metastatic tumors. A bispecific anti-VEGF-A/Ang2 nanobody was able to reduce significantly number and volume of metastatic tumors in a mouse model (70).

Lung cancer is the leading cause of cancer death worldwide and, among the different subtypes, non-small cell lung cancer (NSCLC) accounts for about 85% of the whole cases (71). The trifunctional bispecific antibody Catumaxomab based on Quadroma (Hybrid Hybridoma) technology, which combines the EpCAM rat antibody (IgG2b) and CD3 mouse antibody (IgG2a) into a bispecific molecule (56), was the first multispecific antibody approved by EMA (2009) for the treatment of NSCLC malignant ascites ( Figure 2 ). Other not exclusive biomarkers, such epidermal growth factor receptor (EGFR), are often considered as therapeutic targets in NSCLC because highly overexpressed and often mutated. An anti-EGFR nanobody combined with photosensitizer and catalase succeeded in improving tumor hypoxia and consequently killed A549 primary tumor and inhibited lung metastasis, prolonging mouse survival (72). Also anti-EGFR nanobodies functionalized with a cell-penetration poly-arginine peptide were highly cytotoxic to the same human adenocarcinomic alveolar basal epithelial cells A549 (73).

About 33% of patients with NSCLC tumors and epidermal growth factor receptor mutations develop brain metastasis (74). Osimertinib is effective against mutated EGFR but drug resistance raises in 1 to 2 years (75). To overcome this limitation, dual-targeting liposomes were generated that display an anti-PD-L1 nanobody and a transferrin receptor-binding peptide. This construct is still able to pass the blood-brain barrier and mediate simvastatin/gefitinib delivery to the NSCLC-EGFR^mut metastatic tumor (76).

Nanobodies were also exploited to assess alternative biomarkers and therapeutic options. A technetium-99 m-labeled anti-CEA nanobody was used to prove that the carcinoembryonic antigen (CEA) was a potentially interesting marker for NSCLC (77), whereas bright nanoprobes based on quantum dots conjugated to anti-HER2 nanobodies provided better immunolabeling of lung cancer cell lines than dyes Alexa Fluor 488 and Alexa Fluor 568 (78). Multivalent anti-CCR8 nanobodies fused to Fc were also effective in NSCLC treatment by eliciting antitumor immunity through tumor-promoting Treg cells depletion and ADCC activation (79). Since angiogenesis is critical in tumor development, VEGFR2 was targeted by an anti-VEGFR2 nanobody conjugated with the enzyme urease that can convert endogenous urea into ammonia, a product toxic to tumor cells (80).

Breast cancer is the second most common cancer in women and the one causing more deaths. According to the genetic and biochemical characteristics, breast tumors are grouped into three major classes (Estrogen/Progesterone receptor positive, Her2 positive, Triple Negative - TN), with further stratification describing the subtypes of Triple Negative (81). About 20% of breast cancers are characterized by amplification of the HER-2/neu gene (82). HER2 is a tyrosine kinase receptor the overexpression of which causes increased tumor cell proliferation, tumor invasiveness, accelerated angiogenesis, and reduced apoptosis (83). Anti-HER2 monoclonal IgG antibodies trastuzumab (Herceptin) and pertuzumab, which target independent epitopes, are widely used alone or in combination in clinical treatment of HER2-positive breast cancers (84, 85). Nanobodies that bind to the same epitopes recognized by such IgGs have a potential therapeutic interest (36, 42, 86), whereas those that do not interfere with their binding because target further epitopes are suited for imaging or can be used as complementary theranostic reagents (87–90).

Since nanobodies specific for Her2 are simple to generate (43) and as recombinant proteins are simple to engineer, anti-Her2 nanobodies have been exploited to develop an enormous variety of reagents, such as bivalent and biparatopic molecules (25), immunotoxins (91, 92), activated nanoparticles (25, 93–95), biosensor immunocapture surfaces and other nanostructures suitable for receptor detection (96–101) and have been also in silico modeled to increase their biophysical features (102). As a general rule, multivalent formats provide higher apparent affinity for their antigen due to the avidity effect. Furthermore, multispecific formats are often more effective in inhibiting cell proliferation because of their capacity to block contemporarily alternative activation pathways (19, 103). The number of nanobodies available for TN breast cancers is extremely lower. TN positive cells have been successfully targeted with anti-EGFR-activated Quantum-Dot theranostic micelles, anti-STAT3 and anti-Protein C Receptor nanobodies and anti-TNFα nanobodies used alone or fused to a recognition peptide binding to the αvβ3 receptor on tumor cell membranes (104–108).The common limit of these approaches is that the targeted biomarkers are not exclusive of TN cells and therefore their clinical usefulness remains to be demonstrated. There is no report of nanobodies used for the diagnostic or the therapy of Estrogen/Progesterone receptor positive breast tumors.

Ovarian cancer is characterized by tumor heterogeneity and by controversial diagnostic methodology (109). Nanobody-activated nanospheres were successfully used for developing a highly sensitive (detection limit of 0.560 pg mL^-1) photoelectrochemical biosensor able to detect the serum biomarker Human Epididymis Protein 4 (HE4) and clearly distinguish ovarian cancer patients from healthy individuals (110). Mucins are type I membrane O-glycoproteins with single transmembrane domains that are usually highly upregulated during tumorigenesis and could represent a therapeutic target (111). In this perspective, Li et al. (27) designed a bispecific construct (Muc1-Bi) constituted by two nanobodies, one specific for Muc1 and the second for CD16 that could be applied for ovarian cancer treatment. This reagent can recruit NK cells and drive them to Muc1-overexpressing tumor cells and, in a xenograft model, significantly suppressed tumor growth.

Despite being expressed in several tumors, CEA is often used as a biomarker of colon carcinoma (112). CdSe/ZnS quantum dots conjugated to an anti CEA nanobody have been used as efficient two-photon excitation probes for imaging colon carcinoma tissue (113), whereas a bispecific construct formed by linking anti-CEA and anti-CD16a nanobodies succeeded in engaging NK cells and inhibiting CEA-overexpressing tumor growth in vivo (30) ( Figure 1 ). A novel bispecific nanobody with dual PD-L1/TIGIT demonstrated high inhibitory activity towards both PD-1/PD-L1 and TIGIT/CD155 interactions. Its application synergistically enhanced T cell activity in vitro compared to that of the two parental nanobodies (38) and such strategy for treating tumors might improve the reliability of therapies aiming at immune checkpoint blockade.

Inflammatory bowel disease (IBD) is a multifactorial disorder characterized by chronic and relapsing intestinal inflammation. IBD includes ulcerative colitis (UC) and Crohn’s disease (CD) that affects the ileum, rectum, and colon. Nowadays, inflammatory bowel disease cannot be completely cured, but anti-TNF-α monoclonal antibodies, such as infliximab and adalimumab, have made a significant breakthrough by enabling the delay of IBD progression (121). Moreover, their rapid mucosal healing ability has improved the response and remission rate of patients with IBD, especially for CD treatment (122). Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that represents a critical mediator of the autoimmune process, playing a key role in several inflammatory diseases, including rheumatoid arthritis (RA), ulcerative colitis, and CD. An innovative therapeutic approach for treatment of chronic colitis considered the in situ secretion of anti-TNF nanobodies by orally administered L. lactis bacteria engineered to secrete monovalent and bivalent anti-TNF nanobodies that neutralized TNF in vivo (115). TNF is the target of further inhibitory nanobodies. A trispecific anti-TNF construct could effectively inhibit the TNF/TNFR1 signaling pathway and its inhibitory activity was successfully tested ex vivo using colon biopsies of CD patients (117). In another study, three anti-TNF nanobodies with sub-nanomolar affinity for their antigen were selected and the crystal structures of the TNF–nanobody complexes showed that they targeted (partially) overlapping epitopes (119). Nevertheless, bivalent molecules showed increased blocking activity due to the fact that, differently from conventional antibodies, these constructs can bind simultaneously to two independent receptor binding sites of the trimeric TNF.

Recent studies have shown that IL-23 mediates the over-proliferation of T(H)-17 cells and the resulting accumulation of IL-17 and IL-22 pro-inflammatory cytokines promotes dermal inflammation and CD pathogenesis (123). Anti-human IL-23 nanobodies with low nanomolar affinity for hIL-23 and targeting independent epitopes were assembled together with an anti-human serum albumin nanobody into multivalent constructs. They showed prolonged in vivo half-life and improved hIL-23 neutralization capacity in vitro and in vivo with respect to the single monomeric nanobodies (118).

The above described anti-IL23 multivalent nanobodies might be suitable for treating psoriasis as well. Furthermore, current studies suggest that another effective treatment method would be the inhibition of IL-17 (120). M1095 is a trivalent bispecific-nanobody that can effectively neutralize the pro-inflammatory cytokines IL-17A and IL-17F as well as bind to human serum albumin. The initial clinical trials to evaluate the safety and effectiveness of the immunoreagent in patients with moderate to severe psoriasis showed that M1095 was well tolerated whereas psoriasis-related inflammatory markers were significantly decreased (120).

Rheumatoid Arthritis (RA) is a common chronic autoimmune disease. Tumor necrosis factor-α (TNF-α), as a pleiotropic cytokine, induces adverse pro-inflammatory and cytotoxic effects in the course of RA. A preliminary work showed that antagonistic anti-TNF nanobodies linked to an anti-serum albumin nanobody were 500 times more effective than monovalent nanobodies in controlling rheumatoid arthritis development in a mouse model (124). The bispecific nanobody format showed also higher antagonistic potency than the commercial IgG antibodies infliximab and adalimumab. On the base of such experience, it was designed the compound ATN-103 (ozoralizumab), a trivalent bispecific albumin-conjugated nanobody that targets TNF-α. In Phase II clinical trials it showed to cause no immunogenic response (116). Taisho Pharmaceuticals performed an apparently successful Phase III with this multimeric nanobody and recently submitted an application for approval to manufacture and market the immunodrug ( Table 1 ).

Since also interleukin 6 plays a key role in the pathogenesis of RA, such cytokine was targeted by the bispecific nanobody ALX-0061 that binds as well to human serum albumin, recruited to extend the construct half-life. In cynomolgus monkeys, ALX-0061 induced a dose-dependent inhibition of IL-6-induced inflammatory parameters (125).

The lack of specific treatment for sepsis leads to the worldwide incidence of 31.5 million of cases and to 5.3 million deaths per year. Sepsis severity is associated with plasma levels of matrix metalloproteinase-8 (MMP8) and tumor necrosis factor receptor (TNFR1) and, therefore, the effect of a bispecific nanobody able to block simultaneously MMP8 and HTNFR1 was evaluated (135). The results obtained in mouse model indicated that the nanobody-dependent neutralization of MMP8 and HTNFR1 had a beneficial effect in terms of survival rate. Different combinations of biparatopic nanobodies conferred 100% survival upon prophylactic or up to 24 hour post-infection administration in pneumonia mouse models challenged with Pseudomonas aeruginosa (150).

Viral infectious diseases have been one of the leading killers in the history of mankind. Influenza A virus is the main pathogen causing human influenza, it can infect a variety of animals and cause cross-species infection (151). Influenza virus neuraminidase (NA) plays an important role in the release and spread of the virus as well as in the cellular infection and consequently is a potentially interesting therapeutic target (152). Cardoso et al. (2014) isolated a set of anti-H5N1 NA nanobodies and generated bivalent molecules either connecting two single-domains with a flexible linker or by exploiting the dimerization properties of a mouse IgG2a-Fc domain fused to each single domain (153). The results showed that bivalent nanobodies had an in vitro antiviral potency 30- to 240-fold higher than monovalent nanobodies and protected BALB/c mice from H5N1 infections when used as a prophylactic therapy. Multimeric constructs were also conceived to protect simultaneously from different viruses. Specifically, Hultberg et al. (2011) prepared a trimeric construct linking nanobodies specific for H5N1 Influenza (able to neutralize both clade1 and 2), Respiratory Syncytial Virus and Rabies virus that protected from any of the three viruses (128). The authors also demonstrated that playing on the format (different bivalent and biparatopic combinations of single nanobodies), it was possible to increase the potency of the neutralizing anti-viral reagents and concluded that multimerization of nanobody fragments targeting multiple epitopes on viral trimeric spike proteins is a powerful tool for anti-viral therapy with broader neutralization capacity. The trivalent nanobody construct ALX-0171, designed for the inhalation treatment of respiratory syncytial virus infection, had a remarkable capacity to reduce escape mutant selection and showed promising results in animal models (154, 155) but was finally unable to improve clinical course in patients with established infection in the lower respiratory tract (156). Based on the results of such Phase IIb dose-ranging study, the Sponsor decided to discontinue ALX-0171 trials.

Enterovirus A71 (EVA71) is a major cause of viral encephalitis and severe hand, foot, and mouth disease (HFMD) in young children worldwide. Neither preventive not therapeutic treatments are available for limiting EVA71 infection. Huang et al. (2020) isolated a nanobody (F1), which inhibited EVA71 infection both in vitro and in vivo. The neutralizing activity was improved when multivalent formats were used and the most effective constructs were the ones in which the structure geometry enabled to maximally exploit the avidity effect (141).

Gastroenteritis induced by rotavirus infection is a major health problem in development countries. Bispecific nanobody constructs targeting independent rotavirus epitopes were used to transform Lactobacilli. Next, Lactobacilli cultures were used to deliver the neutralizing nanobodies to the intestinal lumen and such posology showed high anti-virus efficiency (129).

Acquired immunodeficiency syndrome (AIDS) causes about 1.8 million AIDS-related deaths each year (157) and is caused by infection with HIV (Human Immunodeficiency Virus). HIV leads to extensive destruction of T-helper cells, macrophages, dendritic cells, and other cellular components associated with cell-mediated immunity, eventually leading to the destruction of the immune system. Consequently, the organism becomes the target of many opportunistic diseases (158). At the present, the effective antiretroviral therapy (HAART) renders HIV a chronic disease (159) but this treatment is expensive and has many adverse effects (160). Therefore, there is still an urgent demand for effective and low-cost treatment of HIV infection.

Experiments made with combinations of patient-derived HIV neutralizing antibodies targeting complementary epitopes demonstrated that the inhibition effect was increased when more virus epitopes were blocked contemporarily (161). A contribution to the effort of isolating antibodies with complementary characteristics was the method described by Koh et al. that allowed the recovery of nanobodies with distinct binding features (162). However, no further studies with multispecific constructs built using the selected binders were published, despite the encouraging results showing that nanobody homo- or heteromultimers could neutralize a wide array of virus subtype (163). A bivalent nanobody targeting the proximal external region of gp120 had a neutralization capacity 20 times higher than the monovalent ligand (164) and a bispecific construct targeting gp41 and gp120 epitopes possessed a neutralizing potency up to 1400-fold higher than the mixture of the individual nanobodies (139). Liposomes displaying nanobodies were also proposed to increase avidity, but the results were deceiving because the low nanobody density impaired the simultaneous binding to more than one target protein (133). More promising it seems the alternative of using neutralizing nanobodies displayed on the surface of Lactobacillus rhamnosus cells (165).

An alternative approach considered targeting the CXCR4 receptor that participates in the viral uptake (134). Bivalent constructs were constructed by linking nanobodies to a human IgG1 antibody Fc domain and were compared with their monovalent counterparts. The Nb-Fc constructs had higher binding affinity, blocked more efficiently the CXCR4-mediated HIV entry and induced ADCC- and CDC-mediated cell-death of CXCR4-overexpressing cells, a condition that renders such molecules attractive to treat also CXCR4-overexpressing tumors.

SARS-CoV-2 is a single-stranded RNA virus that belongs to the CoVs family. The receptor-binding domain (RBD) of the spike protein binds to the host cell surface receptor angiotensin-converting enzyme 2 (ACE2). Several anti-spike/anti-RBD neutralizing antibodies have been isolated from both patients and by in vitro selection, some of them have entered clinical trials and cocktails of ligands binding to different epitopes have been proposed to overcome resistance due to the virus mutations (127, 166). Multispecific antibody fragments are an alternative solution to prevent mutation-dependent resistance, as it has been already summarized in recent reviews (34, 127, 167). Since the research on anti-SARS-CoV-2 is particularly active and new publications appear constantly, here we report only those relevant to the subject of the present review and published in the first 2021 quarter.

Heterodimers formed by nanobodies recognizing independent epitopes and connected by fusion with the human Fc fragment exhibited the strongest RBD-binding affinity and neutralizing ability against SARS-CoV-2 pseudoviruses (143). In this case, the nanobodies were isolated from a naïve library but the same strategy of using nanobody-based Fc-dependent heterodimers to increase both the apparent affinity and the neutralizing activity of the immune reagents was successfully exploited by another group that used immunized alpaca as the source of the ligands (149). The recognized advantage of targeting independent epitopes was also used to change paradigm in the preparation of multivalent constructs: instead of assembling randomly isolated, not overlapping nanobodies, these were chosen according to the available structural information (168). This strategy enabled to design biparatopic constructs with apparent affinity in the pM range starting from nanobodies with 10-100 times lower affinities (169, 170).

The other actual acute problem posed by the pandemic is the emergency of virus variants. Specifically targeting the single mutations might be extremely demanding, but a recent work (171) demonstrated that it is possible to have a large array of neutralizing nanobodies specific for several independent epitopes of the conserved regions, the combined use of which should offer protection options even in the case of highly mutated virus forms.

In the case of toxin infection, nanobodies have the pivotal advantage over IgG that most of them can be expressed as functional intrabodies and therefore can be directly produced inside host mammalian cells as a protective antidote. The toxin neutralizing capacity of a prophylactic gene therapy has been successfully demonstrated in the case of Bacillus anthracis infection, the causative agent of anthrax (132). The protective antigen (PA) is the common component of B. anthracis toxins. The sequences of two anti-PA nanobodies targeting two independent antigen epitopes were cloned in an adenovirus vector to produce a bispecific immunoreagent. Mice were injected with the vector and the resulting nanobody construct that accumulated in their sera protected the animals by infections with anthrax toxins and spores.

Clostridium difficile is a problematic nosocomial pathogen that can cause diarrhea, pseudomembrane colitis and even death due to the effect of the virulence factors TcdA and TcdB toxins (172). Monomeric nanobodies targeting different TcdB epitopes were incapable of preventing TcdB-induced cytotoxicity in cell-based assays, despite their very high-affinity. However, the toxic effect inhibition was achieved when nanobodies were prepared into Fc-dependent bivalent constructs (137).

Shiga toxin-producing Escherichia coli (STEC) are a subset of potentially lethal pathogens. Tandem repeats of nanobodies targeting the Shiga toxin-2a B subunit provided toxin neutralization capacity 100 times higher than the monovalent constructs (138). For the neutralization of tetanus neurotoxins, the best results were obtained by fusing an anti-toxin nanobody to another specific for Mac-1, a surface integrin receptor expressed on most innate immune cells that plays an essential role in the elimination of complement opsonized microorganisms (130). The bispecific construct allowed mice to survive a 10-fold lethal dose with respect to monomeric anti-toxin and outperformed a sheep anti-toxin polyclonal IgG. Another bispecific nanobody construct alleviated ricin toxin effects by promoting its aggregation and by modifying the dynamics of ricin uptake and its cellular trafficking (131).

Multivalent/multispecific nanobody constructs were also beneficially used for diagnostic goals. Bispecific nanobodies were used to construct an electrochemical biosensor to detect the scorpion venom toxins AahI and AahII (136). In the case of human toxocariasis, the most effective ELISA test was obtained using bivalent nanobodies for antigen capture (140). The Alternaria mycotoxin tenuazonic acid was conveniently detected with high sensitivity using a one-step bioluminescent enzyme immunoassay that required the bifunctional fusion formed by nanobody and nanoluciferase (173).

Type 2 diabetes mellitus is a chronic metabolic disorder disease characterized by hyperglycemia and associated comorbidities (179). Glucagon-like peptide-1 (GLP-1) plays an essential role in glucose homeostasis by binding to and activating the GLP-1 receptor but its extremely short half-life (3 min) limits the effect its therapeutic administration. Everestmab is a tri-functional construct developed to overcome this drawback. It is a fusion protein consisting of a mutated GLP-1, necessary for the biological activation of the GLP-1 receptor, an anti-GLP-1 nanobody suitable for the construct targeted delivery, and an anti-HSA nanobody to prolong its circulation time of the construct to several days. Everestmab treatments produced promising results in animal models (176).

Neovascular age-related macular degeneration and diabetic retinopathy are major causes of visual impairment and blindness, requiring frequent intravitreal injections of anti-angiogenesis biotherapeutics (180). The trispecific nanobody construct BI-X was designed for targeting simultaneously both the angiogenesis factors VEGF-A and Ang-2 and human albumin to increase the molecule half-life (178). BI-X showed superior efficacy and significant half-life extension when tested in cynomolgus monkeys after intravitreal injection. However, intravitreal injection is a burden for patients and has potential complications, such as infections and topic applications will be tested.

Nanobodies have been highly used to study the structure and the development of protein aggregates involved in the progression of neurodegenerative diseases (52, 181–183). A recent review illustrates how multimeric constructs based on antibody fragments can be used as effective intrabodies (184) for re-targeting antigen-antibody complexes. Specifically, nanobodies were engineered to favor PEST-dependent degradation of α-Synuclein (α-Syn) (185, 186). Nanobody bioconjugates were also successfully used as imaging probes able to cross the blood brain barrier and label amyloid-beta deposits after intravenous injection (187) as well as to functionalize gadolinium-based nanoparticles that allowed the visualization of amyloid fibril deposits in pathological tissues (188). Anti-α-Syn nanobodies fused to a fluorescent probe enabled to monitor the cytosolic presence of the antigen and to reveal the presence of transmittable αSyn in human cerebrospinal fluid (189).

Gelsolin amyloidosis, also known as familial amyloidosis of the Finnish type, is an autosomal dominantly inherited systemic disorder with ophthalmologic, neurologic, and dermatologic symptoms (190). A single point mutation (D187N) results in a pathological proteolytic cascade with the formation of amyloidogenic peptides which aggregate in multiple tissues and cause disease-associated symptoms. Nanobodies exclusively selective for one of the amyloidogenic fragment, but not for the wild type protein, acted as molecular chaperones and mitigated the aggregation process (174). Their effect in vivo was significantly improved when the animals were treated with a bispecific molecule obtained by coupling the anti-gelsolin nanobody with an anti-albumin nanobody. The same group demonstrated the protective effect of such construct when it was directly expressed by mutant mice that underwent adenovirus-based gene therapy (175).

A nanobody inhibiting the proprotein convertase subtilisin/kexin type 9 (PCSK9) was expressed fused to an Fc domain and effectively reduced the production of low density lipoprotein (LDL) and cholesterol in a rat model (177). The results were comparable to those obtained with the approved monoclonal evolocumab but at extremely lower production costs.

The bispecific nanobody ALX-0962 that targets both IgE and human serum albumin can neutralize soluble IgE as well as displace preformed IgE-FcϵRI complexes and therefore was considered for the treatment of allergic asthma (191).