Targeted therapy of angiogenesis using anti-VEGFR2 and anti-NRP-1 nanobodies.

PURPOSE: Targeted therapy in cancer researches is a promising approach that can resolve drawbacks of systematic therapeutics. Nanobodies are potent therapeutics due to their high specificity and affinity to the target. METHODS: In this study, we evaluated the effect of the combination of anti-vascular endothelial growth factor receptor 2 (anti-VEGFR2) and anti-neuropilin-1 (anti-NRP1) nanobodies both in vitro (MTT, and tube formation assay) and in vivo (chick chorioallantoic membrane (CAM), and Nude mice treatment assay). RESULTS: Our results showed that the combination of two nanobodies (anti-VEGFR2/NRP-1 nanobodies) significantly inhibited proliferation as well as tube formation of human endothelial cells effective than a single nanobody. In addition, the mixture of both nanobodies inhibited vascularization of chick chorioallantoic membrane ex ovo CAM assay as compared to a single nanobody. Moreover, the mixture of both nanobodies significantly inhibited tumor growth of the mice (tumor volume and weight) higher than individual nanobodies (P < 0.05). CONCLUSION: Our results offer a promising role of combination therapies in cancer therapy as well as angiogenesis.

Caplacizumab Model-Based Dosing Recommendations in Pediatric Patients With Acquired Thrombotic Thrombocytopenic Purpura.

Acquired thrombotic thrombocytopenic purpura (aTTP) is a rare and life-threatening autoimmune thrombotic microangiopathy. Caplacizumab, evaluated in phase II and III studies in adults, shortens the time to platelet count response and reduces aTTP exacerbations, has a favorable safety profile, and can potentially reduce refractoriness and mortality associated with aTTP. Since no children with aTTP were enrolled in these clinical trials, caplacizumab has been initially approved for use only in adult patients with aTTP (10 mg). Pediatric dosing recommendations were developed using model-based simulations. A semimechanistic pharmacokinetic/pharmacodynamic population model has been developed describing the interaction between caplacizumab and von Willebrand factor antigen (vWF:Ag) following intravenous and subcutaneous administration of caplacizumab in different adult populations, at various dose levels, using nonlinear mixed-effects modeling. Based on the allometrically scaled pharmacokinetic/pharmacodynamic model, different dosing regimens were simulated in 8000 children (aged 2-18 years). Simulated caplacizumab exposures and vWF:Ag levels across different age categories were compared to an adult reference group. A simulated daily dose of 5 mg in children weighing <40 kg and of 10 mg in children weighing ≥40 kg resulted in similar exposures and vWF:Ag suppression across age and weight groups. Despite the lack of pediatric clinical data, the results of this modeling and simulation analysis constituted the basis for the European extension of indication for caplacizumab (10 mg) to adolescents aged >12 years and with a body weight ≥40 kg. This represents a rare case in which regulatory authorities have deemed a modeling and simulation study robust enough to approve a variation of indication.

Effectiveness of Caplacizumab Nanobody in Acquired Thrombotic Thrombocytopenic Purpura Refractory to Conventional Treatment.

Acquired thrombocytopenic thrombotic purpura (aTTP) is an autoantibody-mediated disease against the enzyme A Disintegrin and Metalloprotease domain with ThromboSpondin-1 type motif 13, which until now has been treated with plasma exchange (PEX) and corticosteroids. A 29-year-old female patient, who presented with aTTP in the context of pregnancy, has developed multiple relapses after treatment with PEX, corticosteroids, and rituximab. Recently, caplacizumab, a nanobody against von Willebrand factor, has been approved for the treatment of aTTP. In our patient, caplacizumab achieved better disease control, with a lower platelet count restoration time, days of PEX and hospitalization duration, as compared to standard therapy, reproducing the results of clinical trials. Caplacizumab represents a significant advance in the treatment of aTTP, especially in cases of recurrent relapses.

Enzymatic ligation of an antibody and arginine 9 peptide for efficient and cell-specific siRNA delivery.

A fusion protein comprising an antibody and a cationic peptide, such as arginine-9 (R9), is a candidate molecule for efficient and cell-specific delivery of siRNA into cells in order to reduce the side effects of nucleic acid drugs. However, their expression in bacterial hosts, required for their development, often fails, impeding research progress. In this study, we separately prepared anti-EGFR nanobodies with the K-tag sequence MRHKGS at the C-terminus and R9 with the Q-tag sequence LLQG at the N-terminus, and enzymatically ligated them in vitro by microbial transglutaminase to generate Nanobody-R9, which is not expressed as a fused protein in E. coli. Nanobody-R9 was synthesized at a maximum binding efficiency of 85.1%, without changing the binding affinity of the nanobody for the antigen. Nanobody-R9 successfully delivered siRNA into the cells, and the cellular influx of siRNA increased with increase in the ratio of Nanobody-R9 to siRNA. We further demonstrated that the Nanobody-R9-siRNA complex, at a 30:1 ratio, induced an approximately 58.6% reduction in the amount of target protein due to RNAi in mRNA compared to lipofectamine.

A class II MHC-targeted vaccine elicits immunity against SARS-CoV-2 and its variants.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHHMHCII-SpikeRBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.

Nasal delivery of single-domain antibody improves symptoms of SARS-CoV-2 infection in an animal model.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the disease COVID-19 can lead to serious symptoms, such as severe pneumonia, in the elderly and those with underlying medical conditions. While vaccines are now available, they do not work for everyone and therapeutic drugs are still needed, particularly for treating life-threatening conditions. Here, we showed nasal delivery of a new, unmodified camelid single-domain antibody (VHH), termed K-874A, effectively inhibited SARS-CoV-2 titers in infected lungs of Syrian hamsters without causing weight loss and cytokine induction. In vitro studies demonstrated that K-874A neutralized SARS-CoV-2 in both VeroE6/TMPRSS2 and human lung-derived alveolar organoid cells. Unlike other drug candidates, K-874A blocks viral membrane fusion rather than viral attachment. Cryo-electron microscopy revealed K-874A bound between the receptor binding domain and N-terminal domain of the virus S protein. Further, infected cells treated with K-874A produced fewer virus progeny that were less infective. We propose that direct administration of K-874A to the lung could be a new treatment for preventing the reinfection of amplified virus in COVID-19 patients.

Engineering an Antibody V Gene-Selective Vaccine.

The ligand-binding surface of the B cell receptor (BCR) is formed by encoded and non-encoded antigen complementarity determining regions (CDRs). Genetically reproducible or 'public' antibodies can arise when the encoded CDRs play deterministic roles in antigen recognition, notably within human broadly neutralizing antibodies against HIV and influenza virus. We sought to exploit this by engineering virus-like-particle (VLP) vaccines that harbor multivalent affinity against gene-encoded moieties of the BCR antigen binding site. As proof of concept, we deployed a library of RNA bacteriophage VLPs displaying random peptides to identify a multivalent antigen that selectively triggered germline BCRs using the human VH gene IGVH1-2*02. This VLP selectively primed IGHV1-2*02 BCRs that were present within a highly diversified germline antibody repertoire within humanized mice. Our approach thus provides methodology to generate antigens that engage specific BCR configurations of interest, in the absence of structure-based information.

Liposome-based nanocarriers loaded with anthrax lethal factor and armed with anti-CD19 VHH for effectively inhibiting MAPK pathway in B cells.

OBJECTIVE: One of the vital signaling pathways in cancer development and metastasis is mitogen-activated protein kinases (MAPKs). Bacillus anthracis Lethal Toxin (LT) is a potent MAPK signaling inhibitor. This toxin is comprised of two distinct domains, Lethal Factor (LF), MAPK inhibitor, and Protective Antigen (PA). To enter various cell lines, LF must be associated with the protective antigen (PA), which facilitates LF delivery. In the current study, to block MAPK signaling, LF was loaded into anti-CD19 immunoliposomes nanoparticle to deliver the cargo to Raji B cells. METHODS: The liposome nanoparticle was prepared using classical lipid film formation, then conjugated to anti-CD19 VHH. The binding efficiency was measured through flow cytometry. The targeted cytotoxicity of LF immunoliposome was confirmed by BrdU lymphoproliferation assay. This was followed by Real-Time PCR to assess the effect of formulation on pro-apoptotic genes. The inhibitory effect of LF on MAPK signaling was confirmed by western blot. RESULTS: Liposome nano-formulation was optimized to reach the maximum LF encapsulation and targeted delivery. Next, phosphorylation of MAPK pathway mediators like MEK1/2, P38 and JNK were inhibited following the treatment of Raji cells with LF-immunoliposome. The treatment also upregulated caspase genes, clearly illustrating cell death induced by LF through pyroptosis and caspase-dependent apoptosis. CONCLUSIONS: In conclusion, anti-CD19 VHH immunoliposome was loaded with LF, a potent MAPK inhibitor targeting B cells, which curbs proliferation and ushers B cells toward apoptosis. Thus, immunoliposome presents as a versatile nanoparticle for delivery of LF to block aberrant MAPK activation. To use LF as a therapy, it would be necessary to materialize LF without PA. In the current study, PA was substituted with anti-CD19 immunoliposome to make it targeted to CD19+ while keeping the normal cells intact.

A potent SARS-CoV-2 neutralising nanobody shows therapeutic efficacy in the Syrian golden hamster model of COVID-19.

SARS-CoV-2 remains a global threat to human health particularly as escape mutants emerge. There is an unmet need for effective treatments against COVID-19 for which neutralizing single domain antibodies (nanobodies) have significant potential. Their small size and stability mean that nanobodies are compatible with respiratory administration. We report four nanobodies (C5, H3, C1, F2) engineered as homotrimers with pmolar affinity for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. Crystal structures show C5 and H3 overlap the ACE2 epitope, whilst C1 and F2 bind to a different epitope. Cryo Electron Microscopy shows C5 binding results in an all down arrangement of the Spike protein. C1, H3 and C5 all neutralize the Victoria strain, and the highly transmissible Alpha (B.1.1.7 first identified in Kent, UK) strain and C1 also neutralizes the Beta (B.1.35, first identified in South Africa). Administration of C5-trimer via the respiratory route showed potent therapeutic efficacy in the Syrian hamster model of COVID-19 and separately, effective prophylaxis. The molecule was similarly potent by intraperitoneal injection.

Pharmacokinetics of Single Domain Antibodies and Conjugated Nanoparticles Using a Hybrid near Infrared Method.

Iron oxide nanoparticles and single domain antibodies from camelids (VHHs) have been increasingly recognized for their potential uses for medical diagnosis and treatment. However, there have been relatively few detailed characterizations of their pharmacokinetics (PK). The aim of this study was to develop imaging methods and pharmacokinetic models to aid the future development of a novel family of brain MRI molecular contrast agents. An efficient near-infrared (NIR) imaging method was established to monitor VHH and VHH conjugated nanoparticle kinetics in mice using a hybrid approach: kinetics in blood were assessed by direct sampling, and kinetics in kidney, liver, and brain were assessed by serial in vivo NIR imaging. These studies were performed under "basal" circumstances in which the VHH constructs and VHH-conjugated nanoparticles do not substantially interact with targets nor cross the blood brain barrier. Using this approach, we constructed a five-compartment PK model that fits the data well for single VHHs, engineered VHH trimers, and iron oxide nanoparticles conjugated to VHH trimers. The establishment of the feasibility of these methods lays a foundation for future PK studies of candidate brain MRI molecular contrast agents.

Development and assessment of the efficacy and safety of human lung-targeting liposomal methylprednisolone crosslinked with nanobody.

Glucocorticoid (GC) hormone has been commonly used to treat systemic inflammation and immune disorders. However, the side effects associated with long-term use of high-dose GC hormone limit its clinical application seriously. GC hormone that can specifically target the lung might decrease the effective dosage and thus reduce GC-associated side effects. In this study, we successfully prepared human lung-targeting liposomal methylprednisolone crosslinked with nanobody (MPS-NSSLs-SPANb). Our findings indicate that MPS-NSSLs-SPANb may reduce the effective therapeutic dosage of MPS, achieve better efficacy, and reduce GC-associated side effects. In addition, MPS-NSSLs-SPANb showed higher efficacy and lower toxicity than conventional MPS.

Development of a 99mTc-Labeled Single-Domain Antibody for SPECT/CT Assessment of HER2 Expression in Breast Cancer.

Accurate determination of human epidermal growth factor receptor 2 (HER2) expression is essential for HER2-targeted therapy in patients with cancer. HER2 expression in a complex environment, such as in a heterogeneous tumor, makes the precise assessment of the HER2 status difficult using current methods. In this study, we developed a novel 99mTc-labeled anti-HER2 single-domain antibody (99mTc-NM-02) as a molecular imaging tracer for the noninvasive detection of HER2 expression and investigated its safety, radiation dosimetry, biodistribution, and tumor-targeting potential in 10 patients with breast cancer. Our data showed that no drug-related adverse reactions occurred. The tracer mainly accumulated in the kidneys and liver with mild uptake in the spleen, intestines, and thyroid; however, only background tracer levels were observed in other organs where primary tumors and metastases typically occurred. The mean effective dose was 6.56 × 10-3 mSv/MBq, and tracer uptake was visually observed in the primary tumors and metastases. A maximal standard uptake value of 1.5 was determined as a reasonable cutoff for identifying HER2 positivity using SPECT/CT imaging. Our 99mTc-NM-02 tracer is safe for use in breast cancer imaging, with reasonable radiation doses, favorable biodistribution, and imaging characteristics. 99mTc-NM-02 SPECT imaging may be an accurate and noninvasive method to detect the HER2 status in patients with breast cancer.

Perspectives on metals-based radioimmunotherapy (RIT): moving forward.

Radioimmunotherapy (RIT) is FDA-approved for the clinical management of liquid malignancies, however, its use for solid malignancies remains a challenge. The putative benefit of RIT lies in selective targeting of antigens expressed on the tumor surface using monoclonal antibodies, to systemically deliver cytotoxic radionuclides. The past several decades yielded dramatic improvements in the quality, quantity, recent commercial availability of alpha-, beta- and Auger Electron-emitting therapeutic radiometals. Investigators have created new or improved existing bifunctional chelators. These bifunctional chelators bind radiometals and can be coupled to antigen-specific antibodies. In this review, we discuss approaches to develop radiometal-based RITs, including the selection of radiometals, chelators and antibody platforms (i.e. full-length, F(ab')2, Fab, minibodies, diabodies, scFv-Fc and nanobodies). We cite examples of the performance of RIT in the clinic, describe challenges to its implementation, and offer insights to address gaps toward translation.

Inhalable Nanobody (PiN-21) prevents and treats SARS-CoV-2 infections in Syrian hamsters at ultra-low doses.

Globally, there is an urgency to develop effective, low-cost therapeutic interventions for coronavirus disease 2019 (COVID-19). We previously generated the stable and ultrapotent homotrimeric Pittsburgh inhalable Nanobody 21 (PiN-21). Using Syrian hamsters that model moderate to severe COVID-19 disease, we demonstrate the high efficacy of PiN-21 to prevent and treat SARS-CoV-2 infection. Intranasal delivery of PiN-21 at 0.6 mg/kg protects infected animals from weight loss and substantially reduces viral burdens in both lower and upper airways compared to control. Aerosol delivery of PiN-21 facilitates deposition throughout the respiratory tract and dose minimization to 0.2 mg/kg. Inhalation treatment quickly reverses animals' weight loss after infection, decreases lung viral titers by 6 logs leading to drastically mitigated lung pathology, and prevents viral pneumonia. Combined with the marked stability and low production cost, this innovative therapy may provide a convenient and cost-effective option to mitigate the ongoing pandemic.

IL17A/F nanobody sonelokimab in patients with plaque psoriasis: a multicentre, randomised, placebo-controlled, phase 2b study.

BACKGROUND: Sonelokimab (also known as M1095) is a novel trivalent nanobody comprised of monovalent camelid-derived (ie, from the Camelidae family of mammals, such as camels, llamas, and alpacas) nanobodies specific to human interleukin (IL)-17A, IL-17F, and human serum albumin. Nanobodies are a novel class of proprietary therapeutic proteins based on single-domain, camelid, heavy-chain-only antibodies. We assessed the efficacy, safety, and tolerability of sonelokimab across four dosage regimens compared with placebo in patients with plaque-type psoriasis. Secukinumab served as an active control. METHODS: This multicentre, randomised, placebo-controlled, phase 2b trial was done at 41 clinics and research sites in Bulgaria, Canada, Czech Republic, Germany, Hungary, Poland, and the USA. Participants (aged 18-75 years) with stable moderate to severe plaque-type psoriasis (defined as an Investigator's Global Assessment [IGA] score of ≥3, a body surface area involvement of ≥10%, and a Psoriasis Area and Severity Index score of ≥12) for more than 6 months before randomisation, who were candidates for systemic biological therapy were included. Participants previously treated with more than two biologics or any therapy targeting IL-17 were excluded. Randomisation was stratified by weight (≤90 kg or >90 kg) and previous use of biologics. Investigators, participants, and vendors remained masked for the duration of the study, with the exception of each site's study drug administrator (who did not complete any other assessments in the study) and a study monitor who only assessed drug preparation, administration, and accountability. The study sponsor remained masked until all week 24 data were clean and locked. Participants were randomly assigned (1:1:1:1:1:1) using a centralised interactive response technology system to one of six parallel treatment groups: placebo group, sonelokimab 30 mg group, sonelokimab 60 mg group, sonelokimab 120 mg normal load group, sonelokimab 120 mg augmented load group, or secukinumab 300 mg group. All participants underwent a 4-week screening period, a 12-week placebo-controlled induction period, a 12-week dose maintenance or escalation period, and a 24-week response assessment or dose-holding period. During the placebo-controlled induction period (weeks 0-12), participants received either placebo (at weeks 0, 1, 2, 3, 4, 6, 8, and 10), sonelokimab 30 mg, 60 mg, or 120 mg normal load (at weeks 0, 2, 4, and 8), sonelokimab 120 mg augmented load (at weeks 0, 2, 4, 6, 8, and 10), or secukinumab 300 mg (at weeks 0, 1, 2, 3, 4, and 8), with placebo given at weeks 1, 3, 6, and 10 in the sonelokimab 30 mg, 60 mg, and 120 mg normal load groups, at weeks 1 and 3 in the sonelokimab 120 mg augmented load group, and at weeks 6 and 10 in the secukinumab 300 mg group. During the dose maintenance or escalation period (weeks 12-24), participants assigned to the placebo group received sonelokimab 120 mg (at weeks 12, 14, 16, and then every 4 weeks); those assigned to sonelokimab 30 mg or 60 mg groups with an IGA score of more than 1 were escalated to 120 mg and then every 4 weeks, and those with an IGA score of 1 or less stayed on the assigned dose at week 12 and then every 4 weeks; those assigned to the sonelokimab 120 mg groups received sonelokimab 120 mg at week 12 and then every 8 weeks (normal load group) or every 4 weeks (augmented load); and those assigned to the secukinumab 300 mg group received secukinumab 300 mg at week 12 and then every 4 weeks. During this period, placebo was given at week 14 in all groups, except in participants who initially received placebo, and at week 16 in the sonelokimab 120 mg normal load group. In the response assessment with dose-holding period (weeks 24-48), participants in the sonelokimab 30 mg or 60 mg groups who had dose escalation to 120 mg remained on the same regimen regardless of the IGA score at week 24. Participants in the secukinumab 300 mg group also remained on the same regimen regardless of IGA score at week 24. Participants in the sonelokimab 30 mg and 60 mg groups without dose escalation, and all participants in the two sonelokimab 120 mg groups (including placebo rollover patients) were eligible to stop the study drug at week 24. Those participants with an IGA score of 0 at week 24 received placebo; these participants resumed the previous dose of sonelokimab every 4 weeks when they had an IGA score of 1 or more (assessed every 4 weeks). Participants in these groups with an IGA score of 1 or more at week 24 continued on the same dosage. All study treatments were administered as subcutaneous injections. The final dose in all groups was given at week 44. The primary outcome was the proportion of participants in the sonelokimab groups with an IGA of clear or almost clear (score 0 or 1) at week 12 compared with the placebo group. The primary outcome and safety outcomes were assessed on an intention-to-treat basis. The study was not powered for formal comparisons between sonelokimab and secukinumab groups. This trial is registered with ClinicalTrials.gov, NCT03384745. FINDINGS: Between Aug 15, 2018, and March 27, 2019, 383 patients were assessed for eligibility, 313 of whom were enrolled and randomly assigned to the placebo group (n=52), the sonelokimab 30 mg group (n=52), the sonelokimab 60 mg group (n=52), the sonelokimab 120 mg normal load group (n=53), the sonelokimab 120 mg augmented load group (n=51), or the secukinumab 300 mg group (n=53). Baseline characteristics of participants were similar among the groups. At week 12, none (0·0% [95% CI 0·0-6·8]) of the 52 participants in the placebo group had an IGA score of 0 or 1 versus 25 (48·1% [34·0-62·4], p<0·0001) of 52 participants in the sonelokimab 30 mg group, 44 (84·6% [71·9-93·1], p<0·0001) of 52 participants in the sonelokimab 60 mg group, 41 (77·4% [63·8-87·7], p<0·0001) of 53 participants in the sonelokimab 120 mg normal load group, 45 (88·2% [76·1-95·6], p<0·0001) of 51 participants in the sonelokimab 120 mg augmented load group, and 41 (77·4% [63·8-87·7], p<0·0001) of 53 participants in the secukinumab 300 mg group. During the placebo-controlled induction period, 155 (49·5%) of 313 participants had one or more mostly mild to moderate adverse event; the most frequent adverse events in all participants on sonelokimab during weeks 0-12 were nasopharyngitis (28 [13·5%] of 208 participants), pruritus (14 [6·7%] participants), and upper respiratory tract infection (nine [4·3%] participants). One patient from all sonelokimab-containing groups had Crohn's disease that developed during weeks 12-52. Over 52 weeks, sonelokimab safety was similar to secukinumab, with the possible exception of manageable Candida infections (one [1·9%] of 53 participants in the secukinumab group had a Candida infection vs 19 [7·4%] of 257 participants in all sonelokimab-containing groups). INTERPRETATION: Treatment with sonelokimab doses of 120 mg or less showed significant clinical benefit over placebo, with rapid onset of treatment effect, durable improvements, and an acceptable safety profile. FUNDING: Avillion.

Nanobody Conjugates for Targeted Cancer Therapy and Imaging.

Conventional antibody-based targeted cancer therapy is one of the most promising avenues of successful cancer treatment, with the potential to reduce toxic side effects to healthy cells surrounding tumor cells. However, the full potential of antibodies is severely limited due to their large size, low stability, slow clearance, and high immunogenicity. Alternatively, recently discovered nanobodies, which are the smallest naturally occurring antigen-binding format, have shown great potential for addressing these limitations. Bioconjugation of nanobodies to functional groups such as toxins, enzymes, radionucleotides, and fluorophores can improve the efficacy and potency of nanobodies, enhance their in vivo pharmacokinetics, and expand the range of potential applications. Herein, we review the superior characteristics of nanobodies in comparison to conventional antibodies and provide insight into recent developments in nanobody conjugates for targeted cancer therapy and imaging.

Immunogenicity Risk Profile of Nanobodies.

Nanobodies (Nbs), the variable domains of camelid heavy chain-only antibodies, are a promising class of therapeutics or in vivo imaging reagents entering the clinic. They possess unique characteristics, including a minimal size, providing fast pharmacokinetics, high-target specificity, and an affinity in the (sub-)nanomolar range in conjunction with an easy selection and production, which allow them to outperform conventional antibodies for imaging and radiotherapeutic purposes. As for all protein theranostics, extended safety assessment and investigation of their possible immunogenicity in particular are required. In this study, we assessed the immunogenicity risk profile of two Nbs that are in phase II clinical trials: a first Nb against Human Epidermal growth factor Receptor 2 (HER2) for PET imaging of breast cancer and a second Nb with specificity to the Macrophage Mannose Receptor (MMR) for PET imaging of tumor-associated macrophages. For the anti-HER2 Nb, we show that only one out of 20 patients had a low amount of pre-existing anti-drug antibodies (ADAs), which only marginally increased 3 months after administering the Nb, and without negative effects of safety and pharmacokinetics. Further in vitro immunogenicity assessment assays showed that both non-humanized Nbs were taken up by human dendritic cells but exhibited no or only a marginal capacity to activate dendritic cells or to induce T cell proliferation. From our data, we conclude that monomeric Nbs present a low immunogenicity risk profile, which is encouraging for their future development toward potential clinical applications. One Sentence Summary: Nanobodies, the recombinant single domain affinity reagents derived from heavy chain-only antibodies in camelids, are proven to possess a low immunogenicity risk profile, which will facilitate a growing number of Nanobodies to enter the clinic for therapeutic or in vivo diagnostic applications.