Targeting of human fibroblast growth factor receptor 2 by a novel specific nanobody.

Inhibition of FGFR2 signaling is promising in targeted therapy of FGFR2-related tumors. In this study, anti-FGFR2 nanobodies (Nbs) were isolated through screening of an immune camelid phage display library. Four rounds of biopanning were carried out with commercial human FGFR2 antigen and enrichment was assessed by ELISA and phage titration. The gene of Nb was sub-cloned into the expression vector, and the recombinant vector was transformed into Escherichia coli WK6 cells. The recombinant protein was purified using Ni-NTA affinity chromatography. The anti-FGFR2 Nb (C13) was characterized by SDS-PAGE, western blotting, competitive inhibition ELISA, flow cytometry, MTT, and migration assay. C13 Nb recognized FGFR2 with high specificity and no cross-reactivity was observed with other tested antigens. The affinity of C13 Nb was calculated to be 1.5 × 10-9 M. Results of cytotoxicity showed that C13 Nb (10 µg/ml) inhibited 85% of the proliferation of T-47D cells (p < 0.001). In addition, C13 inhibited the migration of 68% of T-47D toward the source of the growth factor (p < 0.01). The flow cytometry showed that C13 Nb bound to the surface of FGFR2+ cells, T-47D cell line (96%). Results indicate the potential of anti-FGFR2 Nb for targeted therapy of FGFR2-overexpressing tumors after complementary investigations.

Development of a highly specific and sensitive VHH-based sandwich immunoassay for the detection of the SARS-CoV-2 nucleoprotein.

The current COVID-19 pandemic illustrates the importance of obtaining reliable methods for the rapid detection of SARS-CoV-2. A highly specific and sensitive diagnostic test able to differentiate the SARS-CoV-2 virus from common human coronaviruses is therefore needed. Coronavirus nucleoprotein (N) localizes to the cytoplasm and the nucleolus and is required for viral RNA synthesis. N is the most abundant coronavirus protein, so it is of utmost importance to develop specific antibodies for its detection. In this study, we developed a sandwich immunoassay to recognize the SARS-CoV-2 N protein. We immunized one alpaca with recombinant SARS-CoV-2 N and constructed a large single variable domain on heavy chain (VHH) antibody library. After phage display selection, seven VHHs recognizing the full N protein were identified by ELISA. These VHHs did not recognize the nucleoproteins of the four common human coronaviruses. Hydrogen Deuterium eXchange-Mass Spectrometry (HDX-MS) analysis also showed that these VHHs mainly targeted conformational epitopes in either the C-terminal or the N-terminal domains. All VHHs were able to recognize SARS-CoV-2 in infected cells or on infected hamster tissues. Moreover, the VHHs could detect the SARS variants B.1.17/alpha, B.1.351/beta, and P1/gamma. We propose that this sandwich immunoassay could be applied to specifically detect the SARS-CoV-2 N in human nasal swabs.

Large-Scale Production of Anti-RNase A VHH Expressed in pyrG Auxotrophic Aspergillus oryzae.

Nanobodies, also referred to as VHH antibodies, are the smallest fragments of naturally produced camelid antibodies and are ideal affinity reagents due to their remarkable properties. They are considered an alternative to monoclonal antibodies (mAbs) with potential utility in imaging, diagnostic, and other biotechnological applications given the difficulties associated with mAb expression. Aspergillus oryzae (A. oryzae) is a potential system for the large-scale expression and production of functional VHH antibodies that can be used to meet the demand for affinity reagents. In this study, anti-RNase A VHH was expressed under the control of the glucoamylase promoter in pyrG auxotrophic A. oryzae grown in a fermenter. The feature of pyrG auxotrophy, selected for the construction of a stable and efficient platform, was established using homologous recombination. Pull-down assay, size exclusion chromatography, and surface plasmon resonance were used to confirm the binding specificity of anti-RNase A VHH to RNase A. The affinity of anti-RNase A VHH was nearly 18.3-fold higher (1.9 nM) when expressed in pyrG auxotrophic A. oryzae rather than in Escherichia coli. This demonstrates that pyrG auxotrophic A. oryzae is a practical, industrially scalable, and promising biotechnological platform for the large-scale production of functional VHH antibodies with high binding activity.

Tuning Nanobodies' Bioactivity: Coupling to Ultrasmall Gold Nanoparticles Allows the Intracellular Interference with Survivin.

Nanobodies are highly affine binders, often used to track disease-relevant proteins inside cells. However, they often fail to interfere with pathobiological functions, required for their clinical exploitation. Here, a nanobody targeting the disease-relevant apoptosis inhibitor and mitosis regulator Survivin (SuN) is utilized. Survivin's multifaceted functions are regulated by an interplay of dynamic cellular localization, dimerization, and protein-protein interactions. However, as Survivin harbors no classical "druggable" binding pocket, one must aim at blocking extended protein surface areas. Comprehensive experimental evidence demonstrates that intracellular expression of SuN allows to track Survivin at low nanomolar concentrations but failed to inhibit its biological functions. Small angle X-ray scattering of the Survivin-SuN complex locates the proposed interaction interface between the C-terminus and the globular domain, as such not blocking any pivotal interaction. By clicking multiple SuN to ultrasmall (2 nm) gold nanoparticles (SuN-N), not only intracellular uptake is enabled, but additionally, Survivin crosslinking and interference with mitotic progression in living cells are also enabled. In sum, it is demonstrated that coupling of nanobodies to nanosized scaffolds can be universally applicable to improve their function and therapeutic applicability.

The Search for Single-Domain Antibodies Interacting with the Receptor-Binding Domain of SARS-CoV-2 Surface Protein.

We performed a search for nanoantibodies that specifically interact with the receptor-binding domain (RBD) of the SARS-CoV-2 surface protein. The specificity of single-domain antibodies from the blood sera of a llama immunized with RBD of SARS-CoV-2 surface protein S (variant B.1.1.7 (Alpha)) was analyzed by ELISA. Recombinant trimers of the SARS-CoV-2 spike protein were used as antigens. In this work, a set of single-domain antibodies was obtained that specifically bind to the RBD of the SARS-CoV-2 virus.

Characterization of Foot-and-Mouth Disease Virus Serotype O-Specific Single Domain Antibody Expressed in the pET Expression System.

Foot and mouth disease (FMD) is an extremely contagious disease of cloven-hoofed domesticated and wild animals, resulting in significant economic losses in many parts of the world. FMD virus (FMDV) serotype O is responsible for approximately 70% of global outbreaks. For detection of FMDV antigen or antibody, ELISAs are used worldwide and have several limitations, such as batch-to-batch variation in generating immunobiologicals, high production cost and ethical concerns over animal sacrifice. The use of single domain antibody (sdAb) or variable N-terminal domain of the heavy chain of heavy chain antibody (VHH) found naturally in camels has proven their effectiveness in diagnostics and therapeutics. In the present study, the anti-FMDV serotype O-specific VHH-C1 gene sequence (Accession no. KJ751546) was retrieved from the NCBI database. The gene was synthesized commercially in the pBluescript KS+ cloning vector and expressed in E. coli BL21 (DE3) cells using the pET303/CT-His expression system with a C-terminal 6X-His tag. The expressed sdAb, verified by SDS‒PAGE and western blotting, was purified by Ni-chelate chromatography and used as a coating antibody in double antibody sandwich (DAS) ELISA for FMDV detection and typing. The sdAb exhibited a high binding affinity for FMDV serotype O, without any cross-reactivity toward serotypes A and Asia-1. It exhibited better thermostability up to 85 °C than conventional rabbit polyclonal anti-FMDV sera. The potential of sdAbs thus produced without sacrificing lab animals could be explored for replacing polyclonal sera in DAS-ELISA as well as for developing biosensors or lateral flow devices for FMDV type O detection.

The role of single-domain antibodies (or nanobodies) in SARS-CoV-2 neutralization.

The severe acute respiratory syndrome (SARS-CoV-2), a newly emerging of coronavirus, continues to infect humans in the absence of a viable treatment. Neutralizing antibodies that disrupt the interaction of RBD and ACE2 has been under the spotlight as a way of developing the COVID-19 treatment. Some animals, such as llamas, manufacture heavy-chain antibodies that have a single variable domain (VHH) instead of two variable domains (VH/VL) as opposed to typical antibodies. Nanobodies are antigen-specific, single-domain, changeable segments of camelid heavy chain-only antibodies that are recombinantly produced. These types of antibodies exhibit a wide range of strong physical and chemical properties, like high solubility, and stability. The VHH's high-affinity attachment to the receptor-binding domain (RBD) allowed the neutralization of SARS-CoV-2. To tackle COVID-19, some nanobodies are being developed against SARS-CoV-2, some of which have been recently included in clinical trials. Nanobody therapy may be useful in managing the COVID-19 pandemic as a potent and low-cost treatment. This paper describes the application of nanobodies as a new class of recombinant antibodies in COVID-19 treatment.

3D Visualization of Human Blood Vascular Networks Using Single-Domain Antibodies Directed against Endothelial Cell-Selective Adhesion Molecule (ESAM).

High-quality three-dimensional (3D) microscopy allows detailed, unrestricted and non-destructive imaging of entire volumetric tissue specimens and can therefore increase the diagnostic accuracy of histopathological tissue analysis. However, commonly used IgG antibodies are oftentimes not applicable to 3D imaging, due to their relatively large size and consequently inadequate tissue penetration and penetration speed. The lack of suitable reagents for 3D histopathology can be overcome by an emerging class of single-domain antibodies, referred to as nanobodies (Nbs), which can facilitate rapid and superior 2D and 3D histological stainings. Here, we report the generation and experimental validation of Nbs directed against the human endothelial cell-selective adhesion molecule (hESAM), which enables spatial visualization of blood vascular networks in whole-mount 3D imaging. After analysis of Nb binding properties and quality, selected Nb clones were validated in 2D and 3D imaging approaches, demonstrating comparable staining qualities to commercially available hESAM antibodies in 2D, as well as rapid and complete staining of entire specimens in 3D. We propose that the presented hESAM-Nbs can serve as novel blood vessel markers in academic research and can potentially improve 3D histopathological diagnostics of entire human tissue specimens, leading to improved treatment and superior patient outcomes.

Development of a platform process for the production and purification of single-domain antibodies.

Single-domain antibodies (sdAbs) offer the affinity and therapeutic value of conventional antibodies, with increased stability and solubility. Unlike conventional antibodies, however, sdAbs do not benefit from a platform manufacturing process. While successful production of a variety of sdAbs has been shown in numerous hosts, purification methods are often molecule specific or require affinity tags, which generally cannot be used in clinical manufacturing due to regulatory concerns. Here, we have developed a broadly applicable production and purification process for sdAbs in Komagataella phaffii (Pichia pastoris) and demonstrated the production of eight different sdAbs at a quality appropriate for nonclinical studies. We developed a two-step, integrated purification process without the use of affinity resins and showed that modification of a single process parameter, pH of the bridging buffer, was required for the successful purification of a variety of sdAbs. Further, we determined that this parameter can be predicted based only on the biophysical characteristics of the target molecule. Using these methods, we produced nonclinical quality sdAbs as few as 5 weeks after identifying the product sequence. Nonclinical studies of three different sdAbs showed that molecules produced using our platform process conferred protection against viral shedding of rotavirus or H1N1 influenza and were equivalent to similar molecules produced in Escherichia coli and purified using affinity tags.

Interaction standards for biophysics: anti-lysozyme nanobodies.

There is a significant demand in the molecular biophysics community for robust standard samples. They are required by researchers, instrument developers and pharmaceutical companies for instrumental quality control, methodological development and in the design and validation of devices, diagnostics and instrumentation. To-date there has been no clear consensus on the need and type of standards that should be available and different research groups and instrument manufacturers use different standard systems which significantly hinders comparative analysis. One of the major objectives of the Association of Resources for Biophysical Research in Europe (ARBRE) is to establish a common set of standard samples that can be used throughout the biophysics community and instrument developers. A survey was circulated among ARBRE members to ascertain the requirements of laboratories when using standard systems and the results are documented in this article. In summary, the major requirements are protein samples which are cheap, relatively small, stable and have different binding strengths. We have developed a panel of sdAb's or 'nanobodies' against hen-egg white lysozyme with different binding strengths and suitable stability characteristics. Here we show the results of the survey, the selection procedure, validation and final selection of a panel of nanobody interaction standards.

A Single-Domain Antibody-Based Anti-PSMA Recombinant Immunotoxin Exhibits Specificity and Efficacy for Prostate Cancer Therapy.

Prostate cancer (PCa) is the second most common cancer in men, causing more than 300,000 deaths every year worldwide. Due to their superior cell-killing ability and the relative simplicity of their preparation, immunotoxin molecules have great potential in the clinical treatment of cancer, and several such molecules have been approved for clinical application. In this study, we adopted a relatively simple strategy based on a single-domain antibody (sdAb) and an improved Pseudomonas exotoxin A (PE) toxin (PE24X7) to prepare a safer immunotoxin against prostate-specific membrane antigen (PSMA) for PCa treatment. The designed anti-PSMA immunotoxin, JVM-PE24X7, was conveniently prepared in its soluble form in an Escherichia coli (E. coli) system, avoiding the complex renaturation process needed for immunotoxin preparation by the conventional strategy. The product was very stable and showed a very strong ability to bind the PSMA receptor. Cytotoxicity assays showed that this molecule at a very low concentration could kill PSMA-positive PCa cells, with an EC50 value (concentration at which the cell viability decreased by 50%) of 15.3 pM against PSMA-positive LNCaP cells. Moreover, this molecule showed very good killing selectivity between PSMA-positive and PSMA-negative cells, with a selection ratio of more than 300-fold. Animal studies showed that this molecule at a very low dosage (5 × 0.5 mg/kg once every three days) completely inhibited the growth of PCa tumors, and the maximum tolerable dose (MTD) was more than 15 mg/kg, indicating its very potent tumor-treatment ability and a wide therapeutic window. Use of the new PE toxin, PE24X7, as the effector moiety significantly reduced off-target toxicity and improved the therapeutic window of the immunotoxin. The above results demonstrate that the designed anti-PSMA immunotoxin, JVM-PE24X7, has good application value for the treatment of PCa.

Active Targeting of Dendritic Polyglycerols for Diagnostic Cancer Imaging.

Active tumor targeting involves the decoration of nanomaterials (NMs) with oncotropic vector biomolecules that selectively recognize certain antigens on malignant cells or in the tumor microenvironment. This strategy can facilitate intracellular uptake of NM through specific interactions such as receptor-mediated endocytosis and can lead to prolonged retention in the malignant tissues by preventing rapid efflux from the tumor. Here, the design of actively targeting, renally excretible bimodal dendritic polyglycerols (dPGs) for diagnostic cancer imaging is described. Single-domain antibodies (sdAbs) specifically binding to the epidermal growth factor receptor (EGFR) are employed herein as targeting warheads owing to their small size and high affinity for their corresponding antigen. The dPGs equipped with EGFR-targeting feature are compared head-to-head with their nontargeting counterparts in terms of interaction with EGFR-overexpressing cells in vitro as well as accumulation at receptor-positive tumors in vivo. Experimental results reveal a higher specificity and preferential tumor accumulation for the α-EGFR dPGs, resulting from the introduction of active targeting capabilities on their backbone. These results highlight the potential for improving the tumor uptake properties of dPGs by strategic use of sdAb functionalization, which can ultimately prove useful to the development of ultrasmall NM with highly specific tumor accumulation.

Camelid single-domain antibodies raised by DNA immunization are potent inhibitors of EGFR signaling.

Up-regulation of epidermal growth factor receptor (EGFR) is a hallmark of many solid tumors, and inhibition of EGFR signaling by small molecules and antibodies has clear clinical benefit. Here, we report the isolation and functional characterization of novel camelid single-domain antibodies (sdAbs or VHHs) directed against human EGFR. The source of these VHHs was a llama immunized with cDNA encoding human EGFR ectodomain alone (no protein or cell boost), which is notable in that genetic immunization of large, outbred animals is generally poorly effective. The VHHs targeted multiple sites on the receptor's surface with high affinity (KD range: 1-40 nM), including one epitope overlapping that of cetuximab, several epitopes conserved in the cynomolgus EGFR orthologue, and at least one epitope conserved in the mouse EGFR orthologue. Interestingly, despite their generation against human EGFR expressed from cDNA by llama cells in vivo (presumably in native conformation), the VHHs exhibited wide and epitope-dependent variation in their apparent affinities for native EGFR displayed on tumor cell lines. As fusions to human IgG1 Fc, one of the VHH-Fcs inhibited EGFR signaling induced by EGF binding with a potency similar to that of cetuximab (IC50: ∼30 nM). Thus, DNA immunization elicited high-affinity, functional sdAbs that were vastly superior to those previously isolated by our group through protein immunization.

Structural Insights into the Mechanism of a Nanobody That Stabilizes PAI-1 and Modulates Its Activity.

Plasminogen activator inhibitor-1 (PAI-1) is the main physiological inhibitor of tissue-type (tPA) and urokinase-type (uPA) plasminogen activators (PAs). Apart from being critically involved in fibrinolysis and wound healing, emerging evidence indicates that PAI-1 plays an important role in many diseases, including cardiovascular disease, tissue fibrosis, and cancer. Targeting PAI-1 is therefore a promising therapeutic strategy in PAI-1 related pathologies. Despite ongoing efforts no PAI-1 inhibitors were approved to date for therapeutic use in humans. A better understanding of the molecular mechanisms of PAI-1 inhibition is therefore necessary to guide the rational design of PAI-1 modulators. Here, we present a 1.9 Å crystal structure of PAI-1 in complex with an inhibitory nanobody VHH-s-a93 (Nb93). Structural analysis in combination with biochemical characterization reveals that Nb93 directly interferes with PAI-1/PA complex formation and stabilizes the active conformation of the PAI-1 molecule.

From atoms to physiology: what it takes to really understand inflammasomes.

Rapid inflammatory responses to cytosolic threats are mediated by inflammasomes - large macromolecular signalling complexes that control the activation of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-18, as well as cell death by pyroptosis. Different inflammasome sensors are activated by diverse direct and indirect signals, and subsequently nucleate the polymerization of the adaptor molecule ASC to form signalling platforms macroscopically observed as ASC specks. Caspase-1 is autocatalytically activated at these sites and subsequently matures pro-inflammatory cytokines and the pore-forming effector molecule gasdermin D. While most molecules and basic assembly principles have been deduced from reductionist experimental systems, we still lack fundamental information on the structure and regulation of these complexes in their physiological environment and in the interplay with other signalling pathways. In this review, novel experimental approaches are proposed, including some that rely on nanobodies and single domain antibodies, to understand inflammasome assembly and regulation in the context of the relevant tissues or cells.

Screening and purification of nanobodies from E. coli culture supernatants using the hemolysin secretion system.

BACKGROUND: The hemolysin (Hly) secretion system of E. coli allows the one-step translocation of hemolysin A (HlyA) from the bacterial cytoplasm to the extracellular medium, without a periplasmic intermediate. In this work, we investigate whether the Hly secretion system of E. coli is competent to secrete a repertoire of functional single-domain VHH antibodies (nanobodies, Nbs), facilitating direct screening of VHH libraries and the purification of selected Nb from the extracellular medium. RESULTS: We employed a phagemid library of VHHs obtained by immunization of a dromedary with three protein antigens from enterohemorrhagic E. coli (EHEC), namely, the extracellular secreted protein A (EspA), the extracellular C-terminal region of Intimin (Int280), and the translocated intimin receptor middle domain (TirM). VHH clones binding each antigen were enriched and amplified by biopanning, and subsequently fused to the C-terminal secretion signal of HlyA to be expressed and secreted in a E. coli strain carrying the Hly export machinery (HlyB, HlyD and TolC). Individual E. coli clones were grown and induced in 96-well microtiter plates, and the supernatants of the producing cultures directly used in ELISA for detection of Nbs binding EspA, Int280 and TirM. A set of Nb sequences specifically binding each of these antigens were identified, indicating that the Hly system is able to secrete a diversity of functional Nbs. We performed thiol alkylation assays demonstrating that Nbs are correctly oxidized upon secretion, forming disulphide bonds between cysteine pairs despite the absence of a periplasmic intermediate. In addition, we show that the secreted Nb-HlyA fusions can be directly purified from the supernatant of E. coli cultures, avoiding cell lysis and in a single affinity chromatography step. CONCLUSIONS: Our data demonstrate the Hly secretion system of E. coli can be used as an expression platform for screening and purification of Nb binders from VHH repertories.

Generation and Application of Fluorescent Anti-Human ß2-Microglobulin VHHs via Amino Modification.

The functionalization of VHHs enables their application in almost every aspect of biomedical inquiry. Amino modification remains a common strategy for protein functionalization, though is considered to be inferior to site-specific methods and cause protein property changes. In this paper, four anti-ß2M VHHs were selected and modified on the amino group by NHS-Fluo. The impacts of amino modification on these VHHs were drastically different, and among all th examples, the modified NB-1 maintained the original stability, bioactivity and homogeneity of unmodified NB-1. Specific recognition of VHHs targeting ß2M detected by fluorescence imaging explored the possible applications of VHHs. Via this study, we successfully functionalized the anti-ß2M VHHs through amino modification and the results are able to instruct the simple and fast functionalization of VHHs in biomedical researches.

Cryo-electron Microscopy Structures of Expanded Poliovirus with VHHs Sample the Conformational Repertoire of the Expanded State.

By using cryo-electron microscopy, expanded 80S-like poliovirus virions (poliovirions) were visualized in complexes with four 80S-specific camelid VHHs (Nanobodies). In all four complexes, the VHHs bind to a site on the top surface of the capsid protein VP3, which is hidden in the native virus. Interestingly, although the four VHHs bind to the same site, the structures of the expanded virus differ in detail in each complex, suggesting that each of the Nanobodies has sampled a range of low-energy structures available to the expanded virion. By stabilizing unique structures of expanded virions, VHH binding permitted a more detailed view of the virus structure than was previously possible, leading to a better understanding of the expansion process that is a critical step in infection. It is now clear which polypeptide chains become disordered and which become rearranged. The higher resolution of these structures also revealed well-ordered conformations for the EF loop of VP2, the GH loop of VP3, and the N-terminal extensions of VP1 and VP2, which, in retrospect, were present in lower-resolution structures but not recognized. These structural observations help to explain preexisting mutational data and provide insights into several other stages of the poliovirus life cycle, including the mechanism of receptor-triggered virus expansion. IMPORTANCE: When poliovirus infects a cell, it undergoes a change in its structure in order to pass RNA through its protein coat, but this altered state is short-lived and thus poorly understood. The structures of poliovirus bound to single-domain antibodies presented here capture the altered virus in what appear to be intermediate states. A careful analysis of these structures lets us better understand the molecular mechanism of infection and how these changes in the virus lead to productive-infection events.

Combining Structural Aggregation Propensity and Stability Predictions To Redesign Protein Solubility.

The aggregation propensity of each particular protein seems to be shaped by evolution according to its natural abundance in the cell. The production and downstream processing of recombinant polypeptides implies attaining concentrations that are orders of magnitude above their natural levels, often resulting in their aggregation; a phenomenon that precludes the marketing of many globular proteins for biomedical or biotechnological applications. Therefore, there is a huge interest in methods aimed to increase the proteins solubility above their natural limits. Here, we demonstrate that an updated version of our AGGRESCAN 3D structural aggregation predictor, that now takes into account protein stability, allows for designing mutations at specific positions in the structure that improve the solubility of proteins without compromising their conformation. Using this approach, we have designed a highly soluble variant of the green fluorescent protein and a human single-domain VH antibody displaying significantly reduced aggregation propensity. Overall, our data indicate that the solubility of unrelated proteins can be easily tuned by in silico-designed nondestabilizing amino acid changes at their surfaces.

Noninvasive imaging of immune responses.

At their margins, tumors often contain neutrophils, dendritic cells, and activated macrophages, which express class II MHC and CD11b products. The interplay between stromal cells, tumor cells, and migratory cells such as lymphocytes creates opportunities for noninvasive imaging of immune responses. We developed alpaca-derived antibody fragments specific for mouse class II MHC and CD11b products, expressed on the surface of a variety of myeloid cells. We validated these reagents by flow cytometry and two-photon microscopy to obtain images at cellular resolution. To enable noninvasive imaging of the targeted cell populations, we developed a method to site-specifically label VHHs [the variable domain (VH) of a camelid heavy-chain only antibody] with (18)F or (64)Cu. Radiolabeled VHHs rapidly cleared the circulation (t1/2 ≈ 20 min) and clearly visualized lymphoid organs. We used VHHs to explore the possibility of imaging inflammation in both xenogeneic and syngeneic tumor models, which resulted in detection of tumors with remarkable specificity. We also imaged the infiltration of myeloid cells upon injection of complete Freund's adjuvant. Both anti-class II MHC and anti-CD11b VHHs detected inflammation with excellent specificity. Given the ease of manufacture and labeling of VHHs, we believe that this method could transform the manner in which antitumor responses and/or infectious events may be tracked.