99mTc‐anti‐epidermal growth factor receptor nanobody for tumor imaging

Nanobodies are important biomolecules for tumor targeting. In this study, we synthesized and labeled anti‐epidermal growth factor receptor (EGFR) nanobody OA‐cb6 with ^99mTc(CO)₃⁺ and evaluated its characteristics for targeting the EGFR in the A431 human epidermal carcinoma cell line. Nanobody radiolabeling was achieved with high yield and radiochemical purity, and the radioconjugate was stable. Biodistribution results in nude mice exhibited a favorable tumor‐to‐muscle ratio at 4‐hr postinjection, and tumor location was visualized at 4 hr after injection of radiolabeled nanobody. Our result showed that the OA‐cb6‐^99mTc‐tricarbonyl radiolabeled nanobody is a promising radiolabeled biomolecule for tumor imaging in cancers with high EGFR overexpression.     

Cryo-EM structure determination of small proteins by nanobody-binding scaffolds (Legobodies)

We describe a general method that allows structure determination of small proteins by single-particle cryo-electron microscopy (cryo-EM). The method is based on the availability of a target-binding nanobody, which is then rigidly attached to two scaffolds: 1) a Fab fragment of an antibody directed against the nanobody and 2) a nanobody-binding protein A fragment fused to maltose binding protein and Fab-binding domains. The overall ensemble of ∼120 kDa, called Legobody, does not perturb the nanobody–target interaction, is easily recognizable in EM images due to its unique shape, and facilitates particle alignment in cryo-EM image processing. The utility of the method is demonstrated for the KDEL receptor, a 23-kDa membrane protein, resulting in a map at 3.2-Å overall resolution with density sufficient for de novo model building, and for the 22-kDa receptor-binding domain (RBD) of SARS-CoV-2 spike protein, resulting in a map at 3.6-Å resolution that allows analysis of the binding interface to the nanobody. The Legobody approach thus overcomes the current size limitations of cryo-EM analysis.

Single-particle electron cryo-microscopy (cryo-EM) has become the method of choice for the determination of protein structures. Cryo-EM analysis has several advantages over X-ray crystallography or NMR (1), but the method becomes increasingly challenging for smaller proteins. Large molecules are relatively easy to identify in noisy low-dose images of vitrified samples and have sufficient contrast and features to determine their orientation and position for alignment and averaging. The structural analysis of small particles (∼100 kDa or less) is much more difficult. Small targets often lack recognizable shape features that can facilitate initial image alignment at low resolution. Without symmetry, small particles require optimal conditions, such as a highly homogeneous sample, rigid protein conformation, and random particle distribution in thin ice, conditions that are difficult to achieve with most samples (2). However, structure determination of small proteins is of great interest, as most proteins have sizes below 100 kDa and ∼50% are smaller than 50 kDa, including many membrane proteins and proteins of medical importance. It is thus a major goal in the field to expand the use of cryo-EM to the routine analysis of small proteins.One approach to employ cryo-EM for small proteins is based on phase contrast methods, such as the use of Volta phase plates. This method has been used to determine the structure of streptavidin, a protein of 52 kDa, at 3.2-Å resolution (3). However, the structure of this protein could be determined even without phase plates (4), likely because streptavidin forms rigid tetramers and the particles display a near-perfect distribution in very thin ice, which greatly facilitates structural analysis.An alternative strategy is to make the target protein larger, either by fusing it to another protein or by using a binding partner. In either case, high rigidity of the added scaffold itself and its rigid connection to the target protein are required to facilitate particle alignment and averaging in cryo-EM images.The fusion approach has been tried with different scaffolds. For example, in a recent study, the BRIL domain was fused into a loop of a small GPCR protein by extending helices on both sides of the fusion point; the size of the scaffold was further increased by a Fab directed against the BRIL domain (5). However, this approach is limited to proteins containing suitable α-helices; their extension has to be customized for each new target to generate a rigid connection, which is difficult to achieve without prior knowledge of the target structure.More promising is the use of a binding partner that can be selected with a screening platform, such as modified ankyrin repeat proteins (DARPins), Fab fragments of antibodies, or nanobodies. In recent studies, DARPins selected against GFP were grafted onto large scaffolds and used to visualize GFP by cryo-EM (6, 7). However, the intrinsic conformational heterogeneity of DARPins limits their potential to achieve high-resolution structures of small proteins (7), and so far only a few DARPins have been selected against membrane proteins. Fab fragments can be used as a fiducial marker to facilitate image alignment in cryo-EM images (8), but they have been mainly used in X-ray crystallography. Only a few examples of their application for cryo-EM analysis have been reported (9–11), in part because the selection of appropriate Fabs is not trivial. In addition, the size of the Fabs (∼50 kDa) and the existence of a somewhat flexible hinge region between the two subdomains still make structural analysis challenging.Nanobodies, derived from single-chain antibodies of camelids, are also becoming popular as versatile binding partners of target proteins. Nanobodies have several attractive features. They form rigid structures that can bind to diverse shapes of target proteins, such as loops, convex surfaces, and cavities (12). They can bind to small exposed surfaces, which may not be accessible to Fab fragments. Nanobodies can be selected from immunized camelids or from large in vitro libraries displayed by phages, yeast cells, or on ribosomes (12, 13), and can be produced in large quantities in a fairly short time. They often lock a protein into a fixed conformation, particularly in the case of membrane proteins, and have been used extensively to determine X-ray structures. The small size of nanobodies (∼12 to 15 kDa) limits their direct application in cryo-EM, but the problem might be overcome if one could increase their size with the rigid attachment of a large scaffold. One reported approach is to fuse a scaffold into a loop of the nanobody, generating a “megabody” (14). However, the linker consisted of β-strands between the nanobody and scaffold, which caused some flexibility and limited the use of the scaffold for particle alignment in cryo-EM analysis.Here, we describe a versatile method that allows cryo-EM analysis of even the smallest protein once a tightly binding nanobody is available. The size of the nanobody is increased to ∼120 kDa by two rigidly attached scaffolds. The overall design is reminiscent of a Lego construction, so we propose to call the scaffolds/nanobody ensemble “Legobody.” The utility of the Legobody method is demonstrated by structures of two small proteins (22 kDa and 23 kDa) that are asymmetric monomers and have a size well below the estimated limit for direct cryo-EM single-particle analysis (∼40 kDa) (15). The Legobody approach can easily be applied to any target protein and should greatly expand the use of cryo-EM single-particle analysis by overcoming the current size limitations.     

TAFIa inhibiting nanobodies as profibrinolytic tools and discovery of a new TAFIa conformation

Summary. Background: Because activated thrombin activatable fibrinolysis inhibitor (TAFIa) has very powerful antifibrinolytic properties, co‐administration of t‐PA and a TAFIa inhibitor enhances t‐PA treatment. Objective: We aimed to generate nanobodies specifically inhibiting the TAFIa activity and to test their effect on t‐PA induced clot lysis. Results: Five nanobodies, raised towards an activated more stable TAFIa mutant (TAFIa A¹⁴⁷‐C³⁰⁵‐I³²⁵‐I³²⁹‐Y³³³‐Q³³⁵), are described. These nanobodies inhibit specifically TAFIa activity, resulting in an inhibition of up to 99% at a 16‐fold molar excess of nanobody over TAFIa, IC₅₀’s range between 0.38‐ and > 16‐fold molar excess. In vitro clot lysis experiments in the absence of thrombomodulin (TM) demonstrate that the nanobodies exhibit profibrinolytic effects. However, in the presence of TM, one nanobody exhibits an antifibrinolytic effect whereas the other nanobodies show a slight antifibrinolytic effect at low concentrations and a pronounced profibrinolytic effect at higher concentrations. This biphasic pattern was highly dependent on TM and t‐PA concentration. The nanobodies were found to bind in the active‐site region of TAFIa and their time‐dependent differential binding behavior during TAFIa inactivation revealed the occurrence of a yet unknown intermediate conformational transition. Conclusion: These nanobodies are very potent TAFIa inhibitors and constitute useful tools to accelerate fibrinolysis. Our data also demonstrate that the profibrinolytic effect of TAFIa inhibition may be reversed by the presence of TM. The identification of a new conformational transition provides new insights into the conformational inactivation of the unstable TAFIa.     

Generation and characterization of nanobodies targeting PSMA for molecular imaging of prostate cancer

Nanobodies show attractive characteristics for tumor targeting in cancer diagnosis and therapy. A radiolabeled nanobody binding the prostate‐specific membrane antigen (PSMA) could offer a noninvasive strategy to select prostate cancer patients eligible for PSMA‐targeted therapies. We here describe the generation, production and in vivo evaluation of anti‐PSMA nanobodies. Nanobodies were derived from heavy‐chain‐only antibodies, raised in immunized dromedaries. Binding characteristics were evaluated through ELISA and flow cytometry. Selected nanobodies were radiolabeled with ^99mTc at their hexahistidine tail, after which cell binding capacity and internalization were evaluated on PSMA^pos LNCaP and PSMA^neg PC3 cell lines. In vivo tumor targeting was analyzed in both LNCaP and PC3 xenografted mice through SPECT/microCT and tissue sampling. A panel of 72 generated clones scored positive on ELISA, all contributing to three nanobody groups, of which group 3 dominated with 70 clones. ELISA and FACS analysis led to the selection of two dominant nanobodies. ^99mTc‐labeled PSMA6 and PSMA30 both showed specific binding on LNCAP cells, but not on PC3 cells. ^99mTc‐PSMA30 internalized significantly more in LNCaP cells compared to ^99mTc‐PSMA6. Higher absolute tumor uptake and tumor‐to‐normal organ ratios were observed for ^99mTc‐PSMA30 compared with ^99mTc‐PSMA6 and a ^99mTc‐control nanobody in LNCaP but not in PC3 tumor‐bearing mice. PSMA30 nanobody has improved targeting characteristics both in vitro as well as in vivo compared with PSMA6 and the control nanobody, and was therefore selected as our in‐house‐developed lead compound for PSMA targeting. Copyright © 2014 John Wiley & Sons, Ltd.     

Sortase A‐mediated site‐specific labeling of camelid single‐domain antibody‐fragments: a versatile strategy for multiple molecular imaging modalities

A generic site‐specific conjugation method that generates a homogeneous product is of utmost importance in tracer development for molecular imaging and therapy. We explored the protein‐ligation capacity of the enzyme Sortase A to label camelid single‐domain antibody‐fragments, also known as nanobodies. The versatility of the approach was demonstrated by conjugating independently three different imaging probes: the chelating agents CHX‐A"‐DTPA and NOTA for single‐photon emission computed tomography (SPECT) with indium‐111 and positron emission tomography (PET) with gallium‐68, respectively, and the fluorescent dye Cy5 for fluorescence reflectance imaging (FRI). After a straightforward purification process, homogeneous single‐conjugated tracer populations were obtained in high yield (30–50%). The enzymatic conjugation did not affect the affinity of the tracers, nor the radiolabeling efficiency or spectral characteristics. In vivo, the tracers enabled the visualization of human epidermal growth factor receptor 2 (HER2) expressing BT474M1‐tumors with high contrast and specificity as soon as 1 h post injection in all three imaging modalities. These data demonstrate Sortase A‐mediated conjugation as a valuable strategy for the development of site‐specifically labeled camelid single‐domain antibody‐fragments for use in multiple molecular imaging modalities. Copyright © 2016 John Wiley & Sons, Ltd.     

Uniform Orientation of Biotinylated Nanobody as an Affinity Binder for Detection of Bacillus thuringiensis (Bt) Cry1Ac Toxin

Nanobodies are the smallest natural fragments with useful properties such as high affinity, distinct paratope and high stability, which make them an ideal tool for detecting target antigens. In this study, we generated and characterized nanobodies against the Cry1Ac toxin and applied them in a biotin-streptavidin based double antibodies (nanobodies) sandwich-ELISA (DAS-ELISA) assay. After immunizing a camel with soluble Cry1Ac toxin, a phage displayed library was constructed to generate Nbs against the Cry1Ac toxin. Through successive rounds of affinity bio-panning, four nanobodies with greatest diversity in CDR3 sequences were obtained. After affinity determination and conjugating to HRP, two nanobodies with high affinity which can recognize different epitopes of the same antigen (Cry1Ac) were selected as capture antibody (Nb61) and detection antibody (Nb44). The capture antibody (Nb61) was biotinylated in vivo for directional immobilization on wells coated with streptavidin matrix. Both results of specificity analysis and thermal stability determination add support for reliability of the following DAS-ELISA with a minimum detection limit of 0.005 μg·mL⁻¹ and a working range 0.010–1.0 μg·mL⁻¹. The linear curve displayed an acceptable correlation coefficient of 0.9976. These results indicated promising applications of nanobodies for detection of Cry1Ac toxin with biotin-streptavidin based DAS-ELISA system.     

Therapeutic bispecific antibody formats: a patent applications review (1994-2017)

Introduction: Bispecific antibodies have become increasingly of interest by enabling new therapeutic applications such as retargeting cellular immunity towards tumor cells. About 23 bispecific antibody platforms have therefore been developed, generating about 62 molecules which are currently being evaluated for potential treatment of a variety of indications, such as cancer and inflammatory diseases, among which three molecules were approved. This class of drugs will represent a multi-million-dollar market over the coming years. Many companies have consequently invested in the development of bispecific antibody platforms, creating an important patent activity in this field.

Areas covered: The present review gives an overview of the patent literature over the period 1994–2017 of different immunoglobulin gamma-based bispecific antibody platforms and the molecules approved or in clinical trials.

Expert opinion: Bispecific antibodies are progressively accepted as potentially superior therapeutic molecules in a broad range of diseases. This frantic activity creates a maze of hundreds of patents that pose considerable legal risks for both newcomers and established companies. It can consecutively be anticipated that the number of patent conflicts will increase. Nevertheless, it can be expected that patents related to the use of a bispecific antibody will have tremendous commercial value.     

Localization,mechanism and reduction of renal retention of technetium‐99m labeled epidermal growth factor receptor‐specific nanobody in mice

Background

Nanobodies are single‐domain antigen binding fragments derived from functional heavy‐chain antibodies elicited in Camelidae. They are powerful probes for radioimmunoimaging, but their renal uptake is relatively high. In this study we have evaluated the role of megalin on the renal uptake of anti‐EGFR ^99mTc‐7C12 nanobody and the potency of gelofusine and/or lysine to reduce renal uptake of ^99mTc‐7C12.

Methods

First we compared the renal uptake of ^99mTc‐7C12 in megalin‐deficient and megalin‐wild‐type mice using pinhole SPECT/microCT and ex vivo analysis. The effect of gelofusine and lysine administration on renal accumulation of ^99mTc‐7C12 was analyzed in CD‐1 mice divided into lysine preload at 30 min before tracer injection (LysPreload), LysPreload + gelofusine coadministration (LysPreload + GeloCoad), lysine coadministration (LysCoad), gelofusine coadministration (GeloCoad) and LysCoad + GeloCoad. The combined effect of gelofusine and lysine on tumor uptake was tested in mice xenografts.

Results

Renal uptake of ^99mTc‐7C12 was 44.22 ± 3.46% lower in megalin‐deficient compared with megalin‐wild‐type mice. In CD‐1 mice, lysine preload had no effect on the renal retention whereas coinjection of lysine or gelofusine with the tracer resulted in 25.12 ± 2.99 and 36.22 ± 3.07% reduction, respectively. The combined effect of gelofusine and lysine was the most effective, namely a reduction of renal retention of 45.24 ± 2.09%. Gelofusine and lysine coadministration improved tumor uptake.

Conclusion

Megalin contributes to the renal accumulation of ^99mTc‐7C12. Gelofusine and lysine coinjection with the tracer reduces the renal uptake while tumor uptake is improved. Although this methodology allows for optimization of imaging protocol using nanobodies, further improvements are needed before using these molecules for radionuclide therapy. Copyright © 2010 John Wiley & Sons, Ltd.     

VHH212 nanobody targeting the hypoxia-inducible factor 1α suppresses angiogenesis and potentiates gemcitabine therapy in pancreatic cancer in vivo

Objective:We aimed to develop a novel anti-HIF-1α intrabody to decrease gemcitabine resistance in pancreatic cancer patients.Methods:Surface plasmon resonance and glutathione S-transferase pull-down assays were conducted to identify the binding affinity and specificity of anti-HIF-1α VHH212 [a single-domain antibody (nanobody)]. Molecular dynamics simulation was used to determine the protein-protein interactions between hypoxia-inducible factor-1α (HIF-1α) and VHH212. The real-time polymerase chain reaction (PCR) and Western blot analyses were performed to identify the expressions of HIF-1α and VEGF-A in pancreatic ductal adenocarcinoma cell lines. The efficiency of the VHH212 nanobody in inhibiting the HIF-1 signaling pathway was measured using a dual-luciferase reporter assay. Finally, a PANC-1 xenograft model was developed to evaluate the anti-tumor efficiency of combined treatment. Immunohistochemistry analysis was conducted to detect the expressions of HIF-1α and VEGF-A in tumor tissues.Results:VHH212 was stably expressed in tumor cells with low cytotoxicity, high affinity, specific subcellular localization, and neutralization of HIF-1α in the cytoplasm or nucleus. The binding affinity between VHH212 and the HIF-1α PAS-B domain was 42.7 nM. Intrabody competitive inhibition of the HIF-1α heterodimer with an aryl hydrocarbon receptor nuclear translocator was used to inhibit the HIF-1/VEGF pathway in vitro. Compared with single agent gemcitabine, co-treatment with gemcitabine and a VHH212-encoding adenovirus significantly suppressed tumor growth in the xenograft model with 80.44% tumor inhibition.Conclusions:We developed an anti-HIF-1α nanobody and showed the function of VHH212 in a preclinical murine model of PANC-1 pancreatic cancer. The combination of VHH212 and gemcitabine significantly inhibited tumor development. These results suggested that combined use of anti-HIF-1α nanobodies with first-line treatment may in the future be an effective treatment for pancreatic cancer.