Nick Devoogdt

Specific Cell Targeting with Nanobody Conjugated Branched Gold Nanoparticles for Photothermal Therapy

Branched gold nanoparticles are potential photothermal therapy agents because of their large absorption cross section in the near-infrared window. Upon laser irradiation they produce enough heat to destroy tumor cells. In this work, branched gold nanoparticles are biofunctionalized with nanobodies, the smallest fully functional antigen-binding fragments evolved from the variable domain, the VHH, of a camel heavy chain-only antibody. These nanobodies bind to the HER2 antigen which is highly expressed on breast and ovarian cancer cells. Flow cytometric analysis and dark field images of HER2 positive SKOV3 cells incubated with anti-HER2 conjugated branched gold nanoparticles show specific cell targeting. Laser irradiation studies reveal that HER2 positive SKOV3 cells exposed to the anti-HER2 targeted branched gold nanoparticles are destroyed after five minutes of laser treatment at 38 W/cm(2) using a 690 nm continuous wave laser. Starting from a nanoparticle optical density of 4, cell death is observed, whereas the control samples, nanoparticles with anti-PSA nanobodies, nanoparticles only, and laser only, do not show any cell death. These results suggest that this new type of bioconjugated branched gold nanoparticles are effective antigen-targeted photothermal therapeutic agents for cancer treatment.

Nanobody-Based Targeting of the Macrophage Mannose Receptor for Effective In Vivo Imaging of Tumor-Associated Macrophages

Tumor-associated macrophages (TAM) are an important component of the tumor stroma and exert several tumor-promoting activities. Strongly pro-angiogenic TAMs that reside in hypoxic tumor areas highly express macrophage mannose receptor (MMR, CD206). In this study, we targeted MMR+ TAMs using nanobodies, which are single-domain antigen-binding fragments derived from Camelidae heavy-chain antibodies. MMR-specific nanobodies stained TAMs in lung and breast tumor single-cell suspensions in vitro, and intravenous injection of 99mTc-labeled anti-MMR nanobodies successfully targeted tumor in vivo. Retention of the nanobody was receptor-specific and absent in MMR-deficient mice. Importantly, co-injection of excess unlabeled, bivalent anti-MMR nanobodies reduced nanobody accumulation in extratumoral organs to background levels, without compromising tumor uptake. Within tumors, the 99mTc-labeled nanobodies specifically labeled MMR+ TAMs, as CCR2-deficient mice that contain fewer TAMs showed significantly reduced tumor uptake. Further, anti-MMR nanobodies accumulated in hypoxic regions, thus targeting pro-angiogenic MMR+ TAMs. Taken together, our findings provide preclinical proof of concept that anti-MMR nanobodies can be used to selectively target and image TAM subpopulations in vivo.

Preclinical screening of anti-HER2 nanobodies for molecular imaging of breast cancer

Accurate determination of tumor human epidermal growth factor receptor 2 (HER2)‐status in breast cancer patients is possible via noninvasive imaging, provided adequate tracers are used. In this study, we describe the generation of a panel of 38 nanobodies, small HER2‐binding fragments that are derived from heavy‐chain‐only antibodies raised in an immunized dromedary. In search of a lead compound, a subset of nanobodies was biochemically characterized in depth and preclinically tested for use as tracers for imaging of xenografted tumors. The selected compound, 2Rs15d, was found to be stable and to interact specifically with HER2 recombinant protein and HER2‐expressing cells in ELISA, surface plasmon resonance, flow cytometry, and radioligand binding studies with low nanomolar affinities, and did not compete with anti‐HER2 therapeutic antibodies trastuzumab and pertuzumab. Single‐photon‐emission computed tomography (SPECT) imaging quantification and biodistribution analyses showed that 99mTc‐labeled 2Rs15d has a high tumor uptake in 2 HER2+ tumor models, fast blood clearance, low accumulation in nontarget organs except kidneys, and high concomitant tumor‐to‐blood and tumor‐to‐muscle ratios at 1 h after intravenous injection. These values were dramatically lower for an irrelevant control 99mTc‐nanobody and for 99mTc‐2Rs15d targeting a HER2– tumor.—Vaneycken, I., Devoogdt, N., Van Gassen, N., Vincke, C., Xavier, C., Wernery, U., Muyldermans, S., Lahoutte, T., Caveliers, V. Preclinical screening of anti‐HER2 nanobodies for molecular imaging of breast cancer. FASEB J. 25, 2433–2446 (2011). www.fasebj.org

Nanobodies and their potential applications

Nanobodies are recombinant, antigen-specific, single-domain, variable fragments of camelid heavy chain-only antibodies. The innate supremacy of nanobodies as a renewable source of affinity reagents, together with their high production yield in a broad variety of expression systems, minimal size, great stability, reversible refolding and outstanding solubility in aqueous solutions, and ability to specifically recognize unique epitopes with subnanomolar affinity, have combined to make them a useful class of biomolecules for research and various medical diagnostic and therapeutic applications. This article speculates on a number of technological innovations that might be introduced in the nanobody identification platform to streamline the generation of more potent nanobodies and to expand their application range.

Nanobodies Targeting Mouse/Human VCAM1 for the Nuclear Imaging of Atherosclerotic Lesions

Rationale: A noninvasive tool allowing the detection of vulnerable atherosclerotic plaques is highly needed. By combining nanomolar affinities and fast blood clearance, nanobodies represent potential radiotracers for cardiovascular molecular imaging. Vascular cell adhesion molecule-1 (VCAM1) constitutes a relevant target for molecular imaging of atherosclerotic lesions. Objective: We aimed to generate, radiolabel, and evaluate anti-VCAM1 nanobodies for noninvasive detection of atherosclerotic lesions. Methods and Results: Ten anti-VCAM1 nanobodies were generated, radiolabeled with technetium-99m, and screened in vitro on mouse and human recombinant VCAM1 proteins and endothelial cells and in vivo in apolipoprotein E–deficient (ApoE−/−) mice. A nontargeting control nanobody was used in all experiments to demonstrate specificity. All nanobodies displayed nanomolar affinities for murine VCAM1. Flow cytometry analyses using human human umbilical vein endothelial cells indicated murine and human VCAM1 cross-reactivity for 6 of 10 nanobodies. The lead compound cAbVCAM1-5 was cross-reactive for human VCAM1 and exhibited high lesion-to-control (4.95±0.85), lesion-to-heart (8.30±1.11), and lesion-to-blood ratios (4.32±0.48) (P<0.05 versus control C57Bl/6J mice). Aortic arch atherosclerotic lesions of ApoE−/− mice were successfully identified by single-photon emission computed tomography imaging. 99mTc-cAbVCAM1-5 binding specificity was demonstrated by in vivo competition experiments. Autoradiography and immunohistochemistry further confirmed cAbVCAM1-5 uptake in VCAM1-positive lesions. Conclusions: The 99mTc-labeled, anti-VCAM1 nanobody cAbVCAM1-5 allowed noninvasive detection of VCAM1 expression and displayed mouse and human cross-reactivity. Therefore, this study demonstrates the potential of nanobodies as a new class of radiotracers for cardiovascular applications. The nanobody technology might evolve into an important research tool for targeted imaging of atherosclerotic lesions and has the potential for fast clinical translation.

Immuno-imaging using nanobodies

Immuno-imaging is a developing technology that aims at studying disease in patients using imaging techniques such as positron emission tomography in combination with radiolabeled immunoglobulin derived targeting probes. Nanobodies are the smallest antigen-binding antibody-fragments and show fast and specific targeting in vivo. These probes are currently under investigation as therapeutics but preclinical studies indicate that nanobodies could also become the next generation of magic bullets for immuno-imaging. Initial data show that imaging can be performed as early as 1 hour post-injection enabling the use of short-lived radio-isotopes. These unique properties should enable patient friendly and safe imaging protocols. This review focuses on the current status of radiolabeled nanobodies as targeting probes for immuno-imaging.

Nanobodies as Tools for In Vivo Imaging of Specific Immune Cell Types

Nanobodies are single-domain antigen-binding fragments derived from heavy-chain antibodies that are devoid of light chains and occur naturally in Camelidae. We have shown before that their small size and high affinity and specificity for their target antigen make Nanobodies ideal probes for in vivo tumor imaging. In the present study, we have evaluated the use of Nanobodies as a generic method for imaging the in vivo biodistribution of specific immune cell types, using myeloid cells as an example. Methods: The cellular specificity of Nanobodies raised against murine bone marrow–derived dendritic cells was verified using flow cytometry on a range of myeloid and nonmyeloid cell types. The Nanobodies were then labeled with 99mTc and their biodistribution was analyzed using SPECT. The biodistribution was also assessed by measuring radioactivity in various organs and tissues. To verify whether the observed biodistribution was due to specific targeting through the antigen-binding loops, rather than retention in organs because of effects of the framework regions, we genetically grafted the antigen-binding loops of the Nanobodies onto the framework region of a Nanobody scaffold that by itself showed low background retention in the periphery. The cellular specificity and biodistribution of these grafted Nanobodies were determined as before. Results: Nb-DC2.1, which recognizes a wide range of myeloid cells, targets most strongly to the liver, spleen, and lungs. Nb-DC1.8, which recognizes immature bone marrow–derived dendritic cells in vitro, gives a much smaller signal in the liver and spleen than does Nb-DC2.1 but mainly targets to the lungs and gives a pronounced signal in the skin. Grafting of the antigen-binding loops of Nb-DC1.8 or Nb-DC2.1 to the scaffold of Nb-BCII10 alters the observed biodistribution of the Nanobodies to resemble that of the Nanobody from which the antigen-binding loops have been derived. Conclusion: The observed in vivo biodistribution of the Nanobodies reflects the main in vivo locations of the cells recognized by the Nanobodies and is determined by the antigen-binding loops of the Nanobodies. Thus, Nanobodies represent elegant targeting probes for imaging the in vivo biodistribution of specific immune cell types.

Synthesis, Preclinical Validation, Dosimetry, and Toxicity of 68Ga-NOTA-Anti-HER2 Nanobodies for iPET Imaging of HER2 Receptor Expression in Cancer

Nanobodies are the smallest fully functional antigen-binding antibody fragments possessing ideal properties as probes for molecular imaging. In this study we labeled the anti–human epidermal growth factor receptor type 2 (HER2) Nanobody with 68Ga via a 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) derivative and assessed its use for HER2 iPET imaging. Methods: The 2Rs15dHis6 Nanobody and the lead optimized current-good-manufacturing-practice grade analog 2Rs15d were conjugated with S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SCN-Bn-NOTA) to enable fast and efficient 68Ga labeling. Biodistribution and PET/CT studies were performed on HER2-positive and -negative tumor xenografts. The effect of injected mass on biodistribution was evaluated. The biodistribution data were extrapolated to calculate radiation dose estimates for the adult female using OLINDA software. A single-dose extended-toxicity study for NOTA-2Rs15d was performed on healthy mice up to a dose of 10 mg/kg. Results: Radiolabeling was quantitative (>97%) after 5 min of incubation at room temperature; specific activity was 55–200 MBq/nmol. Biodistribution studies showed fast and specific uptake (percentage injected activity [%IA]) in HER2-positive tumors (3.13 ± 0.06 and 4.34 ± 0.90 %IA/g for 68Ga-NOTA-2Rs15dHis6 and 68Ga-NOTA-2Rs15d, respectively, at 1 h after injection) and high tumor-to-blood and tumor-to-muscle ratios at 1 h after injection, resulting in high-contrast PET/CT images with high specific tumor uptake. A remarkable finding of the biodistribution studies was that kidney uptake was reduced by 60% for the Nanobody lacking the C-terminal His6 tag. The injected mass showed an effect on the general biodistribution: a 100-fold increase in NOTA-2Rs15d mass decreased liver uptake from 7.43 ± 1.89 to 2.90 ± 0.26 %IA/g whereas tumor uptake increased from 2.49 ± 0.68 to 4.23 ± 0.99 %IA/g. The calculated effective dose, based on extrapolation of mouse data, was 0.0218 mSv/MBq, which would yield a radiation dose of 4 mSv to a patient after injection of 185 MBq of 68Ga-NOTA-2Rs15d. In the toxicity study, no adverse effects were observed after injection of a 10 mg/kg dose of NOTA-2Rs15d. Conclusion: A new anti-HER2 PET tracer, 68Ga-NOTA-2Rs15d, was synthesized via a rapid procedure under mild conditions. Preclinical validation showed high-specific-contrast imaging of HER2-positive tumors with no observed toxicity. 68Ga-NOTA-2Rs15d is ready for first-in-human clinical trials.

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 (99m)Tc-7C12 nanobody and the potency of gelofusine and/or lysine to reduce renal uptake of (99m)Tc-7C12. METHODS First we compared the renal uptake of (99m)Tc-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 (99m)Tc-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 (99m)Tc-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 (99m)Tc-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.

In Vitro Analysis and In Vivo Tumor Targeting of a Humanized, Grafted Nanobody in Mice Using Pinhole SPECT/Micro-CT

Nanobodies are a novel type of immunoglobulinlike, antigen-binding protein with beneficial pharmacologic and pharmacokinetic properties that are ideally suited to targeting cellular antigens for molecular imaging or therapeutic purposes. However, because of their camelid, nonhuman origin, the possible immunogenicity of Nanobodies when used in the clinic is a concern. Here we present a new strategy to quickly generate humanized Nanobodies for molecular imaging purposes. Methods: We genetically grafted the antigen-binding loops of NbCEA5, a Nanobody with specificity for the colon carcinoma marker carcinoembryonic antigen (CEA), onto the framework of a humanized Nanobody scaffold. This scaffold has been previously characterized in our laboratory as a stable Nanobody that can serve as a universal loop acceptor for antigen-binding loops from donor Nanobodies and has been additionally mutated at about 10 crucial surface-exposed sites to resemble the sequence of human variable immunoglobulin domains. The 3 recombinant Nanobodies (NbCEA5, humanized scaffold, and humanized CEA5 graft) were produced in bacteria and purified. Unlabeled and 99mTc-labeled Nanobodies were biochemically characterized in vitro and tested as probes for SPECT/CT of xenografted tumors. Results: The success of loop-grafting was confirmed by comparing these Nanobodies for their capacity to recognize soluble CEA protein in enzyme-linked immunosorbent assay and by surface plasmon resonance and to bind to CEA-positive LS174T colon carcinoma cells and CEA-transfected but not untransfected Chinese hamster ovary cells in flow cytometry. Specificity of binding was confirmed by competition studies. All Nanobodies were heat-stable, could be efficiently labeled with 99mTc, and recognized both soluble and membrane-bound CEA protein in binding studies. Finally, biodistribution experiments were performed with intravenously injected 99mTc-labeled Nanobodies in LS174T tumor–bearing mice using pinhole SPECT/micro-CT. These in vivo experiments revealed specificity of tumor targeting and rapid renal clearance for all Nanobodies, with low signals in all organs besides the kidneys. Conclusion: This study shows the potency of antigen-binding loop-grafting to efficiently generate humanized Nanobodies that retain their targeting capacities for noninvasive in vivo imaging of tumors.