Ilse Vaneycken

Comparison of the Biodistribution and Tumor Targeting of Two 99mTc-Labeled Anti-EGFR Nanobodies in Mice, Using Pinhole SPECT/Micro-CT

Camelidae possess an unusual class of antibodies devoid of light chains. Nanobodies are intact antigen-binding fragments that are stable, easily generated against different targets, and fully functional. Their rapid clearance from the blood circulation favors their use as imaging agents. We compared the in vivo tumor uptake and biodistribution of 2 anti–epidermal growth factor receptor (anti-EGFR) Nanobodies, 99mTc-7C12 and 99mTc-7D12. Methods: Nanobodies were labeled via their hexahistidine tail with 99mTc-tricarbonyl (99mTc(CO)3) generated from a kit. Mice bearing subcutaneous A431 (EGFR-positive) and R1M (EGFR-negative) xenografts were intravenously injected with 99mTc-7C12 and 99mTc-7D12 on separate days. Pinhole SPECT/micro-CT images were acquired at 1 h after administration to assess noninvasively the biodistribution and tumor targeting of the labeled compounds. Pinhole SPECT and micro-CT images from the same mouse were automatically fused on the basis of a mathematic rigid-body-transformation algorithm using six 57Co sources. Images were quantified, and tracer uptake was expressed as percentage injected activity per gram per cubic centimeter (%IA/cm3) of tissue. Ex vivo biodistribution of mice bearing A431 injected with either 99mTc-7C12 or 99mTc-7D12 was also assessed; activity in the tumor and organs was recorded and expressed as percentage injected activity per gram (%IA/g). Results: Binding of both tracers was receptor-specific. Image analysis showed high and similar tumor uptake values for both 99mTc-7C12 and 99mTc-7D12 (4.55 ± 0.24 %IA/cm3 and 4.62 ± 0.36 %IA/cm3, respectively) in A431 xenografts, whereas the uptake in the negative tumor (R1M) was low (1.16 ± 0.14 for 99mTc-7C12 and 1.49 ± 0.60 for 99mTc-7D12). 99mTc-7C12 showed significantly higher kidney uptake (63.48 ± 2.36 vs. 56.25 ± 2.46 %IA/cm3) and lower liver uptake (2.55 ± 0.26 vs. 4.88 ± 0.86 %IA/cm3) than did 99mTc-7D12. The ex vivo analysis confirmed the image quantification with high tumor-to-background ratio; however, 99mTc-7C12 showed higher tumor uptake (9.11 ± 1.12 %IA/g) than did 99mTc-7D12 (6.09 ± 0.77 %IA/g). 99mTc-7D12 demonstrated significantly higher blood activity than did 99mTc-7C12, but both showed short plasma half-lives (<10 min).Conclusion: The Nanobody fragments used here show high tumor uptake, low liver uptake, and rapid blood clearance. Nanobodies are promising probes for noninvasive radioimmunodetection of specific targets early after administration. On the basis of its favorable biodistribution, 99mTc-7C12 was selected for further studies.

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

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.

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.

Phase I Study of 68Ga-HER2-Nanobody for PET/CT Assessment of HER2 Expression in Breast Carcinoma

Human epidermal growth factor receptor 2 (HER2) status is one of the major tumor characteristics in breast cancer to guide therapy. Anti-HER2 treatment has clear survival advantages in HER2-positive breast carcinoma patients. Heterogeneity in HER2 expression between primary tumor and metastasis has repeatedly been described, resulting in the need to reassess HER2 status during the disease course. To avoid repeated biopsy with potential bias due to tumor heterogeneity, Nanobodies directed against HER2 have been developed as probes for molecular imaging. Nanobodies, which are derived from unique heavy-chain-only antibodies, are the smallest antigen-binding antibody fragments and have ideal characteristics for PET imaging. The primary aims were assessment of safety, biodistribution, and dosimetry. The secondary aim was to investigate tumor-targeting potential. Methods: In total, 20 women with primary or metastatic breast carcinoma (score of 2+ or 3+ on HER2 immunohistochemical assessment) were included. Anti-HER2-Nanobody was labeled with 68Ga via a NOTA derivative. Administered activities were 53–174 MBq (average, 107 MBq). PET/CT scans for dosimetry assessment were obtained at 10, 60, and 90 min after administration. Physical evaluation and blood analysis were performed for safety evaluation. Biodistribution was analyzed for 11 organs using MIM software; dosimetry was assessed using OLINDA/EXM. Tumor-targeting potential was assessed in primary and metastatic lesions. Results: No adverse reactions occurred. A fast blood clearance was observed, with only 10% of injected activity remaining in the blood at 1 h after injection. Uptake was seen mainly in the kidneys, liver, and intestines. The effective dose was 0.043 mSv/MBq, resulting in an average of 4.6 mSv per patient. The critical organ was the urinary bladder wall, with a dose of 0.406 mGy/MBq. In patients with metastatic disease, tracer accumulation well above the background level was demonstrated in most identified sites of disease. Primary lesions were more variable in tracer accumulation. Conclusion: 68Ga-HER2-Nanobody PET/CT is a safe procedure with a radiation dose comparable to other routinely used PET tracers. Its biodistribution is favorable, with the highest uptake in the kidneys, liver, and intestines but very low background levels in all other organs that typically house primary breast carcinoma or tumor metastasis. Tracer accumulation in HER2-positive metastases is high, compared with normal surrounding tissues, and warrants further assessment in a phase II trial.

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.

PET Imaging of Macrophage Mannose Receptor–Expressing Macrophages in Tumor Stroma Using 18F-Radiolabeled Camelid Single-Domain Antibody Fragments

Tumor-associated macrophages constitute a major component of the stroma of solid tumors, encompassing distinct subpopulations with different characteristics and functions. We aimed to identify M2-oriented tumor-supporting macrophages within the tumor microenvironment as indicators of cancer progression and prognosis, using PET imaging. This can be realized by designing 18F-labeled camelid single-domain antibody fragments (sdAbs) specifically targeting the macrophage mannose receptor (MMR), which has been identified as an important biomarker on this cell population. Methods: Cross-reactive anti-MMR sdAbs were generated after immunization of an alpaca with the extracellular domains of both human and mouse MMR. The lead binder was chosen on the basis of comparisons of binding affinity and in vivo pharmacokinetics. The PET tracer 18F-fluorobenzoate (FB)-anti-MMR sdAb was developed using the prosthetic group N-succinimidyl-4-18F-fluorobenzoate (18F-SFB), and its biodistribution, tumor-targeting potential, and specificity in terms of macrophage and MMR targeting were evaluated in mouse tumor models. Results: Four sdAbs were selected after affinity screening, but only 2 were found to be cross-reactive for human and mouse MMR. The lead anti-MMR 3.49 sdAb, bearing an affinity of 12 and 1.8 nM for mouse and human MMR, respectively, was chosen for its favorable in vivo biodistribution profile and tumor-targeting capacity. 18F-FB-anti-MMR 3.49 sdAb was synthesized with a 5%–10% radiochemical yield using an automated and optimized protocol. In vivo biodistribution analyses showed fast clearance via the kidneys and retention in MMR-expressing organs and tumor. The kidney retention of the fluorinated sdAb was 20-fold lower than a 99mTc-labeled counterpart. Compared with MMR- and C-C chemokine receptor 2–deficient mice, significantly higher uptake was observed in tumors grown in wild-type mice, demonstrating the specificity of the 18F tracer for MMR and macrophages, respectively. Conclusion: Anti-MMR 3.49 was denoted as the lead cross-reactive MMR-targeting sdAb. 18F radiosynthesis was optimized, providing an optimal probe for PET imaging of the tumor-promoting macrophage subpopulation in the tumor stroma.

Development of 177Lu‐nanobodies for radioimmunotherapy of HER2‐positive breast cancer: evaluation of different bifunctional chelators

Nanobodies show favourable pharmacokinetic characteristics for tumor targeting, including high tumor-to-background-ratios. Labelled with a therapeutic radionuclide, nanobodies could be used as an adjuvant treatment option for HER2-overexpressing minimal residual disease. The therapeutic radionuclide Lutetium-177 is linked to the nanobody using a bifunctional chelator. The choice of the bifunctional chelator could affect the in vivo behaviour of the radiolabeled nanobody. Consequently, we compared four different bifunctional chelators - p-SCN-Bn-DOTA, DOTA-NHS-ester, CHX-A-DTPA or 1B4M-DTPA - in order to select the optimal chemical link between Lutetium-177 and a HER2 targeting nanobody. MS results revealed different degrees of chelator-conjugation. High stability in time was observed, together with nanomolar affinities on HER2-expressing tumor cells. Ex vivo biodistributions as well as SPECT/micro-CT analyses showed high activities in tumors expressing medium HER2 levels with low background activity except for the kidneys. The 1B4M-DTPA-coupled conjugate was further evaluated in a high HER2-expressing tumor model. Here, tumor uptake values of 5.99u2009±u20090.63, 5.12u2009±u20090.17, 2.83u2009±u20090.36 and 2.47u2009±u20090.38 %IA/g were obtained at 1, 3, 24 and 48h p.i., which coincided with exceptionally low background values, except for the kidneys, and unprecedented tumor-to-background ratios. No specific binding was observed in a HER2-negative model. In conclusion, the in-house developed anti-HER2 nanobody 2Rs15dHIS can be successfully labeled with (177) Lu using different bifunctional chelators. Both macrocyclic and acyclic chelators show high stability in time. High specific tumor uptake combined with the lowest background uptake was measured using the 1B4M-DTPA-based conjugate.

Site-Specific Labeling of His-Tagged Nanobodies with 99m Tc: A Practical Guide

99mTc-tricarbonyl chemistry provides an elegant technology to site-specifically radiolabel histidine-tagged biomolecules. Considering their unique biochemical properties, this straightforward technology is particularly suited for Nanobodies. This chapter gives a detailed guide to generate highly specific Nanobody-derived radiotracers for both in vitro binding studies and in vivo molecular imaging.

18F-nanobody for PET imaging of HER2 overexpressing tumors

INTRODUCTIONnRadiolabeled nanobodies are exciting new probes for molecular imaging due to high affinity, high specificity and fast washout from the blood. Here we present the labeling of an anti-HER2 nanobody with (18)F and its validation for in vivo assessment of HER2 overexpression.nnnMETHODSnThe GMP grade anti-HER2 nanobody was labeled with the prosthetic group, N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]-SFB), and its biodistribution, tumor targeting and specificity were evaluated in mouse and rat tumor models.nnnRESULTSn[(18)F]FB-anti-HER2 nanobody was prepared with a 5-15% global yield (decay corrected) and a specific activity of 24.7 ± 8.2 MBq/nmol. In vivo studies demonstrated a high specific uptake for HER2 positive xenografts (5.94 ± 1.17 and 3.74 ± 0.52%IA/g, 1 and 3h p.i.) with high tumor-to-blood and tumor-to-muscle ratios generating high contrast PET imaging. The probe presented fast clearance through the kidneys (4%IA/g at 3h p.i.). [(18)F]FB-anti-HER2 nanobody is able to image HER2 expressing tumors when co-administered with the anti-HER2 therapeutic antibody trastuzumab (Herceptin), indicating the possibility of using the tracer in patients undergoing Herceptin therapy.nnnCONCLUSIONSnThe GMP grade anti-HER2 nanobody was labeled with (18)F. This new PET probe for imaging HER2 overexpression in tumors has ample potential for clinical translation.