Phase II Trial Assessing the Repeatability and Tumor Uptake of [68Ga]Ga-HER2 Single-Domain Antibody PET/CT in Patients with Breast Carcinoma.

Human epidermal growth factor receptor 2 (HER2) status is used for decision-making in breast carcinoma treatment. The status is obtained through immunohistochemistry or in situ hybridization. These two methods have the disadvantage of necessitating tissue sampling, which is prone to error due to tumor heterogeneity or interobserver variability. Whole-body imaging might be a solution to map HER2 expression throughout the body. Methods: Twenty patients with locally advanced or metastatic breast carcinoma (5 HER2-positive and 15 HER2-negative patients) were included in this phase II trial to assess the repeatability of uptake quantification and the extended safety of the [68Ga]Ga-NOTA-anti-HER2 single-domain antibody (sdAb). The tracer was injected, followed by a PET/CT scan at 90 min. Within 8 d, the procedure was repeated. Blood samples were taken for antidrug antibody (ADA) assessment and liquid biopsies. On available tissues, immunohistochemistry, in situ hybridization, and mass spectrometry were performed to determine the correlation of HER2 status with uptake values measured on PET. If relevant preexisting [18F]FDG PET/CT images were available (performed as standard of care), a comparison was made. Results: With a repeatability coefficient of 21.8%, this imaging technique was repeatable. No clear correlation between PET/CT uptake values and pathology could be established, as even patients with low levels of HER2 expression showed moderate to high uptake. Comparison with [18F]FDG PET/CT in 16 patients demonstrated that in 7 patients, [68Ga]Ga-NOTA-anti-HER2 shows interlesional heterogeneity within the same patient, and [18F]FDG uptake did not show the same heterogeneous uptake in all patients. In some patients, the extent of disease was clearer with the [68Ga]Ga-NOTA-anti-HER2-sdAb. Sixteen adverse events were reported but all without a clear relationship to the tracer. Three patients with preexisting ADAs did not show adverse reactions. No new ADAs developed. Conclusion: [68Ga]Ga-NOTA-anti-HER2-sdAb PET/CT imaging shows similar repeatability to [18F]FDG. It is safe for clinical use. There is tracer uptake in cancer lesions, even in patients previously determined to be HER2-low or -negative. The tracer shows potential in the assessment of interlesional heterogeneity of HER2 expression. In a subset of patients, [68Ga]Ga-NOTA-anti-HER2-sdAb uptake was seen in lesions with no or low [18F]FDG uptake. These findings support further clinical development of [68Ga]Ga-NOTA-anti-HER2-sdAb as a PET/CT tracer in breast cancer patients.

Phase I Study of [68Ga]Ga-Anti-CD206-sdAb for PET/CT Assessment of Protumorigenic Macrophage Presence in Solid Tumors (MMR Phase I).

Macrophages play an important role throughout the body. Antiinflammatory macrophages expressing the macrophage mannose receptor (MMR, CD206) are involved in disease development, ranging from oncology to atherosclerosis and rheumatoid arthritis. [68Ga]Ga-NOTA-anti-CD206 single-domain antibody (sdAb) is a PET tracer targeting CD206. This first-in-human study, as its primary objective, evaluated the safety, biodistribution, and dosimetry of this tracer. The secondary objective was to assess its tumor uptake. Methods: Seven patients with a solid tumor of at least 10 mm, an Eastern Cooperative Oncology Group score of 0 or 1, and good renal and hepatic function were included. Safety was evaluated using clinical examination and blood sampling before and after injection. For biodistribution and dosimetry, PET/CT was performed at 11, 90, and 150 min after injection; organs showing tracer uptake were delineated, and dosimetry was evaluated. Blood samples were obtained at selected time points for blood clearance. Metabolites in blood and urine were assessed. Results: Seven patients were injected with, on average, 191 MBq of [68Ga]Ga-NOTA-anti-CD206-sdAb. Only 1 transient adverse event of mild severity was considered to be possibly, although unlikely, related to the study drug (headache, Common Terminology Criteria for Adverse Events grade 1). The blood clearance was fast, with less than 20% of the injected activity remaining after 80 min. There was uptake in the liver, kidneys, spleen, adrenals, and red bone marrow. The average effective dose from the radiopharmaceutical was 4.2 mSv for males and 5.2 mSv for females. No metabolites were detected. Preliminary data of tumor uptake in cancer lesions showed higher uptake in the 3 patients who subsequently progressed than in the 3 patients without progression. One patient could not be evaluated because of technical failure. Conclusion: [68Ga]Ga-NOTA-anti-CD206-sdAb is safe and well tolerated. It shows rapid blood clearance and renal excretion, enabling high contrast-to-noise imaging at 90 min after injection. The radiation dose is comparable to that of routinely used PET tracers. These findings and the preliminary results in cancer patients warrant further investigation of this tracer in phase II clinical trials.

Lyophilization of NOTA-sdAbs: First step towards a cold diagnostic kit for 68Ga-labeling.

Lyophilization is commonly used in the production of pharmaceutical compounds to increase the stability of the Active Pharmaceutical Ingredient (API) by removing solvents. This study investigates the possibility to lyophilize an anti-HER2 and an anti-MMR single-domain antibody fragment (sdAb)-based precursor as a first step in the development of a diagnostic kit for PET imaging. METHODS: NOTA-sdAb precursors have been lyophilized with the following formulation: 100 µg NOTA-sdAb in 0.1 M NaOAc (NaOAc), 5% (w/v%) mannitol-sucrose mix at a 2:1 ratio and 0.1 mg/mL polysorbate 80. During development of the formulation and drying cycle, factors such as cake appearance, glass transition temperature and residual moisture were analyzed to ensure qualitative and stable lyophilized samples. Stability studies of lyophilized precursor were conducted up to 18 months after storage at 2-8 °C by evaluating the precursor integrity, aggregation, functionality and 68Ga-labeling efficiency. A comparative biodistribution study (lyophilized vs non-lyophilized precursor) was conducted in wild type mice (n = 3) and in tumor bearing mice (n = 6). RESULTS: The lyophilized NOTA-anti-HER2 precursor shows consistent stability data in vitro for up to 12 months at 2-8 °C in three separate batches, with results indicating stability even for up to T18m. No aggregation, degradation or activity loss was observed. Radiochemical purity after 68Ga-labeling is consistent over a period of 12 months (RCP ≥ 95% at T12m). In vivo biodistribution analyses show a typical [68Ga]Ga-NOTA-anti-HER2 sdAb distribution profile and a comparable tumor uptake for the lyophilized compound vs non-lyophilized (5.5% vs 5.7 %IA/g, respectively). In vitro results of lyophilized NOTA-anti-MMR precursor indicates stability for up to 18 months, while in vivo data show a comparable tumor uptake (2.5% vs 2.8 %IA/g, respectively) and no significant difference in kidney retention (49.4% vs 47.5 %IA/g, respectively). CONCLUSION: A formulation and specific freeze-drying cycle were successfully developed to lyophilize NOTA-sdAb precursors for long-term storage at 2-8 °C. In vivo data show no negative impact of the lyophilization process on the in vivo behavior or functionality of the lyophilized precursor. These results highlight the potential to develop a kit for the preparation of 68Ga-sdAb-based radiopharmaceuticals.

Phase I Trial of 131I-GMIB-Anti-HER2-VHH1, a New Promising Candidate for HER2-Targeted Radionuclide Therapy in Breast Cancer Patients.

131I-GMIB-anti-human epidermal growth factor receptor type 2 (HER2)-VHH1 is a targeted radionuclide theranostic agent directed at HER2-expressing cancers. VHH1 is a single-domain antibody covalently linked to therapeutic 131I via the linker N-succinimidyl 4-guanidino-methyl-3-iodobenzoate (SGMIB). The phase I study was aimed at evaluating the safety, biodistribution, radiation dosimetry, and tumor-imaging potential of 131I-GMIB-anti-HER2-VHH1 in healthy volunteers and breast cancer patients. Methods: In a first cohort, 6 healthy volunteers were included. The biodistribution of 131I-GMIB-anti-HER2-VHH1 was assessed using whole-body (anterior and posterior) planar images obtained at 40 min and at 2, 4, 24, and 72 h after intravenously administered (38 ± 9 MBq) 131I-GMIB-anti-HER2-VHH1. Imaging data were analyzed using OLINDA/EXM software to determine the dosimetry. Blood and urine samples were obtained over 72 h. In the second cohort, 3 patients with metastatic HER2-positive breast cancer were included. Planar whole-body imaging was performed at 2 and 24 h after injection. Additional SPECT/CT images were obtained after the whole-body images at 2 and 24 h if there was relevant uptake in known cancer lesions. Results: No drug-related adverse events were observed throughout the study. The biologic half-life of 131I-GMIB-anti-HER2-VHH1 in healthy subjects was about 8 h. After intravenous administration, the compound was eliminated from the blood with a 2.5-h half-life. The drug was eliminated primarily via the kidneys. The drug was stable in circulation, and there was no increased accumulation in the thyroid or stomach. The absorbed dose to the kidneys was 1.54 ± 0.25 mGy/MBq, and to bone marrow it was 0.03 ± 0.01 mGy/MBq. SPECT/CT imaging in patients with advanced breast cancer showed focal uptake of 131I-GMIB-anti-HER2-VHH1 in metastatic lesions. Conclusion: Because of its favorable toxicity profile and its uptake in HER2-positive lesions, this radiopharmaceutical can offer new therapeutic options to patients who have progressed on trastuzumab, pertuzumab, or trastuzmab emtansine, given its difference in mode-of-action. A dose escalation is planned in a subsequent phase I/II study to assess the therapeutic window of this compound (NCT04467515).

(18)F-nanobody for PET imaging of HER2 overexpressing tumors.

INTRODUCTION: Radiolabeled 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. METHODS: The 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. RESULTS: [(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. CONCLUSIONS: The 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.

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

UNLABELLED: 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 (68)Ga 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: (68)Ga-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.

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

UNLABELLED: 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 (18)F-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 (18)F-fluorobenzoate (FB)-anti-MMR sdAb was developed using the prosthetic group N-succinimidyl-4-(18)F-fluorobenzoate ((18)F-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. (18)F-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 (99m)Tc-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 (18)F tracer for MMR and macrophages, respectively. CONCLUSION: Anti-MMR 3.49 was denoted as the lead cross-reactive MMR-targeting sdAb. (18)F radiosynthesis was optimized, providing an optimal probe for PET imaging of the tumor-promoting macrophage subpopulation in the tumor stroma.

Synthesis, preclinical validation, dosimetry, and toxicity of 68Ga-NOTA-anti-HER2 Nanobodies for iPET imaging of HER2 receptor expression in cancer.

UNLABELLED: 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 (68)Ga 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 (68)Ga 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 (68)Ga-NOTA-2Rs15dHis6 and (68)Ga-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 (68)Ga-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, (68)Ga-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. (68)Ga-NOTA-2Rs15d is ready for first-in-human clinical trials.

Site-specific labeling of his-tagged Nanobodies with 99mTc: 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.

Molecular imaging using Nanobodies: a case study.

Molecular imaging is a noninvasive method to measure specific biological processes in animal models and patients using imaging. In recent years there has been a tremendous evolution in hardware and software for imaging purposes. This progress has created an urgent need for new labeled targeted molecular probes. The unique physicochemical and pharmacokinetic properties of Nanobodies match the requirements of the ideal molecular imaging tracer. Preclinical studies show strong and specific targeting in vivo with rapid clearance of unbound probe resulting in high contrasted images at early time points after intravenous administration. These data suggest that the Nanobody platform might become a generic method for the development of next generation molecular imaging probes.

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 5.99 ± 0.63, 5.12 ± 0.17, 2.83 ± 0.36 and 2.47 ± 0.38 %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.

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.

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 (99m)Tc-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 (99m)Tc-nanobody and for (99m)Tc-2Rs15d targeting a HER2(-) tumor.

In vitro analysis and in vivo tumor targeting of a humanized, grafted nanobody in mice using pinhole SPECT/micro-CT.

UNLABELLED: 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 (99m)Tc-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 (99m)Tc, and recognized both soluble and membrane-bound CEA protein in binding studies. Finally, biodistribution experiments were performed with intravenously injected (99m)Tc-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.

Comparison of the biodistribution and tumor targeting of two 99mTc-labeled anti-EGFR nanobodies in mice, using pinhole SPECT/micro-CT.

UNLABELLED: 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, (99m)Tc-7C12 and (99m)Tc-7D12. METHODS: Nanobodies were labeled via their hexahistidine tail with (99m)Tc-tricarbonyl ((99m)Tc(CO)(3)) generated from a kit. Mice bearing subcutaneous A431 (EGFR-positive) and R1M (EGFR-negative) xenografts were intravenously injected with (99m)Tc-7C12 and (99m)Tc-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 (57)Co sources. Images were quantified, and tracer uptake was expressed as percentage injected activity per gram per cubic centimeter (%IA/cm(3)) of tissue. Ex vivo biodistribution of mice bearing A431 injected with either (99m)Tc-7C12 or (99m)Tc-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 (99m)Tc-7C12 and (99m)Tc-7D12 (4.55+/-0.24 %IA/cm(3) and 4.62+/-0.36 %IA/cm(3), respectively) in A431 xenografts, whereas the uptake in the negative tumor (R1M) was low (1.16+/-0.14 for (99m)Tc-7C12 and 1.49+/-0.60 for (99m)Tc-7D12). (99m)Tc-7C12 showed significantly higher kidney uptake (63.48+/-2.36 vs. 56.25+/-2.46 %IA/cm(3)) and lower liver uptake (2.55+/-0.26 vs. 4.88+/-0.86 %IA/cm(3)) than did (99m)Tc-7D12. The ex vivo analysis confirmed the image quantification with high tumor-to-background ratio; however, (99m)Tc-7C12 showed higher tumor uptake (9.11+/-1.12 %IA/g) than did (99m)Tc-7D12 (6.09+/-0.77 %IA/g). (99m)Tc-7D12 demonstrated significantly higher blood activity than did (99m)Tc-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, (99m)Tc-7C12 was selected for further studies.