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.

Preclinical Evaluation of a Radiotheranostic Single-Domain Antibody Against Fibroblast Activation Protein α.

Fibroblast activation protein α (FAP) is highly expressed on cancer-associated fibroblasts of epithelial-derived cancers. Breast, colon, and pancreatic tumors often show strong desmoplastic reactions, which result in a dominant presence of stromal cells. FAP has gained interest as a target for molecular imaging and targeted therapies. Single-domain antibodies (sdAbs) are the smallest antibody-derived fragments with beneficial pharmacokinetic properties for molecular imaging and targeted therapy. Methods: We describe the generation, selection, and characterization of a sdAb against FAP. In mice, we assessed its imaging and therapeutic potential after radiolabeling with tracer-dose 131I and 68Ga for SPECT and PET imaging, respectively, and with 131I and 225Ac for targeted radionuclide therapy. Results: The lead sdAb, 4AH29, exhibiting picomolar affinity for a distinct FAP epitope, recognized both purified and membrane-bound FAP protein. Radiolabeled versions, including [68Ga]Ga-DOTA-4AH29, [225Ac]Ac-DOTA-4AH29, and [131I]I-guanidinomethyl iodobenzoate (GMIB)-4AH29, displayed radiochemical purities exceeding 95% and effectively bound to recombinant human FAP protein and FAP-positive GM05389 human fibroblasts. These radiolabeled compounds exhibited rapid and specific accumulation in human FAP-positive U87-MG glioblastoma tumors, with low but specific uptake in lymph nodes, uterus, bone, and skin (∼2-3 percentage injected activity per gram of tissue [%IA/g]). Kidney clearance of unbound [131I]I-GMIB-4AH29 was fast (<1 %IA/g after 24 h), whereas [225Ac]Ac-DOTA-4AH29 exhibited slower clearance (8.07 ± 1.39 %IA/g after 24 h and 2.47 ± 0.18 %IA/g after 96 h). Mice treated with [225Ac]Ac-DOTA-4AH29 and [131I]I-GMIB-4AH29 demonstrated prolonged survival compared with those receiving vehicle solution. Conclusion: [68Ga]Ga-DOTA-4AH29 and [131I]I-GMIB-4AH29 enable precise FAP-positive tumor detection in mice. Therapeutic [225Ac]Ac-DOTA-4AH29 and [131I]I-GMIB-4AH29 exhibit strong and sustained tumor targeting, resulting in dose-dependent therapeutic effects in FAP-positive tumor-bearing mice, albeit with kidney toxicity observed later for [225Ac]Ac-DOTA-4AH29. This study confirms the potential of radiolabeled sdAb 4AH29 as a radiotheranostic agent for FAP-positive cancers, warranting clinical evaluation.

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.

Preclinical Evaluation of 225Ac-Labeled Single-Domain Antibody for the Treatment of HER2pos Cancer.

Human epidermal growth factor receptor type 2 (HER2) is overexpressed in various cancers; thus, HER2-targeting single-domain antibodies (sdAb) could offer a useful platform for radioimmunotherapy. In this study, we optimized the labeling of an anti-HER2-sdAb with the α-particle-emitter 225Ac through a DOTA-derivative. The formed radioconjugate was tested for binding affinity, specificity and internalization properties, whereas cytotoxicity was evaluated by clonogenic and DNA double-strand-breaks assays. Biodistribution studies were performed in mice bearing subcutaneous HER2pos tumors to estimate absorbed doses delivered to organs and tissues. Therapeutic efficacy and potential toxicity were assessed in HER2pos intraperitoneal ovarian cancer model and in healthy C57Bl/6 mice. [225Ac]Ac-DOTA-2Rs15d exhibited specific cell uptake and cell-killing capacity in HER2pos cells (EC50 = 3.9 ± 1.1 kBq/mL). Uptake in HER2pos lesions peaked at 3 hours (9.64 ± 1.69% IA/g), with very low accumulation in other organs (<1% IA/g) except for kidneys (11.69 ± 1.10% IA/g). α-camera imaging presented homogeneous uptake of radioactivity in tumors, although heterogeneous in kidneys, with a higher signal density in cortex versus medulla. In mice with HER2pos disseminated tumors, repeated administration of [225Ac]Ac-DOTA-2Rs15d significantly prolonged survival (143 days) compared to control groups (56 and 61 days) and to the group treated with HER2-targeting mAb trastuzumab (100 days). Histopathologic evaluation revealed signs of kidney toxicity after repeated administration of [225Ac]Ac-DOTA-2Rs15d. [225Ac]Ac-DOTA-2Rs15d efficiently targeted HER2pos cells and was effective in treatment of intraperitoneal disseminated tumors, both alone and as an add-on combination with trastuzumab, albeit with substantial signs of inflammation in kidneys. This study warrants further development of [225Ac]Ac-DOTA-2Rs15d.

The Road to Personalized Myeloma Medicine: Patient-specific Single-domain Antibodies for Anti-idiotypic Radionuclide Therapy.

To this day, multiple myeloma remains an incurable cancer. For many patients, recurrence is unavoidably a result of lacking treatment options in the minimal residual disease stage. This is due to residual and treatment-resistant myeloma cells that can cause disease relapse. However, patient-specific membrane-expressed paraproteins could hold the key to target these residual cells responsible for disease recurrence. Here, we describe the therapeutic potential of radiolabeled, anti-idiotypic camelid single-domain antibody fragments (sdAbs) as tumor-restrictive vehicles against a membrane-bound paraprotein in the syngeneic mouse 5T33 myeloma model and analogously assess the feasibility of sdAb-based personalized medicine for patients with multiple myeloma. Llamas were immunized using extracts containing paraprotein from either murine or human sera, and selective sdAbs were retrieved using competitive phage display selections of immune libraries. An anti-5T33 idiotype sdAb was selected for targeted radionuclide therapy with the ß--particle emitter 177Lu and the α-particle emitter 225Ac. sdAb-based radionuclide therapy in syngeneic mice with a low 5T33 myeloma lesion load significantly delayed tumor progression. In five of seven patients with newly diagnosed myeloma, membrane expression of the paraprotein was confirmed. Starting from serum-isolated paraprotein, for two of three selected patients anti-idiotype sdAbs were successfully generated.

The Prognostic Value of CD206 in Solid Malignancies: A Systematic Review and Meta-Analysis.

An increased presence of CD206-expressing tumor associated macrophages in solid cancers was proposed to be associated with worse outcomes in multiple types of malignancies, but contradictory results are published. We performed a reproducible systematic review and meta-analysis to provide increased evidence to confirm or reject this hypothesis following the Preferred Reporting Items for Systematic Reviews and Meta-analyses statement. The Embase, Web of Science, and MEDLINE-databases were systematically searched for eligible manuscripts. A total of 27 papers studying the prognostic impact of CD206 in 14 different tumor types were identified. Meta-analyses showed a significant impact on the overall survival (OS) and disease-free survival (DFS). While no significant differences were revealed in progression-free survival (PFS) and disease-specific survival (DSS), a shift towards negative survival was correlated with increased CD206-expresion. As a result of the different tumor types, large heterogeneity was present between the different tumor types. Subgroup analysis of hepatocellular carcinoma and gastric cancers revealed no heterogeneity, associated with a significant negative impact on OS in both groups. The current systematic review displays the increased presence CD206-expressing macrophages as a significant negative prognostic biomarker for both OS and DFS in patients diagnosed with solid cancers. Because a heterogenous group of tumor types was included in the meta-analysis, the results cannot be generalized. These results can, however, be used to further lead follow-up research to validate the specific prognostic value of CD206 in individual tumor types and therapeutic approaches.

68Ga-Labeling: Laying the Foundation for an Anti-Radiolytic Formulation for NOTA-sdAb PET Tracers.

During the preparation of [68Ga]Ga-NOTA-sdAb at high activity, degradation of the tracers was observed, impacting the radiochemical purity (RCP). Increasing starting activities in radiolabelings is often paired with increased degradation of the tracer due to the formation of free radical species, a process known as radiolysis. Radical scavengers and antioxidants can act as radioprotectant due to their fast interaction with formed radicals and can therefore reduce the degree of radiolysis. This study aims to optimize a formulation to prevent radiolysis during the labeling of NOTA derivatized single domain antibody (sdAbs) with 68Ga. Gentisic acid, ascorbic acid, ethanol and polyvinylpyrrolidone were tested individually or in combination to find an optimal mix able to prevent radiolysis without adversely influencing the radiochemical purity (RCP) or the functionality of the tracer. RCP and degree of radiolysis were assessed via thin layer chromatography and size exclusion chromatography for up to three hours after radiolabeling. Individually, the radioprotectants showed insufficient efficacy in reducing radiolysis when using high activities of 68Ga, while being limited in amount due to negative impact on radiolabeling of the tracer. A combination of 20% ethanol (VEtOH/VBuffer%) and 5 mg ascorbic acid proved successful in preventing radiolysis during labeling with starting activities up to 1-1.2 GBq of 68Ga, and is able to keep the tracer stable for up to at least 3 h after labeling at room temperature. The prevention of radiolysis by the combination of ethanol and ascorbic acid potentially allows radiolabeling compatibility of NOTA-sdAbs with all currently available 68Ge/68Ga generators. Additionally, a design is proposed to allow the incorporation of the radioprotectant in an ongoing diagnostic kit development for 68Ga labeling of NOTA-sdAbs.

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.

Decorating sdAbs with Chelators: Effect of Conjugation on Biodistribution and Functionality.

Single domain antibodies (sdAbs) have proven to be valuable probes for molecular imaging. In order to produce such probes, one strategy is the functionalization of the reactive amine side chain of lysines with a chelator, resulting in a mixture of compounds with a different degree of conjugation. In this study, we implemented anion exchange chromatography (AEX) to separate the different compounds or fractions that were further characterized and evaluated to study the impact of the conjugation degree on pharmacokinetic properties and functionality. Anti-HER2 and anti-MMR sdAbs were functionalized with NOTA or DTPA chelator. Anion exchange chromatography was performed using 0.02 mol/L Tris pH 7.5 as the first solvent and 0.25 M or 0.4 M NaCl (in case of NOTA chelator or DTPA chelator, respectively) as the second solvent applied as a gradient. The fractions were characterized via mass spectrometry (MS), surface plasmon resonance (SPR), and isoelectric focusing gel electrophoresis (IEF), while in vivo studies were performed after radiolabeling with either 68Ga (NOTA) or 111In (DTPA) to assess the impact of the conjugation degree on pharmacokinetics. AEX could successfully be applied to separate fractions of (chelator)n-anti-HER2 and (chelator)n-anti-MMR sdAb constructs. MS confirmed the identity of different peaks obtained in the separation process. SPR measurement suggests a small loss of affinity for (chelator)3-anti-sdAb, while IEF revealed a correlated decrease in isoelectric point (pI) with the number of conjugated chelators. Interestingly, both the reduction in affinity and in pI was stronger with the DTPA chelator than with NOTA for both sdAbs. In vivo data showed no significant differences in organ uptake for any construct, except for (DTPA)n-anti-MMR, which showed a significantly higher liver uptake for (DTPA)1-anti-MMR compared to (DTPA)2-anti-MMR and (DTPA)3-anti-MMR. For all constructs in general, high kidney uptake was observed, due to the typical renal clearance of sdAb-based tracers. The kidney uptake showed significant differences between fractions of a same construct and indicates that a higher conjugation degree improves kidney clearance. AEX allows the separation of sdAbs with a different degree of conjugation and provides the opportunity to further characterize individual fractions. The conjugation of a chelator to sdAbs can alter some properties of the tracers, such as pI; however, the impact on the general biodistribution profile and tumor targeting was minimal.

Immunogenicity Risk Profile of Nanobodies.

Nanobodies (Nbs), the variable domains of camelid heavy chain-only antibodies, are a promising class of therapeutics or in vivo imaging reagents entering the clinic. They possess unique characteristics, including a minimal size, providing fast pharmacokinetics, high-target specificity, and an affinity in the (sub-)nanomolar range in conjunction with an easy selection and production, which allow them to outperform conventional antibodies for imaging and radiotherapeutic purposes. As for all protein theranostics, extended safety assessment and investigation of their possible immunogenicity in particular are required. In this study, we assessed the immunogenicity risk profile of two Nbs that are in phase II clinical trials: a first Nb against Human Epidermal growth factor Receptor 2 (HER2) for PET imaging of breast cancer and a second Nb with specificity to the Macrophage Mannose Receptor (MMR) for PET imaging of tumor-associated macrophages. For the anti-HER2 Nb, we show that only one out of 20 patients had a low amount of pre-existing anti-drug antibodies (ADAs), which only marginally increased 3 months after administering the Nb, and without negative effects of safety and pharmacokinetics. Further in vitro immunogenicity assessment assays showed that both non-humanized Nbs were taken up by human dendritic cells but exhibited no or only a marginal capacity to activate dendritic cells or to induce T cell proliferation. From our data, we conclude that monomeric Nbs present a low immunogenicity risk profile, which is encouraging for their future development toward potential clinical applications. One Sentence Summary: Nanobodies, the recombinant single domain affinity reagents derived from heavy chain-only antibodies in camelids, are proven to possess a low immunogenicity risk profile, which will facilitate a growing number of Nanobodies to enter the clinic for therapeutic or in vivo diagnostic applications.

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).

Therapeutic Efficacy of 213Bi-labeled sdAbs in a Preclinical Model of Ovarian Cancer.

Targeted alpha-particle therapy (TAT) might be a relevant therapeutic strategy to circumvent resistance to conventional therapies in the case of HER2-positive metastatic cancer. Single-domain antibody fragments (sdAb) are promising vehicles for TAT because of their excellent in vivo properties, high target affinity, and fast clearance kinetics. This study combines the cytotoxic α-particle emitter bismuth-213 (213Bi) and HER2-targeting sdAbs. The in vitro specificity, affinity, and cytotoxic potency of the radiolabeled complex were analyzed on HER2pos cells. Its in vivo biodistribution through serial dissections and via Cherenkov and micro-single-photon emission computed tomography (CT)/CT imaging was evaluated. Finally, the therapeutic efficacy and potential associated toxicity of [213Bi]Bi-DTPA-2Rs15d were evaluated in a HER2pos tumor model that manifests peritoneal metastasis. In vitro, [213Bi]Bi-DTPA-2Rs15d bound HER2pos cells in a HER2-specific way. In mice, high tumor uptake was reached already 15 min after injection, and extremely low uptake values were observed in normal tissues. Co-infusion of gelofusine resulted in a 2-fold reduction in kidney uptake. Administration of [213Bi]Bi-DTPA-2Rs15d alone and in combination with trastuzumab resulted in a significant increase in median survival. We describe for the very first time the successful labeling of an HER2-sdAb with the α-emitter 213Bi, and after intravenous administration, revealing high in vivo stability and specific accumulation in target tissue and resulting in an increased median survival of these mice especially in combination with trastuzumab. These results indicate the potential of [213Bi]Bi-DTPA-sdAb as a new radioconjugate for TAT, alone and as an add-on to trastuzumab for the treatment of HER2pos metastatic cancer.

Preclinical Targeted α- and ß--Radionuclide Therapy in HER2-Positive Brain Metastasis Using Camelid Single-Domain Antibodies.

HER2-targeted therapies have drastically improved the outcome for breast cancer patients. However, when metastasis to the brain is involved, current strategies fail to hold up to the same promise. Camelid single-domain antibody-fragments (sdAbs) have been demonstrated to possess favorable properties for detecting and treating cancerous lesions in vivo using different radiolabeling methods. Here we evaluate the anti-HER2 sdAb 2Rs15d, coupled to diagnostic γ- and therapeutic α- and ß--emitting radionuclides for the detection and treatment of HER2pos brain lesions in a preclinical setting. 2Rs15d was radiolabeled with 111In, 225Ac and 131I using DTPA- and DOTA-based bifunctional chelators and Sn-precursor of SGMIB respectively and evaluated in orthotopic tumor-bearing athymic nude mice. Therapeutic efficacy as well as systemic toxicity were determined for 131I- and 225Ac-labeled sdAbs and compared to anti-HER2 monoclonal antibody (mAb) trastuzumab in two different HER2pos tumor models. Radiolabeled 2Rs15d showed high and specific tumor uptake in both HER2pos SK-OV-3-Luc-IP1 and HER2pos MDA-MB-231Br brain lesions, whereas radiolabeled trastuzumab was unable to accumulate in intracranial SK-OV-3-Luc-IP1 tumors. Administration of [131I]-2Rs15d and [225Ac]-2Rs15d alone and in combination with trastuzumab showed a significant increase in median survival in 2 tumor models that remained largely unresponsive to trastuzumab treatment alone. Histopathological analysis revealed no significant early toxicity. Radiolabeled sdAbs prove to be promising vehicles for molecular imaging and targeted radionuclide therapy of metastatic lesions in the brain. These data demonstrate the potential of radiolabeled sdAbs as a valuable add-on treatment option for patients with difficult-to-treat HER2pos metastatic cancer.

Size and affinity kinetics of nanobodies influence targeting and penetration of solid tumours.

A compound's intratumoural distribution is an important determinant for the effectiveness of molecular therapy or imaging. Antibodies (Abs), though often used in the design of targeted compounds, struggle to achieve a homogenous distribution due to their large size and bivalent binding mechanism. In contrast, smaller compounds like nanobodies (Nbs) are expected to distribute more homogenously, though this has yet to be demonstrated in vivo at the microscopic level. We propose an intravital approach to evaluate the intratumoural distribution of different fluorescently labeled monomeric and dimeric Nb tracers and compare this with a monoclonal antibody (mAb). Monomeric and dimeric formats of the anti-HER2 (2Rb17c and 2Rb17c-2Rb17c) and control (R3B23 and R3B23-R3B23) Nb, as well as the dimeric monovalent Nb 2Rb17c-R3B23 were generated and fluorescently labeled with a Cy5 fluorophore. The mAb trastuzumab-Cy5 was also prepared. Whole-body biodistribution of all constructs was investigated in mice bearing subcutaneous xenografts (HER2+ SKOV3) using in vivo epi-fluorescence imaging. Next, for intravital experiments, GFP-expressing SKOV3 cells were grown under dorsal window chambers on athymic nude mice (n = 3/group), and imaged under a fluorescence stereo microscope immediately after intravenous injection of the tracers. Consecutive fluorescence images within the tumour were acquired over the initial 20 min after injection and later, single images were taken at 1, 3 and 24 h post-injection. Additionally, two-photon microscopy was used to investigate the colocalization of GFP (tumour cells) and Cy5 fluorescence (tracers) at higher resolution. Whole-body images showed rapid renal clearance of all Nbs, and fast tumour targeting for the specific Nbs. Specific tumour uptake of the mAb could only be clearly distinguished from background after several hours. Intravital imaging revealed that monomeric Nb tracers accumulated rapidly and distributed homogenously in the tumour mere minutes after intravenous injection. The dimeric compounds initially achieved lower fluorescence intensities than the monomeric. Furthermore, whereas the HER2-specific dimeric bivalent compound remained closely associated to the blood vessels over 24 h, the HER2-specific dimeric monovalent tracer achieved a more homogenous tumour distribution from 1 h post-injection onwards. Non-specific tracers were not retained in the tumour. Trastuzumab had the most heterogenous intratumoural distribution of all evaluated compounds, while -due to the long blood retention- achieving the highest overall tumour uptake at 24 h post-injection. In conclusion, monomeric Nbs very quickly and homogenously distribute through tumour tissue, at a rate significantly greater than dimeric Nbs and mAbs. This underlines the potential of monomeric Nb tracers and therapeutics in molecular imaging and targeted therapies.

Stromal-targeting radioimmunotherapy mitigates the progression of therapy-resistant tumors.

Radioimmunotherapy (RIT) aims to deliver a high radiation dose to cancer cells, while minimizing the exposure of normal cells. Typically, monoclonal antibodies are used to target the radionuclides to cancer cell surface antigens. However, antibodies face limitations due to their poor tumor penetration and suboptimal pharmacokinetics, while the expression of their target on the cancer cell surface may be gradually lost. In addition, most antigens are expressed in a limited number of tumor types. To circumvent these problems, we developed a Nanobody (Nb)-based RIT against a prominent stromal cell (stromal-targeting radioimmunotherapy or STRIT) present in nearly all tumors, the tumor-associated macrophage (TAM). Macrophage Mannose Receptor (MMR) functions as a stable molecular target on TAM residing in hypoxic areas, further allowing the delivery of a high radiation dose to the more radioresistant hypoxic tumor regions. Since MMR expression is not restricted to TAM, we first optimized a strategy to block extra-tumoral MMR to prevent therapy-induced toxicity. A 100-fold molar excess of unlabeled bivalent Nb largely blocks extra-tumoral binding of 177Lu-labeled anti-MMR Nb and prevents toxicity, while still allowing the intra-tumoral binding of the monovalent Nb. Interestingly, three doses of 177Lu-labeled anti-MMR Nb resulted in a significantly retarded tumor growth, thereby outcompeting the effects of anti-PD1, anti-VEGFR2, doxorubicin and paclitaxel in the TS/A mammary carcinoma model. Together, these data propose anti-MMR STRIT as a valid new approach for cancer treatment.

Beyond the Barrier: Targeted Radionuclide Therapy in Brain Tumors and Metastases.

Brain tumors are notoriously difficult to treat. The blood-brain barrier provides a sanctuary site where residual and metastatic cancer cells can evade most therapeutic modalities. The delicate nature of the brain further complicates the decision of eliminating as much tumorous tissue as possible while protecting healthy tissue. Despite recent advances in immunotherapy, radiotherapy and systemic treatments, prognosis of newly diagnosed patients remains dismal, and recurrence is still a universal problem. Several strategies are now under preclinical and clinical investigation to optimize delivery and maximize the cytotoxic potential of pharmaceuticals with regards to brain tumors. This review provides an overview of targeted radionuclide therapy approaches for the treatment of primary brain tumors and brain metastases, with an emphasis on biological targeting moieties that specifically target key biomarkers involved in cancer development.

Labeling of Anti-HER2 Nanobodies with Astatine-211: Optimization and the Effect of Different Coupling Reagents on Their in Vivo Behavior.

The use of nanobodies (Nbs) as vehicles in targeted alpha therapy (TAT) has gained great interest because of their excellent properties. They combine high in vivo affinity and specificity of binding with fast kinetics. This research investigates a novel targeted therapy that combines the α-particle emitter astatine-211 (211At) and the anti-HER2 Nb 2Rs15d to selectively target HER2+ cancer cells. Two distinctive radiochemical methodologies are investigated using three different coupling reagents. The first method uses the coupling reagents, N-succinimidyl 4-(1,2-bis-tert-butoxycarbonyl)guanidinomethyl-3-(trimethylstannyl)benzoate (Boc2-SGMTB) and N-succinimidyl-3-(trimethylstannyl)benzoate (m-MeATE), which are both directed to amino groups on the Nb, resulting in random conjugation. The second method aims at obtaining a homogeneous tracer population, via a site-specific conjugation of the N-[2-(maleimido)ethyl]-3-(trimethylstannyl)benzamide (MSB) reagent onto the carboxyl-terminal cysteine of the Nb. The resulting radioconjugates are evaluated in vitro and in vivo. 2Rs15d is labeled with 211At using Boc2-SGMTB, m-MeATE, and MSB. After astatination and purification, the binding specificity of the radioconjugates is validated on HER2+ cells, followed by an in vivo biodistribution assessment in SKOV-3 xenografted mice. α-camera imaging is performed to determine uptake and activity distribution in kidneys/tumors. 2Rs15d astatination resulted in a high radiochemical purity >95% for all radioconjugates. The biodistribution studies of all radioconjugates revealed comparable tumor uptake (higher than 8% ID/g at 1 h). [211At]SAGMB-2Rs15d showed minor uptake in normal tissues. Only in the kidneys, a higher uptake was measured after 1 h, but decreased rapidly after 3 h. Astatinated Nbs consisting of m-MeATE or MSB reagents revealed elevated uptake in lungs and stomach, indicating the presence of released 211At. α-Camera imaging of tumors revealed a homogeneous activity distribution. The radioactivity in the kidneys was initially concentrated in the renal cortex, while after 3 h most radioactivity was measured in the medulla, confirming the fast washout into urine. Changing the reagents for Nb astatination resulted in different in vivo biodistribution profiles, while keeping the targeting moiety identical. Boc2-SGMTB is the preferred reagent for Nb astatination because of its high tumor uptake, its low background signals, and its fast renal excretion. We envision [211At]SAGMB-2Rs15d to be a promising therapeutic agent for TAT and aim toward efficacy evaluation.

Clinical Translation of [68Ga]Ga-NOTA-anti-MMR-sdAb for PET/CT Imaging of Protumorigenic Macrophages.

PURPOSE: Macrophage mannose receptor (MMR, CD206) expressing tumor-associated macrophages (TAM) are protumorigenic and was reported to negatively impact therapy responsiveness and is associated with higher chances of tumor relapse following multiple treatment regimens in preclinical tumor models. Since the distribution of immune cells within the tumor is often heterogeneous, sampling "errors" using tissue biopsies will occur. In order to overcome this limitation, we propose positron emission tomography (PET)/X-ray computed tomography (CT) imaging using 68Ga-labeled anti-MMR single-domain antibody fragment (sdAb) to assess the presence of these protumorigenic TAM. PROCEDURES: Cross-reactive anti-MMR-sdAb was produced according to good manufacturing practice (GMP) and conjugated to p-SCN-Bn-NOTA bifunctional chelator for 68Ga-labeling. Biodistribution and PET/CT studies were performed in wild-type and MMR-deficient 3LL-R tumor-bearing mice. Biodistribution data obtained in mice were extrapolated to calculate radiation dose estimates for the human adult using OLINDA software. A 7-day repeated dose toxicity study for NOTA-anti-MMR-sdAb was performed in healthy mice up to a dose of 1.68 mg/kg. RESULTS: [68Ga]Ga-NOTA-anti-MMR-sdAb was obtained with 76 ± 2 % radiochemical yield, 99 ± 1 % radiochemical purity, and apparent molar activity of 57 ± 11 GBq/µmol. In vivo biodistribution analysis showed fast clearance via the kidneys and retention in MMR-expressing organs and tumor, with tumor-to-blood and tumor-to-muscle ratios of 6.80 ± 0.62 and 5.47 ± 1.82, respectively. The calculated effective dose was 0.027 mSv/MBq and 0.034 mSv/MBq for male and female, respectively, which means that a proposed dose of 185 MBq in humans would yield a radiation dose of 5.0 and 6.3 mSv to male and female patients, respectively. In the toxicity study, no adverse effects were observed. CONCLUSIONS: Preclinical validation of [68Ga]Ga-NOTA-anti-MMR-sdAb showed high specific uptake of this tracer in MMR-expressing TAM and organs, with no observed toxicity. [68Ga]Ga-NOTA-anti-MMR-sdAb is ready for a phase I clinical trial.

Evaluation of an Anti-HER2 Nanobody Labeled with 225Ac for Targeted α-Particle Therapy of Cancer.

Human epidermal growth factor receptor type 2 (HER2) is overexpressed in numerous carcinomas. Nanobodies (Nbs) are the smallest antibody-derived fragments with beneficial characteristics for molecular imaging and radionuclide therapy. Therefore, HER2-targeting nanobodies could offer a valuable platform for radioimmunotherapy, especially when labeled with α-particle emitters, which provide highly lethal and localized radiation to targeted cells with minimal exposure to surrounding healthy tissues. In this study, the anti-HER2 2Rs15d-nanobody was conjugated with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid ( p-SCN-Bn-DOTA) and radiolabeled with an α-emitter 225Ac with a high yield (>90%) and a radiochemical purity above 95%. The 225Ac-DOTA-Nb binding affinity was 4.12 ± 0.47 nM with an immunoreactive fraction above 80%. Binding to low HER2-expressing MDA-MB-231 cells was negligible, whereas HER2-overexpressing SKOV-3 cells could be blocked with an excess of unlabeled nanobody, confirming the specificity of binding. Noncompeting binding to HER2 was observed in the presence of an excess of trastuzumab. The cell-associated fraction of 225Ac-DOTA-Nb was 34.72 ± 16.66% over 24 h. In vitro, the radioconjugate was toxic in an HER2-mediated and dose-dependent manner, resulting in IC50 values of 10.2 and 322.1 kBq/mL for 225Ac-DOTA-Nb and the 225Ac-DOTA control, respectively, on SKOV-3 cells, and 282.2 kBq/mL for 225Ac-DOTA-Nb on MDA-MB-231 cells. Ex vivo biodistribution studies, performed in mice bearing subcutaneous HER2-overexpressing and low HER2-expressing tumors, showed a fast uptake in SKOV-3 tumors compared to MDA-MB-231 (4.01 ± 1.58% ID/g vs 0.49 ± 0.20% ID/g after 2 h), resulting also in high tumor-to-normal tissue ratios. In addition, coinjection of 225Ac-DOTA-Nb with Gelofusine reduced kidney retention by 70%. This study shows that 225Ac-DOTA-Nb is a promising new radioconjugate for targeted α-particle therapy and supports its further development.