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.

Vismodegib for treatment of periocular basal cell carcinoma - 6-year experience from a tertiary cancer center

Abstract Background: The treatment of advanced periocular basal cell carcinomas becomes a challenge as surgery may involve highly mutilating procedures. Vismodegib is the first selective hedgehog inhibitor approved for the treatment of locally advanced tumors or metastatic disease. Objective: Analyze the results of treatment with vismodegib for advanced periocular basal cell carcinomas in a real-life setting of a reference center between 2014 and 2020. Methods: Retrospective longitudinal study. The patient's demographic profile, comorbidities, tumor characteristics, and treatment outcomes were analyzed. Results: A total of 13 patients were included. Median follow-up and treatment duration were 15.9 and 10.5 months, respectively. Objective clinical response rate was 76.9%: 30.8% had a complete response and 46.2% a partial response. The median duration of response was 13 months. Progressive disease was observed in 38.5% of cases, with a median of 19 months after the beginning of treatment. Eighty-four percent of the patients had at least one adverse event, and 61.54% needed to interrupt treatment temporarily or permanently to increase tolerability. Study limitations: Being a retrospective study in a real-life setting, the evaluation of objective clinical response was subjective to physician appreciation. Conclusion: Vismodegib is a safe and effective treatment for locally advanced basal cell carcinoma. To prevent recurrences, the drug should be used continually when tolerated. The role of neoadjuvant vismodegib before surgery is being investigated and might add an important step in searching for a definitive treatment for these cases.

Vismodegib for treatment of periocular basal cell carcinoma - 6-year experience from a tertiary cancer center.

BACKGROUND: The treatment of advanced periocular basal cell carcinomas becomes a challenge as surgery may involve highly mutilating procedures. Vismodegib is the first selective hedgehog inhibitor approved for the treatment of locally advanced tumors or metastatic disease. OBJECTIVE: Analyze the results of treatment with vismodegib for advanced periocular basal cell carcinomas in a real-life setting of a reference center between 2014 and 2020. METHODS: Retrospective longitudinal study. The patient's demographic profile, comorbidities, tumor characteristics, and treatment outcomes were analyzed. RESULTS: A total of 13 patients were included. Median follow-up and treatment duration were 15.9 and 10.5 months, respectively. Objective clinical response rate was 76.9%: 30.8% had a complete response and 46.2% a partial response. The median duration of response was 13 months. Progressive disease was observed in 38.5% of cases, with a median of 19 months after the beginning of treatment. Eighty-four percent of the patients had at least one adverse event, and 61.54% needed to interrupt treatment temporarily or permanently to increase tolerability. STUDY LIMITATIONS: Being a retrospective study in a real-life setting, the evaluation of objective clinical response was subjective to physician appreciation. CONCLUSION: Vismodegib is a safe and effective treatment for locally advanced basal cell carcinoma. To prevent recurrences, the drug should be used continually when tolerated. The role of neoadjuvant vismodegib before surgery is being investigated and might add an important step in searching for a definitive treatment for these cases.

Site-Specific Radiolabeling of a Human PD-L1 Nanobody via Maleimide-Cysteine Chemistry.

Immune checkpoint inhibitors targeting the programmed cell death-1 (PD-1) and its ligand PD-L1 have proven to be efficient cancer therapies in a subset of patients. From all the patients with various cancer types, only 20% have a positive response. Being able to distinguish patients that do express PD-1/PD-L1 from patients that do not allows patients to benefit from a more personalized and efficient treatment of tumor lesion(s). Expression of PD-1 and PD-L1 is typically assessed via immunohistochemical detection in a tumor biopsy. However, this method does not take in account the expression heterogeneity within the lesion, nor the possible metastasis. To visualize whole-body PD-L1 expression by PET imaging, we developed a nanobody-based radio-immunotracer targeting PD-L1 site-specifically labeled with gallium-68. The cysteine-tagged nanobody was site-specifically conjugated with a maleimide (mal)-NOTA chelator and radiolabeling was tested at different nanobody concentrations and temperatures. Affinity and specificity of the tracer, referred to as [68Ga]Ga-NOTA-mal-hPD-L1 Nb, were assayed by surface plasmon resonance and on PD-L1POS or PD-L1NEG 624-MEL cells. Xenografted athymic nude mice bearing 624-MEL PD-L1POS or PD-L1NEG tumors were injected with the tracer and ex vivo biodistribution was performed 1 h 20 min post-injection. Ideal 68Ga-labeling conditions were found at 50 °C for 15 min. [68Ga]Ga-NOTA-mal-hPD-L1 Nb was obtained in 80 ± 5% DC-RCY with a RCP > 99%, and was stable in injection buffer and human serum up to 3 h (>99% RCP). The in vitro characterization showed that the NOTA-functionalized Nb retained its affinity and specificity. Ex vivo biodistribution revealed a tracer uptake of 1.86 ± 0.67% IA/g in the positive tumors compared with 0.42 ± 0.04% IA/g in the negative tumors. Low background uptake was measured in the other organs and tissues, except for the kidneys and bladder, due to the expected excretion route of Nbs. The data obtained show that the site-specific 68Ga-labeled NOTA-mal-hPD-L1 Nb is a promising PET radio-immunotracer due to its ease of production, stability and specificity for PD-L1.

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.

Iris melanocytoma and secondary glaucoma.

Iris melanocytoma (IM) is a rare variant of iris nevus with distinctive clinical and histopathological features. A 66-year-old woman, with a history of right eye pigmented iris nevus, presented to us with a recent onset of visual acuity decrease in that eye. She had a melanocytic iris lesion with iridocorneal angle invasion, peripheral corneal adhesion, pupil corectopia, sectorial cataract and high intraocular pressure. Ultrasound biomicroscopy did not exclude malignant transformation, so excisional biopsy was performed revealing the presence of IM without signs of atypia. Subsequently, the patient underwent cataract surgery combined with iridoplasty and later an ab externo trabeculectomy. Most cases of IM remain stable and require no intervention, but in cases of unusual clinical course, with rapid growth or secondary glaucoma, surgical treatment is indicated as a diagnostic and therapeutic measure. This case report highlights the importance of a timely and multidisciplinary ophthalmological approach for a better visual outcome.

Anti-human PD-L1 Nanobody for Immuno-PET Imaging: Validation of a Conjugation Strategy for Clinical Translation.

Immune checkpoints, such as programmed death-ligand 1 (PD-L1), limit T-cell function and tumor cells use this ligand to escape the anti-tumor immune response. Treatments with monoclonal antibodies blocking these checkpoints have shown long-lasting responses, but only in a subset of patients. This study aims to develop a Nanobody (Nb)-based probe in order to assess human PD-L1 (hPD-L1) expression using positron emission tomography imaging, and to compare the influence of two different radiolabeling strategies, since the Nb has a lysine in its complementarity determining region (CDR), which may impact its affinity upon functionalization. The Nb has been conjugated with the NOTA chelator site-specifically via the Sortase-A enzyme or randomly on its lysines. [68Ga]Ga-NOTA-(hPD-L1) Nbs were obtained in >95% radiochemical purity. In vivo tumor targeting studies at 1 h 20 post-injection revealed specific tumor uptake of 1.89 ± 0.40%IA/g for the site-specific conjugate, 1.77 ± 0.29%IA/g for the random conjugate, no nonspecific organ targeting, and excretion via the kidneys and bladder. Both strategies allowed for easily obtaining 68Ga-labeled hPD-L1 Nbs in high yields. The two conjugates were stable and showed excellent in vivo targeting. Moreover, we proved that the random lysine-conjugation is a valid strategy for clinical translation of the hPD-L1 Nb, despite the lysine present in the CDR.

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.

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.

Evaluating a Single Domain Antibody Targeting Human PD-L1 as a Nuclear Imaging and Therapeutic Agent.

The PD-1:PD-L1 immune checkpoint axis is central in the escape of cancer cells from anticancer immune responses. Monoclonal antibodies (mAbs) specific for PD-L1 have been approved for treatment of various cancer types. Although PD-L1 blockade has proven its merit, there are still several aspects that require further attention to fully capitalize on its potential. One of these is the development of antigen-binding moieties that enable PD-L1 diagnosis and therapy. We generated human PD-L1 binding single domain antibodies (sdAbs) and selected sdAb K2, a sdAb with a high affinity for PD-L1, as a lead compound. SPECT/CT imaging in mice following intravenous injection of Technetium-99m (99mTc)-labeled sdAb K2 revealed high signal-to-noise ratios, strong ability to specifically detect PD-L1 in melanoma and breast tumors, and relatively low kidney retention, which is a unique property for radiolabeled sdAbs. We further showed using surface plasmon resonance that sdAb K2 binds to the same epitope on PD-L1 as the mAb avelumab, and antagonizes PD-1:PD-L1 interactions. Different human cell-based assays corroborated the PD-1:PD-L1 blocking activity, showing enhanced T-cell receptor signaling and tumor cell killing when PD-1POS T cells interacted with PD-L1POS tumor cells. Taken together, we present sdAb K2, which specifically binds to human PD-L1, as a new diagnostic and therapeutic agent in cancer management.

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.

Translational molecular imaging in exocrine pancreatic cancer.

Effective treatment for pancreatic cancer remains challenging, particularly the treatment of pancreatic ductal adenocarcinoma (PDAC), which makes up more than 95% of all pancreatic cancers. Late diagnosis and failure of chemotherapy and radiotherapy are all too common, and many patients die soon after diagnosis. Here, we make the case for the increased use of molecular imaging in PDAC preclinical research and in patient management.

Site-Specific Radioactive Labeling of Nanobodies.

Single-domain antibody fragments, also called nanobodies (Nbs), are increasingly being used as targeting molecular tools for imaging and/or targeted radionuclide therapy. To translate these tools to the clinic, it is preferred to obtain a homogeneous, well-defined, and well-characterized product. It has been shown that Sortase A, a transpeptidase found in Staphylococcus aureus, catalyzes the site-specific conjugation between a recognition oligopeptide (LPXTG, known as sortag) and an oligoglycine functionalized probe. This versatile technique manages to couple various molecular reagents, such as biotin, fluorophores, bifunctional chelators, etc., to the target protein containing the sortag. This chapter focuses on the site-specific coupling of a bifunctional chelator (e.g., CHX-A"-DTPA) to a Nb equipped with a C-terminal sortag. The chelator conjugated to the Nb can be radiolabeled with 111In or 177Lu for SPECT imaging or targeted radionuclide therapy, respectively.

Direct fluorine-18 labeling of heat-sensitive biomolecules for positron emission tomography imaging using the Al18F-RESCA method.

Positron emission tomography (PET) is a quickly expanding, non-invasive molecular imaging technology, and there is high demand for new specific imaging probes. Herein, we present a generic protocol for direct radiolabeling of heat-sensitive biomolecules with the positron-emitting radioisotope fluorine-18 (18F) using the aluminum fluoride restrained complexing agent (Al18F-RESCA) method. The Al18F-RESCA method combines the chemical advantages of a chelator-based radiolabeling method with the unique physical properties of the radionuclide of choice, fluorine-18. Proteins of interest can be conjugated to RESCA via amine coupling using (±)-H3RESCA-TFP, followed by purification using size-exclusion chromatography (SEC). Next, RESCA-derivatized biomolecules can be labeled in one step, at room temperature (~20 °C) in an aqueous medium with aluminum fluoride (Al18F). Al18F-labeled proteins can be obtained with moderate (12-17 GBq/µmol) to good (80-85 GBq/µmol) apparent molar activity, depending on the starting activity of 18F-. In addition, satisfactory radiochemical yields (35-55%, non-decay corrected) and high radiochemical purity (>98%, using gel filtration or solid-phase purification) are obtained. The mild radiolabeling procedure takes 0.5 h to complete and can be used for direct labeling of vector molecules such as peptides, protein scaffolds, and engineered antibody fragments.

Pharmacokinetics of radiolabeled dimeric sdAbs constructs targeting human CD20.

Single-domain antibody fragments (sdAbs) are the smallest functional antigen-binding fragments, derived from heavy chain-only camelid antibodies. When designed as radiolabeled monomeric probes for imaging and therapy of cancer, their fast and specific targeting results in high tumor-to-background ratios early after injection. However, their moderate absolute uptake into tumors might not always be sufficient to treat cancerous lesions. We have evaluated the pharmacokinetics of seven constructs derived from a CD20-targeting monomeric sdAb (αCD20). The constructs differed in affinity or avidity towards CD20 (dimeric αCD20-αCD20 and αCD20 fused to a non-targeting control sdAb, referred to as αCD20-ctrl) and blood half-lives (αCD20 fused to an albumin-targeting sdAb (αAlb) = αCD20-αAlb). The constructs were radiolabeled with 111In (imaging) and 177Lu (therapy) using the bifunctional chelator CHX-A"-DTPA and evaluated in vitro and in vivo. In mice, tumor uptake of 177Lu-DTPA-αCD20 decreased from 4.82 ±â€¯1.80 to 0.13 ±â€¯0.05% IA/g over 72 h. Due to its rapid blood clearance, tumor-to-blood (T/B) ratios of >100 were obtained within 24 h. Although in vitro internalization indicated that dimeric 177Lu-DTPA-αCD20-αCD20 was superior in terms of total cell-associated radioactivity, this was not confirmed in vivo. Blood clearance was slower and absolute tumor uptake became significantly higher for αCD20-αAlb. Blood levels of 177Lu-DTPA-αCD20-αAlb decreased from 68.30 ±â€¯10.53 to 3.58 ±â€¯0.66% IA/g over 120 h, while tumor uptake increased from 6.21 ±â€¯0.94 to 24.90 ±â€¯2.83% IA/g, resulting in lower T/B ratios. Taken together, these results indicate that the increased size of dimeric αCD20-αCD20 or the fusion of monomeric αCD20 to an albumin-targeting moiety (αAlb) counterbalance their improved tumor targeting capacity compared to monomeric αCD20.

A nanobody-based tracer targeting DPP6 for non-invasive imaging of human pancreatic endocrine cells.

There are presently no reliable ways to quantify endocrine cell mass (ECM) in vivo, which prevents an accurate understanding of the progressive beta cell loss in diabetes or following islet transplantation. To address this unmet need, we coupled RNA sequencing of human pancreatic islets to a systems biology approach to identify new biomarkers of the endocrine pancreas. Dipeptidyl-Peptidase 6 (DPP6) was identified as a target whose mRNA expression is at least 25-fold higher in human pancreatic islets as compared to surrounding tissues and is not changed by proinflammatory cytokines. At the protein level, DPP6 localizes only in beta and alpha cells within the pancreas. We next generated a high-affinity camelid single-domain antibody (nanobody) targeting human DPP6. The nanobody was radiolabelled and in vivo SPECT/CT imaging and biodistribution studies were performed in immunodeficient mice that were either transplanted with DPP6-expressing Kelly neuroblastoma cells or insulin-producing human EndoC-ßH1 cells. The human DPP6-expressing cells were clearly visualized in both models. In conclusion, we have identified a novel beta and alpha cell biomarker and developed a tracer for in vivo imaging of human insulin secreting cells. This provides a useful tool to non-invasively follow up intramuscularly implanted insulin secreting cells.

Theranostic Radiolabeled Anti-CD20 sdAb for Targeted Radionuclide Therapy of Non-Hodgkin Lymphoma.

Anti-CD20 radioimmunotherapy is an effective approach for therapy of relapsed or refractory CD20pos lymphomas, but faces limitations due to poor tumor penetration and undesirable pharmacokinetics of full antibodies. Camelid single-domain Ab fragments (sdAb) might circumvent some of the limitations of radiolabeled full antibodies. In this study, a set of hCD20-targeting sdAbs was generated, and their capacity to bind hCD20 was evaluated in vitro and in vivo A lead sdAb, sdAb 9079, was selected on the basis of its specific tumor targeting and significant lower kidney accumulation compared with other sdAbs. SdAb 9079 was then radiolabeled with 68Ga and 177Lu for PET imaging and targeted therapy. The therapeutic potential of 177Lu-DTPA-sdAb was compared with that of 177Lu-DTPA-rituximab and unlabeled rituximab in mice bearing hCD20pos tumors. Radiolabeled with 68Ga, sdAb 9079 showed specific tumor uptake, with very low accumulation in nontarget organs, except kidneys. The tumor uptake of 177Lu-DTPA-sdAb 9079 after 1.5 h was 3.4 ± 1.3% ID/g, with T/B and T/M ratios of 13.3 ± 4.6 and 32.9 ± 15.6. Peak tumor accumulation of 177Lu-DTPA-rituximab was about 9 times higher, but concomitantly with high accumulation in nontarget organs and very low T/B and T/M ratios (0.8 ± 0.1 and 7.1 ± 2.4). Treatment of mice with 177Lu-DTPA-sdAb 9079 significantly prolonged median survival compared with control groups and was as effective as treatment with rituximab or its 177Lu-labeled variant. Taken together, sdAb 9079 displays promising features as a theranostic drug to treat CD20pos lymphomas. Mol Cancer Ther; 16(12); 2828-39. ©2017 AACR.