Development and evaluation of a single domain antibody targeting folate receptor alpha for radioligand therapy.

Background: Folate receptor alpha (FRα) overexpression is seen in many cancers. Radioligand therapy (RLT) has emerged as a promising tool to target FRα and has been investigated previously, but further progression was limited due to high kidney retention and, subsequently, toxicity. To circumvent this, we present here the development of a [I]I-GMIB-conjugated anti-human FRα (hFRα) single-domain antibody (sdAb), with intrinsically fast renal clearance and concomitant low kidney retention.

Automated radiofluorination of HER2 single domain antibody: the road towards the clinical translation of [F]FB-HER2 sdAb.

Background: With the next generation of Human Epidermal Growth Factor Receptor 2 (HER2) -targeting therapies, such as antibody-drug conjugates, showing benefit in "HER2 low" and even "HER2 ultralow" patients, the need for novel methods to quantify HER2 expression accurately becomes even more important for clinical decision making. A HER2 PET/CT imaging assessment could evaluate HER2 positive disease locations while improving patient care, reducing the need for invasive biopsies. A single-domain antibody (sdAb)-based PET tracer could combine the high specificity of sdAbs with short-lived radionuclides such as fluorine-18 (F) and gallium-68 (Ga).

Optimizing the Therapeutic Index of sdAb-Based Radiopharmaceuticals Using Pretargeting.

Single-domain antibodies (sdAbs) demonstrate favorable pharmacokinetic profiles for molecular imaging applications. However, their renal excretion and retention are obstacles for applications in targeted radionuclide therapy (TRT). Using a click-chemistry-based pretargeting approach, we aimed to reduce kidney retention of a fibroblast activation protein α (FAP)-targeted sdAb, 4AH29, for Lu-TRT.

Generic semi-automated radiofluorination strategy for single domain antibodies: [F]FB-labelled single domain antibodies for PET imaging of fibroblast activation protein-α or folate receptor-α overexpression in cancer.

Background: Radiofluorination of single domain antibodies (sdAbs) via N-succinimidyl-4-[F]fluorobenzoate ([F]SFB) has shown to be a promising strategy in the development of sdAb-based PET tracers. While automation of the prosthetic group (PG) [F]SFB production, has been successfully reported, no practical method for large scale sdAb labelling has been reported. Therefore, we optimized and automated the PG production, enabling a subsequently efficient manual conjugation reaction to an anti-fibroblast activation protein (FAP)-α sdAb (4AH29) and an anti-folate receptor (FR)-α sdAb (2BD42).

Phase II Trial Assessing the Repeatability and Tumor Uptake of [Ga]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.

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.

Phase I Study of [Ga]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. [Ga]Ga-NOTA-anti-CD206 single-domain antibody (sdAb) is a PET tracer targeting CD206.

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.

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.

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.

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

Unlabelled: 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.

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

During the preparation of [Ga]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.

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.

Immunogenicity Risk Profile of Nanobodies.

Unlabelled: Nanobodies (Nbs), the variable domains of camelid heavy chain-only antibodies, are a promising class of therapeutics or 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.

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

I-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 I via the linker -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 I-GMIB-anti-HER2-VHH1 in healthy volunteers and breast cancer patients.

Therapeutic Efficacy of Bi-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 properties, high target affinity, and fast clearance kinetics. This study combines the cytotoxic α-particle emitter bismuth-213 (Bi) and HER2-targeting sdAbs.

Preclinical Targeted α- and β-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.

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.

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.

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.

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 (At) and the anti-HER2 Nb 2Rs15d to selectively target HER2+ cancer cells.

Clinical Translation of [Ga]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 Ga-labeled anti-MMR single-domain antibody fragment (sdAb) to assess the presence of these protumorigenic TAM.

Evaluation of an Anti-HER2 Nanobody Labeled with Ac 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.