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Abstract 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 (18F) and gallium-68 (68Ga). SdAb-based PET tracers have clinically been used via a 68Ga-chelator approach. However, the distribution of 68Ga-labelled pharmaceuticals to peripheral PET centres is more challenging to organize due to the short half-life of 68Ga, most certainly when the available activity is limited by a generator. Cyclotron produced 68Ga has removed this limitation. Distribution of 18F-labelled pharmaceuticals remains less challenging due to its slightly longer half-life, and radiofluorination of sdAbs via N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) has shown to be a promising strategy for developing sdAb-based PET tracers. Although [18F]SFB automation has been reported, automating protein conjugation proves challenging. Herein we report the fully automated, cartridge-based production of [18F]FB-HER2 sdAb on a single synthesis module. Results [18F]FB-HER2 sdAb (> 6 GBq) was obtained after a fully automated production (95 min), with a RCP > 95%, apparent molar activity > 20 GBq/µmol and decay-corrected radiochemical yield (RCY d.c.) of 14 ± 2% (n = 4). Further upscaling amounted to production batches of 16 GBq with an apparent molar activity > 40 GBq/µmol and RCY d.c. of 8 ± 1% (n = 4). Ex vivo biodistribution and PET imaging showed specific HER2-positive tumour targeting and low kidney retention. Conclusion The [18F]FB-HER2 sdAb tracer was produced with clinically relevant activities using a fully automated production method. The automated production method was designed to ease the translation to the clinic and has the potential to be used not only in mono-centre but also multi-centre clinical trials with one central production site. [18F]FB-HER2 sdAb showed a favourable biodistribution pattern and could be a valuable alternative to 68Ga-labelled sdAb-based PET tracers in the clinic.

Abstract Background Radiofluorination of single domain antibodies (sdAbs) via N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) has shown to be a promising strategy in the development of sdAb-based PET tracers. While automation of the prosthetic group (PG) [18F]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). Both the alpha isoform of FAP and the FR are established tumour markers. FAP-α is known to be overexpressed mainly by cancer-associated fibroblasts in breast, ovarian, and other cancers, while its expression in normal tissues is low or undetectable. FR-α has an elevated expression in epithelial cancers, such as ovarian, brain and lung cancers. Non-invasive imaging techniques, such as PET-imaging, using tracers targeting specific tumour markers can provide molecular information over both the tumour and its environment, which aides in the diagnosis, therapy selection and assessment of the cancer treatment. Results [18F]SFB was synthesized using a fully automated three-step, one-pot reaction. The total procedure time was 54 min and results in [18F]SFB with a RCP > 90% and a RCY d.c. of 44 ± 4% (n = 13). The manual conjugation reaction after purification produced [18F]FB-sdAbs with a RCP > 95%, an end of synthesis activity > 600 MBq and an apparent molar activity > 10 GBq/µmol. Overall RCY d.c., corrected to the trapping of [18F]F− on the QMA, were 9% (n = 1) and 5 ± 2% (n = 3) for [18F]FB-2BD42 and [18F]FB-4AH29, respectively. Conclusion [18F]SFB synthesis was successfully automated and upscaled on a Trasis AllInOne module. The anti-hFAP-α and anti-hFR-α sdAbs were radiofluorinated, yielding similar RCYs d.c. and RCPs, showing the potential of this method as a generic radiofluorination strategy for sdAbs. The radiofluorinated sdAbs showed a favourable biodistribution pattern and are attractive for further characterization as new PET tracers for FAP-α and FR-α imaging.

In cancer molecular imaging, selecting binders with high specificity and affinity for biomarkers is paramount for achieving high-contrast imaging within clinical time frames. Nanobodies have emerged as potent candidates, surpassing antibodies in pre-clinical imaging due to their convenient production, rapid renal clearance, and deeper tissue penetration. Multimerization of nanobodies is a popular strategy to enhance their affinity and pharmacokinetics; however, traditional methods are laborious and may yield heterogeneous products. In this study, we employ a Holliday junction (HJ)-like nucleic acid-based scaffold to create homogeneous nanostructures with precise multivalent and multiparatopic nanobody displays. The plug-and-play assembly allowed the screening of several nanobody multimer configurations for the detection of the breast cancer biomarker, human epidermal growth factor receptor 2 (HER2). In vitro studies demonstrated significant improvements in binding avidity, particularly with the biparatopic construct exhibiting high sensitivity, surpassing that of traditional antibody-based cell binding. Furthermore, our HJ platform allowed for adaptation from fluorescence-based to nuclear imaging, as demonstrated in xenografted mice, thereby allowing for future in vivo applications. This work highlights the potential of nucleic acid-mediated multimerization to markedly enhance nanobody binding, by exploring synergistic combinations and offering versatility for both in vitro diagnostics and cancer molecular imaging with prospects for future theranostic applications.

The precise delivery of cytotoxic radiation to cancer cells through the combination of a specific targeting vector with a radionuclide for targeted radionuclide therapy (TRT) has proven valuable for cancer care. TRT is increasingly being considered a relevant treatment method in fighting micro-metastases in the case of relapsed and disseminated disease. While antibodies were the first vectors applied in TRT, increasing research data has cited antibody fragments and peptides with superior properties and thus a growing interest in application. As further studies are completed and the need for novel radiopharmaceuticals nurtures, rigorous considerations in the design, laboratory analysis, pre-clinical evaluation, and clinical translation must be considered to ensure improved safety and effectiveness. Here, we assess the status and recent development of biological-based radiopharmaceuticals, with a focus on peptides and antibody fragments. Challenges in radiopharmaceutical design range from target selection, vector design, choice of radionuclides and associated radiochemistry. Dosimetry estimation, and the assessment of mechanisms to increase tumor uptake while reducing off-target exposure are discussed.

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.

Intraoperative guidance using targeted fluorescent tracers can potentially provide surgeons with real-time feedback on the presence of tumor tissue in resection margins. To overcome the limited depth penetration of fluorescent light, combining fluorescence with SPECT/CT imaging and/or gamma-ray tracing has been proposed. Here, we describe the design and preclinical validation of a novel bimodal nanobody-tracer, labeled using a “multifunctional single attachment point” (MSAP) label, integrating a Cy5 fluorophore and a diethylenetriaminepentaacetic acid (DTPA) chelator into a single structure. After conjugation of the bimodal MSAP to primary amines of the anti-HER2 nanobody 2Rs15d and 111In-labeling of DTPA, the tracer’s characteristics were evaluated in vitro. Subsequently, its biodistribution and tumor targeting were assessed by SPECT/CT and fluorescence imaging over 24 h. Finally, the tracer’s ability to identify small, disseminated tumor lesions was investigated in mice bearing HER2-overexpressing SKOV3.IP1 peritoneal lesions. [111In]In-MSAP.2Rs15d retained its affinity following conjugation and remained stable for 24 h. In vivo SPECT/CT and fluorescence images showed specific uptake in HER2-overexpressing tumors with low background. High tumor-to-muscle ratios were obtained at 1h p.i. and remained 19-fold on SPECT/CT and 3-fold on fluorescence images over 24 h. In the intraperitoneally disseminated model, the tracer allowed detection of larger lesions via nuclear imaging, while fluorescence enabled accurate removal of submillimeter lesions. Bimodal nuclear/fluorescent nanobody-tracers can thus be conveniently designed by conjugation of a single-molecule MSAP-reagent carrying a fluorophore and chelator for radioactive labeling. Such tracers hold promise for clinical applications.

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.

Since atherosclerotic plaques are small and sparse, their non-invasive detection via PET imaging requires both highly specific radiotracers as well as imaging systems with high sensitivity and resolution. This study aimed to assess the targeting and biodistribution of a novel fluorine-18 anti-VCAM-1 Nanobody (Nb), and to investigate whether sub-millimetre resolution PET imaging could improve detectability of plaques in mice. The anti-VCAM-1 Nb functionalised with the novel restrained complexing agent (RESCA) chelator was labelled with [18F]AlF with a high radiochemical yield (>75%) and radiochemical purity (>99%). Subsequently, [18F]AlF(RESCA)-cAbVCAM1-5 was injected in ApoE−/− mice, or co-injected with excess of unlabelled Nb (control group). Mice were imaged sequentially using a cross-over design on two different commercially available PET/CT systems and finally sacrificed for ex vivo analysis. Both the PET/CT images and ex vivo data showed specific uptake of [18F]AlF(RESCA)-cAbVCAM1-5 in atherosclerotic lesions. Non-specific bone uptake was also noticeable, most probably due to in vivo defluorination. Image analysis yielded higher target-to-heart and target-to-brain ratios with the β-CUBE (MOLECUBES) PET scanner, demonstrating that preclinical detection of atherosclerotic lesions could be improved using the latest PET technology.

CD38 is a multifunctional transmembrane glycoprotein found in multiple tissues and overexpressed in many cancer cells, notably in hematological malignancies such as leukemia and multiple myeloma (MM). Therefore, targeting CD38 remains an attractive strategy for cancer treatment in hematological malignancies as well as in solid tumors. It plays a critical role in the progression of these diseases through its ADP-ribosyl cyclase and cADPR-hydrolase activities. Its importance has led to the development of various anti-CD38 monoclonal antibodies (mAbs), including daratumumab and isatuximab, approved for MM treatment. These mAbs exert their anti-tumor effects through Fc-dependent immune mechanisms and immunomodulation, enhancing T-cell and NK-cell-mediated responses. However, resistance mechanisms arise during the treatment with daratumumab, creating the necessity for new therapies. This review explains current knowledge about the role of CD38 as a target in oncology and aims to delineate the use of single domain antibodies (sdAbs) as innovative theranostic tools in nuclear medicine. For diagnostic purposes, PET radionuclides like 68 Ga, 64Cu, and SPECT radionuclides like 99mTc and 111In, are commonly used. Significant progress has been made in anti-CD38 radioligand therapy (RLT), with anti-CD38 antibodies providing insights into tumor biology and treatment efficacy. In terms of therapy, RLT is a promising approach that offers precise targeting of malignant cells while minimizing exposure to healthy tissue. This involves the use of radionuclides emitting α particles, like 225Ac, 212Pb or 211At, and β−-particles like 90Y, 131I, or 177Lu, to exert cytotoxic effects. Derived from Camelidae heavy chain antibodies, sdAbs offer advantages over conventional mAbs such as small size, high stability, specificity, and ability to recognize hidden epitopes. CD38-specific sdAbs, such as sdAb 2F8, characterized by our laboratory, showing excellent tumor targeting and their engineered constructs, such as biparatopic antibodies and chimeric antibodies, represent a new generation of theranostic agents for diagnosis and treatment CD38-expressing malignancies. [ABSTRACT FROM AUTHOR]