Thuy A. Tran

Background: Antibody-drug conjugates (ADCs) are gaining rapidly expanding clinical utility, with a growing number of approved indications across compounds that differ in target antigen, payload mechanism, and linker design. However, treatment response remains heterogeneous, and predictive biomarkers to identify patients most likely to benefit are limited. Given that ADC efficacy depends on sufficient target antigen expression and distribution across tumor lesions, positron emission tomography (PET) imaging offers a unique opportunity to non-invasively assess whole-body target availability. We therefore conducted a systematic literature review to evaluate the relationship between PET-measured tumor antigen expression and the therapeutic response to ADCs targeting the same antigen. Method: A systematic comprehensive search of PubMed, EMBASE and Web of Science databases was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, using terms related to targeted PET, ADC treatment and response assessment. Article screening, eligibility assessment, and data extraction were performed independently using predefined criteria by two reviewers. Disagreements were resolved by consensus. Results: A total of 5628 records were identified. After removing duplicates, 4323 records were screened by title and Fifty-eight underwent full-text review and seven were included in the final review. Four trials investigated human epidermal growth factor receptor 2 (HER2)-targeted therapy, one Nectin-4, one mesothelin and one STEAP1. In all HER2- and Nectin-4-related trials, patients with a higher uptake on targeted PET had a higher probability of responding to targeted treatment. Included trials were generally small and had a moderate risk of bias. Conclusion: Targeted PET imaging showed potential to predict treatment response in trials evaluating clinically active agents. Additional clinical trials across a broader range of targets, along with standardized acquisition protocols and harmonized study designs, are needed to enable the integration of this technique into clinical practice and ultimately translate its benefits to patients.

Background: Bispecific antibodies exploiting receptor-mediated transcytosis offer a promising strategy to overcome limited blood-brain barrier permeability in Alzheimer’s disease (AD) therapy and imaging. Lecanemab-Fab8D3 (Lec-Fab8D3), a bispecific anti-amyloid beta (Aβ) antibody engineered for enhanced brain delivery, holds potential as a companion immunoPET imaging diagnostic with the novel lecanemab immunotherapy. This study aimed to compare three radionuclides—zirconium-89 ( ⁸⁹ Zr), copper-64 ( ⁶⁴ Cu), and iodine-124 ( ¹²⁴ I)—for PET imaging with Lec-Fab8D3 to study its in vivo brain distribution and evaluate its potential as an AD companion diagnostic. Methods: Lec-Fab8D3 was conjugated to DFO* or NODAGA for ⁸⁹ Zr and ⁶⁴ Cu radiolabeling, respectively, or directly radioiodinated with ¹²⁴ I. PET imaging was performed in the Tg-ArcSwe mouse model of Aβ pathology and wild-type (WT) littermates at multiple time points post administration of the radiolabeled antibody, followed by ex vivo biodistribution, autoradiography, and Aβ quantification to assess brain uptake, specificity, and distribution of the radiolabeled Lec-Fab8D3. Results: Radiolabeled Lec-Fab8D3 variants showed retained binding properties with high radiochemical purity and yields. PET imaging demonstrated cortical brain uptake of all three tradiotracers in Tg-ArcSwe mice, with [ ⁸⁹ Zr]Zr-DFO*-Lec-Fab8D3 and [ ¹²⁴ I]I-Lec-Fab8D3 showing the best discrimination between Tg-ArcSwe and WT mice at 48–72 h post-injection. The highest absolute brain retention, combined with a lower brain-to-cerebellum ratio, was observed in both Tg-ArcSwe and WT mice that received the radiometal-labeled ( ⁸⁹ Zr and ⁶⁴ Cu) antibody, likely due to the residualizing nature of radiometals. Ex vivo analyses confirmed PET findings, and immunostaining demonstrated co-localization of Lec-Fab8D3 with Aβ deposits. Conclusions: ImmunoPET imaging with bispecific Lec-Fab8D3 enables specific detection of brain Aβ pathology in an AD mouse model. ⁸⁹ Zr was superior to ⁶⁴ Cu due to a more compatible half-life, while ¹²⁴ I displayed higher regional contrast than both radiometals, despite lower overall brain signal. The combined findings from radiometal- and iodine-based immunoPET will enhance our understanding of intra-brain distribution of bispecific antibodies. Furthermore, this highlights the importance of the choice of radiolabeling strategy and how it will impact the outcome of immunoPET with bispecific Aβ antibodies.

Background: In vivo monitoring of cell biodistribution using positron emission tomography (PET) provides a quantitative non-invasive method to further optimize cell therapies and related new developments in the field. Our group has earlier optimized and evaluated the in vitro properties of two radiotracers,[⁸⁹Zr]Zr-(oxinate)₄ and [⁸⁹Zr]Zr-DFO-NCS, for the radiolabelling of different cell types. Here, we performed a microPET study to assess the in vivo biodistribution of cells in rats using these two radiotracers. Human decidual stromal cells (hDSC) and rat macrophages (rMac) were radiolabelled with [⁸⁹Zr]Zr-(oxinate)₄ or [⁸⁹Zr]Zr-DFO-NCS. Rats were intravenously injected with radiolabelled cells, and the in vivo biodistribution was monitored with microPET/CT imaging for up to day 7. Organ uptake was evaluated and presented as a percentage of injected activity per gram tissue (%IA/g) and total absorbed organ doses (mSv/MBq). Results: The biodistribution in vivo showed an immediate uptake in the lungs. Thereafter, [⁸⁹Zr]Zr-(oxinate)₄ labelled cells migrated to the liver, while the signal from [⁸⁹Zr]Zr-DFO-NCS labelled cells lingered in the lungs. The differences in the in vivo behaviour for the same cell type appeared related to the radiotracer labelling. After 24 h, [⁸⁹Zr]Zr-(oxinate)₄ labelled cells had over 70% higher liver uptake for both hDSC and rMac compared to [⁸⁹Zr]Zr-DFO-NCS labelled cells, whereas [⁸⁹Zr]Zr-DFO-NCS labelled cells showed over 60% higher uptake in the lungs compared to [⁸⁹Zr]Zr-(oxinate)₄ labelled cells. This difference in both lung and liver uptake continued until day 7. Dosimetry calculations showed a higher effective dose (mSv/MBq) for [⁸⁹Zr]Zr-DFO-NCS compared to [⁸⁹Zr]Zr-(oxinate)₄, for both cell types. Although the bone uptake was higher for [⁸⁹Zr]Zr-(oxinate)₄ labelled cells, the prolonged uptake in the lungs contributed to a significant crossfire to bone marrow resulting in a higher bone dose. Conclusion: The [⁸⁹Zr]Zr-DFO-NCS labelled cells suggest a prolonged accumulation in the lungs, while [⁸⁹Zr]Zr-(oxinate)₄ suggests quicker clearance of the lungs followed by accumulation in the liver. Accumulation of radiolabelled cells in the liver corresponds to other cell-tracking methods. Further studies are required to determine the actual location of the [⁸⁹Zr]Zr-DFO-NCS labelled cell.

Metastatic castration-resistant prostate cancer is today incurable. Conventional imaging methods have limited detection, affecting their ability to give an accurate outcome prognosis, and current therapies for metastatic prostate cancer are insufficient. This inevitably leads to patients relapsing with castration-resistant prostate cancer. Targeting prostate-specific antigens whose expression is closely linked to the activity in the androgen receptor pathway, and thus the pathogenesis of prostate cancer, is a possible way to increase specificity and reduce off-target effects. We have humanized and evaluated radioimmunoconjugates of a previously murine antibody, m5A10, targeting PSA intended for theranostics of hormone-refractory prostate cancer. The humanized antibody h5A10 was expressed in mammalian HEK293 cells transfected with the nucleotide sequences for the heavy and light chains of the antibody. Cell culture medium was filtered and purified by Protein G chromatography, and the buffer was changed to PBS pH 7.4 by dialysis. Murine and humanized 5A10 were conjugated with p-SCN-Bn-CHX-A”-DTPA. Surface plasmon resonance was used to characterize the binding to PSA of the immunoconjugates. Immunoconjugates were labeled with either indium-111 or lutetium-177. Biodistribution studies of murine and humanized 5A10 were performed in mice with LNCaP xenografts. 5A10 was successfully humanized, and in vivo targeting showed specific binding in xenografts. The results thus give an excellent platform for further theranostic development of humanized 5A10 for clinical applications.

By using solid targets in medical cyclotrons, it is possible to produce large amounts of ⁶⁸GaCl₃. Purification of Ga³⁺ from metal ion impurities is a critical step, as these metals compete with Ga³⁺ in the complexation with different chelators, which negatively affects the radiolabeling yields. In this work, we significantly lowered the level of iron (Fe) impurities by adding ascorbate in the purification, and the resulting ⁶⁸GaCl₃could be utilized for high-yield radiolabeling of clinically relevant DOTA-based tracers. ⁶⁸GaCl₃ was cyclotron-produced and purified with ascorbate added in the wash solutions through the UTEVA resins. The ⁶⁸Ga eluate was analyzed for radionuclidic purity (RNP) by gamma spectroscopy, metal content by ICP-MS, and by titrations with the chelators DOTA, NOTA, and HBED. The ⁶⁸GaCl₃eluate was utilized for GMP-radiolabeling of the DOTA-based tracers DOTATOC and FAPI-46 using an automated synthesis module. DOTA chelator titrations gave an apparent molar activity (AMA) of 491 ± 204 GBq/µmol. GMP-compliant syntheses yielded up to 7 GBq/batch [⁶⁸Ga]Ga-DOTATOC and [⁶⁸Ga]Ga-FAPI-46 (radiochemical yield, RCY ~ 60%, corresponding to ten times higher compared to generator-based productions). Full quality control (QC) of ⁶⁸Ga-labelled tracers showed radiochemically pure and stable products at least four hours from end-of-synthesis.

Background: The humanized monoclonal antibody (mAb) hu5A10 specifically targets and internalizes prostate cancer cells by binding to prostate specific antigen (PSA). Preclinical evaluations have shown that hu5A10 is an excellent vehicle for prostate cancer (PCa) radiotheranostics. We studied the impact of different chelates and conjugation ratios on hu5A10′s target affinity, neonatal fc-receptor interaction on in vivo targeting efficacy, and possible enhanced therapeutic efficacy. Methods: In our experiment, humanized 5A10 (hu5A10) was conjugated with DOTA or DTPA at a molar ratio of 3:1, 6:1, and 12:1. Surface plasmon resonance (SPR) was used to study antigen and FcRn binding to the antibody conjugates. [¹¹¹In]hu5A10 radio-immunoconjugates were administered intravenously into BALB/c mice carrying subcutaneous LNCaP xenografts. Serial Single-photon emission computed tomography (SPECT) images were obtained during the first week. Tumors were harvested and radionuclide distribution was analyzed by autoradiography along with microanatomy and immunohistochemistry. Results: As seen by SPR, the binding to PSA was clearly affected by the chelate-to-antibody ratio. Similarly, FcRn (neonatal fc-receptor) interacted less with antibodies conjugated at high ratios of chelator, which was more pronounced for DOTA conjugates. The autoradiography data indicated a higher distribution of radioactivity to the rim of the tumor for lower ratios and a more homogenous distribution at higher ratios. Mice injected with ratio 3:1 ¹¹¹In-DOTA-hu5A10 showed no significant difference in tumor volume when compared to mice given vehicle over a time period of 3 weeks. Mice given a similar injection of ratio 6:1 ¹¹¹In-DOTA-hu5A10 or 6:1 ¹¹¹In-DTPA-hu5A10 or 12:1 ¹¹¹In-DTPA-hu5A10 showed significant tumor growth retardation. Conclusions: The present study demonstrated that the radiolabeling strategy could positively modify the hu5A10′s capacity to bind PSA and complex with the FcRn-receptor, which resulted in more homogenous activity distribution in tumors and enhanced therapy efficacy.

Background: There is a need to better characterise cell-based therapies in preclinical models to help facilitate their translation to humans. Long-term high-resolution tracking of the cells in vivo is often impossible due to unreliable methods. Radiolabelling of cells has the advantage of being able to reveal cellular kinetics in vivo over time. This study aimed to optimise the synthesis of the radiotracers [⁸⁹Zr]Zr-oxine (8-hydroxyquinoline) and [⁸⁹Zr]Zr-DFO-NCS (p-SCN-Bn-Deferoxamine) and to perform a direct comparison of the cell labelling efficiency using these radiotracers. Procedures: Several parameters, such as buffers, pH, labelling time and temperature, were investigated to optimise the synthesis of [⁸⁹Zr]Zr-oxine and [⁸⁹Zr]Zr-DFO-NCS in order to reach a radiochemical conversion (RCC) of >95 % without purification. Radio-instant thin-layer chromatography (iTLC) and radio high-performance liquid chromatography (radio-HPLC) were used to determine the RCC. Cells were labelled with [⁸⁹Zr]Zr-oxine or [⁸⁹Zr]Zr-DFO-NCS. The cellular retention of ⁸⁹Zr and the labelling impact was determined by analysing the cellular functions, such as viability, proliferation, phagocytotic ability and phenotypic immunostaining. Results: The optimised synthesis of [⁸⁹Zr]Zr-oxine and [⁸⁹Zr]Zr-DFO-NCS resulted in straightforward protocols not requiring additional purification. [⁸⁹Zr]Zr-oxine and [⁸⁹Zr]Zr-DFO-NCS were synthesised with an average RCC of 98.4 % (n = 16) and 98.0 % (n = 13), respectively. Cell labelling efficiencies were 63.9 % (n = 35) and 70.2 % (n = 30), respectively. ⁸⁹Zr labelling neither significantly affected the cell viability (cell viability loss was in the range of 1–8 % compared to its corresponding non-labelled cells, P value > 0.05) nor the cells’ proliferation rate. The phenotype of human decidual stromal cells (hDSC) and phagocytic function of rat bone-marrow-derived macrophages (rMac) was somewhat affected by radiolabelling. Conclusions: Our study demonstrates that [⁸⁹Zr]Zr-oxine and [⁸⁹Zr]Zr-DFO-NCS are equally effective in cell labelling. However, [⁸⁹Zr]Zr-oxine was superior to [⁸⁹Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency.

We report herein an efficient and fully automated protocol for the radiosynthesis of [¹⁸F]FAPI‐74, a new positron emission tomography (PET) radiopharmaceutical for in vivo detection of the fibroblast activation protein. [¹⁸F]FAPI‐74 was synthesized via a rapid [¹⁸F]aluminum fluoride coordination reaction, which was first developed on the flexible GE TRACERLab FX2N (FXN) platform and later translated to the cassette‐based module Trasis AllInOne (AIO). The results obtained with both modules were comparable in terms of yield and reproducibility. Automation of [¹⁸F]FAPI‐74 radiosynthesis on the FXN was carried out in 35 min with a radiochemical yield (RCY) of 18.5 ± 2.5% (n = 5, relative to starting [¹⁸F]fluoride). Method transfer to the AIO platform following minor optimizations allowed for the production of [¹⁸F]FAPI‐74 in an isolated RCY of 20 ± 2.5% [n = 3] with an overall synthesis time of 40 min. The radiochemical purity was greater than 95% for [¹⁸F]FAPI‐74, obtained from both modules. Overall, the protocol reliably provides a sterile and pyrogen‐free good manufacturing practice (GMP) compliant product of [¹⁸F]FAPI‐74 suitable for clinical PET imaging.

¹³⁵ La has favorable nuclear and chemical properties for Auger-based targeted internal radiotherapy. Here we present detailed investigations of the production, emissions, and dosimetry related to ¹³⁵ La therapy. ¹³⁵ La was produced by 16.5 MeV proton irradiation of metallic ^nat Ba on a medical cyclotron, and was isolated and purified by trap-and-release on weak cation-exchange resin. The average production rate was 407 ± 19 MBq µ A ⁻¹ (saturation activity), and the radionuclidic purity was 98% at 20 h post irradiation. Chemical separation recovered > 98 % of the ¹³⁵ La with an effective molar activity of 70 ± 20 GBq µ mol ⁻¹ . To better assess cellular and organ dosimetry of this nuclide, we have calculated the x-ray and Auger emission spectra using a Monte Carlo model accounting for effects of multiple vacancies during the Auger cascade. The generated Auger spectrum was used to calculate cellular S -factors. ¹³⁵ La was produced with high specific activity, reactivity, radionuclidic purity, and yield. The emission spectrum and the dosimetry are favorable for internal radionuclide therapy.