Logo: to the web site of Uppsala University

B7-H3 (CD276), an immune checkpoint protein, is overexpressed in malignant tumors, while its expression in normal tissues is low, and several B7-H3-targeting therapies are under clinical evaluation. Radionuclide molecular imaging offers a non-invasive method for determining B7-H3 expression levels and may aid in improved patient selection. The feasibility of the use of Affibody molecules for the visualization of B7-H3 was demonstrated earlier. The selection of an approach for routine labeling providing high radiochemical yields and reproducibility is, however, critical for successful clinical translation. The optimal combination of a targeting protein, chelator/linker, and radionuclide should provide high-contrast visualization. In this study, we evaluated an acyclic chelator, tris(3,4-hydroxypyridinone) (THP), for labeling of the Affibody molecule Z_{B7-H3_2} with ⁶⁸Ga and compared its impact on radiolabeling efficiency and targeting properties with the impact of the cyclic chelator 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA). Maleimide derivatives of THP and NOTA were site-specifically coupled to the C-terminal cysteine of Z_{B7-H3_2}. THP-Z_{B7-H3_2} was successfully labeled with ⁶⁸Ga within 5 min of incubation at room temperature, achieving a 100% radiochemical yield. NOTA-Z_B7-H3 required heating at 60 °C for 10 min, and the radiochemical yield was lower. Both radioconjugates exhibited specific binding to B7-H3-expressing cells with similar binding strength, and both tracers demonstrated similar tumor uptake (p > 0.05) in a murine model. The biodistribution was similar, although [⁶⁸Ga]Ga-NOTA-Z_{B7-H3_2} provided slightly but significantly higher tumor-to-liver and tumor-to-spleen ratios. Nonetheless, the advantages of THP include rapid and mild radiolabeling with high efficiency, eliminating the need for heating and a post-purification step, which suggests a potential for streamlined clinical translation of Z_{B7-H3_2}.

Background:

Trastuzumab deruxtecan (T-DXd) is effective in HER2-expressing metastatic breast cancer (mBC), yet inter-patient benefit varies. The current reference biomarker—HER2 immunohistochemistry (IHC) on tumor biopsies—is insufficient as a sole predictor of response. HER2-targeted PET/CT offers non-invasive, whole-body, real-time assessment of target expression. We hypothesize that HER2-targeted PET with [68Ga]Ga-ABY-025 improves prediction of T-DXd outcomes and supports individualized treatment planning.

Methods:

HER2-Ex PET is a multicenter, phase II open-label diagnostic trial enrolling patients with HER2-low mBC who are candidates for T-DXd under current approvals (EU CT 2024-512721-89-00; NCT06830382). All participants undergo baseline HER2-PET with [68Ga]Ga-ABY-025 and a tumor biopsy. Patients with biopsy-confirmed HER2 expression (IHC 1–2+; Cohort 1) receive T-DXd and repeat HER2-PET after 3–4 cycles; others receive physician’s-choice systemic therapy (Cohort 2). The primary endpoint is the association between baseline HER2-PET signal—defined as the mean SUVmax across the five most avid lesions—and objective response per RECIST v1.1 after 3–4 cycles of T-DXd. A total sample size of 70 provides 80% power (α=0.05), allowing for attrition and technical failures. Secondary endpoints include health-economic outcomes and translational analyses of tumor biology and heterogeneity. The study is open at Karolinska University Hospital (Stockholm, Sweden), with Uppsala and Skåne University Hospitals planned for activation in Q1 2026.

Discussion:

Demonstrating a robust correlation between HER2-PET signal and early radiologic response would validate imaging-based patient selection for T-DXd, facilitate adaptive treatment decisions, and enhance biological understanding of intra- and inter-patient HER2 heterogeneity. Key considerations include standardization of imaging protocols across sites, potential temporal discordance between biopsy and imaging, and the non-randomized design. Positive results would justify incorporation of HER2-targeted PET into clinical pathways and inform the design of subsequent randomized trials testing PET-guided T-DXd strategies.

Clinical Trial Registration:

https://clinicaltrials.gov/study/NCT06830382, identifier NCT06830382.

Objective: Development of companion diagnostics for targeted radionuclide therapy is critical, especially for full-size antibodies with prolonged circulation times. Engineering antibodies to modify their in-vivo pharmacokinetics, such as incorporating neonatal Fc receptor (FcRn)-binding mutations, can potentially enable earlier imaging timing and improved patient stratification. This study aimed to evaluate the impact of FcRn-binding mutations on the in-vitro binding characteristics and in-vivo biodistribution and imaging performance of a CD44v6-targeting full-size antibody, UU-40, labeled with different radionuclides, and to assess its potential as a companion diagnostic.

Methods: The study involved engineering UU-40 with LALA and IAHA mutations, evaluating specific binding, internalization, and affinity using in-vitro cell assays. Biodistribution and imaging studies [PET and single-photon emission computed tomography (SPECT)] were conducted in mice carrying human tumor xenografts in a dual-nuclide setting.

Results: The FcRn mutations (LALA/IAHA) did not affect antibody specificity or affinity, which was target-specific and affinity remained in the subnanomolar range. Biodistribution studies demonstrated that the residualizing radiometal label (¹⁷⁷Lu) resulted in higher liver and spleen uptake compared with the nonresidualizing ¹²⁵I-label, leading to reduced tumor-to-organ ratios. Tumor uptake was higher in A431 xenografts, with peak accumulation at 24 h postinjection. SPECT and PET imaging confirmed superior contrast at later time points (∼24 h) with ¹²⁵I-UU-40_LALA/IAHA, while earlier imaging with ⁶⁸Ga was hindered by increased nonspecific accumulation.

Conclusion: FcRn-binding mutations in full-size antibodies significantly alter their in-vivo pharmacokinetics without affecting binding affinity or specificity. Introducing these mutations enables earlier imaging time points, enhancing the potential for companion diagnostics in clinical settings. 

Targeted radionuclide therapy is an emerging potent therapeutic strategy in oncology. The cell surface antigen CD44v6 is a potential pan-cancer target for radionuclide therapy. This study aimed to evaluate the therapeutic efficacy, biodistribution, dosimetry, and safety profile of AKIR001, an antibody targeting CD44v6 labeled with ¹⁷⁷Lu.

Methods: The biodistribution and preclinical dosimetry of [¹⁷⁷Lu]Lu-AKIR001 were calculated in the highly CD44v6-expressing A431 murine xenograft model, with subsequent extrapolation to predict human dosimetry. Therapeutic efficacy was evaluated across 3 xenograft models, 2 with high and 1 with moderate levels of CD44v6, using multiple dosing levels, fractionation regimens, and combinations with cisplatin. Preclinical toxicology was evaluated in a cross-reactive rabbit model and complemented by a PET imaging study using ⁶⁸Ga-labeled AKIR001 in a cynomolgus macaque.

Results: Biodistribution studies confirmed the high and selective tumor uptake of [¹⁷⁷Lu]Lu-AKIR001, resulting in favorable dosimetry predictions for clinical application. Therapeutic evaluations demonstrated significant dose-dependent efficacy in all tested xenograft models, with fractionated dosing (2 doses) resulting in complete tumor regression in 80% of the animals in a radioresistant xenograft model. Biodistribution in rabbits demonstrated low uptake in normal tissues, and a good-laboratory-practice study using an excessive dose of AKIR001 was well tolerated, with no signs of adverse effects. PET imaging in a cynomolgus macaque corroborated these findings.

Conclusion: Collectively, these data strongly support the therapeutic efficacy, safety, and dosimetry of [¹⁷⁷Lu]Lu-AKIR001, justifying its advancement into clinical trials. A phase 1 clinical trial of [¹⁷⁷Lu]Lu-AKIR001for CD44v6-positive solid cancers (NCT06639191) is currently recruiting patients.

Aim:

This study aimed to improve the efficacy of the CD44v6-targeting antibody UU-40 for molecular radiotherapy through affinity maturation and IgG subclass optimization.

M&M:

A panel of affinity-matured antibody candidates was generated and characterized as both human IgG4 and IgG1 with LALA mutations. Surface plasmon resonance and LigandTracer analyses identified several candidates with superior affinity and off-rates compared to the parental UU-40. Biodistribution studies in xenograft models using Lutetium-177 (177Lu)-labeled antibodies showed improved tumor retention for selected candidates, particularly UU-A-155. Species cross-reactivity assays confirmed binding to cynomolgus and rabbit v6-peptides, supporting future toxicity studies.

Results:

The IgG1 LALA format demonstrated reduced binding to Fc gamma receptors, potentially improving the safety profile. UU-A-155 emerged as the lead candidate for clinical translation, showing superior performance in both affinity and tumor retention. Our findings highlight the importance of comprehensive in vitro and in vivo assessments in antibody development, and provides valuable insights into optimizing antibody-based molecular radiotherapy.

Anaplastic thyroid cancer (ATC) is a rare but severe form of thyroid cancer responsible for approximately 50% of thyroid cancer deaths. Consequently, the identification of innovative therapies remains crucial for the effective treatment of ATC. Molecular radiotherapy is a rapidly growing field within oncology, and the cell surface antigen CD44v6, which is overexpressed in several cancers, is a plausible target for molecular radiotherapy of ATC. IHC of 39 patient samples with ATC was evaluated for CD44v6 expression. Biodistribution and dosimetry of iodine-125 (¹²⁵I)–/lutetium-177 (¹⁷⁷Lu)–labeled UU-40, a CD44v6-specific antibody, followed by in vivo efficacy in two ATC xenograft models with varying target expression levels (ACT-1 and BHT-101), accompanied by single-photon emission computed tomography (SPECT) imaging, evaluated radiolabeled UU-40 for therapeutic efficiency in ATC xenografts. The IHC revealed CD44v6 immunoreactivity in 46% of patient samples with ATC. The biodistribution favored ¹⁷⁷Lu-labeled UU-40 over the ¹²⁵I-labeled antibody and confirmed the in vivo specificity of both radioconjugates. The in vivo efficacy and accompanied SPECT imaging of a moderate CD44v6-expressing xenograft model (BHT-101) verified the tumor specificity, as well as the target-specific effect of ¹⁷⁷Lu-labeled UU-40 on tumor growth and survival. A 100% complete response rate was demonstrated as a result of therapy using a single dose of 16 MBq ¹⁷⁷Lu-labeled UU-40 in a high CD44v6-expressing xenograft model (ACT-1), and SPECT imaging revealed excellent tumor uptake of the radioconjugate at 14 days after injection. This study verifies the expression of CD44v6 in ATC and strengthens the superiority and promise of ¹⁷⁷Lu-labeled UU-40 over ¹³¹I-labeled UU-40 for antibody-based molecular radiotherapy of CD44v6-positive ATC.

Objective. The cell surface antigen CD44v6 is a promising target for several cancers, with favorable in vivo characteristics such as high affinity and suitable biodistribution. In normal tissues, expressions are restricted to basal epithelial cells in skin and mucosa. Consequently, previous clinical studies have reported varying degrees of toxicity in these tissues. To support new radioimmunotherapies (RIT), we developed small-scale internal dosimetry models for abdominal skin and esophageal mucosa. Using published biodistribution data, we compared absorbed doses to epithelial tissues and bone marrow from four clinically relevant radionuclides: rhenium-186, lutetium-177, terbium-161, and actinium-225. Approach. From the Genotype-Tissue Expression database, 288 H&E-stained sections of abdominal skin and esophageal mucosa were obtained from donors aged 21-70 and segmented to generate voxelized models. Monte Carlo simulations were conducted for the radionuclides across multiple source/target combinations. A compartment model generated serum, skin, and bone marrow biodistributions to estimate AD to the epithelium and bone marrow. Main results. Absorbed dose estimates to the basal layer of the skin were highest for 161Tb at 23.7 Gy GBq-1, followed by 177Lu and 186Re, with values of 9.5 and 5.3 Gy GBq-1, respectively, whereas the alpha emitter 225Ac delivered a dose of 4.9 Gy MBq-1. For the beta-emitters, three methods produced absorbed-dose estimates for the red marrow (RM) consistent within 5% of each other, with the highest values in the hip bone: 0.36, 0.25, and 0.39 Gy GBq-1 for 186Re, 177Lu, and 161Tb, respectively, compared to 0.66 Gy MBq-1 for 225Ac. Significance. This study presents a dosimetry framework for CD44v6-targeted RIT, demonstrating that, at clinically relevant administered activities, short-ranged emitters like 161Tb and 225Ac deliver substantially higher doses to basal epithelial layers compared to 186Re. RM doses were comparable for 161Tb and 186Re, lower for 177Lu, and highly uncertain for 225Ac due to daughter redistribution. Overall, these results support the use of 177Lu for initial clinical trials.

The immune checkpoint protein B7–H3 (CD276) is overexpressed in various cancers and is an attractive target for the treatment of malignant tumors. Radionuclide molecular imaging of B7–H3 expression using engineered scaffold proteins such as Affibody molecules is a promising strategy for the selection of potential responders to B7–H3-targeted therapy. Feasibility of B7–H3 imaging was demonstrated using two ^99mTc-labeled probes, AC12 and an affinity-matured SYNT179 using a [^99mTc]Tc-GGGC label. This study aimed to evaluate whether the use of a residualizing ¹¹¹In-based label provides better imaging contrast compared with a nonresidualizing label. To do that, SYNT179 and AC12-GGGC Affibody molecules were labeled with ¹¹¹In using (4,10-bis-carboxymethyl-7-{[2-(2,5-dioxo-3-thioxo-pyrrolidin-1-yl)-ethylcarbamoyl]-methyl}-1,4,7,10-tetraaza-cyclododec-1-yl)-acetic acid (maleimide-DOTA) chelator, site-specifically coupled to the C-terminus of Affibody molecules. The binding affinities of the ¹¹¹In-labeled conjugates to B7–H3-expressing living cells were higher compared with the affinities of the ^99mTc-labeled variants. In mice with B7–H3-expressing xenografts, the tumor uptake of ¹¹¹In-labeled proteins (3.6 ± 0.3 and 1.8 ± 0.5%ID/g for [¹¹¹In]In-SYNT179-DOTA and [¹¹¹In]In-AC12-DOTA, respectively) was significantly (p < 0.05, ANOVA) higher than those for ^99mTc-labeled counterparts (1.6 ± 0.2%ID/g and 0.8 ± 0.2%ID/g for [^99mTc]Tc-SYNT179 and [^99mTc]Tc-AC12-GGGC, respectively). The best variant, [¹¹¹In]In-SYNT179-DOTA, provided a tumor-to-blood ratio of 31.1 ± 2.9, which was twice higher than that for [^99mTc]Tc-SYNT179 and 7-fold higher than that for [^99mTc]Tc-AC12-GGGC. Both ¹¹¹In-labeled Affibody molecules had higher renal retention compared with ^99mTc-labeled ones, but the hepatobiliary excretion of ¹¹¹In-labeled proteins was appreciably lower, potentially improving the imaging of abdominal metastases. Overall, [¹¹¹In]In-SYNT179-DOTA is the most promising tracer for visualization of B7–H3 expression.

Background: Activated hepatic stellate cells (aHSCs) are the key cell population in the injured liver driving fibrogenesis. aHSCs express platelet-derived growth factor receptor beta (PDGFRβ), which is absent from quiescent HSCs. PDGFRβ is therefore an attractive target of PET tracers for imaging of fibrogenesis. Here, we present the pharmacological characterization of [⁶⁸Ga]Ga-DOTA-Cys-ATH001 in preparation for clinical translation and further confirm PDGFRβ as a biomarker of activated HSCs in liver disease by single cell sequencing.

Methods: The expression of PDGFRβ in subpopulations of HSCs was evaluated in scRNAseq datasets from both a mouse and human liver samples. DOTA-Cys-ATH001 was evaluated for affinity and mechanism of binding to PDGFRβ. [⁶⁸Ga]Ga-DOTA-Cys-ATH001 was evaluated for binding in vitro in mouse and human liver biopsies.The in vivo stability, biodistribution, pharmacokinetics, dosimetry and microdosing toxicology were evaluated in rats and pigs.

Results: PDGFRβ expression was specifically upregulated in activated HSCs. [⁶⁸Ga] Ga-DOTA-Cys-ATH001 could differentiate fibrotic liver from healthy liver.The binding co-localized with tissue areas positive for collagen deposition and PDGFRβ immunostaining. Based on the microdosing toxicology study the no observed adverse effect level was at least 1000 μg/kg, suggesting that the intended clinical PET scan dose is safe for use. Dosimetry calculations of [⁶⁸Ga]Ga-DOTA-Cys-ATH001 predicted an effective dose in human amenable to repeated examinations.

Conclusions: The data presented here suggests that PDGFRβ PET imaging with [⁶⁸Ga] Ga-DOTA-Cys-ATH001 has potential for non-invasive detection of activated HSCs. Clinical translation of [⁶⁸Ga]Ga-DOTA-Cys-ATH001 is ongoing.

Background: Lung diseases such as idiopathic pulmonary fibrosis and acute respiratory distress syndrome (ARDS) are associated with significant morbidity and mortality, with limited treatment options. Platelet-derived growth factor receptor beta (PDGFRβ) signaling pathway is a key driver of fibrogenesis in different organs. In the lungs, pericytes have a high PDGFRβ expression, and their role as immune regulators and progenitors of myofibroblasts is increasingly recognized. Non-invasive techniques to assess active lung tissue remodeling are needed to improve disease monitoring and treatment evaluation. This study aimed to evaluate [¹⁸F]TZ-Z09591, targeting PDGFRβ, for imaging pulmonary injuries in human biopsies, and in vivo in animal models of lung injury.

Results: [¹⁸F]TZ-Z09591 demonstrated high and specific binding to PDGFRβ-expressing cells. Autoradiography confirmed tracer uptake in lung injuries, including fibrotic foci, from human, rat, and pig lung tissues. In vivo positron emission tomography (PET) imaging of bleomycin-induced lung fibrosis in rats and an ARDS pig model showed significantly increased uptake in diseased lung segments compared to controls, especially in pulmonary injuries with collagen deposition, despite moderate background uptake.

Conclusions: This study demonstrated that [¹⁸F]TZ-Z09591 can assess PDGFRβ expression in pulmonary injuries, supporting its potential for non-invasive assessment of lung tissue remodeling. PET imaging targeting PDGFRβ could improve disease monitoring, and provide new insights into pulmonary fibrosis progression.