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Non-invasive assessment of fibrogenic activity, rather than fibrotic scars, could significantly improve the management of fibrotic diseases and the development of anti-fibrotic drugs. This study explores the potential of an Affibody molecule (Z09591) labeled with the Al(18)F-restrained complexing agent (RESCA) method as a tracer for the non-invasive detection of fibrogenic cells. Z09591 was functionalized with the RESCA chelator for direct labeling with [ 18 F]AlF. In vivo positron emission tomography/magnetic resonance imaging scans on U-87 tumor-bearing mice exhibited high selectivity of the resulting radiotracer, [ 18 F]AlF-RESCA-Z09591, for platelet-derived growth factor receptor β (PDGFRβ), with minimal non-specific background uptake. Evaluation in a mouse model with carbon tetrachloride-induced fibrotic liver followed by a disease regression phase, revealed the radiotracer's high affinity and specificity for fibrogenic cells in fibrotic livers (standardized uptake value [SUV] 0.43 ± 0.05), with uptake decreasing during recovery (SUV 0.29 ± 0.03) ( p  < 0.0001). [ 18 F]AlF-RESCA-Z09591 accurately detects PDGFRβ, offering non-invasive assessment of fibrogenic cells and promising applications in precise liver fibrogenesis diagnosis, potentially contributing significantly to anti-fibrotic drug development., Competing Interests: B.M. and O.E. are employees of Antaros Medical AB. F.Y.F. is an employee of Affibody AB. O.K. and O.E. are shareholders of Antaros Tracer AB., (© 2024 The Authors.)

Rheumatoid arthritis (RA) is the most common inflammatory joint disease, and early diagnosis is key for effective disease management. CD69 is one of the earliest cell surface markers seen at the surface of activated immune cells, and CD69 is upregulated in synovial tissue in patients with active RA. In this study, we evaluated the performance of a CD69-targeting PET agent, [ 68 Ga]Ga-DOTA-Z CAM241 , for early disease detection in a model of inflammatory arthritis. Methods: A model of inflammatory arthritis was induced by transferring splenocytes from KRN T-cell receptor transgenic B6 mice into T-cell-deficient I-A g7 major histocompatibility complex class II-expressing recipient mice. The mice were examined longitudinally by [ 68 Ga]Ga-DOTA-Z CAM241 PET/CT before and 3, 7, and 12 d after induction of arthritis. Disease progression was monitored by clinical parameters, including measuring body weight and scoring the swelling of the paws. The uptake of [ 68 Ga]Ga-DOTA-Z CAM241 in the paws was analyzed and expressed as SUV mean Tissue biopsy samples were analyzed for CD69 expression by flow cytometry or immunostaining for a histologic correlate. A second group of mice was examined by a nonbinding, size-matched Affibody molecule as the control. Results: Clinical symptoms appeared 5-7 d after induction of arthritis. The uptake of [ 68 Ga]Ga-DOTA-Z CAM241 in the joints was negligible at baseline but increased gradually after disease induction. An elevated PET signal was found on day 3, before the appearance of clinical symptoms. The uptake of [ 68 Ga]Ga-DOTA-Z CAM241 correlated with the clinical score and disease severity. The presence of CD69-positive cells in the joints and lymph nodes was confirmed by flow cytometry and immunostaining. The uptake of the nonbinding tracer that was the negative control also increased gradually with disease progression, although to a lesser extent than with [ 68 Ga]Ga-DOTA-Z CAM241 Conclusion: The uptake of [ 68 Ga]Ga-DOTA-Z CAM241 in the inflamed joints preceded the clinical symptoms in the KRN T-cell transfer model of inflammatory arthritis, in accordance with immunostaining for CD69. [ 68 Ga]Ga-DOTA-Z CAM241 is thus a promising PET imaging marker of activated immune cells in tissue during RA onset., (© 2024 by the Society of Nuclear Medicine and Molecular Imaging.)

Background: Platelet-derived growth factor receptor beta (PDGFRβ) is a receptor overexpressed on activated hepatic stellate cells (aHSCs). Positron emission tomography (PET) imaging of PDGFRβ could potentially allow the quantification of fibrogenesis in fibrotic livers. This study aims to evaluate a fluorine-18 radiolabeled Affibody molecule ([ 18 F]TZ-Z09591) as a PET tracer for imaging liver fibrogenesis., Results: In vitro specificity studies demonstrated that the trans-Cyclooctenes (TCO) conjugated Z09591 Affibody molecule had a picomolar affinity for human PDGFRβ. Biodistribution performed on healthy rats showed rapid clearance of [ 18 F]TZ-Z09591 through the kidneys and low liver background uptake. Autoradiography (ARG) studies on fibrotic livers from mice or humans correlated with histopathology results. Ex vivo biodistribution and ARG revealed that [ 18 F]TZ-Z09591 binding in the liver was increased in fibrotic livers (p = 0.02) and corresponded to binding in fibrotic scars., Conclusions: Our study highlights [18F]TZ-Z09591 as a specific tracer for fibrogenic cells in the fibrotic liver, thus offering the potential to assess fibrogenesis clearly., (© 2023. Springer Nature Switzerland AG.)

Gastrin-releasing peptide receptor (GRPR) is overexpressed in the majority of prostate cancers. This study aimed to investigate the potential of 64 Cu (radionuclide for late time-point PET-imaging) for imaging of GRPR expression using NOTA-PEG 2 -RM26 and NODAGA-PEG 2 -RM26. Methods: NOTA/NODAGA-PEG 2 -RM26 were labeled with 64 Cu and evaluated in GRPR-expressing PC-3 cells. Biodistribution of [ 64 Cu]Cu-NOTA/NODAGA-PEG 2 -RM26 was studied in PC-3 xenografted mice and compared to the biodistribution of [ 57 Co]Co-NOTA/NODAGA-PEG 2 -RM26 at 3 and 24 h p.i. Preclinical PET/CT imaging was performed in tumor-bearing mice. NOTA/NODAGA-PEG 2 -RM26 were stably labeled with 64 Cu with quantitative yields. In vitro, binding of [ 64 Cu]Cu-NOTA/NODAGA-PEG 2 -RM26 was rapid and GRPR-specific with slow internalization. In vivo, [ 64 Cu]Cu-NOTA/NODAGA-PEG 2 -RM26 bound specifically to GRPR-expressing tumors with fast clearance from blood and normal organs and displayed generally comparable biodistribution profiles to [ 57 Co]Co-NOTA/NODAGA-PEG 2 -RM26; tumor uptake exceeded normal tissue uptake 3 h p.i.. Tumor-to-organ ratios did not increase significantly with time. [ 64 Cu]Cu-NOTA-PEG 2 -RM26 had a significantly higher liver and pancreas uptake compared to other agents. 57 Co-labeled radioconjugates showed overall higher tumor-to-non-tumor ratios, compared to the 64 Cu-labeled counterparts. [ 64 Cu]Cu-NOTA/NODAGA-PEG 2 -RM26 was able to visualize GRPR-expression in a murine PC model using PET. However, [ 55/57 Co]Co-NOTA/NODAGA-PEG 2 -RM26 provided better in vivo stability and overall higher tumor-to-non-tumor ratios compared with the 64 Cu-labeled conjugates.

Prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) are promising targets for molecular imaging of prostate cancer (PCa) lesions. Due to the heterogenic overexpression of PSMA and GRPR in PCa, a heterodimeric radiotracer with the ability to bind to both targets could be beneficial. Recently, our group reported the novel heterodimer BQ7800 consisting of a urea-based PSMA inhibitor, the peptide-based GRPR antagonist RM26 and NOTA chelator. The study reported herein, aimed to improve the affinity of BQ7800 towards PSMA by changing the composition of the two linkers connecting the PSMA- and GRPR-targeting motifs. Three novel heterodimeric analogues were synthesized by incorporation of phenylalanine in the functional linker of the PSMA-binding motif and/or shortening the PEG-linker coupled to RM26. The heterodimers were labeled with indium-111 and evaluated in vitro. In the competitive binding assay, BQ7812, featuring phenylalanine and shorter PEG-linker, demonstrated a nine-fold improved affinity towards PSMA. In the in vivo biodistribution study of [ 111 In]In-BQ7812 in PC3-pip tumor-bearing mice (PSMA and GRPR positive), the activity uptake was two-fold higher in the tumor and three-fold higher in kidneys than for [ 111 In]In-BQ7800. Herein, we showed that the affinity of a bispecific PSMA/GRPR heterodimer towards PSMA could be improved by linker modification.

HER3-binding affibody molecules are a promising format for visualization of HER3 expression. Cobalt-55, a positron-emitting isotope, with a half-life of 17.5 h, allows for next-day imaging. We investigated the influence of the charge of the radiocobalt-chelator complex on the biodistribution of anti-HER3 affibody molecule (HE) 3 -Z HER3 and compared the best radiocobalt-labeled variant with a recently optimized gallium-labeled variant. Affibody conjugates (HE) 3 -Z HER3 -X (X = NOTA, NODAGA, DOTA, DOTAGA) were labeled with [ 57 Co]Co (surrogate for 55 Co). Affinity measurements, binding specificity and cellular processing were studied in two HER3-expressing cancer cell lines. Biodistribution was studied 3 and 24 h post-injection (pi) in mice with HER3-expressing BxPC-3 xenografts and compared to [ 68 Ga]Ga-(HE) 3 -Z HER3 -NODAGA. Micro-single-photon emission tomography/computed tomography (microSPECT/CT) and micro-positron emission tomography/computed tomography (microPET/CT) imaging was performed 3 and 24 h pi. Stably labeled conjugates bound to HER3 with subnanomolar affinity. [ 57 Co]Co-(HE) 3 -Z HER3 -DOTA had the best tumor retention and a significantly lower concentration in blood than other conjugates, leading to superior tumor-to-blood and tumor-to-liver ratios 24 h pi. Compared to [ 68 Ga]Ga-(HE) 3 -Z HER3 -NODAGA 3 h pi, [ 57 Co]Co-(HE) 3 -Z HER3 -DOTA provided superior imaging contrast in liver 24 h pi. Concluding, the composition and charge of the [ 57 Co]Co-chelator complex influenced the uptake in tumors and normal tissue. [ 57 Co]Co-(HE) 3 -Z HER3 -DOTA provided the best imaging properties among the cobalt-labeled conjugates. Delayed imaging of HER3 expression with [ 57 Co]Co-(HE) 3 -Z HER3 -DOTA improved imaging contrast compared to early-time-point imaging with [ 68 Ga]Ga-(HE) 3 -Z HER3 -NODAGA.

Background: Radionuclide molecular imaging of Gastrin-Releasing Peptide Receptor (GRPR) expression promises unparalleled opportunities for visualizing subtle prostate tumors, which due to small size, adjacent benign tissue, or a challenging location would otherwise remain undetected by conventional imaging. Achieving high imaging contrast is essential for this purpose and the molecular design of any probe for molecular imaging of prostate cancer should be aimed at obtaining as high tumor-to-organ ratios as possible., Objective: This short review summarizes the key imaging modalities currently used in prostate cancer, with a special focus on radionuclide molecular imaging. Emphasis is laid mainly on the issue of radiometals labeling chemistry and its influence on the targeting properties and biodistribution of radiolabeled GRPR antagonists for imaging of disseminated prostate cancer., Methods: A comprehensive literature search of the PubMed/MEDLINE, and Scopus library databases was conducted to find relevant articles., Results: The combination of radionuclide, chelator and required labeling chemistry was shown to have a significant influence on the stability, binding affinity and internalization rate, off-target interaction with normal tissues and blood proteins, interaction with enzymes, activity uptake and retention in excretory organs and activity uptake in tumors of radiolabeled bombesin antagonistic analogues., Conclusion: Labeling chemistry has a very strong impact on the biodistribution profile of GRPRtargeting peptide based imaging probes and needs to be considered when designing a targeting probe for high contrast molecular imaging. Taking into account the complexity of in vivo interactions, it is not currently possible to accurately predict the optimal labeling approach. Therefore, a detailed in vivo characterization and optimization is essential for the rational design of imaging agents., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Gastrin-releasing peptide receptors (GRPRs) are overexpressed in prostate cancer and are suitable for targeted radionuclide therapy (TRT). We optimized the bombesin-derived GRPR-antagonist PEG 2 -RM26 for labeling with 177 Lu and further determined the effect of treatment with 177 Lu-labeled peptide alone or in combination with the anti-HER2 antibody trastuzumab in a murine model. The PEG 2 -RM26 analog was coupled to NOTA, NODAGA, DOTA and DOTAGA chelators. The peptide-chelator conjugates were labeled with 177 Lu and characterized in vitro and in vivo. A preclinical therapeutic study was performed in PC-3 xenografted mice. Mice were treated with intravenous injections (6 cycles) of (A) PBS, (B) DOTAGA-PEG 2 -RM26, (C) 177 Lu-DOTAGA-PEG 2 -RM26, (D) trastuzumab or (E) 177 Lu-DOTAGA-PEG 2 -RM26 in combination with trastuzumab. 177 Lu-DOTAGA-PEG 2 -RM26 demonstrated quantitative labeling yield at high molar activity (450 GBq/μmol), high in vivo stability (5 min pi >98% of radioligand remained when coinjected with phosphoramidon), high affinity to GRPR (K D = 0.4 ± 0.2 nM), and favorable biodistribution (1 hr pi tumor uptake was higher than in healthy tissues, including the kidneys). Therapy with 177 Lu-DOTAGA-PEG 2 -RM26 induced a significant inhibition of tumor growth. The median survival for control groups was significantly shorter than for treated groups (Group C 66 days, Group E 74 days). Trastuzumab together with radionuclide therapy significantly improved survival. No treatment-related toxicity was observed. In conclusion, based on in vitro and in vivo characterization of the four 177 Lu-labeled PEG 2 -RM26 analogs, we concluded that 177 Lu-DOTAGA-PEG 2 -RM26 was the most promising analog for TRT. Radiotherapy using 177 Lu-DOTAGA-PEG 2 -RM26 effectively inhibited tumor growth in vivo in a murine prostate cancer model. Anti-HER2 therapy additionally improved survival., (© 2019 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

Upregulation of the human epidermal growth factor receptor type 3 (HER3) is a common mechanism to bypass HER-targeted cancer therapy. Affibody-based molecular imaging has the potential for detecting and monitoring HER3 expression during treatment. In this study, we compared the imaging properties of newly generated 68 Ga-labeled anti-HER3 affibody molecules (HE) 3 -Z HER3 -DOTA and (HE) 3 -Z HER3 -DOTAGA with previously reported [ 68 Ga]Ga-(HE) 3 -Z HER3 -NODAGA. We hypothesized that increasing the negative charge of the gallium-68/chelator complex would reduce hepatic uptake, which could lead to improved contrast of anti-HER3 affibody-based PET-imaging of HER3 expression. (HE) 3 -Z HER3 -X (X = DOTA, DOTAGA) were produced and labeled with gallium-68. Binding of the new conjugates was specific in HER3 expressing BxPC-3 and DU145 human cancer cells. Biodistribution and in vivo specificity was studied in BxPC-3 xenograft bearing Balb/c nu/nu mice 3 h pi. DOTA- and DOTAGA-containing conjugates had significantly higher concentration in blood than [ 68 Ga]Ga-(HE) 3 -Z HER3 -NODAGA. Presence of the negatively charged 68 Ga-DOTAGA complex reduced the unspecific hepatic uptake, but did not improve overall biodistribution of the conjugate. [ 68 Ga]Ga-(HE) 3 -Z HER3 -DOTAGA and [ 68 Ga]Ga-(HE) 3 -Z HER3 -NODAGA had similar tumor-to-liver ratios, but [ 68 Ga]Ga-(HE) 3 -Z HER3 -NODAGA had the highest tumor uptake and tumor-to-blood ratio among the tested conjugates. In conclusion, [ 68 Ga]Ga-(HE) 3 -Z HER3 -NODAGA remains the favorable variant for PET imaging of HER3 expression.

Gastrin-releasing peptide receptors (GRPRs) are promising targets in oligometastatic prostate cancer. We have recently used 55 Co (T 1/2 =  17.5 h) as a label for next day PET imaging of GRPR expression obtaining high imaging contrast. The radionuclide-chelator combination can significantly influence the biodistribution of radiopeptides. Therefore, in this study, we hypothesized that the properties of 55 Co-labeled PEG 2 -RM26 can be improved by identifying the optimal macrocyclic chelator. All analogues (X-PEG 2 -RM26, X = NOTA,NODAGA,DOTA,DOTAGA) were successfully labeled with radiocobalt with high yields and demonstrated high stability. The radiopeptides bound specifically and with picomolar affinity to GRPR and their cellular processing was characterized by low internalization. The best binding capacity was found for DOTA-PEG 2 -RM26. Ex vivo biodistribution in PC-3 xenografted mice was characterized by rapid blood clearance via renal excretion. Tumor uptake was similar for all conjugates at 3 h pi, exceeding the uptake in all other organs. Higher kidney uptake and longer retention were associated with N-terminal negative charge (DOTAGA-containing conjugate). Tumor-to-organ ratios increased over time for all constructs, although significant chelator-dependent differences were observed. Concordant with affinity measurements, DOTA-analog had the best retention of activity in tumors, resulting in the highest tumor-to-blood ratio 24 h pi, which translated into high contrast PET/CT imaging (using 55 Co).

Carbonic anhydrase IX (CAIX) is a cancer-associated molecular target for several classes of therapeutics. CAIX is overexpressed in a large fraction of renal cell carcinomas (RCC). Radionuclide molecular imaging of CAIX-expression might offer a non-invasive methodology for stratification of patients with disseminated RCC for CAIX-targeting therapeutics. Radiolabeled monoclonal antibodies and their fragments are actively investigated for imaging of CAIX expression. Promising alternatives are small non-immunoglobulin scaffold proteins, such as affibody molecules. A CAIX-targeting affibody ZCAIX:2 was re-designed with the aim to decrease off-target interactions and increase imaging contrast. The new tracer, DOTA-HE 3 -ZCAIX:2, was labeled with 111 In and characterized in vitro. Tumor-targeting properties of [ 111 In]In-DOTA-HE 3 -ZCAIX:2 were compared head-to-head with properties of the parental variant, [ 99m Tc]Tc(CO) 3 -HE 3 -ZCAIX:2, and the most promising antibody fragment-based tracer, [ 111 In]In-DTPA-G250(Fab') 2 , in the same batch of nude mice bearing CAIX-expressing RCC xenografts. Compared to the 99m Tc-labeled parental variant, [ 111 In]In-DOTA-HE 3 -ZCAIX:2 provides significantly higher tumor-to-lung, tumor-to-bone and tumor-to-liver ratios, which is essential for imaging of CAIX expression in the major metastatic sites of RCC. [ 111 In]In-DOTA-HE 3 -ZCAIX:2 offers significantly higher tumor-to-organ ratios compared with [ 111 In]In-G250(Fab') 2 . In conclusion, [ 111 In]In-DOTA-HE 3 -ZCAIX:2 can be considered as a highly promising tracer for imaging of CAIX expression in RCC metastases based on our results and literature data.

Simultaneous targeting of the prostate-specific membrane antigen (PSMA) and gastrin-releasing peptide receptor (GRPR) could improve the diagnostic accuracy in prostate cancer (PCa). The aim of this study was to develop a PSMA/GRPR-targeting bispecific heterodimer for SPECT and positron emission tomography (PET) diagnostic imaging of PCa. The heterodimer NOTA-DUPA-RM26 was produced by manual solid-phase peptide synthesis. NOTA-DUPA-RM26 was labeled with 111 In and 68 Ga, with yields >98%, and demonstrated a high stability and binding specificity to PSMA and GRPR. IC 50 values for nat In-NOTA-DUPA-RM26 were 4 ± 1 nM towards GRPR and 824 ± 230 nM towards PSMA. An in vivo binding specificity 1 h pi of 111 In-NOTA-DUPA-RM26 in PC3-PIP-xenografted mice demonstrated partially blockable tumor uptake when co-injected with an excess of PSMA- or GRPR-targeting agents. Simultaneous co-injection of both agents induced pronounced blocking. The biodistribution of 111 In-NOTA-DUPA-RM26 and 68 Ga-NOTA-DUPA-RM26 revealed fast activity clearance from the blood and normal organs via the kidneys. Tumor uptake exceeded normal organ uptake for both analogs 1 h pi. 68 Ga-NOTA-DUPA-RM26 had a significantly lower tumor uptake (8 ± 2%ID/g) compared to 111 In-NOTA-DUPA-RM26 (12 ± 2%ID/g) 1 h pi. Tumor-to-organ ratios increased 3 h pi, but decreased 24 h pi, for 111 In-NOTA-DUPA-RM26. MicroPET/CT and microSPECT/CT scans confirmed biodistribution data, suggesting that 68 Ga-NOTA-DUPA-RM26 and 111 In-NOTA-DUPA-RM26 are suitable candidates for the imaging of GRPR and PSMA expression in PCa shortly after administration.