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Background

Gastrin releasing peptide receptor (GRPR)-directed radiopharmaceuticals for targeted radionuclide therapy may be a very promising addition in prostate and breast cancer patient management. Aiming to provide a GRPR-targeting theranostic pair, we have utilized the Tc-99m/Re-188 radiometal pair, in combination with two bombesin based antagonists, maSSS-PEG2-RM26 and maSES-PEG2-RM26. The two main aims of the current study were (i) to elucidate the influence of the radiometal-exchange on the biodistribution profile of the two peptides and (ii) to evaluate the feasibility of using the [^99mTc]Tc labeled counterparts for the dosimetry estimation for the [¹⁸⁸Re]Re-labeled conjugates.

Results

Both peptides were successfully labeled with Re-188 and evaluated both in vitro and in vivo. In GRPR expressing PC-3 cells, both [¹⁸⁸Re]Re-labeled peptides displayed high cellular uptake (8.5 ± 0.1% and 5 ± 0.3% of added activity, respectively), heavily GRPR-driven, while retaining the radioantagonistic profile with slow internalization rates. Both agents demonstrated high receptor affinity when loaded with ^natRe (7.5 nM and 8 nM, respectively). When tested in vivo in GRPR expressing PC-3 xenografts, both radioantagonists demonstrated high tumor accumulation (6.3 ± 0.5%IA/g and 5 ± 1%IA/g at 1 h pi, respectively), with good retention over time (4 ± 2%IA/g and 3.1 ± 0.1%IA/g at 4 h pi, respectively). In addition, their biodistribution profiles were closely mimicking their [^99mTc]Tc-labeled counterparts. Statistically significant lower tumor uptake was found for both conjugates labeled with Tc-99m, which may result in underestimation of the dose delivered to the tumor.

Conclusions

All the results indicate that Tc-99 m could be used for dosimetry evaluation for the two [¹⁸⁸Re]Re-labeled radioligands, with minimal alterations in their biodistribution pattern and tumor targeting capabilities.

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.

Radionuclide imaging of prostate-specific membrane antigen (PSMA) expression can be used for staging prostate cancer. The pseudo-peptide [99mTc]Tc-BQ0413 demonstrated high affinity and specificity to PSMA in preclinical evaluation. The purpose of this study was to clinically evaluate the safety and tolerability of a single administration of [99mTc]Tc-BQ0413 as well as study its biodistribution using SPECT to estimate dosimetry. [99mTc]Tc-BQ0413 was studied in a single-center diagnostic Phase I open-label exploratory study. Whole-body planar scintigraphy and SPECT/CT imaging were performed 2, 4, and 6 h after administration of 50, 100, or 150 mu g (680 +/- 140 MBq) of [99mTc]Tc-BQ0413 in five PCa patients per injected mass (NCT05839990). All injections of [99mTc]Tc-BQ0413 were well tolerated. The elimination of [99mTc]Tc-BQ0413 was predominantly renal. The stable physiological uptake of [99mTc]Tc-BQ0413 was observed in the lacrimal and salivary glands, liver, spleen, and kidneys for all tested peptide-injected masses. The average effective doses were 0.007 +/- 0.001, 0.0049 +/- 0.0003, and 0.0062 +/- 0.0008 mSv/MBq for 50, 100, and 150 mu g/injection, respectively. The radionuclide-associated dose burden per patient was 4-7 mSv/study for the given activity. Uptake of [99mTc]Tc-BQ0413 in primary tumors was identified in all patients and increased with the peptide-injected mass. Uptake in lymph nodes and bone metastases was the highest at 100 mu g/injected mass. The highest tumor lesion/background ratios were observed 6 h after the administration of 100 mu g of [99mTc]Tc-BQ0413. The results of the Phase I study showed that injections of [99mTc]Tc-BQ0413 were well tolerated, safe, and associated with low absorbed doses. Imaging using [99mTc]Tc-BQ0413 enabled the visualization of primary prostate cancer lesions as well as metastases in lymph nodes and bones.

Background: The gastrin-releasing peptide receptor (GRPR) is overexpressed in the majority of primary prostate cancer lesions, with persistent expression in lymph nodes and bone metastases, making it a legitimate molecular target for diagnostic imaging and staging. This study presents the synthesis and preclinical evaluation of [18F]MeTz-PEG2-RM26, a GRPR antagonist which utilises the Inverse Electron Demand Diels-Alder (IEDDA) reaction for 18F-labelling. This click-chemistry approach allows for site-specific incorporation of fluorine-18 under mild conditions, preserving the peptide's structural integrity and biological activity. Receptor specificity and affinity of [18F]MeTz-PEG2-RM26 were evaluated in vitro using GRPR-expressing PC-3 cells. Furthermore, the biodistribution profile of [18F]MeTz-PEG2-RM26 was assessed in NMRI mice and its tumour-targeting capability was investigated in mice bearing PC-3 xenografts.

Results: The labelling of TCO-PEG2-RM26 precursor involved three steps: (1) synthesis of an 18F-labelled activated ester on a quaternary methyl ammonium (QMA) cartridge, (2) conjugation of the labelled ester to a tetrazine amine, and (3) attachment to TCO-PEG2-RM26 via an IEDDA click reaction. This production method of [18F]MeTz-PEG2-RM26 afforded a high apparent molar activity of 3.5-4.3 GBq/mu mol and radiochemical purity exceeding 98%, with 43-70 MBq activity incorporation, while the entire synthesis was completed within 75 min. Both in vitro and in vivo studies confirmed the specific binding of [18F]MeTz-PEG2-RM26 to GRPR, with a significant reduction in activity uptake observed upon receptor saturation. The radioligand exhibited rapid blood clearance and minimal bone uptake, confirming the stability of the fluorine-carbon bond. However, high hepatic uptake (12-13% IA/g at 1 h post-injection) indicated predominant hepatobiliary excretion. Receptor-mediated uptake was observed in the tumours and pancreatic tissue, although the overall activity uptake in tumours was low, likely due to the rapid hepatobiliary clearance of [18F]MeTz-PEG2-RM26.

Conclusions:  These findings demonstrate the effectiveness of the IEDDA click reaction for fluorine-18 labelling of GRPR-targeting PET tracers. Future studies should focus on increasing the hydrophilicity of the imaging probe to improve the targeting properties and biodistribution profile of the radioligand.

The outcome of clinical trials evaluating drugs targeting the human epidermal growth factor receptor 3 (HER3) has been poor, with primary concerns related to lack of efficacy. HER3 is considered a difficult target since its overexpression on tumors is relatively low and there is normal expression in many different organs. However, a significant number of patients across different cancer indications have overexpression of HER3 and the development of novel modalities targeting HER3 is therefore warranted. Here, we have investigated the properties of affibody-based drug conjugates targeting HER3. The HER3-targeting affibody molecule ZHER3 was fused in a mono- and bivalent format to an engineered albumin-binding domain (ABD) for in vivo half-life extension and was coupled to the cytotoxic drug DM1 via a non-cleavable maleimidocaproyl (mc) linker. In vivo, a moderate uptake was observed for [99mTc]Tc-labeled ZHER3-ABD-ZHER3-mcDM1 in HER3 expressing BxPC3 tumors (3.5 ± 0.3%IA/g) at 24 h after injection, and clearance was predominately renal-mediated. Treatment of mice with BxPC3 human pancreatic cancer xenografts showed that a combination of ZHER3-ABD-ZHER3-mcDM1 and its cytostatic analog ZHER3-ABD-ZHER3 was efficacious and superior to treatment with only ZHER3-ABD-ZHER3, providing tumor growth inhibition and longer median survival (90 d) in comparison to monotherapy (68 d) and vehicle control (49 d). ZHER3-ABD-ZHER3-mcDM1 was found to be a potent drug conjugate for the treatment of HER3-expressing tumors in mice.

HER2 status determination is a necessary step for the proper choice of therapy and selection of patients for the targeted treatment of cancer. Targeted radiotracers such as radiolabeled DARPins provide a noninvasive and effective way for the molecular imaging of HER2 expression. This study aimed to evaluate tumor-targeting properties of three ^99mTc-labeled DARPin G3 variants containing Gly-Gly-Gly-Cys (G₃C), (Gly-Gly-Gly-Ser)₃-Cys ((G₃S)₃C), or Glu-Glu-Glu-Cys (E₃C) amino acid linkers at the C-terminus and conjugated to the HYNIC chelating agent, as well as to compare them with the clinically evaluated DARPin G3 labeled with ^99mTc(CO)₃ using the (HE)₃-tag at the N-terminus. The labeling of DARPin G3-HYNIC variants provided radiochemical yields in the range of 50–80%. Labeled variants bound specifically to human HER2-expressing cancer cell lines with affinities in the range of 0.5–3 nM. There was no substantial influence of the linker and HYNIC chelator on the binding of ^99mTc-labeled DARPin G3 variants to HER2 in vitro; however, [^99mTc]Tc-G3-(G₃S)₃C-HYNIC had the highest affinity. Comparative biodistribution of [^99mTc]Tc-G3-G₃C-HYNIC, [^99mTc]Tc-G3-(G₃S)₃C-HYNIC, [^99mTc]Tc-G3-E₃C-HYNIC, and [^99mTc]Tc-(HE)₃-G3 in healthy CD1 mice showed that there was a strong influence of the linkers on uptake in normal tissues. [^99mTc]Tc-G3-E₃C-HYNIC had an increased retention of activity in the liver and the majority of other organs compared to the other conjugates. The tumor uptake of [^99mTc]Tc-G3-(G₃S)₃C-HYNIC and [^99mTc]Tc-(HE)₃-G3 in Nu/j mice bearing SKOV-3 xenografts was similar. The specificity of tumor targeting in vivo was demonstrated for both tracers. [^99mTc]Tc-G3-(G₃S)₃C-HYNIC provided comparable, although slightly lower tumor-to-lung, tumor-to spleen and tumor-to-liver ratios than [^99mTc]Tc-(HE)₃-G3. Radiolabeling of DARPin G3-HYNIC conjugates with ^99mTc provided the advantage of a single-step radiolabeling procedure; however, the studied HYNIC conjugates did not improve imaging contrast compared to the ^99mTc-tricarbonyl-labeled DARPin G3. At this stage, [^99mTc]Tc-(HE)₃-G3 remains the most promising candidate for the clinical imaging of HER2-overexpressing cancers.

Background

Radionuclide molecular imaging can be used to visualize the expression levels of molecular targets. Affibody molecules, small and high affinity non-immunoglobulin scaffold-based proteins, have demonstrated promising properties as targeting vectors for radionuclide tumour imaging of different molecular targets. B7-H3 (CD276), an immune checkpoint protein belonging to the B7 family, is overexpressed in different types of human malignancies. Visualization of overexpression of B7-H3 in malignancies enables stratification of patients for personalized therapies. Affinity maturation of anti-B7-H3 Affibody molecules as an approach to improve the binding affinity and targeting properties was recently investigated. In this study, we tested the hypothesis that a dimeric format may be an alternative option to increase the apparent affinity of Affibody molecules to B7-H3 and accordingly improve imaging contrast.

Results

Two dimeric variants of anti-B7-H3 Affibody molecules were produced (designated Z_AC12*-Z_AC12*-GGGC and Z_AC12*-Z_{Taq_3}-GGGC). Both variants were labelled with Tc-99m (^99mTc) and demonstrated specific binding to B7-H3-expressing cells in vitro. [^99mTc]Tc-Z_AC12*-Z_AC12*-GGGC showed subnanomolar affinity (K_D1=0.28 ± 0.10 nM, weight = 68%), which was 7.6-fold higher than for [^99mTc]Tc-Z_AC12*-Z_{Taq_3}-GGGC (K_D=2.1 ± 0.9 nM). Head-to-head biodistribution of both dimeric variants of Affibody molecules compared with monomeric affinity matured SYNT-179 (all labelled with ^99mTc) in mice bearing B7-H3-expressing SKOV-3 xenografts demonstrates that both dimers have lower tumour uptake and lower tumour-to-organ ratios compared to the SYNT-179 Affibody molecule.

Conclusion

The improved functional affinity by dimerization does not compensate the disadvantage of increased molecular size for imaging purposes.

Purpose

Fusion of Affibody molecules with an albumin-binding domain (ABD) provides targeting agents, which are suitable for radionuclide therapy. To facilitate clinical translation, the low immunogenic potential of such constructs with targeting properties conserved is required.

Methods

The HER2-targeting Affibody molecule ZHER2:2891 was fused with a deimmunized ABD variant and DOTA was conjugated to a unique C-terminal cysteine. The novel construct, PEP49989, was labelled with ¹⁷⁷Lu. Affinity, specificity, and in vivo targeting properties of [¹⁷⁷Lu]Lu-PEP49989 were characterised. Experimental therapy in mice with human HER2-expressing xenografts was evaluated.

Results

The maximum molar activity of 52 GBq/µmol [¹⁷⁷Lu]Lu-PEP49989 was obtained. [¹⁷⁷Lu]Lu-PEP49989 bound specifically to HER2-expressing cells in vitro and in vivo. The HER2 binding affinity of [¹⁷⁷Lu]Lu-PEP49989 was similar to the affinity of [¹⁷⁷Lu]Lu-ABY-027 containing the parental ABD035 variant. The renal uptake of [¹⁷⁷Lu]Lu-PEP49989 was 1.4-fold higher, but hepatic and splenic uptake was 1.7-2-fold lower than the uptake of [¹⁷⁷Lu]Lu-ABY-027. The median survival of xenograft-bearing mice treated with 21 MBq [¹⁷⁷Lu]Lu-PEP49989 (> 90 days) was significantly longer than the survival of mice treated with vehicle (38 days) or trastuzumab (45 days). Treatment using a combination of [¹⁷⁷Lu]Lu-PEP49989 and trastuzumab increased the number of complete tumour remissions. The renal and hepatic toxicity was minimal to mild.

Conclusion

In preclinical studies, [¹⁷⁷Lu]Lu-PEP49989 demonstrated favourable biodistribution and a strong antitumour effect, which was further enhanced by co-treatment with trastuzumab.

Targeting the gastrin-releasing peptide receptor (GRPR) with the bombesin analogue RM26, a 9 aa peptide, has been a promising strategy for cancer theranostics, with recent success in radionuclide imaging of prostate cancer. However, therapeutic application of the short peptide RM26 would require a longer half-life to prevent fast clearance from the circulation. Conjugation to an albumin-binding domain (ABD) is a viable strategy to extend the in vivo half-life of peptides and proteins. We previously reported an ABD-fused RM26 peptide targeting GRPR (ABD-RM26 Gen 1) that showed prolonged and stable tumor uptake over 144 h; however, the observed high kidney uptake indicated that the conjugate’s binding to albumin was reduced and that this could be an obstacle for its use as a delivery system for targeted therapy, especially for radiotherapy. Here, we have designed, produced, and preclinically evaluated a series of novel ABD-RM26 conjugates with the aim of improving the conjugate’s binding to albumin and decreasing the kidney uptake. We developed three second-generation constructs with varying formats, differing in the relative positions of the targeting moieties and the radionuclide chelator. The produced conjugates were radiolabeled with indium-111 and evaluated in vitro and in vivo. All constructs displayed improved biophysical characteristics, biodistribution, and lower kidney uptake compared to previously reported first-generation molecules. The ABD-RM26 Gen 2A conjugate showed the best biodistribution profile with a nearly 6-fold reduction in kidney uptake. However, the ABD-RM26 Gen 2A conjugate’s binding to GRPR was compromised. This conjugate’s assembly of albumin- and GRPR-binding moieties might be used for further development of drug conjugates for targeted therapy/radiotherapy of GRPR-expressing cancers.