Half-life extension via ABD-fusion leads to higher tumor uptake of an affibody-drug conjugate compared to PAS- and XTENylation.

A critical parameter during the development of protein therapeutics is to endow them with suitable pharmacokinetic and pharmacodynamic properties. Small protein drugs are quickly eliminated by kidney filtration, and in vivo half-life extension is therefore often desired. Here, different half-life extension technologies were studied where PAS polypeptides (PAS300, PAS600), XTEN polypeptides (XTEN288, XTEN576), and an albumin binding domain (ABD) were compared for half-life extension of an anti-human epidermal growth factor receptor 2 (HER2) affibody-drug conjugate. The results showed that extension with the PAS or XTEN polypeptides or the addition of the ABD lowered the affinity for HER2 to some extent but did not negatively affect the cytotoxic potential. The half-lives in mice ranged from 7.3 h for the construct including PAS300 to 11.6 h for the construct including PAS600. The highest absolute tumor uptake was found for the construct including the ABD, which was 60 to 160% higher than the PASylated or XTENylated constructs, even though it did not have the longest half-life (9.0 h). A comparison of the tumor-to-normal-organ ratios showed the best overall performance of the ABD-fused construct. In conclusion, PASylation, XTENylation, and the addition of an ABD are viable strategies for half-life extension of affibody-drug conjugates, with the best performance observed for the construct including the ABD.

Preclinical Evaluation of HER2-Targeting DARPin G3: Impact of Albumin-Binding Domain (ABD) Fusion.

Designed ankyrin repeat protein (DARPin) G3 is an engineered scaffold protein. This small (14.5 kDa) targeting protein binds with high affinity to human epidermal growth factor receptor 2 (HER2). HER2 is overexpressed in several cancers. The use of the DARPin G3 for radionuclide therapy is complicated by its high renal reabsorption after clearance via the glomeruli. We tested the hypothesis that a fusion of the DARPin G3 with an albumin-binding domain (ABD) would prevent rapid renal excretion and high renal reabsorption resulting in better tumour targeting. Two fusion proteins were produced, one with the ABD at the C-terminus (G3-ABD) and another at the N-terminus (ABD-G3). Both variants were labelled with 177Lu. The binding properties of the novel constructs were evaluated in vitro and their biodistribution was compared in mice with implanted human HER2-expressing tumours. Fusion with the ABD increased the retention time of both constructs in blood compared with the non-ABD-fused control. The effect of fusion with the ABD depended strongly on the order of the domains in the constructs, resulting in appreciably better targeting properties of [177Lu]Lu-G3-ABD. Our data suggest that the order of domains is critical for the design of targeting constructs based on scaffold proteins.

Influence of Molecular Design on the Tumor Targeting and Biodistribution of PSMA-Binding Tracers Labeled with Technetium-99m.

Previously, we designed the EuK-based PSMA ligand BQ0413 with an maE3 chelator for labeling with technetium-99m. It showed efficient tumor targeting, but our preclinical data and preliminary clinical results indicated that the renal excretion levels need to be decreased. We hypothesized that this could be achieved by a decrease in the ligand's total negative charge, achieved by substituting negatively charged glutamate residues in the chelator with glycine. The purpose of this study was to evaluate the tumor targeting and biodistribution of two new PSMA inhibitors, BQ0411 and BQ0412, compared to BQ0413. Conjugates were radiolabeled with Tc-99m and characterized in vitro, using PC3-pip cells, and in vivo, using NMRI and PC3-pip tumor-bearing mice. [99mTc]Tc-BQ0411 and [99mTc]Tc-BQ0412 demonstrated PSMA-specific binding to PC3-pip cells with picomolar affinity. The biodistribution pattern for the new conjugates was characterized by rapid excretion. The tumor uptake for [99mTc]Tc-BQ0411 was 1.6-fold higher compared to [99mTc]Tc-BQ0412 and [99mTc]Tc-BQ0413. [99mTc]Tc-BQ0413 has demonstrated predominantly renal excretion, while the new conjugates underwent both renal and hepatobiliary excretion. In this study, we have demonstrated that in such small targeting ligands as PSMA-binding EuK-based pseudopeptides, the structural blocks that do not participate in binding could have a crucial role in tumor targeting and biodistribution. The presence of a glycine-based coupling linker in BQ0411 and BQ0413 seems to optimize biodistribution. In conclusion, the substitution of amino acids in the chelating sequence is a promising method to alter the biodistribution of [99mTc]Tc-labeled small-molecule PSMA inhibitors. Further improvement of the biodistribution properties of BQ0413 is needed.

Comparison of approaches for increasing affinity of affibody molecules for imaging of B7-H3: dimerization and affinity maturation.

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 ZAC12*-ZAC12*-GGGC and ZAC12*-ZTaq_3-GGGC). Both variants were labelled with Tc-99m (99mTc) and demonstrated specific binding to B7-H3-expressing cells in vitro. [99mTc]Tc-ZAC12*-ZAC12*-GGGC showed subnanomolar affinity (KD1=0.28 ± 0.10 nM, weight = 68%), which was 7.6-fold higher than for [99mTc]Tc-ZAC12*-ZTaq_3-GGGC (KD=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.

Two Novel [68Ga]Ga-Labeled Radiotracers Based on Metabolically Stable [Sar11]RM26 Antagonistic Peptide for Diagnostic Positron Emission Tomography Imaging of GRPR-Positive Prostate Cancer.

Gastrin releasing peptide receptor (GRPR) is overexpressed in prostate cancer (PC-3) and can be used for diagnostic purposes. We herein present the design and preclinical evaluation of two novel NOTA/NODAGA-containing peptides suitable for labeling with the positron emission tomography (PET) radionuclide Ga-68. These analogs are based on the previously reported GRPR-antagonist DOTAGA-PEG2-[Sar11]RM26, developed for targeted radiotheraostic applications. Both NOTA-PEG2-[Sar11]RM26 and NODAGA-PEG2-[Sar11]RM26 were successfully labeled with Ga-68 and evaluated in vitro and in vivo using PC-3 cell models. Both, [68Ga]Ga-NOTA-PEG2-[Sar11]RM26 and [68Ga]Ga-NODAGA-PEG2-[Sar11]RM26 displayed high metal-chelate stability in phosphate buffered saline and against the EDTA-challenge. The two [68Ga]Ga-labeled conjugates demonstrated highly GRPR-mediated uptake in vitro and in vivo and exhibited a slow internalization over time, typical for radioantagonistis. The [natGa]Ga-loaded peptides displayed affinity in the low nanomole range for GRPR in competition binding experiments. The new radiotracers demonstrated biodistribution profiles suitable for diagnostic imaging shortly after administration with fast background clearance. Their high tumor uptake (13 ± 1 and 15 ± 3% IA/g for NOTA and NODAGA conjugates, respectively) and high tumor-to-blood ratios (60 ± 10 and 220 ± 70, respectively) 3 h pi renders them promising PET tracers for use in patients. Tumor-to-normal organ ratios were higher for [68Ga]Ga-NODAGA-PEG2-[Sar11]RM26 than for the NOTA-containing counterpart. The performance of the two radiopeptides was further supported with the PET/CT images. In conclusion, [68Ga]Ga-NODAGA-PEG2-[Sar11]RM26 is a promising PET imaging tracer for visualization of GRPR-expressing lesions with high imaging contrast shortly after administration.

Evaluation of Approaches for the Assessment of HER2 Expression in Breast Cancer by Radionuclide Imaging Using the Scaffold Protein [99mTc]Tc-ADAPT6.

Due to its small size and high affinity binding, the engineered scaffold protein ADAPT6 is a promising targeting probe for radionuclide imaging of human epidermal growth factor receptor type 2 (HER2). In a Phase I clinical trial, [99mTc]Tc-ADAPT6 demonstrated safety, tolerability and capacity to visualize HER2 expression in primary breast cancer. In this study, we aimed to select the optimal parameters for distinguishing between breast cancers with high and low expression of HER2 using [99mTc]Tc-ADAPT6 in a planned Phase II study. HER2 expression was evaluated in primary tumours and metastatic axillary lymph nodes (mALNs). SPECT/CT imaging of twenty treatment-naive breast cancer patients was performed 2 h after injection of [99mTc]Tc-ADAPT6. The imaging data were compared with the data concerning HER2 expression obtained by immunohistochemical evaluation of samples obtained by core biopsy. Maximum Standard Uptake Values (SUVmax) afforded the best performance for both primary tumours and mALNs (areas under the receiver operating characteristic curve (ROC AUC) of 1.0 and 0.97, respectively). Lesion-to-spleen ratios provided somewhat lower performance. However, the ROC AUCs were still over 0.90 for both primary tumours and mALNs. Thus, lesion-to-spleen ratios should be further evaluated to find if these could be applied to imaging using stand-alone SPECT cameras that do not permit SUV calculations.

GRPR-Antagonists Carrying DOTAGA-Chelator via Positively Charged Linkers: Perspectives for Prostate Cancer Theranostics.

Gastrin-releasing peptide receptor (GRPR)-antagonists have served as motifs in the development of theranostic radioligands for prostate cancer. Our efforts have been focused on the development of radiolabeled RM26 (H-DPhe6-Gln7-Trp8-Ala9-Val10-Gly11-His12-Sta13-Leu14-NH2) analogs, such as [111In]In-DOTAGA-PEG2-RM26. We recently showed that its Gly11/Sar11-substituted version, [111In]In-AU-RM26-M1, resisted degradation by neprilysin (NEP) while in circulation and achieved higher tumor uptake in mice. We herein introduce the following three new AU-RM26-M1 mimics labeled with In-111, with basic residues in the linker: (i) AU-RM26-M2 (PEG2-Pip), (ii) AU-RM26-M3 (PEG2-Arg), and (iii) AU-RM26-M4 (Arg-Arg-Pip). These analogs were compared in PC-3 cells and animal models vs. AU-RM26-M1 (reference). The new analogs showed high affinity and specificity for the GRPR, exhibiting an uptake and distribution pattern in PC-3 cells typical for a radiolabeled GRPR-antagonist. They showed high stability in peripheral mice blood, except for [111In]In-AU-RM26-M3. AU-RM26-M4 achieved the highest tumor uptake and promising background clearance, followed by [111In]In-RM26-M2, showing lower background levels. These findings were confirmed for [111In]In-AU-RM26-M2 and [111In]In-AU-RM26-M4 by micro-SPECT/CT at 4 and 24 h post-injection. Hence, the type of positively charged residues in the linker of AU-RM26-M1 mimics strongly influenced biological behavior. The analogs with Pip next to DPhe6 demonstrated the best overall characteristics and warrant further investigation.

Comparative Preclinical Evaluation of HYNIC-Modified Designed Ankyrin Repeat Proteins G3 for the 99mTc-Based Imaging of HER2-Expressing Malignant Tumors.

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.

Preclinical evaluation of new GRPR-antagonists with improved metabolic stability for radiotheranostic use in oncology.

BACKGROUND: The gastrin-releasing peptide receptor (GRPR) has been extensively studied as a biomolecular target for peptide-based radiotheranostics. However, the lack of metabolic stability and the rapid clearance of peptide radioligands, including radiolabeled GRPR-antagonists, often impede clinical application. Aiming at circumventing these drawbacks, we have designed three new GRPR-antagonist radioligands using [99mTc]Tc-DB15 ([99mTc]Tc-N4-AMA-DIG-DPhe-Gln-Trp-Ala-Val-Sar-His-Leu-NHEt; AMA: p-aminomethylaniline; DIG: diglycolate) as a motif, due to its high GRPR-affinity and stability to neprilysin (NEP). The new analogues carry the DOTAGA-chelator (1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid) through different linkers at the N-terminus to allow for labeling with the theranostic radionuclide pair In-111/Lu-177. After labeling with In-111 the following radioligands were evaluated: (i) [111In]In-AU-SAR-M1 ([111In]In-DOTAGA-AMA-DIG-DPhe-Gln-Trp-Ala-Val-Sar-His-Leu-NHEt), (ii) [111In]In-AU-SAR-M2 ([111In]In-[DOTAGA-Arg]AU-SAR-M1) and (iii) [111In]In-AU-SAR-M3 ([111In]In-[DOTAGA-DArg]AU-SAR-M1). RESULTS: These radioligands were compared in a series of in vitro assays using prostate adenocarcinoma PC-3 cells and in murine models. They all displayed high and GRPR-specific uptake in PC-3 cells. Analysis of mice blood collected 5 min post-injection (pi) revealed similar or even higher metabolic stability of the new radioligands compared with [99mTc]Tc-DB15. The stability could be further increased when the mice were treated with Entresto® to in situ induce NEP-inhibition. In PC-3 xenograft-bearing mice, [111In]In-AU-SAR-M1 displayed the most favourable biodistribution profile, combining a good tumor retention with the highest tumor-to-organ ratios, with the kidneys as the dose-limiting organ. CONCLUSIONS: These findings strongly point at AU-SAR-M1 as a promising radiotherapeutic candidate when labeled with Lu-177, or other medically appealing therapeutic radiometals, especially when combined with in situ NEP-inhibition. To this goal further investigations are currently pursued.

Multifunctional Core/Shell Diamond Nanoparticles Combining Unique Thermal and Light Properties for Future Biological Applications.

We report the development of multifunctional core/shell chemical vapor deposition diamond nanoparticles for the local photoinduced hyperthermia, thermometry, and fluorescent imaging. The diamond core heavily doped with boron is heated due to absorbed laser radiation and in turn heats the shell of a thin transparent diamond layer with embedded negatively charged SiV color centers emitting intense and narrowband zero-phonon lines with a temperature-dependent wavelength near 738 nm. The heating of the core/shell diamond nanoparticle is indicated by the temperature-induced spectral shift in the intensive zero-phonon line of the SiV color centers embedded in the diamond shell. The temperature of the core/shell diamond particles can be precisely manipulated by the power of the incident light. At laser power safe for biological systems, the photoinduced temperature of the core/shell diamond nanoparticles is high enough to be used for hyperthermia therapy and local nanothermometry, while the high zero-phonon line intensity of the SiV color centers allows for the fluorescent imaging of treated areas.

Preclinical Evaluation of a Novel High-Affinity Radioligand [99mTc]Tc-BQ0413 Targeting Prostate-Specific Membrane Antigen (PSMA).

Radionuclide imaging using radiolabeled inhibitors of prostate-specific membrane antigen (PSMA) can be used for the staging of prostate cancer. Previously, we optimized the Glu-urea-Lys binding moiety using a linker structure containing 2-napththyl-L-alanine and L-tyrosine. We have now designed a molecule that contains mercaptoacetyl-triglutamate chelator for labeling with Tc-99m (designated as BQ0413). The purpose of this study was to evaluate the imaging properties of [99mTc]Tc-BQ0413. PSMA-transfected PC3-pip cells were used to evaluate the specificity and affinity of [99mTc]Tc-BQ0413 binding in vitro. PC3-pip tumor-bearing BALB/C nu/nu mice were used as an in vivo model. [99mTc]Tc-BQ0413 bound specifically to PC3-pip cells with an affinity of 33 ± 15 pM. In tumor-bearing mice, the tumor uptake of [99mTc]Tc-BQ0413 (38 ± 6 %IA/g in PC3-pip 3 h after the injection of 40 pmol) was dependent on PSMA expression (3 ± 2 %IA/g and 0.9 ± 0.3 %IA/g in PSMA-negative PC-3 and SKOV-3 tumors, respectively). We show that both unlabeled BQ0413 and the commonly used binder PSMA-11 enable the blocking of [99mTc]Tc-BQ0413 uptake in normal PSMA-expressing tissues without blocking the uptake in tumors. This resulted in an appreciable increase in tumor-to-organ ratios. At the same injected mass (5 nmol), the use of BQ0413 was more efficient in suppressing renal uptake than the use of PSMA-11. In conclusion, [99mTc]Tc-BQ0413 is a promising probe for the visualization of PSMA-positive lesions using single-photon emission computed tomography (SPECT).

177Lu-labeled PSMA targeting therapeutic with optimized linker for treatment of disseminated prostate cancer; evaluation of biodistribution and dosimetry.

Introduction: Prostate specific membrane antigen (PSMA), highly expressed in metastatic castration-resistant prostate cancer (mCRPC), is an established therapeutic target. Theranostic PSMA-targeting agents are widely used in patient management and has shown improved outcomes for mCRPC patients. Earlier, we optimized a urea-based probe for radionuclide visualization of PSMA-expression in vivo using computer modeling. With the purpose to develop a targeting agent equally suitable for radionuclide imaging and therapy, the agent containing DOTA chelator was designed (BQ7876). The aim of the study was to test the hypothesis that 177Lu-labeled BQ7876 possesses target binding and biodistribution properties potentially enabling its use for radiotherapy. Methods: BQ7876 was synthesized and labeled with Lu-177. Specificity and affinity of [177Lu]Lu-BQ7876 to PSMA-expressing PC3-pip cells was evaluated and its processing after binding to cells was studied. Animal studies in mice were performed to assess its biodistribution in vivo, target specificity and dosimetry. [177Lu]Lu-PSMA-617 was simultaneously evaluated for comparison. Results: BQ7876 was labeled with Lu-177 with radiochemical yield >99%. Its binding to PSMA was specific in vitro and in vivo when tested in antigen saturation conditions as well as in PSMA-negative PC-3 tumors. The binding of [177Lu]Lu-BQ7876 to living cells was characterized by rapid association, while the dissociation included a rapid and a slow phase with affinities KD1 = 3.8 nM and KD2 = 25 nM. The half-maximal inhibitory concentration for natLu-BQ7876 was 59 nM that is equal to 61 nM for natLu-PSMA-617. Cellular processing of [177Lu]Lu-BQ7876 was accompanied by slow internalization. [177Lu]Lu-BQ7876 was cleared from blood and normal tissues rapidly. Initial elevated uptake in kidneys decreased rapidly, and by 3 h post injection, the renal uptake (13 ± 3%ID/g) did not differ significantly from tumor uptake (9 ± 3%ID/g). Tumor uptake was stable between 1 and 3 h followed by a slow decline. The highest absorbed dose was in kidneys, followed by organs and tissues in abdomen. Discussion: Biodistribution studies in mice demonstrated that targeting properties of [177Lu]Lu-BQ7876 are not inferior to properties of [177Lu]Lu-PSMA-617, but do not offer any decisive advantages.

Phase I clinical evaluation of 99mTc-labeled Affibody molecule for imaging HER2 expression in breast cancer.

The determination of tumor human epidermal growth factor receptor type 2 (HER2) status is of increasing importance with the recent approval of more efficacious HER2-targeted treatments. There is a lack of suitable methods for clinical in vivo HER2 expression assessment. Affibody molecules are small affinity proteins ideal for imaging detection of receptors, which are engineered using a small (molecular weight 6.5 kDa) nonimmunoglobulin scaffold. Labeling of Affibody molecules with positron emitters enabled the development of sensitive and specific agents for molecular imaging. The development of probes for SPECT would permit the use of Affibody-based imaging in regions where PET is not available. In this first-in-human study, we evaluated the safety, biodistribution, and dosimetry of the 99mTc-ZHER2:41071 Affibody molecule developed for SPECT/CT imaging of HER2 expression. Methods: Thirty-one patients with primary breast cancer were enrolled and divided into three cohorts (injected with 500, 1000, or 1500 µg ZHER2:41071) comprising at least five patients with high (positive) HER2 tumor expression (IHC score 3+ or 2+ and ISH positive) and five patients with low (IHC score 2+ or 1+ and ISH negative) or absent HER2 tumor expression. Patients were injected with 451 ± 71 MBq 99mTc-ZHER2:4107. Planar scintigraphy was performed after 2, 4, 6 and 24 h, and SPECT/CT imaging followed planar imaging 2, 4 and 6 h after injection. Results: Injections of 99mTc-ZHER2:41071 were well tolerated and not associated with adverse events. Normal organs with the highest accumulation were the kidney and liver. The effective dose was 0.019 ± 0.004 mSv/MBq. Injection of 1000 µg provided the best standard discrimination between HER2-positive and HER2-low or HER2-negative tumors 2 h after injection (SUVmax 16.9 ± 7.6 vs. 3.6 ± 1.4, p < 0.005). The 99mTc-ZHER2:41071 uptake in HER2-positive lymph node metastases (SUVmax 6.9 ± 2.4, n = 5) was significantly (p < 0.05) higher than that in HER2-low/negative lymph nodes (SUVmax 3.5 ± 1.2, n = 4). 99mTc-ZHER2:41071 visualized hepatic metastases in a patient with liver involvement. Conclusions: Injections of 99mTc-ZHER2:41071 appear safe and exhibit favorable dosimetry. The protein dose of 1000 µg provides the best discrimination between HER2-positive and HER2-low/negative expression of HER2 according to the definition used for current HER2-targeting drugs.

Evaluation of affinity matured Affibody molecules for imaging of the immune checkpoint protein B7-H3.

B7-H3 (CD276), an immune checkpoint protein, is a promising molecular target for immune therapy of malignant tumours. Sufficient B7-H3 expression level is a precondition for successful therapy. Radionuclide molecular imaging is a powerful technique for visualization of expression levels of molecular targets in vivo. Use of small radiolabelled targeting proteins would enable high-contrast radionuclide imaging of molecular targets if adequate binding affinity and specificity of an imaging probe could be provided. Affibody molecules, small engineered affinity proteins based on a non-immunoglobulin scaffold, have demonstrated an appreciable potential in radionuclide imaging. Proof-of principle of radionuclide visualization of expression levels of B7-H3 in vivo was demonstrated using the [99mTc]Tc-AC12-GGGC Affibody molecule. We performed an affinity maturation of AC12, enabling selection of clones with higher affinity. Three most promising clones were expressed with a -GGGC (triglycine-cysteine) chelating sequence at the C-terminus and labelled with technetium-99m (99mTc). 99mTc-labelled conjugates bound to B7-H3-expressing cells specifically in vitro and in vivo. Biodistribution in mice bearing B7-H3-expressing SKOV-3 xenografts demonstrated improved imaging properties of the new conjugates compared with the parental variant [99mTc]Tc-AC12-GGGC. [99mTc]Tc-SYNT-179 provided the strongest improvement of tumour-to-organ ratios. Thus, affinity maturation of B7-H3 Affibody molecules could improve biodistribution and targeting properties for imaging of B7-H3-expressing tumours.

Synthesis, 123I-Radiolabeling Optimization, and Initial Preclinical Evaluation of Novel Urea-Based PSMA Inhibitors with a Tributylstannyl Prosthetic Group in Their Structures.

Prostate-specific membrane antigen (PSMA) has been identified as a target for the development of theranostic agents. In our current work, we describe the design and synthesis of novel N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-(S)-L-lysine (DCL) urea-based PSMA inhibitors with a chlorine-substituted aromatic fragment at the lysine ε-nitrogen atom, a dipeptide including two phenylalanine residues in the L-configuration as the peptide fragment of the linker, and 3- or 4-(tributylstannyl)benzoic acid as a prosthetic group in their structures for radiolabeling. The standard compounds [127I]PSMA-m-IB and [127I]PSMA-p-IB for comparative and characterization studies were first synthesized using two alternative synthetic approaches. An important advantage of the alternative synthetic approach, in which the prosthetic group (NHS-activated esters of compounds) is first conjugated with the polypeptide sequence followed by replacement of the Sn(Bu)3 group with radioiodine, is that the radionuclide is introduced in the final step of synthesis, thereby minimizing operating time with iodine-123 during the radiolabeling process. The obtained DCL urea-based PSMA inhibitors were radiolabeled with iodine-123. The radiolabeling optimization results showed that the radiochemical yield of [123I]PSMA-p-IB was higher than that of [123I]PSMA-m-IB, which were 74.9 ± 1.0% and 49.4 ± 1.2%, respectively. The radiochemical purity of [123I]PSMA-p-IB after purification was greater than 99.50%. The initial preclinical evaluation of [123I]PSMA-p-IB demonstrated a considerable affinity and specific binding to PC-3 PIP (PSMA-expressing cells) in vitro. The in vivo biodistribution of this new radioligand [123I]PSMA-p-IB showed less accumulation than [177Lu]Lu-PSMA-617 in several normal organs (liver, kidney, and bone). These results warrant further preclinical development, including toxicology evaluation and experiments in tumor-bearing mice.

Preclinical Characterization of a Stabilized Gastrin-Releasing Peptide Receptor Antagonist for Targeted Cancer Theranostics.

Radiolabeled gastrin-releasing peptide receptor (GRPR) antagonists have shown great promise for the theranostics of prostate cancer; however, their suboptimal metabolic stability leaves room for improvements. It was recently shown that the replacement of Gly11 with Sar11 in the peptidic [D-Phe6,Leu13-NHEt,des-Met14]BBN(6-14) chain stabilized the [99mTc]Tc-DB15 radiotracer against neprilysin (NEP). We herein present DOTAGA-PEG2-(Sar11)RM26 (AU-RM26-M1), after Gly11 to Sar11-replacement. The impact of this replacement on the metabolic stability and overall biological performance of [111In]In-AU-RM26-M1 was studied using a head-to-head comparison with the unmodified reference [111In]In-DOTAGA-PEG2-RM26. In vitro, the cell uptake of [111In]In-AU-RM26-M1 could be significantly reduced in the presence of a high-excess GRPR-blocker that demonstrated its specificity. The cell uptake of both radiolabeled GRPR antagonists increased with time and was superior for [111In]In-AU-RM26-M1. The dissociation constant reflected strong affinities for GRPR (500 pM for [111In]In-AU-RM26-M1). [111In]In-AU-RM26-M1 showed significantly higher stability in peripheral mice blood at 5 min pi (88 ± 8% intact) than unmodified [111In]In-DOTAGA-PEG2-RM26 (69 ± 2% intact; p < 0.0001). The administration of a NEP inhibitor had no significant impact on the Sar11-compound (91 ± 2% intact; p > 0.05). In vivo, [111In]In-AU-RM26-M1 showed high and GRPR-mediated uptake in the PC-3 tumors (7.0 ± 0.7%IA/g vs. 0.9 ± 0.6%IA/g in blocked mice) and pancreas (2.2 ± 0.6%IA/g vs. 0.3 ± 0.2%IA/g in blocked mice) at 1 h pi, with rapid clearance from healthy tissues. The tumor uptake of [111In]In-AU-RM26-M1 was higher than for [111In]In-DOTAGA-PEG2-RM26 (at 4 h pi, 5.7 ± 1.8%IA/g vs. 3 ± 1%IA/g), concordant with its higher stability. The implanted PC-3 tumors were visualized with high contrast in mice using [111In]In-AU-RM26-M1 SPECT/CT. The Gly11 to Sar11-substitution stabilized [111In]In-DOTAGA-PEG2-(Sar11)RM26 against NEP without negatively affecting other important biological features. These results support the further evaluation of AU-RM26-M1 for prostate cancer theranostics after labeling with clinically relevant radionuclides.

Human Epidermal Growth Factor Receptor 2-Targeting [68Ga]Ga-ABY-025 PET/CT Predicts Early Metabolic Response in Metastatic Breast Cancer.

Imaging using the human epidermal growth factor receptor 2 (HER2)-binding tracer 68Ga-labeled ZHER2:2891-Cys-MMA-DOTA ([68Ga]Ga-ABY-025) was shown to reflect HER2 status determined by immunohistochemistry and in situ hybridization in metastatic breast cancer (MBC). This single-center open-label phase II study investigated how [68Ga]Ga-ABY-025 uptake corresponds to biopsy results and early treatment response in both primary breast cancer (PBC) planned for neoadjuvant chemotherapy and MBC. Methods: Forty patients with known positive HER2 status were included: 19 with PBC and 21 with MBC (median, 3 previous treatments). [68Ga]Ga-ABY-025 PET/CT, [18F]F-FDG PET/CT, and core-needle biopsies from targeted lesions were performed at baseline. [18F]F-FDG PET/CT was repeated after 2 cycles of therapy to calculate the directional change in tumor lesion glycolysis (Δ-TLG). The largest lesions (up to 5) were evaluated in all 3 scans per patient. SUVs from [68Ga]Ga-ABY-025 PET/CT were compared with the biopsied HER2 status and Δ-TLG by receiver operating characteristic analyses. Results: Trial biopsies were HER2-positive in 31 patients, HER2-negative in 6 patients, and borderline HER2-positive in 3 patients. The [68Ga]Ga-ABY-025 PET/CT cutoff SUVmax of 6.0 predicted a Δ-TLG lower than -25% with 86% sensitivity and 67% specificity in soft-tissue lesions (area under the curve, 0.74 [95% CI, 0.67-0.82]; P = 0.01). Compared with the HER2 status, this cutoff resulted in clinically relevant discordant findings in 12 of 40 patients. Metabolic response (Δ-TLG) was more pronounced in PBC (-71% [95% CI, -58% to -83%]; P < 0.0001) than in MBC (-27% [95% CI, -16% to -38%]; P < 0.0001), but [68Ga]Ga-ABY-025 SUVmax was similar in both with a mean SUVmax of 9.8 (95% CI, 6.3-13.3) and 13.9 (95% CI, 10.5-17.2), respectively (P = 0.10). In multivariate analysis, global Δ-TLG was positively associated with the number of previous treatments (P = 0.0004) and negatively associated with [68Ga]Ga-ABY-025 PET/CT SUVmax (P = 0.018) but not with HER2 status (P = 0.09). Conclusion: [68Ga]Ga-ABY-025 PET/CT predicted early metabolic response to HER2-targeted therapy in HER2-positive breast cancer. Metabolic response was attenuated in recurrent disease. [68Ga]Ga-ABY-025 PET/CT appears to provide an estimate of the HER2 expression required to induce tumor metabolic remission by targeted therapies and might be useful as an adjunct diagnostic tool.

Direct Intra-Patient Comparison of Scaffold Protein-Based Tracers, [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3, for Imaging of HER2-Positive Breast Cancer.

Previous Phase I clinical evaluations of the radiolabelled scaffold proteins [99mTc]Tc-ADAPT6 and DARPin [99mTc]Tc-(HE)3-G3 in breast cancer patients have demonstrated their safety and indicated their capability to discriminate between HER2-positive and HER2-negative tumours. The objective of this study was to compare the imaging of HER2-positive tumours in the same patients using [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3. Eleven treatment-naïve female patients (26-65 years) with HER2-positive primary and metastatic breast cancer were included in the study. Each patient was intravenously injected with [99mTc]Tc-ADAPT6, followed by an [99mTc]Tc-(HE)3-G3 injection 3-4 days later and chest SPECT/CT was performed. All primary tumours were clearly visualized using both tracers. The uptake of [99mTc]Tc-ADAPT6 in primary tumours (SUVmax = 4.7 ± 2.1) was significantly higher (p < 0.005) than the uptake of [99mTc]Tc-(HE)3-G3 (SUVmax = 3.5 ± 1.7). There was no significant difference in primary tumour-to-contralateral site values for [99mTc]Tc-ADAPT6 (15.2 ± 7.4) and [99mTc]Tc-(HE)3-G3 (19.6 ± 12.4). All known lymph node metastases were visualized using both tracers. The uptake of [99mTc]Tc-ADAPT6 in all extrahepatic soft tissue lesions was significantly (p < 0.0004) higher than the uptake of [99mTc]Tc-(HE)3-G3. In conclusion, [99mTc]Tc-ADAPT6 and [99mTc]Tc-(HE)3-G3 are suitable for the visualization of HER2-positive breast cancer. At the selected time points, [99mTc]Tc-ADAPT6 has a significantly higher uptake in soft tissue lesions, which might be an advantage for the visualization of small metastases.

Radionuclide Therapy of HER2-Expressing Xenografts Using [177Lu]Lu-ABY-027 Affibody Molecule Alone and in Combination with Trastuzumab.

ABY-027 is a scaffold-protein-based cancer-targeting agent. ABY-027 includes the second-generation Affibody molecule ZHER2:2891, which binds to human epidermal growth factor receptor type 2 (HER2). An engineered albumin-binding domain is fused to ZHER2:2891 to reduce renal uptake and increase bioavailability. The agent can be site-specifically labeled with a beta-emitting radionuclide 177Lu using a DOTA chelator. The goals of this study were to test the hypotheses that a targeted radionuclide therapy using [177Lu]Lu-ABY-027 could extend the survival of mice with HER2-expressing human xenografts and that co-treatment with [177Lu]Lu-ABY-027 and the HER2-targeting antibody trastuzumab could enhance this effect. Balb/C nu/nu mice bearing HER2-expressing SKOV-3 xenografts were used as in vivo models. A pre-injection of trastuzumab did not reduce the uptake of [177Lu]Lu-ABY-027 in tumors. Mice were treated with [177Lu]Lu-ABY-027 or trastuzumab as monotherapies and a combination of these therapies. Mice treated with vehicle or unlabeled ABY-027 were used as controls. Targeted monotherapy using [177Lu]Lu-ABY-027 improved the survival of mice and was more efficient than trastuzumab monotherapy. A combination of therapies utilizing [177Lu]Lu-ABY-027 and trastuzumab improved the treatment outcome in comparison with monotherapies using these agents. In conclusion, [177Lu]Lu-ABY-027 alone or in combination with trastuzumab could be a new potential agent for the treatment of HER2-expressing tumors.

The GRPR Antagonist [99mTc]Tc-maSSS-PEG2-RM26 towards Phase I Clinical Trial: Kit Preparation, Characterization and Toxicity.

Gastrin-releasing peptide receptors (GRPRs) are overexpressed in the majority of primary prostate tumors and in prostatic lymph node and bone metastases. Several GRPR antagonists were developed for SPECT and PET imaging of prostate cancer. We previously reported a preclinical evaluation of the GRPR antagonist [99mTc]Tc-maSSS-PEG2-RM26 (based on [D-Phe6, Sta13, Leu14-NH2]BBN(6-14)) which bound to GRPR with high affinity and had a favorable biodistribution profile in tumor-bearing animal models. In this study, we aimed to prepare and test kits for prospective use in an early-phase clinical study. The kits were prepared to allow for a one-pot single-step radiolabeling with technetium-99m pertechnetate. The kit vials were tested for sterility and labeling efficacy. The radiolabeled by using the kit GRPR antagonist was evaluated in vitro for binding specificity to GRPR on PC-3 cells (GRPR-positive). In vivo, the toxicity of the kit constituents was evaluated in rats. The labeling efficacy of the kits stored at 4 °C was monitored for 18 months. The biological properties of [99mTc]Tc-maSSS-PEG2-RM26, which were obtained after this period, were examined both in vitro and in vivo. The one-pot (gluconic acid, ethylenediaminetetraacetic acid, stannous chloride, and maSSS-PEG2-RM26) single-step radiolabeling with technetium-99m was successful with high radiochemical yields (>97%) and high molar activities (16-24 MBq/nmol). The radiolabeled peptide maintained its binding properties to GRPR. The kit constituents were sterile and non-toxic when tested in living subjects. In conclusion, the prepared kit is considered safe in animal models and can be further evaluated for use in clinics.