Development and preclinical characterization of a novel radiotheranostic EphA2-targeting bicyclic peptide.

Erythropoietin-producing hepatocellular receptor A2 (EphA2), is a receptor tyrosine kinase involved in cell-cell interactions. It is known to be overexpressed in various tumors and is associated with poor prognosis. EphA2 has been proposed as a target for theranostic applications. Low molecular weight peptide-based scaffolds with low nanomolar affinities have been shown to be ideal in such applications. Bicyclic peptides have emerged as an alternative to traditional peptides for this purpose, offering affinities comparable to antibodies due to their constrained nature, along with high tissue penetration, and improved stability compared to linear counterparts. This study presents the development and comprehensive in vitro and in vivo preclinical evaluation of BCY18469, a novel EphA2-targeting bicyclic peptide-based radiotheranostic agent. Methods: The EphA2-targeting Bicycle® peptide BCY18469 was identified through phage-display and chemically optimized. BCY18469 was radiolabeled with 68Ga, 177Lu and 111In. The physicochemical properties, binding affinity and internalization as well as specificity of the peptide were evaluated in vitro. In vivo PET/MR and SPECT/CT imaging studies were performed using [68Ga]Ga-BCY18469 and [111In]In-BCY18469, respectively, along with biodistribution of [177Lu]Lu-BCY18469 up to 24 h post injection in HT1080- and PC-3-tumor bearing BALB/c nu/nu EphA2-overexpressing xenograft mouse models. Results: The EphA2-targeting bicyclic peptide BCY18469 showed high binding affinity toward human and mouse EphA2 (1.9 and 3.8 nM, respectively). BCY18469 specifically bound and internalized into EphA2-expressing HT1080 cells. Imaging studies showed high tumor enrichment at early time-points (SUV of 1.7 g/mL at 1 h p.i. and 1.2 g/mL at 2 h p.i. in PET/MRI, HT1080 xenograft) with tumor contrast as early as 5 min p.i. and kidney-mediated clearance. Biodistribution studies revealed high early tumor uptake (19.5 ± 3.5 %ID/g at 1 h p.i., HT1080 xenograft) with SPECT/CT imaging further confirming these findings (5.7 ± 1.5 %ID/g at 1 h p.i., PC-3 xenograft). Conclusion: BCY18469 demonstrated high affinity, specific targeting of EphA2, a favorable biodistribution profile, and clearance through renal pathways. These findings underscore the potentially important role of bicyclic peptides in advancing radiotheranostic approaches and encourage additional translational research.

Chelation of [111In]In3+ with the dual-size-selective macrocycles py-macrodipa and py2-macrodipa.

Indium-111 (111In) is a diagnostic radiometal that is important in nuclear medicine for single-photon emission computed tomography (SPECT). In order to apply this radiometal, it needs to be stably chelated and conjugated to a targeting vector that delivers it to diseased tissue. Identifying effective chelators that are capable of binding and retaining [111In]In3+in vivo is an important research area. In this study, two 18-membered macrocyclic chelators, py-macrodipa and py2-macrodipa, were investigated for their ability to form stable coordination complexes with In3+ and to be effectively radiolabeled with [111In]In3+. The In3+ complexes of these two chelators were characterized by NMR spectroscopy, X-ray crystallography, and density functional theory calculations. These studies show that both py-macrodipa and py2-macrodipa form 8-coordinate In3+ complexes and attain an asymmetric conformation, consistent with prior studies on this ligand class with small rare earth metal ions. Spectrophotometric titrations were carried out to determine the thermodynamic stability constants (log KML) of [In(py-macrodipa)]+ and [In(py2-macrodipa)]+, which were found to be 18.96(6) and 19.53(5), respectively, where the values in parentheses are the errors of the last significant figures obtained from the standard deviation from three independent replicates. Radiolabeling studies showed that py-macrodipa and py2-macrodipa can quantitatively be radiolabeled with [111In]In3+ at 25 °C within 5 min, even at ligand concentrations as low as 1 µM. The in vitro stability of the radiolabeled complexes was investigated in human serum at 37 °C, revealing that ∼90% of [111In][In(py-macrodipa)]+ and [111In][In(py2-macrodipa)]+ remained intact after 7 days. The biodistribution of these radiolabeled complexes in mice was investigated, showing lower uptake in the kidneys, liver, and blood at the 24 h mark compared to [111In]InCl3. These results demonstrate the potential of py-macrodipa and py2-macrodipa as chelators for [111In]In3+, suggesting their value for SPECT radiopharmaceuticals.

Synthesis and Evaluation of a Cathepsin B-Recognizing Trifunctional Chelating Agent to Improve Tumor Retention of Radioimmunoconjugates.

Cathepsin B (CTSB) is a lysosomal protease that is overexpressed in tumor cells. Radioimmunoconjugates (RICs) composed of CTSB-recognizing chelating agents are expected to increase the molecular weights of their radiometabolites by forming conjugates with CTSB in cells, resulting in their improved retention in tumor cells. We designed a novel CTSB-recognizing trifunctional chelating agent, azide-[111In]In-DOTA-CTSB-substrate ([111In]In-ADCS), to synthesize a RIC, trastuzumab-[111In]In-ADCS ([111In]In-TADCS), and evaluated its utility to improve tumor retention of the RIC. [111In]In-ADCS and [111In]In-TADCS were synthesized with satisfactory yield and purity. [111In]In-ADCS was markedly stable in murine plasma until 96 h postincubation. [111In]In-ADCS showed binding to CTSB in vitro, and the conjugation was blocked by the addition of CTSB inhibitor. In the internalization assay, [111In]In-TADCS exhibited high-level retention in SK-OV-3 cells, indicating the in vitro utility of the CTSB-recognizing unit. In the biodistribution assay, [111In]In-TADCS showed high-level tumor accumulation, but the retention was hardly improved. In the first attempt to combine a CTSB-recognizing unit and RIC, these findings show the fundamental properties of the CTSB-recognizing trifunctional chelating agent to improve tumor retention of RICs.

Indium-111 radiolabelling of a brain-penetrant Aß antibody for SPECT imaging.

Background: The development of bispecific antibodies that can traverse the blood-brain barrier has paved the way for brain-directed immunotherapy and when radiolabelled, immunoPET imaging. The objective of this study was to investigate how indium-111 (111In) radiolabelling with compatible chelators affects the brain delivery and peripheral biodistribution of the bispecific antibody RmAb158-scFv8D3, which binds to amyloid-beta (Aß) and the transferrin receptor (TfR), in Aß pathology-expressing tg-ArcSwe mice and aged-matched wild-type control mice. Methods: Bispecific RmAb158-scFv8D3 (biAb) was radiolabelled with 111In using CHX-A"-DTPA, DOTA, or DOTA-tetrazine (DOTA-Tz). Affinity toward TfR and Aß, as well as stability, was investigated in vitro. Mice were then intravenously administered with the three different radiolabelled biAb variants, and blood samples were collected for monitoring pharmacokinetics. Brain concentration was quantified after 2 and 72 h, and organ-specific retention was measured at 72 h by gamma counting. A subset of mice also underwent whole-body Single-photon emission computed tomography (SPECT) scanning at 72 h after injection. Following post-mortem isolation, the brains of tg-ArcSwe and WT mice were sectioned, and the spatial distribution of biAb was further investigated with autoradiography. Results: All three [111In]biAb variants displayed similar blood pharmacokinetics and brain uptake at 2 h after administration. Radiolabelling did not compromise affinity, and all variants showed good stability, especially the DOTA-Tz variant. Whole-body SPECT scanning indicated high liver, spleen, and bone accumulation of all [111In]biAb variants. Subsequent ex vivo measurement of organ retention confirmed SPECT data, with retention in the spleen, liver, and bone - with very high bone marrow retention. Ex vivo gamma measurement of brain tissue, isolated at 72 h post-injection, and ex vivo autoradiography showed that WT mice, despite the absence of Aß, exhibited comparable brain concentrations of [111In]biAb as those found in the tg-ArcSwe brain. Conclusions: The successful 111In-labelling of biAb with retained binding to TfR and Aß, and retained ability to enter the brain, demonstrated that 111In can be used to generate radioligands for brain imaging. A high degree of [111In]biAb in bone marrow and intracellular accumulation in brain tissue indicated some off-target interactions or potential interaction with intrabrain TfR resulting in a relatively high non-specific background signal.

A Phase 0 Study to Assess the Biodistribution and Pharmacokinetics of a Radiolabeled Antibody Targeting Human Kallikrein 2 in Participants with Metastatic Castration-Resistant Prostate Cancer.

Despite the inclusion of multiple agents within the prostate cancer treatment landscape, new treatment options are needed to address the unmet need for patients with metastatic castration-resistant prostate cancer (mCRPC). Although prostate-specific membrane antigen is the only cell-surface target to yield clinical benefit in men with advanced prostate cancer, additional targets may further advance targeted immune, cytotoxic, radiopharmaceutical, and other tumor-directed therapies for these patients. Human kallikrein 2 (hK2) is a novel prostate-specific target with little to no expression in nonprostate tissues. This first-in-human phase 0 trial uses an 111In-radiolabeled anti-hK2 monoclonal antibody, [111In]-DOTA-h11B6, to credential hK2 as a potential target for prostate cancer treatment. Methods: Participants with progressive mCRPC received a single infusion of 2 mg of [111In]-DOTA-h11B6 (185 MBq of 111In), with or without 8 mg of unlabeled h11B6 to assess antibody mass effects. Sequential imaging and serial blood samples were collected to determine [111In]-DOTA-h11B6 biodistribution, dosimetry, serum radioactivity, and pharmacokinetics. Safety was assessed within a 2-wk follow-up period from the time of [111In]-DOTA-h11B6 administration. Results: Twenty-two participants received [111In]-DOTA-h11B6 and are included in this analysis. Within 6-8 d of administration, [111In]-DOTA-h11B6 visibly accumulated in known mCRPC lesions, with limited uptake in other organs. Two treatment-emergent adverse events unrelated to treatment occurred, including tumor-related bleeding in 1 patient, which led to early study discontinuation. Serum clearance, biodistribution, and tumor targeting were independent of total antibody mass (2 or 10 mg). Conclusion: This first-in-human study demonstrates that tumor-associated hK2 can be identified and targeted using h11B6 as a platform as the h11B6 antibody selectively accumulated in mCRPC metastases with mass-independent clearance kinetics. These data support the feasibility of hK2 as a target for imaging and hK2-directed agents as potential therapies in patients with mCRPC.

Fundamental evaluation regarding the relationship between albumin-binding and tumor accumulation of PSMA-targeting radioligands.

OBJECTIVE: The marked success of prostate-specific membrane antigen (PSMA)-targeting radioligands with albumin binder (ALB) is attributed to the improvement of blood retention and tumor accumulation. [111In]In-PNT-DA1, our PSMA-targeting radioligand with ALB, also achieved improved tumor accumulation due to its prolonged blood retention. Although the advantage of ALBs is related to their reversible binding to albumin, the relationship between albumin-binding and tumor accumulation of PSMA-targeting radioligands remains unclear because of the lack of information about radioligands with stronger albumin-binding than ALBs. In this study, we designed and synthesized [111In]In-PNT-DM-HSA, a new radioligand that consists of a PSMA-targeting radioligand covalently bound to albumin. The pharmacokinetics of [111In]In-PNT-DM-HSA was compared with those of [111In]In-PNT-DA1 and [111In]In-PSMA-617, a non-ALB-conjugated radioligand, to evaluate the relationship between albumin-binding and tumor accumulation. METHOD: The [111In]In-PNT-DM-HSA was prepared by incubation of [111In]In-PNT-DM, a PSMA-targeting radioligand including a maleimide group, and human serum albumin (HSA). The ability of [111In]In-PNT-DM-HSA was evaluated by in vitro assays. A biodistribution study using LNCaP tumor-bearing mice was conducted to compare the pharmacokinetics of [111In]In-PNT-DM-HSA, [111In]In-PNT-DA1, and [111In]In-PSMA-617. RESULTS: The [111In]In-PNT-DM-HSA was obtained at a favorable radiochemical yield and high radiochemical purity. In vitro assays revealed that [111In]In-PNT-DM-HSA had fundamental characteristics as a PSMA-targeting radioligand interacting with albumin covalently. In a biodistribution study, [111In]In-PNT-DM-HSA and [111In]In-PNT-DA1 showed higher blood retention than [111In]In-PSMA-617. On the other hand, the tumor accumulation of [111In]In-PNT-DA1 was much higher than [111In]In-PNT-DM-HSA and [111In]In-PSMA-617. CONCLUSIONS: These results indicate that the moderate reversible binding of ALB with albumin, not covalent binding, may play a critical role in enhancing the tumor accumulation of PSMA-targeting radioligands.

Reduction of the hepatic radioactivity levels of [111In]In-DOTA-labeled antibodies via cleavage of a linkage metabolized in lysosomes.

INTRODUCTION: Radiolabeled antibodies are promising tools for cancer diagnosis using nuclear medicine. A DOTA-chelating system is useful for preparing immuno-positron emission tomography and immuno-single-photon emission computed tomography probes with various radiometals. Radiolabeled antibodies are generally metabolized in the reticuloendothelial system, producing radiometabolites after proteolysis in hepatic lysosomes. Because of the bulkiness and extremely high hydrophilicity of DOTA, radiometabolites containing a radiometal-DOTA complex typically exhibit high and persistent localization in hepatic lysosomes. Radioactivity in the liver impairs the accurate diagnosis of cancer surrounding the liver and liver metastasis, and a high tumor/liver ratio is desirable. In this study, we reduced the hepatic radioactivity of radiometal-labeled antibodies containing a DOTA-chelating system. A cleavable linkage was inserted to liberate the radiometabolite, which exhibited a short residence time in hepatocytes. METHODS: Using indium-111 (111In)-labeled antibodies, we prepared 111In-labeled galactosyl-neoglycoalbumins (NGAs) because they are useful for evaluating the residence time of radiometabolites in the liver. An 111In-labeled NGA with a cleavable linkage ([111In]In-DO3AiBu-Bn-FGK-NGA) was administered to normal mice, and biodistribution studies and metabolic analyses of urinary and fecal samples were performed with comparison to an 111In-labeled NGA prepared by a conventional method ([111In]In-DOTA-Bn-SCN-NGA). Then, 111In-labeled antibodies ([111In]In-DO3AiBu-Bn-FGK-IgG and [111In]In-DOTA-Bn-SCN-IgG) were prepared using a procedure similar to that for 111In-labeled NGAs. In vitro plasma stability and biodistribution were investigated for both 111In-labeled antibodies in U87MG tumor-bearing mice. RESULTS: Through the liberation of radiometabolites including [111In]In-DO3AiBu-Bn-F, [111In]In-DO3AiBu-Bn-FGK-NGA was cleared more rapidly from the liver than [111In]In-DOTA-Bn-SCN-NGA (4.07 ± 1.54%ID VS 71.68 ± 3.03%ID at 6 h postinjection). [111In]In-DO3AiBu-Bn-FGK-IgG exhibited lower tumor accumulation (8.83 ± 1.48%ID/g) but a significantly higher tumor/liver ratio (2.21 ± 0.53) than [111In]In-DOTA-Bn-SCN-IgG (11.65 ± 2.17%ID/g in the tumor and a tumor/liver ratio of 0.85 ± 0.18) at 72 h after injection. CONCLUSION: A molecular design that reduces the high and persistent hepatic radioactivity of radiolabeled antibodies by liberating radiometabolites with a short hepatic residence time in lysosomes would be applicable for radiometal-labeled antibodies using a DOTA-chelating system.

Development of Novel 111In/225Ac-Labeled Agent Targeting PSMA for Highly Efficient Cancer Radiotheranostics.

Prostate-specific membrane antigen (PSMA) is a promising target for metastatic castration-resistant prostate cancer. We previously reported the effectiveness of PSMA-DA1 as a PSMA-targeting radiotheranostic agent containing an albumin binder moiety. To further enhance tumor uptake, we newly designed PSMA-NAT-DA1 (PNT-DA1) by the introduction of a lipophilic linker into PSMA-DA1. The PSMA affinity of [111In]In-PNT-DA1 was increased (Kd = 8.20 nM) compared with that of [111In]In-PSMA-DA1 (Kd = 89.4 nM). [111In]In-PNT-DA1 showed markedly high tumor accumulation (131.6% injected dose/g at 48 h post-injection), and [111In]In-PNT-DA1 enabled the visualization of the tumor clearly at 24 h post-injection with SPECT/CT imaging. The administration of [225Ac]Ac-PNT-DA1 (2.5 kBq) led to shrinkage of the tumor without marked toxicity, and the antitumor effects of [225Ac]Ac-PNT-DA1 were superior to those of [225Ac]Ac-PSMA-DA1 and [225Ac]Ac-PSMA-617, which is the current gold standard for PSMA-targeting 225Ac-endoradiotherapy. These results suggest that the combination of [111In]In-PNT-DA1 and [225Ac]Ac-PNT-DA1 comprises a promising method of PSMA-targeting radiotheranostics.

Favorable tumor uptake and nuclear transport of Auger electrons by nuclear targeting with 111In-trastuzumab in an intraperitoneal tumor mouse model.

OBJECTIVES: The 111In-labeled anti-HER2 antibody trastuzumab modified with a nuclear-localizing sequence (NLS) peptide (111In-trastuzumab-NLS) is a radiopharmaceutical candidate for Auger electron radioimmunotherapy (AE-RIT). However, in-vivo action of 111In-trastuzumab-NLS is poorly understood in intraperitoneal tumors. We aimed to elucidate the nuclear targeting activity of 111In-trastuzumab-NLS in a mouse model of intraperitoneal tumors. METHODS: Trastuzumab, trastuzumab-NLS-S with shorter NLS peptides, and trastuzumab-NLS-L with longer NLS peptides were tested in an intraperitoneal tumor xenograft. The AE-emitting radionuclide 111In was labeled with these antibodies. The cell-binding activity, nuclear importation, and cytotoxicity of those radiolabeled antibodies were examined in human cancer cell lines. Analyses of the biodistribution and in-vivo nuclear importation of 111In were conducted in a mouse model. RESULTS: The two111In-trastuzumab-NLS variants delivered the radionuclide into the nucleus more efficiently and had a comparable cytotoxicity to 111In-trastuzumab against human gastric cancer cells, although had a lower cell binding affinity. 111In-trastuzumab-NLS-L exhibited both a superior tumor uptake and in vivo nuclear transportation of the radionuclide than 111In-trastuzumab. CONCLUSION: Nuclear targeting using 111In-trastuzumab-NLS promotes a more efficient tumor cell uptake and subsequent nuclear translocation of the 111In AE-emitting radionuclide in vivo. This radio-immunoconjugate will likely be an effective agent for HER2-targeting by AE-RIT.

Development of a novel Indium-111 radiolabeled mogamulizumab targeting CCR4 for imaging adult T-cell leukemia/lymphoma in vivo.

OBJECTIVE: Adult T-cell leukemia/lymphoma (ATL), caused by human T-cell lymphotropic virus type I (HTLV-1) infection, is among the most aggressive categories and has the worst prognosis among T-cell lymphomas. Mogamulizumab, an anti-CC chemokine receptor 4 (CCR 4), has been shown to be effective in the treatment of ATL; however, some ATL cases are often resistant, particularly the lymphoma-type ATL. To evaluate drug delivery in vivo and identify the distribution of CCR4-positive cells in the body, we developed a novel mogamulizumab tracer labeled with Indium-111 (111In) via diethylenetriaminepentaacetic acid (DTPA) for single-photon emission computerized tomography (SPECT), named [111In]In-DTPA-mogamulizumab, and evaluated its potential for visualizing CCR4 expression in vivo. METHODS: [111In]In-DTPA-mogamulizumab was added to HCT116/CCR4 or HCT116/empty vector (EV) cells, and their radioactivity was measured 1 h after administration. A blocking study was additionally performed by treating HCT116/CCR4 cells with excess mogamulizumab in addition to [111In]In-DTPA-mogamulizumab. The biodistribution and SPECT imaging of [111In]In-DTPA-mogamulizumab in HCT116/CCR4 and HCT116/EV dual-xenografted BALB/c-nu mice were evaluated for 72 h after intravenous injection. RESULTS: [111In]In-DTPA-mogamulizumab was acquired with a radiochemical purity > 95%. The cellular uptake level of [111In]In-DTPA-mogamulizumab by HCT116/CCR4 cells was significantly higher than that by HCT116/EV cells (HCT116/CCR4: 0.951 ± 0.069, HCT116/EV: 0.006 ± 0.001%dose/mg protein, p < 0.01), and the uptake was significantly suppressed by co-incubation with excess mogamulizumab (0.013 ± 0.003%dose/mg protein, p < 0.01). In the in vivo study, the radioactivity of the HCT116/CCR4 tumor tissue was significantly higher than that of the HCT116/EV tumor tissue at 72 h after the administration of [111In]In-DTPA-mogamulizumab (HCT116/CCR4: 20.5 ± 5.4, HCT116/EV: 5.7 ± 1.0%ID/g), and HCT116/CCR4 tumors were clearly and specifically visualized on SPECT imaging. CONCLUSIONS: We have successfully developed a novel SPECT imaging tracer targeting CCR4, [111In]In-DTPA-mogamulizumab, which showed good specificity and pharmacokinetics, indicating potential in visualizing CCR4 expression in vivo.

111In-Pentetreotide Uptake Due to COVID-19 Vaccination.

ABSTRACT: A 72-year-old woman was referred for whole-body 111In-pentetreotide scintigraphy with SPECT/CT. There was increased uptake of lymphadenopathy in the left axilla and left deltoid muscle. The patient's history revealed that the patient received the first dose of the COVID-19 vaccine 3 days before the 111In-pentetreotide scintigraphy with SPECT/CT. This case demonstrates that the COVID-19 vaccine can cause 111In-pentetreotide uptake in the lymph nodes and the deltoid muscle.

SPECT/CT imaging of inflammation and calcification in human carotid atherosclerosis to identify the plaque at risk of rupture.

BACKGROUND: Calcification and inflammation are atherosclerotic plaque compositional biomarkers that have both been linked to stroke risk. The aim of this study was to evaluate their co-existing prevalence in human carotid plaques with respect to plaque phenotype to determine the value of hybrid imaging for the detection of these biomarkers. METHODS: Human carotid plaque segments, obtained from endarterectomy, were incubated in [111In]In-DOTA-butylamino-NorBIRT ([111In]In-Danbirt), targeting Leukocyte Function-associated Antigen-1 (LFA-1) on leukocytes. By performing SPECT/CT, both inflammation from DANBIRT uptake and calcification from CT imaging were assessed. Plaque phenotype was classified using histology. RESULTS: On a total plaque level, comparable levels of calcification volume existed with different degrees of inflammation and vice versa. On a segment level, an inverse relationship between calcification volume and inflammation was evident in highly calcified segments, which classify as fibrocalcific, stable plaque segments. In contrast, segments with little or no calcification presented with a moderate to high degree of inflammation, often coinciding with the more dangerous fibrous cap atheroma phenotype. CONCLUSION: Calcification imaging alone can only accurately identify highly calcified, stable, fibrocalcific plaques. To identify high-risk plaques, with little or no calcification, hybrid imaging of calcification and inflammation could provide diagnostic benefit.

Assessment of Cell Cytotoxicity and Comet Assay on HER2/neu Positive Cell Line Due to 111In Auger Electrons as DNA-Targeting Radioimmunoconjugate.

BACKGROUND: Breast cancer Auger electron therapy is a growing field of study in radioimmunotherapy and oncology research. Trastuzumab, a high affinity-binding monoclonal antibody against HER2/neu is which is over-expressed in breast tumors, is used in radiopharmaceutical development. OBJECTIVES: In this work, the lethal effects of 111In3+, 111In-DTPA-trastuzumab and 111In-trastuzumab coupled-nuclear localizing sequence peptide (111In-DTPA-NLS-trastuzumab) on malignant cells were studied in vitro. METHODS: DTPA-NLS-trastuzumab was prepared using sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) conjugation with NLS peptide in the first step, followed by conjugation with diethylenetriaminepentaacetic acid (DTPA). Both DTPA-trastuzumab and DTPA- NLS-trastuzumab were labeled with 111In followed by purification and quality control techniques. Sk-Br-3 (a HER2/neu+ cell line), was used in the cell viability assessment assay for 111In, 111In-DTPA-trastuzumab and 111In-DTPA-NLS-trastuzumab (3.7 MBq) at 37 ºC. The cytotoxicity of the three species was studied using MTT and comet assay was utilized DNA damage detection. RESULTS: A significant radiochemical purity for 111In-DTPA-NLS-trastuzumab (99.36% ± 0.30%, ITLC) at the DTPA:antibody ratio of 6.90 ± 0.34:1, was obtained. Significant cell viability difference was found for 111In-DTPA-NLS-trastuzumab compared to the other treatments at two-time points. In addition, comet assay demonstrated significant DNA damage at 144 h using 111In-DTPA- NLS-trastuzumab. CONCLUSION: The results of cell viability and cell death using MTT assay and comet assay, respectively, demonstrate the NLS-peptide effectively facilitates 111In-trastuzumab transport into the HER2/neu positive cancer cell nuclei to impose the radiotherapeutic effects of Auger electrons on DNA leading to cell death.

Discovery, optimization and biodistribution of an Affibody molecule for imaging of CD69.

Due to the wide scale of inflammatory processes in different types of disease, more sensitive and specific biomarkers are required to improve prevention and treatment. Cluster of differentiation 69 (CD69) is one of the earliest cell surface proteins expressed by activated leukocytes. Here we characterize and optimize potential new imaging probes, Affibody molecules targeting CD69 for imaging of activated immune cells. Analysis of candidates isolated in a previously performed selection from a Z variant E. coli library to the recombinant extracellular domain of human CD69, identified one cross-reactive Z variant with affinity to murine and human CD69. Affinity maturation was performed by randomization of the primary Z variant, followed by selections from the library. The resulting Z variants were evaluated for affinity towards human and murine CD69 and thermal stability. The in vivo biodistribution was assessed by SPECT/CT in rats following conjugation of the Z variants by a DOTA chelator and radiolabeling with Indium-111. A primary Z variant with a Kd of approximately 50 nM affinity to human and murine CD69 was identified. Affinity maturation generated 5 additional Z variants with improved or similar affinity. All clones exhibited suitable stability. Radiolabeling and in vivo biodistribution in rat demonstrated rapid renal clearance for all variants, while the background uptake and washout varied. The variant ZCD69:4 had the highest affinity for human and murine CD69 (34 nM) as well as the lowest in vivo background binding. In summary, we describe the discovery, optimization and evaluation of novel Affibody molecules with affinity for CD69. Affibody molecule ZCD69:4 is suitable for further development for imaging of activated immune cells.

A novel 111indium-labeled dual carbonic anhydrase 9-targeted probe as a potential SPECT imaging radiotracer for detection of hypoxic colorectal cancer cells.

Tumor hypoxia is a common feature in colorectal cancer (CRC), and is associated with resistance to radiotherapy and chemotherapy. Thus, a specifically targeted probe for the detection of hypoxic CRC cells is urgently needed. Carbonic anhydrase 9 (CA9) is considered to be a specific marker for hypoxic CRC diagnosis. Here, a nuclear imaging Indium-111 (111In)-labeled dual CA9-targeted probe was synthesized and evaluated for CA9 detection in in vitro, in vivo, and in human samples. The CA9-targeted peptide (CA9tp) and CA9 inhibitor acetazolamide (AAZ) were combined to form a dual CA9-targeted probe (AAZ-CA9tp) using an automatic microwave peptide synthesizer, which then was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for radioisotope (111In) labeling (111In-DOTA-AAZ-CA9tp). The assays for cell binding, stability, and toxicity were conducted in hypoxic CRC HCT15 cells. The analyses for imaging and biodistribution were performed in an HCT15 xenograft mouse model. The binding and distribution of 111In-DOTA-AAZ-CA9tp were detected in human CRC samples using microautoradiography. AAZ-CA9tp possessed good CA9-targeting ability in hypoxic HCT15 cells. The dual CA9-targeted radiotracer showed high serum stability, high surface binding, and high affinity in vitro. After exposure of 111In-DOTA-AAZ-CA9tp to the HCT15-bearing xenograft mice, the levels of 111In-DOTA-AAZ-CA9tp were markedly and specifically increased in the hypoxic tumor tissues compared to control mice. 111In-DOTA-AAZ-CA9tp also targeted the areas of CA9 overexpression in human colorectal tumor tissue sections. The results of this study suggest that the novel 111In-DOTA-AAZ-CA9tp nuclear imaging agent may be a useful tool for the detection of hypoxic CRC cells in clinical practice.

[Factors influencing platelets labelling with indium-111 oxine]. / Facteurs influençant le marquage des plaquettes à l'oxinate d'indium-111.

Autologous indium-111 labelled platelets can be used for kinetic studies in patients with autoimmune thrombocytopenic purpura [AITP]. The objective of this study was to evaluate some biological and clinical factors influencing the labeling efficiency. METHODS: We studied incubation media (Plasma media [MP] or dry media [MS]), platelet concentration [NP], mean platelet volume [VPM], hemoglobin level and pathology associated with AITP. RESULTS: This was a retrospective study of 93 platelets labelling (43 in MS and 50 in MP), 38 primary AITP (41%) and 55 secondary AITP (59%). The labeling efficiency was 72% (78% in MS versus 53% in MP; p < 0.0001). The labeling efficiency was correlated with VPM (p = 0.0004), NP (p = 0.03), hemoglobin level (p = 0.037) and type of AITP (p = 0.0036). The incubation medium, hemoglobin level and type of the AITP have an independent predictive value on the labeling efficiency. CONCLUSION: These data confirm the influence of the incubation medium on the labeling efficiency and identify two other predictive criteria, hemoglobin level and type of AITP.

Molecular Imaging and Preclinical Studies of Radiolabeled Long-Term RGD Peptides in U-87 MG Tumor-Bearing Mice.

The Arg-Gly-Asp (RGD) peptide shows a high affinity for αvß3 integrin, which is overexpressed in new tumor blood vessels and many types of tumor cells. The radiolabeled RGD peptide has been studied for cancer imaging and radionuclide therapy. We have developed a long-term tumor-targeting peptide DOTA-EB-cRGDfK, which combines a DOTA chelator, a truncated Evans blue dye (EB), a modified linker, and cRGDfK peptide. The aim of this study was to evaluate the potential of indium-111(111In) radiolabeled DOTA-EB-cRGDfK in αvß3 integrin-expressing tumors. The human glioblastoma cell line U-87 MG was used to determine the in vitro binding affinity of the radiolabeled peptide. The in vivo distribution of radiolabeled peptides in U-87 MG xenografts was investigated by biodistribution, nanoSPECT/CT, pharmacokinetic and excretion studies. The in vitro competition assay showed that 111In-DOTA-EB-cRGDfK had a significant binding affinity to U-87 MG cancer cells (IC50 = 71.7 nM). NanoSPECT/CT imaging showed 111In-DOTA-EB-cRGDfK has higher tumor uptake than control peptides (111In-DOTA-cRGDfK and 111In-DOTA-EB), and there is still a clear signal until 72 h after injection. The biodistribution results showed significant tumor accumulation (27.1 ± 2.7% ID/g) and the tumor to non-tumor ratio was 22.85 at 24 h after injection. In addition, the pharmacokinetics results indicated that the 111In-DOTA-EB-cRGDfK peptide has a long-term half-life (T1/2λz = 77.3 h) and that the calculated absorbed dose was safe for humans. We demonstrated that radiolabeled DOTA-EB-cRGDfK may be a promising agent for glioblastoma tumor imaging and has the potential as a theranostic radiopharmaceutical.

IMPROVEMENTS OF 111IN SPECT IMAGES RECONSTRUCTED WITH SPARSELY ACQUIRED PROJECTIONS BY DEEP LEARNING GENERATED SYNTHETIC PROJECTIONS.

The aim was to improve single-photon emission computed tomography (SPECT) quality for sparsely acquired 111In projections by adding deep learning generated synthetic intermediate projections (SIPs). Method: The recently constructed deep convolutional network for generating synthetic intermediate projections (CUSIP) was used for improving 20 sparsely acquired 111In-octreotide SPECTs. Reconstruction was performed with 120 (120P) or 30 (30P) projections, or 120 projections with 90 SIPs generated from 30 projections (30-120SIP). The SPECT reconstructions were performed with attenuation, scatter and collimator response corrections. Postfiltered 30P reconstructed SPECT was also analyzed. Image quality were quantitatively evaluated with root-mean-square error, peak signal-to-noise ratio and structural similarity index metrics. Result: The 30-120SIP reconstructed SPECT had statistically significant improved image quality parameters compared to 30P reconstructed SPECT with and without post filtering. The images visual appearance was similar to slightly filtered 120P SPECTs. Thereby, substantial acquisition time reduction with SIPs seems possible without image quality degradation.

EVALUATION OF THE SPATIAL RESOLUTION IN MONTE CARLO-BASED SPECT/CT RECONSTRUCTION OF 111IN-OCTREOTIDE IMAGES.

The purpose was to evaluate the spatial resolution in 111In-octreotide single-photom emission computed tomography (SPECT)/computed tomography (CT) imaging following reconstructions with three different ordered subset expectation maximizations (OSEM) reconstruction algorithms; attenuation corrected (AC) OSEM, AC OSEM with resolution recovery (ACRR OSEM) and Monte Carlo-based OSEM reconstruction (MC OSEM). SPECT/CT imaging of a triple line phantom containing 111In in air and water was performed. The spatial resolution, represented by the full width at half maximum (FWHM) of a line profile, was determined for each line, for X and Y direction and for all reconstructions. The mean FWHM was 12.2 mm (±standard deviation [SD] 3.7 mm) for AC OSEM, 9.3 mm (±SD 2.5 mm) for ACRR OSEM and 8.2 mm (±SD 2.0 mm) for MC OSEM. MC-based SPECT/CT reconstruction clearly improves the spatial resolution in 111In-octreotide imaging and since MC simulations can be performed for all photon energies MC OSEM has the potential to improve SPECT/CT imaging overall.

A Radiolabeled Self-assembled Nanoparticle Probe for Diagnosis of Lung-Metastatic Melanoma.

Melanoma is a highly malignant skin cancer that frequently metastasizes to the lung, bone, and brain at an early phase. Therefore, noninvasive detection of metastasized melanoma could be beneficial to determine suitable therapeutic strategies. We previously reported a biocompatible ternary anionic complex composed of plasmid DNA (pDNA), polyethyleneimine (PEI), and γ-polyglutamic acid (γ-PGA) based on an electrostatic interaction, which was highly taken up by melanoma cells (B16-F10), even if it was negatively charged. Here, we developed a radiolabeled γ-PGA complex by using indium-111 (111In)-labeled polyamidoamine dendrimer (4th generation; G4) instead of pDNA and iodine-125 (125I)-labeled PEI instead of native PEI, and evaluated its effectiveness as a melanoma-targeted imaging probe. This ternary complex was synthesized at a theoretical charge ratio; carboxyl groups of 111In-diethylenetriaminepentaacetic acid (DTPA)-G4 : amino groups of 125I-PEI : carboxyl groups of γ-PGA was 1 : 8 : 16, and the size and zeta potential were approximately 29 nm and -33 mV, respectively. This complex was taken up by B16-F10 cells with time. Furthermore, a biodistribution study, using normal mice, demonstrated its accumulation in the liver, spleen, and lung, where macrophage cells are abundant. Almost the same level of radioactivity derived from both 111In and 125I was observed in these organs at an early phase after probe injection. Compared with the normal mice, significantly higher lung-to-blood ratios of radioactivity were observed in the B16-F10-lung metastatic cancer model. In conclusion, the radiolabeled γ-PGA complex would hold potentialities for nuclear medical imaging of lung metastatic melanoma.