Evaluation of aptamers for targeted radiotherapy: Binding specificity and labelling with natural lutetium

Many cancer patients suffer from serious side effects when treated with external beam radiotherapy or chemotherapy because of damage to healthy tissues by lack of selectivity. In this regard, specific targeting of tumours through radiopharmaceuticals is increasingly considered as a promising strategy in oncology. Radiopharmaceuticals consist of a radionuclide coupled to a vector that specifically targets cancer-related molecules. Aptamers are small (5-15 kDa) synthetic oligonucleotides (DNA or RNA) that possess several advantages compared to other vectors, such as an relatively easy and cheap chemical synthesis allowing the introduction of different chemical modifications, a selection possible against almost every target, a non-immunogenicity and a good tumour penetration. That is why aptamers are regarded as promising molecules for the development of radiopharmaceuticals. An aptamer targeting the Human Epidermal growth factor Receptor 3 (HER3), which plays an important role in cancer development and progression, was chosen for the development of aptamer-based radiopharmaceuticals

Potential Biomarkers for Noninfectious Scleritis Identified by Serum and Tear Fluid Proteomics

Purpose: Scleritis is an extremely painful and potentially blinding inflammation of the sclera with unknown pathogenesis and unpredictable course. To gain insight in its disease process and identify biomarker candidates, we performed extensive proteomics in serum and tear fluid. Design: Prospective multicenter cohort study. Participants: A total of 121 patients with noninfectious scleritis (of which 39 active cases), 30 healthy controls, and 23 disease controls (uveitis and rheumatoid arthritis) were enrolled in the Netherlands from 2020 to 2022. Methods: Serum, tear fluid of both eyes, and clinical data were gathered. The level of 368 inflammatory proteins was measured using proximity extension assays. Results were validated in an independent cohort of 15 patients with scleritis, and using addressable laser bead immunoassay, or enzyme-linked immunoassays. In addition, we studied an extended panel of matrix metalloproteinases in tear fluid of necrotizing scleritis with addressable laser bead immunoassay. Main Outcome Measures: Statistically significant differences in the level of inflammatory proteins between patients with scleritis and control groups.Results:Proteomics revealed 18 significantly upregulated or downregulated serum proteins in active scleritis cases compared with all control groups in both the discovery cohort and the validation cohort. The most upregulated protein was nuclear migration protein nudC (NudC; P = 0.0032), a protein involved in neurogenesis. The other significant hits included proteins involved in T-cell activation, apoptosis, epithelial barrier maintenance, and angiogenesis. Our tear fluid analysis showed matrix metalloproteinase 9 (MMP9) to be upregulated in the tear fluid of patients with scleral necrosis. Conclusions: The results of our proteomics analysis suggest a role for neurogenesis, T-cell activation, disruption of epithelial barrier, and angiogenesis in the pathogenesis of scleritis, and highlight MMP9 and NudC as biomarkers with potential clinical relevance. </p

TBX2, a Novel Regulator of Labour

Background and Objectives: Therapeutic interventions targeting molecular factors involved in the transition from uterine quiescence to overt labour are not substantially reducing the rate of spontaneous preterm labour. The identification of novel rational therapeutic targets are essential to prevent the most common cause of neonatal mortality. Based on our previous work showing that Tbx2 (T-Box transcription factor 2) is a putative upstream regulator preceding progesterone withdrawal in mouse myometrium, we now investigate the role of TBX2 in human myometrium. Materials and Methods: RNA microarray analysis of (A) preterm human myometrium samples and (B) myometrial cells overexpressing TBX2 in vitro, combined with subsequent analysis of the two publicly available datasets of (C) Chan et al. and (D) Sharp et al. The effect of TBX2 overexpression on cytokines/chemokines secreted to the myometrium cell culture medium were determined by Luminex assay. Results: Analysis shows that overexpression of TBX2 in myometrial cells results in downregulation of TNFα- and interferon signalling. This downregulation is consistent with the decreased expression of cytokines and chemokines of which a subset has been previously associated with the inflammatory pathways relevant for human labour. In contrast, CXCL5 (C-X-C motif chemokine ligand 5), CCL21 and IL-6 (Interleukin 6), previously reported in relation to parturition, do not seem to be under TBX2 control. The combined bioinformatical analysis of the four mRNA datasets identifies a subset of upstream regulators common to both preterm and term labour under control of TBX2. Surprisingly, TBX2 mRNA levels are increased in preterm contractile myometrium. Conclusions: We identified a subset of upstream regulators common to both preterm and term labour that are activated in labour and repressed by TBX2. The increased TBX2 mRNA expression in myometrium collected during a preterm caesarean section while in spontaneous preterm labour compared to tissue harvested during iatrogenic preterm delivery does not fit the bioinformatical model. We can only explain this by speculating that the in vivo activity of TBX2 in human myometrium depends not only on the TBX2 expression levels but also on levels of the accessory proteins necessary for TBX2 activity

Study protocol of the TIRED study:A randomised controlled trial comparing either graded exercise therapy for severe fatigue or cognitive behaviour therapy with usual care in patients with incurable cancer

Background: Fatigue is a common and debilitating symptom for patients with incurable cancer receiving systemic treatment with palliative intent. There is evidence that non-pharmacological interventions such as graded exercise therapy (GET) or cognitive behaviour therapy (CBT) reduce cancer-related fatigue in disease-free cancer patients and in patients receiving treatment with curative intent. These interventions may also result in a reduction of fatigue in patients receiving treatment with palliative intent, by improving physical fitness (GET) or changing fatigue-related cognitions and behaviour (CBT). The primary aim of our study is to assess the efficacy of GET or CBT compared to usual care (UC) in reducing fatigue in patients with incurable cancer. Methods: The TIRED study is a multicentre three-armed randomised controlled trial (RCT) for incurable cancer patients receiving systemic treatment with palliative intent. Participants will be randomised to GET, CBT, or UC. In addition to UC, the GET group will participate in a 12-week supervised exercise programme. The CBT group will receive a 12-week CBT intervention in addition to UC. Primary and secondary outcome measures will be assessed at baseline, post-intervention (14 weeks), and at follow-up assessments (18 and 26 weeks post-randomisation). The primary outcome measure is fatigue severity (Checklist Individual Strength subscale fatigue severity). Secondary outcome measures are fatigue (EORTC-QLQ-C30 subscale fatigue), functional impairments (Sickness Impact Profile total score, EORTC-QLQ-C30 subscale emotional functioning, subscale physical functioning) and quality of life (EORTC-QLQ-C30 subscale QoL). Outcomes at 14 weeks (primary endpoint) of either treatment arm will be compared to those of UC participants. In addition, outcomes at 18 and 26 weeks (follow-up assessments) of either treatment arm will be compared to those of UC participants. Discussion: To our knowledge, the TIRED study is the first RCT investigating the efficacy of GET and CBT on reducing fatigue during treatment with palliative intent in incurable cancer patients. The results of this study will provide information about the possibility and efficacy of GET and CBT for severely fatigued incurable cancer patients

Fatigue and its associated psychosocial factors in cancer patients on active palliative treatment measured over time

PURPOSE: Fatigue is a frequently reported symptom by patients with advanced cancer, but hardly any prospective information is available about fatigue while on treatment in the palliative setting. In a previous cross-sectional study, we found several factors contributing to fatigue in these patients. In this study, we investigated the course of fatigue over time and if psychosocial factors were associated with fatigue over time. METHODS: Patients on cancer treatment for incurable solid tumors were observed over 6 months. Patients filled in the Checklist Individual Strength monthly to measure the course of fatigue. Baseline questionnaires were used to measure disease acceptance, anxiety, depressive mood, fatigue catastrophizing, sleeping problems, discrepancies in social support, and self-reported physical activity for their relation with fatigue over time. RESULTS: At baseline 137 patients and after 6 months 89 patients participated. The mean duration of participation was 4.9 months. At most time points, fatigue scores were significantly higher in the group dropouts in comparison with the group participating 6 months (completers). Overall fatigue levels remained stable over time for the majority of participants. In the completers, 42 % never experienced severe fatigue, 29 % persisted being severely fatigued, and others had either an increasing or decreasing level. Of the investigated factors, low reported physical activity and non-acceptance of cancer were associated significantly to fatigue. CONCLUSION: A substantial number of participants never experienced severe fatigue and fatigue levels remained stable over time. For those who do experience severe fatigue, non-acceptance of having incurable cancer and low self-reported physical activity may be fatigue-perpetuating factors

Preclinical development of 68Ga-labelled aptamers for molecular cancer imaging

Breast cancer is the most common cancer in women worldwide. About 1 in 8 women will develop invasive breast cancer during their lifetime. Breast cancer is the second leading cause of cancer death in women, after lung cancer. Approximately 20-25% of breast cancer cases overexpress the HER2 receptor. Currently, the HER2 status of a tumour is assessed by tumour biopsy and subsequent immunohistochemistry. However, small biopsies may not be representative of the entire tumour mass or metastases (due to tumour heterogeneity), risking misclassification and selection of suboptimal therapies. Molecular imaging of HER2 could potentially overcome these limitations by providing information at the whole body-level in a fast and non-invasive way.This PhD thesis aimed at developing novel aptamer-based radiopharmaceuticals for molecular imaging of HER2-positive cancer at the preclinical level. Aptamers are short, synthetic oligonucleotides that can interact with a specific target through their 3D- structure. We selected novel HER2 aptamers through the systematic evolution of ligands by exponential enrichment (SELEX) process. Various settings of the SELEX process were investigated in order to obtain HER2 aptamers with high affinity and specificity for the HER2 receptor. Subsequently, the selected HER2 aptamers were developed into radiopharmaceuticals for PET imaging. We established an easy and reliable protocol for the bioconjugation to a chelator (NOTA) and radiolabelling with 68Ga in order to obtain pure and stable products. Finally, we evaluated the 68Ga-radiolabelled HER2 aptamers in a mouse model bearing HER2-positive and HER2-negative tumours in both hind flanks. Ex vivo biodistribution analysis and PET/MRI scanning revealed high uptake in the tumours and in the main organs, except the brain, which could be overcome by blood perfusion. Our findings suggest that the aptamers bind to blood proteins in a non-specific way. Further research is warranted to achieve decreased blood residence time in order to gain better contrast for imaging. This PhD research was a first step towards the preclinical development of aptamer-based radiopharmaceuticals for molecular cancer imaging

Improved Aptamers for the Diagnosis and Potential Treatment of HER2-Positive Cancer

Aptamers provide a potential source of alternative targeting molecules for existing antibody diagnostics and therapeutics. In this work, we selected novel DNA aptamers targeting the HER2 receptor by an adherent whole-cell SELEX approach. Individual aptamers were identified by next generation sequencing and bioinformatics analysis. Two aptamers, HeA2_1 and HeA2_3, were shown to bind the HER2 protein with affinities in the nanomolar range. In addition, both aptamers were able to bind with high specificity to HER2-overexpressing cells and HER2-positive tumor tissue samples. Furthermore, we demonstrated that aptamer HeA2_3 is being internalized into cancer cells and has an inhibitory effect on cancer cell growth and viability. In the end, we selected novel DNA aptamers with great potential for the diagnosis and possible treatment of HER2-positive cancer

Development of aptamer-based radiopharmaceuticals for targeted cancer imaging and therapy

This SELEX experiment, for the selection of HER2 targeting aptamers, resulted in 26 selected RNA aptamers for further individual evaluation. Up till now, we were able to identify two aptamers that show binding to HER2 overexpressing SK-OV-3 cancer cells. In addition, we were able to generate silenced SK-OV-3 cells with minimal remaining HER2 levels, which will be used to obtain more information regarding the binding specificity of the selected aptamers

Synthesis of lutetium-carrying oligonucleotides for targeted cancer therapy and imaging

In this study, different techniques were evaluated to test the binding properties of an anti-HER3 aptamer to its target on cells or as pure protein. Furthermore, the aptamer was successfully coupled to DOTA-NHS ester and labelled with natural occurring lutetium. However, for a better yield, the coupling and labelling reactions need further optimization