Title of the project
Evaluation of novel candidates for radioligand therapy in solid tumours
NTS Identifier
NTS-NO-592627 v.1, 21-03-2024
NTS National Identifier
Field will not be published.
EU Submission
Field will not be published.
Yes
Project duration expressed in months.
48
Keywords
Targeted Radiopharmaceuticals
Purpose(s) of the project
Translational and applied research: Human Cancer
Describe the objectives of the project (for example, addressing certain scientific unknowns, or scientific or clinical needs).
The aim of the project is to develop a novel radioligand therapy (RLT) for solid cancers that are not curable with current available treatment. The novel radiopharmaceuticals will use an alpha-emitting radionuclide (212Pb/212Bi) bound to a vector molecule. The vector molecule (small molecule) will selectively target the tumour cells, where the radionuclide will deposit its radiation and thereby killing the cancer cells, while minimising exposure to normal tissues. RLT with alpha emitters may deliver potent and local radiation more selectively to cancer cells than beta emitters and has recently gained remarkable interest. The
process of developing a novel, successful RLT involves the selection of best candidates, found through experiments using tumour cell lines (in vitro) and the testing of these candidates in pilot experiments using mice (in vivo) to select the most promising radioligands. In this regard, the objectives of the current application are:
1) To establish subcutaneous tumour xenograft models of solid cancers in mice for the evaluation of derivative candidates of a novel radioligand molecule targeting breast and other cancers (3 derivative radioligand candidates). Up to 8 cell lines representative of different solid tumour indications will be investigated.
2) To explore the biodistribution of the novel derivative radioligand candidates in relevant solid tumour models (up to three tumour models in total; selected from the model establishment studies)
3) To assess the therapeutic efficacy and short- term toxicity of the most promising derivative radioligand candidate (selected from biodistribution studies).
4) Compare the biodistribution achieved with a Lu-177 labelled version of the lead compound to the biodistribution of a Pb-212 labelled version of the lead compound. The planned studies are necessary to verify efficacy of the radioligands and to identify potential dose-limiting organs/toxicities, as well as to terminate further development of candidates that show low efficacy or unfavourable toxicity profiles.
What are the potential benefits likely to derive from this project? Explain how science could be advanced, or humans, animals or environment may ultimately benefit from the project. Where applicable, differentiate between short-term benefits (within the duration of the project) and long-term benefits (which may accrue after the project is finished).
Solid cancers accounts for approximately 1.4 million new cases and around 700 000 deaths globally per year and its incidence is gradually increasing. This project will allow the identification and selection of novel radioligands for the treatment of solid cancers. The selected radioligands will be further studied in therapy and long-term toxicity studies.
The studies will allow the selection of the best candidates for cancer treatment and will be used for regulatory authorization of the main candidates for clinical study protocols. If successful, the radioligand therapy will be of great importance to patients with diverse solid cancers both for improving their quality of life and for prolonging their life expectancy.
In what procedures will the animals typically be used (for example, injections, surgical procedures)? Indicate the number and duration of these procedures.
The procedures reported in this section are described in detail in Attachment "02 Animal procedures". Ear marking: Animals will be ear marked with an ear punch before the experiment Frequency: 1 time. Duration: 1-2 min. Subcutaneous inoculation of cells: Mice will be inoculated subcutaneously in left and/or right flank with 1–10 x 10^6 cancer cells in sterile culture medium (without supplements) only, or in culture medium (without supplements) mixed 1:1 with Matrigel Matrix in a total volume of 100–200 μl. Frequency: 1 time. Duration: 2-5 min.
Mice will be weighed, and tumour size will be monitored using an electronic calliper 2–3 times per week.
Intravenous injection: Mice will be placed in a warmer environment (e.g. at 28–30°C) for 5- 15 minutes to stimulate dilation of the tail vein. The mice will be restrained and radiopharmaceuticals will be injected intravenously (0.05–0.15 ml) to the tail vein. Frequency: 1 time. Duration of restrain and injection: 5 min.
Blood collection from the lateral saphenous vein: This will be performed at the start of the therapy/ toxicity study (base line), and thereafter at 2-week intervals until endpoint on euthanasia day when the mice reach experimental or humane endpoints. A mouse will be placed in a restraining tube, so its head is covered and its hind legs are free. This can cause stress and therefore the duration of restraint will be minimized (max 5 min).
Less than 10 % of the circulating blood volume (100 µl) will be collected. Frequency: once every 2 weeks and up to 6 months from initiation of experiment. Duration: 5 min.
Euthanasia day: Before euthanasia of the mice, their blood (up to 0.8 ml) will be collected by heart puncture under gas anaesthesia following SOP 6931- Gassanestesi- Radiumhospitalet This will be collected for radioactivity measurements (Biodistribution study) or for analysis of clinical haematology and chemistry parameters (Therapy/ toxicity study). In biodistribution studies, up to 19 organs will be harvested (Table 5 in “FOTS 30669 Summary”) for radioactivity measurements. In model establishment and therapy studies, multiple organs including tumour, spleen, kidneys, liver, femur, salivary glands, stomach and intestines will be collected for histopathology analysis.
Mice will be euthanized by cervical dislocation.
What are the expected impacts/adverse effects on the animals, for example pain, weight loss, inactivity/reduced mobility, stress, abnormal behaviour, and the duration of those effects?
The cumulative severity anticipated to be experienced by the mice throughout the experimental lifetime is herein classified as moderate as per the provisions in the EU directive 2010/63/eu Annex VIII. The mice are likely to experience short-term distress with procedures that are likely to cause mild to moderate pain and impairment to their wellbeing.
Ear-tagging, subcutaneous and intravenous injections and blood sampling from the saphenous vein are short-term procedures classified as causing mild distress. The animals are expected to recover fast after these procedures without any significant long-term health issues. Following proper techniques and hygiene, these procedures fall under the mild severity category. Mice will be closely monitored for 10 minutes after every procedure for any unexpected adverse effects.
Subcutaneous tumour growth may cause moderate distress to mice by affecting normal behaviour and/or mobility especially when the tumour diameters reaches 15 mm (two tumours/mouse) or 20 mm (one tumour/ mouse). Weight loss related to the rate of tumour growth can occur. This corresponds to moderate severity. The mice will be monitored frequently to ensure humane endpoints are respected.
The in vivo metastatic potential of the listed cell lines in subcutaneous xenograft models has not been well reported in the literature. We will monitor for signs of metastases during model establishment and other studies, and will not proceed further with studies where we believe high metastases may be occurring. Progressive body weight loss (rapid reduction from previous recorded weight) and increased clinical signs of disease not corresponding to the volume of the subcutaneous tumour are indicative of metastatic tumour growth. In the event that these is observed, the mice will be monitored daily and the overall condition recorded in the score sheet (Attachment 1) respecting the humane endpoint.
Administration of radioligands will be at doses below the maximum tolerated doses (MTD) so as not to cause any significant adverse clinical effects. The MTD of Pb-212 and Lu-177 is known both from previous experience and literature review. The provisions categorise this procedure as moderate severity. A transient weight loss (with recovery expected within one week) and slight reduction in some haematological parameters, although not manifesting in clinical signs, can occur.
What species and numbers of animals are expected to be used? What are the expected severities and the numbers of animals in each severity category (per species)?
Species
Total number
Estimated numbers per severity
Non recovery
Mild
Moderate
Severe
What will happen to the animals kept alive at the end of the procedure?
Species
Estimated numbers of animals to be reused, to be returned to habitat/husbandry system or to be rehomed
Reused
Returned
Rehomed
Please provide reasons for the planned fate of the animals after the procedure.
All mice used in this project will be euthanised by cervical dislocation when a predetermined criteria is attained.
Model establishment studies: Mice will be euthanised when tumours reach 15 mm in any direction (if 2 tumors/ mouse) or 20 mm (if only take of 1 tumor/mice). Mice without tumour take will be euthanized once the studies are finished as they can't be re-used due to the uncertainty of their tumour status. Autopsies will be performed on all the mice and multiple organs collected for histopathology assessment. This assessment will provide information on whether the subcutaneously xenografted tumours of the different cell lines have metastatic potential within the period prior to reaching a humane endpoint. It is desirable to work with xenograft models that do not have metastatic potential and if any is reported then the model will be excluded from the downstream planned experiments.
Biodistribution studies: Xenografted animals will be euthanized at pre-defined timepoints to study the uptake of the radioligands in different organs and tumour. After euthanasia, tumours and organs will be collected, including blood by cardio puncture while the animal is under anesthesia.
Therapy studies: Animals will be treated with radioligands and monitored frequently for
the health status. If a predetermined limit of suffering or distress is reached, the animals will be euthanized to ensure that they do not suffer unnecessarily. The animals will be autopsied and multiple organs collected for histopathology assessment. If any of the compounds can completely cure the mice (no tumour progression), the mice will be kept and followed until 6 months from experiment start, and organs will then be harvested for histopathology to document any long-term effects of the relevant treatment(s) on selected organs.
1. Replacement
State which non-animal alternatives are available in this field and why they cannot be used for the purposes of the project.
In vitro studies with the main candidates have been performed in order to select the top 2 Pb212 radioligand candidates and the top Lu177 radioligand candidate for the target. Further evaluation of the candidates needs to be assessed in vivo, as there is currently no replacement for biodistribution and therapy/short-term toxicity studies. These types of studies are of key importance when developing and optimising radioligand therapy for cancer treatment. The studies assess the tumour targeting potential and identify key organs which can be related to treatment toxicity in future studies. Therefore, the studies are fundamental to select the best radioligand candidate to move forward for further preclinical evaluation and later, into clinical stage.
2. Reduction
Explain how the numbers of animals for this project were determined. Describe steps that have been taken to reduce the number of animals to be used, and principles used to design studies. Where applicable, describe practices that will be used throughout the project to minimise the number of animals used consistent with scientific objectives. Those practices may include e.g. pilot studies, computer modelling, sharing of tissue and reuse.
The number of groups and mice in each group have been reduced as much as possible considering the importance of obtaining significant data within each group and statistical significance. The model establishment studies will be used to select the tumour models with the best tumour take to reduce the number of required mice for further studies. We will also assess if Matrigel can increase the tumour take. The number of mice needed for the biodistribution and therapy studies assume a tumour take of 75% but this number will be reduced if the tumour takes of the selected models are higher. In literature, the cells listed in this application have successfully generated xenograft models in different strains of mice including athymic nude mice. However, depending on several factors including environmental factors, there could be variations in tumour take and growth. Models resulting in tumour take less than 75% will not be used as the variability in tumour development will affect the scientific outcome of the studies.
3. Refinement
Give examples of the specific measures (e.g., increased monitoring, post-operative care, pain management, training of animals) to be taken, in relation to the procedures, to minimise welfare costs (harms) to the animals. Describe the mechanisms to take up emerging refinement techniques during the lifetime of the project.
Our aim is to cause minimal pain and distress to the mice during experiments by frequently monitoring the animals and sound selection of humane end points.We will use our experience to handle the mice humanely and in accordance with the laws and regulations. Experienced and well-trained researchers will handle the mice during the studies. The welfare of the animals will be frequently monitored and a score sheet (see Attachments "01 Score sheet") will be used to monitor the health of the animals. If a predetermined limit of suffering or distress is reached, the animals will be euthanized to ensure that they do not suffer unnecessarily. To prevent additional distress to the animals, we will follow aseptic techniques when generating injectable solutions and use sterile single-use consumables e.g syringes and needles, for manipulating the mice during drug administration and blood sampling. On completion of every handling procedure, the mice will be monitored for atleast 10 minutes to confirm that they display normal behaviour. Animals will be housed in groups to maintain social interaction, with nesting material and plastic/ cardboard houses as enrichment to reduce stress induced behaviour. We will work in close collaboration with the personnel at the Department of Comparative Medicine that do daily inspection of the cages and have great experience assessing the mice welfare.
Explain the choice of species and the related life stages.
Athymic nude mice lack T-cells and are therefore immunodeficient. This makes the model suitable for xenografting of several cancer cell lines. Additionally, this strain has functional DNA repair mechanisms, giving it a higher tolerance for radiation when compared to other immunodeficient mouse strains. This genetic characteristic makes this strain suitable for biodistribution, therapy and short-term toxicity studies using radioligands. Nude mice are cost-effective and less aggressive compared to other mouse strains, making them easier to handle. It is also easier to quantify the growth of subcutaneous tumours on nude mice due to their hairless phenotype. In literature, the selected cell lines are usually established as subcutaneous models in female mice. This is primarily because majority of the cancers associated with these cell lines harbour unique molecular and genetic mechanisms that increase the susceptibility for these cancers in females than in males. For this reason, including male mice in this study will require an increased number of mice as the sexes must be compared head-to-head for all treatments given to the mice in order to generate significant data across board. The success of tumour take in nude mice is documented to be high in mice aged 3 – 10 weeks. Mice between 3 and 5 weeks of age will be delivered and acclimated at least 7 days before the experiment begins. The age of the mice at study start will be 4–8 weeks when tumour implantation has a higher success rate.