Preventive treatment of lung metastases

Abstract

The present invention relates to a preventive treatment against lung metastases. In particular, a nebulized BCG composition is administered directly to the lungs to increase uptake therein.

Description

[0001] Preventive treatment of lung metastases

[0002] Technical field of the invention

[0003] The present invention relates to a preventive treatment against lung metastases. In particular, a nebulized BCG composition is administered directly to the lungs to increase uptake therein.

[0004] Background of the invention

[0005] Pulmonary metastases represent a significant challenge in cancer treatment, profoundly impacting societal health. Metastasis, the spread of cancer cells from the primary tumor to distant organs, is responsible for the majority of cancer-related deaths. Lung metastases, in particular, are associated with poor prognosis and high mortality rates. This condition not only reduces patients' quality of life and life expectancy but also imposes a substantial economic burden on healthcare systems due to the need for complex and prolonged treatments.

[0006] Treating lung metastases is particularly challenging due to several factors. The metastatic tumors often exhibit resistance to conventional therapies, including chemotherapy and radiation therapy, making them difficult to eradicate. Additionally, the presence of multiple metastatic sites complicates surgical interventions. The heterogeneity of cancer cells within metastases further complicates treatment, as different cells may respond differently to the same therapy. These challenges necessitate the development of more effective and targeted treatment strategies.

[0007] One approach that has been explored is the use of Bacillus Calmette-Guerin (BCG) therapy, which has shown promise in treating certain types of cancer, such as bladder cancer. BCG works by stimulating the immune system to attack cancer cells. However, its application in treating lung metastases has been limited by several shortcomings. The effectiveness of BCG in lung cancer, or lung metastases per se, is not well- established, and the treatment can cause significant side effects, including severe inflammation and infection. Additionally, the response to BCG therapy can be highly variable among patients, making it an unreliable option for widespread use.

[0008] Accordingly, there is a great need to provide an effective method for combatting lung metastases.

[0009] In particular, it would be advantageous to provide a treatment that prevents or inhibits the development of lung metastases from the primary tumour.

[0010] P96598PC Summary of the invention

[0011] The present invention addresses the challenges of treating lung metastases by introducing a novel approach that utilizes nebulized Bacillus Calmette-Guerin (BCG) therapy. This invention is based on the realization that administering nebulized BCG early, before significant metastasis occurs, can effectively prevent the development of metastases. By delivering BCG directly to the lungs in a nebulized form, the treatment stimulates the immune system to target and eliminate cancer cells at an early stage, thereby inhibiting their spread. This method enhances treatment efficacy, reduces side effects, and offers a promising preventive strategy against lung metastases, ultimately improving patient outcomes and reducing the overall burden of cancer on healthcare systems.

[0012] Accordingly, an object of the present invention relates to a preventive treatment of lung metastases in a subject.

[0013] Thus, an aspect of the present invention relates to a Bacillus Calmette-Guerin (BCG) composition for use in the prevention or inhibition of lung metastases in a subject, wherein the BCG composition is administered in nebulized form.

[0014] An embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered to the lungs.

[0015] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is first administered before any lung metastases have been identified.

[0016] Brief description of the figures

[0017] Figure 1 shows the tumour load as measured by tumour cell luminescence (photons / second) in the lungs in each mouse at experimental endpoint. (A) Macroscopic photo (upper) and IVIS (lower) of untreated controls. (B) Macroscopic photo (upper) and IVIS (lower) of BCG nebulized mice.

[0018] Figure 2A shows the metastatic burden (tumour area, % of total lung area) in the lungs of each mouse at experimental endpoint. Untreated control mice are shown as white circles, intranasally BCG-treated mice as light grey circles and the BCG-Nebulized mice are shown as dark grey circles (Unpaired t test; BCG Neb vs Untreated Cntrl p=0,0011 (**), BCG Neb vs BCG IN Cntrl p=0.0342 (*), Untreated Cntrl vs BCG IN Cntrl

[0019] P96598PC p=0.4699 (n.s.). ns = non statistically different, * =p<0.05, ** = p<0.01, ***=p<0.001.

[0020] Figure 2B shows the number of metastatic foci (tumour foci count) on one side of the lung surface in each mouse at experimental endpoint. Untreated control mice are shown as white circles, intranasally BCG-treated mice as light grey circles and the BCG- Nebulized mice are shown as dark grey circles (Unpaired t test; BCG Neb vs Untreated Cntrl p=0.0004 (***), BCG Neb vs BCG IN Cntrl p=0.0092 (**), Untreated Cntrl vs BCG IN Cntrl p=0.3258 (ns).

[0021] Figure 2C shows the tumour load as measured by tumour cell luminescence (photons / second) in the lungs in each mouse at experimental endpoint. Untreated control mice are shown as white circles, intranasally BCG-treated mice as light grey circles and the BCG-Nebulized mice are shown as dark grey circles (Unpaired t test; BCG Neb vs Untreated Cntrl p=0.0074 (**), BCG Neb vs BCG IN Cntrl p=0.0698 (ns), Untreated Cntrl vs BCG IN Cntrl p=0.0425 (*).

[0022] Figure 3 shows the tumour load as measured by tumour cell luminescence (photons / second) in the lungs in each mouse at experimental endpoint. Mice in the BCG Nebulization group (1, 2 and 3) show a reduced tumour load compared to the untreated control mice (4 and 5). The difference is visible both macroscopically (upper picture) and by IVIS (lower picture).

[0023] The present invention will in the following be described in more detail.

[0024] Detailed description of the invention

[0025] Definitions

[0026] Prior to outlining the present invention in more details, a set of terms and conventions is first defined:

[0027] Lung metastases

[0028] In the present context, the term "lung metastases" refers to secondary tumours located in the lungs which have spread from a primary tumour, such as a primary solid tumour. Typically, lung metastases are located in the lung parenchyma or pleura.

[0029] The terms "lung metastases" and "pulmonary metastases" may be used interchangeably.

[0030] P96598PC Prevention

[0031] In the present context, the term "prevention" refers to a treatment that aims to prevent or slow down the development of a disease, i.e. before it causes significant health issues. This is in contrast to a therapeutic treatment which aims to mitigate the effects of an already established and / or diagnosed condition or disease.

[0032] Thus, a preventive treatment is directed at maintaining health and preventing onset of disease, such as the development of lung metastases.

[0033] Preventive treatment includes also the inhibition of the development of lung metastases, e.g. preventive treatment can slow the progression of metastatic disease in the lungs and thereby reduce the metastatic burden and delay onset of significant disease.

[0034] Primary solid tumour

[0035] In the present context, the term "primary solid tumour" refers to an abnormal mass of tissue that grows uncontrollably. The primary solid tumour is the original, or first, in the body. The primary solid tumour may spread to other parts of the body and form secondary tumours called metastases. These metastases are the same type of cancer as the primary solid tumour.

[0036] The primary solid tumour may be either benign or malignant. For the treatment disclosed herein, the primary solid tumour is typically malignant as these tumours are more likely to metastasize than benign tumours.

[0037] The primary solid tumour is to be distinguished from liquid tumours that circulate around the body through the bloodstream.

[0038] Sarcoma

[0039] In the present context, the term "sarcoma" refers to a malignant tumour of transformed cells of mesenchymal origin (connective tissue). The affected mesenchymal cells may be present in different types of connective tissue, such as bone, cartilage, fat, or vascular tissue. Sarcomas may be divided into the two overall groups of bone sarcomas and soft-tissue sarcomas.

[0040] Sarcomas are primary solid tumours that may give rise to metastases elsewhere in the body, such as the lungs.

[0041] P96598PC Melanoma

[0042] In the present context, the term "melanoma" refers to a cancer which develops in melanin-producing cells called melanocytes. Melanocytes are a type of mesenchymal cells which are derived from neural crest cells. Accordingly, melanoma is to be considered a sarcoma.

[0043] Carcinoma

[0044] In the present context, the term "carcinoma" refers to a malignant tumour of transformed cells of epithelial origin. Carcinomas forms in a tissue that lines the inner or outer surfaces of the body and include, but is not limited to, breast cancer, prostate cancer, lung cancer and colorectal cancer.

[0045] Carcinomas may be categorised according to their histological types as adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, anaplastic carcinoma, large cell carcinoma, and small cell carcinoma.

[0046] In the context of the present treatment, carcinomas are considered primary solid tumours that may give rise to metastases elsewhere in the body, such as the lungs.

[0047] Bacillus Calmette-Guerin (BCG) composition

[0048] In the present context, the term "BCG composition" refers to any composition comprising live attenuated Mycobacterium bovis BCG as active ingredient. The BCG composition may comprise one or more pharmaceutically acceptable carriers, excipients and / or diluents so that it is suitable for administration to a subject, such as a human patient. Administration of the BCG composition can be done in nebulized form.

[0049] The BCG composition may be a commercially available BCG vaccine.

[0050] Pharmaceutically acceptable

[0051] In the present context, the term "pharmaceutically acceptable" refers to molecular entities and compositions that are suitable for use with human patients without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit / risk ratio.

[0052] Nebulization and nebulized

[0053] In the present context, the term "nebulization" refers to the process of converting a liquid composition to a fine spray, aerosols or mist. Thus, a nebulized composition is in the form of a fine spray, aerosol or mist.

[0054] P96598PC Nebulization may be achieved using a nebulizer, such as a jet nebulizer, ultrasonic nebulizer, or mesh nebulizer.

[0055] Mechanical ventilation

[0056] In the present context, the term "mechanical ventilation" refers to the use of a ventilator to fully or partially provide artificial ventilation.

[0057] The terms "mechanical ventilation", "assisted ventilation" and "intermittent mandatory ventilation" may be used interchangeably.

[0058] About

[0059] Wherever the term "about" is employed herein in the context of amounts, for example absolute amounts, such as numbers, purities, concentrations, weights, sizes, etc., or relative amounts (e.g. percentages, equivalents or ratios), timeframes, and parameters such as temperatures, pressure, etc., it will be appreciated that such variables are approximate and as such may vary by ±10%, for example ± 5% and preferably ± 2% (e.g. ± 1%) from the actual numbers specified. This is the case even if such numbers are presented as percentages in the first place (for example 'about 10%' may mean ±10% about the number 10, which is anything between 9% and 11%).

[0060] Prevention of lung metastases

[0061] Preventing lung metastases is highly preferred over treating already established lung metastases due to the significant challenges associated with the latter. In particular, established lung metastases are associated with resistance to treatment, limited treatment options, severe side effects and toxicity, and poorer prognosis and survival rates. However, achieving effective prevention is difficult due to the complex nature of cancer metastasis, which involves multiple biological pathways and mechanisms.

[0062] Herein it is found that early administration of a BCG composition can inhibit or prevent the formation of lung metastases. The mechanism of action of BCG in the treatment of cancer is broad and not fully elucidated. Mycobacterium bovis Bacillus Calmette-Guerin (BCG) is a live-attenuated vaccine, initially established to protect against childhood meningitis and disseminated tuberculosis (TB). However, BCG has been demonstrated to hold the potential for increasing the capacity of the immune system to combat other pathogens than TB, e.g. by boosting non-specific responses in both T-cell mediated adaptive responses and innate immune responses.

[0063] P96598PC Accordingly, without being bound by theory, it is contemplated herein that production of pro-inflammatory cytokines by tissue-resident phagocytic cells (innate immune system) after exposure to BCG stimulates the adaptive immune system locally and prevents the colonization and growth of tumour cells into metastases.

[0064] It has surprisingly been found that administration of the BCG composition can slow or prevent development of lung metastases, and ultimately significantly reduce tumour burden compared to non-treated patients.

[0065] Thus, an aspect of the present invention relates to a Bacillus Calmette-Guerin (BCG) composition for use in the prevention or inhibition of lung metastases in a subject, wherein the BCG composition is administered in nebulized form.

[0066] Another aspect of the present invention relates to a Bacillus Calmette-Guerin (BCG) composition for use in the treatment, inhibition, amelioration or prevention of lung metastases in a subject, wherein the BCG composition is administered in nebulized form.

[0067] In the present context treatment may be construed as the elimination or reduction of already established lung metastases, i.e. with the goal of curing the disease. Amelioration is covering slowing down disease progression, e.g. delaying how fast disease is spreading and new symptoms manifest themselves. Inhibition can be construed as the stopping of disease progression.

[0068] The result of the preventive treatment may manifest itself in one or more positive outcomes. The early administration of BCG can efficiently reduce the occurrence of lung metastases, and lower the metastatic burden as quantified by a diminished volume of the lung metastases compared to untreated individuals. Early administration of BCG can also reduce metastatic foci, i.e. the number of lung metastases formed. In total, the preventive treatment will improve the disease prognosis, increase the quality of life, and prolong the life expectancy of the human patient.

[0069] Therefore, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein prevention and / or inhibition means that fewer metastases or less metastatic tissue has formed in the lungs of the subject compared to if no BCG composition is administered to the subject.

[0070] P96598PC Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein prevention and / or inhibition means that the metastatic burden of the subject is reduced compared to a subject that does not receive the BCG composition.

[0071] Yet another embodiment of the present invention relates to the BCG composition for use as described herein, wherein prevention and / or inhibition means that the metastatic burden of the subject is reduced.

[0072] A further embodiment of the present invention relates to the BCG composition for use as described herein, wherein prevention and / or inhibition means that the number of metastatic foci of the subject is reduced compared to a subject that does not receive the BCG composition.

[0073] A still further embodiment of the present invention relates to the BCG composition for use as described herein, wherein prevention and / or inhibition means that the number of metastatic foci of the subject is reduced.

[0074] The nebulized BCG composition may advantageously be administered to the lungs. This may be accomplished by inhalation, e.g. through mouth and / or nose via a nebulizer. Some nebulizers are designed to deliver medication through both the nose and mouth, which can be beneficial for treating conditions affecting the upper respiratory tract. This administration can deliver the BCG composition to the lungs, but herein it is preferred to administer the BCG composition specifically through the mouth to ensure that the medication reaches the lungs and are not upheld in the mucous membranes of the nasal cavity. Administration exclusively through the mouth may be facilitated by inhalation of the nebulized BCG composition through a mouthpiece or mask.

[0075] Thus, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered to the lungs.

[0076] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered via inhalation.

[0077] A further embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered via inhalation through the mouth and / or nose.

[0078] P96598PC A still further embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered via inhalation through the mouth.

[0079] To further guide the BCG composition directly to the target site in the lungs, the administration may entirely circumvent the upper respiratory tract and be performed by intubation. By delivery of the BCG composition directly to the lungs via intubation, the mucosa of the upper airways (nose, throat, etc.) are bypassed, ensuring that none of the active ingredient is retained in the mucosa of the nasal and oral cavity. Intubation of the patient may be executed via any conventional means.

[0080] Accordingly, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered to the lungs via a tracheal tube.

[0081] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the tracheal tube is an endotracheal tube.

[0082] Yet another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the route of administration is orotracheal or nasotracheal, preferably orotracheal.

[0083] As a further means for distributing the BCG composition directly to the lungs, the administration may be performed while the patient is under conditions of mechanical ventilation. Mechanical ventilation refers to the use of a ventilator to fully or partially provide artificial ventilation. Non-invasive mechanical ventilation may be accomplished for patients that are conscious and can use a face or nasal mask. Invasive mechanical ventilation relates to the situation wherein placement of an instrument to create an airway that is placed inside the trachea, such as an endotracheal tube or nasotracheal tube, is utilised.

[0084] Administering aerosolized medications to a patient under mechanical ventilation may enhance distribution to the lungs, i.e. to achieve higher concentration at the target site. Furthermore, mechanical ventilation can improve patient comfort in that it allows co-administration of sedatives and analgesics to keep patients that are otherwise uncomfortable with the situation calm and relaxed.

[0085] P96598PC The two main types of mechanical ventilations are positive pressure ventilation in which air is pushed into the lungs through the airways, and negative pressure ventilation in which air is pulled into the lungs. It is contemplated herein that positive pressure ventilation is particularly advantageous for delivery of the BCG composition to pulmonary metastases.

[0086] Positive pressure ventilation may be enacted by increasing the patient's airway pressure through an endotracheal tube. The positive pressure allows air to flow into the airway until the ventilator stream is stopped. Then, the airway pressure drops to zero, and the elastic recoil of the chest wall and lungs push the breath out through passive exhalation.

[0087] Without being bound by theory, it is contemplated herein that positive pressure ventilation is favourable because the distal areas of the lungs are better inflated and therefore the BCG composition is more completely distributed in the lungs.

[0088] Therefore, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered under conditions of mechanical ventilation of the subject.

[0089] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered under conditions of positive pressure ventilation.

[0090] The active ingredients intended to assert their effect at a target site in the lungs of patients are delivered in the form of a nebulized composition, such as an aerosol. The nebulized composition may be prepared by any conventional means.

[0091] Thus, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is nebulized by pneumatic, mechanical or electrical means.

[0092] The BCG composition described herein is best administered as early as possible to maximise the preventive effect on development of lung metastases. Thus, as soon as a primary tumour is identified it is diligent to administer the BCG composition.

[0093] P96598PC Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is first administered upon initial diagnosis of a primary tumour.

[0094] Yet another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is first administered within 14 days of the initial diagnosis of a primary tumour, such as within 12 days, such as within 10 days, such as within 7 days, such as within 5 days, such as within 4 days, such as within 3 days, such as within 2 days, such as within 1 day.

[0095] A further embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is first administered immediately following diagnosis of a primary tumour.

[0096] A still further embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is first administered before any lung metastases have been identified.

[0097] Identifying lung metastases typically involves one or more imaging techniques, including, but not limited to, X-ray, CT scanning, MRI and PET scanning. Chest X- rays are often the first step, providing a basic view of the lungs to detect any obvious abnormalities. Upon identification of at least one lung metastasis, a formal diagnosis of lung metastasis may be made. However, small metastases can be missed on X- rays, so more detailed imaging is usually required. Computed Tomography (CT) scanning is more precise and can reveal the size, shape, and position of lung metastases. This method involves taking multiple X-ray images from different angles and combining them to create a detailed cross-sectional view of the lungs. Positron Emission Tomography (PET) scanning is also commonly used and involves injecting a small amount of radioactive sugar into the bloodstream. Cancer cells, which consume more sugar than normal cells, will appear as bright spots on the scan. PET scanning is often combined with CT scanning (PET / CT) to provide both metabolic and anatomical information.

[0098] Therefore, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein identification of lung metastases is performed by one or more techniques selected from the group consisting of X-ray, CT scanning, MRI, PET scanning, and intraoperative visualization.

[0099] P96598PC Cancer diseases are notoriously known to be difficult to prognosticate, and the extent of treatment regimens varies from patient to patient. Pulmonary metastases are no different, the required amount of treatment is difficult to predict. However, the vast majority of patients require a repeated administration of effective doses to obtain a positive clinical outcome, namely sustained preventive or inhibitory effect on the development of lung metastases. The BCG composition herein may be administered repeatedly, if necessary, without any severe adverse effects.

[0100] Thus, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered as a single effective dose or as multiple effective doses.

[0101] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the number of effective doses of the BCG composition administered is at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 10, such as at least 15, such as at least 20, such as at least 30, such as at least 40, such as at least 50.

[0102] A further embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is administered daily, every other day, three times a week, weekly, bi-weekly or monthly.

[0103] Lung metastases are secondary tumours that have spread to the lungs of a subject from another primary tumour site in the body. Primary tumours are most commonly solid and may occur in most tissues of the body, including, but not limited to, bone, breast, lung, prostate, colon, bladder, and kidney. In particular malign primary solid tumours are conducive of metastasizing.

[0104] Therefore, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the lung metastases are derived from a primary tumour.

[0105] If possible, it is desirable to treat patients as early as possible in the progression of their cancer disease. At the early stage of the cancer disease likelihood of lung metastases is low and administration of the BCG composition may therefore mitigate the spread, development and / or growth of lung metastases more effectively. This means that the BCG composition may advantageously be administered to patients with an early-stage primary tumour.

[0106] P96598PC An early-stage primary tumour may be characterised by the TNM (tumour, nodes, metastasis) system which is an internationally recognized method for describing the extent and severity of cancer. The system is maintained by the Union for International Cancer Control (UICC) and the American Joint Committee on Cancer (AJCC). In the present context, early-stage primary tumour according to the TNM system means T=1 or 2, N = 0 or 1, M=0.

[0107] Therefore, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the primary tumour is an early-stage primary tumour.

[0108] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the primary tumour is characterized by a score according to the TNM system of:

[0109] - T is less than or equal to 2,

[0110] - N is less than or equal to 1, and

[0111] M is 0.

[0112] Yet another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the primary tumour is malignant.

[0113] Still another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the primary tumour is a primary solid tumour.

[0114] Nearly all primary tumours may give rise to metastases, independent of the origin of the primary tumour. Thus, lung metastases may be derived from a broad variety of types of cancer.

[0115] Accordingly, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the primary solid tumour is selected from the group consisting of sarcoma, carcinoma, lymphoma, and carcinosarcoma.

[0116] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the primary solid tumour is a sarcoma selected from the group consisting of melanoma, soft tissue sarcoma, fibrosarcoma, hemangiopericytoma, peripheral nerve sheet tumour, liposarcoma, myxosarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial cell sarcoma, gastrointestinal stromal

[0117] P96598PC tumour, histiocytic sarcoma, hemangiosarcoma, lymphangiosarcoma, chondrosarcoma, osteosarcoma, and osteochondrosarcoma.

[0118] A further embodiment of the present invention relates to the BCG composition for use as described herein, wherein the primary solid tumour is a melanoma.

[0119] An even further embodiment of the present invention relates to the BCG composition for use as described herein, wherein primary solid tumour is a carcinoma selected from the group consisting of squamous cell carcinoma, nasal adenocarcinoma, thyroid carcinomas and adenocarcinomas, parathyroid carcinomas and adenocarcinomas, gastrointestinal carcinoma and adenocarcinomas, oesophageal carcinoma, gastric carcinoma, small intestinal carcinoma, large intestinal carcinoma, rectal carcinoma, bronchioalveolar carcinomas, pulmonary adenocarcinomas, hepatocellular carcinomas, pancreatic carcinomas and adenocarcinomas, renal cell carcinomas, transitional cell carcinomas, nephroblastoma, prostatic adenocarcinomas, uterine carcinomas and adenocarcinomas, Leydig cell tumour, Sertoli cell tumour, seminoma, granulosa cell tumour, teratocarcinoma, adenocarcinomas, oligodendrocytoma, glioblastoma, and meningiomas.

[0120] Nearly all tumours have the potential to metastasize, but whether they do depend on several factors, including the type, size, and location of the primary tumour. Some cancers are more likely to spread than others. For example, aggressive cancers like melanoma have a higher propensity to metastasize. The propensity to metastasize may be reported as the metastatic potential, i.e. the likelihood or ability of cancer cells to spread from the original tumour site to other parts of the body.

[0121] The preventive treatment described herein is particularly relevant to patients that suffer from a tumour with high metastatic potential as the treatment may mitigate the higher likelihood for development of lung metastases.

[0122] Therefore, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the primary tumour has metastatic potential.

[0123] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the primary tumour has high metastatic potential.

[0124] The metastatic potential of a primary tumour is affected by several factors, including how advanced the tumour is. Higher-grade and higher-stage tumours are more likely

[0125] P96598PC to metastasize due to their aggressive nature and advanced spread. For example, higher-grade tumours (Grade 3 and 4) have more abnormal cells (are more malignant) and are more likely to metastasize.

[0126] Moreover, the gene mutation and / or expression profiles can indicate how aggressive the tumour is. The expression of certain prognostic immune indicators or biomarkers, such as those involved in cell adhesion, invasion, and migration, can indicate a higher likelihood of metastasis. For example, overexpression of genes like MMPs (matrix metalloproteinases) and VEGF (vascular endothelial growth factor) is associated with increased metastatic potential.

[0127] Therefore, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein high metastatic potential means that the primary tumour has an increased risk of spread from its original site to other parts of the body, wherein high metastatic potential is characterised by one or both of the following indicators:

[0128] - a tumour grade of at least grade 3; and

[0129] - a tumour stage of at least stage III.

[0130] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein high metastatic potential means that the primary tumour has an increased risk of spread from its original site to other parts of the body, wherein high metastatic potential is characterised by increased levels of one or more prognostic biomarkers selected from the group consisting of immunohistochemical markers, flow-cytometric markers, genetic markers and serum markers.

[0131] A further embodiment of the present invention relates to the BCG composition for use as described herein, wherein high metastatic potential means that the primary tumour has an increased risk of spread from its original site to other parts of the body, wherein high metastatic potential is characterised by increased levels of vascular endothelial growth factor (VEGF) and / or matrix metalloproteinases (MMPs).

[0132] Without being bound by theory, it is contemplated that the preventive effect of the BCG composition against lung metastases may be caused by its ability to act as an immune stimulating component that efficiently drive maturation of immune cells, such as dendritic cells, macrophages, cytotoxic T-cells and NK cells, and mobilise them at the target site to prevent establishment of cancerous tissue. The BCG composition may

[0133] P96598PC comprise any Mycobacterium bovis strain that induces this maturation, i.e. the present BCG composition is not limited to one particular bacterial strain.

[0134] Therefore, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition comprises live attenuated Mycobacterium bovis BCG.

[0135] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein BCG composition comprises a bacterial strain or substrain selected from the group consisting of Pasteur 1173 P2, Danish 1331, Glaxo 1077, Tokyo 172-1, Russian BCG-I, and Moreau RDJ strains.

[0136] A further embodiment of the present invention relates to the BCG composition for use as described herein, wherein the attenuated live Mycobacterium bovis BCG is Danish strain 1331.

[0137] The BCG composition may function with conventional pharmaceutically acceptable carriers, excipients and diluents and is not limited to any particular one. However, some conventional carriers, excipients and diluents are known to specifically work well with BCG, including, but not limited to, those comprised in BCG vaccines. Thus, the BCG composition may comprise or consist of a commercially available BCG vaccine. Such vaccines are known to be safe and without serious adverse effects.

[0138] Accordingly, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition comprises one or more pharmaceutically acceptable carriers, excipients and / or diluents.

[0139] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the one or more pharmaceutically acceptable carriers, excipients and / or diluents are selected from the group consisting of sodium glutamate, magnesium sulphate heptahydrate, dipotassium phosphate, citric acid monohydrate, L-asparagine monohydrate, ferric ammonium citrate, and glycerol.

[0140] Other carrier solutions include, but are not limited to, buffered saline solutions, such as Hank's balanced salt solution, Tris buffered saline, HEPES buffered solutions, and others.

[0141] P96598PC A preferred embodiment of the present invention relates to the BCG composition for use as described herein, wherein the BCG composition is a BCG vaccine.

[0142] As for many other cancer treatments, the present administration of the BCG composition may be combined with administration of another immunotherapeutic composition. The immunotherapeutic composition may be an already commercially available composition or product. The combination treatment may be administered concomitantly or separated in time.

[0143] Thus, an embodiment of the present invention relates to the BCG composition for use as described herein, further comprising administration of an immunotherapeutic composition.

[0144] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein the immunotherapeutic composition is administered before, simultaneously, or after administration of the BCG composition.

[0145] Alternatively, the BCG composition is combined with treatment directed at the primary tumour. Such treatments may be any conventional treatment, including surgery, radiation therapy, chemotherapy, hormone therapy, immunotherapy, stem cell treatment. The BCG composition may be combined with personalized treatments, such as advanced therapy medicinal products (ATMPs), hereunder gene therapy, cell therapy, and tissue engineering.

[0146] Accordingly, an embodiment of the present invention relates to the BCG composition for use as described herein, wherein said BCG composition is administered as part of a combination therapy that comprises administering a treatment targeting the primary tumour.

[0147] Another embodiment of the present invention relates to the BCG composition for use as described herein, wherein said treatment targeting the primary tumour is selected from the group consisting of surgery, radiation therapy, chemotherapy, hormone therapy, immunotherapy, gene therapy, cell therapy, such as CAR-T cell therapy or stem cell therapy, or combinations thereof.

[0148] An aspect of the present invention relates to a method of treatment, inhibition, amelioration or prevention of lung metastases in a subject, the method comprising administration of a BCG composition in nebulized form.

[0149] P96598PC An embodiment of the present invention relates to the method as described herein, wherein the method is for prevention or inhibition of lung metastases in a subject.

[0150] The listing or discussion of an apparently prior published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0151] Preferences, options and embodiments for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences, options and embodiments for all other aspects, features and parameters of the invention. This is especially true for the description of the medical use of the BCG composition, which embodiments may readily be part of the method of treatment described herein. Embodiments and features of the present invention are also outlined in the following items.

[0152] Items

[0153] XI. A Bacillus Calmette-Guerin (BCG) composition for use in the treatment, inhibition, amelioration or prevention of lung metastases in a subject, wherein the BCG composition is administered in nebulized form.

[0154] X2. The BCG composition for use according to item XI, wherein the use is for prevention or inhibition of lung metastases in a subject.

[0155] X3. The BCG composition for use according to any one of items XI or X2, wherein the use is for prevention of lung metastases in a subject.

[0156] X4. The BCG composition for use according to any one of items XI or X2, wherein the use is for inhibition of lung metastases in a subject.

[0157] X5. The BCG composition for use according to item XI, wherein the use is for amelioration of lung metastases in a subject.

[0158] X6. The BCG composition for use according to item XI, wherein the use is for treatment of lung metastases in a subject.

[0159] X7. The BCG composition for use according to any one of items X1-X4, wherein prevention and / or inhibition means that fewer metastases or less metastatic tissue has

[0160] P96598PC formed in the lungs of the subject compared to if no BCG composition is administered to the subject.

[0161] X8. The BCG composition for use according to any one of items X1-X4, wherein prevention and / or inhibition means that the metastatic burden of the subject is reduced compared to a subject that does not receive the BCG composition.

[0162] X9. The BCG composition for use according to any one of items X1-X4, wherein prevention and / or inhibition means that the metastatic burden of the subject is reduced.

[0163] X10. The BCG composition for use according to any one of items X1-X4, wherein prevention and / or inhibition means that the number of metastatic foci of the subject is reduced compared to a subject that does not receive the BCG composition.

[0164] XI 1. The BCG composition for use according to any one of items X1-X4, wherein prevention and / or inhibition means that the number of metastatic foci of the subject is reduced.

[0165] X12. The BCG composition for use according to any one of the preceding items, wherein the subject is a human.

[0166] X13. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered to the lungs.

[0167] X14. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered via inhalation.

[0168] X15. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered via inhalation through the mouth and / or nose.

[0169] X16. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered via inhalation through the mouth.

[0170] X17. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered to the lungs via a tracheal tube.

[0171] P96598PC X18. The BCG composition for use according to item X17, wherein the tracheal tube is an endotracheal tube.

[0172] X19. The BCG composition for use according to any one of the preceding items, wherein the route of administration is orotracheal or nasotracheal, preferably orotracheal.

[0173] X20. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered under conditions of mechanical ventilation of the subject.

[0174] X21. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered under conditions of positive pressure ventilation.

[0175] X22. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is nebulized by pneumatic, mechanical or electrical means.

[0176] X23. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is first administered upon initial diagnosis of a primary tumour.

[0177] X24. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is first administered within 14 days of the initial diagnosis of a primary tumour, such as within 12 days, such as within 10 days, such as within 7 days, such as within 5 days, such as within 4 days, such as within 3 days, such as within 2 days, such as within 1 day.

[0178] X25. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is first administered immediately following diagnosis of a primary tumour.

[0179] X26. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is first administered before any lung metastases have been identified.

[0180] X27. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is first administered before any visible or macroscopic lung metastases have been identified.

[0181] P96598PC X28. The BCG composition for use according to any one of items X26 or X27, wherein identification of lung metastases is performed by one or more techniques selected from the group consisting of X-ray, CT scanning, MRI, PET scanning, and intraoperative visualization.

[0182] X29. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered as a single effective dose or as multiple effective doses.

[0183] X30. The BCG composition for use according to any one of the preceding items, wherein the number of effective doses of the BCG composition administered is at least 2, such as at least 3, such as at least 4, such as at least 5, such as at least 6, such as at least 7, such as at least 10, such as at least 15, such as at least 20, such as at least 30, such as at least 40, such as at least 50.

[0184] X31. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is administered daily, every other day, three times a week, weekly, bi-weekly or monthly.

[0185] X32. The BCG composition for use according to any one of the preceding items, wherein the lung metastases are derived from a primary tumour.

[0186] X33. The BCG composition for use according to any one of items 23-32, wherein the primary tumour is an early-stage primary tumour.

[0187] X34. The BCG composition for use according to any one of items 23-33, wherein the primary tumour is characterized by a score according to the TNM system of:

[0188] - T is less than or equal to 2,

[0189] - N is less than or equal to 1, and

[0190] M is 0.

[0191] X35. The BCG composition for use according to any one of items 23-34, wherein the primary tumour has metastatic potential.

[0192] X36. The BCG composition for use according to any one of items X23-35, wherein the primary tumour has high metastatic potential.

[0193] P96598PC X37. The BCG composition for use according to item X36, wherein high metastatic potential means that the primary tumour has an increased risk of spread from its original site to other parts of the body, wherein high metastatic potential is characterised by one or more of the following indicators:

[0194] - a tumour grade of at least grade 3; and

[0195] - a tumour stage of at least stage III.

[0196] X38. The BCG composition for use according to item X36, wherein high metastatic potential means that the primary tumour has an increased risk of spread from its original site to other parts of the body, wherein high metastatic potential is characterised by increased levels of one or more prognostic biomarkers selected from the group consisting of immunohistochemical markers, flow-cytometric markers, genetic markers and serum markers.

[0197] X39. The BCG composition for use according to item X36, wherein high metastatic potential means that the primary tumour has an increased risk of spread from its original site to other parts of the body, wherein high metastatic potential is characterised by increased levels of vascular endothelial growth factor (VEGF) and / or matrix metalloproteinases (MMPs).

[0198] X40. The BCG composition for use according to any one of items X23-X39, wherein the primary tumour is malignant.

[0199] X41. The BCG composition for use according to any one of items X23-X40, wherein the primary tumour is a primary solid tumour.

[0200] X42. The BCG composition for use according to item X41, wherein the primary solid tumour is selected from the group consisting of sarcoma, carcinoma, lymphoma, and carcinosarcoma.

[0201] X43. The BCG composition for use according to any one of items X41 or X42, wherein the primary solid tumour is a sarcoma selected from the group consisting of melanoma, soft tissue sarcoma, fibrosarcoma, hemangiopericytoma, peripheral nerve sheet tumour, liposarcoma, myxosarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial cell sarcoma, gastrointestinal stromal tumour, histiocytic sarcoma, hemangiosarcoma, lymphangiosarcoma, chondrosarcoma, osteosarcoma, and osteochondrosarcoma.

[0202] P96598PC X44. The BCG composition for use according to any one of items X41-X43, wherein the primary solid tumour is a melanoma.

[0203] X45. The BCG composition for use according to any one of items X41 or X42, wherein primary solid tumour is a carcinoma selected from the group consisting of squamous cell carcinoma, nasal adenocarcinoma, thyroid carcinomas and adenocarcinomas, parathyroid carcinomas and adenocarcinomas, gastrointestinal carcinoma and adenocarcinomas, oesophageal carcinoma, gastric carcinoma, small intestinal carcinoma, large intestinal carcinoma, rectal carcinoma, bronchioalveolar carcinomas, pulmonary adenocarcinomas, hepatocellular carcinomas, pancreatic carcinomas and adenocarcinomas, renal cell carcinomas, transitional cell carcinomas, nephroblastoma, prostatic adenocarcinomas, uterine carcinomas and adenocarcinomas, Leydig cell tumour, Sertoli cell tumour, seminoma, granulosa cell tumour, teratocarcinoma, adenocarcinomas, oligodendrocytoma, glioblastoma, and meningiomas.

[0204] X46. The BCG composition for use according to any one of the preceding items, wherein the BCG composition comprises live attenuated Mycobacterium bovis BCG.

[0205] X47. The BCG composition for use according to any one of the preceding items, wherein BCG composition comprises a bacterial strain or sub-strain selected from the group consisting of Pasteur 1173 P2, Danish 1331, Glaxo 1077, Tokyo 172-1, Russian BCG- I, and Moreau RDJ strains.

[0206] X48. The BCG composition for use according to any one of items X46 or X47, wherein the attenuated live Mycobacterium bovis BCG is Danish strain 1331.

[0207] X49. The BCG composition for use according to any one of the preceding items, wherein the BCG composition comprises one or more pharmaceutically acceptable carriers, excipients and / or diluents.

[0208] X50. The BCG composition for use according to item X49, wherein the one or more pharmaceutically acceptable carriers, excipients and / or diluents are selected from the group consisting of sodium glutamate, magnesium sulphate heptahydrate, dipotassium phosphate, citric acid monohydrate, L-asparagine monohydrate, ferric ammonium citrate, and glycerol.

[0209] X51. The BCG composition for use according to any one of the preceding items, wherein the BCG composition is a BCG vaccine.

[0210] P96598PC X52. The BCG composition for use according to any one of the preceding items, further comprising administration of an immunotherapeutic composition.

[0211] X53. The BCG composition for use according to item X52, wherein the immunotherapeutic composition is administered before, simultaneously, or after administration of the BCG composition.

[0212] X54. The BCG composition for use according to any one of items X23-X53, wherein said BCG composition is administered as part of a combination therapy that comprises administering a treatment targeting the primary tumour.

[0213] X55. The BCG composition for use according to item X54, wherein said treatment targeting the primary tumour is selected from the group consisting of surgery, radiation therapy, chemotherapy, hormone therapy, immunotherapy, gene therapy, cell therapy, such as CAR-T cell therapy or stem cell therapy, or combinations thereof.

[0214] Yl. A method of treatment, inhibition, amelioration or prevention of lung metastases in a subject, the method comprising administration of a BCG composition in nebulized form.

[0215] Y2. The method according to item Yl, wherein the method is for prevention or inhibition of lung metastases in a subject.

[0216] Examples

[0217] Example 1: Induction of lung metastasis in a melanoma mouse model and treatment scheme

[0218] This example describes establishment of a tumour model for determining the effect of early / preventive treatment on development of lung metastases. Tumour cells were injected in a mouse recipient to simulate a primary tumour metastasizing, and treatment was initiated shortly thereafter to evaluate the effect of preventive treatment, i.e. before lung metastases had developed or fully established themselves.

[0219] Method

[0220] Tumour cells

[0221] B16-F10 Iuc2 (CRL-6475 LUC2) cells were cultured in DMEM +10% FBS (foetal bovine serum, heat inactivated), Penicillin-streptomycin and Blasticidin (10 ug / mL). For

[0222] P96598PC tumour cell inoculation, the cells were harvested, washed in DPBS and counted. The cells were resuspended in DPBS to a final concentration of 2xl06cells / mL. Tumour cells were transported to the animal facility on ice.

[0223] Tumour cell inoculation

[0224] Mice were gently warmed under a heat lamp for 5-10 minutes to make the tail vein dilated. The tumour cells were carefully resuspended by using a ImL syringe (without needle). The mice were inoculated with 100 pL tumour cell suspension (2xl05cells / mouse) by intravenous injection in the lateral tail vein using a 27g needle. Control mice did not receive any injection.

[0225] Anaesthesia

[0226] All mice in all groups (including untreated controls) were anesthetized with ZRF cocktail (3.3 mg zolazepam, 3.3 mg tiletamine, 0.5 mg xylazine and 2.6 pg fentanyl per 1 mL 0.9% NaCI). ZRF was administered by intraperitoneal (i.p.) injection (max 100 pL / lOg). The mice were kept on a heating mat until awake.

[0227] Preparation of BCG for administration

[0228] BCG was resuspended in DPBS so that one vial of BCG gives 10 doses (one vial Mycobacterium Bovis BCG has 2-8xl06CFU attenuated BCG (Bacillus Calmette-Guerin, Danish strain 1331). One dose BCG has 2-8xl05CFU BCG. For the nebulization of BCG, one vial was resuspended in 2000 pL to give 10 doses of 200 pL each. For the intranasal (IN) administration of BCG, one vial was resuspended in 200 pL to give 10 doses of 20 pL.

[0229] Administration of BCG by nebulization

[0230] Mice were under anaesthesia during the whole procedure. Endotracheal intubation was performed before BCG administration. The intubated mice were connected to the ventilator with settings: 120 bpm priority press, 15 cm H2O pressure, 0,145 mL tidal volume, 17 mL / min minute volume (SomnoSuite® Low-Flow Anaesthesia System with the RoVent® Automatic Ventilator Module, Kent scientific). The BCG dose (200 pL) was added to the Nebulizer (Aeroneb Lung Instillation Nebulizer Delivery System) and the mice received the nebulized BCG directly into the lungs via the endotracheal tube. The mice were disconnected from the ventilator system and the endotracheal tube removed when the nebulization chamber was empty (indication that the whole dose was administered). A total of 9 mice received BCG by nebulization.

[0231] Administration of BCG by intranasal delivery

[0232] P96598PC Mice were under anaesthesia during the whole procedure. To receive the BCG dose intranasally (i.n.), the mice were held ventral side up with the head slightly downwards. The 20 pL dose was then administered as 5-6 drops (ca 4-5 pL each) on the nostrils waiting until the mouse had inhaled the drop before adding a new drop. A total of 9 mice received BCG by intranasal delivery.

[0233] Untreated controls

[0234] Untreated controls received no treatment. Only anaesthesia was administered. A total of 8 mice were in the control group and received no treatment.

[0235] Treatment regimen

[0236] All mice were intravenously injected with tumour cells on Day 0. On Days 3, 7 and 11, each mouse in the two treatment groups (nebulization and intranasal) were administered BCG as described above. On Day 14 the experiment was terminated and lung tissue was harvested for further examination.

[0237] Example 2: Quantitative assessment of lung metastases by luminescence and macroscopic analysis.

[0238] This example describes the analysis of data acquired from the experiments of example 1.

[0239] Method

[0240] Preparation of mice before euthanasia and lung harvest

[0241] Before euthanasia, the mice were injected with luciferin (D-Luciferin sodium salt, GoldBio). Luciferin was administered intraperitoneally (i.p.) at the standard 150mg / kg injection dose. The mice were injected with 200 pL 15 mg / ml luciferin solution (3 mg for ~20g mouse).

[0242] Euthanasia and Lung harvest

[0243] Mice were euthanised by cervical dislocation. For maximum signal plateau during imaging (plateau at 10-20 min), the mice were euthanized 8 minutes after luciferin injection and the pictures taken at 10-12 minutes post luciferin injection. The lungs were removed from the mice, rinsed in DPBS and placed on the black plastic surface for IVIS imaging. A paper note was placed below the lung during imaging to indicate mouse number.

[0244] Measurement of luminescence, IVIS imaging

[0245] P96598PC Quantification of tumour load in the lungs was done by measuring luminescence from the lung tissue ex v / vo at 10-12 minutes after luciferin injection into the live animal (signal plateau at 10-20 min). The plastic mat with the lungs was placed in the IVIS imaging machine (PerkinElmer IVIS spectrum) and a picture taken with standard settings and auto-exposure. The total flux for each metastasis was quantified by manually drawing a region of interest (Rol) around each bioluminescent signal and summing the total recorded signal.

[0246] Photography of the lung tissue

[0247] After the imaging procedure in the IVIS machine was finished, the lungs were placed on a white plastic board for photography of the lungs. The lungs were carefully spread out on the board to get the largest surface area. A paper note below each lung was used to indicate the mouse number. One side of the lung was photographed.

[0248] Measurement of tumour area in the lungs

[0249] The tumour area in the lung was measured by ImageJ software (area in % of lung surface). A threshold was applied to the images to select areas of dark pigmentation (tumour tissues). The images were then converted to black-and-white images (8-bit). The selected area corresponding to tumour tissues was then quantified using the area measurement tool (ImageJ) and reported as a percentage of the total lung area.

[0250] Counting of lung metastases (metastatic foci)

[0251] The number of lung metastases (metastatic foci) was counted using ImageJ software. The number of foci was counted manually using the point tool to ensure all foci were counted and to avoid duplicated counts.

[0252] Results

[0253] Mice tolerated the handling well and the model consistently resulted in mice developing lung metastases if not treated (figure 1A). Upon preventive treatment with BCG nebulization there was a visual reduction in prevalence and size of lung metastases as recorded by IVIS (figure IB).

[0254] The effect of the BCG nebulization was quantified as the metastatic burden (figure 2A), count of metastatic foci (figure 2B), and tumour load (total flux, figure 2C). Apparent from the quantitative data is that BCG nebulization had a significant preventive effect on formation of metastases compared to untreated mice. Moreover, a direct comparison to another route of administration (i.e. intranasal) showed that

[0255] P96598PC nebulization of BCG had a significantly better preventive effect on development of lung metastases.

[0256] Conclusion

[0257] The Example demonstrates the advantage of utilizing BCG nebulization as a treatment route for preventing or inhibiting the development of lung metastases from a primary tumour.

[0258] Example 3: Evaluation of preventive treatment in melanoma model

[0259] This example describes an experiment where the establishment of a prevention model was based on injecting a reduced amount of B16-F10 Iuc2 (CRL-6475 LUC2) cells (IxlO5cells / mouse), for a slower development of the disease.

[0260] Method

[0261] This example was performed with same method as described in Examples 1 and 2, except for the following differences:

[0262] Tumour cell inoculation - Reduced amount of tumour cells inoculated

[0263] Mice were gently warmed under a heat lamp for 5-10 minutes to make the tail vein dilated. The tumour cells were carefully resuspended by using a ImL syringe (without needle). The mice were inoculated with 100 pL tumour cell suspension (IxlO5cells / mouse) by intravenous injection in the lateral tail vein using a 27g needle. Control mice did not receive any injection.

[0264] Untreated controls

[0265] Untreated controls (N = 2) received no treatment and no anaesthesia, and were euthanized on day 25 (mouse 4 and 5).

[0266] Healthy controls

[0267] Healthy controls (N=2) were not inoculated with B16-F10 Iuc2 cells and did not receive treatment and anaesthesia. These mice were euthanized on the same day as for the other groups.

[0268] Treatment regimen - BCG Nebulization

[0269] All mice were intravenously injected with tumour cells on Day 0. The treatment group received 2 BCG Nebulization Treatments on day 14, 18 and 22, and the experiment was terminated on day 25 (mouse 1, 2, 3). Lung tissue was harvested for further examination.

[0270] P96598PC Results

[0271] In line with the previous experiment, preventive treatment with BCG nebulization showed a visual reduction in prevalence and size of lung metastases, both macroscopically and by IVIS, compared to control mice (figure 3).

[0272] Conclusion

[0273] Early administration of BCG prevents development of lung metastases in a melanoma model where development of metastases is more slowly induced (less tumour cells inoculated) and treatment started at a later time point (day 14).

[0274] P96598PC

Claims

Claims1. A Bacillus Calmette-Guerin (BCG) composition for use in the prevention or inhibition of lung metastases in a subject, wherein the BCG composition is administered in nebulized form.

2. The BCG composition for use according to claim 1, wherein the subject is a human.

3. The BCG composition for use according to any one of claims 1 or 2, wherein the BCG composition is administered to the lungs.

4. The BCG composition for use according to any one of the preceding claims, wherein the BCG composition is administered via inhalation.

5. The BCG composition for use according to any one of the preceding claims, wherein the BCG composition is first administered before any lung metastases have been identified.

6. The BCG composition for use according to any one of the preceding claims, wherein the BCG composition is first administered upon initial diagnosis of a primary tumour.

7. The BCG composition for use according to any one of the preceding claims, wherein the BCG composition is first administered within 14 days of the initial diagnosis of a primary tumour, such as within 12 days, such as within 10 days, such as within 7 days, such as within 5 days, such as within 4 days, such as within 3 days, such as within 2 days, such as within 1 day.

8. The BCG composition for use according to any one of the preceding claims, wherein the lung metastases are derived from a primary tumour.

9. The BCG composition for use according to any one of claims 6-8, wherein the primary tumour is an early-stage primary tumour.

10. The BCG composition for use according to any one of claims 6-9, wherein the primary tumour is characterized by a score according to the TNM system of:- T is less than or equal to 2,- N is less than or equal to 1, andM is 0.P96598PC11. The BCG composition for use according to any one of claims 6-10, wherein the primary tumour has metastatic potential.

12. The BCG composition for use according to any one of claims 6-11, wherein the primary tumour is a primary solid tumour.

13. The BCG composition for use according to claim 12, wherein the primary solid tumour is selected from the group consisting of sarcoma, carcinoma, lymphoma, and carcinosarcoma.

14. The BCG composition for use according to any one of claims 12 or 13, wherein the primary solid tumour is a sarcoma selected from the group consisting of melanoma, soft tissue sarcoma, fibrosarcoma, hemangiopericytoma, peripheral nerve sheet tumour, liposarcoma, myxosarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial cell sarcoma, gastrointestinal stromal tumour, histiocytic sarcoma, hemangiosarcoma, lymphangiosarcoma, chondrosarcoma, osteosarcoma, and osteochondrosarcoma.

15. The BCG composition for use according to any one of the preceding claims, wherein prevention and / or inhibition means that the metastatic burden of the subject is reduced compared to a subject that does not receive the BCG composition.

16. The BCG composition for use according to any one of the preceding claims, wherein the BCG composition comprises live attenuated Mycobacterium bovis BCG.

17. The BCG composition for use according to any one of the preceding claims, wherein the BCG composition is a BCG vaccine.

18. The BCG composition for use according to any one of claims 6-17, wherein said BCG composition is administered as part of a combination therapy that comprises administering a treatment targeting the primary tumour.

19. The BCG composition for use according to claim 18, wherein said treatment targeting the primary tumour is selected from the group consisting of surgery, radiation therapy, chemotherapy, hormone therapy, immunotherapy and stem cell treatment, or combinations thereof.P96598PC

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