Method of treating cancer by intratumoral deposition of radioactive microparticle

Abstract

To provide a method of microbrachytherapy which provides one advantage as described in paragraph [0007] of the description.SOLUTION: Provided is a composition comprising microparticles comprising a non-radioactive isotope and a radioactive isotope, and an injection vehicle, for use in the intratumoral treatment of cancer, by combined antitumoral effect of radioactivity and intratumoral microparticle deposition, in which the antitumoral effect provided by the cytotoxicity of radioactivity is enhanced by modulation of the tumoral immune response induced by microparticle deposition inside the tumor, thereby decreasing the dose of radioactivity required to treat solid tumors. Provided is a method of treating solid tumors using a sub-optimal tumor-volume coverage dose of radioactivity combined with intratumoral microparticle deposition as means of tumor control.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THE PRESENT APPLICATION The present invention relates to a method for the treatment of solid tumors by the combined antitumor effect of radiation and in situ microparticle deposition, where the antitumor effect provided by the cytotoxicity of radiation is enhanced by the modulation of the tumor immune response induced by microparticle deposition inside the tumor, thereby reducing the dose of radiation required to treat the solid tumor.The present invention relates to a method for the treatment of solid tumors with a sub-optimal tumor-volume coverage dose of radiation combined with in situ microparticle deposition as a means of tumor control. [Background technology]

[0002] Malignant brain tumors and brain metastases have an extremely poor prognosis and are often considered fatal in adults due to frequent tumor recurrence. Glioblastoma is the most common primary brain tumor in adults. Besides glioblastoma, brain metastases from other primary tumors, including those from lung cancer, skin cancer, and breast cancer, also represent a significant number of central nervous system (CNS) tumors. It has been reported that 15-30% of patients with metastatic breast cancer and metastatic lung cancer develop brain metastases. Radiation therapy (RT) is the state-of-the-art treatment for brain tumors and brain metastases. Even with treatments including surgical intervention, radiation therapy (RT), and chemotherapy, only a portion of these patients with malignant brain tumors / malignant brain metastases survive more than 2 years after diagnosis. Radioresistance of glioblastoma is a major problem that makes it impossible to completely treat with current therapeutic methods.

[0003] Solid tumors develop within a complex microenvironment (tumor stroma) that includes non-neoplastic cells (tumor stromal cells) that significantly affect tumor growth, tumor transformation, and tumor metastasis. Among these non-neoplastic cells, tumor-associated macrophages (TAMs), classified into M1 phenotype that suppresses cancer progression and M2 phenotype that promotes it, are key regulators of tumorigenesis. This is especially true in the case of the most common brain tumors (gliomas such as glioblastomas), where the majority of stromal cells are TAMs (macrophages / microglia). Glioblastoma infiltration is an obstacle to its complete resection, and the presence of TAMs has an important role in resistance to radiotherapy. Therefore, combining radiotherapy with TAM targeting is beneficial for the treatment of common brain tumors such as glioblastomas.

[0004] Brachytherapy is a type of internal radiation therapy in which radioactive implants are placed in or near tumors. Direct intratumoral injection of radioactive microparticles (micro brachytherapy) is a promising therapeutic approach for cancer treatment. Holmium (Ho-165 / Ho-166) microparticles under various forms have been particularly studied, as holmium offers several advantages for therapy, including its suitability for in vivo imaging. 165-Ho is stable, and 166-Ho is a pure, high-energy β-emitter (Emax=1.86MeV, t1 / 2=26.8h, γ-emission=80keV6.7%), with limited tissue penetration of β-particles (maximum of 8mm). 166-Ho is also a gamma-emitter making it suitable for SPECT (single photon emission computed tomography) imaging. Moreover, holmium is highly paramagnetic and has a high mass attenuation coefficient, which makes the atom visible in magnetic resonance imaging (MRI) and computed tomography (CT), respectively.

[0005] The therapeutic efficacy of intratumoral injection of holmium microparticles has been studied in various cancers. Ho-166 chitosan conjugates have been tested in a prostate cancer model in rats (Kwak, Cheol Hong et al., European Journal of Nuclear Medicine and Molecular Imaging 2005, 32, 1400-1405), in a phase IIb clinical trial of liver cancer (Kim et al., Clinical Cancer Research 2006, 12, 543-548) and in malignant gliomas in rats (Ryoong Huh et al., Yonsei Med.J. 2005, 46(1), 51-60). Ho-166 acetylacetonate microspheres (HoAcAcMS) have been tested in a renal cancer model (Bult et al., PLoS One.8(1) 2013, e52178.doi:10.1371), a mouse model of kidney cancer (W. Bult, Holmium microparticles for intratumoral radioablation, Thesis, University of Utrecht, 2010), oral squamous cell carcinoma in cats (Nimwegen et al., Vet. Comp. Oncol., 2017, DOI:10.1111 / vco.l2319) and head and neck squamous cell carcinoma (Bakker et al., Nuclear Medicine communications, 2018, 39, 213-221).

[0006] Promising results were observed. However, the therapeutic effect was minimal (human and livestock studies), especially for large tumors. This was explained by incomplete tumor coverage by the effective radiation dose, since it is recognized in the field (Barani et al., Current Understanding and Treatment of Gliomas pp. 49-73 - Springer Edition, 2015) that complete coverage of the tumor volume and surrounding infiltrated areas by the effective radiation dose (considered to be >60 Gy to achieve tumor necrosis) is essential to obtain a therapeutic effect. It was reported that technical difficulties in intratumoral administration of microparticles resulted in incomplete tumor coverage by an efficient radiation dose. Therefore, current research aims to meet this recognized requirement of complete tumor coverage by an effective radiation dose, for example by improving the microparticle administration technique. There is also a potential risk of toxicity with this technique, since leakage of radioactive microparticles outside the tumor was often reported.

[0007] A more efficient method of microbachytherapy that combines therapeutic efficacy with a lack of toxicity would be an advance for the treatment of solid tumors. Moreover, it would be desirable to have a method of microbrachytherapy that is effective in treating tumors associated with tumor-promoting macrophage activation, such as major and common brain tumors, including glioblastoma. It is an object of the present invention to provide a method of microbrachytherapy that provides such advantages. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] FR 2 930 890 [Patent Document 2] US2008 / 0166297 [Non-patent literature]

[0009] [Non-Patent Document 1] Kwak, Cheol Hong et al., European Journal of Nuclear Medicine and Molecular Imaging 2005, 32, 1400~1405 [Non-Patent Document 2] Kim et al., Clinical Cancer Research, 2006, 12, 543-548 [Non-Patent Document 3] Ryoong Huh et al., Yonsei Med.J., 2005, 46(1), 51-60 [Non-Patent Document 4] Bult et al., PLoS One.8(1)2013, e52178.doi:10.1371 [Non-Patent Document 5] W. Bult, Holmium microparticles for intratumoral radioablation, Thesis, University of Utrecht, 2010 [Non-Patent Document 6] Nimwegen et al., Vet.Comp.Oncol., 2017, DOI:10.1111 / vco.l2319 [Non-Patent Document 7] Bakker et al., Nuclear Medicine communications, 2018, 39, 213–221 [Non-Patent Document 8] Barani et al., Current Understanding and Treatment of Gliomas, pp. 49-73 - Springer Edition, 2015 [Non-Patent Document 9] Marcon et al., J. Nanomed. Nanotechnol., 2017, 8, 5; DOI:10.4172 / 2157~7439.1000460) [Non-Patent Document 10] Zielhuis et al., Int. J Pharm., 2006, 311, 69-74 [Non-Patent Document 11] Bult et al., Pharm. Res., 2012, 29, 827-836 [Non-Patent Document 12] Miller et al., Br.Med.J. (Clin.Res.Ed.), 1983, 286:266~ [Non-Patent Document 13] Urbanska et al., Termedia Publishing, 2014, 18, 307-12 [Non-Patent Document 14] Houben et al., Ned.Tij dschr Geneeskd.2005, 149, 2268~72 [Non-Patent Document 15] Schwartzbaum et al., Nat. Clin. Pract. Neurol., 2006, 2, 494~503 [Non-Patent Document 16] Hovinga et al., J.Neurooncol.2005, 74, 99~103 [Non-Patent Document 17] Naidu et al., J.Radiat.Res.2010, 51, 393~404 [Non-Patent Document 18] Selek et al., J. Neurosci. Methods., 2014, 221, 159~165 [Non-Patent Document 19] Khoshnevis et al., J.Neurosc.i Methods.Elsevier, 2017, 282, 61~68 [Non-Patent Document 20] Srinivas et al., Evol.Comput.MIT Press;1994, 2, 221~248 [Non-Patent Document 21] Frey B et al., Immunol Rev. 2017;280:231-248. Summary of the Invention

[0010] SUMMARY OF THE PRESENT APPLICATION The present invention relates to a method for treating solid tumors by the combined antitumor effect of radiation and in situ microparticle deposition. The invention is based on the observation by the inventors that the antitumor effect of microbrachytherapy provided by the cytotoxicity of radiation is enhanced by the modulation of tumor immune response induced by microparticle deposition inside the tumor. As a result of this enhancement, the dose of radiation required to treat solid tumors is reduced, thereby allowing the use of suboptimal tumor volume coverage doses of radiation in cancer treatment, with the direct consequence of sparing surrounding healthy tissue from radiation-induced toxicity. The inventors have studied the therapeutic effect of intratumoral injection of Ho-165 / Ho-166 microparticles in a human glioblastoma minipig model. Surprisingly, the inventors have found that suboptimal tumor volume coverage doses of radiation result in efficient tumor control and increased survival in the absence of visible toxic effects in treated animals (Example 6; Figure 1, Table 2, Table 3, Table 4). The method advantageously involves intratumoral deposition of radioactive microparticles.

[0011] In particular, the present invention relates to the use of compositions comprising microparticles containing a radioactive isotope, and optionally a non-radioactive isotope, optionally derived from the same chemical element, and an injection vehicle, for intratumoral treatment of cancer through the combined antitumor effects of radioactivity and intratumoral microparticle deposition.

[0012] In some embodiments, intratumoral microparticle deposition induces the elimination of tumor-promoting macrophages (tumor-associated macrophages (TAMs) having an M2 phenotype or M2-TAMs) from the treated tumor.

[0013] In some embodiments, intratumoral microparticle deposition induces an anti-tumor immune response.

[0014] In some embodiments, the radioisotope is suitable for use in radiation therapy, particularly internal radiation therapy. In some embodiments, the radioisotope is Holmium-166 (Ho-166 or 166Ho), Lutetium-177 (Lu-177 or 177 Lu), rhenium-186 or rhenium-188 (Re-186( 186 Re) or Re-188( 188 Re), Gold-198 (Au-198 or 198 Au), Yttrium-90 (Y-90 or 90 Y), Thorium-227 (Th-227 or 227 Th), Actinium-225 (Ac-225 or 225 Ac) and combinations thereof, preferably holmium-166. In some embodiments, the microparticles comprise holmium-165 and holmium-166. Preferably, the microparticles comprise at least 20% (w / w) total holmium, more preferably at least 25% (w / w) total holmium.

[0015] In some embodiments, the microparticles comprise or consist of polysiloxane, acetlyacetonate (AcAc), poly-L-lactic acid (PLLA), alginate, chitosan, resin, glass, or combinations thereof. In some preferred embodiments, the microparticles comprise or consist of polysiloxane.

[0016] In some embodiments, the microparticles, preferably Ho-165 / Ho-166 microparticles such as Ho-165 / Ho-166 polysiloxane microparticles, have an average or median diameter of 0.1 μm to 10 μm, preferably 0.1 μm to 1 μm, more preferably 0.3 μm to 0.6 μm, and even more preferably 0.4 μm to 0.5 μm.

[0017] In some embodiments, the microparticles, preferably Ho-165 / Ho-166 microparticles, such as Ho-165 / Ho-166 polysiloxane microparticles, have a specific activity of at least 1 megabecquerel (MBq) per milligram, for example, 1-10 MBq per milligram, particularly such as 2-3 MBq per milligram.

[0018] In some embodiments, the composition comprises a particulate suspension having a density of at least 1 g / mL, for example between 1 g / mL and 1.5 g / mL, preferably a Ho-165 / Ho-166 particulate suspension, such as a Ho-165 / Ho-166 polysiloxane particulate suspension.

[0019] In some embodiments, the composition comprises a particulate suspension having a viscosity of at least 3 mPa.s, preferably from 3 mPa.s to 12 mPa.s, more preferably from 6 mPa.s to 10 mPa.s, preferably a Ho-165 / Ho-166 particulate suspension, such as a Ho-165 / Ho-166 polysiloxane particulate suspension.

[0020] In some embodiments, the composition comprises an aqueous suspension of microparticles, preferably Ho-165 / Ho-166 microparticles such as Ho-165 / Ho-166 polysiloxane microparticles, in an injection vehicle comprising water and finally a small amount of ethanol, preferably less than 5% (v / v).

[0021] In some embodiments, the use of the composition comprises intratumoral administration of a suboptimal tumor volume coverage dose of radioactivity. In some preferred embodiments, the use of the composition comprises intratumoral administration of an absorbed dose of 100 Gray or more in less than 95%, 85%, 75%, 65%, 55%, 45%, or 35% of the tumor volume, preferably 35%-55% of the tumor volume. In other preferred embodiments, the use of the composition comprises intratumoral administration of an absorbed dose of 60 Gray or more in less than 95%, 85%, 75%, 65%, 55%, or 35% of the tumor volume, preferably 35%-55% of the tumor volume.

[0022] In some embodiments, the cancer is selected from the group including bladder cancer, brain cancer, breast cancer, colon cancer, stomach cancer, eye cancer, head and neck cancer, skin cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, oral cancer, prostate cancer, small intestine cancer, soft tissue cancer, testicular cancer, thyroid cancer and uterine cancer. Preferably, the cancer is brain cancer, particularly glioblastoma. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Detailed Description of the Invention The present invention relates to a method for the treatment of solid tumors by the combined antitumor effect of radioactivity and in situ microparticle deposition, which allows the use of suboptimal tumor volume coverage doses of radioactivity to treat solid tumors, with the direct consequence of sparing the surrounding healthy tissue from radiation-induced toxicity. The method advantageously involves intratumoral deposition of radioactive microparticles.

[0024] As used herein, the term "microparticle" or "microsphere" refers to particles of any material to which isotopes (radioactive and non-radioactive) can be attached to produce particles loaded or doped with radioactive and non-radioactive isotopes. The microparticles of the present invention are biocompatible and biopersistent.

[0025] As used herein, unless the context clearly dictates otherwise, the term "radioactive microparticles" refers to microparticles that include non-radioactive and radioactive isotopes as defined in the present invention.

[0026] "Biocompatible" means non-toxic to the body (pharmaceutical acceptable).

[0027] "Biopersistence" refers to a material that, when injected into a tissue or organ, such as a tumor, of an individual, is retained at the injection site and remains at the injection site for at least the duration of the treatment. Biopersistence includes biodurability, which is stability or resistance to chemical processes in vivo, such as dissolution or leaching. Biopersistence also includes stability or resistance to physical / mechanical processes in vivo, such as translocation, diffusion, fragmentation or destruction.

[0028] As used herein, the term "microparticle size" refers to the mean, median, or mode reflecting particle size distribution by number, surface, volume, or frequency as determined by conventional particle size measurement techniques known to those of skill in the art, such as laser diffraction, dynamic light scattering, photon correlation spectroscopy, sedimentation field flow fractionation, disk centrifugation, or electrical detection methods. Size values ​​may also include the width of the distribution (standard deviation, variance).

[0029] As used herein, the terms "patient", "individual" or subject refer to mammals including humans, domestic animals such as bovine (cattle and the like), ovine (sheep and the like), caprine (goats and the like) and equine (horses and the like), and commercial animals such as, but not limited to, dogs and cats. The patient is preferably a human.

[0030] As used herein, the term "treat" or "treatment" as used herein means reversing, alleviating, or inhibiting the progression of the disorder or condition to which such term applies, or reversing, alleviating, or inhibiting the progression of one or more symptoms of the disorder or condition to which such term applies.

[0031] As used herein, the term "tumor" refers to cancerous tumors, including primary tumors and metastases.

[0032] As used herein, the term "cancer" refers to any type of solid cancer, also referred to as a solid tumor, solid tumor cancer, or solid cancer tumor.

[0033] As used herein, the term "suboptimal tumor volume coverage dose of radiation" refers to a dose of radiation that is lower than the estimated minimum absorbed dose (i.e., effective radiation dose) that achieves necrosis of the treated tumor. The estimated minimum absorbed dose is between 60 Gray and 100 Gray in the entire tumor volume (100%).

[0034] As used herein, the terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, a "radioisotope" as used herein is understood to represent one or more radioisotopes. Thus, the terms "a" (or "an"), "one or more," or "at least one" may be used interchangeably herein.

[0035] In particular, the present invention relates to the use of compositions comprising microparticles containing non-radioactive and radioactive isotopes and an injection vehicle for intratumoral treatment of cancer through the combined antitumor effect of radioactivity and intratumoral deposition of the microparticles.

[0036] Radioactivity destroys tumor cells by delivering localized high-energy radiation. The direct cytotoxic effect of radioactivity on tumor cells is used to treat solid tumors. The effect of radioactivity is considered as an alternative to physical ablation, as well as surgical resection. In the present invention, localized microparticle deposition inside the tumor provides an additional antitumor effect to the direct cytotoxic effect of radioactivity by inducing modulation of tumor immune response. Thus, the overall antitumor effect is enhanced using the method of the present invention. As a result of this enhancement, the dose of radioactivity required to treat solid tumors is reduced, allowing the use of suboptimal tumor volume coverage doses of radioactivity in cancer treatment, with the direct consequence of sparing surrounding healthy tissue from radiation-induced toxicity.

[0037] In some embodiments, intratumoral microparticle deposition induces the removal of tumor-promoting macrophages (M2-TAM) from the treated tumor. Proinflammatory macrophages (M2-TAM) present in the tumor microenvironment are attracted to non-radioactive / radioactive microparticles and are eliminated by a direct killing effect. This effect is persistent over time beyond the radiation effect. The application of radioactivity to induce a local tumor killing effect by physical means is combined with the removal of tumor-promoting macrophages from the treated tumor induced by local (in situ) microparticle deposition.

[0038] In some embodiments, intratumoral microparticle deposition induces antitumor immune responses. In particular, ionizing radiation delivered by microparticles induces antitumor immune mechanisms by recruiting external macrophages and / or dendritic cells and / or activating T-lymphocyte production. This significantly alters the tumor microenvironment and acts synergistically in combination with the direct cytotoxicity mechanism of radioactivity against tumor cells. More specifically, in the present invention, the local deposition of said radioactive microparticles exerts a combined antitumor effect of local tumor killing by physical means (radioactivity), removal of tumor-promoting macrophages (TAM) from the treated tumor, and activation of antitumor immune cell recruitment.

[0039] The present invention may be considered as part of the emerging field of immuno-oncology (IO) therapy, which utilizes the body's own immune system to fight disease. The goal of IO therapy is to restore the immune system's ability to eliminate cancer cells by directly activating the immune system, by inhibiting tumor-inhibitory mechanisms, or by modulating / suppressing those mechanisms that favor tumor growth and aggressiveness. Besides the effects of radiation on tumors and macrophages, the microparticles used in the present invention surprisingly exert a direct killing effect on tumor-associated macrophages (TAMs), which is prolonged due to the persistence of the microparticles as stable implants in tumor tissue. This effect is sustained over time beyond the transient effect induced by radiation. This induces continuous recruitment and direct toxic effects on TAMs over time, thereby ensuring long-term antitumor effects. These recruitments are responsible for tumor shrinkage and induction of antitumor immunity.

[0040] Thus, the present invention relates to a novel combination of temporary suboptimal lethal radiation exposure and in situ persistence of anti-TAM microparticles that synergistically controls cancer cell proliferation, limiting the sequelae of radiotherapeutic ablation of healthy tissues induced by current radiotherapy approaches. Said combination is capable of inducing a prolonged antitumor response in addition to its ability to disrupt tumor cell cycle by delivery of ionizing radiation. For example, treatment with Ho-166 microparticles in a human xenograft glioblastoma pig tumor model was observed to increase survival rate in the absence of any visible adverse effects on treated animals. In addition to the local radiotherapeutic effect, by deposition in situ, the radioactive particles according to the present invention, i.e., such Ho-166 / Ho-165 microparticles, allow a continuous and stable internal anti-TAM effect with a more constant and sustained tumor control. In this context, the antitumor effect of radiotherapy is enhanced by the persistence of microparticles in tumor tissue.

[0041] Thus, the present invention provides a tumor therapy using a suboptimal tumor volume coverage dose of radioactivity combined with microparticle deposition as a means of tumor control. In this therapy, the direct cytotoxic effect of ionizing radiation on tumor cells is enhanced by the removal of tumor-promoting macrophages from the treated tumor induced by local microparticle deposition, and the induction of antitumor immune mechanisms by recruitment of external macrophages and dendritic cells and activation of T-cell lymphopoiesis. This approach can produce a widespread antitumor effect both within the tumor mass and the surrounding infiltrating regions. The combined antitumor effect of radioactivity, removal of tumor-promoting macrophages from the treated tumor, and activation of antitumor immune cell recruitment can reduce the total absorbed dose of radioactivity required for tumor killing effect, with the direct consequence of sparing surrounding healthy tissue from radiation-induced toxicity, compared to standard external beam radiation therapy (EBRT) treatment.

[0042] Radioactive microparticles for use in accordance with the present invention are made from materials that can be loaded or doped with radioactive and non-radioactive isotopes to form biocompatible and biopersistent radioactive microparticles. For example, isotopes may be loaded onto the microparticles by impregnation, absorption or covalent attachment, either directly or via suitable linkers or chelators, using standard techniques well known in the art.

[0043] The radioactive microparticles for use according to the invention have good tissue penetration and good fixation in the tumor after injection without migration out of the tumor. In particular, the radioactive microparticles for use according to the invention are biopersistent and remain at the injection site in the tumor without leaching any radioisotope from the treated patient in healthy tissue. Thus, potential damage to the surrounding tissue and toxic effects on the treated patient are limited by the radioactive microparticles according to the invention. The radioactive microparticles for use according to the invention are capable of inducing the removal of tumor-promoting macrophages from the treated tumor after their local deposition inside the tumor. The tumor-promoting macrophage removal effect is also present for non-radioactive microparticles (non-activated microparticles containing non-radioactive isotopes). The radioactive microparticles for use according to the invention also have very good colloidal stability, favorable for injectability, and are not altered by neutron activation. These properties depend on the intrinsic properties of the microparticle suspension, in particular the concentration, stability, particle size distribution and / or viscosity. These properties can be readily determined using standard methods well known in the art and can be adapted by one of ordinary skill in the art to obtain radioactive microparticles having the desired properties.

[0044] Non-limiting examples of microparticles for use in the present invention include acetotriacetonates (AcAc), resins, ceramics including glass, plastics, natural or synthetic polymers, other materials, and combinations thereof. Examples of polymers for use in the present invention include, but are not limited to, polysiloxanes, poly-L-lactic acid (PLLA), alginates, chitosan, and combinations thereof. Polysiloxane particles are disclosed, for example, in patent FR 2 930 890 and Marcon et al., J. Nanomed. Nanotechnol., 2017, 8, 5; DOI: 10.4172 / 2157-7439.1000460). PLLA particles are disclosed, for example, in Zielhuis et al., Int. J Pharm., 2006, 311, 69-74; Bult et al., Pharm. Res., 2012, 29, 827-836. Chitosan particles are described, for example, in US2008 / 0166297. In some embodiments, the microparticles comprise or consist of polysiloxane, acetoacetate (AcAc), poly-L-lactic acid (PLLA), alginate, chitosan, resin, glass, or combinations thereof. In some preferred embodiments, the microparticles comprise or consist of polysiloxane.

[0045] Particle size distribution is an important parameter of microparticles. In fact, particle size is closely related to the distribution of microparticles in vivo, but also to their disappearance. Microparticles usually have a mean or median diameter of 0.1 μm to 60 μm. In some embodiments, microparticles have a mean or median diameter of 0.1 μm to 10 μm, preferably 0.1 μm to 1 μm, more preferably 0.3 μm to 0.6 μm, and even more preferably 0.4 μm to 0.5 μm. In some preferred embodiments, microparticles are microspheres with a mean diameter of 0.1 μm to 60 μm, preferably 0.1 μm to 10 μm or 0.1 μm to 1 μm, more preferably 0.3 μm to 0.6 μm, and even more preferably 0.4 μm to 0.5 μm.

[0046] In some embodiments, the radioisotope is a beta-negative emitting radioisotope or an alpha emitting radioisotope or a combination thereof, preferably a short half-life and high energy beta-negative emitting radioisotope or a combination thereof. Examples of radioisotopes that can be used in the present invention include holmium-166 (Ho-166 or 166 Ho), Lutetium-177 (Lu-177 or 177 Lu), rhenium-186 or rhenium-188 (Re-186( 186 Re) or Re-188( 188 Re), Gold-198 (Au-198 or 198 Au), Yttrium-90 (Y-90 or 90 Y), Thorium-227 (Th-227 or 227 Th), Actinium-225 (Ac-225 or 225 Ac) and combinations thereof.

[0047] [Table 1]

[0048] In some preferred embodiments, the radioisotope is holmium-166. Ho-166 has a short half-life and high β - It is of particular interest for use in the present invention because it offers a very good compromise between combining energies. - It is both a β- and γ-emitter and therefore combines high therapeutic efficacy with the possibility of detection by single photon emission computed tomography (SPECT), which allows suitable nuclear imaging for individualized patient treatment, in particular for dose calculation. - Compared with the β- and γ-emitters (Lu-177; Re-186 and Re-188), Ho-166 is a more suitable β-emitter than lutetium (Lu-177) for localized radiotherapy (brachytherapy) applications. -Due to the localized tissue penetration of its β-particles, Ho-166 allows for much higher localized radiation doses (typically 200 up to over 1000 Gy in the immediate vicinity of the deposition site, which can reach up to tens of thousands of cells) compared to external beam radiation therapy (EBRT).

[0049] In some embodiments, the microparticles contain radioactive and non-radioactive isotopes derived from the same chemical element, such as holmium-165 and holmium-166. The radioactive isotopes are advantageously produced by standard neutron activation methods well known in the art, such as the use of a neutron activator to allow activation of a portion of the microparticle by incorporation of neutrons. Neutron activation is disclosed, for example, in patent FR 2 930 890. The microparticles are stable during neutron activation.

[0050] In some preferred embodiments, the microparticles, such as polysiloxane microparticles, comprise holmium-165 and holmium-166. Preferably, the microparticles, such as polysiloxane microparticles, comprise at least 20% (w / w) total holmium, more preferably at least 25% (w / w) total holmium. Total holmium content refers to the total holmium content, meaning holmium-165 and holmium-166. Holmium content is determined by standard analytical methods, such as, for example, inductively coupled plasma mass spectrometry (ICP-MS). High holmium content potentially allows for the delivery of high radioactive doses to tumor cells.

[0051] Ho-165, a stable natural isotope that gives Ho-166 upon neutron activation, is monoisotopic and therefore easier to control for both activation and imaging / dosimetry, compared to biisotope-containing natural rhenium, which upon neutron activation produces two radioisotopes with different properties (Re-186 and Re-188). Furthermore, Ho-165 remains predominant in the microparticles after irradiation. Moreover, holmium is highly paramagnetic and has a high mass attenuation (X-ray absorption) coefficient that makes this atom detectable by magnetic resonance imaging (MRI), ultrasound and computed tomography (CT) techniques. Hence, multimodal imaging is possible with Ho-165 / Ho-166 microparticles, which facilitates the determination of the optimal distribution of microparticles per injection and also the estimation of dosimetry. Thus, Ho-165 / Ho-166 microparticles, such as Ho-165 / Ho-166 polysiloxane microparticles, advantageously combine multimodal imaging and therapeutic efficacy.

[0052] In some embodiments, the microparticles, preferably Ho-165 / Ho-166 microparticles, such as Ho-165 / Ho-166 polysiloxane microparticles, have a specific activity of at least 1 megabecquerel (MBq) per milligram, for example, 1-10 MBq per milligram, particularly such as 2-3 MBq per milligram.

[0053] The composition for use according to the invention is a pharmaceutical composition comprising a therapeutically effective amount of microparticles, preferably Ho-165 / Ho-166 microparticles, such as Ho-165 / Ho-166 polysiloxane microparticles, suspended in a pharma- ceutical acceptable infusion vehicle, which are suspended in the infusion vehicle to form a stable, injectable suspension.

[0054] The amount or concentration of microparticles in a composition for use according to the invention is reflected by its dry matter (w / v) content or its density (w / v). A high density or value of dry matter refers to a highly concentrated composition. Dry matter is related to the concentration of microparticles obtained after synthesis and dispersion in an aqueous solution. The concentration of particles is directly related to the specific radioactivity generated during activation and therefore to the therapeutic activity induced after injection. These parameters can be easily determined using standard methods well known in the art and can be adapted by the skilled artisan to obtain a pharmaceutical composition with the desired therapeutic activity.

[0055] The microparticle concentration, sedimentation rate, and viscosity of the pharmaceutical composition are adapted to formulate a stable, injectable suspension. The sedimentation rate provides information about the colloidal stability for proper injection of the suspension. Viscosity is an important parameter for assessing the correct circulation of the suspension flow in the fluid lines of the injector system during intratumoral injection.

[0056] In the context of this invention, a therapeutically effective amount refers to a dose sufficient to reverse, alleviate, or inhibit the progression of the disorder or condition to which such term applies, or to reverse, alleviate, or inhibit the progression of one or more symptoms of the disorder or condition to which such term applies.

[0057] Effective dosage treatment regimens will be determined and adjusted according to factors such as the composition used, the route of administration, the physical characteristics of the individual under consideration, e.g., sex, age, and weight, concurrent medications, and other factors that will be recognized by those of ordinary skill in the medical arts.

[0058] Treatment doses are adjusted to the characteristics of solid tumors such as their shape, bulk size and location within the body, especially if they are located in "organs at risk".

[0059] A "pharmaceutically acceptable vehicle" refers to a vehicle that does not produce adverse, allergic or other untoward reactions when administered to a mammal, especially a human, as appropriate.

[0060] The pharmaceutical compositions include pharma- ceutically acceptable vehicles for injectable formulations. These may be liquid carriers, particularly aqueous carriers, such as sterile water, isotonic solutions, sterile solutions, salt solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, and the like, or mixtures of such salts), dextrose solutions, mannitol, such as Ringer's solution, Ringer-lactacte, Hank's solution, and other physiologically balanced salt solutions. Suitable pharmaceutical forms for injectable use include, in particular, sterile aqueous suspensions. The suspensions may contain additives compatible with the radioactive particles, such as substances that enhance isotonicity and chemical stability. In all cases, the form must be sterile and must be fluid to the extent that easy injectability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0061] The pharmaceutical compositions of the present invention have a pH and osmolality consistent with what is tolerated by the patient being treated, for example, the pH may be from about 6 to about 9, and the osmolality may be from about 100 mOsm to about 300 mOsm.

[0062] In some embodiments, the composition comprises an aqueous suspension of radioactive microparticles, preferably a Ho-165 / Ho-166 microparticle suspension, such as a Ho-165 / Ho-166 polysiloxane microparticle suspension, in an injection vehicle comprising water and finally a small amount of ethanol, preferably less than 5% (v / v).

[0063] In some embodiments, the particulate suspension, preferably a Ho-165 / Ho-166 particulate suspension, such as a Ho-165 / Ho-166 polysiloxane particulate suspension, has a density of at least 1 g / mL, for example from 1 g / mL to 1.5 g / mL. Alternatively or additionally, the particulate suspension may have a dry matter content of from about 400 g / L to about 700 g / L.

[0064] In some embodiments, the microparticle suspension, preferably the Ho-165 / Ho-166 microparticle suspension, has a viscosity of at least 3 mPa.s, preferably between 3 mPa.s and 12 mPa.s, more preferably between 6 mPa.s and 10 mPa.s. Alternatively or additionally, the microparticle suspension may have colloidal stability characterized by less than 15% settling after 1 hour or less than 25% settling after 24 hours as determined by the Westergren method described in Miller et al., Br. Med. J. (Clin. Res. Ed.), 1983, 286:266-.

[0065] The microparticle suspension is stable in vitro and in vivo. In particular, the microparticle suspension is stable in vitro for several hours during the intratumoral administration phase to a patient. The microparticle suspension is stable in vivo and persists at the injection site within the tumor for several months, as shown in the examples of the present application.

[0066] The pharmaceutical composition of the present invention is administered to a patient by intratumoral administration (injection directly into a tumor) according to known procedures at a dose and duration effective to induce a therapeutic effect in the patient. The composition may be administered through single or multiple injection sites of suitable injection volumes at suitable injection rates using suitable injection devices well known in the art. For safety and efficacy of treatment, intratumoral administration of radioactive microparticle suspension is performed and monitored in a controlled manner. For example, stereotactic injection may be combined with image-guided administration using computed tomography (CT) or ultrasound. The average radiation dose (gray) absorbed in the tumor is calculated from tumor imaging data using suitable software well known in the art, such as the Monte Carlo simulation software Geant4 Application for Tomographic Emission (GATE) and the like.

[0067] In some embodiments, the invention involves administration of a suboptimal tumor volume coverage dose of radiation. In some preferred embodiments, the invention involves intratumoral administration of an absorbed dose of 100 Gray or more in less than 95%, 85%, 75%, 65%, 55%, 45%, or 35% of the tumor volume, preferably 35%-55% of the tumor volume. In other preferred embodiments, the invention involves intratumoral administration of an absorbed dose of 60 Gray or more in less than 95%, 85%, 75%, 65%, 55%, or 35% of the tumor volume, preferably 35%-55% of the tumor volume.

[0068] As shown in the examples of the present application, the suboptimal tumor volume coverage dose of radioactivity delivered by the composition of the present invention is sufficient to obtain a therapeutic effect in the treated cancer patient. The therapeutic effect against cancer is characterized by a complete remission or stable disease. As used herein, the term "complete remission" refers to the disappearance of the tumor and the absence of clinical signs in the treated cancer patient. As used herein, the term stable disease refers to a stable reduction in tumor mass over time with limited or no clinical signs in the treated cancer patient.

[0069] The entire tumor is treated by a combined antitumor effect of direct killing by the absorbed dose of ionizing radiation, elimination of tumor-promoting macrophages (TAM), and activation of antitumor immune mechanisms (recruitment of external macrophages, dendritic cells, and production of T-cell lymphocytes).The method invention allows for the use of a suboptimal tumor volume coverage dose of radiation, so that peripheral regions of the tumor receive a lower dose of radiation than the estimated minimum absorbed dose (60Gy-100Gy), preventing damage to healthy tissue surrounding the tumor.

[0070] In some embodiments of the invention, the cancer treatment comprises a single intratumoral administration of the composition or multiple intratumoral administrations over time. As shown in the examples of the present application, a single administration of a suboptimal tumor volume coverage dose of radioactivity delivered by the compositions of the present invention is sufficient to obtain a therapeutic effect in the treated cancer patient.

[0071] In some embodiments, intratumoral administration of the composition is used in combination with one or more other anti-cancer treatments, such as surgery, external radiation therapy, chemotherapy, and immunotherapy, where the treatments are administered simultaneously, separately, and / or sequentially.

[0072] Treatment of cancer according to the present invention includes one or more of inhibiting solid tumor growth, inhibiting solid tumor cell growth, inhibiting solid tumor metastasis, reducing the tumorigenicity of solid tumor cells, reducing the frequency of cancer stem cells or tumor initiating cells within solid tumors, and producing a broad anti-tumor effect both within the tumor mass and surrounding invasive areas.

[0073] The solid tumors treated with the radioactive microparticles of the present invention may be defined by their grade and may be low grade solid tumors, intermediate grade solid tumors or high grade solid tumors.

[0074] In some embodiments, the solid cancer is a tumor that has a macrophage component, such as, but not limited to, glioblastoma, glioma, Kaposi's sarcoma, breast cancer, ovarian cancer, lymphoma, colon cancer, gastric cancer, lung cancer, or prostate cancer.

[0075] In some embodiments, the solid cancer is a solid tumor that is unresectable or does not respond to current radiation or chemotherapy, such as, but not limited to, glioblastoma, glioma, Kaposi's sarcoma, and the like.

[0076] In some embodiments, the cancer is selected from the group including bladder cancer, brain cancer, breast cancer, colon cancer, stomach cancer, eye cancer, head and neck cancer, skin cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, oral cancer, prostate cancer, small intestine cancer, soft tissue cancer, testicular cancer, thyroid cancer and uterine cancer. Preferably, the cancer is brain cancer.

[0077] Brain cancers include primary brain tumors, metastatic brain tumors, and base of skull cancers. Brain cancers include low-grade to high-grade gliomas, such as astrocytoma, oligodendroglioma, ependydoma, and glioblastoma; meningioma; primitive neuroectodermal tumor; pituitary tumor; pineal tumor; choroid plexus tumor; teratoma; neurocytoma; dysembroplastic neuroepithelial tumor; lipoma; hemangioblastoma; hamartoma; neuroma; schwannoma; neurofibroma; metastatic brain tumors, especially brain metastatic adenocarcinoma; carcinomatous meningitis; and mixed brain tumors. Base of skull cancers include chondroma, chordoma, sarcoma, gliosarcoma, chondrosarcoma, and rhabdomyodarcoma. In some preferred embodiments, the brain cancer is glioblastoma or a metastatic brain tumor, such as a brain metastatic adenocarcinoma.

[0078] One aspect of the present invention relates to a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition as described above.

[0079] In the above embodiment, the microparticles are preferably Ho-165 / Ho-166 microparticles, in particular Ho-165 / Ho-166 polysiloxane microparticles, preferably comprising at least 20% (w / w) total holmium, more preferably at least 25% (w / w) total holmium, and / or having a mean or median diameter preferably between 0.1 μm and 1 μm, more preferably between 0.3 μm and 0.6 μm, even more preferably between 0.4 μm and 0.5 μm, and / or having a specific activity preferably of at least 1 megabecquerel (MBq) per milligram, for example between 1 and 10 MBq per milligram, in particular between 2 and 3 MBq per milligram. Preferably, the composition comprises a microparticle suspension having a density of at least 1 g / mL, for example between 1 g / mL and 1.5 g / mL, and / or a viscosity of at least 3 mPa.s, preferably between 3 mPa.s and 12 mPa.s, more preferably between 6 mPa.s and 10 mPa.s. Preferably, the composition comprises an aqueous suspension of microparticles in an injection vehicle comprising water and finally a small amount of ethanol, preferably less than 5% (v / v).

[0080] In the above embodiment, the microparticles, preferably Ho-165 / Ho-166 microparticles, in particular Ho-165 / Ho-166 polysiloxane microparticles such as those defined above, are preferably used for the treatment of brain cancer, in particular glioblastoma, as defined above.

[0081] The practice of the present invention will employ, unless otherwise indicated, conventional techniques within the skill of the art, which techniques are explained fully in the literature.

[0082] The invention will now be illustrated by the following non-limiting examples with reference to the accompanying drawings. Drawing legend [Brief description of the drawings]

[0083] [Figure 1]Kaplan-Meier survival curves for group 1 (Ho-166 / Ho-165), group 2 (Ho-165) and the control group are shown. The graph shows that the survival time for group 1 was significantly longer than the other two groups. [Diagram 2] Representative examples of images obtained after D14 (after injection of contrast agent): A. Tumor size 1.5 cm just before injection of Holmium (Ho-166 / Ho-165) microparticle suspension; B. Presence of Holmium-166 just after intratumoral injection of Holmium (Ho-166 / Ho-165) microparticle suspension at D14 in the center of a glioblastoma (GB); C. Tumor status 15 days after intratumoral injection of Holmium (Ho-166 / Ho-165) microparticle suspension. The tumor size has regressed; D. Tumor 25 days after intratumoral injection of Holmium (Ho-166 / Ho-165) microparticle suspension. The tumor size is still regressing; E. Tumor status 65 days after injection of Holmium (Ho-166 / Ho-165) microparticle suspension just before euthanasia of the animals at the end of the observation period (2 months after injection of Holmium). A small tumor volume is observable, F. Tumor status 65 days after intratumoral injection of Holmium (Ho-166 / Ho-165) microparticle suspension just prior to euthanasia of the animals at the end of the observation period. This image was obtained from a minipig in which the tumor was no longer observable 45 days after injection of Holmium (Ho-166 / Ho-165) microparticle suspension, suggesting a complete response, G. CT scan of an animal (group 1) that was injected with Holmium (Ho-166 / Ho-165) microparticle suspension outside the tumor. Tumor growth continued on the side opposite the area of ​​Holmium-166 injection (arrow), H. CT scan of a minipig from group 2 (Homium-165 microparticle suspension) taken prior to euthanasia. The tumor volume had increased, I. CT scan of a minipig from the control group taken prior to euthanasia. The tumor volume had increased.

[0084] Example 1: Synthesis of Holmium-165 (165Ho) Siloxane Particles Nanostructured Ho203 precursor (0.397 mol) was slurried with acetic acid (0.262 mol) and Si-EDTA (0.05 mol) in 1.5 L of ethanol and refluxed. It was cooled to room temperature and then transferred into a 200 mL plastic centrifuge bottle. The particles were washed in ethanol twice by centrifugation cycles at 4,100 rpm for 10 min and then resuspended in 160 mL of mQ water. The particle size was homogenized by stirring the suspension at 30° C. for 4 days. The aim was to synthesize particles with high holmium-165 content. In this study, the particle mean diameter was 470 nm. The dry matter concentration (i.e., solid phase once completely dried) was 550±50 g / L, consisting of a holmium mass content of 28±3%. This small mass increase of holmium allows the delivery of highly activated holmium-166 doses to tumor cells in a low and adequate volumetric unit treatment. The density of the final holmium-165 microparticle suspension was 1.38 g / ml, measured in triplicate at 25° C. by weighing 1 ml of the suspension.

[0085] Example 2: Activation of Holmium-166 Microparticle Suspensions A volume of 1 ml of the holmium-165 suspension for one tumor was drawn from the initial vial and loaded into a suitable thermoplastic polyetheretherketone (PEEK) capsule for the activation process. Each capsule was weighed before and after the activation process. Holmium-166 was then generated in the suspension of microparticles with a neutron activator (Advanced Accelerator Applications) linked to a 70 MeV cyclotron (IBA, C70 at the GIP Arronax facility). The activation principle consisted of the generation of a neutron flux generated by the interaction of a proton beam (70 MeV) with a metal target. The neutrons then interacted with the suspension of microparticles, and some of the stable atoms of holmium (holmium-165) were transformed into activated holmium (holmium-166) by neutron capture. In capturing the incident neutron, the nucleus also emits gamma rays to compensate for the excess energy provided by the neutron and return to its basic energy state, so in nuclear physics this neutron capture reaction is described as Holmium-165(n,γ) Holmium-166. Samples were generated to obtain specific activities upon injection of 2-3 MBq / mg of microparticles.

[0086] Example 3: Direct cytotoxic effect of cold microparticles on monocytes / macrophages To evaluate the possible effect of the cold microparticles themselves on the potential activation of the immune system, Ho-165 microparticles (prepared as described in Example 1) were tested undiluted and diluted at the following concentrations (1:10, 1:100, 1:1000) to evaluate their immunotoxic effects in whole heparinized blood of healthy volunteers. After incubation with whole blood, immunophenotyping analysis was performed by FACS. The following cell surface markers were evaluated: CD3 (T cell marker), CD4 (helper T cell marker), CD8 (cytotoxic T cell marker), CD14 (monocyte differentiation antigen), CD19 (B lymphocyte antigen), CD45 (leukocyte common antigen (LCA)) and CD69 (Very Early Activation Antigen (VEA)). Ho-165 microparticles diluted 1:10, 1:100 and 1:1000 were incubated in whole blood for 4 hours at 37°C. A reduction in monocytes was observed after incubation with Ho-165 microparticles diluted 1:10, whereas PMN and lymphocytes were unaffected. Leukocyte combinations were also examined. Both helper T cells (CD3+CD4+) and cytotoxic T cells (CD3+CD8+) as well as activated subpopulations (CD4+CD69+ and CD8+CD69+) were unaffected by treatment with Ho-165 microparticles (1:10 and 1:100). Treatment of whole blood with Ho-165 microparticles 1:10 and 1:100 had no effect on normal cells (CD3-CD19+) and activated B cells (CD3-CD19+CD69+), whereas dilution 1:10 was able to reduce the percentage of monocytes. These results indicate that the cold microparticles themselves have a direct effect on monocytes / macrophages.

[0087] Example 4: Cold particles activate the complement pathway Cold Ho-165 microparticles (prepared as described in Example 1) were tested for the evaluation of blood interactions to examine possible changes in coagulation, platelet activation and complement activation. For coagulation studies, WB was incubated with Ho-165 microparticles diluted 1:10, 1:100 and 1:1000 for 4 hours at 37°C. Ho-165 microparticles did not induce any changes in thrombin-antithrombin complex (TAT) levels quantified by ELISA. The effect of Ho-particles on platelet activation was evaluated by measuring the surface expression of two activation markers (CD62P and AnnexinV) after 30 minutes of incubation at 37°C of platelet-rich plasma obtained by centrifugation of WB and Ho-165 microparticles (1:10, 1:100 and 1:1000). No changes in platelet activation were recorded after incubation with Ho-165 microparticles. The effect of complement system activation was evaluated after 1 hour incubation of human serum obtained from WB and Ho-165 microparticles (1:10, 1:100 and 1:1000) at 37° C. Ho microparticles at the highest concentration tested (1:10) induced activation of the complement pathway.

[0088] These in vitro data on monocytes suggest that cold Ho-165 microparticles have an antitumor effect through suppression of TAMs and / or activation of antitumor macrophages.

[0089] Example 5: Particulate retention at injection site To evaluate the persistence of holmium particles in situ and to perform a preliminary toxicity evaluation of holmium particles in rats, the tolerability of holmium particles (formulated as described in Example 1) was investigated in Sprague-Dawley rats after a 4-week and 12-week observation period after a single intramuscular administration of 55 mg of microparticles. Daily clinical signs, body weights, blood cell counts and clinical chemistry examinations, gross observations, and organ weighing were performed in all animals. Histopathological evaluation of the injection site was also performed. No deaths occurred and no signs of systemic toxicity were observed in animals sacrificed after the 4-week or 12-week non-treatment period. However, there were clear signs of a granulomatous reaction associated with fibrosis, indicating that the injected particles remained at the injection site. Based on the results obtained in this study, it can be concluded that holmium particles did not induce any systemic toxicity at the doses tested, but were poorly tolerated locally.

[0090] The aim of the present invention is to use activated / cold microparticles for the treatment of solid tumors such as glioblastoma. It is known from the literature that radiation can affect the inflammatory response. In particular, low dose irradiation induced mainly anti-inflammatory activation of macrophages, whereas high dose irradiation was more prone to enhance the pro-inflammatory properties of macrophages. The combined effect of cold radioactive particles can be used in the treatment of tumors with a robust macrophage microenvironment and was investigated in an in vivo model of glioblastoma in pigs.

[0091] Example 6: Therapeutic efficacy of Ho-166 / Ho-165 microparticle suspension against induced glioblastoma in a miniature pig tumor model Glioblastoma (GB) invasion is a major problem that makes complete treatment of this tumor impossible with current therapeutic methods (Urbanska et al., Termedia Publishing, 2014, 18, 307-12) (Houben et al., Ned.Tij dschr Geneeskd.2005, 149, 2268-72). In this study, the therapeutic efficacy of a holmium-166 (Ho-166 / Ho-165) microparticle suspension was evaluated after its intratumoral injection in a GB minipig model. This radioactive source was chosen due to its reasonable half-life (26.8 hours) and its ability to emit both low-energy gamma rays (used for imaging) and high-energy beta particles (suitable for tumor treatment). Beta particles have limited penetration in tissues, thereby minimizing irradiation of healthy tissue (Schwartzbaum et al., Nat. Clin. Pract. Neurol., 2006, 2, 494-503).

[0092] Two weeks after (D14) implantation of tumor cells (U87 glioblastoma cell line) using a stereotactic frame connected to a preclinical injection system, the Ho-166 / Ho-165 microparticle suspension was administered intratumorally in a group of minipigs. The results were compared with two other groups of minipigs, one of which was treated with a non-activated microparticle suspension (holmium-165) and one of which was not given any treatment (control group) under the same operating procedure. The criteria for treatment efficacy were regression (inhibition) of tumor growth and overall survival after intratumoral injection of holmium-166 microparticles.

[0093] Materials and Methods U87 cell culture U87 cells (a human primary glioblastoma (GB) cell line officially known as U-87MG (Uppsala 87 malignant glioma)) were chosen because U87 is one of the most radioresistant (Hovinga et al., J. Neurooncol. 2005, 74, 99-103; Naidu et al., J. Radiat. Res. 2010, 51, 393-404). They were prepared as previously reported to induce GB in Landrace pigs (Selek et al., J. Neurosci. Methods. 2014, 221, 159-165) and Yucatan minipigs (Khoshnevis et al., J. Neurosc.i Methods. Elsevier, 2017, 282, 61-68). Briefly, 35–50 × 10 cells were cultured from two T175 tissue culture flasks after 1 week in growth medium (minimal essential medium (MEM) supplemented with 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, 2 mM l -glutamine, and 0.25% g / ml amphotericin B) with three medium changes. 6 U87 cells were harvested. Cells were harvested by treatment with 0.25% trypsin-EDTA, then washed twice in PBS and pelleted at 3000 g for 4 min in 2 ml Eppendorf vials immediately before inoculation. The final cell concentration in each Eppendorf vial was approximately 1.7 x 10 6 For each subject, 40 μl of cells were injected into the right hemisphere.

[0094] animal Experiments were performed using 12 Yucatan minipigs as a large animal glioblastoma (GB) model. The minipigs were 3-4 months old and of both sexes. The minipigs were housed under conventional conditions (temperature = 19 °C, humidity > 35%, ventilation 10 times / h) with free access to tap water and were fed pelleted food twice daily. A 2-week isolation period served as an acclimation period before transplantation of U87 cells.

[0095] Preparation of Holmium-165 Microparticles A suspension composed of Holmium-165 (stable isotope) microparticles was synthesized according to the experimental procedure described in Example 1.

[0096] Activation of Holmium-166 Microparticle Suspensions Holmium-166 was then generated in the suspension of the microparticles by a neutron activator according to the experimental procedure described in Example 2.

[0097] Stereotaxic Frame A large animal stereotaxic instrument (Model 68901) from RWD Life Science was selected for intracerebral implantation of U87 cells followed by intratumoral injection of radioactive holmium. Prior to any injection, the tumor location is obtained by preoperative CT scan.

[0098] Treatment Plan Treatment plans (TP) covered different percentages (%) (35%, 45%, 50%, 60%, 80% and 97%) of the tumor volume (TV) with at least 100 Gy (considered the tumor-killing dose). In all injected minipigs, TP was performed based on a preoperative CT scan obtained on Day 14 (D14) after implantation, 30 min before the injection of the holmium suspension. Performing a pre-CT scan as late as possible meant that planning was performed with the most up-to-date information on the size, shape and location of the tumor. To obtain the CT, product contrast was used to easily distinguish the tumor from healthy tissue. Tumor segmentation was then performed by open source software (3D Slicer). Once segmentation was completed, TPS was performed with an optimization algorithm (non-dominated selection genetic algorithm II) (Srinivas et al., Evol. Comput. MIT Press; 1994, 2, 221-248). The volume of each injection unit of treatment (UoT) varied between 5 and 8 μl depending on the tumor size and as a result of calculations to deliver the targeted absorbed dose and volume coverage while reducing the number of injections and decreasing the operation time (Table 1).

[0099] immunosuppressive treatment The animals were immunosuppressed to prevent rejection of the transplanted cells over the course of the experiment. This works by weakening the immune system so that the transplanted cells are accepted as if they were the animal's own. Cyclosporine solution (Neoral® 100 mg / ml) was administered orally twice daily (25 mg / kg). Cyclosporine treatment was continued daily until the last day of the experiment.

[0100] Tumor cell implantation and intratumoral injection of holmium microparticles At D0, 12 Yucatan minipigs were anesthetized and tumor cells were injected intracerebrally in the striatal region in the right hemisphere. 14 days after tumor cell implantation, the minipigs were divided into two groups: group 1 received an injection of Ho-166 / Ho-165 suspension, and group 2 received an injection of Ho-165 suspension (non-radioactive). In the control group, no injection was performed after tumor cell implantation. For the three different groups, there were 6 minipigs in group 1, 3 in group 2, and 3 in the control group. All suspension preparations and intratumoral injection procedures were identical between radioactive and non-radioactive holmium injections, and the radioactive injections took into account the necessary radiation protection protocols. One pig in group 1 (positive failure pig) was excluded from group 1 after a needle positioning error that led to the injection of the radioactive suspension outside the tumor. A needle was inserted into the brain according to the tumor location as determined by the preoperative CT image obtained immediately before holmium injection. Using the coordinates given by the TP, several treatment units were injected inside the tumor at different sites. The number of injections varied between tumors and the percentage of tumors requiring treatment with a dose of more than 100 Gy.

[0101] CT Scan Imaging All minipigs were scanned periodically by CT scans to study the distribution of holmium inside the tumor and to evaluate the development of tumor size over time. CT scanner (GE BrightSpeed ​​16) to obtain all CTs. After cell implantation, tumor growth was evaluated in all minipigs by imaging on Day 7, Day 10 and Day 14. In 9 treated minipigs (excluding 3 in the control group), pre-operative and post-operative CT imaging was performed on the day of holmium suspension injection (D14) to evaluate the tumor positioning and to confirm the distribution and location of the injected holmium, respectively. After treatment, the first post-injection CT scan was performed 5 days after the intervention and then every 10 days thereafter to study the changes in the injected holmium suspension and the therapeutic effect of this radiotherapy. The follow-up CT obtained for the control group minipigs was the same as that for Groups 1 and 2. In all minipigs, manual segmentation of the tumor was performed after each post-operative CT. In this way it was possible to study changes in tumor size over time.

[0102] Follow-up survey A standard follow-up period of 2 months after treatment was used, during which all animals were monitored daily for clinical side effects. Clinical parameters observed included food intake, body temperature, weight, alertness, level of consciousness, posture, and gait.

[0103] Histology and immunochemistry Histological and immunohistochemical analyses were performed. Samples were collected from brain tumors, heart, liver, lungs, and kidneys, fixed in 4% formalin, routinely processed, and embedded in paraffin. For each sample, one 4 μm section was obtained and stained with hematoxylin-eosin (HE). For each brain sample, seven sections were cut and used for immunohistochemistry. Immunohistochemical staining was performed by the avidin-biotin-peroxidase complex method. Lymphocyte infiltration was characterized using anti-CD3 epsilon (diluted 1:100, Bio Rad, Hercules, CA, USA) for T lymphocytes and anti-Pax-5 (clone 24 / Pax5 (undiluted), BD Transduction laboratories, Lexington, KY, USA) for B lymphocytes. To detect macrophage / dendritic cell (DC) populations, the following antibodies were applied: anti-Iba-1, a pan-marker for macrophages / DCs (ab5076, diluted 1:500, Abcam, Tokyo, Japan); anti-CD204 (MSR-1, clone SRA-E5, diluted 1:50, Abnova, Taipei, Taiwan), which labels conventional macrophages / monocytes but not DCs; anti-DC-lamp (CD208, clone 1010E1,01, diluted 1:200, Dendritics, Lyon, France), which labels conventional antigen-presenting DCs but not macrophages; and anti-CD303 (BRDA-2, clone 124B3, diluted 1:200, Dendritics, Lyon, France), which labels plasmacytoid DCs. For all applied antibodies, except for anti-DC-lamp, antigen retrieval was performed by heating in citrate tampon (pH 6) at 90°C for 40 min followed by cooling. Antigen retrieval for anti-DC-lamp was performed in pH 9 solution (Dako target retrieval solution (pH 9), Dako, Carpinteria, CA) at 90°C for 40 min. Labeling was amplified with the final step product of the ultraTek HRP (anti-polyvalent) ready-to-use kit (ScyTek) (30 min at 20°C) and revealed with the Vector NovaRED Peroxidase (HRP) Substrate kit (5 min).Hematoxylin counterstain (10 seconds) was applied and then sections were dehydrated and mounted.

[0104] For each antibody, a negative control was used, characterized by replacing the first antibody with a PBS solution, and a positive control for each applied antibody, represented by a 4 μm section of a spleen obtained from a pig of the control group.

[0105] Scoring and pattern identification in light microscopy Histological analysis of the brain was used to confirm the findings of the CT images and to observe the tumor size and necrosis after treatment. For autopsy, histological analysis was performed during which tumor-bearing sections of the brain, kidney, heart, lung and liver were promptly collected and fixed in 4% formalin. Small sections of the tumor and other organs were processed and embedded in paraffin. One 4 μm section was stained with hematoxylin-eosin and examined histologically. In the HE-stained sections of the brain, the following parameters were scored: presence / absence of tumor, presence / absence of Ho crystals, tumor necrosis, lymphocytic inflammation and granulomatous inflammation. For tumor necrosis, the following semiquantitative scores were applied: (-)=absence, (+)=1-30% of the tumor area, (++)=31-60% of the tumor area, (+++)=61-100% of the tumor area. Tumor lymphocytic and granulomatous inflammation was assessed semiquantitatively, ie, (-)=absent, (+)=mild, (++)=moderate, (+++)=severe.

[0106] Lymphocytic inflammation was then characterized using anti-CD3-epsilon to label T lymphocytes and anti-Pax-5 for B lymphocytes. The presence of plasmacytoid DCs was recorded based on anti-CD303 labeling within the tumor. Macrophage / DC infiltration was evaluated based on positive labeling for anti-Iba-1, anti-CD204 and anti-DC-lamp in different locations, i.e., around (peritumoral) and inside (intramyocellular), around coagulation necrosis and around Ho-associated necrosis, and within lymphocytic infiltrates. To semiquantitatively detect the amount of positive cells in each location for each antibody, the following scores were applied: (-)=absence of labeled cells, (+)=mild amount of labeled cells, (++)=moderate amount of labeled cells, (+++)=heavy amount of labeled cells. All histological and immunohistochemical parameters were scored blindly.

[0107] statistical analysis Survival data were analyzed using the log-rank test (Mantel-Cox). Statistical analysis was performed using this non-parametric test, which is widely used in preclinical and clinical trials to establish the efficacy of new treatments compared to control treatments. A value of p<0.01 was considered to indicate statistical significance.

[0108] result Intratumoral injection of holmium and survival rate Intratumoral injections of Ho-166 / Ho-165 microparticle suspension and Ho-165 microparticle suspension were performed in groups 1 and 2, respectively. In the control group, no treatment or injection was performed after tumor implantation. The number of injections per tumor depended on the size and shape of the tumor and the percentage of tumors treated with more than 100 Gy (Table 1).

[0109] [Table 2]

[0110] To calculate the survival time of the animals in each group, the experimental minipigs were kept alive for 2 months after the treatment date. The Kaplan-Meier survival curve (Figure 1) was the longest in group 1. For group 2 and the control group, the survival curves were almost the same.

[0111] The survival time of all animals and the median survival time of each group were calculated from the date of holmium injection on post-implantation day 14 (D14). The survival time of group 1 was 63 days (Table 2), which was statistically higher (p<0.01) than the survival time of group 2 (8.6 days) and the control group (7.3 days), which were not statistically significant with each other (p=0.09).

[0112] [Table 3]

[0113] In group 2 and the control group, all minipigs showed severe neurological signs 6-9 days after D14, which resulted in their euthanasia. For the "positive failure pigs", non-treatable neurological symptoms were observed, which led to the animals being sacrificed 15 days after treatment.

[0114] In group 1, all pigs (5 / 5) survived until the end of the observation period (2 months after D14). At the time they were euthanized, they were healthy in good general condition without any clinical or clinically relevant signs of disease.

[0115] CT imaging During the tumor growth period, several CT scans were obtained before and after injection of the holmium suspension in groups 1 and 2. At D14, all implanted tumors had a well-defined border and a multilobed morphology, as well as an increased density compared to the surrounding brain parenchyma. Postoperative CT scans in groups 1 and 2 showed substantial accumulation of particulates at the injection site, indicating successful intratumoral injection of the holmium suspension in all but one minipig in group 1. In this animal, a significant amount of the Ho-166 / Ho-165 suspension was injected outside the outer part of the tumor.

[0116] One of the most important points in microbrachytherapy is the retention of the injected suspension within the injection area over time to avoid its leakage outside the tumor. As can be seen in Figure 2, the Ho-166 / Ho-165 suspension was well circumscribed inside the tumor and the following scan showed good retention of the holmium particles at the injection site up to 2 months after injection. This is a very important point for the prevention of damage to normal brain tissue after microbrachytherapy.

[0117] A positive response to treatment was obtained in all treated animals of group 1 (Table 3). This means that the Ho-166 / Ho-165 microparticles provided good tumor control, with no evidence of tumor remaining in CT scans of three of the animals from 45 days after injection (Figure 2). Two other animals showed a significant reduction in tumor size at the end of the two-month post-treatment observation period. The five minipigs treated with Ho-166 / Ho-165 microparticles were in good general and nutritional condition until the day of termination. This confirms the absence of local and systemic toxic effects after intratumoral administration of the holmium-166 suspension. In contrast, tumor growth continued in group 2 and the control group, leading to the sacrifice of the animals.

[0118] [Table 4]

[0119] Dosimetry The main observation from the dosimetry results (Table 4) is that independent of tumor coverage with adequate absorbed dose, the efficacy of treatment was the same for a range of volumetric coverages, including between 43 and 99.3% for a minimum absorbed dose of 60 Gy. This surprising observation encompasses an additive therapeutic effect compared to that resulting from the cytotoxic effect delivered by Ho-166 ionizing radiation. This effect therefore results from a combination of microparticles present in the tumor that contribute to additive immune modification, as observed in immunohistochemistry sections. The "positive failure pigs," which received higher tumor coverage absorbed doses than some of the pigs in Group 1, but failed to receive microparticles within the tumor, further demonstrates that the additive effect of enhancing the cytotoxicity of ionizing radiation requires the presence of microparticles within the tumor.

[0120] [Table 5]

[0121] Histological examination Tumors were detected in all animals in Group 2 and the control group. Tumors were clearly visible in the "positive failure pigs." In Group 1, no adequate tumors were identified in any of the five pigs due to severe necrosis and inflammation.

[0122] Holmium crystals were observed in almost all animals in groups 1 and 2 (5 / 5 group 1, 3 / 3 group 2) but none in control animals. Holmium was visible as irregular, angular, tan crystals 30 μm in size located freely within the necrotic areas of the tumor.

[0123] In group 1, necrosis was severe and replaced by tumor, which was no longer identifiable in all animals. It represented 60-100% of the lesions in all animals and was liquefied necrosis, which was associated with granulomatous inflammation. The severity and type of necrosis indicated a holmium-166 effect (Table 5). In contrast, necrosis was absent or mild (<30% of lesions) in controls or mild to moderate (30-60% of lesions) in group 2.

[0124] Lymphocytic inflammation was slightly more severe in group 1 when compared with group 2 and the control group (Table 5).

[0125] Granulomatous inflammation was evaluated in HE slides as the presence of recognizable macrophages, epithelioid cells, and multinucleated giant cells. In group 1, moderate to severe granulomatous reaction was observed (Table 5). It was located around the necrotic area, suggesting that it was an immune response to antigens released by holmium-induced necrosis. Since mild granulomatous reaction was also observed in group 2, it was possible that holmium crystals themselves were involved in inducing this type of inflammation as a foreign body reaction, independent of radioactivity.

[0126] In contrast, inflammatory infiltrates were absent in all animals in the control group and mild in all animals in group 2 (Table 5).

[0127] [Table 6]

[0128] immunohistochemistry In this study, lymphocytic infiltration, macrophage infiltration, and DC infiltration were evaluated to characterize the immune microenvironment and to better understand the mechanism of action of holmium-166. Immune surveillance encompasses natural killer T lymphocytes and cytotoxic T lymphocytes (CTLs), both of which are responsible for cancer cell death by cytotoxicity. Both of them are boosted by interferon I, which is secreted mainly by plasmacytoid DCs. Conventional DCs present antigens by major histocomptability (MHC) class II to induce adaptive responses that stimulate CD4 T lymphocytes, and by MHC I to stimulate CTLs (Frey B et al., Immunol Rev. 2017;280:231-248.). These cells are necessary to develop acquired specific immune responses, which are of particular interest when targeting tumor cells. Furthermore, tumor-associated macrophages (TAMs), including tumor-promoting (M1 phenotype) and anti-tumor (M2 phenotype) TAMs, are important regulators of tumorigenesis.

[0129] Lymphocytes were characterized with anti-CD3 epsilon (T lymphocytes) and anti-Pax-5 (B lymphocytes) antibodies specific for porcine lymphocytes, as confirmed by control experiments. Plasmacytoid DCs were detected by the specific marker CD303. For identification of macrophages and DCs, anti-Iba-1 was used as a pan-marker recognizing both cell types, whereas anti-CD204 and anti-DC-Lamp were used to identify macrophages and conventional DCs, respectively. In positive control spleens, anti-Iba-1 antibody labeled splenic periarteriolar macrophages, reticular cells, and T- and B-antigen-presenting DCs, showing its large spectrum in the identification of macrophages / DCs in pigs. The anti-CD204 antibody used was shown to be specific for porcine macrophages, and the anti-DC-lamp used was specific for porcine DCs but not for macrophages.

[0130] Lymphocytic infiltrates, composed mainly of T lymphocytes, were present in all animals, but did not differ significantly between groups. Because antibodies directed against paraffin-embedded porcine tissue were not available, it was not possible to identify CTL or NK cells. However, the consistency of the presence of lymphocytes between groups, independent of holmium treatment, indicates that they are not holmium-166 treatment related.

[0131] Plasmacytoid DCs were present in all brain lesions but did not differ between groups, suggesting that their presence is tumor-associated but not related to the holmium effect. Their insufficient numbers indicate that their role in the immune system tumor microenvironment may be less prominent.

[0132] Based on the lack of differences in lymphocytic infiltrates and the presence of plasmacytoid DCs between the different groups, it was speculated that the observed necrosis in holmium-166-treated animals was not dependent on cytotoxic lymphocytic activity, despite their presence.

[0133] Within the peritumoral locations and necrotic periphery, Iba-1+ macrophages / DCs were more abundant in Groups 1 and 2 than in the control group, indicating a holmium effect. Within the tumors, large amounts of Iba-1+ macrophages / DCs were detected in all animals in all groups, indicating that immune infiltration is related to the presence of tumors and not to a holmium effect. Within lymphoid aggregates, no differences in the amount of Iba-1+ cells were observed between groups (Table 6).

[0134] In brain samples, CD204+ macrophages were more abundant in peritumoral locations in Groups 1 and 2 than in the control group, indicating that their presence was associated with holmium treatment. Intratumoral and peri-necrotic locations, their presence was comparable in all groups, indicating that CD204+ macrophage infiltration in these locations was related to the presence of tumor and not to the holmium effect (Table 6).

[0135] In brain samples, DC-lamp+DCs were scarce in the control group, sparsely present in group 2, and abundant in group 1, indicating that holmium-166 was associated with severe DC-lamp+DC infiltration (Table 6).

[0136] In conclusion, immunohistochemical analysis showed that in peritumoral locations, moderate to severe macrophage / DC infiltration was observed in groups 1 and 2, but was minimal in the control group. This infiltration consisted of macrophages and DCs, which were more abundant in group 1 than in group 2.

[0137] Intratumoral infiltration was severe in all groups and consisted of macrophages, but also DCs, especially in group 1. Necrosis was also associated with moderate to severe macrophage / DC infiltration, consisting of macrophages in all groups and also DCs in group 1. Moderate amounts of macrophages / DCs were detected within lymphoid aggregates in all groups. They were mainly composed of DCs, but macrophages were also present.

[0138] Thus, holmium-166 was associated with highly infiltrating conventional DCs around and within the tumor, as well as within the necrotic periphery and lymphocytic infiltrates.

[0139] DCs were scarce in Group 2 and control animals, but abundant around and within the tumors and around holmium-associated necrosis. These cells are necessary to mount acquired specific immune responses that are of particular interest when targeting tumor cells.

[0140] In addition to DCs, increased numbers of macrophages were found in peritumoral locations in holmium-treated animals, which, since it was observed in holmium-166- and holmium-165-treated animals, was interpreted as a response to holmium particles rather than a response to radioactivity alone.

[0141] [Table 7]

[0142] conclusion This study demonstrated excellent tumor control efficiency and absence of toxic effects of Ho-166 / Ho-165 microbrachytherapy in a human glioblastoma model in minipigs using one of the most radioresistant cell lines (U87).In light of these encouraging results, intratumoral treatment with Ho-166 / Ho-165 microparticles could be considered as a promising therapeutic approach for the treatment of glioblastoma and other solid tumors.

[0143] The observed antitumor effect cannot be explained by a single effect of ionizing radiation, since the percentage of tumor volume receiving >60 Gy or >100 Gy (considered to be the filling dose in the literature) does not seem to correlate with response (tumor volume reduction). These results show that besides the direct effect of ionizing radiation, microparticles delivered inside the tumor induced antitumor immune mechanisms by recruiting external macrophages and dendritic cells, but activating the production of T-cell lymphocytes. This last response is prolonged by the persistence of the microparticles (as stable implants) in the tumor tissue. This may lead to the localized antitumor effect induced by the combination of radiotherapy and direct TAM toxicity resulting in a long-range effect extending to the entire tumor mass and the surrounding infiltrated areas.

[0144] Other advantages of this novel Ho-166 / Ho-165 microbrachytherapy compared to other treatment modalities are its single-session approach, its minimal invasiveness, and the lack of severe secondary effects that often occur with chemotherapy and other radiotherapy methods. In particular, the flexibility allowed in terms of dosimetry is totally opposite to the requirements present in external beam radiotherapy (EBRT), which here represents an innovative new treatment modality paving the way for better treatment efficacy.

Claims

1. 1. A composition comprising microparticles comprising a radioisotope suitable for use in internal radiotherapy and an injection vehicle for use in the treatment of cancer by the combined antitumor effect of radioactivity and intratumoral microparticle deposition, The composition is administered by intratumoral injection, advantageously at a suboptimal tumor volume coverage dose of radioactivity.

2. 2. The composition for use according to claim 1, wherein an absorbed dose of 60 Gray or greater is administered in less than 95%, less than 85%, less than 75%, less than 65%, less than 55% or less than 35% of the tumor volume, preferably in 35% to 55% of the tumor volume.

3. 3. The composition for use according to claim 1 or 2, wherein an absorbed dose of 100 Gray or more is administered in less than 95%, less than 85%, less than 75%, less than 65%, less than 55% or less than 35% of the tumor volume, preferably in 35% to 55% of the tumor volume.

4. A composition for use according to any one of claims 1 to 3, wherein intratumoral microparticle deposition induces removal of tumor-promoting macrophages from the treated tumor.

5. 5. A composition for use according to any one of claims 1 to 4, wherein intratumoral microparticle deposition induces an antitumor immune response.

6. 6. The composition for use according to any one of claims 1 to 5, wherein the radioisotope is selected from the group comprising holmium-166, lutetium-177, rhenium-186 or rhenium-188, gold-198, yttrium-90, thorium-227, actinium-225 and combinations thereof, advantageously holmium-166.

7. 7. The composition for use according to any one of claims 1 to 6, wherein the microparticles optionally further comprise a non-radioactive isotope derived from the same chemical element.

8. 8. The composition for use according to claim 7, wherein the microparticles comprise holmium-165 and holmium-166, preferably at least 20% (w / w) total holmium, more preferably at least 25% (w / w) total holmium.

9. 9. The composition for use according to any one of claims 1 to 8, wherein the microparticles comprise or consist of polysiloxane, acetotriacetonate (AcAc), poly-L-lactic acid (PLLA), alginate, chitosan, resin, glass and combinations thereof, advantageously polysiloxane.

10. 10. The composition for use according to any one of claims 1 to 9, wherein the microparticles have a mean or median diameter of from 0.1 μm to 10 μm, preferably from 0.1 μm to 1 μm, more preferably from 0.3 μm to 0.6 μm, even more preferably from 0.4 μm to 0.5 μm.

11. The composition for use according to any one of claims 1 to 10, wherein the microparticles have a specific activity of at least 1 megabecquerel (MBq) per milligram, preferably between 1 and 10 MBq per milligram, more preferably between 2 and 3 MBq per milligram.

12. The composition for use according to any one of claims 1 to 11, wherein the composition comprises a particulate suspension having a density of at least 1 g / mL, preferably between 1 g / mL and 1.5 g / mL.

13. 13. A composition for use according to any one of claims 1 to 12, wherein the composition comprises a particulate suspension having a viscosity of at least 3 mPa.s, preferably between 3 mPa.s and 12 mPa.s, more preferably between 6 mPa.s and 10 mPa.s.

14. 14. A composition for use according to any one of claims 1 to 13, wherein the composition comprises an aqueous suspension of radioactive microparticles in an injection vehicle comprising water and finally a small amount of ethanol, preferably less than 5% (v / v).

15. 15. The composition for use according to any one of claims 1 to 14, wherein the cancer is selected from the group comprising bladder cancer, brain cancer, breast cancer, colon cancer, stomach cancer, eye cancer, head and neck cancer, skin cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, oral cancer, prostate cancer, small intestine cancer, soft tissue cancer, testicular cancer, thyroid cancer and uterine cancer, preferably brain cancer, in particular glioblastoma or metastatic brain tumours, such as brain metastatic adenocarcinoma.

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