EA040587B1 - METHOD FOR MALIGNANT NEOPLASMS TREATMENT - Google Patents

Description

Field of invention

The present invention relates to the field of human health, and more specifically, relates to nanoparticles for use as a therapeutic vaccine, alone or included in the composition, in the context of radiation therapy administered to an individual suffering from a malignant neoplasm, in particular a metastatic malignant neoplasm or hemoblastosis. Nanoparticles, the use of which is presented in the present description, consist of a material having a density of at least 7 g/cm ³ and an atomic number (Z) of at least 25. Each nanoparticle is coated with a biocompatible coating, allowing them to be stable at pH from 6.5 up to 7.5 in physiological fluid. When irradiated, these nanoparticles are able to enhance the destruction of malignant cells due to the optimized stimulation of the immune system of the individual itself, compared with the destruction of malignant cells achieved by radiation therapy alone, in particular in an individual suffering from metastatic malignant neoplasm, preferably an individual suffering from metastatic malignant neoplasm and undergoing palliative radiotherapy, an individual suffering from metastatic cancer in which (radical) radiation therapy was rejected, an individual suffering from a malignant neoplasm that is not (conventionally) treated with radiation therapy, and an individual suffering from hemoblastosis. With the present invention, the doses of radiation to which an individual is subjected in the context of radiotherapy, preferably in the context of fractionated radiotherapy, are significantly more effective in destroying malignant cells without increasing the adverse effect on surrounding healthy tissue. In addition, the present description presents new compositions and kits, as well as their use.

State of the art

Malignant neoplasms are the leading cause of death worldwide, accounting for 8.2 million deaths in 2012. It is expected that the annual number of cases of malignant neoplasms will increase from 14 million in 2012 to 22 over the next two decades (according to WHO). A malignant neoplasm can develop locally and / or spread systemically by lymphogenous or hematogenous routes.

Surgery, radiation therapy and pharmaceuticals, in particular chemotherapy drugs, are of primary importance in the treatment of malignant neoplasms, and each of these methods can be used alone or in combination to affect all sites where the disease can develop, depending on the type of cancer being treated. A malignant neoplasm to be treated must be controlled at the local and systemic levels.

Despite recent advances in the identification and treatment of some cancers, metastasis remains common, accounting for approximately 80-90% of cancer deaths. The standard treatment for metastatic disease in most cancer patients is systemic cytotoxic chemotherapy and hormone deprivation (Dhara M. MacDermed et al., Journal of Surgical Oncology 2008). There is an urgent need to develop new methods of radical therapy for the treatment of patients with metastatic malignancy.

Some specific tumors/malignant neoplasms have the property of spreading to certain organs. The most common sites of metastasis are the lungs (eg, if the primary malignancy is a sarcoma), liver (eg, if the primary malignancy is gastrointestinal malignancy), bones (eg, if the primary malignancy is breast or prostate cancer) ) and the brain (for example, if the primary malignancy is lung cancer, kidney cancer, or melanoma).

Radiation therapy is the second most used cancer treatment, preceded by pharmaceuticals, with approximately half of all newly diagnosed cancer patients receiving radiation therapy at some point during their treatment of their disease. Radiation therapy uses high energy particles or waves such as x-rays, gamma rays, electron beams, or protons to destroy or damage malignant cells.

The therapeutic use of local ionizing radiation is largely guided by a strategy aimed at achieving effective removal of malignant cells. Radiation therapy as the only therapeutic intervention can provide the possibility of functional preservation of the organ, for example, in relation to cancer of the bladder and larynx. As adjuvant therapy, radiation therapy may facilitate resection when used before surgery, or promote the treatment of microscopic residual disease when used after surgery, such as breast-sparing treatment after lumpectomy. A new development is the use of stereotaxic

- 1 040587 radiotherapy (SBRT) for ablation of sites of oligometastatic disease. SBRT enables largely focal cancer treatment using a single fraction or a small amount of high energy radiation fractions (typically 5 to 25 Gy). The existence of an oligometastatic state is based on an intermediate state between well-localized lesions and extensive metastatic lesions. Typically, 1 to 5, such as 1, 2, 3, 4, or 5, metastases or sites of metastasis are considered in the case of SBRT (Dhara M. MacDermed et al., Journal of Surgical Oncology 2008; Ralph R. Weichselbaum Nat. Rev. Clin Oncol 2011).

However, metastatic malignancies with multiple distant metastases (typically more than 5, preferably more than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 metastases) are associated with poor prognosis (Ralph R. Weichselbaum Nat. Rev. Clin. Oncol. 2011).

Generally, oncologists do not consider patients with advanced systemic diseases (patients who have multiple metastases also identified as polymetastatic patients) as being treatable with existing regional therapies such as radiation therapy. On the other hand, palliative radiotherapy is used for temporary relief of symptoms and is different from radiotherapy given as curative treatment (Sonam Sharma et al., Seminars in Oncology 2014). Palliative radiotherapy is an effective treatment for many of the symptoms of locally advanced or metastatic tumors, even in patients with a short life expectancy (Sonam Sharma et al., Seminars in Oncology 2014). In this case, radiation therapy may be an effective treatment for pain, neurological symptoms, relief of obstructive symptoms (such as obstruction of the bile ducts or urinary tract), and bleeding or ulceration. Palliative radiotherapy can also be used to maintain local tumor control in an area that is likely to be damaged and become symptomatic. Typical dose fractionation schemes for palliative radiotherapy are as follows:

1x8 Gy, 5-6x4 Gy, 10x3 Gy in case of uncomplicated bone metastases, for 1 day to 2 weeks;

5x4 Gy, 10x3 Gy, 15x2.5 Gy in case of whole brain radiotherapy, for 1 to 3 weeks;

2x7.5-8.5 Gy, 10x3 Gy, 15x2.5 Gy in case of advanced lung cancer causing airway obstruction, superior vena cava syndrome, for 1 to 3 weeks;

1x8-10 Gy, 5x4 Gy, 10x3 Gy, 15-30x2-3 Gy in case of visceral metastases causing pain, symptoms of obstruction, bleeding, within 1 day to 6 weeks;

1-5x6-24 Gy in the case of stereotactic radiotherapy, usually given to patients with good general condition, long expected prognosis and/or low metastasis, for 1 to 5 days (Sonam Sharma et al., Seminars in Oncology 2014, table 3).

In addition, the available evidence suggests that local irradiation at clinically therapeutic doses induces some activation of innate and adaptive immunity. Irradiation has been shown to induce immunogenic cell death (ICD), potentially converting a tumor into an in situ vaccine, specifically characterized by three molecular signals that facilitate the capture of dying cells by dendritic cells, the cross-presentation of tumor-derived antigens to T cells, and the activation of antitumor T cells, including activation of cytotoxic CD8+ T cells: exposure of calreticulin (CRT) on the surface of tumor cells, release of high mobility group B1 protein (HMGB1), and release of ATP (Oliver Kepp et al., Oncoimmunology 2014).

It is also known that tumor response to irradiation involves DNA damage and that recognition of tumor-derived DNA can trigger IFN production and induce antitumor T-cell responses to immunogenic tumors (see, e.g., Theresa L. Whiteside et al., 2016).

Radiation is a complex modifier of the tumor microenvironment, and is rarely enough by itself to induce a therapeutically significant antitumor immune response, since it can also activate the immune suppressor pathway, causing harm. The proportion of tumor cells undergoing ICD and remodeling the tumor microenvironment after irradiation is variable. The state of this balance ultimately determines the ability of irradiation to convert dying malignant cells into an effective vaccine in situ (Sandra Demaria and Silvia C. Formenti, 2012).

Combinations of radiation and immunotherapy have been shown to stimulate an immune response. Cytokines [interleukin-2 (IL-2) and interferon-alpha (IFNa)] have been used as a therapeutic approach for decades. Numerous strategies for overcoming tumor immune evasion are currently under development. Examples of immunotherapeutic approaches under clinical evaluation include (1) T-cell checkpoint inhibitors or

- 2 040587 T cell activation pathway agonists, (2) new cytokines such as IL-12 and IL-15, (3) a therapeutic vaccine, (4) removal of immunosuppressive cells, and (5) other tools and approaches designed to enhance functioning of immune cells (Scott J. Antonia et al., 2014; Theresa L. Whiteside et al., 2016).

However, obtaining effective therapeutic vaccines remains difficult. To be effective, cancer vaccines must achieve two goals. First, they must stimulate specific immune responses against the correct target cells (ie malignant cells). Second, immune responses must be strong enough to overcome the barriers used by these cells to protect themselves from attack by the host's immune system, typically B cells and natural killer T cells.

The essence of the invention

Further in the present description, the authors of the present invention describe the preferred use of a nanoparticle and/or an aggregate of nanoparticles as a therapeutic vaccine, alone or included in a composition (generally being a therapeutic composition, in particular a vaccine composition), in relation to an individual suffering from a malignant neoplasm and undergoing radiation therapy. A nanoparticle and/or an aggregate of nanoparticles, as well as a composition containing a nanoparticle and/or an aggregate of nanoparticles, as a rule, are intended for use in the treatment of malignant neoplasms. The combination of such nanoparticles with radiotherapy significantly enhances the antitumor immune response of the host and optimizes the overall treatment, in particular in the context of fractionated radiotherapy, including at least one stage of radiation, where the dose of ionizing radiation is in the range from 1.8 to 30 Gray (Gy ), preferably from 1.8 to 20 Gray (Gy), typically from 2 to 15 Gray (Gy). The present invention is particularly effective against metastatic malignant neoplasm, where the malignant neoplasm has developed into an advanced systemic disease (as defined herein above), as a result of the abscopal effect made possible by this technology, and against hemoblastosis, and promotes complete and permanent antitumor activity.

The abscopal effect is a phenomenon seen in the treatment of metastatic cancer where localized irradiation of a specific area of the tumor elicits a response at a site distant from the irradiated volume. Radiation therapy can help reverse tolerance to weakly immunogenic tumor-associated antigens to elicit an antitumor immune response. However, the abscopal effect remains a rare clinical event when radiotherapy alone is used. Kobe Reynders et al. report 23 clinical cases of abscopal effects after radiotherapy between 1973 and 2013. (Kobe Reynders et al., Cancer Treatment Review 2015).

With the present invention, local irradiation is able to induce a sustained antitumor response compared to radiation therapy alone, thus enhancing local and systemic tumor control.

The examples first presented in the present description by the present inventors demonstrate the in vitro enhancement of damage-associated molecular structures (DAMPs) induced by dying cancer cells [in the present description, cancer cell lines including radiosensitive cell lines (such as a colorectal cancer cell line) were tested. human HCT 116) and radioresistant cell lines (such as the human glioblastoma cell line 42 MG BA or the human pancreatic cancer cell line PANC-1)], using the nanoparticle and/or nanoparticle aggregate as a therapeutic vaccine.

Of greatest interest is that vaccination of immunocompetent mice with CT-26 mouse colorectal cancer cells treated with nanoparticles and/or aggregates of nanoparticles of the present invention and irradiated in vitro significantly prevents tumor formation when healthy/live animals are injected 7 days later. malignant CT-26 cells compared to radiotherapy alone. These results surprisingly indicate that the combination of the nanoparticles of the invention and radiation therapy enhances the immune response and converts dying cancer cells into an effective cancer vaccine.

In a preferred aspect, the products of the invention (a nanoparticle and/or aggregate of nanoparticles and compositions comprising such a nanoparticle and/or aggregate of nanoparticles) provided herein are preferably for use in an individual selected from an individual suffering from metastatic cancer undergoing palliative radiotherapy. therapy, an individual suffering from a metastatic malignant neoplasm in which (radical) radiotherapy was rejected, an individual suffering from a malignant neoplasm that is not (conventionally) treated with radiotherapy, and an individual suffering from hemoblastosis, and are preferably intended for use in relation to an individual selected from an individual suffering from a metastatic malignant neoplasm and undergoing palliative radiotherapy, an individual suffering from a metastatic malignant neoplasm, in the case of whom (radical) radiotherapy was rejected, and an individual suffering from hemoblastosis.

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An individual suffering from a metastatic malignant neoplasm is generally an individual, as defined herein, suffering from a malignant neoplasm having multiple distant metastases, typically developing into a widespread systemic disease. Individuals with multiple distant metastases are also referred to herein as polymetastatic individuals or patients.

Thus, provided herein is a nanoparticle and/or aggregate of nanoparticles for use as a therapeutic vaccine, or a nanoparticle and/or aggregate of nanoparticles for use in preparing a therapeutic composition, typically a vaccine composition, for use (typically for use in the treatment of cancer). neoplasms) in relation to an individual suffering from a malignant neoplasm, usually a metastatic malignant neoplasm [metastatic malignant neoplasm, generally defined in the present description as including many distant metastases and, as a rule, considered as a widespread systemic disease], a malignant neoplasm that are not (conventionally) treated with radiotherapy, or hemoblastosis, in the context of fractionated radiotherapy, including at least one stage of radiation, where the dose of ionizing radiation is in the range of 1.8 d about 30 Gray (Gy), preferably from 1.8 to 20 Gray (Gy), typically from 2 to 15 Gray (Gy), and where each nanoparticle consists of a material having a density of at least 7 g/cm ³ and an atomic number (Z) of at least 25, and each nanoparticle or nanoparticle aggregate is coated with a biocompatible coating allowing it to be stable at pH 6.5 to 7.5 in a physiological fluid.

The nanoparticle and/or aggregate of nanoparticles is generally intended for use in the treatment of cancer, preferably in a selected group of individuals/patients, typically individuals suffering from metastatic cancer and undergoing palliative radiotherapy, individuals suffering from metastatic cancer, if whom (radical) radiotherapy has been rejected, individuals suffering from malignancy who are not being (conventionally) treated with radiotherapy, or individuals suffering from hemoblastosis.

Also provided herein is a vaccine composition for use, typically for use in the treatment of cancer, in an individual suffering from a cancer, typically a metastatic cancer (particularly a metastatic cancer where the cancer develops into an advanced systemic disease). ), or hemoblastosis, in the context of radiotherapy, preferably in the context of fractionated radiotherapy.

An exemplary composition is a vaccine composition comprising a nanoparticle and/or an aggregate of nanoparticles for use in the treatment of cancer in an individual suffering from metastatic cancer and undergoing palliative radiation therapy, an individual suffering from metastatic cancer in which (radical) radiation therapy has been rejected, an individual suffering from a malignant neoplasm who is not being (conventionally) treated with radiation therapy, or an individual suffering from hemoblastosis, where the treatment includes exposing the individual to fractionated radiotherapy, including at least one stage of radiation, where the dose of ionizing radiation is in the range from 1.8 to 30 Gray (Gy), preferably 1.8 to 20 Gray (Gy), and where each nanoparticle consists of a material having a density of at least 7 g/cm ³ and an atomic number (Z) of at least 25 , and each nanoparticle and or aggregate of nanoparticles are coated with a biocompatible coating allowing them to be stable at pH 6.5 to 7.5 in physiological fluid.

The vaccine composition contains a nanoparticle or aggregate of nanoparticles as described herein, preferably together with a pharmaceutically acceptable carrier or agent. The particular vaccine composition further comprises at least one immunotherapeutic agent and optionally a cancer therapeutic agent.

Also provided herein is the use of a nanoparticle, or an aggregate of nanoparticles, or a therapeutic composition, as defined herein, for the treatment of cancer in an individual, preferably in a selected group of individuals/patients, typically an individual suffering from metastatic cancer and undergoing palliative radiotherapy. therapy, an individual suffering from a metastatic malignant neoplasm in which (radical) radiation therapy was rejected, an individual suffering from a malignant neoplasm that is not (conventionally) treated with radiation therapy, or an individual suffering from hemoblastosis, as well as corresponding methods of treating malignant neoplasm in an individual in need, comprising the step of administering to said individual a nanoparticle, or an aggregate of nanoparticles, or a therapeutic composition as defined herein.

The present invention further relates to a kit, typically a vaccine kit, comprising (i) a nanoparticle or nanoparticle aggregate as described herein, or a composition comprising such a nanoparticle or nanoparticle aggregate, preferably together with (ii) at least at least one immunotherapeutic agent and/or therapeutic agent for the treatment of malignancy.

Brief description of the drawings

Fig. 1: HMGB1 released in the concentrated supernatant of cells treated or untreated with the HfO2 nanoparticle (NP) suspension from Example 1 and irradiated or not (5 Gy per fraction) is shown as a fold change compared to the untreated control.

Fig. 2: HMGB1 released in the concentrated supernatant of cells treated or untreated with the HfO2 nanoparticle (NP) suspension from Example 1 and irradiated or not (10 Gy as a single fraction) is shown as a fold change compared to the untreated control.

Fig. 3: HMGB1 released in the concentrated supernatant of cells treated or untreated with the HfO ₂ (NP) nanoparticle suspension of Example 1 and irradiated or not treated as a single fraction is shown as a fold change compared to the untreated control. A) The malignant cell line is the human colorectal cancer cell line HCT 116, and the radiation doses correspond to 4 and 6 Gy; C) the malignant cell line is a 42 MG BA human glioblastoma cell line, and the radiation doses correspond to 10 and 15 Gy; C) The malignant cell line is the PANC-1 human pancreatic cancer cell line and the radiation dose is 8 Gy.

It is well known to adapt the dose in vitro (as a single fraction) to the radiosensitivity of malignant cells. A radiosensitive cancer cell line, such as the HCT 116 cell line, can be exposed to a lower dose of radiation compared to more radioresistant cancer cell lines, such as the 42MG BA and PANC-1 cell lines. The increased production of DAMP observed in vitro precedes the enhanced immune response in vivo.

Fig. 4: ATP secretion from HCT 116 malignant cells treated or untreated with the HfO ₂ (NP) nanoparticle suspension of Example 1 and irradiated or not (6 Gy per fraction) is presented as a fold change compared to the untreated control.

Fig. 5: illustration of a vaccination assay protocol performed using the mouse colorectal cancer cell line CT-26 in immunocompetent mice, representing the injection schedule for three groups, group 1 (control group), group 2 (irradiated group: 6 Gy as one fraction) and group 3 (NP HfO2 from example 1, irradiated: 6 Gy as a single fraction)

Fig. 6: Vaccination analysis protocol where the percentage of tumor-free mice in group 1 (control), group 2 (RTx, 6 Gy) and group 3 (NP HfO ₂ from Example 1+RTx, 6 Gy) is plotted as a function of days post-vaccination.

Detailed description of the invention

Radiation therapy.

A nanoparticle and/or aggregate of nanoparticles, as well as any composition containing such a nanoparticle and/or aggregate of nanoparticles, are provided herein for use as a therapeutic composition or vaccine (composition) in relation to an individual suffering from a malignant neoplasm, in particular a metastatic malignant neoplasm, preferably a metastatic malignancy, where the malignancy develops into a widespread systemic disease, or hemoblastosis, in the context of radiotherapy, i.e. in relation to an individual who is injected with nanoparticles and who is then subjected to radiation therapy. In other words, to be useful as a therapeutic vaccine, the nanoparticles must be exposed to ionizing radiation, which means that they are used in combination with radiation therapy.

A malignant neoplasm developing into a widespread systemic disease, as a rule, includes many distant metastases, usually more than 5, preferably more than 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19 or 20 metastases, and in most cases is associated with a poor prognosis (see Ralph R. Weichselbaum Nat. Rev. Clin. Oncol. 2011). An individual suffering from such a malignant neoplasm may additionally have, in addition to the foci/sites of metastasis associated with said malignant neoplasm, other sites/foci of malignant cells, which are foci of primary and/or metastatic malignant neoplasm associated with another primary malignant neoplasm/tumor.

A specific composition provided herein is a vaccine composition comprising a nanoparticle and/or an aggregate of nanoparticles for use in the treatment of cancer in an individual suffering from metastatic cancer and undergoing palliative radiotherapy, an individual suffering from metastatic cancer, in which radiotherapy has been rejected (generally rejected as a (general) radical treatment), an individual suffering from a malignancy who is not being (conventionally/classically) treated with radiation therapy, or an individual suffering from hemoblastosis, where the treatment involves exposing the individual to fractionated radiation therapy, including at least one stage of irradiation, where the dose of ionizing radiation is in the range from 1.8 to 30 Gray (Gy), preferably from 1.8 to 20 Gray (Gy), and where each nanoparticle consists of a material having o a density of at least 7 g/cm ³ and an atomic number (Z) of at least 25, and each nanoparticle or aggregate of nanoparticles is coated with a biocompatible coating allowing them to be stable at pH 6.5 to 7.5 in a physiological fluid.

In the context of the present invention, the individual or patient is a mammal. In a particular embodiment, the mammal is a human of any age or gender. The individual suffers from a malignant neoplasm.

The preferred individual who is likely to benefit from the invention typically suffers from a solid cancer or hemoblastosis.

In a specific aspect, the individual is suffering from a cancer undergoing conventional treatment with radiation therapy, or where radiation therapy is the conventional treatment or the most appropriate treatment for a particular individual, or where radiation therapy may be given as indicated.

In another specific and preferred aspect, the individual who will benefit from the present invention is an individual suffering from a malignant neoplasm, where radiation therapy will not be considered as a treatment option (or, in other words, not considered as a conventional treatment or a possible curative treatment for the considered an individual, or is used exclusively against one or a small number, typically less than five, sites/foci of malignant cells that are metastatic or primary foci of malignant neoplasm among the many sites/foci of malignant cells present in an individual) or is no longer considered as an option (radical) treatment, typically when the individual is undergoing palliative care, or when radiation therapy has been rejected. If palliative treatment is performed, the individual is still subjected to radiation therapy, but said radiation therapy can no longer be considered as radical radiation therapy.

In this context, the individual is preferably an individual suffering from a metastatic cancer with multiple distant metastases (i.e., widespread metastases), typically more than 5, preferably more than 6, 7, 8, 9, 10, 11, 12, 13, 14 , 15, 16, 17, 18, 19 or 20 metastases as described above.

In other words, the nanoparticles and/or aggregates of nanoparticles of the present invention, when exposed to ionizing radiation, may represent a solution for patient groups in which the oncologist does not consider radiation therapy as a definitive treatment.

Now, through the present invention, such specific individuals offer the option of radical treatment of malignant tumors.

An exemplary cancer subject who is likely to benefit from the invention is selected from a metastatic cancer subject undergoing palliative radiotherapy, a metastatic cancer subject in whom (radical) radiotherapy was rejected, an individual suffering from a malignant neoplasm, which is not treated (conventionally/classically) by radiation therapy, and an individual suffering from hemoblastosis. Preferably, the subject is selected from an individual suffering from metastatic cancer and undergoing palliative radiation therapy, an individual suffering from metastatic cancer in which (radical) radiation therapy was rejected, and an individual suffering from hemoblastosis.

In this field and in the context of the present invention, the term radical treatment or radical therapy refers to a treatment or therapy, in particular radiation therapy, providing the individual being treated with a radical solution for the treatment of malignancies from which he/she suffers, intended for the general treatment of said individual (primary tumors, as well as the corresponding foci of metastasis).

As one of skill in the art would know, palliative radiotherapy is used to relieve symptoms and is different from radiotherapy, i.e. radiation therapy, carried out as radical treatment (also defined in the present description as radical radiation therapy). In fact, experts consider palliative radiotherapy to be an effective treatment for many of the symptoms caused by locally advanced or metastatic tumors, even in patients with a short life expectancy.

Typically, a metastatic malignancy involves (i) the connective tissue

- 6 040587 tissue, and it is preferably selected from fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, malignant fibrous histiocytoma, (ii) endothelial or mesothelial tissue, and it is preferably selected from hemangiosarcoma, angiosarcoma, lymphangiosarcoma and mesothelioma, (iii) muscle tissue and it is preferably selected from leiomyosarcoma and rhabdomyosarcoma, (iv) epithelial tissue and it is preferably selected from adenocarcinoma, squamous cell carcinoma and epidermoid carcinoma, (v) nervous tissue and it is preferably selected from glioblastoma multiforme, glioma, neuroblastoma, medulloblastoma , meningiomas, neurofibrosarcomas and schwannomas, and (vi) the APUD system, and is preferably selected from thyroid cancer, pancreatic cancer, gastric cancer and colon cancer. In another preferred embodiment, the metastatic cancer is melanoma.

The metastatic malignancy may be a malignancy selected from, for example, skin cancer, central nervous system malignancy, head and neck cancer, lung cancer, kidney cancer, breast cancer, gastrointestinal malignancy (GIST), prostate cancer , liver cancer, colon cancer, rectal cancer, anal cancer, esophageal cancer, malignant neoplasm of the male genitourinary system, malignant neoplasm of the female reproductive system, cancer of the adrenal glands and cancer of the retroperitoneal space, bone and soft tissue sarcomas, malignant neoplasms of childhood, neuroblastoma, malignant neoplasm of the central nervous system and Ewing's sarcoma, or may originate from it.

As a rule, hemoblastosis affects the blood or lymphoid tissue. It is usually selected from leukemia, myeloma and lymphoma.

The individual may have a tumor. Unless otherwise indicated herein, a tumor is a malignant tumor.

In another embodiment, the subject is suffering from hemoblastosis and radiation therapy is prescribed by an oncologist (eg, in the case of lymphoma).

Preferably, the radiation therapy to which the subject is to be subjected is fractionated radiation therapy, preferably fractionated radiation therapy, comprising at least one radiation stage (also referred to herein as fractionated treatment), usually several radiation stages, where the dose of ionizing radiation is in in the range of 1.8 to 30 Gray (Gy), preferably 1.8 to 20 Gray (Gy), preferably 1.8 to 15 Gray (Gy) per irradiation step.

In the context of fractionated radiotherapy, the total dose of ionizing radiation is divided into several smaller doses over a period of several days. This allows maximizing the effect of radiation on malignant neoplasms and minimizing negative side effects on healthy cells. For typical fractionation regimens, the total dose is divided into 30 units/fractions administered each weekday for 6 weeks, although current studies are looking at the beneficial effects of accelerated fractionation (2 administrations per day and/or additional administrations on weekends).

The term ionizing radiation refers to high energy particles or waves that can ionize an atom or molecule. The ability to ionize depends on the energy of individual particles or waves, and not on their number. A large stream of particles or waves, in most cases, will not cause ionization unless the individual particles or waves have sufficient energy. Typical ionizing radiation is radiation with an energy of at least 1.8 keV.

In a preferred embodiment, the dose of ionizing radiation per irradiation step is selected from 1.8, 2, 2.2, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3, 1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4, 4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9, 5, 10, 11, 12, 13, 14, 15, 20, 25 and 30 Gy per fractionated treatment. The dose of ionizing radiation is preferably selected from 1.8, 2, 2.4, 2.5, 3, 3.2, 3.6, 4, 4.5, 5, 5.5, 6, 7, 8, 10, 15, 20, 25 and 30 Gy per fractionated treatment, even more preferably from 2, 3, 5, 6, 7, 8, 10, 15, 20, 25 and 30 Gy.

Preferred fractionated radiotherapy can be selected from 25 x 2 Gy fractions (total: 50 Gy), 30 x 2 Gy fractions (total: 60 Gy), 35 x 2 Gy fractions (total: 70 Gy), 40 x 2 Gy fractions (total: 70 Gy), : 80 Gy), 5 x 3 Gy fractions (total: 15 Gy), 10 x 3 Gy fractions (total: 30 Gy), 15 x 3 Gy fractions (total: 45 Gy), 20 x 3 Gy fractions (total: 60 Gy), 25 x 3 Gy fractions (total: 75 Gy), 3 x 4 Gy fractions (total: 12 Gy), 5 x 4 Gy fractions (total: 20 Gy), 8 x 4 Gy fractions (total: 32 Gy) , 10 x 4 Gy Fractions (Total: 40 Gy), 15 x 4 Gy Fractions (Total: 60 Gy), 20 x 4 Gy Fractions (Total: 80 Gy), 2 x 5 Gy Fractions (Total: 10 Gy), 3 5 Gy fractions (total: 15 Gy), 4 x 5 Gy fractions (total: 20 Gy), 5 x 5 Gy fractions (total: 25 Gy), 6 x 5 Gy fractions (total: 30 Gy), 8 fractions 5 Gy each (total: 40 Gy), 10 fractions of 5 Gy (total: 50 Gy), 1 fraction of 6 Gy (total: 6 Gy), 2 fractions of 6 Gy (total: 12 Gy), 3 fractions of 6 Gy (total: 18 Gy), 4 x 6 Gy fractions (total: 24 Gy), 5 x 6 Gy fractions (total: 30 Gy), 6 fractions of 6 Gy (total: 36 Gy), 10 fractions of 6 Gy (total: 60 Gy), 1 fraction of 7 Gy (total: 7 Gy), 2 fractions of 7 Gy (total: 14 Gy), 3 fractions of 7 Gy (total: 21 Gy), 4 x 7 Gy fractions (total: 28 Gy), 5 x 7 Gy fractions (total: 35 Gy), 1 x 8 Gy fractions (total: 8 Gy), 2 x 8 Gy fractions (total: 16 Gy), 3 fractions of 8 Gy (total: 24 Gy), 4

- 7 040587 fractions of 8 Gy (total: 32 Gy), 5 fractions of 8 Gy (total: 40 Gy), 1 fraction of 9 Gy (total: 9 Gy), 2 fractions of 9 Gy (total: 18 Gy), 3 x 9 Gy Fractions (Total: 27 Gy), 4 x 9 Gy Fractions (Total: 36 Gy), 5 x 9 Gy Fractions (Total: 45 Gy), 1 x 10 Gy Fractions (Total: 10 Gy), 2 fractions of 10 Gy (total: 20 Gy), 3 fractions of 10 Gy (total: 30 Gy), 4 fractions of 10 Gy (total: 40 Gy), 1 fraction of 15 Gy (total: 15 Gy), 2 fractions of 15 Gy (Total: 30 Gy), 3 Fractions of 15 Gy (Total: 45 Gy), 4 Fractions of 15 Gy (Total: 60 Gy), 1 Fraction of 20 Gy (Total: 20 Gy), 2 Fractions of 20 Gy (total: 40 Gy), 3 x 20 Gy fractions (total: 60 Gy), 1 x 25 Gy fractions (total: 25 Gy), 2 x 25 Gy fractions (total: 50 Gy), 3 x 25 Gy fractions (total: : 75 Gy), 1 x 30 Gy fractions (total: 30 Gy), and 2 x 30 Gy fractions (total: 60 Gy).

In a particularly preferred aspect, the subject is an individual suffering from metastatic cancer and undergoing palliative radiation therapy, an individual suffering from metastatic cancer in which radiation therapy was rejected, or an individual suffering from a malignant neoplasm who is not being treated with radiation therapy, and fractionated radiotherapy selected from 1 x 6 Gy fraction (total: 6 Gy), 2 x 6 Gy fractions (total: 12 Gy), 3 x 6 Gy fractions (total: 18 Gy), 4 x 6 Gy fractions (total: 2 4 Gy), 5 x 6 Gy fractions (total: 30 Gy), 1 x 7 Gy fractions (total: 7 Gy), 2 x 7 Gy fractions (total: 14 Gy), 3 x 7 Gy fractions (total: 21 Gy) , 4 x 7 Gy fractions (total: 28 Gy), 1 x 8 Gy fractions (total: 8 Gy), 2 x 8 Gy fractions (total: 16 Gy), 3 x 8 Gy fractions (total: 24 Gy), 4 fractions of 8 Gy (total: 32 Gy), 1 fraction of 9 Gy (total: 9 Gy), 2 fractions of 9 Gy (total: 18 Gy), 3 fractions of 9 Gy (total: 18 Gy), 3 fractions of 9 Gy (total: o: 27 Gy), 1 x 10 Gy fractions (total: 10 Gy), 2 x 10 Gy fractions (total: 20 Gy), 3 x 10 Gy fractions (total: 30 Gy), 1 x 15 Gy fractions (total: 15 Gy), 2 x 15 Gy fractions (total: 30 Gy), 1 x 20 Gy fractions (total: 20 Gy), 2 x 20 Gy fractions (total: 40 Gy), 1 x 25 Gy fractions (total: 25 Gy) and 1 fraction of 30 Gy (total: 30 Gy).

Nanoparticle.

The nanoparticle used in the context of the invention preferably consists of a material having a density of at least 7 g/cm ³ and an atomic number (Z) of at least 25. The nanoparticle or aggregate of nanoparticles is coated with a biocompatible coating, allowing them to be stable at a pH of 6, 5 to 7.5 in physiological fluid.

In the present description, the term nanoparticle refers to a product, in particular a synthetic product, with a size in the nanometer range, typically from 1 to 500 nm.

The term aggregate of nanoparticles refers to the collection of nanoparticles, strongly, usually covalently associated with each other.

Transmission electron microscopy (TEM) can be used to measure the size of nanoparticles. In addition, dynamic light scattering (DLS) can be used to measure the hydrodynamic diameter of nanoparticles in solution. These two methods can additionally be used one after the other to compare the measured sizes and confirm the specified size. The preferred method is DLS (see International Standard ISO22412 Particle Size Analysis - Dynamic Light Scattering, International Organization for Standardization (ISO) 2008). The largest nanoparticle size as defined herein is typically about 4 to about 250 nm, preferably about 4 or 10 to about 100 nm or about 200 nm, even more preferably about 20 to about 150 nm.

Since the shape of a particle can affect its biocompatibility, a particle having a fairly uniform shape is preferred. Thus, for pharmacokinetic reasons, nanoparticles that are substantially spherical, round or ovoid in shape are preferred. This shape also favors the interaction or capture of nanoparticles by cells. A spherical or round shape is particularly preferred.

Typically, the largest size is the diameter of a round or spherical nanoparticle or the largest size of an ovoid or oval nanoparticle.

The inorganic material of the nanoparticle present in the composition preferably has a theoretical (bulk) density of at least 7, and may be selected from any material exhibiting this property and presented in the table from the Physical Constants of Inorganic Compounds, shown on pages 4-43 in Handbook of Chemistry and Physics (David R. Lide Editor-in-Chief, ^88th Edition 2007-2008).

The inorganic material constituting the nanoparticle is preferably a material having an effective atomic number (Z _eff ) of at least 25, preferably at least 40 or 41, more preferably at least 50 or 51, more preferably at least 60, 61, 62 or even 63.

The term effective atomic number is similar to atomic number, but is used for compounds (such as water) and mixtures of different materials (such as tissue and bone) rather than atoms. The effective atomic number is used to calculate the average atomic number for a compound or mixture of materials. It is abbreviated as Z _eff .

The effective atomic number is calculated by multiplying the mass fraction of each atom in the compound by the atomic number of the atom. The formula for the effective atomic number Z _eff is:

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where fn is the fraction of the total number of electrons associated with each element, and

Zn is the atomic number of each element.

The atomic number (also known as the proton number) is the number of protons found in the nucleus of an atom. It is commonly denoted by the symbol Z. Atomic number uniquely defines a chemical element. In an atom with a neutral charge, the atomic number is equal to the number of electrons.

An example is water (H ₂ O) consisting of two hydrogen atoms (Z=1) and one oxygen atom (Z=8). The total number of electrons is 1+1+8=10. The proportion of electrons corresponding to two hydrogen atoms is 2/10, and the proportion of electrons corresponding to a single oxygen atom is (8/10). Thus Z _eff of water is:

Ze _)} = = 7.42

Z _eff contributes to the ability of nanoparticles to absorb incident radiation.

The inorganic material constituting the nanoparticle is typically selected from oxide, metal, sulfide, and any mixture thereof.

If the inorganic material constituting the nanoparticle is an oxide, the oxide is preferably selected from cerium (IV) oxide (CeO ₂ ), neodymium (III) oxide (Nd ₂ O ₃ ), samarium (III) oxide (Sm ₂ O ₃ ), oxide europium (III) (Eu2O ₃ ), gadolinium (III) oxide (Gd ₂ O ₃ ), terbium (III) oxide (Tb ₂ O ₃ ), dysprosium (III) oxide (Dy ₂ O ₃ ), holmium oxide (Ho ₂ O ₃ ), erbium oxide (Er ₂ O ₃ ), thulium (III) oxide (Tm ₂ O ₃ ), ytterbium oxide (Yb ₂ O ₃ ), lutetium oxide (Lu ₂ O ₃ ), hafnium oxide (IV) (HfO ₂ ), tantalum (V) oxide (Ta ₂ O ₅ ), rhenium (IV) oxide (ReO ₂ ), bismuth (III) oxide (Bi ₂ O ₃ ). In the context of the present invention, a mixture of inorganic oxides can also be used to obtain the nanoparticles of the invention.

If the inorganic material constituting the nanoparticle is a metal, the metal is preferably selected from gold (Au), silver (Ag), platinum (Pt), palladium (Pd), tin (Sn), tantalum (Ta), ytterbium (Yb), zirconium (Zr), hafnium (Hf), terbium (Tb), thulium (Tm), cerium (Ce), dysprosium (Dy), erbium (Er), europium (Eu), holmium (Ho), iron (Fe), lanthanum (La), neodymium (Nd), praseodymium (Pr), lutetium (Lu). In the context of the present invention, a mixture of metals is also possible. In the context of the present invention, a mixture of an inorganic oxide and a metal can also be used to obtain the nanoparticles of the invention.

If the inorganic material constituting the nanoparticle is a sulfide, this sulfide is preferably silver sulfide (Ag ₂ S).

In a preferred embodiment, the nanoparticle used in the context of the present invention to obtain a composition of interest can be coated with a biocompatible material selected from a low visibility agent. In fact, when the nanoparticles of the present invention are administered to an individual by the intravenous (IV) route, a biocompatible coating with a material selected from a low visibility agent is particularly advantageous in order to optimize the biological distribution of the nanoparticles. This coating is responsible for the so-called low visibility property of the nanoparticle.

The low visibility agent may be a steric group exhibiting agent. Such a group can be selected from, for example, polyethylene glycol (PEG); polyethylene oxide; polyvinyl alcohol; polyacrylate; polyacrylamide (poly(N-isopropylacrylamide)); polyurea; biopolymer; a polysaccharide such as dextran, xylan and cellulose; collagen; a zwitterionic compound such as polysulfobetaine; etc.

In another preferred embodiment, the nanoparticles can be coated with a biocompatible material selected from a means that allows interaction with the biological target. Such a tool, as a rule, can impart a positive or negative charge to the surface of the nanoparticle. This charge can be determined by zeta potential measurements, typically carried out on suspensions of nanoparticles, the concentration of which varies from 0.2 to 10 g/l, suspended in an aqueous medium with a pH of 6 to 8.

The agent that forms a positive charge on the surface of the nanoparticle may be, for example, aminopropyltriethoxysilane or polylysine. The agent that forms a negative charge on the surface of the nanoparticle may be, for example, a phosphate (eg, polyphosphate, metaphosphate, pyrophosphate, etc.), a carboxylate (eg, citrate or dicarboxylic acid, in particular succinic acid) or sulfate.

A complete biocompatible coating of the nanoparticle or aggregate may be preferred, in particular for intravenous (IV) administration, to avoid interaction of the particle surface with any recognition element (macrophage, opsonins, etc.). The term full coverage implies the presence of a very high compactness of biocompatible molecules capable of creating at least a complete monolayer on the particle surface.

- 9 040587

The biocompatible coating enables, in particular, the stability of the nanoparticle in a liquid, such as a physiological fluid (blood, plasma, serum, etc.) or any isotonic media or physiological media necessary for pharmaceutical administration.

Stability can be confirmed by calculating the dry residue using a drying oven and measured in a suspension of nanoparticles before and after filtration, typically on a 0.22 or 0.45 µm filter.

Preferably, the coating maintains the integrity of the particle in vivo, provides or improves its biocompatibility, and facilitates its optional functionalization (eg, using spacer molecules, biocompatible polymers, targeting agents, proteins, etc.).

The particular nanoparticle of the present invention may further comprise a targeting agent to enable it to interact with the recognition element present on the target cell. Such a targeting agent generally operates after the accumulation of nanoparticles in the target site. The targeting agent may be any biological or chemical entity that exhibits affinity for molecules present in the human or animal body. For example, it may be a peptide, oligopeptide or polypeptide, protein, nucleic acid (DNA, RNA, miRNA, tRNA, miRNA, etc.), hormone, vitamin, enzyme, ligand of a molecule expressed by a pathological cell, in particular a ligand of a tumor antigen , hormone receptor, cytokine receptor or growth factor receptor. Said targeting agents may be selected, for example, from the group consisting of LHRH, EGF, folate, anti-B-FN antibody, E-selectin/P-selectin, anti-IL-2R antibody, GHRH, etc.

Composition.

In the present description, the authors of the present invention also describe a therapeutic composition, usually a vaccine composition, for use in relation to an individual suffering from a malignancy, as defined herein, preferably a metastatic malignancy or hemoblastosis, in the context of radiation therapy, usually fractionated beam radiation. therapy as defined herein, wherein the composition comprises (i) a nanoparticle or aggregate of nanoparticles, each nanoparticle consisting of a material having a density of at least 7 g/cm ³ and an atomic number (Z) of at least 25, and each the nanoparticle or aggregate of nanoparticles is coated with a biocompatible coating allowing the nanoparticle to be stable at pH 6.5 to 7.5 in a physiological fluid, preferably together with (ii) a pharmaceutically acceptable carrier or agent.

In a specific embodiment, a vaccine composition comprising a nanoparticle and/or an aggregate of nanoparticles is described for use in the treatment of cancer in an individual suffering from metastatic cancer and undergoing palliative radiation therapy, an individual suffering from metastatic cancer in which (radical) radiation therapy was rejected, an individual suffering from a malignant neoplasm who is not being treated with radiation therapy, or an individual suffering from hemoblastosis, where the treatment includes exposing the individual to fractionated radiotherapy, including at least one stage of radiation, where the dose of ionizing radiation is in the range of 1, 8 to 30 Gray (Gy), preferably 1.8 to 20 Gray (Gy), and where each nanoparticle consists of a material having a density of at least 7 g/cm ³ and an atomic number (Z) of at least 25, and each nanoparticle and whether the aggregate of nanoparticles is coated with a biocompatible coating allowing them to be stable at pH 6.5 to 7.5 in physiological fluid. In addition to the nanoparticle and/or aggregate of nanoparticles, the composition may contain a pharmaceutically acceptable carrier or agent.

The pharmaceutically acceptable carrier or agent may be any base conventional to those skilled in the art such as, for example, saline, isotonic, sterile, buffered, non-aqueous carrier solutions, and the like. A typical carrier is isotonic media or physiological media containing NaCl, PBS and/or glucose. The carrier may contain, for example, glucose (5%) or dextrose (5%) and/or NaCl (0.9%).

The composition may also contain stabilizers, sweeteners, surfactants, polymers, and the like.

The composition may be in the form of a solid, liquid (particles in suspension), aerosol, gel, paste, and the like. Preferred compositions are in the form of a liquid or gel. Particularly preferred compositions are in the form of a liquid.

They can be formulated, for example, in the form of an ampoule, syringe, aerosol, bottle, container, tablet, capsule, by methods known to the person skilled in the art for preparing pharmaceutical formulations.

Typically, a composition in the form of a liquid or gel contains from about 0.05 g/L to about 450 g/L of nanoparticles or aggregates of nanoparticles, from about 0.05 g/L to about 250 g/L of nanoparticles, preferably at least about 10, 11, 12, 13, 14, 15, 16, 17,

- 10 040587

18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,

78, 79, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350 or 400 g/l nanoparticles.

The concentration of nanoparticles in the composition can be measured by dry residue. The dry residue is theoretically measured after the step of drying the suspension containing the nanoparticles in a drying oven.

In a particular embodiment, the composition further comprises at least one immunotherapeutic agent and optionally an additional cancer therapeutic agent.

As used herein, the term immunotherapeutic agent generally means any molecule, drug, cell-based or cell-based vaccine, oncolytic virus, DNA-based vaccine, peptide-based vaccine, toll-like receptor agonist, vesicle derived from a cell, and also any combination thereof capable of stimulating the immune system of the individual and known to the person skilled in the art.

The molecule or drug, for example, can be selected from a monoclonal antibody, a cytokine, and combinations thereof.

The drug, as a rule, can be an inhibitor of indolamine-2,3-dioxygenase (IDO), such as 1-methyl-D-tryptophan.

In a preferred embodiment, the monoclonal antibody inhibits the CTLA-4 molecule or the interaction between PD-1 and its ligands. The monoclonal antibody is preferably selected from anti-CTLA-4, anti-PD-1, anti-PD-L1, anti-PD-L2. The monoclonal antibody, for example, can be selected from ipilimumab, tremelimumab, nivolumab, pembrolizumab, pidilizumab, and lambrolizumab.

In another preferred embodiment, the monoclonal antibody enhances CD27 signaling, CD137 signaling, OX-40 signaling, GITR signaling and/or MHCII signaling and/or activates CD40. The monoclonal antibody, for example, can be selected from dacetuzumab, lucatumumab, and urelumab.

In a further embodiment, the monoclonal antibody inhibits TGF-β signaling or KIR signaling. The monoclonal antibody, for example, can be selected from fresolimumab and lirimumab.

The cytokine can preferably be selected from granulocyte-macrophage colony-stimulating factor (GM-CSF), FMS-like tyrosine kinase ligand 3 (FLT3L), IFNa, IFNα2b, IFNy, IL-2, IL-7, IL-10 and IL-15.

In another preferred embodiment, the immunotherapeutic agent is an immunocytokine, such as L19-IL2 immunocytokine (Nicolle H. Rekers Radiotherapy and Oncology 2015).

The cell used as an immunotherapeutic agent is typically an immune cell presenting a tumor antigen or sensitized with a tumor antigen, preferably a tumor antigen specific for the cancer to be treated, such as a dendritic cell or a T cell; a cell secreting an immunogenic molecule; or a dead tumor cell or a dying tumor cell undergoing immunogenic cell death, ie. a cell expressing CRT and/or producing HMGB1 and/or producing ATP in a typical ICD amount, eg a dying or dead tumor cell exposed to radiation therapy. The cell may be an autologous cell or an allogeneic cell. Preferably the cell is an autologous cell isolated from the individual to be treated. The dead or dying tumor cell may be a mature tumor cell or a tumor stem cell.

The toll-like receptor agonist is preferably selected from a TLR 2/4 agonist, a TRL 7 agonist, a TRL 7/8 agonist, and a TRL 9 agonist. .

A preferred combination of immunotherapeutic agents, for example, can be selected from a cytokine, a monoclonal antibody, a toll-like receptor agonist, and a peptide-based vaccine.

As used herein, the term cancer therapeutic agent generally means an agent used in the conventional treatment of cancer, such as a biological compound, a small molecule targeted therapeutic agent, or a cytotoxic compound.

The biological compound, for example, is an antibody, preferably a monoclonal antibody (mAb), such as alemtuzumab, brentuximab vedotin, catumaxomab, denosumab, gemtuzumab ozogamicin, ibritumomab tiuxetan, pertuzumab, ofatumumab, bevacizumab, rituximab, trastuzumab, cetuximab, panatimumab, or tositumumab.

A small molecule targeted therapeutic agent typically inhibits enzymatic domains on a mutated, overexpressed, or other critical protein (potentially

- 11 040587 targets for the treatment of malignant tumors) in malignant cells. Some therapeutic agents include agents that target cell division (eg, an Aurora kinase inhibitor or a cyclin-dependent kinase inhibitor) as well as other biological mechanisms such as protein metabolism and chromatin modification (eg, a histone deacetylase inhibitor). Small molecule targeted therapeutics, for example, can be selected from imatinib, rapamycin, gefitinib, erlotinib, sorafenib, sunitinib, nilotinib, dasatinib, lapatinib, bortezomib, and atorvastatin, and the like.

The cytotoxic compound, for example, is a DNA modifying agent such as an anthracycline (such as dexamethasone, daunorubicin, idarubicin or methotrexate) or an antimitotic agent (spindle poison such as vincristine or vinblastine); a taxane such as docetaxel, larotaxel, cabazitaxel, paclitaxel (PG-paclitaxel and DHA-paclitaxel), orthotaxel, tesetaxel or taxoprexin; gemcitabine; etoposide; mitomycin C; an alkylating agent (eg melphalan or temozolomide); a platinum-based agent such as oxaliplatin or carboplatin; a TLR (toll-like receptor)-3 ligand; and a prodrug.

The prodrug (eg, capecitabine or irinotecan) is metabolized to its active form in vivo, producing the expected therapeutic effect.

Other exemplary cytotoxic compounds are typically selected from chemotherapeutic agents, as described herein or known to those skilled in the art of oncology.

The vaccine composition provided herein, comprising at least one immunotherapeutic agent, optionally in combination with at least one cancer therapeutic agent, can be administered to the individual being treated, simultaneously or separately from nanoparticles or nanoparticle aggregates, as presented in the present description.

Kit.

In the present description, the authors of the present invention also describe a vaccine kit containing (i) a nanoparticle or an aggregate of nanoparticles, consisting of a material having a density of at least 7 g/cm ³ and an atomic number (Z) of at least 25, with each nanoparticle or the nanoparticle aggregate is coated with a biocompatible coating allowing the nanoparticle to be stable at pH 6.5 to 7.5 in a physiological fluid or vaccine composition as described herein preferably together with (ii) at least one immunotherapeutic agent and/or therapeutic agent for the treatment of malignancy, as described herein, and, optionally, (iii) a brochure with instructions for the implementation of vaccination in the context of radiation therapy.

Protocol.

Nanoparticles or nanoparticle aggregates as described herein, or a composition containing such nanoparticles or nanoparticle aggregates, are preferably contacted with cancer cells prior to radiation therapy, typically fractionated radiation therapy. Contact between nanoparticles and malignant cells can be done ex vivo after biopsy or blood sampling, or in vivo by administering them to the individual being treated, systemically or directly into the tumor, tumor bed (after tumor resection through surgery) or tumor metastases.

When administered in vivo, the nanoparticles of the invention can be administered to an individual using various possible routes, such as topical [intratumoral (IT), intra-arterial (IA)], subcutaneous, intravenous (IV), intradermal, respiratory (inhalation), intraperitoneal , intramuscular, intraarticular, intrathecal, intraocular, or oral route (per os) preferably using IT, IV, or IA.

If necessary, repeated injections or injections of nanoparticles can be carried out.

In a particular embodiment, the nanoparticle or aggregate of nanoparticles is preferably administered to the individual to be treated along with at least one immunotherapeutic agent. A nanoparticle, or an aggregate of nanoparticles, or a composition containing such a nanoparticle or aggregate of nanoparticles and at least one immunotherapeutic agent, can be administered to an individual simultaneously or separately.

In a particular embodiment, if the cancer is a metastatic cancer and/or a cancer that is not (conventionally) treated with radiation therapy, at least one irradiation step is typically performed in vivo on one but not more than two , tumor sites of a metastatic malignant neoplasm containing nanoparticles or aggregates of nanoparticles. Thanks to nanoparticles or aggregates of nanoparticles used as a vaccine, it is possible to observe the antitumor effect of radiation therapy outside the irradiated area or site due to the effective mobilization of the immune system of the individual.

In general, in the present description, the authors of the present invention demonstrate that a significant increase in HMGB1 released by dying malignant cells is observed when using

- 12 040587 the use of nanoparticles or aggregates of nanoparticles according to the invention exposed to ionizing radiation, compared with irradiation alone in radiosensitive HCT 116 (human colorectal cancer cell lines) and radioresistant 42 MG BA (human glioblastoma cell lines) and PANC-1 (line human pancreatic cancer cells) using a single dose of radiation. In addition, a significant increase in ATP secretion by dying malignant cells is observed using the nanoparticles or nanoparticle aggregates of the invention exposed to ionizing radiation compared to irradiation alone in the HCT 116 malignant cell line.

These DAMPs are a sign of immunogenic death of malignant cells, and the nanoparticles or nanoparticle aggregates of the present invention, when exposed to radiation therapy, can increase their secretion and release.

In addition, the results of a vaccination assay performed on immunocompetent mice indicate the ability of nanoparticles or nanoparticle aggregates according to the invention to effectively mobilize the immune system of animals, preventing the appearance of tumors, if the animals were first vaccinated with irradiated malignant cells and then injected with viable malignant cells 7 days later: 66 % of animals were tumor-free in the group vaccinated with malignant cells treated with nanoparticles and 6 Gy, while only 33% of animals were tumor-free when vaccinated with malignant cells only irradiated at 6 Gy.

These data strongly support the applicability of these nanoparticles or nanoparticle aggregates as a vaccine to achieve the antitumor effect of radiation therapy outside the irradiated area or site due to the effective mobilization of the individual's immune system.

In a preferred embodiment, a nanoparticle or aggregate of nanoparticles of the invention, or a composition of the invention comprising such a nanoparticle or aggregate of nanoparticles, allows alteration or destruction of metastatic cancer cells present in the lungs (e.g., if the primary malignancy is a sarcoma, bladder cancer, breast cancer, colon cancer, kidney cancer, or prostate cancer), liver (eg, if the primary malignancy is gastrointestinal malignancy, breast cancer, colon cancer, lung cancer, or skin cancer), bone (eg. if the primary malignancy is breast cancer, prostate cancer, or lung cancer) and/or the brain (eg, if the primary malignancy is lung cancer, kidney cancer, melanoma, or breast cancer).

In another specific embodiment, at least one irradiation step is performed ex vivo on a cancer sample of an individual containing nanoparticles or aggregates of nanoparticles, and ex vivo lethally irradiated cancer cells together with at least a portion of the cell supernatant from the cancer sample, at least least partially administered to the individual repeatedly before any optional subsequent in vivo treatment of malignancy in the specified individual.

Cell supernatant from an irradiated cancer sample typically contains immunogenic molecules such as HMGB1, ATP, various chaperones from the heat shock protein (HSP) family, namely 70 kDa heat shock protein (HSP70) and 90 kDa heat shock protein (HSP90 ), immunostimulatory cytokines like interferon a (IFN), metabolites of sphingomyelin, a breakdown product of the extracellular matrix, etc. In the context of the present invention, increased release of immunogenic molecules in the cell supernatant from an irradiated cancer sample containing nanoparticles or a nanoparticle aggregate compared to an irradiated cancer sample without nanoparticles or a nanoparticle aggregate (see Example 4) can further enhance cancer treatment.

Re-administration to the individual being treated is typically by subcutaneous or intradermal injection.

If the malignancy is hemoblastosis, at least one irradiation step is typically performed ex vivo on the individual's hemoblastosis sample containing the nanoparticles or nanoparticle aggregates, and the irradiated hemoblastosis sample is at least partially reintroduced to the individual before any optional subsequent in vivo treatment of malignant neoplasm in the specified individual.

Re-administration to the individual being treated may be by subcutaneous or intradermal injection. It can also be carried out by intra-arterial (IA), intravenous (IV) or intraperitoneal (IP) injection.

The hemoblastosis sample is typically a blood sample or whole blood volume of the individual being treated. If the individual's whole blood volume is to be irradiated, it can be performed ex vivo by extracorporeal perfusion, with the whole irradiated whole blood volume reintroduced to the individual.

- 13 040587

It is noteworthy that Y. Suzuki et al. (2012) reported that tumor antigen-specific T-cell responses were observed in 38% of patients with esophageal squamous cell carcinoma (ESCC) after chemotherapy and radiotherapy, and that these responses were accompanied by elevated serum HMGB1 concentrations in these patients. HMGB1 in the tumor microenvironment was significantly upregulated in ESCC patients with preoperative chemoradiotherapy, but not in patients without chemoradiotherapy, and the amount of HMGB1 was positively correlated with patient survival.

In a preferred embodiment, the lethally irradiated ex vivo cancer cells or irradiated hemoblastosis specimen is at least partially reintroduced to the individual, together with at least one additional immunotherapeutic agent and/or cancer therapeutic agent, as described herein.

The lethally irradiated ex vivo cancer cells or irradiated hemoblastosis specimen and at least one additional immunotherapeutic agent and/or at least one cancer therapeutic agent may be administered to the individual simultaneously or separately.

Other aspects and advantages of the invention will be apparent from the following examples, given for illustrative purposes, but not by way of limitation.

Experimental section

Example 1 Synthesis and characterization of functionalized hafnium oxide (HfO ₂ ) nanoparticles.

A solution of tetramethylammonium hydroxide (TMAOH) is added to 40 g of HfCl ₄ solution. The addition of the TMAOH solution is carried out until the pH of the final suspension is between 7 and 13. A white precipitate is obtained.

The precipitate is then transferred to an autoclave and heated at between 120 and 300° C. to effect crystallization. After cooling, the suspension is washed with deionized water.

The peptization step is carried out to obtain a stable suspension of nanoparticles or aggregates of nanoparticles.

The sodium hexametaphosphate suspension is then added to the peptized solution (the amount of sodium hexametaphosphate is below LD50/5) and the pH of the suspension is adjusted to pH 6.5 to 7.5.

In the case of in vitro experiments, the sterilization step is carried out at this step, for example using a 0.22 µm filter.

In the case of in vivo experiments, the formulation step using 5% glucose can be performed before or after the sterilization step.

The following table presents the main characteristics of the suspension of biocompatible nanoparticles or aggregates of nanoparticles thus obtained.

Density Morphology Specific surface area (SS) in ^m2 /g Average hydrodynamic diameter (F) in nm 8.3 Spherical in shape 20<SS<60 15<Ф< 200

Example 2. Synthesis of gold nanoparticles and physicochemical characterization of gold nanoparticles of different sizes.

Gold nanoparticles are obtained by reducing gold chloride with sodium citrate in an aqueous solution. Method adapted from G. Frens Nature Physical Science 241 (1973) 21.

In a typical experiment, a solution of HAuCl44 is heated to boiling. Then sodium citrate solution is added. The resulting solution is kept boiling for another 5 minutes.

The nanoparticle size is adjusted from 15 to 105 nm by carefully modifying the ratio of citrate and gold compound precursor (see Table 1).

The suspensions of gold nanoparticles thus obtained are then concentrated using an ultrafiltration device (Amicon model 8400 stirred cell from Millipore) using a 30 kDa cutoff cellulose membrane.

Finally, the resulting suspensions are filtered through a 0.22 µm membrane filter (PES membrane from Millipore) in a fume hood and stored at 4°C.

Particle size is determined using transmission electron microscopy (TEM) by counting more than 200 particles, taking the largest nanoparticle size as its size.

- 14 040587

Table 1

Samples Size particles (nm) Synthesis Citrate HAiS1 ₄ Gold-15 15±2 (1σ) 2 0 ml 3 0 mM 500 ml 0.25 mM Gold-30 32±10 (1σ) 7.5 ml 40 mM 500 ml 0.25 mM Gold-60 60±10 (1σ) 2 ml 8 5 mM 500 ml 0.25 mM Gold-80 80±10(1σ) 1.2 ml 4 3 mM 200 ml 0.30 mM Gold-105 105±25 (1σ) 1.2 ml 3 9 mM 200 ml 0.33 mM

Example 3 Suspension of nanoparticles containing a gold material at least partially coated with a hafnium oxide material.

A solution of tetramethylammonium hydroxide (TMAOH) is added to a solution of hafnium chloride (HfCl ₄ ). The addition of the TMAOH solution is carried out until the pH of the final suspension is between 7 and 13. A white precipitate is obtained.

The suspension of gold particles from example 2 is slowly added to the white precipitate with vigorous stirring.

Then the resulting precipitate is transferred to an autoclave and heated at a temperature of from 100 to 300°C. After cooling, the suspension is washed with water.

The peptization step is carried out to obtain a stable suspension of nanoparticles containing the gold material at least partially enclosed in the hafnium oxide material.

Then, a suspension of sodium hexametaphosphate is added to the peptized solution and the pH of the suspension is adjusted to pH 6 to 8.

Example 4 HMGB1 Release by Dying Cancer Cells.

The release of HMGB1 by dying malignant cells was investigated using the human glioblastoma cell line 42 MG BA. The 42-MG-BA cell line was purchased from Deutsche Sammlung von Mikroorganismenund Zelkulturen GmbH, German Collection of Microorganism and Cell Cultures (Braunschweig, Germany).

Cells were distributed into T25 flasks in an amount ranging from 1.5 x 10 ⁶ to 2 x 10 ⁶ cells/flask. When the cells attached to the surface, a suspension of HfO ₂ nanoparticles from example 1 at a concentration of 400 μm was added overnight (12-15 hours) to the cells before using the radiation dose. Cells were cultured with antibiotics (Penistrepto).

A single X-ray dose of 5 or 10 Gy was applied at a dose rate of 1.26 Gy-min ^-1 using an X-ray source (200 kV, 15 mA, 0.2 mm copper filter).

Cells were cultured for 96 h at 37°C and 5% CO ₂ in a humid chamber. After 96 h, the cell supernatant was collected and concentrated using Centricon filters.

HMGB1 was quantified in concentrated cell supernatants using a human HMGB1 enzyme-linked immunosorbent assay (ELISA) kit.

In FIG. 1 shows HMGB1 released by dying cancer cells. HMGB1 released in the concentrated supernatant of cells treated or untreated with the HfO2 nanoparticle suspension from Example 1 and treated or not exposed to radiation therapy (5 Gy as 1 fraction) is presented as a fold change compared to the untreated control (i.e. cell line 42 MG VA without irradiation).

In FIG. 2 shows HMGB1 released by dying cancer cells. HMGB1 released in the concentrated supernatant of cells treated or untreated with the HfO2 nanoparticle suspension from Example 1 and treated or not irradiated (10 Gy as 1 fraction) is shown as a fold change compared to the untreated control (i.e. cell line 42 MG BA without irradiation).

Conclusions.

A significant increase in HMGB1 released by dying malignant cells is observed in the concentrated supernatant of cells treated with hafnium oxide nanoparticles from example 1 when exposed to a single dose of radiation of 5 or 10 Gy compared to irradiation alone. These results support the concept of using these nanoparticles or nanoparticle aggregates as a therapeutic vaccine in the context of radiotherapy.

Example 5 HMGB1 Release by Dying Cancer Cells in Various Cell Lines.

HMGB1 release by dying cancer cells was examined using the human colorectal cancer cell line HCT 116, the human glioblastoma cell line 42 MG BA and the human pancreatic cancer cell line PANC-1. Cell lines were purchased from the American Type Culture Collection (ATCC) (HCT 116, cat. no. CCL-247 and PANC-1, cat. no. CRL-1469) or Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (42 MG BA, cat. No. ACC 431).

Cells were dispensed into T25 vials at 1.10 ⁶ cells/vial. When the cells were attached to the surface, a suspension of HfO2 nanoparticles from example 1 at a concentration of 800 μM (in the case of HBT

116) or 400 μM (in the case of PANC-1 and 42 MG) was added overnight (12-15 hours) to the cells before using the radiation dose. Cells were cultured with antibiotics (medium with 1% penicillin streptomycin).

X-ray doses were applied at a dose rate of 1 Gy-min' ¹ using an X-ray source (320 kV, X-RAD 320). The dose of radiation for each cell line is given in table. 3.

Table 3. Radiation doses for each cell line

cell line NST 116 42MG BA PANC-1 Radiation doses 4 Gr 6 Gr 10 Gr 15 Gr 8 Gr

Cell line HCT 116: cells were cultured for 72 h at 37°C and 5% CO ₂ in a humid chamber. Cell supernatant was collected after 72 hours.

Cell lines 42 MG BA and PANC-1: cells were cultured for 96 h at 37°C and 5% CO ₂ in a humid chamber. After 96 h, the cell supernatant was collected and concentrated using Centricon filters.

HMGB1 was quantified in cell supernatants using a human HMGB1 enzyme-linked immunosorbent assay (ELISA) kit (such as the HMGB1 ELISA kit from IBL International, cat. no. ST51011).

In FIG. 3 shows HMGB1 released by dying cancer cells. HMGB1 released in the supernatant of cells treated or untreated with the HfO2 nanoparticle suspension of Example 1 and treated or not treated with radiation therapy (as 1 fraction) is shown as a fold change compared to the untreated control.

In FIG. 3A shows HMGB1 released by dying HCT 116 malignant cells. Radiation doses were 4 and 6 Gy. Data are presented as the average of 2 independent experiments performed in triplicate and pooled.

In FIG. 3B shows HMGB1 released by dying 42 MG BA cancer cells. The radiation doses were 10 and 15 Gy. Data are presented as the average of 2 independent experiments performed in triplicate and pooled.

In FIG. 3C shows HMGB1 released by dying PANC-1 cancer cells. The radiation dose was equal to 8 Gy. Data are presented as the average of 2 independent experiments performed in triplicate and pooled.

Conclusions.

A significant increase in HMGB1 released by dying malignant cells is observed in the supernatant of cells treated with hafnium oxide nanoparticles from Example 1 when exposed to a single dose of radiation compared to irradiation alone. These results support the concept of using these nanoparticles or nanoparticle aggregates as a therapeutic vaccine in the context of radiotherapy among a wide range of malignancies.

Example 6 Secretion of ATP by dying malignant cells.

ATP secretion from dying cancer cells was examined using the human colorectal cancer cell line HCT 116. The HCT 116 cell line was purchased from the American Type Culture Collection (ATCC) (HCT 116, cat. no. CCL-247).

Cells were distributed into T25 vials at a concentration of 2x10 ⁶ cells/vial. When the cells were attached to the surface, the HfO ₂ nanoparticles from example 1 at a concentration of 800 μm was added overnight (1215 hours) to the cells before using the radiation dose. Cells were cultured with antibiotics (medium containing 1% penicillin-streptomycin).

A single dose of x-rays of 6 Gy was applied at a dose rate of 1 Gy-min ^l 1 using an x-ray source (320 kV, X-RAD 320).

Cells were cultured for 9 h at 37°C and 5% CO2 in a humid chamber. Cell supernatant was collected after 9 hours.

ATP secretion was quantified in cell supernatants using a bioluminescent assay kit for measuring ATP (such as the ENLITEN® ATP Assay System from Promega, cat. no. FF2000).

In FIG. 4 shows ATP secretion by dying HCT 116 malignant cells. ATP secretion in the supernatant of cells treated or untreated with the HfO ₂ nanoparticle suspension from Example 1 and treated or not exposed to radiation therapy (6 Gy as 1 fraction) is presented as a fold change compared to untreated control (ie cell line HCT 116 without irradiation). Data are presented as the average of 3 independent experiments carried out in triplicate and pooled.

Conclusions.

A significant increase in ATP secretion by dying malignant cells is observed in

- 16 040587 supernatant of cells treated with hafnium oxide nanoparticles from example 1, when exposed to a single dose of radiation compared to irradiation separately. These results support the concept of using these nanoparticles or nanoparticle aggregates as a therapeutic vaccine in the context of radiotherapy.

Example 7 Vaccination Assay.

As stated in an article titled Consensus guidelines for the detection of immunogenic cell death (Oliver Kepp et al., Oncolmmunology 2014), the gold standard approach for evaluating the ability of a particular stimulus to induce a true ICD is based on vaccination assays. Under these conditions, selected mouse malignant cells are exposed in vitro to an immunogenic cell death (ICD) inducer and ultimately injected subcutaneously (s.c.) into the flank (vaccination site) of immunocompetent syngeneic mice (theoretically 5-10 per group). One week later, mice were injected with live malignant cells of the same type, inoculated with s.c. in the opposite side (injection site). Tumor incidence and growth were routinely monitored at both injection sites for 1-2 months. The development of neoplastic lesions at the site of vaccination indicates that the stimulus under investigation is not capable of inducing cell death (under the conditions tested) to a degree compatible with the induction of adaptive immunity. Conversely, in the absence of tumors at the vaccination site, the ability of a test stimulus to stimulate a true ICD is inversely correlated with the number of neoplastic lesions that develop at the injection site.

In this case, CT26 mouse colorectal cancer cells were selected for vaccination analysis. The cell line was purchased from the American Type Culture Collection (ATCC) (CT26, cat. no. CRL2638).

Cells were dispensed into T300 vials at a concentration of 10 x 106 cells/vial. When the cells attached to the surface, a suspension of HfO ₂ nanoparticles from example 1 at a concentration of 400 μm was added overnight (12-15 hours) to the cells before using the radiation dose. Cells were cultured with antibiotics (medium with 1% penicillin-streptomycin).

X-ray doses were applied at a dose rate of 1 Gy-min' ¹ using an X-ray source (320 kV, X-RAD 320). One fraction of 6 Gy was applied to cells treated or untreated with NP HfO ₂ from Example 1.

Cells were cultured for 48 h at 37°C and 5% CO ₂ in a humid chamber. After 48 hours, cells were harvested, washed with PBS prior to trypsinization, and 1.106 viable cells were injected subcutaneously into the left flank of immunocompetent Balb/c mice. In the case of the control group, 100 μl of PBS was injected subcutaneously into the left flank of the mouse.

Seven days later, mice were injected with untreated live CT26 cells: 3.105 cells were injected subcutaneously into the right flank of the mice (FIG. 5). The incidence and growth of tumors were monitored twice a week at both injection sites for 47 days (FIG. 6).

Conclusions.

Forty-seven (47) days post-vaccination, 66% of mice were tumor-free in the 6 Gy NP HfO ₂ treated group of Example 1 compared to 33% of mice exposed to 6 Gy alone. A significant increase in the number of tumor free mice was observed when vaccinated with cells treated with the hafnium oxide nanoparticles of Example 1 and exposed to a single dose of radiation compared to irradiation alone. These results support the concept of using these nanoparticles or nanoparticle aggregates as a therapeutic vaccine in the context of radiotherapy.

These data demonstrate the ability of the nanoparticles or nanoparticle aggregates of the invention, when subjected to radiation therapy, to elicit an effective mobilization of the immune system of an individual compared to radiation therapy alone.

Such an effective immune response triggered by the use of nanoparticles or nanoparticle aggregates of the invention upon exposure to radiation therapy is of particular interest to selected groups of patients, typically individuals suffering from metastatic cancer and/or undergoing palliative radiotherapy, individuals suffering from metastatic cancer. , in which radiation therapy was rejected, individuals suffering from malignancy, which are not treated (conventionally) with radiation therapy, or individuals suffering from hemoblastoses.

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Claims (15)

Links - Dhara M. MacDermed et al. A rationale for the targeted treatment of oligometastases with radiotherapy. Journal of Surgical Oncology 2008. 98 202-206. - Ralph R. Weichselbaum et al. Oligometastases revisited. Nat. Rev. Clin. oncol. 2011. 8, 378-382. - Sonam Sharma et al. Palliative radiotherapy: current status and future directions. Seminars on Oncology 2014. 41 (6) 751-763. - Sandra Demaria and Silvia C. Formenti. Radiation as an immunological adjuvant: current evidence on dose and fractionation. Frontiers in Oncology. October 2012 Volume 2 Article 153 1-7. - Oliver Kepp. Consensus guidelines for the detection of immunogenic cell death. Oncoimmunology 2014 3(9) e955691. - Kobe Reynders et al. The abscopal effect of local radiotherapy: using immunotherapy to make a rare event clinically relevant. Cancer Treatment Review 2015 41(6), 503510. - Scott J. Antonia et al. Immuno-oncology combinations: a review of clinical experience and future prospects. Clinical cancer research; 20(24) 2014 6258-6268. - Theresa L. Whiteside et al. Emerging opportunities and challenges in cancer immunotherapy. Clin Cancer Res 2016. 22(8) 1845-1855. - Nicolle H. Rekers et al. Combination of radiotherapy with the immunocytokine L19-IL2: additive effect in a NK cell dependent tumor model. Radiotherapy and Oncology 2015, 116 438442 . - Y. Suzuki et al. Immunogenic Tumor cell death induced by chemoradiotherapy in patients with esophageal squamous cell carcinoma (ESCC). Cancer Res. 2012.72(16) 3967-76. CLAIM 1. Method for the treatment of malignant neoplasms due to the abscopal effect by promoting the immunogenic death of malignant cells in a patient, which is a human suffering from an extensive metastatic malignant neoplasm and undergoing palliative radiation therapy, a patient, which is a human suffering from an extensive metastatic malignant neoplasm, in which case radiation therapy has been discontinued, or a human patient suffering from an extensive metastatic malignant neoplasm who is not being treated with radiation therapy, or a human patient suffering from hemoblastosis, comprising the step of administering a vaccine composition containing a nanoparticle and/or an aggregate of nanoparticles to the patient , representing a person, or in his sample of malignant neoplasm, and the stage of exposure of the patient, representing a person, or his sample of malignancy a given neoplasm containing a nanoparticle and / or an aggregate of nanoparticles, fractionated radiotherapy, including at least one stage of irradiation, where the dose of ionizing radiation is in the range from 1.8 to 30 Gray (Gy), and where each nanoparticle consists of a material having a density of at least 7 g/cm ³ and an atomic number (Z) of at least 25, and each nanoparticle or aggregate of nanoparticles is coated with a biocompatible coating allowing them to be stable at pH 6.5 to 7.5 in a physiological fluid, and when the cancer sample is subjected to fractionated radiotherapy, the step of administering at least partially the irradiated sample to a human patient. 2. The method according to claim 1, where the dose of ionizing radiation is in the range from 1.8 to 20 Gray (Gy). - 18 040587 3. The method of claim 1 or 2, wherein if the cancer is a metastatic cancer, at least one irradiation step is performed in vivo on one, but not more than two, tumor sites of the metastatic cancer containing nanoparticles or aggregates of nanoparticles. 4. The method of claim 1 or 2, wherein if the cancer is a metastatic cancer, at least one irradiation step is performed ex vivo on the individual's cancer sample containing the nanoparticles or nanoparticle aggregates and the ex vivo lethally irradiated cancer cells, and at least a portion of the cell supernatant from the cancer sample is at least partially re-administered to the individual prior to any optional subsequent in vivo cancer treatment in said individual. 5. The method of claim 1 or 2, wherein if the malignancy is hemoblastosis, at least one irradiation step is performed ex vivo on the individual's hemoblastosis sample containing nanoparticles or aggregates of nanoparticles, and the irradiated hemoblastosis sample is at least partially administered to the individual again prior to any optional subsequent in vivo cancer treatment in said individual. 6. The method of claim 5, wherein the hemoblastosis sample is a blood sample or whole blood volume of an individual. 7. The method according to any one of claims 4 to 6, wherein the lethally irradiated ex vivo malignant cells or the irradiated sample of hemoblastosis is at least partially re-administered to the individual together with at least one additional immunotherapeutic agent, while the lethally irradiated ex vivo malignant cells or an irradiated sample of hemoblastosis and at least one additional immunotherapeutic agent is administered to the individual simultaneously or separately. 8. The method according to any one of claims 1-7, where the dose of ionizing radiation is selected from 1.8, 2, 2.4, 2.5, 3, 3.2, 3.6, 4, 4.5, 5, 5.5, 6, 7, 8, 10, 15 and 20 Gy per fractional treatment. 9. The method according to any one of claims 1 to 8, wherein the metastatic malignancy involves (i) connective tissue and is selected from fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, malignant fibrous histiocytoma, (ii) endothelial or mesothelial tissue , and it is selected from hemangiosarcoma, angiosarcoma, lymphangiosarcoma, and mesothelioma, (iii) muscle tissue, and it is selected from leiomyosarcoma and rhabdomyosarcoma, (iv) epithelial tissue, and it is selected from adenocarcinoma, squamous cell carcinoma, and epidermoid carcinoma, (v) nervous tissue , and it is selected from glioblastoma multiforme, glioma, neuroblastoma, medulloblastoma, meningioma, neurofibrosarcoma, and schwannoma, and (vi) the APUD system, and it is selected from thyroid cancer, pancreatic cancer, gastric cancer, and colon cancer; or where the metastatic malignancy is melanoma. 10. The method of claim 9 wherein the metastatic cancer is or develops from a cancer selected from skin cancer, central nervous system cancer, head and neck cancer, lung cancer, kidney cancer, breast cancer, gastrointestinal cancer tract (GIST), prostate cancer, liver cancer, colon cancer, rectal cancer, anal cancer, esophageal cancer, malignant neoplasm of the male genitourinary system, malignant neoplasm of the female reproductive system, adrenal and retroperitoneal cancer, bone tissue sarcoma and soft tissues, malignant neoplasms of childhood, neuroblastoma, malignant neoplasms of the central nervous system and Ewing's sarcoma. 11. The method according to any one of claims 1 to 8, wherein the hemoblastosis affects the blood or lymphoid tissue and is selected from leukemia, myeloma and lymphoma. 12. The method according to any one of claims 1 to 11, where the nanoparticle or aggregate of nanoparticles is administered to the individual being treated, together with at least one immunotherapeutic agent, while the nanoparticle or aggregate of nanoparticles and at least one immunotherapeutic agent is administered to the individual simultaneously or separately . 13. The method according to any one of claims 1 to 12, wherein the composition further comprises a pharmaceutically acceptable carrier. 14. The method of claim 13, wherein the composition further comprises at least one immunotherapeutic agent. 15. The method of claim 14, wherein the at least one immunotherapeutic agent is selected from a monoclonal antibody, a cytokine, and a combination thereof. -