WO2024236048A1 - Combination therapy for treating tumors with radiotherapy - Google Patents

Abstract

The disclosure relates to a method of treating cancer in a subject in need thereof, said method comprising (i) a priming phase, and (ii) a treatment phase, wherein said priming phase comprises administering an efficient amount of said nanoparticles in combination with a low-dose radiotherapy, and said treatment phase comprises administering an efficient amount of said nanoparticles in combination with radiotherapy, optionally in combination with an immune checkpoint blocker, wherein said nanoparticles are nanoparticles containing an element with an atomic Z number higher than 20, preferably higher than 50, for example between 40 and 83, more preferably gadolinium or bismuth.

Description

COMBINATION THERAPY FOR TREATING TUMORS WITH RADIOTHERAPY

Technical Domain

The disclosure relates to a method of treating cancer in a subject in need thereof, said method comprising

(i) a priming phase, followed by

(ii) a treatment phase, wherein said priming phase comprises administering a therapeutically effective amount of nanoparticles in combination with a low-dose radiotherapy, and said treatment phase comprises either

(i) administering a therapeutically effective amount of nanoparticles in combination with radiotherapy, and optionally one or more immune check point blockers, or

(ii) administering a therapeutically effective amount of one or more immune checkpoint blockers, wherein said nanoparticles are nanoparticles containing an element with an atomic Z number higher than 20, preferably higher than 50, for example between 20 and 83, more preferably gadolinium and/or bismuth.

Background

In 2018, the American Association for Cancer Research (AACR) has estimated in its cancer progress report, that by 2035, the number of cancer cases will increase by 37%, from 1 ,735,350 cases to 2,387,304 cases (www.aacr.org), thus pointing out the urgent need to improve the efficacy of conventional cancer treatments and to identify novel anticancer therapeutic strategies. Radiotherapy is a cornerstone of anticancer therapies, which is used in among almost 60% of newly diagnosed cancer patients (1 ). Radiotherapy acts directly on cancer cells by inducing DNA lesions (such as single-strand breaks (SSB) and double-strand breaks (DSB)) (2) and indirectly through the generation of free radicals and reactive oxygen species (ROS) (3), both leading to the induction of cell death and/or senescence modalities (4- 6). Efficient DNA sensing and repair require cell cycle arrest and are essential to maintain cellular homeostasis and physiological functions (7). By overcoming these cellular functions, radiotherapy increases the genomic instability of cancer cells (8) and drives the accumulation of genomic DNA in the cytosol of irradiated cells (9-11 ). Furthermore, radiotherapy has also been shown to promote antitumor immune-mediated responses by inducing immunogenic cell death (12, 13), enhancing tumor antigen presentation (12, 14, 15), remodeling the tumor microenvironment (TME) (16) and eliciting systemic (abscopal) antitumor effects (17, 18). Recently, radiotherapy was shown to stimulate cGAS/STING-dependent signaling pathway after the production and the recognition of cytosolic nucleic acids (such as double-stranded DNA or micronuclei), leading to type I interferon (IFN-I) secretion and to the upregulation of interferon stimulated genes (ISGs) (10, 19, 20). However, radiotherapy still faces some limitations caused by the acute and chronic toxic side effects. Several applications of nanomedicine (such as metallic nanoparticles) have been developed to improve the therapeutic index of radiation therapy and to minimize its side effects on neighboring healthy tissues.

The AGulX (Activation and Guidance of Irradiation by X-ray) nanoparticles, which have a hydrodynamic diameter of approximately 5 nm, are composed of a polysiloxane matrix and Gadolinium (Gd) chelates. Considering their unique magnetic properties, AGulX have been initially used as an MRI contrast agent providing in addition to anatomic and structural information, imaging features (21 ) and supporting image-guided radiotherapy (22). The nanoparticles AGulX with the high atomic number of Gd (Z=64) can also be used to potentiate incident ionizing radiation into tumors, thus increasing the radiosensitivity of treated tumors. This process enhances the accumulation of DNA lesions and the production of ROS in cancer cells and leads to the destruction of numerous solid tumors (such as melanoma, glioblastoma, breast and lung carcinomas) (23, 24). Accordingly, AGulX is now used in numerous clinical trials for the treatment of solid tumor cancers (including brain metastases (NCT02820454, NCT03818386, NCT04899908), pancreatic cancer and lung tumors (NCT04789486), and advanced cervical cancer (NCT03308604)).

The discovery of immune checkpoint blockers has revolutionized antitumor strategies in clinic and changed our understanding of cancer biology. However, therapeutic response to immune checkpoint blockers varies among patients and tumor types (25). The abrogation of immune checkpoint blockers resistance mechanisms is now identified as the main challenge for several innovative antitumor therapeutic strategies. The combination of radiotherapy with immunotherapies has been extensively explored for its potential to amplify antitumor immune response and overcome treatment resistances (26). Radiotherapy was shown to modulate antitumor immune response through the release of immunogenic damage-associated molecular patterns (DAMPs) (such as the high mobility group box 1 protein (HMGB1 ) and ATP) by irradiated tumor cells, the stimulation of the antigen presentation by dendritic cells, the increase of CD8+ T cell and NK cell infiltration and their cytotoxicity activities into tumor bed, the enhanced expression of PD-L1 on tumor cells and the stimulation of type I IFN secretion by tumor cells and/or by distinct subsets of immune cells (such as dendritic cells and macrophages) (20, 27). Many studies investigating the impact of combining immune checkpoint blockers and radiotherapy on antitumor responses revealed the importance of IFN- mediated signaling pathway for the induction of an antitumor immune response. The secretion of type I IFN was recently involved for the optimal therapeutic response and the abscopal effect in mice that were treated by radiotherapy in combination with anti-CTLA-4 antibodies (19). Similarly, STING depletion was shown to hamper the abscopal effect elicited by the irradiation of primary tumors in presence of anti-CTLA-4 antibodies, and reduced mice overall survival (10). In this context, various therapeutic strategies currently combine metallic and/or multifunctional nanoparticles to radiotherapy and immune checkpoint blockers to overcome suppressive TME and optimize immune responses against tumors (28-31 ).

Despite the growing interest of combining AGulX with ionizing radiations for tumor radiosensitization, there is still a need to provide efficient and safe therapeutic methods for treating cancers combining radiotherapy and nanoparticles, in particular to overcome resistance to immunotherapies, and/or enhancing an anti-tumor response in certain cancers.

In the present disclosure, the inventors surprisingly revealed that a synergistic antitumor effect was induced by the combined administration of nanoparticles such as AGulX and radiotherapy, when low-dose of ionizing radiations is administered prior to a treatment phase consisting in ionizing radiations with said nanoparticles and/or immune checkpoint blockers. The results further demonstrated the ability of the nanoparticles, such as AGulX in combination with a low- dose radiotherapy as a priming phase to significantly reduce resistance to immune checkpoint blocker such as anti-PD1 acquired inhibitor resistance.

The present disclosure thus relates in particular to novel clinical irradiation plan that would integrate a combinatorial strategy with one or a few low-dose irradiations concomitantly with nanoparticles administration during the early time points of clinical protocol followed by the treatment phase using such nanoparticles in combination with either radiotherapy and/or immune checkpoint blockers.

Brief Description

Accordingly, a first aspect of the present disclosure relates to a method of treating cancer in a subject in need thereof, said method comprising

(i) a priming phase, followed by

(ii) a treatment phase, wherein said priming phase comprises administering a therapeutically effective amount of said nanoparticles in combination with a low-dose radiotherapy, and said treatment phase comprises either

(i) administering a therapeutically effective amount of said nanoparticles in combination with radiotherapy, and optionally one or more immune check point blockers, or

(ii) administering a therapeutically effective amount of one or more immune checkpoint blockers, wherein said nanoparticles are nanoparticles containing an element with an atomic Z number higher than 20, preferably higher than 50, for example between 20 and 83, more preferably gadolinium and/or bismuth.

The present disclosure also relates to a method for enhancing a systemic anti-tumoral response, typically a type I interferon response, in a subject in need thereof, said method comprising a priming phase, wherein said priming phase comprises administering a therapeutically effective amount of said nanoparticles in combination with a low-dose radiotherapy.

Legends to Figures

Figure 1. AGulX treatment in combination with ionizing radiation induces DNA damage and micronuclei accumulation in cancer cells. (A, B) Percentage of cells with micronuclei detected on Caco-2 (A) or CT26 (B) cells after 24 hours of treatment with control, 0.6 mM AGulX, 1.2 mM AGulX, 6 Gy IR, 0.6 mM AGulX combined with 6 Gy IR or 1.2 mM AGulX combined with 6 Gy IR are shown. (C, D) Percentages of cells with micronuclei (C) or with y- H2AX⁺ foci (D) detected on Caco-2 cells at 24 hours after treatment with control, 0.6 mM AGulX, 1.2 mM AGulX, 6 Gy IR, 0.6 mM AGulX combined with 6 Gy IR, or 1.2 mM AGulX combined with 6 Gy IR, combined with control or 10 pM RO-3306 are shown. (E, F) Percentages of cells with micronuclei (E) or with y-H2AX⁺ foci (F) detected on Caco-2 cells at 24 hours after treatment with control, 0.6 mM AGulX, 1 .2 mM AGulX, 6 Gy IR, 0.6 mM AGulX combined with 6 Gy IR, or 1.2 mM AGulX combined with 6 Gy IR, combined with control or 100 pM N-acetyl cysteine (NAC) are shown. Data are means ± S.E.M from three independent experiments. P-values (****p< 0.0001 , ***p< 0.001 and *P<0.05) were calculated by using two- way AN OVA test. Figure 2. AGulX treatment in combination with ionizing radiation promotes cGAS stabilization and localization to the micronuclei. (A, B) Percentages of cells with cGAS⁺ micronuclei detected on Caco-2 (A) or CT26 (B) cells after 24 hours of treatment with control, 0.6 mM AGulX, 1.2 mM AGulX, 6 Gy IR, 0.6mM AGulX combined with 6 Gy IR or 1.2 mM AGulX combined with 6 Gy IR are shown. (C, D) cGAS expression levels were determined using western blots on Caco-2 (C) or CT26 (D) cells after 24 hour treatment with control, 75 nM AGulX, 150 nM AGulX, 300 nM AGulX, 0.6 mM AGulX, 1.2 mM AGulX, 6 Gy IR, 75 nM AGulX combined with 6 Gy IR, 150 nM AGulX combined with 6 Gy IR, 300 nM AGulX combined with 6 Gy IR, 0.6 mM AGulX combined with 6 Gy IR, 1.2 mM AGulX combined with 6 Gy IR. Representative western blots are shown. GAPDH is used as loading control. Data are means ± S.E.M from two (C) or three (D) independent experiments. P-values (***p< 0.001 , **P< 0.01 ) were calculated by using two-way ANOVA test.

Figure 3. AGulX nanoparticles enhance the stimulation of IR-mediated cGAS/STINGZ Type I IFN signaling pathway. Expression levels of STINGS365*, STING, TBK1 S172* and TBK1 were determined using western blots on CT26 cells at day 6 after treatment with control, 75 nM AGulX, 150 nM AGulX, 300 nM AGulX, 0.6 mM AGulX, 1.2 mM AGulX, 6 Gy IR, 75 nM AGulX combined with 6 Gy IR, 150 nM AGulX combined with 6 Gy IR, 300 nM AGulX combined with 6 Gy IR, 0.6 mM AGulX combined with 6 Gy IR, 1 .2 mM AGulX combined with 6 Gy IR. Representative western blots are shown. GAPDH is used as loading control.

Figure 4. AGulX treatment in combination with high ionizing radiation in CT26 tumorbearing mice. (A-D) Tumor growth curves of CT26 tumor-bearing BALB/c mice and treated when tumors reached an average of 60 mm³ with control (A), AGulX (B), 8 Gy IR (C) or AGulX combined to 8 Gy IR (D) are shown. (E) Survival curves of CT26 tumor-bearing BALB/c mice treated with control, AGulX, 8 Gy IR or AGulX combined to 8 Gy IR. (F) Weight curves of CT26 tumor-bearing BALB/c mice treated with control, AGulX, 8 Gy IR or AGulX combined to 8 Gy IR are shown. Data are means from one (E and F) experiment (n=6 mice per group).

Figure 5. AGulX treatment in combination with ionizing radiation induces a CD8⁺ T celldependent antitumor response. (A-D) Tumor growth curves of CT26 tumor-bearing BALB/c mice that were treated when tumors reached an average of 60 mm³ with control (A), AGulX (B), 4 Gy IR (C) or 420 mg/kg AGulX combined to 4 Gy IR (D) are shown. (E) Survival curves of CT26 tumor-bearing BALB/c mice that were treated when tumors reached an average of 60 mm³ with control, AGulX, 4 Gy IR or AGulX combined to 4 Gy IR are shown. (F) Percentages of CD8⁺ T cells detected by flow cytometry in tumors after treatment with control, AGulX, 4 Gy IR or AGulX combined to 4 Gy IR are shown. (G, H) Tumor growth curves of CT26 tumorbearing BALB/c mice that were treated when tumors reached an average of 60 mm³ with control and anti-IgG antibodies (H), control and anti-CD8 antibodies (H), AGulX combined to 4 Gy IR and anti-IgG antibodies (G) or AGulX combined to 4 Gy IR and anti-CD8 antibodies (G) are shown. (I) Means of tumor growth curves presented in (G, H) are shown. (J) Survival curves of CT26 tumor-bearing BALB/c mice that were treated when tumors reached an average of 60 mm³ with control and anti-IgG antibodies, control and anti-CD8 antibodies, AGulX combined to 4 Gy and anti-IgG antibodies or AGulX combined to 4 Gy IR and anti-CD8 antibodies. (K) Weight curves of CT26 tumor-bearing BALB/c mice treated with control, AGulX, 4 Gy IR or AGulX combined to 4 Gy IR are shown. (L) Percentages of CD8⁺ T cells detected by flow cytometry in tumors treated with anti-IgG and anti-CD8 antibodies are shown. (M) Weight curves of CT26 tumor-bearing BALB/c mice and treated with control + anti-IgG antibodies, control + anti-CD8 antibodies, AGulX combined to 4 Gy IR + anti-IgG antibodies or AGulX combined to 4 Gy IR + anti-CD8 antibodies. Data are means ± S.E.M from three independent experiments and n=8-11 mice per group. P-values (****p< 0.0001 , ***p< 0.001 , **P< 0.01 and *P< 0.05) were calculated by using one-way ANOVA test (F, I), Mantel-cox survival test (E, J) or unpaired t-test (L).

Figure 6. Tumor growth and survival curves of PD1 -sensitive CT26 tumor-bearing mice treated with ionizing radiations, AGulX, anti-PD1 antibodies or combinatorial strategies. (A-H) Tumor growth curves of CT26 tumor-bearing BALB/c mice and treated when tumors reached 60 mm³ with control + anti-IgG antibodies (A), AGulX + anti-IgG antibodies (B), 4 Gy single-dose irradiation + anti-IgG antibodies (C), AGulX combined to 4 Gy single-dose irradiation + anti-IgG antibodies (D), control + anti-PD1 antibodies (E), AGulX + anti-PD1 antibodies (F), 4 Gy single-dose irradiation + anti-PD1 antibodies (G) or AGulX combined to 4 Gy single-dose irradiation + anti-PD1 antibodies (H) are shown. (I) Survival curves of CT26 tumor-bearing BALB/c mice treated with control + anti-IgG antibodies, AGulX + anti-IgG antibodies, 4 Gy single-dose irradiation + anti-IgG antibodies, AGulX combined to 4 Gy single-dose irradiation + anti-IgG antibodies, control + anti-PD1 antibodies, AGulX + anti- PD1 antibodies, 4 Gy localized single-dose irradiation + anti-PD1 antibodies or AGulX combined to 4 Gy localized single-dose irradiation + anti-PD1 antibodies .(J) Weight curves of CT26 tumor-bearing BALB/c mice treated with control + anti-IgG antibodies, AGulX and anti-IgG antibodies, 4 Gy IR + anti-IgG antibodies, AGulX combined to 4 Gy IR and anti-IgG antibodies, control and anti-PD1 antibodies, AGulX and anti-PD1 antibodies, 4 Gy IR and anti- PD1 antibodies or AGulX combined to 4 Gy IR and anti-PD1 antibodies are shown. Data are from one experiment with n=3-5 mice per group. P-values (**P<0.01 and *P< 0.05) were calculated by using Mantel-cox survival test (I). Figure 7. The combination of ionizing radiation and AGulX sensitizes tumors to PD1 immunotherapy. (A-H) Tumor growth curves of CT26 tumor-bearing BALB/c mice that were treated when tumors reached 100 mm³ with control + anti-IgG antibodies (A), AGulX and anti- IgG antibodies (B), 4 Gy IR and anti-IgG antibodies (C), AGulX combined to 4 Gy IR and anti- IgG antibodies (D), control and anti-PD1 antibodies (E), AGulX and anti-PD1 antibodies (F), 4 Gy IR and anti-PD1 antibodies (G) or AGulX combined to 4 Gy IR and anti-PD1 antibodies are shown (H). (I) Survival curves of CT26 tumor-bearing BALB/c mice that were treated with control + anti-IgG antibodies, AGulX and anti-IgG antibodies, 4 Gy IR + anti-IgG antibodies, AGulX combined to 4 Gy IR and anti-IgG antibodies, control and anti-PD1 antibodies, AGulX and anti-PD1 antibodies, 4 Gy IR and anti-PD1 antibodies or AGulX combined to 4 Gy IR and anti-PD1 antibodies are shown. (J) Weight curves of CT26 tumor-bearing BALB/c mice treated with control, AGulX and anti-IgG antibodies, 4 Gy IR + anti-IgG antibodies, AGulX combined to 4 Gy IR and anti-IgG antibodies, control and anti-PD1 antibodies, AGulX and anti-PD1 antibodies, 4 Gy IR and anti-PD1 antibodies or AGulX combined to 4 Gy IR and anti-PD1 antibodies are shown. Data are means from two independent experiments (n=8-11 mice per group). P-values (*P< 0.05) were calculated by using Wilcoxon survival test (I).

Detailed Description

The present disclosure follows in part from the surprising discovery as shown by the inventors of a synergistic antitumor effect induced by the combined administration of nanoparticles and radiotherapy, when low-dose of ionizing radiations is administered. The inventors also further demonstrated the ability of nanoparticles in combination with immunotherapy to significantly improve the tumor control of anti-PD1 resistant cancer cells.

These findings led the inventors to design novel clinical irradiation plans that would integrate combinatorial strategies with a few low dose irradiations concomitantly with nanoparticles administration during the early time points of clinical protocol followed by (i) standard of care radiotherapy treatment in combination with such nanoparticles, (ii) low-dose radiotherapy treatment optionally with the administration of immune checkpoint blockers, or (iii) standard of care immunotherapy using immune checkpoint blockers.

General Definitions

The use of the articles “a”, “an”, and “the” in both the description and claims are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising”, “having”, “being of’, “including", and “containing” are to be construed as open terms (i.e., meaning “including but not limited to”) unless otherwise noted. Additionally, whenever “comprising” or another open-ended term is used in an embodiment, it is to be understood that the same embodiment can be more narrowly claimed using the intermediate term “consisting essentially of” or the closed term “consisting of.

The term “about” or “ca.” has herein the meaning that the following value may vary for ± 20%, preferably ± 10%, more preferably ± 5%, even more preferably ± 2%, even more preferably ± 1 %.

As used herein, the term "treating" or "treatment" refers to one or more of (1 ) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and/or symptomatology); and (2) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of disease or reducing or alleviating one or more symptoms of the disease. In particular, with reference to the treatment of a tumor, the term “treatment” may refer to the inhibition of the growth of the tumor, or the reduction of the size of the tumor.

As used herein in the context of the disclosed combination therapy, the term “treatment” encompasses the administration of a therapeutically effective amount of the nanoparticles, optionally in combination with radiotherapy and/or immune checkpoint blockers. Such treatment may therefore comprise one or more administrations of the nanoparticles over a determined period. According to the present methods, the term “treatment” may encompass a “priming phase” and a “treatment phase” as described in detail herein.

The terms “tumor” and “cancer” are used interchangeably herein, e.g., both terms encompass solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term “cancer” or “tumor” includes premalignant, as well as malignant cancers and tumors and benign cancers. The term "cancer" as used herein includes primary malignant cells or tumors (e.g., those whose cells have not migrated to sites in the subject's body other than the site of the original malignancy or tumor) and secondary malignant cells or tumors (e.g., those arising from metastasis, the migration of malignant cells or tumor cells to secondary sites that are different from the site of the original tumor).

As used herein, the phrase “therapeutically effective amount” of a compound (e.g. AGulX) refer to an amount of the compound that will elicit a desired therapeutic response in at least a sub- population of subjects, for example, ameliorate the symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, at a reasonable benefit/risk ratio applicable to any medical treatment. It is understood, however, that the full therapeutic effect does not necessarily occur by administration of one dose, and may occur only after administration of a series of doses, and in combination with other treatment such as radiotherapy and immunotherapy as disclosed herein. The amount of active agent administered to the subject will depend on the type and severity of the disease or condition and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the desired effect, whether at the priming phase or at the treatment phase as disclosed herein. The skilled artisan will be able to determine appropriate dosages depending on these and other factors.

As used herein, the term "radiotherapy" is used interchangeably with “radiation therapy” for the treatment of diseases of oncological nature with irradiation corresponding to ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow.

The term "subject" or "patient" as used herein is intended to include animals, which are capable of suffering from or afflicted with a cancer or any disorder involving, directly or indirectly, a cancer. Examples of subjects include mammals, e.g., humans, apes, monkeys, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats. In an embodiment, the subject is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from cancers.

“Combination therapy” refers to therapies where a first active principle, e.g. the nanoparticles as disclosed herein (preferably AGu IX), is administered with either a combination partner, e.g. another drug as explained below, such as an immune checkpoint blocker, and/or with radiotherapy. These therapies may be administered concurrently or separately, i.e. separately within time intervals, especially where these time intervals allow that the combination partners and/or combination radiotherapies show a cooperative effect with the nanoparticles, e.g. synergistic effect (i.e. concomitantly). The single components may be packaged in a kit or separately. One or both of the components (e.g., powders or liquids) may be reconstituted or diluted to a desired dose prior to administration.

The terms “co-administration” or “combined administration” or the like as utilized herein are also meant to encompass administration of the selected combination partners, e.g. the nanoparticles and the immune checkpoint blocker, to a single subject in need thereof (e.g. a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time.

In the chemical formulae, the wavy line

represents the attachment point of the moiety.

The nanoparticles for use in the methods of the disclosure

The nanoparticles for use in the combination therapy of the present disclosure are nanoparticles containing an element with an atomic Z number higher than 20, preferably higher than 50, for example between 20 and 83, more preferably gadolinium and/or bismuth.

As used herein, the term “Z element” will refer to an element having a Z atomic number higher than 20.

As used herein, the term “high-Z element” will refer to an element having a Z atomic number higher than 50.

In specific embodiments, said Z element is selected in the group consisting of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, stannum, palladium, silver, zirconium, gold and bismuth, preferably in the group consisting of gadolinium and bismuth.

The Z elements are preferably cationic elements, comprised as complexes with chelating agents, such as organic chelating agents.

In specific preferred embodiments, the nanoparticles have a density inferior to 7 g/cm3, preferably inferior to 3g/cm3, for example comprised between 1 and 3 g/cm3. As used herein, the density refers to the mean density as measured according to the method of ISO 12154:2014 (en) “Determination of density by volumetric displacement - skeleton density by gas pycnometry”.

In other embodiments, the nanoparticles have a density of at least 7g/cm3.

In specific embodiments, the nanoparticles may further comprise a biocompatible coating allowing the nanoparticle stability between pH 6.5 and 7.5 in a physiological fluid. Agent suitable for such biocompatible coating includes without limitation biocompatible polymers, such as polyethylene glycol, polyethyleneoxide, polyacrylamide, biopolymers, polysaccharides, or polysiloxane. The nanoparticles may be a synthetic product with a mean hydrodynamic diameter between 1 nm and 500 nm, for example between about 4 and about 250 nm.

The size distribution of the nanoparticles is, for example, measured using a commercial particle sizer, such as a Malvern Zetasizer Nano-S particle sizer or equivalent based on PCS (Photon Correlation Spectroscopy). For the purposes of the invention, the term “mean hydrodynamic diameter” or “mean diameter” is defined as the mode value issued from the as-measured volume particle size distribution. Another method for measuring this parameter is described in standard ISO 13321 :1997.

A small size of the nanoparticles, between 1 and 10 nm, enables rapid renal elimination after injection and passive targeting to solid tumors after systemic administration. Accordingly, in preferred embodiments, said nanoparticles have a mean hydrodynamic diameter below 10 nm, for example between 1 nm and 8 nm and more preferably between 2 and 6 nm.

The shape of the nanoparticles is preferably spheroidal. The nanoparticles may comprise inorganic material, typically selected from an oxide, metal, a sulfide and any mixtures thereof or organic and inorganic material, or organic material.

In specific embodiments, said Z element is selected in the group consisting of lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, tin (stannum), palladium, silver, zirconium, gold and bismuth, preferably in the group consisting of gadolinium and bismuth.

The Z elements are preferably cationic elements, comprised as complexes with chelating agents, such as organic chelating agents.

The nanoparticles preferably have radiosensitizing properties, with a Z atomic number higher than 50 and/or contrast agent properties for imaging by magnetic resonance imaging, CT, SPCCT.

In one particular embodiment, the nanoparticles that can be used according to the disclosure are characterized in that they comprise at least a Z atomic number higher than 50, and at least one imaging or contrast agent suitable for one of the following imaging techniques:

PET or SPECT scintigraphy,

- fluorescence in the visible range or in the near-infrared range, In other particular embodiments, the nanoparticles may further comprise a radioactive isotope, suitable for its use in scintigraphy, such as PET/SPECT imaging.

Such radioactive isotope may be chosen from the group consisting of the radioactive isotopes of In, Tc, Ga, Cu, Zr, Y or Lu, for example:¹¹¹1n, "mTc,⁶⁷Ga,⁶⁸Ga,⁶⁴Cu,⁸⁹Zr,⁹⁰Y or¹⁷⁷Lu.

For fluorescence in the near-infrared range, the nanoparticles may additionally comprise a lanthanide chosen from Nd, Yb or Er.

For fluorescence in the visible range, the nanoparticles may additionally comprise a lanthanide chosen from Eu or Tb can be used.

For fluorescence in the near-infrared range, the nanoparticles may additionally comprise an organic fluorophore chosen from Cyanine 5.5, Cyanine 7, Alexa 680, Alexa 700, Alexa 750, Alexa 790, Bodipy, ICG.

In specific embodiments, said nanoparticles comprise, as Z element, a heavy metal, or a mixture of heavy metals, typically gadolinium, bismuth, or a mixture thereof.

In preferred embodiments, said nanoparticles comprise

• a biocompatible matrix,

• chelates covalently bound to said biocompatible matrix,

• high-Z elements complexed by the chelates.

Suitable biocompatible matrix includes without limitation biocompatible polymers, such as polyethylene glycol, polyethyleneoxide, polyacrylamide, biopolymers, polysaccharides, or polysiloxane. In specific embodiments, the nanoparticle comprises a silica-based matrix, for example essentially consisting of polyorganosiloxane.

In specific embodiments, the preferred nanoparticles for use as disclosed herein comprise chelating agents complexed to metal ions, wherein the chelating agents are covalently bonded to a biocompatible matrix, e.g. polyorganosiloxane.

In specific embodiments which may be combined with any of the previous embodiments, said nanoparticles comprise polyorganosiloxane, chelating agents covalently bound to said polyorganosiloxane, high-Z elements complexed by the chelating agents, Z being higher than 50.

As used herein, the term “chelating agent” or “chelates” refers to a compound or moiety capable of complexing one or more metal ions.

Exemplary chelating agents include, but not limited to, 1 ,4,7-triazacyclononanetriacetic acid (NOTA), l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), 1 ,4,7- triazacyclononane-l-glutaric acid-4, 7-diacetic acid (NODAGA), ethylene diamine tetra-acetic acid (EDTA), diethylene triaminepentaacetic acid (DTPA), cyclohexyl-l,2-diaminetetraacetic acid (CDTA), ethyleneglycol-0,0'- bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N- bis(hydroxybenzyl)- ethylenediamine-N,N'-diacetic acid (HBED), triethylene tetramine hexaacetic acid (TTHA), hydroxyethyidiamine tnacetic acid (HEDTA), 1 ,4,8,11- tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (TETA), and 1 , 4,7,10-tetraaza- 1,4,7,10- tetra-(2-carbamoyl methyl)-cyclododecane (TCMC) and 1 ,4,7,10-tetraazacyclododececane,1- (glutaric acid)-4,7,10-triacetic acid (DOTAGA), desferrioxamine (DFO).

In some embodiments, said nanoparticles comprise a chelating agent selected among the following:

wherein the wavy bond indicates the bond connecting the chelating moiety to a linking group of a biocompatible matrix forming the nanoparticle.

In specific embodiments, at least 65% of the chelating agent present in the nanoparticle is chelated to at least one Z element, preferably at least 75% and more preferably at least 80%.

In a specific embodiment, that may be preferably combined with the previous embodiment, said chelating agent is complexed to a metal ion, and more specifically to gadolinium and/or bismuth, preferably DOTA or DOTAGA chelating Gd³⁺ and/or Bi³⁺.

In specific and preferred embodiments, the mean number of Z element per nanoparticle, for example the number of rare earth elements, e.g. gadolinium (optionally as chelated with DOTAGA) per nanoparticle, is between 4 and 200, preferably between 4 and 80, typically around 15.

In certain embodiments, the method of treatment as disclosed herein is carried out with nanoparticles which comprise or essentially consist of:

• polyorganosiloxane with a silicon weight ratio of at least 8% of the total weight of the nanoparticle, preferably between 8% and 50%,

• chelating agents covalently bound to said polyorganosiloxane, in a proportion comprising between 4 and 200, preferably between 4 and 80 per nanoparticle, and,

• at least one element having a Z of at least 20 chelated to at least a part of the chelating agents, for example, an atomic number of at least 50.

In a specific embodiment, said nanoparticles are gadolinium-chelated polyorganosiloxane nanoparticles of the following formula:

wherein POS is a matrix of polyorganosiloxane, and, n is comprised between 0 and 100, m is comprised between 0 and 100, provided that n+m is comprised between 4 and 200, preferably 4 and 80, and wherein said nanoparticle has a mean hydrodynamic diameter comprised between 1 and 8 nm, preferably 2 and 6 nm.

In another specific embodiment, said nanoparticles are gadolinium-chelated polyorganosiloxane nanoparticles of the following formula:

wherein POS is a matrix of polyorganosiloxane, and, n is comprised between 4 and 200, preferably 4 and 80, and wherein said nanoparticles have a mean hydrodynamic diameter comprised between 1 and 8 nm, preferably between 2 and 6 nm.

More specifically, said gadolinium-chelated polysiloxane nanoparticles as described in the above formula are ultrafine nanoparticles, more preferably AGulX nanoparticles as described in the next section.

Ultrafine nanoparticles that can be used according to the methods of the disclosure may be obtained or obtainable by a top-down synthesis route comprising the steps of: a) obtaining a metal (M) oxide core, wherein M is an element with an atomic number Z as described previously, preferably gadolinium, b) adding a polyorganosiloxane shell around the M oxide core, for example via a sol gel process, c) grafting a chelating agent to the polyorganosiloxane shell, so that the chelating agent is bound to said polyorganosiloxane shell by an -Si-C- covalent bond, thereby obtaining a core-shell precursor nanoparticle, and, d) transferring the core-shell precursor nanoparticle in an aqueous solution for dissolution of the metal oxide core and purifying, wherein the grafted agent is in sufficient amount to dissolve the metal (M) oxide core at step d. and to complex the cationic form of (M) thereby reducing the mean hydrodynamic diameter of the resulting hybrid nanoparticle to a mean hydrodynamic diameter less than 10 nm, for example, between 1 and 8 nm, typically between 2 and 6 nm. These nanoparticles obtained according to the mode described above do not comprise a core of metal oxide encapsulated by at least one coating. More details regarding the synthesis of these nanoparticles are given hereafter.

This top-down synthesis method results in observed sizes typically of between 1 and 8 nm, more specifically between 2 and 6 nm. The term then used herein is ultrafine nanoparticles.

Alternatively, another "one-pot” synthesis method is described hereafter to prepare said core- free nanoparticles with a mean hydrodynamic diameter less than 10 nm, for example, between 1 and 8 nm, typically between 2 and 6 nm.

Further details regarding these ultrafine or core-free nanoparticles, the processes for synthesizing them and their uses are described in patent application WO2011/135101 , WO2018/224684 or WO2019/008040, which is incorporated by way of reference.

Generally, those skilled in the art will be able to easily produce nanoparticles used according to the invention. More specifically, the following elements will be noted:

For nanoparticles of core-shell type, based on a core of lanthanide oxide or oxyhydroxide, use may be made of a production process using an alcohol as solvent, as described for example in P. Perriat et aL, J. Coll. Int. Sci, 2004, 273, 191 ; O. Tillement et aL, J. Am. Chem. Soc., 2007, 129, 5076 and P. Perriat et aL, J. Phys. Chem. C, 2009, 113, 4038.

For the PCS matrix, several techniques can be used, derived from those initiated by Stoeber (Stoeber, W; J. Colloid Interf Sci 1968, 26, 62). Use may also be made of the process used for coating as described in Louis et al. (Louis et al., 2005, Chemistry of Materials, 17, 1673-1682) or international application WO 2005/088314.

In practice, synthesis of ultrafine nanoparticles is for example described in Mignot et al. Chem. Eur. J. 2013, 19, 6122-6136: Typically, a precursor nanoparticle of core/shell type is formed with a lanthanide oxide core (via the modified polyol route) and a polyorganosiloxane shell (via sol/gel); this object has, for example, a mean hydrodynamic diameter of around 5-10 nm. A lanthanide oxide core of very small size (adjustable less than 10 nm) can be produced in an alcohol by means of one of the processes described in the following publications: P. Perriat et aL, J. Coll. Int. Sci, 2004, 273, 191 ; O. Tillement et aL, J. Am. Chem. Soc., 2007, 129, 5076 and P. Perriat et aL, J. Phys. Chem. C, 2009, 113, 4038. These cores can be coated with a layer of polyorganosiloxane according to, for example, a protocol described in the following publications: C. Louis et al., Chem. Mat., 2005, 17, 1673 and O. Tillement et al., J. Am. Chem. Soc., 2007, 129, 5076.

Chelating agents specific for the intended metal cations (for example DOTAGA for Gd³⁺) are grafted to the surface of the polyorganosiloxane; it is also possible to insert a part thereof inside the layer, but the control of the formation of the polyorganosiloxane is complex and simple external grafting gives, at these very small sizes, a sufficient proportion of grafting.

The core is destroyed by dissolution (for example by modifying the pH or by introducing complexing molecules into the solution). This destruction of the core then allows a diffusion and a rearrangement of the polyorganosiloxane layer (according to a mechanism of slow corrosion or collapse), which makes it possible to finally obtain a polyorganosiloxane object with a complex morphology, the characteristic dimensions of which are of the order of magnitude of the thickness of the polyorganosiloxane layer, i.e. smaller than the objects produced up until now. Furthermore, this operation makes it possible to increase the number of M (e.g. gadolinium) per nm³ in comparison with a theoretical polyorganosiloxane nanoparticle of the same size but comprising M (e.g. gadolinium) only at the surface. The mean number of M per nanoparticle can be evaluated by virtue of the M/Si atomic ratio measured by EDX or by ICP/MS. Typically, this number of M per ultrafine nanoparticle may be comprised between 4 and 200, preferably 4 and 80.

The nanoparticles may be separated from the synthesis residues by means of a method of dialysis or of tangential filtration, for example on a membrane comprising pores of appropriate size.

Removing the core thus makes it possible to decrease from a particle size of approximately 5- 10 nanometers in mean hydrodynamic diameter to a size below 8 nm, for example between 2- 8 nm.

Alternatively, the nanoparticles for use in the method of treatment according to the present disclosure may be obtained or obtainable by a synthesis method (“one-pot synthesis method”) comprising the mixing of at least one hydroxysilane or alkoxysilane which is negatively charged at physiological pH and at least one chelating agent chosen from polyamino polycarboxylic acids with at least one hydroxysilane or alkoxysilane which is neutral at physiological pH, and/or at least one hydroxysilane or alkoxysilane which is positively charged at physiological pH and comprises an amino function, wherein:

- the molar ratio A of neutral silane(s) to negatively charged silane(s) is defined as follows: 0 < A < 6, preferably 0.5 < A < 2;

- the molar ratio B of positively charged silane(s) to negatively charged silane(s) is defined as follows: 0 < B < 5, preferably 0.25 < B < 3;

- the molar ratio C of neutral and positively charged silanes to negatively charged silane(s) is defined as follows 0 < C < 8, preferably 1 < C < 4.

According to a more specific embodiment of such one pot synthesis method, the method comprises the mixing of at least one alkoxysilane which is negatively charged at physiological pH, said alkoxysilane being chosen among APTES-DOTAGA, TANED, CEST and mixtures thereof, with

- at least alkoxysilane which is neutral at physiological pH, said alkoxysilane being chosen among TMOS, TEOS and mixtures thereof, and/or

- APTES which is positively charged at physiological pH, wherein:

- the molar ratio A of neutral silane(s) to negatively charged silane(s) is defined as follows: 0 < A < 6, preferably 0.5 < A < 2;

- the molar ratio B of positively charged silane(s) to negatively charged silane(s) is defined as follows: 0 < B < 5, preferably 0.25 < B < 3;

- the molar ratio C of neutral and positively charged silanes to negatively charged silane(s) is defined as follows 0 < C < 8, preferably 1 < C < 4.

According to a specific embodiment, the one-pot synthesis method comprises the mixing of APTES-DOTAGA which is negatively charged at physiological pH with

- at least one alkoxysilane which is neutral at physiological pH, said alkoxysilane being chosen among TMOS, TEOS and mixtures thereof, and/or

- APTES which is positively charged at physiological pH, wherein:

- the molar ratio A of neutral silane(s) to negatively charged silane(s) is defined as follows: 0 < A < 6, preferably 0.5 < A < 2;

- the molar ratio B of positively charged silane(s) to negatively charged silane(s) is defined as follows: 0 < B < 5, preferably 0.25 < B < 3; - the molar ratio C of neutral and positively charged silanes to negatively charged silane(s) is defined as follows 0 < C < 8, preferably 1 < C < 4.

Nanoparticles with targeting molecules for targeting tumors

The nanoparticles may further be functionalized with molecules which allow targeting of the nanoparticles to specific tissues, more particularly specific tumors. Said agents can be coupled to the nanoparticle by covalent couplings, or trapped by non-covalent bonding, for example by encapsulation or hydrophilic/hydrophobic interaction or using a chelating agent.

For example, in such embodiment, each nanoparticle comprises a chelating agent which has an acid function, for example DOTA or DOTAGA. The acid function of the nanoparticle may be activated for example using EDC/NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide / N- hydrosuccinimide) in the presence of an appropriate amount of targeting molecules. The nanoparticles thus grafted are then purified, for example by tangential filtration.

AGulX Nanoparticles

In a more particularly preferred embodiment, said gadolinium-chelated polyorganosiloxane based nanoparticle is the AGulX nanoparticle of the formula below

wherein POS is polyorganosiloxane and n is between 4 and 200, preferably between 4 and 80, and having a mean hydrodynamic diameter of 5 ± 3 nm and a mean mass of between 5 and 250 kDa, preferably between 5 and 100kDa.

In specific embodiments, said AGulX nanoparticle can also be described by the average chemical formula: (GdSi3-8C24-34 N₅-80i5-3oH4o-6o, nbWJx, where x is between 4 and 200, preferably between 4 and 80.

Methods for preparing AGulX nanoparticles is described for example in G. Le Due, S. Roux, A. Paruta-Tuarez, S. Dufort, E. Brauer, A. Marais, C. Truillet, L. Sancey, P. Perriat, F. Lux, O. Tillement, « Advantages of gadolinium based ultrasmall nanoparticles vs molecular gadolinium chelates for radiotherapy guided by MRI for glioma treatment », Cancer Nanotechnology, 2014, 5, 4.

Pharmaceutical formulations of the nanoparticles for use according to the disclosed methods

When employed as pharmaceuticals, the compositions comprising said nanoparticles for use as provided herein can be administered in the form of pharmaceutical formulation of a suspension of nanoparticles. These formulations can be prepared as described herein or elsewhere, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated.

In particular, said pharmaceutical formulations for use as described herein, contain, as the active ingredient, a suspension of nanoparticles, as provided herein, in combination with one or more pharmaceutically acceptable carriers (excipients). In making a pharmaceutical formulation provided herein, the nanoparticles composition may be, for example, mixed with an excipient or diluted by an excipient. When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier, or medium for the nanoparticle composition.

Thus, the pharmaceutical formulations can be in the form of powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), sterile injectable solutions, sterile packaged powders, and the like.

In specific embodiments, said pharmaceutical formulation for use as described herein, is sterile lyophilized powder, contained in a pre-filled vial to be reconstituted, for example in an aqueous solution for intravenous injection. In specific embodiments, said lyophilized powder comprises, as the active ingredient, an efficient amount of said nanoparticles, typically gadolinium- chelated polysiloxane based nanoparticles, and more specifically AGulX nanoparticles as described herein. In certain specific embodiments, said lyophilized powder contains either about between 200 mg and 15 g per vial, for example between 280 and 320 mg of AGulX per vial, typically 300 mg of AGulX per vial, or about between 800 mg and 1200 mg, for example 1000 mg of AGulX per vial, or about between 2000 mg and 3000 mg, for example 2500 mg of AGulX per vial. Such powder may further contain one or more additional excipients, and in particular CaCh, for example between 1 and 8 mg of dehydrated CaCh per g of AGulX, typically 4.4 mg of dehydrated CaCh per g of AGulX. Said lyophilized powder may be reconstituted in an aqueous solution, typically water for injection. Accordingly, in specific embodiments, said pharmaceutical solution for use according to the present disclosure is a solution for injection, comprising, as the active ingredient, an efficient amount of said high-Z containing nanoparticles, typically gadolinium-chelated polysiloxane based nanoparticles, and more specifically AGulX nanoparticles as described herein. For example, said solution for injection for use in the methods as disclosed herein is a solution of gadolinium-chelated polysiloxane based nanoparticles, typically AGulX nanoparticles, between 15 and 150 mg/mL, for example 50 and 120 mg/mL, typically about 100 mg/mL, optionally comprising one or more additional pharmaceutically acceptable excipient, for example between 0.1 and 0.3 mg/mL of CaCh, typically about 0.22 mg/mL of CaCh.

In specific embodiments, said pharmaceutical formulation for use as described herein, is sterile liquid formulation contained in a vial ready to be injected, for example intravenously. In specific embodiments, said liquid pharmaceutical formulation comprises, as the active ingredient, an efficient amount of said nanoparticles, typically gadolinium-chelated polysiloxane based nanoparticles, and more specifically AGulX nanoparticles as described herein. In certain specific embodiments, said lyophilized powder contains either about between 200 mg and 15 g per vial, for example between 280 and 320 mg of AGulX per vial, typically 300 mg of AGulX per vial, or about between 800 mg and 1200 mg, for example 1000 mg of AGulX per vial, or about between 2000 mg and 3000 mg, for example 2500 mg of AGulX per vial. Such liquid formulation may further contain one or more additional excipients, and in particular CaCh, for example between 1 and 8 mg of dehydrated CaCh per g of AGulX, typically 4.4 mg of dehydrated CaCh per g of AGulX. . Accordingly, in specific embodiments, said pharmaceutical liquid formulation for use according to the present disclosure contains as the active ingredient, an efficient amount of said high-Z containing nanoparticles, typically gadolinium-chelated polysiloxane based nanoparticles, and more specifically AGulX nanoparticles as described herein. For example, said liquid formulation for injection for use in the methods as disclosed herein is a solution of gadolinium-chelated polysiloxane based nanoparticles, typically AGulX nanoparticles, between 15 and 150 mg/mL, for example 50 and 120 mg/mL, typically about 100 mg/mL, optionally comprising one or more additional pharmaceutically acceptable excipient, for example between 40 and 80 mg of NaCI per g of AGulX, typically about 60 mg of NaCI per g of AGulX.

Methods of use of the present disclosure

The present disclosure relates to a method of inducing or enhancing a systemic anti-tumoral response in a subject in need thereof, typically a type I interferon response, said method comprising a priming phase combining (i) nanoparticles administration (e.g. AGulX or other nanoparticles as described above) and (ii) low-dose radiotherapy in said subject as disclosed above. Said method may advantageously precede anti-cancer therapies, including immunotherapies with immune checkpoint blockers or radiotherapy, whether in combination with nanoparticles (e.g. AGulX) or not.

The present disclosure also relates to a method of treating a cancer in a subject in need thereof, comprising

(i) a priming phase, followed by

(ii) a treatment phase, wherein said priming phase comprises administering a therapeutically effective amount of said nanoparticles in combination with a low-dose radiotherapy, and said treatment phase comprises either

(iii) administering a therapeutically effective amount of said nanoparticles in combination with radiotherapy, and optionally one or more immune check point blockers, or

(iv) administering a therapeutically effective amount of one or more immune checkpoint blockers, wherein said nanoparticles are nanoparticles containing an element with an atomic Z number higher than 20, preferably higher than 50, for example between 20 and 83, more preferably gadolinium and/or bismuth.

The present disclosure relates to nanoparticles containing an element with an atomic Z number higher than 20, preferably higher than 50, for example between 20 and 83, more preferably gadolinium and/or bismuth, for use in a method of treating a cancer in a subject in need thereof, comprising

(i) a priming phase, followed by

(ii) a treatment phase, wherein said priming phase comprises administering a therapeutically effective amount of said nanoparticles in combination with a low-dose radiotherapy, and said treatment phase comprises either (i) administering a therapeutically effective amount of said nanoparticles in combination with radiotherapy, and optionally one or more immune check point blockers, or

(ii) administering a therapeutically effective amount of one or more immune checkpoint blockers.

The disclosure further relates to nanoparticles for use in the manufacture of a medicament for the treatment of a cancer in a subject in need thereof, method comprising

(i) a priming phase, followed by

(ii) a treatment phase, wherein said priming phase comprises administering a therapeutically effective amount of said nanoparticles in combination with a low-dose radiotherapy, and said treatment phase comprises either

(i) administering a therapeutically effective amount of said nanoparticles in combination with radiotherapy, and optionally one or more immune check point blockers, or

(ii) administering a therapeutically effective amount of one or more immune checkpoint blockers, wherein said nanoparticles are nanoparticles containing an element with an atomic Z number higher than 20, preferably higher than 50, for example between 20 and 83, more preferably gadolinium and/or bismuth.

In specific embodiments, said cancer is a cancer with a solid tumor, preferably selected from the group consisting of cancer of head and neck, skin cancer, liver cancer, colon cancer, pancreatic cancer, breast cancer, ovary cancer, uterus cancer, cervical cancer, brain cancer, thyroid cancer, bladder cancer and renal cancer. In preferred embodiments, said cancer is selected from the group consisting of lung cancer, head and neck cancer, colorectal cancer, cervical cancer and melanoma.

In preferred embodiments, the method of the present disclosure for treating cancer comprises at least two steps of administering an efficient amount of said nanoparticles to said subject, including at least one step of nanoparticles administration at the priming phase, and at least one step of nanoparticles administration at the treatment phase, as described hereafter.

The priming phase The priming phase is a combined therapy characterized by a concomitant administration of an efficient amount of nanoparticles with a low-dose radiotherapy, with the objective of triggering a CD8+ T cell-dependent anti-tumor response. The inventors indeed showed that a synergistic effect of nanoparticles administration and radiotherapy may be observed in triggering a IFN type I response and CD8+ T cell-dependent anti-tumor response, when the subject is exposed to a reduced irradiation dose (low-dose radiotherapy).

In specific embodiments, the priming phase may therefore advantageously precede a treatment phase, consisting of either

(i) a low-dose radiotherapy in combination with nanoparticles (e.g. AGulX) therapy, optionally with immunotherapy with immune checkpoint blockers, such as PD1/PD-L1 blockers,

(ii) a standard of care radiotherapy in combination with nanoparticles (e.g. AGulX) therapy, optionally with immunotherapy with immune checkpoint blockers, such as PD1/PD-L1 blockers, or

(iii) an immunotherapy with immune checkpoint blockers, such as PD1/PD-L1 inhibitors, (without additional radiotherapy), optionally in combination with nanoparticles (e.g. AGulX).

In specific embodiments, an efficient amount of nanoparticles are administered intravenously at the priming phase. In a specific embodiment, an efficient amount of nanoparticles is an amount sufficient to trigger the interferon type I response in a subject in need thereof in combination with low-dose radiotherapy. The skilled person will determine such amount depending on the age, sex, pathologies or other conditions of the subject. In specific embodiments, said nanoparticles are administered once, at the priming phase at an amount ranging from 15 to 150mg/kg, e.g. 100 mg/kg. In specific embodiments, said nanoparticles are administered at an amount ranging from 15 - 40 mg/kg, from 40 - 80 mg/kg or from 80- 150mg/kg.

The priming phase also comprises exposing the tumor to be treated to an efficient low-dose of ionizing radiations, wherein said ionizing radiations are typically selected from X-Rays, gamma-Rays, electron beams (electrons), and ion beams (such as protons). X-rays are particularly preferred ionizing radiations. Linear accelerators (LINAC) and betatrons produce X-rays of increasingly greater energy. The use of machines to focus radiation (such as X-rays) on a cancer site is called external beam radiotherapy.

Ionizing radiations are typically of about 2 KeV to about 25 000 KeV, in particular of about 2 KeV to about 6000 KeV. The low-dose radiotherapy is preferably initiated the same day as the first administration of said nanoparticles. For example, low-dose irradiation is performed after the step of administering the nanoparticles (e.g. AGulX), for example 1 hour to 24 hours, preferably 1 to 12 hours, typically 1 hour to 8 hours, e.g. 4 hours after the first administration of the nanoparticles (e.g. AGulX).

In specific embodiments, low-dose radiotherapy at the priming phase comprises 1 to 3 fractions of ionizing radiations at a dose ranging from 0.2 to 2 Gy per fraction, for example from 0.5 to 1 .8 Gy, or from 0.5 to 1 .5 Gy, prior to the treatment phase. In preferred embodiments, low-dose radiotherapy at the priming phase comprises 1 to 3 fractions of ionizing radiations at a dose above or equal to 0.2 Gy but below 1 .8 Gy, for example from 0.5 to 1 .5 Gy.

In specific embodiments, said priming phase comprises

(i) a single administration of nanoparticles as herein defined, preferably AGulX, at an amount ranging from 15 to 150 mg/kg, for example 100 mg/kg, and

(ii) concomitantly irradiating the subject with low-dose radiotherapy consisting of 1 to 3 fractions of ionizing radiations at a dose ranging from 0.2 to 2 Gy, preferably below 1.8Gy, for example the first fraction being administered between 1 to 24 hours, for example between 1 to 12 hours, after the administration of the nanoparticles.

In specific embodiments, the total amount of ionizing radiations at the priming phase is below 6Gy, for example below 5.4 Gy, for example between 0.5Gy and 2Gy, or even below 1 ,8Gy.

The disclosure also relates to a method of inducing or enhancing a systemic anti-tumoral response in a subject in need thereof, typically a type I interferon response, said method comprising a priming phase combining nanoparticles administration (e.g. AGulX) and low-dose radiotherapy as disclosed above.

Said type I interferon response may be characterized in particular in tumor biopsies, either through an immune infiltration analysis evaluating an increase in CD8⁺ T-cell tumor infiltration and pro-inflammatory reprograming of tumor macrophages through immunohistochemistry, multiplexed imaging, flow cytometry, single cell RNA sequencing or spatial transcriptomics, or by RT-qPCR assessing the increase of type I interferon RNA expression, as compared to the level prior to said priming phase.

Said method of inducing or enhancing a systemic anti-tumoral response according to the present disclosure may be appropriate for patients eligible to any cancer therapy, and more specifically to immunotherapy with immune checkpoint blockers, such as PD1/PD-L1 inhibitors, or radiotherapy (optionally in combination with other anti-tumor agents, such as chemotherapy).

Such method may be particularly beneficial in subjects which are not eligible to standard of care radiotherapy but only to low-dose radiotherapy or to immunotherapy without radiotherapy.

It may also be particularly advantageous in subjects which are resistant or refractory to immune checkpoint blockers therapy, in particular to prior PD1/PD-L1 inhibitors therapy.

In preferred embodiments, said method of inducing or enhancing a systemic anti-tumoral response according to the present disclosure may be advantageously selected in combination with the “treatment phase” as disclosed in the next section.

Specific treatment phases according to the present disclosure

The benefits of the priming phase are particularly expected with the following treatment phases, comprising administration of a therapeutically effective amount of nanoparticles (e.g. AGulX) in combination with either:

(i) a standard of care radiotherapy, optionally with an immunotherapy with immune checkpoint blockers, such as PD1/PD-L1 inhibitors,

(ii) a low-dose radiotherapy, optionally with immunotherapy with immune checkpoint blockers, such as PD1/PD-L1 inhibitors, or

(iii) an immunotherapy with immune checkpoint blockers, such as PD1/PD-L1 inhibitors, (without additional radiotherapy).

In specific embodiments, in order to maximize the anti-tumoral effect of the priming phase, said treatment phase is initiated at least 48 hours after the last low-dose irradiation of the priming phase, preferably between 7 days and 28 days after last low-dose irradiation of the priming phase.

For example, the subject receives a single administration of nanoparticles (e.g. AGulX) concomitantly with 1 -3 fractions of low-dose ionizing radiations, and a second administration of nanoparticles for the treatment phase, at least 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days, or 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27 or 28 days after said first single administration at the priming phase. Subsequent administrations of nanoparticles may be carried out for the treatment phase. The nanoparticles at the treatment phase when used in combination with radiotherapy preferably comprises an element with an atomic number Z higher than 50 for obtaining radiosensitizing properties.

As used herein, the term “radiosensitizing” means the capacity to interact with ionizing radiation to enhance the generation of chemical reactive species and thus trigger biological damages and/or dysregulation.

The nanoparticles (e.g. AGulX) can be administered to the subject using different possible routes such as local (intra-tumoral (IT), intra-arterial (IA)), subcutaneous, intravenous (IV), intradermic, airways (inhalation), intra-peritoneal, intramuscular, intra-thecal, intraocular or oral route.

In preferred embodiments, said treatment phase comprises administering said nanoparticles, e.g. AGulX, intravenously to the subject.

The amount of the nanoparticles will be determined to optimize the anti-tumor efficacy, for example when used as radiosentizing agent in combination with radiotherapy, or as immunotherapeutic agent in combination with immune checkpoint blockers. In specific embodiments, said treatment phase comprises administering the nanoparticles one or more times at a dose ranging from 50 mg/kg to 150 mg/kg, typically, from 80 to 120 mg/kg, for example 100 mg/kg.

In specific embodiments, said treatment phase comprises administering the nanoparticles (e.g. AGulX) the same day as the first irradiation of the treatment phase, or 1 or 2 days before.

Typically, when using fractionated radiation therapy, the nanoparticles (e.g. AGulX) may be further injected once every week during multiple sessions of radiation therapy. For example, in a specific embodiment, the treatment phase as disclosed herein further comprises at least one additional step of injecting a therapeutically effective amount of the nanoparticles (e.g AGulX) within 5-10 days after one or more sessions of fractionated radiation therapy, for example 7 days after said injecting step for the first session of fractionated radiation therapy.

In another particular embodiment, the treatment phase also comprises a step of pre-filling the tumor with the nanoparticles (e.g. AGulX).

Such pre-filling step comprises administering an efficient amount of nanoparticles (e.g. AGulX) in said subject in need thereof within a period between 2 and 10 days, preferably 2 and 7 days, prior to the first exposure to radiation therapy in the treatment phase. Said efficient amount for the pre-filling step may be comprised between 50 mg/kg and 150 mg/kg, typically, between 80 and 120 mg/kg, for example 100 mg/kg.

Indeed, considering the remanence of the nanoparticle in the tumor, preferably AGu IX, it may be advantageous to “pre-fiH” the tumor with the nanoparticles with the period between 2 and 10 days, and then administering again the nanoparticles e.g. , 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours), prior to the administration of the first session of radiotherapy, to the subject with the tumor to be treated.

Hence, in a particular embodiment, the treatment phase comprises

(i) injecting a first effective amount of said nanoparticles (e.g. AGulX) in said subject in need thereof within a period between 2 and 10 days, preferably between 2 and 7 days, prior to the first irradiation of the tumor,

(ii) injecting a second effective amount of said nanoparticles within a period between 1 hour to 12 hours prior to the first irradiation of the tumor, and,

(iii) exposing the subject to one or more sessions of radiation therapy, for example with standard of care radiotherapy as described below.

Such protocol is also described in WO2021/018898 which content is incorporated herein by reference in its entirety.

Three distinct specific embodiments for the treatment phase will now be described hereafter.

Standard of care radiotherapy (optionally with immunotherapy)

In one embodiment, said treatment phase comprises a standard-of-care radiotherapy in combination with administering a therapeutically effective amount of said nanoparticles, optionally in combination with administering a therapeutically effective amount of one or more immune checkpoint blocker.

As used herein, examples of standard of care radiotherapy are described without limitation for example in:

Clinical practice guidelines published by European Society for Medical Oncology (ESMO), American Society for Clinical Oncology (ASCO), European Society for Radiation Therapy Oncology (ESTRO), American Society for Radiation Therapy Oncology (ASTRO). For example: • Brain metastases: Radiation Therapy for Brain Metastases: An ASTRO Clinical Practice Guideline. Practical Radiation Oncology, Volume 12, ISSUE 4, P265-282, July 2022

• Gliomas: High-Grade Malignant Glioma: ESMO Clinical Practice Guidelines Published in 2014 Ann Oncol (2014) 25 (suppl 3): iii93-iii101 . Authors: R. Stupp, M. Brada, M. J. van den Bent, J.-C. Tonn, G. Pentheroudak.

• Early and locally advanced non-small-cell lung cancer (NSCLC): Definitive radiation therapy in locally advanced non-small cell lung cancer: Executive summary of an American Society. Practical Radiation Oncology (2015), Volume 5, ISSUE 3, P141-148 and its updates. ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of Oncology 28 (Supplement 4): ivl-iv21 , 2017, and its updates.

• Cancer of the pancreas: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Annals of Oncology 26 (Supplement 5): v56-v68, 2015, and its updates.

• Melanoma: ESMO consensus conference recommendations on the management of locoregional melanoma: under the auspices of the ESMO Guidelines Committee. O. Michelin et al. Annals of Oncology Volume 31 , ISSUE 11 , P1449-1461 , November 2020

• Head & Neck cancers: Radiation therapy for oropharyngeal squamous cell carcinoma: Executive summary of an ASTRO Evidence-Based Clinical Practice Guideline. David J. Scher et al. Practical Radiation Oncology, Special Article Volume 7, ISSUE 4, P246- 253, July 2017

• Rectal cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and followup. Annals of Oncology 28 (Supplement 4): iv22-iv40, 2017

• Cervical cancer: Radiation Therapy for Cervical Cancer: An ASTRO Clinical Practice Guideline. Junzo Chino et al. Practical Radiation Oncology ractical (2020) 10, 220-234

Additional clinical guidelines may be obtained on the National Comprehensive Cancer Network website: https://www.nccn.org/guidelines, notably for indications listed above under the names:

• Central Nervous System Cancers

• Cervical Cancers

• Cutaneous melanoma

• Head & Neck Cancers

• Non Small Cell Lung Cancers

• Pancreatic Adenocarcinoma

• Rectal Cancers The amount of radiation used in radiation therapy is measured in gray (Gy), and varies depending on the type and stage of cancer being treated.

For standard of care radiotherapy, the typical total dose for a solid tumor ranges from 10 to 120 Gy. Many other factors are considered by radiation oncologists when selecting a dose, including whether the patient is receiving chemotherapy, patient co-morbidities, whether radiation therapy is being administered before or after surgery, and the degree of success of surgery.

In specific embodiments, the total dose is preferably fractionated (spread out over time). Amount and schedules (planning and delivery of ionizing radiations, fraction dose, fraction delivery schema, total dose alone or in combination with other anti-cancer agents etc) is defined for any disease/anatomical site/disease stage patient setting/age and constitutes the standard of care for any specific situation. The primary and/or the metastatic tumors may be targeted with the radiotherapy.

In other specific embodiments, said standard-of-care radiotherapy at the treatment phase comprises exposing the subject with a total dose of radiations comprised between 15 and 80 Gy, for example at a dose per fraction comprised between 1.8 and 10 Gy, for example fractionated in 5 to 40 fractions, and for example, with 5 fractions per week.

A typical conventional fractionation schedule for adults for the methods of the present disclosure may be 2.5 to 3.5 Gy per day, five days a week, for example for 2 to 3 consecutive weeks. In specific embodiments, said radiotherapy consists of exposing the subject to a total dose of ionizing radiations between 20 and 35 Gy, for example 30 Gy.

In other specific embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 2 to 8 Gy, and the total dose is administered preferably in a maximum of 10 fractions, for example 10 fractions of 3 Gy, 5 days a week for two weeks. This dosing may be suitable for example for whole brain radiotherapy (WBRT) for treating brain metastases.

In certain embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 4 to 10 Gy, and the total dose is ranging from 20-30 Gy, administered preferably in a maximum of 5 fractions, for example in 5 or 6 days. This dosing may be suitable for example for stereotactic radiotherapy for treating brain metastases.

In certain embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 1 ,5 to 2 Gy, for example 1 ,8 Gy, and the total dose is ranging from 40-60 Gy, administered preferably in 25 fractions, for example in 4-6 weeks. This dosing may be suitable for example for stereotactic radiotherapy for treating cervical cancer.

In certain embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 1 ,5 to 4 Gy, for example 1 ,8 Gy, and the total dose is ranging from 60-90 Gy, for example 80 Gy, administered preferably in 30-50 fractions, for example in 6-10 weeks. This dosing may be suitable for example for External Beam Radiation Therapy (EBRT) for treating prostate cancer.

In certain embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 1 ,5 to 4 Gy, for example 2 Gy, and the total dose is ranging from 35-70 Gy, for example 60 Gy, administered preferably in 10-35 fractions, for example in 2-6 weeks. This dosing may be suitable for example for External Beam Radiation Therapy for treating glioblastoma.

In certain embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 6 to 10 Gy, for example 8 Gy, and the total dose is ranging from 30-50 Gy, for example 40 Gy, administered preferably in 5-6 fractions, for example in 5 consecutive days. This dosing may be suitable for example for Stereotactic Magnetic resonance-guided Adaptive Radiation Therapy (SMART) for treating pancreatic cancer.

In certain embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 1.5 to 3 Gy, for example 2 Gy, and the total dose is ranging from 30-60 Gy, for example 54 Gy, administered preferably in 5 to 6 weeks. This dosing may be suitable for example for Stereotactic Body Radiation Therapy (SBRT) for treating pancreatic cancer.

In certain embodiments, the subject is exposed to a dose of ionizing radiations per fraction of about 8 to 12 Gy, for example 10 Gy, and the total dose is ranging from 40-60 Gy, for example 50 Gy, administered preferably in 5-6 fractions, for example in 5 consecutive days. This dosing may be suitable for example for SMART for treating non-small lung cell cancer.

In specific embodiments, magnetic resonance image-guided radiation therapy may be used in the standard of care radiotherapy.

As used herein, the term “magnetic resonance image-guided radiation therapy” refers to the combined use of a magnetic resonance imaging unit with a radiation therapy unit, allowing real-time imaging of target volumes and organs at risk before and during treatment delivery with replanning as necessary. Magnetic resonance image-guided radiation therapy is particularly useful in sites affected by inter- and intrafraction motion such as the thorax, abdomen and pelvis. Typically, organ at risk and target visualization can be improved with magnetic resonance image-guided radiation therapy compared to cone beam computerized tomography, which can permit plan adaptation and reduction of toxicity. This technique may use automated beam gating for precise and accurate dosing. When the tumor moves, the beam automatically stops. Clinicians can therefore shrink margins with confidence while escalating dose.

Any MR-Linac for image-guided radiation therapy may be used in the methods of treatment of the present disclosure.

The MR-Linac as currently used includes perpendicular beam-field systems (e.g. Elekta and Viewray) which are now commercial products. Other systems include inline orientation (Aurora-RT) and both perpendicular and inline orientation (Australian). The field strength of the current MR-Linac systems vary from 0.35 T; for example the MRIdian (Viewray), 0.5T (Aurora- RT, MagneTx), and 1.5T (Unity, Elekta). Further details of the MR-Linac systems and their mode of use are described in Liney et al Clinical Oncology 30 (2018) 686-691.

In preferred embodiment, a MR-Linac used in the treatment phase of the present disclosure has a magnetic field strength of 0.5 T or lower, for example 0.35 T. Such embodiment is particularly preferred with AGulX nanoparticles as described in the previous sections.

Examples of such method of image-guided radiation therapy in combination with nanoparticles (e.g. AGulX) are also disclosed in WO2021/228867.

Low-dose radiotherapy at the treatment phase, optionally with immunotherapy with immune checkpoint blockers, such as PD1/PD-L1 inhibitors

In one embodiment, said treatment phase comprises a low-dose radiotherapy in combination with administering a therapeutically effective amount of said nanoparticles, optionally in combination with administering a therapeutically effective amount of one or more immune checkpoint blocker.

As used herein, low-dose radiotherapy may be described for example in Herrera FG et al. Low- Dose Radiotherapy Reverses Tumor Immune Desertification and Resistance to Immunotherapy. Cancer Discov. 2022 Jan;12(1 ):108-133. doi: 10.1158/2159-8290. CD-21- 0003. Epub 2021 Sep 3. PMID: 34479871 ; PMCID: PMC9401506.

For low-dose radiotherapy used in the treatment phase, the typical total dose for a solid tumor ranges from 2 to 20 Gy, preferably with a dose per fraction which does not exceed 2 Gy, preferably below 1 .8 Gy. In specific embodiments, said low-dose radiotherapy at the treatment phase comprises 3 to 12 fractions of ionizing radiations at a dose ranging from 0.2 to 2 Gy per fraction, for example from 0.5 to 1 .8 Gy, or from 0.5 to 1 .5 Gy, for example once every two weeks for 6-24 weeks.

Such treatment phase in combination with the priming phase is particularly suitable to subjects which are not eligible to the standard-of-care radiotherapy as described in the previous section. For example, such patients are not eligible after having been evaluated as being at high risk to undergo a standard-of-care treatment with radiotherapy, or as having a high risk of intolerance, or after determination of their benefit/risk ratio.

Such patient may be typically a patient with physical inactivity (with overweight or obesity), or with exposure to etiological agents (such as infectious agents) or with familial or hereditary risk factors.

In specific embodiments, said subject is having a paediatric cancer, for example a child below 12 years old, more specifically in child suffering from high grade glioma.

Immunotherapy with immune checkpoint blockers, such as PD1/PD-L1 inhibitors (with or without radiotherapy)

The inventors have also shown that the priming phase may overcome resistance to immunotherapy.

Hence, the treatment phase following the priming phase advantageously comprises administering a therapeutically effective amount of an immune checkpoint blocker, such as PD1/PD-L1 inhibitor, in combination with the nanoparticles (e.g. AGulX) either, with, or without radiotherapy as described above.

Examples of preferred immune checkpoint blockers include without limitation anti-PD-1 or anti- PD-L1 antibodies, (e.g. nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, durvalumab or atezolizumab) or anti-CTLA-4 antibodies (e.g. ipilimumab), and the like.

In specific embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as KEYTRUDA®, lambrolizumab, and MK-3475; see WO2008/156712), PDR001 (Novartis; also known as spartalizumab; see WO 2015/112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012/145493), cemiplimab (Regeneron; also known as REGN-2810; see WO 2015/112800), JS001 (TAIZHOU JUNSHI PHARMA; see Si-Yang Liu et al., J. Hematol. Oncol. 70: 136 (2017)), BGB-A317 ("Tislelizumab" Beigene; see WO 2015/35606 and US 2015/0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015/085847; Si-Yang Liu et al., ./. Hematol. Oncol. 70: 136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011 ; see WO2014/179664), GLS-010 (Wuxi/Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang Liu et al., J. Hematol. Oncol. 70: 136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO 2014/194302), AGEN2034 (Agenus; see WO 2017/040790), MGA012 (Macrogenics, see WO 2017/19846), IBI308 (Innovent; see WO 2017/024465, WO 2017/025016, WO 2017/132825, and WO 2017/133540), and BCD- 100 (Biocad)).

In specific embodiments suitable unit dose for intravenous administration of a PD-1 inhibitor can be selected from 100 to 2000 mg (e.g. preferably selected from the group consisting of nivolumab, pembrolizumab, avelumab, durvalumab, cemiplimab, pidilizumab, dostarlimab, or atezolizumab).

The immune checkpoint blockers are preferably administered according to the prescribing information and standard -of-care therapies.

In specific embodiments, said immune checkpoint blocker is an anti-PD-1 antibody, for example Nivolumab or Pembrolizumab, and said anti-PD-1 antibody is administered at the treatment phase by intravenous injection, at a unit dose ranging from 200 - 240 mg, for example every two weeks, or 400-480 mg every four weeks, for example a first administration concomitant with the first administration of the nanoparticles at the treatment phase or with the first irradiation.

Other Combination therapy with the treatment phase

The nanoparticles for use as disclosed herein may be further administered in combination to, other drugs e.g cytotoxic, anti-proliferative, or other anti-tumor agents, e.g. for the treatment or prevention of cancer disorders, as mentioned above.

Suitable cytotoxic, anti-proliferative or anti-tumor agents may include without limitation cisplatin, doxorubicin, taxol, etoposide, irinotecan, topotecan, paclitaxel, docetaxel, epothilones, tamoxifen, 5-fluorouracil, methotrexate, temozolomide, cyclophosphamide, tipifarnib, gefitinib, erlotinib, imatinib, gemcitabine, uracil mustard, chlormethine, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, busulfan, carmustine, lomustine, streptozocin, dacarbazine, floxuridine, cytarabine, 6-mercaptopurine, 6- thioguanine, fludarabine phosphate, oxaliplatin, folinic acid, pentostatin, vinblastine, vincristine, vindesine, bleomycin, dactinomycin, daunorubicin, epirubicin, idarubicin, mithramycin, deoxycoformycin, mitomycin-C, L-asparaginase, teniposide.

In some embodiments, the additional cytotoxic, anti-proliferative or anti-tumor agent is administered simultaneously with the nanoparticles provided herein. In some embodiments, the cytotoxic, anti-proliferative or anti-tumor agent is administered after administration of the nanoparticles provided herein. In some embodiments, the cytotoxic, anti-proliferative or antitumor agent is administered prior to administration of the nanoparticles herein.

The additional therapeutic agents provided herein can be effective over a wide dosage range and are generally administered in an effective amount. It will be understood, however, that the amount of the therapeutic agent administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual subject, the severity of the subject's symptoms, and the like.

Other aspects and advantages of the method of the disclosure will become apparent in the following examples, which are given for purposes of illustration only.

Specific Embodiments

Embodiment 1 : Nanoparticles for use in a method of treating cancer in a subject in need thereof, said method comprising

(i) a priming phase, followed by

(ii) a treatment phase, wherein said priming phase comprises administering a therapeutically effective amount of said nanoparticles in combination with a low-dose radiotherapy, and said treatment phase comprises either

(i) administering a therapeutically effective amount of said nanoparticles in combination with radiotherapy, and optionally one or more immune check point blockers, or

(ii) administering a therapeutically effective amount of one or more immune checkpoint blockers, wherein said nanoparticles are nanoparticles containing an element with an atomic Z number higher than 20, preferably higher than 50, for example between 20 and 83, more preferably gadolinium and/or bismuth.

Embodiment 2: The nanoparticles for use according to Embodiment 1 , wherein said low-dose radiotherapy at the priming phase comprises 1 to 3 fractions of ionizing radiations at a dose ranging from 0.2 to 2 Gy per fraction, for example equal or above 0.5 Gy and below 1 .8 Gy, or from 0.5 to 1.5 Gy.

Embodiment 3: The nanoparticles for use according to Embodiment 1 or 2, wherein said low- dose radiotherapy at the priming phase comprises 1 fraction of ionizing radiations at a dose ranging from 0.2 to 2 Gy per fraction, for example from 0.5 to 1 .8 Gy, or from 0.5 to 1 .5 Gy.

Embodiment 4: The nanoparticles for use according to any one of Embodiments 1 -3, wherein said low-dose radiotherapy at the priming phase is initiated the same day as the administration of said nanoparticles.

Embodiment 5: The nanoparticles for use according to any one of Embodiments 1 -4, wherein said treatment phase is initiated at least 48 hours after the last fraction of ionizing radiations of the priming phase, preferably between 7 days and 28 days after last fraction of ionizing radiations of the priming phase.

Embodiment 6: The nanoparticles for use of any one of Embodiments 1 -5, wherein said nanoparticles are injected intravenously.

Embodiment 7: The nanoparticles for use of any one of Embodiments 1 -6, wherein said nanoparticles have a mean hydrodynamic diameter below 10 nm, for example between 1 and 8 nm, more preferably between 2 and 6 nm.

Embodiment 8: The nanoparticles for use of any one of Embodiments 1 -7, wherein said nanoparticles comprise, as Z element, a heavy metal, or a mixture of heavy metals.

Embodiment 9: The nanoparticles for use of any one of Embodiments 1 -8, wherein said nanoparticles comprise, as Z element, gadolinium, bismuth, or a mixture thereof.

Embodiment 10: The nanoparticles for use of any one of Embodiments 1 -9, wherein said nanoparticles comprise chelates of Z element, for example chelates of rare earth elements, and more preferably chelates of gadolinium, bismuth, or a mixture thereof. Embodiment 11 : The nanoparticles for use of any one of Embodiments 1 -10, wherein said nanoparticles comprise between 5 and 50 % by weight of said Z element, preferably between 8 and 30%.

Embodiment 12: The nanoparticles for use of any one of Embodiments 1 -11 , wherein said nanoparticles comprise

• polyorganosiloxane,

• chelates covalently bound to said polyorganosiloxane,

• high-Z elements complexed to the chelates.

Embodiment 13: The nanoparticles for use of any one of Embodiments 1 -12, wherein said nanoparticles comprise

• polyorganosiloxane with a silicon weight ratio of at least 8% of the total weight of the nanoparticle, preferably between 8% and 50%,

• chelates covalently bound to said polyorganosiloxane, in a proportion comprising between 4 and 200, preferably between 4 and 80 per nanoparticle, and,

• high-Z elements complexed to the chelates.

Embodiment 14: The nanoparticles for use of any one of Embodiments 1 -13, wherein said nanoparticles comprise chelates for complexing the Z elements, obtained by grafting one or more of the following chelating agents on said nanoparticles: 1 ,4,7-triazacyclononanetriacetic acid (NOTA), l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), 1 ,4,7- triazacyclononane-l-glutaric acid-4, 7-diacetic acid (NODAGA), ethylene diamine tetra-acetic acid (EDTA), diethylene triaminepentaacetic acid (DTPA), cyclohexyl-l,2-diaminetetraacetic acid (CDTA), ethyleneglycol-0,0'- bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N- bis(hydroxybenzyl)- ethylenediamine-N,N'-diacetic acid (HBED), triethylene tetramine hexaacetic acid (TTHA), hydroxyethyidiamine tnacetic acid (HEDTA), 1 ,4,8,11- tetraazacyclotetradecane-N,N^,,N",N'"-tetraacetic acid (TETA), and 1 , 4,7,10-tetraaza- 1,4,7,10- tetra-(2-carbamoyl methyl)-cyclododecane (TCMC) and 1 ,4,7,10-tetraazacyclododececane,1- (glutaric acid)-4,7,10-triacetic acid (DOTAGA), desferrioxamine (DFO).

Embodiment 15: The nanoparticles for use of any one of Embodiments 1 -14, wherein said nanoparticles comprise chelates for complexing the Z elements selected from the group consisting of

wherein the wavy bond indicates the bond connecting the chelate to a linking group of the nanoparticle.

Embodiment 16: The nanoparticles for use of any one of Embodiments 1-15, wherein said nanoparticles are gadolinium and/or bismuth-chelated polyorganosiloxane nanoparticles of the following formula:

wherein POS is a matrix of polyorganosiloxane, and, n is comprised between 0 and 100, m is comprised between 0 and 100, provided that n+m is comprised between 4 and 200, preferably 4 and 80, and wherein said nanoparticle has a mean hydrodynamic diameter comprised between 1 and 8 nm, preferably 2 and 6 nm.

Embodiment 17: The nanoparticles for use of any one of Embodiments 1-16, wherein said nanoparticles are gadolinium-chelated polyorganosiloxane nanoparticles of the following formula

wherein POS is a matrix of polyorganosiloxane, and, n is comprised between 4 and 200, preferably 4 and 80, more preferably 4 and 20, and wherein the hydrodynamic diameter is comprised between 1 and 8 nm, preferably between 2 and 6 nm.

Embodiment 18: The nanoparticles for use of any one of Embodiments 1 -17, wherein said nanoparticles are AGulX nanoparticles.

Embodiment 19: The nanoparticles for use of any one of Embodiments 1 -18, wherein said nanoparticles are administered once at the priming phase at an amount ranging from 15 to 150 mg/kg, e.g. 100 mg/kg.

Embodiment 20: The nanoparticles for use of any one of Embodiments 1 -19, wherein said treatment phase comprises administering the nanoparticles one or more times at a unit dose ranging from 50 mg/kg to 150mg/kg, typically, from 80 to 120 mg/kg, for example 100 mg/kg.

Embodiment 21 : The nanoparticles for use according to any one of Embodiments 1-20, wherein said treatment phase comprises a standard-of-care radiotherapy in combination with administering a therapeutically effective amount of said nanoparticles, optionally in combination with administering a therapeutically effective amount of one or more immune checkpoint blocker.

Embodiment 22: The nanoparticles for use according to Embodiment 21 , wherein said standard-of-care radiotherapy at the treatment phase comprises exposing the subject with a total dose of radiations comprised between 15 and 80 Gy, for example at a dose comprised between 1.8 and 10 Gy per fraction, for example fractionated in 5 to 40 fractions, and for example, with 5 fractions per week.

Embodiment 23: The nanoparticles for use according to any one of Embodiments 1-20, wherein said treatment phase comprises a low-dose radiotherapy in combination with administering a therapeutically effective amount of said nanoparticles, in combination with administering a therapeutically effective amount of one or more immune checkpoint blockers.

Embodiment 24: The nanoparticles for use according to Embodiment 23, wherein said low- dose radiotherapy at the treatment phase comprises 3 to 12 fractions of ionizing radiations at a dose ranging from 0.2 to 2 Gy per fraction, for example from 0.5 to 1 .8 Gy, or from 0.5 to 1 .5 Gy, for example once every two weeks for 6-24 weeks. Embodiment 25: The nanoparticles for use according to any one of Embodiments 1-20, wherein said treatment phase comprises administering a therapeutically effective amount of one or more immune checkpoint blocker without radiotherapy.

Embodiment 26: The nanoparticles for use of any one of Embodiments 23-25, wherein the subject is selected from subjects ineligible to standard-of-care treatment involving radiotherapy.

Embodiment 27: The nanoparticles for use of any one of Embodiments 23-26, wherein the cancer is a pediatric cancer.

Embodiment 28: The nanoparticles for use of any one of Embodiments 1 -27, wherein said cancer is a cancer with solid tumor, preferably selected from the group consisting of cancer of head and neck, skin cancer, liver cancer, colon cancer, pancreatic cancer, breast cancer, ovary cancer, uterus cancer, cervical cancer, brain cancer, thyroid cancer, bladder cancer and renal cancer.

Embodiment 29: The nanoparticles for use of any one of Embodiments 1 -28, wherein said cancer is selected from the group consisting of lung cancer, head and neck cancer and melanoma.

Embodiment 30: The nanoparticles for use of any one of Embodiments 1 -29, wherein said subject is selected from subjects resistant or refractory to immune checkpoint blocker therapy, such as PD-1 inhibitor therapy.

Embodiment 31 : The nanoparticles for use of any one of Embodiments 1 -30, wherein treatment phase comprises administering a therapeutically effective amount of an immune checkpoint blocker.

Embodiment 32: The nanoparticles for use of Embodiment 31 , wherein said immune checkpoint blocker is selected from the group consisting of anti-PD-1 or anti-PD-L1 antibodies, e.g. nivolumab, pembrolizumab, avelumab, cemiplimab, durvalumab or atezolizumab, or anti- CTLA-4 antibodies (e.g. ipilimumab).

Embodiment 33: The nanoparticles for use of any one of Embodiments 1 -32, wherein the treatment phase further comprises a step of imaging the tumor by magnetic resonance imaging (MRI) after administering said nanoparticles, wherein said nanoparticles is used as a T1 contrast agent for said MRI. Embodiment 34: The nanoparticles for use of any one of Embodiments 1 -33, wherein said priming phase induces or enhances a systemic anti-tumoral response at the treatment phase.

Embodiment 35: A method for enhancing a systemic anti-tumoral response, typically a type I interferon response, in a subject in need thereof, said method comprising

(i) a priming phase, and wherein said priming phase comprises administering a therapeutically effective amount of said nanoparticles in combination with a low-dose radiotherapy.

Embodiment 36: A method for treating cancer in a subject in need thereof, said method comprising

(i) a priming phase, followed by

(ii) a treatment phase, wherein said priming phase comprises administering a therapeutically effective amount of said nanoparticles in combination with a low-dose radiotherapy to said subject, and said treatment phase comprises either

(i) administering a therapeutically effective amount of said nanoparticles in combination with radiotherapy, and optionally one or more immune check point blockers, or

(ii) administering a therapeutically effective amount of one or more immune checkpoint blockers, wherein said nanoparticles are nanoparticles containing an element with an atomic Z number higher than 20, preferably higher than 50, for example between 20 and 83, more preferably gadolinium and/or bismuth.

Embodiment 37: The method of Embodiment 35 or 36, wherein said low-dose radiotherapy at the priming phase comprises 1 to 3 fractions of ionizing radiations at a dose ranging from 0.2 to 2 Gy per fraction, for example equal or above 0.5 Gy and below 1 .8 Gy, or from 0.5 to 1 .5 Gy. Embodiment 38: The method of Embodiment 35, 36 or 37, wherein said low-dose radiotherapy at the priming phase comprises 1 fraction of ionizing radiations at a dose ranging from 0.2 to 2 Gy per fraction, for example from 0.5 to 1 .8 Gy, or from 0.5 to 1 .5 Gy.

Embodiment 39: The method of any one of Embodiments 35-38, wherein said low-dose radiotherapy at the priming phase is initiated the same day as the administration of said nanoparticles.

Embodiment 40: The method of any one of Embodiments 36-39, wherein said treatment phase is initiated at least 48 hours after the last fraction of ionizing radiations of the priming phase, preferably between 7 days and 28 days after the last fraction of ionizing radiations of the priming phase.

Embodiment 41 : The method of Embodiments 36-40, wherein said nanoparticles are injected intravenously.

Embodiment 42: The method of any one of Embodiments 36-41 , wherein said nanoparticles have a mean hydrodynamic diameter below 10 nm, for example between 1 and 8 nm, more preferably between 2 and 6 nm.

Embodiment 43: The method of any one of any one of Embodiments 36-42, wherein said nanoparticles comprise, as element with a Z atomic number higher than 20, a heavy metal, or a mixture of heavy metals.

Embodiment 44: The method of any one of Embodiments 36-43, wherein said nanoparticles comprise, as element with a Z atomic number higher than 20, gadolinium, bismuth, and/or a mixture thereof.

Embodiment 45: The method of any one of Embodiments 36-44, wherein said nanoparticles comprise chelates of elements with a Z atomic number higher than 20 and , for example chelates of rare earth elements, and more preferably chelates of gadolinium, bismuth, or a mixture thereof.

Embodiment 46: The method of any one of Embodiments 36-45, wherein said nanoparticles comprise between 5 and 50 % by weight of said element with a Z atomic number higher than 20, preferably between 8 and 30%.

Embodiment 47: The method of any one of Embodiments 36-46, wherein said nanoparticles comprise polyorganosiloxane, chelates covalently bound to said polyorganosiloxane, high-Z elements complexed to the chelates.

Embodiment 48: The method of any one of Embodiments 36-47, wherein said nanoparticles comprise

• polyorganosiloxane with a silicon weight ratio of at least 8% of the total weight of the nanoparticle, preferably between 8% and 50%,

• chelates covalently bound to said polyorganosiloxane, in a proportion comprising between 4 and 200, preferably between 4 and 80 per nanoparticle, and,

• high-Z elements complexed to the chelates.

Embodiment 49: The method of any one of Embodiments 36-48, wherein said nanoparticles comprise chelates for complexing the elements, obtained by grafting one or more of the following chelating agents on said nanoparticles: 1 ,4,7-triazacyclononanetriacetic acid (NOTA), l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid (DOTA), 1 ,4,7- triazacyclononane-l-glutaric acid-4, 7-diacetic acid (NODAGA), ethylene diamine tetra-acetic acid (EDTA), diethylene triaminepentaacetic acid (DTPA), cyclohexyl-l,2-diaminetetraacetic acid (CDTA), ethyleneglycol-0,0'- bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid (EGTA), N,N- bis(hydroxybenzyl)- ethylenediamine-N,N'-diacetic acid (HBED), triethylene tetramine hexaacetic acid (TTHA), hydroxyethyidiamine tnacetic acid (HEDTA), 1 ,4,8,11- tetraazacyclotetradecane-N,N',N",N'"-tetraacetic acid (TETA), and 1 , 4,7,10-tetraaza- 1,4,7,10- tetra-(2-carbamoyl methyl)-cyclododecane (TCMC) and 1 ,4,7,10-tetraazacyclododececane,1- (glutaric acid)-4,7,10-triacetic acid (DOTAGA), desferrioxamine (DFO).

Embodiment 50: The method of any one of Embodiments 36-49, wherein said nanoparticles comprise chelates for complexing the elements having a Z atomic number higher than 20 selected from the group consisting of

wherein the wavy bond indicates the bond connecting the chelate to a linking group of the nanoparticle.

Embodiment 51 : The method of any one of Embodiments 36-50, wherein said nanoparticles are gadolinium and/or bismuth-chelated polyorganosiloxane nanoparticles of the following formula:

wherein POS is a matrix of polyorganosiloxane, and, n is comprised between 0 and 100, m is comprised between 0 and 100, provided that n+m is comprised between 4 and 200, preferably 4 and 80, and wherein said nanoparticle has a mean hydrodynamic diameter comprised between 1 and 8 nm, preferably 2 and 6 nm.

Embodiment 52: The method of any one of Embodiments 36-51 , wherein said nanoparticles are gadolinium-chelated polyorganosiloxane nanoparticles of the following formula

wherein POS is a matrix of polyorganosiloxane, and, n is comprised between 4 and 200, preferably 4 and 80, more preferably 4 and 20, and wherein the hydrodynamic diameter is comprised between 1 and 8 nm, preferably between 2 and 6 nm.

Embodiment 53: The method of any one of Embodiments 36-52, wherein said nanoparticles are AGulX nanoparticles. Embodiment 54: The method of any one of Embodiments 36-53, wherein said nanoparticles are administered once at the priming phase at an amount ranging from 15 to 150 mg/kg, e.g. 100 mg/kg.

Embodiment 55: The method of any one of Embodiments 36-54, wherein said treatment phase comprises administering the nanoparticles one or more times at a unit dose ranging from 50 mg/kg to 150mg/kg, typically, from 80 to 120 mg/kg, for example 100 mg/kg.

Embodiment 56: The method of any one of Embodiments 36-55, wherein said treatment phase comprises a standard-of-care radiotherapy in combination with administering a therapeutically effective amount of said nanoparticles, optionally in combination with administering a therapeutically effective amount of one or more immune checkpoint blocker.

Embodiment 57: The method of Embodiment 56, wherein said standard-of-care radiotherapy at the treatment phase comprises exposing the subject with a total dose of radiations comprised between 15 and 80 Gy, for example at a dose comprised between 1.8 and 10 Gy per fraction, for example fractionated in 5 to 40 fractions, and for example, with 5 fractions per week.

Embodiment 58: The method of any one of Embodiments 1-55, wherein said treatment phase comprises a low-dose radiotherapy in combination with administering a therapeutically effective amount of said nanoparticles, in combination with administering a therapeutically effective amount of one or more immune checkpoint blockers.

Embodiment 59: The method of Embodiment 58, wherein said low-dose radiotherapy at the treatment phase comprises 3 to 12 fractions of ionizing radiations at a dose ranging from 0.2 to 2 Gy per fraction, for example from 0.5 to 1 .8 Gy, or from 0.5 to 1 .5 Gy, for example once every two weeks for 6-24 weeks.

Embodiment 60: The method of any one of Embodiments 36-55, wherein said treatment phase comprises administering a therapeutically effective amount of one or more immune checkpoint blockers without radiotherapy.

Embodiment 61 : The method of any one of Embodiments 36-60, wherein the subject is selected from subjects ineligible to standard-of-care treatment involving radiotherapy.

Embodiment 62: The nanoparticles for use of any one of Embodiments 58-61 , wherein the cancer is a pediatric cancer. Embodiment 63: The method of any one of Embodiments 36-62, wherein said cancer is a cancer with solid tumor, preferably selected from the group consisting of cancer of head and neck, skin cancer, liver cancer, colon cancer, pancreatic cancer, breast cancer, ovary cancer, uterus cancer, cervical cancer, brain cancer, thyroid cancer, bladder cancer and renal cancer.

Embodiment 64: The method of any one of Embodiments 36-63, wherein said cancer is selected from the group consisting of lung cancer, head and neck cancer and melanoma.

Embodiment 65: The method of any one of Embodiments 36-64, wherein said subject is selected from subjects resistant or refractory to immune checkpoint blocker therapy, such as PD-1 inhibitor therapy.

Embodiment 66: The method of any one of Embodiments 36-65, wherein treatment phase comprises administering a therapeutically effective amount of an immune checkpoint blocker.

Embodiment 67: The method of Embodiment 65, wherein said immune checkpoint blocker is selected from the group consisting of anti-PD-1 or anti-PD-L1 antibodies, e.g. nivolumab, pembrolizumab, avelumab, cemiplimab, durvalumab or atezolizumab, or anti-CTLA-4 antibodies (e.g. ipilimumab).

Embodiment 68: The method of any one of Embodiments 36-66, wherein the treatment phase further comprises a step of imaging the tumor by magnetic resonance imaging (MRI) after administering said nanoparticles, wherein said nanoparticles is used as a T1 contrast agent for said MRI.

Embodiment 69: The nanoparticles for use of any one of Embodiments 36-67, wherein said priming phase induces or enhances a systemic anti-tumoral response at the treatment phase.

EXAMPLES

Materials and Methods

Cells and reagents

The Caco-2 cells were obtained from Pr. Guido Kroemer (Institut Gustave Roussy, France) and cultured in Eagle’s Minimum Essential Medium (EMEM, ATCC® 30-2003™) with 20% fetal bovine serum (FBS) (#CVFSVF0001 , Eurobio), 100 U/mL penicillin (#15140130, Life Technologies), and 100 pg/mL streptomycin (#15140130, Life Technologies at 37°C. The CT26 cells (ATCC-CRL-2638) were purchased from ATCC and cultured in Roswell Park Memorial Institute Medium (RMPI-1640-Glutamax #61870044, Life Technologies) with 10% FBS, 100 U/mL penicillin, and 100 pg/mL streptomycin at 37°C.

Primary cells

Six-to-eight-week-old female wild type BALB/c were purchased from Janvier (BALB/cJRj). After euthanasia, skin and muscles of the lower extremities were dissected and femurs were isolated. Bones were cleaned from muscles residues and bone marrow was flushed using a 1 mL syringe (BD #303172) with a 26-gauge needle filled with sterile cold phosphate buffer saline (PBS) containing 5% FBS (5% FBS-PBS). After filtration through 70 pm cell strainers (#130-110-916, Miltenyi), cells were washed with cold 5% FBS-PBS, resuspended at 2x10⁶ cells/mL and cultured at 37°C with 5% CO2 in Roswell Park Memorial Institute Medium (RMPI- 1640-Glutamax #61870044, Life Technologies) containing 10% FBS, 100 U/mL penicillin, and 100 pg/mL streptomycin supplemented with 20 ng/mL of granulocyte-macrophage colonystimulating factor (GM-CSF) (#415-ML-02/CF, R&D systems). At day 2 and 6, half the medium was removed and replaced with a fresh medium supplemented with 20 ng/mL GM-CSF. At day 9 post-differentiation, cells were harvested by scrapping and characterized by flow cytometry, or co-cultured with indicated cancer cells.

Ionizing Radiation and combination with AGulX nanoparticles

Cells were seeded and left to adhere for 24 hours before the treatment. At day 1 , cells were washed with PBS and incubated for 1 hour at 37°C with the indicated concentrations of AGulX in Hanks' Balanced Salt Solution (HBSS) (14025100, Life Technologies). Then, cells were irradiated with X-ray irradiator (1 Gy/min, 200 keV, 15 mA, 2 mm copper thickness, X-RAD 320, Precision X-Ray) and harvested at indicated time points after irradiation for subsequent experiments.

In vivo mouse tumor model

Experiments were performed in accordance with French government and institutional guidelines, regulations and approved by the French Animal Experimentation Ethic Committee N°26 (APAFIS#27337-20190924150307v3). Six-to-seven old wild type female BALB/c mice were purchased from Janvier and were maintained in the animal facility of Gustave Roussy Cancer Campus. To generate syngeneic tumor models, 5x10⁵ CT26 cells were injected subcutaneously on the right flank of the mice in 50 pL of cold PBS. On day 9 or on day 11 , when tumors reached an average volume of 60 mm³ or 100 mm³ respectively, animals were randomly assigned to different treatment groups. AGulX nanoparticles diluted in DNase/RNase free water were administered in tumor-bearing mice by intravenous route at a concentration of 420 mg/Kg. Then, mice received localized radiotherapy to the tumor within 5 to 15 minutes after AGulX administration. Irradiation was delivered to mice using a Varian Tube NDI 226 (X- ray machine; 250 kV; tube current, 15 mA; beam filter, 0.2 mm Cu), with a dose rate of 1.08 Gy/min with a single dose of 4 or 8 Gy. Anti-mouse PD1 monoclonal antibodies (#BE0146, BioXCell) or its isotype control monoclonal antibodies (#BE0089, BioXCell) were administered i.p. (100 pg per mouse) on day 11 , 14 and 17. Anti-mouse CDS monoclonal antibodies (#BE0061 , BioXCell) or its isotype control monoclonal antibodies (#BE0089, BioXCell) were administered i.p. (100 pg per mouse) on day 7, 8, 9, 11 and then, once a week until the end of the experiment. The antibodies are described in Key Resources Table. Tumors were measured twice weekly, and the estimation of the tumor volume was calculated as (length x 2 3 width )/2. Mice were euthanized when the tumor size was > 2000 mm or boundary points were reached, according to the French and European laws and regulations for the use of mice for scientific purposes.

Flow cytometry

For flow-cytometry analysis, tumors were weighed then cut into small pieces (1 -2 mm) and digested using the Tumor dissociation kit (#130-096-730, Miltenyi) for 30 minutes at 37°C, according to the manufacturer’s instructions. Cells were then filtered through a 70 pm cell strainer (#130-110-916, Miltenyi), red blood cells were lysed with ACK buffer (#A1049201 , Life technologies) during 10 minutes on ice. Cells were then counted and resuspended at 1x10⁶ cells/mL in RPMI. Fc receptors were blocked for 10 minutes at 4°C using a purified anti-mouse aCD16/32 (#BLE101302, Biolegend). Cell staining was performed with cell surface antibody mix incubated with cells for 20 minutes at 4 °C. Then, cells were fixed with 4% PFA. Antibody characteristics and panels are described in Key Resources Table. CD8⁺ T cells were identified as CD45⁺CD3⁺CD8⁺ cells. Cells were also analyzed for cellular DNA content following cell staining with propidium iodide (PI) (Sigma, #P4864). At indicated time points, cells were detached with trypsin (#2500054, Life Technologies), fixed for 30 minutes with a cold solution of 70% ethanol at -20°C. Then, cells were washed extensively twice with DPBS and incubated for 30 minutes at 37°C in DPBS containing 20 pg/mL of PI, 100 pg/mL of DNase-free RNase A (Macherey-Nagel, #740505) and 20 mM EDTA before FACS analysis. Samples were acquired using the Fortessa LSR II (BD biosciences) and analyzed using FlowJo v10.7.1 software.

Western blots

Total cellular proteins were lysed in appropriated buffer containing 0.1 % NP-40, 5 mM EDTA, 20 mM HEPES, 10 mM KCI, 1 % glycerol and the protease and phosphatase inhibitors (Roche)). Then, 10-30 pg of protein extracts were separated by 4-12% Bis-Tris NuPAGE gels (NP0336BOX, Life Technologies) and were transferred at 4°C onto a nitrocellulose membrane (0.2 micrometer). After 2 hours of saturation at room temperature with 5% bovine serum albumin (BSA) in Tris-buffered saline and 0.1 % Tween 20, nitrocellulose membranes were incubated with primary antibody at 4°C overnight. Horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit (SouthernBiotech) antibodies were then incubated for 1 hour and revealed with the enhanced ECL detection system (GE Healthcare). The primary antibodies are described in Key Resources Table.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 8.0 (GraphPad). Statistical tests and statistical significances (*P< 0.05, **P< 0.01 , ***p< 0.001 , and ****p < 0.0001 ) are indicated for each figure in the corresponding figure legend.

Example 1 : Gadolinium-based nanoparticles AGulX enhance ionizing radiation-induced micronuclei formation. We evaluated the ability of ionizing radiation + AGulX combination to enhance the formation of ionizing radiation-induced micronuclei, which are cytosolic events gathering genomic instability and antitumor immune response (11 ). Using fluorescence microscopy, we thus analyzed the accumulation of micronuclei in human colorectal adenocarcinoma Caco-2 cells and murine colorectal carcinoma CT26 cells that were exposed to a single-dose of 6 Gy IR in presence or in absence of 0.6 mM or 1.2 mM of AGulX. Twenty-four hours after irradiation, micronuclei are detected in the cytosol of irradiated Caco-2 cells (Figure 1A) and CT26 cells (Figure 1 B). Moreover, the combination of ionizing radiation with 0.6 mM and 1 .2 mM of AGulX significantly enhanced the frequency of cells showing micronuclei in their cytosol, as compared to control or irradiated cancer cells (Figures 1A-1 B). Although micronuclei formation occurs from chromosome lagging or fragmentation after the induction of DNA lesions and the progression through mitosis (10), we simultaneously detected the presence of DSB marker, the nuclear foci containing the phosphorylated form of the histone variant H2AX on serine 139 (H2AXS139*), also known as y-H2AX⁺ foci, in Caco-2 cells exposed to control, ionizing radiation alone or combinatorial strategies (data not shown). Twenty-four hours after treatment with ionizing radiation + AGulX combination, the frequency of cells showing nuclear y-H2AX⁺ foci increased, as compared to control cells, but at the same extent with cells that were subjected to ionizing radiation alone (data not shown), demonstrating that DSB DNA lesions are induced in both ionizing radiation alone and IR + AGulX-treated cells. We then analyzed the impact of ionizing radiation + AGulX combination on cell cycle distribution and evaluated the effect of cell cycle arrest on micronuclei formation. Interestingly, the treatment of Caco-2 cells with the cyclin-dependent kinase 1 (CDK1 ) inhibitor, RO-3306, which induced a cell cycle arrest and inhibited cell mitotic entry prevented the formation of micronuclei in Caco-2 cells that were subjected to 6 Gy single-dose irradiation or to ionizing radiation + AGulX combination (Figure 1 C). The frequency of cells showing y-H2AX⁺ foci after ionizing radiation + AGulX combination remained unchanged following RO-3306 treatment, as compared to irradiated cells (Figure 1 D). These results demonstrate that the formation of micronuclei in ionizing radiation + AGulX-treated cancer cells is induced after the induction of double strand DNA breaks and by the cell cycle progression through mitosis. Importantly, the increased frequency of cells showing micronuclei after ionizing radiation + AGulX treatment also reveals the ability of this combinatorial strategy to enhance the genomic instability of irradiated cells. Considering that reactive oxygen species (ROS) mediates DNA damage and genomic instability after IR (32, 33), we then evaluated the effect of the ROS scavenger, N-acetyl cysteine (NAC) on micronuclei formation and DSB lesions. Caco-2 cells were pre-treated during 24 hours with NAC and irradiated alone (or not) or in presence of AGulX (0.6 mM AGulX or 1 .2 mM AGulX). Intriguingly, NAC abrogated micronuclei formation following IR or combined treatment with AGulX (Figure 1 E) but did not affect the frequency of y-H2AX⁺ foci (Figure 1 F), thus indicating that ROS generated in response to IR alone or combined treatment with AGulX contributes to micronuclei formation. Altogether, these results reveal the ability of ionizing radiation + AGulX combination to trigger irradiated cancer cells to cell cycle progression through mitosis after the induction of DSB DNA lesions and consecutively, leading to the formation of micronuclei in their cytosol.

Example 2: Gadolinium-based nanoparticles AGulX increase cGAS recruitment to ionizing radiation -elicited micronuclei.

Considering that the recognition of micronuclei by cGAS is a critical event for the immune response to cancer (11 , 34), we then determined whether cGAS can be recruited to micronuclei and can be stabilized in Caco-2 cells (Figure 2A) and CT26 cells (Figure 2B) that were exposed to a single-dose of 6 Gy in presence or in absence of 0.6 mM or 1 .2 mM of AGulX. We observed using fluorescence microscopy that 24 hours after irradiation, cGAS localizes to micronuclei in the cytosol of irradiated Caco-2 cells and CT26 cells (Data not shown). The combination of IR with 0.6 mM and 1 .2 mM of AGulX significantly enhanced the frequency of cells showing the relocalization of cGAS to micronuclei (cGAS⁺ micronuclei), as compared to control or irradiated cancer cells (Figures 2A-2B). The increased recruitment of cGAS at the formed micronuclei was observed in both cell types regardless the absence or the presence of cGAS nuclear expression and localization. Accordingly, increased cGAS protein expression was also observed in Caco-2 cells (Figure 2C) and CT26 cells (Figure 2D) that were exposed to a singledose of 6 Gy in presence or in absence of AGulX at 24 hours after irradiation. Importantly, a significant increase of cGAS protein expression levels was detected in Caco-2 cells (Figure 2E) and CT26 cells (Figure 2F) that were irradiated in presence of 0.6 mM and/or 1.2 mM AGulX, as compared to control or irradiated cells.

Altogether, these results demonstrate that the ionizing radiation + AGulX combination also enhances the recruitment of cGAS DNA sensor to the ionizing radiation - induced micronuclei.

Example 3: Gadolinium-based nanoparticles AGulX enhance the stimulation of IR- driven cGAS/STING/Type I interferon signaling pathway.

We then investigated the impact of ionizing radiation + AGulX combination on signaling pathways shown induced after the recognition of micronuclei by cGAS (11 ). According to the fact that inflammatory responses have a delayed onset with respect to the recognition of micronuclei by cytosolic DNA sensors (10), the protein expression levels of central effectors of type I interferon pathway, the proteins STING and TBK1 , and their respective activating phosphorylation on serine 365 (STINGS365*) and on serine 172 (TBK1 S172*) at day 6 after the exposure of CT26 cells to a single-dose of 6 Gy and to a combined treatment with AGulX were analyzed. We observed that CT26 cells treated with ionizing radiation + AGulX combination exhibited an enhanced activation of STINGS365* and TBK1S172* after 6 days of irradiation, as compared to control and irradiated cells (Figure 3). Altogether, these results reveal the ability of AGulX to enhance the stimulation of IR-mediated cGAS/STING/Type I interferon signaling pathway.

Example 4: The combination of ionizing radiation with Gadolinium-based nanoparticles AGulX triggers a CD8⁺ T cell-dependent antitumor response using low-dose irradiation.

Considering the contribution of cGAS/STING-dependent immune response on IR-mediated antitumor activity (10, 20), we investigated the effects of ionizing radiation + AGulX combination on tumor growth of colon carcinoma CT26 tumor-bearing BALB/c mice. We first evaluated the antitumor response induced by a single-dose of 8 Gy, 420 mg/Kg of AGulX or combined treatment and observed significant inhibition of tumor growth and increase of overall survival tumor-bearing mice that were irradiated only or combined treatment with AGulX, as compared to control or AGulX alone (Figure 4A-F). At this dose of irradiation, no toxicity of the treatment was observed (Figure 4F) and no synergic antitumor effect (Figure 4C, D) and increase of the overall survival of mice (Figure 4E) that were treated with ionizing radiation + AGulX combination, were observed, as compared to 8 Gy-irradiated tumors.

Considering the ability of high dose irradiation to attenuate immunogenicity of radiotherapy (19), we then evaluated the antitumor effect observed after reducing the dose of radiotherapy used in the combinatorial strategy. Using a single-dose of 4 Gy, we demonstrated a significant delay in the tumor growth of mice treated with ionizing radiation + AGulX combination, as compared to ionizing radiation alone (Figures 5A-D). Moreover, the overall survival of ionizing radiation + AGulX-treated mice was significantly increased, as compared to control, AGulX- and IR-treated mice (Figure 5E) and out of 11 tumors, the growth of 2 tumors was inhibited (Figures 5D, E), thus demonstrating the potential of a therapeutic approach using AGulX (420 mg/Kg) and a reduced irradiation dose of 4 Gy. This process occurs in absence of detectable toxicity (Figure 5K). To explore the antitumor response elicited by IR + AGulX combination, we analyzed the infiltration of CD8⁺ T cells in tumors that were treated with control, AGulX, single dose of 4 Gy irradiation and combined treatment, and revealed a significant increase of CD8⁺ T cell infiltration in tumors that were treated with IR + AGulX, as compared to control, AGulX or IR (Figure 5F). To evaluate the contribution of CD8⁺ T cells on IR + AGulX-driven antitumor response, we intra-peritoneally injected anti-CD8 antibodies to CT26-tumor bearing mice that were treated with control or combined treatment of ionizing radiation + AGulX. The injection of anti-CD8 antibodies resulted in a complete depletion of CD8⁺ T cells in tumors (Figure 5L) and did not cause toxicity in mice, as compared to IgG-treated mice (Figure 5M). More importantly, we observed that the depletion of CD8 T cells significantly abrogated the antitumor effect detected in mice that were treated with ionizing radiation + AGulX combination, as compared to mice that were treated with ionizing radiation + AGulX combination and injected with control IgG (Figure 5G-I). Accordingly, this process is associated with a significant decrease in the overall survival of mice that were treated with ionizing radiation + AGulX and injected with anti- CD8 antibodies (Figure 5J), thus demonstrating that ionizing radiation + AGulX combination promotes a CD8⁺ T cell-dependent antitumor response. These findings indicate that the combination of AGulX with reduced irradiation dose efficiently stimulates an antitumor immune response that mainly depends on CD8⁺ T cells.

Example 5: The combination of gadolinium-based nanoparticle AGulX, IR and anti-PD1 blockade overcomes resistance to immunotherapy.

The combination of radiotherapy with ICBs has emerged as a new therapeutic strategy to improve antitumor efficacy of radiotherapy (26, 36) and to overcome immune resistance mechanisms (35). To determine the antitumor effect of a combinatorial approach involving IR, AGulX and anti-PD1 antibodies, BALB/c mice bearing subcutaneous CT26 tumors were first treated when the tumor volume reached approximately 60 mm³. The intravenous injection of AGulX (420 mg/Kg) was followed by the treatment with a single dose of 4 Gy irradiation and 3 injections of anti-PD1 antibodies. In agreement with our previous results (Figures 5A-D), CT26 tumor-bearing BALB/c mice that were treated with IR + AGulX combination and injected with control IgG exhibited a delayed tumor growth, as compared to mice that were not treated, treated with IR alone or AGulX alone, and injected with control IgG (Figures 6A-D). Consistently, IR + AGulX-treated mice also revealed an increase of their overall survival (Figures 6I). However, we observed in tumor-bearing mice that were treated with anti-PD1 antibodies, a complete response to anti-PD1 antibodies (Figure 6E-H) and a significant increased survival of mice that were treated with anti-PD1 antibodies (Figure 6I). Despite the fact that the combination of AGulX with IR increased the antitumor effect of IR, their association with anti-PD1 antibodies did not exhibit a synergic antitumor effect and appeared less effective to elongate the survival of treated mice, as compared to mice treated with IR and anti-PD1 antibodies (Figure 6I). These results suggest the absence of interest for combining IR, AGulX and anti-PD1 antibodies for the treatment of tumors sensitive to anti-PD1 antibodies. Considering the impact of tumor volume on clinical responses to RT and/or ICBs (37, 38), we also evaluated the antitumor effect of this combinatorial strategy on BALB/c mice bearing subcutaneous CT26 tumors with a tumor volume of approximately 100 mm³. According to the previously described experimental procedure, CT26 tumor-bearing BALB/c mice that were treated with IR or IR + AGulX combination and injected with control IgG exhibited a significant delayed tumor growth, as compared to mice that were not treated or treated with AGulX alone, and injected with control IgG (Figures 7A-D). We also detected a significant tumor growth delay between CT26 tumor-bearing BALB/c mice that were treated with IR alone or with IR + AGulX combination (Figures 7C, D), thus revealing that AGulX also exhibit a synergic antitumor effect on 100 mm³ tumors when combined with IR. Nevertheless, we did not observe a significant tumor growth delay (Figure 7E) and an increase of the overall survival of mice (Figure 7I) that were treated with anti-PD1 antibodies, thus revealing acquired immune resistance of 100 mm³ tumors. Interestingly, we observed that CT26 tumor-bearing mice that were treated with IR (Figures 7G, I) or IR + AGulX combination (Figure 7H, I) and anti-PD1 antibodies (Figures 7G- I) showed significant tumor growth delays (Figures 7G,H) and the increased survival of treated mice (Figure 7I), as compared to CT26 tumor-bearing mice treated with anti-PD1 antibodies (Figures 7E, I) or AGulX and anti-PD1 antibodies (Figures 7F,I). More important, the triple therapy combining IR, AGulX and anti-PD1 antibodies resulted in a significant tumor growth delay (Figure 7H) and a significant increase of the survival of treated mice (Figure 7I), as compared to tumor bearing mice treated with anti-PD1 antibodies, the combination of AGulX with anti-PD1 antibodies or the combination of IR with anti-PD1 antibodies. In addition, a complete remission of 6/11 treated mice was observed, as compared to the remission of 2/9 mice that were treated with IR and anti-PD1 antibodies (Figures 7A-I). Altogether, these results clearly indicate the beneficial outcome of the association of IR + AGulX combination and anti- PD1 therapy, by improving antitumor responses of IR and anti-PD1 therapy, and by overcoming resistance to anti-PD1 therapy.

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Claims

1. Nanoparticles for use in a method of treating cancer in a subject in need thereof, said method comprising

(i) a priming phase, followed by

(ii) a treatment phase, wherein said priming phase comprises administering a therapeutically effective amount of said nanoparticles in combination with a low-dose radiotherapy, and said treatment phase comprises either

(i) administering a therapeutically effective amount of said nanoparticles in combination with radiotherapy, and optionally one or more immune check point blockers, or

(ii) administering a therapeutically effective amount of one or more immune checkpoint blockers, wherein said nanoparticles are nanoparticles containing an element with an atomic Z number higher than 20, preferably higher than 50, for example between 20 and 83, more preferably gadolinium or bismuth.

2. The nanoparticles for use according to Claim 1 , wherein said low-dose radiotherapy at the priming phase comprises 1 to 3 fractions of ionizing radiations at a dose ranging from 0.2 to 2 Gy per fraction, for example equal or above 0.5 Gy and below 1 .8 Gy, or from 0.5 to 1.5 Gy.

3. The nanoparticles for use according to Claim 1 or 2, wherein said low-dose radiotherapy at the priming phase comprises 1 fraction of ionizing radiations at a dose ranging from 0.2 to 2 Gy per fraction, for example from 0.5 to 1 .8 Gy, or from 0.5 to 1 .5 Gy.

4. The nanoparticles for use according to any one of Claims 1-3, wherein said low-dose radiotherapy at the priming phase is initiated the same day as the administration of said nanoparticles.

5. The nanoparticles for use according to any one of Claims 1-4, wherein said treatment phase is initiated at least 48 hours after the last fraction of ionizing radiations of the priming phase, preferably between 7 days and 28 days after last fraction of ionizing radiations of the priming phase.

6. The nanoparticles for use of any one of Claims 1-5, wherein said nanoparticles have a mean hydrodynamic diameter below 10 nm, for example between 1 and 8 nm, more preferably between 2 and 6 nm.

7. The nanoparticles for use of any one of Claims 1-6, wherein said nanoparticles comprise

• polyorganosiloxane,

• chelates covalently bound to said polyorganosiloxane,

• high-Z elements complexed to the chelates.

8. The nanoparticles for use of any one of Claims 1-7, wherein said nanoparticles are gadolinium-chelated polyorganosiloxane nanoparticles of the following formula

wherein POS is a matrix of polyorganosiloxane, and, n is comprised between 4 and 200, preferably 4 and 80, more preferably 4 and 20, and wherein the hydrodynamic diameter is comprised between 1 and 8 nm, preferably between 2 and 6 nm.

9. The nanoparticles for use of any one of Claims 1-8, wherein said nanoparticles are AGulX nanoparticles.

10. The nanoparticles for use of any one of Claims 1-9, wherein said nanoparticles are administered once at the priming phase at an amount ranging from 15 to 150 mg/kg, e.g. 100 mg/kg.

11. The nanoparticles for use according to any one of Claims 1-10, wherein said treatment phase comprises a standard-of-care radiotherapy in combination with administering a therapeutically effective amount of said nanoparticles, optionally in combination with administering a therapeutically effective amount of one or more immune checkpoint blocker.

12. The nanoparticles for use according to any one of Claims 1-11 , wherein said treatment phase comprises administering a therapeutically effective amount of one or more immune checkpoint blocker without radiotherapy.

13. The nanoparticles for use of any one of Claims 1 -12, wherein said cancer is a cancer with solid tumor, preferably selected from the group consisting of cancer of head and neck, skin cancer, liver cancer, colon cancer, pancreatic cancer, breast cancer, ovary cancer, uterus cancer, cervical cancer, brain cancer, thyroid cancer, bladder cancer and renal cancer, preferably lung cancer, cervical cancer, colorectal cancer, head and neck cancer and melanoma.

14. The nanoparticles for use of any one of Claims 1-13, wherein said subject is selected from subjects resistant or refractory to immune checkpoint blocker therapy, such as PD-1 inhibitor therapy.

15. The nanoparticles for use of any one of Claims 1-14, wherein said treatment phase comprises administering a therapeutically effective amount of an immune checkpoint blocker, preferably selected from the group consisting of anti-PD-1 or anti-PD-L1 antibodies, e.g. nivolumab, pembrolizumab, avelumab, cemiplimab, durvalumab or atezolizumab, or anti-CTLA-4 antibodies (e.g. ipilimumab).