Lipid microbubbles for targeted delivery of active ingredients

Abstract

The present invention relates to optimized microbubbles, in particular lipid microbubbles, which can be used in the prevention and treatment of diseases or in labeling. The invention relates in particular to a lipid microbubble, comprising at least one cationic compound selected from lipophosphoramidates, histidylated polyethylenimines, and any mixture thereof. The microbubble preferably further comprises at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof. The invention further relates to a method for producing this microbubble. The present invention also relates to a composition, in particular a pharmaceutical composition, and a kit, comprising at least one of these microbubbles. The present invention further relates to the use of these microbubbles, composition, kit, as a medicament or as a labeling agent.

Description

Title of the invention: Lipid microbubbles for the targeted delivery of active ingredients Field of invention

[0001] The present invention falls within the field of targeted delivery of active ingredients, for therapy and / or labeling, as well as in the field of microbubbles, in particular functionalized microbubbles.

[0002] The present invention relates in particular to optimized microbubbles, in particular lipid microbubbles, which can be used in the prevention and treatment of diseases or even labeling.

[0003] The invention relates in particular to a lipid microbubble, comprising at least one cationic compound chosen from lipophosphoramidates, histidylated polyethylenimines, and any mixture thereof. The microbubble preferably further comprising at least one agent chosen from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof.

[0004] The invention further relates to a method of producing this microbubble.

[0005] The present invention also relates to a composition, in particular a pharmaceutical composition, and a kit, comprising at least one of these microbubbles. The present invention further relates to the use of these microbubbles, composition, kit, as a medicament or as a marking agent. State of the Art

[0006] Initially developed for diagnostic purposes, microbubbles (MB) can also be used as a vector for therapeutic purposes. Indeed, thanks to the great diversity of molecules that can compose the envelope, it is thus possible to encapsulate drugs or to complex nucleic acids. Generally, MB serving as vectors have a functionalizable lipid envelope, allowing either to embed the therapeutic molecules between the lipids, or to dissolve them in a drop of oil inside the envelope. In addition, by using cationic lipids, it is possible to complex nucleic acids by electrostatic interactions. Other lipids can also be used to serve as attachments to nanoparticles or antibodies, using streptavidin-biotin interactions or be functionalized in order to carry out click chemistry.

[0007] Some studies have allowed the creation of MB as vectors of molecules of interest. Thus Zhu et al., Scientific Reports, 2016, describe MB loaded with chemotherapeutic agent (paclitaxel). Fan et al., Biomaterials, 2013, reported the development of microbubbles loaded with an antineoplastic agent. (l,3-bis(2-chloroethyl)-l-nitrosourea or BCNU). However, drugs complexed with these MBs are limited to anticancer chemical compounds. In particular, the possibility of delivering nucleic acids with these microbubbles has not been demonstrated. However, more and more emerging therapies, particularly anticancer therapies, use nucleic acids. These molecules offer flexibility unmatched by chemical compounds, allowing for example personalized therapies.

[0008] Delalande et al., Bioscience Reports, 2017, describe cationic MBs complexed with plasmid DNA. However, the delivery efficiency of nucleic acids and the specificity of targeting by these MBs remain limited. These aspects are particularly critical in the treatment of brain pathologies. In addition, one of the major obstacles to treatments concerns the passage of the blood-brain barrier (BBB). For these reasons, the molecules to be delivered into the central nervous system were until now chosen for their ability to passively cross the BBB. This implies a low delivery efficiency of nucleic acids, as well as a virtual absence of targeting, which can be the cause of many harmful side effects.

[0009] It is therefore essential to develop new effective and specific means of delivering therapeutic agents to the brain, in particular nucleic acids, capable of crossing the BBB without being degraded.

[0010] In this context, microbubbles represent a promising system. Thus, Fan et al., 2016, produced MBs composed of folate and complexing nucleic acids for the purpose of targeted transfection at the brain level.

[0011] These cationic microbubbles are based on the use of DSTAP or DPT AP lipids, using a trimethylammonium function as a cationic charge for complexation. The main disadvantage of these formulations is that they are not effective enough to be used in gene therapy protocols. In addition, these formulations have been shown to be toxic.

[0012] There is therefore still a need to develop microbubbles capable of effectively and targetedly delivering therapeutic agents, such as nucleic acids, to specific organs or even cells.

[0013] The present invention makes it possible to meet this need, by describing microbubbles based on new cationic formulations whose advantages are as follows:

[0014] - Better compaction of nucleic acids, - Lower toxicity, - Use of the endosomal escape system (“proton sponge effect”) to increase transfection and avoid the lysosome pathway, - Better transfection efficiency.

[0015] Thus, the microbubbles developed by the present inventors have the capacity to transport drugs, in particular nucleic acids, stably in the bloodstream; to cross the blood-brain barrier actively; to deliver drugs in a targeted manner, in particular towards antigens of interest. The technology developed here makes it possible to transiently open the BBB and effectively deliver nucleic acid-type active agents through it. It also makes it possible to permeabilize the vessels, in order to deliver molecules of interest. Description of the invention

[0016] In the context of the present invention, the inventors have developed innovative microbubbles, in particular innovative lipid microbubbles, capable of transporting drugs, in particular nucleic acids, in a stable manner in the blood circulation; of crossing the blood-brain barrier (BBB) in an active manner; and of delivering drugs in a targeted manner, in particular towards antigens of interest.

[0017] The inventors have notably shown that, surprisingly, the lipid microbubbles thus developed have significantly improved stability, unlike the microbubbles described in the prior art. Remarkably, the data also reveal that these optimized microbubbles are capable of delivering agents of interest, in particular nucleic acids, more efficiently than the microbubbles described in the prior art. These microbubbles are notably capable of actively crossing the vessels, as well as the BBB, or even the tumor microenvironment. The inventors have also demonstrated that the localized application of ultrasound makes it possible to target these optimized microbubbles very precisely towards the area to be treated, including very difficult-to-access areas such as the central nervous system, the vessels, and the tumor microenvironment.These data thus reveal the therapeutic potential of these lipid microbubbles to treat numerous pathologies in a targeted manner, including pathologies of the central nervous system.

[0018] The data also show that these optimized microbubbles are detection and imaging tools.

[0019] The present invention therefore provides both powerful and broad-spectrum treatment methods for pathologies, as well as effective and reliable diagnostic methods. Detailed description of the invention Summary of the invention

[0020] The present invention relates in particular to lipid microbubbles, which can be used both in the prevention and treatment of pathologies, and in detection and imaging, in particular medical.

[0021] The present invention relates in particular to a lipid microbubble, comprising at least one cationic compound chosen from lipophosphoramidates, polyethyl- histidylated lenimines, and any mixture thereof.

[0022] The present invention also relates to a pharmaceutical composition comprising at least one microbubble as defined above, and, optionally, a pharmaceutically acceptable excipient, the concentration of microbubbles in the composition preferably ranging from 106 to 1014 microbubbles / ml, more preferably from 107 to 1013 microbubbles / ml, more preferably from 108 to 1012 microbubbles / ml, more preferably from 109 to 1011 microbubbles / ml, more preferably the concentration of microbubbles in the composition being approximately 1010 microbubbles / ml.

[0023] The present invention also relates to a kit, comprising:

[0024] a) at least one microbubble as defined above, in a first container;

[0025] b) at least one therapeutic agent, in a second container;

[0026] c) optionally, at least one targeting agent in a third container;

[0027] d) optionally, at least one marking agent in a fourth container;

[0028] e) optionally, instructions for preparation and / or use;

[0029] the therapeutic agent and / or the targeting agent and / or the labeling agent preferably being chosen from:

[0030] 1. a nucleic acid; 2. a fat-soluble active ingredient; 3. a chemotherapeutic agent, such as a cytotoxic agent and / or a cytostatic agent; 4. an antibody; 5. a protein; 6. an antigen; 7. a toxin; 8. a receiver 9. an enzyme; 10. a hormone; 11. a ligand; 12. a viral vector; 13. a nanoparticle, preferably comprising at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof; 14. any derivative of 1) to 13), preferably any functional derivative thereof; 15. any fragment of 1) to 14), preferably any functional fragment thereof; And 16. any combination of 1) to 15);

[0031] more preferably among a nucleic acid, a liposoluble active agent, a chemotherapeutic agent, an antibody, an antibody derivative, a functional antibody fragment or its derivative, a protein, a protein fragment, a nanoparticle, and any combination thereof.

[0032] According to another aspect, the present invention relates to a microbubble, a pharmaceutical composition, or a kit, as defined above, for its use as a medicament or as a marking agent.

[0033] According to another aspect, the present invention relates to a method for producing at least one microbubble as defined above, comprising the following steps:

[0034] a. Mixture, in a container, of cationic compounds selected from lipophosphoramidates, histidylated polyethyleimines, and any mixtures thereof; ethanol; and optionally at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof; b. Evaporation of the mixture obtained in step a) to obtain a lipid film and rehydration of the lipid film to form a liposomal suspension; this entire step can also be carried out by microfluidics; c. Lyophilization of the liposomal suspension obtained in step b); d. Replacement of the air contained in the container containing the lyophilisate obtained in step c), by a biocompatible gas, the gas preferably being chosen from a perfluorobutane (C4F10), a perfluoropropane (C3F8), dinitrogen (N2), a sulfur hexafluoride (SF6), a nitrogen oxide (NO), hydrogen, dioxygen, helium, xenon, argon, nitrous oxide (N2O), and any mixture thereof; e. Rehydration of the lyophilisate from step d) to obtain a solution; f. Agitation of the solution obtained in step d) to form microbubbles; g. Optionally, functionalization of the microbubbles by at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof; and / or functionalization of the microbubbles by adding at least one functional group allowing binding to at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof. Definitions

[0035] The terms “at least one” are here considered synonymous with the terms “one or more”, or with the terms “one or more”.

[0036] In this document, the term "microbubble" designates a bubble whose diameter ranges from approximately one micrometer to several tens of micrometers (up to approximately one hundred micrometers), generally from approximately one micrometer to ten of micrometers. A microbubble comprises a shell surrounding a filler material. The shell can be made of lipids, proteins, sugars, ionic compounds, or mixtures of these. In the case of a shell made of lipids or made primarily of lipids, it is called a lipid microbubble.

[0037] Among the compounds that can be used to form a microbubble envelope according to the present invention, we find in particular:

[0038] - Cationic compounds, such as lipophosphoramidates, histidylated polyethylenimines, and any mixture thereof; and / or - lipids, in particular chosen from the group consisting of a dimyristoyl-glycero-phosphocholine, a distearoyl-glycero-phosphocholine, a di-myristoyl-glycero-phosphoethanolamine-polyethylene glycol, a distearoyl-glycero-phosphoethanolamine-polyethylene glycol 2000, a distearoyl-glycero-phosphoethanolamine-[biotinyl(polyethylene glycol)], a cholesterol, a beta-sitosterol, a l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), a l-oleoyl-2-[6-[(7-nitro-2-l,3-benzoxadiazol-4-yl)amino]hexanoyl]-3-trimethylammonium propane (DOTAP), a 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), clickable lipids DBCO, Tetrazine, Methyl tetrazine, NHS (DSPE-PEG2000-X), and any combination thereof; - and any combination thereof.

[0039] The filling material of the microbubble may in particular be a gas of any type. The filling gas is advantageously biocompatible (i.e. it is well tolerated by a living organism). The filling gas may in particular be chosen from a perfluorobutane (C4F10), a perfluoropropane (C3F8), dinitrogen (N2), a sulfur hexafluoride (SF6), a nitrogen oxide (NO), hydrogen, dioxygen, helium, xenon, argon, nitrous oxide (N2O), and any mixture thereof.

[0040] By "lipid microbubble" is meant a microbubble whose envelope is essentially made up of lipids, or is made up of lipids. The terms "envelope essentially made up of lipids" mean here an envelope comprising 55% lipids or more, preferably 60% lipids or more, preferably 70% lipids or more, preferably 80% lipids or more, preferably 90% lipids or more, preferably 91% lipids or more, preferably 92% lipids or more, preferably 93% lipids or more, preferably 94% lipids or more, preferably 95% lipids or more, preferably 96% lipids or more, preferably 97% lipids or more, preferably 98% lipids or more, more preferably 99% lipids or more, the percentage being expressed as mass of lipids relative to the total mass of the envelope.

[0041] Advantageously, the lipid microbubble comprises at least one cationic compound, in particular a cationic lipid or a lipid coupled to at least one cationic polymer.

[0042] The envelope of the lipid microbubble may comprise a cationic lipid or a lipid coupled to at least one cationic polymer, or consist essentially of cationic lipids or lipids coupled to at least one cationic polymer, or consist of cationic lipids and / or lipids coupled to at least one cationic polymer.

[0043] Advantageously, the envelope comprises cationic lipids and / or lipids coupled to at least one cationic polymer, and fusogenic lipids.

[0044] According to one implementation, the lipid envelope of the microbubble comprises from 2 to 50% of cationic lipids, preferably at least 2% of cationic lipids, preferably at least 10% of cationic lipids, preferably at least 20% of cationic lipids, preferably at least 30% of cationic lipids, preferably at least 40% of cationic lipids, preferably 50% of cationic lipids, the percentage being expressed in moles of cationic lipids relative to the number of moles of total lipids constituting the envelope.

[0045] By "cationic compound" is meant a compound comprising at least one cation and whose overall charge in solution is positive. Examples of cationic compounds (cationic lipids) which can be used to form a microbubble envelope include, in particular, without limitation: lipophosphoramidates, histidylated polyethylenimines, and any mixture thereof. By "cationic lipid" is therefore meant a lipid comprising at least one cation and whose overall charge in solution is positive.

[0046] By "lipophosphoramidate" is meant a bioinspired amphiphilic phospholipid which carries a cationic charge on its polar head.

[0047] Lipophosphoramidates include, in particular, dimyristoyl phosphoramidates (such as dimyristoyl bromide phosphoramidates, and dimyristoyl histamine phosphoramidates), and dioleyl phosphoramidates (such as dioleyl methylimidazolium phosphoramidates).

[0048] By "polyethylenimine", or "PEI", or "polyaziridine", is meant an organic polymer of chemical formula H[CH2-CH2-NH-]„H. By "histidylated polyethylenimine" is meant a polyethylenimine comprising at least one histidyl group. Advantageously, the histidylated polyethylenimine used to form the microbubble is coupled to a fatty acid. Examples of fatty acids that can be coupled to a histidylated polyethylenimine include in particular stearic acid, myristic acid, palmitic acid, oleic acid, and any combination thereof.

[0049] By "compound / agent exposed to the surface of the microbubble" is meant a compound or agent conjugated / coupled / bound to the external surface of the microbubble (for example conjugated / coupled / bound to the envelope of the microbubble) and in contact with the environment external to the microbubble.

[0050] By "compound / agent embedded in the lipid envelope of the microbubble" is meant a compound or agent partially or totally integrated / incorporated in the layer of compounds (in particular lipids) forming the envelope of the microbubble. Thus, a compound or agent totally integrated / incorporated in the envelope is only in contact with the compounds (in particular lipids) forming the envelope of the microbubble: in this case, the compound or agent is therefore in contact neither with the external environment nor with the internal environment of the microbubble. A compound or agent partially integrated / incorporated in the envelope can, on the other hand, be either with the external environment of the microbubble, or with the internal environment of the microbubble, or with both the external environment and the internal environment of the microbubble ("passing through" compound).

[0051] By "compound / agent incorporated inside the microbubble" is meant a compound or agent located in the internal medium of the microbubble (i.e. in the medium / cavity formed by the envelope of the microbubble). This compound or agent may be in contact with the internal surface of the microbubble (for example with the internal surface of the envelope of the microbubble). In particular, it may be conjugated / coupled / linked to the internal surface of the microbubble (for example to the internal surface of the envelope of the microbubble).

[0052] By "therapeutic agent" or "therapeutic compound" is meant any agent or compound or molecule presented as having curative or preventive properties with regard to human or animal pathologies or diseases. A therapeutic agent or a therapeutic compound therefore includes any agent or compound which can be used in humans or animals or administered to them for the purpose of establishing a medical diagnosis or restoring, correcting or modifying their physiological functions by exerting a pharmacological, immunological and / or metabolic action. The therapeutic agent can therefore be a pharmacological agent.

[0053] The therapeutic agent or compound may be of any nature or type and is independent of its origin. The therapeutic agent may be chemically synthesized, naturally occurring, recombinantly produced (and optionally purified), or synthetically designed and produced. Examples include a small molecule, a nucleic acid, a peptide (including a post-translationally modified peptide), a polypeptide (including a post-translationally modified polypeptide), a protein (including a post-translationally modified protein), a chemical compound, a cancer chemotherapy agent (such as a cytostatic or cytological agent), an antibody, a toxin, an antigen, a hormone, an enzyme, a ligand, a receptor, an antiviral compound, an antibiotic compound, an antifungal compound, an antibacterial compound, a nanoparticle, or any fragment thereof (preferably a functionally active fragment), or any derivative thereof (preferably a functionally active derivative), such as a peptidomimetic, an antibody mimetic, a chemical derivative, among others, etc.The therapeutic agent or compound may, for example, comprise, or consist essentially of, or consist of, a nucleic acid, a lipid-soluble active, a chemotherapeutic agent (such as a cytotoxic agent and / or a cytostatic agent), an antibody, an antibody derivative, a functional fragment of an antibody or its derivative, a protein (including a post-translationally modified protein), a protein fragment (such as a peptide, an antigen, an epitope, a protein functional domain, and any combination thereof; including a post-translationally modified protein fragment), a nanoparticle, etc.

[0054] By "targeting agent" or "targeting compound" is meant any agent or compound or molecule presented as being capable of recognizing and / or binding to another molecule (called "target molecule"), preferably in a specific manner. These terms therefore also include "binding agents" or "binding compounds". The terms "binding agents" or "binding compounds" designate any agent or compound or molecule capable of binding to another molecule (called "target molecule"), said binding preferably being a specific binding.

[0055] The targeting / binding agent recognizes a defined site, domain, region, pocket, epitope, spatial configuration, conformation, chemical grouping, or any combination thereof of the target molecule. The targeting / binding agent may be of any nature or type and is independent of its origin. The targeting / binding agent may be chemically synthesized, naturally occurring, recombinantly produced (and optionally purified), or synthetically designed and produced.It may, in particular, be a small molecule, a nucleic acid, a peptide (including a post-translationally modified peptide), a polypeptide (including a post-translationally modified polypeptide), a protein (including a post-translationally modified protein), a chemical compound, an antibody, a toxin, an antigen, an epitope, a hormone, an enzyme, a ligand, a receptor, a nanoparticle, or any fragment thereof (preferably a functionally active fragment), or any derivative thereof (preferably a functionally active derivative), such as a peptidomimetic, a mimetic antibody, a chemical derivative, among others, etc. The targeting / binding agent may, for example, comprise, or consist essentially of, or consist of, . a nucleic acid, a lipid-soluble active, a chemotherapeutic agent (such as a cytotoxic agent and / or a cytostatic agent), an antibody, an antibody derivative, a functional fragment of an antibody or its derivative, a protein (including a post-translationally modified protein), a protein fragment (such as a peptide, an antigen, an epitope, a functional domain of a protein, and any combination thereof; including a post-translationally modified protein fragment), a nanoparticle, etc.

[0056] The target molecule may be of any nature or type and does not depend on its origin. The target molecule may in particular be a pathogenic agent (such as a virus, a bacterium, a parasite, etc.) or a fragment thereof (for example a nucleic acid, a protein, a polypeptide, a peptide, an epitope, a lipid, a sugar, etc.).

[0057] By "labeling agent" or "label" is meant any agent or compound or molecule presented as being capable of labeling another molecule (preferably specifically) and of being detected by any means. The means for detecting the labeling agent are well known to the person skilled in the art, who is quite capable of selecting the appropriate technique depending on the labeling agent used. The means for detecting the labeling agent include in particular the following techniques, without being limited to: optical detection techniques (such as techniques using fluorescence, absorbance, diffraction, light scattering, interferometry, reflectometry, ellipsometry, surface plasmon resonance (SPR), spectroscopy, magnetic particles, etc.), mechanical detection techniques (such as techniques using microbalances, microbeams, etc.), electrical detection techniques (such as techniques using electrodes, electrical sensors, etc.). The labeling agent may therefore be a contrast agent.

[0058] The labeling agent may be of any nature or type and is independent of its origin. The targeting / binding agent may be chemically synthesized, naturally occurring, recombinantly produced (and optionally purified), or synthetically designed and produced.It may, in particular, be a small molecule, a nucleic acid, a peptide (including a post-translationally modified peptide), a polypeptide (including a post-translationally modified polypeptide), a protein (including a post-translationally modified protein), a chemical compound, an antibody, a toxin, an antigen, an epitope, a hormone, an enzyme, a ligand, a receptor, a nanoparticle, or any fragment thereof (preferably a functionally active fragment), or any derivative thereof (preferably a functionally active derivative), such as a peptidomimetic, an antibody mimetic, a chemical derivative, among others. etc. The labeling agent may, for example, comprise, or consist essentially of, or consist of, a nucleic acid, a lipid-soluble active, a chemotherapeutic agent (such as a cytotoxic agent and / or a cytostatic agent), an antibody, an antibody derivative, a functional fragment of an antibody or its derivative, a protein (including a post-translationally modified protein), a protein fragment (such as a peptide, an antigen, an epitope, a protein functional domain, and any combination thereof; including a post-translationally modified protein fragment), a nanoparticle, etc.

[0059] The labeling agent may for example comprise, or consist essentially of, or consist of, a fluorophore (for example fluorescein or luciferase), a fluorescent protein / polypeptide / peptide (for example GFP and its variants, such as RFP, CFP, YFP, etc.), a radioisotope (particularly suitable for scintigraphy, for example 99mTc), a label recognizable by an antibody (for example c-Myc protein or a poly-histidine tag), an affinity tag (for example biotin, streptavidin, etc.), an enzyme (for example horseradish peroxidase), a contrast agent, a peptide tag, etc.

[0060] The term "derivative" generally refers to a component or species (protein, antibody, protein fragment, polypeptide, polynucleotide, oligonucleotide, nucleoside, nucleotide, vector, virus, etc.) having one or more modifications compared to a reference component (for example, the wild-type component as found in nature as originally identified, i.e., the corresponding "original" component called the original component). A derivative may, in particular, be a fragment, a part, a variant, a mutant, a synthetic version (for example, manufactured, in particular in vitro), a mimetic, or a combination thereof, of the original component or species. The terms "variant" or "mutant" can be used interchangeably to generally refer to a component or species (protein, antibody, protein fragment, polypeptide, polynucleotide, oligonucleotide, nucleoside, nucleotide, vector, virus, etc.) having one or more modifications compared to a reference component (e.g., the wild-type component as found in nature as originally identified, i.e., the corresponding "original" component called the original component). A nucleotide or nucleoside variant may have a modified base and / or a modified sugar and / or a modified linkage. With respect to polypeptide, polynucleotide, and antibody variants, any modification may be considered, including substitution, insertion, deletion, and any combination thereof, of one or more nucleotide / amino acid residues. The variant may be of natural or artificial origin (e.g., mutated and / or engineered).

[0061] When several mutations are envisaged, they may concern consecutive residues and / or non-consecutive residues. Preferred are variants (for example, respectively, protein variants, peptide variants, antibody variants, virus variants, etc.) which retain a degree of sequence identity of at least 80% with the reference component (for example, respectively, the corresponding "original" protein, the corresponding "original" protein fragment, the corresponding "original" polynucleotide). For example, "at least 80% identity" means 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, at least 80% identity also includes 100% identity.

[0062] By "functional fragment", or "functionally active fragment", is meant any fragment of a molecule, an agent, a species, a compound, exhibiting at least one of the original functions of the molecule, the agent, the species, the compound, from which said fragment originates.Preferably, the functional fragment performs said function with an efficiency equal to at least 30% of that of said peptide or said protein, preferably at least 40%, preferably at least 45%, preferably at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99%, preferably at least 100% of the efficiency of said molecule, said agent, said species, said compound, from which said fragment is derived. .

[0063] By "identity" or "sequence identity" is meant an exact sequence match between two polypeptides or amino acids, or between two nucleic acid molecules or oligonucleotides. The percentages of identity referred to in the context of the disclosure of the present invention are determined after optimal global alignment of the sequences to be compared, which may therefore comprise one or more additions, deletions, truncations and / or substitutions. This percentage of identity can be calculated by any sequence analysis method well known to those skilled in the art. The percentage of identity is determined after global alignment of the sequences to be compared taken in their entirety, over their entire length. In addition to manually, it is possible to determine the global alignment of sequences using the algorithm of Needleman and Wunsch (1970).

[0064] In particular, for the nucleotide sequences, the comparison of the sequences can be carried out using any software well known to those skilled in the art, such as for example the Needle software. The parameters used can in particular be the following: “Gap Open” equal to 10.0, “Gap Extend” equal to 0.5 and the EDNAFULL matrix (EMBOSS version of NCBI NUC4.4).

[0065] For amino acid sequences, the comparison of the sequences can be carried out using any software well known to those skilled in the art, such as for example the Needle software. The parameters used can in particular be the following: “Gap Open” equal to 10.0, “Gap Extend” equal to 0.5 and the BLOSUM62 matrix.

[0066] For illustrative purposes, "at least 80% sequence identity" as used herein includes, but is not limited to, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.

[0067] The terms 'polynucleotide', 'nucleic acid molecule' and 'nucleic acid' are used interchangeably herein and refer to a polymeric or oligomeric macromolecule consisting of nucleotide monomers (preferably at least 5 nucleotide monomers, also referred to as nucleotide residues). The nucleotide monomers are composed of a nucleobase, a five-carbon sugar (such as, but not limited to, ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, a polynucleotide is formed by phosphodiester bonds between the individual nucleotide monomers. Nucleic acid molecules include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and mixtures thereof, such as RNA-DNA hybrids (mixed polyribo-polydeoxyribonucleotides).These terms encompass single or double-stranded, linear or circular, natural or synthetic, unmodified or modified versions thereof (e.g., genetically engineered polynucleotides; optimized polynucleotides), sense or antisense polynucleotides, chimeric mixture (e.g., RNA-DNA hybrids). In addition, a polynucleotide may comprise nucleotides of non-natural origin and may be interrupted by non-nucleotide components. Examples of DNA nucleic acids include, but are not limited to, complementary DNA (cDNA), genomic DNA, plasmid DNA, vector DNA, viral DNA (e.g., viral genomes, viral vectors), oligonucleotides, probes, primers, satellite DNA, microsatellite DNA, coding DNA, non-coding DNA, antisense DNA, and any mixture thereof.Exemplary RNA nucleic acids include, but are not limited to, messenger RNA (mRNA), precursor messenger RNA (pre-mRNA), small interfering RNA (siRNA), short hairpin RNA (snRNA), microRNA (miRNA), vector RNA, viral RNA, guide RNA (gRNA), antisense RNA, coding RNA, non-coding RNA, antisense RNA, satellite RNA, small cytoplasmic RNA, small nuclear RNA, etc. The polynucleotides described herein may be synthesized by standard methods known in the art, for example, using an automated DNA synthesizer (such as those that . are commercially available from Biosearch, Applied Biosystems, etc.) or obtained from a natural source (e.g., a genome, cDNA, etc.) or an artificial source (such as a commercially available library, a plasmid, etc.) using molecular biology techniques well known in the art (e.g., cloning, PCR, etc.). Nucleic acids may, for example, be chemically synthesized, e.g., by the phosphotriester method (see, e.g., Uhlmann, E. & Peyman, A. (1990) Chemical Reviews, 90, 543-584).

[0068] The nucleic acid may comprise at least one modified nucleotide (i.e. a nucleotide which is not a nucleotide of a natural DNA or RNA). These modified nucleotides may in particular be used to increase the resistance of the nucleic acid to degradation by nucleases. This is particularly advantageous for RNAs, which are generally more sensitive to nucleases than DNA aptamers. An RNA comprising at least one modified nucleotide is called modified RNA. A DNA comprising at least one modified nucleotide is called modified DNA.

[0069] The nucleic acid may also comprise at least one additional group, in addition to the nucleotides constituting its nucleic acid sequence. Thus, the nucleic acid may be linked to at least one additional group.

[0070] By "modified DNA" is meant a DNA comprising at least one modified nucleotide. A modified DNA may in particular be a DNA in which the backbone of the nucleic acid is modified, in whole or in part, in particular to make it resistant to hydrolytic degradation, in particular due to the action of nucleases. The DNA may be modified in whole (i.e. each nucleotide which constitutes it is modified) or in part (i.e. only a part of the nucleotides which constitute it is modified). When the DNA is modified in part, it is possible to choose to modify all or part of the purines and / or all or part of the pyrimidines.

[0071] By "modified RNA" is meant an RNA comprising at least one modified nucleotide. A modified RNA may in particular be an RNA in which the backbone of the nucleic acid is modified, in whole or in part, in particular to make it resistant to hydrolytic degradation, in particular due to the action of nucleases. The RNA may be modified in whole (i.e. each nucleotide which constitutes it is modified) or in part (i.e. only a part of the nucleotides which constitute it is modified). When the RNA is modified in part, it is possible to choose to modify all or part of the purines and / or all or part of the pyrimidines.

[0072] The modifications of a DNA or an RNA (and / or a nucleotide) are well known to those skilled in the art and may in particular be chosen from: the modification of the OH function on the carbon in the 2' position of the ribose by methylation; the substitution of the OH function on the carbon in the 2' position of the ribose by an O-Methoxyethyl group; the substitution of the OH function on the carbon in the 2' position of the ribose by an amino group; the substitution of the OH function on the carbon in the 2' position of the ribose by a halogen (in particular by fluorine); the replacement of the phos-phodiester (PO) by a phosphorothioate (PS) group (this is then referred to as a phosphorothioate skeleton); the use of a locked nucleic acid (LNA) structure, i.e. the formation of a methylene bridge in order to lock the ribose in the C3'-endo (N-type) conformation; the use of a peptide nucleic acid (PNA) structure, i.e. the replacement of the sugar-phosphate skeleton by a peptide skeleton; and any combination thereof.

[0073] By "vector" is meant a vehicle, preferably a nucleic acid molecule or a viral particle, which contains the elements necessary to enable the administration, propagation and / or expression of one or more nucleic acid molecule(s) in a host cell or organism.

[0074] From a functional point of view, this term encompasses vectors for maintenance (cloning vectors), vectors for expression in various host cells or organisms (expression vectors), extrachromosomal vectors (e.g., multicopy plasmids), or integrating vectors (e.g., designed to integrate into the genome of a host cell and produce additional copies of the nucleic acid molecule therein when the host cell replicates). This term also encompasses shuttle vectors (e.g., functioning in both prokaryotic and / or eukaryotic hosts) and transfer vectors (e.g., for the transfer of nucleic acid molecule(s) into the genome of a host cell).

[0075] From a structural point of view, vectors can be natural, synthetic or artificial genetic sources, or a combination of natural and artificial genetic elements.

[0076] Thus, in the context of the invention, the term "vector" should be understood broadly to include plasmid (or plasmid) and viral vectors.

[0077] A "plasmid" as used herein means a replicable DNA construct. Typically, plasmid vectors contain selection marker genes that allow host cells carrying the plasmid to be identified and / or selected positively or negatively in the presence of the compound corresponding to the selection marker. A variety of positive or negative selection marker genes are known in the art. For illustration, an antibiotic resistance gene can be used as a positive selection marker gene to select a host cell in the presence of the corresponding antibiotic.

[0078] The term "viral vector" as used herein refers to a nucleic acid vector that comprises at least one element of a virus genome and can be packaged into a viral particle or a viral particle. Viral vectors may be replication-competent or selective (e.g., designed to replicate better or selectively in specific host cells), or may be genetically disabled so as to be defective or deficient in replication.

[0079] By "polypeptide", "protein", "protein fragment" and "peptide" are meant polymers of amino acid residues that comprise at least nine amino acids linked by peptide bonds. The polymer may be linear, branched or cyclic. The polymer may comprise naturally occurring amino acids and / or amino acid analogues and may be interrupted by non-amino acid residues. As a general indication and without being bound herein, if the polymer of amino acids contains more than 50 amino acid residues, it is preferably referred to as a polypeptide or a protein, whereas if the polymer consists of 50 or fewer amino acids, it is preferably referred to as a "peptide". The reading and writing directions of an amino acid sequence of a polypeptide, a protein and a peptide as used herein are the conventional reading and writing directions.The reading and writing convention for amino acid sequences of a polypeptide, protein, and peptide places the amino terminus on the left, with the sequence then being written and read from the amino terminus (N-terminus) to the carboxyl terminus (C-terminus), from left to right.

[0080] The amino acids constituting the polypeptides, proteins and peptides include in particular the so-called “standard” amino acids (also called “natural amino acids”, of which Table 1 below provides a non-exhaustive list), as well as the non-standard amino acids (also called “rare amino acids”, including for example pyrrolysine (symbolized by the letter O), selenocysteine (symbolized by the letter U), alloisoleucine, allothreonine, omithine, etc.)

[0081] Table 1: Standard amino acids

[0082] [Tables 1] Name 3 letters 1 letter alanine Ala A arginine Arg R asparagine Asn N Aspartate or aspartic acid Asp D cysteine Cys C Glutamate or glutamic acid Glu E glutamine Gin Q glycine Gly G histidine His H isoleucine Ile I leucine Leu L lysine Lys K methionine Met M phenylalanine Phe F proline Pro P serine Ser S threonine Thr T tryptophan Trp W tyrosine Tyr Y valine Val V

[0083] By "peptide or protein fragment" or "part of peptide or protein" is meant a portion of a peptide or protein, i.e. a portion of the sequence of consecutive amino acids composing said peptide or said protein (called peptide or protein from which the fragment is derived). The peptide or protein fragment preferably comprises at least 10 consecutive amino acids of the peptide or protein from which it is derived; more preferably at least 12 consecutive amino acids, more preferably at least 15 consecutive amino acids, more preferably at least 20 consecutive amino acids, more preferably at least 30 consecutive amino acids of the peptide or protein from which it is derived. The peptide or protein fragment preferably has a three-dimensional structure, under non-denaturing conditions (for example, conditions that are usually non-denaturing for proteins, in particular in the absence of denaturing and / or chaotropic agents).

[0084] As used herein, the term "post-translational modification" refers to a naturally occurring or non-naturally occurring chemical or enzymatic modification of a protein or protein fragment, after or concomitantly with translation of the protein (e.g., biological or biochemical synthesis, e.g., using cellular machinery), or after or concomitantly with synthesis of the protein (e.g., artificial and / or chemical synthesis). This means that at least one of the naturally occurring amino acids of the protein or protein fragment is modified by the addition of at least one chemical group and / or the modification (including, but not limited to, removal) of at least one chemical group of the naturally occurring amino acid.Examples of such chemical or enzymatic modifications include, but are not limited to, glycosylation, phosphorylation, acylation, carboxylation, acetylation, biotinylation, hydroxylation, lipoylation, amidation, ubiquitination, sumoylation, deamination, etc. By "post-translationally modified protein" is meant herein a protein having at least one post-translational modification. By "post-translationally modified protein fragment" is meant a protein fragment having at least one post-translational modification.

[0085] By "liposoluble active" is meant any agent or compound or molecule which solubilizes in a fatty substance, and having a biological activity, in particular a therapeutic, pharmacological, targeting, or marking activity, as defined above with regard to therapeutic, targeting, or marking agents. Liposoluble actives include in particular actives which solubilize in lipids or their derivatives, for example in oils, butters, oily esters, and other ingredients comprising lipids or their derivatives.

[0086] By "chemotherapeutic agent" is meant any agent or compound or molecule having chemotherapeutic activity. The chemotherapeutic agent includes in particular agents having anticancer activity (in particular agents capable of eliminating cancer cells and / or tumors, and / or of inducing / stimulating the elimination of cancer cells and / or tumors) as well as agents having anti-autoimmune disease activity. The chemotherapeutic agent may therefore be a cytotoxic agent and / or a cytostatic agent. Examples of chemotherapeutic agents include in particular, but are not limited to, paclitaxel, doxorubicin, gencitabin (eg Gemzar), temozolomide, etc.

[0087] By "antibody" is meant a protein or a glycoprotein belonging to the su Immunoglobulin perfamily; the terms antibody and immunoglobulin are used interchangeably. In mammals, antibodies are mostly secreted by cells derived from B lymphocytes: plasma cells. They are used in particular by the immune system to specifically detect and neutralize foreign bodies (including pathogens, such as bacteria, viruses, parasites, etc.). Antibodies also include autoantibodies (produced, for example, in the case of an autoimmune disease). The antibody recognizes a unique part of the foreign target, its antigen.

[0088] Antibodies have a structure formed of 4 polypeptide chains (150,000 amu or dalton): two identical heavy chains (H for "heavy", of 50,000 amu each) and two identical light chains (L for "light", of 25,000 amu each) which are linked together by a variable number of disulfide bridges ensuring the cohesion of the molecule. These chains form a Y-shaped structure (each light chain constitutes half an arm of the Y) and are made up of immunoglobulin domains which can comprise approximately 110 amino acids. Each light chain is made up of a constant domain (called CL) and a variable domain (called VL); the heavy chains are composed of a variable domain (called VH) and, depending on the isotype, three or four constant domains respectively called CH1, CH2, CH3, (CH4). For a given antibody, the two heavy chains are identical, as are the two light chains.Constant domains are characterized by a very similar amino acid sequence from one antibody to another, characteristic of the species and isotype. Constant domains are generally not involved in antigen recognition, but are involved in the activation of the complement system, as well as in the elimination of immune complexes (antibody bound to its antigen) by immune cells possessing constant fragment receptors (cFRs). An antibody has four variable domains located at the ends of the two "arms". The association between a variable domain carried by a heavy chain (VH) and the adjacent variable domain carried by a light chain (VL) constitutes the recognition site (or paratope) of the antigen. Thus, an immunoglobulin molecule has two antigen-binding sites, one at the end of each arm.These two sites are identical (but intended for different epitopes), hence the possibility of binding two antigen molecules by antibody. The antigen recognition site (or paratope) comprises 6 regions called complementarity-determining regions (or CDRs). Each VH has 3 CDRs and each VL also has 3.

[0089] Specific enzymatic cleavage allows the isolation of different fragments:

[0090] - the Fc fragment (Crystallizable Fragment). It is the support of the biological properties of the immunoglobulin, in particular its capacity to be recognized by effectors of immunity or to activate complement. It consists of the constant fragments of the heavy chains (CH2) beyond the hinge region. It generally does not recognize the antigen; - the Fv fragment (Variable Fragment). This is the smallest fragment that retains the properties of the antibody that immunoglobulin possesses. It is made up of only the variable regions VL and VH, so it binds the antigen with the same affinity as the complete antibody and is monovalent; - the Fab fragment (Fragment antigen-binding). This fragment has the same affinity for the antigen as the complete antibody. The Fab fragment is formed of the entire light chain (VL+CL) and part of the heavy chain (VH+CH1). It is monovalent; - the F(ab')2 fragment. It corresponds to the association of two Fab fragments linked by a small part of the constant parts of the heavy chains, the hinge region. It has the same affinity as the antibody for the antigen and is divalent.

[0091] As used herein, the term antibody encompasses native antibodies and their functional derivatives, (e.g., mutated and / or modified antibodies as well as mimetic antibodies), provided that such a derivative is capable of specifically binding to an antigen (referred to as "functional antibody derivatives"). As used herein, the term "antibody derivatives" also encompasses antibody fragments. Preferably, an "antibody fragment" is capable of specifically binding to an antigen (referred to as a "functional antibody fragment").

[0092] The antibodies may be produced by various systems known to those skilled in the art. Antibody production systems include, for example, animal systems (such as rodents, camelids, etc.), hybridoma systems, mammalian cell systems (including, but not limited to, CHO cell lines (Chinese hamster ovary cells; for example, CHO-K1, CHO-DG44, etc.), mouse myeloma cell lines (for example, NS0), baby hamster kidney cell lines (for example, BHK), human embryonic kidney cell lines (for example, HEK293), etc.), yeast systems (including, but not limited to, yeasts improved for glycolization), insect cell systems (including, but not limited to, insect cell lines improved for glycolization), plant cell systems (including, but not limited to, plant cell lines improved for glycolization), etc.

[0093] Preferably, the antibody is an antibody of an animal, preferably a mammalian antibody, more preferably a human or humanized antibody. Advantageously, the antibody is humanized.

[0094] The term “antibody” encompasses native antibodies and their derivatives (e.g., mutated and / or modified antibodies as well as mimetic antibodies), preferably provided that this derivative is capable of binding specifically to an antigen.

[0095] The different categories of antibodies and their production methods are well known to those skilled in the art, who may in particular refer to reference works in the field (such as Thomas D. Pollard, William C. Earnshaw, Jennifer Lippincott-Schwartz, Graham Johnson Cell Biology E-Book, Elsevier Health Sciences, 1 Nov. 2016; Mohammed Zourob, Recognition Receptors in Biosensors, DOI 10.1007 / 978-1-4419-0919-0, Springer-Verlag New York 2010; Abbas, Lichtman, Pillai, Cellular and Molecular Immunology E-Book, Elsevier Health Sciences, 22 Aug 2014; Bayer V., An overview of monoclonal antibodies. Semin Oncol Nurs. 2019 Sep 2:150927; Wang W, Wang EQ, Balthasar JP. Pharmacokinetics and pharmacodynamics of monoclonal antibodies. Clin Pharmacol Ther. 2008 Nov;84(5):548-58).

[0096] The term "functional antibody fragment", as used herein, refers to one or more part(s) or fragment(s) of an antibody, retaining the ability to specifically bind to an antigen. Examples of binding fragments encompassed by the term "functional antibody fragment" include, but are not limited to, an antigen-binding fragment (Fab), a Fab' fragment, an F(ab')2 fragment, a variable fragment (Fv), a single chain variable fragment (scFv) (corresponding to the VH and VL regions fused using a linker peptide), a dsFv fragment (disulfide-bond stabilized Fv), a ds-scFv fragment (disulfide-bond stabilized scFv), a VH domain, a VL domain, a di-scFv (divalent scFv, consisting of the association of two scFvs), a diabody (consisting of the covalent or non-covalent association of two scFvs), a single chain diabody, a triple body, a minibody,consisting of the fragments VL-VH-CH3), a nanobody (“nanobody”), a single domain antibody (sdAb), a single chain antibody fragment (scAb), a heavy chain antibody (HcAb), a VHH, a VNAR (variable new antigen receptor), an immunoglobulin novel antigen receptor (IgNAR), a bispecific T cell engager (BITEs), a dual affinity retargeting molecule (DART), and any combination thereof (e.g., a fusion protein thereof).

[0097] The term "single domain antibody," or "sdAb," as used herein, refers to antibody fragments consisting of a single monomeric variable domain of an antibody. These antibodies comprise only the monomeric variable regions of the heavy chain of heavy chain antibodies produced in particular by camelids or cartilaginous fish. Due to their different origins, they are also referred to as VHH (camelid) or VNAR (variable new antigen receptor; cartilaginous fish) fragments. Single domain antibodies are also referred to as Nanobodies. Single-domain antibodies can also be obtained by monomerizing the variable domains of conventional mouse or human antibodies through genetic engineering. They have a molecular mass of approximately 12–15 kDa and are therefore the smallest antibody fragments capable of recognizing an antigen.

[0098] The term "diabody" as used herein refers to a fusion protein or bivalent antibody that can bind to different antigens. A diabody is composed of two unique protein chains that comprise fragments of an antibody, namely variable fragments. Diabodies comprise a heavy chain variable (VH) domain linked to a light chain variable (VL) domain on the same polypeptide chain (VH-VL, or VL-VH). By using a short peptide linking the two variable domains, the domains are forced to pair with the complementary domain of another chain and thus create two antigen binding sites. Diabodies can target the same antigen (monospecific) or different antigens (bispecific).

[0099] The terms "antibody mimetic" or "antibody mimetic" as used herein, refer to compounds that can specifically bind to antigens, similar to an antibody, but are not structurally related to antibodies. Typically, antibody mimetics are artificial peptides or proteins having a molecular weight of about 3 to 20 kDa that include one, two, or more exposed domains that specifically bind to an antigen. Examples of antibody mimetics include, but are not limited to, LACLD1 (lipoprotein-associated inhibitor of coagulation); affilins, e.g., human ubiquitin or human γ B-crystallin; cystatin; Sac7D from Sulfolobus acido-caldarius; lipocalins and lipocalin-derived anticalins; DARPins (Designed Ankyrin Repeat Proteins); the SH3 domain of Fyn; the Kunits domains of protease inhibitors;monobodies, e.g., the 10th type III domain of fibronectin; ad-nectins; knottins (cysteine knot miniproteins); atrimers; evibodies; affibodies, e.g., the three-helix bundle of the Z domain of Staphylococcus aureus protein A; Trans-bodies, e.g., human transferrin; tetranectins, e.g., the monomeric or trimeric domain of human C-type lectin; microbodies (microbodies), e.g., trypsin inhibitor-II; armadillo-like repeat proteins; etc. Nucleic acids and small molecules can also be considered antibody mimetics (e.g., aptamers), but not artificial antibodies, antibody fragments, and fusion proteins composed of them. Common advantages over antibodies are better solubility, better pe; penetration into tissues, stability to heat and enzymes, and comparatively low production costs.

[0100] The terms "DARPin" or "Designed Ankyrin Repeat Protein" herein refer to engineered antibody mimetic proteins that generally exhibit highly specific and high affinity binding to the target protein. They are derived from natural ankyrin repeat proteins, one of the most common classes of binding proteins in nature, which are responsible for various functions such as cell signaling, regulation and structural integrity of the cell. DARPins comprise, or consist essentially of, or consist of, at least three repeating motifs or modules, of which the most N- and most C-terminal modules are called "caps", as they protect the hydrophobic core of the protein. The number of internal modules is indicated by a number (e.g. NIC, N2C, N3C,...) while the caps are indicated by "N" or "C", respectively.

[0101] By "antigen" we mean a natural or synthetic molecule which, recognized by antibodies or cells of the immune system of an organism, is capable of triggering an immune response in the latter. Thus any foreign substance, any microbe, introduced into the body, can behave as an antigen, that is to say cause the production of special proteins, antibodies which have the property of neutralizing the harmful effects of the foreign substance. Antigens are generally peptides, proteins, sugars (such as polysaccharides or polyosides) and their lipid derivatives (lipids). Antigens can also be nucleic acids, or haptens (i.e. fragments of antigens). Antigens, as markers of agents foreign to the organism, are the basis of the adaptive immune response.It is the recognition of the antigen by immunocompetent cells, directly or via antigen-presenting cells (APCs), that activates specific immunity. In the case of protein antigens, the part of the antigen recognized by an antibody or a lymphocyte receptor is called an "epitope" or "antigenic determinant." The same antigen can contain several epitopes (identical or different) and thus induce a varied immune response. There are sequential epitopes, corresponding to a sequence of amino acids, and conformational epitopes, linked to the structure of the protein and therefore sensitive to denaturation. Recognition of the antigen by lymphocytes depends on the nature of the epitope. B lymphocytes bind directly to conformational epitopes thanks to the immunoglobulins in their membrane. T lymphocytes recognize the sequential epitopes presented by antigen-presenting cells.The antigen can be exogenous, that is, it is foreign to the individual (in this case, it can be allogenic: from an individual of the same species; or xenogeneic: from other species), or it can be endogenous, that is, an antigen specific to the host. (self-antigens). The antigen is preferably an antigen of a microorganism, plant, algae, microalgae, bacterium, virus, parasite, yeast, fungus, insect, animal, or tumor; preferably an antigen of a eukaryotic or prokaryotic pathogen, or of a cancer; preferably a protein antigen, a lipid, or a sugar of a bacterium, virus, parasite, yeast, fungus, or tumor.

[0102] The term antigen encompasses native antigens and their derivatives (e.g., mutated and / or modified antigens), preferably provided that such derivative is capable of being the target of an immune response.

[0103] The different categories of antigens are well known to those skilled in the art, who may in particular refer to reference works in the field (such as GJV Nossal, GL Ada, Antigens, Lymphoid Cells and the Immune Response, Academie Press, 1971; Marc HV Van Regenmortel, Structure of Antigens, Volume 3 CRC Press, Dec. 20, 1995; Edouard Drouhet, Garry T. Cole, Louis De Repentigny, Jean Latge, Fungal Antigens: Isolation, Purification, and Detection, Springer Science & Business Media, Nov. 11, 2013; Graziano DF, Finn OJ (2005) Tumor Antigens and Tumor Antigen Discovery. In: Khleif SN (eds) Tumor Immunology and Cancer Vaccines. Cancer Treatment and Research, vol. 123. Springer, Boston, MA; Wang M, Claesson MH, Methods Mol Biol. 2014;1184:309-17.Classification of human leukocyte antigen (HLA) supertypes; as well as specialized databases as described in Galperin, Fernandez-Suarez, Rigden, The 24th annual Nucleic Acids Research database issue: a look back and upcoming changes, NAR, Volume 45, Issue Dl, January 2017, Pages Dl-Dl 1; in particular the PMAPP database, a database of human autoantigens (available in particular on the website aagatlas.ncpsb.org).

[0104] As used herein, an "antigen fragment" is any portion of an antigen, preferably provided that such fragment / portion is capable of being the target of an immune response (e.g., epitopes, immunogenic domains, etc.). In the case of a protein antigen, the antigenic fragment preferably comprises at least 6 consecutive amino acid residues of the antigen (preferably at least 8 consecutive amino acid residues of the antigen, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the antigen).

[0105] By "toxin" we mean a substance that is toxic to one or more living organisms. A toxin is typically synthesized by a living organism (bacteria, poisonous mushroom, insect or poisonous snake), to which it confers its pathogenic power. Toxins produced by bacteria are called bacteriotoxins, those by fungi are called mycotoxins, those produced by plants are called phytotoxins, those produced by algae are called phycotoxins, those by animals are called animal toxins. The toxin can be a chemical molecule, a peptide, a protein, a glycoprotein, a sugar, an oside, a lipid, a nucleic acid, or any combination of these. Several families of bacteria secrete biotoxins (exotoxins) in the tissues they colonize. Other bacteria (Gram negative) retain most of the toxic compounds within themselves, which are only released during cell lysis, under the action of chemical, physical or mechanical means (endotoxins). Toxic plants produce toxins via their secondary metabolites: these are molecules which, unlike primary toxins (proteins, lipids, carbohydrates, amino acids, etc.) are produced outside the metabolic pathways necessary to ensure survival (therefore primary metabolites).Plant toxins can be classified into three groups: phenols, nitrogenous toxins and terpenes. The toxin can be a neurotoxin (a toxin acting on the nervous system), a myotoxin (acting on muscle contraction, including cardiotoxins on the heart and others such as strychnine on the respiratory muscles), a hemotoxin (acting on the blood), a cytotoxin (acting on cells), a dermatotoxin (acting on the skin and mucous membranes), a hepatotoxin (acting on the liver), a nephrotoxin (acting on the kidney), an enterotoxin (acting on the digestive tract) etc. The toxin can be an anatoxin; that is to say a toxin that has been treated in such a way as to retain its antigenic power and lose its toxic power.The toxin is preferably a toxin of a microorganism, plant, algae, microalgae, bacterium, virus, parasite, yeast, fungus, insect, animal, or tumor; preferably a toxin of a eukaryotic or prokaryotic pathogen, or of a cancer.

[0106] The different categories of toxins are well known to those skilled in the art, who may in particular refer to reference works in the field (such as Michael W. Parker, Protein Toxin Structure, Springer Science & Business Media, June 29, 2013; Michael R. Dobbs, Clinical Neurotoxicology E-Book: Syndromes, Substances, Environments, Elsevier Health Sciences, July 22, 2009; Walker AA, Robinson SD, Yeates DK, Jin J, Baumann K, Dobson J, Fry BG, King GF. Entomo-venomics: The evolution, biology and biochemistry of insect venoms. Toxicon. 2018 Nov; 154:15-27; Vilarino N, Louzao MC, Abal P, Cagide E, Carrera C, Vieytes MR, Botana LM. Human Poisoning from Marine Toxins: Unknowns for Optimal Consumer Protection. Toxins (Basel).2018 Aug 9;10(8); as well as specialized databases as described in Galperin, Fernandez-Suarez, Rigden, The 24th annual Nucleic Acids Research database issue: a look back and upcoming changes, NAR, Volume 45, Issue Dl, January 2017, Pages Dl-Dll; in particular the Comparative Toxicogenomics Database, as described in Davis, Grondin. Murphy, Johnson, Lay, Lennon-Hopkins, Saraceni-Richards, Sciaky, King, Ro-senstein, Wiegers, Mattingly, The Comparative Toxicogenomics Database: update 2013, NAR, Volume 41, Issue Dl, 1 January 2013, Pages D1104-D1114 (available in particular on the website ctdbase.org)).

[0107] As used herein, a "toxin fragment" is any part of a toxin, preferably provided that such fragment / part is capable of being toxic to an organism and / or cell. In the case of a protein toxin, the toxin fragment preferably comprises at least 6 consecutive amino acid residues of the toxin (preferably at least 8 consecutive amino acid residues of the toxin, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the toxin).

[0108] By "receptor" is meant a molecule of the cell membrane or of the cytoplasm or of the cell nucleus which binds specifically to a specific factor (a ligand, such as a neurotransmitter, a hormone, or another substance), inducing a cellular response to this ligand. The modifications of the behavior of the receptor induced by the ligand lead to physiological modifications which constitute the "biological effects" of the ligand. The receptors can comprise at least: a peptide, a protein, a glycoprotein, a sugar, an oside, a lipid, a nucleic acid, or any combination thereof. The receptors are generally proteins or mixed proteins (proteins modified and / or associated with another molecule).The receptor may be a receptor on the outer part of the plasma membrane, a transmembrane receptor embedded in the lipid bilayer of cell membranes (usually a transmembrane protein, acting for example as a receptor for hormones and neurotransmitters - These receptors are either coupled to a G protein or carry enzymatic or ion channel activity that allow the activation of metabolic pathways of signal transduction in response to ligand binding), or an intracellular receptor (these receptors can sometimes penetrate into the cell nucleus to modulate the expression of specific genes, in response to ligand activation).The receptor is preferably a receptor of a microorganism, plant, algae, microalgae, bacterium, virus, parasite, yeast, fungus, insect, animal, or tumor; preferably a receptor of a eukaryotic or prokaryotic pathogen, or of a cancer; preferably a protein or glycoprotein receptor of a bacterium, virus, parasite, yeast, fungus or tumor. The different categories of receptors are well known to those skilled in the art, who may in particular refer to reference works in the field (such as Thomas D. Pollard, William C. Earnshaw, Jennifer Lippincott-Schwartz, Graham Johnson Cell Biology E-Book, Elsevier Health Sciences, Nov. 1, 2016; Mohammed Zourob, Recognition Receptors in . Biosensors, DOI 10.1007 / 978-1-4419-0919-0, Springer-Verlag New York 2010; Abbas, Lichtman, Pillai, Cellular and Molecular Immunology E-Book, Elsevier Health Sciences, August 22, 2014; as well as specialized databases as described in Galperin, Fernandez-Suarez, Rigden, The 24th annual Nucleic Acids Research database issue: a look back and upcoming changes, NAR, Volume 45, Issue Dl, January 2017, Pages Dl-Dl 1; in particular the GPCRdb database, as described in Isberg V., Mordalski S., Munk C., Rataj K., Harpsoe K., Hauser AS, Vroling B., Bojarski AJ, Vriend G., Gloriam DE. GPCRdb: an information system for G protein-coupled receptors. Nucleic Acids Res. 2016; 44:D356-D364 (available in particular on the site gpcrdb.org)).

[0109] As used herein, a "receptor fragment" is any portion of a receptor, preferably provided that such fragment / portion is capable of specifically binding to a specific factor (e.g., a ligand, a hormone, or another substance). In the case of a protein receptor, the receptor fragment preferably comprises at least 6 consecutive amino acid residues of the receptor (preferably at least 8 consecutive amino acid residues of the receptor, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the receptor).

[0110] An "enzyme" is a protein with catalytic properties. Virtually all biomolecules capable of catalyzing chemical reactions in cells are enzymes; however, some catalytic biomolecules are made of RNA and are therefore distinct from enzymes: these are ribozymes. An enzyme acts by lowering the activation energy of a chemical reaction, thereby increasing the reaction rate. The enzyme is not modified during the reaction. The initial molecules are the substrates of the enzyme, and the molecules formed from these substrates are the products of the reaction. Enzymes are notably characterized by their very high specificity. In addition, an enzyme has the characteristic of being reusable.

[0111] Enzymes are generally globular proteins that act alone or in complexes of several enzymes or subunits. Like all proteins, enzymes consist of one or more polypeptide chains folded to form a three-dimensional structure corresponding to their native state.

[0112] Enzymes are molecules much larger than their substrates. Their size can vary from about fifty to one hundred residues to more than 2,000 residues. Only a very small part of the enzyme — between two and four residues most often, sometimes more — is directly involved in catalysis, the so-called catalytic site (or catalytic domain). The latter can be located near one or more binding sites, at which the substrate(s) is (are) bound and oriented in order to allow the chemical reaction to be catalyzed. The catalytic site and the binding sites form the active site of the enzyme.

[0113] Enzymes perform a large number of functions in living beings. For example, they may be involved in signal transduction mechanisms and regulation of cellular processes, in the generation of movements, in active transmembrane transport, in digestion, in metabolism, in the immune system, in mechanisms for digesting or cleaving nucleic acids or even producing nucleic acids (hereinafter referred to as "nucleic acid-acting enzymes"), in prodrug conversion mechanisms (conversion of prodrug into drug). The enzyme is preferably a prokaryotic, eukaryotic or viral enzyme, preferably an enzyme of an animal, a plant, an algae, a microalgae, an insect, a microorganism, a bacterium, a parasite, a yeast, a fungus or a virus, more preferably a mammalian enzyme, such as a human enzyme.The different categories of enzymes are well known to those skilled in the art, who may in particular refer to reference works in the field (such as Schomburg D., Schomburg L, Springer Handbook of Enzymes. 2 edn. Heidelberg: Springer; 2001-2009; Liébecq C., IUPAC-IUBMB Joint Commission on Biochemical Nomenclature (JCBN) and Nomenclature Committee of IUBMB (NC-IUBMB) Biochem. Mol. Biol. Int. 1997;43:1151-1156; IUBMB (1992), Enzyme Nomenclature 1992, Academie Press, San Diego; as well as specialized databases such as those described in Schomburg D, Schomburg I. Methods Mol Biol. 2010;609:113-28. Enzyme databases; in particular the BRENDA database (available in particular on the site brenda-enzymes.org), as described for example by Chang A, Schomburg I, Placzek S, Jeske L, Ulbrich M, Xiao M, Sensen CW, Schomburg D, Nucleic Acids Res. 2015 Jan;43. Epub 2014 Nov 5. BRENDA in 2015: exciting developments in its 25th year of existence). .

[0114] As used herein, an "enzyme fragment" is any portion of an enzyme, preferably provided that such fragment / portion is capable of having enzymatic activity. In the case of a protein enzyme, the enzyme fragment preferably comprises at least 6 consecutive amino acid residues of the enzyme (and is preferably a catalytic site of the enzyme) (preferably at least 8 consecutive amino acid residues of the enzyme, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the enzyme).

[0115] By "enzymatic activity" or "catalytic activity" or even "activity" of an enzyme, we mean the efficiency of an enzyme in converting a substrate into a product in a given environment. The efficiency of the enzyme here takes into account the speed of conversion of the substrate into product by the enzyme and the rate of conversion of the substrate into product by the enzyme. By "rate of conversion of the substrate into product by the enzyme" is meant here the ratio between the quantity of final product obtained compared to the initial quantity of substrate for a defined quantity of enzyme. For example, an enzymatic activity within the meaning of the invention can be expressed as the quantity of phloroglucinol produced in a given volume (in g / L).

[0116] By "hormone" we mean a biologically active chemical substance, generally synthesized by a glandular cell (usually following stimulation) and secreted into the internal environment where it circulates (via the blood, lymph or sap). It transmits a message in chemical form (generally by acting on specific receptors of a target cell) and therefore plays a messenger role in the body. It is capable of acting at very low doses.

[0117] The hormone is advantageously a plant or animal hormone. Plant hormones are also called phytohormones or growth factors. They often have the function of ensuring the growth of the plant or its morphogenesis. Animal hormones are in most cases produced by the endocrine system (an endocrine gland or endocrine tissue).

[0118] Advantageously, the hormone is a vertebrate hormone, preferably chosen from the following chemical classes:

[0119] - Amine-derived hormones, which consist of a single amino acid (tyrosine or tryptophan) but in a derived form. - Peptide hormones, which are chains of amino acids, therefore proteins, called peptides for the shortest ones. - Steroid hormones, which are steroids derived from cholesterol. - Lipid and phospholipid-based hormones.

[0120] The hormone is preferably chosen from peptide or protein hormones, amine-derived hormones, steroid hormones and lipid hormones. The hormone is preferably a hormone of an animal or a plant, preferably a mammalian hormone, preferably a human hormone. The different categories of hormones are well known to those skilled in the art, who may in particular refer to reference works in the field (such as Davies PJ (2010) The Plant Hormones: Their Nature, Occurrence, and Functions. In: Davies PJ (eds) Plant Hormones. Springer, Dordrecht; AW Norman, G Litwack, Hormones, Academie Press, 1997; A Kastin, Handbook of biologically active peptides, Academie Press, 2013).

[0121] As used herein, a "hormone fragment" is any portion of a hormone, preferably provided that such fragment / portion is capable of stimulating and / or inhibiting a biological process. In the case of a protein hormone, the hormone fragment preferably comprises at least 6 consecutive amino acid residues of the hormone (preferably at least 8 consecutive amino acid residues of the hormone, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the hormone).

[0122] The term "ligand" generally refers to a substance that binds to a receptor on a cell and induces a biological signal. The term ligand includes, but is not limited to, the terms "addressing or targeting or transport signal", "signaling molecule", "signal", and "cellular signal". Examples of ligands include peptide and protein addressing sequences, oligosaccharides, molecules that enable cellular transport and / or internalization, neurotransmitters, receptor ligands (receptors being as defined above), and cell recognition molecules such as Toll Like receptor ligands or C-type lectin receptor ligands. An addressing sequence is a short sequence of amino acids, generally located at the N-terminus of the protein, used to designate the proteins to be addressed, and indicate their destination. Thus,the addressing or targeting or transport signal may be an addressing or targeting or transport signal to / from the nucleus; an addressing or targeting or transport signal to / from the cytoplasm; an addressing or targeting or transport signal to / from the cytosol; an addressing or targeting or transport signal to / from the cell membrane; an addressing or targeting or transport signal to / from the mitochondria; an addressing or targeting or transport signal to / from the peroxisomes; an addressing or targeting or transport signal to / from the lysosomes; an addressing or targeting or transport signal to / from the endoplasmic reticulum; an addressing or targeting or transport signal to the secretory pathways; and a ligand of a receptor, preferably a membrane or transmembrane receptor, preferably a membrane or transmembrane receptor of a membrane chosen from a cell membrane,an extracellular membrane, a cytoplasmic membrane or a nuclear membrane. The addressing or targeting or transport signal may comprise at least one of: a peptide, a protein, a glycoprotein, a sugar, an oside, a lipid, a nucleic acid, or any combination thereof. Preferably, the addressing or targeting or transport signal comprises at least one of peptide, a protein, a glycoprotein, or a nucleic acid. The signal is preferably a prokaryotic, eukaryotic or viral signal, preferably a signal from an animal, a plant, an algae, a microalgae, a microorganism, a bacterium, a parasite, a yeast, a fungus, an insect, a virus, or a cancer; more preferably a mammalian signal, such as a human signal. The different categories of signals are well known to those skilled in the art, who may in particular refer to the reference works in the , field (such as Thomas D. Pollard, William C. Eamshaw, Jennifer Lippincott-Schwartz, Graham Johnson Cell Biology E-Book, Elsevier Health Sciences, Nov. 1, 2016; Mohammed Zourob, Recognition Receptors in Biosensors, DOI 10.1007 / 978-1-4419-0919-0, Springer-Verlag New York 2010; Abbas, Lichtman, Pillai, Cellular and Molecular Immunology E-Book, Elsevier Health Sciences, August 22, 2014).

[0123] A "ligand fragment" is any portion of a ligand, preferably provided that such fragment / portion is capable of binding to a receptor of a cell and inducing a biological signal. In the case of a protein ligand, the ligand fragment preferably comprises at least 6 consecutive amino acid residues of the ligand (preferably at least 8 consecutive amino acid residues of the ligand, preferably at least 10, preferably at least 15, preferably at least 20, preferably at least 30 amino acid residues of the ligand).

[0124] By "nanoparticle" is meant an object whose three dimensions are on the nanometric scale, that is to say a particle whose nominal diameter is less than approximately 100 nm (for example as defined by the ISO TS / 27687 standard).

[0125] By "functional group" or "functional group" is meant a reactive group, i.e. having the capacity to form at least one chemical, biological, biochemical, enzymatic reaction, or any combination thereof, with another molecule. By "functional group allowing binding to an agent" is meant a functional group having the capacity to form at least one chemical, biological, biochemical, enzymatic reaction, or any combination thereof, with an agent (in particular chosen from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof).

[0126] The functional group may, for example, comprise, or consist essentially of, or consist of, a peptide tag, a chemical group (such as a clickable function, a crosslinking group, and any combination thereof), an antibody, an antibody derivative, a functional fragment of an antibody or its derivative, an affinity tag (e.g., biotin, streptavidin, chitin-binding protein (CBP), maltose-binding protein (MBP), Strep-tag, glutathione-S-transferase (GST), poly(His) tag, etc.), or any combination thereof.

[0127] By "clickable function", or "click chemistry" or even "rapid bio-orthogonal chemistry", we mean a chemical group capable of reacting with another chemical group, in the absence of solvent, at a physiological pH, without formation of residue or by-product. Examples of clickable functions include in particular, but are not limited to, azide groups, alkyne groups (eg acetylene), and any combination thereof. The clickable function can in particular be selected from an N-hydroxysuccinimide (NHS), dibenzocyclooctyne (DBCO), tetrazine, methyl-tetrazine group, and any combination thereof.

[0128] As used herein, the terms "peptide tag", or "peptide label", or "peptide tag", refer to a peptide sequence of between 6 and 400 amino acids (preferably between 8 and 300 amino acids, more preferably between 10 and 200 amino acids, more preferably between 12 and 82 amino acids). Examples of peptide tags include affinity peptide tags, solubilization peptide tags, chromatography peptide tags, epitope peptide tags, fluorescence peptide tags, etc. Affinity peptide tags are generally added to proteins so that they can be purified from their raw biological source using an affinity technique. These include chitin-binding protein (CBP), maltose-binding protein (MBP), Strep-tag, glutathione-S-transferase (GST), poly(His) tag, etc.Solubilizing peptide tags are particularly used for proteins expressed in chaperone-deficient species, such as E. coli, to aid in proper protein folding and prevent precipitating. These include thioredoxin (TRX) and poly(NANP). Some affinity peptide tags have a dual role as solubilizing agents, such as MBP and GST. Chromatographic tags are used to modify the chromatographic properties of the protein to allow different resolution in a particular separation technique. They often consist of polyanionic amino acids, such as the FLAG tag. Epitope tags are short peptide sequences chosen because high-affinity antibodies can be reliably produced in many different species. They are usually derived from viral genes.Epitope tags include ALFA tag, V5 tag, Myc tag, HA tag, Spot tag, T7 tag, NE tag, etc. Fluorescence tags are used to provide a visual readout of a protein. GFP and its variants are the most commonly used fluorescence tags. Peptide tags can allow for specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with FlAsH-EDT2 for fluorescence imaging). Peptide tags can be combined, particularly to connect proteins to several other components. Peptide tags also include covalent peptide tags. Examples of covalent peptide tags include, but are not limited to: .

[0129] - Isopeptag (covalently binding to pilin-C protein); - SpyTag (covalently binding to the SpyCatcher protein); - SnoopTag (covalently binding to the SnoopCatcher protein); - SnoopTagJr (covalently binding to SnoopCatcher protein or DogTag protein (mediated by SnoopLigase)); - DogTag (covalently binding to SnoopTagJr protein, mediated by SnoopLigase), - SdyTag (covalently binding to the SdyCatcher protein); - Any variation of these.

[0130] The variants of the tag peptides are well described in the literature, are available to those skilled in the art, and do not need to be described in detail herein.

[0131] By "disease" or "condition" or "disorder" or "pathology" (these terms are here considered to be synonymous), we mean an alteration of the functions or health of a living organism. This includes both disease, referring to all alterations of health, and a disease, which then designates a particular entity characterized by its own causes, symptoms, evolution and therapeutic possibilities.

[0132] By "prevention" or "prevention of a disease" or "prevention of the occurrence of a disease" is meant the reduction of the risk of occurrence, development or amplification of a disease, the causes of a disease, the symptoms of a disease, the effects (or consequences, preferably the harmful, deleterious effects / consequences) of a disease, or any combination thereof; and / or delaying the occurrence, development or amplification of a disease, the causes of a disease, the symptoms of a disease, the effects (or consequences, preferably the deleterious effects / consequences) of a disease, or any combination thereof. Prevention includes in particular preventive treatments.

[0133] By "treatment" or "treatment of a disease" is meant the reduction, inhibition and / or disappearance of a disease, the causes of a disease, the symptoms of a disease, the effects (or consequences, preferably the harmful, deleterious effects / consequences) of a disease, or any combination thereof. The treatment is preferably a curative treatment.

[0134] A "treatment" or "therapy" includes, but is not limited to, one or more molecules and / or drugs (including any type of molecule or drug, such as chemical or biological compounds, antibodies, antigens, gene therapy, cell therapy, immunotherapy, chemotherapy, any combination thereof, etc.), and / or other treatments (such as radiotherapy, immunotherapy, chemotherapy, surgery, endoscopy, interventional radiology, physical oncology, phototherapy, light therapy, ultrasound therapy, thermotherapy, cryotherapy, electrotherapy, electroconvulsive therapy, oxygen therapy, assisted ventilation, hydrotherapy massage, organ / tissue transplantation / of fluids, implantation, and any combination thereof, etc.) A therapy may be administered by various modes of administration. One skilled in the art knows how to select the most appropriate mode(s) of administration depending on the therapy, the disease, and the subject to be treated. For example, modes of administration include, but are not limited to, oral administration; administration by injection into a vein (intravenous, IV), into a muscle (intramuscular, IM), into the space around the spinal cord (intrathecal), under the skin (subcutaneous, sc); sublingual administration; buccal administration; rectal administration; vaginal administration; ocular administration; otic administration; nasal administration; by inhalation; by nebulization; cutaneous, topical, or systemic administration; transdermal administration.

[0135] "Medicine" or "drug" means any substance or composition presented as having curative or preventive properties with respect to human or animal diseases. A medicine therefore includes any substance or composition which can be used in humans or animals or administered to them for the purpose of establishing a medical diagnosis or restoring, correcting or modifying their physiological functions by exerting a pharmacological, immunological or metabolic action. The term medicine includes in particular vaccines.

[0136] The terms "therapeutic" or "therapeutic uses" in the context of the present invention cover the uses "prevention", "treatment" and "vaccine".

[0137] A "therapeutically effective amount" is the amount of each active entity that is sufficient to produce a beneficial health result. An "immunologically effective amount" is the amount of each active entity that is sufficient to produce a detectable immune response. The active entity is, for example, an active ingredient, a therapeutic agent, a targeting agent, a labeling agent, or any combination thereof.

[0138] As used herein, a "pharmaceutically acceptable excipient" or "pharmaceutically acceptable carrier" is intended to include all carriers, solvents, diluents, excipients, adjuvants, vehicles, dispersion media, coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like, compatible with administration to a subject, especially an animal, and in particular a human. Suitable carriers for use herein are well known in the art (see, for example, the most recent edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins). Non-limiting examples of excipients include water, NaCl, saline solutions, saccharide solutions (e.g., glucose, trehalose, sucrose, dextrose, etc.), lactated Ringer's, alcohols, oils, gelatins, carbohydrates such as lactose, amylose or starch, fatty acid esters, . hydroxymethylcellulose, etc. Examples of adjuvants that may be mentioned include swelling agents such as, for example, a sugar such as lactose, sucrose, trehalose, sorbitol, glucose, raffinose, mannitol, preferably lactose, sucrose, trehalose, glucose, or mannitol, an amino acid such as arginine, glycine, or histidine, preferably glycine, or polymers of the dextran or polyethylene glycol type, or mixtures thereof.

[0139] By "subject" or "patient" is meant a human individual or an animal other than a human. The subject is for example a human or an animal likely to contract a disease, likely to be affected by a disease, or suffering from a disease. The subject is preferably a human being. The subject may be a child (human subject aged 16 years or younger) or an adult (human subject aged over 16 years). By "healthy subject" is meant a subject who does not suffer from the disease in question. In the context of the present invention, a healthy subject is preferably a subject who does not suffer from any disease. By "reference subject" is meant a subject who suffers from a known disease, at a known stage.

[0140] By "biological sample" or "sample" from a subject is meant a whole organ or tissue or part of such organ or tissue, fluid or fraction of such fluid, cells or cellular components, obtained from that subject, as well as a homogenate, lysate or extract prepared therefrom. In particular, a "biological sample" or "sample" is preferably any tissue (preferably portions or fractions thereof) that can be used to detect a disease, including, but not limited to, plasma, blood, lymph, serum, urine, mucus, saliva, a central nervous system (CNS) sample; such as a brain sample or a spinal cord sample, etc.), a respiratory tract sample (such as a lung sample, etc.), a salivary gland sample, a nasopharyngeal sample, an oropharyngeal sample, a digestive system sample (e.g., colon, intestine, etc.), a skin sample, an organ sample (e.g. liver, kidney, spleen, etc.), etc.

[0141] The biological sample may have been previously obtained by any technique known in the art. These techniques include, for example, collection using a swab, needle or syringe, surgery (such as stereotaxic surgery), puncture, explant, excision, biopsy. By "excision" is meant a surgical procedure consisting of cutting (excising) a more or less wide or deep part of the tissue, preferably an abnormality or growth of the tissue. An excision may be performed to remove and / or analyze a cancerous or suspicious tumor. The term "biopsy" herein refers to a sample of cells or tissues taken for analysis. Several types of biopsy procedures are known and practiced in the field. The most common types include (1) inci- ional, in which only a sample of the tissue is taken; (2) excisional biopsy (or surgical biopsy), which involves completely removing a tumor mass, thus performing a therapeutic and diagnostic procedure; and (3) needle biopsy, in which a tissue sample is taken using a needle, which can be large or fine. Other types of biopsy exist, such as smears or curettage, and can also be used to obtain the sample. Therefore, the sample can be, for example, an explant, an excision, a biopsy, etc. The sample is preferably obtained by a minimally invasive procedure, such as stereotaxic surgery.

[0142] In the following detailed description, the embodiments may be taken alone or in any suitable combination by those skilled in the art, and the above definitions apply to all the embodiments described below as well as to their combinations. Lipid microbubble

[0143] In the context of the present invention, the Inventors have developed innovative microbubbles, capable of delivering agents of interest into the body in a precise and targeted manner.

[0144] The inventors have notably shown that, surprisingly, the lipid microbubbles thus developed have significantly improved stability, unlike the microbubbles described in the prior art. Remarkably, the data also reveal that these optimized microbubbles are capable of delivering different types of therapeutic agents, targeting agents and / or labeling agents, including nucleic acids, more efficiently than the microbubbles described in the prior art. These microbubbles are notably capable of actively crossing vessels, the blood-brain barrier (BBB), or even the tumor microenvironment. The inventors have also demonstrated that the localized application of ultrasound makes it possible to target these optimized microbubbles very precisely towards the area to be treated.These data thus reveal the therapeutic potential of these lipid microbubbles to treat numerous pathologies in a targeted manner, including pathologies of the central nervous system.

[0145] The data also show that these optimized microbubbles are tools for labeling, detection and imaging.

[0146] This great flexibility is notably linked to the original formulation of the microbubbles which consists of the use of a mixture of cationic molecules such as lipo-phosphoramidates and / or histidylated polyethyleimine.

[0147] The present invention therefore relates to a lipid microbubble, comprising, or consisting essentially of, or consisting of, at least one cationic compound chosen from lipophosphoramidates, histidylated polyethylenimines, and any mixture of these. Advantageously, the envelope of the lipid microbubble comprises, or consists essentially of, or consists of, at least one cationic compound chosen from lipophosphoramidates, histidylated polyethylenimines, and any mixture thereof. Thus, according to an advantageous embodiment, the present invention relates to a lipid microbubble having an envelope comprising, or consisting essentially of, or consisting of, at least one cationic compound chosen from lipophosphoramidates, histidylated polyethylenimines, and any mixture thereof.

[0148] The lipid microbubble according to the invention may in particular be an ultrasound contrast agent.

[0149] According to a preferred embodiment, the microbubble according to the invention further comprises at least one agent chosen from a therapeutic / pharmacological agent, a targeting agent, a labeling agent, and any combination thereof.

[0150] The therapeutic / pharmacological agent, the targeting agent, the labeling agent, and their combination is (are) preferably:

[0151] i. exposed to the surface of the microbubble, or ii. embedded in the lipid envelope of the microbubble, or iii. incorporated within the microbubble, or iv. any combination of i to iii.

[0152] The therapeutic / pharmacological agent, the targeting agent, and the labeling agent are advantageously as defined in the “Definitions” section above.

[0153] According to one embodiment, said at least one agent comprises, or consists essentially of, or consists of, or is(are) chosen from:

[0154] 1. a nucleic acid; 2. a fat-soluble active ingredient; 3. a chemotherapeutic agent, such as a cytotoxic agent and / or a cytostatic agent; 4. an antibody; 5. a protein; 6. an antigen; 7. a toxin; 8. a receiver 9. an enzyme; 10. a hormone; 11. a ligand; 12. a viral vector; 13. a nanoparticle, preferably comprising at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof; 14. any derivative of 1) to 13), preferably any functional derivative thereof; 15. any fragment of 1) to 14), preferably any functional fragment thereof; And 16. any combination of 1) to 15).

[0155] According to a particularly preferred embodiment, said at least one agent comprises, or consists essentially of, or consists of, or is(are) chosen from:

[0156] a. a nucleic acid; b. a fat-soluble active ingredient; c. a chemotherapeutic agent, such as a cytotoxic agent and / or a cytostatic agent; d. an antibody; e. an antibody derivative; f. a functional fragment of an antibody or its derivative; g. a protein; h. a protein fragment, such as a peptide (eg a cell penetrating peptide (CPP)), an antigen, an epitope, a functional protein domain, etc.; i. a nanoparticle, preferably comprising (which may further comprise) at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof (including a hydrophilic / non-liposoluble agent); and j. any combination of a) to i).

[0157] The different types of agents 1) to 16), and a) to i), as listed above, are advantageously as defined in the “Definitions” section above.

[0158] Preferably, the nucleic acid 1) and / or a) comprises, consists essentially of, or consists of a plasmid, a vector, a complementary DNA (cDNA), a single-stranded DNA, a double-stranded DNA, a DNA comprising a sequence coding for a gene or a gene fragment, a DNA coding for a gene or a gene fragment (preferably a functional fragment), an RNA, a double-stranded RNA, a messenger RNA, a non-coding RNA, a small RNA, etc.

[0159] Preferably, the chemotherapeutic agent 3) or c) is chosen from cytotoxic agents, cytostatic agents, and cytotoxic and cytostatic agents. The chemotherapeutic agent may in particular be chosen, without being limited thereto, from paclitaxel, doxorubicin, gencitabin (eg Gemzar), temozolomide, etc.

[0160] The targeting agent may in particular comprise, consist essentially of, or consist of an antibody, an antibody derivative, a functional fragment of an antibody or its derivative, a protein, a protein fragment (such as a peptide, an antigen, an epitope, a functional domain of a protein, etc.), a nanoparticle (which may in further comprising at least one therapeutic agent, in particular a hydrophilic therapeutic agent), and any combination thereof.

[0161] According to an advantageous embodiment, the microbubble according to the invention further comprises at least one functional group, in particular a functional group allowing binding to at least one agent chosen from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof. Said group is preferably:

[0162] i. exposed to the surface of the microbubble, or ii. embedded in the lipid envelope of the microbubble, or iii. incorporated within the microbubble, or iv. any combination of i to iii.

[0163] The functional group is advantageously as defined in the “Definitions” section above.

[0164] Advantageously, the functional group comprises, or consists essentially of, or consists of, or is chosen from:

[0165] a. a tag peptide; b. a chemical group, preferably selected from a clickable function, a coupling group, and any combination thereof; c. an antibody; d. an antibody derivative; e. a functional fragment of an antibody or its derivative; f. an affinity tag (e.g. biotin, streptavidin, β-protein chitin-binding protein (CBP), maltose-binding protein (MBP), Strep-tag, glutathione-S-transferase (GST), poly(His) tag, etc.); and g. any combination of a) to f).

[0166] According to one embodiment, the lipophosphoramidate comprises, or consists essentially of, or consists of, or is selected from the group consisting of, a dimyristoyl phosphoramidate (preferably selected from a dimyristoyl bromide phosphoramidate (preferably O,O-dimyristoyl-N-[3N-(N methylimidazolium bromide) propylene] phosphoramidate (compound KLN27)), a dimyristoyl histamine phosphoramidate (preferably O,O-dimyristoyl(-N-(histamine)phosphoramidate (compound MM30)), and any combination thereof), a dioleyl phosphoramidate (preferably selected from the group of dioleyl methylimidazolium phosphoramidates (preferably O,O-dioleyl-N-(3 N-(A-methylimidazolium iodide) propylene) phosphoramidate (compound KLN25)), and any mixture of these.

[0167] According to one embodiment, the histidylated polyethyleimine is coupled to a fatty acid, preferably chosen from stearic acid, myristic acid, palmitic acid, oleic acid, and any combination thereof.

[0168] Advantageously, the microbubble further comprises (in particular the envelope of the microbubble further comprises) an additional lipid selected from a dimyristoyl-glycero-phosphocholine (preferably l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)), a distearoyl-glycero-phosphocholine (preferably l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)), a dimyristoyl-glycero-phosphoethanolamine-(polyethylene glycol) (preferably l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DMPE-PEG2000)), a distearoyl-glycero-phosphoethanolamine-(polyethylene glycol) (preferably l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG2000)), and a distearoyl-glycero-phosphoethanolamine-[biotinyl(polyethylene glycol)] (preferably l,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (DSPE-PEG2000-biotin)), a cholesterol, a beta-sitosterol, a l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l-oleoyl-2-[6-[(7-nitro-2-l,3-benzoxadiazol-4-yl)amino]hexanoyl]-3-trimethylammonium propane (DOTAP), l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), and any combination thereof.

[0169] According to a preferred embodiment, the microbubble further contains a biocompatible gas. The biocompatible gas is preferably contained by the envelope of the microbubble (it is therefore inside the microbubble, in the medium / cavity formed by the envelope). The biocompatible gas is preferably chosen from a perfluorobutane (C4F10), a perfluoropropane (C3F8), dinitrogen (N2), a sulfur hexafluoride (SF6), a nitrogen oxide (NO), hydrogen, dioxygen, helium, xenon, argon, nitrous oxide (N2O), and any mixture thereof; more preferably chosen from a perfluorobutane (C4F10), a perfluoropropane (C3F8), dinitrogen (N2), a sulfur hexafluoride (SF6), dioxygen, nitrous oxide (N2O), and any mixture thereof. Particularly preferably, the gas is chosen from the group of perfluorobutanes (C4F10).

[0170] The data obtained by the Inventors reveal that the microbubbles according to the invention, as defined above, are, remarkably, capable of actively crossing the vessels, the blood-brain barrier (BBB) and / or the tumor microenvironment (in particular during the localized application of ultrasound). The Inventors have also demonstrated that the localized application of ultrasound makes it possible to target these microbubbles very precisely towards the area to be treated, including areas that are difficult to access such as the central nervous system, the vessels, and the tumor microenvironment. In addition, the data also show that these microbubbles Optimized bubbles are capable of efficiently delivering different types of therapeutic agents, targeting agents and / or labeling agents, including nucleic acids, to these hard-to-reach areas.

[0171] Thus, according to an advantageous embodiment, the microbubble according to the invention is characterized in that it is capable of actively crossing the blood-brain barrier and / or the tumor microenvironment (in particular during the localized application of ultrasound). Methods for producing a lipid microbubble

[0172] The present invention also relates to a method for producing at least one microbubble according to the invention, as described above, comprising, or consisting essentially of, or consisting of, the following steps:

[0173] a. Mixture, in a container, of cationic compounds selected from lipophosphoramidates, histidylated polyethyleimines, and any mixtures thereof; ethanol; and optionally at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof; b. Evaporation of the mixture obtained in step a) to obtain a lipid film and rehydration of the lipid film to form a liposomal suspension; this entire step can also be carried out by microfluidics; c. Lyophilization of the liposomal suspension obtained in step b); d. Replacement of the air contained in the container containing the lyophilisate obtained in step c), by a biocompatible gas, the gas preferably being chosen from a perfluorobutane (C4F10), a perfluoropropane (C3F8), dinitrogen (N2), a sulfur hexafluoride (SF6), a nitrogen oxide (NO), hydrogen, dioxygen, helium, xenon, argon, nitrous oxide (N2O), and any mixture thereof; e. Rehydration of the lyophilisate from step d) to obtain a solution; f. Agitation of the solution obtained in step d) to form microbubbles; g. Optionally, functionalization of the microbubbles by at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof; and / or functionalization of the microbubbles by adding at least one functional group allowing binding to at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof.

[0174] The microbubble and its constituents, such as lipophosphoramidate, histidylated polyethyleimine, therapeutic agent, targeting agent, labeling agent, biocompatible gas, are advantageously as described in the preceding sections. preceding (in the “Definition” and / or “Lipid microbubble” section).

[0175] The invention further relates to a microbubble obtainable, or obtained, or directly obtained, by the production method described above. Compositions and kits

[0176] The present invention further relates to a kit comprising, or consisting essentially of, at least one microbubble according to the invention, as defined above.

[0177] The present invention also relates to a composition comprising, or consisting essentially of, or consisting of, at least one microbubble according to the invention, as defined above, and, optionally, an excipient.

[0178] The present invention relates in particular to a pharmaceutical composition comprising, or consisting essentially of, or consisting of, at least one microbubble according to the invention, as defined above, and, optionally, a pharmaceutically acceptable excipient.

[0179] Advantageously, the composition, in particular the pharmaceutical composition, comprises a therapeutically effective amount of microbubbles. The concentration of microbubbles in the composition, in particular the pharmaceutical composition, preferably ranges from 106 to 1014 microbubbles / ml, more preferably from 107 to 1013 microbubbles / ml, more preferably from 108 to 1012 microbubbles / ml, more preferably from 109 to 10 11 microbubbles / ml, more preferably the concentration of microbubbles in the composition being approximately 1010 microbubbles / ml.

[0180] According to one embodiment, the composition, in particular the pharmaceutical composition, comprises an amount of pharmaceutically acceptable excipient in the composition which ranges from 5% to 99% by weight relative to the total weight of the composition, preferably from 10 to 97% by weight, preferably from 20 to 95% by weight, preferably from 30 to 90% by weight, preferably from 40 to 85% by weight, preferably from 50 to 80% by weight, preferably from 60 to 70% by weight, relative to the total weight of the composition.

[0181] According to one embodiment, the kit comprises, or consists essentially of, or consists of:

[0182] a) at least one microbubble according to the invention (as defined above) or a composition as defined above, in particular a pharmaceutical composition as defined above, in a first container;

[0183] b) at least one therapeutic agent, in a second container;

[0184] c) optionally, at least one targeting agent in a third container;

[0185] d) optionally, at least one marking agent in a fourth container; and

[0186] e) optionally, instructions for preparation and / or use.

[0187] Advantageously, the kit further comprises means suitable for detecting the presence or absence of the marking agent in a sample and / or in a subject.

[0188] The microbubble and its constituents, such as lipophosphoramidate, histidylated polyethyleimine, therapeutic agent, targeting agent, labeling agent, biocompatible gas, are advantageously as described in the preceding sections (in the section "Definition", and / or "Lipid microbubble", and / or "Methods for producing a lipid microbubble").

[0189] Thus, preferably, the therapeutic agent and / or the targeting agent and / or the labeling agent comprises, consists essentially of, or consists of, or is chosen from:

[0190] 1. a nucleic acid; 2. a fat-soluble active ingredient; 3. a chemotherapeutic agent, such as a cytotoxic agent and / or a cytostatic agent; 4. an antibody; 5. a protein; 6. an antigen; 7. a toxin; 8. a receiver 9. an enzyme; 10. a hormone; 11. a ligand; 12. A viral vector; 13. a nanoparticle, preferably comprising at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof; 14. any derivative of 1) to 13), preferably any functional derivative thereof; 15. any fragment of 1) to 14), preferably any functional fragment thereof; And 16. any combination of 1) to 15);

[0191] more preferably among a nucleic acid, a liposoluble active agent, a chemotherapeutic agent, an antibody, an antibody derivative, a functional fragment of an antibody or its derivative, a protein, a protein fragment, a nanoparticle, and any combination thereof. Uses and Therapeutic Methods

[0192] The inventors have notably shown that, surprisingly, the innovative microbubbles developed here have the capacity to deliver agents of interest into the body in a precise and targeted manner, including in very difficult-to-access areas such as the central nervous system, vessels, and the tumor microenvironment. Indeed, the inventors have notably shown that, surprisingly, Lipid microbubbles are significantly more stable than prior art microbubbles. In particular, they are capable of actively crossing vessels, the blood-brain barrier (BBB), or even the tumor microenvironment. The inventors have also demonstrated that the localized application of ultrasound makes it possible to target these optimized microbubbles very precisely to the area to be treated. These data thus reveal the therapeutic potential of these lipid microbubbles to treat numerous pathologies in a targeted manner, including pathologies of the central nervous system.

[0193] The data also show that these optimized microbubbles are tools for labeling, detection and imaging.

[0194] The present invention therefore provides both powerful and broad-spectrum treatment methods for pathologies, as well as marking methods, particularly for medical imaging.

[0195] The present invention therefore relates to a microbubble according to the invention (as defined above); or a pharmaceutical composition or a kit comprising, or consisting essentially of, at least one microbubble according to the invention (as defined above); or a pharmaceutical composition as defined above; or a kit as defined above; or any combination thereof; for its use as a medicament.

[0196] The present invention also relates to a microbubble according to the invention (as defined above); or a pharmaceutical composition or a kit comprising, or consisting essentially of, at least one microbubble according to the invention (as defined above); or a pharmaceutical composition as defined above; or a kit as defined above; or any combination thereof; for its use as a marking agent, in particular as a contrast agent.

[0197] The present invention also relates to the use of a microbubble according to the invention (as defined above); or of a pharmaceutical composition or a kit comprising, or consisting essentially of, at least one microbubble according to the invention (as defined above); or a pharmaceutical composition as defined above; or a kit as defined above; or any combination thereof; as a medicament.

[0198] The present invention also relates to the use of a microbubble according to the invention (as defined above); or of a pharmaceutical composition or a kit comprising, or consisting essentially of, at least one microbubble according to the invention (as defined above); or a pharmaceutical composition as defined above; or a kit as defined above; or any combination thereof; as a marking agent, in particular as a contrast agent.

[0199] The present invention also relates to the use of a microbubble according to the invention (as defined above); or a pharmaceutical composition or kit comprising, or consisting essentially of, at least one microbubble according to the invention (as defined above); or a pharmaceutical composition as defined above; or a kit as defined above; or any combination thereof; for the manufacture of a medicament.

[0200] The present invention also relates to the use of a microbubble according to the invention (as defined above); or of a pharmaceutical composition or a kit comprising, or consisting essentially of, at least one microbubble according to the invention (as defined above); or a pharmaceutical composition as defined above; or a kit as defined above; or any combination thereof; for the manufacture of a marking agent, in particular a contrast agent.

[0201] The present invention also relates to a method of treatment, comprising administering a microbubble according to the invention (as defined above); or a pharmaceutical composition or kit comprising, or consisting essentially of, at least one microbubble according to the invention (as defined above); or a pharmaceutical composition as defined above; or a kit as defined above; or any combination thereof; to a subject (preferably a subject in need thereof).

[0202] The present invention also relates to a labeling method, comprising administering a microbubble according to the invention (as defined above); or a pharmaceutical composition or kit comprising, or consisting essentially of, at least one microbubble according to the invention (as defined above); or a pharmaceutical composition as defined above; or a kit as defined above; or any combination thereof; to a subject (preferably a subject in need thereof).

[0203] The microbubble and its constituents, such as lipophosphoramidate, histidylated polyethyleimine, therapeutic agent, targeting agent, labeling agent, biocompatible gas, are advantageously as described in the preceding sections (in the section "Definition", and / or "Lipid microbubble", and / or "Methods for producing a lipid microbubble").

[0204] Advantageously, the pharmaceutical composition and / or the kit are as described above in the “Compositions and kits” section.

[0205] According to a preferred embodiment, the microbubble, the composition, the kit, or any combination thereof, is (are) administered to a subject in need thereof, preferably in a therapeutically effective amount.

[0206] The microbubble, composition, kit, or any combination thereof, is (are) preferably formulated to be administered one or more times by the same route or by different routes. All conventional routes of administration are applicable. in the context of the invention, including oral, parenteral and topical routes.

[0207] Parenteral routes are intended for administration by injection or infusion and include systemic routes as well as local routes. Preferably, the microbubble, composition, kit, or any combination thereof, is formulated for one or more parenteral administrations, and preferably intravenously (into a vein), intravascularly (into a blood vessel), intraarterially (into an artery), intradermally (into the dermis), subcutaneously (under the skin), intramuscularly (into the muscle), intraperitoneally (into the peritoneum), or intratumorally (into a tumor). Administration may be as a single bolus dose or may also be by a continuous infusion pump.

[0208] Preferably, the microbubble, composition, kit, or any combination thereof, is formulated to be administered by intravenous infusion.

[0209] Administrations may utilize conventional syringes and needles (e.g., Quadrafuse injection needles) or any compound or device available in the art capable of facilitating or enhancing delivery of a microbubble to the subject (e.g., electroporation to facilitate intramuscular administration). An alternative is the use of a needle-free injection device (e.g., the Biojector TM device). Transdermal patches may also be considered.

[0210] Several doses within the indicated ranges may be administered to the subject. In the case of repeated administrations over several or more days, treatment will generally be continued until observable clinical benefit occurs. These doses may be administered intermittently, for example, daily, every 2 or 3 days, weekly, every 2 weeks, every 3 weeks, or monthly (for example, such that the subject receives from about two to about twenty doses of the composition). The doses may also be adjusted with each administration (for example, one or more higher initial doses followed by one or more lower doses).

[0211] In one embodiment, the microbubble, composition, kit, or any combination thereof, is administered in a "prime boost" approach that includes sequential administrations of one or more priming compositions and one or more boosting compositions. Typically, the priming and boosting compositions may utilize the same active agent (i.e., the microbubble, composition, kit, or any combination thereof), or may utilize a different active agent (i.e., the microbubble, composition, kit, or any combination thereof). Further, the priming and boosting compositions may be administered to the same or a different area of the body, by the same route or by different routes of administration. A preferred priming and boosting approach involves a first injection (e.g., subcutaneous, intramuscular, intradermal, intratumoral, or intravenous) (priming) followed by a second injection (e.g., subcutaneous, intramuscular, intradermal, intratumoral, or intravenous) after an optimal period of time. The present invention encompasses one or more administrations of the priming and / or boosting composition(s), with subcutaneous, intramuscular, intradermal, intratumoral, intranasal, and intravenous routes being preferred. The time period between the priming and boosting administrations varies from one week to 6 months, with one week to one month being preferred, and one to two weeks being preferred.

[0212] The microbubble, composition, kit, or any combination thereof, is (are) preferably administered in combination with the application of ultrasound, preferably the localized application of ultrasound, preferably the localized application of ultrasound on / towards the area to be treated and / or marked. The ultrasound is preferably administered at a frequency ranging from 1kHz to 10MHz, preferably from 10kHz to 9MHz, preferably from 50KHz to 8MHz, preferably from 100kHz to 7MHz, preferably from 150kHz to 6MHz, preferably from 200kHz to 5MHz, preferably from 300kHz to 4MHz, preferably from 400kHz to 3MHz, preferably from 500kHz to 2MHz, preferably from 750kHz to 1.5MHz, preferably from 0.8MHz to 1.2MHz, more preferably at a frequency of about 1MHz.

[0213] The ultrasound is preferably pulsed at a frequency ranging from 1Hz to 10kHz, preferably from 10Hz to 9kHz, preferably from 50Hz to 8kHz, preferably from 100Hz to 7kHz, preferably from 150Hz to 6kHz, preferably from 200Hz to 5kHz, preferably from 300Hz to 4kHz, preferably from 400Hz to 3kHz, preferably from 500Hz to 2kHz, preferably from 750Hz to 1.5kHz, preferably from 0.8kHz to 1.2kHz, more preferably at a frequency of about 1kHz.

[0214] The ultrasound is preferably administered at an acoustic pressure ranging from 100 to 800 kPa (negative peak), preferably from 200 to 700 kPa, preferably from 300 to 600 kPa, preferably from 400 to 500 kPa.

[0215] The ultrasound is preferably administered for 30 to 300 seconds, preferably for 40 to 250 seconds, preferably for 50 to 200 seconds, preferably for 60 to 180 seconds, preferably for 80 to 150 seconds, preferably for 90 to 120 seconds.

[0216] Advantageously, the method of administering the microbubble, the composition, the kit, or any combination thereof, comprises, or consists essentially of, or consists of, the following steps:

[0217] a. Administration of the microbubble, the composition, the kit, or any combination thereof, to a subject, by a suitable mode of administration (in particular as described above, preferably by parenteral route, more preferably intravenously); b. Application of ultrasound to the area to be treated and / or marked (preferably according to the frequency, pulse, acoustic pressure and duration conditions described above).

[0218] The administration method may further comprise additional steps of preparing the microbubble, the composition, the kit, or any combination thereof, prior to step a) of administration; in particular when the microbubbles, the composition, the kit, or any combination thereof, is in a lyophilized form; said additional steps being as follows:

[0219] i. Optionally, suspending the lyophilisate; ii. Optionally, activation of microbubbles; iii. Optionally, mixing the microbubbles with an excipient (including a diluent) and incubating for 0.5 to 15 minutes, preferably 1 to 10 minutes, preferably 1.5 to 8 minutes, preferably 2 to 6 minutes, preferably 1 to 5 minutes, preferably 1 to 3 minutes; iv. Optionally bringing the microbubbles into contact with a therapeutic agent, a targeting agent, a labeling agent, or any combination thereof; the therapeutic agent, the targeting agent, the labeling agent, or any combination thereof; may be premixed with an excipient (in particular a diluent).

[0220] According to one embodiment, the microbubbles are activated using a mechanical stirrer; for example with stirring between 2000 and 5000 revolutions per minute (rpm), preferably at 4000 rpm; for a period between 10 and 120 seconds, preferably for 45 seconds.

[0221] According to one embodiment, when the microbubble, the composition, the kit, or any combination thereof, is used for the purpose of labeling, the method further comprises a step of detecting the presence or absence of the labeling agent. In vitro Uses and Methods

[0222] The data reveal that the microbubble according to the invention can be effectively used as a labeling, detection and imaging tool.

[0223] The present invention therefore provides effective and reliable diagnostic methods. It allows in particular the diagnosis, prognosis, stratification or even monitoring of diseases, or even evaluation of the effectiveness of a treatment.

[0224] The present invention therefore relates to the in vitro use of at least one microbubble according to the invention (as defined above); or of a composition (in particular pharmaceutical) or of a kit comprising, or consisting essentially of, at least one microbubble according to the invention (as defined above); or a composition (in particular pharmaceutical) as defined above; or of a kit such as defined above; or any combination thereof; for:

[0225] i. delivering an agent (therapeutic, targeting, labeling, or any combination thereof) into a cell, or biological sample; ii. the marking of a cell or biological sample; iii. the diagnosis of a disease, in a subject likely to suffer from a disease; iv. monitoring of a subject suffering from an illness; v. the stratification of a subject suffering from a disease; vi. the evaluation of the effectiveness of a treatment (in particular curative) administered to a subject suffering from an illness; vii. detecting the presence or absence of at least one microbubble in a sample, in particular a biological sample; viii. determining the presence or absence, or the quantity, of at least one marking agent (in particular a contrast agent) in a sample, in particular a biological sample; ix. screening for compounds / molecules having an effect in the prevention, treatment, marking, or any combination thereof, of a disease; or x. any combination of i. to ix.

[0226] The present invention relates in particular to the in vitro use of at least one microbubble according to the invention (as defined above); or of a composition (in particular pharmaceutical) or of a kit comprising, or consisting essentially of, at least one microbubble according to the invention (as defined above); or a composition (in particular pharmaceutical) as defined above; or of a kit as defined above; or of any combination thereof; for delivering an agent inside at least one cell, or a biological sample, preferably by sonoporation;

[0227] the agent preferably being selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof;

[0228] the cell preferably being selected from an animal cell, a plant cell, a microorganism cell, and any combination thereof.

[0229] The microbubble, composition, kit, or any combination thereof, is (are) preferably brought into contact with the cell or biological sample (in particular administered to the sample) in combination with the application of ultrasound, preferably the localized application of ultrasound, preferably the localized application of ultrasound on / towards the area to be treated and / or marked. The ultrasound is preferably applied at a frequency ranging from 1kHz to 10MHz, preferably from 10kHz to 9MHz, preferably from 50KHz to 8MHz, preferably from 100kHz to 7MHz, preferably from 150kHz to 6MHz, preferably 200kHz to 5MHz, preferably 300kHz to 4MHz, preferably 400kHz to 3MHz, preferably 500kHz to 2MHz, preferably 750kHz to 1.5MHz, preferably 0.8MHz to 1.2MHz, more preferably at a frequency of about 1MHz.

[0230] The ultrasound is preferably pulsed at a frequency ranging from 1Hz to 10kHz, preferably from 10Hz to 9kHz, preferably from 50Hz to 8kHz, preferably from 100Hz to 7kHz, preferably from 150Hz to 6kHz, preferably from 200Hz to 5kHz, preferably from 300Hz to 4kHz, preferably from 400Hz to 3kHz, preferably from 500Hz to 2kHz, preferably from 750Hz to 1.5kHz, preferably from 0.8kHz to 1.2kHz, more preferably at a frequency of about 1kHz.

[0231] The ultrasound is preferably administered at an acoustic pressure ranging from 100 to 800 kPa (negative peak), preferably from 200 to 700 kPa, preferably from 300 to 600 kPa, preferably from 400 to 500 kPa.

[0232] The ultrasound is preferably administered for 30 to 300 seconds, preferably for 40 to 250 seconds, preferably for 50 to 200 seconds, preferably for 60 to 180 seconds, preferably for 80 to 150 seconds, preferably for 90 to 120 seconds, preferably for 60 seconds.

[0233] Advantageously, the method of administering the microbubble, the composition, the kit, or any combination thereof, comprises, or consists essentially of, or consists of, the following steps:

[0234] a. contacting the microbubble, the composition, the kit, or any combination thereof, with a cell or a biological sample; or administering the microbubble, the composition, the kit, or any combination thereof, to a biological sample, by a suitable mode of administration (in particular as described above, in the section "Therapeutic Uses and Methods"); b. Application of ultrasound to the area to be treated and / or marked (preferably according to the frequency, pulse, acoustic pressure and duration conditions described above).

[0235] The administration method may further comprise additional steps of preparing the microbubble, the composition, the kit, or any combination thereof, prior to step a) of administration; in particular when the microbubbles, the composition, the kit, or any combination thereof, is in a lyophilized form; said additional steps being as follows:

[0236] i. Optionally, suspending the lyophilisate; ii. Optionally, activation of microbubbles; iii. Optionally, mixing the microbubbles with an excipient (including a diluent) and incubating for 0.5 to 15 minutes, preferably 1 to 10 minutes, preferably 1.5 to 8 minutes, preferably 2 to 6 minutes, preferably 1 to 5 minutes, preferably 1 to 3 minutes; iv. Optionally bringing the microbubbles into contact with a therapeutic agent, a targeting agent, a labeling agent, or any combination thereof; the therapeutic agent, the targeting agent, the labeling agent, or any combination thereof; may be premixed with an excipient (in particular a diluent).

[0237] According to one embodiment, when the microbubble, the composition, the kit, or any combination thereof, is used for the purpose of labeling, the method further comprises a step of detecting the presence or absence of the labeling agent in the cell and / or the biological sample.

[0238] The microbubble and its constituents, such as lipophosphoramidate, histidylated polyethyleimine, therapeutic agent, targeting agent, labeling agent, biocompatible gas, are advantageously as described in the preceding sections (in the section "Definition", and / or "Lipid microbubble", and / or "Methods for producing a lipid microbubble").

[0239] Advantageously, the composition and / or the kit are as described above in the “Compositions and kits” section.

[0240] The following examples are intended to illustrate the present invention, and should not be considered limiting. Description of the figures

[0241] [Fig-1] Summary diagram of the formulations developed. The functionalization of the microbubble can depend on different constituents, such as cationic lipids (capable of electrostatic interactions), and biotinylated lipids (capable of binding antibodies in particular by streptavidin-biotin bonds).

[0242] [Fig.2] Chemical structure of cationic (LIPID 1) and fusogenic (LIPID 2) lipids used in lipophosphoramidate-based formulations.

[0243] [Fig.3] Size distribution of the different microbubble formulations developed. The analysis was carried out using microscopy image analysis (Image!® software).

[0244] [Fig.4] Zeta potential measurements (mV) of different formulations. Values represent the mean ± SD of 3 measurements.

[0245] [Fig.5] Complexation gel (0.6% agarose) of different volumes of MBc (cationic MB) with a constant amount of plasmid DNA (Ipg).

[0246] [Fig.6] Confocal imaging of MBc-tPTX gas microbubbles complexing CpG oligonucleotides coupled to FITC.

[0247] [Fig.7] Diagram of the assembly carried out allowing the flow analysis of the targeting of MBs functionalized.

[0248] [Fig.8] A) Graph representing the binding of MB44 on hCMEC / D3 cells stimulated or not for the production of VEGF receptor. Lanes: dot-dash) MBa on stimulated cells; dotted) MBa carrying the anti-VEGFR2 receptor on unstimulated cells; solid line) MBa carrying the anti-VEGFR2 receptor on stimulated cells. B) Graph representing the binding of MBc-tPTX on cells stimulated for the production of the receptor for 24 h. Lanes: dotted) MBc-tPTX without antibody; solid line) MBc-tPTX carrying the antibody. C) Graph representing the binding of MBc-tPTX complexing CpG ODNs on cells stimulated for the production of the receptor for 48 h. Lanes: dotted) MBc-tPTX without antibody; solid line) MBc-tPTX with antibody. Vertical bar: end of injection, start of rinsing.

[0249] [Fig.9] Schematic representing the experimental device of in vivo sonoporation comprising a focused ultrasound probe and a motorized positioning system. The mouse is placed in the cradle after injection of Evan's Blue by IV route the left hemisphere is then targeted, the FUS treatment is carried out 10s after injection of MBs for 60s. Adapted from Celia Si Ahmed, M2 2018, Orléans.

[0250] [Fig. 10] A) Diagram showing the brain partitioning performed for the luciferase activity assay (blue: area treated with FUS). B) Photograph of the brain of a mouse treated with MBc + FUS at 109 kPa, 24 hours post-transfection, the blue spot corresponds to the extravasation of Evan's blue. C) Photograph of a brain included for the production of sections with the Cryostat®.

[0251] [Fig. 11] Graph representing luciferase activity per mg of protein in different brain areas 24 h after sonoporation with a pLuc plasmid complexed with MBc. The area targeted by the ultrasound device is zone 2. Data represent the mean ± SEM. **: p<0.01. Examples

[0252] EXAMPLE: Design and development of microbubbles to deliver active ingredients in a targeted manner 1. Introduction

[0253] In the context of the present invention, a new formulation of gas microbubbles has been developed. These microbubbles are particularly advantageous, since they allow both the encapsulation of different types of active ingredients, such as nucleic acids, and their localized delivery after activation by focused ultrasound ( [Fig.l]). A new device has also been developed, allowing ultrasound to be sent into the brain of the mouse in a targeted manner. This system is coupled with these original gas microbubbles allowing the encapsulation and delivery of active ingredients. The for mulation of gas microbubbles is activated then using an agitator, the active ingredient can be present in the formulation or can be added after activation of the formulation. The device is positioned at the coordinates corresponding to the delivery site desired by the user in a motorized manner. The original microbubbles developed here are capable of crossing the BBB. Thus, the positioning system developed can be implemented with a brain atlas to locate the brain structures to be treated. The gas microbubbles are injected systemically then the ultrasounds are sent. The device can be implemented with a passive cavitation detection system to control in real time the activation of the microbubbles by ultrasounds.

[0254] Gas microbubble formulations make it possible to encapsulate or co-encapsulate different active ingredients:

[0255] i. liposoluble active agents, such as chemotherapeutic agents (eg paclitaxel), in particular on the surface and / or in the envelope of the microbubble; ii. anionic active agents such as nucleic acids (plasmid DNA, small RNA, messenger RNA, etc.; eg for gene therapy), for example using cationic lipids; iii. antibodies, for example using the streptavidin-biotin couple, or click chemistry, or lipids coupled with methyl tetrazine groups; iv. proteins or peptides (such as cell-penetrating peptides (CPPs)), for example using the streptavidin-biotin couple, or click chemistry, or lipids coupled with methyl tetrazine groups.

[0256] This great flexibility is notably linked to the original formulation of the microbubbles which consists of the use of a mixture of cationic molecules such as lipo-phosphoramidates and / or histidylated polyethyleimine.

[0257] Indeed, two types of cationic microbubbles have been developed; one based on lipophosphoramidates, the other based on histidylated polyethyleneimine coupled to a fatty acid.

[0258] These microbubbles have already shown their effectiveness in vitro and in vivo as an ultrasound theranostic (therapy and imaging) agent. The blood-brain barrier was transiently permeabilized without danger to the animal, this was validated by MRI imaging and histology. An expression of a luciferase transgene was detected after the sonoporation protocol. 2. Materials and methods

[0259] 2.1. Production of microbubbles (MB) according to the invention

[0260] The first step in MB production is to mix different lipids depending on the type of microbubble desired. The lipids KLN27 and MM30 ([Fig.2]) come from a collaboration with the University of Brest (Berchel). These are lipids phosphoramidate, LIPID 1 is cationic in nature while LIPID 2 is used as a fusogenic lipid (lipid that can fuse to membranes, notably used to promote endosomal escape). All other lipids used (DMPC, DSPC, DMPE-PEG2000, DSPE-PEG2000, DSPE-PEG2000biot) are from Avanti Polar Lipids (Alabaster, AL, USA). The mixing of the different lipids is carried out in a flask in the presence of absolute ethanol (99.96% pure). During this step, PTX solubilized in absolute ethanol (10 mM, Merck, Germany) is added according to the formulation (Table 2).

[0261] Table 2: List of formulations used. The proportions of the different lipids constituting the envelope are indicated in molar percentage, relative to the total molar quantity of lipids.

[0262] [Tables2] Nature of the lipid Neutral lipid PEGylé lipid Cationiq ue lipid Fused lipid Autre Nom DSP C DMP C DSPE- PEG20 00 DMPE -PEG2 000 DSPE-PEG20 00 biot LIPIDE 1 LIPIDE 2 PTX (pM) Ca tio ni qu e No n ci bl ée Co nt rôl e MBc 22,5 % 10% 45% 22.5% PTX MBc-P TX 22.5% 10% 45% 22.5% 175.66 Ci bl ée (t) Co nt rôl e MBc-t 22.5% 9% 1% 45% 22.5% PTX MBc-t PTX 22.5% 9% 1% 45% 22.5% 175.66 Anionic Ciblée MBa 90% 9% 1%

[0263] For the suite, the mixture is evaporated with the help of a Rotavapor (Büchi, Schwabach, Germany) for 30 min, 20 rpm at 60°C. After this step, a lipid film forms in the flask. The lipid film formed is taken up in 2 mL of HEPES (10 mM, pH 7.4) and then sonicated for 5 min to obtain a homogeneous lipid solution ( [Fig. 10]). The solution is then distributed in equal volume into 4 crimp-top vials (VWR International, Radnor, PA, USA) which will be stored for 1 h at -80°C. Finally, the vials are placed in a lyophilizer (Bioblock Scientific, Illkirch, France) overnight. Once the lyophilizate is collected, the vials are manually crimped and stored at 4°C before being activated. Activation consists of replacing the air in the vial with perfluorobutane (C4F10, F2 Chemical, UK) by overpressure, then rehydrating the lyophilisate with 500 μl of a 10 mM HEPES solution. The vials are shaken for 45 s using a VIALMIX (Bristol Myers Squibb, USA).This step allows the formation of microbubbles in the bottle, however it is necessary to wait 5 minutes after shaking before taking a sample. Once a bottle is activated, it can be kept for a few days at 4°C.

[0264] 2.2. Characterization of microbubbles 2.2.1. Concentration and size

[0265] The microbubbles are observed by an inverted microscope (Nikon Diaphot 300 invert) connected to a computer. This installation allows photography to be taken by a FASTCAM SA7 camera (Photron, USA) and the ICcapture® software. The photos are taken with different objectives (x10, x20, x40) then processed by ImageJ®. The processing consists of an 8-bit conversion of the image followed by thresholding allowing the detection of the contours of the MB. Subsequently, a particle analysis allows counting and obtaining the size. In order to carry out this characterization, the microbubbles are diluted to 10th or 100th in HEPES (10 mM, pH 7.4) then deposited on a Malassez slide. 2.2.2. Measurement of Zeta potential (Ç)

[0266] The Ç potential corresponds to the overall charge of a particle at its shear plane (surface of the particle). This is measured using the Nano partica SZ-100 (Horiba, Japan). In order to carry out the measurements, 30 pL of MB are diluted in 970 pL of 10 mM HEPES pH 7.4. The analysis is carried out at 25°C. 3. Results

[0267] 3.1. Characterization of the microbubbles according to the invention

[0268] The size and concentration of the microbubbles prepared according to Example 2 (MB according to the present invention) are evaluated by optical imaging. [Fig.3] presents the results obtained for the different formulations. The size information collected shows an average diameter of 1.41 pm for MBc and 1.41 pm for MBc-PTX, while it is 1.55 pm for MBc-t. MBc-tPTX have an average diameter of 1.98 pm. Concerning the anionic microbubbles, the MBa have an average size of 1.41 pm. The concentrations of the different formulations are summarized in Table 3. The size and concentration distribution remains homogeneous between the two main categories of microbubbles (cationic and anionic). In general, anionic MBs (similar to commercial MBs) have a similar size to cationic MBs, but a higher concentration. MBc functionalized with PTX appear slightly smaller and in higher concentration than the basic formulation (MBc). MBc-tPTX with biotinylated lipids as well as PTX are those with the largest size as well as the lowest concentration. However, the size distribution of MBs remains below 10 pm, allowing their in vivo injection.

[0269] Table 3: Summary table of concentrations and average sizes of the different microbubble formulations.

[0270] [Tables3] MB Formulation Average diameter (pm) Concentration (MB / mL) Cationic MBc 1.41 8.3 x 108 MBc-PTX 1.41 9.3 x 108 MBc-t 1.55 1.0 x 10' MBc-tPTX 1.98 4.25 x 108 Anionic MBa 1.41 2.38 x 10'

[0271] [Fig.4] shows the Ç potential measurements of the developed formulations. These results do not show any significant change in the overall charge when the MBc are functionalized with PTX, biotin, or both at the same time. These remain positive (MBc average: +28.8 mV). The MBa formulation does not have KLN27 (cationic lipid) and therefore serves as a control, its charge is -23.4 mV.

[0272] 3.2. Complexation of nucleic acids

[0273] The capacity of microbubbles to vectorize nucleic acids is evaluated by gel retardation. [Fig.5] shows the complexation capacity of MBc microbubbles (possessing the cationic lipid KLN27) in the presence of 1 pg of plasmid DNA (plasmid pLuc). When there are no microbubbles, a band migrating at approximately 3 kilobases appears after UV revelation. This band corresponds to the uncomplexed plasmid DNA. The higher the MB concentration, the more the intensity of the 3 kb band decreases, corresponding to a complexation of the pDNA with the mi bubbles. When the pDNA is complexed, it no longer migrates and remains in the deposition wells. By using 10 μL of MB, all of the pDNA is complexed; the 3 kb band is no longer visible, instead labeling is present in the wells.

[0274] This complexing capacity was also confirmed by confocal fluorescence microscopy ([Fig.6]). The MBc-tPTX are complexed for 2 min with CpG oligonucleotides coupled to FITC at the ratio of 1 pg of nucleic acid to 10 pL of microbubbles. The fluorescence observed on the contour of the microbubbles confirms the complexation of the nucleic acids. Fluorescent debris is visible around the microbubbles, probably showing the formation of lipoplexes from fragments of destroyed microbubbles or free lipids in solution.

[0275] 3.3. Evaluation of the targeting of functionalized MBs

[0276] In order to evaluate the targeting capacity of MBs against the VEGF receptor, a culture in Ibidi® of hCMEC / D3 cells stimulated for the production of VEGFR is carried out, then a flow analysis of the MBs by optical microscopy is done. To do this, 6-channel flow culture plates (Ibidi® p-Slide VI0.4, Clinisciences) are seeded at a rate of 18,000 cells / channel. Once the cells are fixed at the bottom of the channels, the wells are filled with culture medium supplemented (or not) with TGF[3 at 5 ng / ml and the cells are incubated for 24 h or 48 h at 37°C under a 5% CO2 atmosphere. The optimal TGF[3 concentration allowing an increase in the number of VEGFR2 receptors on the surface of endothelial cells is determined by flow cytometry.

[0277] Subsequently, the Ibidi® plate is positioned on the inverted microscope equipped with a camera and connected to the computer. The setup used throughout the experiment is shown in [Fig.7].

[0278] In order to analyze the fixation of the microbubbles, a field of observation is chosen. Different types of microbubbles are used (described in Table 2 above). Following the analyses carried out by flow cytometry, certain microbubbles are functionalized with the antibody directed against the VEGF receptor (VEGFR2). To do this, 1.23 μl of streptavidin (15 mM) are incubated in the presence of 0.3465 μL of anti-VEGFR2-Biot antibody (Anti-mouse CD309, 0.5 mg / mL, eBioscience) in order to obtain a ratio of 2.5 mol of antibody per mol of streptavidin. Then, 20 μL of the MB solution are incubated for 10 min in order to functionalize the MB, by preventing the formation of microbubble aggregates by streptavidin-biotin bonds. At the end of 10 min, a 10 mM HEPES solution pH 7.4 is added to reach a final volume of 1 mL.

[0279] The rest of the experiment consists of a succession of washing of the cells in culture. First of all, a first wash of 10 min with PBS takes place, at a flow rate of 0.137 mL / min (0.25 dyn / cm2). Then, the injection of the MB contained in 1 mL is carried out for 15 min at the same flow rate, followed by a first rinse for 10 min with PBS, then a second rinse of 10 min at 0.275 mL / min (0.5 dyn / cm2), to finish with a third rinse of 10 min at 0.550 mL / min (1 dyn / cm2), followed by a final rinse at 1.1 mL / min (2 dyn / cm2). Photographs of the channel are taken every minute from the injection. The series of photos is then processed on ImageJ® to obtain a count of the number of MB per minute.

[0280] The targeting capacity of microbubbles towards the VEGF receptor (VEGFR-2) was evaluated in vitro in real time using a culture chamber allowing the establishment of a flow. The cells used for this analysis are hCMEC / D3 endothelial cells, stimulated or not by TGF[3. Different formulations were tested on these cells in order to evaluate the effect of different constituents on the fixation capacity of the microbubbles ([Fig.8]).

[0281] Figure 8A shows the results obtained for the anionic formulation MBa. The volume of injected MB is the same for each condition tested. This formulation is tested without antibodies on cells stimulated for 24 h (dot-dash), with antibodies on unstimulated cells (dotted lines) and with antibodies on cells stimulated for 24 h (solid line). At t=15 min, the first rinse begins. The results show a very low number of bound MBs concerning the curve with dashes, corresponding to the non-specific interactions of these MBs on the cells, with 42 MBa bound at t=15 min. This number decreases to 27 at the end of the washes. Concerning the dotted curve, corresponding to the specific bindings of MBs on the cells, the number of bound MBs is 112 at t=15 min. This number decreases to 33 at the end of the washes. The solid line curve, corresponding to cells stimulated for VEGFR2 production in the presence of targeted MBs, shows a number of MBs fixed at t=15min of 102.This number decreases to 52 at the end of the washes. This curve therefore records the highest number of fixations over time.

[0282] Figures 8B and 8C show the results obtained for the cationic formulation MBc tPTX (incorporating PTX and biotinylated lipids). This formulation is tested on cells stimulated at 24 h and 48 h. The injection and washing rates are reduced by half compared to Figure 8A, in order not to stress the cells too quickly. When the cationic microbubbles are functionalized with the anti-VEGFR2 antibody, they bind in greater numbers compared to the microbubbles not functionalized for the antibody (Figures 8B, 8C). Without nucleic acid complexation, the number of bound MBs possessing the antibody is approximately 110% higher than the number of bound MBs without antibody. The complexation of CpG oligonucleotides (Figure 8C) by the microbubbles decreases the receptor binding capacity. When the microbubble has the antibody and the nucleic acid, the number of bound MB is approximately 60% higher compared to the control without antibody..

[0283] 3.4. In vivo experimentation 3.4.1. In vivo sonoporation

[0284] In order to perform transfection by MB and FUS, the hairs located on the mouse skull must be removed to allow perfect transmission of US between the skin and the ultrasound probe. Once the mouse is prepared, it is anesthetized using an oxygen / air mixture 1.5% isoflurane (Vetflurane, France) throughout the experiment. A catheter equipped with a 26G needle is placed in the tail vein to allow intravenous injections. An injection of Evan's blue (5%, saline solution 1 mL / kg) is performed. Evan's blue is a dye that binds to circulating albumin and does not naturally pass the BBB, its extravasation is however visible after an opening via MB+FUS, allowing us to obtain information on the location of the area targeted by our protocol once the brain is extracted from the skull.

[0285] Next, the mouse is placed in the cradle of the in vivo sonoporation platform ([Fig.9]). The FUS are applied using a 54 mm diameter single-element focused ultrasound transducer (Précision Acoustics, UK). The transducer is placed in a sealed cylinder filled with degassed water and closed by a membrane. The presence of bubbles is to be avoided to avoid poor propagation of the US. The transducer is positioned on a motorized control platform connected to a computer, allowing its movement using the Repetier software. The targeting of the transfection zone is done visually using a pointer located at the transducer. The chosen zone corresponds to half the eye-ear distance at the level of the left hemisphere (zone 2). Ultrasound conduction gel is applied to the mouse skull to allow transmission of the US.After pointing, the transducer is positioned on the pointed area and then descends into contact with the gel.

[0286] Subsequently, a solution of 30 pg of pDNA diluted in 60 pL of 10 rnM HEPES pH 7.4 and 20 pL of 20% sucrose is prepared. This solution is incubated for 2 min in the presence of 120 pL of MBc and then injected intravenously into the mouse. Ten seconds after the end of the injection, the US is sent for 60 s (1 MHz, 5% duty cycle, pulse duration 1 sec). Two acoustic pressures were tested: 109 kPa and 145 kPa. After application of the US, the mice are awakened.

[0287] 3.4.2. Measurement of luciferase activity (RLU assay)

[0288] 24H after sonoporation, mice transfected with the plasmid encoding luciferase are euthanized and their brains are removed. The brain is sectioned into 6 parts, zones 1, 2, and 3 correspond to the left part of the brain and zones 4, 5, 6 to the right part. The transfection zone targeted by our protocol is located in zone 2 visualized by the extravasation of Evan's blue ([Fig. 10]).

[0289] Each part is then placed in Eppendorf® tubes and then immersed in liquid nitrogen. Subsequently, the sections are manually ground in a mortar in the presence of liquid nitrogen, then the ground materials are solubilized in 500 pL of CCLR lysis buffer for 1h30. Centrifugation is performed (5 min, 12,000 RPM, 4°C), and 150 pL of supernatant is recovered in order to perform a reading using the Lumat LB9507 luminometer (Berthold, Germany). The device is then loaded with a LAR (Luciferase Assay Reagent) solution allowing the measurement of luciferase activity. The rest of the lysate is then used to determine the protein concentration contained in each zone. This quantification is carried out by a BCA assay. The values obtained are then normalized with the luciferase activity values in RLU / mg of protein.

[0290] The efficiency of nucleic acid delivery was tested by measuring the activity of a reporter gene encoding luciferase. This gene was transfected into the brain using cationic microbubbles (cMB) as well as focused ultrasound. Two different acoustic pressures were tested, 109 kPa (n=8) and 145 kPa (n=5).

[0291] The results presented in [Fig.l 1] show a high luciferase activity (between 5.5x103 and 1.05x104 RLU / mg protein) in zones 2 and 3 of the mouse brain, for the two ultrasound powers used. Luciferase expression is very significantly different between zones 2 and 4 as well as for 2 and 5 for a power of 109 kPa. The results obtained at 145 kPa are not significantly different from those obtained at 109 kPa. Luciferase activity is low in zones 1, 4, 5 and 6 of the brain (less than 103 RLU / mg protein). It should be noted that these values are close to those of the background noise of non-transfected tissues. Since targeting is not stereotaxic and the cutting of zones is imprecise, zones 1 and 3 may present luciferase activity.A strong expression of luciferase is therefore essentially measured in the left hemisphere (targeting location) and not in the right hemisphere, confirming the possibility of using focused ultrasound coupled with cationic microbubbles in order to carry out the delivery of nucleic acids. 3.4.3. Immunohistochemistry

[0292] Sections of brains of mice transfected with different plasmids were made in order to study the transfection zone as well as the cell types involved. The transfection zones are determined by observing the extravasation of Evan's Blue on the section.

[0293] In the context of the present invention, the inventors have developed innovative microbubbles, in particular innovative lipid microbubbles, capable of transporting drugs, in particular nucleic acids, in a stable manner in the blood circulation; of crossing the blood-brain barrier (BBB); and of delivering drugs in a targeted manner, in particular towards antigens of interest.

[0294] The inventors have notably shown that, surprisingly, the microbubbles Lipid microbubbles thus developed have significantly improved stability, unlike the microbubbles described in the prior art. Remarkably, the data also reveal that these optimized microbubbles are capable of delivering agents of interest, in particular nucleic acids, more efficiently than the microbubbles described in the prior art. These microbubbles are notably capable of crossing vessels as well as the BBB. The Inventors have also demonstrated that the localized application of ultrasound makes it possible to target these optimized microbubbles very precisely to the area to be treated. These data thus reveal the therapeutic potential of these lipid microbubbles to treat numerous pathologies in a targeted manner, including pathologies of the central nervous system.

[0295] The data also show that these optimized microbubbles are detection and imaging tools.

[0296] The present invention therefore provides both powerful and broad-spectrum treatment methods for pathologies, as well as effective and reliable diagnostic methods.

[0297] 3.5. Discussion

[0298] All of these data show that the innovative microbubbles developed by the presented Inventors are capable of transporting different types of drugs, in particular nucleic acids, in a stable manner in the blood circulation; of crossing the blood-brain barrier (BBB); and of delivering drugs in a targeted manner, in particular towards antigens of interest.

[0299] The inventors have notably shown that, surprisingly, the lipid microbubbles thus developed have significantly improved stability, unlike the microbubbles described in the prior art. Remarkably, the data also reveal that these optimized microbubbles are capable of delivering different types of agents of interest, including nucleic acids, more efficiently than the microbubbles described in the prior art. Particularly interestingly, these microbubbles are capable of crossing the vessels as well as the BBB. The inventors have also demonstrated that the localized application of ultrasound makes it possible to target these optimized microbubbles very precisely towards the area to be treated. These data thus reveal the therapeutic potential of these lipid microbubbles for treating numerous pathologies in a targeted manner, including pathologies of the central nervous system.

[0300] The data also show that these optimized microbubbles are detection and imaging tools.

[0301] The present invention therefore provides both powerful and broad-spectrum treatment methods for pathologies, as well as effective and reliable diagnostic methods. REFERENCES

[0302] Zhu X, Guo J, He C, Geng H, Yu G, Li J, Zheng H, Ji X, Yan F. Ultrasound triggered image-guided drug delivery to inhibit vascular reconstruction via paclitaxel-loaded microbubbles. SciRep. 2016 Feb 22;6:21683. doi:10.1038 / srep21683. PMID: 26899550; PMCID: PMC4761943.

[0303] Fan CH, Ting CY, Liu HL, Huang CY, Hsieh HY, Yen TC, Wei KC, Yeh CK. Anti-angiogenic-targeting drug-loaded microbubbles combined with focused ultrasound for glioma treatment. Biomaterials. 2013 Mar;34(8):2142-55. doi: 10.1016 / j.biomaterials.2012.11.048. Epub 2012 Dec 14. PMID: 23246066.

[0304] Delalande A, Bastié C, Pigeon L, Manta S, Lebertre M, Mignet N, Midoux P, Pichon C. Cationic gas-filled microbubbles for ultrasound-based nucleic acids delivery. Biosci Rep. 2017 Dec 22;37(6):BSR20160619. doi: 10.1042 / BSR20160619. PMID: 29180378; PMCID: PMC5741830.

[0305] Fan CH, Chang EL, Ting CY, Lin YC, Liao EC, Huang CY, Chang YC, Chan HL, Wei KC, Yeh CK. Folate-conjugated gene-carrying microbubbles with focused ultrasound for concurrent blood-brain barrier opening and local gene delivery. Biomaterials. 2016 Nov; 106:46-57. doi: 10.1016 / j.biomaterials.2016.08.017. Epub 2016 Aug 12. PMID: 27544926.

Claims

1.

2.

3. Claims Lipid microbubble, comprising at least one cationic compound chosen from lipophosphoramidates, histidylated polyethylenimines, and any mixture thereof. The microbubble of claim 1, further comprising at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof; said at least one agent preferably being: i. exposed to the surface of the microbubble, or ii. embedded in the lipid envelope of the microbubble, or iii. incorporated within the microbubble, or iv. any combination of i to iii. Microbubble according to claim 1 or 2, characterized in that said at least one agent is chosen from:

1. a nucleic acid; 2. a fat-soluble active ingredient; 3. a chemotherapeutic agent, such as a cytotoxic agent and / or a cytostatic agent; 4. an antibody; 5. a protein; 6. an antigen; 7. a toxin; 8. a receiver 9. an enzyme; 10. a hormone; 11. a ligand; 12. A viral vector; 13. a nanoparticle, preferably comprising at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof; 14. any derivative of 1) to 13), preferably any functional derivative thereof; 15. any fragment of 1) to 14), preferably any functional fragment thereof; and 16. any combination of 1) to 15).

4. A microbubble according to any preceding claim, further comprising at least one functional group enabling binding to at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof; said group preferably being: i. exposed on the surface of the microbubble, or ii. embedded in the lipid envelope of the microbubble, or iii. incorporated within the interior of the microbubble, or iv. any combination of i to iii.

5. Microbubble according to claim 4, the functional group being chosen from: - a tag peptide; - a chemical group, preferably chosen from a clickable function, a coupling group, and any combination thereof; - an antibody; - an antibody derivative; - a functional fragment of an antibody or its derivative; - an affinity tag; and - any combination thereof.

6. A microbubble according to any one of the preceding claims, characterized in that the lipophosphoramidate is selected from the group consisting of a dimyristoyl phosphoramidate, preferably selected from a dimyristoyl bromide phosphoramidate, a dimyristoyl histamine phosphoramidate, and any combination thereof; a dioleyl phosphoramidate, preferably selected from a dioleyl methylimi-dazolium phosphoramidate, and any combination thereof; and any mixture thereof.

7. Microbubble according to any one of the preceding claims, characterized in that the histidylated polyethyleimine is coupled to an acid fatty, the fatty acid preferably being selected from stearic acid, myristic acid, palmitic acid, oleic acid, and any combination thereof.

8. A microbubble according to any preceding claim, further comprising an additional lipid selected from the group consisting of dimyristoyl-glycero-phosphocholine, distearoyl-glycero-phosphocholine, dimyristoyl-glycero-phosphoethanolamine-polyethylene glycol, distearoyl-glycero-phosphoethanolamine-polyethylene glycol, and distearoyl-glycero-phosphoethanolamine-[biotinyl(polyethylene glycol)], cholesterol, beta-sitosterol, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-oleoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-3-trimethylammonium propane (DOTAP), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), and any combination thereof.

9. A microbubble according to any preceding claim, containing a biocompatible gas, the gas preferably being selected from perfluorobutane (C4F10), perfluoropropane (C3F8), nitrogen (N2), sulfur hexafluoride (SF6), nitrogen oxide (NO), hydrogen, oxygen, helium, xenon, argon, nitrous oxide (N2O), and any mixture thereof.

10. A pharmaceutical composition comprising at least one microbubble according to any one of the preceding claims, and, optionally, a pharmaceutically acceptable excipient, the concentration of microbubbles in the composition preferably ranging from 106 to 1014 microbubbles / ml, more preferably from 107 to 1013 microbubbles / ml, more preferably from 108 to 1012 microbubbles / ml, more preferably from 109 to 1011 microbubbles / ml, more preferably the concentration of microbubbles in the composition being approximately 1010 microbubbles / ml.

11. Kit, comprising: a) at least one microbubble according to any one of claims 1 to 9, in a first container; b) at least one therapeutic agent, in a second container; c) optionally, at least one targeting agent in a third container; d) optionally, at least one labeling agent in a fourth container; e) optionally, instructions for preparation and / or use; the therapeutic agent and / or the targeting agent and / or the labeling agent preferably being chosen from:

1. a nucleic acid; 2. a fat-soluble active ingredient; 3. a chemotherapeutic agent, such as a cytotoxic agent and / or a cytostatic agent; 4. an antibody; 5. a protein; 6. an antigen; 7. a toxin; 8. a receiver 9. an enzyme; 10. a hormone; 11. a ligand; 12. A viral vector; 13. a nanoparticle, preferably comprising at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof; 14. any derivative of 1) to 13), preferably any functional derivative thereof; 15. any fragment of 1) to 14), preferably any functional fragment thereof; and 16. any combination of 1) to 15); more preferably among a nucleic acid, a liposoluble active, a chemotherapeutic agent, an antibody, an antibody derivative, a functional fragment of an antibody or its derivative, a protein, a protein fragment, a nanoparticle, and any combination thereof.

12. Microbubble according to any one of claims 1 to 9, pharmaceutical composition according to claim 10, or kit according to claim 11, for use as a medicament or as a labeling agent.

13. Method of producing at least one microbubble according to one of any of claims 1 to 9, comprising the following steps: a. A mixture, in a container, of cationic compounds selected from lipophosphoramidates, histidylated polyethyleimines, and any mixtures thereof; ethanol; and optionally at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof; b. Evaporation of the mixture obtained in step a) to obtain a lipid film and rehydration of the lipid film to form a liposomal suspension; c. Lyophilization of the liposomal suspension obtained in step b); d. Replacing the air contained in the container containing the lyophilisate obtained in step c), with a biocompatible gas, the gas preferably being chosen from a perfluorobutane (C4F10), a perfluoropropane (C3F8), dinitrogen (N2), a sulfur hexafluoride (SF6), a nitrogen oxide (NO), hydrogen, dioxygen, helium, xenon, argon, nitrous oxide (N2O), and any mixture thereof; e. Rehydration of the lyophilisate from step d) to obtain a solution; f. Agitation of the solution obtained in step d) to form microbubbles; g. Optionally, functionalization of the microbubbles by at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof; and / or functionalization of the microbubbles by adding at least one functional group allowing binding to at least one agent selected from a therapeutic agent, a targeting agent, a labeling agent, and any combination thereof.

Images