WO2018178601A1

WO2018178601A1 - Treatment of an infection by the respiratory syncytial virus

Info

Publication number: WO2018178601A1
Application number: PCT/FR2018/050812
Authority: WO
Inventors: Abdessatar Chtourou; Gérald PERRET
Original assignee: Laboratoire Francais Du Fractionnement Et Des Biotechnologies
Priority date: 2017-03-31
Filing date: 2018-03-30
Publication date: 2018-10-04
Also published as: FR3064485A1

Abstract

The invention relates to the use of a composition of immunoglobulins (Ig) hyper-enriched in anti-RSV immunoglobulins in the prophylactic or curative treatment of an infection by the RSV virus in a patient, by administering the composition in a form such that the anti-RSV immunoglobulins neutralise the virus in the lungs.

Description

Treatment of respiratory syncytial virus infection

The present invention relates to the use of a immunoglobulin (Ig) composition hyper-enriched with respiratory syncytial virus immunoglobulin (RSV) immunoglobulin in the prophylactic or curative treatment of RSV infection in a subject, by administering of the composition in a form adapted so that the anti-RSV immunoglobulins bind to the RSV in the lower respiratory tract, preferably in the lungs.

BACKGROUND OF THE INVENTION

Respiratory syncytial virus (RSV) is a common infectious agent of the respiratory tract. RSV generally causes a mild, asymptomatic or weak (cold) infection in immunocompetent individuals, but may be responsible for several infectious episodes per year on the same individual, which may cause absenteeism or job.

In addition, this virus is an important factor in hospital infections in immunocompromised patients, whether on or off transplant, in infants or young children or in the elderly. RSV is the most important cause of bronchiolitis and pneumonia in infants and young children, and is largely responsible for respiratory infections in nursing homes.

Currently, there is no effective vaccine available to prevent RSV infection. Although anti-viral drugs are proposed to treat RSV infections, their efficacy is questionable, especially in children.

There is therefore still a need for preventive and curative treatment capable of neutralizing respiratory syncytial virus (RSV) and thus preventing or treating RSV infection.

SUMMARY OF THE INVENTION

The aim of the invention is to act locally in the lungs, and in a targeted manner, a composition of immunoglobulins (Ig) hyper-enriched with anti-RSV immunoglobulins.

The present invention relates to an immunoglobulin (Ig) composition hyper-enriched with anti-RSV immunoglobulins, for use in the prophylactic or curative treatment of RSV infection in a subject, by administering the composition in a form adapted for anti-RSV immunoglobulins to bind to RSV in the lower respiratory tract. Preferably, the lower airways are the lungs. The composition according to the invention is intended to be administered in the form of particles or droplets with a size of between 0.025 μιη and 10 μιη, preferably between 0.5 μιη and 5 μιη and preferably between 1 μιη and 3 μιη.

According to a particular embodiment, the composition of hyper-enriched Ig anti-RSV immunoglobulin may further comprise a surfactant. According to a particular embodiment, said composition may further comprise a viscosity modifying agent.

According to a particular embodiment, said composition comprises an anti-RSV immunoglobulin concentration of between 10 and 250 g / l, preferably between 10 and 100 g / l.

In a particular embodiment, the useful composition according to the invention is intended to be administered by the pulmonary route. In particular, the composition according to the invention may be administered by the pulmonary route by inhalation of an aerosol, preferably by means of a mouth spray or by nebulization by means of a nebulizer, in particular a sieve nebulizer. Said composition may be administered by the pulmonary route with a delivery volume of between 250 μl and 12 ml, preferably between 250 μl and 10 ml.

In a particular embodiment, the useful composition according to the invention is intended to be administered nasally. In particular, the composition according to the invention can be administered nasally in the form of an aerosol. Said composition may be administered nasally with a delivery volume of less than 20 ml, preferably less than 15 ml, more preferably between 2 ml and 8 ml.

Advantageously, the composition is used in the prophylactic or curative treatment of an RSV infection in a subject, the subject possibly belonging to at least one of the following groups: children, infants, immunocompromised persons, especially persons treated for a transplant especially a lung transplant, premature infants and children or elderly with lung disease especially chronic obstructive pulmonary disease (COPD), cardiac or immune. LEGEND OF FIGURES

Figure 1. Viral replication analysis by in vivo imaging: Preventive administration of the anti-RSV Ig enriched composition in the lungs intranasally. In vivo imaging study of viral replication 5 days after RSV infection in mice given preventively, intranasally: placebo; a composition comprising nonspecific Ig (negative control, "irrelevant antibody"); a composition enriched in anti-RSV Ig at a dose of 1 mg / kg (= LFB 1); a composition enriched in anti-RSV Ig at a dose of 0.1 mg / kg (= 0.1 LFB). The histogram represents the averages of the results obtained for each experimental condition. The results were obtained from 3 mice for the placebo condition and 4 mice for each of the other experimental conditions. Figure 2. Quantification of viral replication (by quantification of luciferase activity on lung lysates), 4 days after RSV infection. Viral replication is analyzed by quantification of luciferase activity in lung lysates of pre-implanted mouse lungs intranasally: placebo; a composition comprising non-specific Ig ("irrelevant antibodies"); a composition enriched in anti-RSV Ig at a dose of 1 mg / kg (= LFB 1); a composition enriched with anti-RSV Ig at a dose of 0.1 mg / kg (= 0.1 LFB). The histogram represents the averages of the results obtained for each experimental condition. The results were obtained from 3 mice for the placebo condition and 4 mice for each of the other experimental conditions. Figure 3. Quantification of viral replication (by RT-Q-PCR), 5 days after RSV infection. The expression of the RSV N nucleocapsid gene is quantified by RT-qPCR from mouse lung lysates that have been preventively delivered to the lungs intranasally: a placebo; a composition comprising non-specific Ig ("irrelevant antibodies"); a composition enriched in anti-RSV Ig at a dose of 1 mg / kg (= LFB 1); a composition enriched with anti-RSV Ig at a dose of 0.1 mg / kg (= 0.1 LFB). The histogram represents the averages of the results obtained for each experimental condition. The results were obtained from 3 mice for the placebo condition and 4 mice for each of the other experimental conditions. DETAILED DESCRIPTION OF THE INVENTION

Treatment

Here is described a method for the prophylactic or curative treatment of respiratory syncytial virus (RSV) infection in a subject, said method comprising the administration of an immunoglobulin (Ig) composition hyper-enriched in anti-inflammatory immunoglobulins. RSV in a form adapted for anti-RSV immunoglobulins to bind to RSV in the lower respiratory tract, preferably in the lungs.

The subject may be any mammal, preferably a human being, regardless of age or sex. This may include a subject at risk for infection with the RSV virus, for example children, infants, or an immunocompromised person, particularly in premature infants and children or elderly with a lung, heart or immune disease. Preferably, the subject, if infected, is asymptomatic, i.e., he has not yet developed symptoms associated with the infection.

In particular, the subject belongs to at least one of the following groups: children, infants, immunocompromised persons including persons treated for a transplant, in particular a lung transplant, premature infants and children or elderly people with a pulmonary disease especially chronic obstructive pulmonary disease (COPD), cardiac or immune. The term "prophylactic treatment" refers to preventing or stopping the development of the infection before it reaches the lower respiratory tract and causes inflammation of the lungs. It prevents the onset of symptoms, namely congestion or runny nose, but especially lung inflammation, especially in premature infants and children with lung, heart or immune disease.

The term "curative treatment" refers to the disappearance or decrease of RSV infection in the lower respiratory tract and / or the disappearance or alleviation of one or more symptoms, namely congestion or runny nose, but especially lung inflammation, especially in premature infants and children with pulmonary, cardiac or immune diseases. Ig composition hyper-enriched with anti-RSV Ig

The immunoglobulin composition useful according to the invention is a hyper-enriched immunoglobulin composition in anti-RSV immunoglobulins which has a neutralizing action vis-à-vis the RSV and has a therapeutic efficacy for the patient. More particularly, the composition is hyper-enriched with immunoglobulins directed specifically against the RSV virus. The term "specific" means that immunoglobulins recognize and bind to an immunogen of said virus, substantially without cross-reactivity with another virus. Throughout the description, the terms "Ig", "immunoglobulin" and "antibody" may be interchangeable.

The immunoglobulin composition comprises antibodies that bind to the same viral immunogen (referred to as a monovalent antibody population), or to several immunogens different from RSV (it may then be referred to as a polyvalent antibody population). Essentially, it is polyclonal antibodies.

Said immunoglobulin composition essentially contains polyvalent human immunoglobulins which may be immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin M (IgM) or immunoglobulin G (IgG). Advantageously, the immunoglobulin composition essentially contains IgG and / or IgM and / or IgA.

In a particular embodiment, the immunoglobulin composition essentially comprises IgG, regardless of their subclass (IgG1, IgG2, IgG3 and IgG4).

Preferably, the composition is suitable for topical administration and preferably contains predominantly IgG and / or IgA, IgA allowing the recruitment of cells such as mucosal cells with alfa receptors.

By "composition of Ig hyper-enriched anti-RSV immunoglobulin" is meant an immunoglobulin composition comprising at least 10%, at least 20%>, at least 30%>, at least 40%>, at least 50% > at least 60%>, at least 65%, at least 70%, at least 75%, at least 80%; at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%>, at least 99% by weight neutralizing immunoglobulins vis-à-vis the RSV. In a particular embodiment of the invention, the immunoglobulin hyper-enriched anti-RSV immunoglobulin concentrate comprises at least 10% by weight of polyvalent immunoglobulins capable of recognizing and / or neutralizing the RSV virus or one of of its parts. In another particular embodiment of the invention, the immunoglobulin concentrate hyper-enriched anti-RSV immunoglobulin comprises at least 30% by weight of polyvalent immunoglobulins capable of recognizing and / or neutralizing the RSV virus or a part thereof. In yet another particular embodiment of the invention, the anti-RSV immunoglobulin hyper-enriched immunoglobulin concentrate comprises at least 60% by weight of polyvalent immunoglobulins capable of recognizing and / or neutralizing the RSV virus or one of its parts.

Immunoglobulins directed specifically against RSV can bind to any of the proteins of the virus, preferably a surface protein, playing a role in the mechanisms of infection, especially during recognition, binding of the particle viral to its host cell, from the fusion of the virus particle to its host cell and from the spread of the host cell host cell virus. There may be mentioned in particular SH protein, protein G or RSV F protein.

The composition is usually concentrated. In particular, the composition may comprise an anti-RSV immunoglobulin concentration of between 10 and 250 g / l, preferably between 10 and 100 g / l.

Methods for preparing the hyper-enriched Ig composition of anti-RSV Ig

Several methods for the preparation of the hyper-enriched Ig composition of anti-RSV Ig are possible. Preferably, a method is used for preparing immunoglobulins from plasma pools, preferably human plasma. Such a method involves concentration operations of the plasma preparations, typically by a factor of 100 to 1000.

According to one particular embodiment, preparations of immunoglobulins derived from plasma or blood plasma fractions are subjected to affinity chromatography using a viral protein as an affinity ligand, to obtain a hyper-enriched immunoglobulin preparation. immunoglobulins capable of recognizing, and advantageously neutralizing, said RSV. This method uses affinity chromatography supports on which are grafted one or more RSV viral proteins, or antigenic fragments thereof. Among the viral proteins that are useful as ligands, there may be mentioned for example the proteins of the envelope: the SH protein, the protein G (highly glycosylated transmembrane glycoprotein responsible for binding to the host cell), the F protein (transmembrane glycoprotein responsible for fusion to the host membrane, entry of the virus into the cell and formation of syncitia). The chromatography uses a matrix comprising a support based on polymer particles, and is preferably in the form of a gel or a resin. These polymer particles are preferably of spherical or oblong shape, it may be in particular beads. The polymer may be natural or non-natural, organic or inorganic, crosslinked or uncrosslinked. The polymer is preferably an organic polymer, preferably crosslinked. In a preferred embodiment, the polymer is cellulose, and the particles are preferably porous cellulose beads. Other types of possible polymers include agarose, dextran, polyacrylates, polystyrene, polyacrylamide, polymethacrylamide, copolymers of styrene and divinylbenzene, or mixtures of these polymers. The particles can provide a chromatography medium that can be used to fill a column, for example.

On the support is grafted an RSV viral protein, or an antigenic fragment thereof, either directly or via a spacer, which covalently binds the ligand to the particles of the chromatographic medium. In a preferred embodiment of the invention, the substrate-grafted protein is an RSV surface protein, for example RSV F protein or RSV G protein, or an antigenic fragment thereof, either directly or via a spacer, which covalently binds the ligand to the particles of the chromatographic medium. In a particular embodiment of the invention, the grafted support has several different proteins and / or several different antigenic fragments belonging to the RSV, in order to obtain an immunoglobulin concentrate with different antigenic targets. A particle can carry several spacers. The bond between the ligand and the spacer may be, for example, an amide bond.

The gel matrix is prepared by conventional addition of a buffer to the ligand-carrying polymer particles, as known to those skilled in the art, so as to obtain a gel matrix suitable for chromatography. affinity. The matrix as defined herein is useful in affinity chromatography binding immunoglobulins. The matrix is particularly useful in mixed bed affinity chromatography. Such a matrix advantageously comprises several different antigens specific for RSV and is therefore capable of binding immunoglobulins directed against several different epitopes of RSV. The anti-RSV immunoglobulins present in the plasma or plasma fraction bind to the matrix, and the adsorbed product is eluted and recovered, enriched in anti-RSV immunoglobulins. Advantageously, the conditions of implementation of the affinity chromatography are adapted by those skilled in the art to ensure the reuse of the matrix while maintaining non-degrading elution conditions for the product of interest. The plasma or plasma fraction at the start of the enrichment process consists of plasma from blood plasma or pools of blood plasma from mammalian subjects (human or non-human) or a fraction of that plasma, being any part or part of the plasma, having been subjected to one or more purification steps. In a particular embodiment, the plasma or plasma fraction initially subjected to the enrichment process advantageously consist of plasma from pools of blood plasma of normal human subjects or a fraction thereof, any part or sub- part of the plasma, having undergone one or more purification steps. The plasma fractions that can be used include the cryoprecipitated plasma supernatant, the plasma cryoprecipitate (resuspended), the fractions I to V obtained by ethanolic fractionation (according to the Cohn or Kistler & Nitschmann method), the supernatant and the precipitate obtained. after precipitation with caprylic acid and / or caprylate, the chromatographic eluates and the non-adsorbed fractions of the chromatography columns, and the filtrates.

Particularly advantageously, the plasma or the initial plasma fraction subjected to the enrichment process come from blood plasma pools of normal human subjects, without prior selection of the donors, in particular, without selecting the donors on the basis of their possible rate. plasma anti-RSV immunoglobulin. The plasma or the initial plasma fraction subjected to the enrichment process thus advantageously comprises an anti-RSV immunoglobulin level similar to or equal to the anti-RSV immunoglobulin level of a plasma derived from the pool of at least 1000 human donors. Normal randomly chosen. The plasma or the starting plasma fraction subjected to the enrichment process is advantageously not enriched with anti-RSV immunoglobulins of interest compared to the anti-RSV immunoglobulin level of a plasma derived from the pool of at least 1000 selected donors. random way.

In a further embodiment, the plasma or plasma starter fraction subjected to the enrichment process consists of plasma from blood plasma or blood plasma pools of non-human, male and / or female mammal subjects, preferably goat , ewe, goat, bison, buffalo, camel, llama, mouse, rat, cow, bull, pig, sow, rabbit, horse, mare. The non-human mammal is advantageously transgenic and has been previously exposed to the RSV virus in order to produce anti-RSV human immunoglobulins in its plasma.

The plasma or starting plasma fraction subjected to the enrichment process contains polyvalent human immunoglobulins which may be immunoglobulin A (IgA), immunoglobulin E (IgE), immunoglobulin M (IgM) or immunoglobulin G (IgG). Advantageously, the plasma or the initial plasma fraction essentially contains IgG irrespective of their subclass (IgG1, IgG2, IgG3 and IgG4) and / or IgA.

The method can typically be an independent method, dedicated to the purification of anti-RSV immunoglobulins, and is then advantageously performed from plasma or a plasma fraction.

In this embodiment, the method advantageously comprises the following steps: providing a plasma sample or a plasma fraction,

passing the plasma sample or a plasma fraction on an immunoaffinity chromatography on the matrix described here,

recovery of the adsorbed fraction on the matrix corresponding to the immunoglobulin composition hyper-enriched in anti-RSV immunoglobulins.

Advantageously, the method may further comprise, after the step of capture on anti-RSV affinity chromatography, a dedicated depletion step of certain immunoglobulins, in particular immunoglobulins E (IgE) and / or immunoglobulins (IgM). , which may be involved in mechanisms of deleterious side effects in patients.

The method advantageously further comprises a step of subjecting the anti-RSV immunoglobulin hyper-enriched immunoglobulin composition to at least one inactivation and / or viral elimination step.

In yet another particular embodiment, the method for obtaining anti-RSV immunoglobulins is coupled to a polyvalent immunoglobulin G method in order to obtain, from the same starting plasma pool, both a multipurpose G immunoglobulin concentrate and a hyper-enriched anti-RSV immunoglobulin concentrate.

The process then typically comprises a prior step of obtaining the plasma fraction, by ethanolic fractionation and / or caprylic fractionation and / or chromatographic separation.

In this embodiment, the method advantageously comprises the following steps: providing a sample of a prepurified plasma fraction obtained by ethanolic fractionation and / or caprylic fractionation and / or chromatographic separation

passage of the prepurified plasma fraction on an immunoaffinity chromatography on the matrix described here, recovery of the adsorbed fraction on the matrix corresponding to the immunoglobulin composition hyper-enriched in anti-RSV immunoglobulins.

By "chromatographic separation" is meant any chromatography step, whether it is ion exchange chromatography (anion exchange and / or cation exchange), mixed mode, affinity (using chemical-type ligands, antibodies, antibody fragments, aptamers). By chromatographic separation is meant both a single chromatography column, or a set of chromatography columns, using or not the same type of support, in series (multicolumn mode for example) or in parallel.

More specifically, the plasma fraction can be obtained by an ethanol fractionation originally developed by Cohn et al (Cohn et al, 1946. J. Am Chem Soc 68, 459, Oncley et al, 1949, J. Am. Soc 71, 541), or by chromatographic separation, as described, for example, in EP 0 703 922 and WO 99/64462, or by caprylic fractionation as described by Steinbuch et al 1969, Arch Biochem. Biophys. 134 (2): 279-84). Particularly preferred methods developed by the Applicant in the patent applications WO 94/29334 and WO 02/092632, and particularly that described in WO 02/092632. In this case, a blood plasma, or an Ig-enriched blood plasma fraction, is subjected to caprylic fractionation (prepurification by precipitation of non-immunoglobulin contaminants), and a single chromatography on an anion exchange resin carrier carried out at pH. alkaline. Viral inactivation treatment can be carried out, preferably carried out by solvent-detergent, as described by Horowitz in US Pat. No. 4,764,369, optionally supplemented by a step of viral elimination by nanofiltration on a 75N, 35N pore size filter. , 20N and / or 15N.

The Ig fraction thus harvested is already concentrated, but can then undergo additional concentration steps by ultrafiltration, and sterilizing filtration.

This concentrate is then subjected to an immunoaffinity chromatographic step on the matrix as described above.

Advantageously, the method may furthermore comprise, preferably after the step of capture on anti-virus affinity chromatography, a dedicated stage of depletion of certain immunoglobulins, in particular immunoglobulins A (IgA), and / or immunoglobulins. E (IgE) and / or immunoglobulin M (IgM), which may be involved in mechanisms of deleterious side effects in patients. The immunoglobulins to be depleted are advantageously selected in particular according to the route of administration chosen for the anti-virus immunoglobulin hyper-enriched immunoglobulin concentrate and / or according to the content of the plasma sample or the starting plasma fraction. The method may further comprise the subsequent steps of adding one or more pharmaceutically acceptable stabilizers; and optionally freeze or lyophilize the concentrate thus obtained. Formulation

Advantageously, an immunoglobulin composition as described above is in the form of a pharmaceutical composition comprising anti-RSV immunoglobulins, in combination with at least one pharmaceutically acceptable excipient. By pharmaceutically acceptable excipient means excipients which are not harmful or toxic vis-à-vis the respiratory tract and more particularly the tracheobronchial epithelium. The formulation of the pharmaceutical composition useful according to the invention is adapted so that the anti-RSV immunoglobulins bind to RSV in the lower respiratory tract, preferably in the lungs.

Thus, the pharmaceutical composition can be prepared according to the usual methods, using appropriate excipients, preferably adapted for administration to reach the lower respiratory tract.

In particular, additives can be used in the formulation, such as

stabilizing agents such as albumin, and / or

antioxidants (such as ascorbic acid, cysteine, alpha tocopherol, metbionme), and / or

sugars and glycols (such as glucose, maltose, mannitol, sorbitol, glycerin, lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitosis, myoinisitol, galactose, galactitol, glycerol, cyclitols (such as Pinositol), and PEG), and / or complexing agents (such as sodium edetate), and / or

preservatives and / or bacteriostats (such as anhydrous ethanol, benzalkonium chloride, benzyl alcohol, chlorobutanol, parabens, phenylethyl alcohol, benzoic acid, EDTA, 2-pyrrolidone, and their derivatives), and / or - viscosity modifiers (such as calcium chloride, magnesium chloride, sodium chloride, sodium bisphosphate, caffeine, theophylline, tyramine, procaine, lidocaine , imidazole, aspartame, saccharin, and acesulfame potassium), and / or

agents that influence osmolarity (such as mannitol, arginine or glycine), and / or surfactants such as nonionic surfactants, such as polysorbates (polysorbate 80 or polysorbate 20), poloxamers (such as poloxamer 188, PF68), Triton ™, sodium dodecyl sulfate (SDS), sodium octyl glycoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, stearyl sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, stearyl sarcosine, linoleyl betaine, myristyl betaine, cetyl betaine, lauroamidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, isostearamidopropyl betaine (such as lauroamidopropyl), myristarnidopropyl-, palmidopropyl- , or isostearamidopropyl-dimethylamine, sodium methyl cocoyl-, or disodium methyl ofeyl-taurate, quaternary phosphate compounds (such as Monaquat ™ series from Mona Industries, Inc., Paterson, NJ), polyethyl glycol, polypropyl glycol, and / or amino acids or their derivatives (such as isoleucine, arginine hydrochloride and lysine hydrochloride), and / or

enzyme inhibitors and / or

pH-influencing agents (such as citrate or phosphate buffer) and / or agents that influence the tonicity of the formulation (such as sodium chloride or arginine glutamate, metalloprotein complexes, polymers biodegradable (especially polyesters), ions capable of forming salts (such as sodium), polyhydric alcohol sugars, amino acids (such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid and threonine), organic sugars or alcohol sugars (such as lactitol, stachyose, mannose , sorbose, xylose, ribose, ribitol, myoinisitose, myoinisitol, galactose, galactitol, glycerol, cyclitols including inositol, polyethylene glycol), sulfur reducing agents (such as urea , glutathione, thioctic acid, sodium thioglycolate, thioglycerol, Γα- monothioglycerol and sodium thiosulfate) and / or

cryoprotectants (such as sucrose or trehalose)

In particular, the composition according to the invention may comprise a surfactant. The term "surfactant" means an amphiphilic molecule having two parts of different polarity, one lipophilic and apolar, the other hydrophilic and polar. A surfactant may be of ionic type (cationic or anionic), zwitterionic or nonionic. The surfactant may advantageously be chosen from nonionic surfactants, such as polysorbates (polysorbate 80 or polysorbate 20), poloxamers (eg poloxamer 188, PF68), Triton ™, sodium dodecyl sulfate (SDS), sodium octyl glycoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, stearyl sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, stearyl sarcosine, linoleyl betaine, myristyl betaine, cetyl betaine, lauroamidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, isostearamidopropyl betaine (such as lauroamidopropyl), myristarnidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine, sodium methyl cocoyl-, or disodium methyl ofeyl-taurate, quaternary phosphate compounds (such as Monaquat ™ series from Mona Industries, Inc., Paterson, NJ), polyethyl glycol, polypropyl glycol.

According to a particular embodiment, the composition according to the invention may comprise a viscosity modifying agent, the viscosity being the set of phenomena of resistance to flow occurring in the mass of a material. For example, the lower the viscosity, the easier the fluid flows. In particular, the viscosity modifier may be a plasticizer. The viscosity modifying agent may be advantageously chosen from calcium chloride, magnesium chloride, sodium chloride, sodium bisphosphate, caffeine, theophylline, tyramine, procaine, lidocaine, imidazole, and the like. aspartame, saccharin, and acesulfame potassium.

In a particular embodiment of the invention, the composition comprises agents which influence the pH (such as citrate buffer or phosphate).

In a particular embodiment of the invention, the composition comprises agents which influence the tonicity of the formulation (such as sodium chloride or arginine glutamate, metalloprotein complexes, biodegradable polymers (in particular polyesters ), ions capable of forming salts (such as sodium), polyhydric alcohol sugars, amino acids (such as alanine, glycine, glutamine, asparagine, histidine, arginine) , lysine, ornithine, leucine, 2-phenylalanine, glutamic acid and threonine), organic sugars or alcohol sugars (such as lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinisitosis, myoinisitol, galactose, galactitol, glycerol, cyclitols including inositol, polyethylene glycol), sulfur reducing agents (such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, Γα-monoth ioglycerol and sodium thiosulfate). In a particular embodiment of the invention, the composition comprises cryoprotectants (such as sucrose or trehalose). In a particular embodiment, said composition is in the form of an inhalation powder which additionally contains suitable physiologically acceptable adjuvants, for example chosen from monosaccharides, disaccharides, oligo- and polysaccharides, polyalcohols, salts and the like. or mixtures of these adjuvants. In a particular embodiment, said composition is dispersed in a propellant gas selected from hydrocarbons, such as n-propane, n-butane or isobutane or halohydrocarbons, such as fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane. For example, the propellant may be trichlorofluoromethane, dichlorodifluoromethane, chlorodifluoromethane or dichlorotetrafluoroethane. These propellants can also act as a solvent because they are liquefied before being sprayed with said composition.

Administration Said immunoglobulin (Ig) composition hyper-enriched with anti-RSV immunoglobulins as described above is intended to be administered in a form adapted so that the anti-RSV immunoglobulins bind to RSV in the lower respiratory tract, preferably in the lungs, particularly at the level of the pulmonary epithelium. The composition according to the invention is administered in the form of particles or droplets of suitable size and morphology for targeting the lower respiratory tract, preferably the lungs, and more particularly the pulmonary epithelium. In particular, the size (also called mass median aerodynamic diameter) of the particles (solid) or droplets (liquid) is adapted to allow the passage thereof through the respiratory tract to the pulmonary alveoli. In particular, the size of the particles or droplets is between 0.025 microns (25 nm) and 10 μιη, preferably between 0.5 μιη and 5 μιη and more preferably between 1 μιη and 3 μιη. Particle size can be measured by any technique known to those skilled in the art, especially laser diffraction techniques. Droplet size may be measured by any technique known to those skilled in the art, including laser diffraction techniques and Doppler-based techniques. In particular, those skilled in the art will be able to carry out their measurements using a device implementing laser diffraction and in particular the Visisize D30, Visisize P15, Visisize N60 and Visisize S200 (Oxford Lasers Inc) models.

In a particular embodiment, said composition is intended to be administered by the pulmonary route. The term "pulmonary administration" is intended to mean any release and / or application of the composition to the lower respiratory tract, and preferably to the lungs, more preferably to the level of the pulmonary epithelium, these surfaces constituting a major territory of the pulmonary epithelium. RSV infection and are therefore of interest in the prophylaxis and curative treatment of RSV infection.

By "lower respiratory tract" or "lower airway" is meant any intra-thoracic duct or cavity such as the trachea, bronchi and lungs (consisting of bronchioles and alveoli). In particular, the administration allows the composition to reach and / or penetrate the pulmonary epithelium, characterized by a surface of 90000 cm ² developed within the pulmonary alveoli. Preferably, the administration makes it possible to preventively and / or curatively treat a pulmonary infection caused by the RSV virus.

In a particular embodiment of the invention, said composition is intended for inhalation.

Said immunoglobulin (Ig) composition which is hyper-enriched with anti-RSV immunoglobulins can thus be administered by the pulmonary route in the form of a powder, a solution, a suspension or an aerosol. Preferably, said composition is administered as an aerosol comprising solid particles or liquid droplets having a size and morphology adapted to target the lower respiratory tract, and preferably the lungs. An aerosol is defined here as the dispersion of solid particles or very fine liquid droplets in a gas. In particular, the aerosol comprises a continuous gaseous phase and, dispersed therein, a discontinuous phase of particles or droplets. Said immunoglobulin (Ig) composition hyper-enriched with anti-RSV immunoglobulins may be administered by the pulmonary route using a dispenser, preferably using an inhaler device such as: a powder inhaler, a pressurized metered dose inhaler, a nebulizer or any other device known in the pharmaceutical literature that makes it possible to inhale a composition.

In the case of the powder inhaler, the composition is in the form of a micronized dry powder, which is inhaled by the subject with the aid of a deep inspiration. In the case of a pressurized metered dose inhaler, said composition may be in the form of a micronized powder suspended in a gas or in the form of a solution dispersed in a gas. More particularly, the aerosol dispenser aerosolizes said composition in suspension or in solution by means of a propellant (such as a hydrofluoroalkane). The metered dose inhaler is advantageously in the form of a mouth spray.

Thus, according to a particular embodiment, said composition is administered by inhalation of an aerosol, preferably by means of an oral spray.

A nebulizer is defined herein as a device capable of aerosolising a liquid material in a dispersed liquid phase. More particularly, the nebulizer makes it possible to transform said liquid composition into "mist" with the aid of a pressurized gas or an air compressor. The nebulizer allows the administration of said composition by means of a mask or tip disposed on the mouth and / or the nose of the subject. In a particular embodiment of the invention, the pharmaceutical compositions according to the invention are administered using a nebulizer chosen from the following group: a jet nebulizer, an ultrasound nebulizer and a sieve nebulizer.

Thus, according to a particular embodiment, said composition is administered by nebulization, in particular by means of a sieve nebulizer.

In the case of administration carried out using a nebuliser the duration of nebulization is adapted according to the subject. Preferably, the duration of nebulization will be a maximum of 20 minutes for an adult subject and a maximum of 10 minutes for a child. Preferably the administration device allows the administration of said composition in aerosol form. Preferably, the delivery device is a nebulizer or a pressurized metered dose inhaler for spraying said composition in aerosol form.

The pharmaceutical composition according to the invention may be in the form of unit doses (the device allows only one administration) or multidose (the device allows multiple administrations). In a particular embodiment of the invention, the pharmaceutical compositions are advantageously in the form of unit doses.

The pharmaceutical composition according to the invention may be administered by the pulmonary route with a delivery volume of between 250 μl and 12 ml, preferably between 250 μl and 10 ml.

By "administration volume" is meant the volume of said composition administered, dose or dose. In a particular embodiment of the invention, said dose or dose has a suitable administration volume so that the anti-RSV immunoglobulins bind to RSV in the lower respiratory tract, preferably in the lungs. Preferably, the administration volume is sufficient to allow the diffusion of said composition in the lower respiratory tract, preferably in the lungs.

In a particular embodiment, said composition is intended to be administered nasally to reach the lower respiratory tract. "Nasal" administration is understood to mean any administration, release and / or application of the composition in the upper respiratory tract via the nasal cavity of a subject. By "upper respiratory tract" or "upper airway" is meant any duct or extra-thoracic cavity in contact with the air such as the nose, mouth, nasal cavity, pharynx or larynx. In particular, nasal administration may allow the composition to reach and / or enter the lower respiratory tract. Preferably, the nasal administration allows the composition to reach and / or penetrate the lungs. Advantageously, the administration of the composition through the nasal route is adapted so that the anti-RSV immunoglobulins bind to RSV in the lower respiratory tract, preferably in the lungs. Preferably, the nasal administration makes it possible to preventatively and / or curatively treat a pulmonary infection caused by the RSV virus.

In a particular embodiment, the composition is administered nasally, in the form of an aerosol. In a particular embodiment of the invention, the immunoglobulin (Ig) composition hyper-enriched with anti-RSV immunoglobulins is administered nasally with a delivery volume of less than 20 ml, preferably less than 15 ml, more preferably between 2 ml and 8 ml. The dosage may be adjusted appropriately to achieve the desired levels of immunoglobulins in the lungs.

In a particular embodiment of the invention, said hyper-enriched Ig anti-RSV immunoglobulin composition is administered once a day. In another particular embodiment of the invention, said hyper-enriched Ig anti-RSV immunoglobulin composition is administered once a week.

The following examples illustrate the invention without limiting its scope.

EXAMPLES EXAMPLE 1 Preparation of a composition enriched in anti-RSV Ig, from plasma fractions

In this example, the sample used is a plasma fraction resulting from the immunoglobulin purification process as described in the patent application WO02092632. The purification process comprises the following steps:

- Recovery of a fraction I + II + III, obtained from ethanol-treated blood plasma Caprylic acid precipitation

Solvent-detergent treatment (Triton / TnBP)

Anion exchange chromatography (TMAE) at basic pH

The eluate of the basic pH anion exchange chromatography is then subjected to immunoaffinity chromatography as described below.

1. Anti-RSV affinity chromatography (F protein)

The RSV-F 11049-V08B protein (Sino Biological Inc), a synthetic protein derived from an HRSV (RSS-2) DNA sequence from Met 1 to Thr 529, containing 518 amino acids, was used as an affinity ligand. cleavage of the propeptide and whose predicted molecular mass is 58 kDa. In SDS-PAGE analysis and reduction conditions, the apparent molecular weights are 45-55 kDa and 18 kDa. The RSV-F 11049-V08B ligand used for grafting is in post-fusion conformation.

2mL of NHS-activated SepharoseTM 4 Fast Flow gel is placed in a 1.1cm diameter chromatography column and the buffers used are those recommended by the gel supplier. After washing (1% HCl at 4 ° C.) and equilibration in coupling buffer, 1.98 mg of protein F to be grafted are added to the gel and the mixture is stirred. The blocking of the reactive groups on the gel is carried out by adding 6 ml of 0.5M ethanolamine buffer, 0.5M NaCl, pH 8.3 (6mL), and the gel is stirred for 12h. The coupled gel is washed with an alternation of 3 volumes of basic and then acidic buffers: 0.1M Tris-HCl pH 8.4 and 0.1M acetate, 0.5M NaCl pH 4, this cycle being repeated three times. The gel is then stored in lOmM citrate buffer, 0.5M NaCl, O.Ig / L sodium azide pH 6.6

The coupling was evaluated at 98%, corresponding to a ligand density of 0.89 mg / ml of grafted gel. The sample is equilibrated in the injection buffer by adding a concentrated solution of NaCl buffer and trisodium citrate qs 0.05M NaCl and 0.01M trisodium citrate. The product is then passed over the column during 3.3min (contact time). After washing, the elution is carried out in 0.1M glycine buffer, 30% propylene glycol at pH 2.57. The eluate is immediately neutralized with 1M Tris-HCl pH 9 buffer to raise the pH to about 6. The gel is then regenerated in order to remove the non-eluted Ig with a 6M guanidine buffer pH 6. The fraction recovered is also neutralized by the addition of 1M Tris-HCl pH9 to raise the pH to 6.

2. Anti-RSV affinity chromatography (G protein)

Alternatively, or in addition, the RSV-G strain A protein (ref 11070-V08H2, SB), a synthetic protein derived from a DNA sequence of hRSV (strain rsbl734) from Asn 66 to Arg 297, containing 242 amino acids after cleavage of the propeptide and with a predicted molecular weight of 26.3 kDa. In SDS-PAGE analysis and reducing conditions, the apparent molecular weight is included because of a high glycosylation between 60 kDa and 90 kDa.

strain B (ref 13029-VO8H, SB), a synthetic protein derived from a hRSV DNA sequence (strain 036633-1) from His 67 to Ala 299, containing 244 amino acids after cleavage of the propeptide and whose mass predicted molecular is 27kDa. In SDS-PAGE analysis and reducing conditions, the apparent molecular weight is included because of a high glycosylation between 80 kDa and 90 kDa.

The coupling is performed according to the same protocol as that used in section 1.

Three columns are made:

gel G-A, corresponding to a density of Protein G strain A of 1 mg / ml of grafted gel, gel GB, corresponding to a density of Protein G strain B of 1 mg / ml of grafted gel, G-AB gel, corresponding to a mixture of 50% GA gel and 50%> GB gel.

The design of the experiment also revealed that the anti-RSV IgG purified on the variant A of the G protein also interacted with the variant B of the G protein and vice versa (anti-varB on variantA), suggesting that there is cross-reactivity between the anti-RSV polyclonal antibodies for both variants of the G protein.

A high proportion of the anti-RSV immunoglobulins bind to the hRSV-G affinity chromatography support, irrespective of the strain, which represents in this test at least 6 μg of anti-RSV immunoglobulin per ml of gel. . The unadsorbed fraction and the wash fraction are depleted of anti-RSV immunoglobulins. Fraction immunoglobulin eluted from the acidic pH support and in the presence of propylene glycol shows a minimum enrichment of 109 times in anti-RSV immunoglobulins.

The assay shows that affinity chromatography using a G protein ligand of A or B strains grafted via NHS chemistry allows the purification of anti-RSV immunoglobulins with a yield of at least 26.2% under the conditions tested, corresponding to a minimum enrichment factor of 109 times.

The composition obtained after affinity chromatography on G protein ligand therefore comprises at least 30% by weight of immunoglobulins specifically directed against at least one epitope of the G protein.

EXAMPLE 2 Study of the Efficacy of a Preventive Treatment by Administration to the Lower Respiratory Tract of the Anti-RSV Ig Enriched Composition

The composition enriched in anti-RSV Ig as prepared in Example 1 above was tested as a prophylactic treatment against RSV virus. The composition was administered to the site of interest (the lung) via the nasal route.

Material and methods

The study is conducted over 7 days (D1 to D5), with female BALB / c mice 8 weeks old and made susceptible to RSV virus. ^1st stage: administration of the prophylactic treatment.

An initial blood sample is taken at the beginning of the study on all the mice tested. Prophylaxis is administered intranasally to the lungs according to

Placebo and the composition comprising nonspecific antibodies serve as negative controls.

The study was performed on 2 groups of 16 mice (4 mice / condition / group).

^2nd stage: Infection with RSV.

The RSV virus infection was carried out after anesthesia of animals on day 10, by intranasal administration of a solution comprising RSV virus genetically modified to express luciferase. When put in the presence of its substrate D-luciferin, luciferase can detect the virus by bioluminescence reaction and thus monitor in real time replication of RSV virus in mice. ^3rd step: Analysis of viral replication in mice

At Day D4: A group of mice was sacrificed to collect the lungs / nostrils / bronchoalveolar fluid to conduct a bio luminescence test on organ broths (based on the detection of luciferase, complexed with the RSV virus) and that quantification of the viral RNA by RT-Q-PCR.

At day 5: The viral replication is analyzed (in the second group of mice) by an in vivo imaging system (IVIS) based on the detection of bioluminescence. Mice were previously anesthetized and D-luciferin administered intranasally just before reading. Results

Analysis of viral replication by in vivo imaging:

5 days after RSV infection, control mice (either placebo or nonspecific Ig) showed significant viral replication in the lungs. Conversely, the viral replication is considerably reduced, or even suppressed (luminescent signal extinguished) in the lungs of the mice which have received, by prevention, by administration to the lung via the nasal route, the composition enriched with anti-RSV Ig. This effect is observed in the mice tested (FIG. 1), regardless of the dose (1 mg / kg or 0.1 mg / kg). These results suggest a protection of the lungs against the RSV virus in the mice which have received the anti-RSV Ig-enriched composition by administration to the lung via the nasal route. These results further confirm that the administration of the composition enriched with anti-RSV Ig, allowed diffusion directly into the pulmonary tract, explaining this protective effect. Analysis of the viral replication by analysis of the luciferase activity (bioluminescence test): The luciferase activity (enzyme expressed by the genetically modified RSV virus) is revealed by bio-luminescence reaction and quantified (FIG. 2), thus making it possible to quantify Indirectly the RSV viral replication in mouse lung lysates, 4 days after infection. Figure 2 demonstrates that viral replication is suppressed in the lungs through intrapulmonary preventive administration via the nasal route of a composition enriched in anti-RSV Ig at a dose of 1 mg / kg. Viral replication is also reduced following intrapulmonary preventive administration via the nasal route of a composition enriched in anti-RSV Ig at a dose of 0.1 mg / kg.

Analysis of viral replication by quantification of viral RNA (RT-qPCR):

Viral RNA was quantified by RT-qPCR (quantification of N nucleocapsid gene expression), 4 days after RSV infection. The quantification results of the viral RNA (FIG. 3) are perfectly consistent with the results of the quantification of the luciferase activity. These results support that nasal intrapulmonary preventive administration of an anti-RSV Ig enriched composition is effective against replication of RSV in the lungs.

EXAMPLE 3 Evaluation of the deposition in the lungs of the enriched anti-RSV Ig composition administered by nebulization

The composition enriched in anti-RSV Ig as prepared in Example 1 above was administered by nebulization in the macaque. Material and methods

The study is conducted on 3 female cynomolgus macaques.

1st step: administration of the composition enriched in anti-RSV Ig

The composition enriched in anti-RSV Ig is labeled with a radioactive tracer (2 x 40 μg anti-RSV Ig (each 40 μg in a final volume of 1 ml) of composition labeled with MBq of Tc99m).

The composition is then administered by nebulization to the animal using a mask, using the Aerogen® Solo device (Aerogen®, Ireland). 2nd step: pulmonary deposition analysis

Pulmonary deposition is determined by scintigraphic imaging of the lung using an ECAM gamma camera (Siemens, Germany).

immediately after administration of the composition enriched in anti-RSV Ig (T0)

and 30 min after administration of the composition enriched in anti-RSV Ig (T30) Bronchoalveolar lavage is also performed after scintigraphy at 30 min to measure the biological activity of the anti-RSV immunoglobulin. The lung of the animal is washed twice with a final volume of 10 ml / kg pre-heated PBS IX at 37 ° C. The 2 washes are then grouped into a single vial for analysis.

Results:

Particle size and activity conservation of nebulized anti-RSV Ig

The size of nebulized anti-RSV Ig particles is measured to verify the possible impact of radioactive tracing:

The results confirm that the radioactive labeling does not impact the size of the nebulized anti-RSV Ig particles, which remains below ΙΟμιη.

In addition, a neutralizing activity test was carried out with the nebulized anti-RSV Ig enriched immunoglobulin immunoglobulin composition, using the same non-nebulized composition as a control. The results confirm that nebulized anti-RSV Ig enriched immunoglobulin composition retains its neutralization capacity after nebulization (with IC50 values between 11 and 49 ng / mL, for a non-nebulized control at 32 ng / ml). mL). The results thus show that the immunoglobulin composition enriched in anti-RSV Ig can be nebulized in the form of particles or droplets of a size smaller than 5 μm and in the form of particles or droplets of a size less than Ιμιη. while maintaining its neutralizing activity. Deposit of anti-RSV PIg nebulized in the lung

The results obtained are as follows:

The results show that the immunoglobulin composition enriched with anti-RSV Ig can be administered by nebulization in the lower respiratory tract, and in particular the lung (target organ).

Thus, the results confirm that immunoglobulins enriched with anti-RSV Ig, despite their size of 150 kDa, can be administered by nebulization in the lower respiratory tract, and in particular the lung (target organ), in the form of particles or droplets smaller than 5μιη and in the form of particles or droplets smaller than Ιμιη and retain their neutralizing activity.

Claims

1. An immunoglobulin (Ig) composition hyper-enriched with anti-RSV immunoglobulins, for use in the prophylactic or curative treatment of RSV infection in a subject, by administering the composition in a form suitable for the anti-RSV immunoglobulins bind to RSV in the lower respiratory tract, said composition being intended to be administered in the form of particles or droplets of a size between 0.025 μιη and 10 μιη.

2. Composition of hyper-enriched Ig anti-RSV immunoglobulins for use according to claim 1, characterized in that the lower respiratory tract consists of the lungs, and preferably the pulmonary epithelium.

3. Composition of hyper-enriched Ig anti-RSV immunoglobulins for use according to claim 1 or 2, said composition being intended to be administered in the form of particles or droplets of a size between 0.5 μιη and 5 μιη. μιη and preferably between 1 μιη and 3 μιη.

4. An anti-RSV immunoglobulin hyper-enriched Ig composition for use as claimed in any one of the preceding claims, said composition further comprising a surfactant and / or a viscosity modifier.

5. Composition of hyper-enriched Ig anti-RSV immunoglobulins for use according to any one of the preceding claims, said composition comprising an anti-RSV immunoglobulin concentration of between 10 and 250 g / l, preferably between 10 and and 100 g / 1.

6. Composition of hyper-enriched Ig anti-RSV immunoglobulins for use according to any one of the preceding claims, the administration being a pulmonary administration, preferably by inhalation of an aerosol, preferably by means of a mouth spray or spray using a nebulizer, and preferably a sieve nebulizer.

7. Composition of hyper-enriched Ig anti-RSV immunoglobulins for use according to any one of the preceding claims, characterized in that it is intended to be administered by the pulmonary route with a delivery volume of between 250 μl and 12 ml, preferably between 250 μl and 10 ml.

8. Composition of hyper-enriched Ig anti-RSV immunoglobulin for use according to any one of claims 1 to 5, characterized in that the administration is a nasal administration, preferably in the form of a aerosol.

9. Composition of hyper-enriched Ig anti-RSV immunoglobulin for use according to claim 8, characterized in that it is intended to be administered nasally with a volume of administration less than 20 ml, preferably lower than to 15 ml, more preferably between 2 ml and 8 ml.

10. Composition of hyper-enriched Ig anti-RSV immunoglobulin for use as claimed in any one of the preceding claims, the subject belonging to at least one of the following groups: children, infants, immunocompromised persons including those treated for a transplant especially a lung transplant, premature infants and children or elderly with lung disease, especially chronic obstructive pulmonary disease (COPD), cardiac or immune.