ES2794449T3 - Liquid formulation comprising a GM-CSF neutralizing compound - Google Patents

Abstract

An aqueous composition comprising: i) a GM-CSF neutralizing compound which is a human monoclonal antibody that binds to GM-CSF comprising a light chain amino acid sequence as set forth in SEQ ID NO. 34 and a heavy chain amino acid sequence as set forth in SEQ ID NO: 35, at a concentration of 100 mg / ml to 180 mg / ml, ii) 5% (w / v) sorbitol, iii) L-histidine 30 mM and iv) has a pH of 5.8.

Description

DESCRIPTION

Liquid formulation comprising a GM-CSF neutralizing compound

Field of the invention

The present invention relates to stable liquid formulations comprising a granulocyte-macrophage colony stimulating factor (GM-CSF) neutralizing compound. The ingredients of the formulations preferably provide stability during repeated long-term freeze-thaw cycles. In a preferred aspect, the formulations are for use in therapies, preferably for use in treating inflammatory and autoimmune disorders, preferably including allergic and psoriatic disorders, as well as arthritic or asthmatic diseases. Furthermore, a kit comprising the formulations of the invention is provided.

Proteins are used in a wide range of applications in the fields of pharmaceuticals, veterinary products, cosmetics and other consumer products, food, feed, diagnostics, industrial chemistry and decontamination. Sometimes these uses have been limited by restrictions inherent to the proteins themselves or imposed by the environment or the media in which they were used. Such restrictions can result in poor protein stability, yield variability, or high costs. Due to the advent of biotechnology, it is possible to produce a wide variety of proteins for therapeutic applications. After production, protein pharmaceuticals are generally stored prior to use. Due to the fact that proteins are generally larger and more complex than "traditional" pharmaceuticals, the formulation and processing of protein pharmaceuticals that are suitable for storage may be particularly promising. For reviews of protein pharmaceutical formulations and process design, see Carpenter et al. (1997), Pharm. Res. 14: 969-975; Wang (2000), Int. J. Pharmaceutics 203: 1-60; and Tang and Pikal (2004), Pharm. Res. 21: 191-200.

Several factors can be considered when designing formulations and procedures for the pharmaceutical production of proteins. The primary concern is the stability of the proteins through any or all of the manufacturing, shipping, and handling steps, which may include compounding, freezing, lyophilization, drying, storage, shipping, reconstitution, freeze / cycles. thawing and storage after reconstitution by the end user. Other possible considerations include ease and economy of manufacture, handling, and distribution; the composition of the final product for administration to the patient; and the ease of use by the end user, including the solubility of the lyophilized formulation after reconstitution. Liquid formulations can satisfy certain objectives. Possible advantages of liquid formulations include their ease of manufacture and economy and their suitability for the end user. Often when stored for long periods, polypeptides are unstable in solution (Manning et al (1989), Pharm. Res. 6: 903-918). As a consequence, other processing steps have been developed to allow a longer shelf life, including drying, e.g. eg, by lyophilization. Freeze-dried formulations can also provide certain benefits. The potential benefits of lyophilization include increased protein stability, as well as ease and economy in shipping and storage. However, lyophilized pharmaceutical compositions may be less convenient for the end user.

In addition to choosing the basic form of the composition (e.g., lyophilized, liquid, frozen, etc.), optimizing a protein formulation generally involves varying the formulation components and their respective concentrations to maximize the stability of the formulation. protein. A variety of factors can affect protein stability, including ionic strength, pH, temperature, freeze / thaw cycles, shear forces, freezing, lyophilization, drying, agitation, and reconstitution. . Protein instability can be caused by physical degradation (eg, denaturation, aggregation, or precipitation) or chemical degradation (eg, deamidation, oxidation, or hydrolysis). The optimization of formulation components and concentrations is based solely on empirical studies and / or rational approaches to overcome sources of instability.

Sometimes, in long-term storage of pharmaceutical compositions containing polypeptides, including aqueous and lyophilized formulations, the active polypeptides can be lost due to aggregation and / or degradation.

Accordingly, typical practices can be addressed to improve the stability of the polypeptide by varying the concentration of elements with the formulation, or by adding excipients to modify the formulation (U.S. Patent Nos. 5,580,856 and 6,171,586 and U.S. Patent applications United States Nos. US 2003/0202972, US 2003/0180287). US 5,580,856 is a prototype patent that describes agents, such as natural polymers, surfactants, sulfated polysaccharides, proteins, and buffers that can be added to stabilize a dry protein during or after dehydration. However, apart from many options, US Patent 5,580,856 does not teach which stabilizer should be added to which protein. Consequently, while the skilled reader realizes that there are many options, he or she would have to find the best conditions for his protein among the many options described by the US document. 5,580,856. US patent application 2003/0202972 describes a stable lyophilized formulation of an anti-Her2 antibody, in which the stabilizer is sugar, trehalose or a buffer. However, although these stabilizers can be useful for an antibody, they cannot be extrapolated to other proteins. US patent application 2003/0180287 is similar to US 2003/0202972 in which a stable solution of an immunoglobulin-like protein is also described, that is, a protein containing an Fc domain. The stabilizer can be sodium phosphate, potassium phosphate, sodium or potassium citrate, maleic acid, ammonium acetate, tris buffer, acetate, diethanolamine, histidine, lysine, or cysteine. Among these chemically distinct stabilizers that could be chosen by the skilled reader, lysine turned out to be suitable. However, as with US 2003/0202972, the specific stabilizer is simply suitable for a specific protein, here an Fc domain containing the protein, and cannot per se be extrapolated to another protein. Consequently, the use of additives cannot be extrapolated from one specific protein to another unrelated protein. In fact, the use of additives, while improving storage, can still result in inactive polypeptides. Furthermore, in the case of lyophilization, the rehydration step can introduce conditions that result in inactivation of the polypeptide by, for example, aggregation or denaturation (Hora et al. (1992), Pharm Res., 9: 33-36 ; Liu et al. (1991) Biotechnol. Bioeng., 37: 177-184). Indeed, polypeptide aggregation is undesirable as it can lead to immunogenicity (Cleland et al. (1993) Crit. Rev. Therapeutic Drug Carrier Systems, 10: 307-377; and Robbins et al. (1987), Diabetes, 36: 838-845).

The maintenance of biological activity during the development and manufacture of pharmaceutical products depends on the inherent stability of the macromolecule, as well as the stabilization techniques employed. There is a range of protein stabilization techniques; including the addition of chemical "stabilizers" to the aqueous solution or suspension of the protein. For example, US Patent 4,297,344 describes the stabilization of coagulation factors II and VIII, antithrombin III and plasminogen against heat by the addition of selected amino acids. US Patent 4,783,441 describes a method of stabilizing proteins by adding surfactants. US Patent 4,812,557 describes a method for stabilizing interleukin-2 using human serum albumin. Freeze / thaw methods in which the preparation is mixed with a cryoprotectant and stored at very low temperatures is another option for stabilizing a protein. However, not all proteins will survive a freeze / thaw cycle. Cold storage with a cryoprotective additive, usually glycerol, is another option. Storage in the form of glass, as described in US Patent 5,098,893 could also be done. In this case, the proteins dissolve in water-soluble or swellable substances that are in an amorphous or glassy state. The most widely used method for protein stabilization is lyophilization. Whenever sufficient stability of the protein in aqueous solution cannot be achieved, lyophilization provides the most viable alternative. One disadvantage of freeze drying is that it requires sophisticated processing, is time consuming, and costly. Furthermore, if lyophilization is not carried out carefully, most preparations are at least partially denatured by the freezing and dehydration steps of the technique. The result is often the irreversible aggregation of a portion of protein molecules, rendering a formulation unacceptable for parenteral administration.

In general terms, the degradation of proteins has been well described in the literature, but the storage and solubility of the compounds neutralize the granulocyte and macrophage colony stimulating factor (better known as GM-CSF), in particular of polypeptides and anti-GM-CSF antibodies, has not been described.

Furthermore, although it was known in the art that there were a multitude of options for protein stabilizing agents, as well as for agents that allow for a high concentration of a protein while remaining stable, until the present invention, it was not recognized in the art that a formulation comprising GM-CSF neutralizing compounds in high concentrations could be unstable and therefore require improvement.

In preparing a pharmaceutical composition comprising a protein, such as an antibody, eg a monoclonal antibody, it is an objective to develop high concentration liquid formulations due to the potential for subcutaneous administration resulting in greater convenience for the patient. However, there is a general consensus that the development of high concentration antibody formulations, in particular monoclonal antibodies, poses serious challenges with respect to the physical and chemical stability of monoclonal antibodies, such as increased formation of soluble aggregates. and insoluble that improve the probability of an immunogenic response, as well as lead to low bioactivity.

Aggregation by a polypeptide during storage of a liquid pharmaceutical composition can adversely affect the biological activity of that polypeptide, resulting in loss of therapeutic efficacy of the pharmaceutical composition. Furthermore, the formation of aggregates can cause other challenges, such as blockage of tubes, membranes or pumps when the pharmaceutical composition containing the polypeptide is administered using an infusion system. In addition, high concentration antibody formulations have been reported to result in increased viscosity, thus creating serious challenges to manufacturability and ease of injection. Highly viscous formulations are difficult to manufacture, syringe, and inject. The use of force in handling viscous formulations leads to excessive foaming, which can lead to denaturation and inactivation of active monoclonal antibodies.

Therefore, there is a great need for a stable highly concentrated protein pharmaceutical composition comprising a compound that neutralizes GM-CSF, e.g. g., an antibody and having a low and workable viscosity suitable for subcutaneous administration, such as in a ready-to-use device. Furthermore, from the point of view of the patient, it would be highly desirable to have stable products at room temperature. At this time, in particular, there are no commercially available antibody formulations in which storage at room temperature is possible throughout the shelf life of the drug product. Typically, increased protein aggregation occurs causing an unacceptably high level of protein-related aggregates and impurities, which can lead to immunogenic reactions. Many of the monoclonal antibody products marketed contain surfactants in their formulation. Typically, surfactants are added to reduce interfacial stress that can induce protein aggregation and particle formation leading to unacceptable product quality. Examples of interfacial stress could be the contact of the protein with i) air ii) container closure material, such as a rubber plunger, piston, glass, pre-filled syringes iii) production-related materials, such as steel tanks, tubes and pumps iv) ice, during freeze / thaw, etc. However, surfactants, such as polysorbates, generally contain a residue of peroxides that can oxidize the protein molecule, which can compromise the quality of the product. Furthermore, from the point of view of manufacture, the addition of polysorbates requires an additional stage in the production since ultra / diafiltration is difficult to perform when the formulation contains said polysorbates. Preventing the formation of oxidized products is a difficult problem, therefore, careful handling of polysorbates is required to control the formation of oxidized products. Therefore, it would also be desirable to design surfactant-free formulations, both from a stability and manufacturing standpoint.

Granulocyte macrophage colony stimulating factor (GM-CSF), initially as a hematopoietic growth factor, has recently been shown to be an important cytokine in inflammation and autoimmunity. Elevated levels of GM-CSF mRNA or protein are measured at a variety of inflammatory sites, including in allergic and psoriatic patients, arthritic patients, and asthmatics. Numerous in vivo studies have shown in recent years that blocking GM-CSF through neutralizing antibodies can prevent or even cure pro-inflammatory diseases in various models of inflammation, including models for experimental autoimmune encephalitis of arthritis, psoriasis, and lung disease. Therefore, it is highly desirable to have available a formulation with a GM-CSF neutralizing compound which is, in particular, stable, containing high amounts of a GM-CSF neutralizing compound and / or can be administered subcutaneously.

Therefore, the technical challenge of the present invention is to meet the needs described above.

The present invention addresses these needs and therefore provides a solution to the technical challenge of formulations embodiments, as well as the methods and uses that apply these formulations in treating subjects suffering from diseases that would benefit from administration of GM-CSF neutralizing compounds. These embodiments are characterized and described herein, illustrated in the Examples, and reflected in the claims.

It should be noted that, as used herein, the singular forms "an / one", "a" and "the / the" include plural references, unless the context clearly indicates otherwise. Thus, for example, reference to "an antibody" includes one or more such other antibodies and reference to "the method" includes reference to equivalent steps and methods known to those of skill in the art that could be modified or substituted for those of methods described in this document.

Unless otherwise indicated, the expression "at least" preceding a series of items should be understood to refer to each item in the series. Those skilled in the art will recognize, or be able to determine using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

Throughout this specification and the claims that follow, unless the context requires otherwise, it will be understood that the word "comprise" and its variations, such as "comprises" and "comprising" imply the inclusion of a whole number or stage or group of established whole numbers or stages, but not the exclusion of any other whole number, or stage or group of whole numbers or stages. When used herein, the term "comprising" it may be replaced by the term "containing" or sometimes when used herein by the term "having" or it may even be substituted for expression "consisting of".

When used herein "consisting of" any element, step or ingredient not specified in the element of the claim is excluded. When used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel features of the claim. In each case herein, any of the terms "consisting essentially of" and "consisting of" may be replaced by either of the other two terms.

As used herein, the conjunctive term "and / or" among the multiple elements cited is understood to encompass both individual and combined options. For example, when two elements are joined by "and / or", a first option refers to the applicability of the first element without the second. A second option concerns the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Either of these options is understood to be within the meaning and therefore satisfies the requirement of the term "and / or" as used in this document. The concurrent applicability of more than one of the options is also understood within the meaning and therefore satisfies the requirement of the term "and / or" as used in this document.

Various aspects and embodiments of the invention are described below.

The present invention relates to an aqueous composition comprising:

i) a GM-CSF neutralizing compound that is a human monoclonal antibody

1. An aqueous composition comprising:

i) a GM-CSF neutralizing compound, which is a human monoclonal antibody that binds to GM-CSF comprising a light chain amino acid sequence as set forth in SEQ ID NO. 34 and a heavy chain amino acid sequence as set forth in SEQ ID NO: 35, at a concentration of 100 mg / ml to 180 mg / ml,

ii) 5% (w / v) sorbitol,

iii) 30 mM L-histidine and

iv) has a pH of 5.8.

Furthermore, the present invention relates to the above composition which is free of additional surfactants or amino acids.

The present invention also relates to the aforementioned composition, which comprises 150 mg / ml of the antibody or its functional fragment that binds to GM-CSF.

Furthermore, the present invention relates to the aforementioned composition which is stable for at least 24 months at about 2-8 ° C or at least 28 days at room temperature.

The aforementioned composition may be for use in therapy.

Furthermore, the present invention relates to the aforementioned composition for intravenous and / or subcutaneous administration.

The composition of the invention can be used in the treatment of inflammatory and autoimmune disorders.

These disorders are selected from allergic, psoriatic, arthritic, and asthmatic disorders.

Furthermore, a kit comprising the composition described above is described.

With the aim in mind of providing a formulation having a high concentration of GM-CSF neutralizing compounds, the present inventors recognized that compounds that neutralize GM-CSF can be unstable at high concentrations and can also be unstable for a prolonged period of time. storage.

In fact, there are many ways that GM-CSF neutralizing compounds, such as proteins, can be unstable. For example, protein instability could be caused by protein aggregation or disaggregation, but also by chemical instability due to deamination, deamidation, oxidation, breaking and formation of disulfide bonds, hydrolysis, succinimidation, non-disulfide crosslinking, deglycosylation or "enzymatic browning" (Maillard reaction) or any combination of these phenomena; see, for example, Wang et al. (1999), In Int. J. Pharm. 185: 129-188). Furthermore, physicochemical parameters such as temperature, pH value, adsorption on surfaces, salts, metal ions, chelating agents, physical forces such as shear forces, protein denaturants, non-aqueous solvents, protein concentration, source and purity of protein, protein morphism, or pressure can influence protein stability. However, there are many factors that can influence the stability of proteins, many steps could be taken to stabilize a protein. For example, a protein can be stabilized internally (by changing amino acids) or externally. External stabilization could be achieved by chelating agents, metal ions, reducing agents, polymers, polyethylene glycols / polyols, serum albumin, surfactants, sugars and polyols, fatty acids and phospholipids, amino acids, buffers, etc .; see, for example, Wang, Y, and Hanson M (1988), J. Parental Sci. & Technology, 42, Supplement: 4-26; Wang et al. (1999), In Int. J. Pharm. 185: 129-188. In In summary, to stabilize GM-CSF neutralizing compounds, such as antibodies in a formulation, experts would have had many options available.

In the present case, the inventors observed that GM-CSF neutralizing compounds may show aggregation and / or may not dissolve at high concentrations. Many different factors can cause aggregation of a protein in a formulation. Typical purification and storage procedures can expose protein formulations to conditions and components that cause the protein to aggregate. For example, proteins in a formulation can aggregate as a result of one or more of the following: storage, exposure to elevated temperatures, the pH of the formulation, the ionic strength of the formulation, and the presence of certain surfactants (eg. , polysorbate-20 and polysorbate-80) and emulsifying agents. Similarly, proteins can aggregate when exposed to shear stress, such as reconstituting a lyophilized protein cake in solution, purifying a protein sample by filtration, freezing-thawing, shaking, or transferring a protein solution via syringe. Aggregation can also occur as a result of interactions of polypeptide molecules in solution and at the liquid-air interfaces within storage vials. Conformational changes in adsorbed polypeptides at the air-liquid and solid-liquid interfaces can occur during compression or extension of the interfaces as a result of agitation during transport. Such agitation can cause the protein in a formulation to aggregate and eventually precipitate with other adsorbed proteins.

Also, exposing a protein formulation to light can cause the protein to clump. The present invention thus provides formulations that allow high concentrations of GM-CSF neutralizing compounds and that reduce aggregation of these compounds. Without being limited to any theory, the reduction of aggregation is believed to be achieved by controlling one or more of the aggregation mechanisms mentioned above. This can result, for example, in better product stability and greater flexibility in manufacturing processes and storage conditions.

The present inventors aimed to provide a formulation with a high concentration of GM-CSF neutralizing compounds, in order, for example, to allow a smaller injection volume that would be adequate to reduce side effects such as pain due to a volume large injection or allow subcutaneous administration with a small volume.

Thus, the present inventors have observed during their studies a certain instability of the compounds that neutralize GM-CSF and therefore aimed to improve this unwanted observation. Consequently, their objective was to concentrate the GM-CSF neutralizing compounds by keeping them in solution, that is, in a dissolved stage. In doing so, they had a multitude of options and alternatives available, however, without any indication that any of them were adequate to solve the target problem.

"Dissolved stage" means that the GM-CSF neutralizing compound, preferably at a concentration of at least about 20 mg / ml, is in solution, that is, dissolved and / or dispersed directly in the aqueous solution (that is, in the aqueous phase) of the formulation. Preferably, the GM-CSF neutralizing compound is homogeneously dissolved or dispersed. Homogeneously means that the GM-CSF neutralizing compound that is dissolved and / or dispersed in the aqueous formulation is almost uniform, preferably it is uniformly distributed in the aqueous formulation so that the concentration ("c") of the GM-CSF neutralizing compound ("n" in case of molar mass or "m" in case of mass) is almost identical, preferably identical in the volume ("v") (or in the whole volume) of the aqueous solution, that is, c = n / voc = m / v, respectively, is almost constant, preferably constant. Preferably there is no concentration gradient within the formulation.

Accordingly, a stable formulation of the present invention comprising a GM-CSF neutralizing compound can preferably be considered as an aqueous solution, in which the GM-CSF neutralizing compound is directly dissolved and / or dispersed therein.

A "solution" is a homogeneous mixture of two or more substances / components. In such a mixture, a solute (in the present invention a GM-CSF neutralizing compound) is dissolved (as described above) in another substance (in the present invention preferably an aqueous formulation), also known as a solvent. Given the above, the GM-CSF neutralizing compound is preferably not dissolved and / or dispersed in the aqueous solution. The term "dissolved state" also includes that the GM-CSF neutralizing compound is essentially and preferably not emulsified or, more preferably, not emulsified at all in the aqueous solution.

Furthermore, the term "dissolved state" includes that the GM-CSF neutralizing compound is not essentially encapsulated and / or entrapped (preferably less than 2%, 1% or 0.5% of the GM-CSF neutralizing compound can be encapsulated and / or entrapped, or more preferably not encapsulated and / or entrapped at all, for example, in liposomes, multilamellar liposomes or the like.

Also encompassed in the present description is a liquid formulation that contains a stable GM-CSF neutralizing compound and that does not undergo the formation of conjugates / aggregates or fragments / degradation products when stored for a long period, and whose formulation is suitable for subcutaneous administration.

Specifically, after testing many different stabilizing agents, the present inventors found that the GM-CSF neutralizing compounds could be stabilized if a tonicity modifier were added to the solution to be stored. Some examples of tonicity modifiers include, but are not limited to, sugars and sugar alcohols. Simple sugars are called monosaccharides and include glucose, fructose, galactose, xylose, ribose, rone, lactulose, allose, altrose, gulose, idosa, talose, arabinose, and lixose. Also encompassed are disaccharides including, for example, sucrose, maltose, lactose, isomalt, trehalose, and cellubose. Sugar alcohols include sorbitol, mannitol, glycerin, erythritol, maltitol, xylitol, polyglycitol. The sugar may not be a non-reducing sugar like sucrose or trehalose. Non-reducing sugars are characterized by the absence of an open chain structure, so they are not susceptible to oxidation-reduction reactions. Therefore, one or more of the non-reducing sugars, such as sucrose or trehalose, or one or more of the sugar alcohols, such as mannitol or sorbitol could be added to the formulation comprising the GM-CSF neutralizing compound. . Combinations of non-reducing sugars and sugar alcohols could also be added to the solution, such as sucrose and mannitol, sucrose and sorbitol, trehalose and mannitol, or trehalose and sorbitol. More preferably the sugar alcohols mannitol and / or sorbitol are added, preferably in their D form, more preferably sorbitol is added to the solution. The concentration of the tonicity modifier, preferably sorbitol, is between about 1% and about 15% (w / v), preferably between about 2% and about 10% (w / v), more preferably between about 3% and about 7% (w / v), more preferably between about 4% and about 6% (w / v), and more preferably about 5% (w / v).

Another specifically preferred substance for stabilizing high concentration GM-CSF neutralizing compounds with respect to long-term storage is a buffer system with a pH of between about 4 and about 10, preferably between about 4 and about 7, more preferably between about 4 and about 6 or between about 5 and about 7, even more preferably between about 5.5 and about 6.5, and more preferably with a pH of about 5.8. The buffer of the invention is a histidine buffer. When referring to this, an amino acid is understood to be an L-amino acid or D-amino acid, where L-amino is preferred. Specifically, histidine or a salt thereof is used for the buffer system. Preferably the salt is a chloride, phosphate, acetate or sulfate, more preferably the salt is a chloride. The pH of the histidine buffer system is between 5 and about 7, preferably between about 5.5 and about 6.5, more preferred is the pH about, or exactly equal to 5.8. The pH can be adjusted by using conventional acids and bases, preferably NaOH. The concentration of the buffer system, specifically the histidine buffer system, is between about 10mM and about 50mM, preferably between about 20mM and about 40mM, more preferably about 30mM. The concentration of the histidine buffer of the present invention is 30 mM.

The combination of the buffer system, specifically the histidine buffer, and the tonicity modifier, specifically sorbitol, is used to stabilize the GM-CSF neutralizing compounds in the solution, in order to avoid aggregation and make the formulation more stable enough for long term storage and / or during one or more freeze / warp cycles. It proved to be preferable in terms of stability to have about 16% (w / v) and higher of sugar alcohol, preferably sorbitol, in the formulation. However, the upper limit for the osmolality of the formulation is set to be approximately 470 mOsm / kg which is still hyperosmotic but similar to the osmolality of an approved product (Synagis; Lm. Administration). Therefore, a compromise had to be found between optimal stability, tonicity, and concentration of the GM-CSF neutralizing compound as described in the examples of the present invention. Therefore, a preferred concentration of sugar alcohol, preferably sorbitol, is between about 3% and about 7% (w / v), more preferably between about 4% and about 6% (w / v) and , more preferably about 5% (w / v).

In some embodiments of the present invention, the formulations or compositions of the invention comprising a GM-CSF neutralizing compound require no further excipients in addition to those disclosed above (i.e., a buffer and a tonicity modifier), such as, for For example, surfactants and amino acids, which are used in traditional formulations to stabilize proteins in solution. Furthermore, the formulations described herein are preferred over standard formulations because they have lower immunogenicity due to the lack of additional agents commonly needed for protein stabilization.

Amino acids are known to be useful in stabilizing proteins at high concentration, among other things, by mediating protein solubility and / or inhibiting protein aggregation. Although threonine (eg, at 250 mM) indicates a minor stabilizing effect, the liquid formulation of the present invention is preferably free of other amino acids.

Furthermore, it is preferred that the present formulation be sodium chloride free or essentially free of it. By "essentially free" it is meant that the concentration of sodium chloride is close to or very close to 0 (zero) mM, eg, less than about 50 mM, preferably less than about 20 mM, more preferably less than about 10 mM, even more preferably less than about 5 mM and more preferably less than about 2 mM or even less than about 1 mM.

In biopharmaceuticals, the addition of surfactants may be useful to reduce protein degradation during storage. Polysorbates 20 and 80 (Tween 20 and Tween 80) are well established excipients for this purpose. However, due to no or negative effects on the stability of GM-CSF neutralizing compounds, the liquid formulation of the present invention preferably does not comprise any surfactant.

The concentration of the respective GM-CSF neutralizing compounds used is at least about 100 mg / ml in the liquid formulation to be stored, frozen / thawed and / or ready for use. Concentrations from about 20 mg / ml to about 200 mg / mg, preferably about 50 mg / ml to about 200 mg / ml, more preferably about 100 mg / ml to about 180 mg / ml, even more preferably about 130 mg / ml at about 170 mg / ml, even more preferably about 135 mg / ml to about 165 mg / ml, and more preferred about 150 mg / ml is used in the present invention.

The shelf life of the liquid formulation produced has a preferred minimum requirement of 24 months between 2 and 8 ° C, preferably 36 months between 2 and 8 ° C, more preferably 48 months between 2 and 8 ° C, preferably 60 months between 2 and 8 ° C, or at least 28 days at room temperature (252C + 22C).

The present invention relates to a stable formulation, preferably a stable liquid formulation that surprisingly allows long-term storage of GM-CSF neutralizing compounds. This formulation is useful, in part, because it is more convenient for the patient to use, as the GM-CSF neutralizing compounds in this formulation are highly concentrated to reduce side effects such as pain due to high volume injections.

Accordingly, one aspect of the invention is based on the discovery that formulations comprising:

- a GM-CSF neutralizing compound,

- a buffer system preferably selected from a histidine buffer, an acetate buffer and / or a citrate buffer with a preferred pH of between 5 and 7,

- and a tonicity modifier preferably selected from non-reducing sugars, such as sucrose or trehalose, or sugar alcohols, such as mannitol or sorbitol,

they become stable enough for long-term storage and / or freeze / thaw cycles and / or shear stresses (stability during stirring). The formulation of the invention has many advantages over standard buffered formulations. In one aspect, the formulation exhibits minimal aggregation behavior on long-term storage without deleterious effects that might be expected with high protein formulations. Other advantages of the formulation according to the invention are: minimal fragmentation of the GM-CSF neutralizing compound and no significant impact on the bioactivity of the GM-CSF neutralizing compound with respect to long-term storage, and a low viscosity of the composition. Finally, in a preferred embodiment, the formulation is free of other excipients such as surfactants, other amino acids and / or sodium chloride.

The present description comprises the following: Formulations, in which the GM-CSF neutralizing compound is a polypeptide, a peptidomimetic, a nucleic acid or a small molecule.

In a preferred description, the GM-CSF neutralizing compound (which is preferably a polypeptide and, more preferably, an antibody or a functional fragment thereof) binds alone or specifically binds to GM-CSF or the GM-CSF receptor. The GM-CSF or GM-CSF receptor is expected to come from an animal, including but not limited to mammals such as laboratory animals (rodents such as rats, guinea pigs, hamsters or mice, non-human primates such as Cynomolgus or Rhesus macaque) , domestic animals or pets (for example, dogs or cats), farm or agricultural animals (for example, cattle, sheep, goats and pigs) and / or humans. Preferably, the GM-CSF receptor or the GM-CSF itself is a human GM-CSF (Homo sapiens) receptor or human GM-CSF, respectively, or a primate non-human GM-CSF or non-GM-CSF receptor. human primate, respectively. Especially preferred variants (homologues) of the primate non-human GM-CSF or primate non-human GM-CSF receptor include those of the gibbon monkey (Nomascus concolor, also known as the western black crested gibbon) and of the monkeys of the macaca family, for example the rhesus monkey (Macaca mulatta) and the cynomolgus monkey (Macaca fascicularis). According to a particularly preferred embodiment of the invention, the compound that binds to GM-CSF or to the GM-CSF receptor (preferably the antibody or its fragments) exhibits cross-reactivity between humans and at least one of the species of monkeys mentioned above. For example, an antibody or a fragment thereof is capable of binding (and neutralizing) both human GM-CSF and Cynomolgus monkey (Macaca fascicularis) GM-CSF. This is especially advantageous for an antibody molecule intended for therapeutic administration in human subjects, as such an antibody will normally have to proceed through a multitude of tests prior to regulatory approval, of which certain initial tests involve non-human animal species. . When conducting such tests, it is generally desirable to use as a non-human species a species that has a high degree of genetic similarity to beings. humans (eg, non-human primates such as the Cynomolgus monkey), as the results thus obtained will generally be highly predictive of the corresponding results that can be expected when the same molecule is administered to humans. However, this predictive power based on animal tests depends, at least partially, on the comparability of the molecule, and is very high when, due to interspecies reactivity, the same therapeutic molecule can be administered to humans and animals. animal models. As in this embodiment of the invention, when an antibody molecule is cross-reactive for the same antigen in humans and in other closely related species, tests can be performed using the same antibody molecule in humans and in this closely related species. related, for example in one of the monkey species mentioned above. This increases both the efficiency of the tests themselves and the predictive power provided by such tests with respect to the behavior of such antibodies in humans, the final species of interest from a therapeutic point of view. It is preferred that the antibody or its functional fragment that binds to GM-CSF or the GM-CSF receptor is a monoclonal antibody or its functional fragment. The same is true for GM-CSF neutralizing compounds, which are not antibodies or derived from antibodies.

In the context of the invention, the GM-CSF neutralizing compound is a human monoclonal antibody.

The compound described as a GM-CSF neutralizer may be an antibody or a functional fragment thereof that binds to an epitope of human and non-human primate GM-CSF. This epitope preferably comprises amino acids 23-27 (RRLLN) and / or amino acids 65-77 (GLR / QGSLTKLkGpL). The variability at position 67 within the stretch of amino acid sequence 65-77 reflects the heterogeneity in this portion of GM-CSF between, on the one hand, human GM-CSF and gibbon (in which position 67 is R) and , on the other hand, monkeys of the macaca family, for example cynomolgus and rhesus monkeys (in which position 67 is Q). If the epitope comprises two stretches of amino acid sequence that are not adjacent, such as 23-27 (RRLLN) and 65-77 (GLR / QGSLTKLKGPL), the epitope can also be called a "discontinuous" epitope. Said GM-CSF epitope or said discontinuous GM-CSF epitope may further comprise amino acids 28-31 (LSRD), amino acids 32-33 (TA) and / or amino acids 21-22 (EA).

The human monoclonal antibody or its functional fragment preferably comprises in its heavy chain variable region a CDR3 comprising an amino acid sequence selected from the group consisting of those established in SEQ ID NOs: 1-13 and 56; preferably the CDR3 heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO. 2.

Any such heavy chain variable region CDR3 sequences can exist even closer together in a heavy chain variable region with the CDR1 heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. 14 and the CDR2 heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO. fifteen.

Furthermore, the human monoclonal antibody or its functional fragment may comprise in its light chain variable region a CDR1 comprising the amino acid sequence established in SEQ ID NO. 16, a CDR2 comprising the amino acid sequence established in SEQ ID NO. 17, and a CDR3 comprising the amino acid sequence established in SEQ ID NO. 18.

In an especially preferred description, the human monoclonal antibody or its functional fragment comprises in its light chain variable region a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO. 16, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO. 17 and a CDR3 comprising an amino acid sequence as set forth in SEQ ID: 18, and in its heavy chain variable region a CDR1 comprising an amino acid sequence as set forth in SEQ ID NO. 14, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO. 15 and a CDR3 comprising an amino acid sequence selected from the group consisting of those set forth in SEQ ID NOs: 1-13 and 56, more preferably SEQ ID NO. 2.

Also described herein is a human monoclonal antibody or its functional fragment comprising in its light chain variable region an amino acid sequence selected from the group consisting of those set forth in SEQ ID NOs: 19, 54 and 55. It is also described a human monoclonal antibody or its functional fragment comprising in its heavy chain variable region an amino acid sequence selected from the group consisting of those established in SEQ ID NOs: 20-33, 52 and 53. The human monoclonal antibody or the su functional fragment may comprise a light chain amino acid sequence as set forth in SEQ ID NO. 34 and / or a heavy chain amino acid sequence selected from the group consisting of any of SEQ ID NOs: 35-48, more preferably SEQ ID NO. 35. Also described is a human monoclonal antibody or its functional fragment that can comprise one or more amino acid sequences that have at least 70%, 80%, 90%, 95%, 98% or 99% homology with the sequence of respective amino acids as established in any of SEQ ID NOs: 1-48 and 52-56, preferably with the respective amino acid sequence as established in any of SEQ ID NOs: 1-18 and 56 and / or with the sequence of amino acids of the framework regions (FR) within the amino acid sequence as established in any of SEQ ID NOs: 19-48 and 52-55. Therefore, a human monoclonal antibody or its functional fragment is described which may comprise one or more amino acid sequences that have at least 70%, 80%, 90%, 95%, 98% or 99% homology with the respective amino acid sequence as set forth in any of SEQ ID NOs: 1-18 and 56.

Alternatively, in any of the amino acid sequences of a CDR established in any of SEQ ID NOs: 1-18 and 56, one, two, three, four, five, six, seven, eight, nine or 10 amino acids can be substituted. Preferably, such a CDR having substitutions is still capable of binding GM-CSF as described herein.

As an alternative or in addition, it is disclosed that the human monoclonal antibody or its functional fragment can comprise one or more amino acid sequences that have at least 70%, 80%, 90%, 95%, 98% or 99% homology with the respective amino acid sequence of a VH, VL, H or L region, respectively, as set forth in any of SEQ ID NOs: 19-48 and 52-55. Homology is described to be with respect to the entire amino acid sequence VH, VL, H or L. In this description, homology is referred to within CDRs as described above or to homology within FRs (or non-CDRs ) of such VH, VL, H or L region as set forth in any of SEQ ID NOs: 19-48 and 52-55. Consequently, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids can be substituted in each of FR. Such a FR substitution variant is still capable of binding GM-CSF as described herein.

Those skilled in the art can easily identify FRs (or non-CDRs) within SEQ ID NOs: 19-48 and 52-55, since SEQ ID NOs: 1-18 and 56 show CDR sequences composed of one or more of the VH, VL, H or L sequences shown in SEQ ID NOs: 19-48 and 52-55. Namely, the sequence listing provides in the sequence identifier <223> the designation of each of the amino acid sequences. Identical designations indicate that these amino acid sequences "belong" together, meaning that a CDR is contained in a VH, VL, H or L region, eg SEQ ID NOs: 16, 17, 18 are amino acid sequences of CDRs found in the amino acid sequence shown in SEQ ID NO. 19 (since they are all designated as "5-306").

By way of further illustration, if amino acids are substituted in one, or more, or all of the CDRs or FRs of the heavy and / or light chain, it is preferred that the "substituted" sequence obtained at that time is at least 70 %, more preferably 80%, even more preferably 90%, especially and preferably 95%, more particularly, preferably 98% or 99% identical with the "original" CDR or FR sequence. This means that it depends on the length of the CDR or FR to what degree it will be homologous with the "substituted" sequence.

Homology is determined by standard sequence alignment programs such as the NTI Vector (InforMax ™, Maryland, USA) or, more preferably, by the BLASTP program, preferably the blastp version 2.2.5 (November 16, 2002; cf. Altschul, SF et al. (1997) Nucl. Acids Res. 25, 3389-3402). Percent homology is based on alignment of all polypeptide sequences (matrix: BLOSUM 62; gap costs: 11.1; cutoff value set to 10-3) using any of the amino acid sequence CDR, VH, VL , H or L for reference in a pairwise comparison. It is calculated as the percentage of "positive" numbers (homologous amino acids) reported as the output of the BLASTP program divided by the total number of amino acids selected by the program for alignment.

When used herein, amino acid or nucleotide sequence homology can be used interchangeably with the term "identity." The term "homology" used in the present invention means the percentage of identical residues in pairs - after homological alignment of a sequence of an amino acid sequence or nucleotide sequence of the present invention with a sequence in question - with respect to the number of residues in the greater stretch of these two sequences. As described above, programs to determine homology (or identity) compare aligned sequences on an amino acid-by-amino acid basis, and can be set at various levels of strigence for comparison (e.g., identical amino acid, conservative amino acid substitution, etc.). As the term is used herein, two amino acids in question are considered "conservative substitutions" for each other if they each belong to the same chemical class, ie, acidic, nonpolar / hydrophobic, uncharged polar, and basic. By way of non-limiting example, two different amino acids belonging to the class of nonpolar amino acids would be considered "conservative substitutions" for each other, even if these two amino acids were not identical, whereas a nonpolar amino acid on one side and a basic amino acid on others would not be considered "conservative substitutions" for each other. Panel 3.1 of "Molecular Biology of the Cell", 4th Edition (2002), by Alberts, Johnson, Lewis, Raff, Roberts and Walter groups amino acids into four main groups: acidic, nonpolar, uncharged polar and basic. Such a grouping can be used to determine, in the context of the present invention, whether or not a given amino acid is a conservative substitution for another amino acid in question. The above-mentioned main groups can be sub-classified, for example, into small non-polar and large non-polar amino acids, large aromatic amino acids, etc. The term "conservative amino acid substitution" also indicates any amino acid substitution for a particular amino acid residue, when the substitute residue is so chemically similar to that of the given residue that there is no substantial decrease in the results of polypeptide function (eg, the Union).

The GM-CSF neutralizing compound is typically formulated as a pharmaceutical composition for parenteral administration, eg, intravenous, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal. to a subject, so subcutaneous administration is preferred. The pharmaceutical composition of the invention is a liquid composition, specifically an aqueous composition.

The concentration of the GM-CSF neutralizing compound in the liquid pharmaceutical composition is at least 100 mg / ml, even more preferably between about 100 mg / ml and about 200 mg / ml, such as approx. 150 mg / ml. When the composition is intended for subcutaneous administration, higher concentrations of the GM-CSF neutralizing compound can be used.

As noted above, the compositions comprise a buffer. As used herein, the term "buffer" refers to an added composition that allows a liquid formulation to resist changes in pH. In certain embodiments, the added buffer allows a liquid formulation to resist changes in pH through the action of its acidic and basic conjugated components. Examples of suitable buffers include, but are not limited to, a buffered histidine, acetate, or citrate system; In the present invention, histidine is used.

The term "specifically binds" or related expressions such as "specific binding", "specifically binds", "specific linker", etc., as used herein, refers to the ability of the GM neutralizing compound. -CSF and preferably the (human) (monoclonal) antibody or its functional fragment to discriminate between its target (for example, GM-CSF or the GM-CSF receptor) and any other potential antigen different from GM-CSF or the GM-CSF receptor to such an extent that, from a plurality of different antigens as possible binding partners, only GM-CSF / GM-CSF receptor is bound, or is significantly bound. In the sense of the invention, a target is "significantly" bound when, among a plurality of different antigens equally accessible as possible binding partners, the target is bound at least 10 times, preferably at least 50 times, preferably at least 100 times. or more frequently (in a kinetic sense) than any antigen other than the target. These kinetic measurements can be performed, for example, using SPR technology, such as a Biacore instrument. As used herein, the terms "binds (specifically) to" or related terms such as "acknowledges (specifically)", "targets", "interacts (specifically) with" and "reacts (specifically) with "means, according to this invention, that a GM-CSF neutralizing compound (eg, an antibody) exhibits appreciable affinity for its target (eg, GM-CSF or the GM-CSF receptor) and, generally, it does not show any significant reactivity with proteins or antigens other than the aforementioned targets. "Appreciable affinity" includes binding with an affinity of about 10-6 M (KD) or stronger, such as 10-7 M or stronger. Preferably, binding is considered specific when the binding affinity is about 10-11 to 10-8 M, preferably about 10-11 to 10-9 M, more preferably about 10-11 to 10 10 M. If a Compound (eg an antibody) reacts specifically, or binds to a target, this can be easily tested, among other things, by comparing the reaction of said compound with its target protein or antigen with the reaction of said compound with proteins or antigens other than your target. Preferably, a compound according to the invention does not bind essentially or is not capable of binding proteins or antigens other than GM-CSF or the GM-CSF receptor. The expression "does not bind essentially" or "is not capable of binding" means that the compounds of the present invention do not show reactivity of more than 30%, preferably more than 20%, more preferably more than 10%, particularly preferably more than 9%, 8%, 7%, 6%, or 5% with proteins or antigens other than GM-CSF or the GM-CSF receptor.

As used herein, "neutralization", "neutralizer", "neutralizer" and the grammatically related variations thereto refer to the partial or complete attenuation of the biological effects of GM-CSF. Said partial or complete attenuation of the biological effects of GM-CSF is the result of the modification, interruption and / or abrogation of processes mediated by GM-CSF, such as signal transduction, as manifested, for example, in intracellular signaling. , cell proliferation or release of soluble substances, up-regulation or down-regulation of intracellular gene activation, resulting, for example, in the expression of surface receptors for ligands other than GM-CSF. As understood by those skilled in the art, there are multiple ways to determine whether a compound, for example an antibody or its functional fragment, should be classified as a neutralizer. For example, this can be achieved by a standard in vitro test performed as follows: In a first proliferation experiment, a cell line, the degree of proliferation of which is known to depend on GM-CSF activity, is incubated with a series of samples with different concentrations of GM-CSF, after which incubation the degree of proliferation of the cell line is measured. From this measurement, the concentration of GM-CSF is determined allowing the maximum media proliferation of the cells. Subsequently, a second proliferation experiment is carried out using the same number of cells used in the first proliferation experiment, the previously determined concentration of GM-CSF and, in this case, varying the numbers of cells in each of the series of samples. concentrations of the compound suspected of being a GM-CSF neutralizer. Cell proliferation is measured again to determine the concentration of the test compound that is sufficient to cause maximum mean growth inhibition. If the graph resulting from the inhibition of growth versus the concentration of the tested compound is sigmoidal in shape, which results in a decrease in cell proliferation with increasing the concentration of the tested compound, then a certain degree of inhibition of the tested compound has been performed. growth, that is, GM-CSF activity has been neutralized to some extent. In such a case, the compound in question can be considered a "neutralizer" in the sense of the present invention. An example of a cell line, whose degree of proliferation known to depend on GM-CSF activity, it is the TF-1 cell line, as described in Kitamura, T. et al. (1989). J Cell Physiol 140, 323-34.

As understood by those skilled in the art, the degree of cell proliferation is not the only parameter by which the GM-CSF neutralizing ability can be established. For example, measurement of the level of signaling molecules (eg, cytokines), the level of secretion of which depends on GM-CSF, can be used to identify a putative GM-CSF neutralizer / GM-CSF inhibitor compound.

Other examples of cell lines that can be used to determine whether a subject compound, such as an antibody or its functional fragment, is a neutralizer of GM-CSF activity, include AML-193 (Lange, B. et al. (1987) Blood 70, 192-9); GF-D8 (Rambaldi, A. et al. (1993). Blood 81, 1376-83); (1990). Experimental Hematology 18, 1108-11); MO7E (Avanzi, G. C. et al. (1990). Journal of Cellular Physiology 145, 458-64); TALL-103 (Valtieri, M. et al. (1987). Journal of Immunology 138, 4042-50); and UT-7 (Komatsu, N. et al. (1991). Cancer Research 51,341-8).

It is understood that the neutralization of GM-CSF, in line with the present description, can be carried out outside the cells that carry the GM-CSF receptors or within said cells. Therefore, the neutralization of GM-CSF by a compound can be an inhibition or prevention of the binding of GM-CSF to its specific receptor or an inhibition of the intracellular signal induced by a binding of cytokines to its receptors. A GM-CSF neutralizing compound can, for example, bind to GM-CSF directly or to the GM-CSF receptor, thus interfering in both cases with the biological effects of GM-CSF.

As defined above, GM-CSF inhibitors can be selected from the group consisting of a polypeptide, a peptidomimetic, a nucleic acid molecule, and a small molecule.

The term "polypeptide", as used herein, describes a group of molecules that generally consists of at least 30 amino acids coupled to each other through a covalent peptide bond. According to the description, the group of polypeptides comprises "proteins" consisting of a single polypeptide or more than one polypeptide. The term "polypeptide" also describes protein fragments as long as these fragments consist of at least 30 amino acids. It is well known in the art that polypeptides can form multimers such as dimers, trimers, and higher oligomers, that is, they consist of more than one polypeptide molecule. These multimers are also included in the definition of the term "polypeptide". The polypeptide molecules that form such dimers, trimers, etc. they may or may not be identical. The corresponding higher order structures of such multimers are therefore called homo- or heterodimers, homo- or heterotrimers, etc. An example of a heteromultimer is an antibody molecule, which, in its natural form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "polypeptide" and "protein" also refer to naturally or unmodified polypeptides / proteins, where the modification is carried out, for example, by post-translational modifications such as glycosylation, acetylation, phosphorylation, disulfide bond formation and the like or by chemical modifications such as PEgylation. Such modifications are common in the art.

The term "nucleic acid" or "polynucleotide" defines, in the context of the invention, polymeric macromolecules consisting of multiple repeating units of phosphoric acid, purine and pyrimidine sugars and bases. Embodiments of these molecules include DNA, RNA, and ANP. The nucleic acid can be single or double stranded, linear or circular. Nucleic acid, in the context of the invention, is an aptamer. Nucleic acid aptamers are DNA or RNA molecules that have been randomly selected based on their ability to bind to other molecules. Aptamers have been selected that bind nucleic acids, proteins, small organic compounds, and even whole organisms. They consist of generally short oligonucleotide chains, typically 50 bases or less.

The term "small molecule" defines a group of organic pharmaceutical compounds having a molecular weight of less than 1000 Daltons, preferably up to 800 Daltons, and more preferably 300 to 700 Daltons. The upper molecular weight limit for a small molecule provides the ability for it to rapidly diffuse through cell membranes so that it can reach intracellular sites of action. Corresponding small molecules can be derived from an at least partially randomized peptide library. Suitable small molecule libraries according to the present invention are common in the art and / or can be purchased from commercial distributors.

The term "peptidomimetic" describes a small protein-like chain designed to mimic peptides. These types of molecules are artificially derived by modifying an existing peptide in order to alter the properties of the molecule. For example, the existing parent peptide is modified to change the stability or biological activity of the molecule. These modifications include the alteration of the structure and the incorporation of unnatural amino acids.

The term "GM-CSF receptor" refers to the physiological cell surface receptor for GM-CSF, which is described in the art as a heteromer of an alpha chain (CD116) and a common beta subunit (beta-c).

A preferred embodiment of a neutralizing polypeptide is an antibody or its functional fragments, more preferably a human antibody or its functional fragments. Techniques for the production of antibodies are common in the art and are described, for example, in Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane "Using Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, 1999.

The definition of the term "antibody" includes such embodiments as monoclonal, chimeric, single chain, humanized, and human antibodies. In addition to full-length antibodies, the definition also includes antibody derivatives and antibody fragments, such as, but not limited to, Fab fragments. Antibody fragments or derivatives further comprise F (ab ') ² , Fv, scFv fragments or single domain antibodies, such as domain antibodies or nanobodies, immunoglobulin single variable domain or single variable domain antibodies that comprise a single variable domain, which could be VHH, VH or VL, that specifically bind to an antigen or epitope, independently of other V regions or domains; see, for example, Harlow and Lane (1988) and (1999), loc. cit .; Kontermann and Dübel, Antibody Engineering, Springer, 2nd ed.

2010 and Little, Recombinant Antibodies for Immunotherapy, Cambridge University Press 2009. This term also includes diabodies or double affinity reorientation antibodies (DART). More single-stranded (bispecific) diabodies, tandem diabodies (Tandab), "minibodies" are expected exemplified by a structure that is as follows: (VH-VL-CH3) ² , (scFv-CH3) ^2, or (scFv-CH3-scFv ) ² , "Fc DART" and "IgG DA ^r T", multibodies as triabodies. Immunoglobulin single variable domains encompass not only an isolated antibody single variable domain polypeptide, but also larger polypeptides comprising one or more monomers of an antibody single variable domain polypeptide sequence.

Furthermore, the term "antibody", as used herein, also refers to derivatives or variants of the antibodies described herein that show the same specificity as the described antibodies. Examples of "antibody variants" include humanized non-human antibody variants, "affinity matured" antibodies (see, for example, Hawkins et al. J. Mol. 254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837 (1991)) and antibody mutants with altered effector functions (see, for example, US Pat. No. 5,648,260, Kontermann and Dübel (2010), loc. Cit. And Little (2009), loc. cit.).

The term "antibody" also encompasses immunoglobulins (Ig) of different classes (ie IgA, IgG, IgM, IgD, and IgE) and subclasses (such as IgG1, IgG2, etc.). Derivatives of antibodies, which also come within the definition of the term antibody in the meaning of the invention, include modifications of molecules such as, for example, glycosylation, acetylation, phosphorylation, disulfide bond formation, farnesylation, hydroxylation, methylation or esterification.

A functional fragment of an antibody includes the domain of an F (ab ') ² fragment, a Fab fragment, scFv, or constructs comprising single immunoglobulin variable domains or single domain antibody polypeptides, eg, single heavy chain variable domains. or single light chain variable domains, as well as other antibody fragments as described herein. The F (ab ') ² or Fab can be designed to minimize or completely eliminate the intermolecular disulfide interactions that occur between the C ^h1 and C ^l domains.

The term "human antibody", as used herein, should be understood in the sense that the antibody or its functional fragment, comprises one or more amino acid sequences contained in the human repertoire of germ antibodies. Therefore, for the purposes of the definition herein, an antibody, or its fragment, may be considered human if it consists of said human germline amino acid sequence (s), that is, if the amino acid sequences of the antibody in question or its functional fragments are identical to the human germline amino acid sequence (s). An antibody or its functional fragment may also be considered human if it consists of one or more sequences that deviate from its closest human germ sequence (s) by no more than would be expected due to the impression of somatic hypermutation. Furthermore, the antibodies of many non-human mammals, for example rodents such as mice and rats, comprise VH CDR3 amino acid sequences that can be expected to exist in the expressed human antibody repertoire as well. Any sequence or sequences of human or non-human origin that can be expected to exist in the expressed human repertoire will also be considered "human" for the purposes of the present invention. Thus, the term "human antibody" includes antibodies that have variable and constant regions that substantially correspond to human germ cell immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (see Kabat et al. (1991) loc. cit.). The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in CDRs and, in particular, in CDR3. The human antibody can have at least one, two, three, four, five or more positions replaced by an amino acid residue that is not encoded by the human germ cell immunoglobulin sequence.

Human antibodies or their functional fragments of the invention are monoclonal. It is particularly difficult to prepare human antibodies that are monoclonal. Unlike fusions of murine B cells with immortalized cell lines, fusions of human B cells with immortalized cell lines are not viable. By Therefore, human monoclonal antibodies are the result of overcoming significant, generally known technical hurdles that exist in the field of antibody technology. The monoclonal nature of antibodies makes them especially suitable for use as therapeutic agents, since such antibodies will exist as a single homogeneous molecular species that can be well characterized and reproducibly manufactured and purified. These factors result in products whose biological activities can be predicted with a high level of precision, very important if these molecules are to gain regulatory approval for therapeutic administration in humans. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies that make up the population are identical, except for possible natural mutations and / or post-translational modifications (eg, isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site. Furthermore, unlike conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous because they are synthesized by the hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as derived from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention can be made by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or can be made by recombinant DNA methods (see , eg, US Pat. No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991), for example.

The monoclonal antibodies or the corresponding functional fragments of the invention are human antibodies or the corresponding functional fragments. When contemplating antibody agents for therapeutic administration to humans, it is highly advantageous that the antibodies are of human origin. After administration to a human patient, the human antibody or its functional fragment most likely will not elicit a strong immunogenic response by the patient's immune system, that is, it will not be recognized as a foreign protein as it is a non-human protein. This means that no antibodies will be generated against the host, that is, of the patient against the therapeutic antibody that would otherwise block the activity of the therapeutic antibody and / or accelerate the elimination of the therapeutic antibody from the patient's body, thus preventing it from being exerted. the desired therapeutic effect.

The human monoclonal antibody or its functional fragment to be used for pharmaceutical purposes exhibits reactivity between the human species and at least one species of monkey. The same cross-species reactivity is also preferred for all other neutralizing / inhibiting compounds derived from GM-CSF antibodies or non-antibodies.

The antibody can be an IgG antibody. An IgG isotype comprises not only the variable antibody regions of the heavy and light chains responsible for the recognition and binding of highly discriminatory antigens, but also the constant regions of the polypeptide chains of heavy and light antibodies normally present in "naturally" produced antibodies. and, in some cases, even modifications to one or more carbohydrate sites. This glycosylation is generally a hallmark of the IgG format, and is found in the constant regions that comprise the so-called Fc region of a full-length antibody that are known to elicit various effector functions in vivo. In addition, the fc region mediates the binding of IgG to the Fc receptor, as well as facilitates the movement of IgG to places with a greater presence of the Fc receptor, for example, towards inflamed tissues. Advantageously, the IgG antibody is an IgG1 antibody or an IgG4 antibody, formats that are preferred since their in vivo mechanism of action is particularly well understood and characterized. This is especially the case for IgG1 antibodies.

The functional antibody fragment may preferably be a scFv, a single domain antibody, a Fv, a VHH antibody, a diabody, a tandem diabody, a Fab, a Fab 'or a F (ab') 2. These formats can generally be divided into two subclasses, namely, those consisting of a single polypeptide chain, and those that comprise at least two polypeptide chains. Members of the above subclass include a scFv (comprising a VH region and a VL region joined into a single polypeptide chain via a polypeptide linker); a single domain antibody (comprising a single antibody variable region) or a VHH antibody (comprising a single VH region). Members of the latter subclass include an Fv (comprising a VH region and a VL region as separate polypeptide chains that are not covalently associated with each other); a diabody (comprising two non-covalently associated polypeptide chains, each of which comprises two antibody variable regions - typically one VH and one VL per polypeptide chain - the two polypeptide chains arranged in a head-to-tail conformation so that a bivalent antibody molecule is produced); a tandem diabody (single chain bispecific Fv antibodies comprising four covalently linked immunoglobulin regions - VH and VL - of two different specificities, forming a homodimer that is twice as large as the diabody described above); an Fab (comprising as one polypeptide chain an entire antibody chain, comprising a VL region and the entire light chain constant region and, as another polypeptide chain, a part of an antibody heavy chain comprising a complete region of VH and part of the constant region of the heavy chain, said two polypeptide chains being intermolecularly connected through an interchain disulfide bond); a Fab '(as a Fab, above, except with additional reduced disulfide bonds made up in the heavy chain of antibodies); and an F (ab) 2 (comprising two Fab 'molecules, each Fab' molecule being linked to the respective other Fab 'molecule via interchain disulfide bonds). In general, functional antibody fragments of the type described above allow great flexibility in configuration, for example, the pharmacokinetic properties of a desired antibody for therapeutic administration to the particular requirements at hand. For example, it may be desirable to reduce the size of the administered antibody in order to increase the degree of tissue penetration when treating tissues that are known to be poorly vascularized (eg, the joints). In some circumstances, it may also be desirable to increase the rate at which the therapeutic antibody is cleared from the body, said rate being generally accelerable by decreasing the size of the administered antibody. An antibody fragment is defined as a functional antibody fragment in the context of the invention, as long as the fragment maintains the specific binding characteristics for the epitope / target of the main antibody, that is, as long as it specifically binds to GM -CSF or GM-CSF receptor.

According to a new embodiment of the invention, said antibody or functional fragment thereof may be present in monospecific monovalent forms; monospecific multivalents, in particular bivalent monospecific; or multispecific multivalents, in particular bispecific bivalents. In general, a multivalent monospecific antibody, in particular monospecific bivalent such as full-length human IgG, as described herein above can bring with it the therapeutic advantage that the neutralization carried out by said antibody is enhanced by avidity effects, that is, the binding by the same antibody to multiple molecules of the same antigen, GM-CSF itself or the GM-CSF receptor. Several monospecific monovalent forms of antibody fragments (eg, a scFv, an Fv, a VHH, or a single domain antibody) have been described previously. Multivalent multispecific forms, particularly bivalent bispecific forms of an antibody, can include a full length IgG in which one binding arm binds to the primate GM-CSF / GM-CSF receptor, while the other binding arm binds to another antigen than GM-CSF / GM-CSF receptor. Another mulespecific multivalent, in particular, bispecific bivalent form may advantageously be a human single chain bispecific antibody, that is, a recombinant human antibody construct comprising two scFv entities as described above, linked into a contiguous polypeptide chain by a spacer of Short intervening polypeptide as is generally known in the art (see for example WO 99/54440 for a bi-specific anti-CD19 x anti-CD3 single chain antibody). In this case, a scFv portion of the compound bispecific single chain antibody within the single chain bispecific antibody will specifically bind to the GM-CSF / GM-CSF receptor as stated above, while the other respective scFv portion of this bispecific single chain antibody will bind will bind to another specific antigen that has therapeutic benefit.

According to a further embodiment, the antibodies or functional fragments thereof can be derived, for example with an organic polymer, for example with one or more molecules of polyethylene glycol ("PEG") and / or polyvinyl pyrrolidone ("PVP"). As is known in the art, such derivatization can be advantageous in modulating the pharmacodynamic properties of antibodies or their functional fragments. Especially preferred are derivatized PEG molecules such as PEG-maleimide, allowing conjugation to the antibody or its functional fragment in a site-specific manner through the sulfhydryl group of a cysteine amino acid. Of these, 20 kD and / or 40 kD PEG-maleimide, either in branched or straight chain form, are especially preferred. It may be especially advantageous to increase the effective molecular weight of smaller human anti-GM-CSF antibody fragments, such as scFv fragments, by coupling the latter to one or more PEG molecules, especially PEG-maleimide.

The antibodies described in this document also include "chimeric" antibodies (immunoglobulins) in which a part of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a class or particular antibody subclass, while the rest of the chain or chains are identical or homologous with respect to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of said antibodies, provided and when they show the desired biological activity (US Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodies of interest here include "primitized" antibodies comprising variable domain antigen binding sequences derived from a non-human primate (eg, Old World Monkey, ape, etc.) and human constant region sequences.

"Humanized" forms of non-human antibodies (eg, murine) are chimeric immunoglobulins, immunoglobulin chains, or their fragments (such as Fv, Fab, Fab ', F (ab') 2, or other antibody antigen subsequences) mostly human sequences, containing a minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (receptor antibodies) in which residues from a hypervariable region (also CDR) from the receptor are replaced by residues from a hypervariable region from a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, human immunoglobulin residues from the Fv framework region (FR) are replaced by non-human residues corresponding. Furthermore, "humanized antibodies" as used herein can also comprise residues that are found neither in the recipient's antibody nor in the donor's antibody. These modifications are made to further refine and optimize the performance of the antibodies. The optimally humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details, see Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992).

As used herein, the numbering of amino acid residues or positions of human and non-human primate GM-CSF refers to that of mature GM-CSF, that is, GM-CSF without its signal sequence 17 amino acids (the total length of mature GM-CSF in the human and non-human primate species described above is 127 amino acids). The sequence of human GM-CSF and gibbon GM-CSF is as follows:

SEQ ID NO: 49

APARSPSPST QPWEHVNAIQ EARRLLNLSR DTAAEMNETV EVISEMFDLQ

EPTCLQTRLE LYKQGLRGSL TKLKGPLTMM ASHYKQHCPP TPETSCATQI

ITFESFKENL KDFLLVIPFD CWEPVQE

The sequence of GM-CSF in certain members of the macaca family of monkeys, such as the Rhesus monkey and the Cynomolgus monkey, is as follows:

SEQ ID NO: 50

APARSPSPGT QPWEHVNAIQ EARRLLN LSR DJAAEMNKTV EVVSEMFDLQ

EPSCLQTRLE LYKQGLQGSL TKLKGPLTMM ASHYKQHCPP TPETSCATQI

ITFQSFKENL KDFLLVIPFD CWEPVQE

The sequence of human GM-CSF is also shown in SEQ ID NO.57. That of gibbon GM-CSF is also shown in SEQ ID NO. 58:

APARSPSPST QPWEHVNAIQ EARRLLN LSR DTAAEMNETV EVISEMFDLQ

EPTCLQTRLE LYKQGLRGSL TKLKGPLTMM ASHYKQHCPP TPETSCATQI

ITFESFKENL KDFLLVIPFD CWEPVQE

(SEQ ID NO.57 and 58)

The GM-CSF sequence in certain members of the macaca family of monkeys such as, for example, the Rhesus monkey (SEQ ID NO.59) and the Cynomolgus monkey (SEQ ID NO.60) is also as follows:

APARSPSPGT QPWEHVNAIQ EARRL LN LSR DTAAEMNKTV EVVSEMFDLQ EPSCLQTRLE LYKQGLQGSL TKLKGPLTMM ASHYKQHCPP TPETSCATQI ITFQSFKENL KDFLLVIPFD CWEPVQE

(SEQ ID NO. 59 and 60)

The minimal epitope, advantageously a discontinuous epitope as described herein, bound by the antibody, preferably a human monoclonal antibody (or its functional fragment) is indicated in the GM-CSF sequence (s) shown above in bold. .

The GM-CSF neutralizing human monoclonal antibody or its functional fragment, which binds to GM-CSF, also binds to a GM-CSF epitope that preferably comprises amino acids 23-27 (RRLLN) and / or amino acids 65- 77 (GLR / QGSLTKLKGPL). These stretches of the amino acid sequence are indicated in the GM-CSF sequence above in bold. The term "epitope" refers to a site on an antigen or a target (eg, GM-CSF) to which a compound, such as an antibody or its functional fragment, specifically binds. Such binding / interaction is also understood to define a "specific recognition". "Epitopes" can be formed by both contiguous amino acids and non-contiguous amino acids juxtaposed by the tertiary folding of a protein. A "linear epitope" is an epitope in which a primary amino acid sequence comprises the recognized epitope. A linear epitope typically includes at least 3, or at least 4, and more generally at least 5, or at least 6, or at least 7, for example, from about 8 to about 10 amino acids or more in a unique sequence. In the context of the present invention, it is preferred that the GM-CSF epitope is a discontinuous epitope. In case the antibody binds to both sequence stretches 23-27 and 65-77, the epitope can be called "discontinuous". In the secondary structure of human GM-CSF, amino acids 15-35 are located in helix A, while residues corresponding to positions 65-77 are part of a loop structure located between helices C and D. A model Three-dimensional folding of the molecule reveals a close steric proximity of these sites with respect to each other (see also WO 2006/111353). As used herein, the term "discontinuous epitope" should be understood as at least two non-adjacent amino acid sequence stretches within a given polypeptide chain, in this case mature human GM-CSF and non-human primates, that are simultaneously and specifically bound by an antibody. According to this definition, said simultaneous specific binding can be of the GM-CSF polypeptide in linear form. In this case, the mature GM-CSF polypeptide can be envisioned forming an extended loop, in a region of which the two sequences indicated in bold above are aligned, for example, more or less parallel and in proximity to each other. In this state, they are specifically and simultaneously bound by the antibody fragment. According to this definition, the simultaneous specific binding of the two sequential stretches of mature GM-CSF indicated above may also take the form of an antibody binding to a conformational epitope. In this case, mature GM-CSF already has its tertiary conformation formed as it normally exists in vivo. In this tertiary conformation, the mature GM-CSF polypeptide chain is folded in such a way that the two stretches of sequence listed above come together, for example on the outer surface of a particular region of folded, mature GM-CSF, where then they are recognized by virtue of their three-dimensional conformation in the context of surrounding polypeptide sequences.

In another more preferred embodiment, the GM-CSF prior epitope or discontinuous epitope above further comprises:

• amino acids 28-31 (LSRD), italicized in the above sequences of human GM-CSF and non-human primate;

• amino acids 32-33 (TA), underlined in the above sequences of human GM-CSF and non-human primate; me

• amino acids 21-22 (EA), underlined in the above sequences of human GM-CSF and non-human primate.

Preferred human monoclonal anti-GM-CSF antibodies or their functional fragments are those specifically described in WO2006 / 111353 under SEQ ID Nos: 1 to 48 and 52 to 56, which represent amino acid sequences of complementarity determining regions heavy and light chains (CDRs) 1-3, as well as variable regions of heavy and light chains and full-length chains.

Especially preferred are anti-GM-CSF antibodies or their functional fragments comprising a CDR1 sequence of the heavy chain variable region as set forth in SEQ ID NO. 14, a CDR2 sequence of the heavy chain variable region as set forth in SEQ ID NO. 15 and a heavy chain variable region CDR3 sequence as set forth in SEQ ID NO. 1; or comprising a CDR1 sequence of the heavy chain variable region as set forth in SEQ ID NO. 14, a CDR2 sequence of the heavy chain variable region as set forth in SEQ ID NO. 15 and a heavy chain variable region CDR3 sequence as set forth in SEQ ID NO. 2; or comprising a CDR1 sequence of the heavy chain variable region as set forth in SEQ ID NO. 14, a CDR2 sequence of the heavy chain variable region as set forth in SEQ ID NO. 15 and a heavy chain variable region CDR3 sequence as set forth in SEQ ID NO. 3; or comprising a CDR1 sequence of the heavy chain variable region as set forth in SEQ ID NO. 14, a CDR2 sequence of the heavy chain variable region as set forth in SEQ ID NO. 15 and a heavy chain variable region CDR3 sequence as set forth in SEQ ID NO. 4; or comprising a CDR1 sequence of the heavy chain variable region as set forth in SEQ ID NO. 14, a CDR2 sequence of the heavy chain variable region as set forth in SEQ ID NO. 15 and a heavy chain variable region CDR3 sequence as set forth in SEQ ID NO. 5; or comprising a CDR1 sequence of the heavy chain variable region as set forth in SEQ ID NO. 14, a CDR2 sequence of the heavy chain variable region as set forth in SEQ ID NO. 15 and a heavy chain variable region CDR3 sequence as set forth in SEQ ID NO. 6; or comprising a CDR1 sequence of the heavy chain variable region as set forth in SEQ ID NO. 14, a CDR2 sequence of the heavy chain variable region as set forth in SEQ ID NO. 15 and a heavy chain variable region CDR3 sequence as set forth in SEQ ID NO. 7; or comprising a CDR1 sequence of the heavy chain variable region as set forth in SEQ ID NO. 14, a CDR2 sequence of the heavy chain variable region as set forth in SEQ ID NO. 15 and a heavy chain variable region CDR3 sequence as set forth in SEQ ID NO. 8; or comprising a CDR1 sequence of the heavy chain variable region as set forth in SEQ ID NO. 14, a CDR2 sequence of the heavy chain variable region as set forth in SEQ ID NO. 15 and a heavy chain variable region CDR3 sequence as set forth in SEQ ID NO. 9; or comprising a CDR1 sequence of the heavy chain variable region as set forth in SEQ ID NO. 14, a CDR2 sequence of the heavy chain variable region as set forth in SEQ ID NO. 15 and a heavy chain variable region CDR3 sequence as set forth in SEQ ID NO. 10; or comprising a heavy chain variable region CDR1 sequence as set forth in SEQ ID NO. 14, a CDR2 sequence of the heavy chain variable region as set forth in SEQ ID NO. 15 and a heavy chain variable region CDR3 sequence as set forth in SEQ ID NO. eleven; or comprising a heavy chain variable region CDR1 sequence as set forth in SEQ ID NO. 14, a CDR2 sequence of the heavy chain variable region as set forth in SEQ ID NO. 15 and a heavy chain variable region CDR3 sequence as set forth in SEQ ID NO. 12; or comprising a heavy chain variable region CDR1 sequence as set forth in SEQ ID NO. 14, a CDR2 sequence of the heavy chain variable region as set forth in SEQ ID NO. 15 and a heavy chain variable region CDR3 sequence as set forth in SEQ ID NO. 13; or comprising a CDR1 sequence of the heavy chain variable region as set forth in SEQ ID NO. 14, a CDR2 sequence of the heavy chain variable region as set forth in SEQ ID NO. 15 and a heavy chain variable region CDR3 sequence as set forth in SEQ ID NO. 56.

Even more preferred, any of the above 14 combinations of the heavy chain CDR1, CDR2 and CDR3 sequences exist in an antibody or its functional fragment that further comprises in its light chain variable region a CDR1 comprising the amino acid sequence established in SEQ ID NO. 16, a CDR2 comprising the amino acid sequence established in SEQ ID NO. 17, and a CDR3 comprising the amino acid sequence established in SEQ ID NO. 18.

An especially preferred anti-GM-CSF antibody or its functional fragment comprises a heavy chain variable region CDR1 sequence, as set forth in SEQ ID NO. 14, a CDR2 sequence of the heavy chain variable region, as set forth in SEQ ID NO. 15 and a CDR3 sequence of the heavy chain variable region, as set forth in SEQ ID NO. 2 and further comprises in its light chain variable region a CDR1 comprising the amino acid sequence established in SEQ ID NO. 16, a CDR2 comprising the amino acid sequence established in SEQ ID NO. 17, and a CDR3 comprising the amino acid sequence established in SEQ ID NO. 18. This anti-GM-CSF antibody is the most preferred compound for neutralizing GM-CSF and is also described in document Wo 2006/111353.

The anti-GM-CSF antibody or its functional fragment comprises in its light chain variable region an amino acid sequence as set forth in SEQ ID NO. 19. An antibody or its functional fragment is described, the light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. twenty; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. twenty-one; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 22; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 2. 3; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 24; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 25; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 26; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 27; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 28; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 29; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 30; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 31; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as stated in SEQ ID NO. 32; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 33; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 52; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 19 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 53.

The anti-GM-CSF antibody or its functional fragment described herein may comprise in its light chain variable region an amino acid sequence as set forth in SEQ ID NO. 54. An antibody or its functional fragment is also described, the light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. twenty; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. twenty-one; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 22; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 2. 3; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 24; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 25; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 26; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 27; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 28; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 29; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 30; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 31; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 32; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 33; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 52; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO.

54 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 53.

The anti-GM-CSF antibody or its functional fragment described herein may comprise in its light chain variable region an amino acid sequence as set forth in SEQ ID NO. 55. An antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 55 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. twenty; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 55 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. twenty-one; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 55 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 22; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 55 and a heavy chain variable region comprising an amino acid sequence as shown set to SEQ ID NO. 2. 3; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 55 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 24; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 55 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 25; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 55 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 26; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 55 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 27; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 55 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 28; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 55 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 29; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 55 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 30; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 55 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 31; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 55 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 32; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 55 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 33; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as established in SEQ ID NO. 55 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 52; or an antibody or its functional fragment, the light chain variable region comprising an amino acid sequence as set forth in SEQ ID NO.

55 and a heavy chain variable region comprising an amino acid sequence as set forth in SEQ ID NO. 53.

The anti-GM-CSF antibody or its functional fragment of the invention comprises in its light chain variable region a CDR1 comprising an amino acid sequence as established in SEQ ID NO. 16, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO. 17 and a CDR3 comprising an amino acid sequence as set forth in SEQ ID NO. 18 and comprises in its heavy chain variable region a CDR1 comprising an amino acid sequence SEQ ID NO. 14, a CDR2 comprising an amino acid sequence as set forth in SEQ ID NO. 15 and a CDR3 comprising an amino acid sequence as established in any of SEQ ID NO. 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 56.

Also described is an antibody that further comprises in its light chain an amino acid sequence as set forth in SEQ ID NO. 34 and in its heavy chain an amino acid sequence as established in SEQ ID NO. 35; or in its light chain an amino acid sequence as established in SEQ ID NO. 34 and in its heavy chain an amino acid sequence as established in SEQ ID NO. 36; or in its light chain an amino acid sequence as established in SEQ ID NO. 34 and in its heavy chain an amino acid sequence as established in SEQ ID NO. 37; or in its light chain an amino acid sequence as established in SEQ ID NO. 34 and in its heavy chain an amino acid sequence as established in SEQ ID NO. 38; or in its light chain an amino acid sequence as established in SEQ ID NO. 34 and in its heavy chain an amino acid sequence as established in SEQ ID NO. 39; or in its light chain an amino acid sequence as established in SEQ ID NO. 34 and in its heavy chain an amino acid sequence as established in SEQ ID NO. 40; or in its light chain an amino acid sequence as established in SEQ ID NO. 34 and in its heavy chain an amino acid sequence as established in SEQ ID NO. 41; or in its light chain an amino acid sequence as established in SEQ ID NO. 34 and in its heavy chain an amino acid sequence as established in SEQ ID NO. 42; or in its light chain an amino acid sequence as established in SEQ ID NO. 34 and in its heavy chain an amino acid sequence as established in SEQ ID NO. 43; or in its light chain an amino acid sequence as established in SEQ ID NO. 34 and in its heavy chain an amino acid sequence as established in SEQ ID NO. 44; or in its light chain an amino acid sequence as established in SEQ ID NO. 34 and in its heavy chain an amino acid sequence as established in SEQ ID NO. Four. Five; or in its light chain an amino acid sequence as established in SEQ ID NO. 34 and in its heavy chain an amino acid sequence as established in SEQ ID NO. 46; or in its light chain an amino acid sequence as established in SEQ ID NO. 34 and in its heavy chain an amino acid sequence as established in SEQ ID NO. 47; or in its light chain an amino acid sequence as established in SEQ ID NO. 34 and in its heavy chain an amino acid sequence as established in SEQ ID NO. 48. Also described is the anti-GM-CSF antibody comprising in its light chain an amino acid sequence as established in SEQ ID NO. 34 and in its heavy chain an amino acid sequence as set forth in SEQ ID NO. 35 and is also described in WO 2006/111353.

Human monoclonal antibody molecules and / or functional fragments of the invention are especially advantageous as neutralizers of human GM-CSF activity. Antibodies or their functional fragments in accordance with these especially preferred embodiments are highly advantageous for several reasons.

First, they recognize human GM-CSF with high specificity. That is, from a mixture of human GM-CSF with other human colony stimulating factors, the binding molecules according to these embodiments are highly discriminant for human GM-CSF, whereas the other colony stimulating factors in the same environment are not recognize. This means that an antibody or its functional fragment according to these embodiments, when administered to a human, is expected to specifically bind and neutralize only the desired target, while other unwanted targets will neither be bound nor neutralized. Ultimately, this leads to a high degree of predictability with respect to the therapeutic mode of action in vivo.

Second, the binders according to these embodiments bind to primate and human GM-CSF with appreciable affinity. "Appreciable affinity" includes binding with an affinity of about 10 ^-6 M (KD) or stronger. Preferably, binding is considered appreciable (or high or specific) when the binding affinity is about 10 ^-12 to 10 ^-8 M, 10 ^-12 to 10 ^-9 M, 10 ^-12 to 10 ^-10 M, of 10 ^-11 to 10 ^-8 M, preferably about 10 ^-11 to 10 ^-9 M. Thus, a binder of the invention preferably has a K ^D within that range. Since K ^d values of about 4 x 10 ^-9 M to as low as about 0.04 x 10 ^-9 M, the latter corresponding to about 40 pM, have been observed for the antibody molecules described herein, it is preferred that the antibody molecules of the invention have a K ^D within that range. However, it is also preferred that the antibody molecules of the invention have a K ^d as described above. Since the association kinetic constant of such molecules in aqueous media is largely controlled by diffusion and therefore cannot be improved beyond what local diffusion conditions will allow under physiological conditions, low K ^D It arises mainly as a result of the dissociation kinetic constant, k ^off , which for the highest affinity antibody binder is approximately 10 ^-5 s ^-1 . This means that once the complex is formed between a human monoclonal antibody or its functional fragment according to any of these embodiments on the one hand and GM-CSF on the other hand, it is not easily separated or at least not separated. separates quickly. For binding molecules intended to be neutralizers of biological activity, these characteristics are highly advantageous since the desirable neutralizing effect will normally last only as long as the molecule, whose biological activity must be neutralized (in this case, primate and human GM-CSF) continues. bound by the neutralizing binding molecule. Therefore, a neutralizing molecule that remains attached to its intended target for a long time will continue to be neutralized for a long time accordingly.

The high binding affinity of antibodies or their functional fragments to primate and human GM-CSF has an additional advantage. Normally, the antibodies or their functional fragments will be cleared from a patient's bloodstream in a size-dependent manner, with smaller molecules being excreted and eliminated earlier than larger ones. Since the complex of the two polypeptides (antibody or antibody fragment and bound GM-CSF) is obviously larger than the antibody alone, the aforementioned low k ^off has the effect that the therapeutic neutralizer is excreted and eliminated from the body of the patient more slowly than would be the case, if it were not bound to GM-CSF. Therefore, not only the magnitude of the neutralizing activity is increased, but also its duration in vivo.

The neutralizing activity determined for binders according to the above embodiments is surprisingly high. As will be described in more detail below, the neutralizing activity of GM-CSF was measured in vitro using a TF-1 growth inhibition assay (Kitamura, T. et al. (1989), J. Cell Physiol.

140, 323-34). As an indication of the neutralizing potential, IC ⁵⁰ , IC ⁵⁰ values representing the concentration of the antibody or its functional fragment were measured according to any of these embodiments necessary to elicit maximum mean inhibition of TF-1 cell proliferation. For human monoclonal anti-GM-CSF antibodies or their specified functional fragments an IC ⁵⁰ value of approximately 3 x 10 ^-10 M, or approximately 0.3 nM was determined. Therefore, the binding molecules are very potent neutralizers of human GM-CSF activity.

Other examples of neutralizing anti-GM-CSF antibodies are the human E10 antibody and the human G9 antibody described in Li et al., (2006) PNAS 103 (10): 3557-3562. E10 and G9 are IgG class antibodies. E10 has a binding affinity of 870 pM for GM-CSF and G9 has an affinity of 14 pM for GM-CSF. Both antibodies are specific for binding to human GM-CSF and show strong neutralizing activity as assessed with a TF-1 cell proliferation assay. Other examples are the human anti-GM-CSF antibodies that are described in WO 2006/122797.

GM-CSF antagonists including antibodies to the alpha chain or beta chain of GM-CSF receptors are also described. An anti-GM-CSF receptor antibody described herein can be in any form of antibody as explained above, for example intact, chimeric, monoclonal, polyclonal, antibody fragment or derivative, single chain, humanized, human, etc. Examples of anti-GM-CSF receptor antibodies are known in the art, for example, high affinity neutralizing antibodies (see, eg, US Pat. No. 5,747,032 and Nicola et al., Blood 82:15, 1724, 1993).

Examples of known anti-GM-CSF antibodies are provided in WO2006 / 122797, WO2007 / 049472, WO2007 / 092939, WO2009 / 134805, WO2009 / 064399, WO2009 / 038760. Antibodies against the GM-C ^s F receptor are provided in WO2007 / 110631.

In summary, anti-GM-CSF antibodies or their functional fragments exhibit a high degree of discrimination for the desired antigen, bind this antigen extremely strongly and for a long time, and exhibit very potent neutralizing activity for a long time while remaining attached. At the same time, the long persistence of the binder-antigen complex slows down the removal of this binder from the body, thus lengthening the duration of the desired therapeutic effect in vivo. The same characteristics preferably also apply for antibodies that recognize the GM-CSF receptor, as described above.

The composition of the present invention is preferably a pharmaceutical composition. In accordance with the present embodiments, the term "pharmaceutical composition" refers to a composition for administration to a patient, preferably a human patient. The pharmaceutical compositions or formulations are generally in a form that allows the biological activity of the active ingredient to be effective and, therefore, that it can be administered to a subject for therapeutic use as described herein. Generally, the pharmaceutical composition comprises suitable (ie pharmaceutically acceptable) formulations of carriers, stabilizers, and / or excipients. In a preferred embodiment, the pharmaceutical composition is a composition for parenteral, transdermal, intraluminal, intraarterial, intrathecal and / or intranasal administration or for direct injection into tissue. In particular, it is envisaged that said composition is administered to a patient by infusion or injection. The administration of suitable compositions can be effected by different routes, for example, intravenously, intraperitoneally, subcutaneously, intramuscularly, topically or intradermally. The composition of the present invention may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are common in the art and include buffered saline solutions, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions, liposomes, etc. Compositions comprising these carriers can be formulated by known conventional methods.

In accordance with the present embodiments, the term "effective amount" refers to an amount of the GM-CSF neutralizing compound that is effective for the treatment of diseases associated with GM-CSF, such as inflammatory and autoimmune disorders.

Preferred doses and preferred methods of administration are such that, after administration, the GM-CSF neutralizing compound is present in the blood in effective doses. The administration schedule can be adjusted by observing disease conditions and analyzing serum levels of the neutralizing compound GM-CSF in laboratory tests followed by extending the administration interval, for example, from twice a week or once a week. to once every two weeks, once every three weeks, once every four weeks, etc., or, alternatively, reducing the corresponding administration at intervals.

The pharmaceutical compositions can be administered to the subject at a suitable dose. The dosage regimen will be determined by the attending physician and by clinical factors. As is well known in the medical arts, the doses for each patient depend on many factors, including the patient's body volume, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health status. , and other medications that are administered simultaneously.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous vehicles include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, sodium dextrose chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelators, inert gases, etc. Furthermore, the pharmaceutical composition according to the present invention could comprise protein carriers, such as, for example, serum albumin or immunoglobulin, preferably of human origin. It is envisaged that the pharmaceutical composition according to the invention may comprise, in addition to the compounds described above, biologically active agents, depending on the intended use of the pharmaceutical composition. Such agents can be drugs that act on the gastro-intestinal system, drugs that act as cytostatic agents, drugs to prevent hyperuricemia, drugs that inhibit immunoreactions (for example, corticosteroids), drugs that modulate the inflammatory response, drugs that act on the circulatory system and / or agents, such as cytokines, known in the art.

To analyze the effect of a GM-CSF neutralizing compound, for example in rheumatoid arthritis (RA), data measures, for example, of pharmacokinetics, immunogenicity and the potential to improve clinical signs and symptoms of RA can be selected. measured according to the DAS28, ACR20 / 50/70 and / or EULAR response criteria, MRI images for synovitis and bone edema, as well as data reported by the patient. ACR is a measure that summarizes the improvement in the number of tender and swollen joints, the pain scale, the evaluation of the improvement by patients and doctors, and certain laboratory markers. ACR 20 describes the percentage of study participants who achieved a 20 percent improvement in clinical signs and symptoms, for example, a 20 percent improvement in the count of tender or swollen joints, as well as an improvement of 20 percent in three other criteria relevant to the disease.

Another major problem in drug development, such as the pharmaceutical composition according to the invention, is the predictable modulation of pharmacokinetic properties. For this purpose, a pharmacokinetic profile of the test drug is established, that is, a profile of the pharmacokinetic parameters that affect the ability of a particular drug to treat a given condition. Drug pharmacokinetic parameters that influence a drug's ability to treat a particular disease entity include, but are not limited to: half-life, volume of distribution, first-pass hepatic metabolism, and degree of binding to blood serum. . The efficacy of a given pharmacological agent can be influenced by each of the parameters mentioned above. The "half-life" means the time that 50% of an administered drug is eliminated through biological processes, eg, metabolism, excretion, etc. By "first-pass hepatic metabolism" is meant the propensity of a drug to be metabolized on first contact with the liver, that is, during its first pass through the liver. "Volume of distribution" means the degree of retention of a drug in the various compartments of the body, such as, for example, intracellular and extracellular spaces, tissues and organs, and so on. and the distribution of the drug within these compartments. "Degree of binding to blood serum" means the propensity of a drug to interact with and bind to serum proteins in the blood, such as albumin, leading to a reduction or loss of the drug's biological activity.

Pharmacokinetic parameters also include bioavailability, delay time (T ^lag ), T ^max , absorption rates and / or C max for a given amount of drug administered. "Bioavailability" means the amount of a drug in the blood compartment. "Delay time" means the delay time between the administration of the drug and its detection and its ability to measure in blood or plasma. "T ^max " is the time after which the maximum blood concentration of the drug is reached, absorption is defined as the movement of a drug from the site of administration into the systemic circulation, and "C ^max " is the maximum blood concentration obtained with a given drug. The time to reach a blood or tissue concentration of the drug that is required for its biological effect is influenced by all parameters.

The term "toxicity", as used herein, refers to the toxic effects of a drug manifested in adverse events or serious adverse events. These secondary events may refer to a lack of ability to tolerate the drug in general and / or a lack of local tolerance after administration. Toxicity could also include teratogenic or carcinogenic effects caused by the drug.

The terms "safety", "in vivo safety" or "drug tolerance", as used herein, define the administration of a drug without inducing serious adverse events directly after administration (local tolerance) and during a longer period of drug application. The "safety", the "in vivo safety" or the "ability to tolerate the drug" can be evaluated, for example, at regular intervals during the treatment and the follow-up period. Measurements include clinical evaluation, for example organ manifestations, and screening for laboratory abnormalities. Clinical evaluation can be carried out and deviated from the normal findings recorded / coded according to the NCI-CTC and / or MedDRA guidelines. Organ manifestations can include criteria such as allergy / immunology, blood / bone marrow, cardiac arrhythmia, coagulation and the like, as set out, for example, in the Common Terminology Criteria for Adverse Events v3.0 (CTCAE). Laboratory parameters that can be tested include, for example, hematology, clinical chemistry, coagulation profile and urinalysis, and examination of other body fluids such as serum, plasma, lymphoid or cerebrospinal fluid, exudates, etc. Therefore, safety can be assessed, for example, by physical examination, diagnostic imaging techniques (i.e. ultrasound, X-rays, CT scans, magnetic resonance imaging (MRI), other measurements with technical devices (i.e. , electrocardiograms), vital signs, measuring laboratory parameters and recording adverse events The term "effective and non-toxic dose", as used herein, refers to a tolerable dose of the GM-neutralizing compound. CSF, preferably the antibody as defined herein, which is high enough to cure or stabilize the disease of interest without causing significant toxic effects, or essentially without them.Such effective and non-toxic doses can be determined, by For example, by dose escalation studies described in the art and should be below the dose that induces serious adverse side events ( dose-limiting toxicity, DLT).

The formulation of the invention (sometimes also known as "composition of matter";"composition", or "solution") is a liquid formulation.

"Liquid formulation", as used herein, refers to a composition of matter that is found as a liquid, characterized by the free movement of the constituent molecules among themselves, but without a tendency to separate at room temperature. . Liquid formulations of the invention include aqueous formulations. An aqueous formulation is a formulation in which the solvent or the main solvent is water, preferably water for injection (WFI). The dissolution of the GM-CSF neutralizing compound in the formulation can be homogeneous or heterogeneous, it being preferred that it be homogeneous as described above.

Non-aqueous liquids are also disclosed that could be employed as long as they provide stability to the formulation. The non-aqueous liquid could be a hydrophilic liquid. Illustrative examples of suitable nonaqueous liquids include: glycerol; dimethylsulfoxide (DMSO); polydimethylsiloxane (PMS); ethylene glycols, such as ethylene glycol, diethylene glycol, triethylene glycol, polyethylene glycol ("PEG") 200, PEG 300, and PEG 400; and propylene glycols, such as dipropylene glycol, tripropylene glycol, polypropylene glycol ("PPG") 425, and PPG 725.

"Mixed aqueous / nonaqueous liquid formulation" as used herein refers to a liquid formulation containing a mixture of water, preferably WFI, and an additional liquid composition. When used herein, a "formulation" or a "composition" is understood to be a mixture of a GM-CSF neutralizing compound (ie, the drug / active substance) and other necessary chemicals and / or additives. for a drug that is preferably in a liquid state. A formulation of the invention includes a pharmaceutical formulation.

Preparation of the formulation includes the process in which different chemicals, including the active drug, are combined to produce a final drug, such as a pharmaceutical composition. The active drug of the formulation of the invention is a GM-CSF neutralizing compound.

In certain embodiments, the GM-CSF neutralizing compound to be formulated is essentially pure and / or essentially homogeneous (i.e., it is substantially free of contaminating substances, e.g., proteins, etc., impurities that may be related to the product, and / or related to the process). The term "essentially pure" means a composition comprising at least about 80%, preferably about 90% or by weight of the compound, preferably at least about 95% by weight of the compound, more preferably at least about 97% by weight of the compound or more preferably at least about 98% by weight of the compound, preferably the compound in a monomeric state. The term "essentially homogeneous" means a composition comprising at least 99% by weight of the compound, preferably the compound in a monomeric state, excluding the mass of various stabilizers and water in solution.

When used herein, the term "about" is understood to mean that there may be variation in the respective value or range (such as pH, concentration, percentage, molarity, number of amino acids, time, etc.) which can be up to 5 %, up to 10%, up to 15% or up to including 20% of the given value. For example, if a formulation comprises about 5 mg / ml of a compound, this is understood to mean that a formulation can have between 4 and 6 mg / ml, preferably between 4.25 and 5.75 mg / ml, more preferably between 4.5 and 5.5 mg / ml and even more preferably between 4.75 and 5.25 mg / ml, with 5 mg / ml being most preferred. As used herein, an interval that is defined as "(from) X to Y" is equivalent to an interval that is defined as "between X and Y". Both ranges specifically include the upper limit and also the lower limit. This means that, for example, a range of "5 mg / ml to 10 mg / ml" or "between 5 mg / ml and 10 mg / ml" includes a concentration of 5, 6, 7, 8, 9 and 10 mg. / ml, as well as any given intermediate value.

A "stable" formulation is a formulation in which the GM-CSF neutralizing compound essentially retains its physical stability and / or chemical stability and / or biological activity on storage and / or shows no substantial signs of aggregation, precipitation, fragmentation, degradation and / or denaturation compared to a control sample, preferably after visual examination for color and / or clarity, or as measured by UV light scattering or size exclusion chromatography. Several additional analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example.

"During storage", as used herein, means a formulation that, once prepared, is not used immediately; rather, after its preparation, it is packaged for storage, either in liquid form, in a frozen state, or in a dry form for later reconstitution in a liquid or other form.

The GM-CSF neutralizing compound is expected to be preferably stable insofar as it does not form substantially aggregates or fragments / degradation products (for example, due to one or more of the aforementioned causes) during storage and / or during or after freeze / thaw stress and / or during or after shear stress (eg agitation). Accordingly, it is preferably anticipated that no more than 10% of the GM-CSF neutralizing compound, more preferably no more 8%, even more preferably not more than 5%, particularly and preferably not more than 2% in relation to the amount of the GM-CSF neutralizing compound at the beginning of storage or before carrying out one or more freeze / thawing or before conducting stirring studies form aggregates and / or fragments. This stability applies preferably for a time interval of at least 1 month, at least 2 months or at least 3 months; preferably 4 months, at least 5 months or at least 6 months; more preferably at least 9 months or at least 12 months; even more preferably at least 18 months or at least 24 months; and most preferably at least 30 months or at least 36 months or at least 48 months or at least 54 months or at least 60 months. Preferred storage temperature conditions are up to room temperature (from about 20 ° C to about 25 ° C), more preferably about 2-8 ° C, the preferred time interval is at least 3 years or even at least 4 years. The number of freeze / thaw cycles during which the compound is preferably stable according to the above parameters is at least one cycle, preferably at least 2, 3, or 4 cycles, more preferably at least 5, 6 or 7 cycles and, more preferably, at least 8, 9 or 10 cycles. The number of days of stirring during which the compound is preferably stable according to the above parameters (for example, at a temperature of 5 ° C 3 ° C, see also example 12d) is at least 1 day, preferably at least 2, 3, or 4 days, more preferably at least 5, 6 or 7 days, even more preferably at least 8, 9, 10 or 11 days, and more preferably at least 12, 13 or 14 days.

Alternatively, it is envisioned that a GM-CSF neutralizing compound is preferably stable insofar as it does not form dimers, oligomers or fragments thereof. Stated differently, the stability of a GM-CSF neutralizing compound can be determined according to the percentage of monomeric protein in the solution, with a low percentage of protein degraded (eg, fragmented) and / or added. For example, a formulation of the invention comprising a GM-CSF neutralizing compound, such as an antibody, may include at least 90%, more preferably at least 92%, even more preferably at least 95%, particularly preferably at least 98% and more preferably at least 99% of the monomer of the GM-CSF neutralizing compound.

By "aggregate" is meant a physical interaction between protein molecules that results in the formation of covalent or non-covalent dimers or oligomers (ie, high molecular weight entities) that can remain soluble, or form insoluble aggregates that precipitate from of the solution. An "aggregate" also includes a degraded and / or fragmented protein.

As mentioned above, a number of different analytical methods can be used to detect the presence and levels of aggregates in a formulation comprising a GM-CSF neutralizing compound. These include, but are not limited to, for example, natural polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), capillary gel electrophoresis (CGE), size exclusion chromatography (SEC ), analytical ultracentrifugation (AUC), field flow fractionation (FFF), light scattering detection, sedimentation rate, UV spectroscopy, differential scanning calorimetry, turbidimetry, nephelometry, microscopy, high-liquid size exclusion chromatography resolution (SE-HPLC), reverse phase high performance liquid chromatography (RP-HPLC), tandem electroSPRay ionization mass spectroscopy (ESI-MS) and tandem RP-HPLC / ESI-MS, flow fractionation technique of static and / or dynamic light field and scattering. These methods can be used alone or in combination. Preferably, analytical methods for detecting the presence and levels of aggregates and / or fragments in a formulation comprising a GM-CSF neutralizing compound are size exclusion chromatography such as SE-HPLc, analytical ultracentrifugation, and flow fractionation. asymmetric field. A method to determine the biological activity of a GM-CSF neutralizing compound is, for example, the measurement of its degree of binding to GM-CSF (or GM-CSF receptor) (immobilized) is the so-called surface plasmon resonance (SPR ).

A common problem with formulations comprising proteins is irreversible accumulation of aggregates by temporary, thermal or shear stress modes. Normally, when aggregates precipitate, they form large particles that are easy to detect. However, smaller, non-covalent soluble aggregates, which are often precursors to large particle precipitation, are more difficult to detect and quantify. Therefore, the methods for detecting and quantifying protein aggregation in a protein formulation must be based on the type of aggregate being evaluated.

Among the above methods, the suggested methods to determine the presence and / or the amounts of soluble and covalent aggregates in a protein formulation are: SEC / light scattering, SDS-PAGE, CGE, RP-HPLC / ESI-MS, FFF and AUC : Suggested methods to determine the presence and / or amounts of soluble and non-covalent aggregates in a protein formulation are: SEC, PAGE, SDS-PAGE, CGE, FFF, AUC and dynamic light scattering. Suggested methods to determine the presence and / or amounts of insoluble and non-covalent aggregates in a protein formulation are: UV spectroscopy, turbidimetry, nephelometry, microscopy, AUC, asymmetric field flow fractionation, and dynamic light scattering.

Furthermore, the formulation of the invention preferably provides better long-term storage such that the GM-CSF neutralizing compound is stable during storage, either in liquid or frozen, preferably in liquid form. As used herein, the phrase "long-term" storage is understood to mean that the formulation can be stored for at least one month, two or three months or more, for six months or more and preferably for a year and more, or even 2 years, 3 years or 4 years or 5 years and more. Long-term storage is also understood to mean that the pharmaceutical composition is stored at 2-82C or at room temperature, preferably at 2-82C, so that the formulation preferably does not lose its biological activity to the extent described herein and / or it does not form aggregates to the extent described herein and / or comprises monomers to the extent described herein. The tests for these properties are described elsewhere in this document.

In one aspect of the invention, the GM-CSF neutralizing compound in the formulations is stable in liquid form for at least 1 month, 2 months, 3 months; at least 4 months, at least 5 months; at least 6 months; at least 12 months; at least 24 months; at least 36 months; at least 48 months, at least 60 months. Intervals to the time periods cited above are also intended to be part of this invention, eg, 9 months, etc. Also, ranges of values that use a combination of any of the above-cited values as upper and / or lower limits are intended to be included. Preferably, the formulation is stable at room temperature (from about 20 ° C to 25 ° C) for at least 1 month and / or stable at about 2-8 ° C for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or more preferably stable at about 2-8 ° C for at least 2 years, at least 3 years, at least 4 years or even at least 5 years.

Another important aspect of stability is that the GM-CSF neutralizing compound within the composition according to the invention maintains its biological activity or potency during storage time and within storage conditions, especially temperature, and with the possible temperature changes. The activity or potency is, for example, reflected by the compound's own neutralizing ability (which can be measured in cell-based assays, as described herein above) or by the ability of the GM-CSF-neutralizing compound to bind to GM-CSF or to the GM-CSF receptor. Binding affinity can be assessed by SPR or other methods common in the art, eg, a Biacore or Scatchard assay.

The formulation of the invention is prepared by adding to a GM-CSF neutralizing compound as described herein, in an aqueous solution, a buffer and a tonicity modifier. Those of skill in the art will understand that the combination of the various components to be included in the formulation can be done in any appropriate order. For example, the buffer can be added first, in the middle, or last, and the tonicity modifier can also be added first, in the middle, or last. It should also be understood by those skilled in the art that some of these chemicals may be incompatible in certain combinations and that consequently they are easily substituted by different chemicals that have similar properties but are compatible in the relevant mixture.

The formulation of the invention comprises a buffer. The term "buffer" or "buffer agent", as used herein, includes those agents that maintain pH in a desired range. A buffer is an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or a weak base and its conjugated acid. It has the property that the pH of the solution changes very little when a small amount of strong acid or base is added. Buffer solutions are used as a means of maintaining the pH at a near constant value in a wide variety of chemical applications. In general, a buffer, when applied in the formulation of the invention, preferably stabilizes the GM-CSF neutralizing compound.

"Amino acid buffers", when used herein, include, for example, amino acid bases, for example histidine and its conjugated salt. An example of an amino acid buffer is histidine chloride or histidine itself. This example preferably applies to the invention.

The preferred pH of a formulation, as described herein, can be chosen from the following ranges: from about 4 to about 10, preferably from about 4 to about 6, or from about 5 to about 7, more preferably from about 5 .5 to about 6.5. The pH of the formulation of the present invention is approximately, or exactly, 5.8. Accordingly, a buffer that can maintain a solution at pH 5 to 7 is preferably used. The term "about", when used in the context of pH value / range, preferably means a numerical value having a range of / - 20% approximately the quoted value. When the pH of the pharmaceutical composition is set at or near physiological levels, patient comfort after administration is maximized. It should be understood that the pH can be adjusted as necessary to maximize the stability and solubility of the GM-CSF neutralizing compound in a particular formulation and, as such, a pH outside the physiological ranges, although it should preferably be tolerable for the patient, it will be within the scope of the invention.

In the present invention, the buffer comprises histidine. Non-limiting examples of other buffers that can be used in a formulation described herein include succinate, gluconate, citrate, arginine, lysine, aspartic acid, glutamic acid, tris (trometamol), Bis-Tris, MOPS, ACES, TES, HEPES, EPPS, ethylenediamine, phosphoric acid, maleic acid / phosphate, 2-morpholinoethanolamine (MES), phosphate, acetate and diethanolamine.

In the formulation of the invention a histidine buffer is applied in the formulation of the invention, preferably between pH 5 and 7, more preferably between pH 5.5 and 6.5, even more preferably at pH 5.8. The buffers applied in the invention are common in the art and are manufactured by known methods and available from commercial suppliers.

The formulation may further comprise sodium hydroxide (NaOH). In particular embodiments, the formulation comprises 1-200 mM, or less than 50 mM, less than 40 mM, less than 35 mM, less than 30 mM, less than 25 mM, less than 20 mM, or less than 15 mM, for example, 10 mM NaOH or less, such as 5 mM or 1 mM sodium hydroxide.

In addition to the GM-CSF neutralizing compound and the buffer, the formulation according to the invention may also contain other substances including, inter alia, stabilizing agents.

Furthermore, the formulation of the invention comprises a stabilizer that can also act as a tonicity modifier. The term "stabilizing agent" refers to an agent that improves or otherwise enhances the stability of the formulation, in particular of the GM-CSF neutralizing compounds. A stabilizing agent, which is a tonicity modifier, is defined as a non-reducing sugar, a sugar alcohol, or a combination thereof. Tonicity modifiers in liquid compositions ensure that the tonicity, i.e. osmolarity, of the solution is essentially the same as that of normal physiological fluids and thus prevents post-administration swelling or rapid absorption. of the composition due to the differential ion concentrations between the composition and the physiological fluids. Generally, the nature of the formulation, as described herein, is such that the osmolality of the formulation is between about 240 and about 470 mOsmoI / kg, more preferably, between about 300 and about 400 mOsmoI / kg, more preferably about 350 mOsmoI / kg. A stabilizing / modifying agent for tonicity is described that can be one or more of non-reducing sugars, such as sucrose or trehalose or one or more sugar alcohols, such as mannitol or sorbitol, also combinations of non-reducing sugars and alcohols of sugar. In the present invention, the concentration of the stabilizing / tonicity modifying agent in the composition is about 5% (w / v). The stabilizer / tonicity modifier applied in the formulation of the invention is sorbitol, 5% (p / v).

In a preferred embodiment, the formulation described herein does not comprise further excipients, such as amino acids (except when the buffer is chosen from an amino acid buffer, such as a histidine buffer) or surfactants.

In less preferred embodiments, the formulation described herein comprises other excipients. Preferably the excipient is selected from the group consisting of an amino acid, a cryoprotectant, a lyoprotectant, a surfactant, a bulking agent, an antioxidant, and combinations thereof.

Excipients, also known as chemical additives, co-solutes or co-solvents, which preferentially stabilize the GM-CSF neutralizing compounds while in solution (also in dry or frozen forms) can be added to a formulation of the present disclosure. Preferably, the excipients contribute to the stability of the GM-CSF neutralizing compounds, but it should be understood that the excipients may otherwise contribute to the physical, chemical and biological properties of the formulation. Excipients are common in the art and are manufactured by known methods available from commercial suppliers.

Examples of excipients include among other sugars / polyols such as: sucrose, lactose, glycerol, xylitol, sorbitol, mannitol, maltose, inositol, trehalose, glucose; polymers such as: serum albumin (bovine serum albumin (BSA), human SA or recombinant HA), dextran, PVA, hydroxypropyl methylcellulose (HPMC), polyethyleneimine, gelatin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC), hydroxyethyl starch (HEC), hydroxyethyl starch (HEC), hydroxyethyl starch non-aqueous solvents such as: polyhydric alcohols (eg, PEG, ethylene glycol, and glycerol), dimethylsulfoxide (DMSO), and dimethylformamide (DMF); amino acids such as: threonine, serine, proline, alanine, valine, glutamine, methionine, cysteine, isoleucine, aspartic acid, glutamic acid, arginine, glycine, histidine, lysine, phenylalanine, leucine, asparagine, tryptophan and tyrosine; surfactants such as: Tween-80, Tween-20, SDS, polysorbate, polyoxyethylene copolymer; and various excipients such as: potassium phosphate, sodium acetate, ammonium sulfate, magnesium sulfate, sodium sulfate, trimethylamine N-oxide, betaine, metal ions (for example, zinc, copper, calcium, manganese, and magnesium), CHAPS, monolaurate, 2-O-beta-mannogIicerate or any combination of the above.

"Cryoprotectants" include substances that provide stability to frozen protein during production, freezing, storage, handling, distribution, reconstitution, or use. In a particular aspect, "cryoprotectants" include substances that protect protein from stresses. induced by the freezing process. Cryoprotectants may have lyoprotective effects. Non-limiting examples of cryoprotectants include sugars, such as sucrose, glucose, trehalose, mannitol, sorbitol, mannose, and lactose; polymers, such as dextran, hydroxyethyl starch, polyvinylpyrrolidone (PVP) and polyethylene glycol; surfactants, such as polysorbates (for example, PS-20 or PS-80); and amino acids, such as glycine, arginine, leucine, and serine. Generally, a cryoprotectant is used that has low toxicity in systems biological.

A disaccharide, as described herein, can act as a lyoprotectant or cryoprotectant. "Lyoprotectants" include substances that prevent or reduce the chemical or physical instability of a protein against lyophilization and subsequent storage. In one aspect, the lyoprotectant prevents or reduces chemical or physical instabilities in the protein as water is lost in the composition during the drying process. In another aspect, the lyoprotectant stabilizes the protein by helping to maintain the proper conformation of the protein through hydrogen bonding.

Accordingly, in one aspect, a disaccharide, as described herein, can serve to stabilize GM-CSF neutralizing compounds during freezing. Since protection during freezing may depend on the absolute concentration of the disaccharide (Carpenter et al., Pharm. Res. (1997), 14: 969-975), concentrations greater than 5% may be necessary to maximize stability.

A lyoprotectant can be added to the formulation described in this document. The term "lyoprotectant" as used herein includes agents that provide stability to the protein during the lyophilization or dehydration process (primary and secondary lyophilization cycles), providing an amorphous glassy matrix and binding to the protein. through hydrogen bonds, replacing the water molecules that are removed during the drying process. This helps maintain protein conformation, minimize protein degradation during the lyophilization cycle, and improve long-term product stability. The lyoprotectant is added to the previously lyophilized formulation in a "lyoprotective amount" which means that after lyophilization of the GM-CSF neutralizing compounds in the presence of the lyoprotective amount of the lyoprotectant, the compounds essentially retain their physical and chemical stability. and integrity under lyophilization and storage.

Non-limiting examples of lyoprotectants include sugars, such as sucrose or trehalose; an amino acid, such as monosodium glutamate, glycine, or histidine; a methylamine, such as betaine; a lyotropic salt, such as magnesium sulfate; a polyol, such as trihydric or higher sugar alcohols, for example arabitol, xylitol, sorbitol and mannitol; polyethylene glycol; pluronics; and their combinations. Preferably, the lyoprotectants are as described above for stabilizing agents. The amount of lyoprotectant added to the formulation is generally an amount that does not lead to an unacceptable degree of degradation / aggregation of the protein when the protein formulation is lyophilized.

Another excipient is a surfactant. The term "surfactant" generally includes those agents that protect GM-CSF neutralizing compounds from air / solution interface induced stresses and solution / surface induced stresses. For example, surfactants can protect the protein from aggregation.

Examples of surfactants include, but are not limited to, nonionic surfactants, such as polysorbates (eg, polysorbate 80 or polysorbate 20); poloxamers (eg, poloxamer 188); Triton, sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearylsulfobetaine, IauriI-sarcosine, myristyl-sarcosine, linoIeiI-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamidopropyl-beta-propyl IinoIeamidopropyl-betaine, myristamidopropyl betaine, palmidopropyl betaine, isostearamidopropyl betaine (eg, lauroamidopropyl), myristamidopropyl-, palmidopropyl-, or isostearamidopropyl dimethylamine; sodium methyl cocoyl, or disodium methyl phenyl taurate; and the Monaquat series (Mona Industries, Inc., Paterson, NJ.), polyethylglycol, polypropylglycol, and copolymers of ethylene and propyleneglycol (eg, pluronics, PF68). The amount of surfactant added is such that it maintains protein aggregation at an acceptable level as analyzed using, for example, SEC-HPLC, or light obscuration measurements to determine the percentage of high molecular weight (HMW) species. or low molecular weight (LMW) species, and minimizes the formation of particles of the protein formulation described herein.

The bulking agent can also be an excipient. The term "bulking agent", as used herein, includes agents that provide the structure of a lyophilized product without directly interacting with the pharmaceutical product. In addition to providing a pharmaceutically elegant cake, bulking agents can also impart useful qualities with respect to modifying collapse temperature, provide protection against freeze-thaw processes, and improve protein stability on long-term storage. term. Non-limiting examples of bulking agents include mannitol, glycine, lactose, and sucrose. Bulking agents can be crystalline (such as glycine, mannitol, or sodium chloride) or amorphous (such as dextran, hydroxyethyl starch) and are generally used in protein formulations in an amount of 0.5 to 10 %.

Preferably, the bulking agent applied in the formulation can promote the formation of a cake that is aesthetically acceptable, uniform, or mechanically strong. The bulking agents also preferentially promote the formation of an open pore structure and ease and speed in reconstitution. Bulking agents also preferably reduce or prevent cake collapse, eutectic melt or residual moisture retention. In another aspect, the bulking agents preferably help protect the GM-CSF neutralizing compounds against stresses (eg, physical and chemical stresses) and help maintain protein activity.

An antioxidant can also be an excipient. As used herein, an "antioxidant" is a molecule capable of slowing or preventing the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons from a substance to an oxidizing agent. For use in the composition, physiologically acceptable antioxidants are of interest. Such antioxidants include, without limitation, reducing agents, ascorbic acid (vitamin C), lipoic acid, melatonin, uric acid, carotenes, retinols, tocopherols and tocotrienols, for example, a-tocopherol (vitamin E), ubiquinone (coenzyme Q), etc.

The formulation can optionally contain a preservative. A "preservative" is a compound that can be added to formulations in this case to reduce bacterial activity. The addition of a preservative can, for example, facilitate the production of a multipurpose (multiple dose) formulation. Examples of possible preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides, in which the alkyl groups are long chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohols, alkyl parabens such as methyl- or propyl-paraben, catechol, resorcinol, cyclohexanol, 3-pentanoI, and m-cresol. The most preferred preservative herein is benzyl alcohol.

Consequently, the formulation of the present invention is a liquid, specifically aqueous, formulation for long-term storage comprising between approximately 100 mg / ml and 200 mg / ml of a GM-CSF neutralizing compound and a histidine buffer, at a pH of about 5 to about 7, and the formulation further comprises sorbitol as a stabilizer / tonicity modifier.

In particularly preferred embodiments, a formulation of the invention is a liquid, specifically aqueous, formulation for long-term storage comprising between about 100 mg / ml and 200 mg / ml of a GM-CSF neutralizing compound, more preferably about 150 mg / ml, and a histidine buffer at a concentration of about 20mM to about 40mM, specifically about 30mM, at a pH of about 5.5 to about 6.5, specifically 5.8, and the formulation further comprises from about 4% to about 6%, specifically about 5% (w / v) sorbitol and optionally without additional excipients such as surfactants, amino acids and / or NaCl.

It is to be understood that certain components of the composition can be interchanged with alternatives known in the art. However, those skilled in the art will also understand that the inclusion of certain components will preclude the use of other components, concentrations or methods of preparation of the formulation, for reasons including, but not limited to, chemical compatibility, pH, tonicity and stability.

It is beneficial for the liquid formulation of the invention to have a low and feasible viscosity that is suitable for subcutaneous administration, as in a ready-to-use device. The viscosity of the formulation according to the invention is therefore preferably less than 20 mPa * s at a shear rate between about 50 and about 1000 [1 / s] and at a temperature of about 20 ° C. More preferably, the viscosity is less than 15 mPa * s under the above conditions.

As mentioned herein, this application generally relates to the discovery that the addition of various substances to a formulation comprising the neutralizing compounds of GM-CSF can reduce the aggregation and / or degradation / fragmentation of these compounds in a formulation. Regardless of what causes a GM-CSF neutralizing compound in a formulation to degrade or degrade, the addition of the substances as described herein reduces aggregation / fragmentation of the GM-CSF neutralizing compounds in the formulation. . In certain embodiments, the addition of a disclosed substance reduces aggregation / degradation in a formulation caused, for example, by storage, exposure to elevated temperatures, exposure to light, exposure to shear stress, the presence of surfactants, ionic and pH conditions, and any combination thereof. The measures found by the present inventors can be used to decrease the aggregation and / or degradation / fragmentation of the GM-CSF neutralizing compounds formulated, in particular, in liquid and frozen form. Therefore, reduced aggregation / fragmentation is preferably observed in a liquid formulation. It is assumed that reduced aggregation / degradation can also be observed when stored directly in liquid form for later use, stored frozen and thawed prior to use, or prepared in dry form, such as a lyophilized, dried form Air or spray dried, for subsequent reconstitution in a liquid or other form before use.

Therefore, it is envisioned that the formulation described herein may be stored by any method known to those of skill in the art. Non-limiting examples include cooling, freezing, lyophilization and spray drying of the formulation, with storage by cooling being preferred.

In some cases, protein formulations are frozen for storage. Consequently, it is desirable that the formulation be relatively stable under such conditions, including freeze-thaw cycles. One method of determining this suitability of a formulation is to subject a sample of the formulation to at least one, for example, one, three, five, seven, and up to ten freeze cycles and thawing (for example, a freeze at about -802C 10 ° C overnight and rapid thawing at room temperature or slow thawing on ice, for example for 6 hours), determining the amount of low molecular weight (LMW) species and / or high molecular weight (HMW) species that accumulate after the freeze-thaw cycles and comparing it with the amount of LMW species or HMW species present in the sample before the freeze-thaw procedure. An increase in LMW or HMW species indicates a decreased stability of a protein stored as part of the formulation. Size exclusion high performance liquid chromatography (SE-HPLC) can be used to determine the presence of LMW and HMW species.

Preferably, the protein formulations can be stored as a liquid. Consequently, as described herein, it is desirable that the liquid formulation be stable under such conditions, even at various temperatures. For example, one method of determining the suitability of a formulation is to store the sample formulation at various temperatures (such as 2-8 ° C, 15 ° C, 20 ° C, 25 ° C, 30 ° C, 35 ° C , 40 ° C and 50 ° C) and monitoring the amount of HMW and / or LMW species that accumulate over time. The lower the amounts of HMW and / or LMW species that accumulate over time, the better the storage condition of the formulation. Additionally, the protein loading profile can be monitored by cation exchange high performance liquid chromatography (CEX-HPLC). Alternatively, the formulations can be stored after lyophilization. Furthermore, it is desirable that the formulation be stable under the conditions of shear stress. One method of determining the suitability of the formulation is to shake the formulation in a container on a shaker, for example a vertical shaker, at the desired temperature (such as 2-8 ° C, 15 ° C, 20 ° C, 25 ° C, 30 ° C, 35 ° C, 40 ° C and 50 ° C, preferably 5 ° C 3 ° C). The receiver (eg, a vial) can be stored on a shaker for a desired period of time, eg, up to 14 days or more, versus a control that is stored without shaking at the same temperature. Data point collection can be carried out, for example, after 0, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14 and 14 days. The amount of low molecular weight (LMW) species and / or high molecular weight (HMW) species that accumulate after shake storage can be determined and compared to the amount of LMW species or HMW species present in the sample. not agitated. An increase in LMW or HMW species indicates a decreased stability of a protein stored as part of the formulation. Size exclusion high performance liquid chromatography (SE-HPLC) can be used to determine the presence of LMW and HMW species.

In some cases, the formulation is spray dried and then stored. For spray drying, a liquid formulation is sprayed in the presence of a stream of dry gas. Water is removed from the formulation droplets in the gas stream, resulting in dry particles of the pharmaceutical formulation. Excipients can be included in the formulation to (i) protect the protein during spray drying dehydration, (ii) protect the protein during storage after spray drying, and / or iii) give the solution properties suitable for aerosolization. The method is similar to that described above for freezing, except that the sample formulation is spray dried rather than frozen, reconstituted in a diluent, and the reconstituted formulation is tested for the presence of LMW species and / or HMW species. An increase in LMW or HMW species in the spray-dried sample compared to a corresponding sample formulation that was not lyophilized indicates a decrease in stability in the spray-dried sample.

The term "iiofiiized" includes a state of a substance that has been subjected to a drying process such as lyophilization, where at least 90% preferably 95%, more preferably 98% of the moisture has been removed. Consequently, the term "lyophilization", as used herein, refers to a process whereby the material to be dried is first frozen followed by removal of the ice or frozen solvent by sublimation in an environment empty. An excipient (for example, the lyoprotectant) can be included in the formulations that must be lyophilized in order to improve the stability of the lyophilized product at the time of storage. The term "reconstituted formulation", as used herein, refers to a formulation that has been prepared by dissolving a lyophilized protein formulation in a diluent in such a way that the GM-CSF neutralizing compound is dispersed in the diluent.

The term "diluent," as used herein, is a substance that is pharmaceutically acceptable (safe and non-toxic for administration to a human) and that is useful for the preparation of a liquid formulation, such as a reconstituted formulation after lyophilization. Non-limiting examples of diluents include sterile water, bacteriostatic water for injection (BWFI), a buffered pH solution (eg, phosphate saline), sterile saline, Ringer's solution, dextrose solution, or aqueous solutions of salts and / or buffers.

In another aspect of the invention, the formulation is designed for use in therapy. Consequently, the invention provides for a pharmaceutical composition (or medicine) comprising the formulation described herein.

In yet another embodiment, the invention provides a method of treatment to a subject comprising administering a therapeutically effective amount of the formulation described herein, wherein the subject has a disease or disorder that can be beneficially treated with a neutralizing compound. by GM-CSF.

Preferably, the formulation described herein is applied in the prophylaxis and / or treatment of a disease that can be prevented and / or treated and / or ameliorated with GM-CSF neutralizing compounds.

The term "subject" is intended to include living organisms. Examples of subjects include mammals, eg, humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and non-human transgenic animals. In preferred embodiments of the invention, the subject is a human.

The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or, at least partially, achieve the desired effect. The term "therapeutically effective dose" is defined as an amount sufficient to cure or, at least partially, stop the disease and its complications in a patient who has already suffered from the disease. Amounts effective for this use will depend on the severity of the medical condition and the general condition of the subject's own immune system. The term "patient" includes human subjects and other mammals receiving prophylactic or therapeutic treatment.

The appropriate dose, or therapeutically effective amount, of the formulation will depend on the condition to be treated, the severity of the condition prior to treatment, and the patient's medical history and response to the therapeutic agent. The appropriate dose can be adjusted according to the judgment of the attending physician such that it can be administered to the patient once or in a series of administrations. The pharmaceutical composition can be administered as a single therapeutic agent or in combination with additional therapies as necessary. The pharmaceutical compositions of this invention are particularly useful for parenteral administration, that is, subcutaneously, intramuscularly, intravenously, intraarticularly, and / or intrasynovial, where subcutaneous administration is preferred. Parenteral administration can be carried out by bolus injection or continuous infusion.

Injectable pharmaceutical compositions may be presented in unit dose form, eg, in ampoules or in multidose containers, with another preservative. In addition, several recent drug delivery approaches have been developed and the pharmaceutical compositions of the present invention are suitable for administration using these new methods, for example, Inject-ease, Genject, injection pens like Genen, and needle-free devices like MediJector. and BioJector. The present pharmaceutical composition can also be adapted for administration methods yet to be discovered. See also Langer, 1990, Science, 249: 1527-1533.

The lyophilized pharmaceutical compositions can preferably be presented in a vial containing the active ingredient. In one embodiment, the pharmaceutical compositions further comprise a solution for rebuilding.

The pharmaceutical composition may further comprise other pharmaceutically acceptable components. Other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in the 16th edition of the Remington Pharmaceutical Sciences manual, Osol, A. Ed. (1980), may also be included in a protein formulation such as that described herein, provided that they do not adversely affect the desired characteristics of the formulation. As used herein, "pharmaceutically acceptable carrier" means any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is common in the art. Acceptable carriers, excipients, or stabilizers are not toxic to receptors at the doses and concentrations employed and include: tonicity modifiers; additional buffering agents; preservatives; co-solvents; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (eg, Zn-protein complexes); biodegradable polymers, such as polyesters; salt-forming counter ions, such as sodium, polyhydric sugar alcohols; amino acids, such as alanine, glycine, asparagine, 2-phenylalanine, and threonine; sugars or sugar alcohols, such as lactitol, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoinysitose, myoinisitol, galactose, galactitol, glycerol, cyclitols (eg, inositol), polyethylglycol; sulfur-containing reducing agents, such as glutathione, thioctic acid, sodium thioglycolate, thioglycerol, [alpha] -monothioglycerol, and sodium thio-sulfate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; and hydrophilic polymers, such as polyvinylpyrrolidone.

The formulations described in this document are useful as pharmaceutical compositions in the treatment and / or prevention and / or amelioration of a disease, or disorder in a patient in need thereof. The term "treatment" refers to both therapeutic treatment and prophylactic or preventive measures. Treatment includes applying or administering the formulation to the body, an isolated tissue, or a cell of a patient having a disease / disorder, a symptom of a disease / disorder, or a predisposition toward a disease / disorder, for the purpose of cure, heal, alleviate, alter, remedy, improve, enhance or affect the disease, the symptom of the disease or the predisposition towards the disease.

Those who "need treatment" include those already with the disorder, as well as those in whom the disorder must be prevented. The term "disorder" is any condition that would benefit from treatment with the protein formulation described in this document. This includes chronic and acute disorders or diseases, including those pathological conditions that predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include inflammatory and autoimmune disorders, preferably including allergic and psoriatic disorders, as well as arthritic and asthmatic disorders, e.g. eg, arthritis, rheumatoid arthritis (RA), autoimmune encephalitis, psoriasis, multiple sclerosis, lung disease such as asthma, chronic obstructive pulmonary disease (COPD), and acute respiratory distress syndrome (ARDS); Crohn's disease, idiopathic pulmonary fibrosis (IPF), inflammatory bowel disease (IBD), uveitis, macular degeneration, colitis, Wallerian degeneration, antiphospholipid syndrome (APS), acute coronary syndrome, restinosis, atherosclerosis, relapsing polychondritis (RP), acute hepatitis or chronic, failed orthopedic implants, glomerulonephritis, lupus, atopic dermatitis, and inflammatory, arthritic and osteoarthritic pain.

An allergic disorder is any disorder caused by an allergy or allergic reaction. An allergy is a hypersensitivity disorder of the immune system. It occurs when a person's immune system reacts, or overreacts, to normally harmless foreign substances (allergens), such as food, pollen, mold, house dust, animal hair, dust mites.

Psoriasis is an autoimmune disease that mainly affects the skin. The growth cycle of skin cells is accelerated due to erroneous signals sent by the immune system. There are five types of psoriasis: plaque, guttate, inverse, pustular, and erythrodermic. The most common form, plaque psoriasis, commonly appears as reddish and whitish tones of scaly patches that appear on the first upper layer of the epidermis (skin). Some patients, however, do not have dermatological signs or symptoms. The disorder is a chronic recurring condition that ranges in severity from minor localized spots to full body coverage. Fingernails and toenails are frequently affected (psoriatic nail dystrophy) and can be seen as an isolated sign. Psoriasis can also cause inflammation of the joints; what is known as psoriatic arthritis.

Arthritis is a form of joint disorder that involves inflammation of one or more joints. There are more than 100 forms of arthritis. The most common form, osteoarthritis (degenerative joint disease), is the result of trauma to the joints, infection of the joints, or age. Other forms of arthritis include rheumatoid arthritis, psoriatic arthritis, and other related autoimmune diseases. Septic arthritis is caused by an infection of the joints.

Asthma is a common chronic inflammatory disorder of the airways in which many cells and cellular elements play a role. Asthma is associated with hypersensitivity in the airways leading to recurrent episodes of wheezing, coughing, chest tightness, and shortness of breath. These episodes are generally associated with a generalized, but variable obstruction of airflow within the lung that is often reversible, either spontaneously or with treatment. Asthma can be classified according to the frequency of symptoms, the forced expiratory volume in one second, and the maximum expiratory flow rate. Asthma can also be classified as atopic (extrinsic) or non-atopic (intrinsic).

In addition to the neutralizing compounds for GM-CSF, the pharmaceutical composition of the invention may comprise additional therapeutic or biologically active agents. For example, therapeutic factors useful in the treatment of a particular indication such as osteoarthritis may be present (e.g., one or more inhibitors that are involved in the destruction of articular cartilage or selected synovial components, but are not limited to antimethaloproteinases, compounds of cyclin, cytokine antagonists, corticosteroids, TNF inhibitors, IL inhibitors, anti-angiogenic substances, aggrecanase inhibitors, p38 kinase inhibitors, apoptosis inhibitors, hyaluronidase inhibitors, and proteolytic enzyme inhibitors). Factors that control inflammation, including infliximab, etanercerpt, adalimulab, nerelimonmab, lenercerpt, etc., or combinations thereof, can be part of the composition. It is also envisioned that the pharmaceutical composition may include components of the extracellular matrix such as hyaluronic acid or a derivative thereof, including salts, ester derivatives, inner ester and sulphates, preferably a partial ester of hyaluronic acid.

In another embodiment, the present invention relates to a kit (or article of manufacture) or container, containing a formulation of the invention. The formulation may preferably already be in a liquid state. However, alternatively, it may preferably be in a lyophilized state. It can also be in a frozen, lyophilized, or spray-dried state. Consequently, if the formulation is in a state other than liquid, it can be prepared by the practitioner as an aqueous (liquid) pharmaceutical composition. For example, the formulation can be lyophilized and then have to be reconstituted. Accordingly, the kit may further comprise means for reconstitution of a frozen, lyophilized, or spray-dried formulation and / or means for diluting the formulation and / or means for administering the formulation or pharmaceutical composition, respectively, such as a syringe, a pump, an infuser, a needle or the like. The kit may comprise one or more vials containing the formulation of the invention. The kit can also be accompanied by instructions for use.

Therefore, an article of manufacture is provided that contains a formulation described herein and preferably provides instructions for its use. The article of manufacture comprises a suitable container to contain the formulation. Suitable containers include, but are not limited to, vials, vials (eg, dual chamber vials), syringes (eg, single or double chamber syringes), test tubes, nebulizers, inhalers (eg, dose inhalers). measures or dry powder inhalers) or reservoirs. The container can be formed from a variety of materials, such as glass, metal, or plastic (eg, polycarbonate, polystyrene, polypropylene, polyolefin). The container contains the formulation, and the label on or attached to the container may indicate instructions for its reconstitution and / or use. The label may further indicate that the formulation is useful or that it is intended for subcutaneous administration. The container containing the formulation can be a multipurpose vial, allowing for repeated administrations (eg, 2-6 administrations) of the formulation. The article of manufacture may further comprise a second container comprising a suitable diluent (eg, WFI, 0.9% NaCl, BWFI, phosphate buffered saline). When the article of manufacture comprises a lyophilized version of a GM-CSF neutralizing compound formulation, mixing a diluent with the lyophilized formulation will provide a desired final protein concentration in the reconstituted formulation. The article of manufacture may further include other commercially and user-desirable materials, including other buffers, diluents, filters, needles, syringes, and package inserts with corresponding instructions for use.

Also included in the present description are devices that can be used to deliver the formulation of the present disclosure. Examples of these devices include, but are not limited to, a syringe, a pen, an implant, a needleless injection device, an inhalation device, and a patch.

The invention is further illustrated with figures and examples which are merely illustrative and are not construed as limiting the scope of the present invention. Figures show:

Figure 1: Effect of storage time, storage temperature and protein concentration on the monomer level of the anti-GM-CSF antibody.

Figure 2: Pareto diagram of the standardized effect with osmolality [mOsmol / kg] as variable.

The following Examples illustrate the present invention.

Example 1: Materials

The following examples were carried out with a human IgG1 monoclonal antibody (hereinafter referred to as "the antibody") that binds and neutralizes human GM-CSF with high affinity and specificity and is described in document ^w O 2006/111353 . Their generation is described in Example 2 of WO 2006/111353. More specifically, the antibody comprises the CDR light chain and heavy chain sequences, as shown in SEQ ID NO: 16, 17, 18, 14, 15 and 2. These CDR sequences are composed of the variable domain of the heavy and light chain, respectively, shown in SEQ ID NO: 34 and 35, respectively. GM-CSF is aberrantly and overproduced in a multitude of pro-inflammatory and autoimmune human diseases, and the addition of recombinant GM-CSF has been found to aggravate these diseases. Possible pathological indications for treatment with a neutralizing antibody to GM-CSF include rheumatoid arthritis (RA), asthma and other forms of lung inflammation, multiple sclerosis (MS), and psoriasis.

The antibody was produced in a bioreactor using a medium without serum and proteins. Inoculum for fermented production was prepared from a single vial of the antibody producing clone. After the fermentation processes are complete, the recovery containing the secreted antibody is processed by filtration to separate the cells and the debris from the supernatant. Recovery purification relies on common chromatographic approaches to reduce HCPs, DNA, and potential viruses. A comprehensive virus inactivation step was an additional part of the subsequent process. To produce the formulation, a buffer concentration and exchange step was performed.

Example 2: Test methods

Size Exclusion High Performance Liquid Chromatography (SE-HPLC) was established to determine the degree of aggregation of the antibody (HPLC: Agilent 1100 Chemstation; column: Tosoh Biosep TSKgeI G4000SWXL). The SE-HPLC method was scored by performing a nine-point interval test, from which precision (six injections per replicate) and linearity (triplicate standard curves) were determined and tested. All assays were performed using 100 mM KH2PO4, 200 mM Na2SO4, pH 6.6 as a running buffer.

The method of surface plasmon resonance (SPR) was established to determine the degree of binding activity of the antibodies against immobilized GM-CSF (Biacore 3000 / CHIP sensor CM5 / HBS-EP operating buffer). The SPR method was scored by performing a nine-point interval test, from which precision (six injections per replicate) and linearity (triplicate standard curves) were determined and tested.

Reduction and non-reduction SDS-PAGE was performed for the detection of degradation products (fragments) and antibody aggregates.

Example 3: Effect of stress by pH and temperature

A study was carried out to evaluate the stability of the antibody in a low ionic strength selection buffer (LISSB: 2 mM glycine, 2 mM citric acid, 2 mM HEPES, 2 mM MES, and 2 mM Tris) at values pH ranging from 3 to 10. The antibody samples were stored in the respective solutions at 55 ° C for a period of 14 days. At T0, T7 and T14 (days) the samples were analyzed. SPR analysis indicated that the antibody was more stable at pH 4-7. When analyzed by SE-HPLC the monomer of the antibody was found to be more stable at pH 4-6 (T14). Both the non-reducing and reducing SDS-PAGE of the T14 samples show only minimal formation of degradation products and aggregates at pH 4-6.

Example 4: Effect of stress by ionic strength and temperature

A study was conducted to evaluate the stability of the antibody in low ionic strength screening buffer (LISSB) in the presence of 0, 10, 100 and 500 mM NaCl. Antibody samples were dialyzed in LISSB solutions (pH 4.5 and pH 7.5) with more NaCl at a concentration of approximately 1 mg / ml and stored at 55 ° C for a period of 14 days. At T0, T7 and T14 (days) the samples were analyzed by SE-HPLC and SPR.

HPLC and SPR studies indicate that the addition of salts impairs the stability of the antibody and that the effect is more pronounced at pH 4.5 than at pH 7.5. Furthermore, the HPLC data shows that destabilization of the antibody with high salt concentrations (> 100 mM) at pH 4.5 mainly results in the accumulation of antibody aggregates. At pH 7.5, destabilization of the antibody at high salt concentrations (500 mM) primarily results in the accumulation of antibody degradation products. In LISSB at pH 4.5; 500 mM NaCl, the precipitation level (T7 and T14) of the antibody was very high.

Example 5: Effect of tampons

A study was conducted to evaluate the stability of the antibody in various buffers at pH 5-7. Antibody samples were dialyzed in 20 mM citrate buffer solutions at pH 5, 6 and 7; 20 mM phosphate buffer, pH 6 and pH 7; 20 mM succinate buffer, pH 6 and pH 7, 20 mM histidine buffer, pH 6 and pH 7; and 20 mM acetate buffer, pH 5 and pH 6 at a concentration of approximately 1 mg / ml and stored at 55 ° C for a period of 14 days. At T0, T7 and T14 (days) the samples were analyzed by SE-HPLC, SPR, and reducing and non-reducing SDS-PAGE.

The SPR study, HPLC and aggregate monomer data, and reducing and non-reducing SDS-PAGE indicate that the antibody is more stable in acetate buffer pH 5, histidine buffer pH 6 and citrate buffer pH 5, 6 and 7 (data T14). . Example 6: Effect of amino acids

A study was carried out to evaluate the stability of the antibody in low ionic strength selection buffer (LISSB) with the addition of different amino acids. Antibody samples were stored in the solution at pH 6 with 250 mM of the respective amino acids at 55 ° C for a period of 14 days. At T0, T7 and T14 (days) the samples were analyzed. The SPR study indicates a slightly stabilizing effect especially of glutamic acid, trionine, lysine and valine (T14). HPLC data indicate a significant stabilizing effect of glutamic acid, threonine and alanine resulting in approximately 2-3% more intact monomer and approximately 30%, 31% and 20% less aggregates, respectively, compared to the reference to when no amino acid is added. Example 7: Effect of sugars and surfactants

A study was conducted to evaluate the stability of the antibody in the low ionic strength selection buffer (LISSB) with the addition of various sugars or surfactants. The antibody samples were dialyzed in LISSB solutions at pH 6.0 at a concentration of approximately 1 mg / ml with an additional 6% (w / v) of sugars (D-mannitol, D-sorbitol, D-mannose sucrose, D-maltose, D-trehalose, D-glucose), 0.05% (v / v) of Tween 20 or 0.02% (v / v) of Tween 80, and stored at 55 ° C for a period of 14 days. At T0, T7 and T14 (days) the samples were analyzed by SE-HPLC, SPR and reducing and non-reducing SDS-PAGE.

SPR and HPLC studies indicate that D-sorbitol and D-mannitol improve antibody stability by approximately 20% / 3% (SPR / HPLC data, T14) and 14% / 4% (SPR / HPLC data, T14), respectively. . Trehalose and sucrose have worse effects at 7% / 0.7% (SPR / HPLC, T14) and 6% / 2% (SPR / HPLC, T14), respectively. Furthermore the HPLC data indicate that D-sorbitol and D-mannitol reduce the formation of antibody aggregates by 43% and 50%, respectively, which is also supported by the data obtained by SDS-PAGE. Neither of the 0.05% (v / v) Tween 20 or 0.02% (v / v) Tween 80 appeared to improve the stability of the antibody.

Example 8: Effect of combinations of buffers, amino acids and sugars

Based on previous studies, a study was conducted to evaluate the stability of 1 mg / ml of the antibody formulated in combinations of buffers, amino acids and sugars. Previous studies indicated stabilizing effects of: acetate 20 mm pH 5; histidine 20 mm pH 6; 250 mM glutamic acid; threonine 250 mm, 6% (w / v) sorbitol and 6% (w / v) mannitol. The antibody was formulated at a concentration of 5.4 mg / ml in solutions of combinations of 20 mM buffer, 250 mm amino acid and 6% (v / v) sugar as indicated in Table 1, and stored at 55 ° C for a period of 14 days. The antibody at 1xbp was included as a control. At T0, T7 and T14 (days), the samples were analyzed by SE-HPLC and SPR.

In addition, samples at T0 comprising the antibody (5.4 mg / ml) at 1xPBS were analyzed for freezing and thawing ability. The storage vials were slowly frozen at -20 ° C in the freezer. After completion of freezing, the samples were thawed at room temperature. This procedure was repeated until five freeze-thaw cycles had been completed. Each sample was examined for SPR and SE-HPLC recovery after two and five cycles.

Table 1: Combination of buffers, sugars and amino acids (Ac = acetate buffer; His = histidine buffer; M = mannitol; S = sorbitol)

The SPR data shows that the binding activities in samples 5-16 at T14 / 55 ° C are> 93% relative to T0. For the antibody in 1xPBS (sample 17) and samples 1-4 (all without mannitol or sorbitol), the numbers are 62%, 19%, 26%, 91%, and 85%, respectively. The SPR data, therefore, indicate that the addition of mannitol or sorbitol improves the stability of binding activity. Furthermore, samples 1-4 appeared yellowish at T7 and T14 indicating oxidation. SE-HPLC data shows that the lowest levels of aggregates (3.5% - 5.6%) and degradation products (4.2% - 5.4%) are found in samples 5-8, 11 -12 and 15-16 (data T14 / 55 ° C), supporting the stabilizing effect of mannitol and sorbitol on the antibody monomer and indicating a possible minor effect when adding 250 mM threonine. The effect of threonine is, however, at least minimally judged by the levels of monomers found in samples 5-8, 11-12 and 15-16 (91.2%, 89.8%, 91.5%, 90.8%, 89.8%, 89.0%, 91.4% and 90.9%, respectively) -for sample 17 (PBS) the number is 80.3% (data T14 / 55 ° C) . 250 mM glutamic acid together with sorbitol or mannitol had no negative effect on the stability of the antibody monomer, especially in the acetate buffer. In conclusion, the monomer of the antibody seems to be the most stable in combination of the 20 mM acetate buffer, at pH 5 or 20 mM histidine buffer at pH 6 and 6% (w / v) of sorbitol or 6% mannitol (w / v ).

SE-HPLC data from freeze / thaw experiments indicate a minor superior effect of sorbitol over mannitol in stabilizing the antibody monomer. In samples 6 and 8, the monomer content is 98.6% and 98.4%, respectively, after 5 rounds of freeze / thaw compared to the monomer content in samples 5 and 7 of 96, 9% and 96.0%.

Example 9: Effect of combinations of histidine or acetate, sorbitol or mannitol, and Tween 20 or Tween 80 Based on stability data from previous studies (showing a particular stabilizing effect of 20 mM acetate pH 5, 20 mM histidine pH 6, 6% (w / v) sorbitol and 6% (w / v) mannitol), a new study was conducted to evaluate the stability of 10 mg / ml of the antibody formulated in combinations of histidine buffers or acetate, sorbitol or mannitol, and Tween 20 or Tween 80. Due to the antibody concentrations at 10 mg / ml, the addition of 0.02% (w / v) of Tween 20 and 0.02% ( w / v) of Tween 80 to determine its effect on aggregation.

The antibody was formulated at a concentration of 10 mg / ml in solutions of 20 mM acetate buffer pH 5.0 or 20 mM histidine buffer pH 6.0 and with the additives indicated in Table 2, and stored at 55 ° C for a period of 14 days. At T0, T7 and T14 (days), the samples were analyzed by SE-HPLC and SPR.

In addition, antibody samples were tested for freezing and thawing ability. The antibody was slowly frozen to a concentration of 10 mg / ml at 20 ° C in the freezer. After the freezing was complete, the samples were thawed at room temperature. This procedure was repeated until three and / or five freeze-thaw cycles had been completed. Each sample was examined for SPR and SE-HPLC recovery after three and / or five freeze-thaw cycles.

Table 2: Combination of buffers, sugars and detergents (Ac = acetate buffer; His = histidine buffer; M = mannitol; S = sorbitol; T20 = Tween 20; T80 = Tween 80)

The HPLC data clearly showed that 0.02% (w / v) of Tween 20 and 0.02% (w / v) of Tween 80 had a negative effect on the stability of the antibody monomer. After 14 days of storage at 55 ° C, approximately 89-90% of the formulated antibody monomer remained without detergents and approximately 83-88% of the formulated antibody monomer with added detergents remained. The loss of monomer from the antibody is mainly causing higher levels of antibody aggregates (approximately 7.6% -10.3%) when detergents are added. Without Tween 20 and Tween 80, the antibody aggregates make up approximately 5.2% -6.4%. Although the difference between acetate and histidine as a buffer system is minor, histidine appears at least as good as acetate.

Freeze-thaw experiments indicate that there is at least no positive effect of detergents on the antibody monomer level after five rounds of freeze-thaw. However, the monomer of the antibody is more stable when 6% (w / v) sorbitol is added to the formulation compared to 6% (w / v) mannitol. With the mannitol added, the antibody monomer is added to a higher degree after five rounds of freezing and thawing. HPLC data from the freeze-thaw experiment does not show any difference between the use of acetate and histidine.

In conclusion, a preformulation containing 20 mM histidine, pH 6.0 and 6% (w / v) sorbitol appears optimal for the stability of the antibody monomer.

Example 10: Evaluation of the short-term stability of a pre-formulation of 20 mM histidine, 6% (w / v) D-sorbitol

Based on the identification of 20 mM histidine at pH 6.0 and sorbitol 6% (w / v) as optimal preformulation, a short-term stability test was performed to evaluate this preformulation at higher concentrations of the antibody.

The antibody was formulated in the above solution at concentrations of approximately 22, 36, 42, 83 and 118 mg / ml. The formulated antibody was stored at 5 ° C and 25 ° C for a period of up to 4 weeks. At T0, T14 and T28 (days), the samples were analyzed by SE-HPLC.

SE-HPLC data clearly show that the antibody is stable at 20 mM histidine pH 6.0, 6% (w / v) sorbitol. The antibody monomer concentration detected after 28 days of storage at 5 ° C or 25 ° C was always above 97%, with the exception of the highest antibody concentration tested (118 mg / ml) at 25 ° C, where the antibody monomer concentration was approximately 96.5%. The results are shown in FIG. 1.

Example 11: Fine tuning of final excipients

Based on the data generated in the previous experiments, sorbitol and histidine have been chosen to stabilize the antibody. In the next step, the amount of excipients, as well as the pH value were adjusted for antibody concentrations between 10 and 100 mg / ml using the "Design of the Experiment" (DOE), including 3 central points and resulting in 30 reviews. individual. The randomized experimental plan was carried out with the following parameters:

Antibody: 10-55-100 mg / ml

pH: 5-6-7

Histidine: 10-30-50 mM

Sorbitol: 2-6-10% (p / v)

To allow short-term evaluation, the samples were stressed under accelerated conditions of 50 ° C for 14 days. In addition, three thaw cycles (-20 ° C) were performed to reproduce the storage conditions of the antibody.

Results:

Osmolality was determined to ensure the physiological conditions for formulation. For an i.v. or s.c. For the antibody, an osmolality between 250 and 450 mOsmol / kg is an acceptable range. As shown in FIG. 2, osmolality is mainly controlled by the amount of sorbitol in the formulation. Both histidine at low concentrations (10-50 mM) and the antibody itself have only minimal effects on osmolality. The dependence of osmolality on the concentration of sorbitol and histidine can be represented in a contour blot. This transfer shows that to maintain osmolality in the physiological range, a concentration between 3% and 7% (w / v) of sorbitol is necessary. The data generated allude to an optimal sorbitol concentration of approximately 6%, which results in a fairly high osmolality value (> 400 mOsmol / kg). Since a certain reduction in sorbitol does not affect the stability of the antibody, the sorbitol concentration can be lowered to 5% to achieve an osmolality of about 350 mOsmol / kg and thus to increase convenience for the patient.

The optimal histidine concentration was established at 30 mM based on the data generated. In a concentration range between 10 mM and 50 mM histidine, no impact on aggregation or fragmentation was detected, as measured by HP-SEC.

The aggregation of the antibody is mainly concentration dependent. Increasing antibody concentrations results in higher aggregation level and increased clarity values during freeze / thaw and under accelerated temperature stress conditions. Furthermore, the pH value affects the monomer content of the antibody. While accelerated stress at pH <6 leads to increased antibody fragmentation, freeze / thaw stability is not affected, but slightly better stability is observed. At pH values of pH> 6, both for accelerated temperature and for freeze / thaw treatment, the reduced monomer content could be measured by HP-SEC and clarity analysis. In order to balance the contrary effects, a pH of 5.8 seems to be more suitable for the formulation of antibodies.

This resulted in a formulation with the following parameters:

Antibody 10mg / ml-55mg / ml-100mg / ml

D-sorbitol 5% (w / v)

30 mM L-Histidine Buffer (Hydrochloride Monohydrate)

pH 5.8 (adjusted with 2M sodium hydroxide)

The conditions or parameters of this composition can also be conveyed to a composition with higher concentrations of antibodies, as will be shown in the following examples.

Example 12: Stability studies

a) Long-term studies

The study was designed for a trial period of up to 60 months. During this period, antibody samples were stored at 5 ° C + 3 ° C in DIN R2 glass vials. In addition, an accelerated study at 25 ° C 2 ° C and a stress study at 40 ° C + 2 ° C were performed in DIN R2 glass vials for a maximum of 12 and 6 months, respectively. The tests were carried out using the antibody at a concentration of 106 mg / ml and 145 mg / ml, in a formulation with 30 mM histidine monohydrochloride and 5% (w / v) sorbitol at pH 5.8.

The following parameters were measured: pH, osmolality, concentration (OD280), percentage of monomers, aggregates and fragments by SE-HPLC, potency (cell-based assay). The results are shown in the following Tables 3-8.

Table 3: Stability test at 5 ° C ± 3 ° C 106 m / ml anti-leather

Table 4: Stability test at 5 ° C ± 3 ° C 145 m / ml antibody

T l Pr ili 2 ° 2 ° 1 m ml ni r

Table 6: Stability tests at 25 ° C 2 ° C (145 mg / ml antibody)

Table 7: Stability tests at 40 ° C 2 ° C (106 mg / ml antibody)

Table 8: Stability tests at 402C 2 ° C (145 mg / ml antibody)

b) Accelerated / stress studies

To demonstrate the comparability of the drug substance after extension, a stability study was performed under accelerated (25 ° C) and stressed (40 ° C) conditions using two batches of drug substances with antibody concentrations of 165 mg / ml and 171 mg / ml formulated in a solution of 30 mM histidine, 5% sorbitol, pH 5.8.

The following parameters were measured: pH, osmolality, concentration (OD280), percentage of monomers, aggregates and fragments by SE-HPLC, potency (cell-based assay). Results of the 171 mg / ml antibody concentration are shown in Tables 9 and 10 below.

Table 9: Stability tests at 25 ° C 2 ° C (171 mg / ml antibody)

Table 10: Stability tests at 40 ° C ± 2 ° C (171 mg / ml antibody)

c) Freeze / thaw studies

Freeze / thaw stability was tested for antibody concentrations of 106 mg / ml and 145 mg / ml formulated in a 30 mM solution of histidine pH 5.8 and 5% sorbitol. The antibody was frozen at -80 ° C ± 10 ° C for at least overnight. Thawing was carried out for> 6 h at room temperature. Several freeze / thaw cycles 0, 1,3, 5, 7 and 10 were carried out.

The following parameters were measured: pH, osmolality, concentration (OD280), percentage of monomers, aggregates and fragments by SE-HPLC, potency (cell-based assay). The results are shown in tables 11 and 12.

Table 11: Freeze / thaw stability tests at -80 ° C ± 10 ° C (106 mg / ml antibody)

Table 12: Freeze / thaw stability tests at -80 ° C ± 10 ° C (145 mg / ml antibody)

d) Stirring stability

This study was initiated to obtain information on the impact of shear stress on the antibody during the procedure, from filling to administration to the patient (eg, refilling, packaging, shipping). Therefore, the antibody at a concentration of 106 mg / ml and 145 mg / ml and formulated in a 30 mM solution of histidine at pH 5.8 and 5% sorbitol was passed on a vertical shaker in primary packaging material. (DIN R2 glass vials). The vials were stored at 5 ° C 3 ° C for up to 14 days on a shaker compared to a control (stored without shaking at 5 ° C 3 ° C). Data point collection was carried out after 0, 1, 2, 3, 7 and 14 days.

The following parameters were measured: pH, osmolality, concentration (OD280), percentage of monomers, aggregates and fragments by SE-HPLC and potency (cell-based assay). The results are shown in the following Tables 13-16.

Table 13. Shaking stability tests at 5 ° C 3 ° C (106 mg / ml antibody) with shaking

Table 14: Shaking stability tests at 5 ° C 3 ° C (106 mg / ml antibody) without shaking

Table 15. Shaking stability tests at 5 ° C 3 ° C (145 mg / ml antibody) with shaking

Table 16: Stirring Stability Tests at 52C 32C (145mg / ml Antibody) with Shaking

e) Results and conclusions

A long-term stability study including accelerated and stressed conditions was conducted for up to 60 months for antibody concentrations of approximately 106 mg / ml and approximately 145 mg / ml. Additional stability studies were also performed under accelerated and stressed conditions for antibody concentrations up to about 171 mg / ml.

During the 60-month period, samples stored at 5 ° C ± 3 ° C for both concentrations showed less than 2% aggregation and had the same potency compared to the reference. After 12 months of storage under accelerated conditions (+ 25 ° C ± 2 ° C), both concentrations (106 mg / ml and 145 mg / ml) showed less than 2% aggregation and had the same potency as compared to the reference. After 6 months of storage under stress conditions (+ 40 ° C ± 2 ° C), both concentrations showed an aggregation of less than 5% and had approximately the same potency compared to the reference.

Data from the other accelerated / stress studies showed stability for 3 months and 1 month at 25 ° C and 40 ° C, respectively. This indicates that stability at 2-8 ° C can be expected to be comparable to that obtained for antibody concentrations of 106 mg / ml and 145 mg / ml, as explained above.

Furthermore, the antibody is stable for at least 10 freeze / thaw cycles at -80 ° C ± 10 ° C at both tested antibody concentrations of approximately 106 mg / ml and approximately 145 mg / ml. Finally, the shaking stability study was set up for a period of 14 days on a vertical shaker at 5 ° C ± 3 ° C versus a control shaker. During this period, no trends could be detected on parameters to evaluate pH, osmolality, degradation, aggregation, fragmentation, concentration or potency. Agitation appears to have no impact on the stability of the antibodies.

Based on the above data, a shelf life of at least 60 months can be proposed for concentrations up to approximately 171 mg / ml at 5 ° C ± 3 ° C.

Example 13: Viscosity evaluation

Viscosity was determined with a rheometer. A rheometer was used for fluids that could not be defined by a single viscosity value and therefore required more parameters to be set and measured than a viscometer.

For some fluids, the viscosity is a constant over a wide range of shear rates (Newtonian fluids). Fluids without a constant viscosity (non-Newtonian fluids) cannot be described by a single number. Non-Newtonian fluids exhibit a variety of different correlations between shear stress and shear rate. Therefore, the viscosity depends on the temperature, as well as the shear rate of non-Newtonian fluids. Viscosity is indicated in milli Pascal * seconds (mPas * s) at a given temperature and at a given shear rate ^y [1 / s].

The viscosity of the formulation with about 150 mg / ml of antibody is less than 12 mPas * s at a shear rate between 50 and about 1000 [1 / s] at a temperature of 20 ° C.

The viscosity of the formulation with about 150 mg / ml of antibody is less than 20 mPas * s at a shear rate between 50 and about 1000 [1 / s] at a temperature of 5 ° C.

Claims (8)

1. An aqueous composition comprising: i) a GM-CSF neutralizing compound which is a human monoclonal antibody that binds to GM-CSF comprising a light chain amino acid sequence as set forth in SEQ ID NO. 34 and a heavy chain amino acid sequence as set forth in SEQ ID NO: 35, at a concentration of 100 mg / ml to 180 mg / ml, ii) 5% (w / v) sorbitol, iii) 30 mM L-histidine and iv) has a pH of 5.8. 2. The composition according to claim 1, which is free of surfactants or other amino acids. The composition according to claim 1 or 2, comprising 150 mg / ml of the antibody that binds to GM-CSF. The composition according to any one of the preceding claims, which is stable for at least 24 months at about 2-8 ° C or at least 28 days at room temperature. The composition according to any of the preceding claims for use in therapy. 6. The composition for use according to claim 5, wherein the composition is administered intravenously and / or subcutaneously. The composition for use according to claim 5 or 6 in the treatment of inflammatory and autoimmune disorders. The composition for use according to claim 7, wherein the disorders are selected from allergic, psoriatic, arthritic and asthmatic disorders.