Stable aqueous compositions of ENGRAILED proteins

Abstract

The present invention relates to a formulation of an Engrail (EN) protein, and more particularly to an aqueous formulation of an Engrail (EN) protein capable of inhibiting or minimizing aggregation of the EN protein. In some embodiments, the formulations of the present invention are capable of inhibiting or minimizing dimerization and / or oligomerization of an EN protein, which is a monomer.

Description

Technical Field

[0001] The present invention relates to specific formulations for obtaining stable aqueous protein compositions. In particular, the present invention relates to stable aqueous compositions of Engrailed (EN) proteins comprising a specific combination of stabilizing compounds. Technical Background

[0002] Neurodegenerative diseases are a group of irreversible neurological disorders caused by the loss of neurons in the brain and spinal cord, and primarily include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, and others. Most neurodegenerative diseases still lack effective treatments, so finding effective methods to prevent, delay, and treat these diseases is an urgent issue.

[0003] Amyotrophic lateral sclerosis (ALS) is a progressive neurological disease that affects nerve cells in the brain and spinal cord, leading to loss of muscle control. It is sporadic or familial in origin and results in the death of motor neurons. The initial symptoms of ALS are, for example, weakness in the hands, dyskinesia of the fingers, and fasciculations in the upper limbs. Thereafter, ALS causes muscle atrophy and / or muscle weakness, bulbar palsy, and fasciculations, and ultimately leads to respiratory failure and death within 3 to 5 years of disease onset.

[0004] The proposed treatments cannot reverse the damage of ALS, but can only slow the progression of symptoms, prevent complications, and make those who need it more comfortable and independent.

[0005] To date, the anti-glutamate small molecule riluzole, the antioxidant edaravone, and more recently sodium phenylbutyrate and taurursodiol have been approved for the treatment of ALS, with modest efficacy and doses limited by significant side effects such as hepatotoxicity and fatigue (Jaiswal MK., Med Res Rev, 2019; 39(2), 733-748).

[0006] Therefore, there remains a need for new strategies to treat ALS, and more specifically to prevent and / or delay motor neuron death.

[0007] A major issue in developing drugs for the treatment of central nervous diseases (CNS), such as ALS, is ensuring the level of efficacy of the drug in the brain and spinal cord. Therefore, in terms of pharmacokinetics, the drug under consideration needs to be able to cross the blood-brain barrier, which restricts access to neuronal cells, and reach the motor neurons in sufficient concentrations and within a reasonable time period. Therefore, the delivery route and formulation of the drug need to be suitable for this purpose. In ALS, the degeneration of motor neurons occurs in the cortex, brainstem, and spinal cord, so direct intrathecal or intraventricular administration of the drug is the preferred delivery route for patients with ALS.

[0008] Homeobox proteins or homeobox proteins are transcription factors that play an important role in the cell migration and differentiation process involved in morphogenesis. They are characterized by the presence of 60 amino acid sequences, i.e., homeobox domains, which are DNA binding domains with spiral / turn / helical structures. Studies have shown that the separation domains of the antennapedia mutant proteins of Drosophila pass through neuronal membranes in culture, assemble in the nucleus and promote neurite outgrowth (EP0485578). Homeobox domains are highly conservative and give the internalization characteristics of a large number of homeobox proteins (Spatazza et al., 2013, Pharmacol. Rev. 65, 90-104).

[0009] Engrailed proteins (Engrailed-1 and Engrailed-2) are homologous proteins with similar biological activities, collectively referred to as Engrailed below (EN for humans and En1 / 2 for mice). In newborns and adults, Engrailed is expressed in cerebellar granule cells and midbrain dopaminergic (DA) nuclei, including the substantia nigra pars compacta (SNpc, which degenerates in Parkinson's disease) and the ventral tegmental area (VTA). En1 / 2 plays an important role in the development of mesencephalic dopaminergic (mDA) neurons in the midbrain and mesencephalon (Joyner, 1996, Trends Genet 12, 15-20). En1 / 2 also plays a redundant role in the survival of adult mDA neurons in the SNpc and VTA located in the ventral part of the midbrain (Alberi et al., 2004, Development 131, 3229-3236), and therefore has been proposed for the prevention or treatment of DA neuron loss in Parkinson's disease. In WO2013 / 128239, it was reported that local administration of En1 / 2 by infusion in the midbrain increased DA synthesis and related motor activity of DA neurons. Prochiantz et al. (2011, FEBS Letters, 278, 52 (Abstract, Brunet et al., Nature 438:94-98, 2005 and Alvarez-Fisher et al., Nature Neurosci 14:1260-1266, 2011) reported that Engrailed is not only a transcription factor but also a translation regulator that enhances the translation of mitochondrial mRNA transcribed in the nucleus, and showed that Engrailed transduction upregulated the translation of two proteins Ndufs1 and Ndufs3 of mitochondrial complex I and increased ATP synthesis (see also Alvarez-Fischer et al., 2011, Nature Neuroscience, 14, 1260-1266, Stettler et al., 2011, Nature Neuroscience, 14, 1260-1266). al. 2012). Alternatively, in WO2007 / 099227, it was shown that systemic administration of En1 / 2 to mice induced an increase in DA turnover in the striatum, as reflected by increased production of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), without changes in dopamine levels.

[0010] US20210379144 describes that the accumulation of DNA damage is associated with the aging process and the onset of age-related diseases, including neurodegenerative diseases such as ataxia, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease and Parkinson's disease (Canugovi et al., 2013, 12, 578-587; Madabhushi et al., 2014, Neuron 83, 266-282)".

[0011] Vargas Abonce et al. 2020 (bioRxiv 734020, https: / / doi.org / 10.1101 / 734020) showed that EN protein injection promoted motor neuron survival and motor function and could be used as a therapy to alleviate the consequences of α-motor neuron decline.

[0012] The present invention is based on the fact that EN proteins aggregate in liquid formulations of purified EN proteins. However, according to FDA guidance, it is crucial for manufacturers of therapeutic protein products to minimize protein aggregation to reduce the likelihood and risks associated with immune responses, which may pose problems for patient safety and product efficacy.

[0013] Thus, among other aspects, the present invention provides formulations of Engrailed (EN) proteins, more specifically aqueous formulations of Engrailed (EN) proteins, that inhibit or minimize EN protein aggregation. In some embodiments, the formulations of the present invention inhibit or minimize EN protein dimerization and / or oligomerization, wherein the EN protein is a monomer.

[0014] Protein aggregation refers to the process in which protein molecules are assembled into a complex consisting of two or more proteins, wherein a single protein is a monomer. Protein aggregation is usually driven by forces and interactions such as van der Waals and hydrophobic forces, hydrogen bonds, and electrostatic attraction. The various types of interactions that occur between the amino acids in a protein and cause it to fold also occur between the amino acids of adjacent proteins, resulting in protein aggregation. According to the present invention, "protein aggregation," "protein dimerization," or "protein oligomerization" are synonyms.

[0015] In some embodiments, formulations of Engrailed (EN) proteins of the present invention delay the formation of EN dimers, oligomers, and / or aggregates upon prolonged storage.

[0016] Thus, in a preferred embodiment, the present invention relates to stable aqueous compositions of Engrailed (EN) proteins.

[0017] Advantageously, the compositions of the Engrailed (EN) proteins of the present invention can be stored for extended periods of time without the EN proteins losing their biological activity or becoming excessively dimerized, oligomerized, and / or aggregated. In certain embodiments, the compositions of the Engrailed (EN) proteins of the present invention can be stored at temperatures up to at least -65°C for at least 6 months.

[0018] Preferably, the present invention relates to an injectable composition of an Engrailed (EN) protein for non-systemic administration to a subject in need thereof.

[0019] In a preferred embodiment, the compositions of the Engrailed (EN) proteins of the present invention are suitable for treating diseases or conditions associated with neuronal, preferably motor neuronal, death. In an even more preferred embodiment, the compositions of the present invention are suitable for intrathecal or intracerebroventricular administration to a subject in need thereof.

[0020] The present invention also relates to methods for treating a subject suffering from a disease or condition associated with neuronal death, preferably motor neuron death, using compositions of the Engrailed (EN) proteins of the present invention. More specifically, the present invention provides methods for treating a subject suffering from amyotrophic lateral sclerosis (ALS). The present invention also relates to methods for preventing and / or delaying motor neuron death. According to the present invention, the method of treatment comprises administering a composition of the Engrailed (EN) protein of the present invention.

[0021] The present invention also relates to compositions of the Engrailed (EN) proteins of the present invention for use in treating a subject suffering from a disease or condition associated with neuronal death, preferably motor neuron death.

[0022] The present invention also relates to the use of a composition of Engrailed (EN) proteins of the present invention in the manufacture of a medicament for treating a subject suffering from a disease or condition associated with neuronal death, preferably motor neuron death.

[0023] The present invention also relates to a method for increasing the stability of an EN protein in an aqueous solution and / or reducing the aggregation of an EN protein in an aqueous solution, the method comprising mixing the EN protein with a specific formulation, wherein the formulation increases the stability of the EN protein in an aqueous solution and / or reduces the aggregation of the EN protein in an aqueous solution; and wherein the method provides a stable aqueous composition of the EN protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present disclosure will be more readily understood by reference to the following detailed description when considered in conjunction with the accompanying drawings, in which:

[0025] Figure 1: Effect of pH on EN1 protein oligomerization as determined by non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-page electrophoresis) stained with Coomassie Brilliant Blue, lane 20: gradient; lane 21, EN1 in 5 mM citrate / phosphate 250 mM D-glucose, 2,13 mM MgCl2, pH 4 after dialysis; lane 22, EN1 in 5 mM citrate / phosphate 250 mM D-glucose, 2,13 mM MgCl2, pH 4 after small-scale ultrafiltration; lane 23, EN1 in 5 mM citrate / phosphate 250 mM D-glucose, 2,13 mM MgCl2, pH 5 after dialysis; lane 24, EN1 in 5 mM citrate / phosphate 250 mM D-glucose, 2,13 mM MgCl2, pH 5 after small-scale ultrafiltration. 5; lane 25, EN1 in 5 mM citrate / phosphate 250 mM D-glucose, 2.13 mM MgCl2, pH 5 after dialysis; lane 26, EN1 in 5 mM citrate / phosphate 250 mM D-glucose, 2.13 mM MgCl2, pH 5 after small-scale ultrafiltration.

[0026] Figure 2 : Effect of pH on EN1 protein oligomerization determined by SEC-HPLC

[0027] Curve 1: 5 mM citrate / phosphate 250 mM D-glucose, 2.13 mM MgCl2 pH 6; Curve 2: 5 mM citrate / phosphate 250 mM D-glucose, 2.13 mM MgCl2 pH 4; Curve 3: 5 mM citrate / phosphate 250 mM D-glucose, 2.13 mM MgCl2 pH 5.

[0028] Figure 3 : Effect of glutathione on EN1 protein oligomerization determined by SEC HPLC and high molecular weight (~300 kDa) detected at 280 nm.

[0029] Curve 1: 5 mM citrate / phosphate 250 mM D-glucose, 2,13 mM MgCl2 pH 5; Curve 2: 5 mM citrate / phosphate 250 mM D-glucose, 2,13 mM MgCl2 pH 5, 50 μM glutathione.

[0030] Figure 4 : Effect of glutathione dose on EN1 protein oligomerization measured by SEC HPLC and high molecular weight (HMW) detected at 280 nm.

[0031] Curve 1: 5 mM citrate / phosphate, 250 mM D-glucose, 2.13 mM MgCl2, pH 5, 50 μM glutathione; Curve 2: 5 mM citrate / phosphate, 250 mM D-glucose, 2.13 mM MgCl2, pH 5, 1 μM glutathione; Curve 3: 5 mM citrate / phosphate, 250 mM D-glucose, 2.13 mM MgCl2, pH 5, 100 μM glutathione;

[0032] Figure 5 : Effect of glutathione dosage on EN1 protein oligomerization as determined by non-reducing SDS-page electrophoresis with Coomassie brilliant blue staining.

[0033] Lane 17, EN1 in 5 mM citrate / phosphate, 250 mM D-glucose, 2.13 mM MgCl2 pH 5, glutathione 50 μM; lane 18, EN1 in 5 mM citrate / phosphate, 250 mM D-glucose, 2.13 mM MgCl2 pH 5, glutathione 1 μM; lane 19, EN1 in 5 mM citrate / phosphate, 250 mM D-glucose, 2.13 mM MgCl2 pH 5, glutathione 100 μM; lane 21: blank; lane 22: gradient. DETAILED DESCRIPTION

[0034] Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings showing exemplary embodiments of the present invention. However, the scope of the present invention should not be limited by the specific embodiments described herein. In order to enable those skilled in the art to more clearly understand and practice the present invention, the following embodiments are provided.

[0035] Many approved protein drugs and protein drugs in clinical trials are manufactured and stored in liquid form, although historically more products have been developed in the form of solid formulations that are reformulated into a liquid state before injection. Proteins inherently tend to form aggregates over time, which may cause problems from the perspective of product quality, especially product safety, efficacy, delivery or dosage, and marketability, and are therefore considered harmful from the perspective of drug quality and performance (see, for example, Lundahl et al., 2021, RCS Chem.Biol., 2, 1004-1020). A major achievement in the development of protein formulations is the identification of inactivation, aggregation, and / or degradation pathways of the corresponding proteins. It is well known in the art that homeobox transcription factors such as EN proteins can dimerize (Perez-Villamil et al., 2004, JBC 279, 38062-38071, Papadopoulos et al., 2012, Dev. Biol., 367, 78-89) and the inventors have observed that recombinant human EN protein oligomerizes at room temperature during downstream production processing, leading to aggregation of the protein in aqueous solution (protein oligomerization / aggregation was detected by non-reducing SDS-Page electrophoresis).

[0036] Against this backdrop, the inventors carefully designed a formulation capable of inhibiting or reducing the aggregation of EN protein in aqueous solution and developed a stable aqueous composition of Engrailed (EN) protein. Advantageously, the composition developed by the inventors is compatible with intrathecal administration.

[0037] In an embodiment, the present invention provides a stable aqueous composition of an Engrailed (EN) protein comprising an effective amount or effective dose of an EN protein, a stabilizing concentration of reduced glutathione, an osmotic pressure regulator, and a buffer that provides the formulation with a pH of less than 7, preferably less than 6, more preferably less than 5, and advantageously about 4.

[0038] Unexpectedly, the present inventors observed that aggregation of EN protein in aqueous compositions can be inhibited or reduced in the presence of reduced glutathione.

[0039] In preferred embodiments, the stable aqueous compositions of Engrailed (EN) proteins of the present invention comprise reduced glutathione at a concentration of about 10 μM to about 100 μM. In embodiments, the compositions of the present invention contain about 50 μM reduced glutathione. In preferred embodiments, the reduced glutathione is reduced L-glutathione.

[0040] Furthermore, the present inventors have surprisingly demonstrated that the presence of an osmotic pressure regulating agent in an aqueous EN protein composition improves EN protein stabilization, ie, inhibits or reduces EN protein aggregation.

[0041] In embodiments, the stable aqueous composition of Engrailed (EN) protein has an osmotic pressure in the range of about 250 mOsmol / L to about 350 mOsmol / L, more specifically in the range of about 270 mOsmol / L to about 320 mOsmol / L. Examples of suitable osmotic pressure regulators include, but are not limited to, sugars such as glucose (e.g., D-glucose), poly(glucose), fructose, dextran, glycerol, sorbitol, mannitol, trehalose, mannose, polyanions, and the like, and combinations thereof. Other osmotic pressure regulators that may be non-sugar agents and have equivalent functions may serve as viable alternatives, such as small molecule amino acids.

[0042] In a preferred embodiment, the stable aqueous composition of the Engrailed (EN) protein is for intrathecal administration, and the osmotic pressure regulator is preferably D-glucose. In a preferred embodiment, the osmotic pressure regulator is D-glucose. In an embodiment, the stable aqueous composition of the Engrailed (EN) protein of the present invention comprises D-glucose at a concentration of about 220 mM to about 320 mM, advantageously about 240 mM to about 290 mM. In a preferred embodiment, the D-glucose is about 250 mM.

[0043] Advantageously, the present inventors have also discovered that the addition of a magnesium salt can significantly reduce aggregation of EN compositions. Thus, in one embodiment, the stable aqueous composition of an Engrailed (EN) protein of the present invention comprises a magnesium salt, preferably MgCl. In a preferred embodiment, the magnesium salt is present at a concentration of about 0.1 mM to about 3 mM. In a preferred embodiment, the stable aqueous composition of an Engrailed (EN) protein of the present invention comprises about 2.13 mM MgCl.

[0044] Advantageously, the aqueous EN protein composition of the present invention comprises dextrose in combination with magnesium chloride.

[0045] Advantageously, the inventors have also discovered that the addition of low levels (i.e., less than 0.2%) of nonionic surfactants can significantly reduce adhesion to the support and help stabilize the EN protein. Therefore, in one embodiment, the stable aqueous composition of the Engrailed (EN) protein of the present invention comprises a stabilizing concentration of a nonionic surfactant. Nonionic surfactants that can be used in the compositions of the present invention are known in the art and include, but are not limited to, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and various poloxamers or pluronics (including Pluronic F-68), or mixtures thereof. In a preferred embodiment, the nonionic surfactant is polysorbate 20. In certain embodiments, the surfactant is present at a concentration of about 0% to about 0.2%. In a preferred embodiment, the surfactant is present at a concentration of about 0% to about 0.01%. In another preferred embodiment, the surfactant is present at a concentration of about 0.005%.

[0046] In an embodiment, the present invention provides a stable aqueous composition of an Engrailed (EN) protein comprising:

[0047] An effective amount or dosage of EN protein;

[0048] about 10 μM to about 100 μM reduced glutathione;

[0049] About 240 to about 290 mM of an osmotic pressure regulator, preferably D-glucose;

[0050] About 0.1 mM to about 3 mM MgCl2;

[0051] 0 to 0.01% polysorbate 20; and

[0052] • Buffering agent to maintain a pH of approximately 4.

[0053] In embodiments, the present invention provides a stable aqueous composition of an Engrailed (EN) protein comprising an effective amount or effective dose of about 0.05 mg / mL to about 1 mg / mL of EN protein. In some embodiments, the EN is present in the compositions provided herein at a concentration of about 0.05 mg / mL to about 0.9 mg / mL. In some embodiments, the EN is present at a concentration of about 0.1 mg / mL to about 0.9 mg / mL. In some embodiments, the EN may be present at about 0.6 mg / mL.

[0054] In embodiments, the buffer is sodium acetate / acetic acid.

[0055] In an advantageous aspect, the present invention provides a stable aqueous composition of an Engrailed (EN) protein comprising:

[0056] About 0.6 mg / ml of EN protein;

[0057] About 250 mM D-glucose;

[0058] About 2.13 mM MgCl2;

[0059] About 50 μM reduced glutathione;

[0060] About 0.005% polysorbate 20; and

[0061] • About 5 mM sodium acetate / acetic acid to maintain a pH of about 4.

[0062] As used herein, the term "EN" refers to an Engrailed protein. In the context of the present invention, EN proteins encompass any EN protein from mammals (such as primates, humans, monkeys, rabbits, pigs, cattle or rodents, preferably humans), such as Engrailed 1 (EN1) or Engrailed 2 (EN2) and mixtures thereof, as well as biologically active derivatives thereof. Active mutants and variant EN proteins, as well as functional fragments and fusion proteins of EN proteins are also encompassed. Exemplary EN1 proteins or polypeptides include, but are not limited to, human EN1 proteins or polypeptides having a primary amino acid sequence annotated by Genbank accession number AAA 53502.2 or NCBI NP_001417.3. Exemplary EN2 proteins or polypeptides include, but are not limited to, human EN2 proteins or polypeptides having a primary amino acid sequence annotated by Genbank accession number AAA 53504.2 or NCBI NP_001418.2. In certain embodiments of the formulations provided herein, the EN protein is human EN or recombinant human EN protein, or a biologically active derivative or fragment thereof. In one embodiment, the EN protein is human EN1.

[0063] As used herein, the term "biologically active derivative" refers to any polypeptide that has substantially the same biological function as an EN. The polypeptide sequence of a biologically active derivative may contain one or more amino acid deletions, additions, and / or substitutions, where the deletion, presence, and / or substitution of these amino acids, respectively, does not have any substantial negative impact on the biological activity of the polypeptide.

[0064] As used herein, the terms "EN" and "biologically active derivatives" also include polypeptides obtained by recombinant DNA technology or synthesis, respectively. Recombinant EN (e.g., recombinant human EN) can be produced by any method known in the art. This includes any method known in the art for: (i) generating recombinant DNA by genetic engineering (e.g., by reverse transcription of RNA and / or amplification of DNA), (ii) introducing the recombinant DNA into prokaryotic or eukaryotic cells by transfection (i.e., by electroporation or microinjection), (iii) culturing the transformed cells, e.g., continuously or in batches, (iv) expressing the EN, e.g., continuously or upon induction, and (v) isolating the EN, e.g., from the culture medium or by harvesting the transformed cells, so as to (vi) obtain substantially purified recombinant EN, e.g., by anion exchange chromatography or affinity chromatography.

[0065] As used herein, "effective amount or effective dose" or "sufficient amount or sufficient dose" refers to the amount of the compound that produces the effect being administered. The exact amount will depend on the purpose of the treatment and will be determined by those skilled in the art using known techniques.

[0066] As used herein, the term "about" refers to an approximate range of plus or minus 10% from a specified value. For example, the expression "about 20%" encompasses a range of 18-22%. As used herein, about also includes an exact amount. Thus, "about 20%" means "about 20%" and also means "20%."

[0067] As used herein, the expression "aqueous composition" refers to a composition comprising water as a solvent.

[0068] As used herein, "storage" means that once the composition of the present invention is prepared, it is not immediately administered to a subject, but rather is stored for a period of time under specific conditions (e.g., a specific temperature, etc.) before use. For example, a liquid or lyophilized composition can be stored at different temperatures (such as frozen (=<-65°C or -15°C to -25°C), refrigerated (0° to 10°C), or room temperature (e.g., a temperature up to 32°C)) for days, weeks, months, or years before administration to a subject.

[0069] According to specific embodiments, the compositions of the present invention reduce or delay dimerization, oligomerization, and / or aggregation of EN proteins over time. In one embodiment, the stable aqueous compositions of Engrailed (EN) proteins of the present invention are stable when stored at a temperature of at least about -65°C for at least about 6 months. In other embodiments, the compositions provided herein retain significant EN activity upon long-term storage.

[0070] As used herein, a "stable composition of a protein" or a "stable protein in a composition" means that the EN protein in the composition maintains the same oligomerization state, preferably remains as a monomer and / or does not form aggregates. A person skilled in the art will know how to determine the oligomerization / aggregation state of a protein, for example, by non-reducing SDS-Page electrophoresis or size exclusion chromatography (SEC). In a preferred embodiment, a "stable composition of an EN protein" means that the EN protein dimers, oligomers and / or aggregates in the composition are less than about 5%, preferably less than about 3% of the total amount of EN protein.

[0071] In a related aspect, the present invention provides a stable lyophilized composition of an Engrailed (EN) protein, wherein the formulation is lyophilized from a stable aqueous composition of an Engrailed (EN) protein provided herein. Lyophilization can be performed according to methods in the art.

[0072] Generally, the stable aqueous compositions of Engrailed (EN) proteins provided herein are suitable for pharmaceutical administration.

[0073] In preferred embodiments, the EN compositions provided herein are sterile and comprise low endotoxin levels (according to the European Pharmacopoeia).

[0074] In some embodiments, the EN compositions provided herein may further comprise one or more pharmaceutically acceptable excipients, carriers and / or diluents. In addition, the compositions provided herein may further comprise other agents, carriers, adjuvants, diluents, tissue penetration enhancers and solubilizers, etc. Methods for preparing compositions and formulations for pharmaceutical administration are known to those skilled in the art.

[0075] The stable aqueous composition of the Engrailed (EN) protein provided herein can be formulated for administration by known methods, as a bolus or by continuous infusion over a period of time. It is particularly suitable for administration by the intrathecal or intracerebroventricular route.

[0076] The stability of aqueous compositions of engrailed (EN) proteins can be determined by one or more biophysical properties of the EN protein in the formulation. Non-limiting examples of properties that can be used to assess stability include the degree of monodispersity or polydispersity of the protein, the degree of dimerization, oligomerization, or aggregation of the EN protein. Those skilled in the art will readily appreciate that other stability measurement methods that can be used to assess the stability of EN formulations include, but are not limited to, size exclusion chromatography (SEC), dynamic or static light scattering, RP-HPLC, ion exchange chromatography, polyacrylamide gel electrophoresis, and non-reducing SDS-Page electrophoresis, etc.

[0077] In certain embodiments, aqueous compositions of Engrailed (EN) proteins of the present invention can be stable for extended periods of time when stored at a particular temperature, such as at about -65°C, -20°C, 4°C, 18°C, room temperature, 25°C, 30°C, 35°C, 37°C, 40°C, or higher. In some embodiments, the extended period is at least about one week. In other embodiments, the extended period can include at least about 2 weeks, or at least about 3 weeks, or at least about 1 month, or at least about 2 months, or at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, or 18 months. In yet other embodiments, the compositions of the present invention can be stable for at least about 2, 3, 4, 5 years, or longer.

[0078] In an embodiment, the present invention relates to a stable aqueous composition of Engrailed (EN) protein that is substantially free of aggregated EN protein, dimeric EN protein, oligomeric EN protein, or mixtures thereof. In a preferred embodiment, the stable aqueous composition of Engrailed (EN) protein is substantially free of aggregated EN protein, dimeric EN protein, oligomeric EN protein, or mixtures thereof when the composition contains less than 5%, preferably less than 3%, or less than 2%, or less than 1% of aggregated EN protein, dimeric EN protein, oligomeric EN protein, or mixtures thereof.

[0079] In another embodiment of the present invention, a stable aqueous composition of an Engrailed (EN) protein of the present invention has an EN protein population consisting of at least about 95% EN protein monomers after long-term storage. In other embodiments, the EN protein composition has at least about 97% EN protein monomers, or at least about 98%, 99%, or a higher percentage of EN protein monomers.

[0080] In some embodiments, the EN proteins used in the formulations provided herein can be expressed, produced, or purified according to methods well known in the art.

[0081] Recombinant EN protein can be produced by expression in any suitable prokaryotic or eukaryotic host system. In one embodiment, the EN protein can be expressed in bacterial cells, yeast cells, insect cells, avian cells and mammalian cells. Examples of eukaryotic cells include, but are not limited to, mammalian cells such as CHO, COS, HEK 293, BHK, SK-Hep-1 and HepG2; insect cells such as SF9 cells, SF21 cells, S2 cells and High Five cells; and yeast cells such as Saccharomyces, Pichia pastoris or Schizosaccharomyces cells.

[0082] In one embodiment, the cell can be any bacterial cell that can be cultured, preferably cultured during the manufacturing process (ie, at least 1 liter), to produce the desired EN protein. In a preferred embodiment, the cell line is an E. coli cell line.

[0083] A variety of vectors can be used to express EN protein (for example, EN1) and can be selected from eukaryotic and prokaryotic expression vectors. In certain embodiments, in expressing EN protein (for example, EN1), it is considered to use a plasmid vector. The plasmid will include a nucleotide sequence encoding EN protein (for example, EN1), which is operably connected to one or more control sequences, such as a promoter, for example, an inducible promoter. According to certain embodiments, a viral vector is used to introduce the nucleotide sequence encoding EN protein (for example, EN1) into a host cell for expression. The viral vector will include a nucleotide sequence encoding EN protein (for example, EN1), which is operably connected to one or more control sequences (for example, a promoter). The non-limiting example of the viral vector that can be used to deliver nucleic acid includes adenovirus vectors, AAV vectors and retroviral vectors. The non-limiting example of the vector for prokaryotic expression includes plasmids, such as pRSET, pET, pBAD etc., wherein the promoter used in the prokaryotic expression vector includes lac, trc, trp, recA, araBAD etc. Examples of vectors for eukaryotic expression include: (i) for expression in yeast, vectors such as pAO, pPIC, pYES, pMET, using promoters such as AOX1, GAP, GAL1, AUG1, etc.; (ii) for expression in insect cells, vectors such as pMT, pAc5, pIB, pMIB, pBAC, etc., using promoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh, etc.; and (iii) for expression in mammalian cells, vectors such as pSVL, pCMV, pRc / RSV, pcDNA3, pBPV, etc., and vectors derived from viral systems (such as vaccinia virus, adeno-associated virus, herpes virus, retrovirus, etc.), using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV and β-actin.

[0084] In certain embodiments, EN expression can include the use of a cell culture system operated in batch or continuous operation mode. For example, when utilizing batch cell culture, they can operate in single batch, fed batch or repeated batch mode. Similarly, continuous cell culture can be carried out under, for example, perfusion, turbidostat or chemostat mode. Batch and continuous cell culture can be carried out under suspension or adhesion conditions. When operating under suspension conditions, cells will be freely suspended and mixed in culture medium. Alternatively, under adhesion conditions, cells will be attached to a solid phase, such as microcarriers, porous microcarriers, disc carriers, ceramic shells, hollow fibers, flat sheets and gel matrix etc.

[0085] Batch culture is typically large-scale cell culture, in which the cell inoculum is cultivated to maximum density in a tank or fermentor tank and harvested and processed as a single batch. Fed-batch culture is typically batch culture, which is supplied with fresh nutrients (e.g., growth-restricted substrates) or additives (e.g., product precursors) to maintain cell growth and reach higher biomass. The feed solution is typically highly concentrated to avoid the dilution of a bioreactor. In repeated batch culture, cells are placed in a culture medium and grown to the required cell density. In order to avoid the beginning of the decline phase and cell death, before cells reach their maximum concentration, the culture is diluted with complete growth medium. The amount and frequency of dilution vary greatly and depend on the growth characteristics of the cell line and the convenience of the culture process. This process can be repeated many times as needed, and unless cells and culture medium are discarded when subculture, the volume of the culture will progressively increase with each dilution. The volume increased can be processed by using a reactor of sufficient size to allow dilution in a container or the diluted culture to be divided into several containers. The basic principle of this type of culture is to maintain the cell in an exponential growth state. In certain embodiments, the EN protein can be recovered after harvesting the supernatant of the batch culture.

[0086] Methods for expression and purification of recombinant EN proteins are also known in the art.

[0087] In addition, another aspect of the present invention relates to a purification method for recombinant EN protein. In one embodiment, the recombinant EN protein is expressed in recombinant bacteria and purified from the resulting conditioned medium through a series of chromatography and filtration / ultrafiltration steps.

[0088] In one embodiment, a method for providing an EN protein (e.g., EN1) composition is provided, comprising the steps of: (a) culturing cells containing a nucleic acid encoding an EN protein (e.g., EN1) in a culture medium; (b) performing a lysis step to release the EN protein into a supernatant n; (c) clarifying the supernatant containing the EN protein; (d) purifying the EN protein by a series of chromatography and filtration / ultrafiltration steps; and e) formulating an EN protein (e.g., EN1) composition according to the formulation provided herein, thereby providing an EN protein (e.g., EN1) formulation.

[0089] In one embodiment, the present invention provides a method for making a stable aqueous composition of an Engrailed (EN) protein, the method comprising the steps of: (i) expressing an EN protein (e.g., EN1) or a biologically active derivative thereof by culturing cells containing a nucleic acid encoding the EN protein (e.g., EN1) in cells cultured in a culture medium; (ii) performing a lysis step to release the EN protein into a supernatant; (iii) clarifying the supernatant containing the EN protein; (iv) purifying the EN protein (e.g., EN1); and (v) preparing a composition comprising an effective amount or effective dose of the EN protein, an osmotic pressure regulator, a stabilizing concentration of reduced glutathione, and a buffer that provides a pH of less than 7, preferably less than 6, more preferably less than 5, and advantageously about 4.

[0090] In one embodiment, the present invention provides a method for making a stable aqueous composition of an Engrailed (EN) protein, the method comprising the steps of: (i) expressing an EN protein (e.g., EN1) or a biologically active derivative thereof by culturing cells containing a nucleic acid encoding the EN protein (e.g., EN1) in cells cultured in a culture medium; (ii) performing a lysis step to release the EN protein into a supernatant; (iii) clarifying the supernatant containing the EN protein; (iv) purifying the EN protein (e.g., EN1); and (v) preparing a composition comprising (a) about 0.6 mg / ml EN protein; (b) about 250 mM D-glucose; (c) about 50 μM reduced glutathione; (d) about 2.13 mM MgCl2; (e) 0.005% polysorbate 20; and (f) a buffer for maintaining a pH of less than 7, preferably less than 6, more preferably less than 5, and advantageously about 4.

[0091] Administration and treatment methods

[0092] The compositions of the present invention can be used for therapeutic treatment or preventive treatment. Typically, for therapeutic applications, a therapeutically effective dose of the formulation is administered to a patient suffering from a disease or condition related to neuronal death, preferably motor neuron death, preferably amyotrophic lateral sclerosis (ALS). The formulation and amount effective for these uses will depend on the severity of the disease or condition and the overall health of the patient. The formulation can be administered in a single or multiple doses, depending on the dosage and frequency required and tolerated by the patient.

[0093] For the purposes of the present invention, "patient" or "subject" includes humans and other animals, particularly mammals. The compositions, formulations, and methods are suitable for both human therapy and veterinary applications. In specific embodiments, the patient is a mammal, preferably a human. Other known treatments and therapies for conditions associated with neuronal death, preferably motor neuron death, can be used in combination with the formulations and methods provided herein.

[0094] The preferred route of delivery is intrathecal or intracerebroventricular administration, and the more preferred route of delivery is intrathecal delivery. The volume of the intrathecal injection is in the range of 0.1 mL to 20 mL.

[0095] EN protein can be administered intrathecally or intracerebrally at a dose ranging from 0.01 mg to 18 mg, more specifically intrathecally to patients suffering from a disease or condition associated with neuronal death, preferably motor neuron death, preferably amyotrophic lateral sclerosis (ALS).

[0096] According to specific embodiments, the stable aqueous composition of Engrailed (EN) protein can be administered monthly or bimonthly.

[0097] According to a specific embodiment, the stable aqueous composition of an Engrailed (EN) protein (preferably an EN1 protein) of the present invention is administered to a patient suffering from a disease or condition associated with neuronal death, preferably motor neuron death, preferably amyotrophic lateral sclerosis (ALS). According to a specific embodiment, administering the stable aqueous composition of an Engrailed (EN) protein (preferably an EN1 protein) of the present invention to a patient suffering from a disease or condition associated with neuronal death, preferably motor neuron death, preferably amyotrophic lateral sclerosis (ALS) results in controlling the progression of motor neuron death, preferably preventing motor neuron death.

[0098] In another aspect of the present invention, a test kit for treating a disease or condition associated with neuronal death, preferably motor neuron death, more preferably amyotrophic lateral sclerosis (ALS) is provided. In one embodiment, the test kit comprises the EN protein composition as provided above. In some embodiments, the test kit provided herein can contain one or more dosages of a liquid or lyophilized composition as provided herein. According to a special embodiment, for example, a test kit with a lyophilized EN protein composition, the test kit also contains a suitable liquid for reconstructing a liquid formulation, such as artificial CSF or a pharmaceutically acceptable buffer. In some embodiments, the test kit can comprise the EN protein composition prepackaged in a syringe, for use by a healthcare professional or for family use.

[0099] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that those skilled in the art may make various modifications or changes based thereon, and that such modifications or changes should be included within the spirit and scope of this application and the scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

[0100] Example

[0101] The buffer exchange step has been demonstrated to promote oligomerization of EN proteins during downstream protein production.

[0102] Buffer exchange small-scale models (dialysis and ultrafiltration centrifuge tubes) have been selected and used in order to screen different formulations of EN protein and EN protein oligomerization levels.

[0103] Two main analytical methods have been used to measure EN protein oligomerization: size exclusion chromatography (SEC) HPLC and non-reducing electrophoresis SDS-Page with Coomassie Brilliant Blue staining.

[0104] Example 1: Acidic pH Effect

[0105] The effect of acidic pH on EN1 protein oligomerization was analyzed in two different test models (dialysis or small-scale ultrafiltration) with different EN1 protein compositions. Figure 1 ):

[0106] -EN1 protein composition contains 5mM citrate / phosphate, 250mM D-glucose, 2.13mM MgCl2, pH 4 after dialysis;

[0107] -EN1 protein composition contains 5mM citrate / phosphate, 250mM D-glucose, 2.13mM MgCl2, and has a pH of 4 after small-scale ultrafiltration;

[0108] -EN1 protein composition contains 5mM citrate / phosphate, 250mM D-glucose, 2.13mM MgCl2, and the pH after dialysis is 5;

[0109] -EN1 protein composition contains 5mM citrate / phosphate, 250mM D-glucose, 2.13mM MgCl2, and has a pH of 5 after small-scale ultrafiltration;

[0110] -EN1 protein composition contains 5mM citrate / phosphate, 250mM D-glucose, 2.13mM MgCl2, pH 6 after dialysis;

[0111] -EN1 protein composition contains 5mM citrate / phosphate, 250mM D-glucose, 2.13mM MgCl2, and has a pH of 6 after small-scale ultrafiltration.

[0112] The oligomerization of EN protein has been determined using non-reducing SDS-Page electrophoresis. Figure 1 It was shown that a composition of pH 4 resulted in limited oligomerization of the EN1 protein compared to pH 5 and pH 6.

[0113] Similarly, EN protein oligomerization has been determined in dialyzed compositions using the SEC-HPLC method (see above), and high molecular weight (HMW) ( Figure 2 The results demonstrated that the composition with a pH of 4 had a lower oligomer peak and was more effective in preventing EN1 protein oligomerization than the compositions with pHs 5 and 6.

[0114] Example 2 Glutathione Effect

[0115] EN1 protein oligomerization was measured by SEC-HPLC in a composition comprising EN1 protein, 5 mM citrate / phosphate, 250 mM D-glucose, 2.13 mM MgCl2, pH 5, in the absence or presence of 50 μM reduced glutathione ( Figure 3 ).

[0116] Figure 3 It was shown that the amount of EN1 oligomers was drastically reduced in the presence of glutathione in the EN1 protein composition.

[0117] Example 3 Effect of Glutathione Concentration

[0118] EN1 protein oligomerization was measured by SEC-HPLC using a composition containing EN1 protein, 5 mM citrate / phosphate, 250 mM D-glucose, 2.13 mM MgCl2, pH 5, and different concentrations of reduced glutathione (1 μM, 50 μM, or 100 μM) ( Figure 4 ).

[0119] Figure 4 This indicates that the presence of 50 μM or 100 μM glutathione in the composition can prevent EN1 protein oligomerization.

[0120] Similar analysis and conclusions have been observed using non-reducing SDS-Page electrophoresis.

Claims

1. A stable aqueous composition of an Engrailed (EN) protein, comprising an effective amount or effective dose of the EN protein, a stabilizing concentration of reduced glutathione, an osmotic pressure regulator, and a buffer, wherein the buffer provides a pH of less than 7.

2. The composition according to claim 1, wherein The stabilizing concentration of reduced glutathione is selected from about 10 μM to about 100 μM.

3. The composition according to claim 1 or 2, wherein The osmolarity of the composition is in the range of about 250 mOsmol / L to about 350 mOsmol / L.

4. The composition according to any one of claims 1 to 3, wherein The concentration of the osmotic pressure regulating agent is about 220 mM to about 320 mM.

5. The composition according to any one of claims 1 to 4, wherein The osmotic pressure regulator is D-glucose.

6. The composition according to any one of claims 1 to 5, wherein The composition comprises a magnesium salt.

7. The composition according to claim 6, wherein The concentration of the magnesium salt is from about 0.1 mM to about 3 mM.

8. The composition according to any one of claims 1 to 7, wherein The composition comprises a nonionic detergent.

9. The composition according to claim 8, wherein The nonionic surfactant is polysorbate 20.

10. The composition according to claim 8 or 9, wherein The concentration of the nonionic surfactant is from about 0% to about 0.2%.

11. The composition according to any one of claims 1 to 10, wherein The composition comprises: An effective amount or dosage of EN protein; about 10 μM to about 100 μM reduced glutathione; About 240 mM to about 290 mM of an osmotic pressure regulator; About 0.1 mM to about 3 mM MgCl2; 0 to 0.01% polysorbate 20; and • Buffering agent to maintain a pH of approximately 4.

12. The composition according to claim 11, wherein The effective amount or effective dosage of EN protein is about 0.05 mg / mL to about 1 mg / mL.

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