Stabilized Engrailed protein aqueous composition

Abstract

The present invention relates to formulations of Engrailed (EN) proteins, more particularly aqueous formulations of Engrailed (EN) proteins, that can inhibit or minimize aggregation of the EN proteins. In some embodiments, the formulations of the present invention can inhibit or minimize dimerization and / or oligomerization of the EN proteins, wherein the EN proteins are monomeric.

Description

[Technical Field]

[0001] The present invention relates to a specific formulation for obtaining a stable aqueous protein composition. In particular, the present invention relates to a stabilized aqueous composition of Engrailed (EN) protein, which comprises a specific combination of stabilizing compounds. [Background technology]

[0002] Neurodegenerative diseases are a group of irreversible neurological disorders caused by the loss of nerve cells in the brain and spinal cord, and mainly include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, etc. Since most degenerative diseases of the nervous system still lack effective treatments, finding effective methods to prevent, delay, and treat the diseases is an urgent problem to be solved.

[0003] Amyotrophic lateral sclerosis (ALS) is a progressive neurological disease that affects nerve cells in the brain and spinal cord, causing loss of muscle control. It can be sporadic or familial in origin and results in the death of motor neurons. ALS begins with early symptoms such as hand weakness, impaired finger movement, and fasciculations of the upper limbs. ALS then leads to muscle atrophy and / or weakness, bulbar paralysis, and muscle fasciculations, ultimately leading to respiratory failure and death within 3–5 years of disease onset.

[0004] The proposed treatments cannot reverse the damage caused by amyotrophic lateral sclerosis, but can only slow the progression of symptoms, prevent complications, and make those who need them more comfortable and independent.

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

[0006] Therefore, there remains a need for new strategies that can treat ALS, and more specifically, prevent and / or delay the death of motor neurons.

[0007] One of the major problems in developing drugs for treating central nervous system (CNS) diseases such as ALS is to ensure therapeutic levels of the drug in the brain and spinal cord.Therefore, the pharmacokinetic aspect requires that the drug in question be able to pass through the blood-brain barrier, which restricts access to nerve cells, and reach motor neurons at sufficient concentrations over a reasonable period of time.Therefore, the drug delivery route and drug formulation must be suitable for the purpose.In ALS, the deterioration of motor neurons occurs in the cortex, brainstem, and spinal cord, so direct intrathecal or intracerebroventricular administration of drugs is the preferred delivery route in ALS patients.

[0008] Homeoproteins, or homeodomain proteins, are transcription factors that play a key role in cell migration and differentiation processes involved in morphogenesis. They are characterized by the presence of a homeodomain, a DNA-binding domain with a 60-amino acid sequence, i.e., a helix-turn-helix structure. Isolated domains of the Drosophila Antennapedia protein have been shown to cross the membranes of cultured neurons, accumulate in the nucleus, and promote neurite outgrowth (European Patent No. 0485578). Homeodomains are highly conserved and confer internalization properties to many homeoproteins (Spatazza et al., 2013, Pharmacol. Rev. 65, 90-104).

[0009] The Engrailed proteins (Engrailed-1 and Engrailed-2) are homeoproteins with similar biological activities and are collectively referred to as Engrailed (EN in humans or En1 / 2 in mice). In neonates 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 midbrain development and the generation of mesencephalic dopaminergic (mDA) neurons in the midbrain (Joyner, 1996, Trends Genet 12, 15-20). En1 / 2 also plays a redundant role in the survival of adult mDA neurons located in the SNpc and VTA in the ventral part of the midbrain (Alberi et al., 2004, Development 131, 3229-3236). Therefore, it has been proposed to prevent or treat the loss of DA neurons in Parkinson's disease. International Publication No. 2013 / 128239 reported that local administration of En1 / 2 by injection into the midbrain increased DA synthesis by DA neurons and associated motor activity. 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 translational regulator that enhances the translation of nuclear-transcribed mitochondrial mRNAs. They showed that transduction of Engrailed upregulates the translation of Ndufs1 and Ndufs3, two proteins of mitochondrial complex I, and increases ATP synthesis (Alvarez-Fischer et al., 2011, Nature Neuroscience, 14, 1260-1266; see also Stettler et al. 2012).Alternatively, WO 2007 / 099227 showed that systemic administration of En1 / 2 to mice induced an increase in DA turnover in the striatum, reflected by increased production of the DA metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), without changes in dopamine concentrations.

[0010] US Patent Application Publication No. 20210379144 describes that the accumulation of DNA damage is associated with the aging process and the development of age-related diseases, including neurodegenerative disorders 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 (bioRxiv734020, https: / / doi.org / 10.1101 / 734020) showed that EN protein injections could be used as a treatment to promote motor neuron survival and motor function and alleviate the effects of α-motor neuron degeneration.

[0012] The present invention is based on the fact that EN protein aggregates in the liquid preparation of purified EN protein.However, according to FDA guidance, it is important for therapeutic protein product manufacturers to minimize protein aggregation as much as possible, in order to reduce the possibility of immune response, which may cause some problems in both patient safety and product effectiveness, and the risk associated with immune response, which may cause some problems in both patient safety and product effectiveness.

[0013] Thus, the present invention provides, among other aspects, formulations of Engrailed (EN) proteins, more particularly aqueous formulations of Engrailed (EN) proteins, that inhibit or minimize aggregation of the EN proteins. In some embodiments, the formulations of the present invention inhibit or minimize dimerization and / or oligomerization of the EN proteins, and the EN proteins are monomeric.

[0014] Protein aggregation refers to the process by which individual proteins become monomers and protein molecules assemble into complexes composed of two or more proteins. Protein aggregation is typically caused by forces and interactions, such as van der Waals and hydrophobic attractions, hydrogen bonds, and electrostatic attractions. These types of interactions that occur between amino acids within a protein and lead to its folding also exist between amino acids in neighboring proteins, leading to protein aggregation. "Protein aggregation," "protein dimerization," or "protein oligomerization" are synonymous terms according to the present invention.

[0015] In some embodiments, formulations of Engrailed (EN) proteins of the present invention delay the formation of EN dimers, oligomers and / or aggregates when stored for extended periods of time.

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

[0017] Advantageously, Engrailed (EN) protein compositions of the present invention can be stored for extended periods of time without the EN protein losing its biological activity or undergoing excessive dimerization, oligomerization, and / or aggregation. In certain embodiments, Engrailed (EN) protein compositions of the present invention can be stored at temperatures of at least about -65°C for at least 6 months.

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

[0019] In a preferred embodiment, the Engrailed (EN) protein compositions of the present invention are suitable for treating diseases or conditions associated with neuronal death, preferably motor neuron 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 further relates to a method of using the Engrailed (EN) protein composition of the present invention for treating a subject with a disease or condition associated with neuronal death, preferably motor neuron death. More specifically, the present invention provides a method of treating a subject with amyotrophic lateral sclerosis (ALS). The present invention further relates to a method of preventing and / or delaying motor neuron death. According to the present invention, the treatment method comprises administering the Engrailed (EN) protein composition of the present invention.

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

[0023] The present invention further relates to a method for increasing the stability and / or reducing the aggregation of an EN protein in an aqueous solution, comprising mixing the EN protein with a particular formulation, wherein the formulation increases the stability and / or reduces the aggregation of the EN protein in aqueous solution. The method provides a stable aqueous composition of the EN protein. [Brief explanation 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.

[0025] [Figure 1] FIG. 1 shows the effect of pH on the oligomerization of EN1 protein as measured by non-reducing SDS-page electrophoresis with Coomassie blue staining. Lane 20: ladder; lane 21: EN1 in 5 mM citrate / phosphate 250 mM dextrose, 2.13 mM MgCl2, pH 4 after dialysis; lane 22: EN1 in 5 mM citrate / phosphate 250 mM dextrose, 2.13 mM MgCl2, pH 4 after small-scale ultrafiltration; lane 23: EN1 in 5 mM citrate / phosphate 250 mM dextrose, 2.13 mM MgCl2, pH 5 after dialysis; lane 24: EN1 in 5 mM citrate / phosphate 250 mM dextrose, 2.13 mM MgCl2, pH 5 after small-scale ultrafiltration; lane 25: EN1 in 5 mM citrate / phosphate 250 mM dextrose, 2.13 mM MgCl2, pH 5 after dialysis; lane 26: 5 mM citrate / phosphate after small-scale ultrafiltration. EN1 in 250 mM dextrose, 2.13 mM MgCl2 pH 5. [Figure 2] Figure 2 shows the effect of pH on the oligomerization of EN1 protein as measured by SEC-HPLC. Curve 1: 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl, pH 6; Curve 2: 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl, pH 4; Curve 3: 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl, pH 5. [Figure 3] Figure 3 shows the effect of glutathione on the oligomerization of EN1 protein as measured by SEC HPLC and its high molecular weight (approximately 300 kDa) detected at 280 nm. Curve 1: 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl, pH 5; Curve 2: 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl, pH 5, 50 μM glutathione. [Figure 4] Figure 4 shows the effect of glutathione dose on EN1 protein oligomerization as measured by SEC HPLC and high molecular weight (HMW) detected at 280 nm. Curve 1: 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl, pH 5, 50 μM glutathione; Curve 2: 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl, pH 5, 1 μM glutathione; Curve 3: 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl, pH 5, 100 μM glutathione. [Figure 5] Figure 5 shows the effect of glutathione dosage on the oligomerization of EN1 protein as measured by non-reducing SDS-page electrophoresis with Coomassie blue staining. Lane 17: EN1 in 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl2, pH 5, and 50 μM glutathione; Lane 18: EN1 in 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl2, pH 5, and 1 μM glutathione; Lane 19: EN1 in 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl2, pH 5, and 100 μM glutathione; Lane 21: blank; Lane 22: ladder. DETAILED DESCRIPTION OF THE INVENTION

[0026] Detailed Description The present invention will now be described in more detail with reference to the accompanying drawings, which show exemplary embodiments of the invention. However, the present invention is not limited in scope by the specific embodiments described herein. The following embodiments are presented to enable those skilled in the art to more clearly understand and practice the present invention.

[0027] While many approved protein drugs, as well as those in clinical trials, are manufactured and stored as liquids, historically, more products were developed as solid formulations that were reconstituted into a liquid state immediately prior to injection. Proteins inherently tend to form aggregates over time, which can pose problems for product quality—specifically, product safety, efficacy, delivery or administration, and marketability—and are therefore considered harmful from the perspective of pharmaceutical quality and performance (see, e.g., Lundahl et al., 2021, RCS Chem. Biol., 2, 1004-1020). One of the key outcomes of protein drug development is the identification of the inactivation, aggregation, and / or degradation pathways for each protein. It is well known in the art that homeobox transcription factors, such as EN proteins, can dimerize (Perez-Villamil et al., 2004, JBC279, 38062-38071; Papadopoulos et al., 2012, Dev. Biol., 367, 78-89). We observed that recombinant human EN protein oligomerizes during downstream production processing at room temperature, leading to protein aggregation in aqueous solution (protein oligomerization / aggregation was detected by non-reducing SDS-Page electrophoresis assay).

[0028] In this regard, the present inventors have devised a formulation that can inhibit or reduce the aggregation of EN protein in aqueous solution and have developed a stabilized aqueous composition of Engrailed (EN) protein. Advantageously, the composition developed by the present inventors is suitable for intrathecal administration.

[0029] In one embodiment, the present invention provides a stabilized aqueous composition of Engrailed (EN) protein comprising an effective amount or dose of EN protein, a stabilizing concentration of reduced glutathione, a osmolality adjusting agent, and a buffering agent that provides the formulation with a pH of less than 7, preferably less than 6, more preferably less than 5, advantageously about 4.

[0030] Unexpectedly, the present inventors have observed that aggregation of EN proteins in aqueous compositions can be inhibited or reduced in the presence of reduced glutathione.

[0031] In a preferred embodiment, the stabilized aqueous composition of Engrailed (EN) protein of the present invention contains reduced glutathione at a concentration of about 10 μM to about 100 μM. In one embodiment, the composition of the present invention contains reduced glutathione at about 50 μM. In a preferred embodiment, the reduced glutathione is reduced L-glutathione.

[0032] The present inventors have also surprisingly shown that the presence of an osmotic agent in an EN protein aqueous composition improves the stabilization of the EN protein, ie, inhibits or reduces aggregation of the EN protein.

[0033] In one embodiment, the stabilized aqueous composition of Engrailed (EN) protein has an osmolality in the range of about 250 to about 350 mOsmol / L, more specifically in the range of about 270 mOsmol / L to about 320 mOsmol / L. Examples of suitable osmolality adjusters include, but are not limited to, sugars such as glucose (e.g., dextrose), poly(glucose), fructose, dextran, glycerol, sorbitol, mannitol, trehalose, mannose, polyanions, and combinations thereof. Other osmolality adjusters may be viable alternatives, such as small amino acids, which are non-sugar agents that can function as equivalents.

[0034] In a preferred embodiment, the stabilized aqueous composition of Engrailed (EN) protein is for intrathecal administration, and the osmolality modifier is preferably dextrose. In a preferred embodiment, the osmolality modifier is dextrose. In one embodiment, the stabilized aqueous composition of Engrailed (EN) protein of the present invention comprises dextrose at a concentration of about 220 to about 320 mM, advantageously about 240 mM to about 290 mM. In a preferred embodiment, it is about 250 mM.

[0035] Advantageously, the present inventors have also found that the inclusion of a magnesium salt substantially reduces aggregation of the EN composition. Thus, in one embodiment, the stabilized aqueous composition of Engrailed (EN) protein of the present invention comprises a magnesium salt, preferably MgCl2. 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 stabilized aqueous composition of Engrailed (EN) protein of the present invention comprises about 2.13 mM MgCl2.

[0036] Advantageously, the aqueous EN protein composition of the present invention comprises dextrose in combination with magnesium chloride.

[0037] Advantageously, the inventors have also found that the inclusion of a low concentration (i.e., less than 0.2%) of a nonionic surfactant substantially reduces adhesion to the support and aids in stabilizing the EN protein. Thus, in one embodiment, the stabilized aqueous composition of the Engrailed (EN) protein of the present invention includes a stabilizing concentration of a nonionic detergent. 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, various poloxamers or pluronics, including polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and 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%.

[0038] In one embodiment, the present invention provides an effective amount or dose of an EN protein; Approximately 10 μM to approximately 100 μM of reduced glutathione, about 240 to about 290 mM of an osmolality adjuster, preferably dextrose; About 0.1 mM to about 3 mM MgCl2, 0-0.01% polysorbate 20, a buffering agent to maintain a pH of about 4; The present invention provides a stabilized aqueous composition of Engrailed (EN) protein, comprising:

[0039] In one embodiment, the present invention provides a stabilized aqueous composition of Engrailed (EN) protein comprising an effective amount or dose of EN protein of about 0.05 mg / mL to about 1 mg / mL. In some embodiments, 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, EN is present at a concentration of about 0.1 mg / mL to about 0.9 mg / mL. In some embodiments, EN may be present at about 0.6 mg / mL.

[0040] In one embodiment, the buffer is sodium acetate / acetic acid.

[0041] In an advantageous aspect, the present invention provides a method for producing a medicament for the treatment of a medicament comprising: Approximately 0.6 mg / ml of EN protein; Approximately 250mM dextrose, Approximately 2.13 mM MgCl2, Approximately 50 μM reduced glutathione, Approximately 0.005% polysorbate 20, about 5 mM sodium acetate / acetic acid to maintain a pH of about 4; The present invention provides a stabilized aqueous composition of Engrailed (EN) protein, comprising:

[0042] As used herein, the term "EN" refers to Engrailed protein. In the context of the present invention, EN protein includes any EN protein from a mammal, such as a primate, human, monkey, rabbit, pig, cow, or rodent, preferably a human, such as Engrailed 1 (EN1) or Engrailed 2 (EN2), and mixtures thereof, as well as biologically active derivatives thereof. Active mutants and variants of EN proteins are also encompassed, as are functional fragments and fusion proteins of EN proteins. Exemplary EN1 proteins or polypeptides include, but are not limited to, the human EN1 protein or polypeptide having the primary amino acid sequence annotated under GenBank Accession No. AAA53502.2 or NCBI NP_001417.3. Exemplary EN2 proteins or polypeptides include, but are not limited to, the human EN2 protein or polypeptide having the primary amino acid sequence annotated under GenBank Accession No. AAA53504.2 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.

[0043] As used herein, the term "biologically active derivative" refers to any polypeptide having substantially the same biological function as EN. The polypeptide sequence of a biologically active derivative may contain one or more amino acid deletions, additions, and / or substitutions, the absence, presence, and / or substitution of which does not substantially adversely affect the biological activity of the polypeptide.

[0044] As used herein, the terms "EN" and "biologically active derivative" also include polypeptides obtained by recombinant DNA technology or synthetically obtained polypeptides, 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) producing recombinant DNA by genetic engineering (e.g., by reverse transcription of RNA and / or amplification of DNA), (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by transfection, i.e., electroporation or microinjection, (iii) culturing the transformed cells (e.g., continuously or batchwise), (iv) expressing EN (e.g., constitutively or upon induction), and (v) isolating the EN (e.g., from the culture medium or by harvesting the transformed cells), and (vi) obtaining substantially purified recombinant EN (e.g., by anion exchange chromatography or affinity chromatography).

[0045] As used herein, "effective amount or dose" or "sufficient amount or dose" refers to the amount of a compound for which it is administered to produce the effect. The exact amount will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques.

[0046] As used herein, the term "about" indicates an approximate range of plus or minus 10% from a specified value. For example, the term "about 20%" encompasses a range of 18-22%. As used herein, about also includes the exact amount. Thus, "about 20%" also means "about 20%" and "20%."

[0047] As used herein, the expression "aqueous composition" refers to a composition that includes water as a solvent.

[0048] As used herein, "storage" means that the compositions of the invention are not administered to a subject immediately upon preparation, but are maintained under specified conditions (e.g., at a specified temperature) for a period of time prior to use. For example, liquid or lyophilized compositions can be maintained under various temperatures, such as frozen (≦−65°C or −15°C to −25°C), refrigerated (0°C to 10°C), or room temperature (e.g., up to 32°C), for days, weeks, months, or years before administration to a subject.

[0049] According to particular embodiments, the compositions of the present invention reduce or delay dimerization, oligomerization, and / or aggregation of EN proteins over time. In one embodiment, the stabilized aqueous Engrailed (EN) protein compositions of the present invention are stable when stored at temperatures up to at least about -65°C for at least about 6 months. In other embodiments, the compositions provided herein retain significant EN activity when stored for extended periods of time.

[0050] As used herein, a "stabilized composition of a protein" or a "stable protein in a composition" means that the EN protein maintains the same oligomerization state, preferably remains as a monomer, and / or does not form aggregates in the composition. Those 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 "stabilized composition of an EN protein" means that the amount of EN protein dimers, oligomers, and / or aggregates in the composition is less than about 5%, preferably less than about 3%, of the total EN protein.

[0051] In a related aspect, the present invention provides a stabilized lyophilized composition of EN protein, wherein the formulation is lyophilized from a stabilized aqueous composition of Engrailed (EN) protein provided herein. Lyophilization can be performed according to methods known in the art.

[0052] Generally, the stabilized aqueous compositions of Engrailed (EN) proteins provided herein are suitable for pharmaceutical administration.

[0053] In preferred embodiments, the EN compositions provided herein are sterile and contain low endotoxin levels (according to the European Pharmacopoeia).

[0054] In some embodiments, the EN compositions provided herein may further comprise one or more pharmaceutically acceptable excipients, carriers, and / or diluents. Additionally, the compositions provided herein may further comprise other drugs, carriers, adjuvants, diluents, tissue penetration enhancers, solubilizers, etc. Methods for preparing compositions and formulations for pharmaceutical administration are known to those skilled in the art.

[0055] The stabilized aqueous compositions of Engrailed (EN) protein provided herein can be formulated for administration in known manner, as a bolus or by continuous infusion over a period of time, and are particularly adapted for administration by intrathecal or intracerebroventricular routes.

[0056] The stability of aqueous compositions of Engrailed (EN) protein can be measured by one or more biophysical properties of the EN protein in the formulation.Non-limiting examples of properties that can be used to evaluate 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 know other stability measurement methods that can be used to evaluate the stability of EN formulations, including but not limited to size exclusion chromatography (SEC), dynamic light scattering or static light scattering, RP-HPLC, ion exchange chromatography, polyacrylamide gel electrophoresis, non-reducing SDS-Page electrophoresis, etc.

[0057] In certain embodiments, aqueous compositions of Engrailed (EN) proteins of the invention may be stable for extended periods of time when stored at certain temperatures, e.g., 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, an extended period of time is at least about one week. In other embodiments, an extended period of time may include at least about two weeks, or at least about three weeks, or at least about one month, or at least about two months, or at least about three, four, five, six, seven, eight, nine, ten, eleven, twelve, fourteen, sixteen, or eighteen months. In still other embodiments, compositions of the invention may be stable for at least about two, three, four, five, or more years.

[0058] In one embodiment, the present invention relates to a stabilized 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 stabilized aqueous composition of Engrailed (EN) protein is substantially free of aggregated EN protein, dimeric EN protein, oligomeric EN protein, or mixtures thereof when it contains less than 5%, preferably less than 3%, of aggregated EN protein, dimeric EN protein, oligomeric EN protein, or mixtures thereof, or less than 2%, or less than 1% of aggregated EN protein, dimeric EN protein, oligomeric EN protein, or mixtures thereof.

[0059] In another embodiment of the invention, the stabilized aqueous Engrailed (EN) protein compositions of the invention have an EN protein population consisting of at least about 95% EN protein monomers after extended storage, hi other embodiments, the EN protein compositions have at least about 97% EN protein monomers, or at least about 98%, 99%, or more EN protein monomers.

[0060] 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.

[0061] The 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, mammalian cells, etc. Examples of eukaryotic cells include, but are not limited to, mammalian cells such as CHO, COS, HEK293, BHK, SK-Hep-1, and HepG2, insect cells such as SF9, SF21, S2, and High Five cells, and yeast cells such as Saccharomyces, Pichia, or Schizosaccharomyces.

[0062] In one embodiment, the cells can be any bacterial cell that can be cultured to produce the desired EN protein, preferably in a manufacturing process (i.e., at least 1 liter). In a preferred embodiment, the cell line is an E. coli cell line.

[0063] A wide variety of vectors can be used for the expression of an EN protein (e.g., EN1), and can be selected from eukaryotic and prokaryotic expression vectors. In certain embodiments, a plasmid vector is intended for use in expressing an EN protein (e.g., EN1). The plasmid comprises a nucleotide sequence encoding an EN protein (e.g., EN1) operably linked to one or more control sequences, such as a promoter, e.g., an inducible promoter. According to certain embodiments, a viral vector is used to introduce a nucleotide sequence encoding an EN protein (e.g., EN1) into a host cell for expression. The viral vector comprises a nucleotide sequence encoding an EN protein (e.g., EN1) operably linked to one or more control sequences, e.g., a promoter. Non-limiting examples of viral vectors that can be used to deliver nucleic acids include adenoviral vectors, AAV vectors, and retroviral vectors. Non-limiting examples of vectors for prokaryotic expression include plasmids such as pRSET, pET, and pBAD, and promoters used in prokaryotic expression vectors include lac, trc, trp, recA, and araBAD. Examples of vectors for eukaryotic expression include: (i) vectors such as pAO, pPIC, pYES, and pMET, which use promoters such as AOX1, GAP, GAL1, and AUG1 for expression in yeast; (ii) vectors such as pMT, pAc5, pIB, pMIB, and pBAC, which use promoters such as PH, p10, MT, Ac5, OpIE2, gp64, and polh for expression in insect cells; and (iii) vectors such as pSVL, pCMV, pRc / RSV, pcDNA3, and pBPV for expression in mammalian cells, as well as vectors derived from viruses such as vaccinia virus, adeno-associated virus, herpes virus, and retrovirus, which use promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin.

[0064] In certain embodiments, EN expression may involve the use of cell culture systems operated in batch or continuous operation modes. For example, when batch cell cultures are utilized, they may be operated in single-batch, fed-batch, or repeated-batch modes. Similarly, continuous cell cultures may be operated, for example, under perfusion, turbodostat, or chemostat modes. Batch and continuous cell cultures may be performed under either suspension or adherent conditions. When operating under suspension conditions, cells are freely suspended and mixed within the culture medium. Alternatively, under adherent conditions, cells are attached to a solid phase, such as microcarriers, porous microcarriers, disk carriers, ceramic cartridges, hollow fibers, flat sheets, and gel matrices.

[0065] Batch culture is typically a large-scale cell culture in which a cell inoculum is grown to maximum density in a tank or fermenter, harvested, and processed as a single batch. Fed-batch culture is typically a batch culture in which either fresh nutrients (e.g., growth-limiting substrates) or additives (e.g., product precursors) are supplied to maintain cell growth and reach higher biomass. The feed solution is usually highly concentrated to avoid dilution of the bioreactor. In repeated batch culture, cells are placed in culture medium and grown to the desired cell density. To avoid the onset of a decline phase and cell death, the culture is then diluted with complete growth medium before the cells reach maximum density. The amount of dilution and frequency of dilution vary widely and depend on the growth characteristics of the cell line and the convenience of the culture process. This process can be repeated as many times as necessary, and the volume of the culture increases stepwise with each dilution, unless cells and medium are discarded during subculture. The increase in volume can be accommodated by having a reactor of sufficient size to allow dilution within the vessel or by splitting the diluted culture into several vessels. The rationale for this type of culture is to maintain cells in an exponentially growing state. In certain embodiments, the EN protein may be recovered after harvesting the supernatant of the batch culture.

[0066] Methods for expression and purification of recombinant EN proteins are also known in the art.

[0067] Yet another aspect of the present invention relates to a method for purifying recombinant EN protein, in one embodiment, the recombinant EN protein is expressed in recombinant bacteria and purified from the resulting conditioned media by a series of chromatography and filtration / ultrafiltration steps.

[0068] In one embodiment, a method for providing an EN protein (e.g., EN1) composition is provided, comprising: (a) culturing cells having a nucleic acid encoding an EN protein (e.g., EN1) in a culture medium; (b) performing a lysis step to release the EN protein in supernatant n; (c) clarifying the supernatant containing the EN protein; (d) purifying the EN protein by a series of chromatography steps and filtration / ultrafiltration steps; and e) formulating the EN protein (e.g., EN1) composition according to the formulations provided herein, thereby providing an EN protein (e.g., EN1) formulation.

[0069] In one embodiment, the present invention provides a method for producing a stabilized aqueous composition of 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 having a nucleic acid encoding the EN protein (e.g., EN1) in a culture medium; (ii) performing a lysis step to release the EN protein in the 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 dose of EN protein, an osmotic agent, a stabilizing concentration of reduced glutathione, and a buffering agent that provides the formulation with a pH of less than 7, preferably less than 6, more preferably less than 5, and advantageously about 4.

[0070] In one embodiment, the present invention provides a method for producing a stabilized aqueous composition of 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 having a nucleic acid encoding the EN protein (e.g., EN1) in a culture medium; (ii) performing a lysis step to release the EN protein in the 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 dextrose; (c) about 50 μM reduced glutathione; (d) about 2.13 mM MgCl2; (e) 0.005% polysorbate 20; and (f) a buffering agent to maintain a pH of less than 7, preferably less than 6, more preferably less than 5, and advantageously about 4.

[0071] Administration and Treatment Methods The composition of the present invention can be administered for therapeutic or preventive treatment.Generally, for therapeutic application, the preparation is administered to patients with diseases or conditions related to neuronal death, preferably motor neuron death, preferably amyotrophic lateral sclerosis (ALS) in a therapeutically effective dose.The effective preparation and amount for these uses depend on the severity of the disease or condition and the general state of the patient's health.Single or multiple administrations of the preparation can be administered according to the dosage and frequency that the patient requires and tolerates.

[0072] For the purposes of the present invention, a "patient" or "subject" includes both humans and other animals, particularly mammals. The compositions, formulations and methods are applicable to both human therapy and veterinary use. In certain 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 by the present invention.

[0073] The preferred delivery route is intrathecal or intraventricular administration, with intrathecal being the more preferred route of delivery. The volume of intrathecal injection ranges from 0.1 to 20 mL.

[0074] Doses ranging from 0.01 mg to 18 mg of EN protein may be administered intrathecally or intracerebrally, more specifically, intrathecally to patients with a disease or condition associated with neuronal death, preferably motor neuron death, preferably amyotrophic lateral sclerosis (ALS).

[0075] According to particular embodiments, the stabilized aqueous composition of Engrailed (EN) protein may be administered monthly or bimonthly.

[0076] According to one particular embodiment, a stabilized aqueous composition of an Engrailed (EN) protein (preferably an EN1 protein) of the present invention is administered to a patient having a disease or condition associated with neuronal death, preferably motor neuron death, preferably amyotrophic lateral sclerosis (ALS). According to a particular embodiment, administration of a stabilized aqueous composition of an Engrailed (EN) protein (preferably an EN1 protein) of the present invention to a patient having a disease or condition associated with neuronal death, preferably motor neuron death, preferably amyotrophic lateral sclerosis (ALS), results in the control of the progression of motor neuron death, preferably the cessation of motor neuron death.

[0077] In another aspect of the present invention, a 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 kit comprises an EN protein composition as described above. In some embodiments, the kits provided herein may comprise one or more doses of the liquid or lyophilized compositions provided herein. According to particular embodiments (e.g., kits comprising a lyophilized EN protein composition), the kit further comprises a liquid suitable for reconstituting the liquid formulation, such as artificial CSF or a pharmaceutically acceptable buffer. In some embodiments, the kit may comprise the EN protein composition pre-packaged in a syringe for administration by a medical professional or for home use.

[0078] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or variations in light thereof will be suggested to those skilled in the art and are within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. [Example]

[0079] The buffer exchange step has been shown to promote oligomerization of the EN protein during downstream protein production processes.

[0080] To screen formulations with different EN proteins and oligomerization levels of EN proteins, buffer exchange small-scale models (dialysis tubing and ultrafiltration centrifuge tubes) have been selected and used.

[0081] Two main analytical methods have been used to measure the oligomerization of EN proteins: size exclusion chromatography (SEC) HPLC and non-reducing electrophoresis SDS-Page with Coomassie blue staining.

[0082] Example 1: Effect of acidic pH The effect of acidic pH on the oligomerization of EN1 protein was analyzed in two different test models (dialysis or small-scale ultrafiltration) and with different compositions of EN1 protein (Figure 1): - EN1 protein composition containing 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl2, pH 4 after dialysis; - EN1 protein composition containing 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl2, pH 4 after small-scale ultrafiltration; - EN1 protein composition containing 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl2, pH 5 after dialysis; - EN1 protein composition containing 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl2, pH 5 after small-scale ultrafiltration; - EN1 protein composition containing 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl2, pH 6 after dialysis; - an EN1 protein composition comprising 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl2 pH 6 after small-scale ultrafiltration, Oligomerization of EN1 proteins was assessed using non-reducing SDS-Page electrophoresis. Figure 1 shows that a pH 4 composition limits EN1 protein oligomerization compared to pH 5 and pH 6.

[0083] Similarly, oligomerization of EN1 protein in the dialysis composition (see above) was evaluated using SEC-HPLC with high molecular weight (HMW) detected at 280 nm (Figure 2). The results confirmed that the pH 4 composition had a lower oligomer peak and prevented EN1 protein oligomerization more efficiently than the pH 5 and pH 6 compositions.

[0084] Example 2: Effect of glutathione The oligomerization of EN1 protein was measured by SEC-HPLC in a composition containing EN1 protein, 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl2, pH 5, in the absence or presence of 50 μM reduced glutathione (Figure 3).

[0085] FIG. 3 shows that in the presence of glutathione in the EN1 protein composition, the amount of EN1 oligomers is dramatically reduced.

[0086] Example 3: Effect of glutathione concentration The oligomerization of EN1 protein was measured by SEC-HPLC in a composition containing EN1 protein, 5 mM citrate / phosphate, 250 mM dextrose, 2.13 mM MgCl, pH 5, and various concentrations of reduced glutathione (1 μM, 50 μM, or 100 μM) (Figure 4).

[0087] FIG. 4 shows that the presence of 50 μM or 100 μM glutathione in the composition prevents oligomerization of the EN1 protein.

[0088] Similar analyses and conclusions were observed using non-reducing SDS-Page electrophoresis.

Claims

1. A stabilized aqueous composition of Engrailed (EN) protein, comprising an effective amount or effective dose of EN protein, a stabilizing concentration of reduced glutathione, an osmotic agent, and a buffer exhibiting a pH of less than 7.

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

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

4. 4. The composition of claim 1, wherein the concentration of the osmotic agent is from about 220 to about 320 mM.

5. 5. The composition of claim 1, wherein the osmolality adjusting agent is dextrose.

6. The composition of claim 1 , wherein the composition comprises a magnesium salt.

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

8. The composition of claim 1 , wherein the composition comprises a non-ionic detergent.

9. 9. The composition of claim 8, wherein the nonionic surfactant is polysorbate 20.

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

11. The composition comprises: an effective amount or dose of an EN protein; - about 10 μM to about 100 μM reduced glutathione; about 240 to about 290 mM of an osmolality adjusting agent; about 0.1 mM to about 3 mM MgCl ２ and, 0 to 0.01% polysorbate 20, a buffer to maintain a pH of about 4; 11. The composition of claim 1, comprising:

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

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