Pharmaceutical composition for treating, preventing or ameliorating GM1 ganglioside deposition or Morquio syndrome B and method of administration thereof

Abstract

The invention relates to a beta-galactosidase 1 (GLB1) recombinant protein with a truncated C terminal and a preparation method of the beta-galactosidase 1 (GLB1) recombinant protein. A pharmaceutical composition for the treatment, prevention or amelioration of GM1 ganglioside deposition or Morquio Syndrome B, the pharmaceutical composition comprising the above-mentioned protein, and a pharmaceutical composition for the treatment, prevention or amelioration of GM1 ganglioside deposition or Morquio Syndrome B; a method of treating, preventing or ameliorating GM1 ganglioside deposition or Morquio Syndrome B, the method comprising administering the pharmaceutical composition described above to a subject; and a method for producing the protein. In one embodiment of the present disclosure, C-terminally truncated recombinant GLB1 proteins do not have the problem of heterogeneity protein truncation, exhibit enzymatic activity equivalent to full-length GLB1 proteins, and increase GLB1 enzymatic activity in patient blood. The disclosure shows that the protein can be used for treating, preventing or improving GM1 ganglioside deposition or Morquio syndrome B.

Description

Technical Field

[0001] The present disclosure relates to a C-terminally truncated β-galactosidase-1 (GLB1) recombinant protein; a pharmaceutical composition for treating, preventing or improving GM1 gangliosidosis or Morquio syndrome B, the pharmaceutical composition comprising the above protein; and a method for treating, preventing or improving GM1 gangliosidosis or Morquio syndrome B, the method comprising administering the above pharmaceutical composition to a subject. Background Art

[0002] GM1 gangliosidosis is an inherited central nervous system (CNS) disease that destroys nerve cells in the central and peripheral nerves. It is caused by mutations in the GLB1 gene that result in impaired GLB1 enzyme activity in the lysosomes. The genetic cause of GM1 gangliosidosis is an autosomal recessive mutation in the GLB1 gene, which produces β-galactosidase-1. Due to insufficient β-galactosidase-1 enzyme activity in the lysosomes, GM1 gangliosidosis impedes the degradation of oligosaccharides, ultimately leading to the accumulation of galactose-rich oligosaccharides and keratan sulfate in the brain and internal organs, causing the disease.

[0003] GM1 gangliosidosis can be divided into three phenotypes based on the age of onset: infantile, juvenile, and adult. Infantile GM1 gangliosidosis (type I) presents with symptoms such as hepatosplenomegaly, facial swelling, thick upper lip, maxillary hyperplasia, gingival hyperplasia, and macroglossia. At about six months of age, changes in the skeletal system (e.g., spine) can be observed, similar to those seen in Hurler syndrome. Juvenile GM1 gangliosidosis (type II) has no distinguishing external features. Patients usually appear normal until the age of one year, but then they gradually lose control, exhibit autistic behavior, and / or become retarded. These symptoms may progress to ataxia, seizures, and / or spastic paralysis. If untreated, patients usually die before the age of ten and may develop symptoms such as vertebral fractures. Adult GM1 gangliosidosis (type III) has no distinguishing external features. This form develops during childhood and typically presents with extrapyramidal symptoms resembling dystonia, Parkinson disease, and ataxia associated with atypical cerebellar atrophy, as well as severe dementia.

[0004] The incidence of GM1 gangliosidosis is estimated to be 1 in 100,000 to 200,000 live births, with the infantile form (type I) reported more frequently than the other phenotypes.

[0005] Morquio syndrome is an autosomal recessive genetic disorder. Morquio syndrome type B is caused by a deficiency of β-galactosidase, resulting in a defect in the degradation of keratan sulfate. The symptoms of Morquio syndrome become apparent at 18 to 24 months of age, and if left untreated, various complications can lead to the death of the patient, mainly before the age of 20.

[0006] The physical characteristics of patients with Morquio syndrome include, for example, short stature, rough face, low nasal bridge, wide nasal wings, thick lips, macrocephaly, corneal opacity, short neck, glaucoma, retinal degeneration, and hearing loss. In addition, patients with Morquio syndrome may have skeletal dysplasia, resulting in a protruding sternum, abnormal chest shape, and / or severe kyphosis due to spinal abnormalities, which may cause respiratory failure or spinal cord compression. Other symptoms of Morquio syndrome also include inguinal hernia, hepatomegaly, and dental caries due to hypoplasia of the odontoid process.

[0007] Currently, there is no effective treatment for GM1 gangliosidosis and Morquio syndrome, and only symptomatic treatment can be carried out. Therefore, the treatment needs for GM1 gangliosidosis and Morquio syndrome are urgent.

[0008] Enzyme replacement therapy (ERT) is a method of treating diseases by systematically administering natural or recombinant proteins and / or enzymes to a subject, and is considered one of the methods for treating diseases caused by enzyme defects or deficiencies.

[0009] In this context, the present disclosure relates to a C-terminally truncated GLB1 recombinant protein (hereinafter referred to as "GLB1S"), which does not have the problem of heterologous protein truncation and has high enzyme activity. The present disclosure demonstrates that when the recombinant protein GLB1S is administered to patients, it has significant effects in treating, preventing, or improving GM1 gangliosidosis or Morquio syndrome B. Summary of the Invention

[0010] The present disclosure relates to a GLB1 recombinant protein (GLB1S) having a C-terminal truncation compared to GLB1. In one embodiment, the present disclosure relates to a polynucleotide encoding the recombinant protein, an expression vector containing the polynucleotide, and / or a host cell containing the polynucleotide or the expression vector.

[0011] The present disclosure aims to provide a pharmaceutical composition for treating, preventing, and / or improving GM1 gangliosidosis or Morquio syndrome B or at least one of its symptoms, the pharmaceutical composition containing the above recombinant protein (GLB1S).

[0012] The present disclosure relates to a method for treating, preventing or ameliorating GM1 gangliosidosis or Morquio syndrome B or at least one symptom thereof, the method comprising administering a pharmaceutical composition comprising the recombinant protein (GLB1S).

[0013] In one embodiment of the present disclosure, the GLB1 recombinant protein (GLB1S) with C-terminal truncation does not have the problem of protein truncation during the protein manufacturing process. In one embodiment, the recombinant truncated GLB1 has an enzyme activity equivalent to that of the full-length GLB1 protein. In one embodiment, the recombinant truncated GLB1 has an enzyme activity at least equivalent to that of the full-length GLB1 protein. In one embodiment, the short form of the recombinant GLB1 protein (GLB1S) may increase the enzyme activity of GLB1 in the patient's blood. In one embodiment of the present disclosure, compared with the full-length GLB1 protein, the short form of the recombinant GLB1 protein (GLB1S) or a pharmaceutical composition comprising the same can be used as a drug with better effectiveness for treating, preventing or improving GM1 gangliosidosis or Morquio syndrome B.

[0014] The present disclosure relates to a method for producing a protein comprising an amino acid sequence as shown in SEQ ID NO: 1, the method comprising: a) introducing a target gene encoding a protein comprising an amino acid sequence as shown in SEQ ID NO: 1 into a vector; b) transfecting a host cell with the vector; c) culturing the transfected cells and collecting the cell culture supernatant; d) purifying the supernatant to obtain the protein.

[0015] In one embodiment of the present disclosure, the host cell is a Chinese Hamster Ovary (CHO) cell.

[0016] The present disclosure relates to a protein produced by the above method for producing a protein comprising the amino acid sequence shown in SEQ ID NO: 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the detailed workflow for GLB1S purification.

[0018] Figure 2 The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified recombinant GLB1S obtained by 50 L scale culture of CHO-K1 cells are shown.

[0019] Figure 3A and 3B The results of size exclusion-high performance liquid chromatography (SEC-HPLC) analysis of purified recombinant GLB1S obtained by 50 L scale cultivation of CHO-K1 cells.

[0020] Figure 4Aand 4B This is the result of reverse phase-high performance liquid chromatography (RP-HPLC) analysis of purified recombinant GLB1S obtained by 50 L scale cultivation of CHO-K1 cells.

[0021] Figure 5 Shown are the genotyping results of GLB1 KO mice obtained by PCR screening.

[0022] Figures 6A to 6F The results of analyzing the keratan sulfate content in the liver tissue of mice in each group (G2 to G7) on days 7, 14, and 28 are shown.

[0023] Figure 7 The results of analyzing the keratan sulfate content in the liver tissues of mice in groups G2 to G7 on day 7 are shown.

[0024] Figure 8 The results of analyzing the keratan sulfate content in the liver tissues of mice in groups G2 to G7 on day 14 are shown. DETAILED DESCRIPTION

[0025] The various embodiments, aspects or embodiments of the present disclosure are intended to illustrate and / or explain the present application, and are not intended to be limiting. The present disclosure includes various modifications, equivalents and alternatives to each embodiment, aspect or embodiment described in the present application, and any possible combination of all or part of each embodiment, aspect or embodiment described in the present application. Those of ordinary skill in the art will understand and recognize the above-mentioned modifications, equivalents and alternatives or the combinations. The scope of rights of the present disclosure is not limited to the various embodiments, aspects or embodiments described below, nor is it limited to the specific description of the embodiments, aspects or embodiments.

[0026] Unless otherwise defined, all technical and scientific terms used in this application generally have the common meaning understood by those of ordinary skill in the art. All terms used in this application are used to describe and / or explain the present disclosure and are not intended to limit the scope of the present disclosure. Certain terms are discussed in this application to provide additional guidance for describing and / or explaining the compositions and methods of the present disclosure.

[0027] definition

[0028] In the present disclosure, unless the context clearly indicates otherwise, the singular forms "a", "an", "the" and singular words include plural references, and the singular forms and words listed in the claims also apply. For example, "a compound" includes a plurality of such compounds, and "a compound A" includes a plurality of compound A.

[0029] The term "and / or" means any one or more, any combination or all of the items associated therewith. The terms "include," "including," "have," "contain," or variations thereof are interpreted as open ended in a manner similar to the term "comprising." The terms "include," "including," "have," "contain," and variations thereof used in this disclosure are understood to be open ended terms, implying that other embodiments may be included unless otherwise expressly stated. The term "consisting of," and variations thereof used in this disclosure are understood to be closed ended terms, excluding any components not listed in the description of the embodiment (i.e., "including and limited to").

[0030] All ranges listed in this application may include any and all possible subranges and combinations thereof. The ranges listed include each specific value, integer or decimal within the range. It will be readily appreciated by those of ordinary skill in the art that any range listed fully describes and / or supports its subranges, including but not limited to equal parts, one-third, one-quarter, one-fifth or one-tenth of the range.

[0031] The terms "greater than," "more than," "at least," "or more," "less than," "up to," and the like are inclusive of the numbers recited, and these terms refer to ranges that can be broken down into sub-ranges as described above. The specific values ​​listed for a range are for illustrative purposes only and are not intended to be limiting, nor do they exclude other values ​​within the range.

[0032] Unless otherwise stated, the terms "about" and "approximately" generally include values ​​close to the listed range or value, which are within an acceptable error range known to those skilled in the art. An acceptable error range can be determined based on the nature or precision of the measurement or manufacturing process. In one embodiment, an acceptable error range can be determined based on the functional equivalence of a composition or method or embodiment. In one embodiment, in the context of the numerical values ​​or ranges listed in the present disclosure, the terms "about" or "approximately" may refer to a change of ±25%, ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1% of the provided value. In another embodiment, particularly in biological systems, the terms "about" and "approximately" may refer to a value within an order of magnitude, within 9 times, within 8 times, within 7 times, within 6 times, within 5 times, within 4 times, within 3 times or within 2 times of a given value. Unless otherwise stated, all numerical values ​​provided in this application are approximate values. In addition, these numerical values ​​inherently contain the variability that is necessarily generated by their measurement.

[0033] In the present disclosure, the term "pharmaceutical composition" may refer to a pharmaceutical preparation form that allows the biological activity of the active ingredients contained in the composition to be effectively exerted. In one embodiment, the pharmaceutical composition is a composition comprising one or more active ingredients that can be administered to a patient, and does not further comprise unacceptable toxic components.

[0034] In the present disclosure, the term "pharmaceutically acceptable" may refer to the property of a substance that can be used to prepare a generally safe, non-toxic and biologically or otherwise desirable pharmaceutical composition, and is suitable for human pharmaceutical use and / or veterinary use. In one embodiment, the term "pharmaceutically acceptable" means approved or approvable by a governmental regulatory agency, or listed in a recognized pharmacopoeia for use in humans and / or animals.

[0035] In the present disclosure, the term "pharmaceutically acceptable excipient" refers to any ingredient that is not therapeutically active (inert) and non-toxic. In one embodiment, a pharmaceutically acceptable excipient may include, but is not limited to, for example, a binder, a filler, a solvent, a buffer, an osmotic pressure regulator, a stabilizer, an antioxidant, a surfactant, or a lubricant, which are configured to formulate a drug product.

[0036] As used herein, the term "pharmaceutically acceptable carrier" refers to a component other than the active ingredient in a pharmaceutical composition that is non-toxic to a subject. In one embodiment, the pharmaceutically acceptable carrier may include, but is not limited to, for example, a buffer, an excipient, a stabilizer or a preservative.

[0037] In the present disclosure, the terms "pharmaceutically effective dose", "pharmaceutically effective amount", "administered dose", "administered amount", "therapeutically effective dose", "therapeutically effective amount", "effective dose" or "effective amount" of a pharmaceutical composition may refer to an amount of the pharmaceutical composition (administered for a desired duration) sufficient to achieve a therapeutic response or effect, a desired local or systemic therapeutic result, or a desired preventive result. In one embodiment, the above terms refer to an amount of a pharmaceutical composition that, when administered to a subject, (i) treats or prevents a disease, condition, or disorder described herein, (ii) alleviates, reduces, stops, attenuates, improves, or eliminates one or more symptoms of a disease, condition, or disorder described herein, or (iii) prevents or delays the onset of one or more symptoms of a disease, condition, or disorder described herein.

[0038] In the present disclosure, the term "subject" refers to a mammal. Mammals may include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In one embodiment, the subject may be a human.

[0039] One aspect of the present disclosure relates to a protein comprising an amino acid sequence as shown in SEQ ID NO: 1. The amino acid sequence as shown in SEQ ID NO: 1 in the present disclosure is a recombinant protein (GLB1S) truncated at the C-terminus of β-galactosidase-1 (GLB1) protein.

[0040] One aspect of the present disclosure provides a protein comprising an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% homology to the amino acid sequence as shown in SEQ ID NO: 1. In one embodiment, the protein may include, but is not limited to, any amino acid sequence having homology to the amino acid sequence as shown in SEQ ID NO: 1 and having the same or substantially the same or corresponding activity as the protein comprising the amino acid sequence as shown in SEQ ID NO: 1. In one embodiment, the protein may include any amino acid sequence having one or more amino acid deletions, modifications, substitutions or additions, as long as the amino acid sequence has homology to the amino acid sequence as shown in SEQ ID NO: 1; the amino acid sequence has one or more activities of the protein comprising the amino acid sequence as shown in SEQ ID NO: 1; or these deletions, modifications, substitutions or additions do not significantly affect the function of the protein. For example, one or more amino acids may be deleted, modified, substituted or added to optimize and / or facilitate the protein expression and extraction process.

[0041] In the present disclosure, "homology" may refer to the degree of identity of nucleic acid or amino acid residues in an amino acid sequence or a gene nucleic acid sequence encoding a protein, after aligning the two sequences as closely as possible within a specific comparison region. If the homology between two genes is high enough, the expression products of the two genes may have the same or similar activity. The percentage (%) of the sequence identity can be determined using a known sequence comparison program (e.g., Blast (NCBI)).

[0042] In one embodiment of the present application, the protein can be expressed and produced by known methods in the field of the present disclosure. Host cells suitable for expressing and producing the protein described in the present disclosure include, but are not limited to, bacterial cells, such as Escherichia coli, Streptomyces, and Salmonella typhimurium; yeast cells, such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris; insect cells, such as Drosophila, Spodoptera Sf9 cells; animal cells, such as CHO, COS, NS0, 293, Bowes melanoma cells; or plant cells.

[0043] In one embodiment, host cells suitable for expressing and producing proteins described in the present disclosure include vertebrate host cells and eukaryotic cells described herein. Examples of mammalian host cell lines include, but are not limited to, monkey kidney cells (CV1), monkey kidney CV1 cells transformed by SV40 (COS-7), human embryonic kidney cells (293 cells), baby hamster kidney cells (BHK), Chinese hamster ovary cells (CHO), mouse supporting cells (TM4), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), dog kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse breast tumor cells (MMT 060562), TRI cells, MRC 5 cells, and FS4 cells.

[0044] In one embodiment, the host cells used to produce the proteins described in the present disclosure can be cultured in various culture media. Commercial culture media that can be used for host cell culture include, but are not limited to, for example, Ham F10 culture medium, minimum essential medium (MEM), RPMI-1640, Dulbecco's modified Eagle medium (DMEM), and any other culture media known to those skilled in the art. Any of these culture media can be supplemented with one or more hormones and / or growth factors (e.g., insulin, transferrin, or epidermal growth factor), one or more salts (e.g., sodium chloride, calcium, magnesium, or phosphate), one or more buffers (e.g., HEPES), one or more nucleotides (e.g., adenosine or thymidine), one or more antibiotics (e.g., gentamicin), one or more trace elements, glucose, and / or one or more equivalent energy sources as needed. Any other necessary / auxiliary supplements of appropriate concentrations known to those skilled in the art may also be included. Other culture conditions, including but not limited to temperature, pH, and CO2 The concentration can be set to appropriate conditions known to those skilled in the art according to the host cell selected for protein expression.

[0045] In one embodiment, the protein described in the present disclosure can be purified by any suitable protein purification method known in the art to which the present disclosure relates. Known protein purification methods include, but are not limited to, hydrophobic interaction chromatography (HIC), immunoaffinity or ion exchange column fractionation, ethanol precipitation, reversed-phase high performance liquid chromatography (HPLC), silica gel chromatography, anion exchange resin or cation exchange resin chromatography, chromatofocusing, SDS-polyacrylamide gel electrophoresis (SDS-PAGE), ammonium sulfate precipitation, gel filtration, genetic engineering methods (e.g., tags) and / or other similar purification methods, and any combination thereof (e.g., to improve the purity of the protein). The protein purified by the above method may have a high purity that can be applied to animals (particularly humans) in the present disclosure for experimental, clinical and therapeutic purposes.

[0046] According to one embodiment of the present disclosure, a protein comprising an amino acid sequence as shown in SEQ ID NO: 1 (GLB1S protein) exhibits GLB1 activity at high purity and does not have a protein truncation problem. In addition, the enzymatic activity of the GLB1S protein is at least equivalent to that of the full-length GLB1 protein, because the enzymatic activity of the GLB1S protein is about 10% to about 20% higher than that of the full-length GLB1 protein. The enzyme activity is measured by a β-galactosidase activity assay known in the art. Specifically, the enzyme activity is measured by detecting the fluorescence of 4-methylumbelliferyl (4-MU), which is generated when the substrate 4-methylumbelliferyl-β-galactoside (MUG) is cut by the GLB1 or GLB1S enzyme.

[0047] The above-mentioned protein truncation problem refers to the deletion of the N-terminal or C-terminal part of the protein. For GLB1, random cleavage is observed at the C-terminus (all or part of R637 to V654 in the amino acid sequence of the full-length GLB1 protein, which contains the amino acid sequence shown in SEQ ID NO: 2) when the protein is expressed. If variants with different C-terminal sequences are produced, problems may arise in quality control and potential drug approval processes. For example, during the approval process, the following issues may arise: (i) whether there are differences in efficacy between variants, (ii) if the ratio of variants in the drug product changes, whether there are differences in stability and / or effect, and (iii) how to uniformly manage the ratio of variants in the drug product. The GLB1S protein disclosed herein, which contains the amino acid sequence shown in SEQ ID NO: 1, can solve these problems.

[0048]

[0049]

[0050] In order to solve the above problems, a method such as treating the full-length GLB1 protein with trypsin can be considered to remove the irregular C-terminus, but this method has the problems of significantly low protein yield, high cost and incomplete purification. According to one embodiment of the present invention, the GLB1S protein comprising the amino acid sequence shown in SEQ ID NO: 1 does not require the use of a proteolytic enzyme (such as trypsin), so the protein purification of GLB1S is easier, and a high-purity protein can be obtained.

[0051] Another aspect of the present disclosure provides a polynucleotide encoding a protein comprising an amino acid sequence as shown in SEQ ID NO: 1. According to one embodiment of the present disclosure, the nucleic acid sequence comprising the above polynucleotide may be in the form of a nucleic acid sequence encoding the amino acid sequence as shown in SEQ ID NO: 1. In another embodiment of the present disclosure, one or more nucleic acid sequences encoding various amino acid sequences that can be added to the N-terminus or C-terminus of the amino acid sequence as shown in SEQ ID NO: 1 may be added to the 5' end or 3' end of the nucleic acid sequence encoding the amino acid sequence as shown in SEQ ID NO: 1.

[0052] In one embodiment of the present disclosure, the above-mentioned polynucleotides can be modified by replacement, deletion or insertion of one or more nucleic acids or any combination thereof. If the nucleotide sequence is produced by chemical synthesis, synthetic methods well known in the art can be used, including but not limited to the methods described in, for example, Engels and Uhlmann, Angew Chem Int Ed Engl., 37:73-127, 1988; triester, phosphite, phosphoramidite and H-phosphoester methods; PCR and other automatic primer methods; and oligonucleotide synthesis on a solid support.

[0053] One aspect of the present disclosure provides an expression vector comprising the above-mentioned polynucleotide encoding the protein with the amino acid sequence shown in SEQ ID NO: 1. In the present disclosure, the term "expression vector" refers to a gene construct comprising a gene insert fragment for expressing a target protein in a suitable host cell and comprising necessary regulatory elements operably linked so that the gene insert fragment is expressed in the host cell.

[0054] In the present disclosure, "vector" refers to any tool for cloning and / or transferring nucleic acid to a host cell. A vector can be a replicon to which another DNA fragment can be attached to achieve replication of the attached fragment. "Replicon" refers to any genetic element (e.g., plasmid, bacteriophage, cosmid, chromosome, virus) that acts as an autonomous DNA replication unit in vivo, i.e., can replicate under its own control. The term "vector" may include viral and non-viral tools for introducing nucleic acid into an organism (e.g., a host cell) in vitro, in vitro, or in vivo. The term "vector" may also include miniature circular DNA, transposons (Izsvak et al. J. Mol. Biol. 302: 93-102 (2000)) or artificial chromosomes.

[0055] Examples of conventional vectors may include natural or recombinant plasmids, cosmids, viruses, and phages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A may be used as phage vectors or cosmid vectors, but are not limited thereto. As plasmid vectors, pBR type, pUC type, pBluescriptII type, pGEM type, pTZ type, pCL type, and pET type may all be used, but are not limited thereto.

[0056] The above-mentioned expression vector includes regulatory factors, such as start codon, stop codon, promoter and operator. The start and stop codons are generally considered to be part of the nucleotide sequence encoding the polypeptide, and must function in the individual to whom the gene construct is administered and maintain a reading frame consistent with the coding sequence (in frame with). The promoter of the vector can be constitutive or inducible.

[0057] In the present disclosure, the term "operably connected" refers to a functional connection between a nucleic acid sequence encoding a target protein or RNA and an expression control sequence so that these sequences encode the desired functions. For example, the nucleic acid sequence of the target protein or RNA is operably connected to its promoter and / or control sequence so that the connected promoter and / or control sequence functionally controls the expression of the coding sequence. Operably connected elements can be adjacent or non-adjacent. Operably connected to an expression vector can use gene recombination techniques well known in the art, and enzymes generally known in the art can be used for site-specific DNA cutting and connection.

[0058] In one embodiment of the present disclosure, the expression vector can introduce a polynucleotide encoding a protein comprising an amino acid sequence as shown in SEQ ID NO: 1 into CHO-K1 (CHO cell line), thereby converting CHO-K1 into a cell line capable of producing the above protein. Another aspect of the present disclosure provides a host cell comprising the above polynucleotide or the above expression vector.

[0059] The expression vector can be introduced into a host cell, which can then be transformed to express one or more polynucleotides contained in the expression vector and produce the protein of the present disclosure. The host cells into which the above-mentioned expression vector can be introduced include, but are not limited to, the cells mentioned above, as long as they are capable of expressing the above-mentioned polynucleotides to produce the protein of the present disclosure.

[0060] Cell transformation can be performed by a variety of methods. In one embodiment of the present disclosure, the transformation method includes but is not limited to CaCl 2 Precipitation method; Hanahan method, which involves the use of CaCl 2 The reducing agent DMSO is used in the precipitation method to improve the efficiency; the heat shock method; the electroporation method; the calcium phosphate precipitation method; the protoplast fusion method; the stirring method using silicon carbide fiber / whisker; the Agrobacterium-mediated transformation (AMT) method; the polyethylene glycol (PEG)-mediated transformation method; the dextran sulfate-mediated transformation method; the liposome-mediated transformation method; the drying / inhibition-mediated transformation method and other transformation methods that can enable host cells to produce the protein of the present invention.

[0061] One aspect of the present disclosure provides a pharmaceutical composition for treating, preventing or improving GM1 gangliosidosis or Morquio syndrome B, the pharmaceutical composition comprising a protein comprising an amino acid sequence as shown in SEQ ID NO: 1. One aspect of the present disclosure provides a method for treating, preventing or improving GM1 gangliosidosis or Morquio syndrome B.

[0062] In the present disclosure, the term "improving" a disease state may refer to any action that reduces the extent of at least one parameter (e.g., symptom) associated with the disease state. In the context of GM1 gangliosidosis or Morquio syndrome B, the term "improving" the disease may include any action that reduces the extent of at least one parameter associated with GM1 gangliosidosis or Morquio syndrome B by administering the above-mentioned pharmaceutical composition. The term "preventing" a disease state may refer to reducing the risk of acquiring a disease, condition, or disorder, or preventing a subject who may be exposed to or susceptible to the disease but has not yet experienced or displayed symptoms of the disease from experiencing at least one clinical symptom. In the present disclosure, the term "preventing" GM1 gangliosidosis or Morquio syndrome B may include any action that inhibits or delays the onset of GM1 gangliosidosis or Morquio syndrome B by administering the pharmaceutical composition, or preventing a subject from experiencing at least one clinical symptom. The term "treatment" as used herein includes alleviating, alleviating or ameliorating a disease, condition or disorder or a symptom thereof, preventing other symptoms, ameliorating or preventing the underlying cause of a symptom, inhibiting a disease, condition or disorder (e.g., preventing the development of a disease, condition or disorder), relieving a disease, condition or disorder, causing a disease, condition or disorder to regress, alleviating a condition caused by a disease, condition or disorder, or preventing a symptom of a disease, condition or disorder prophylactically and / or therapeutically. The term "treating" GM1 gangliosidosis or Morquio syndrome B may include any action that alleviates, alleviates or ameliorates GM1 gangliosidosis or Morquio syndrome B or a symptom of a disease that has occurred or is suspected to occur in a subject by administering the pharmaceutical composition.

[0063] The pharmaceutical composition of the present disclosure may comprise a pharmaceutically effective amount of the protein of the present disclosure, and may further comprise a pharmaceutically acceptable carrier.

[0064] In the present disclosure, GM1 gangliosidosis refers to a hereditary central nervous system disease that destroys nerve cells in the central and peripheral nerves and is caused by insufficient β-galactosidase-1 enzyme activity in lysosomes due to mutations in the GLB1 gene. Morquio syndrome type B in the present disclosure refers to a disease caused by accumulation of keratan sulfate and its subsequent defective degradation due to deficiency of β-galactosidase.

[0065] In one embodiment, the protein comprising the amino acid sequence as shown in SEQ ID NO: 1 can be included in a pharmaceutical composition at a concentration of about 0.001 to about 500 mg / ml, a concentration of about 1 to about 50 mg / ml, a concentration of about 1 to about 10 mg / ml, a concentration of about 3 to about 7 mg / ml, a concentration of about 5 mg / ml, a concentration of about 10 to about 50 mg / ml, a concentration of about 20 to about 40 mg / ml, or a concentration of about 30 mg / ml, without limitation. In one embodiment, the protein comprising the amino acid sequence as shown in SEQ ID NO: 1 can be included in a pharmaceutical composition for intravenous injection at a concentration of about 1 to about 10 mg / ml, without limitation. In one embodiment, the protein comprising the amino acid sequence as shown in SEQ ID NO: 1 can be included in a pharmaceutical composition for intraventricular injection at a concentration of about 10 to about 50 mg / ml, without limitation.

[0066] In one embodiment of the present disclosure, the pharmaceutical composition comprising the above-mentioned protein can be formulated and applied in a manner consistent with medical and clinical practice. Factors considered in this regard include, but are not limited to, the disease being treated, the specific animal being treated, the clinical condition of the individual subject, the cause of the disease, the delivery site of the substance, the method of administration, the administration plan, and other factors known to clinicians. In one embodiment, the preparation of the pharmaceutical composition may include, but is not limited to, a liquid preparation or a lyophilized powder preparation. In one embodiment, the pharmaceutical composition of the present disclosure can be manufactured in the form of an ampoule, a vial, a bottle, a cartridge, a reservoir, a lyophilized syringe, or a prefilled syringe. In one embodiment, the pharmaceutical composition can be manufactured in a single dose form or a multi-dose form.

[0067] In the present disclosure, "pharmaceutically effective dose", "pharmaceutically effective amount", "administered dose", "administered amount", "therapeutically effective dose", "therapeutically effective amount", "effective dose" and / or "effective amount" of a protein administered to a subject refers to the minimum amount required to prevent, improve or treat a specific disease (e.g., GM1 gangliosidosis or Morquio syndrome B), and can be determined based on the above considerations. In one embodiment, the "pharmaceutically effective dose", "pharmaceutically effective amount", "administered dose", "administered amount", "therapeutically effective dose", "therapeutically effective amount", "effective dose" and / or "effective amount" of the above-mentioned protein can be, but are not limited to, for example, about 0.0001 mg / kg to about 100 mg / kg per dose, about 0.001 mg / kg to about 100 mg / kg per dose, about 0.01 mg / kg to about 100 mg / kg per dose, about 0.1 mg / kg to about 100 mg / kg per dose, about 0.1 mg / kg to about 30 mg / kg per dose, about 0.1 mg / kg to about 10 mg / kg per dose, about 1 mg / kg to about 30 mg / kg per dose, or about 1 mg / kg to about 5 mg / kg per dose. Therefore, a single dose of the pharmaceutical composition of the present disclosure may contain about 0.003 to about 3000 mg, about 30 mg to about 900 mg, or about 30 mg to about 150 mg of the protein of the present disclosure (e.g., GLB1S protein). In one embodiment, the pharmaceutical composition of the present disclosure can be injected intravenously at a concentration of about 0.1 mg / kg / week to about 10 mg / kg / week, about 1 mg / kg / week to about 5 mg / kg / week or about 2 mg / kg / week, and is not limited thereto. In one embodiment, the pharmaceutical composition of the present disclosure can be injected intraventricularly at a concentration of about 5 mg / person / two weeks to about 30 mg / person / two weeks, about 15 mg / person / two weeks to about 30 mg / person / two weeks, about 15 mg / person / two weeks, about 5 mg / person / month to about 30 mg / person / month, about 15 mg / person / month to about 30 mg / person / month or about 15 mg / person / month, and is not limited thereto.

[0068] The pharmaceutical compositions of the present disclosure may be administered regularly (including but not limited to, for example, once a day, three times a week, twice a week, once a week, once every two weeks, three times a month, twice a month, or once a month) according to the judgment of an experienced clinician, and irregularly, including but not limited to, for example, in cases of acute progression of the disease.

[0069] In one embodiment of the present application, the pharmaceutical composition of the present disclosure can be prepared using standard methods known to those skilled in the art, such as mixing a protein having the desired purity with a pharmaceutically or physiologically acceptable carrier, excipient or stabilizer. In one embodiment, acceptable carriers include, but are not limited to, for example, saline or buffer (e.g., phosphate, citrate and other organic acids); antioxidants (e.g., ascorbic acid); low molecular weight polypeptides (including less than about 10 amino acid residues); proteins (e.g., serum albumin, gelatin or immunoglobulins); hydrophilic polymers (e.g., polyvinyl pyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, arginine or lysine); monosaccharides, disaccharides and other carbohydrates (e.g., glucose, mannose or dextrin); chelating agents (e.g., EDTA); sugar alcohols (e.g., mannitol or sorbitol); salt-forming counterions (e.g., sodium); and / or nonionic surfactants (e.g., TWEEN TM 、PLURONICS TM or PEG).

[0070] In one embodiment, the pharmaceutical composition of the present disclosure may include a pharmaceutically acceptable salt at a concentration of about physiological concentration. Alternatively, the preparation of the pharmaceutical composition according to one embodiment of the present disclosure may include a pharmaceutically acceptable preservative. Specifically, in one embodiment, the preservative concentration may be about 0.1% to about 2.0% (usually v / v). In one embodiment, the preservative may be a preservative generally known in the pharmaceutical industry, and the preservative may be, for example, but not limited to, benzyl alcohol, phenol, metacresol, methylparaben, propylparaben, or any combination thereof. In one embodiment, the pharmaceutical composition may include a pharmaceutically acceptable surfactant. Specifically, in one embodiment, the surfactant concentration may be about 0.005% to about 0.02%.

[0071] The pharmaceutical composition of the present disclosure may further comprise one or more active compounds required for treating, preventing or improving GM1 gangliosidosis or Morquio syndrome B. Specifically, the pharmaceutical composition may further comprise one or more active compounds with complementary activities, which do not adversely affect the protein of the present disclosure, and / or are not affected by the protein of the present disclosure. The pharmaceutical composition may comprise an effective amount of the active compound to achieve the intended purpose of the pharmaceutical composition.

[0072] In one embodiment of the present disclosure, the pharmaceutical composition can be administered to a human or animal subject via the intracerebroventricular (ICV), intravenous (IV), intramuscular (IM), intraperitoneal (IP), intrathecal, subcutaneous (SC), intra-articular, intrasynovial, intrathecal, oral, topical or inhalation routes, according to one or more methods known in the art. In one embodiment, the pharmaceutical composition can be administered via the intracerebroventricular (ICV) or intravenous (IV) route. Specifically, the pharmaceutical composition can be administered intracerebroventricularly (ICV) via an intracerebroventricular catheter system comprising a reservoir and a catheter connected to the reservoir.

[0073] According to one embodiment of the present disclosure, when the pharmaceutical composition described in the present application is administered to a patient, the GLB1 enzyme activity in the patient's blood may increase by at least 50%.

[0074] According to one embodiment of the present disclosure, when the pharmaceutical composition described in the present application is administered to a patient, the content of keratan sulfate in one or more internal organs (such as the brain, liver, etc.) of the patient may be reduced.

[0075] One aspect of the present disclosure provides a method for producing a protein comprising the amino acid sequence shown in SEQ ID NO:1. In one embodiment, the method comprises: a) introducing a target gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO:1 into a vector; b) transfecting a host cell with the vector; c) culturing the transfected cell and collecting the cell culture supernatant; d) purifying the supernatant to obtain the protein. In one embodiment, the host cell is a Chinese hamster ovary (CHO) cell.

[0076] One aspect of the present disclosure provides a protein produced by a method for producing a protein comprising the amino acid sequence shown in SEQ ID NO:1.

[0077] The work and experimental examples described herein explain in detail the compositions, methods and effects of the present disclosure, but these examples are for illustrative purposes only to help understand the present disclosure, and the categories and / or scope of the present disclosure are not limited by these examples. Comparative (control) example 1: Preparation of a full-length recombinant β-galactosidase (GLB1) protein expression vector

[0078] The target gene, i.e., GLB1 (SEQ ID NO: 2), was cloned into a mammalian expression vector to construct a plasmid. The target gene was synthesized and inserted into the upstream MCS region of the pGenHT1.0-DGV vector. pGenHT1.0-DGV is a mammalian expression vector for the CHOGS system, designed and synthesized by GenScript. The vector contains an MCS for protein target gene expression. The vector also contains a glutamine synthetase (GS) gene for the selection of positive clones. Finally, the prepared plasmid concentration was 1 μg / μl.

[0079] Comparative (control) example 2: Construction of GLB1 protein expression cell line and development of GLB1 protein expression process

[0080] CHOK1-GenS authorized from ECACC (European Collection of Certified Cell Cultures) was used as the host cell. The plasmid described in the above comparative example 1 was transfected into CHOK1-GenS cells. 48 hours after transfection, the supernatant was collected for Western blot detection. The transfected cells were inoculated into a 24-well plate and pool screening was performed using a selective culture medium. After screening, the supernatant of all cell pools was analyzed by dot blotting. Based on the signal intensity, mixed cell pools M1 and M2 were selected, and single cell clone screening was performed using the limiting dilution method. Cell Metrics was used. TM The CLD imager records and confirms the monoclonality of single cell clones. 43 clones with high titers and good confluence are selected. Finally, the top clones showing the best productivity and growth performance are selected.

[0081] The selected cells were subcultured every 72 hours. The total number of generations (i.e., the number of times the culture was subcultured) was 20, and the generations were named P0 to P20. The cells at P0, P17, and P20 were revived for fed-batch culture. The cells at P0 and P20 were collected for target sequence detection. Cell growth live cell density, viability, GLB1 expression titer, and corresponding protein quality proved that the top clones remained stable during subculture for more than 60 days.

[0082] The supernatant of P0 fed-batch culture was collected on the last day for purification and sequence analysis. Specifically, the C-terminal sequence of the protein was analyzed by mass spectrometry (MS). Table 1-3 shows the results of mass spectrometry analysis of the C-terminal sequence of the GLB1 protein.

[0083] Table 1 Mass spectrometry analysis results of the C-terminal sequence of the GLB1 protein produced by clone #1

[0084]

[0085] Table 2 Mass spectrometry analysis results of the C-terminal sequence of the GLB1 protein produced by clone #2

[0086]

[0087]

[0088] Table 3 Mass spectrometry analysis results of the C-terminal sequence of the GLB1 protein produced by clone #3

[0089]

[0090] As shown in Tables 1 to 3, it was observed that the C-terminus of the GLB1 protein was severely truncated, as shown by the gradual cleavage of C-terminal amino acids of different lengths. In all three top clones developed in the cell line, 3-4 variants accounting for more than 10% were found, but none of the variants could be considered as the main product. This observation triggered the subsequent GLB1S project, which could potentially rescue this situation by artificially removing the C-terminus.

[0091] Example 1: Preparation of recombinant β-galactosidase (GLB1S) plasmid

[0092] The target gene, GLB1S (SEQ ID NO: 1), was optimized and synthesized for CHO host cells. The target gene was inserted into the upstream multiple cloning site (MCS) region of the pGenHT1.0-DGV vector. The concentration of the prepared plasmid was 1 μg / μl. The transfected plasmid was named NP4K_pGenHT1.0-DGV.

[0093] Example 2: Construction of GLB1S protein expression cell line and development of GLB1S protein expression process

[0094] The plasmid prepared as described in Example 1 above was transfected into CHOK1-GenS host cells. 48 hours after transfection, the supernatant was collected for fluorescent β-galactosidase detection and Western blot test. The transfected cells were seeded into 24-well plates and cell pools were screened using selection medium. After screening, the supernatants of all cell pools were analyzed by dot blot. Based on the signal intensity, mixed cell pools M1, M2, M3 and M4 were selected for single cell clone screening using the limiting dilution method. CellMetric TM The CLD imager records and confirms the monoclonality of single cell clones. 24 clones with high titers and good confluence are selected. Finally, the top clones showing the best productivity and growth performance are selected.

[0095] The selected top clones were cultured in a 3L bioreactor in a fed-batch format and variables such as pH, dissolved oxygen (DO), agitation, and temperature were monitored. The top clones were cultured according to the conditions and parameters of the GenScript cell culture process development platform using the materials and reagents listed in Table 4. The culture supernatant was collected on day 15 or when the cell viability was 70% or less, whichever was earlier.

[0096] Table 4 Materials and reagents used for 3 L scale culture of top clones expressing GLB1S protein

[0097]

[0098] The titer of the collected cell culture supernatant was measured by 4-methylumbelliferyl-β-galactoside (MUG) assay to be 2.409 g / L.

[0099] Subsequently, the selected top clones were cultured in a 50L bioreactor in a fed-batch culture, and variables such as pH, dissolved oxygen (DO), agitation, and temperature were monitored to demonstrate the scalability of the process. The top clones were cultured using the materials and reagents listed in Table 5 according to the conditions and parameters of the GenScript cell culture process development platform. The culture supernatant was collected on day 15 or when the cell viability was 70% or less, whichever was earlier.

[0100] Table 5 Materials and reagents used for 50 L scale culture of top clones expressing GLB1S protein

[0101]

[0102]

[0103] The titer of the collected cell culture supernatant was measured by 4-methylumbelliferyl-β-galactoside (MUG) assay to be 2.061 g / L.

[0104] In addition, the products harvested from the bioreactor are purified by the developed purification process. After obtaining the culture supernatant, the protein is purified by depth filtration (DF), solvent / detergent treatment (S / D), anion exchange chromatography (AEX), cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC) and ultrafiltration / diafiltration (UF / DF) in sequence. The detailed purification process flow is as follows Figure 1 The purified protein product was analyzed by reverse phase high performance liquid chromatography (RP-HPLC) and size exclusion high performance liquid chromatography (SEC-HPLC). The analysis results of RP-HPLC and SEC-HPLC are shown in Table 6.

[0105] Table 6 RP-HPLC and SEC-HPLC analysis results of GLB1S protein

[0106]

[0107] As shown in Table 6, RP-HPLC and SEC-HPLC showed that the product had high purity. The main peak in RP-HPLC indicated the target protein GLB1S, and the front peak indicated the fragment. RP-HPLC measurement showed that the ratio of the front peak of the product was very low. SEC-HPLC showed that the main peak ratio (GLB1S) of the product was higher than 99%, and the ratio of the high molecular weight (HMW) and low molecular weight (LMW) of the product was also within an acceptable range.

[0108] Example 3: Confirmation of GLB1S protein expression by SDS-PAGE and SEC-HPLC

[0109] The GLB1S protein was obtained by 50 L culture and purification as described in Example 2 above.

[0110] The purified protein was analyzed by SDS-PAGE, SEC-HPLC and RP-HPLC. Figure 2 , as shown in Figure 3 and Figure 4. Figure 2 As shown, GLB1S protein (molecular weight between 50 kDa and 75 kDa markers) appears as the major product. Figure 3A and 3B As shown, the GLB1S protein with a purity of 97.26% was obtained. Figure 4A and 4B As shown, the RP-HPLC purity of the GLB1S protein was 99.57%.

[0111] Example 4: Confirmation of GLB1S protein activity

[0112] The enzyme activities of GLB1S (rhGLB1S), rhGLB1 (GenScript) and rhGLB1-Fc (AbClon) were measured. In order to obtain rhGLB1S, CHO-K1 cells expressing GLB1S protein were cultured on a 10L scale according to the method described in Examples 1 and 2 above, and the culture supernatant was purified in sequence by anion exchange chromatography (AEX), cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC) and ultrafiltration / diafiltration. Specifically, a 1 mg / ml solution of GLB1S was used for activity analysis. For rhGLB1-Fc, the hinge region, CH2 and CH3 (SEQ ID NO: 3) of the immunoglobulin gamma-1 heavy chain were used as Fc. 0.5 mM 4-methylumbelliferyl-β-galactoside (MUG, 0.17 mg / ml) dissolved in 0.1 M sodium acetate-acetic acid buffer (pH 4.0) containing 5.84 mg / ml sodium chloride (NaCl, 0.1 M) was used as the substrate solution and was stably stored at 4°C or -20°C for several months.

[0113] For leukocytes, the analysis requires 0.020-0.030 mg of protein, and for fibroblasts and amniocytes, 0.010-0.020 mg of protein. In order to achieve the required protein concentration, 0.10 ml of diluted tissue lysate was placed in a 12 x 75 mm test tube, and duplicates were prepared for each sample. Then, 0.10 ml of the above substrate solution was added to the test tube to start the reaction, and the test tube was incubated at 37 ° C for 20 minutes. Subsequently, the reaction was stopped by adding 1.30 mL of 0.17 M glycine-carbonate buffer (pH 9.8). For the blank control (blank tube), only the substrate solution was cultured, and then the reaction was stopped by adding glycine-carbonate buffer. Next, a control lysate preparation diluted to an appropriate protein concentration was added. The fluorescence value of the prepared sample was read at an excitation wavelength of 360 nm and an emission wavelength of 415 nm, and the average value of the duplicate readings was taken. Activity was calculated by the following formula, and the results are shown in Table 7.

[0114]

[0115] Table 7 Enzyme activities of GLB1, GLB1S and GLB1-Fc

[0116] protein 1:1000 dilution 1:2000 dilution rhGLB1S 416,953 486,007 rhGLB1 371,604 436,399 rhGLB1-Fc 11,946 -

[0117] As shown in Table 7, under the condition of 1:1000 dilution, the activity of the rhGLB1S protein disclosed in the present invention was determined to be 416953 nmol / hr / mg, which was about 12.2% higher than the full-length rhGLB1 protein (371604 nmol / hr / mg). Under the condition of 1:2000 dilution, the activity of the rhGLB1S protein was about 11.4% higher than that of the rhGLB1 protein. In other words, the activity of the GLB1S protein, a truncated version of the GLB1 protein (i.e., C-terminal truncation) is higher than that of the full-length GLB1 protein or the antibody-coupled GLB1 protein (GLB1-Fc). The above data show that the GLB1S protein can be used as a drug with equivalent efficacy (compared with the GLB1 protein) for the treatment, prevention or improvement of GM1 gangliosidosis or Morquio syndrome B.

[0118] Example 5: Preparation of GLB1 KO (gene knockout) mouse model

[0119] The GLB1 KO mouse model was prepared using CRISPR (clustered regularly interspaced short palindromic repeats) gene editing technology. Exon 6 of the mouse GLB1 gene was edited, and the preparation work was completed by Macrogen. In order to confirm the knockout (KO) status of the GM1 gangliosidosis or Morquio syndrome B model mouse (GLB1 KO mouse), genotyping was performed. 22 F0 transgenic mice were screened for GLB1 mutations, and the presence of KO mutations was determined in F0#1, #5, and #19 mice. Among them, F0#1 mice were selected for breeding and used in subsequent experiments ( Figure 5 ).

[0120] Example 6: Confirmation of the effect of GLB1S administration in the GLB1 KO mouse model

[0121] 1. Control group and test group

[0122] Table 8 Control group and test group

[0123]

[0124] G1: Wild-type mouse group (wild-type control group)

[0125] G2: KO mouse group (negative control group)

[0126] G3-G7: KO mouse group (test group)

[0127] 2. Administration of vehicle or GLB1S

[0128] Healthy mice were weighed, sorted, and then randomly assigned to groups according to Table 8. This was done to ensure that the mean body weight and gender distribution between groups was as even as possible.

[0129] After fixing the mouse using a fixing device or a fixing appliance, the vehicle or GLB1S was intravenously injected once (single administration) using an insulin syringe.

[0130] 3. Body Weight and Organ Assessment

[0131] 3-1. Evaluation items and sampling

[0132] After administration of vehicle or GLB1S, the general symptoms and body weight of mice were evaluated. From the start of administration to the entire observation period, the death of each mouse and the type and severity of general symptoms were observed and recorded at least once a day. The day of administration was designated as day 0.

[0133] Body weights of mice were measured at multiple time points, including upon acquisition of mice, grouping, administration of vehicle or GLB1S, autopsy, and once a week during the observation period.

[0134] Regarding sampling, 5 mice were sampled on the 7th, 14th and 28th day after the administration of the vehicle or GLB1S. The mice were perfused on the scheduled dissection day and then dissected to obtain the organs. The harvested samples were then stored at -70°C. Since one mouse was removed from the G3 group, the experiment of the G3 group was completed with 14 mice (i.e., 5 mice were sampled on the 7th and 14th day, and 4 mice were sampled on the 28th day).

[0135] Statistical analysis

[0136] Medical statistical analysis was performed using SPSS Statistics 12.0K. The normal control group (G1) was compared with the negative control group (G2), and the negative control group (G2) was compared with the test groups (G3-G7). Since the number of mice in each group was 5 or less, the nonparametric Kruskal-Wallis test was used, followed by the Mann-Whitney post hoc test and the Bonferroni correction was applied. The statistical significance level for all analyses was set at p < 0.05.

[0137] 3-3. Evaluation results

[0138] (1) Mortality

[0139] In the 0.1 mg / kg dose group (G3), one mouse died on day 27. However, no other deaths were observed in the remaining experimental groups (G1, G2, G4, G5, G6, and G7).

[0140] (2) General symptoms

[0141] All groups (G1-G7) showed no adverse reactions.

[0142] (3)Weight

[0143] From the start of drug administration to the entire observation period, no difference in the body weight of mice was observed in all groups (G1-G7).

[0144] Table 9 weight

[0145]

[0146] Data are presented as mean ± standard error (SE). Results were statistically analyzed by Kruskal-Wallis test.

[0147] (4) Organ weight

[0148] No statistically significant differences in organ weights were observed in all tested groups. However, on day 7, liver and spleen weights in the negative control group were more than 32% higher than those in the normal control group. Liver weights in all GLB1S-treated groups (G3-G7) were more than 22% lower than those in the negative control group. None of the above differences in liver and spleen weights were statistically significant.

[0149] Table 10 Organ weight (Day 7)

[0150]

[0151]

[0152] Data are presented as mean ± standard error (SE). Results were statistically analyzed by Kruskal-Wallis test.

[0153] 4. Determination of Keratan Sulfate Content in Mouse Liver Tissue

[0154] 4-1. Determination method of keratan sulfate content

[0155] The level of keratan sulfate was evaluated by collecting liver tissues of the control group and the test group on days 7, 14, and 28. The description of the negative control group and the test group and the number of mice in each group are shown in Table 11 below. The collected mouse liver tissues were homogenized, liver tissue lipids were removed, and enzymes were treated to generate keratan sulfate in a disaccharide form. Subsequently, keratan sulfate was selectively extracted using PGC-SPE, and its content was quantified using LC-MS / MS.

[0156] Table 11 Liver tissue collection information for keratan sulfate measurement

[0157]

[0158] 4-2. Results of keratan sulfate content in mouse liver tissue

[0159] Figures 6A-6FThe results of the analysis of keratan sulfate content in each group on the 7th, 14th and 28th days are shown. Figures 6A-6F As shown, keratan sulfate levels increased over time in all groups except G2, which served as a negative control group. This suggests that GLB1S significantly suppressed keratan sulfate levels within 14 days after administration. Reaccumulation of keratan sulfate was observed in all treatment groups on day 28 after GLB1S administration.

[0160] Figure 7 is a graph showing the results of analysis of keratan sulfate content 7 days after administration of vehicle or GLB1S. Figure 7 As shown, the keratan sulfate levels in all GLB1S-treated groups were significantly decreased compared with those in G2 in a dose-dependent manner.

[0161] Figure 8 is a graph showing the results of keratan sulfate assay after 14 days of vehicle or GLB1S treatment. Figure 8 As shown, significantly decreased keratan sulfate content was observed in G5, G6 and G7 (corresponding to treatment groups receiving doses above 1.0 mg / kg) compared to G2.

[0162] Based on the above results, a significant enzyme replacement therapy effect can be expected when GLB1S is administered once a week or once every two weeks.

[0163] For rhGLB1 (full-length protein), it is difficult to obtain uniform protein during the manufacturing process due to the heterogeneous protein truncation observed in rhGLB1. This becomes an obstacle to the production of therapeutic proteins using large-scale production facilities. Additional processing or purification processes may be required after the protein is expressed from the transformed cells to obtain a protein that meets the standards for drug marketing approval. Due to the heterogeneity between the expressed proteins, the use of host cells expressing the full-length rhGLB1 protein cannot ensure the predictable quality of the drug, which also raises drug safety issues.

[0164] According to one embodiment of the present disclosure, the GLB1S protein does not exhibit the above-mentioned problems of the full-length GLB1 protein (e.g., heterogeneous protein truncation), and for the first time confirms that the GLB1S protein has equivalent enzymatic activity compared to the full-length GLB1 protein. The present disclosure demonstrates the production of GLB1S, significantly improves the predictability of protein manufacturing conditions and drug quality on a commercial scale, and indicates that the GLB1S protein can be used as a drug with better efficacy for the treatment, prevention or improvement of GM1 gangliosidosis or Morquio syndrome B.

Claims

1. A protein comprising an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

2. The protein according to claim 1, comprising the amino acid sequence shown in SEQ ID NO:

1.

3. A polynucleotide encoding the protein according to claim 1 or 2. An expression vector comprising the polynucleotide according to claim 3 .

5. A host cell, comprising the polynucleotide according to claim 3 or the expression vector according to claim 4.

6. A method for treating, preventing or ameliorating GM1 gangliosidosis or Morquio syndrome B, comprising: A therapeutically effective amount of a pharmaceutical composition comprising the protein of claim 1 or 2 is administered to a patient suffering from GM1 gangliosidosis or Morquio syndrome B.

7. The method of claim 6, wherein the intensity, severity, or frequency of at least one symptom of GM1 gangliosidosis or Morquio syndrome B is reduced, or the onset of at least one symptom is delayed, following administration of a therapeutically effective amount of the pharmaceutical composition.

8. The method of claim 6, wherein the therapeutically effective amount of the pharmaceutical composition is administered at a dose of 0.1 mg / kg to 100 mg / kg.

9. The method of claim 6, wherein the therapeutically effective amount of the pharmaceutical composition is administered via intracerebroventricular (ICV) administration or intravenous (IV) administration.

10. The method of claim 9, wherein the intracerebroventricular (ICV) administration comprises administering a therapeutically effective amount of the pharmaceutical composition to a patient via an intraventricular catheter system comprising a reservoir and a catheter connected to the reservoir.

11. The method of claim 6, wherein the protein has an enzymatic activity equivalent to that of a full-length GLB1 protein.

12. The method of claim 6, wherein the therapeutically effective amount of the pharmaceutical composition is configured to increase GLB1 enzyme activity in the patient's blood by at least 50%.

13. The method of claim 6, wherein the therapeutically effective amount of the pharmaceutical composition is administered once a week, once every two weeks, twice a month, or once a month.

14. The method of claim 6, wherein the therapeutically effective amount of the pharmaceutical composition reduces keratan sulfate levels in one or more internal organs of the patient.

15. A pharmaceutical composition for treating, preventing or improving GM1 gangliosidosis or Morquio syndrome B, comprising the protein according to claim 1 or 2.

16. The pharmaceutical composition of claim 15, wherein the protein has an enzymatic activity equivalent to that of the full-length GLB1 protein.

17. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition is configured to increase GLB1 enzyme activity in the patient's blood by at least 50%.

18. The pharmaceutical composition of claim 15, wherein the pharmaceutical composition reduces keratan sulfate levels in one or more internal organs of the patient.

19. A method for producing a protein comprising the amino acid sequence shown in SEQ ID NO: 1, comprising: a) introducing a target gene encoding a protein comprising the amino acid sequence shown in SEQ ID NO: 1 into a vector; b) transfecting a host cell with the vector; c) culturing the transfected cells and collecting the cell culture supernatant; and d) purifying the supernatant to obtain the protein.

20. The method of claim 19, wherein the host cell is a Chinese Hamster Ovary (CHO) cell.

21. A protein produced by the method of claim 19 or 20.

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