Pharmaceutical composition for treating, preventing, or ameliorating GM1 gangliosidosis or Morquio syndrome type B and its administration method

Abstract

The present disclosure relates to a recombinant GLB1 protein having a truncated C-terminus; a pharmaceutical composition comprising the protein for treating, preventing, or ameliorating GM1 gangliosidosis or Morquio syndrome type B; and a method for treating, preventing, or ameliorating GM1 gangliosidosis or Morquio syndrome type B, comprising administering a pharmaceutical composition comprising the protein. According to one aspect of the disclosure, a recombinant GLB1 protein having a truncated C-terminus does not encounter problems with heterologous protein truncation during the protein production process, exhibits enzymatic activity equivalent to that of the full-length GLB1 protein, and increases GLB1 enzymatic activity in the blood of patients. The present disclosure demonstrates that the protein can be used to treat, prevent, or ameliorate GM1 gangliosidosis or Morquio syndrome type B.

Description

[Technical Field]

[0001] The present disclosure relates to a recombinant protein of beta-galactosidase-1 (GLB1) having a truncated C-terminus; a pharmaceutical composition for treating, preventing, or ameliorating GM1 gangliosidosis or Morquio syndrome type B, comprising the protein; and a method for treating, preventing, or ameliorating GM1 gangliosidosis or Morquio syndrome type B, comprising administering a pharmaceutical composition comprising the protein. [Background technology]

[0002] GM1 gangliosidosis is a hereditary central nervous system (CNS) disorder caused by a lack of intralysosomal beta-galactosidase-1 (GLB1) enzyme activity due to a GLB1 gene mutation, resulting in the destruction of nerve cells in the central and peripheral nervous system. The causative gene for GM1 gangliosidosis is the GLB1 gene, which produces beta-galactosidase-1, and is inherited in an autosomal recessive manner. GM1 gangliosidosis occurs when oligosaccharide breakdown is hindered due to the lack of intralysosomal beta-galactosidase-1 enzyme activity, resulting in the accumulation of galactose-rich oligosaccharides and keratan sulfate in the brain and internal organs, leading to the disease.

[0003] GM1 gangliosidosis is divided into three phenotypes, infantile, juvenile, and adult, depending on the age of onset. Infantile GM1 gangliosidosis (Type I) manifests with hepatosplenomegaly, a puffy face, a broad upper lip, maxillary overgrowth, hypertrophic gingiva, and macroglossia. Skeletal changes, including vertebral changes resembling Hurler syndrome, become noticeable at approximately six months of age. Juvenile GM1 gangliosidosis (Type II) has no externally recognizable features. Patients are usually normal until age one, after which they lose control, exhibit autistic behavior, and become obtuse. After one year of age, symptoms may progress to ataxia, epilepsy, and spastic paralysis. Death usually occurs before the age of ten, and changes such as vertebral breaking may occur. Adult GM1 gangliosidosis (Type III) also has no externally distinguishing features, but it begins in childhood and manifests as dystonia, Parkinson's disease, and extrapyramidal symptoms similar to atypical cerebellar contractile ataxia, and usually presents with severe dementia.

[0004] GM1 gangliosidosis is estimated to occur in 1 in 100,000–200,000 newborns, with the infantile form (Type I) being reported more frequently than other phenotypes.

[0005] Morquio syndrome is an autosomal recessive genetic disorder, and Morquio syndrome type B is caused by a deficiency of the enzyme beta-galactosidase, which impairs the breakdown of keratan sulfate. Symptoms of Morquio syndrome become apparent between 18 and 24 months of age, and due to various complications, patients usually die before the age of 20.

[0006] Physical characteristics include short stature, a coarse face, a low or wide nose, thick lips, macrocephaly, corneal opacity, a short neck, glaucoma, retinal degeneration, and hearing loss. Skeletal dysplasia also appears, usually with a prominent sternum and an abnormal chest shape, and spinal abnormalities can lead to severe kyphosis, which can lead to respiratory failure or spinal cord compression. Other conditions may include inguinal prolapse, liver enlargement, and dental caries due to odontoid hypoplasia.

[0007] There is no fundamental treatment for GM1 gangliosidosis and Morquio syndrome, and treatment is given symptomatically, so there is a need for a treatment.

[0008] Meanwhile, enzyme replacement therapy (ERT) is a therapeutic method achieved by systemically administering natural or recombinant proteins and / or enzymes to a subject, and is considered one of the treatments for diseases caused by enzyme deficiency or deficiency.

[0009] In this situation, the present inventors developed a C-terminal truncated recombinant protein of beta-galactosidase-1 (GLB1) (hereinafter referred to as "GLB1S") that is free from the protein truncation issue and has high enzymatic activity, and confirmed the surprising effect of treating GM1 gangliosidosis or Morquio syndrome type B when this protein is administered to patients, thereby completing the present invention. Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention relates to a recombinant protein of beta-galactosidase-1 (GLB1) having a truncated C-terminus; a pharmaceutical composition for treating, preventing, or ameliorating GM1 gangliosidosis or Morquio syndrome type B, comprising the protein; a method for treating, preventing, or ameliorating GM1 gangliosidosis or Morquio syndrome type B, comprising administering the pharmaceutical composition to a subject; and a method for producing the protein. [Means for solving the problem]

[0011] In one aspect of the present disclosure, which aims to solve the above-mentioned problems, the present invention provides a recombinant protein of GLB1 (GLB1S) that has a truncated C-terminus compared to GLB1. In one embodiment of the present disclosure, the present invention provides a polynucleotide encoding the recombinant protein, an expression vector containing the polynucleotide, and / or a host cell containing the polynucleotide or the expression vector.

[0012] In one aspect of solving the above-mentioned problems, the present invention provides a pharmaceutical composition for treating, preventing, and / or ameliorating GM1 gangliosidosis or Morquio syndrome type B, or at least one symptom thereof, comprising the recombinant protein (GLB1S).

[0013] In another aspect for solving the above-mentioned problems, the present invention provides a method for treating, preventing, and / or ameliorating GM1 gangliosidosis or Morquio syndrome type B, or at least one symptom thereof, which comprises administering a pharmaceutical composition containing the recombinant protein (GLB1S).

[0014] In one embodiment of the present disclosure, a recombinant GLB1 protein (GLB1S) with a truncated C-terminus does not have the problem of protein cleavage during the protein production process. In one embodiment, the recombinant truncated GLB1 also has equivalent enzymatic activity compared to the full-length GLB1 protein. In one embodiment, the recombinant truncated GLB1 has enzymatic activity at least equivalent to the full-length GLB1 protein. In one embodiment, the short form recombinant GLB1 protein (GLB1S) can increase GLB1 enzyme activity in a patient's blood. In one embodiment of the present disclosure, the short form recombinant GLB1 protein (GLB1S) or a pharmaceutical composition comprising the same can be used as a drug with superior efficacy compared to the full-length GLB1 protein for treating, preventing, or ameliorating GM1 gangliosidosis or Morquio syndrome type B.

[0015] In another aspect of solving the above problem, the present invention provides a method for producing a protein comprising the amino acid sequence of SEQ ID NO: 1, comprising the steps of: a) introducing a target gene encoding a protein comprising the amino acid sequence of SEQ ID NO: 1 into a vector; b) transfecting host cells with the vector; c) culturing the transfected cells and harvesting a cell culture supernatant; and d) purifying the supernatant to obtain the protein.

[0016] In one embodiment of the present disclosure, the host cell is a Chinese hamster ovary (CHO) cell.

[0017] In another aspect of solving the above problems, the present invention provides a protein produced by the method for producing a protein comprising the amino acid sequence of SEQ ID NO:1. [Effects of the Invention]

[0018] The GLB1S protein does not exhibit the problems of the full-length GLB1 protein (e.g., cleavage of heterologous proteins) and has equivalent enzymatic activity to the full-length GLB1 protein. The present disclosure demonstrates a GLB1S production method that greatly improves the predictability of protein production conditions and drug quality on a commercial scale, and demonstrates that the GLB1S protein can be used as a drug with superior efficacy for the treatment, prevention, or amelioration of GM1 gangliosidosis or Morquio syndrome type B. [Brief explanation of the drawings]

[0019] [Figure 1] FIG. 1 is a detailed workflow diagram for the purification of GLB1S. [Figure 2] This shows the results of SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) analysis of purified recombinant GLB1S obtained by culturing CHO-K1 cells at a 50 L scale. [Figure 3A]1 shows the results of SEC-HPLC (size exclusion-high performance liquid chromatography) analysis of purified recombinant GLB1S obtained by culturing CHO-K1 cells at a 50 L scale. [Figure 3B] 1 shows the results of SEC-HPLC (size exclusion-high performance liquid chromatography) analysis of purified recombinant GLB1S obtained by culturing CHO-K1 cells at a 50 L scale. [Figure 4A] This shows the results of RP-HPLC (reverse phase-high performance liquid chromatography) analysis of purified recombinant GLB1S obtained by culturing CHO-K1 cells at a 50 L scale. [Figure 4B] This shows the results of RP-HPLC (reverse phase-high performance liquid chromatography) analysis of purified recombinant GLB1S obtained by culturing CHO-K1 cells at a 50 L scale. [Figure 5] This shows the results of genotyping of GLB1KO mice obtained by PCR screening. [Figure 6A] The keratan sulfate content analyzed from the liver tissue of mice in each group (G2) on days 7, 14, and 28 is shown. [Figure 6B] The keratan sulfate content analyzed from the liver tissue of mice in each group (G3) on days 7, 14, and 28 is shown. [Figure 6C] The keratan sulfate content analyzed from the liver tissue of mice in each group (G4) on days 7, 14, and 28 is shown. [Figure 6D] The keratan sulfate content analyzed from the liver tissue of mice in each group (G5) on days 7, 14, and 28 is shown. [Figure 6E] The keratan sulfate content analyzed from the liver tissue of mice in each group (G6) on days 7, 14, and 28 is shown. [Figure 6F] The keratan sulfate content analyzed from the liver tissue of mice in each group (G7) on days 7, 14, and 28 is shown. [Figure 7] 1 shows the keratan sulfate content analyzed from the liver tissues of mice from groups G2 to G7 on day 7. [Figure 8] 1 shows the keratan sulfate content analyzed from the liver tissues of mice from groups G2 to G7 on day 14. DETAILED DESCRIPTION OF THE INVENTION

[0020] The various embodiments, aspects, or examples of the present disclosure are illustrated for the purpose of explaining the technical idea of ​​the present disclosure, and the scope of the rights of the present disclosure is not limited to the embodiments, aspects, or specific descriptions thereof presented below. The present disclosure includes various modifications, equivalents, and alternatives of each embodiment, aspect, or example described in the present application, as well as any possible combination of all or part of each embodiment, aspect, or example described in the present application. A person of ordinary skill in the art to which the present disclosure pertains will understand and recognize the modifications, equivalents, and alternatives or combinations described above. The scope of the rights of the present disclosure is not limited to the various embodiments, aspects, or examples presented below; or the specific descriptions of the embodiments, aspects, or examples.

[0021] Unless otherwise specified, all technical and scientific terms used in this application generally have the ordinary meanings understood by those of ordinary skill in the art to which this disclosure belongs. All terms used in this application are selected to describe and / or explain the present disclosure and are not selected to limit the scope of rights under the present disclosure. Specific terms are discussed in this application to provide additional guidance in describing and / or explaining the compositions and methods of the present disclosure.

[0022] definition As used in this disclosure, the singular forms ("a," "an," and "the") and singular words include the plural forms unless the context clearly dictates otherwise, and this also applies to the singular forms and words presented in the claims. For example, "a compound" includes a plurality of such compounds, and "compound A" includes a plurality of compound A.

[0023] The term "and / or" means one or more of an item, any combination of items, or any of the items associated with this term. The terms "containing," "contain," "contains," "including," "include," "includes," "having," "have," "has," "with," or variations thereof, are to be construed as inclusive in a manner similar to the term "comprising." The terms "comprising," "including," "having," "containing," and variations thereof are to be understood in this disclosure as open-ended terms implying the possibility of including other embodiments, unless otherwise specified. The term "consisting of" and variations thereof are to be understood in this disclosure as closed-ended terms (i.e., "including, including") excluding any element not recited in the description of the embodiment.

[0024] All ranges mentioned in this application may include any and all possible subranges and combinations of subranges. A mentioned range includes each specific value, integer or fraction within the range. A person of ordinary skill in the art will readily understand that any mentioned range fully describes and / or allows for its subranges, including, but not limited to, one-half, one-third, one-quarter, one-fifth, or one-tenth of the mentioned range.

[0025] The terms "greater than," "more than," "at least," "or more," "less than," "up to," and the like, are inclusive of the numbers referenced, and such terms refer to ranges that may be subdivided into subranges, as described above. Particular values ​​referenced in ranges are for illustrative purposes only and are not intended to be limiting and do not exclude other values ​​within the range.

[0026] Unless otherwise specified, the terms "about" and "approximately" generally include values ​​near the stated range or within an acceptable error, as is well known to those of ordinary skill in the art. The acceptable degree of error may be determined taking into account the nature or precision of the measurement or manufacturing process. In one embodiment, the acceptable degree of error may be determined based on equivalence in terms of the functionality of a composition, method, or example. In one embodiment, in the context of a numerical value or range provided in this disclosure, the term "about" or "approximately" may refer to a variation of ±25%, ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the provided value. In yet other embodiments, particularly in biological systems, the terms "about" and "approximately" can refer to a value that is within 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, or 2-fold of the given value. All numerical values ​​provided in this application are approximations unless otherwise specified and inherently contain variability necessarily resulting from measurement.

[0027] In this disclosure, the term "pharmaceutical composition" refers to a pharmaceutical formulation that allows the biological activity of the active ingredients contained in the composition to be effective. In one embodiment, a pharmaceutical composition is a composition containing one or more active ingredients that can be administered to a patient and that does not further contain unacceptable toxic ingredients.

[0028] In this disclosure, the term "pharmaceutically acceptable" can refer to the properties of a substance that is generally safe, non-toxic, biologically or otherwise useful in the manufacture of pharmaceutical compositions, and acceptable for human pharmaceutical and / or veterinary use. In one embodiment, the term "pharmaceutically acceptable" means approved or approvable by a government regulatory agency or listed in a generally recognized pharmacopoeia for use in humans and / or other animals.

[0029] In the present disclosure, the term "pharmaceutically acceptable excipient" refers to any ingredient that is not therapeutically active (inactive) and non-toxic. In one embodiment, pharmaceutically acceptable excipients may include, but are not limited to, binders, fillers, solvents, buffers, tonicity agents, stabilizers, antioxidants, surfactants, or lubricants configured for use in formulating pharmaceutical products.

[0030] As used herein, the term "pharmaceutically acceptable carrier" refers to an ingredient of a pharmaceutical composition other than the active ingredient, which is non-toxic to a subject. In one embodiment, a pharmaceutically acceptable carrier may include, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[0031] In the present disclosure, the terms "pharmaceutically effective dose," "pharmaceutically effective amount," "administered dose," "dosage," "therapeutically effective dose," "therapeutically effective amount," "effective dosage," or "effective amount" of a pharmaceutical composition may refer to the amount of the pharmaceutical composition (administered for the required period of time) sufficient to achieve a therapeutic response or effect, a desired local or systemic therapeutic result, or a desired preventative result. In one embodiment, the term refers to the amount of the pharmaceutical composition that, when administered to a subject, (i) treats or prevents one or more symptoms of a disease, condition, or disorder described herein, (ii) alleviates, reduces, halts, attenuates, ameliorates, or eliminates one or more symptoms thereof, or (iii) prevents or delays the onset of one or more symptoms thereof.

[0032] 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.

[0033] One aspect of the present disclosure provides a protein comprising the amino acid sequence of SEQ ID NO: 1. In the present disclosure, the amino acid sequence of SEQ ID NO: 1 is a C-terminally truncated recombinant protein (GLB1S) of beta-galactosidase-1 (GLB1) protein.

[0034] In addition to the amino acid sequence of SEQ ID NO: 1, one aspect of the present disclosure may include amino acid sequences 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 SEQ ID NO: 1. In one embodiment, such homologous sequences include, without limitation, amino acid sequences that represent proteins having substantially the same or corresponding activity as a protein comprising the amino acid sequence of SEQ ID NO: 1. Furthermore, proteins may include any amino acid sequence that is homologous to the amino acid sequence of SEQ ID NO: 1; that has one or more activities of a protein comprising the amino acid sequence of SEQ ID NO: 1; or that has one or more deletions, modifications, substitutions, or additions of amino acids, so long as such deletions, modifications, substitutions, or additions do not substantially affect the function of the protein. For example, one or more amino acids may be deleted, modified, substituted, or added for optimization and / or convenience of the protein expression and extraction process.

[0035] In the present disclosure, "homology" refers to the degree of identity of bases or amino acid residues between sequences of the amino acid or nucleic acid sequences of a protein-encoding gene after aligning the two sequences to the greatest extent possible in a specific comparison region. If the homology is sufficiently high, the expression product of the gene may have the same or similar activity. The percentage (%) of sequence identity may be determined using a known sequence comparison program (e.g., Blast (NCBI), etc.).

[0036] In the present disclosure, proteins may be expressed and produced by methods known in the art to which the present disclosure pertains. Host cells are not particularly limited as long as they are capable of expressing and producing the proteins, and may include 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 and Spodoptera Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bowes melanoma cells; or plant cells.

[0037] In the present disclosure, host cells suitable for expressing and producing proteins include vertebrate host cells and eukaryotic cells as described herein. Examples of mammalian host cells include, but are not limited to, monkey kidney cells (CV1), SV40-transformed monkey kidney CV1 line (COS-7), human embryonic kidney line (293 cells), baby hamster kidney cells (BHK), Chinese hamster ovary cells (CHO), mouse Sertoli cells (TM4), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor (MMT 060562), TRI cells, MRC5 cells, and FS4 cells.

[0038] The host cells used to produce the proteins of the present disclosure may be cultured in a variety of media. Commercially available media, such as Ham's F10 medium, minimal essential medium, RPMI-1640, Dulbecco's modified Eagle's medium (DMEM), and any other media used in the field of the technology to which the present disclosure pertains, can be used for host cell culture. Any of these media may contain, as needed, hormones and / or growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., gentamicin), etc. (商標) The culture medium may be supplemented with various nutrients, such as drugs, trace elements, and glucose or an equivalent energy source. Any other essential / auxiliary supplements may also be included at appropriate concentrations known to those of ordinary skill in the relevant art. Other culture conditions, such as temperature, pH, and CO2 concentration, may be set to appropriate conditions known in connection with the host cell selected for protein expression, which will be apparent to those of ordinary skill in the relevant art.

[0039] In the present disclosure, proteins may be purified by any protein purification method known in the art. For example, various known purification methods can be used, including, but not limited to, hydrophobic interaction chromatography (HIC), fractionation on immunoaffinity or ion-exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on anion or cation-exchange resins, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration, or genetic engineering methods (e.g., tagging). Multiple purification methods can be used in any combination to increase protein purity. In the present disclosure, proteins purified by the above methods may have a purity high enough to allow administration to animals, particularly humans, for experimental, clinical, and therapeutic purposes.

[0040] According to one embodiment of the present disclosure, a protein comprising the amino acid sequence of SEQ ID NO: 1 (GLB1S protein) exhibits GLB1 activity with high purity without the problem of protein truncation. Furthermore, the enzymatic activity of GLB1S is approximately 10% to 20% higher than that of the full-length GLB1 protein, and the enzymatic activity of the GLB1S protein is at least equivalent to that of the full-length GLB1 protein. The enzymatic activity was measured using a beta-galactosidase activity assay commonly used in the art. Specifically, the enzymatic activity was measured by detecting the fluorescence of 4-methylumbelliferone (4-MU), which was produced when the substrate 4-methylumbelliferyl-β-galactopyranoside (MUG) was cleaved by the GLB1 or GLB1S enzyme.

[0041] The term "protein truncation" refers to the removal of the N- or C-terminus of a protein. In GLB1, random truncation is observed at the C-terminus (R637 to V654 in the amino acid sequence of the full-length GLB1 protein, including the amino acid sequence of SEQ ID NO: 2) during protein expression. The emergence of variants with different C-terminal sequences can lead to problems in terms of drug quality control and approval. For example, during the approval process, issues such as (i) differences in efficacy between variants, (ii) differences in stability and / or efficacy when the ratio of variants in the product changes, and (iii) how to uniformly control the ratio of variants in the product can arise. However, these issues do not arise when using GLB1S of SEQ ID NO: 1 of the present invention.

[0042] SEQ ID NO:1: 1 MPGFLVRILL LLLVLLLLGP TRGLRNATQR MFEIDYSRDS FLKDGQPFRY ISGSIHYSRV 61 PRFYWKDRLL KMKMAGLNAI QTYVPWNFHE PWPGQYQFSE DHDVEYFLRL AHELGLLVIL 121 RPGPYICAEW EMGGLPAWLL EKESILLRSS DPDYLAAVDK WLGVLLPKMK PLLYQNGGPV 181 ITVQVENEYG SYFACDFDYL RFLQKRFRHH LGDDVVLFTT DGAHKTFLKC GALQGLYTTV 241 DFGTGSNITD AFLSQRKCEP KGPLINSEFY TGWLDHWGQP HSTIKTEAVA SSLYDILARG 301 ASVNLYMFIG GTNFAYWNGA NSPYAAQPTS YDYDAPLSEA GDLTEKYFAL RNIIQKFEKV 361 PEGPIPPSTP KFAYGKVTLE KLKTVGAALD ILCPSGPIKS LYPLTFIQVK QHYGFVLYRT 421 TLPQDCSNPA PLSSPLNGVH DRAYVAVDGI PQGVLERNNV ITLNITGKAG ATLDLLVENM 481 GRVNYGAYIN DFKGLVSNLT LSSNILTDWT IFPLDTEDAV RSHLGGWGHR DSGHHDEAWA 541 HNSSNYTLPA FYMGNFSIPS GIPDLPQDTF IQFPGWTKGQ VWINGFNLGR YWPARGPQLT 601 LFVPQHILMT SAPNTITVLE LEWAPCSSDD PELCAVTFVD RPVIGSSVTY DHPSKPVEK SEQ ID NO:2: 1 MPGFLVRILL LLLVLLLLGP TRGLRNATQR MFEIDYSRDS FLKDGQPFRY ISGSIHYSRV 61 PRFYWKDRLL KMKMAGLNAI QTYVPWNFHE PWPGQYQFSE DHDVEYFLRL AHELGLLVIL 121 RPGPYICAEW EMGGLPAWLL EKESILLRSS DPDYLAAVDK WLGVLLPKMK PLLYQNGGPV 181 ITVQVENEYG SYFACDFDYL RFLQKRFRHH LGDDVVLFTT DGAHKTFLKC GALQGLYTTV 241 DFGTGSNITD AFLSQRKCEP KGPLINSEFY TGWLDHWGQP HSTIKTEAVA SSLYDILARG 301 ASVNLYMFIG GTNFAYWNGA NSPYAAQPTS YDYDAPLSEA GDLTEKYFAL RNIIQKFEKV 361 PEGPIPPSTP KFAYGKVTLE KLKTVGAALD ILCPSGPIKS LYPLTFIQVK QHYGFVLYRT 421 TLPQDCSNPA PLSSPLNGVH DRAYVAVDGI PQGVLERNNV ITLNITGKAG ATLDLLVENM 481 GRVNYGAYIN DFKGLVSNLT LSSNILTDWT IFPLDTEDAV RSHLGGWGHR DSGHHDEAWA 541 HNSSNYTLPA FYMGNFSIPS GIPDLPQDTF IQFPGWTKGQ VWINGFNLGR YWPARGPQLT 601 LFVPQHILMT SAPNTITVLE LEWAPCSSDD PELCAVTFVD RPVIGSSVTY DHPSKPVEKR LMPPPPQKNK DSWLDHV To solve this problem, one may consider treating the produced full-length GLB1 protein with trypsin to remove the irregular C-terminus, but this has drawbacks such as a significant decrease in protein production yield, excessive costs, and problems such as incomplete purification. When GLB1S of SEQ ID NO: 1 of the present invention is used, there is no need to use degradative enzymes such as trypsin, which makes protein purification easier and allows for the production of highly pure proteins.

[0043] Another aspect of the present disclosure provides a polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 1. In the present disclosure, the nucleotide sequence constituting the polynucleotide may be a nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 1, or a nucleotide sequence encoding various amino acid sequences that can be added to the N-terminus or C-terminus of the amino acid sequence of SEQ ID NO: 1, added to the 5'-terminus or 3'-terminus of the nucleotide sequence capable of encoding the amino acid sequence of SEQ ID NO: 1.

[0044] On the other hand, the polynucleotide may be mutated by substitution, deletion, insertion, or a combination thereof of one or more bases. When the nucleotide sequence is produced by chemical synthesis, synthesis methods widely known in the art, such as those described in the literature (Engels and Uhlmann, Angew Chem Int Ed Engl., 37:73-127, 1988), can be used, including triester, phosphite, phosphoramidate, and H-phosphate methods, PCR and other autoprimer methods, and oligonucleotide synthesis on a solid support.

[0045] Yet another aspect of the present disclosure provides an expression vector comprising the polynucleotide encoding a protein comprising the amino acid sequence of SEQ ID NO: 1. In the present disclosure, the term "expression vector" refers to a genetic construct that contains a gene insert capable of expressing a protein of interest in a suitable host cell, and that contains the necessary regulatory elements operably linked to express the gene insert.

[0046] In the present disclosure, the term "vector" refers to any vehicle for cloning and / or transferring bases into a host cell. A vector may be a replication unit to which other DNA segments can be attached, resulting in replication of the attached segments. Here, "replication unit" refers to any genetic unit (e.g., a plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replicating under its own control. The vector may include viral and non-viral vehicles for introducing bases into an organism, e.g., a host cell, in vitro, or in vivo, and may include mini-globular DNA, transposons such as Sleeping Beauty (Izsvak et al. J. MoI. Biol. 302:93-102 (2000)), or artificial chromosomes.

[0047] Commonly used vectors include, but are not limited to, natural or recombinant plasmids, cosmids, viruses, and bacteriophages. Specific examples of phage or cosmid vectors that can be used include pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A. Examples of plasmid vectors that can be used include, but are not limited to, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET.

[0048] The expression vector contains expression control elements such as an initiation codon, a termination codon, a promoter, and an operator. The initiation codon and termination codon are generally considered to be part of the nucleotide sequence encoding a polypeptide, and must be present in frame with the coding sequence to be functional in an individual when the gene construct is administered. The promoter of the vector may be constitutive or inducible.

[0049] In the present disclosure, "operably linked" refers to a state in which a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein or RNA of interest are functionally linked together so as to have their general functions. For example, a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect the expression of the coding sequence. Operable linkage with an expression vector can be produced using recombinant gene technology well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art.

[0050] In the present disclosure, the expression vector can be used to introduce a polynucleotide encoding a protein comprising SEQ ID NO: 1 of the present disclosure into a CHO cell line, CHO-K1, to transform the CHO-K1 into a cell line capable of producing the protein. Yet another aspect of the present disclosure provides a host cell comprising the polynucleotide or the expression vector.

[0051] The expression vector can be introduced into a host cell and then transformed to produce a transformant, which can be used to express the polynucleotide contained in the expression vector and produce the protein of the present disclosure. The host cell into which the expression vector can be introduced is not particularly limited as long as it can express the polynucleotide and produce the protein of the present disclosure, and specific host cells are the same as those described above.

[0052] Cell transformation may be carried out by various methods, including, but not limited to, CaCl precipitation, the Hanahan method in which the efficiency of CaCl precipitation is increased by using a reducing agent called DMSO (dimethyl sulfoxide), heat shock, electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, PEG-mediated transformation, dextran sulfate, lipofectamine, and desiccation / quenching-mediated transformation, as long as the protein of the present disclosure can be produced.

[0053] Yet another aspect of the present disclosure provides a pharmaceutical composition for treating, preventing, or ameliorating GM1 gangliosidosis or Morquio syndrome type B, comprising a protein comprising the amino acid sequence of SEQ ID NO: 1. Yet another aspect of the present disclosure provides a method for treating, preventing, or ameliorating GM1 gangliosidosis or Morquio syndrome type B.

[0054] In the present disclosure, "amelioration" of a disease state refers to any action that reduces the severity of at least one parameter (e.g., symptom) associated with the disease state. In the present disclosure, "amelioration" refers to any action that at least reduces the severity of a parameter (e.g., symptom) associated with the GM1 gangliosidosis or Morquio B condition by administering the pharmaceutical composition. In the present disclosure, "prevention" of a disease state refers to reducing the risk of acquiring a disease, condition, or disorder, or preventing the occurrence of at least one clinical symptom of the disease in a subject who may be exposed to or predisposed to the disease but who has not yet experienced or displayed symptoms of the disease. In the present disclosure, "prevention" of GM1 gangliosidosis or Morquio B refers to any action that inhibits or delays the onset of GM1 gangliosidosis or Morquio B by administering the pharmaceutical composition. In this disclosure, "treatment" includes alleviating, attenuating, or ameliorating a disease, condition, disorder, or symptoms thereof; preventing additional symptoms; ameliorating or preventing the underlying cause of a symptom; inhibiting a disease, condition, or disorder (e.g., preventing the onset of the disease, condition, or disorder); relieving a disease, condition, or disorder; inducing regression of a disease, condition, or disorder; alleviating conditions caused by a disease, condition, or disorder; or prophylactically and / or therapeutically halting symptoms of a disease, condition, or disorder. In this disclosure, "treatment" of GM1 gangliosidosis or Morquio syndrome type B refers to any action that improves or beneficially alters the symptoms of an individual suspected of or affected with GM1 gangliosidosis or Morquio syndrome type B by administering the pharmaceutical composition.

[0055] A pharmaceutical composition of the present disclosure comprises a pharmaceutically effective amount of a protein of the present disclosure and may further comprise a pharmaceutically acceptable carrier.

[0056] In the present disclosure, GM1 gangliosidosis is a genetic central nervous system disease caused by a deficiency in beta-galactosidase-1 enzyme activity in lysosomes due to a GLB1 gene mutation, resulting in the destruction of nerve cells in the central and peripheral nervous system.In the present disclosure, Morquio syndrome type B is a disease caused by a deficiency in the beta-galactosidase enzyme, which leads to an impairment of keratan sulfate degradation.

[0057] In the present disclosure, the protein comprising the amino acid sequence of SEQ ID NO: 1 may be contained in a composition at a concentration of, but not limited to, about 0.001 to about 500 mg / ml, about 1 to about 50 mg / ml, about 1 to about 10 mg / ml, about 3 to about 7 mg / ml, about 5 mg / ml, about 10 to about 50 mg / ml, about 20 to about 40 mg / ml, or about 30 mg / ml. The protein comprising the amino acid sequence of SEQ ID NO: 1 may be contained in a pharmaceutical composition for intravenous administration at a concentration of, but not limited to, about 1 to about 10 mg / ml. The protein comprising the amino acid sequence of SEQ ID NO: 1 may be contained in a pharmaceutical composition for intracerebroventricular administration at a concentration of, but not limited to, about 10 to about 50 mg / ml.

[0058] In the present disclosure, pharmaceutical compositions containing the proteins may be formulated and administered in a manner consistent with medical practice. Factors to be considered in this regard include the disease being treated, the particular animal being treated, the clinical condition of the individual subject, the cause of the disease, the site of delivery of the substance, the method of administration, the administration schedule, and other factors known to clinicians. In the present disclosure, dosage forms may include liquid dosage forms, lyophilized powder dosage forms, etc. Pharmaceutical compositions of the present disclosure may be packaged in ampoules, vials, bottles, cartridges, reservoirs, Lyo-Ject, etc. (登録商標) or pre-filled syringes, and may be manufactured in single or multi-dose forms.

[0059] The "pharmaceutically effective dose," "pharmaceutical effective amount," "administered dose," "administered amount," "therapeutically effective dose," "therapeutically effective amount," "effective dose," and / or "effective amount" of the administered protein may be determined by the above considerations and is the minimum amount necessary to prevent, ameliorate, or treat a particular disease (e.g., GM1 gangliosidosis or Morquio syndrome type B). In the present disclosure, the terms "pharmaceutically effective dose," "pharmaceutically effective amount," "administered dose," "administration amount," "therapeutically effective dose," "therapeutically effective amount," "effective dose," and / or "effective amount" of the protein are, for example, but not limited to, about 0.0001 mg / kg to about 100 mg / kg per administration, about 0.001 mg / kg to about 100 mg / kg per administration, about 0.01 mg / kg to about 100 mg / kg per administration, about 0.1 mg / kg to about 100 mg / kg per administration, about 0.1 mg / kg to about 30 mg / kg per administration, about 0.1 mg / kg to about 10 mg / kg per administration, about 1 mg / kg to about 30 mg / kg per administration, or about 1 mg / kg to about 5 mg / kg per administration. Thus, the composition of the present disclosure may contain a single dose of 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 (e.g., GLB1S protein). The pharmaceutical composition of the present disclosure may be administered intravenously at a concentration of, but not limited to, 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. The pharmaceutical composition of the present disclosure may be administered intracerebroventricularly at a concentration of, but not limited to, about 5 mg / head / every other week to about 30 mg / head / every other week, about 15 mg / head / every other week to about 30 mg / head / every other week, about 15 mg / head / every other week, about 5 mg / head / month to about 30 mg / head / month, about 15 mg / head / month to about 30 mg / head / month, or about 15 mg / head / month.

[0060] The pharmaceutical compositions of the present disclosure may be administered periodically, such as once daily, three times a week, twice a week, once a week, three times a month, twice a month, or once a month, at the discretion of an experienced clinician, or may be administered non-periodically in situations such as, but not limited to, acute progression of disease.

[0061] In the present disclosure, pharmaceutical compositions may be prepared by mixing the protein having a desired degree of purity with pharmaceutically or physiologically acceptable carriers, excipients, or stabilizers using standard methods known in the art. Acceptable carriers include saline or buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides (containing less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, PLURONICS®, or PEG.

[0062] The pharmaceutical composition of the present disclosure may contain a pharmaceutically acceptable salt at approximately physiological concentrations. Optionally, the dosage form of the present disclosure may contain a pharmaceutically acceptable preservative. Specifically, in one embodiment, the preservative concentration may be about 0.1% to about 2.0% (generally v / v). In the present disclosure, the preservative may be one known in the pharmaceutical industry, specifically, benzyl alcohol, phenol, m-cresol, methylparaben, propylparaben, or a combination thereof. The pharmaceutical composition of the present disclosure may contain a pharmaceutically acceptable surfactant at a concentration of about 0.005 to about 0.02%.

[0063] In accordance with the present disclosure, the pharmaceutical composition may further contain one or more active compounds necessary for the treatment of GM1 gangliosidosis or Morquio syndrome type B, particularly active compounds with complementary activities to the protein that do not adversely affect each other. The compounds may be included in the composition in an amount effective to achieve the intended purpose.

[0064] In the present disclosure, the pharmaceutical composition may be administered to a human or animal subject by known methods, such as intracerebroventricular, intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, subcutaneous, or inhalation routes. Specifically, the pharmaceutical composition may be administered intracerebroventricularly or intravenously, more specifically, intracerebroventricularly through an intracerebroventricular catheter system comprising a reservoir and a catheter connected to the reservoir.

[0065] According to one embodiment of the present disclosure, when the pharmaceutical composition is administered to a patient, the GLB1 enzyme activity in the patient's blood can be increased by 50% or more.

[0066] According to one embodiment of the present disclosure, when the pharmaceutical composition is administered to a patient, the amount of keratan sulfate in one or more internal organs of the patient, such as the brain, liver, etc., may be reduced.

[0067] Yet another aspect of the present disclosure provides a method for producing a protein comprising the amino acid sequence of SEQ ID NO: 1. In one embodiment, the method includes the steps of: a) introducing a target gene encoding a protein comprising the amino acid sequence of SEQ ID NO: 1 into a vector; b) transfecting host cells with the vector; c) culturing the transfected cells and harvesting the cell culture supernatant; and d) purifying the supernatant to obtain the protein. In one embodiment, the host cells are Chinese hamster ovary (CHO) cells.

[0068] Yet another aspect of the present disclosure provides a protein produced by the method for producing a protein, the protein comprising the amino acid sequence of SEQ ID NO:1. [Example]

[0069] The configurations and effects of the present disclosure will be described in more detail below with reference to examples, however, these examples are provided for illustrative purposes only to aid in understanding the present disclosure and are not intended to limit the scope of the present disclosure.

[0070] Comparative Example 1 (Control Group): Construction of recombinant beta-galactosidase full-length protein (GLB1) expression vector The target gene, 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 CHO GS system designed and synthesized by GenScript. The vector contains an MCS for expression of the protein target gene. The vector also contains a glutamine synthetase (GS) gene for selection of positive clones. Finally, the plasmid was prepared at a concentration of 1 μg / μl.

[0071] Comparative Example 2 (control group): Construction of GLB1 protein-expressing cell line and development of GLB1 protein expression process CHOK1-GenS cells were used as host cells, licensed from the European Collection of Authenticated Cell Cultures (ECACC). Plasmids as described in Comparative Example 1 above were transfected into CHOK1-GenS cells. Forty-eight hours after transfection, supernatants were harvested for Western blot analysis. For pool screening, transfected cells were seeded in 24-well plates with selective medium. After screening, supernatants from all cell pools were analyzed by dot blot. Based on signal intensity, mixed cell pools M1 and M2 were selected for single-cell clone screening by limiting dilution method. Cell Metric (商標) The monoclonality of single cell clones was recorded and confirmed using a CLD Imager. 43 single clones with higher titers and superior growth performance were selected. Finally, the top clones showing the best productivity and growth performance were selected.

[0072] The selected cells were subcultured every 72 hours. The total number of passages, i.e., the number of times the culture was subcultured, was 20, and the passages were designated P0-P20, respectively. Cells at P0, P17, and P20 were harvested for fed-batch culture. Cells at P0 and P20 were collected for target sequence detection. Cell growth survival, cell density, viability, GLB1 expression titer, and corresponding protein quality demonstrated that the top clones were stable over a 60-day subculture period.

[0073] The supernatant from the P0 fed-batch culture was harvested on the final day for purification and sequence analysis. Specifically, the C-terminal sequence of the protein was analyzed by mass spectrometry (MS). Tables 1 to 3 show the MS analysis results for the C-terminal sequence of the GLB1 protein.

[0074] [Table 1]

[0075] [Table 2]

[0076] [Table 3]

[0077] As shown in Tables 1 to 3, severe C-terminal truncation of the GLB1 protein was observed in a progressive truncation pattern with C-terminal amino acids of different lengths. Three to four mutants with percentages greater than 10% were found in all three clones, and no mutants were considered to be major products, confirming severe C-terminal truncation of the GLB1 protein.

[0078] Example 1. Plasmid construction for recombinant beta-galactosidase (GLB1S) The target gene, GLB1S (SEQ ID NO: 1), was synthesized and optimized for CHO host cells. The target gene was inserted into the upstream MCS region of the pGenHT1.0-DGV vector. The plasmid was prepared at a concentration of 1 μg / μl. The transfection plasmid was named NP4K_pGenHT1.0-DGV.

[0079] Example 2. Construction of GLB1S protein-expressing cell line and development of GLB1S protein expression process The plasmids prepared as described in Example 1 above were transfected into CHOK1-GenS host cells. 48 hours after transfection, the supernatants were harvested for Western blot analysis. The transfected cells were seeded into 24-well plates with selective medium for pool screening. After screening, the supernatants of all cell pools were analyzed by dot blot. Based on signal intensity, mixed cell pools M1, M2, M3, and M4 were selected for single-cell clone screening by limiting dilution method. Cell Metric (商標) The monoclonality of single cell clones was recorded and confirmed using a CLD Imager. 24 single clones with higher titers and superior growth performance were selected. Finally, the top clones showing the best productivity and growth performance were selected.

[0080] The selected top clones were cultured in a 3-L bioreactor in a fed-batch manner, controlling and monitoring variables such as pH, dissolved oxygen (DO), agitation, and temperature. The top clones were cultured according to the GenScript cell culture process development platform conditions and parameters using the materials and reagents listed in Table 4. The culture supernatant was harvested on day 15 of culture or when the viability was 70% or less, whichever was earlier.

[0081] [Table 4]

[0082] The titer of the harvested cell culture supernatant was determined to be 2.409 g / L by 4-MU-β-galactopyranoside (MUG) assay.

[0083] The selected top clones were then applied to fed-batch cultivation in a 50-L bioreactor while controlling and monitoring variables such as pH, dissolved oxygen (DO), agitation, and temperature to demonstrate process scalability. The top clones were cultured according to GenScript's cell culture process development platform conditions and parameters using the materials and reagents listed in Table 5. Culture supernatants were harvested on day 15 of culture or when viability was 70% or less, whichever was earlier.

[0084] [Table 5]

[0085] The titer of the harvested cell culture supernatant was determined to be 2.061 g / L by 4-MU-β-galactopyranoside (MUG) assay.

[0086] The product harvested from the bioreactor was also purified using the developed purification process. After obtaining the culture supernatant, the protein was purified sequentially through depth filtration (DF), solvent / detergent (S / D), anion exchange chromatography (AEX), cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), and ultrafiltration / diafiltration. A detailed purification process flow is shown in Figure 1. The purified protein product was analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC) and size-exclusion high-performance liquid chromatography (SEC-HPLC), and the results are shown in Table 6.

[0087] [Table 6]

[0088] As shown in Table 6, the purity of the product confirmed by RP-HPLC and SEC-HPLC was high. The main peak in RP-HPLC represented the target protein GLB1S, and the pre-peak represented a fragment. It can be seen that the pre-peak ratio of the product measured by RP-HPLC was very low. The main peak ratio of the product (GLB1S) as measured by SEC-HPLC was over 99%, and the high molecular weight (HMW) and low molecular weight (LMW) ratios of the product were also acceptable.

[0089] Example 3. Confirmation of GLB1S protein expression by SDS-PAGE and SEC-HPLC As shown in Example 2, the GLB1S protein was obtained by 50 L cultivation and purification.

[0090] The purified protein was subjected to SDS-PAGE, SEC-HPLC, and RP-HPLC analyses, and the results are shown in Figures 2 to 4. As shown in Figure 2, the GLB1S protein (having a molecular weight between the 50 kDa marker and the 75 kDa marker) appears as the major product. As shown in Figures 3A and 3B, a 97.26% pure GLB1S protein was obtained. As shown in Figures 4A and 4B, the RP-HPLC purity of the GLB1S protein was 99.57%.

[0091] Example 4. Confirmation of GLB1S protein activity The enzyme activities of GLB1S (rhGLB1S), rhGLB1 (GenScript), and rhGLB1-Fc (AbClone) were measured. To obtain rhGLB1S, CHO-K1 cells expressing GLB1S protein were cultured at a 10 L scale using the methods described in Examples 1 and 2. The culture supernatant was purified sequentially by anion exchange chromatography (AEX), cation exchange chromatography (CEX), hydrophobic interaction chromatography (HIC), and ultrafiltration / diafiltration. Specifically, a 1 mg / ml GLB1S solution was used for activity analysis. For rhGLB1-Fc, the hinge, CH2, and CH3 of the heavy chain of immunoglobulin gamma-1 (SEQ ID NO: 3) were used as Fc. The substrate used was 0.5 mM 4-MU-β-galactopyranoside (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), which is stable for several months at 4°C or -20°C.

[0092] The tissues used for activity analysis were leukocytes, fibroblasts, and amniotic fluid cells. For this analysis, 0.020–0.030 mg protein was required for leukocytes, and 0.010–0.020 mg protein for fibroblasts and amniotic fluid cells. To achieve the required protein concentrations, 0.10 ml of diluted tissue lysate was placed in a 12 x 75 mm test tube, and duplicates of each sample were prepared. 0.10 ml of substrate solution was then added, followed by incubation at 37°C for 20 minutes. The reaction was then stopped by adding 1.30 ml of 0.17 M glycine-carbonate buffer (pH 9.8). For a blank, the substrate alone was incubated, the reaction was stopped by adding glycine-carbonate buffer, and a control lysate preparation diluted to the appropriate protein concentration range was added. Fluorescence readings for the prepared samples were taken at an excitation wavelength of 360 nm and an emission wavelength of 415 nm, and the duplicate readings were averaged. The activity was also calculated using the following formula, and the results are shown in Table 7.

[0093]

number

[0094] [Table 7]

[0095] As can be seen from Table 7, when diluted at a ratio of 1:1000, the activity of the rhGLB1S of the present invention was 416,953 nmol / hr / mg, which was approximately 12.2% higher than the activity of the full-length rhGLB1 protein, 371,604 nmol / hr / mg. When diluted at a ratio of 1:2000, the activity of rhGLB1S was approximately 11.4% higher than that of rhGLB1. In other words, the activity of GLB1S, in which the C-terminus is truncated from GLB1, was superior to that of the full-length GLB1 protein and GLB1-Fc conjugated to an antibody, confirming that it can be used as a drug with superior efficacy for the amelioration, prevention, or treatment of GM1 gangliosidosis or Morquio syndrome type B.

[0096] Example 5. Preparation of GLB1KO (Knock-out) mouse model GLB1KO mice were generated using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genetic scissors technology. GLB1KO mice were generated by targeting exon 6 of mouse GLB1. The specific generation was outsourced to Macrogen. Genotyping was performed to confirm the presence or absence of knockout (KO) in GM1 gangliosidosis or Morquio syndrome type B disease model mice (GLB1KO mice). KO mice were identified through genotyping. Specifically, the presence or absence of GlB1 mutations was confirmed in 22 F0 mutant mice. KO mutations were confirmed in F0 #1, #5, and #19. F0 #1 was selected, maintained, and subsequently used for further experiments (Figure 5).

[0097] Example 6: Confirmation of the effect of GLB1S administration in a GLB1KO (Knock-out) mouse model 1. Composition of the control group and the experimental group

[0098] [Table 8]

[0099] G1: wild-type mice group (wild-type control group); G2: KO mouse group (negative control group) G3-G7: KO mouse group (experimental group) 2. Administration of vehicle or GLB1S Healthy mice were weighed and ranked, and randomly allocated according to the structure of Table 8 so that the average weight and sex of each group were distributed as evenly as possible.

[0100] After the mice were corrected using a corrector, vehicle or GLB1S was administered intravenously to the mice once using an insulin syringe (single administration).

[0101] 3. Non-clinical trials 3-1. Non-clinical trial evaluation items and sample collection After vehicle or GLB1S administration, general symptoms and body weight were evaluated. During the administration and observation period, the presence or absence of death and the type and severity of general symptoms were observed at least once a day and recorded for each individual. The day of administration was designated Day 0.

[0102] The body weight of the mice was measured at the time of obtaining the mice, at the time of group separation, at the time of vehicle or GLB1S administration, at autopsy, and once a week during the observation period.

[0103] Five mice were administered vehicle or GLB1S and sampled on days 7, 14, and 28, respectively. On the scheduled necropsy day, the mice were perfused, then necropsied and organs were removed. The removed samples were stored at -70°C. One mouse was dropped out of group G3, and the experiment for group G3 was completed with 14 mice (i.e., five mice sampled on days 7 and 14, and four mice sampled on day 28).

[0104] 3-2.Statistical analysis Statistical analysis was performed using SPSS Statistics 12.0K for medical use. Comparisons were made between the normal control group (G1) and the negative control group (G2), and between the negative control group (G2) and the experimental groups (G3-G7). Non-parametric comparisons were performed because the number of subjects to be compared in each group was 5 or less. Non-parametric comparison procedures included Kruskal-Wallis, Mann-Whitney post-hoc tests, and Bonferroni correction. All statistical analyses were considered statistically significant when P<0.05.

[0105] 3-3. Non-clinical trial results (1)Dead animals One mouse in the 0.1 mg / kg GLB1S administration group (G3) died on day 27. No mice died in the other groups (G1, G2, G4, G5, G6, and G7).

[0106] (2) General symptoms No abnormal symptoms were observed in any of the groups (G1 to G7).

[0107] (3) Weight No differences in body weight were observed among all groups (G1 to G7) during the administration and observation period.

[0108] [Table 9]

[0109] Data were expressed as mean ± SE. Results were statistically analyzed by Kruskal-Wallis.

[0110] (4) Organ weight No statistically significant differences were observed in body and organ weights among all groups. However, on day 7, the negative control group showed a 32% or greater increase in liver and spleen weight compared to the normal control group, and all GLB1S-treated groups (G3 to G7) showed a 22% or greater decrease in liver weight compared to the negative control group. These differences in liver and spleen weight were not statistically significant.

[0111] [Table 10]

[0112] Data were expressed as mean ± SE. Results were statistically analyzed by Kruskal-Wallis.

[0113] 4. Measurement of Keratan Sulfate Content in Mouse Liver Tissue 4-1. Keratan sulfate content measurement method Keratan sulfate levels were measured by collecting liver tissue from each control and experimental group on days 7, 14, and 28 after vehicle or GLB1S administration. The description of the negative control and experimental groups and the number of individuals in each group are shown in Table 11 below. Liver tissue collected from mice was homogenized, and lipids in the liver tissue were removed. Disaccharide keratan sulfate was then produced by enzymatic treatment. Keratan sulfate was then selectively extracted using PGC-SPE, and the keratan sulfate content was measured using LC-MS / MS.

[0114] [Table 11]

[0115] 4-2. Measurement of keratan sulfate content in mouse liver tissue Figures 6A-6F are graphs showing the results of analyzing the keratan sulfate content in each group on days 7, 14, and 28. As can be seen in Figures 6A-6F, the keratan sulfate content increased over time in all groups except for the negative control group G2, indicating that GLB1S can suppress keratan sulfate levels up to day 14 after administration. On day 28 after GLB1S administration, re-accumulation of keratan sulfate was observed in all treatment groups.

[0116] Figure 7 is a graph showing the results of analyzing keratan sulfate content 7 days after vehicle or GLB1S administration. As can be seen in Figure 7, keratan sulfate content was significantly reduced in a dose-dependent manner in all GLB1S-treated groups compared to G2.

[0117] Figure 8 is a graph showing the results of analyzing the keratan sulfate content 14 days after vehicle or GLB1S administration. As can be seen from Figure 8, the keratan sulfate content was significantly reduced in G5, G6, and G7, which were administered at a dose of >1.0 mg / kg, compared to G2.

[0118] The above results show that significant enzyme replacement therapy effects can be expected when GLB1S is administered weekly or every other week.

[0119] In the case of the full-length protein rhGLB1, it is difficult to obtain a homogeneous protein due to protein truncation during the process of producing the protein for use as a drug. Using large-scale production facilities to commercialize such full-length proteins is not economically viable. This is because additional processing steps beyond simply expressing the protein from transformed host cells are required to obtain a protein that meets the standards for use as a final drug. Furthermore, using host cells that express the full-length protein poses the problem of not being able to guarantee predictability in drug quality.

[0120] The present invention has resolved the problems (e.g., protein cleavage) that can occur when using a full-length protein as described above, and has confirmed for the first time that the use of a GLB1S protein exhibits enzyme activity equivalent to that of the full-length GLB1 protein. The present invention has secured a GLB1S that significantly improves the predictability of protein production conditions and drug quality on a commercial scale, and has confirmed that such a protein can be used as a drug with superior efficacy for the amelioration, prevention, or treatment of GM1 gangliosidosis or Morquio syndrome type B.

Claims

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

1.

2. The protein of claim 1, comprising the amino acid sequence of SEQ ID NO:

1.

3. A polynucleotide encoding the protein of claim 1 or 2.

4. An expression vector comprising the polynucleotide of claim 3.

5. A host cell comprising the polynucleotide of claim 3 or the expression vector of claim 4.

6. A method for treating, preventing, or ameliorating GM1 gangliosidosis or Morquio syndrome type B, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising the protein of claim 1 or 2 to a patient with GM1 gangliosidosis or Morquio syndrome type B.

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

8. 7. 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. 7. The method of claim 6, wherein a therapeutically effective amount of the pharmaceutical composition is administered intracerebroventricularly (ICV) or intravenously.

10. 10. The method of claim 9, wherein ICV administration comprises administering a therapeutically effective amount of the pharmaceutical composition to the patient through 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 the full-length GLB1 protein.

12. 7. 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. 7. The method of claim 6, wherein the therapeutically effective amount of the pharmaceutical composition is administered once weekly, once every two weeks, twice monthly, or once monthly.

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

15. A pharmaceutical composition for treating, preventing, or ameliorating GM1 gangliosidosis or Morquio syndrome type B, comprising the protein of 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. 16. The pharmaceutical composition of claim 15, which increases GLB1 enzyme activity in the patient's blood by 50% or more.

18. 16. The pharmaceutical composition of claim 15, which reduces the amount of keratan sulfate in one or more internal organs of a patient.

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

20. 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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