WO2024080304A1 - Protéine de fusion d'albumine sérique et de protéine physiologiquement active - Google Patents
Protéine de fusion d'albumine sérique et de protéine physiologiquement active Download PDFInfo
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- C12N15/62—DNA sequences coding for fusion proteins
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Definitions
- the present invention relates to a fusion protein in which serum albumin (SA) is bound to a protein having physiological activity (biologically active protein) and a method for producing the same.
- SA serum albumin
- biologically active protein a protein having physiological activity
- Such a fusion protein relates, for example, to one in which the C-terminus of SA is bound to the N-terminus of a biologically active protein.
- Some biologically active proteins have low expression levels and/or low activity when expressed as a recombinant protein by introducing a gene encoding the biologically active protein into a host cell such as a mammalian cell, particularly when expressed so that the recombinant protein is secreted from the cell and accumulated in the culture medium.
- the present invention relates to such a fusion protein of SA and a biologically active protein that can be efficiently produced as a highly active recombinant protein, and also relates to a method for producing the fusion protein.
- a biologically active protein to be fused to SA, and any biologically active protein can be made into a fusion protein with SA.
- the present invention relates in particular to a fusion protein in which serum albumin (SA) is bound to a lysosomal enzyme, for example, the C-terminus of SA is bound to the N-terminus of a lysosomal enzyme, or the C-terminus of a lysosomal enzyme is bound to the N-terminus of SA, either directly or via a linker.
- SA serum albumin
- Some lysosomal enzymes have low expression levels and/or low activity when expressed as recombinant proteins by introducing a gene encoding the lysosomal enzyme into a host cell such as a mammalian cell, particularly when the recombinant protein is expressed so as to be secreted from the cell and accumulated in the culture medium.
- the present invention relates to such a fusion protein of a lysosomal enzyme and SA that can be efficiently produced as a highly active recombinant protein, and also relates to a method for producing the fusion protein.
- a physiologically active protein to be fused to the lysosomal enzyme, and any lysosomal enzyme can be made into a fusion protein with SA.
- the present invention also relates to a fusion protein in which serum albumin (SA) and galactosylceramidase (GALC) are bound, for example, by binding the C-terminus of SA to the N-terminus of GALC, or the C-terminus of GALC to the N-terminus of SA, either directly or via a linker.
- SA serum albumin
- GALC galactosylceramidase
- a gene encoding GALC is introduced into a host cell such as a mammalian cell and the protein is expressed as a recombinant protein, the expression level and/or activity of GALC may be low, particularly when the recombinant protein is expressed so as to be secreted from the cell and accumulated in the culture medium.
- the present invention relates to such a fusion protein of GALC and SA that can be efficiently produced as a highly active recombinant protein, and to a method for producing the fusion protein.
- the present invention also relates to a fusion protein in which serum albumin (SA) and glucocerebrosidase (GBA) are bound, for example, by binding the C-terminus of SA to the N-terminus of GBA, or the C-terminus of GBA to the N-terminus of SA, either directly or via a linker.
- SA serum albumin
- GBA glucocerebrosidase
- the present invention relates to a fusion protein in which serum albumin (SA) and a cytokine are bound, for example, by binding the C-terminus of SA to the N-terminus of a cytokine, or the C-terminus of a cytokine to the N-terminus of SA, either directly or via a linker.
- SA serum albumin
- Some cytokines have low expression levels and/or low activity when expressed as a recombinant protein by introducing a gene encoding the cytokine into a host cell such as a mammalian cell, particularly when expressed so that the recombinant protein is secreted from the cell and accumulated in the culture medium.
- the present invention relates to such a fusion protein of cytokine and SA that can be efficiently produced as a highly active recombinant protein, and to a method for producing the fusion protein.
- physiologically active protein to be fused with the cytokine, and any cytokine can be made into a fusion protein with SA.
- the present invention relates in particular to a fusion protein in which serum albumin (SA) and an interleukin are bound, for example, by binding the C-terminus of SA to the N-terminus of an interleukin, or the C-terminus of an interleukin to the N-terminus of SA, either directly or via a linker.
- SA serum albumin
- Some interleukins have low expression levels and/or low activity when expressed as a recombinant protein by introducing a gene encoding the interleukin into a host cell such as a mammalian cell, particularly when the recombinant protein is expressed so as to be secreted from the cell and accumulated in the culture medium.
- the present invention relates to such a fusion protein of an interleukin and SA that can be efficiently produced as a highly active recombinant protein, and also relates to a method for producing the fusion protein.
- a physiologically active protein to be fused with the interleukin, and all interleukins can be made into a fusion protein with SA.
- the present invention relates in particular to a fusion protein in which serum albumin (SA) and interleukin 10 (IL-10) are bound, for example, by binding the C-terminus of SA to the N-terminus of IL-10, or the C-terminus of IL-10 to the N-terminus of SA, either directly or via a linker.
- SA serum albumin
- IL-10 interleukin 10
- IL-10 is expressed as a recombinant protein by introducing a gene encoding IL-10 into a host cell such as a mammalian cell
- the expression level and/or activity of IL-10 may be low, particularly when the recombinant protein is expressed so as to be secreted from the cell and accumulated in the culture medium.
- the present invention relates to such a fusion protein of IL-10 and SA that can be efficiently produced as a highly active recombinant protein, and also to a method for producing said fusion protein.
- the present invention relates to a fusion protein in which serum albumin (SA) and a neurotrophic factor are bound, for example, by binding the C-terminus of SA to the N-terminus of a neurotrophic factor, or the C-terminus of a neurotrophic factor to the N-terminus of SA, either directly or via a linker.
- SA serum albumin
- Some neurotrophic factors have low expression levels and/or low activity when expressed as recombinant proteins by introducing a gene encoding the neurotrophic factor into a host cell such as a mammalian cell, particularly when expressed so that the recombinant protein is secreted from the cell and accumulated in the culture medium.
- the present invention relates to such a fusion protein of a neurotrophic factor and SA that can be efficiently produced as a highly active recombinant protein, and also relates to a method for producing the fusion protein.
- physiologically active protein to be fused to the neurotrophic factor, and any neurotrophic factor can be made into a fusion protein with SA.
- the present invention relates in particular to a fusion protein in which serum albumin (SA) and brain-derived neurotrophic factor (BDNF) are bound, for example, by binding the C-terminus of SA to the N-terminus of BDNF, or the C-terminus of BDNF to the N-terminus of SA, either directly or via a linker.
- SA serum albumin
- BDNF brain-derived neurotrophic factor
- BDNF brain-derived neurotrophic factor
- SA serum albumin
- BDNF brain-derived neurotrophic factor
- the present invention also relates to a fusion protein in which serum albumin (SA) and nerve growth factor (NGF) are bound, for example, by binding the C-terminus of SA to the N-terminus of NGF, or the C-terminus of NGF to the N-terminus of SA, either directly or via a linker.
- SA serum albumin
- NGF nerve growth factor
- the present invention relates to such a fusion protein of NGF and SA that can be efficiently produced as a highly active recombinant protein, and to a method for producing the fusion protein.
- the present invention also relates to a fusion protein in which serum albumin (SA) and neurotrophin 3 (NT-3) are bound, for example, by binding the C-terminus of SA to the N-terminus of NT-3, or the C-terminus of NT-3 to the N-terminus of SA, either directly or via a linker.
- SA serum albumin
- NT-3 neurotrophin 3
- NT-3 is expressed as a recombinant protein by introducing a gene encoding NT-3 into a host cell such as a mammalian cell, the expression level and/or activity may be low, particularly when the recombinant protein is expressed so as to be secreted from the cell and accumulated in the culture medium.
- the present invention relates to such a fusion protein of NT-3 and SA that can be efficiently produced as a highly active recombinant protein, and to a method for producing said fusion protein.
- the present invention also relates to a fusion protein in which serum albumin (SA) and neurotrophin 4 (NT-4) are bound, for example, by binding the C-terminus of SA to the N-terminus of NT-4, or the C-terminus of NT-4 to the N-terminus of SA, either directly or via a linker.
- SA serum albumin
- NT-4 neurotrophin 4
- the present invention relates to such a fusion protein of NT-4 and SA that can be efficiently produced as a highly active recombinant protein, and to a method for producing said fusion protein.
- Krabbe disease a type of lysosomal disease, is also known as galactosylceramide lipidosis or globoid cell leukodystrophy. It is a genetic disease caused by a genetic abnormality that reduces the activity or deficiency of galactosylceramidase (galactocerebrosidase, GALC), which is necessary for the breakdown of sphingolipids in lysosomes.
- GALC galactosylceramidase
- GALC galactosylceramidase uses galactosylsphingosine, galactocerebroside, and other substrates as catalysts for the hydrolysis of the galactose ester bonds in the molecules of these substrates.
- Krabbe disease is classified into infantile type, which begins at about 3 to 6 months of age and is characterized by irritability and regression, and often results in death within 2 to 3 years; late infantile type, which begins at about 6 months to 3 years of age and is characterized by irritability, delayed psychomotor development, and regression; juvenile type, which begins at about 3 to 10 years of age and progresses slowly, and is characterized by visual impairment, gait disturbance, ataxia, etc.; and adult type, which begins at about 10 years of age or older and is characterized by psychiatric symptoms, etc.
- Gaucher disease a type of lysosomal disease, is a genetic disease caused by a genetic abnormality that results in a decrease in the activity or deficiency of glucocerebrosidase ( ⁇ -glucosidase, GBA), an enzyme required for the breakdown of the biological glycolipid glucocerebroside in the lysosome.
- GBA glucocerebrosidase
- GBA Glucocerebrosidase
- Glucocerebroside accumulates in macrophages, particularly in the liver, spleen, and bones, causing anemia and thrombocytopenia due to decreased splenic function, as well as hepatosplenomegaly, bone pain, fractures, and central nervous system disorders. It is believed that the central nervous system disorders are caused by the accumulation of glucosylsphingosine, the lyso form of glucocerebroside, in the brain.
- Gaucher disease is classified according to the presence or absence and severity of neurological symptoms into the mildest type I (non-neurological type), which occurs at ages ranging from infancy to adulthood and is characterized by slowly progressing symptoms such as enlargement of the liver and spleen, anemia, decreased platelets, and fractures without accompanying neurological symptoms; the most severe type II (acute neurological type), which begins in infancy and, in addition to the symptoms of type I, rapidly progresses with neurological symptoms such as psychomotor developmental delay, convulsions, and neck retroversion, resulting in death from oxygen deficiency by the age of two; and the progressive type III (subacute neurological type), which develops gradually during infancy and progresses more slowly than type II.
- Lysosomal diseases other than Krabbe disease and Gaucher disease are also caused by genetic deficiencies in lysosomal enzymes.
- enzyme replacement therapy is performed in which the genetically deficient enzyme is produced as a recombinant enzyme using genetic engineering technology and administered to the patient.
- Non-Patent Document 1 The gene encoding human GALC (hGALC) was isolated in 1993 (Non-Patent Document 1). However, there are no drugs for use as enzyme replacement therapy for Krabbe disease that contain recombinant human GALC (rhGALC) produced using this gene as the active ingredient.
- Non-Patent Document 2 The gene encoding human GBA (hGBA) was isolated in 1986 (Non-Patent Document 2). However, there are no drugs for use as enzyme replacement therapy for Gaucher disease that contain recombinant human GBA (rhGBA) produced using this gene as the active ingredient.
- IL-10 a type of cytokine, is an anti-inflammatory cytokine produced by Th2 cells and can inhibit cytokine production by Th1 cells, suppressing immune responses. Due to its anti-inflammatory effect, IL-10 is expected to be effective against many inflammatory diseases, namely, neuropathic pain, multiple sclerosis, spinal cord injury, ALS, neuroinflammation, symptoms associated with arthritis and other joint diseases, and autoimmune diseases. In addition to its immunosuppressive effect, it has also been reported that IL-10 may have an anti-cancer effect (Non-Patent Document 3). The gene encoding human IL-10 was isolated in 1991 (Non-Patent Document 4). However, there are no pharmaceuticals for use as a therapeutic agent for inflammatory diseases or cancer that contain recombinant human IL-10 (rhIL-10) produced using this gene as an active ingredient.
- rhIL-10 recombinant human IL-10
- BDNF neurotrophic factor
- TrkB a specific receptor on the surface of target cells and regulates the growth of nerve cells, such as the survival and growth of nerve cells and the enhancement of synaptic function. Due to its neurodevelopmental effect, BDNF is expected to be developed as a treatment for a variety of diseases, including neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, spinal degenerative diseases such as amyotrophic lateral sclerosis, as well as diabetic neuropathy, cerebral ischemic disease, developmental disorders such as Rett syndrome, schizophrenia, depression, and Rett syndrome.
- neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease
- spinal degenerative diseases such as amyotrophic lateral sclerosis, as well as diabetic neuropathy, cerebral ischemic disease, developmental disorders such as Rett syndrome, schizophrenia, depression, and Rett syndrome.
- Non-Patent Documents 5 and 6 The gene encoding human BDNF was isolated in 1993 (Non-Patent Documents 5 and 6). However, there are no pharmaceuticals containing recombinant human BDNF (rhBDNF) produced using this gene as an active ingredient for use as a treatment for neurodegenerative diseases, etc.
- rhBDNF recombinant human BDNF
- NGF neurotrophic factor
- a type of neurotrophic factor is a protein that promotes the survival and growth of sympathetic nerve cells and spinal sensory neurons in the peripheral nervous system, and promotes the survival and differentiation of cholinergic nerve cells in the central nervous system, especially in the basal forebrain.
- NGF is expected to be developed as a treatment for neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, but it is also expected to be effective in preventing aging and degeneration of brain function, improving brain function, preventing and treating dementia, improving memory and learning ability by building neural networks, and enhancing the action of neurotransmitters.
- Non-Patent Document 7 a gene encoding at least the ⁇ subunit was isolated in 1990 (Non-Patent Document 7).
- rhNGF recombinant human NGF
- NT-3 a type of neurotrophic factor, is known to be involved in promoting the survival and growth of nerve cells and glial cells and neurogenesis by transmitting signals through Trk receptors, particularly TrkC, and is attracting attention for its ability to promote neurotransmission and nerve repair, particularly the differentiation and regeneration of photoreceptor cells.
- Trk receptors particularly TrkC
- NT-3 is therefore expected to be developed as a treatment for neurodegenerative diseases.
- the gene encoding human NT-3 was isolated in 1991 (Non-patent Document 8). However, there are no pharmaceuticals for use as a treatment for neurodegenerative diseases that contain recombinant human NT-3 (rhNT-3) as an active ingredient, which is produced using the gene encoding human NT-3.
- NT-4 a type of neurotrophic factor, transmits signals through Trk receptors, particularly TrkB, and promotes the growth and survival of peripheral and central nervous system neurons, similar to NT-3.
- TrkB Trk receptors
- NT-4 is therefore expected to be developed as a treatment for neurodegenerative diseases.
- the gene encoding human NT-4 was isolated in 1992 (Non-patent Document 9). However, there are no pharmaceuticals for use as a treatment for neurodegenerative diseases that contain recombinant human NT-4 (rhNT-4) as an active ingredient, which is produced using the gene encoding human NT-4.
- Non-Patent Document 10 A method for producing growth hormone, which is rapidly degraded and loses its activity when administered to the body, as a fusion protein with serum albumin.
- Growth hormone fused to serum albumin has increased stability in the body. Therefore, although growth hormone is normally administered subcutaneously every day, by making it into a fusion protein with serum albumin, the number of times it is administered can be reduced.
- One of the objects of the present invention is to provide a physiologically active substance that is usually expressed in a low amount and/or has a low activity when expressed as a recombinant protein using a host cell such as a CHO cell, in the form of a fusion protein with serum albumin (SA).
- SA serum albumin
- Such a fusion protein can be efficiently produced as a highly active recombinant protein.
- Also provided is a method for producing the fusion protein.
- Another object of the present invention is to provide a lysosomal enzyme that is expressed in a low amount and/or has low activity when expressed as a recombinant protein using a host cell such as a CHO cell, particularly when expressed so that the recombinant protein is secreted from the cell and accumulated in the culture medium, in the form of a fusion protein with serum albumin.
- a fusion protein can be efficiently produced as a highly active recombinant protein.
- a method for producing the fusion protein is also provided.
- the lysosomal enzyme include hGALC and hGBA.
- a DNA sequence encoding a leader peptide is placed in frame on the 5' side of the gene encoding the recombinant protein. This allows the expressed recombinant protein to be secreted from cells.
- the leader peptide is preferably an SA leader peptide when an SA is located at the N-terminus of the recombinant protein, a lysosomal enzyme leader peptide when a lysosomal enzyme is located at the N-terminus of the recombinant protein, a cytokine leader peptide when a cytokine is located at the N-terminus of the recombinant protein, or a neurotrophic factor leader peptide when a neurotrophic factor is located at the N-terminus of the recombinant protein.
- the leader peptide may be a leader peptide of a heterologous protein, such as a growth hormone leader peptide, or an artificial leader peptide.
- Another object of the present invention is to provide a cytokine that is expressed in a low amount and/or has low activity when expressed as a recombinant protein using a host cell such as a CHO cell, particularly when expressed so that the recombinant protein is secreted from the cell and accumulated in a culture medium, in the form of a fusion protein with serum albumin.
- a fusion protein can be efficiently produced as a highly active recombinant protein.
- a method for producing the fusion protein is also provided.
- the cytokine is, for example, an interleukin, particularly hIL-10.
- Another object of the present invention is to provide a neurotrophic factor that is expressed in a low amount and/or has low activity when expressed as a recombinant protein using a host cell such as a CHO cell, particularly when expressed so that the recombinant protein is secreted from the cell and accumulates in the culture medium, in the form of a fusion protein with serum albumin.
- a fusion protein can be efficiently produced as a highly active recombinant protein.
- a method for producing the fusion protein is also provided.
- Examples of neurotrophic factors include hBDNF, hNGF, hNT-3, and hNT-4.
- the inventors conducted extensive investigations and found that, as a result of the discovery, when a fusion protein in which HSA is bound directly or via a linker to the N-terminus or C-terminus of human lysosomal enzymes hGALC or hGBA, as described in detail in this specification, is expressed by culturing a host cell into which an expression vector incorporating a gene encoding the fusion protein has been introduced, the expression level of the recombinant fusion protein (converted to the expression level of the portion of the recombinant fusion protein corresponding to the wild-type human lysosomal enzyme) is significantly increased compared to the expression level of the recombinant wild-type human lysosomal enzyme when the recombinant fusion protein is expressed by culturing a host cell into which an expression vector incorporating a gene encoding the wild-type human lysosomal enzyme has been introduced, thereby completing the present invention
- the present inventors conducted extensive investigations and found that, as a result of culturing and expressing a fusion protein in which HSA is bound directly or via a linker to the N-terminus or C-terminus of the human cytokine hIL-10, as described in detail herein, in a host cell into which an expression vector incorporating a gene encoding the fusion protein has been introduced, the activity of the recombinant fusion protein is significantly increased compared to the activity of recombinant wild-type hIL-10 in which the recombinant fusion protein is expressed in a host cell into which an expression vector incorporating a gene encoding wild-type hIL-10 has been introduced, thereby completing the present invention.
- a fusion protein in which HSA is bound directly or via a linker to the N-terminus or C-terminus of human neurotrophic factors hBDNF, hNGF, hNT-3, or hNT-4, as described in detail in this specification is expressed by culturing a host cell into which an expression vector incorporating a gene encoding the fusion protein has been introduced, the activity of the recombinant fusion protein is significantly increased compared to the activity of recombinant wild-type human neurotrophic factor when expressed by culturing a host cell into which an expression vector incorporating a gene encoding a wild-type human neurotrophic factor has been introduced, thereby completing the present invention.
- the present invention includes the following.
- a fusion protein comprising a lysosomal enzyme and serum albumin (SA).
- SA serum albumin
- SA human serum albumin
- the hGALC has 90% or more identity to wild-type hGALC having the amino acid sequence shown in SEQ ID NO:1
- the HSA has 90% or more identity to wild-type HSA having the amino acid sequence shown in SEQ ID NO:3. 6.
- the hGALC comprises the amino acid sequence of wild-type hGALC shown in SEQ ID NO:1
- the HSA comprises the amino acid sequence of wild-type human serum albumin shown in SEQ ID NO:3. 15.
- the linker is a peptide chain consisting of 1 to 150 amino acids.
- the linker comprises an amino acid sequence selected from the group consisting of the following (a) to (g): (a) Gly; (b) Ser; (c) GlySer; (d) GlyGlySer; (e) the amino acid sequence set forth in SEQ ID NO:9; (f) the amino acid sequence set forth in SEQ ID NO: 10; and (g) The amino acid sequence shown in SEQ ID NO:11. 21.
- linker comprises an amino acid sequence in which an amino acid sequence selected from the group consisting of the following (a) to (g) is repeated 2 to 10 times: (a) Gly; (b) Ser; (c) GlySer; (d) GlyGlySer; (e) the amino acid sequence set forth in SEQ ID NO:9; (f) the amino acid sequence set forth in SEQ ID NO: 10; and (g) The amino acid sequence shown in SEQ ID NO:11. 22.
- linker comprises an amino acid sequence in which an amino acid sequence selected from the group consisting of the following (a) to (g) is repeated 2 to 6 times: (a) Gly; (b) Ser; (c) GlySer; (d) GlyGlySer; (e) the amino acid sequence set forth in SEQ ID NO:9; (f) the amino acid sequence set forth in SEQ ID NO: 10; and (g) The amino acid sequence shown in SEQ ID NO:11. 23.
- the fusion protein according to claim 19, wherein the linker comprises an amino acid sequence in which an amino acid sequence selected from the group consisting of the following (a) to (g) is repeated 3 to 5 times: (a) Gly; (b) Ser; (c) GlySer; (d) GlyGlySer; (e) the amino acid sequence set forth in SEQ ID NO:9; (f) the amino acid sequence set forth in SEQ ID NO: 10; and (g) The amino acid sequence shown in SEQ ID NO:11.
- the linker comprises the amino acid sequence Gly Ser. 25.
- the fusion protein according to 18 above which comprises an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:5.
- the fusion protein according to claim 26 which comprises an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted and/or added to the amino acid sequence shown in SEQ ID NO:5.
- the fusion protein according to claim 26 which comprises an amino acid sequence in which 1 to 5 amino acids have been substituted, deleted and/or added to the amino acid sequence shown in SEQ ID NO:5.
- the fusion protein according to claim 26 which comprises an amino acid sequence in which 1 to 3 amino acids have been substituted, deleted and/or added relative to the amino acid sequence shown in SEQ ID NO:5. 30.
- the fusion protein according to claim 18, which comprises the amino acid sequence shown in SEQ ID NO:5.
- the fusion protein according to 17 above which comprises an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO:7.
- the fusion protein according to 17 above which comprises an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:7.
- the fusion protein according to 32 above which comprises an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted and/or added to the amino acid sequence shown in SEQ ID NO:7. 34.
- the fusion protein according to 32 above which comprises an amino acid sequence in which 1 to 5 amino acids have been substituted, deleted and/or added to the amino acid sequence shown in SEQ ID NO:7.
- fusion protein according to 32 above which comprises an amino acid sequence in which 1 to 3 amino acids have been substituted, deleted and/or added to the amino acid sequence shown in SEQ ID NO:7.
- 36. The fusion protein according to claim 17, comprising the amino acid sequence shown in SEQ ID NO:7.
- 37. The fusion protein according to any one of 4 to 36 above, which has a specific activity of 10% or more compared with the specific activity of normal wild-type hGALC. 38.
- hGBA has 90% or more identity to wild-type hGBA having the amino acid sequence shown in SEQ ID NO:37
- said HSA has 90% or more identity to wild-type HSA having the amino acid sequence shown in SEQ ID NO:3. 40.
- hGBA comprises an amino acid sequence in which one amino acid has been substituted with respect to the amino acid sequence of wild-type hGBA shown in SEQ ID NO:37.
- amino acid substitution is within an amino acid family in which the side chain is an amino acid that can be hydroxylated.
- HSA comprises an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted and/or added to the amino acid sequence of wild-type HSA shown in SEQ ID NO:3.
- the fusion protein according to claim 38 wherein said hGBA comprises the amino acid sequence of wild-type hGBA shown in SEQ ID NO:37, and said HSA comprises the amino acid sequence of wild-type HSA shown in SEQ ID NO:12. 50.
- linker comprises an amino acid sequence in which an amino acid sequence selected from the group consisting of the following (a) to (g) is repeated 2 to 10 times: (a) Gly; (b) Ser; (c) GlySer; (d) GlyGlySer; (e) the amino acid sequence set forth in SEQ ID NO:9; (f) the amino acid sequence set forth in SEQ ID NO: 10; and (g) The amino acid sequence shown in SEQ ID NO:11. 56.
- linker comprises an amino acid sequence in which an amino acid sequence selected from the group consisting of the following (a) to (g) is repeated 2 to 6 times: (a) Gly; (b) Ser; (c) GlySer; (d) GlyGlySer; (e) the amino acid sequence set forth in SEQ ID NO:9; (f) the amino acid sequence set forth in SEQ ID NO: 10; and (g) The amino acid sequence shown in SEQ ID NO:11. 57.
- the fusion protein according to the above 53, wherein the linker consists of the amino acid sequence represented by Gly Ser. 59.
- the fusion protein according to 52 above comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO:39. 60.
- the fusion protein according to 52 above which comprises an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:39. 61.
- the fusion protein according to 60 above which comprises an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted and/or added to the amino acid sequence shown in SEQ ID NO:39. 62.
- the fusion protein according to 60 above which comprises an amino acid sequence in which 1 to 5 amino acids have been substituted, deleted and/or added to the amino acid sequence shown in SEQ ID NO:39. 63.
- the fusion protein according to 60 above which comprises an amino acid sequence in which 1 to 3 amino acids have been substituted, deleted and/or added to the amino acid sequence shown in SEQ ID NO:39.
- 64. The fusion protein according to claim 51, comprising the amino acid sequence shown in SEQ ID NO:41.
- 65 The fusion protein according to claim 51, comprising an amino acid sequence having 80% or more identity to the amino acid sequence shown in SEQ ID NO:41.
- 66. The fusion protein according to the above item 51, comprising an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO:41. 67.
- the fusion protein according to 66 above which comprises an amino acid sequence in which 1 to 10 amino acids have been substituted, deleted and/or added to the amino acid sequence shown in SEQ ID NO:41. 68.
- the fusion protein according to 66 above which comprises an amino acid sequence in which 1 to 5 amino acids have been substituted, deleted and/or added relative to the amino acid sequence shown in SEQ ID NO:41. 69.
- the fusion protein according to 66 above which comprises an amino acid sequence in which 1 to 3 amino acids have been substituted, deleted and/or added relative to the amino acid sequence shown in SEQ ID NO:41. 70.
- the fusion protein according to claim 51 comprising the amino acid sequence shown in SEQ ID NO:41. 71.
- the fusion protein according to any one of 38 to 70 above which has a specific activity of 10% or more compared with the specific activity of normal wild-type hGBA.
- 72. A DNA comprising a gene encoding the fusion protein according to any one of 1 to 71 above.
- An expression vector comprising the DNA according to 72 above.
- 74. A mammalian cell transformed with the expression vector according to 73 above.
- 75. A method for producing a fusion protein, comprising the step of culturing the mammalian cell according to 74 above in a serum-free medium.
- 76. A conjugate of the fusion protein according to any one of 1 to 170 above with an antibody.
- 77. Conjugates of lysosomal enzymes, serum albumin and antibodies.
- the conjugate according to claim 77 which is selected from the following (1) to (6): (1) A conjugate in which serum albumin is bound to the C-terminus of the lysosomal enzyme directly or via a linker, and the antibody is further bound to the C-terminus of the serum albumin directly or via a linker; (2) A conjugate in which an antibody is bound to the C-terminus of the lysosomal enzyme directly or via a linker, and the serum albumin is further bound to the C-terminus of the antibody directly or via a linker; (3) A conjugate in which the lysosomal enzyme is bound to the C-terminus of the serum albumin directly or via a linker, and the antibody is further bound to the C-terminus of the lysosomal enzyme directly or via a linker; (4) A conjugate in which the antibody is bound to the C-terminus of the serum albumin, either directly or via a linker, and the lysosomal enzyme is further bound to the C-terminus of
- the receptor on a vascular endothelial cell is selected from the group consisting of an insulin receptor, a transferrin receptor, a leptin receptor, a lipoprotein receptor, and an IGF receptor.
- the receptor on vascular endothelial cells is a transferrin receptor.
- the antibody is any one of a Fab antibody, an F(ab') 2 antibody, an F(ab')2 antibody, a single domain antibody, a single chain antibody, a VHH, or an Fc antibody.
- the fusion protein is bound to either the C-terminus or the N-terminus of the heavy chain of the antibody.
- the linker sequence comprises an amino acid sequence selected from the group consisting of one glycine, one serine, the amino acid sequence Gly-Ser, the amino acid sequence Ser-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence of SEQ ID NO:9, the amino acid sequence of SEQ ID NO:10, the amino acid sequence of SEQ ID NO:11, and an amino acid sequence consisting of 1 to 10 consecutive amino acids of these amino acid sequences.
- a DNA comprising a gene encoding the conjugate according to any one of 76 to 87 above.
- An expression vector comprising the DNA according to 88 above.
- a method for producing a conjugate of an antibody and a fusion protein of a physiologically active protein and SA comprising the step of culturing the mammalian cell according to 90 above in a serum-free medium.
- a human lysosomal enzyme that is relatively difficult to express as an active recombinant protein can be provided in the form of a fusion protein with HSA. Since such a fusion protein can be efficiently produced as a highly active recombinant protein, it can be stably supplied to medical institutions as a drug used for enzyme replacement therapy for patients with lysosomal diseases in which the lysosomal enzyme is deficient.
- a cytokine that is relatively difficult to express as an active recombinant protein can be provided in the form of a fusion protein with HSA.
- a fusion protein can be efficiently produced as a recombinant protein, it can be stably supplied to medical institutions as a drug.
- a human neurotrophic factor that is relatively difficult to express as an active recombinant protein can be provided in the form of a fusion protein with HSA. Since such a fusion protein can be efficiently produced as a recombinant protein, it can be stably supplied to medical institutions as a drug. Note that the effects of the present invention are not limited to these.
- the figure shows a schematic diagram of a single-chain polypeptide fusion protein having, in order from the N-terminus, HSA, a linker, and hGALC.
- the linker is a peptide linker
- the fusion protein is such that the C-terminus of HSA is bound to the N-terminus of the linker by a peptide bond, and the C-terminus of the linker is bound to the N-terminus of hGALC by a peptide bond.
- the figure shows a schematic diagram of a single-chain polypeptide fusion protein having HSA, a linker, and hGBA in that order from the N-terminus.
- the linker is a peptide linker
- the fusion protein is such that the C-terminus of HSA is bound to the N-terminus of the linker by a peptide bond, and the C-terminus of the linker is bound to the N-terminus of hGBA by a peptide bond.
- the figure shows a schematic diagram of a single-chain polypeptide fusion protein having, in order from the N-terminus, hGALC, a linker, and HSA.
- the linker is a peptide linker
- the fusion protein is such that the C-terminus of hGALC is bound to the N-terminus of the linker by a peptide bond, and the C-terminus of the linker is bound to the N-terminus of HSA by a peptide bond.
- the figure shows a schematic diagram of a single-chain polypeptide fusion protein having, in order from the N-terminus, hGBA, a linker, and HSA.
- the linker is a peptide linker
- the fusion protein is such that the C-terminus of hGBA is bound to the N-terminus of the linker by a peptide bond, and the C-terminus of the linker is bound to the N-terminus of HSA by a peptide bond.
- the figure shows a schematic diagram of a single-chain polypeptide fusion protein having, in order from the N-terminus, HSA, a linker, and hIL-10.
- the linker is a peptide linker
- the fusion protein is such that the C-terminus of HSA is bound to the N-terminus of the linker by a peptide bond, and the C-terminus of the linker is bound to the N-terminus of hIL-10 by a peptide bond.
- the figure shows a schematic diagram of a single-chain polypeptide fusion protein having, in order from the N-terminus, hIL-10, a linker, and HSA.
- the linker is a peptide linker
- the fusion protein is such that the C-terminus of hIL-10 is bound to the N-terminus of the linker by a peptide bond, and the C-terminus of the linker is bound to the N-terminus of HSA by a peptide bond.
- the figure shows a schematic diagram of a single-chain polypeptide fusion protein having, in order from the N-terminus, HSA, a linker, and hBDNF.
- the linker is a peptide linker
- the fusion protein is such that the C-terminus of HSA is bound to the N-terminus of the linker by a peptide bond, and the C-terminus of the linker is bound to the N-terminus of hBDNF by a peptide bond.
- the figure shows a schematic diagram of a single-chain polypeptide fusion protein having HSA, a linker, and hNGF in that order from the N-terminus.
- the linker is a peptide linker
- the fusion protein is such that the C-terminus of HSA is bound to the N-terminus of the linker by a peptide bond, and the C-terminus of the linker is bound to the N-terminus of hNGF by a peptide bond.
- the figure shows a schematic diagram of a single-chain polypeptide fusion protein having, in order from the N-terminus, HSA, a linker, and hNT-3.
- the linker is a peptide linker
- the fusion protein is such that the C-terminus of HSA is bound to the N-terminus of the linker by a peptide bond, and the C-terminus of the linker is bound to the N-terminus of hNT-3 by a peptide bond.
- the figure shows a schematic diagram of a single-chain polypeptide fusion protein having, in order from the N-terminus, HSA, a linker, and hNT-4.
- the linker is a peptide linker
- the fusion protein is such that the C-terminus of HSA is bound to the N-terminus of the linker by a peptide bond, and the C-terminus of the linker is bound to the N-terminus of hNT-4 by a peptide bond.
- the figure shows a schematic diagram of a single-chain polypeptide fusion protein having, in order from the N-terminus, hBDNF, a linker, and HSA.
- the linker is a peptide linker
- the fusion protein is such that the C-terminus of hBDNF and the N-terminus of the linker are bound by a peptide bond, and the C-terminus of the linker and the N-terminus of HSA are bound by a peptide bond.
- the figure shows a schematic diagram of a single-chain polypeptide fusion protein having, in order from the N-terminus, hNGF, a linker, and HSA.
- the linker is a peptide linker
- the fusion protein is such that the C-terminus of hNGF and the N-terminus of the linker are bound by a peptide bond, and the C-terminus of the linker and the N-terminus of HSA are bound by a peptide bond.
- the figure shows a schematic diagram of a single-chain polypeptide fusion protein having, in order from the N-terminus, hNT-3, a linker, and HSA.
- the linker is a peptide linker
- the fusion protein is such that the C-terminus of hNT-3 and the N-terminus of the linker are bound by a peptide bond, and the C-terminus of the linker and the N-terminus of HSA are bound by a peptide bond.
- the figure shows a schematic diagram of a single-chain polypeptide fusion protein having, in order from the N-terminus, hNT-4, a linker, and HSA.
- the linker is a peptide linker
- the fusion protein is such that the C-terminus of hNT-4 and the N-terminus of the linker are bound by a peptide bond, and the C-terminus of the linker and the N-terminus of HSA are bound by a peptide bond.
- the vertical axis of the bar graph in the upper row (a) indicates the expression level of the enzyme contained in the culture supernatant in terms of enzyme activity ( ⁇ M/hour).
- the black bars indicate the enzyme activity of wild-type hGALC
- the white bars indicate the enzyme activity of HSA-hGALC
- the diagonal line bars indicate the enzyme activity of hGALC-HSA.
- the middle row (b) shows the results of an analysis of the culture supernatant by SDS-page
- the lower row (c) shows the results of an analysis of the culture supernatant by Western blotting, showing the positions of the bands corresponding to wild-type hGALC and the fusion protein of HSA and hGALC. From the left, the analysis results are shown 6, 7, and 8 days after the start of culture in transient expression.
- the figure shows the elution profiles of wild-type hGALC, HSA-hGALC, and hGALC-HSA obtained by transient expression in SE-HPLC analysis.
- the vertical dashed line indicates the position of the peak derived from the buffer, and the vertical straight line indicates the positions of the peaks corresponding to the HSA-hGALC and hGALC-HSA monomers.
- FIG. 1 Schematic diagram showing the structure of the pCI MCS-modified vector (plasmid) Schematic diagram showing the structure of Dual(+)pCI-neo vector (plasmid)
- the figure shows the results of an experiment to confirm the expression levels of wild-type hGALC, Fab-HSA-hGALC, HSA-hGALC-Fab, Fab-hGALC-HSA, and hGALC-HSA-Fab by transient expression.
- the vertical axis shows the expression level of the enzyme contained in the culture supernatant in terms of enzyme activity ( ⁇ M/hour).
- FIG. 1 shows the elution profiles of transiently expressed Fab-HSA-hGALC, HSA-hGALC-Fab, Fab-hGALC-HSA, and hGALC-HSA-Fab in SE-HPLC analysis.
- the vertical dashed line indicates the position of the peak derived from the buffer.
- FIG. 1 Schematic diagram showing the structure of pEmIGS-hGBA vector (plasmid) Schematic diagram showing the structure of pCIneo-hGBA vector (plasmid) Schematic diagram showing the structure of pCIneo-HSA-hGBA vector (plasmid)
- the figure shows the results of an experiment (activity measurement) to confirm the expression levels of wild-type hGBA, HSA-hGBA, and hGBA-HSA by transient expression.
- the vertical axis shows the expression level of the enzyme contained in the culture supernatant in terms of enzyme activity ( ⁇ M/hour).
- FIG. 1 shows the results of an experiment (SDS-PAGE) to confirm the expression levels of wild-type hGBA, HSA-hGBA, and hGBA-HSA by transient expression. The positions of the bands corresponding to wild-type hGBA and the fusion protein of HSA and hGBA are shown.
- the figure shows the results of an experiment (ELISA) to confirm the expression levels of wild-type mIL-10, mIL-10-MSA, and MSA-mIL-10 by transient expression.
- the vertical axis shows the expression level of each protein contained in the culture supernatant as a concentration (mol/L).
- FIG. 1 shows the results of an experiment (SDS-PAGE) to confirm the expression levels of wild-type mIL-10, mIL-10-MSA, and MSA-mIL-10 by transient expression, in which the positions of the bands corresponding to wild-type mIL-10 and the fusion protein of MSA and mIL-10 are shown.
- This figure shows the results of an experiment (ELISA) to confirm the expression levels of wild-type human neurotrophic factors and fusion proteins of HSA and human neurotrophic factors by transient expression.
- the vertical axis shows the expression level of each protein contained in the culture supernatant as concentration (mol/L).
- the black bars show the concentration of each wild-type human neurotrophic factor
- the white bars show the concentration of each human neurotrophic factor-HSA fusion protein
- the shaded bars show the concentration of each HSA-human neurotrophic factor fusion protein.
- Lane (a) corresponds to wild-type human neurotrophic factor
- lane (b) to human neurotrophic factor-HSA fusion protein
- lane (c) to HSA-human neurotrophic factor fusion protein.
- the positions of the bands corresponding to wild-type human neurotrophic factor and the fusion protein of HSA and human neurotrophic factor are shown.
- There are four types of human neurotrophic factors and from the left in the figure, the results for hBDNF, hNGF, hNT-4, and hNT-4 are shown.
- Lane (a) corresponds to wild-type human neurotrophic factor
- lane (b) to human neurotrophic factor-HSA fusion protein
- lane (c) to HSA-human neurotrophic factor fusion protein.
- the positions of the bands corresponding to wild-type human neurotrophic factor and the fusion protein of HSA and human neurotrophic factor are shown.
- the protein should be one that has a low expression level and/or low activity when the gene encoding the protein is introduced into a host cell and expressed as a recombinant protein.
- examples of such proteins include lysosomal enzymes, cytokines, interleukins, and neurotrophic factors, or fusion proteins of these with antibodies. Interleukins belong to the cytokine category, and are a general term for cytokines distributed in particular from helper T cells.
- Lysosomal enzymes include, in particular, galactosylceramidase (GALC) and glucocerebrosidase (GBA), or fusion proteins of these with antibodies or ligands.
- GALC galactosylceramidase
- GBA glucocerebrosidase
- lysosomal enzymes are not limited to GALC and GBA.
- Other lysosomal enzymes that can increase the expression level and/or activity when expressed as recombinant proteins by binding with SA, particularly when expressed so that the recombinant proteins are secreted from cells and accumulated in the culture medium, are also included in the lysosomal enzymes to be bound with SA. The same applies to fusion proteins of these other lysosomal enzymes with antibodies or ligands.
- the present invention can be applied to lysosomal enzymes that are easy to produce as recombinant proteins. That is, the lysosomal enzyme is not particularly limited, and examples thereof include iduronate-2-sulfatase, ⁇ -L-iduronidase, ⁇ -galactosidase, GM2 activator protein, ⁇ -hexosaminidase A, ⁇ -hexosaminidase B, N-acetylglucosamine 1-phosphotransferase, ⁇ -mannosidase, ⁇ -mannosidase, saposin C, arylsulfatase A, ⁇ -L-fucosidase, aspartylglucosaminidase, ⁇ -N-acetylgalactosaminidase, acid sphingomyelinase, and the like.
- It may be enzyme, ⁇ -galactosidase A, ⁇ -glucuronidase, heparan N-sulfatase, ⁇ -N-acetylglucosaminidase, acetyl-CoA ⁇ -glucosaminide N-acetyltransferase, N-acetylglucosamine 6-sulfate sulfatase, acid ceramidase, amylo-1,6-glucosidase, sialidase, palmitoyl protein thioesterase-1, tripeptidyl peptidase-1, hyaluronidase-1, acid ⁇ -glucosidase, CLN1, or CLN2.
- Cytokines include interleukins, such as IL-10, and fusion proteins of IL-10 with antibodies or ligands.
- the cytokines are not limited to IL-10.
- the present invention can also be applied to cytokines that are easy to produce as recombinant proteins.
- fusion proteins also include fusion proteins of cytokines other than IL-10 with SA, and further fusion proteins of these with antibodies.
- the cytokine is not particularly limited, but may be, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, and IL-19 to IL-36.
- the neurotrophic factors include, in particular, BDNF, NGF, NT-3, and NT-4, and fusion proteins of these with antibodies.
- the neurotrophic factors are not limited to these.
- Other neurotrophic factors that can increase the expression level and/or activity when expressed as a recombinant protein by binding with SA, particularly when expressed so that the recombinant protein is secreted from cells and accumulated in the culture medium, are also included in the neurotrophic factors to be bound with SA.
- the present invention can be applied to neurotrophic factors that are easy to produce as recombinant proteins.
- the neurotrophic factors are not particularly limited, and may be, for example, glial cell line neurotrophic factor (GDNF) or NT-5.
- the biological species of the protein to be fused with SA there are no particular limitations on the biological species of the protein to be fused with SA, but it is preferably derived from humans, such as human lysosomal enzymes, human cytokines, and human growth and nutritional factors.
- Fusion proteins of serum albumin (SA) and lysosomal enzymes can be used as therapeutic agents in enzyme replacement therapy for lysosomal diseases.
- SA serum albumin
- lysosomal enzymes For example, glucocerebrosidase (GBA) fused with SA can be used as a therapeutic agent for Gaucher disease, and galactosylceramidase (GALC) can be used as a therapeutic agent for Krabbe disease.
- human lysosomal enzyme when used, it includes not only normal wild-type human lysosomal enzymes, but also mutants of human lysosomal enzymes in which one or more amino acid residues have been substituted, deleted, and/or added (in this specification, "addition" of an amino acid residue means adding a residue to the end or inside of the sequence) to the amino acid sequence of the wild-type human lysosomal enzyme, as long as the mutants have the function of a human lysosomal enzyme, such as having the enzymatic activity corresponding to the type of human lysosomal enzyme. The same applies to lysosomal enzymes of animal species other than humans.
- a human lysosomal enzyme when a human lysosomal enzyme is said to have the function of a human lysosomal enzyme, it means that the human lysosomal enzyme has a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of a normal wild-type human lysosomal enzyme is taken as 100%.
- specific activity refers to the enzyme activity per mass of protein.
- the specific activity of a fusion protein of a human lysosomal enzyme and another protein is calculated as the enzyme activity per mass of the portion of the fusion protein that corresponds to the human lysosomal enzyme.
- the specific activity of the human lysosomal enzyme in the fusion protein is calculated by multiplying the enzyme activity of the human lysosomal enzyme per unit mass of the fusion protein ( ⁇ M/hour/mg protein) by (the molecular weight of the fusion protein/the molecular weight of the portion of the fusion protein that corresponds to the human lysosomal enzyme).
- amino acid residues in the amino acid sequence of a wild-type human lysosomal enzyme are replaced with other amino acid residues, the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- amino acid residues in the amino acid sequence of a wild-type human lysosomal enzyme are deleted, the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- a mutant human lysosomal enzyme consisting of an amino acid sequence in which one amino acid residue is deleted from the N-terminus or C-terminus of a wild-type human lysosomal enzyme a mutant human lysosomal enzyme consisting of an amino acid sequence in which two amino acid residues are deleted from the N-terminus or C-terminus of a wild-type human lysosomal enzyme, etc. are also human lysosomal enzymes.
- a mutation that combines the substitution and deletion of these amino acid residues can be added to the amino acid sequence of a wild-type human lysosomal enzyme. The same can be said for lysosomal enzymes of animal species other than humans.
- amino acid residues When adding amino acid residues to the amino acid sequence of a wild-type human lysosomal enzyme, one or more amino acid residues are added into the amino acid sequence of the human lysosomal enzyme or to the N-terminus or C-terminus of the amino acid sequence.
- the number of amino acid residues added in this case is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- Mutations that combine the addition of these amino acid residues with the above-mentioned substitutions can also be added to the amino acid sequence of a wild-type human lysosomal enzyme, and mutations that combine the addition of these amino acid residues with the above-mentioned deletions can also be added to the amino acid sequence of a wild-type human lysosomal enzyme. The same can be said for lysosomal enzymes of animal species other than humans.
- a combination of the above three types of mutations, substitution, deletion, and addition of amino acid residues can be added to the amino acid sequence of a wild-type human lysosomal enzyme.
- a human lysosomal enzyme is obtained by deleting 1 to 10 amino acid residues from the amino acid sequence of a wild-type human lysosomal enzyme, substituting 1 to 10 amino acid residues with other amino acid residues, and adding 1 to 10 amino acid residues
- a human lysosomal enzyme is obtained by deleting 1 to 5 amino acid residues from the amino acid sequence of a wild-type human lysosomal enzyme, substituting 1 to 5 amino acid residues with other amino acid residues, and adding 1 to 5 amino acid residues
- a human lysosomal enzyme is obtained by deleting 1 to 3 amino acid residues from the amino acid sequence of a wild-type human lysosomal enzyme, substituting 1 to 3 amino acid residues with other amino acid residues, and adding 1 to 5 amino acid residues
- Human lysosomal enzymes are also those in which 1 or 2 amino acid residues have been deleted from the amino acid sequence of a wild-type human lysosomal enzyme, 1 or 2 amino acid residues have been replaced with another amino acid residue, and 1 or 2 amino acid residues have been added. Human lysosomal enzymes are also those in which 1 amino acid residue has been deleted from the amino acid sequence of a wild-type human lysosomal enzyme, 1 amino acid residue has been replaced with another amino acid residue, and 1 amino acid residue has been added. The same can be said about lysosomal enzymes of animal species other than humans.
- each mutation in a human lysosomal enzyme mutant compared to a normal wild-type human lysosomal enzyme can be easily confirmed by aligning the amino acid sequences of both human lysosomal enzymes. The same can be said for lysosomal enzymes of animal species other than humans.
- the amino acid sequence of the human lysosomal enzyme mutant preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more or 99% or more identity, to the amino acid sequence of a normal wild-type human lysosomal enzyme. The same can be said for lysosomal enzyme mutants of animal species other than humans.
- the identity between the amino acid sequence of a wild-type human lysosomal enzyme and the amino acid sequence of a mutant human lysosomal enzyme can be easily calculated using well-known homology calculation algorithms. Examples of such algorithms include BLAST (Altschul S F. J Mol. Biol. 215. 403-10, (1990)), the similarity search method of Pearson and Lipman (Proc. Natl. Acad. Sci. USA. 85. 2444 (1988)), and the local homology algorithm of Smith and Waterman (Adv. Appl. Math. 2. 482-9 (1981)). The same can be said for the lysosomal enzymes of non-human animal species, GALC, GBA, MSA, and non-human animal species SA. These algorithms can also be applied throughout this specification as homology calculation algorithms between the wild-type amino acid sequence of another protein and the amino acid sequence of a mutant of that other protein.
- human galactosylceramidase “human galactocerebrosidase”, or “hGALC”
- hGALC human galactosylceramidase
- mutants of hGALC in which one or more amino acid residues have been substituted, deleted, and/or added (as used herein, "addition" of an amino acid residue means adding a residue to the end or inside of the sequence) to the amino acid sequence as shown in SEQ ID NO: 1, as long as they have the function of hGALC, such as the enzyme activity capable of decomposing sphingolipids such as galactocerebroside and/or galactosylsphingosine.
- Wild-type hGALC is, for example, encoded by a gene having the base sequence as shown in SEQ ID NO: 2.
- mouse galactosylceramidase when simply referring to "mouse galactosylceramidase”, “mouse galactocerebrosidase”, or “mGALC”, it includes, without distinction, not only the normal wild-type mGALC having the amino acid sequence shown in SEQ ID NO: 14, but also mGALC mutants in which one or more amino acid residues have been substituted, deleted, and/or added (as used herein, "addition” of an amino acid residue means adding a residue to the end or inside of the sequence) to the amino acid sequence shown in SEQ ID NO: 14, as long as they have the function of mGALC, such as having the enzymatic activity to degrade sphingolipids such as galactocerebroside and/or galactosylsphingosine.
- Wild-type mGALC is, for example, encoded by a gene having the base sequence shown in SEQ ID NO: 15.
- hGALC When hGALC is said to have the function of hGALC, it means that hGALC preferably retains a specific activity of 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, of the specific activity of normal wild-type hGALC taken as 100%.
- specific activity refers to the enzyme activity per mass of protein.
- the specific activity of a fusion protein of hGALC and another protein is calculated as the enzyme activity per mass of the portion of the fusion protein that corresponds to hGALC. The same can be said for mGALC.
- the specific activity of hGALC in a fusion protein is calculated by multiplying the enzyme activity of hGALC per unit mass of the fusion protein ( ⁇ M/hour/mg protein) by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein that corresponds to hGALC).
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- hGALC mutants consisting of 642 amino acid residues with one amino acid residue deleted from the N-terminus or C-terminus of wild-type hGALC, and hGALC mutants consisting of 641 amino acid residues with two amino acid residues deleted from the N-terminus or C-terminus of wild-type hGALC are also hGALC. Mutations that combine the substitution and deletion of these amino acid residues can also be added to the amino acid sequence of wild-type hGALC. The same can be said for GALC of animal species other than humans.
- amino acid residues When amino acid residues are added to the amino acid sequence of wild-type hGALC, one or more amino acid residues are added into the amino acid sequence of hGALC or to the N-terminus or C-terminus of the amino acid sequence.
- the number of amino acid residues added in this case is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- Mutations that combine the addition of these amino acid residues with the above-mentioned substitutions can also be added to the amino acid sequence of wild-type hGALC, and mutations that combine the addition of these amino acid residues with the above-mentioned deletions can also be added to the amino acid sequence of wild-type hGALC. The same can be said for GALC of animal species other than humans.
- amino acid sequence of wild-type hGALC can be obtained by deleting 1 to 10 amino acid residues, substituting 1 to 10 amino acid residues with other amino acid residues, and adding 1 to 10 amino acid residues.
- amino acid sequence of wild-type hGALC shown in SEQ ID NO:1 can be obtained by deleting 1 to 5 amino acid residues, substituting 1 to 5 amino acid residues with other amino acid residues, and adding 1 to 5 amino acid residues.
- the amino acid sequence of wild-type hGALC shown in SEQ ID NO:1 can be obtained by deleting 1 to 3 amino acid residues, hGALC also includes those in which one to three amino acid residues have been replaced with other amino acid residues and one to three amino acid residues have been added, and those in which one or two amino acid residues have been deleted from the wild-type hGALC amino acid sequence shown in SEQ ID NO:1, one or two amino acid residues have been replaced with other amino acid residues, and one or two amino acid residues have been added, and those in which one amino acid residue has been deleted from the wild-type hGALC amino acid sequence shown in SEQ ID NO:1, one amino acid residue has been replaced with other amino acid residue, and one amino acid residue has been added.
- the same can be said for GALCs of animal species other than humans.
- each mutation in the hGALC mutant compared to the normal wild-type hGALC can be easily confirmed by aligning the amino acid sequences of both hGALCs. The same can be said for GALCs of animal species other than humans.
- the amino acid sequence of the hGALC mutant preferably has 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, to the amino acid sequence of the normal wild-type hGALC shown in SEQ ID NO: 1. The same can be said for GALC of animal species other than humans.
- human glucocerebrosidase “human ⁇ -glucosidase,” or “hGBA”
- hGBA human ⁇ -glucosidase
- Wild-type hGBA is, for example, encoded by a gene having the base sequence as shown in SEQ ID NO: 38.
- mouse glucocerebrosidase when simply referring to "mouse glucocerebrosidase”, “mouse ⁇ -glucosidase”, or “mGBA”, it includes, without distinction, not only the normal wild-type mGBA having the amino acid sequence shown in SEQ ID NO: 43, but also mGBA mutants in which one or more amino acid residues have been substituted, deleted, and/or added (in this specification, “addition” of an amino acid residue means adding a residue to the end or inside of the sequence) to the amino acid sequence shown in SEQ ID NO: 43, as long as they have the function of mGBA, such as having the enzymatic activity to degrade glycolipids such as galactocerebroside and/or galactosylsphingosine.
- Wild-type mGBA is, for example, encoded by a gene having the base sequence shown in SEQ ID NO: 44.
- hGBA When hGBA is said to have the function of hGBA, it means that hGBA has a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of normal wild-type hGBA is taken as 100%.
- specific activity refers to the enzyme activity per mass of protein.
- the specific activity of a fusion protein of hGBA and another protein is calculated as the enzyme activity per mass of the portion of the fusion protein that corresponds to hGBA.
- the specific activity of hGBA in the fusion protein is calculated by multiplying the enzyme activity of hGBA per unit mass of the fusion protein ( ⁇ M/hour/mg protein) by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein that corresponds to hGBA). The same can be said for GBA of animal species other than humans.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- hGBA mutants consisting of 496 amino acid residues with one amino acid residue deleted from the N-terminus or C-terminus of wild-type hGBA, and hGBA mutants consisting of 495 amino acid residues with two amino acid residues deleted from the N-terminus or C-terminus of wild-type hGBA are also hGBA. Mutations that combine the substitution and deletion of these amino acid residues can also be added to the amino acid sequence of wild-type hGBA. The same can be said for GBAs of animal species other than humans.
- amino acid residues When amino acid residues are added to the amino acid sequence of wild-type hGBA, one or more amino acid residues are added into the amino acid sequence of hGBA or to the N-terminus or C-terminus of the amino acid sequence.
- the number of amino acid residues added in this case is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2. Mutations that combine the addition of these amino acid residues with the above-mentioned substitutions can also be added to the amino acid sequence of wild-type hGBA, and mutations that combine the addition of these amino acid residues with the above-mentioned deletions can also be added to the amino acid sequence of wild-type hGBA. The same can be said for GBAs of animal species other than humans.
- an hGBA is obtained by deleting 1 to 10 amino acid residues from the amino acid sequence of wild-type hGBA shown in SEQ ID NO:37, substituting 1 to 10 amino acid residues with other amino acid residues, and adding 1 to 10 amino acid residues.
- An hGBA is also obtained by deleting 1 to 5 amino acid residues from the amino acid sequence of wild-type hGBA shown in SEQ ID NO:37, substituting 1 to 5 amino acid residues with other amino acid residues, and adding 1 to 5 amino acid residues.
- hGBA is also obtained by deleting 1 to 3 amino acid residues from the amino acid sequence of wild-type hGBA shown in SEQ ID NO:37
- hGBA also includes the amino acid sequence of wild-type hGBA shown in SEQ ID NO: 37 in which one or two amino acid residues have been deleted, one or two amino acid residues have been replaced with other amino acid residues, and one or two amino acid residues have been added.
- hGBA also includes the amino acid sequence of wild-type hGBA shown in SEQ ID NO: 37 in which one amino acid residue has been deleted, one amino acid residue has been replaced with other amino acid residue, and one amino acid residue has been added. The same can be said for GBAs of animal species other than humans.
- each mutation in the hGBA mutant compared to the normal wild-type hGBA can be easily confirmed by aligning the amino acid sequences of both hGBAs. The same can be said for GBAs of animal species other than humans.
- the amino acid sequence of the hGBA mutant preferably has 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, to the amino acid sequence of normal wild-type hGBA shown in SEQ ID NO: 37. The same is true for GBA of animal species other than humans.
- SA serum albumin
- HSA human serum albumin
- human serum albumin or "HSA” includes, without distinction, wild-type human serum albumin consisting of 585 amino acids having the amino acid sequence shown in SEQ ID NO: 3, as well as mutants of HSA in which one or more amino acid residues have been substituted, deleted, and/or added to the amino acid sequence shown in SEQ ID NO: 3 (as used herein, “addition” of an amino acid residue means adding a residue to the end or inside of the sequence).
- Wild-type HSA is, for example, encoded by a gene having the base sequence shown in SEQ ID NO: 4.
- human serum albumin When human serum albumin is expressed as a recombinant protein using host cells such as CHO as a fusion protein bound to a wild-type human lysosomal enzyme, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the expression amount of at least one type of wild-type human lysosomal enzyme as a human lysosomal enzyme can be increased compared to when the wild-type human lysosomal enzyme is expressed as a recombinant protein by a similar method.
- human serum albumin when human serum albumin is expressed as a recombinant protein using host cells such as CHO as a fusion protein bound to wild-type hGALC or wild-type hGBA, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the expression amount of the enzyme can be increased compared to when these wild-type enzymes are expressed as recombinant proteins by a similar method.
- the human serum albumin here is preferably one that has the functions of human serum albumin, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited thereto.
- mouse serum albumin or “MSA” includes, without distinction, wild-type mouse serum albumin having the amino acid sequence shown in SEQ ID NO: 16, as well as mutants of MSA in which one or more amino acid residues have been substituted, deleted, and/or added (as used herein, "addition" of an amino acid residue means adding a residue to the end or inside of the sequence) to the amino acid sequence shown in SEQ ID NO: 16.
- Wild-type MSA is encoded, for example, by a gene having the base sequence shown in SEQ ID NO: 17.
- MSA is preferably one that has the functions of MSA, such as the function of binding and transporting endogenous substances in blood and exogenous substances such as drugs, but is not limited to this.
- serum albumin refers to a comprehensive concept including serum albumin of mammals other than humans, including mouse serum albumin (MSA) and bovine serum albumin (BSA).
- MSA mouse serum albumin
- BSA bovine serum albumin
- serum albumin when serum albumin is expressed as a recombinant protein using host cells such as CHO as a fusion protein bound to wild-type hGALC or wild-type hGBA, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, it is possible to increase the expression amount of the enzyme, compared to when these wild-type enzymes are expressed as recombinant proteins by the same method.
- Human serum albumin is known to have several wild-type variants. Human serum albumin Redhill is one of them. Human serum albumin Redhill differs from the above-mentioned normal human serum albumin amino acid sequence, which consists of 585 amino acids, in that the 320th amino acid residue from the N-terminus is threonine instead of alanine, and an arginine residue is added to the N-terminus, and consists of 586 amino acids as shown in SEQ ID NO: 12.
- the above-mentioned change of alanine to threonine creates a sequence represented by Asn-Tyr-Thr in the amino acid sequence of human serum albumin Redhill, and the Asn (asparagine) residue in this sequence is N-linked glycosylated.
- This human serum albumin Redhill is also a wild-type HSA.
- HSA mutant human serum albumin (HSA-A320T) consisting of 585 amino acids as shown in SEQ ID NO:13, in which the 320th amino acid residue from the N-terminus of the wild-type HSA amino acid sequence as shown in SEQ ID NO:3 is replaced with threonine.
- HSA mutant is one in which the 319th amino acid residue, tyrosine, is replaced with an amino acid other than proline while preserving the 318th amino acid residue from the N-terminus of the HSA mutant as shown in SEQ ID NO:13.
- mutations that can be made to the wild-type HSA amino acid sequence that are permissible in one embodiment of the present invention, but the mutations can also be applied to human serum albumin Redhill, the HSA mutant as shown in SEQ ID NO:13, or serum albumin of animal species other than humans.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- HSA mutants in which one amino acid residue is deleted at the N-terminus or C-terminus of wild-type or HSA-A320T HSA, and HSA mutants in which two amino acid residues are deleted at the N-terminus or C-terminus of wild-type or HSA-A320T HSA are also HSA. Mutations that combine the substitution and deletion of these amino acid residues can also be added to the amino acid sequence of wild-type or HSA-A320T HSA. The same can be said about SA in non-human animal species.
- amino acid residues When amino acid residues are added to the amino acid sequence of wild-type HSA or HSA-A320T, one or more amino acid residues are added to the amino acid sequence of HSA or to the N-terminus or C-terminus of the amino acid sequence.
- the number of amino acid residues added in this case is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- HSA mutants in which one amino acid residue is added to the N-terminus or C-terminus of wild-type or HSA-A320T HSA, and HSA mutants in which two amino acid residues are added to the N-terminus or C-terminus of wild-type or HSA-A320T HSA, etc. are also HSA.
- mutations that combine the addition of these amino acid residues and the above-mentioned substitutions can also be added to the amino acid sequence of wild-type or HSA-A320T HSA
- mutations that combine the addition of these amino acid residues and the above-mentioned deletions can also be added to the amino acid sequence of wild-type or HSA-A320T HSA. The same can be said for SA of animal species other than humans.
- HSA can be obtained by deleting 1 to 10 amino acid residues from the amino acid sequence of wild-type HSA shown in SEQ ID NO:3, substituting 1 to 10 amino acid residues with other amino acid residues, and adding 1 to 10 amino acid residues
- HSA can be obtained by deleting 1 to 5 amino acid residues from the amino acid sequence of wild-type HSA shown in SEQ ID NO:3, substituting 1 to 5 amino acid residues with other amino acid residues, and adding 1 to 5 amino acid residues
- HSA can be obtained by deleting 1 to 3 amino acid residues from the amino acid sequence of wild-type HSA shown in SEQ ID NO:3, HSA is also one in which 1 to 3 amino acid residues have been replaced with other amino acid residues and 1 to 3 amino acid residues have been added, HSA is also one in which 1 or 2 amino acid residue
- the amino acid sequence of the HSA mutant preferably has 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, to the amino acid sequence of normal wild-type HSA shown in SEQ ID NO: 3. The same is true for SA of animal species other than humans.
- the expression amount of at least one type of wild-type human lysosomal enzyme as a human lysosomal enzyme in the culture supernatant can be at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or enzyme activity, for example, 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, etc., compared to when the wild-type human lysosomal enzyme is expressed as a recombinant protein by a similar method.
- the amount of hGALC expressed in the culture supernatant can be at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or enzyme activity, compared to when wild-type hGALC is expressed as a recombinant protein by a similar method.
- the amount of hGBA expressed in the culture supernatant can be at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or enzyme activity, for example, 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, 10 to 20 times, etc., compared to when wild-type hGBA is expressed as a recombinant protein by a similar method.
- the term "conservative amino acid substitution” refers to a substitution of an amino acid within a family of amino acids that are related in their side chains and chemical properties. Substitutions within such an amino acid family are not expected to significantly alter the function of the original protein.
- amino acid families include the following (1) to (12): (1) The acidic amino acids aspartic acid and glutamic acid, (2) the basic amino acids histidine, lysine, and arginine; (3) the aromatic amino acids phenylalanine, tyrosine, and tryptophan; (4) Serine and threonine, which are amino acids having a hydroxyl group (hydroxyamino acids), (5) the hydrophobic amino acids methionine, alanine, valine, leucine, and isoleucine, (6) The neutral hydrophilic amino acids cysteine, serine, threonine, asparagine, and glutamine, (7) Glycine and proline, which are amino acids that affect the orientation of peptide chains.
- Asparagine and glutamine which are amide amino acids (polar amino acids), (9) The aliphatic amino acids alanine, leucine, isoleucine, and valine, (10) Amino acids with small side chains: alanine, glycine, serine, and threonine; (11) Alanine and glycine, which are amino acids with particularly small side chains. (12) The branched chain amino acids valine, leucine, and isoleucine, (13) Proline and serine, which are amino acids whose side chains can be hydroxylated. The same can be said for the lysosomal enzymes GALC, GBA, and SA of non-human animal species.
- substitution of an amino acid in the amino acid sequence of wild-type human lysosomal enzymes and HSA with another amino acid is preferably a conservative amino acid substitution.
- the above conservative amino acid substitutions apply to the wild-type forms of hGALC and hGBA.
- the above wild-type or mutant human lysosomal enzymes such as hGALC or hGBA, whose constituent amino acids are modified with sugar chains are also human lysosomal enzymes.
- the above wild-type or mutant human lysosomal enzymes whose constituent amino acids are modified with phosphate are also human lysosomal enzymes. Those modified with something other than sugar chains and phosphate are also human lysosomal enzymes.
- the above wild-type or mutant human lysosomal enzymes whose constituent amino acid side chains have been converted by substitution reactions or the like are also human lysosomal enzymes. Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said for the lysosomal enzymes, GALC, and GBA of animal species other than humans.
- human lysosomal enzymes modified with sugar chains such as hGALC or hGBA
- human lysosomal enzymes modified with phosphate are included in human lysosomal enzymes with the original amino acid sequence.
- Those modified with things other than sugar chains and phosphate are also included in human lysosomal enzymes with the original amino acid sequence.
- Human lysosomal enzymes in which the side chains of the amino acids constituting the human lysosomal enzymes have been converted by substitution reactions or the like are also included in human lysosomal enzymes with the original amino acid sequence.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said for lysosomal enzymes of animal species other than humans, GALC, and GBA.
- the above wild-type or mutant HSA in which the constituent amino acids are modified with sugar chains is also HSA.
- the above wild-type or mutant HSA in which the constituent amino acids are modified with phosphate is also HSA.
- HSA modified with something other than sugar chains and phosphate is also HSA.
- the above wild-type or mutant HSA in which the side chains of the constituent amino acids have been converted by substitution reactions or the like is also HSA.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said for SA of animal species other than humans.
- HSA modified with sugar chains is included in HSA with the original amino acid sequence.
- HSA modified with phosphate is included in HSA with the original amino acid sequence.
- HSA modified with things other than sugar chains and phosphate is also included in HSA with the original amino acid sequence.
- HSA with the original amino acid sequence also includes HSA with the original amino acid sequence when the side chains of the amino acids that make up HSA have been changed by substitution reactions, etc. Such changes include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said for SA of animal species other than humans.
- the present invention relates to a fusion protein in which a polypeptide containing the amino acid sequence of a wild-type or mutant human lysosomal enzyme is bound to a polypeptide containing the amino acid sequence of a wild-type or mutant SA.
- binding polypeptides refers to binding different polypeptides by covalent bond, either directly or indirectly via a linker.
- the SA is preferably HSA.
- the human lysosomal enzyme is, for example, hGALC or hGBA.
- a common method for linking two different polypeptides is, for example, to create an expression vector incorporating a DNA fragment in which a gene encoding one polypeptide is linked in-frame downstream of a gene encoding the other polypeptide, and then to express the polypeptide as a recombinant protein by culturing a host cell transformed with this expression vector.
- the resulting recombinant protein is a single-chain polypeptide in which the two polypeptides are peptide-linked, either directly or via different amino acid sequences.
- SA-human lysosomal enzyme fusion protein or "SA-human lysosomal enzyme” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of a human lysosomal enzyme is linked to the C-terminus of the amino acid sequence of SA, either directly or via a linker, and which has the function of a human lysosomal enzyme.
- animal species of the SA there is no particular limitation on the animal species of the SA, but it is preferably a mammalian SA such as a primate, mouse, or cow, more preferably a primate SA, and even more preferably HSA.
- the human lysosomal enzyme in the SA-human lysosomal enzyme fusion protein has the function of a human lysosomal enzyme, it means that the human lysosomal enzyme preferably retains a specific activity of 10% or more, more preferably a specific activity of 20% or more, even more preferably a specific activity of 50% or more, and even more preferably a specific activity of 80% or more, when the specific activity of a normal wild-type human lysosomal enzyme is taken as 100%.
- the specific activity of the human lysosomal enzyme in the SA-human lysosomal enzyme fusion protein is calculated by multiplying the enzymatic activity of the human lysosomal enzyme per unit mass of the fusion protein ( ⁇ M/hour/mg protein) by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein that corresponds to the human lysosomal enzyme).
- the term "HSA-human lysosomal enzyme fusion protein" or "HSA-human lysosomal enzyme” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of a human lysosomal enzyme is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of HSA, and which has the function of a human lysosomal enzyme.
- the definition of the SA-human lysosomal enzyme fusion protein described above can be applied.
- the SA-human lysosomal enzyme fusion protein it is preferable that the SA has a function as an SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- a fusion protein having an amino acid sequence in which the N-terminus of a lysosomal enzyme of a non-human animal species is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of SA of a non-human animal species, and which has the function of a human lysosomal enzyme is referred to as a "(non-human animal species) SA-(non-human animal species) lysosomal enzyme fusion protein.”
- a fusion protein of mouse SA and mouse lysosomal enzyme is referred to as an "MSA-mouse lysosomal enzyme fusion protein" or "MSA-mouse lysosomal enzyme.”
- mutations When mutations are added to an HSA-human lysosomal enzyme fusion protein, which is a fusion protein of wild-type HSA and wild-type human lysosomal enzyme, mutations can be added only to the HSA portion and not to the human lysosomal enzyme portion, mutations can be added only to the human lysosomal enzyme portion without mutations to the HSA portion, or mutations can be added to both the HSA portion and the human lysosomal enzyme portion.
- the amino acid sequence of the portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above.
- the amino acid sequence of the portion is the amino acid sequence of human lysosomal enzyme obtained by adding a mutation to the wild-type human lysosomal enzyme described above.
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above
- the amino acid sequence of the human lysosomal enzyme portion is the amino acid sequence of human lysosomal enzyme obtained by adding a mutation to the wild-type human lysosomal enzyme described above.
- the same can be said about fusion proteins between wild-type SA (including wild-type MSA) of non-human animal species and wild-type human lysosomal enzymes.
- HSA-human lysosomal enzyme fusion proteins in which the amino acids constituting the protein are modified with sugar chains are also HSA-human lysosomal enzyme fusion proteins.
- HSA-human lysosomal enzyme fusion proteins in which the amino acids constituting the protein are modified with phosphate are also HSA-human lysosomal enzyme fusion proteins.
- HSA-human lysosomal enzyme fusion proteins modified with anything other than sugar chains and phosphate are also HSA-human lysosomal enzyme fusion proteins.
- HSA-human lysosomal enzyme fusion proteins in which the side chains of the amino acids constituting the protein are converted by substitution reactions or the like are also HSA-human lysosomal enzyme fusion proteins.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine.
- the same can be said about fusion proteins of SA of a non-human animal species and a human lysosomal enzyme (SA-human lysosomal enzyme), and fusion proteins of SA of a non-human animal species and a lysosomal enzyme of a non-human animal species, such as MSA-mouse lysosomal enzyme fusion proteins.
- HSA-human lysosomal enzyme fusion proteins modified with sugar chains are included in HSA-human lysosomal enzyme fusion proteins having the original amino acid sequence.
- HSA-human lysosomal enzyme fusion proteins modified with phosphate are included in HSA-human lysosomal enzyme fusion proteins having the original amino acid sequence.
- those modified with something other than sugar chains and phosphate are included in HSA-human lysosomal enzyme fusion proteins having the original amino acid sequence.
- those in which the side chains of the amino acids constituting the HSA-human lysosomal enzyme fusion proteins have been converted by substitution reactions or the like are included in HSA-human lysosomal enzyme fusion proteins having the original amino acid sequence.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine.
- SA-human lysosomal enzyme fusion proteins of SA of animal species other than human and lysosomal enzyme of animal species other than human, such as MSA-mouse lysosomal enzyme fusion protein.
- HSA-human lysosomal enzyme fusion proteins in which the human lysosomal enzyme that constitutes it is a precursor of human lysosomal enzyme, are also HSA-human lysosomal enzyme fusion proteins.
- precursor refers to a type that is biosynthesized as an HSA-human lysosomal enzyme fusion protein, and after the biosynthesis, the part that functions as a human lysosomal enzyme is separated from the fusion protein during the manufacturing process or in the living body when administered to the living body, and can function as a human lysosomal enzyme by itself.
- the HSA-human lysosomal enzyme fusion protein may be biosynthesized, it may be cleaved at a specific site by a hydrolase or the like, and the part containing HSA and the part containing the mature human lysosomal enzyme may be separated.
- the obtained human lysosomal enzyme is not a fusion protein with HSA, but the HSA-human lysosomal enzyme fusion protein is synthesized once during the process of manufacturing the human lysosomal enzyme. Therefore, when human lysosomal enzyme is manufactured by such a method, the manufacturing method is included in the manufacturing method of HSA-human lysosomal enzyme fusion protein.
- SA-human lysosomal enzyme fusion proteins of SA of non-human animal species and human lysosomal enzyme
- fusion proteins of SA of non-human animal species and lysosomal enzyme of non-human animal species such as MSA-mouse lysosomal enzyme fusion protein.
- SA-hGALC fusion protein or "SA-hGALC” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of hGALC is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of SA, and which has the function of hGALC.
- SA animal species of the SA, but it is preferably a mammalian SA, more preferably a primate SA, and even more preferably HSA.
- hGALC in the SA-hGALC fusion protein has the function of hGALC
- hGALC preferably retains a specific activity of 10% or more, more preferably a specific activity of 20% or more, even more preferably a specific activity of 50% or more, and even more preferably a specific activity of 80% or more, when the specific activity of normal wild-type hGALC is taken as 100%.
- the specific activity of hGALC in the SA-hGALC fusion protein is calculated by multiplying the enzyme activity of hGALC per unit mass of the fusion protein ( ⁇ M/hour/mg protein) by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein corresponding to hGALC).
- the term "HSA-hGALC fusion protein" or "HSA-hGALC” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of hGALC is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of HSA, and which has the function of hGALC.
- HSA-hGALC fusion protein has the function of hGALC
- the definition of the SA-hGALC fusion protein above can be applied.
- a fusion protein having an amino acid sequence in which the N-terminus of GALC of a non-human animal species is linked, directly or via a linker, to the C-terminus of the amino acid sequence of SA of a non-human animal species, and which has the function of GALC is designated as a "(non-human animal species) SA-(non-human animal species) GALC fusion protein.”
- a fusion protein of mouse SA and mouse GALC is designated as an "MSA-mouse GALC fusion protein" or "MSA-mouse GALC.”
- the definition of the SA-hGALC fusion protein described above can be applied.
- the SA has the function of SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but this is not limited thereto.
- a preferred HSA-hGALC fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 5.
- the HSA-hGALC fusion protein shown in SEQ ID NO: 5 is obtained by linking wild-type hGALC to the C-terminus of wild-type HSA via the linker sequence Gly-Ser.
- the HSA-hGALC fusion protein shown in SEQ ID NO: 5 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 6.
- HSA-hGALC fusion proteins include those having an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 5 have been replaced with other amino acid residues, deleted, or mutated by addition, so long as they have the function of hGALC. It is preferable that the HSA-hGALC fusion protein has the function of human serum albumin, such as the function of binding and transporting endogenous substances in blood and exogenous substances such as drugs, but is not limited thereto.
- MSA-mGALC fusion protein refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of mGALC is linked, directly or via a linker, to the C-terminus of the amino acid sequence of MSA, and which has the function of mGALC.
- a preferred MSA-mGALC fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 18.
- the MSA-mGALC fusion protein shown in SEQ ID NO: 18 is a fusion protein in which wild-type mGALC is linked to the C-terminus of wild-type MSA via the linker sequence Gly-Ser.
- the MSA-mGALC fusion protein shown in SEQ ID NO: 18 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 19.
- MSA-mGALC fusion proteins also include those having an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 18 have been replaced with other amino acid residues, deleted, or mutated by addition, as long as they have the function of mGALC. It is preferable that the MSA-mGALC fusion protein has the functions of mouse serum albumin, such as binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- mutations When mutations are introduced into the HSA-hGALC fusion protein, which is a fusion protein of wild-type HSA and wild-type hGALC, mutations can be introduced only in the HSA portion and not in the hGALC portion, mutations can be introduced only in the hGALC portion without mutations in the HSA portion, or mutations can be introduced in both the HSA portion and the hGALC portion.
- mutations When mutations are introduced only in the HSA portion, the amino acid sequence of the portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above.
- the amino acid sequence of the portion is the amino acid sequence of hGALC obtained by adding a mutation to the wild-type hGALC described above.
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above
- the amino acid sequence of the hGALC portion is the amino acid sequence of hGALC obtained by adding a mutation to the wild-type hGALC described above.
- MSA-mGALC fusion proteins including those having the amino acid sequence shown in SEQ ID NO: 18.
- the MSA-mGALC fusion protein having the amino acid sequence shown in SEQ ID NO: 18 is encoded by a gene having the base sequence shown in SEQ ID NO: 19, for example.
- the same can be said about fusion proteins of wild-type SA (including wild-type MSA) of animal species other than humans and wild-type hGALC.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- an amino acid residue is added, one or more amino acid residues are added to the amino acid sequence shown in SEQ ID NO:5 or to the N-terminus or C-terminus of the amino acid sequence.
- the HSA-hGALC fusion protein may be a combination of these substitutions, deletions, and additions of amino acid residues.
- the mutation may be made only to the HSA portion, only to the hGALC portion, or to both portions.
- SA-hGALC fusion proteins between SA of a non-human animal species and hGALC
- GALC fusion proteins between SA of a non-human animal species and GALC of a non-human animal species, such as the fusion protein MSA-mouse GALC.
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:5 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:5.
- HSA-hGALC fusion proteins in which the amino acids constituting the protein are modified with sugar chains are also HSA-hGALC fusion proteins.
- HSA-hGALC fusion proteins in which the amino acids constituting the protein are modified with phosphate are also HSA-hGALC fusion proteins.
- HSA-hGALC fusion proteins modified with anything other than sugar chains and phosphate are also HSA-hGALC fusion proteins.
- HSA-hGALC fusion proteins in which the side chains of the amino acids constituting the protein are modified by substitution reactions or the like are also HSA-hGALC fusion proteins.
- Such modifications include, but are not limited to, the conversion of cysteine residues to formylglycine.
- SA-hGALC fusion proteins of SA of non-human animal species and hGALC
- fusion proteins of SA of non-human animal species and GALC of non-human animal species such as the fusion protein of MSA-mouse lysosomal enzyme.
- HSA-hGALC fusion proteins modified with sugar chains are included in HSA-hGALC fusion proteins having the original amino acid sequence.
- HSA-hGALC fusion proteins modified with phosphate are included in HSA-hGALC fusion proteins having the original amino acid sequence.
- those modified with things other than sugar chains and phosphate are included in HSA-hGALC fusion proteins having the original amino acid sequence.
- those in which the side chains of the amino acids constituting the HSA-hGALC fusion protein have been changed by substitution reactions or the like are included in HSA-hGALC fusion proteins having the original amino acid sequence.
- Such changes include, but are not limited to, the conversion of cysteine residues to formylglycine.
- fusion proteins of SA of a non-human animal species and hGALC SA-hGALC
- fusion proteins of SA of a non-human animal species and GALC of a non-human animal species such as the fusion protein MSA-mouse GALC.
- HSA-hGALC fusion proteins in which the constituent hGALC is a precursor of hGALC are also HSA-hGALC fusion proteins.
- the term "precursor” here refers to a type of protein that is biosynthesized as an HSA-hGALC fusion protein, and in which the portion that functions as hGALC after the biosynthesis is cleaved from the fusion protein during the manufacturing process or in the living body when administered to the living body, and can function as hGALC by itself.
- the HSA-hGALC fusion protein after the HSA-hGALC fusion protein is biosynthesized, it may be cleaved at a specific site by a hydrolase or the like, and the portion containing HSA and the portion containing mature hGALC may be separated.
- the obtained hGALC is not a fusion protein with HSA, but the HSA-hGALC fusion protein is synthesized once during the process of synthesizing the hGALC. Therefore, when hGALC is produced by such a method, the production method is included in the method for producing HSA-hGALC fusion protein.
- SA-hGALC non-human animal species SA and hGALC
- GALC MSA-mouse GALC
- SA-hGBA fusion protein or "SA-hGBA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of hGBA is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of SA, and which has the function of hGBA.
- SA animal species of the SA, but it is preferably a mammalian SA, more preferably a primate SA, and even more preferably HSA.
- hGBA in the SA-hGBA fusion protein has the function of hGBA
- hGBA retains a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of normal wild-type hGBA is taken as 100%.
- the specific activity of hGBA in the SA-hGBA fusion protein is calculated by multiplying the enzyme activity of hGBA per unit mass of the fusion protein ( ⁇ M/hour/mg protein) by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein corresponding to hGBA).
- the term "HSA-hGBA fusion protein" or "HSA-hGBA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of hGBA is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of HSA, and which has the function of hGBA.
- HSA-hGBA fusion protein has the function of hGALC
- the definition of the SA-hGBA fusion protein described above can be applied.
- a fusion protein having an amino acid sequence in which the N-terminus of GBA of a non-human animal species is linked, directly or via a linker, to the C-terminus of the amino acid sequence of SA of a non-human animal species, and which has the function of GBA is designated as a "(non-human animal species) SA-(non-human animal species) GBA fusion protein.”
- a fusion protein of mouse SA and mouse GBA is designated as an "MSA-mouse GBA fusion protein" or "MSA-mouse GBA.”
- a preferred HSA-hGBA fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 39.
- the HSA-hGBA fusion protein shown in SEQ ID NO: 39 is obtained by linking wild-type hGBA to the C-terminus of wild-type HSA via a linker sequence having the amino acid sequence shown in SEQ ID NO: 9.
- the HSA-hGBA fusion protein shown in SEQ ID NO: 39 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 40.
- HSA-hGBA fusion proteins include those having an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 39 have been replaced with other amino acid residues, deleted, or mutated by addition, as long as they have the function of hGBA. It is preferable that the HSA-hGBA fusion protein has the function of human serum albumin, such as the function of binding and transporting endogenous substances in blood and exogenous substances such as drugs, but is not limited thereto.
- MSA-mGBA fusion protein refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of mGBA is linked, directly or via a linker, to the C-terminus of the amino acid sequence of MSA, and which has the function of mGBA.
- a preferred MSA-mGBA fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 45.
- the MSA-mGBA fusion protein shown in SEQ ID NO: 45 is a fusion protein in which wild-type mGBA is linked to the C-terminus of wild-type MSA via a linker sequence having the amino acid sequence shown in SEQ ID NO: 9.
- the MSA-mGBA fusion protein shown in SEQ ID NO: 45 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 46.
- MSA-mGBA fusion proteins also include those having an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 45 are replaced with other amino acid residues, deleted, or mutated by addition, as long as they have the function of mGBA. It is preferable that the MSA-mGBA fusion protein has mouse serum albumin functions, such as binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- mutations When mutations are introduced into an HSA-hGBA fusion protein, which is a fusion protein of wild-type HSA and wild-type hGBA, mutations can be introduced only in the HSA portion and not in the hGBA portion, mutations can be introduced only in the hGBA portion without mutations in the HSA portion, or mutations can be introduced in both the HSA portion and the hGBA portion.
- the amino acid sequence of the portion is the amino acid sequence of HSA obtained by mutating the wild-type HSA described above.
- mutations are introduced only in the hGBA portion
- the amino acid sequence of the portion is the amino acid sequence of hGBA obtained by mutating the wild-type hGBA described above.
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by mutating the wild-type HSA described above
- the amino acid sequence of the hGBA portion is the amino acid sequence of hGBA obtained by mutating the wild-type hGBA described above.
- the same can be said about MSA-mGBA fusion proteins, including those having the amino acid sequence shown in SEQ ID NO: 45.
- the MSA-mGBA fusion protein having the amino acid sequence shown in SEQ ID NO: 45 is encoded by a gene having the base sequence shown in SEQ ID NO: 46, for example. The same can be said about fusion proteins of wild-type SA (including wild-type MSA) of animal species other than humans and wild-type hGBA.
- the following is an example of a case where a mutation is made to an HSA-hGBA fusion protein having the amino acid sequence shown in SEQ ID NO:39.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- an amino acid residue is added, one or more amino acid residues are added to the amino acid sequence shown in SEQ ID NO:39 or to the N-terminus or C-terminus of the amino acid sequence.
- the HSA-hGBA fusion protein may be a combination of these substitutions, deletions, and additions of amino acid residues.
- the mutation may be made only to the HSA portion, only to the hGBA portion, or to both portions.
- SA-hGBA fusion proteins of SA of a non-human animal species and hGBA
- GBA fusion protein of a non-human animal species
- MSA-mouse GBA fusion protein MSA-mouse GBA
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:39 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:39.
- HSA-hGBA fusion proteins in which the amino acids constituting the protein are modified with sugar chains are also HSA-hGBA fusion proteins.
- HSA-hGBA fusion proteins in which the amino acids constituting the protein are modified with phosphate are also HSA-hGBA fusion proteins.
- HSA-hGBA fusion proteins modified with anything other than sugar chains and phosphate are also HSA-hGBA fusion proteins.
- HSA-hGBA fusion proteins in which the side chains of the amino acids constituting the protein have been converted by substitution reactions or the like are also HSA-hGBA fusion proteins.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said for fusion proteins of SA and hGBA of non-human animal species (SA-hGBA), and fusion proteins of SA and GBA of non-human animal species, such as the fusion protein MSA-mouseGBA.
- HSA-hGBA fusion proteins modified with sugar chains are included in HSA-hGBA fusion proteins having the original amino acid sequence.
- HSA-hGBA fusion proteins modified with phosphate are included in HSA-hGBA fusion proteins having the original amino acid sequence.
- those modified with things other than sugar chains and phosphate are included in HSA-hGBA fusion proteins having the original amino acid sequence.
- those in which the side chains of the amino acids constituting the HSA-hGBA fusion protein have been converted by substitution reactions or the like are included in HSA-hGBA fusion proteins having the original amino acid sequence.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine.
- SA-hGBA non-human animal species
- GBA fusion protein MSA-mouse GBA
- HSA-hGBA fusion proteins in which the hGBA that constitutes them is a precursor of hGBA are also HSA-hGBA fusion proteins.
- precursor refers to a type that is biosynthesized as an HSA-hGBA fusion protein, and in which the portion that functions as hGBA after the biosynthesis is separated from the fusion protein during the manufacturing process or in the living body when administered to the living body, and can function as hGBA by itself.
- the HSA-hGBA fusion protein after the HSA-hGBA fusion protein is biosynthesized, it may be cleaved at a specific site by a hydrolase or the like, and the portion containing HSA and the portion containing mature hGBA may be separated.
- the obtained hGBA is not a fusion protein with HSA, but the HSA-hGBA fusion protein is synthesized once during the process of synthesizing the hGBA. Therefore, when hGBA is produced by such a method, the production method is included in the method for producing HSA-hGBA fusion protein.
- SA-hGBA fusion proteins of SA of a non-human animal species and hGBA
- GBA fusion proteins of SA of a non-human animal species, such as the MSA-mouse GBA fusion protein.
- human lysosomal enzyme-SA fusion protein or "human lysosomal enzyme-SA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of SA is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of a human lysosomal enzyme, and which has the function of a human lysosomal enzyme.
- the human lysosomal enzyme in a human lysosomal enzyme-SA fusion protein is said to have the function of a human lysosomal enzyme, it means that the human lysosomal enzyme preferably retains a specific activity of 10% or more, more preferably a specific activity of 20% or more, even more preferably a specific activity of 50% or more, and even more preferably a specific activity of 80% or more, when the specific activity of a normal wild-type human lysosomal enzyme is taken as 100%.
- the specific activity of the human lysosomal enzyme in the human lysosomal enzyme-SA fusion protein is calculated by multiplying the enzymatic activity of the human lysosomal enzyme per unit mass of the fusion protein ( ⁇ M/hour/mg protein) by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein that corresponds to the human lysosomal enzyme).
- human lysosomal enzyme-HSA fusion protein or "human lysosomal enzyme-HSA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of HSA is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of a human lysosomal enzyme, and which has the function of a human lysosomal enzyme.
- human lysosomal enzyme-HSA fusion protein has the function of a human lysosomal enzyme
- the definition of human lysosomal enzyme-SA above can be applied.
- the SA has a function as an SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- the human lysosomal enzyme-HSA fusion protein it is preferable that the SA has a function as an SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- the human lysosomal enzyme-HSA fusion protein it is preferable that the SA has a function as an SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- the human lysosomal enzyme-HSA fusion protein it is preferable that the SA has a function as an SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- the human lysosomal enzyme-HSA fusion protein it is prefer
- a fusion protein having an amino acid sequence in which the N-terminus of SA of a non-human animal species is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of a lysosomal enzyme of a non-human animal species, and which has the function of a human lysosomal enzyme is referred to as a "(non-human animal species) lysosomal enzyme-(non-human animal species) SA fusion protein.”
- a fusion protein of mouse lysosomal enzyme and mouse SA is referred to as a "mouse lysosomal enzyme-MSA fusion protein" or "mouse lysosomal enzyme-MSA.”
- a mutation When a mutation is introduced into a human lysosomal enzyme-HSA fusion protein, which is a fusion protein of wild-type human lysosomal enzyme and wild-type HSA, a mutation can be introduced only into the human lysosomal enzyme portion and not into the HSA portion, a mutation can be introduced only into the HSA portion without introducing a mutation into the human lysosomal enzyme portion, or a mutation can be introduced into both the human lysosomal enzyme portion and the HSA portion.
- the amino acid sequence of the portion is the amino acid sequence of the human lysosomal enzyme obtained by introducing a mutation into the wild-type human lysosomal enzyme described above.
- the amino acid sequence of the portion is the amino acid sequence of the HSA obtained by introducing a mutation into the wild-type HSA described above.
- the amino acid sequence of the human lysosomal enzyme portion is the amino acid sequence of the human lysosomal enzyme obtained by introducing a mutation into the wild-type human lysosomal enzyme described above
- the amino acid sequence of the HSA portion is the amino acid sequence of the HSA obtained by introducing a mutation into the wild-type HSA described above. The same can be said about fusion proteins between wild-type human lysosomal enzymes and wild-type SA (including wild-type MSA) of non-human animal species.
- Human lysosomal enzyme-HSA fusion proteins in which the amino acids constituting the protein are modified with sugar chains are also human lysosomal enzyme-HSA fusion proteins.
- Human lysosomal enzyme-HSA fusion proteins in which the amino acids constituting the protein are modified with phosphate are also human lysosomal enzyme-HSA fusion proteins.
- Human lysosomal enzyme-HSA fusion proteins modified with anything other than sugar chains and phosphate are also human lysosomal enzyme-HSA fusion proteins.
- Human lysosomal enzyme-HSA fusion proteins in which the side chains of the amino acids constituting the protein have been converted by a substitution reaction or the like are also human lysosomal enzyme-HSA fusion proteins.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine.
- the same can be said about fusion proteins of human lysosomal enzyme and SA of a species of animal other than human (human lysosomal enzyme-SA) and fusion proteins of lysosomal enzyme and SA of a species of animal other than human, such as mouse lysosomal enzyme-MSA.
- a human lysosomal enzyme-HSA fusion protein modified with a sugar chain is included in the human lysosomal enzyme-HSA fusion protein having the original amino acid sequence.
- a human lysosomal enzyme-HSA fusion protein modified with phosphate is included in the human lysosomal enzyme-HSA fusion protein having the original amino acid sequence.
- a fusion protein modified with something other than sugar chains and phosphate is also included in the human lysosomal enzyme-HSA fusion protein having the original amino acid sequence.
- a human lysosomal enzyme-HSA fusion protein in which the side chains of the amino acids constituting the human lysosomal enzyme-HSA fusion protein have been converted by a substitution reaction or the like is also included in the human lysosomal enzyme-HSA fusion protein having the original amino acid sequence.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said about a fusion protein of a human lysosomal enzyme and SA of an animal species other than human (human lysosomal enzyme-SA), and a fusion protein of a lysosomal enzyme of an animal species other than human and SA of an animal species other than human.
- a human lysosomal enzyme-HSA fusion protein in which the human lysosomal enzyme that constitutes it is a precursor of a human lysosomal enzyme is also a human lysosomal enzyme-HSA fusion protein.
- hGALC-SA fusion protein or "hGALC-SA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of SA is linked, directly or via a linker, to the C-terminus of the amino acid sequence of hGALC, and which has the function of hGALC.
- hGALC in an hGALC-SA fusion protein is said to have the function of hGALC, it means that hGALC retains a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of normal wild-type hGALC is taken as 100%.
- the specific activity of hGALC in the hGALC-SA fusion protein is calculated by multiplying the enzyme activity of hGALC per unit mass of the fusion protein ( ⁇ M/hour/mg protein) by (the molecular weight of the fusion protein/the molecular weight of the portion of the fusion protein corresponding to hGALC).
- hGALC-HSA fusion protein or "hGALC-HSA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of HSA is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of hGALC, and which has the function of hGALC.
- the definition of the hGALC-SA fusion protein described above can be applied.
- a fusion protein having an amino acid sequence in which the N-terminus of SA of a non-human animal species is linked, directly or via a linker, to the C-terminus of the amino acid sequence of GALC of a non-human animal species, and which has the function of GALC is designated as a "(non-human animal species) GALC-(non-human animal species) SA fusion protein.”
- a fusion protein of mouse GALC and mouse SA is designated as a "mouse GALC-MSA fusion protein" or "mouse GALC-MSA.”
- the definition of the hGALC-HSA fusion protein described above can be applied.
- the SA has the function of SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but this is not limited thereto.
- a preferred hGALC-HSA fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 7.
- the hGALC-HSA fusion protein shown in SEQ ID NO: 7 is obtained by linking wild-type HSA to the C-terminus of wild-type hGALC via the linker sequence Gly-Ser.
- the hGALC-HSA fusion protein shown in SEQ ID NO: 7 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 8.
- the hGALC-HSA fusion protein also includes an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 7 have been replaced with other amino acid residues, deleted, or mutated by addition, so long as it has the function of hGALC.
- the hGALC-HSA fusion protein is preferably one that has the function of human serum albumin, such as the function of binding and transporting endogenous substances in blood and exogenous substances such as drugs, but is not limited thereto.
- mGALC-MSA fusion protein refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of MSA is linked, directly or via a linker, to the C-terminus of the amino acid sequence of mGALC, and which has the function of mGALC.
- a preferred mGALC-MSA fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 20.
- the mGALC-MSA fusion protein shown in SEQ ID NO: 20 is a fusion protein in which wild-type MSA is linked to the C-terminus of wild-type mGALC via the linker sequence Gly-Ser.
- the mGALC-MSA fusion protein shown in SEQ ID NO: 20 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 21.
- mGALC-MSA fusion proteins also include those having an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 20 have been replaced with other amino acid residues, deleted, or mutated by addition, as long as they have the function of mGALC. It is preferable that the mGALC-MSA fusion protein has mouse serum albumin functions, such as binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- mutations When mutations are introduced into the hGALC-HSA fusion protein, which is a fusion protein of wild-type hGALC and wild-type HSA, mutations can be introduced only in the hGALC portion and not in the HSA portion, mutations can be introduced only in the HSA portion without mutations in the hGALC portion, or mutations can be introduced in both the hGALC portion and the HSA portion.
- the amino acid sequence of the portion is the amino acid sequence of hGALC obtained by mutating the wild-type hGALC described above.
- mutations are introduced only in the HSA portion, the amino acid sequence of the portion is the amino acid sequence of HSA obtained by mutating the wild-type HSA described above.
- the amino acid sequence of the hGALC portion is the amino acid sequence of hGALC obtained by mutating the wild-type hGALC described above
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by mutating the wild-type HSA described above.
- the same can be said about mGALC-MSA fusion proteins, including those having the amino acid sequence shown in SEQ ID NO: 20.
- the mGALC-MSA fusion protein having the amino acid sequence shown in SEQ ID NO: 20 is encoded by a gene having the base sequence shown in SEQ ID NO: 21, for example. The same can be said about fusion proteins of wild-type hGALC and wild-type SA (including wild-type MSA) of animals other than humans.
- mutations When mutations are introduced into the hGALC-HSA fusion protein, which is a fusion protein of wild-type hGALC and wild-type HSA, mutations can be introduced only in the HSA portion and not in the hGALC portion, mutations can be introduced only in the hGALC portion without mutations in the HSA portion, or mutations can be introduced in both the HSA portion and the hGALC portion.
- mutations When mutations are introduced only in the HSA portion, the amino acid sequence of the portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above.
- the amino acid sequence of the portion is the amino acid sequence of hGALC obtained by adding a mutation to the wild-type hGALC described above.
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above
- the amino acid sequence of the hGALC portion is the amino acid sequence of hGALC obtained by adding a mutation to the wild-type hGALC described above.
- mGALC-MSA fusion proteins including those having the amino acid sequence shown in SEQ ID NO: 20.
- the mGALC-MSA fusion protein having the amino acid sequence shown in SEQ ID NO: 20 is encoded by a gene having the base sequence shown in SEQ ID NO: 21, for example.
- the same can be said about a fusion protein of wild-type hGALC and wild-type SA (hGALC-SA) of an animal other than human.
- the following is an example of a case where a mutation is made to an hGALC-HSA fusion protein having the amino acid sequence shown in SEQ ID NO:7.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- an amino acid residue is added, one or more amino acid residues are added to the amino acid sequence shown in SEQ ID NO:7 or to the N-terminus or C-terminus of the amino acid sequence.
- the hGALC-HSA fusion protein may be a combination of these substitutions, deletions, and additions of amino acid residues.
- the mutation may be made only to the HSA portion, only to the hGALC portion, or to both portions.
- SA-GALC non-human animal species
- fusion proteins between SA and GALC of non-human animal species such as the fusion protein MSA-mouse GALC.
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:7 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:7.
- An hGALC-HSA fusion protein in which the amino acids constituting the protein are modified with sugar chains is also an hGALC-HSA fusion protein.
- An hGALC-HSA fusion protein in which the amino acids constituting the protein are modified with phosphate is also an hGALC-HSA fusion protein.
- An hGALC-HSA fusion protein modified with something other than sugar chains and phosphate is also an hGALC-HSA fusion protein.
- An hGALC-HSA fusion protein in which the side chains of the amino acids constituting the protein have been changed by a substitution reaction or the like is also an hGALC-HSA fusion protein.
- Such a change includes, but is not limited to, the conversion of a cysteine residue to formylglycine.
- the same can be said about a fusion protein of GALC and SA of a non-human animal species (GALC-SA), and a fusion protein of GALC of a non-human animal species and SA of a non-human animal species, for example, a fusion protein of mouse GALC and MSA.
- hGALC-HSA fusion proteins modified with sugar chains are included in the hGALC-HSA fusion proteins having the original amino acid sequence.
- hGALC-HSA fusion proteins modified with phosphate are included in the hGALC-HSA fusion proteins having the original amino acid sequence.
- those modified with things other than sugar chains and phosphate are included in the hGALC-HSA fusion proteins having the original amino acid sequence.
- hGALC-HSA fusion proteins in which the side chains of the amino acids constituting the hGALC-HSA fusion protein have been changed by substitution reactions or the like are included in the hGALC-HSA fusion proteins having the original amino acid sequence.
- Such changes include, but are not limited to, the conversion of cysteine residues to formylglycine.
- the same can be said for fusion proteins of GALC and SA of a non-human animal species (GALC-SA), and fusion proteins of GALC of a non-human animal species and SA of a non-human animal species.
- an hGALC-HSA fusion protein in which the hGALC that constitutes it is a precursor of hGALC is also an HSA-hGALC fusion protein.
- hGBA-SA fusion protein or "hGBA-SA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of SA is linked, directly or via a linker, to the C-terminus of the amino acid sequence of hGBA, and which has the function of hGBA.
- animal species of the SA it is preferably a mammalian SA, more preferably a primate SA, and even more preferably HSA.
- hGBA in an hGBA-SA fusion protein has the function of hGBA
- hGBA retains a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of normal wild-type hGBA is taken as 100%.
- the specific activity of hGBA in the hGBA-SA fusion protein is calculated by multiplying the enzyme activity of hGBA per unit mass of the fusion protein ( ⁇ M/hour/mg protein) by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein corresponding to hGBA).
- hGBA-HSA fusion protein or "hGBA-HSA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of HSA is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of hGBA, and which has the function of hGBA.
- the definition of the SA-hGBA fusion protein described above can be applied.
- a fusion protein having an amino acid sequence in which the N-terminus of SA of a non-human animal species is linked, directly or via a linker, to the C-terminus of the amino acid sequence of GBA of a non-human animal species, and which has the function of GBA is designated as a "(non-human animal species) GBA-(non-human animal species) SA fusion protein.”
- a fusion protein of mouse GBA and mouse SA is designated as a "mouse GBA-MSA fusion protein" or "mouse GBA-MSA.”
- the definition of the hGBA-HSA fusion protein described above can be applied.
- the SA has the function of SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but this is not limited thereto.
- a preferred hGBA-HSA fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 41.
- the hGBA-HSA fusion protein shown in SEQ ID NO: 41 is obtained by linking wild-type HSA to the C-terminus of wild-type hGBA via a linker sequence having the amino acid sequence shown in SEQ ID NO: 9.
- the hGBA-HSA fusion protein shown in SEQ ID NO: 41 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 42.
- hGBA-HSA fusion proteins also include those having an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 41 have been replaced with other amino acid residues, deleted, or mutated by addition, as long as they have the function of hGBA. It is preferable that the hGBA-HSA fusion protein has the function of human serum albumin, such as the function of binding and transporting endogenous substances in blood and exogenous substances such as drugs, but is not limited thereto.
- mGBA-MSA fusion protein or "mGBA-MSA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of MSA is linked, directly or via a linker, to the C-terminus of the amino acid sequence of mGBA, and which has the function of mGBA.
- a preferred mGBA-MSA fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 47.
- the mGBA-MSA fusion protein shown in SEQ ID NO: 47 is a fusion protein in which wild-type MSA is linked to the C-terminus of wild-type mGBA via a linker sequence having the amino acid sequence shown in SEQ ID NO: 9.
- the mGBA-MSA fusion protein shown in SEQ ID NO: 47 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 48.
- mGBA-MSA fusion proteins also include those having an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 47 have been replaced with other amino acid residues, deleted, or mutated by addition, as long as they have the function of mGBA. It is preferable that the mGBA-MSA fusion protein has mouse serum albumin functions, such as binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- mutations When mutations are introduced into the hGBA-HSA fusion protein, which is a fusion protein of wild-type hGBA and wild-type HSA, mutations can be introduced only in the hGBA portion and not in the HSA portion, mutations can be introduced only in the HSA portion without mutations in the hGBA portion, or mutations can be introduced in both the hGBA portion and the HSA portion.
- the amino acid sequence of the portion is the amino acid sequence of hGBA obtained by mutating the wild-type hGBA described above.
- mutations are introduced only in the HSA portion, the amino acid sequence of the portion is the amino acid sequence of HSA obtained by mutating the wild-type HSA described above.
- the amino acid sequence of the hGBA portion is the amino acid sequence of hGBA obtained by mutating the wild-type hGBA described above
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by mutating the wild-type HSA described above.
- the same can be said about mGBA-MSA fusion proteins, including those having the amino acid sequence shown in SEQ ID NO: 47.
- the mGBA-MSA fusion protein having the amino acid sequence shown in SEQ ID NO: 47 is encoded by a gene having the base sequence shown in SEQ ID NO: 48, for example.
- the same can be said about fusion proteins of wild-type hGBA and wild-type SA (including wild-type MSA) of an animal species other than human.
- the following is an example of a case where a mutation is made to an hGBA-HSA fusion protein having the amino acid sequence shown in SEQ ID NO:41.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- an amino acid residue is added, one or more amino acid residues are added to the amino acid sequence shown in SEQ ID NO:41 or to the N-terminus or C-terminus of the amino acid sequence.
- the hGBA-HSA fusion protein may be a combination of these substitutions, deletions, and additions of amino acid residues.
- the mutation may be made only to the HSA portion, only to the hGBA portion, or to both portions.
- fusion proteins of hGBA and SA of non-human animal species hGBA-SA
- fusion proteins of GBA of non-human animal species and SA of non-human animal species such as mouse GBA-MSA fusion protein.
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:41 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:41.
- An hGBA-HSA fusion protein in which the amino acids constituting the protein are modified with sugar chains is also an hGBA-HSA fusion protein.
- An hGBA-HSA fusion protein in which the amino acids constituting the protein are modified with phosphate is also an hGBA-HSA fusion protein.
- An hGBA-HSA fusion protein modified with something other than sugar chains and phosphate is also an hGBA-HSA fusion protein.
- An hGBA-HSA fusion protein in which the side chains of the amino acids constituting the protein have been converted by a substitution reaction or the like is also an hGBA-HSA fusion protein.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine.
- a fusion protein of hGBA and SA of a non-human animal species hGBA-SA
- a fusion protein of GBA of a non-human animal species and SA of a non-human animal species such as mouse GBA-MSA fusion protein.
- hGBA-HSA fusion proteins modified with sugar chains are included in the hGBA-HSA fusion proteins having the original amino acid sequence.
- hGBA-HSA fusion proteins modified with phosphate are included in the hGBA-HSA fusion proteins having the original amino acid sequence.
- those modified with things other than sugar chains and phosphate are included in the hGBA-HSA fusion proteins having the original amino acid sequence.
- those in which the side chains of the amino acids constituting the hGBA-HSA fusion proteins have been changed by substitution reactions or the like are included in the hGBA-HSA fusion proteins having the original amino acid sequence.
- Such changes include, but are not limited to, the conversion of cysteine residues to formylglycine.
- hGBA-SA non-human animal species
- GBA fusion proteins of non-human animal species and SA of non-human animal species, such as mouse GBA-MSA fusion protein.
- an hGBA-HSA fusion protein in which the hGBA that constitutes it is a precursor of hGBA is also an HSA-hGALC fusion protein.
- fusion protein of SA and lysosomal enzyme include both the above-mentioned “SA-lysosomal enzyme fusion protein” and “lysosomal enzyme-SA fusion protein”.
- fusion protein of SA and GALC include both the above-mentioned “SA-GALC fusion protein” and "GALC-SA fusion protein”.
- fusion protein of SA and GBA include both the above-mentioned “SA-GBA fusion protein” and "GBA-SA fusion protein”.
- SA is HSA
- lysosomal enzyme is human lysosomal enzyme
- GALC is hGALC
- GBA hGBA
- SA and lysosomal enzymes taking as examples a fusion protein of HSA and hGALC and a fusion protein of HSA and hGBA, and the manufacturing method of the fusion protein, but these descriptions can also be applied to fusion proteins in which the SA is non-human, and the lysosomal enzyme is other than GALC and GBA.
- they also apply to fusion proteins of SA of a non-human animal species and hGALC or hGBA, fusion proteins of SA of a non-human animal species and GALC or GBA of a non-human animal species, and fusion proteins of MSA and mGALC or mGBA.
- the SA and the lysosomal enzyme are linked directly or via a linker.
- linker refers to a structure used to link two types of proteins, or a portion that does not belong to either protein when two or more types of proteins are fused.
- Linkers include peptide linkers and non-peptide linkers.
- a peptide linker that constitutes part of a fusion protein can also be simply called a linker.
- the amino acid sequence of a peptide linker can also be called a linker sequence.
- the linker refers to a portion that does not belong to either the amino acid sequence of SA or the amino acid sequence of the lysosomal enzyme.
- the linker is a peptide chain that is interposed between the SA and the lysosomal enzyme. Linkers have various functions.
- the functions include a function between SA and lysosomal enzyme that binds SA and lysosomal enzyme, a function to reduce mutual interference between SA and lysosomal enzyme by increasing the distance between them within the fusion protein molecule, and a function to act as a hinge between SA and lysosomal enzyme that connects SA and lysosomal enzyme and gives flexibility to the three-dimensional structure of the fusion protein.
- the linker exerts at least one of these functions. This is the same, for example, when the SA is HSA, and when the lysosomal enzyme is a human lysosomal enzyme, such as hGALC or hGBA.
- the amino acid sequence of the linker is not particularly limited as long as it functions as a linker in the fusion protein molecule.
- the length of the peptide linker is also not particularly limited as long as it functions as a linker in the fusion protein molecule.
- the peptide linker is composed of one or more amino acids. When the peptide linker is composed of multiple amino acids, the number of amino acids is preferably 2 to 50, more preferably 5 to 30, and even more preferably 10 to 25.
- Suitable examples of peptide linkers include those consisting of Gly-Ser, Gly-Gly-Ser, or amino acid sequences shown in SEQ ID NOs: 9 to 11 (collectively referred to as basic sequences), and those containing these.
- the peptide linker is one that contains an amino acid sequence in which the basic sequence is repeated 2 to 10 times, one that contains an amino acid sequence in which the basic sequence is repeated 2 to 6 times, and one that contains an amino acid sequence in which the basic sequence is repeated 3 to 5 times.
- One or more amino acids in these amino acid sequences may be deleted, replaced with other amino acids, added, etc. When deleting an amino acid, the number of amino acids to be deleted is preferably 1 or 2.
- the number of amino acids to be substituted is preferably 1 or 2.
- the number of amino acids to be added is preferably 1 or 2.
- the desired amino acid sequence of the linker portion can be obtained by combining these deletions, substitutions, and additions of amino acids.
- the peptide linker may be composed of one amino acid, and the amino acid constituting the linker is, for example, glycine or serine. This is the same, for example, when the SA is HSA, and when the lysosomal enzyme is, for example, a human lysosomal enzyme, such as hGALC or hGBA.
- a fusion protein of HSA and hGALC can be produced as a recombinant protein by preparing an expression vector incorporating a DNA fragment in which a gene encoding hGALC is linked in-frame downstream or upstream of a gene encoding HSA, and culturing a host cell transformed by introducing this expression vector.
- a fusion protein of HSA and hGBA can be produced as a recombinant protein by preparing an expression vector incorporating a DNA fragment in which a gene encoding hGBA is linked in-frame downstream or upstream of a gene encoding HSA, and culturing a host cell transformed by introducing this expression vector.
- the fusion protein produced as a recombinant protein in this way consists of a single-chain polypeptide.
- a fusion protein produced as a recombinant protein is called a recombinant fusion protein.
- a gene encoding hGALC When preparing an HSA-hGALC fusion protein as a recombinant fusion protein, a gene encoding hGALC can be linked in-frame downstream of a gene encoding HSA to obtain a fusion protein having the amino acid sequence of hGALC at the C-terminus of the amino acid sequence of HSA. Conversely, a gene encoding hGALC can be linked in-frame upstream of a gene encoding HSA to obtain an hGALC-HSA fusion protein having the amino acid sequence of hGALC at the N-terminus of the amino acid sequence of HSA.
- a gene encoding hGBA can be linked in-frame downstream of a gene encoding HSA to obtain an HSA-hGBA fusion protein having the amino acid sequence of hGBA at the C-terminus of the amino acid sequence of HSA.
- a gene encoding hGBA can be linked in-frame upstream of a gene encoding HSA to obtain an hGBA-HSA fusion protein having the amino acid sequence of hGBA at the N-terminus of the amino acid sequence of HSA.
- the fusion protein prepared as a recombinant fusion protein is a single-chain polypeptide.
- single-chain polypeptide refers to a polypeptide that has one N-terminus and one C-terminus and has no branched peptide chains. As long as this condition is met, those that have intramolecular disulfide bonds and those that are modified with sugar chains, lipids, phospholipids, etc. are also single-chain polypeptides. In addition, when single-chain polypeptides form complexes such as dimers through non-covalent bonds, each peptide chain that forms the complex is understood to be a single-chain polypeptide, and the complex itself is understood to be an assembly of single-chain polypeptides.
- FIG. 1 shows a schematic diagram of a single-chain polypeptide HSA-hGALC fusion protein having HSA, a linker, and hGALC in this order from the N-terminus.
- FIG. 2 also shows a schematic diagram of a single-chain polypeptide HSA-hGBA fusion protein having HSA, a linker, and hGBA in this order from the N-terminus.
- HSA-hGBA fusion protein the C-terminus of HSA and the N-terminus of the linker are bound by a peptide bond, and the C-terminus of the linker and the N-terminus of hGBA are bound by a peptide bond.
- FIG. 3 shows a single-chain polypeptide hGALC-HSA fusion protein having hGALC, a linker, and HSA in this order from the N-terminus.
- FIG. 4 shows a single-chain polypeptide hGBA-HSA fusion protein having hGBA, a linker, and HSA in this order from the N-terminus.
- the C-terminus of hGBA and the N-terminus of the linker are bound by a peptide bond
- the C-terminus of the linker and the N-terminus of HSA are bound by a peptide bond.
- a fusion protein of HSA and hGALC refers to a fusion protein characterized in that when expressed in host cells as a recombinant protein, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the expression amount of hGALC in the culture supernatant is at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or enzyme activity, compared to when wild-type hGALC is expressed in host cells as a recombinant protein under the same conditions.
- the fusion protein of HSA and hGBA refers to a fusion protein characterized in that when expressed in a host cell as a recombinant protein, particularly when expressed so that the recombinant protein is secreted from the cell and accumulated in the culture medium, the amount of hGBA expressed in the culture supernatant is at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, for example, 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, 10 to 20 times, etc., in terms of concentration or enzyme activity, compared to when wild-type hGBA is expressed in a host cell as a recombinant protein under the same conditions.
- "under the same conditions” means that the expression vector, host cell, culture conditions, etc. are the same.
- the preferred host cells used in this case are mammalian cells such as CHO cells and NS/0 cells, and particularly CHO cells.
- Preferred embodiments of the fusion protein of HSA and hGALC include the following (1) and (2): (1) A polypeptide having an amino acid sequence shown in SEQ ID NO:5, in which the N-terminus of the amino acid sequence of wild-type hGALC shown in SEQ ID NO:1 is linked to the C-terminus of the amino acid sequence of wild-type HSA shown in SEQ ID NO:3 via a linker represented by Gly-Ser; (2) Having the amino acid sequence shown in SEQ ID NO: 7, in which the N-terminus of the amino acid sequence of wild-type HSA shown in SEQ ID NO: 3 is linked to the C-terminus of the amino acid sequence of wild-type hGALC shown in SEQ ID NO: 1 via a linker represented by Gly-Ser.
- Preferred embodiments of the fusion protein of HSA and hGBA include the following (1) and (2): (1) having the amino acid sequence shown in SEQ ID NO: 39, in which the N-terminus of the amino acid sequence of wild-type hGBA shown in SEQ ID NO: 37 is linked to the C-terminus of the amino acid sequence of wild-type HSA shown in SEQ ID NO: 3 via a linker having the amino acid sequence shown in SEQ ID NO: 9; (2) Having the amino acid sequence shown in SEQ ID NO: 41, in which the N-terminus of the amino acid sequence of wild-type HSA shown in SEQ ID NO: 3 is linked to the C-terminus of the amino acid sequence of wild-type hGBA shown in SEQ ID NO: 37 via a linker having the amino acid sequence shown in SEQ ID NO: 9.
- the fusion protein of HSA and hGALC is characterized in that when expressed in a host cell as a recombinant protein, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the expression amount of hGALC in the culture supernatant is increased in terms of concentration or enzyme activity compared to when wild-type hGALC is expressed in a host cell as a recombinant protein under the same conditions. Therefore, when the fusion protein of HSA and hGALC is produced as a recombinant protein, the production efficiency can be increased compared to wild-type hGALC, and production costs can be reduced.
- the fusion protein of HSA and hGBA is characterized in that when expressed in a host cell as a recombinant protein, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the expression amount of hGBA in the culture supernatant is increased in terms of concentration or enzyme activity compared to when wild-type hGBA is expressed in a host cell as a recombinant protein under the same conditions. Therefore, when the fusion protein of HSA and hGBA is produced as a recombinant protein, the production efficiency can be increased compared to wild-type hGBA, and production costs can be reduced.
- a fusion protein of HSA and hGALC expressed in a host cell as a recombinant protein with wild-type hGALC expressed in a host cell under the same conditions, it is preferable to compare the GALC enzyme activity of the expressed protein rather than the mass of the expressed protein.
- the expression levels of a fusion protein of HSA and hGBA, a fusion protein of SA of a non-human animal species and hGALC or hGBA, and a fusion protein of SA of a non-human animal species and GALC or GBA of a non-human animal species for example, a fusion protein of MSA and mGALC or mGBA.
- HSA/hGALC fusion protein and HSA/hGBA fusion protein can be produced as recombinant proteins by culturing host cells transformed with an expression vector incorporating a gene encoding the fusion protein.
- the host cells used in this case are not particularly limited as long as they are capable of expressing a fusion protein of HSA and hGALC or a fusion protein of HSA and hGBA by introducing such an expression vector, and may be any eukaryotic cell such as a mammalian cell, yeast, plant cell, or insect cell, although mammalian cells are particularly preferred.
- mammalian cells When mammalian cells are used as host cells, there is no particular limitation on the type of mammalian cell, but cells derived from humans, mice, or Chinese hamsters are preferred, and in particular CHO cells derived from Chinese hamster ovary cells or NS/0 cells derived from mouse myeloma are preferred.
- the expression vector used to incorporate and express a DNA fragment containing a gene encoding a fusion protein of HSA and hGALC or a fusion protein of HSA and hGBA can be used without particular limitation as long as it brings about expression of the gene when introduced into a mammalian cell.
- the gene incorporated in the expression vector is placed downstream of a DNA sequence (gene expression control site) that can regulate the frequency of gene transcription in mammalian cells.
- a DNA sequence gene expression control site
- gene expression control sites include a promoter derived from cytomegalovirus, an SV40 early promoter, a human elongation factor-1 ⁇ (EF-1 ⁇ ) promoter, and a human ubiquitin C promoter.
- GGS glutamine synthetase
- IVS internal ribosome entry site
- the term "internal ribosome binding site” refers to a region (structure) present within an mRNA strand to which a ribosome can directly bind and initiate translation independent of a cap structure, or a region (structure) of a DNA strand that generates the region by being transcribed.
- the term "gene encoding an internal ribosome binding site” refers to a region (structure) of a DNA strand that generates the region by being transcribed.
- IRES internal ribosome entry sites
- viruses such as Picornaviridae viruses (poliovirus, rhinovirus, mouse encephalomyocarditis virus, etc.), foot and mouth disease virus, hepatitis A virus, hepatitis C virus, coronavirus, bovine enteric virus, Theiler's murine encephalomyelitis virus, and Coxsackie B virus, as well as in the 5' untranslated regions of genes such as human immunoglobulin heavy chain binding protein, Drosophila antennapedia, and Drosophila ultravithorax.
- viruses such as Picornaviridae viruses (poliovirus, rhinovirus, mouse encephalomyocarditis virus, etc.), foot and mouth disease virus, hepatitis A virus, hepatitis C virus, coronavirus, bovine enteric virus, Theiler's murine encephalomyelitis virus, and Coxsackie B virus, as well as in the 5' untranslated regions of genes such as human immuno
- the IRES is a region of approximately 450 bp that exists in the 5' untranslated region of the mRNA.
- the "5' untranslated region of the virus” refers to the 5' untranslated region of the viral mRNA, or the region (structure) of the DNA strand that produces said region by being transcribed.
- an expression vector for expressing a target protein which comprises a first gene expression control site, a gene encoding the protein downstream of the first gene expression control site, an internal ribosome binding site further downstream, and a gene encoding glutamine synthetase further downstream, and further comprises a dihydrofolate reductase gene or a drug resistance gene downstream of the first gene expression control site or a second gene expression control site different from the first gene expression control site, can be suitably used for producing a fusion protein of HSA and hGALC or a fusion protein of HSA and hGBA.
- the first gene expression control site or the second gene expression control site can be suitably used with a promoter derived from cytomegalovirus, an SV40 early promoter, a human elongation factor-1 ⁇ promoter (hEF-1 ⁇ promoter), or a human ubiquitin C promoter, with the hEF-1 ⁇ promoter being particularly suitable.
- a promoter derived from cytomegalovirus an SV40 early promoter, a human elongation factor-1 ⁇ promoter (hEF-1 ⁇ promoter), or a human ubiquitin C promoter, with the hEF-1 ⁇ promoter being particularly suitable.
- the internal ribosome binding site those derived from the 5' untranslated region of the genome of a virus selected from the group consisting of a virus of the picornavirus family (including mouse encephalomyocarditis virus), foot and mouth disease virus, hepatitis A virus, hepatitis C virus, coronavirus, bovine enteric virus, Theiler's murine encephalomyelitis virus, and Coxsackie B virus, or a gene selected from the group consisting of the human immunoglobulin heavy chain binding protein gene, the Drosophila antennapedia gene, and the Drosophila ultravithorax gene, are preferably used, but an internal ribosome binding site derived from the 5' untranslated region of the mouse encephalomyocarditis virus genome is particularly preferable.
- a virus of the picornavirus family including mouse encephalomyocarditis virus
- foot and mouth disease virus hepatitis A virus, hepatitis C virus, coronavirus,
- the drug resistance gene preferably used is a puromycin or neomycin resistance gene, and more preferably a puromycin resistance gene.
- an expression vector for expressing a target protein which contains a human elongation factor-1 ⁇ promoter, a gene encoding the protein downstream thereof, an internal ribosome binding site derived from the 5' untranslated region of the mouse encephalomyocarditis virus genome further downstream thereof, and a gene encoding glutamine synthetase further downstream thereof, and further contains another gene expression control site and a dihydrofolate reductase gene downstream thereof, in which the internal ribosome binding site is an internal ribosome binding site in which some of the multiple initiation codons contained in the wild-type internal ribosome binding site have been destroyed, can be suitably used for producing a fusion protein of HSA and hGALC or a fusion protein of HSA and hGBA.
- expression vectors include the expression vectors described in WO2013/161958.
- an expression vector for expressing a target protein which contains a human elongation factor-1 ⁇ promoter, a gene encoding the protein downstream thereof, an internal ribosome binding site derived from the 5' untranslated region of the mouse encephalomyocarditis virus genome further downstream thereof, and a gene encoding glutamine synthetase further downstream thereof, and further contains another gene expression control site and a drug resistance gene downstream thereof, in which the internal ribosome binding site is an internal ribosome binding site in which some of the multiple initiation codons contained in the wild-type internal ribosome binding site have been destroyed, can be suitably used for producing a fusion protein of HSA and hGALC or a fusion protein of HSA and hGBA.
- expression vectors include pE-mIRES-GS-puro described in WO2012/063799 and pE-mIRES-GS-mNeo described in WO2013/161958.
- the 3' end of the internal ribosome binding site derived from the 5' untranslated region of the wild-type murine encephalomyocarditis virus genome contains three initiation codons (ATG).
- the above pE-mIRES-GS-puro and pE-mIRES-GS-mNeo are expression vectors that have an IRES in which some of the initiation codons have been destroyed.
- the fusion protein of HSA and hGALC or the fusion protein of HSA and hGBA can be expressed in cells or in a medium by culturing a host cell into which an expression vector incorporating a gene encoding the protein has been introduced.
- the method of expressing the fusion protein of HSA and hGALC or the fusion protein of HSA and hGBA when the host cell is a mammalian cell is described in detail below.
- a serum-free medium used as a medium for producing recombinant proteins is preferably one containing, for example, 3 to 700 mg/L amino acids, 0.001 to 50 mg/L vitamins, 0.3 to 10 g/L monosaccharides, 0.1 to 10,000 mg/L inorganic salts, 0.001 to 0.1 mg/L trace elements, 0.1 to 50 mg/L nucleosides, 0.001 to 10 mg/L fatty acids, 0.01 to 1 mg/L biotin, 0.1 to 20 ⁇ g/L hydrocortisone, 0.1 to 20 mg/L insulin, 0.1 to 10 mg/L vitamin B12, 0.01 to 1 mg/L putrescine, 10 to 500 mg/L sodium pyruvate and a water-soluble iron compound. If desired, thy
- DMEM/F12 medium (mixture of DMEM and F12) may be used as a basic medium, and these media are well known to those skilled in the art.
- DMEM(HG)HAM Improved (R5) medium which contains sodium bicarbonate, L-glutamine, D-glucose, insulin, sodium selenite, diaminobutane, hydrocortisone, ferrous sulfate (II), asparagine, aspartic acid, serine, and polyvinyl alcohol, may be used.
- serum-free media such as CD OptiCHO TM medium, CHO-S-SFM II medium, or CD CHO medium (Thermo Fisher Scientific), EX-CELL TM 302 medium, or EX-CELL TM 325-PF medium (SAFC Biosciences), may be used as a basic medium.
- the fusion protein of HSA and hGALC is characterized in that when host cells into which an expression vector incorporating a gene encoding the fusion protein has been introduced are cultured in the above-mentioned serum-free medium to be expressed as a recombinant protein, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the amount of hGALC expressed in the culture supernatant is at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or enzyme activity, compared to when wild-type hGALC is expressed as a recombinant protein in host cells under the same conditions.
- the expression amount of hGBA in the culture supernatant is at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, for example, 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, 10 to 20 times, etc., in terms of concentration or enzyme activity, compared to when wild-type hGBA is expressed as a recombinant protein in a host cell under the same conditions.
- the host cell used in this case is preferably a mammalian cell, particularly a normal cell used for producing recombinant proteins, such as a CHO cell or an NS/0 cell.
- wild-type hGALC When wild-type hGALC is expressed as a recombinant using a host cell transformed with an expression vector incorporating a gene encoding it, it is difficult to efficiently produce it as a recombinant due to reasons such as a decrease in the viability of the host cell. However, by expressing hGALC as a fusion protein with HSA, the decrease in the viability of the host cell observed when wild-type hGALC is expressed is suppressed.
- a fusion protein of HSA and hGALC When a fusion protein of HSA and hGALC is expressed as a recombinant protein by culturing mammalian cells into which an expression vector incorporating a gene encoding it has been introduced in a serum-free medium, especially when the recombinant protein is expressed so that it is secreted from the cells and accumulated in the culture medium, the expression amount increases compared to when wild-type hGALC is expressed as a recombinant protein under the same conditions. It is considered that the suppression of the decrease in viability contributes to this increase in the expression amount.
- the host cell used for expressing the fusion protein is preferably a mammalian cell, particularly a normal cell used for producing recombinant proteins such as CHO cells and NS/0 cells. That is, one embodiment of the present invention is a recombinant fusion protein between SA and GALC, in particular a recombinant fusion protein between HSA and hGALC.
- the decrease in the survival rate of host cells when hGALC and hGBA are expressed means that the number of dead cells increases.
- the contents of dead cells leak out, becoming contaminants, and a process for removing the contaminants is required in the subsequent purification process of the expressed fusion protein.
- the number of dead cells generated during culture can be reduced, and contaminants can be reduced, making it easier to purify the expressed fusion protein. This also increases the purification efficiency during purification. It also reduces the amount of contaminants contained in the purified fusion protein.
- a host cell By culturing a host cell encoding a fusion protein of HSA and hGALC or a fusion protein of HSA and hGBA, it can be expressed in cells or in a medium. These fusion proteins can be separated from impurities and purified by methods such as column chromatography.
- the purified fusion protein of HSA and hGALC or fusion protein of HSA and hGBA can be used as a pharmaceutical composition.
- the fusion protein of HSA and hGALC can be used as a pharmaceutical composition for treating Krabbe disease (galactosylceramide lipidosis or globoid cell leukodystrophy).
- the fusion protein of HSA and hGBA can be used as a pharmaceutical composition for treating Gaucher disease.
- pharmaceutical composition refers to a composition that contains a fusion protein as an active ingredient as well as a pharma- ceutical acceptable excipient.
- a pharmaceutical composition containing the fusion protein of HSA and hGALC or the fusion protein of HSA and hGBA as an active ingredient can be administered intravenously, intramuscularly, intraperitoneally, subcutaneously, or intracerebroventricularly as an injection.
- injections can be supplied as freeze-dried preparations or aqueous liquid preparations.
- an aqueous liquid preparation it may be in the form of being filled in a vial, or it can be supplied as a prefilled preparation in which it is filled in a syringe in advance.
- a freeze-dried preparation it is dissolved in an aqueous medium before use and reconstituted for use.
- the ratio of monomer to total protein is preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, for example 95% or more.
- the same can be said for a solution obtained by dissolving a freeze-dried preparation in an aqueous medium and reconstituting it.
- the fusion protein of HSA and hGALC or the fusion protein of HSA and hGBA can be further bound to an antibody or a ligand.
- the fusion protein of HSA and hGALC in one embodiment of the present invention can be bound to an antibody or a ligand capable of specifically binding to a receptor on cerebrovascular endothelial cells.
- the fusion protein of HSA and hGBA in one embodiment of the present invention can be bound to an antibody or a ligand capable of specifically binding to a receptor on cerebrovascular endothelial cells.
- the fusion protein of HSA and hGALC or the fusion protein of HSA and hGBA can bind to a receptor on cerebrovascular endothelial cells by being bound to an antibody or a ligand capable of specifically binding to a receptor on cerebrovascular endothelial cells.
- the fusion protein bound to the receptor on cerebrovascular endothelial cells can pass through the blood-brain barrier (BBB) and reach the tissues of the central nervous system (CNS). Therefore, by combining a fusion protein of HSA and hGALC or a fusion protein of HSA and hGBA with such an antibody or ligand, it is possible to pass through the blood-brain barrier (BBB) and exert its function in the central nervous system (CNS).
- BBB blood-brain barrier
- CNS central nervous system
- the term "ligand" refers to a substance having affinity for a specific substance, particularly a protein having affinity for a specific substance.
- such a ligand is a substance, particularly a protein, having affinity for a receptor present on cerebrovascular endothelial cells.
- receptors present on cerebrovascular endothelial cells include insulin receptors, transferrin receptors, leptin receptors, lipoprotein receptors, and IGF receptors, particularly, but not limited to, transferrin receptors.
- the receptors are preferably human-derived receptors.
- Ligands for insulin receptors, transferrin receptors, leptin receptors, lipoprotein receptors, and IGF receptors are insulin, transferrin, leptin, lipoprotein, and IGF (IGF-1 and IGF-2), respectively. These ligands may be full-length or fragments thereof as long as they have affinity for the receptors, and may be wild-type or mutants in which substitutions, deletions, and/or additions have been introduced into the wild-type.
- hGALC When hGALC is said to have the function of hGALC in a conjugate of an antibody and a fusion protein of HSA and hGALC, it means that hGALC has a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, for example 90% or more, 95% or more, when the specific activity of normal wild-type hGALC is taken as 100%.
- the specific activity of hGALC in the conjugate is calculated by multiplying the enzyme activity of hGALC per unit mass of the conjugate ( ⁇ M/hour/mg protein) by (molecular weight of the conjugate/molecular weight of the portion of the conjugate corresponding to hGALC).
- hGBA in a conjugate of an antibody and a fusion protein of HSA and hGBA is said to have the function of hGBA, it means that, when the specific activity of normal wild-type hGBA is taken as 100%, hGBA has a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, for example 90% or more, 95% or more.
- the specific activity of hGBA in the conjugate is calculated by multiplying the enzyme activity of hGBA per unit mass of the conjugate ( ⁇ M/hour/mg protein) by (molecular weight of the conjugate/molecular weight of the portion of the conjugate corresponding to hGBA).
- a conjugate of an antibody and a fusion protein of HSA and hGALC refers to a conjugate characterized in that when the conjugate is expressed as a recombinant protein in host cells, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the expression amount of hGALC in the culture supernatant is at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or enzyme activity, compared to when wild-type hGALC is expressed as a recombinant protein in host cells under the same conditions.
- a conjugate of an antibody and a fusion protein of HSA and hGBA is characterized in that when the conjugate is expressed as a recombinant protein in a host cell, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the amount of hGBA expressed in the culture supernatant is at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or enzyme activity, compared to when wild-type hGBA is expressed as a recombinant protein in a host cell under the same conditions, such as 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, 10 to 20 times, etc.
- cytokines particularly interleukins
- IL-10 is one such interleukin.
- One embodiment of the present invention is a recombinant protein in which such a cytokine that is difficult to produce as a recombinant protein is fused with SA.
- SA a recombinant protein in which such a cytokine that is difficult to produce as a recombinant protein is fused with SA.
- Such a recombinant protein can be mass-produced more easily than the corresponding wild-type cytokine using host cells into which an expression vector incorporating a gene encoding the cytokine is introduced.
- the animal species of the cytokine to be bound to SA, but it is preferably a human cytokine.
- the animal species of the SA to be bound to the cytokine, but it is preferably HSA.
- human cytokine when used simply, it includes not only normal wild-type human cytokines, but also mutant human cytokines in which one or more amino acid residues have been substituted, deleted, and/or added to the amino acid sequence of a wild-type human cytokine (as used herein, "addition" of an amino acid residue means adding a residue to the end or inside of the sequence), without any particular distinction, so long as the mutant has the function of a human cytokine, such as having physiological activity corresponding to the type of human cytokine in question.
- cytokines of animal species other than humans The same also applies to interleukins.
- a human cytokine When a human cytokine is said to have the function of a human cytokine, it means that the human cytokine preferably retains a specific activity of 10% or more, more preferably a specific activity of 20% or more, even more preferably a specific activity of 50% or more, and even more preferably a specific activity of 80% or more, when the specific activity of a normal wild-type human cytokine is taken as 100%.
- specific activity refers to the physiological activity per mass of the protein.
- the specific activity of a fusion protein of a human cytokine and another protein is determined as the physiological activity per mass of the portion of the fusion protein that corresponds to the human cytokine.
- the specific activity of the human cytokine in the fusion protein is calculated by multiplying the physiological activity of the human cytokine per unit mass of the fusion protein by (the molecular weight of the fusion protein/the molecular weight of the portion of the fusion protein that corresponds to the human cytokine).
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- a human cytokine mutant consisting of an amino acid sequence in which one amino acid residue is deleted from the N-terminus or C-terminus of a wild-type human cytokine a human cytokine mutant consisting of an amino acid sequence in which two amino acid residues are deleted from the N-terminus or C-terminus of a wild-type human cytokine, and the like are also human cytokines.
- a mutation that combines the substitution and deletion of these amino acid residues can be added to the amino acid sequence of a wild-type human cytokine. The same applies to cytokines of animal species other than humans. The same applies to interleukins.
- amino acid residues When adding amino acid residues to the amino acid sequence of a wild-type human cytokine, one or more amino acid residues are added into the amino acid sequence of the human cytokine or to the N-terminus or C-terminus of the amino acid sequence.
- the number of amino acid residues added in this case is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- Mutations that combine the addition of these amino acid residues with the above-mentioned substitutions can also be added to the amino acid sequence of a wild-type human cytokine, and mutations that combine the addition of these amino acid residues with the above-mentioned deletions can also be added to the amino acid sequence of a wild-type human cytokine.
- cytokines of animal species other than humans The same applies to interleukins.
- a combination of the three types of mutations mentioned above, namely substitution, deletion, and addition of amino acid residues, can be added to the amino acid sequence of a wild-type human cytokine.
- a human cytokine is one in which 1 to 10 amino acid residues are deleted from the amino acid sequence of a wild-type human cytokine, 1 to 10 amino acid residues are substituted with other amino acid residues, and 1 to 10 amino acid residues are added;
- a human cytokine is one in which 1 to 5 amino acid residues are deleted from the amino acid sequence of a wild-type human cytokine, 1 to 5 amino acid residues are substituted with other amino acid residues, and 1 to 5 amino acid residues are added;
- a human cytokine is one in which 1 to 3 amino acid residues are deleted from the amino acid sequence of a wild-type human cytokine, 1 to 3 amino acid residues are substituted with other amino acid residues, and 1 to 3 amino acid residues are added;
- each mutation in a human cytokine mutant compared to a normal wild-type human cytokine can be easily confirmed by aligning the amino acid sequences of both human cytokines. The same is true for cytokines of non-human animal species, and also for interleukins.
- the amino acid sequence of the human cytokine mutant preferably has 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more or 99% or more identity, to the amino acid sequence of a normal wild-type human cytokine.
- identity preferably has 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more or 99% or more identity, to the amino acid sequence of a normal wild-type human cytokine.
- cytokines of animal species other than humans The same also applies to interleukins.
- human interleukin 10 when simply referring to "human interleukin 10", “human IL-10”, or “hIL-10”, it includes not only the normal wild-type hIL-10 consisting of 160 amino acid residues as shown in SEQ ID NO:24, but also includes, without any particular distinction, mutants of hIL-10 in which one or more amino acid residues have been substituted, deleted, and/or added (as used herein, “addition” of an amino acid residue means adding a residue to the end or inside of the sequence) to the amino acid sequence as shown in SEQ ID NO:24, so long as they have the functions of hIL-10, such as having physiological activity as an inhibitory cytokine that calms the immune response.
- Wild-type hIL-10 is encoded, for example, by a gene having the base sequence as shown in SEQ ID NO:49. The same applies to IL-10 of animal species other than humans.
- hIL-10 when hIL-10 is said to have the function of hIL-10, it means that hIL-10 retains a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of normal wild-type hIL-10 is taken as 100%.
- specific activity refers to the physiological activity per mass of the protein.
- the specific activity of a fusion protein of hIL-10 and another protein is calculated as the physiological activity per mass of the portion of the fusion protein that corresponds to hIL-10.
- the specific activity of hIL-10 in the fusion protein is calculated by multiplying the physiological activity of hIL-10 per unit mass of the fusion protein by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein that corresponds to hIL-10).
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- a mutant hIL-10 consisting of 159 amino acid residues obtained by deleting one amino acid residue at the N-terminus or C-terminus of wild-type hIL-10, and a mutant hIL-10 consisting of 158 amino acid residues obtained by deleting two amino acid residues at the N-terminus or C-terminus of wild-type hIL-10, etc. are also hIL-10.
- a mutation that combines the substitution and deletion of these amino acid residues can be added to the amino acid sequence of wild-type hIL-10. The same applies to IL-10 of animal species other than humans.
- amino acid residues When amino acid residues are added to the amino acid sequence of wild-type hIL-10, one or more amino acid residues are added into the amino acid sequence of hIL-10 or to the N-terminus or C-terminus of the amino acid sequence.
- the number of amino acid residues added in this case is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2. Mutations that combine the addition of these amino acid residues with the above-mentioned substitutions can also be added to the amino acid sequence of wild-type hIL-10, and mutations that combine the addition of these amino acid residues with the above-mentioned deletions can also be added to the amino acid sequence of wild-type hIL-10. The same applies to IL-10 from animal species other than humans.
- a combination of the above three types of mutations, substitution, deletion, and addition of amino acid residues can be added to the amino acid sequence of wild-type hIL-10.
- a wild-type hIL-10 amino acid sequence shown in SEQ ID NO:24 in which 1 to 10 amino acid residues have been deleted, 1 to 10 amino acid residues have been replaced with other amino acid residues, and 1 to 10 amino acid residues have been added is also hIL-10
- a wild-type hIL-10 amino acid sequence shown in SEQ ID NO:24 in which 1 to 5 amino acid residues have been deleted, 1 to 5 amino acid residues have been replaced with other amino acid residues, and 1 to 5 amino acid residues have been added is also hIL-10
- a wild-type hIL-10 amino acid sequence shown in SEQ ID NO:24 in which 1 to 3 amino acid residues have been deleted hIL-10 also includes the wild-type hIL-10 amino acid sequence shown in SEQ ID NO:24 in which one or two amino acid residues have been deleted, one
- hIL-10 also includes the wild-type hIL-10 amino acid sequence shown in SEQ ID NO:24 in which one amino acid residue has been deleted, one amino acid residue has been replaced with other amino acid residue, and one amino acid residue has been added. The same applies to IL-10 from animal species other than humans.
- each mutation in the hIL-10 mutant compared to normal wild-type hIL-10 can be easily confirmed by aligning the amino acid sequences of both hIL-10. The same is true for IL-10 from animal species other than humans.
- the amino acid sequence of the hIL-10 mutant preferably has 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, to the amino acid sequence of normal wild-type hIL-10 shown in SEQ ID NO: 24.
- substitution of an amino acid in the amino acid sequence of a wild-type human cytokine with another amino acid is preferably conservative amino acid substitutions.
- substitution of an amino acid in the amino acid sequence of a wild-type human cytokine with another amino acid is preferably conservative amino acid substitutions.
- substitution of an amino acid in the amino acid sequence of a wild-type human cytokine with another amino acid are preferably conservative amino acid substitutions.
- the above wild-type or mutant human cytokines in which the constituent amino acids are modified with sugar chains are also human cytokines.
- the above wild-type or mutant human cytokines in which the constituent amino acids are modified with phosphate are also human cytokines. Those modified with something other than sugar chains and phosphate are also human cytokines.
- the above wild-type or mutant human cytokines in which the side chains of the constituent amino acids have been converted by substitution reactions or the like are also human cytokines.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine.
- human cytokines modified with sugar chains are included in human cytokines with the original amino acid sequence.
- Human cytokines modified with phosphate are included in human cytokines with the original amino acid sequence.
- Cytokines modified with substances other than sugar chains and phosphate are also included in human cytokines with the original amino acid sequence.
- Cytokines in which the side chains of the amino acids that make up human cytokines have been changed by substitution reactions or the like are also included in human cytokines with the original amino acid sequence.
- Such changes include, but are not limited to, the conversion of cysteine residues to formylglycine.
- a wild-type human cytokine is produced as a recombinant protein fused with HSA.
- a fusion protein is expressed as a recombinant protein using a host cell such as CHO, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium
- the amount of expression of the human cytokine in the culture supernatant can be at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, compared to when the wild-type human cytokine is expressed as a recombinant protein by a similar method. For example, 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, 3 to 4 times, etc.
- hIL-10 is produced as a recombinant protein fused with HSA.
- a fusion protein is expressed as a recombinant protein using a host cell such as CHO, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium
- the amount of expression of hIL-10 in the culture supernatant can be at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, compared to when wild-type hIL-10 is expressed as a recombinant protein by a similar method. For example, 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, 3 to 4 times, etc.
- one embodiment of the present invention is a fusion protein in which a polypeptide containing the amino acid sequence of a cytokine is bound to a polypeptide containing the amino acid sequence of an SA.
- binding polypeptides refers to binding different polypeptides by covalent bonding, either directly or indirectly via a linker.
- the cytokine and SA are preferably of human origin.
- Another embodiment of the present invention is a fusion protein in which a polypeptide containing the amino acid sequence of IL-10 is bound to a polypeptide containing the amino acid sequence of SA.
- binding polypeptides refers to binding different polypeptides by covalent bonding, either directly or indirectly via a linker.
- IL-10 and SA are preferably of human origin.
- a common method for linking two different polypeptides is, for example, to create an expression vector incorporating a DNA fragment in which a gene encoding one polypeptide is linked in-frame downstream of a gene encoding the other polypeptide, and then to express the resulting protein as a recombinant protein by culturing a host cell transformed with this expression vector.
- the resulting recombinant protein is a single-chain polypeptide in which the two polypeptides are peptide-linked, either directly or via different amino acid sequences.
- SA-human cytokine fusion protein or "SA-human cytokine” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of a human cytokine is linked to the C-terminus of the amino acid sequence of SA, either directly or via a linker, and which has the function of a human cytokine.
- the animal species of the SA is not particularly limited, but it is preferably a mammalian SA, more preferably a primate SA, and even more preferably HSA.
- a human cytokine in an SA-human cytokine fusion protein has the function of a human cytokine
- the human cytokine has a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of a normal wild-type human cytokine is taken as 100%.
- the specific activity of the human cytokine in the SA-human cytokine fusion protein is calculated by multiplying the physiological activity of the human cytokine per unit mass of the fusion protein by (the molecular weight of the fusion protein/the molecular weight of the portion of the fusion protein corresponding to the human cytokine).
- the term "HSA-human cytokine fusion protein" or "HSA-human cytokine” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of a human cytokine is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of HSA, and which has the function of a human cytokine.
- HSA-human cytokine fusion protein has the function of a human cytokine
- the definition of the SA-human cytokine fusion protein described above can be applied.
- the SA has a function as an SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- the HSA-human lysosomal enzyme fusion protein it is preferable that the SA has a function as an SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- the HSA-human lysosomal enzyme fusion protein is preferable that the SA has a function as an SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- the HSA-human lysosomal enzyme fusion protein it is preferable that the SA has a function as an SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- the HSA-human lysosomal enzyme fusion protein is preferable that
- mutations When mutations are added to an HSA-human cytokine fusion protein, which is a fusion protein of wild-type HSA and wild-type human cytokine, mutations can be added only to the HSA portion and not to the human cytokine portion, mutations can be added only to the human cytokine portion without mutations to the HSA portion, or mutations can be added to both the HSA portion and the human cytokine portion.
- the amino acid sequence of the portion is the amino acid sequence of the wild-type HSA with a mutation added.
- mutations are added only to the human cytokine portion, the amino acid sequence of the portion is the amino acid sequence of the wild-type human cytokine with a mutation added.
- the amino acid sequence of the HSA portion is the amino acid sequence of the wild-type HSA with a mutation added
- the amino acid sequence of the human cytokine portion is the amino acid sequence of the wild-type human cytokine with a mutation added. The same can be said for fusion proteins of wild-type SA of animal species other than humans and human cytokine.
- HSA-human cytokine fusion proteins in which the amino acids constituting the protein are modified with sugar chains are also HSA-human cytokine fusion proteins.
- HSA-human cytokine fusion proteins in which the amino acids constituting the protein are modified with phosphate are also HSA-human cytokine fusion proteins.
- HSA-human cytokine fusion proteins modified with something other than sugar chains and phosphate are also HSA-human cytokine fusion proteins.
- HSA-human cytokine fusion proteins in which the side chains of the amino acids constituting the protein have been converted by substitution reactions or the like are also HSA-human cytokine fusion proteins.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said for fusion proteins of SA of animal species other than humans and human cytokines (SA-human cytokine).
- HSA-human cytokine fusion proteins modified with sugar chains are included in HSA-human cytokine fusion proteins having the original amino acid sequence.
- HSA-human cytokine fusion proteins modified with phosphate are included in HSA-human cytokine fusion proteins having the original amino acid sequence.
- those modified with things other than sugar chains and phosphate are also included in HSA-human cytokine fusion proteins having the original amino acid sequence.
- those in which the side chains of the amino acids constituting the HSA-human cytokine fusion proteins have been converted by substitution reactions or the like are also included in HSA-human cytokine fusion proteins having the original amino acid sequence.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine.
- SA-human cytokines The same can be said for fusion proteins of SA of other animals and human cytokines (SA-human cytokines).
- SA-human cytokines The same can be said for fusion proteins of SA of animal species other than humans and human cytokines (SA-human cytokines).
- HSA-human cytokine fusion proteins in which the human cytokine that constitutes it is a precursor of the human cytokine are also HSA-human cytokine fusion proteins.
- the term "precursor” here refers to a type that is biosynthesized as an HSA-human cytokine fusion protein, and after the biosynthesis, the part that functions as a human cytokine is separated from the fusion protein during the manufacturing process or in the living body when administered to the living body, and can function as a human cytokine by itself.
- the HSA-human cytokine fusion protein after the HSA-human cytokine fusion protein is biosynthesized, it may be cleaved at a specific site by a hydrolase or the like, and the part containing HSA and the part containing the mature human cytokine may be separated.
- the human cytokine obtained is not a fusion protein with HSA, but the HSA-human cytokine fusion protein is synthesized once during the process of manufacturing the cytokine. Therefore, when human cytokine is manufactured by such a method, the manufacturing method is included in the manufacturing method of HSA-human cytokine.
- SA-human cytokine a fusion protein of SA of an animal species other than human and a human cytokine
- MSA-mouse cytokine fusion protein a fusion protein of SA of an animal species other than human and a human cytokine of an animal species other than human
- SA-hIL-10 fusion protein or "SA-hIL-10” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of hIL-10 is linked to the C-terminus of the amino acid sequence of SA, either directly or via a linker, and which has the function of hIL-10.
- SA animal species of the SA, but it is preferably a mammalian SA, more preferably a primate SA, and even more preferably HSA.
- hIL-10 in the SA-hIL-10 fusion protein has the function of hIL-10
- hIL-10 retains a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of normal wild-type hIL-10 is taken as 100%.
- the specific activity of hIL-10 in the SA-hIL-10 fusion protein is calculated by multiplying the physiological activity of hIL-10 per unit mass of the fusion protein by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein corresponding to hIL-10).
- the term "HSA-hIL-10 fusion protein" or "HSA-hIL-10” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of hIL-10 is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of HSA, and which has the function of hIL-10.
- HSA-hIL-10 fusion protein has the function of hIL-10
- the definition of the SA-hIL-10 fusion protein described above can be applied.
- the SA has the function of SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but this is not limited to this.
- a preferred HSA-hIL-10 fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 50.
- the HSA-hIL-10 fusion protein shown in SEQ ID NO: 50 is a protein in which wild-type hIL-10 is directly bound to the C-terminus of wild-type HSA.
- the HSA-hIL-10 fusion protein shown in SEQ ID NO: 50 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 51.
- HSA-hIL-10 fusion proteins also include those having an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 50 have been replaced with other amino acid residues, deleted, or mutated by addition, as long as they have the function of hIL-10. It is preferable that the HSA-hIL-10 fusion protein has the function of human serum albumin, such as the function of binding and transporting endogenous substances in blood and exogenous substances such as drugs, but is not limited thereto.
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:50 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:50.
- mutations When mutations are added to an HSA-hIL-10 fusion protein, which is a fusion protein of wild-type HSA and wild-type hIL-10, mutations can be added only to the HSA portion and not to the hIL-10 portion, mutations can be added only to the hIL-10 portion without mutations to the HSA portion, or mutations can be added to both the HSA portion and the hIL-10 portion.
- mutations When mutations are added only to the HSA portion, the amino acid sequence of the portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above.
- the amino acid sequence of the portion is the amino acid sequence of hIL-10 obtained by adding a mutation to the wild-type hIL-10 described above.
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above
- the amino acid sequence of the hIL-10 portion is the amino acid sequence of hIL-10 obtained by adding a mutation to the wild-type hIL-10 described above.
- the HSA-hIL-10 fusion protein having the amino acid sequence shown in SEQ ID NO: 50.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- an amino acid residue is added, one or more amino acid residues are added to the amino acid sequence shown in SEQ ID NO:50 or to the N-terminus or C-terminus of the amino acid sequence.
- the HSA-hIL-10 fusion protein may be a combination of these substitutions, deletions, and additions of amino acid residues.
- the mutation may be made only to the HSA part, only to the hIL-10 part, or to both parts.
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:50 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:50.
- HSA-hIL-10 fusion proteins in which the amino acids constituting the protein are modified with sugar chains are also HSA-hIL-10 fusion proteins.
- HSA-hIL-10 fusion proteins in which the amino acids constituting the protein are modified with phosphate are also HSA-hIL-10 fusion proteins.
- HSA-hIL-10 fusion proteins modified with anything other than sugar chains and phosphate are also HSA-hIL-10 fusion proteins.
- HSA-hIL-10 fusion proteins in which the side chains of the amino acids constituting the protein are changed by substitution reactions, etc. Examples of such changes include, but are not limited to, the conversion of cysteine residues to formylglycine.
- SA-hIL-10 The same can be said for fusion proteins of SA and hIL-10 of other animals (SA-hIL-10).
- SA-hIL-10 The same can be said for fusion proteins of SA and hIL-10 of animal species other than humans
- HSA-hIL-10 fusion proteins modified with sugar chains are included in HSA-hIL-10 fusion proteins having the original amino acid sequence.
- HSA-hIL-10 fusion proteins modified with phosphate are included in HSA-hIL-10 fusion proteins having the original amino acid sequence.
- those modified with something other than sugar chains and phosphate are included in HSA-hIL-10 fusion proteins having the original amino acid sequence.
- HSA-hIL-10 fusion proteins in which the side chains of the amino acids constituting the HSA-hIL-10 fusion proteins have been changed by substitution reactions or the like are included in HSA-hIL-10 fusion proteins having the original amino acid sequence.
- Such changes include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said for fusion proteins of SA and hIL-10 of animal species other than humans (SA-hIL-10).
- HSA-hIL-10 fusion proteins in which the constituent hIL-10 is a precursor of hIL-10 are also HSA-hIL-10 fusion proteins.
- precursor refers to a type that is biosynthesized as an HSA-hIL-10 fusion protein, and after the biosynthesis, the part that functions as hIL-10 is cleaved from the fusion protein during the manufacturing process or in the living body when administered to the living body, and can function as hIL-10 by itself.
- the HSA-hIL-10 fusion protein after the HSA-hIL-10 fusion protein is biosynthesized, it may be cleaved at a specific site by a hydrolase or the like, and the part containing HSA and the part containing mature hIL-10 may be separated.
- the obtained hIL-10 is not a fusion protein with HSA, but the HSA-hIL-10 fusion protein is synthesized once during the process of synthesizing the hIL-10. Therefore, when hIL-10 is produced by such a method, the production method is included in the method for producing HSA-hIL-10 fusion protein. The same can be said about the fusion protein of SA and hIL-10 from non-human animal species (SA-hIL-10).
- human cytokine-SA fusion protein or "human cytokine-SA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of SA is linked, directly or via a linker, to the C-terminus of the amino acid sequence of a human cytokine, and which has a function as a human cytokine.
- a human cytokine in a human cytokine-SA fusion protein When a human cytokine in a human cytokine-SA fusion protein is said to have a function as a human cytokine, it means that the human cytokine retains a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of a normal wild-type human cytokine is taken as 100%.
- the specific activity of the human cytokine in the human cytokine-SA fusion protein is calculated by multiplying the physiological activity of the human cytokine per unit mass of the fusion protein by (the molecular weight of the fusion protein/the molecular weight of the portion of the fusion protein corresponding to the human cytokine).
- human cytokine-HSA fusion protein or "human cytokine-HSA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of HSA is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of a human cytokine, and which has the function of a human cytokine.
- the definition of human cytokine-SA above can be adopted.
- the SA has a function as an SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- the same is true for the human cytokine-HSA fusion protein.
- mutations When mutations are introduced into a human cytokine-HSA fusion protein, which is a fusion protein of a wild-type human cytokine and a wild-type HSA, mutations can be introduced only into the human cytokine portion and not into the HSA portion, mutations can be introduced only into the HSA portion without mutations in the human cytokine portion, or mutations can be introduced into both the human cytokine portion and the HSA portion.
- the amino acid sequence of the portion is the amino acid sequence of the human cytokine obtained by mutating the wild-type human cytokine described above.
- the amino acid sequence of the portion is the amino acid sequence of the HSA obtained by mutating the wild-type HSA described above.
- the amino acid sequence of the human cytokine portion is the amino acid sequence of the human cytokine obtained by mutating the wild-type human cytokine described above
- the amino acid sequence of the HSA portion is the amino acid sequence of the HSA obtained by mutating the wild-type HSA described above. The same can be said about a fusion protein between a wild-type human cytokine and the wild-type SA of an animal species other than human.
- a human cytokine-HSA fusion protein in which the amino acids constituting the protein are modified with sugar chains is also a human cytokine-HSA fusion protein.
- a human cytokine-HSA fusion protein in which the amino acids constituting the protein are modified with phosphate is also a human cytokine-HSA fusion protein.
- a human cytokine-HSA fusion protein modified with something other than sugar chains and phosphate is also a human cytokine-HSA fusion protein.
- a human cytokine-HSA fusion protein in which the side chains of the amino acids constituting the protein have been modified by a substitution reaction or the like is also a human cytokine-HSA fusion protein.
- Such a conversion includes, but is not limited to, the conversion of a cysteine residue to formylglycine. The same can be said about a fusion protein of a human cytokine and SA of an animal species other than human (human cytokine-SA).
- a human cytokine-HSA fusion protein modified with a sugar chain is included in the human cytokine-HSA fusion protein having the original amino acid sequence.
- a human cytokine-HSA fusion protein modified with phosphate is included in the human cytokine-HSA fusion protein having the original amino acid sequence.
- a human cytokine-HSA fusion protein modified with something other than a sugar chain or phosphate is also included in the human cytokine-HSA fusion protein having the original amino acid sequence.
- a human cytokine-HSA fusion protein in which the side chains of the amino acids constituting the human cytokine-HSA fusion protein have been changed by a substitution reaction or the like is also included in the human cytokine-HSA fusion protein having the original amino acid sequence.
- Such a change includes, but is not limited to, the change of a cysteine residue to formylglycine. The same can be said about a fusion protein of a human cytokine and SA of an animal species other than human (human cytokine-SA).
- a human cytokine-HSA fusion protein in which the constituent human cytokine is a precursor of a human cytokine is also a human cytokine-HSA fusion protein.
- the same can be said for a fusion protein of a human cytokine and SA of an animal species other than human (human cytokine-SA).
- hIL-10-SA fusion protein or "hIL-10-SA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of SA is linked, directly or via a linker, to the C-terminus of the amino acid sequence of hIL-10, and which has the function of hIL-10.
- hIL-10 in the hIL-10-SA fusion protein is said to have the function of hIL-10, it means that hIL-10 retains a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of normal wild-type hIL-10 is taken as 100%.
- the specific activity of hIL-10 in the hIL-10-SA fusion protein is calculated by multiplying the physiological activity of hIL-10 per unit mass of the fusion protein by (the molecular weight of the fusion protein/the molecular weight of the portion of the fusion protein corresponding to hIL-10).
- hIL-10-HSA fusion protein or "hIL-10-HSA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of HSA is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of hIL-10, and which has the function of hIL-10.
- the definition of hIL-10-SA above can be applied.
- the SA has the function of SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but this is not limited to this.
- a preferred hIL-10-HSA fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 52.
- the hIL-10-HSA fusion protein shown in SEQ ID NO: 52 is a fusion protein in which wild-type HSA is directly bound to the C-terminus of wild-type hIL-10.
- the hIL-10-HSA fusion protein shown in SEQ ID NO: 52 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 53.
- the hIL-10-HSA fusion protein also includes an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 52 are replaced with other amino acid residues, deleted, or mutated by addition, as long as it has the function of hIL-10.
- the hIL-10-HSA fusion protein is preferably one that has the function of human serum albumin, such as the function of binding and transporting endogenous substances in blood and exogenous substances such as drugs, but is not limited thereto.
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:52 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:52.
- mutations When mutations are introduced into the hIL-10-HSA fusion protein, which is a fusion protein of wild-type hIL-10 and wild-type HSA, mutations can be introduced only in the HSA portion and not in the hIL-10 portion, mutations can be introduced only in the hIL-10 portion without mutations in the HSA portion, or mutations can be introduced in both the HSA portion and the hIL-10 portion.
- the amino acid sequence of the portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above.
- the amino acid sequence of the portion is the amino acid sequence of hIL-10 obtained by adding a mutation to the wild-type hIL-10 described above.
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above
- the amino acid sequence of the hIL-10 portion is the amino acid sequence of hIL-10 obtained by adding a mutation to the wild-type hIL-10 described above.
- the hIL-10-HSA fusion protein having the amino acid sequence shown in SEQ ID NO: 52.
- the same is true for the fusion protein of wild-type hIL-10 and wild-type SA (hIL-10-SA) of an animal species other than human.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- an amino acid residue is added, one or more amino acid residues are added to the amino acid sequence shown in SEQ ID NO:52 or to the N-terminus or C-terminus of the amino acid sequence.
- the hIL-10-HSA fusion protein may be a combination of these substitutions, deletions, and additions of amino acid residues.
- the mutation may be made only to the HSA portion, only to the hIL-10 portion, or both portions.
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:52 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:52.
- hIL-10-HSA fusion proteins in which the amino acids constituting the fusion protein are modified with sugar chains are also hIL-10-HSA fusion proteins.
- hIL-10-HSA fusion proteins in which the amino acids constituting the fusion protein are modified with phosphate are also hIL-10-HSA fusion proteins.
- hIL-10-HSA fusion proteins modified with anything other than sugar chains and phosphate are also hIL-10-HSA fusion proteins.
- hIL-10-HSA fusion proteins in which the side chains of the amino acids constituting the fusion protein are changed by a substitution reaction or the like are also hIL-10-HSA fusion proteins.
- Such changes include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said about fusion proteins of SA and hIL-10 of animal species other than humans (SA-hIL-10).
- hIL-10-HSA fusion proteins modified with sugar chains are included in the hIL-10-HSA fusion proteins having the original amino acid sequence.
- hIL-10-HSA fusion proteins modified with phosphate are included in the hIL-10-HSA fusion proteins having the original amino acid sequence.
- those modified with something other than sugar chains and phosphate are also included in the hIL-10-HSA fusion proteins having the original amino acid sequence.
- those in which the side chains of the amino acids constituting the hIL-10-HSA fusion protein have been changed by a substitution reaction or the like are also included in the hIL-10-HSA fusion proteins having the original amino acid sequence.
- Such changes include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said for fusion proteins of SA and hIL-10 of animal species other than humans (SA-hIL-10).
- a hIL-10-HSA fusion protein in which the constituent hIL-10 is a precursor of hIL-10 is also a hIL-10-HSA fusion protein.
- SA and hIL-10 of a non-human animal species SA-hIL-10
- SA-hIL-10 a fusion protein of SA and hIL-10 of a non-human animal species
- SA-hIL-10 a fusion protein of SA and IL-10 of a non-human animal species
- a fusion protein of SA and IL-10 of a non-human animal species such as a fusion protein of MSA-mouse IL-10.
- the terms “HSA and human cytokine fusion protein”, “human cytokine and HSA fusion protein”, or “human serum albumin and human cytokine fusion protein” include both the above-mentioned “HSA-human cytokine fusion protein” and “human cytokine HSA fusion protein”.
- the terms “HSA and hIL-10 fusion protein”, “hIL-10 and HSA fusion protein”, or “human serum albumin and human cytokine 10 fusion protein” include both the above-mentioned "HSA-hIL-10 fusion protein” and "hIL-10-HSA fusion protein”.
- fusion protein of HSA and hIL-10 The manufacturing method and other details of the fusion protein of HSA and hIL-10 are described below, but these descriptions can also be applied when the cytokine is other than IL-10, when the SA is from an animal species other than human, or when the cytokine is from an animal species other than human. For example, they also apply to a fusion protein of SA of an animal species other than human and hIL-10, and a fusion protein of SA of an animal species other than human and IL-10 of an animal species other than human.
- the SA and the cytokine are linked directly or via a linker.
- linker refers to a portion that does not belong to either the amino acid sequence of the SA or the amino acid sequence of the cytokine.
- the linker is a peptide chain that is interposed between the SA and the cytokine.
- the linker has various functions.
- These functions include a function between the SA and the cytokine to link the SA and the cytokine, a function to reduce mutual interference between the SA and the cytokine by increasing the distance between them within the fusion protein molecule, and a function between the SA and the cytokine to act as a hinge that connects the SA and the cytokine and gives flexibility to the three-dimensional structure of the fusion protein.
- the linker exerts at least one of these functions. This is the same, for example, whether the SA is HSA or whether the cytokine is a human cytokine, such as hIL-10.
- the amino acid sequence of the peptide linker is not particularly limited as long as it functions as a linker in the fusion protein molecule.
- the length of the peptide linker is also not particularly limited as long as it functions as a linker in the fusion protein molecule.
- the peptide linker is composed of one or more amino acids. When the peptide linker is composed of multiple amino acids, the number of amino acids is preferably 2 to 50, more preferably 5 to 30, and even more preferably 10 to 25.
- Suitable examples of peptide linkers include those consisting of Gly-Ser, Gly-Gly-Ser, or amino acid sequences shown in SEQ ID NOs: 9 to 11 (collectively referred to as basic sequences), and those containing these.
- the peptide linker is one that contains an amino acid sequence in which the basic sequence is repeated 2 to 10 times, one that contains an amino acid sequence in which the basic sequence is repeated 2 to 6 times, or one that contains an amino acid sequence in which the basic sequence is repeated 3 to 5 times.
- One or more amino acids in these amino acid sequences may be deleted, replaced with other amino acids, added, etc. When deleting an amino acid, the number of amino acids to be deleted is preferably 1 or 2.
- the number of amino acids to be replaced is preferably 1 or 2.
- the number of amino acids to be added is preferably 1 or 2.
- the amino acid sequence of the desired linker portion can be obtained by combining these deletions, substitutions, and additions of amino acids.
- the peptide linker may be composed of one amino acid, and the amino acid that constitutes the linker is, for example, glycine or serine.
- the fusion protein of HSA and hIL-10 can be produced as a recombinant protein by creating an expression vector incorporating a DNA fragment in which a gene encoding hIL-10 is linked in-frame to the downstream or upstream of a gene encoding HSA, and then culturing a host cell transformed with this expression vector.
- the fusion protein produced as a recombinant protein in this way consists of a single polypeptide chain.
- a gene encoding hIL-10 When producing a fusion protein as a recombinant fusion protein, a gene encoding hIL-10 can be linked in-frame downstream of a gene encoding HSA to obtain an HSA-hIL-10 fusion protein having the amino acid sequence of hIL-10 at the C-terminus of the amino acid sequence of HSA. Conversely, a gene encoding hIL-10 can be linked in-frame upstream of a gene encoding HSA to obtain an hIL-10-HSA fusion protein having the amino acid sequence of hIL-10 at the N-terminus of the amino acid sequence of HSA. In either case, the fusion protein produced as a recombinant fusion protein is a single-chain polypeptide.
- FIG. 5 shows a schematic diagram of an HSA-hIL-10 fusion protein, a single-chain polypeptide having HSA, a linker, and hIL-10 in that order from the N-terminus.
- the C-terminus of HSA is bound to the N-terminus of the linker by a peptide bond
- the C-terminus of the linker is bound to the N-terminus of hIL-10 by a peptide bond.
- FIG. 6 shows a schematic diagram of a hIL-10-HSA fusion protein, which is a single-chain polypeptide having hIL-10, a linker, and HSA in that order from the N-terminus.
- the C-terminus of hIL-10 is bound to the N-terminus of the linker by a peptide bond, and the C-terminus of the linker is bound to the N-terminus of HSA by a peptide bond.
- the fusion protein of HSA and hIL-10 refers to a fusion protein characterized in that when expressed in a host cell as a recombinant protein, particularly when expressed so that the recombinant protein is secreted from the cells and accumulated in the culture medium, the amount of expression of hIL-10 in the culture supernatant is at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, compared to when wild-type hIL-10 is expressed in a host cell as a recombinant protein under the same conditions.
- "under the same conditions” means that the expression vector, host cell, culture conditions, etc. are the same.
- the preferred host cells used in this case are mammalian cells such as CHO cells and NS/0 cells, but particularly CHO cells.
- Preferred embodiments of the fusion protein of HSA and hIL-10 include the following (1) to (4): (1) A substance having an amino acid sequence shown in SEQ ID NO:50, in which the N-terminus of the amino acid sequence of wild-type hIL-10 shown in SEQ ID NO:24 is directly linked to the C-terminus of the amino acid sequence of wild-type HSA shown in SEQ ID NO:3; (2) Having the amino acid sequence shown in SEQ ID NO:52, in which the N-terminus of the amino acid sequence of wild-type HSA shown in SEQ ID NO:3 is directly linked to the C-terminus of the amino acid sequence of wild-type hIL-10 shown in SEQ ID NO:24.
- preferred embodiments of the fusion protein of HSA and hIL-10 include human serum albumin (HSA-A320T) consisting of 585 amino acids as shown in SEQ ID NO:13, in which the 320th amino acid residue from the N-terminus of the amino acid sequence of wild-type HSA as shown in SEQ ID NO:3 is replaced with threonine, and further include the following (5) to (8): (5) A peptide having an amino acid sequence shown in SEQ ID NO:56, in which the N-terminus of the amino acid sequence of wild-type hIL-10 shown in SEQ ID NO:24 is directly linked to the C-terminus of the amino acid sequence of human serum albumin (HSA-A320T) shown in SEQ ID NO:13; (6) The C-terminus of the amino acid sequence of wild-type hIL-10 shown in SEQ ID NO:24 is directly linked to the N-terminus of the amino acid sequence of human serum albumin (HSA-A320T) shown in SEQ ID NO:13, and the overall amino
- the fusion protein of HSA and hIL-10 is characterized in that when expressed in a host cell as a recombinant protein, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the amount of hIL-10 expressed in the culture supernatant is increased in terms of concentration or physiological activity, compared to when wild-type hIL-10 is expressed in a host cell as a recombinant protein under the same conditions. Therefore, when produced as a recombinant protein, the fusion protein of HSA and hIL-10 can increase the production efficiency compared to wild-type hIL-10, and therefore can reduce production costs.
- fusion protein of HSA and hIL-10 expressed in a host cell as a recombinant protein with wild-type hIL-10 expressed in a host cell under the same conditions as a recombinant protein
- the comparison is not based on the mass of the expressed protein, but on the number of molecules of the expressed protein or the hIL-10 physiological activity.
- fusion proteins of SA and hIL-10 from animal species other than human, and fusion proteins of SA and other cytokines.
- the fusion protein of HSA and hIL-10 can be produced using an expression vector, host cell, medium, etc. that can be used to produce the above-mentioned fusion protein of HSA and hGALC or the fusion protein of HSA and hGBA. The same can be said for fusion proteins of SA and cytokines in general. is preferred
- the fusion protein of HSA and hIL-10 is characterized in that when host cells into which an expression vector incorporating a gene encoding this protein has been introduced are cultured in the above-mentioned serum-free medium to be expressed as a recombinant protein, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the amount of expression of hIL-10 in the culture supernatant is at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, compared to when wild-type hIL-10 is expressed as a recombinant protein in host cells under the same conditions.
- the expression vector, host cells, culture conditions, etc. are the same.
- the host cells used in this case are preferably mammalian cells, particularly ordinary cells used for producing recombinant proteins such as CHO cells and NS/0 cells.
- Wild-type hIL-10 tends to have a low expression level when expressed as a recombinant using host cells transformed with an expression vector incorporating a gene encoding it, making it difficult to efficiently mass-produce it as a recombinant.
- the expression level of the recombinant can be increased.
- the fusion protein of HSA and hIL-10 can be expressed more efficiently than when wild-type hIL-10 is expressed as a recombinant protein under the same conditions, making it suitable for mass production.
- the host cells used to express the fusion protein are preferably mammalian cells, especially ordinary cells used for producing recombinant proteins such as CHO cells and NS/0 cells. That is, one embodiment of the present invention is a recombinant fusion protein of SA and IL-10, particularly a recombinant fusion protein of HSA and hIL-10.
- the fusion protein of HSA and hIL-10 By culturing host cells encoding the fusion protein of HSA and hIL-10, it can be expressed in cells or in the medium.
- the fusion protein of HSA and hIL-10 can be purified by separating it from impurities using methods such as column chromatography.
- the purified fusion protein of HSA and hIL-10 can be used as a pharmaceutical composition.
- the fusion protein of HSA and hIL-10 can be used as a pharmaceutical composition targeting inflammatory diseases or cancer.
- a pharmaceutical composition containing a fusion protein of HSA and hIL-10 as an active ingredient can be administered intravenously, intramuscularly, intraperitoneally, subcutaneously, or intracerebroventricularly as an injection.
- injections can be supplied as lyophilized preparations or aqueous liquid preparations.
- an aqueous liquid preparation it may be in the form of a vial filled with the drug, or it can be supplied as a prefilled preparation in a syringe.
- a lyophilized preparation it is dissolved in an aqueous medium and reconstituted before use.
- the fusion protein of HSA and hIL-10 can further be conjugated with an antibody or a ligand.
- the fusion protein of HSA and hIL-10 can be conjugated with an antibody or a ligand capable of specifically binding to a receptor on cerebrovascular endothelial cells.
- the fusion protein of HSA and hIL-10 or the fusion protein of HSA and hGBA can bind to a receptor on cerebrovascular endothelial cells by being conjugated with an antibody or a ligand capable of specifically binding to a receptor on cerebrovascular endothelial cells.
- the fusion protein bound to the receptor on cerebrovascular endothelial cells can pass through the blood-brain barrier (BBB) and reach the tissues of the central nervous system (CNS). Therefore, the fusion protein of HSA and hIL-10 can be conjugated with such an antibody or ligand to pass through the blood-brain barrier (BBB) and exert its function in the central nervous system (CNS).
- BBB blood-brain barrier
- CNS central nervous system
- hIL-10 in a conjugate of an antibody and a fusion protein of HSA and hIL-10 is said to have the function of hIL-10, it means that, when the specific activity of normal wild-type hIL-10 is taken as 100%, hIL-10 has a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, for example 90% or more, 95% or more.
- the specific activity of hIL-10 in the conjugate is calculated by multiplying the physiological activity of hIL-10 per unit mass of the conjugate by (the molecular weight of the conjugate/the molecular weight of the portion of the conjugate corresponding to hIL-10).
- a conjugate of an antibody and a fusion protein of HSA and hIL-10 refers to a conjugate characterized in that when the conjugate is expressed as a recombinant protein in a host cell, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the amount of expression of hIL-10 in the culture supernatant is at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, compared to when wild-type hIL-10 is expressed as a recombinant protein in a host cell under the same conditions.
- neurotrophic factors are relatively difficult to produce in large quantities when expressed as recombinant proteins using host cells introduced with an expression vector incorporating a gene encoding the wild-type neurotrophic factor, due to limited expression levels.
- neurotrophic factors include BDNF (brain-derived neurotrophic factor), NGF (nerve growth factor), NT-3 (neurotrophin-3), NT-4 (neurotrophin-4), and NT-5 (neurotrophin-5).
- BDNF brain-derived neurotrophic factor
- NGF nerve growth factor
- NT-3 neurotrophin-3
- NT-4 neurotrophin-4
- NT-5 neurotrophin-5
- the difference in structure between NT-4 and NT-5 is an interspecies variation, and since these are considered to be the same factor, they may be referred to as NT-4/5, etc., but in this specification, they will be referred to as NT-4.
- One embodiment of the present invention is a recombinant protein in which such a neurotrophic factor, which is relatively difficult to produce as a recombinant protein, is fused with SA.
- a recombinant protein can be produced in large quantities more easily using host cells introduced with an expression vector incorporating a gene encoding the recombinant protein, compared to the corresponding neurotrophic factor.
- SA neurotrophic factor
- there is no particular restriction on the animal species of the neurotrophic factor to be bound to SA but human neurotrophic factor is preferable.
- HSA is preferred.
- human neurotrophic factor when used, it includes not only normal wild-type human neurotrophic factor, but also mutant human neurotrophic factors in which one or more amino acid residues have been substituted, deleted, and/or added (in this specification, "addition" of an amino acid residue means adding a residue to the end or inside of the sequence) to the amino acid sequence of the wild-type human neurotrophic factor, as long as the mutant has the function of a human neurotrophic factor, such as having physiological activity corresponding to the type of human neurotrophic factor. The same applies to neurotrophic factors of animal species other than humans.
- a human neurotrophic factor has a function as a human neurotrophic factor
- the human neurotrophic factor has a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of a normal wild-type human neurotrophic factor is taken as 100%.
- specific activity refers to the physiological activity per mass of the protein.
- the specific activity of a fusion protein of a human neurotrophic factor and another protein is calculated as the physiological activity per mass of the portion of the fusion protein that corresponds to the human neurotrophic factor.
- the specific activity of the human neurotrophic factor in the fusion protein is calculated by multiplying the physiological activity of the human neurotrophic factor per unit mass of the fusion protein by (the molecular weight of the fusion protein/the molecular weight of the portion of the fusion protein that corresponds to the human neurotrophic factor).
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- a human neurotrophic factor mutant consisting of an amino acid sequence in which one amino acid residue is deleted from the N-terminus or C-terminus of wild-type human neurotrophic factor, a human neurotrophic factor mutant consisting of an amino acid sequence in which two amino acid residues are deleted from the N-terminus or C-terminus of wild-type human neurotrophic factor, etc. are also human neurotrophic factors.
- a mutation that combines the substitution and deletion of these amino acid residues can be added to the amino acid sequence of wild-type human neurotrophic factor. The same applies to neurotrophic factors of animal species other than humans.
- amino acid residues When adding amino acid residues to the amino acid sequence of wild-type human neurotrophic factor, one or more amino acid residues are added into the amino acid sequence of human neurotrophic factor or to the N-terminus or C-terminus of the amino acid sequence.
- the number of amino acid residues added in this case is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- Mutations that combine the addition of these amino acid residues with the above-mentioned substitutions can also be added to the amino acid sequence of wild-type human neurotrophic factor, and mutations that combine the addition of these amino acid residues with the above-mentioned deletions can also be added to the amino acid sequence of wild-type human neurotrophic factor. The same applies to neurotrophic factors of animal species other than humans.
- a combination of the above three types of mutations, substitution, deletion, and addition of amino acid residues can be added to the amino acid sequence of wild-type human neurotrophic factor.
- a human neurotrophic factor obtained by deleting 1 to 10 amino acid residues from the amino acid sequence of wild-type human neurotrophic factor, substituting 1 to 10 amino acid residues with other amino acid residues, and adding 1 to 10 amino acid residues is also a human neurotrophic factor
- a human neurotrophic factor obtained by deleting 1 to 5 amino acid residues from the amino acid sequence of wild-type human neurotrophic factor, substituting 1 to 5 amino acid residues with other amino acid residues, and adding 1 to 5 amino acid residues is also a human neurotrophic factor
- a human neurotrophic factor obtained by deleting 1 to 3 amino acid residues from the amino acid sequence of wild-type human neurotrophic factor, and adding 1 to 3 amino acid residues is also a human neurotrophic factor.
- Human neurotrophic factors are those in which an amino acid residue has been replaced with another amino acid residue and one to three amino acid residues have been added. Human neurotrophic factors are also those in which one or two amino acid residues have been deleted from the amino acid sequence of wild-type human neurotrophic factors, one or two amino acid residues have been replaced with another amino acid residue, and one or two amino acid residues have been added. Human neurotrophic factors are also those in which one amino acid residue has been deleted from the amino acid sequence of wild-type human neurotrophic factors, one amino acid residue has been replaced with another amino acid residue, and one amino acid residue has been added. The same applies to neurotrophic factors of animal species other than humans.
- each mutation in a human neurotrophic factor mutant compared to normal wild-type human neurotrophic factor can be easily confirmed by aligning the amino acid sequences of both human neurotrophic factors.
- the amino acid sequence of the human neurotrophic factor mutant preferably has 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, to the amino acid sequence of a normal wild-type human neurotrophic factor.
- substitution of an amino acid in the amino acid sequence of wild-type human neurotrophic factor with another amino acid is preferably a conservative amino acid substitution.
- the above wild-type or mutant human neurotrophic factors in which the constituent amino acids are modified with sugar chains are also human neurotrophic factors.
- the above wild-type or mutant human neurotrophic factors in which the constituent amino acids are modified with phosphate are also human neurotrophic factors.
- human neurotrophic factors modified with something other than sugar chains and phosphate are also human neurotrophic factors.
- the above wild-type or mutant human neurotrophic factors in which the side chains of the constituent amino acids have been converted by a substitution reaction or the like are also human neurotrophic factors.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same applies to neurotrophic factors of animal species other than humans.
- human neurotrophic factors modified with sugar chains are included in human neurotrophic factors with the original amino acid sequence.
- Human neurotrophic factors modified with phosphate are included in human neurotrophic factors with the original amino acid sequence.
- Human neurotrophic factors modified with things other than sugar chains and phosphate are also included in human neurotrophic factors with the original amino acid sequence.
- Human neurotrophic factors in which the side chains of the amino acids that make up human neurotrophic factors have been converted by substitution reactions or the like are also included in human neurotrophic factors with the original amino acid sequence. Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same applies to neurotrophic factors of animal species other than humans.
- human brain-derived neurotrophic factor when simply referring to "human brain-derived neurotrophic factor,” “human BDNF,” or “hBDNF,” it includes not only the normal wild-type hBDNF consisting of 119 amino acid residues as shown in SEQ ID NO: 60, but also includes, without any particular distinction, mutants of hBDNF in which one or more amino acid residues have been substituted, deleted, and/or added (as used herein, “addition” of an amino acid residue means adding a residue to the end or inside of the sequence) to the amino acid sequence as shown in SEQ ID NO: 60, as long as they have the functions of hBDNF, such as binding to the specific receptor TrkB on the surface of target cells and having physiological activity as a neurotrophic factor that promotes the development, growth, maintenance, and regeneration of nerve cells.
- Wild-type hBDNF is, for example, encoded by a gene having the base sequence as shown in SEQ ID NO: 61. The same applies to BDNF of animal species other than humans.
- hBDNF When hBDNF is said to have the function of hBDNF, it means that hBDNF preferably retains a specific activity of 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, of the specific activity of normal wild-type hBDNF taken as 100%.
- specific activity refers to the physiological activity per mass of protein.
- the specific activity of a fusion protein of hBDNF and another protein is determined as the physiological activity per mass of the portion of the fusion protein that corresponds to hBDNF.
- the specific activity of hBDNF in the fusion protein is calculated by multiplying the physiological activity of hBDNF per unit mass of the fusion protein by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein that corresponds to hBDNF). The same applies to BDNF of animal species other than humans.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- hBDNF mutants consisting of 118 amino acid residues obtained by deleting one amino acid residue at the N-terminus or C-terminus of wild-type hBDNF, and hBDNF mutants consisting of 117 amino acid residues obtained by deleting two amino acid residues at the N-terminus or C-terminus of wild-type hBDNF are also hBDNF. Mutations that combine the substitution and deletion of these amino acid residues can also be added to the amino acid sequence of wild-type hBDNF. The same applies to BDNF of animal species other than humans.
- amino acid residues When amino acid residues are added to the amino acid sequence of wild-type hBDNF, one or more amino acid residues are added into the amino acid sequence of hBDNF or to the N-terminus or C-terminus of the amino acid sequence.
- the number of amino acid residues added in this case is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2. Mutations that combine the addition of these amino acid residues with the above-mentioned substitutions can also be added to the amino acid sequence of wild-type hBDNF, and mutations that combine the addition of these amino acid residues with the above-mentioned deletions can also be added to the amino acid sequence of wild-type hBDNF.
- a combination of the above three types of mutations, substitution, deletion, and addition of amino acid residues can be added to the amino acid sequence of wild-type hBDNF.
- a wild-type hBDNF shown in SEQ ID NO:60 in which 1 to 10 amino acid residues have been deleted, 1 to 10 amino acid residues have been replaced with other amino acid residues, and 1 to 10 amino acid residues have been added is also hBDNF
- a wild-type hBDNF shown in SEQ ID NO:60 in which 1 to 5 amino acid residues have been deleted, 1 to 5 amino acid residues have been replaced with other amino acid residues, and 1 to 5 amino acid residues have been added is also hBDNF
- a wild-type hBDNF shown in SEQ ID NO:60 in which 1 to 3 amino acid residues have been deleted, hBDNF is also obtained by deleting one or two amino acid residues from the amino acid sequence of wild-type hBDNF shown in SEQ ID NO:60, substituting one or two amino acid residues
- hBDNF is also obtained by deleting one amino acid residue from the amino acid sequence of wild-type hBDNF shown in SEQ ID NO:60, substituting one amino acid residue with other amino acid residues, and adding one amino acid residue.
- SEQ ID NO:60 amino acid sequence of wild-type hBDNF shown in SEQ ID NO:60
- substituting one amino acid residue with other amino acid residues substituting one amino acid residue with other amino acid residues.
- adding one amino acid residue is also obtained by deleting one amino acid residue from the amino acid sequence of wild-type hBDNF shown in SEQ ID NO:60, substituting one amino acid residue with other amino acid residues, and adding one amino acid residue.
- each mutation in the hBDNF mutant compared to normal wild-type hBDNF can be easily confirmed by aligning the amino acid sequences of both hBDNFs. The same is true for BDNFs of animal species other than humans.
- the amino acid sequence of the hBDNF mutant preferably has 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, to the amino acid sequence of normal wild-type hBDNF shown in SEQ ID NO:60.
- human nerve growth factor when simply referring to "human nerve growth factor”, “human NGF” or “hNGF”, it includes, without distinction, not only the usual wild-type hNGF consisting of the 120 amino acid residues shown in SEQ ID NO: 62, but also mutants of hNGF in which one or more amino acid residues have been substituted, deleted and/or added (in this specification, “addition” of an amino acid residue means adding a residue to the end or inside of the sequence) to the amino acid sequence shown in SEQ ID NO: 62, as long as they have the functions of hNGF, such as the physiological activity as a neurotrophic factor, such as the action of promoting the extension of nerve axons and the synthesis of neurotransmitters, the action of maintaining nerve cells, the action of repairing damaged cells, and the action of promoting the recovery of brain nerve functions and preventing aging.
- Wild-type hNGF is, for example, encoded by a gene having the base sequence shown in SEQ ID NO: 63. The
- hNGF When hNGF is said to have the function of hNGF, it means that hNGF preferably retains a specific activity of 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, of the specific activity of normal wild-type hNGF taken as 100%.
- specific activity refers to the physiological activity per mass of protein.
- the specific activity of a fusion protein of hNGF and another protein is determined as the physiological activity per mass of the portion of the fusion protein that corresponds to hNGF.
- the specific activity of hNGF in the fusion protein is calculated by multiplying the physiological activity of hNGF per unit mass of the fusion protein by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein that corresponds to hNGF). The same applies to NGFs of animal species other than humans.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- hNGF mutants consisting of 119 amino acid residues obtained by deleting one amino acid residue at the N-terminus or C-terminus of wild-type hNGF, and hNGF mutants consisting of 118 amino acid residues obtained by deleting two amino acid residues at the N-terminus or C-terminus of wild-type hNGF, are also hNGF. Mutations that combine the substitution and deletion of these amino acid residues can also be added to the amino acid sequence of wild-type hNGF. The same applies to NGFs of animal species other than humans.
- amino acid residues When amino acid residues are added to the amino acid sequence of wild-type hNGF, one or more amino acid residues are added into the amino acid sequence of hNGF or to the N-terminus or C-terminus of the amino acid sequence.
- the number of amino acid residues added in this case is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- Mutations that combine the addition of these amino acid residues with the above-mentioned substitutions can also be added to the amino acid sequence of wild-type hNGF, and mutations that combine the addition of these amino acid residues with the above-mentioned deletions can also be added to the amino acid sequence of wild-type hNGF. The same applies to NGFs of animal species other than humans.
- a combination of the above three types of mutations, substitution, deletion, and addition of amino acid residues can be added to the amino acid sequence of wild-type hNGF.
- a wild-type hNGF shown in SEQ ID NO:62 in which 1 to 10 amino acid residues have been deleted, 1 to 10 amino acid residues have been replaced with other amino acid residues, and 1 to 10 amino acid residues have been added is also an hNGF;
- a wild-type hNGF shown in SEQ ID NO:62 in which 1 to 5 amino acid residues have been deleted, 1 to 5 amino acid residues have been replaced with other amino acid residues, and 1 to 5 amino acid residues have been added is also an hNGF;
- a wild-type hNGF shown in SEQ ID NO:62 in which 1 to 3 amino acid residues have been deleted, hNGF is also obtained by deleting one or two amino acid residues from the amino acid sequence of wild-type hNGF shown in SEQ ID NO:62, substituting one or two amino acid
- hNGF is also obtained by deleting one amino acid residue from the amino acid sequence of wild-type hNGF shown in SEQ ID NO:62, substituting one amino acid residue with other amino acid residues, and adding one amino acid residue.
- SEQ ID NO:62 the amino acid sequence of wild-type hNGF shown in SEQ ID NO:62
- substituting one amino acid residue with other amino acid residues substituting one amino acid residue with other amino acid residues.
- adding one amino acid residue is also obtained by deleting one amino acid residue from the amino acid sequence of wild-type hNGF shown in SEQ ID NO:62, substituting one amino acid residue with other amino acid residues, and adding one amino acid residue. The same applies to NGFs of animal species other than humans.
- each mutation in the hNGF mutant compared to normal wild-type hNGF can be easily confirmed by aligning the amino acid sequences of both hNGFs. The same is true for NGFs of animal species other than humans.
- the amino acid sequence of the hNGF mutant preferably has 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, to the amino acid sequence of normal wild-type hNGF shown in SEQ ID NO:62.
- human neurotrophin-3 when simply referring to "human neurotrophin-3", “human NT-3”, or “hNT-3”, it includes, without distinction, not only the usual wild-type hNT-3 consisting of the 119 amino acid residues shown in SEQ ID NO: 64, but also mutants of hNT-3 in which one or more amino acid residues have been substituted, deleted, and/or added (as used herein, “addition” of an amino acid residue means adding a residue to the end or inside of the sequence) to the amino acid sequence shown in SEQ ID NO: 64, as long as they have the functions of hNT-3, such as having physiological activity as a nerve growth factor that acts via TrkC and is necessary for maintaining the survival and promoting differentiation of nerve cells.
- Wild-type hNT-3 is, for example, encoded by a gene having the base sequence shown in SEQ ID NO: 65. The same applies to hNT-3 of animal species other than humans.
- hNT-3 When hNT-3 is said to have the function of hNT-3, it means that hNT-3 preferably retains a specific activity of 10% or more, more preferably a specific activity of 20% or more, even more preferably a specific activity of 50% or more, and even more preferably a specific activity of 80% or more, when the specific activity of normal wild-type hNT-3 is taken as 100%.
- specific activity refers to the physiological activity per mass of the protein.
- the specific activity of a fusion protein of hNT-3 and another protein is determined as the physiological activity per mass of the portion of the fusion protein that corresponds to hNT-3.
- the specific activity of hNT-3 in the fusion protein is calculated by multiplying the physiological activity of hNT-3 per unit mass of the fusion protein by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein that corresponds to hNT-3).
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- hNT-3 mutants consisting of 118 amino acid residues obtained by deleting one amino acid residue at the N-terminus or C-terminus of wild-type hNT-3, and hNT-3 mutants consisting of 117 amino acid residues obtained by deleting two amino acid residues at the N-terminus or C-terminus of wild-type hNT-3, etc. are also hNT-3. Mutations that combine the substitution and deletion of these amino acid residues can also be added to the amino acid sequence of wild-type hNT-3. The same applies to hNT-3 of animal species other than humans.
- amino acid residues When amino acid residues are added to the amino acid sequence of wild-type hNT-3, one or more amino acid residues are added into the amino acid sequence of hNT-3 or to the N-terminus or C-terminus of the amino acid sequence.
- the number of amino acid residues added in this case is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2. Mutations that combine the addition of these amino acid residues with the above-mentioned substitutions can also be added to the amino acid sequence of wild-type hNT-3, and mutations that combine the addition of these amino acid residues with the above-mentioned deletions can also be added to the amino acid sequence of wild-type hNT-3. The same applies to hNT-3 from animal species other than humans.
- a combination of the above three types of mutations, substitution, deletion, and addition of amino acid residues can be added to the amino acid sequence of wild-type hNT-3.
- a wild-type hNT-3 shown in SEQ ID NO:64 has 1 to 10 amino acid residues deleted, 1 to 10 amino acid residues replaced with other amino acid residues, and 1 to 10 amino acid residues added to it, which is also an hNT-3;
- a wild-type hNT-3 shown in SEQ ID NO:64 has 1 to 5 amino acid residues deleted, 1 to 5 amino acid residues replaced with other amino acid residues, and 1 to 5 amino acid residues added to it, which is also an hNT-3;
- a wild-type hNT-3 shown in SEQ ID NO:64 has 1 to 3 amino acid residues deleted, 1 to 10 amino acid residues replaced with other amino acid residues, and 1 to 10 amino acid residues added to it, which is also an hNT-3.
- hNT-3 also includes a wild-type hNT-3 amino acid sequence shown in SEQ ID NO:64 in which one or two amino acid residues have been deleted, one or two amino acid residues have been replaced with other amino acid residues, and one or two amino acid residues have been added; and hNT-3 also includes a wild-type hNT-3 amino acid sequence shown in SEQ ID NO:64 in which one amino acid residue has been deleted, one amino acid residue has been replaced with other amino acid residue, and one amino acid residue has been added. The same applies to hNT-3 from animal species other than humans.
- the amino acid sequence of the hNT-3 mutant preferably has 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, to the amino acid sequence of the normal wild-type hNT-3 shown in SEQ ID NO:64.
- human neurotrophin-4 when simply referring to "human neurotrophin-4,” “human NT-4,” or “hNT-4,” it includes, without distinction, not only the usual wild-type hNT-4 consisting of the 130 amino acid residues shown in SEQ ID NO: 66, but also mutants of hNT-4 in which one or more amino acid residues have been substituted, deleted, and/or added (as used herein, “addition” of an amino acid residue means adding a residue to the end or inside of the sequence) to the amino acid sequence shown in SEQ ID NO: 66, as long as they have the functions of hNT-4, such as having physiological activity as a nerve growth factor that acts via TrkB and is necessary for maintaining the survival and promoting differentiation of nerve cells.
- Wild-type hNT-4 is, for example, encoded by a gene having the base sequence shown in SEQ ID NO: 67. The same applies to hNT-4 of animal species other than humans.
- hNT-4 When hNT-4 is said to have the function of hNT-4, it means that hNT-4 preferably retains a specific activity of 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, of the specific activity of normal wild-type hNT-4 taken as 100%.
- specific activity refers to the physiological activity per mass of protein.
- the specific activity of a fusion protein of hNT-4 with another protein is determined as the physiological activity per mass of the portion of the fusion protein that corresponds to hNT-4.
- the specific activity of hNT-4 in the fusion protein is calculated by multiplying the physiological activity of hNT-4 per unit mass of the fusion protein by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein that corresponds to hNT-4). The same applies to hNT-4 from animal species other than humans.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- hNT-4 mutants consisting of 129 amino acid residues obtained by deleting one amino acid residue at the N-terminus or C-terminus of wild-type hNT-4, and hNT-4 mutants consisting of 128 amino acid residues obtained by deleting two amino acid residues at the N-terminus or C-terminus of wild-type hNT-4, etc. are also hNT-4. Mutations that combine the substitution and deletion of these amino acid residues can also be added to the amino acid sequence of wild-type hNT-4. The same applies to hNT-4 of animal species other than humans.
- amino acid residues When amino acid residues are added to the amino acid sequence of wild-type hNT-4, one or more amino acid residues are added into the amino acid sequence of hNT-4 or to the N-terminus or C-terminus of the amino acid sequence.
- the number of amino acid residues added in this case is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2. Mutations that combine the addition of these amino acid residues with the above-mentioned substitutions can also be added to the amino acid sequence of wild-type hNT-4, and mutations that combine the addition of these amino acid residues with the above-mentioned deletions can also be added to the amino acid sequence of wild-type hNT-4. The same applies to hNT-4 from animal species other than humans.
- a combination of the above three types of mutations, substitution, deletion, and addition of amino acid residues can be added to the amino acid sequence of wild-type hNT-4.
- a wild-type hNT-4 amino acid sequence shown in SEQ ID NO:66 in which 1 to 10 amino acid residues have been deleted, 1 to 10 amino acid residues have been replaced with other amino acid residues, and 1 to 10 amino acid residues have been added is also an hNT-4;
- a wild-type hNT-4 amino acid sequence shown in SEQ ID NO:66 in which 1 to 5 amino acid residues have been deleted, 1 to 5 amino acid residues have been replaced with other amino acid residues, and 1 to 5 amino acid residues have been added is also an hNT-4;
- a wild-type hNT-4 amino acid sequence shown in SEQ ID NO:66 in which 1 to 3 amino acid residues have been deleted, hNT-4 also includes a wild-type hNT-4 amino acid sequence shown in SEQ ID NO:66 in which one or two amino acid residues have been
- hNT-4 mutant compared to normal wild-type hNT-4 can be easily confirmed by aligning the amino acid sequences of both hNT-4. The same is true for hNT-4 from animal species other than humans.
- the amino acid sequence of the hNT-4 mutant preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, to the amino acid sequence of normal wild-type hNT-4 shown in SEQ ID NO:66.
- substitutions of amino acids in the amino acid sequence of wild-type hBDNF or wild-type hNGF, wild-type hNT-3, and wild-type hNT-4 are, for example, conservative amino acid substitutions. The same is true for these neurotrophic factors of animal species other than humans.
- the above wild-type or mutant hBDNF, hNGF, hNT-3, or hNT-4, in which the constituent amino acids are modified with sugar chains are also hBDNF, hNGF, hNT-3, or hNT-4, respectively.
- Such changes include, but are not limited to, the conversion of cysteine residues to formylglycine.
- hBDNF, hNGF, hNT-3, and hNT-4 The same applies to these neurotrophic factors of animal species other than humans.
- hBDNF, hNGF, hNT-3, or hNT-4 modified with sugar chains are included in hBDNF, hNGF, hNT-3, or hNT-4 having the original amino acid sequence, respectively.
- these human neurotrophic factors modified with phosphate The same applies to those modified with things other than sugar chains and phosphate.
- the same also applies to those in which the side chains of the amino acids that make up these human neurotrophic factors have been changed by substitution reactions, etc.
- One such conversion is the conversion of a cysteine residue to formylglycine.
- neurotrophic factors of animal species other than humans are examples of neurotrophic factors of animal species other than humans.
- One embodiment of the present invention is a recombinant protein in which a wild-type neurotrophic factor is fused with HSA.
- a fusion protein is expressed as a recombinant protein using a host cell such as CHO, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium
- the amount of expression of human neurotrophic factor in the culture supernatant can be at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, for example, 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, 10 to 20 times, 20 to 30 times, etc., compared to when wild-type human neurotrophic factor is expressed as a recombinant protein by a similar method.
- Another embodiment of the present invention is a recombinant protein in which wild-type hBDNF is fused with HSA.
- a fusion protein is expressed as a recombinant protein using a host cell such as CHO, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium
- the amount of hBDNF expressed in the culture supernatant can be at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, for example, 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, 10 to 20 times, 20 to 30 times, etc., compared to when wild-type hBDNF is expressed as a recombinant protein by a similar method.
- Another embodiment of the present invention is a recombinant protein in which wild-type hNGF is fused with HSA.
- a fusion protein is expressed as a recombinant protein using a host cell such as CHO, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium
- the amount of hNGF expressed in the culture supernatant can be at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, for example, 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, 10 to 20 times, 20 to 30 times, etc., compared to when wild-type hNGF is expressed as a recombinant protein by a similar method.
- Another embodiment of the present invention is a recombinant protein in which wild-type hNT-3 is fused with HSA.
- a fusion protein is expressed as a recombinant protein using a host cell such as CHO, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium
- the amount of expression of hNT-3 in the culture supernatant can be at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, for example, 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, 10 to 20 times, 20 to 30 times, etc., compared to when wild-type hNT-3 is expressed as a recombinant protein by a similar method.
- Another embodiment of the present invention is a recombinant protein in which wild-type hNT-4 is fused with HSA.
- a fusion protein is expressed as a recombinant protein using a host cell such as CHO, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium
- the amount of hNT-4 expressed in the culture supernatant can be at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, for example, 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, 10 to 20 times, 20 to 30 times, etc., compared to when wild-type hNT-4 is expressed as a recombinant protein by a similar method.
- One embodiment of the present invention relates to a fusion protein in which a polypeptide containing the amino acid sequence of a human neurotrophic factor is bound to a polypeptide containing the amino acid sequence of SA.
- binding polypeptides refers to binding different polypeptides by covalent bonding, either directly or indirectly via a linker.
- the neurotrophic factor is preferably human neurotrophic factor.
- SA is preferably HSA.
- the human neurotrophic factor is, for example, hBDNF, hNGF, hNT-3, or hNT-4. The same applies to neurotrophic factors of animal species other than humans.
- a common method for linking two different polypeptides is, for example, to create an expression vector incorporating a DNA fragment in which a gene encoding one polypeptide is linked in-frame downstream of a gene encoding the other polypeptide, and then to express the polypeptide as a recombinant protein by culturing a host cell transformed with this expression vector.
- the resulting recombinant protein is a single-chain polypeptide in which the two polypeptides are peptide-linked, either directly or via different amino acid sequences.
- SA-human neurotrophic factor fusion protein or "SA-human neurotrophic factor” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of human neurotrophic factor is linked to the C-terminus of the amino acid sequence of SA, either directly or via a linker, and which has the function of a human neurotrophic factor.
- the animal species of SA is not particularly limited, but preferably it is a mammalian SA, more preferably it is a primate SA, and even more preferably it is HSA.
- the human neurotrophic factor in the SA-human neurotrophic factor fusion protein has the function of a human neurotrophic factor
- the human neurotrophic factor preferably retains a specific activity of 10% or more, more preferably a specific activity of 20% or more, even more preferably a specific activity of 50% or more, and even more preferably a specific activity of 80% or more, when the specific activity of a normal wild-type human neurotrophic factor is taken as 100%.
- the specific activity of human neurotrophic factor in the SA-human neurotrophic factor fusion protein is calculated by multiplying the physiological activity of human neurotrophic factor per unit mass of the fusion protein by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein that corresponds to human neurotrophic factor).
- the term "HSA-human neurotrophic factor fusion protein" or "HSA-human neurotrophic factor” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of human neurotrophic factor is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of HSA, and which has the function of a human neurotrophic factor.
- HSA-human neurotrophic factor fusion protein has the function of a human neurotrophic factor
- the definition of the SA-neurotrophic factor fusion protein described above can be applied.
- the SA has a function as an SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- the HSA-human neurotrophic factor fusion protein The same is true for the HSA-human neurotrophic factor fusion protein.
- mutations When mutations are added to an HSA-human neurotrophic factor fusion protein, which is a fusion protein of wild-type HSA and wild-type human neurotrophic factor, mutations can be added only to the HSA portion without mutations in the human neurotrophic factor portion, mutations can be added only to the human neurotrophic factor portion without mutations in the HSA portion, or mutations can be added to both the HSA portion and the human neurotrophic factor portion.
- the amino acid sequence of the portion is the amino acid sequence of the wild-type HSA with a mutation added.
- mutations are added only to the human neurotrophic factor portion, the amino acid sequence of the portion is the amino acid sequence of the wild-type human neurotrophic factor with a mutation added.
- the amino acid sequence of the HSA portion is the amino acid sequence of the wild-type HSA with a mutation added
- the amino acid sequence of the human neurotrophic factor portion is the amino acid sequence of the wild-type human neurotrophic factor with a mutation added. The same can be said about fusion proteins between wild-type SA of non-human animal species and wild-type human neurotrophic factor.
- HSA-human neurotrophic factor fusion proteins in which the amino acids constituting the protein are modified with sugar chains are also HSA-human neurotrophic factor fusion proteins.
- HSA-human neurotrophic factor fusion proteins in which the amino acids constituting the protein are modified with phosphate are also HSA-human neurotrophic factor fusion proteins.
- HSA-human neurotrophic factor fusion proteins modified with something other than sugar chains and phosphate are also HSA-human neurotrophic factor fusion proteins.
- HSA-human neurotrophic factor fusion proteins in which the side chains of the amino acids constituting the protein have been converted by a substitution reaction or the like are also HSA-human neurotrophic factor fusion proteins.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said about fusion proteins of SA of animal species other than humans and human neurotrophic factor (SA-human neurotrophic factor).
- HSA-human neurotrophic factor fusion proteins modified with sugar chains are included in HSA-human neurotrophic factor fusion proteins having the original amino acid sequence.
- HSA-human neurotrophic factor fusion proteins modified with phosphate are included in HSA-human neurotrophic factor fusion proteins having the original amino acid sequence.
- those modified with things other than sugar chains and phosphate are included in HSA-human neurotrophic factor fusion proteins having the original amino acid sequence.
- HSA-human neurotrophic factor fusion proteins in which the side chains of the amino acids constituting the HSA-human neurotrophic factor fusion proteins have been converted by substitution reactions or the like are included in HSA-human neurotrophic factor fusion proteins having the original amino acid sequence.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said for fusion proteins of SA and human neurotrophic factor of animal species other than humans (SA-human neurotrophic factor).
- HSA-human neurotrophic factor fusion protein in which the human neurotrophic factor constituting the protein is a precursor of human neurotrophic factor is also an HSA-human neurotrophic factor fusion protein.
- precursor used here refers to a type of protein that is biosynthesized as an HSA-human neurotrophic factor fusion protein, and in which the portion that functions as a human neurotrophic factor after the biosynthesis is cleaved from the fusion protein during the manufacturing process or in the living body when administered to the living body, and can function as a human neurotrophic factor by itself.
- the HSA-human neurotrophic factor fusion protein may be cleaved at a specific site after biosynthesis by a hydrolase or the like, and the portion containing HSA and the portion containing mature human neurotrophic factor may be separated.
- the human neurotrophic factor obtained is not a fusion protein with HSA, but the HSA-human neurotrophic factor fusion protein is synthesized once during the process of manufacturing the neurotrophic factor. Therefore, when human neurotrophic factor is manufactured by such a method, the manufacturing method is included in the manufacturing method of HSA-human neurotrophic factor. The same can be said about fusion proteins of SA from non-human animal species and human neurotrophic factor (SA-human neurotrophic factor).
- SA-hBDNF fusion protein or "SA-hBDNF” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of hBDNF is linked to the C-terminus of the amino acid sequence of SA, either directly or via a linker, and which has the function of hBDNF.
- the animal species of SA is not particularly limited, but it is preferably a mammalian SA, more preferably a primate SA, and even more preferably HSA.
- hBDNF in an SA-hBDNF fusion protein When hBDNF in an SA-hBDNF fusion protein is said to have the function of hBDNF, it means that hBDNF retains a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of normal wild-type hBDNF is taken as 100%.
- the specific activity of hBDNF in the SA-hBDNF fusion protein is calculated by multiplying the physiological activity of hBDNF per unit mass of the fusion protein by (the molecular weight of the fusion protein/the molecular weight of the portion of the fusion protein corresponding to hBDNF).
- the term "HSA-hBDNF fusion protein" or "HSA-hBDNF” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of hBDNF is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of HSA, and which has the function of hBDNF.
- HSA-hBDNF fusion protein has the function of hBDNF
- the definition of the SA-hBDNF fusion protein described above can be applied.
- a preferred HSA-hBDNF fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 68.
- the HSA-hBDNF fusion protein shown in SEQ ID NO: 68 is obtained by linking wild-type hBDNF to the C-terminus of wild-type HSA via the linker sequence Gly-Ser.
- the HSA-hBDNF fusion protein shown in SEQ ID NO: 68 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 69.
- HSA-hBDNF fusion proteins include those having an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 68 have been replaced with other amino acid residues, deleted, or mutated by addition, as long as they have the function of hBDNF. It is preferable that the HSA-hBDNF fusion protein has the function of human serum albumin, such as the function of binding and transporting endogenous substances in blood and exogenous substances such as drugs, but is not limited thereto.
- an HSA-hBDNF fusion protein which is a fusion protein of wild-type HSA and wild-type hBDNF
- a mutation may be added only to the HSA portion and not to the hBDNF portion, or a mutation may be added only to the hBDNF portion without any mutation to the HSA portion, or a mutation may be added to both the HSA portion and the hBDNF portion.
- the amino acid sequence of the portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above.
- the amino acid sequence of the portion is the amino acid sequence of hBDNF obtained by adding a mutation to the wild-type hBDNF described above.
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above
- the amino acid sequence of the hBDNF portion is the amino acid sequence of hBDNF obtained by adding a mutation to the wild-type hBDNF described above.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- an amino acid residue is added, one or more amino acid residues are added to the amino acid sequence shown in SEQ ID NO:68 or to the N-terminus or C-terminus of the amino acid sequence.
- the HSA-hBDNF fusion protein may be a combination of these substitutions, deletions, and additions of amino acid residues.
- the mutation may be made only to the HSA part, only to the hIL-10 part, or to both parts.
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:68 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:68.
- HSA-hBDNF fusion proteins in which the amino acids constituting the protein are modified with sugar chains are also HSA-hBDNF fusion proteins.
- HSA-hBDNF fusion proteins in which the amino acids constituting the protein are modified with phosphate are also HSA-hBDNF fusion proteins.
- HSA-hBDNF fusion proteins modified with something other than sugar chains and phosphate are also HSA-hBDNF fusion proteins.
- HSA-hBDNF fusion proteins in which the side chains of the amino acids constituting the protein have been changed by a substitution reaction or the like are also HSA-hBDNF fusion proteins.
- Such changes include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said about fusion proteins of SA and hBDNF of animal species other than humans (SA-hBDNF).
- HSA-hBDNF fusion proteins modified with sugar chains are included in HSA-hBDNF fusion proteins with the original amino acid sequence.
- HSA-hBDNF fusion proteins modified with phosphate are included in HSA-hBDNF fusion proteins with the original amino acid sequence.
- those modified with things other than sugar chains and phosphate are also included in HSA-hBDNF fusion proteins with the original amino acid sequence.
- those in which the side chains of the amino acids constituting the HSA-hBDNF fusion protein have been converted by substitution reactions or the like are also included in HSA-hBDNF fusion proteins with the original amino acid sequence.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said for fusion proteins of SA and hBDNF of animal species other than humans (SA-hBDNF).
- HSA-hBDNF fusion proteins in which the constituent hBDNF is a precursor of hBDNF are also HSA-hBDNF fusion proteins.
- the term "precursor” refers to a type that is biosynthesized as an HSA-hBDNF fusion protein, and after the biosynthesis, the part that functions as hBDNF is separated from the fusion protein during the manufacturing process or in the living body when administered to the living body, and can function as hBDNF by itself.
- the HSA-hBDNF fusion protein after the HSA-hBDNF fusion protein is biosynthesized, it may be cleaved at a specific site by a hydrolase or the like, and the part containing HSA and the part containing mature hBDNF may be separated.
- the obtained hBDNF is not a fusion protein with HSA, but the HSA-hBDNF fusion protein is synthesized once during the process of manufacturing hBDNF. Therefore, when hBDNF is manufactured by such a method, the manufacturing method is included in the manufacturing method of HSA-hBDNF fusion protein. The same can be said for a fusion protein of SA and hBDNF of an animal species other than human (SA-hBDNF).
- SA-hNGF fusion protein or "SA-hNGF” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of hNGF is linked to the C-terminus of the amino acid sequence of SA, either directly or via a linker, and having the function of hNGF.
- the animal species of SA is not particularly limited, but preferably it is a mammalian SA, more preferably it is a primate SA, and even more preferably it is HSA.
- hNGF in an SA-hNGF fusion protein has the function of hNGF
- hNGF retains a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of normal wild-type hNGF is taken as 100%.
- the specific activity of hNGF in the SA-hNGF fusion protein is calculated by multiplying the physiological activity of hNGF per unit mass of the fusion protein by (the molecular weight of the fusion protein/the molecular weight of the portion of the fusion protein corresponding to hNGF).
- the term "HSA-hNGF fusion protein" or “HSA-hNGF” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of hNGF is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of HSA, and which has the function of hNGF.
- HSA-hNGF fusion protein has the function of hNGF
- the definition of the SA-hNGF fusion protein described above can be applied.
- a preferred HSA-hNGF fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 70.
- the HSA-hNGF fusion protein shown in SEQ ID NO: 70 is obtained by linking wild-type hNGF to the C-terminus of wild-type HSA via the linker sequence Gly-Ser.
- the HSA-hNGF fusion protein shown in SEQ ID NO: 70 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 71.
- HSA-hNGF fusion proteins also include those having an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 70 have been replaced with other amino acid residues, deleted, or mutated by addition, as long as they have the function of hNGF. It is preferable that the HSA-hNGF fusion protein has the function of human serum albumin, such as the function of binding and transporting endogenous substances in blood and exogenous substances such as drugs, but is not limited thereto.
- mutations When mutations are introduced into an HSA-hNGF fusion protein, which is a fusion protein of wild-type HSA and wild-type hNGF, mutations can be introduced only in the HSA portion and not in the hNGF portion, mutations can be introduced only in the hNGF portion without mutations in the HSA portion, or mutations can be introduced in both the HSA portion and the hNGF portion.
- mutations When mutations are introduced only in the HSA portion, the amino acid sequence of the portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above.
- the amino acid sequence of the portion is the amino acid sequence of hNGF obtained by adding a mutation to the wild-type hNGF described above.
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above
- the amino acid sequence of the hNGF portion is the amino acid sequence of hNGF obtained by adding a mutation to the wild-type hNGF described above.
- the following is an example of a case where a mutation is made to an HSA-hNGF fusion protein having the amino acid sequence shown in SEQ ID NO:70.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- an amino acid residue is added, one or more amino acid residues are added to the amino acid sequence shown in SEQ ID NO:70 or to the N-terminus or C-terminus of the amino acid sequence.
- the HSA-hNGF fusion protein may be a combination of these substitutions, deletions, and additions of amino acid residues.
- the mutation may be made only to the HSA portion, only to the hNGF portion, or to both portions. The same applies to a fusion protein of SA and hNGF of an animal species other than human (SA-hNGF).
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:70 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:70.
- HSA-hNGF fusion proteins in which the amino acids constituting the protein are modified with sugar chains are also HSA-hNGF fusion proteins.
- HSA-hNGF fusion proteins in which the amino acids constituting the protein are modified with phosphate are also HSA-hNGF fusion proteins.
- HSA-hNGF fusion proteins modified with anything other than sugar chains and phosphate are also HSA-hNGF fusion proteins.
- HSA-hNGF fusion proteins in which the side chains of the amino acids constituting the protein are modified by substitution reactions, etc. are also HSA-hNGF fusion proteins.
- Such modifications include, but are not limited to, the conversion of cysteine residues to formylglycine. Mutations may be made only to the HSA portion, only to the hNGF portion, or to both portions. The same can be said about fusion proteins of SA and hNGF of animal species other than humans (SA-hNGF).
- HSA-hNGF fusion proteins modified with sugar chains are included in HSA-hNGF fusion proteins having the original amino acid sequence.
- HSA-hNGF fusion proteins modified with phosphate are included in HSA-hNGF fusion proteins having the original amino acid sequence.
- those modified with things other than sugar chains and phosphate are included in HSA-hNGF fusion proteins having the original amino acid sequence.
- HSA-hNGF fusion proteins in which the side chains of the amino acids constituting the HSA-hNGF fusion protein have been changed by substitution reactions or the like are included in HSA-hNGF fusion proteins having the original amino acid sequence.
- Such changes include, but are not limited to, the conversion of cysteine residues to formylglycine. Mutations may be made only to the HSA portion, only to the hNGF portion, or to both portions. The same can be said for fusion proteins of SA and hNGF of animal species other than humans (SA-hNGF).
- HSA-hNGF fusion proteins in which the hNGF constituting the protein is a precursor of hNGF are also HSA-hNGF fusion proteins.
- precursor here refers to a type of protein that is biosynthesized as an HSA-hNGF fusion protein, and after the biosynthesis, the portion that functions as hNGF is separated from the fusion protein during the manufacturing process or in the living body when administered to the living body, and can function as hNGF by itself.
- the HSA-hNGF fusion protein after the HSA-hNGF fusion protein is biosynthesized, it may be cleaved at a specific site by a hydrolase or the like, and the portion containing HSA and the portion containing mature hNGF may be separated.
- the resulting hNGF is not a fusion protein with HSA, but the HSA-hNGF fusion protein is synthesized once during the process of manufacturing hNGF. Therefore, when hNGF is manufactured by such a method, the manufacturing method is included in the manufacturing method of HSA-hNGF fusion protein.
- the mutation may be added only to the HSA portion, only to the hNGF portion, or both portions. The same can be said about fusion proteins of SA and hNGF from non-human animal species (SA-hNGF).
- SA-hNT-3 fusion protein or "SA-hNT-3” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of hNT-3 is linked, directly or via a linker, to the C-terminus of the amino acid sequence of SA, and which has the function of hNT-3.
- SA animal species of SA, but it is preferably a mammalian SA, more preferably a primate SA, and even more preferably HSA.
- hNT-3 in the SA-hNT-3 fusion protein has the function of hNT-3
- hNT-3 preferably retains a specific activity of 10% or more, more preferably a specific activity of 20% or more, even more preferably a specific activity of 50% or more, and even more preferably a specific activity of 80% or more, when the specific activity of normal wild-type hNT-3 is taken as 100%.
- the specific activity of hNT-3 in the SA-hNT-3 fusion protein is calculated by multiplying the physiological activity of hNT-3 per unit mass of the fusion protein by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein corresponding to hNT-3).
- the term "HSA-hNT-3 fusion protein" or "HSA-hNT-3” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of hNT-3 is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of HSA, and which has the function of hNT-3.
- HSA-hNT-3 fusion protein has the function of hNT-3
- the definition of the SA-hNT-3 fusion protein described above can be applied.
- a preferred HSA-hNT-3 fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 72.
- the HSA-hNT-3 fusion protein shown in SEQ ID NO: 72 is obtained by linking wild-type hNT-3 to the C-terminus of wild-type HSA via the linker sequence Gly-Ser.
- the HSA-hNT-3 fusion protein shown in SEQ ID NO: 72 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 73.
- HSA-hNT-3 fusion proteins also include those having an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 72 have been replaced with other amino acid residues, deleted, or mutated by addition, as long as they have the function of hNT-3. It is preferable that the HSA-hNT-3 fusion protein has the function of human serum albumin, such as the function of binding and transporting endogenous substances in blood and exogenous substances such as drugs, but is not limited thereto.
- mutations When mutations are introduced into the HSA-hNT-3 fusion protein, which is a fusion protein of wild-type HSA and wild-type hNT-3, mutations can be introduced only in the HSA portion and not in the hNT-3 portion, mutations can be introduced only in the hNT-3 portion without mutations in the HSA portion, or mutations can be introduced in both the HSA portion and the hNT-3 portion.
- mutations When mutations are introduced only in the HSA portion, the amino acid sequence of the portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above.
- the amino acid sequence of the portion is the amino acid sequence of hNT-3 obtained by adding a mutation to the wild-type hNT-3 described above.
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above
- the amino acid sequence of the hNT-3 portion is the amino acid sequence of hNT-3 obtained by adding a mutation to the wild-type hNT-3 described above.
- the HSA-hNT-3 fusion protein having the amino acid sequence shown in SEQ ID NO: 72.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- an amino acid residue is added, one or more amino acid residues are added to the amino acid sequence shown in SEQ ID NO:72 or to the N-terminus or C-terminus of the amino acid sequence.
- the HSA-hNT-3 fusion protein may be a combination of these substitutions, deletions, and additions of amino acid residues.
- the mutation may be made only to the HSA portion, only to the hGALC portion, or to both portions.
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:72 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:72.
- HSA-hNT-3 fusion proteins in which the amino acids constituting the protein are modified with sugar chains are also HSA-hNT-3 fusion proteins.
- HSA-hNT-3 fusion proteins in which the amino acids constituting the protein are modified with phosphate are also HSA-hNT-3 fusion proteins.
- HSA-hNT-3 fusion proteins modified with something other than sugar chains and phosphate are also HSA-hNT-3 fusion proteins.
- HSA-hNT-3 fusion proteins in which the side chains of the amino acids constituting the protein have been changed by a substitution reaction or the like are also HSA-hNT-3 fusion proteins.
- Such changes include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said about fusion proteins of SA and hNT-3 of animal species other than humans (SA-hNT-3).
- HSA-hNT-3 fusion proteins modified with sugar chains are included in HSA-hNT-3 fusion proteins having the original amino acid sequence.
- HSA-hNT-3 fusion proteins modified with phosphate are included in HSA-hNT-3 fusion proteins having the original amino acid sequence.
- those modified with things other than sugar chains and phosphate are also included in HSA-hNT-3 fusion proteins having the original amino acid sequence.
- HSA-hNT-3 fusion proteins in which the side chains of the amino acids constituting the HSA-hNT-3 fusion proteins have been converted by substitution reactions or the like are also included in HSA-hNT-3 fusion proteins having the original amino acid sequence.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said for fusion proteins of SA and hNT-3 of animal species other than humans (SA-hNT-3).
- HSA-hNT-3 fusion proteins in which the hNT-3 constituting the protein is a precursor of hNT-3 are also HSA-hNT-3 fusion proteins.
- the term "precursor” here refers to a type of protein that is biosynthesized as an HSA-hNT-3 fusion protein, and in which the portion that functions as hNT-3 after the biosynthesis is cleaved from the fusion protein during the manufacturing process or in the living body when administered to the living body, and can function as hNT-3 by itself.
- the HSA-hNT-3 fusion protein after the HSA-hNT-3 fusion protein is biosynthesized, it may be cleaved at a specific site by a hydrolase or the like, and the portion containing HSA and the portion containing mature hNT-3 may be separated.
- the resulting hNT-3 is not a fusion protein with HSA, but the HSA-hNT-3 fusion protein is synthesized once during the process of synthesizing the hNT-3. Therefore, when hNT-3 is manufactured by such a method, the manufacturing method is included in the manufacturing method of HSA-hNT-3 fusion protein. The same can be said about the fusion protein of SA and hNT-3 from non-human animal species (SA-hNT-3).
- SA-hNT-4 fusion protein or "SA-hNT-4" refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of hNT-4 is linked, directly or via a linker, to the C-terminus of the amino acid sequence of SA, and which has the function of hNT-4.
- SA animal species of SA, but it is preferably a mammalian SA, more preferably a primate SA, and even more preferably HSA.
- hNT-4 in the SA-hNT-4 fusion protein has the function of hNT-4, it means that hNT-4 preferably retains a specific activity of 10% or more, more preferably a specific activity of 20% or more, even more preferably a specific activity of 50% or more, and even more preferably a specific activity of 80% or more, when the specific activity of normal wild-type hNT-4 is taken as 100%.
- the specific activity of hNT-4 in the SA-hNT-4 fusion protein is calculated by multiplying the physiological activity of hNT-4 per unit mass of the fusion protein by (molecular weight of the fusion protein/molecular weight of the portion of the fusion protein corresponding to hNT-4).
- the term "HSA-hNT-4 fusion protein" or "HSA-hNT-4" refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of hNT-4 is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of HSA, and which has the function of hNT-4.
- HSA-hNT-4 fusion protein has the function of hNT-4
- the definition of the SA-hNT-4 fusion protein above can be applied.
- a preferred HSA-hNT-4 fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 74.
- the HSA-hNT-4 fusion protein shown in SEQ ID NO: 74 is obtained by linking wild-type hNT-4 to the C-terminus of wild-type HSA via the linker sequence Gly-Ser.
- the HSA-hNT-4 fusion protein shown in SEQ ID NO: 74 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 75.
- HSA-hNT-4 fusion proteins also include those having an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 74 have been replaced with other amino acid residues, deleted, or mutated by addition, as long as they have the function of hNT-4. It is preferable that the HSA-hNT-4 fusion protein has the function of human serum albumin, such as the function of binding and transporting endogenous substances in blood and exogenous substances such as drugs, but is not limited thereto.
- mutations When mutations are introduced into the HSA-hNT-4 fusion protein, which is a fusion protein of wild-type HSA and wild-type hNT-4, mutations can be introduced only in the HSA portion and not in the hNT-4 portion, mutations can be introduced only in the hNT-4 portion without mutations in the HSA portion, or mutations can be introduced in both the HSA portion and the hNT-4 portion.
- mutations When mutations are introduced only in the HSA portion, the amino acid sequence of the portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above.
- the amino acid sequence of the portion is the amino acid sequence of hNT-4 obtained by adding a mutation to the wild-type hNT-4 described above.
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above
- the amino acid sequence of the hNT-4 portion is the amino acid sequence of hNT-4 obtained by adding a mutation to the wild-type hNT-4 described above.
- the HSA-hNT-4 fusion protein having the amino acid sequence shown in SEQ ID NO: 74.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- an amino acid residue is added, one or more amino acid residues are added to the amino acid sequence shown in SEQ ID NO:74 or to the N-terminus or C-terminus of the amino acid sequence.
- the HSA-hNT-4 fusion protein may be a combination of these substitutions, deletions, and additions of amino acid residues.
- the mutation may be made only to the HSA portion, only to the hNT-4 portion, or to both portions. The same can be said about the fusion protein (SA-hNT-4) of SA and hNT-3 from non-human animal species.
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:74 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably shows 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:74.
- HSA-hNT-4 fusion proteins in which the amino acids constituting the protein are modified with sugar chains are also HSA-hNT-4 fusion proteins.
- HSA-hNT-4 fusion proteins in which the amino acids constituting the protein are modified with phosphate are also HSA-hNT-4 fusion proteins.
- HSA-hNT-4 fusion proteins modified with something other than sugar chains and phosphate are also HSA-hNT-4 fusion proteins.
- HSA-hNT-4 fusion proteins in which the side chains of the amino acids constituting the protein have been changed by a substitution reaction or the like are also HSA-hNT-4 fusion proteins.
- Such changes include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said about fusion proteins (SA-hNT-4) of SA and hNT-3 of animal species other than humans.
- HSA-hNT-4 fusion proteins modified with sugar chains are included in HSA-hNT-4 fusion proteins having the original amino acid sequence.
- HSA-hNT-4 fusion proteins modified with phosphate are included in HSA-hNT-4 fusion proteins having the original amino acid sequence.
- those modified with things other than sugar chains and phosphate are also included in HSA-hNT-4 fusion proteins having the original amino acid sequence.
- HSA-hNT-4 fusion proteins in which the side chains of the amino acids constituting the HSA-hNT-4 fusion protein have been converted by substitution reactions or the like are also included in HSA-hNT-4 fusion proteins having the original amino acid sequence.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said for fusion proteins of SA and hNT-3 of animal species other than humans (SA-hNT-4).
- HSA-hNT-4 fusion proteins in which the constituent hNT-4 is a precursor of hNT-4 are also HSA-hNT-4 fusion proteins.
- precursor refers to a type of protein that is biosynthesized as an HSA-hNT-4 fusion protein, and in which the portion that functions as hNT-4 after the biosynthesis is cleaved from the fusion protein during the manufacturing process or in the living body when administered to the living body, and can function as hNT-4 by itself.
- the HSA-hNT-4 fusion protein after the HSA-hNT-4 fusion protein is biosynthesized, it may be cleaved at a specific site by a hydrolase or the like, and the portion containing HSA and the portion containing hNT-4 may be separated.
- the resulting hNT-4 is not a fusion protein with HSA, but the HSA-hNT-4 fusion protein is synthesized once during the process of synthesizing the hNT-4. Therefore, when hNT-4 is manufactured by such a method, the manufacturing method is included in the manufacturing method of HSA-hNT-4 fusion protein. The same can be said about the fusion protein of SA and hNT-4 from non-human animal species (SA-hNT-4).
- human neurotrophic factor-SA fusion protein or “human neurotrophic factor-SA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of SA is linked directly or via a linker to the C-terminus of the amino acid sequence of human neurotrophic factor, and which has the function of human neurotrophic factor.
- the human neurotrophic factor in the human neurotrophic factor-SA fusion protein is said to have the function of human neurotrophic factor, it means that the human neurotrophic factor retains a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of a normal wild-type human neurotrophic factor is taken as 100%.
- the specific activity of human neurotrophic factor in the human neurotrophic factor-SA fusion protein is calculated by multiplying the physiological activity of human neurotrophic factor per unit mass of the fusion protein by (the molecular weight of the fusion protein/the molecular weight of the portion of the fusion protein corresponding to human neurotrophic factor).
- human neurotrophic factor-HSA fusion protein or "human neurotrophic factor-HSA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of HSA is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of human neurotrophic factor, and which has the function of a human neurotrophic factor.
- the definition of the neurotrophic factor-HSA fusion protein described above can be applied.
- the SA has a function as an SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- human neurotrophic factor-HSA fusion protein it is preferable that the SA has a function as an SA, such as the function of binding and transporting endogenous substances in the blood and exogenous substances such as drugs, but is not limited to this.
- human neurotrophic factor-HSA fusion protein The same is true for hBDNF-HSA fusion protein, hNGF-HSA fusion protein, hNT-3-HSA fusion protein, and hNT-4-HSA fusion protein.
- a fusion protein having an amino acid sequence in which the N-terminus of SA of a non-human animal species is linked, directly or via a linker, to the C-terminus of the amino acid sequence of a neurotrophic factor of a non-human animal species, and which has a function as a neurotrophic factor is referred to as a "(non-human animal species) neurotrophic factor-(non-human animal species) SA fusion protein.”
- a fusion protein of mouse neurotrophic factor and mouse SA is referred to as a "mouse neurotrophic factor-MSA fusion protein" or “mouse neurotrophic factor-MSA.”
- the definition of the human neurotrophic factor-SA fusion protein described above can be applied.
- the SA has a function as an SA, such as a function to bind and transport endogenous substances in the blood and
- a mutation When a mutation is added to a human neurotrophic factor-HSA fusion protein, which is a fusion protein of wild-type human neurotrophic factor and wild-type HSA, a mutation can be added only to the human neurotrophic factor portion and not to the HSA portion, a mutation can be added only to the HSA portion without adding a mutation to the human neurotrophic factor portion, or a mutation can be added to both the human neurotrophic factor portion and the HSA portion.
- the amino acid sequence of that portion is the amino acid sequence of the human neurotrophic factor obtained by adding a mutation to the wild-type human neurotrophic factor described above.
- the amino acid sequence of that portion is the amino acid sequence of the HSA obtained by adding a mutation to the wild-type HSA described above.
- the amino acid sequence of the human neurotrophic factor portion is the amino acid sequence of the human neurotrophic factor obtained by adding a mutation to the wild-type human neurotrophic factor described above
- the amino acid sequence of the HSA portion is the amino acid sequence of the HSA obtained by adding a mutation to the wild-type HSA described above. The same can be said about fusion proteins between wild-type human neurotrophic factor and wild-type SA of non-human animal species.
- Human neurotrophic factor-HSA fusion proteins in which the amino acids constituting the protein are modified with sugar chains are also human neurotrophic factor-HSA fusion proteins.
- Human neurotrophic factor-HSA fusion proteins in which the amino acids constituting the protein are modified with phosphate are also human neurotrophic factor-HSA fusion proteins.
- Human neurotrophic factor-HSA fusion proteins modified with something other than sugar chains and phosphate are also human neurotrophic factor-HSA fusion proteins.
- Human neurotrophic factor-HSA fusion proteins in which the side chains of the amino acids constituting the protein have been converted by a substitution reaction or the like are also human neurotrophic factor-HSA fusion proteins.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said about fusion proteins of human neurotrophic factor and SA of animal species other than human (human neurotrophic factor-SA).
- a human neurotrophic factor-HSA fusion protein modified by a sugar chain is included in the human neurotrophic factor-HSA fusion protein having the original amino acid sequence.
- a human neurotrophic factor-HSA fusion protein modified by phosphate is included in the human neurotrophic factor-HSA fusion protein having the original amino acid sequence.
- a human neurotrophic factor-HSA fusion protein modified by something other than a sugar chain or phosphate is also included in the human neurotrophic factor-HSA fusion protein having the original amino acid sequence.
- a human neurotrophic factor-HSA fusion protein in which the side chains of the amino acids constituting the human neurotrophic factor-HSA fusion protein have been converted by a substitution reaction or the like is also included in the human neurotrophic factor-HSA fusion protein having the original amino acid sequence.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said about a fusion protein of human neurotrophic factor and SA of an animal species other than human (human neurotrophic factor-SA).
- a human neurotrophic factor-HSA fusion protein in which the human neurotrophic factor that constitutes it is a precursor of human neurotrophic factor is also a human neurotrophic factor-HSA fusion protein.
- the precursor of human neurotrophic factor does not exhibit the function of human neurotrophic factor, it is necessary that it is processed in vivo or in vitro and converted into one that exhibits the function of human neurotrophic factor.
- hBDNF-SA fusion protein or "hBDNF-SA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of SA is linked, directly or via a linker, to the C-terminus of the amino acid sequence of hBDNF, and which has the function of hBDNF.
- hBDNF in an hBDNF-SA fusion protein is said to have the function of hBDNF, it means that hBDNF retains a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of normal wild-type hBDNF is taken as 100%.
- the specific activity of hBDNF in an hBDNF-SA fusion protein is calculated by multiplying the physiological activity of hBDNF per unit mass of the fusion protein by (the molecular weight of the fusion protein/the molecular weight of the portion of the fusion protein corresponding to hBDNF).
- hBDNF-HSA fusion protein or "hBDNF-HSA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of HSA is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of hBDNF, and which has the function of hBDNF.
- the definition of the hBDNF-SA fusion protein described above can be applied.
- a fusion protein having an amino acid sequence in which the N-terminus of SA of a non-human animal species is linked, directly or via a linker, to the C-terminus of the amino acid sequence of BDNF of a non-human animal species, and which has the function of BDNF is referred to as a "(non-human animal species) BDNF-(non-human animal species) SA fusion protein.”
- a fusion protein of mouse BDNF and mouse SA is referred to as a "mouse BDNF-mouse SA fusion protein" or "mouse BDNF-MSA.”
- a preferred hBDNF-HSA fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 76.
- the hBDNF-HSA fusion protein shown in SEQ ID NO: 76 is obtained by linking wild-type HSA to the C-terminus of wild-type hBDNF via the linker sequence Gly-Ser.
- the hBDNF-HSA fusion protein shown in SEQ ID NO: 76 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 77.
- the hBDNF-HSA fusion protein also includes an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 76 have been replaced with other amino acid residues, deleted, or mutated by addition, as long as it has the function of hBDNF.
- the hBDNF-HSA fusion protein is preferably one that has the function of human serum albumin, such as the function of binding and transporting endogenous substances in blood and exogenous substances such as drugs, but is not limited thereto.
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:76 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:76.
- mutations When mutations are added to an hBDNF-HSA fusion protein, which is a fusion protein of wild-type hBDNF and wild-type HSA, mutations can be added only to the HSA portion and not to the hBDNF portion, mutations can be added only to the hBDNF portion without mutations to the HSA portion, or mutations can be added to both the HSA portion and the hBDNF portion.
- mutations When mutations are added only to the HSA portion, the amino acid sequence of the portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above.
- the amino acid sequence of the portion is the amino acid sequence of hBDNF obtained by adding a mutation to the wild-type hBDNF described above.
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above
- the amino acid sequence of the hBDNF portion is the amino acid sequence of hBDNF obtained by adding a mutation to the wild-type hBDNF described above.
- the following is an example of a case where a mutation is made to an hBDNF-HSA fusion protein having the amino acid sequence shown in SEQ ID NO:76.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- an amino acid residue is added, one or more amino acid residues are added to the amino acid sequence shown in SEQ ID NO:76 or to the N-terminus or C-terminus of the amino acid sequence.
- the hBDNF-HSA fusion protein may be a combination of these substitutions, deletions, and additions of amino acid residues.
- the mutation may be made only to the HSA portion, only to the hBDNF portion, or to both portions. The same applies to a fusion protein of hBDNF and SA of an animal species other than human (hBDNF-SA).
- An hBDNF-HSA fusion protein in which the constituent amino acids are modified with sugar chains is also an hBDNF-HSA fusion protein.
- An hBDNF-HSA fusion protein in which the constituent amino acids are modified with phosphate is also an hBDNF-HSA fusion protein.
- An hBDNF-HSA fusion protein modified with something other than sugar chains and phosphate is also an hBDNF-HSA fusion protein.
- An hBDNF-HSA fusion protein in which the side chains of the constituent amino acids have been converted by a substitution reaction or the like is also an hBDNF-HSA fusion protein.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said about a fusion protein of hBDNF and SA of an animal species other than human (hBDNF-SA).
- hBDNF-HSA fusion proteins modified with sugar chains are included in hBDNF-HSA fusion proteins having the original amino acid sequence.
- hBDNF-HSA fusion proteins modified with phosphate are included in hBDNF-HSA fusion proteins having the original amino acid sequence.
- those modified with things other than sugar chains and phosphate are also included in hBDNF-HSA fusion proteins having the original amino acid sequence.
- hBDNF-HSA fusion proteins in which the side chains of the amino acids constituting the hBDNF-HSA fusion protein have been changed by substitution reactions or the like are also included in hBDNF-HSA fusion proteins having the original amino acid sequence.
- Such changes include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said for fusion proteins of hBDNF and SA of animal species other than human (hBDNF-SA).
- hBDNF-HSA fusion protein in which the hBDNF that constitutes it is a precursor of hBDNF is also an hBDNF-HSA fusion protein.
- the hBDNF-HSA fusion protein in which hBDNF is a precursor does not exhibit the functions of hBDNF, it is necessary that it be processed in vivo or in vitro and converted into one that exhibits the functions of hBDNF.
- hNGF-SA fusion protein or "hNGF-SA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of SA is linked to the C-terminus of the amino acid sequence of hNGF, either directly or via a linker, and which has the function of hNGF.
- hNGF in an hNGF-SA fusion protein is said to have the function of hNGF, it means that hNGF retains a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of normal wild-type hNGF is taken as 100%.
- the specific activity of hNGF in an hNGF-SA fusion protein is calculated by multiplying the physiological activity of hNGF per unit mass of the fusion protein by (the molecular weight of the fusion protein/the molecular weight of the portion of the fusion protein corresponding to hNGF).
- hNGF-HSA fusion protein or "hNGF-HSA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of HSA is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of hNGF, and which has the function of hNGF.
- the definition of the hNGF-SA fusion protein described above can be applied.
- a fusion protein having an amino acid sequence in which the N-terminus of SA of a non-human animal species is linked, directly or via a linker, to the C-terminus of the amino acid sequence of NGF of a non-human animal species, and which has the function of NGF is referred to as a "(non-human animal species) NGF-(non-human animal species) SA fusion protein.”
- a fusion protein of mouse NGF and mouse SA is referred to as a "mouse NGF-mouse SA fusion protein" or "mouse NGF-MSA.”
- a preferred hNGF-HSA fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 78.
- the hNGF-HSA fusion protein shown in SEQ ID NO: 78 is obtained by linking wild-type HSA to the C-terminus of wild-type hNGF via the linker sequence Gly-Ser.
- the hNGF-HSA fusion protein shown in SEQ ID NO: 78 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 79.
- the hNGF-HSA fusion protein also includes an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 78 have been replaced with other amino acid residues, deleted, or mutated by addition, as long as it has the function of hNGF.
- the hNGF-HSA fusion protein is preferably one that has the function of human serum albumin, such as the function of binding and transporting endogenous substances in blood and exogenous substances such as drugs, but is not limited thereto.
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:78 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:78.
- mutations When mutations are introduced into an hNGF-HSA fusion protein, which is a fusion protein of wild-type hNGF and wild-type HSA, mutations can be introduced only in the HSA portion and not in the hNGF portion, mutations can be introduced only in the hNGF portion without mutations in the HSA portion, or mutations can be introduced in both the HSA portion and the hNGF portion.
- mutations When mutations are introduced only in the HSA portion, the amino acid sequence of the portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above.
- the amino acid sequence of the portion is the amino acid sequence of hNGF obtained by adding a mutation to the wild-type hNGF described above.
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above
- the amino acid sequence of the hNGF portion is the amino acid sequence of hNGF obtained by adding a mutation to the wild-type hNGF described above.
- the following is an example of a case where a mutation is made to an hNGF-HSA fusion protein having the amino acid sequence shown in SEQ ID NO:78.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- an amino acid residue is added, one or more amino acid residues are added to the amino acid sequence shown in SEQ ID NO:78 or to the N-terminus or C-terminus of the amino acid sequence.
- the hNGF-HSA fusion protein may be a combination of these substitutions, deletions, and additions of amino acid residues.
- the mutation may be made only to the HSA portion, only to the hNGF portion, or to both portions. The same can be said for fusion proteins of non-human animal species' NGF and HSA (NGF-SA), and fusion proteins of non-human animal species' NGF and non-human animal species' SA.
- An hNGF-HSA fusion protein in which the amino acids constituting the protein are modified with sugar chains is also an hNGF-HSA fusion protein.
- An hNGF-HSA fusion protein in which the amino acids constituting the protein are modified with phosphate is also an hNGF-HSA fusion protein.
- An hNGF-HSA fusion protein modified with something other than sugar chains and phosphate is also an hNGF-HSA fusion protein.
- An hNGF-HSA fusion protein in which the side chains of the amino acids constituting the protein have been changed by a substitution reaction or the like is also an hNGF-HSA fusion protein.
- Such a change includes, but is not limited to, the change of a cysteine residue to formylglycine. The same can be said about a fusion protein (hNGF-SA) between hNGF and SA of an animal species other than human.
- hNGF-HSA fusion proteins modified with sugar chains are included in the hNGF-HSA fusion proteins having the original amino acid sequence.
- hNGF-HSA fusion proteins modified with phosphate are included in the hNGF-HSA fusion proteins having the original amino acid sequence.
- those modified with things other than sugar chains and phosphate are included in the hNGF-HSA fusion proteins having the original amino acid sequence.
- hNGF-HSA fusion proteins in which the side chains of the amino acids constituting the hNGF-HSA fusion proteins have been changed by substitution reactions or the like are included in the hNGF-HSA fusion proteins having the original amino acid sequence.
- Such changes include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said about fusion proteins of hNGF and SA of animals other than humans (hNGF-SA).
- hNGF-HSA fusion protein in which the hNGF that constitutes it is a precursor of hNGF is also an hNGF-HSA fusion protein.
- the hNGF-HSA fusion protein, in which hNGF is a precursor does not function as hNGF, it is necessary that it be processed in vivo or in vitro and converted into one that functions as hNGF. The same can be said for a fusion protein of hNGF and SA of an animal species other than human (hNGF-SA).
- hNT-3-SA fusion protein or "hNT-3-SA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of SA is linked, directly or via a linker, to the C-terminus of the amino acid sequence of hNT-3, and which has the function of hNT-3.
- hNT-3 in an hNT-3-SA fusion protein is said to have the function of hNT-3, it means that hNT-3 retains a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of normal wild-type hNT-3 is taken as 100%.
- the specific activity of hNT-3 in the hNT-3-SA fusion protein is calculated by multiplying the physiological activity of hNT-3 per unit mass of the fusion protein by (the molecular weight of the fusion protein/the molecular weight of the portion of the fusion protein corresponding to hNT-3).
- hNT-3-HSA fusion protein or "hNT-3-HSA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of HSA is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of hNT-3, and which has the function of hNT-3.
- the definition of the hNT-3-SA fusion protein described above can be applied.
- a fusion protein having an amino acid sequence in which the N-terminus of SA of a non-human animal species is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of NT-3 of a non-human animal species, and which has the function of NT-3 is referred to as a "(non-human animal species) NT-3-(non-human animal species) SA fusion protein.”
- a fusion protein of mouse NT-3 and mouse SA is referred to as a "mouse NT-3-mouse SA fusion protein" or "mouse NT-3-MSA.”
- a preferred hNT-3-HSA fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 80.
- the hNT-3-HSA fusion protein shown in SEQ ID NO: 80 is obtained by linking wild-type HSA to the C-terminus of wild-type hNT-3 via the linker sequence Gly-Ser.
- the hNT-3-HSA fusion protein shown in SEQ ID NO: 80 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 81.
- the hNT-3-HSA fusion protein also includes an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 80 have been replaced with other amino acid residues, deleted, or mutated by addition, as long as it has the function of hNT-3.
- the hNT-3-HSA fusion protein is preferably one that has the function of human serum albumin, such as the function of binding and transporting endogenous substances in blood and exogenous substances such as drugs, but is not limited thereto.
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:80 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably exhibits 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:80.
- mutations When mutations are introduced into the hNT-3-HSA fusion protein, which is a fusion protein of wild-type hNT-3 and wild-type HSA, mutations can be introduced only in the HSA portion and not in the hNT-3 portion, mutations can be introduced only in the hNT-3 portion without mutations in the HSA portion, or mutations can be introduced in both the HSA portion and the hNT-3 portion.
- mutations When mutations are introduced only in the HSA portion, the amino acid sequence of the portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above.
- the amino acid sequence of the portion is the amino acid sequence of hNT-3 obtained by adding a mutation to the wild-type hNT-3 described above.
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above
- the amino acid sequence of the hNT-3 portion is the amino acid sequence of hNT-3 obtained by adding a mutation to the wild-type hNT-3 described above.
- the hNT-3-HSA fusion protein having the amino acid sequence shown in SEQ ID NO: 80.
- the same is true for the fusion protein of wild-type hNT-3 and wild-type SA (hNT-3-SA) of an animal species other than human.
- the following is an example of a case where a mutation is made to an hNT-3-HSA fusion protein having the amino acid sequence shown in SEQ ID NO:80.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- an amino acid residue is added, one or more amino acid residues are added to the amino acid sequence shown in SEQ ID NO:80 or to the N-terminus or C-terminus of the amino acid sequence.
- the hNT-3-HSA fusion protein may be a combination of these substitutions, deletions, and additions of amino acid residues.
- the mutation may be made only to the HSA portion, only to the hNT-3 portion, or to both portions. The same can be said about the fusion protein (hNT-3-SA) between hNT-3 and SA of a non-human animal species.
- An hNT-3-HSA fusion protein in which the amino acids constituting the protein are modified with sugar chains is also an hNT-3-HSA fusion protein.
- An hNT-3-HSA fusion protein in which the amino acids constituting the protein are modified with phosphate is also an hNT-3-HSA fusion protein.
- An hNT-3-HSA fusion protein modified with something other than sugar chains and phosphate is also an hNT-3-HSA fusion protein.
- An hNT-3-HSA fusion protein in which the side chains of the amino acids constituting the protein have been converted by a substitution reaction or the like is also an hNT-3-HSA fusion protein.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said about a fusion protein (hNT-3-SA) of hNT-3 and SA of an animal species other than human.
- hNT-3-HSA fusion proteins modified with sugar chains are included in the hNT-3-HSA fusion proteins having the original amino acid sequence.
- hNT-3-HSA fusion proteins modified with phosphate are included in the hNT-3-HSA fusion proteins having the original amino acid sequence.
- those modified with things other than sugar chains and phosphate are included in the hNT-3-HSA fusion proteins having the original amino acid sequence.
- hNT-3-HSA fusion proteins in which the side chains of the amino acids constituting the hNT-3-HSA fusion proteins have been converted by substitution reactions or the like are included in the hNT-3-HSA fusion proteins having the original amino acid sequence.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said for fusion proteins of hNT-3 and SA of animal species other than human (hNT-3-SA).
- hNT-3-HSA fusion protein in which the hNT-3 that constitutes it is a precursor of hNT-3 is also an hNT-3-HSA fusion protein.
- the hNT-3-HSA fusion protein, of which hNT-3 is a precursor does not function as hNT-3, it is necessary that it be processed in vivo or in vitro and converted into one that functions as hNT-3. The same can be said for a fusion protein of hNT-3 and SA of an animal species other than human (hNT-3-SA).
- hNT-4-SA fusion protein or "hNT-4-SA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of SA is linked, directly or via a linker, to the C-terminus of the amino acid sequence of hNT-4, and which has the function of hNT-4.
- hNT-4 in an hNT-4-SA fusion protein is said to have the function of hNT-4, it means that hNT-4 retains a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, when the specific activity of normal wild-type hNT-4 is taken as 100%.
- the specific activity of hNT-4 in the hNT-4-SA fusion protein is calculated by multiplying the physiological activity of hNT-4 per unit mass of the fusion protein by (the molecular weight of the fusion protein/the molecular weight of the portion of the fusion protein corresponding to hNT-4).
- hNT-4-HSA fusion protein or "hNT-4-HSA” refers to a fusion protein having an amino acid sequence in which the N-terminus of the amino acid sequence of HSA is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of hNT-4, and which has the function of hNT-4.
- the definition of the hNT-4-SA fusion protein described above can be applied.
- a fusion protein having an amino acid sequence in which the N-terminus of SA of a non-human animal species is linked, either directly or via a linker, to the C-terminus of the amino acid sequence of NT-4 of a non-human animal species, and which has the function of NT-4 is referred to as a "(non-human animal species) NT-4-(non-human animal species) SA fusion protein.”
- a fusion protein of mouse NT-4 and mouse SA is referred to as a "mouse NT-4-mouse SA fusion protein" or "mouse NT-4-MSA.”
- a preferred hNT-4-HSA fusion protein has, for example, the amino acid sequence shown in SEQ ID NO: 82.
- the hNT-4-HSA fusion protein shown in SEQ ID NO: 82 is obtained by linking wild-type HSA to the C-terminus of wild-type hNT-4 via the linker sequence Gly-Ser.
- the hNT-4-HSA fusion protein shown in SEQ ID NO: 82 is encoded by a gene having, for example, the base sequence shown in SEQ ID NO: 83.
- the hNT-4-HSA fusion protein also includes an amino acid sequence in which one or more amino acid residues in the amino acid sequence shown in SEQ ID NO: 82 have been replaced with other amino acid residues, deleted, or mutated by addition, as long as it has the function of hNT-4.
- the hNT-4-HSA fusion protein is preferably one that has the function of human serum albumin, such as the function of binding and transporting endogenous substances in blood and exogenous substances such as drugs, but is not limited thereto.
- the position and type (deletion, substitution, and addition) of each mutation when the amino acid sequence shown in SEQ ID NO:82 is mutated can be easily confirmed by alignment of the amino acid sequence before and after the mutation.
- the mutated amino acid sequence preferably has 80% or more identity, 85% or more identity, 90% or more identity, or 95% or more identity, for example, 98% or more, or 99% or more identity, with the amino acid sequence shown in SEQ ID NO:82.
- mutations When mutations are introduced into the hNT-4-HSA fusion protein, which is a fusion protein of wild-type hNT-4 and wild-type HSA, mutations can be introduced only in the HSA portion and not in the hNT-4 portion, mutations can be introduced only in the hNT-4 portion without mutations in the HSA portion, or mutations can be introduced in both the HSA portion and the hNT-4 portion.
- mutations When mutations are introduced only in the HSA portion, the amino acid sequence of the portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above.
- the amino acid sequence of the portion is the amino acid sequence of hNT-4 obtained by adding a mutation to the wild-type hNT-4 described above.
- the amino acid sequence of the HSA portion is the amino acid sequence of HSA obtained by adding a mutation to the wild-type HSA described above
- the amino acid sequence of the hNT-4 portion is the amino acid sequence of hNT-4 obtained by adding a mutation to the wild-type hNT-4 described above.
- the hNT-4-HSA fusion protein having the amino acid sequence shown in SEQ ID NO: 82.
- the same is true for the fusion protein of wild-type hNT-4 and wild-type SA (hNT-4-SA) of an animal species other than human.
- the following is an example of a case where a mutation is made to an hNT-4-HSA fusion protein having the amino acid sequence shown in SEQ ID NO:82.
- the number of amino acid residues to be replaced is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- the number of amino acid residues to be deleted is 1 to 10, 1 to 5, or 1 to 3, for example, 1 or 2.
- an amino acid residue is added, one or more amino acid residues are added to the amino acid sequence shown in SEQ ID NO:82 or to the N-terminus or C-terminus of the amino acid sequence.
- the hNT-4-HSA fusion protein may be a combination of these substitutions, deletions, and additions of amino acid residues.
- the mutation may be made only to the HSA portion, only to the hNT-4 portion, or to both portions. The same can be said about the fusion protein of hNT-4 and SA of a non-human animal species (hNT-4-SA).
- An hNT-4-HSA fusion protein in which the amino acids constituting the protein are modified with sugar chains is also an hNT-4-HSA fusion protein.
- An hNT-4-HSA fusion protein in which the amino acids constituting the protein are modified with phosphate is also an hNT-4-HSA fusion protein.
- An hNT-4-HSA fusion protein in which the amino acids constituting the protein are modified with something other than sugar chains and phosphate is also an hNT-4-HSA fusion protein.
- An hNT-4-HSA fusion protein in which the side chains of the amino acids constituting the protein have been converted by a substitution reaction or the like is also an hNT-4-HSA fusion protein.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine. The same can be said for a fusion protein of NT-4 of a non-human animal species and HSA (NT-4-SA), and a fusion protein of NT-4 of a non-human animal species and SA of a non-human animal species.
- hNT-4-HSA fusion proteins modified with sugar chains are included in the hNT-4-HSA fusion proteins having the original amino acid sequence.
- hNT-4-HSA fusion proteins modified with phosphate are included in the hNT-4-HSA fusion proteins having the original amino acid sequence.
- those modified with things other than sugar chains and phosphate are included in the hNT-4-HSA fusion proteins having the original amino acid sequence.
- those in which the side chains of the amino acids constituting the hNT-4-HSA fusion protein have been converted by a substitution reaction or the like are included in the hNT-4-HSA fusion protein having the original amino acid sequence.
- Such conversions include, but are not limited to, the conversion of cysteine residues to formylglycine.
- NT-4-SA non-human animal species and HSA
- NT-4-SA fusion protein of NT-4 of a non-human animal species and SA of a non-human animal species.
- hNT-4-HSA fusion protein in which the hNT-4 that constitutes it is a precursor of hNT-4 is also an hNT-4-HSA fusion protein.
- the hNT-4-HSA fusion protein, of which hNT-4 is a precursor does not exhibit the function of hNT-4, it is necessary that it be processed in vivo or in vitro and converted into one that exhibits the function of hNT-4.
- fusion protein of HSA and human neurotrophic factor include both the above-mentioned “HSA-human neurotrophic factor fusion protein” and “human neurotrophic factor-HSA fusion protein”. These also apply when SA is from an animal species other than human, or when nerve growth factor is from an animal species other than human.
- a fusion protein of HSA and hBDNF we include both the above-mentioned “HSA-hBDNF fusion protein” and "hBDNF-HSA fusion protein.”
- a fusion protein of HSA and hNGF we include both the above-mentioned “HSA-hNGF fusion protein” and "hNGF-HSA fusion protein.”
- a fusion protein of HSA and hNT-3 we include both the above-mentioned “HSA-hNT-3 fusion protein” and "hNT-3-HSA fusion protein.”
- SA is of an animal species other than human
- NT-3 is of an animal species other than human.
- a fusion protein of HSA and hNT-4 we include both the above-mentioned “HSA-hNT-4 fusion protein” and "hNT-4-HSA fusion protein.” These also apply when SA is from an animal species other than human, or when NT-4 is from an animal species other than human.
- the SA and the neurotrophic factor are linked directly or via a linker.
- linker refers to a portion that does not belong to either the amino acid sequence of SA or the amino acid sequence of a neurotrophic factor.
- the linker is a peptide chain that is interposed between the SA and the neurotrophic factor.
- the linker has various functions.
- These functions include a function between the SA and the neurotrophic factor to bind the SA and the neurotrophic factor, a function to reduce mutual interference between the SA and the neurotrophic factor by increasing the distance between them in the fusion protein molecule, and a function between the SA and the neurotrophic factor to act as a hinge that connects the SA and the neurotrophic factor and gives flexibility to the three-dimensional structure of the fusion protein.
- the linker exerts at least one of these functions.
- fusion proteins of SA and neurotrophic factors are described in detail, taking as examples a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, and a fusion protein of HSA and hNT-4.
- the matters described in detail here can be applied when SA is from an animal species other than human, or when BDNF, NGF, NT-3, or NT-4 is from an animal species other than human. They can also be applied to neurotrophic factors other than BDNF, NGF, NT-3, or NT-4.
- a fusion protein of HSA and hBDNF can be produced as a recombinant protein by preparing an expression vector incorporating a DNA fragment in which a gene encoding hBDNF is linked in-frame downstream or upstream of a gene encoding HSA, and culturing a host cell transformed with the expression vector.
- a fusion protein of HSA and hNGF can be produced as a recombinant protein by preparing an expression vector incorporating a DNA fragment in which a gene encoding hNGF is linked in-frame downstream or upstream of a gene encoding HSA, and culturing a host cell transformed with the expression vector.
- a fusion protein of HSA and hNT-3 can be produced as a recombinant protein by preparing an expression vector incorporating a DNA fragment in which a gene encoding hNT-3 is linked in-frame downstream or upstream of a gene encoding HSA, and culturing a host cell transformed with the expression vector.
- a fusion protein of HSA and hNT-4 can be produced as a recombinant protein by preparing an expression vector incorporating a DNA fragment in which a gene encoding hNT-4 is linked in-frame to the downstream or upstream of a gene encoding HSA, and culturing a host cell transformed with the expression vector.
- the fusion protein produced as a recombinant protein in this way consists of a single polypeptide chain.
- a gene encoding hBDNF When preparing a fusion protein as a recombinant fusion protein, a gene encoding hBDNF can be linked in-frame downstream of a gene encoding HSA to obtain a fusion protein having the amino acid sequence of hBDNF at the C-terminus of the amino acid sequence of HSA. Conversely, a gene encoding hBDNF can be linked in-frame upstream of a gene encoding HSA to obtain a fusion protein having the amino acid sequence of hBDNF at the N-terminus of the amino acid sequence of HSA.
- a gene encoding hNGF When preparing a fusion protein as a recombinant fusion protein, a gene encoding hNGF can be linked in-frame downstream of a gene encoding HSA to obtain a fusion protein having the amino acid sequence of hNGF at the C-terminus of the amino acid sequence of HSA. Conversely, a gene encoding hNGF can be linked in-frame upstream of a gene encoding HSA to obtain a fusion protein having the amino acid sequence of hNGF at the N-terminus of the amino acid sequence of HSA.
- a gene encoding hNT-3 When preparing a fusion protein as a recombinant fusion protein, a gene encoding hNT-3 can be linked in-frame downstream of a gene encoding HSA to obtain a fusion protein having the amino acid sequence of hNT-3 at the C-terminus of the amino acid sequence of HSA. Conversely, by linking a gene encoding hNT-3 in frame upstream of the gene encoding HSA, a fusion protein having the amino acid sequence of hNT-3 at the N-terminus of the amino acid sequence of HSA can be obtained.
- a fusion protein produced as a recombinant fusion protein is a single-chain polypeptide.
- the amino acid sequence of HSA when the amino acid sequence of HSA is located on the N-terminal side of the amino acid sequence of hBDNF, hNGF, hNT-3 or hNT-4, the C-terminus of HSA and the N-terminus of hBDNF, hNGF, hNT-3 or hNT-4 are linked directly by a peptide bond or via a linker.
- the "linker” refers to a portion of the single-chain polypeptide that is present between the C-terminus of HSA and the N-terminus of hBDNF, hNGF, hNT-3 or hNT-4 and has an amino acid sequence that does not belong to HSA, hBDNF, hNGF, hNT-3 or hNT-4.
- Figure 7 shows a schematic diagram of a single-chain polypeptide HSA-hBDNF fusion protein that has HSA, a linker and hBDNF in this order from the N-terminus.
- FIG. 8 shows a schematic diagram of a single-chain polypeptide HSA-hNGF fusion protein having HSA, a linker, and hNGF in this order from the N-terminus.
- the C-terminus of HSA is bound to the N-terminus of the linker by a peptide bond
- the C-terminus of the linker is bound to the N-terminus of hNGF by a peptide bond.
- FIG. 9 shows a schematic diagram of a single-chain polypeptide HSA-hNT-3 fusion protein having HSA, a linker, and hNT-3 in this order from the N-terminus.
- the C-terminus of HSA is bound to the N-terminus of the linker by a peptide bond
- the C-terminus of the linker is bound to the N-terminus of hNT-3 by a peptide bond ...
- FIG. 10 shows a schematic diagram of a single-chain polypeptide HSA-hNT-4 fusion protein having HSA, a linker, and hNT-4 in this order from the N-terminus.
- the HSA-hNT-4 fusion protein is composed of the C-terminus of HSA and the N-terminus of the linker bound by a peptide bond, and the C-terminus of the linker and the N-terminus of hNT-4 bound by a peptide bond.
- the amino acid sequence of hBDNF, hNGF, hNT-3 or hNT-4 is located at the N-terminus of the amino acid sequence of HSA in the single-chain polypeptide of the fusion protein, the C-terminus of hBDNF, hNGF, hNT-3 or hNT-4 and the N-terminus of HSA are linked directly by a peptide bond or via a linker.
- the "linker” refers to a portion of the single-chain polypeptide that is present between the C-terminus of hBDNF, hNGF, hNT-3 or hNT-4 and the N-terminus of HSA and has an amino acid sequence that does not belong to HSA, hBDNF, hNGF, hNT-3 or hNT-4.
- Figure 11 shows a schematic diagram of a single-chain polypeptide hBDNF-HSA fusion protein having hBDNF, a linker and HSA in that order from the N-terminus.
- FIG. 12 shows a schematic representation of a single-chain polypeptide hNGF-HSA fusion protein having hNGF, a linker, and HSA in this order from the N-terminus.
- FIG. 13 shows a schematic representation of a single-chain polypeptide hNT-3-HSA fusion protein having hNT-3, a linker, and HSA in this order from the N-terminus.
- FIG. 14 shows a schematic diagram of the hNT-4-HSA fusion protein, which is a single-chain polypeptide having, in order from the N-terminus, hNT-4, a linker, and HSA.
- the C-terminus of hNT-4 and the N-terminus of the linker are bound by a peptide bond
- the C-terminus of the linker and the N-terminus of HSA are bound by a peptide bond.
- the amino acid sequence of the peptide linker is composed of one or more amino acids. When the peptide linker is composed of multiple amino acids, the number of amino acids is preferably 2 to 50, more preferably 5 to 30, and even more preferably 10 to 25. Suitable examples of peptide linkers include those consisting of Gly-Ser, Gly-Gly-Ser, or amino acid sequences shown in SEQ ID NOs: 9 to 11 (collectively referred to as basic sequences), and those containing these.
- the peptide linker is one that contains an amino acid sequence in which the basic sequence is repeated 2 to 10 times, one that contains an amino acid sequence in which the basic sequence is repeated 2 to 6 times, or one that contains an amino acid sequence in which the basic sequence is repeated 3 to 5 times.
- One or more amino acids in these amino acid sequences may be deleted, replaced with other amino acids, added, etc.
- the number of amino acids to be deleted is preferably 1 or 2.
- the number of amino acids to be replaced is preferably 1 or 2.
- the number of amino acids to be added is preferably 1 or 2.
- the amino acid sequence of the desired linker portion can be obtained by combining these deletions, substitutions, and additions of amino acids.
- the peptide linker may be composed of one amino acid, and the amino acid that constitutes the linker is, for example, glycine or serine.
- a fusion protein of HSA and hBDNF refers to a fusion protein characterized in that when expressed in a host cell as a recombinant protein, particularly when expressed so that the recombinant protein is secreted from the cells and accumulated in the culture medium, the amount of hBDNF expressed in the culture supernatant is at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, compared to when wild-type hBDNF is expressed in a host cell as a recombinant protein under the same conditions, such as 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, 10 to 20 times, 20 to 30 times, etc.
- the term "fusion protein of HSA and hNGF” refers to a fusion protein characterized in that, when expressed in a host cell as a recombinant protein, in particular when expressed so that the recombinant protein is secreted from the cells and accumulated in the culture medium, the amount of hNGF expressed in the culture supernatant is at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, compared to when wild-type hNGF is expressed in a host cell as a recombinant protein under the same conditions, such as 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, 10 to 20 times, 20 to 30 times, etc.
- a fusion protein of HSA and hNT-3 refers to a fusion protein characterized in that, when expressed in a host cell as a recombinant protein, particularly when expressed so that the recombinant protein is secreted from the cells and accumulated in the culture medium, the expression amount of hNT-3 in the culture supernatant is at least 1.1 times or more, 1.2 times or more, 1.5 times or more, 2 times or more, 2.5 times or more, or 3.5 times or more, in terms of concentration or physiological activity, compared to when wild-type hNT-3 is expressed in a host cell as a recombinant protein under the same conditions, (4)
- a fusion protein of HSA and hNT-4 refers to a fusion protein characterized in that, when expressed in host cells as a recombinant protein, in particular when expressed so that the recombinant protein is secreted from the cells and accumulated in the culture medium, the expression amount of
- "under the same conditions” means that the expression vector, host cells, culture conditions, etc. are the same.
- the preferred host cells used in this case are mammalian cells such as CHO cells and NS/0 cells, and particularly CHO cells.
- Preferred embodiments of the fusion protein of HSA and hBDNF include the following (1) to (4): (1) A peptide having an amino acid sequence shown in SEQ ID NO:68, in which the N-terminus of the amino acid sequence of wild-type hBDNF shown in SEQ ID NO:60 is linked to the C-terminus of the amino acid sequence of wild-type HSA shown in SEQ ID NO:3 via the linker sequence Gly-Ser; (2) Having the amino acid sequence shown in SEQ ID NO: 76, in which the N-terminus of the amino acid sequence of wild-type HSA shown in SEQ ID NO: 3 is linked to the C-terminus of the amino acid sequence of wild-type hBDNF shown in SEQ ID NO: 60 via the linker sequence Gly-Ser.
- Preferred embodiments of the fusion protein of HSA and hNGF include the following (1) to (4): (1) A peptide having an amino acid sequence shown in SEQ ID NO: 70, in which the N-terminus of the amino acid sequence of wild-type hNGF shown in SEQ ID NO: 62 is linked to the C-terminus of the amino acid sequence of wild-type HSA shown in SEQ ID NO: 3 via the linker sequence Gly-Ser; (2) Having the amino acid sequence shown in SEQ ID NO: 78, in which the N-terminus of the amino acid sequence of wild-type HSA shown in SEQ ID NO: 3 is linked to the C-terminus of the amino acid sequence of wild-type hNGF shown in SEQ ID NO: 62 via the linker sequence Gly-Ser.
- preferred embodiments of the fusion protein of HSA and hNT-3 include the following (1) to (4): (1) A peptide having an amino acid sequence shown in SEQ ID NO: 72, in which the N-terminus of the amino acid sequence of wild-type hNT-3 shown in SEQ ID NO: 64 is linked to the C-terminus of the amino acid sequence of wild-type HSA shown in SEQ ID NO: 3 via the linker sequence Gly-Ser; (2) Having the amino acid sequence shown in SEQ ID NO: 80, in which the N-terminus of the amino acid sequence of wild-type HSA shown in SEQ ID NO: 3 is linked to the C-terminus of the amino acid sequence of wild-type hNT-3 shown in SEQ ID NO: 64 via the linker sequence Gly-Ser.
- Preferred embodiments of the fusion protein of HSA and hNT-4 include the following (1) to (4): (1) A peptide having an amino acid sequence shown in SEQ ID NO: 74, in which the N-terminus of the amino acid sequence of wild-type hNT-4 shown in SEQ ID NO: 66 is linked to the C-terminus of the amino acid sequence of wild-type HSA shown in SEQ ID NO: 3 via the linker sequence Gly-Ser; (2) Having the amino acid sequence shown in SEQ ID NO: 82, in which the N-terminus of the amino acid sequence of wild-type HSA shown in SEQ ID NO: 3 is linked to the C-terminus of the amino acid sequence of wild-type hNT-4 shown in SEQ ID NO: 66 via the linker sequence Gly-Ser.
- the fusion protein of HSA and hBDNF is characterized in that when expressed in a host cell as a recombinant protein, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the amount of hBDNF expressed in the culture supernatant is increased in terms of concentration or physiological activity compared to when wild-type hBDNF is expressed in a host cell as a recombinant protein under the same conditions. Therefore, when produced as a recombinant protein, the fusion protein of HSA and hBDNF can increase production efficiency compared to wild-type hBDNF, thereby reducing production costs.
- neurotrophic factors such as hBDNF, hNGF, hNT-3, and hNT-4.
- fusion protein of HSA and hBDNF expressed in a host cell when comparing the expression levels of a fusion protein of HSA and hBDNF expressed in a host cell as a recombinant protein with wild-type hBDNF expressed in a host cell under the same conditions, the comparison is not based on the mass of the expressed protein, but on the number of molecules of the expressed protein or on the hBDNF biological activity.
- This rule also applies to fusion proteins of HSA and hNGF, fusion proteins of HSA and hNT-3, and fusion proteins of HSA and hNT-4. This rule also applies regardless of the animal species of the SA and nerve growth factor that make up the fusion protein.
- HSA and hBDNF fusion protein, HSA and hNGF fusion protein, HSA and hNT-3 fusion protein, and HSA and hNT-4 fusion protein can be produced as recombinant proteins by culturing host cells transformed with an expression vector incorporating a gene encoding the fusion protein. In this case, they can be produced using an expression vector, host cells, culture medium, etc. that can be used to produce the above-mentioned HSA and hGALC fusion protein or HSA and hGBA fusion protein.
- the protein By culturing host cells encoding a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4, the protein can be expressed in cells or in the medium. These fusion proteins can be separated from impurities and purified by methods such as column chromatography.
- the purified fusion protein of HSA and hBDNF, the fusion protein of HSA and hNGF, the fusion protein of HSA and hNT-3, or the fusion protein of HSA and hNT-4 can be used as a pharmaceutical composition.
- the fusion protein of HSA and hBDNF can be used as a pharmaceutical composition for treating neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, spinal degenerative diseases such as amyotrophic lateral sclerosis, and other diseases such as diabetic neuropathy, cerebral ischemic disease, developmental disorders such as Rett syndrome, schizophrenia, depression, and Rett syndrome.
- the fusion protein of HSA and hNGF can be used as a pharmaceutical composition for treating neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease.
- the fusion protein of HSA and hNT-3 and the fusion protein of HSA and hNT-4 can be used as a pharmaceutical composition for treating neurodegenerative diseases.
- a pharmaceutical composition containing as an active ingredient a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4 can be administered intravenously, intramuscularly, intraperitoneally, subcutaneously, or intracerebroventricularly as an injection.
- injections can be supplied as lyophilized preparations or aqueous liquid preparations.
- an aqueous liquid preparation it may be in the form of a vial filled with the preparation, or it can be supplied as a prefilled preparation in which the preparation is filled in a syringe beforehand.
- a lyophilized preparation it is dissolved in an aqueous medium and reconstituted before use.
- the fusion protein of HSA and hBDNF, the fusion protein of HSA and hNGF, the fusion protein of HSA and hNT-3, or the fusion protein of HSA and hNT-4 can be further bound to an antibody or a ligand to form a conjugate.
- the fusion protein of HSA and hBDNF or a ligand, the fusion protein of HSA and hNGF, the fusion protein of HSA and hNT-3, and the fusion protein of HSA and hNT-4 can each be bound to an antibody or a ligand that can specifically bind to a receptor on cerebrovascular endothelial cells.
- the fusion proteins of HSA and hBDNF, HSA and hNGF, HSA and hNT-3, and HSA and hNT-4 can bind to the receptor on cerebrovascular endothelial cells by being bound to an antibody or a ligand that can specifically bind to the receptor on cerebrovascular endothelial cells.
- the fusion proteins bound to the receptor on cerebrovascular endothelial cells can pass through the blood-brain barrier (BBB) and reach the tissues of the central nervous system (CNS). Therefore, these fusion proteins can be conjugated with such antibodies or ligands to pass through the blood-brain barrier (BBB) and exert their functions in the central nervous system (CNS).
- the SA is from an animal species other than human
- BDNF, NGF, hNT-3, and hNT-4 are from an animal species other than human
- the neurotrophic factors are neurotrophic factors other than BDNF, NGF, hNT-3, and hNT-4.
- hBDNF When hBDNF is said to have the function of hBDNF in a conjugate of a fusion protein of HSA and hBDNF with an antibody or ligand, it means that hBDNF has a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, for example 90% or more, 95% or more, when the specific activity of normal wild-type hBDNF is taken as 100%.
- the specific activity of hBDNF in the conjugate is calculated by multiplying the physiological activity of hBDNF per unit mass of the conjugate by (molecular weight of the conjugate/molecular weight of the portion of the conjugate corresponding to hBDNF).
- hNGF when hNGF is said to have the function of hNGF in a conjugate of a fusion protein of HSA and hNGF with an antibody or ligand, it means that hNGF has a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, for example 90% or more, 95% or more, when the specific activity of normal wild-type hNGF is taken as 100%.
- the specific activity of hNGF in the conjugate is calculated by multiplying the physiological activity of hNGF per unit mass of the conjugate by (molecular weight of the conjugate/molecular weight of the portion of the conjugate corresponding to hNGF).
- hNT-3 in a conjugate of a fusion protein of HSA and hNT-3 with an antibody or ligand is said to have the function of hNT-3, it means that hNT-3 has a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, for example 90% or more, 95% or more, when the specific activity of normal wild-type hNT-3 is taken as 100%.
- the specific activity of hNT-3 in the conjugate is calculated by multiplying the physiological activity of hNT-3 per unit mass of the conjugate by (molecular weight of the conjugate/molecular weight of the portion of the conjugate corresponding to hNT-3).
- hNT-4 when hNT-4 is said to have the function of hNT-4 in a conjugate of a fusion protein of HSA and hNT-4 with an antibody or ligand, it means that hNT-4 has a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, for example 90% or more, 95% or more, when the specific activity of normal wild-type hNT-4 is taken as 100%.
- the specific activity of hNT-4 in the conjugate is calculated by multiplying the physiological activity of hNT-4 per unit mass of the conjugate by (molecular weight of the conjugate/molecular weight of the portion of the conjugate corresponding to hNT-4).
- a conjugate between a fusion protein of HSA and hBDNF and an antibody or ligand is characterized in that when the conjugate is expressed as a recombinant protein in a host cell, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the amount of hBDNF expressed in the culture supernatant is at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, compared to when wild-type hBDNF is expressed as a recombinant protein in a host cell under the same conditions, such as 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, 10 to 20 times, 20 to 30 times, etc.
- a conjugate between a fusion protein of HSA and hNGF and an antibody or ligand is characterized in that when the conjugate is expressed as a recombinant protein in a host cell, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the amount of hNGF expressed in the culture supernatant is at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, compared to when wild-type hNGF is expressed as a recombinant protein in a host cell under the same conditions.
- a conjugate between a fusion protein of HSA and hNT-3 and an antibody or ligand is characterized in that when the conjugate is expressed as a recombinant protein in a host cell, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the expression amount of hNT-3 in the culture supernatant is at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, compared to when wild-type hNT-3 is expressed as a recombinant protein in a host cell under the same conditions.
- a conjugate between a fusion protein of HSA and hNT-4 and an antibody or ligand is characterized in that when the conjugate is expressed as a recombinant protein in a host cell, particularly when the recombinant protein is expressed so as to be secreted from the cells and accumulated in the culture medium, the expression amount of hNT-4 in the culture supernatant is at least 1.1 times, 1.2 times, 1.5 times, 2 times, 2.5 times, or 3.5 times, in terms of concentration or physiological activity, compared to when wild-type hNT-4 is expressed as a recombinant protein in a host cell under the same conditions, and is 1.1 to 4 times, 1.5 to 3.6 times, 2 to 3.6 times, 10 to 20 times, 20 to 30 times, etc.
- Some neurotrophic factors are difficult to produce in large quantities when expressed as recombinant proteins using host cells introduced with an expression vector incorporating a gene encoding the wild-type neurotrophic factor, because the expression level is limited.
- Examples of such neurotrophic factors include BDNF, NGF, NT-3, and NT-4.
- One embodiment of the present invention is a recombinant protein in which such a neurotrophic factor that is difficult to produce as a recombinant protein is fused with SA.
- SA a recombinant protein
- Such a recombinant protein can be produced in large quantities more easily using host cells introduced with an expression vector incorporating a gene encoding the recombinant protein, compared to the corresponding wild-type neurotrophic factor.
- there is no particular restriction on the animal species of the neurotrophic factor to be bound to SA but human neurotrophic factor is preferred.
- antibody primarily refers to human antibodies, mouse antibodies, humanized antibodies, antibodies derived from camelids (including alpacas), chimeric antibodies between human antibodies and antibodies from other mammals, and chimeric antibodies between mouse antibodies and antibodies from other mammals, but is not limited to these as long as it has the property of specifically binding to a specific antigen, and there is also no particular restriction on the animal species of the antibody.
- human antibody refers to an antibody whose entire protein is encoded by a gene of human origin.
- human antibodies also include antibodies encoded by genes in which mutations have been added to the original human gene without changing the original amino acid sequence for the purpose of increasing gene expression efficiency, etc.
- antibodies produced by combining two or more genes encoding human antibodies and replacing a part of a human antibody with a part of another human antibody are also "human antibodies”.
- Human antibodies have three complementarity determining regions (CDRs) in the light chain and three complementarity determining regions (CDRs) in the heavy chain. The three CDRs in the light chain are called CDR1, CDR2, and CDR3, starting from the N-terminus.
- the three CDRs in the heavy chain are also called CDR1, CDR2, and CDR3, starting from the N-terminus.
- Antibodies in which the antigen specificity, affinity, etc. of a human antibody have been modified by replacing the CDR of a certain human antibody with the CDR of another human antibody are also included in human antibodies.
- the term "human antibody” also includes antibodies in which mutations such as substitution, deletion, and addition have been added to the amino acid sequence of the original antibody by modifying the gene of the original human antibody.
- the number of amino acids to be replaced is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3.
- the number of amino acids to be deleted is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3.
- antibodies in which mutations that combine these amino acid substitutions and deletions have been added are also human antibodies.
- amino acids When amino acids are added, preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or the N-terminus or C-terminus of the original antibody.
- Antibodies in which mutations that combine these amino acid additions, substitutions, and deletions have been added are also human antibodies.
- the amino acid sequence of the mutated antibody preferably exhibits 80% or more identity to the amino acid sequence of the original antibody, more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.
- the mutations can be added to the variable region of the antibody.
- the mutations can be added to either the CDR or framework region of the variable region, but are particularly added to the framework region.
- the term "human-derived gene” includes not only the original human-derived gene, but also genes obtained by modifying it.
- humanized antibody refers to an antibody in which the amino acid sequence of a part of the variable region (e.g., all or part of the CDR in particular) is derived from a mammal other than human, and the other regions are derived from humans.
- a humanized antibody can be an antibody produced by replacing the three complementarity determining regions (CDRs) of the light chain and the three complementarity determining regions (CDRs) of the heavy chain that constitute a human antibody with the CDRs of another mammal.
- the species of the other mammal from which the CDRs are grafted to the appropriate positions of the human antibody are derived is not particularly limited as long as it is a mammal other than human, but is preferably a mouse, rat, rabbit, horse, or a non-human primate, more preferably a mouse or rat, and even more preferably a mouse.
- an antibody in which the amino acid sequence of the original humanized antibody is modified with the same mutations as those that can be added to the above-mentioned human antibody is also included in the "humanized antibody".
- chimeric antibody refers to an antibody that is formed by linking fragments of two or more different antibodies derived from two or more different species.
- a chimeric antibody between a human antibody and an antibody of another mammal is an antibody in which a part of a human antibody is replaced by a part of an antibody of a mammal other than human.
- the antibody consists of an Fc region, a Fab region, and a hinge region, which are explained below.
- a specific example of such a chimeric antibody is a chimeric antibody in which the Fc region is derived from a human antibody while the Fab region is derived from an antibody of another mammal.
- the hinge region is derived from either a human antibody or an antibody of another mammal.
- a chimeric antibody in which the Fc region is derived from another mammal while the Fab region is derived from a human antibody is included.
- the hinge region is derived from either a human antibody or an antibody of another mammal.
- an antibody can also be said to be composed of a variable region and a constant region.
- chimeric antibodies include those in which the heavy chain constant region ( CH ) and the light chain constant region ( CL ) are derived from a human antibody, while the heavy chain variable region ( VH ) and the light chain variable region ( VL ) are derived from an antibody of another mammal, and conversely, those in which the heavy chain constant region ( CH ) and the light chain constant region ( CL ) are derived from an antibody of another mammal, while the heavy chain variable region ( VH ) and the light chain variable region ( VL ) are derived from a human antibody.
- the species of the other mammal is not particularly limited as long as it is a mammal other than human, but is preferably a mouse, rat, rabbit, horse, or a non-human primate, such as a mouse.
- an antibody has a basic structure consisting of a total of four polypeptide chains: two immunoglobulin light chains (or simply “light chains”) and two immunoglobulin heavy chains (or simply “heavy chains”).
- an “antibody” in addition to those having this basic structure, (1) Consists of two polypeptide chains, one light chain and one heavy chain, (2) Those consisting of a Fab region in which the Fc region has been deleted from the basic structure of an antibody in the original sense, and those consisting of a Fab region and all or part of the hinge region (including Fab, F(ab') and F(ab') 2 ), (3) A single-chain antibody, which is composed of a light chain with a linker at its C-terminus and a heavy chain at its C-terminus.
- a single-chain antibody which is composed of a heavy chain with a linker at its C-terminus and a light chain at its C-terminus.
- Single domain antibodies described below, are also included in the term "antibody.”
- the antibody in one embodiment of the present invention is an antibody derived from a camelid (including an alpaca). Some camelid antibodies are composed of two heavy chains linked by a disulfide bond. An antibody composed of two heavy chains is called a heavy chain antibody.
- a VHH is an antibody composed of a single heavy chain including the variable region of the heavy chain constituting a heavy chain antibody, or an antibody composed of a single heavy chain lacking the constant region (CH) constituting a heavy chain antibody.
- a VHH is also one of the antibodies in an embodiment of the present invention.
- Another antibody in an embodiment of the present invention is an antibody composed of two light chains linked by a disulfide bond. An antibody composed of two light chains is called a light chain antibody.
- an antibody in one embodiment of the present invention is also an antibody in which a mutation has been added to the amino acid sequence of an antibody of a camelid.
- a mutation is added to the amino acid of an antibody of a camelid, the same mutation as that which can be added to an antibody described in this specification can be added.
- the antibody in one embodiment of the present invention is a shark-derived antibody.
- a shark antibody consists of two heavy chains linked by a disulfide bond. An antibody consisting of these two heavy chains is called a heavy chain antibody.
- a VNAR is an antibody consisting of a single heavy chain that includes the variable region of the heavy chain that constitutes a heavy chain antibody, or an antibody consisting of a single heavy chain that lacks the constant region (CH) that constitutes a heavy chain antibody.
- VNAR is also one of the antibodies in an embodiment of the present invention.
- an antibody in one embodiment of the present invention is also an antibody in one embodiment of the present invention in which mutations have been added to the amino acid sequence of the shark antibody.
- mutations are added to the amino acids of a shark antibody, the same mutations that can be added to the antibodies described in this specification can be added.
- a humanized shark antibody is also one of the antibodies in one embodiment of the present invention.
- An antibody has a basic structure consisting of four polypeptide chains, two light chains and two heavy chains, and has three complementarity determining regions (CDRs) in the variable region of the light chain (V L ) and three complementarity determining regions (CDRs) in the variable region of the heavy chain (V H ).
- the three CDRs in the light chain are called CDR1, CDR2, and CDR3, starting from the N-terminus.
- the three CDRs in the heavy chain are also called CDR1, CDR2, and CDR3, starting from the N-terminus.
- these CDRs are incomplete or absent, they are included in the antibody as long as they have the property of specifically binding to a specific antigen.
- the regions other than the CDRs in the variable regions of the light and heavy chains are called framework regions (FR).
- the FRs are called FR1, FR2, FR3, and FR4, starting from the N-terminus.
- CDRs and FRs are present in the following order from the N-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
- the antibody also includes an antibody in which a mutation such as substitution, deletion, or addition has been added to the amino acid sequence of the original antibody.
- a mutation such as substitution, deletion, or addition has been added to the amino acid sequence of the original antibody.
- the number of amino acids to be replaced is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3.
- the number of amino acids to be deleted is preferably 1 to 20, more preferably 1 to 5, and even more preferably 1 to 3.
- an antibody in which a mutation combining these amino acid substitutions and deletions has been added is also an antibody.
- amino acid sequence of the mutated antibody preferably exhibits 80% or more identity with the amino acid sequence of the original antibody, more preferably 85% or more identity, and even more preferably 90% or more, 95% or more, or 98% or more identity.
- the antibody also includes an antibody in which a mutation such as substitution, deletion, or addition has been added to the amino acid sequence of the variable region of the original antibody.
- a mutation such as substitution, deletion, or addition has been added to the amino acid sequence of the variable region of the original antibody.
- the number of amino acids to be replaced is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
- the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
- an antibody in which a mutation that combines the substitution and deletion of these amino acids has been added is also an antibody.
- an amino acid preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or the N-terminus or C-terminus of the original antibody.
- An antibody in which a mutation has been added is also an antibody in which a mutation that combines the addition, substitution, and deletion of these amino acids has been added is also an antibody.
- the amino acid sequence of the antibody in which the mutation has been added is preferably 80% or more identical to the amino acid sequence of the original antibody, more preferably 85% or more identical, and even more preferably 90% or more, 95% or more, or 98% or more identical.
- mutations may be made in either the CDR or framework region of the variable region, but are particularly made in the framework region.
- the antibody also includes an antibody in which a mutation such as substitution, deletion, or addition has been added to the amino acid sequence of the framework region of the variable region of the original antibody.
- a mutation such as substitution, deletion, or addition has been added to the amino acid sequence of the framework region of the variable region of the original antibody.
- the number of amino acids to be replaced is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
- the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
- an antibody in which a mutation that combines these amino acid substitutions and deletions has been added is also an antibody.
- amino acid sequence of the mutated antibody preferably exhibits 80% or more identity to the amino acid sequence of the original antibody, more preferably 85% or more identity, and even more preferably 90% or more, 95% or more, or 98% or more identity.
- the antibody also includes an antibody in which a mutation such as substitution, deletion, or addition has been added to the amino acid sequence in the CDR region of the variable region of the original antibody.
- a mutation such as substitution, deletion, or addition has been added to the amino acid sequence in the CDR region of the variable region of the original antibody.
- the number of amino acids to be replaced is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 or 2.
- the number of amino acids to be deleted is preferably 1 to 5, more preferably 1 to 3, and even more preferably 1 or 2.
- an antibody in which a mutation combining these amino acid substitutions and deletions has been added is also an antibody.
- amino acid sequence of the mutated antibody preferably exhibits 80% or more identity to the amino acid sequence of the original antibody, more preferably 85% or more identity, and even more preferably 90% or more, 95% or more, or 98% or more identity.
- Fab refers to a molecule in which one light chain including a variable region and a CL region (light chain constant region) and one heavy chain including a variable region and a CH1 region (part 1 of the heavy chain constant region) are bound by disulfide bonds between cysteine residues present in each.
- the heavy chain may further include a part of the hinge region in addition to the variable region and the CH1 region (part 1 of the heavy chain constant region), but in this case, the hinge region lacks cysteine residues present in the hinge region that bind the heavy chains of the antibody.
- the light chain and the heavy chain are bound by disulfide bonds formed between cysteine residues present in the light chain constant region ( CL region) and cysteine residues present in the heavy chain constant region ( CH1 region) or hinge region.
- the heavy chain that forms Fab is called a Fab heavy chain.
- Fab lacks the cysteine residues present in the hinge region that bind the heavy chains of an antibody, and therefore consists of one light chain and one heavy chain.
- the light chain that constitutes Fab contains a variable region and a CL region.
- the heavy chain that constitutes Fab may be composed of a variable region and a CH1 region, or may contain a part of the hinge region in addition to the variable region and the CH1 region.
- the hinge region is selected so as not to contain a cysteine residue that binds the heavy chains, so that a disulfide bond is not formed between the two heavy chains at the hinge region.
- the heavy chain contains the whole or part of the hinge region that contains the cysteine residue that binds the heavy chains, in addition to the variable region and the CH1 region.
- F(ab') 2 refers to a molecule in which two F(ab') are bound by disulfide bonds between the cysteine residues present in the hinge regions of each other.
- the heavy chain that forms F(ab') or F(ab') 2 is called a Fab' heavy chain.
- polymers such as dimers and trimers formed by binding multiple antibodies directly or via a linker are also antibodies.
- any one that contains a part of an antibody molecule and has the property of specifically binding to an antigen is included in the "antibody” as used in the present invention. That is, the term “light chain” includes one that is derived from a light chain and has all or part of the amino acid sequence of its variable region.
- the term “heavy chain” includes one that is derived from a heavy chain and has all or part of the amino acid sequence of its variable region. Therefore, as long as it has all or part of the amino acid sequence of the variable region, for example, one that has deleted the Fc region is also a heavy chain.
- Fc or Fc region herein refers to a region in an antibody molecule that includes a fragment consisting of the CH2 region (part 2 of the heavy chain constant region) and the CH3 region (part 3 of the heavy chain constant region).
- the antibody in one embodiment of the present invention comprises: (7) Also included are scFab, scF(ab'), and scF(ab') 2 , which are single-chain antibodies formed by linking the light chain and the heavy chain constituting Fab, F(ab'), or F(ab') 2 shown in (2) above via a linker.
- scFab, scF(ab'), and scF(ab') 2 may be formed by linking a linker to the C-terminus of the light chain and further linking a heavy chain to the C-terminus, or may be formed by linking a linker to the C-terminus of the heavy chain and further linking a light chain to the C-terminus.
- antibodies also include scFv, which is a single-chain antibody formed by linking a light chain variable region and a heavy chain variable region via a linker.
- scFv is a single-chain antibody formed by linking a light chain variable region and a heavy chain variable region via a linker.
- it may be formed by linking a linker to the C-terminus of the light chain variable region and further linking a heavy chain variable region to the C-terminus, or may be formed by linking a linker to the C-terminus of the heavy chain variable region and further linking a light chain variable region to the C-terminus.
- antibody includes full-length antibodies, as well as those listed in (1) to (7) above, and also includes antigen-binding fragments (antibody fragments) in which a portion of a full-length antibody is deleted, which is a broader concept including (1) to (7).
- Antigen-binding fragments include heavy chain antibodies, light chain antibodies, VHHs, VNARs, and those in which a portion of these antibodies is deleted.
- binding fragment refers to a fragment of an antibody that retains at least a part of the specific binding activity with an antigen.
- binding fragments include Fab, Fab', F(ab') 2 , variable region (Fv), single-chain antibody (scFv) in which the heavy chain variable region ( VH ) and the light chain variable region ( VL ) are linked with an appropriate linker, diabody, which is a dimer of a polypeptide containing a heavy chain variable region ( VH ) and a light chain variable region ( VL ), minibody, which is a dimer of a scFv heavy chain (H chain) bound to a part of the constant region ( CH3 ), other low molecular weight antibodies, etc.
- it is not limited to these molecules as long as it has the ability to bind to an antigen.
- single-chain antibody refers to a protein that can specifically bind to a specific antigen, which is composed of an amino acid sequence containing all or part of the variable region of a light chain, to which a linker is attached at the C-terminus, and to which an amino acid sequence containing all or part of the variable region of a heavy chain is further attached at the C-terminus.
- a protein that can specifically bind to a specific antigen which is composed of an amino acid sequence containing all or part of the variable region of a heavy chain, to which a linker is attached at the C-terminus, and to which an amino acid sequence containing all or part of the variable region of a light chain is further attached at the C-terminus
- a single-chain antibody in which a light chain is attached to the C-terminus of a heavy chain via a linker, the heavy chain usually lacks an Fc region.
- the variable region of the light chain has three complementarity determining regions (CDRs) that are involved in the antigen specificity of the antibody. Similarly, the variable region of the heavy chain also has three CDRs.
- a single chain antibody contains all three CDRs of the heavy chain and all three CDRs of the light chain.
- a single chain antibody can also be one in which one or more CDRs are deleted, so long as the antigen-specific affinity of the antibody is maintained.
- the linker disposed between the light and heavy chains of the antibody is preferably a peptide chain composed of 2 to 50, more preferably 8 to 50, even more preferably 10 to 30, even more preferably 12 to 18 or 15 to 25, for example 15 or 25 amino acid residues.
- amino acid sequence of such a linker is preferably composed of only glycine or glycine and serine, and includes, for example, the amino acid sequence Gly-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly, the amino acid sequence Gly-Gly-Gly, the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 9), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 10), the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 11), or a sequence in which these amino acid sequences are repeated 2 to 10 times or 2 to 5 times.
- a linker containing a total of 15 amino acids corresponding to three consecutive amino acid sequences Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 9) is preferred.
- a single domain antibody refers to an antibody that has the property of specifically binding to an antigen through a single variable region.
- Single domain antibodies include antibodies whose variable region consists only of the variable region of a heavy chain (heavy chain single domain antibodies) and antibodies whose variable region consists only of the variable region of a light chain (light chain single domain antibodies).
- VHH and VNAR are types of single domain antibodies.
- the term "human transferrin receptor” refers to a membrane protein having the amino acid sequence shown in SEQ ID NO: 22.
- the antibody of the present invention specifically binds to the portion of the amino acid sequence shown in SEQ ID NO: 22 from the 89th cysteine residue from the N-terminus to the phenylalanine at the C-terminus (the extracellular domain of the transferrin receptor), but is not limited thereto.
- the most common method for producing antibodies against a desired protein is to produce a recombinant protein using cells that have been introduced with an expression vector incorporating a gene that codes for the protein, and then immunize animals such as mice with this recombinant protein. After immunization, antibody-producing cells against the recombinant protein are extracted from the animal, and these are then fused with myeloma cells to produce hybridoma cells capable of producing antibodies against the recombinant protein.
- mice for immunizing immune system cells obtained from an animal such as a mouse with a desired protein by in vitro immunization
- cells that produce antibodies against the protein can be obtained.
- in vitro immunization there is no particular limitation on the animal species from which the immune system cells are derived, but preferred are mice, rats, rabbits, guinea pigs, dogs, cats, horses, and primates including humans, more preferably mice, rats, and humans, and even more preferably mice and humans.
- splenocytes prepared from mouse spleens can be used as mouse immune system cells.
- human immune system cells cells prepared from human peripheral blood, bone marrow, spleen, etc. can be used.
- human immune system cells are immunized by in vitro immunization, human antibodies against the recombinant protein can be obtained.
- Immune system cells can be immunized by in vitro immunization, and then fused with myeloma cells to produce hybridoma cells capable of producing antibodies. It is also possible to extract mRNA from the immunized cells, synthesize cDNA, and use this cDNA as a template to amplify DNA fragments containing genes encoding the light and heavy chains of immunoglobulins through PCR reactions, which can then be used to artificially reconstruct antibody genes.
- Hybridoma cells obtained as is by the above method also include cells that produce antibodies that recognize non-target proteins as antigens. Furthermore, not all hybridoma cells that produce antibodies against a desired protein necessarily produce antibodies that exhibit the desired properties, such as having high affinity for that protein.
- artificially reconstructed antibody genes also include genes that code for antibodies that recognize untargeted proteins as antigens. Furthermore, not all genes that code for antibodies against a desired protein necessarily code for antibodies that exhibit the desired properties, such as having high affinity for that protein.
- a step is required to select hybridoma cells that produce antibodies with the desired characteristics from the hybridoma cells obtained as described above. Furthermore, in the case of artificially reconstructed antibody genes, a step is required to select genes that code for antibodies with the desired characteristics from the antibody genes.
- the method described in detail below is effective as a method for selecting hybridoma cells that produce antibodies that show high affinity for a desired protein (high affinity antibodies), or genes that code for high affinity antibodies.
- the protein when selecting hybridoma cells that produce antibodies with high affinity to a desired protein, the protein is added to a plate and retained thereon, then the culture supernatant of the hybridoma cells is added, and antibodies that are not bound to the protein are removed from the plate, and the amount of antibody retained on the plate is measured.
- the higher the affinity of the antibody contained in the culture supernatant of the hybridoma cells added to the plate for the protein the greater the amount of antibody retained on the plate. Therefore, the amount of antibody retained on the plate can be measured, and the hybridoma cells corresponding to the plate that retain more antibodies can be selected as cell lines that produce antibodies with relatively high affinity for the protein.
- mRNA can be extracted to synthesize cDNA, and the cDNA can be used as a template to amplify a DNA fragment containing a gene encoding an antibody against the protein using PCR, thereby isolating a gene encoding a high-affinity antibody.
- the artificially reconstructed antibody gene is first incorporated into an expression vector, and this expression vector is introduced into a host cell.
- the cells used as the host cell are not particularly limited, regardless of whether they are prokaryotic or eukaryotic, as long as they can express the antibody gene by introducing an expression vector incorporating the artificially reconstructed antibody gene, but cells derived from mammals such as humans, mice, and Chinese hamsters are preferred, and CHO cells derived from Chinese hamster ovaries or NS/0 cells derived from mouse myeloma are particularly preferred.
- the expression vector used to incorporate and express a gene encoding an antibody gene can be used without any particular limitation as long as it expresses the gene when introduced into a mammalian cell.
- the gene incorporated into the expression vector is located downstream of a DNA sequence (gene expression control site) that can regulate the frequency of gene transcription in a mammalian cell.
- gene expression control sites include a promoter derived from a cytomegalovirus, an SV40 early promoter, a human elongation factor-1 alpha (EF-1 ⁇ ) promoter, and a human ubiquitin C promoter.
- Mammalian cells into which such an expression vector has been introduced will express the above-mentioned artificially reconstructed antibody incorporated in the expression vector.
- a method is used in which the protein is added to a plate and retained thereon, the cell culture supernatant is brought into contact with the protein, and then antibodies that are not bound to the protein are removed from the plate, and the amount of antibody retained on the plate is measured. According to this method, the higher the affinity of the antibody contained in the cell culture supernatant for the protein, the greater the amount of antibody retained on the plate.
- cells corresponding to the plate that retain a greater number of antibodies can be selected as cell lines that produce antibodies with relatively high affinity for the protein, and thus a gene encoding an antibody with high affinity for the protein can be selected. From the cell line thus selected, a DNA fragment containing a gene encoding an antibody against the protein can be amplified using PCR to isolate a gene encoding a high affinity antibody.
- a method for producing a conjugate in which a fusion protein of SA and a lysosomal enzyme, a fusion protein of SA and a cytokine, or a fusion protein of SA and a neurotrophic factor is bound to an antibody is a method in which the fusion protein and the antibody are bound via a non-peptide linker or a peptide linker.
- non-peptide linker polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitins, and hyaluronic acid, or derivatives thereof, or combinations thereof, can be used.
- the peptide linker is a peptide chain or a derivative thereof consisting of 1 to 50 amino acids bound by peptide bonds, and its N-terminus and C-terminus form covalent bonds with either the fusion protein or the antibody, respectively, thereby binding the fusion protein and the antibody.
- the SA, lysosomal enzyme, cytokine, and neurotrophic factor may each be derived from a human or a non-human animal.
- a conjugate of a fusion protein and an antibody is produced by producing the fusion protein and the antibody separately, then reacting them with a non-peptide linker or a peptide linker to bind the fusion protein to one end of the linker and the antibody to the other end.
- the fusion protein and the antibody can each be produced as recombinant proteins.
- a conjugate in which a fusion protein of HSA and a human lysosomal enzyme is bound to an antibody via a non-peptide linker or a peptide linker a conjugate in which a fusion protein of HSA and a human cytokine is bound to an antibody via a non-peptide linker or a peptide linker, or a conjugate in which a fusion protein of HSA and a human neurotrophic factor is bound to an antibody via a non-peptide linker or a peptide linker can be produced.
- a fusion protein of HSA and hGALC a fusion protein of HSA and hGBA, a fusion protein of HSA and hIL-10, a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4 can be obtained in which the fusion protein is bound to an antibody or a ligand via a non-peptide linker or a peptide linker.
- a conjugate formed by binding a fusion protein of SA and a lysosomal enzyme, a fusion protein of SA and a cytokine, or a fusion protein of SA and a neurotrophic factor to an antibody or a ligand can be produced by binding the C-terminus or N-terminus of these fusion proteins to the N-terminus or C-terminus of an antibody or ligand via a peptide bond, either directly or via a linker.
- Such conjugates can be produced as recombinant proteins by the production methods exemplified below.
- an antibody and a fusion protein of HSA and hGALC can be produced by binding the N-terminus or C-terminus of the fusion protein to the C-terminus or N-terminus of the heavy or light chain of the antibody via a peptide bond, either directly or via a linker.
- a conjugate formed by binding an antibody to a fusion protein of HSA and hGALC can be obtained as a conjugate protein by inserting a DNA fragment in which the cDNA encoding the fusion protein of HSA and hGALC is placed in frame at the 3'-terminus or 5'-terminus of the cDNA encoding the heavy or light chain of the antibody, either directly or via a DNA fragment encoding a linker, into an expression vector for mammalian cells, and culturing mammalian cells into which this expression vector has been introduced.
- an expression vector for mammalian cells incorporating a cDNA fragment encoding the light chain of an antibody can also be introduced into the same host cell, and in the case where a DNA fragment encoding a fusion protein of HSA and hGALC is bound to a light chain, an expression vector for mammalian cells incorporating a cDNA fragment encoding the heavy chain of an antibody can also be introduced into the same host cell.
- the conjugate of the antibody and the fusion protein of HSA and hGALC can be obtained as a recombinant protein by incorporating a DNA fragment encoding a single-chain antibody or VHH linked to the 5'-end or 3'-end of the cDNA encoding the fusion protein of HSA and hGALC directly or via a DNA fragment encoding a linker into an expression vector (for mammalian cells, eukaryotic cells such as yeast, or prokaryotic cells such as E. coli) and expressing the DNA fragment in the cells into which the expression vector has been introduced.
- an expression vector for mammalian cells, eukaryotic cells such as yeast, or prokaryotic cells such as E. coli
- Conjugates of the fusion protein of HSA and hGBA and an antibody conjugates of the fusion protein of HSA and hIL-10 and an antibody, conjugates of the fusion protein of HSA and hBDNF and an antibody, conjugates of the fusion protein of HSA and hNGF and an antibody, conjugates of the fusion protein of HSA and hNT-3 and an antibody, and conjugates of the fusion protein of HSA and hNT-4 and an antibody can also be obtained as recombinant proteins in the same manner as the conjugates of the fusion protein of HSA and hGALC and an antibody.
- Antibody, HSA and hGALC can also be conjugated in a form in which an antibody is interposed between HSA and hGALC.
- the N-terminus of the heavy or light chain of the antibody can be bound to the C-terminus of HSA via a linker or directly, and hGALC can be bound to the C-terminus via a peptide bond either via a linker or directly.
- the N-terminus of the heavy or light chain of the antibody can be bound to the C-terminus of hGALC via a linker or directly, and HSA can be bound to the C-terminus via a linker or directly, respectively.
- the antibody is a single-chain antibody or VHH
- the antibody, HSA and hGALC can be made into a fusion protein in a form in which an antibody is interposed between HSA and hGALC.
- the N-terminus of the single-chain antibody or VHH can be bound to the C-terminus of HSA via a linker or directly
- hGALC can be bound to the C-terminus via a peptide bond either via a linker or directly.
- the N-terminus of a single chain antibody or VHH can be bound to the C-terminus of hGALC via a linker or directly
- HSA can be bound to the C-terminus of the N-terminus of the single chain antibody or VHH via a linker or directly, respectively, by peptide bonds.
- Similar conjugates can also be produced for lysosomal enzymes other than hGALC, such as hGBA, cytokines, such as hIL-10, and neurotrophic factors, such as hBDNF, hNGF, hNT-3, and hNT-4.
- conjugates in this manner that involve an antibody are also referred to as fusion proteins of an antibody, another human lysosomal enzyme, and HSA, fusion proteins of an antibody, another human cytokine, and HSA, and fusion proteins of an antibody, another human neurotrophic factor, and HSA.
- the above method can also be applied to the production of conjugates of fusion proteins of other human lysosomal enzymes and HSA, fusion proteins of other human cytokines and HSA, or fusion proteins of other human neurotrophic factors and HSA, and antibodies. It can also be applied to the production of conjugates in which the SA is from an animal species other than human, and conjugates in which the lysosomal enzymes, cytokines, and neurotrophic factors are from an animal species other than human.
- the expression vectors, host cells, media, etc. that can be used to produce the above-mentioned fusion protein of HSA and hGALC or the fusion protein of HSA and hGBA can also be used to produce a conjugate of the above-mentioned fusion protein and antibody.
- Preferred embodiments of the conjugates of antibody, HSA and hGALC include the following (1) to (4): (1) A conjugate in which a fusion protein of HSA and hGALC is bound to the C-terminus of an antibody heavy chain directly or via a linker, and an antibody light chain; (2) A conjugate in which a fusion protein of HSA and hGALC is bound to the N-terminus of an antibody heavy chain directly or via a linker, and an antibody light chain; (3) A conjugate in which a fusion protein of HSA and hGALC is bound to the C-terminus of an antibody light chain directly or via a linker, and an antibody heavy chain; (4) A conjugate in which a fusion protein of HSA and hGALC is bound to the N-terminus of an antibody light chain directly or via a linker, and an antibody heavy chain; (5) A conjugate in which a single-chain antibody or VHH is bound to the C-terminus of HSA directly or via a linker, and hGALC is further bound
- a preferred example of (1) is a conjugate A in which a Fab heavy chain having the amino acid sequence of SEQ ID NO:25 is connected to its C-terminus with a linker having an amino acid sequence in which SEQ ID NO:9 is repeated three times, and further connected to its C-terminus with HSA having the amino acid sequence of SEQ ID NO:3, and further connected to its C-terminus with a linker having the amino acid sequence of SEQ ID NO:9, and further connected to its C-terminus with hGALC having the amino acid sequence of SEQ ID NO:1, and a light chain of SEQ ID NO:23.
- conjugate A has the amino acid sequence of SEQ ID NO:28.
- a suitable example of (2) is a conjugate B in which a linker having the amino acid sequence of SEQ ID NO: 9 is attached to the C-terminus of HSA having the amino acid sequence of SEQ ID NO: 3, hGALC having the amino acid sequence of SEQ ID NO: 1 at its C-terminus, a linker having an amino acid sequence in which SEQ ID NO: 9 is repeated three times at its C-terminus, and a Fab heavy chain having the amino acid sequence of SEQ ID NO: 25 at its C-terminus is attached to the light chain of SEQ ID NO: 23.
- conjugate B has the amino acid sequence of SEQ ID NO: 30.
- a preferred example of (3) is a conjugate C in which a linker having an amino acid sequence of SEQ ID NO: 25, an amino acid sequence in which SEQ ID NO: 9 is repeated three times, and hGALC having the amino acid sequence of SEQ ID NO: 1 at its C-terminus, a linker having the amino acid sequence of SEQ ID NO: 9 at its C-terminus, and HSA having the amino acid sequence of SEQ ID NO: 3 at its C-terminus are bound to the light chain of SEQ ID NO: 23.
- the conjugate C has the amino acid sequence of SEQ ID NO: 32.
- a preferred example of (4) is a conjugate D in which hGALC having the amino acid sequence of SEQ ID NO:1 is connected to its C-terminus with a linker having the amino acid sequence of SEQ ID NO:9, and further connected to its C-terminus with HSA having the amino acid sequence of SEQ ID NO:3, and further connected to its C-terminus with a linker having an amino acid sequence in which SEQ ID NO:9 is repeated three times, and further connected to its C-terminus with a Fab heavy chain having the amino acid sequence of SEQ ID NO:25, and a light chain of SEQ ID NO:23.
- conjugate C has the amino acid sequence of SEQ ID NO:34.
- Preferred embodiments of the conjugates of antibody, HSA and hGBA include the following (1) to (4): (1) A conjugate in which a fusion protein of HSA and hGBA is bound to the C-terminus of an antibody heavy chain directly or via a linker, and an antibody light chain; (2) A conjugate in which a fusion protein of HSA and hGBA is bound to the N-terminus of an antibody heavy chain directly or via a linker, and an antibody light chain; (3) A conjugate in which a fusion protein of HSA and hGBA is bound to the C-terminus of an antibody light chain directly or via a linker, and an antibody heavy chain; (4) A conjugate in which a fusion protein of HSA and hGBA is bound to the N-terminus of an antibody light chain directly or via a linker, and an antibody heavy chain; (5) A conjugate in which a single-chain antibody or VHH is bound to the C-terminus of HSA directly or via a linker, and hGBA is further bound
- Preferred embodiments of the conjugate of an antibody, HSA and hIL-10 include the following (1) to (4): (1) A conjugate in which a fusion protein of HSA and hIL-10 is bound to the C-terminus of an antibody heavy chain directly or via a linker, and an antibody light chain; (2) A conjugate in which a fusion protein of HSA and hIL-10 is bound to the N-terminus of an antibody heavy chain directly or via a linker, and an antibody light chain; (3) A conjugate in which a fusion protein of HSA and hIL-10 is bound to the C-terminus of an antibody light chain directly or via a linker, and an antibody heavy chain; (4) A conjugate in which a fusion protein of HSA and hIL-10 is bound to the N-terminus of an antibody light chain directly or via a linker, and an antibody heavy chain; (5) A conjugate in which a single-chain antibody or VHH is bound to the C-terminus of HSA directly or via a linker, and hIL-10 is further
- Preferred embodiments of the conjugate of an antibody, HSA and hBDNF include the following (1) to (4): (1) A conjugate in which a fusion protein of HSA and hBDNF is bound to the C-terminus of an antibody heavy chain directly or via a linker, and an antibody light chain; (2) A conjugate in which a fusion protein of HSA and hBDNF is bound to the N-terminus of an antibody heavy chain directly or via a linker, and an antibody light chain; (3) A conjugate in which a fusion protein of HSA and hBDNF is bound to the C-terminus of an antibody light chain directly or via a linker, and an antibody heavy chain; (4) A conjugate in which a fusion protein of HSA and hBDNF is bound to the N-terminus of an antibody light chain directly or via a linker, and an antibody heavy chain; (5) A conjugate in which a single-chain antibody or VHH is bound to the C-terminus of HSA directly or via a linker, and hBDNF is further
- Preferred embodiments of the conjugate of antibody, HSA and hNGF include the following (1) to (4): (1) A conjugate in which a fusion protein of HSA and hNGF is bound to the C-terminus of an antibody heavy chain directly or via a linker, and an antibody light chain; (2) A conjugate in which a fusion protein of HSA and hNGF is bound to the N-terminus of an antibody heavy chain directly or via a linker, and an antibody light chain; (3) A conjugate in which a fusion protein of HSA and hNGF is bound to the C-terminus of an antibody light chain directly or via a linker, and an antibody heavy chain; (4) A conjugate in which a fusion protein of HSA and hNGF is bound to the N-terminus of an antibody light chain directly or via a linker, and an antibody heavy chain; (5) A conjugate in which a single-chain antibody or VHH is bound to the C-terminus of HSA directly or via a linker, and hNGF is further bound
- Preferred embodiments of the conjugate of an antibody, HSA and hNT-3 include the following (1) to (4): (1) A conjugate in which a fusion protein of HSA and hNT-3 is bound to the C-terminus of an antibody heavy chain directly or via a linker, and an antibody light chain; (2) A conjugate in which a fusion protein of HSA and hNT-3 is bound to the N-terminus of an antibody heavy chain directly or via a linker, and an antibody light chain; (3) A conjugate in which a fusion protein of HSA and hNT-3 is bound to the C-terminus of an antibody light chain directly or via a linker, and an antibody heavy chain; (4) A conjugate in which a fusion protein of HSA and hNT-3 is bound to the N-terminus of an antibody light chain directly or via a linker, and an antibody heavy chain; (5) A conjugate in which a single-chain antibody or VHH is bound to the C-terminus of HSA directly or via a linker, and hNT-3 is further
- Preferred embodiments of the conjugate of an antibody, HSA and hNT-4 include the following (1) to (4): (1) A conjugate in which a fusion protein of HSA and hNT-4 is bound to the C-terminus of an antibody heavy chain directly or via a linker, and an antibody light chain; (2) A conjugate in which a fusion protein of HSA and hNT-4 is bound to the N-terminus of an antibody heavy chain directly or via a linker, and an antibody light chain; (3) A conjugate in which a fusion protein of HSA and hNT-4 is bound to the C-terminus of an antibody light chain directly or via a linker, and an antibody heavy chain; (4) A conjugate in which a fusion protein of HSA and hNT-4 is bound to the N-terminus of an antibody light chain directly or via a linker, and an antibody heavy chain; (5) A conjugate in which a single-chain antibody or VHH is bound to the C-terminus of HSA directly or via a linker, and hNT-4 is further
- Linkers used when binding antibodies to a fusion protein of SA and a lysosomal enzyme, a fusion protein of SA and a cytokine, or a fusion protein of SA and a neurotrophic factor are described in detail below. These linkers are particularly suitable as linkers when binding antibodies to a fusion protein of HSA and hGALC, a fusion protein of HSA and hGBA, a fusion protein of HSA and hIL-10, a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4.
- the linker disposed between the antibody or ligand and the fusion protein is preferably a peptide chain composed of 1 to 60 or 1 to 50, more preferably 1 to 17, even more preferably 1 to 10, and even more preferably 1 to 5 amino acids, but the number of amino acids constituting the linker can be appropriately adjusted to 1, 2, 3, 1 to 17, 1 to 10, 10 to 40, 20 to 34, 23 to 31, 25 to 29, 27, etc.
- Such a linker is not limited in its amino acid sequence as long as the antibody linked thereto retains affinity for a receptor on cerebrovascular endothelial cells and the fusion protein linked by the linker can exert the physiological activity of the fusion protein under physiological conditions, but is preferably composed of glycine and serine.
- amino acid sequence Gly-Ser amino acid sequence Ser-Ser, the amino acid sequence Gly-Gly-Ser, the amino acid sequence Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 9), the amino acid sequence Gly-Gly-Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 10), the amino acid sequence Ser-Gly-Gly-Gly-Gly (SEQ ID NO: 11), or those containing a sequence consisting of 1 to 10 or 2 to 5 consecutive amino acids of these amino acid sequences.
- those containing the amino acid sequence Gly-Ser can be suitably used as a linker.
- a linker containing a total of 27 amino acids consisting of the amino acid sequence Gly-Ser followed by five consecutive amino acid sequences of Gly-Gly-Gly-Gly-Ser SEQ ID NO: 9 can be preferably used as the linker.
- a linker containing a total of 25 amino acids consisting of five consecutive amino acid sequences of Gly-Gly-Gly-Gly-Ser can also be preferably used as the linker.
- each linker is named, starting from the N-terminus, as the first linker, the second linker, and so on.
- antibodies to be bound to the fusion protein of SA and a lysosomal enzyme, the fusion protein of SA and a cytokine, or the fusion protein of SA and a neurotrophic factor include the following anti-human transferrin receptor antibodies (anti-hTfR antibodies).
- a light chain variable region comprising the amino acid sequence of SEQ ID NO:96 as CDR1, the amino acid sequence of SEQ ID NO:97 as CDR2, and the amino acid sequence of SEQ ID NO:98 as CDR3, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:99 as CDR1, the amino acid sequence of SEQ ID NO:100 as CDR2, and the amino acid sequence of SEQ ID NO:101 as CDR3, respectively; and (2) a light chain variable region comprising the amino acid sequence of SEQ ID NO:96 as CDR1, the amino acid sequence of SEQ ID NO:97 as CDR2, and the amino acid sequence of SEQ ID NO:98 as CDR3, respectively, and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:102 as CDR1, the amino acid sequence of SEQ ID NO:103 as CDR2, and the amino acid sequence of SEQ ID NO:104 as CDR3,
- the present invention is not limited to these, and the above amino acid sequences may be appropriately mutated by substitution, deletion, addition
- the anti-hTfR antibodies (1) and (2) above are particularly suitable as antibodies to be bound to a fusion protein of HSA and hGALC, a fusion protein of HSA and hGBA, a fusion protein of HSA and hIL-10, a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4.
- anti-hTfR antibodies (1) and (2) above with substitutions, additions, deletions, etc. can also be used as antibodies constituting the conjugate, so long as they retain their affinity for the human transferrin receptor.
- the number of amino acids to be replaced is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 or 2.
- the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 or 2. Mutations that combine these amino acid replacements and deletions can also be added.
- amino acids are added to the amino acid sequence of the light chain of the anti-hTfR antibody of (1) and (2) above, preferably 1 to 10 amino acids, more preferably 1 to 5 amino acids, even more preferably 1 to 3 amino acids, and even more preferably 1 or 2 amino acids are added to the amino acid sequence of the light chain or to the N-terminus or C-terminus. Mutations that combine the addition, substitution, and deletion of these amino acids can also be added.
- the amino acid sequence of the mutated light chain preferably has an identity of 80% or more, more preferably an identity of 90% or more, and even more preferably an identity of 95% or more, with the amino acid sequence of the original light chain.
- the number of amino acids to be replaced is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 or 2.
- the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 or 2. Mutations that combine these amino acid substitutions and deletions can also be added.
- amino acids are added to the amino acid sequence of the heavy chain of the anti-hTfR antibody of (1) and (2) above, preferably 1 to 10 amino acids, more preferably 1 to 5 amino acids, even more preferably 1 to 3 amino acids, and even more preferably 1 or 2 amino acids are added to the amino acid sequence of the heavy chain or to the N-terminus or C-terminus. Mutations that combine the addition, substitution, and deletion of these amino acids can also be added.
- the amino acid sequence of the mutated heavy chain preferably has an identity of 80% or more, more preferably an identity of 90% or more, and even more preferably an identity of 95% or more, with the amino acid sequence of the original heavy chain.
- antibodies to be bound to the fusion protein of SA and a lysosomal enzyme, the fusion protein of SA and a cytokine, or the fusion protein of SA and a neurotrophic factor include the following anti-hTfR heavy chain antibody variable region (VHH) peptides (hTfR affinity peptides): (3) An hTfR affinity peptide comprising the amino acid sequence of SEQ ID NO: 105 or SEQ ID NO: 106 as CDR1, the amino acid sequence of SEQ ID NO: 107 or SEQ ID NO: 108 as CDR2, and the amino acid sequence of SEQ ID NO: 109 or SEQ ID NO: 110 as CDR3, and (4) an hTfR affinity peptide comprising the amino acid sequence of SEQ ID NO: 105 or SEQ ID NO: 106 as CDR1, the amino acid sequence of SEQ ID NO: 111 or SEQ ID NO: 112 as CDR2, and the amino acid sequence of SEQ ID NO: 109 or SEQ ID NO: 110
- the hTfR affinity peptides (3) and (4) above are particularly suitable as antibodies to be bound to a fusion protein of HSA and hGALC, a fusion protein of HSA and hGBA, a fusion protein of HSA and hIL-10, a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4.
- hTfR affinity peptides (3) and (4) can also be used as antibodies that contain substitutions, additions, deletions, etc., as long as they retain their affinity for the human transferrin receptor.
- the number of amino acids to be replaced is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 or 2.
- the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, even more preferably 1 to 3, and even more preferably 1 or 2. Mutations that combine these amino acid substitutions and deletions can also be added.
- amino acids are added to the amino acid sequence of the hTfR affinity peptide in (3) and (4) above, preferably 1 to 10 amino acids are added to the amino acid sequence of the light chain or to the N-terminus or C-terminus, more preferably 1 to 5 amino acids, even more preferably 1 to 3 amino acids, and even more preferably 1 or 2 amino acids. Mutations that combine the addition, substitution, and deletion of these amino acids can also be added.
- the amino acid sequence of the mutated light chain preferably has an identity of 80% or more, more preferably 90% or more, and even more preferably 95% or more with the amino acid sequence of the original hTfR affinity peptide.
- the fusion protein of HSA and hGALC can be bound to an antibody or ligand for a receptor on cerebrovascular endothelial cells to form a conjugate that can pass through the blood-brain barrier and function in the brain. Therefore, the conjugate can be used to manufacture a drug for administration into the blood to treat central nervous system diseases caused by GALC deficiency, such as Krabbe disease.
- the conjugate can also be used in a treatment method that includes administering a therapeutically effective amount of the conjugate into the blood (including intravenous injection such as intravenous drip) to a patient for central nervous system diseases caused by GALC deficiency, such as Krabbe disease.
- the conjugate administered into the blood can reach not only the brain, but also other organs and tissues that express GALC.
- the drug can also be used to prevent the onset of the disease.
- the fusion protein of HSA and hGBA can be bound to an antibody or ligand for a receptor on cerebrovascular endothelial cells to form a conjugate that can pass through the blood-brain barrier and function in the brain. Therefore, the conjugate can be used to manufacture a drug for administration into the blood to treat central nervous system diseases caused by GBA deficiency, such as Gaucher disease.
- the conjugate can also be used in a treatment method that includes administering a therapeutically effective amount of the conjugate into the blood (including intravenous injection such as intravenous drip) to a patient for central nervous system diseases caused by GBA deficiency, such as Gaucher disease.
- the conjugate administered into the blood can reach not only the brain but also other organs and tissues.
- the drug can also be used to prevent the onset of the disease.
- the fusion protein of HSA and hIL-10 can be bound to an antibody or ligand for a receptor on cerebrovascular endothelial cells to form a conjugate that can pass through the blood-brain barrier and exert its function in the brain. Therefore, the conjugate can be used to manufacture a drug for administration to the blood for the treatment of a disease of the central nervous system.
- the conjugate can also be used in a treatment method that includes administering a therapeutically effective amount of the disease of the central nervous system to a patient in the blood (including intravenous injection such as intravenous drip).
- the conjugate administered to the blood can reach not only the brain, but also other organs and tissues in which a therapeutic effect can be exerted by IL-10.
- the drug can also be used to prevent the onset of the disease.
- the fusion protein of HSA and hBDNF can be bound to an antibody or ligand for a receptor on cerebrovascular endothelial cells to form a conjugate that can pass through the blood-brain barrier and exert its function in the brain. Therefore, the conjugate can be used to manufacture a drug for administration to the blood for the treatment of a disease of the central nervous system.
- the conjugate can also be used in a treatment method that includes administering a therapeutically effective amount of the disease of the central nervous system to a patient in the blood (including intravenous injection such as intravenous drip).
- the conjugate administered to the blood can reach not only the brain, but also other organs and tissues in which BDNF can exert a therapeutic effect.
- the drug can also be used to prevent the onset of the disease.
- the fusion protein of HSA and hNGF can be bound to an antibody or ligand for a receptor on cerebrovascular endothelial cells to form a conjugate that can pass through the blood-brain barrier and exert its function in the brain. Therefore, the conjugate can be used to manufacture a drug for administration to the blood for the treatment of diseases of the central nervous system.
- the conjugate can also be used in a treatment method that includes administering a therapeutically effective amount of the conjugate to the blood of a patient (including intravenous injection such as intravenous drip infusion).
- the conjugate administered to the blood can reach not only the brain, but also other organs and tissues in which NGF can exert a therapeutic effect.
- the drug can also be used to prevent the onset of the disease.
- the fusion protein of HSA and hNT-3 can be bound to an antibody or ligand for a receptor on cerebrovascular endothelial cells to form a conjugate that can pass through the blood-brain barrier and exert its function in the brain. Therefore, the conjugate can be used to manufacture a drug for administration to the blood for the treatment of diseases of the central nervous system.
- the conjugate can also be used in a treatment method that includes administering a therapeutically effective amount of the drug to a patient in the blood (including intravenous injection such as intravenous drip infusion).
- the conjugate administered to the blood can reach not only the brain, but also other organs and tissues in which NT-3 can exert a therapeutic effect.
- the drug can also be used to prevent the onset of the disease.
- the fusion protein of HSA and hNT-4 can be bound to an antibody or ligand for a receptor on cerebrovascular endothelial cells to form a conjugate that can pass through the blood-brain barrier and exert its function in the brain. Therefore, the conjugate can be used to manufacture a drug for administration to the blood for the treatment of a disease of the central nervous system.
- the conjugate can also be used in a treatment method that includes administering a therapeutically effective amount of the disease of the central nervous system to a patient in the blood (including intravenous injection such as intravenous drip).
- the conjugate administered to the blood can reach not only the brain, but also other organs and tissues in which NT-4 can exert a therapeutic effect.
- the drug can also be used to prevent the onset of the disease.
- HSA-hGALC fusion protein, HSA-hGBA fusion protein, HSA-hIL-10 fusion protein, HSA-hBDNF fusion protein, HSA-hNGF fusion protein, HSA-hNT-3 fusion protein, and HSA-hNT-4 fusion protein can be used as drugs that are administered into the blood and exert their medicinal effects in the central nervous system (CNS) by binding to antibodies or ligands for receptors on cerebrovascular endothelial cells.
- CNS central nervous system
- Such drugs are generally administered to patients by intravenous injection, subcutaneous injection, or intramuscular injection, but there are no particular limitations on the administration route.
- hGALC has the function of hGALC in a conjugate of an antibody and a fusion protein of HSA and hGALC
- hGALC has a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, for example 90% or more or 95% or more, when the specific activity of normal wild-type hGALC is taken as 100%.
- hGALC has the function of hGALC in a conjugate of an antibody and a fusion protein of HSA and hGALC
- hGALC has a specific activity of preferably 10% or more, more preferably 20% or more, even more preferably 50% or more, and even more preferably 80% or more, for example 90% or more or 95% or more, when the specific activity of normal wild-type hGALC is taken as 100%.
- the specific activity of hGALC in the conjugate is calculated by multiplying the enzyme activity of hGALC per unit mass of the conjugate ( ⁇ M/hour/mg protein) by (molecular weight of the conjugate/molecular weight of the portion of the conjugate corresponding to hGALC).
- the same can be said for the conjugate of the fusion protein of HSA and hGBA and an antibody.
- the same can be said for the conjugate of the fusion protein of HSA and hIL-10 and an antibody, the conjugate of the fusion protein of HSA and hBDNF and an antibody, the conjugate of the fusion protein of HSA and hNGF and an antibody, the conjugate of the fusion protein of HSA and hNT-3 and an antibody, and the conjugate of the fusion protein of HSA and hNT-4 and an antibody, although hIL-10, hBDNF, hNGF, hNT-3, and hNT-4 are not enzymes, so the specific activity is based on the physiological activity of each of the proteins hIL-10, hBDNF, hNGF, hNT-3, and hNT-4, rather than on the enzyme activity.
- the fusion protein of SA and a lysosomal enzyme, the fusion protein of SA and a cytokine, or the fusion protein of SA and a neurotrophic factor may be a conjugate with a ligand.
- the SA, the lysosomal enzyme, the cytokine, and the neurotrophic factor may all be derived from humans or from animals other than humans.
- examples of the fusion protein of SA and a lysosomal enzyme include a fusion protein of HSA and hGALC and a fusion protein of HSA and hGBA
- examples of the fusion protein of SA and a cytokine include a fusion protein of HSA and hIL-10
- examples of the fusion protein of SA and a neurotrophic factor include a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, and a fusion protein of HSA and hNT-4.
- the ligand is, for example, one that can specifically bind to a receptor on cerebrovascular endothelial cells, such as the insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and IGF receptor.
- the corresponding ligands for the insulin receptor, transferrin receptor, leptin receptor, lipoprotein receptor, and IGF receptor are insulin, leptin, lipoprotein, and IGF (IGF-1, IGF-2), respectively.
- the ligand may be a wild type or a mutant wild type, so long as it can specifically bind to the target receptor. Also, it may be a fragment of the ligand, so long as it can specifically bind to the target receptor.
- ligands are particularly suitable as ligands to be bound to a fusion protein of HSA and hGALC, a fusion protein of HSA and hGBA, a fusion protein of HSA and hIL-10, a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4.
- the transferrin when the ligand is transferrin (Tf), the transferrin may be human Tf or Tf of an animal species other than human, but is preferably human Tf.
- the transferrin may be wild-type, or may have a mutation introduced therein, so long as it has affinity for the transferrin receptor (TfR).
- the mutation may be, for example, a substitution, addition, or deletion of one or two amino acids.
- the transferrin may be full-length, or may be a fragment thereof, so long as it has affinity for the transferrin receptor (TfR). The same can be said for insulin, leptin, lipoproteins, and IGF (IGF-1, IGF-2).
- a conjugate of a fusion protein of SA and a lysosomal enzyme, a fusion protein of SA and a cytokine, or a fusion protein of SA and a neurotrophic factor, and a ligand can be produced by the following method (1) or (2): (1) A fusion protein and a ligand are conjugated via a non-peptide linker or a peptide linker; (2) The fusion protein and the ligand are produced as a recombinant protein bound via a linker sequence or directly via a peptide bond.
- non-peptide linker examples include polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitins, and hyaluronic acid, or derivatives or combinations of these.
- the peptide linker is a peptide chain or derivatives consisting of 1 to 50 peptide-bonded amino acids, whose N-terminus and C-terminus form covalent bonds with either the fusion protein or the ligand, respectively, thereby binding the fusion protein and the ligand.
- the fusion protein and the ligand are each produced as recombinant proteins.
- the fusion protein and the ligand are produced as a recombinant protein in which the N-terminus of the ligand is bound to the C-terminus of the fusion protein via a linker sequence or directly, or the N-terminus of the fusion protein is bound to the C-terminus of the ligand via a linker sequence or directly.
- the expression vectors, host cells, and media used to produce such recombinant proteins can be those used to produce the above-mentioned fusion protein of HSA and hGALC.
- the fusion protein of SA and a lysosomal enzyme, the fusion protein of SA and a cytokine, or the fusion protein of SA and a neurotrophic factor can be a conjugate with another protein (A) as well as an antibody and a ligand.
- A another protein
- the SA, the lysosomal enzyme, the cytokine, and the neurotrophic factor may be derived from humans or animals other than humans.
- examples of the fusion protein of SA and a lysosomal enzyme include a fusion protein of HSA and hGALC and a fusion protein of HSA and hGBA
- examples of the fusion protein of SA and a cytokine include a fusion protein of HSA and hIL-10
- examples of the fusion protein of SA and a neurotrophic factor include a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, and a fusion protein of HSA and hNT-4.
- a conjugate of a fusion protein of SA and a lysosomal enzyme, a fusion protein of SA and a cytokine, or a fusion protein of SA and a neurotrophic factor, and a protein (A) can be produced by the following method (1) or (2): (1) The fusion protein and protein (A) are conjugated via a non-peptide linker or a peptide linker; (2) The fusion protein and protein (A) are produced as a recombinant protein by binding them via a linker sequence or directly via a peptide bond.
- non-peptide linker examples include polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ether, biodegradable polymers, lipid polymers, chitins, and hyaluronic acid, or derivatives or combinations of these.
- the peptide linker is a peptide chain or derivatives thereof consisting of 1 to 50 peptide-bonded amino acids, and its N-terminus and C-terminus form covalent bonds with either the fusion protein or protein (A), respectively, thereby binding the fusion protein and protein (A).
- the fusion protein and protein (A) are each produced as recombinant proteins.
- the fusion protein and the ligand are produced as a recombinant protein in which the N-terminus of the ligand is bound to the C-terminus of the fusion protein via a linker sequence or directly, or the N-terminus of the fusion protein is bound to the C-terminus of the ligand via a linker sequence or directly.
- the expression vectors, host cells, and media used to produce such recombinant proteins can be those used to produce a fusion protein of HSA and hGALC.
- the conjugate of the fusion protein of HSA and hGALC with an antibody, and the conjugate of the fusion protein of HSA and hGALC with a ligand or protein (A) can both be used as a pharmaceutical composition for the treatment of Krabbe disease (galactosylceramide lipidosis, or globoid cell leukodystrophy).
- Krabbe disease galactosylceramide lipidosis, or globoid cell leukodystrophy
- the conjugate of the fusion protein of HSA and hGBA with an antibody, and the conjugate of the fusion protein of HSA and hGBA with a ligand or protein (A) can both be used as a pharmaceutical composition for the treatment of Gaucher disease.
- the conjugate of the fusion protein of HSA and hIL-10 with an antibody, and the conjugate of the fusion protein of HSA and hIL-10 with a ligand or protein (A) can both be used as pharmaceutical compositions for treating inflammatory diseases, neuropathic pain, multiple sclerosis, spinal cord injury, ALS, neuroinflammation, symptoms associated with arthritis and other joint diseases, and autoimmune diseases, etc.
- the conjugate of the fusion protein of HSA and hBDNF with an antibody, and the conjugate of the fusion protein of HSA and hBDNF with a ligand or protein (A) can both be used as pharmaceutical compositions for treating neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, spinal degenerative diseases such as amyotrophic lateral sclerosis, diabetic neuropathy, cerebral ischemic disease, developmental disorders such as Rett syndrome, schizophrenia, depression, and Rett syndrome, etc.
- neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease
- spinal degenerative diseases such as amyotrophic lateral sclerosis, diabetic neuropathy, cerebral ischemic disease, developmental disorders such as Rett syndrome, schizophrenia, depression, and Rett syndrome, etc.
- the conjugate of the fusion protein of HSA and hNGF with an antibody, and the conjugate of the fusion protein of HSA and hNGF with a ligand or protein (A) can both be used as pharmaceutical compositions for treating neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease.
- the conjugate of the fusion protein of HSA and hNT-3 with an antibody, and the conjugate of the fusion protein of HSA and hNT-3 with a ligand or protein (A) can both be used as a pharmaceutical composition for treating diseases such as neurodegenerative disorders.
- the conjugate of the fusion protein of HSA and hNT-4 with an antibody, and the conjugate of the fusion protein of HSA and hNT-4 with a ligand or protein (A) can both be used as a pharmaceutical composition for treating diseases such as neurodegenerative disorders.
- a pharmaceutical composition containing as an active ingredient a conjugate of an antibody and a fusion protein of HSA and hGALC, or a conjugate of a fusion protein of HSA and hGALC and a ligand or protein (A), can be administered intravenously, intramuscularly, intraperitoneally, subcutaneously, or intracerebroventricularly as an injection. These injections can be supplied as freeze-dried preparations or aqueous liquid preparations. In the case of an aqueous liquid preparation, it may be in the form of a vial filled with the fusion protein, or it can be supplied as a prefilled preparation in a syringe.
- the ratio of monomer to total protein is preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, for example 95% or more. The same can be said for a solution obtained by dissolving a freeze-dried preparation in an aqueous medium and reconstituting it.
- compositions comprising, as an active ingredient, a conjugate of a fusion protein of HSA and hGBA and an antibody, or a conjugate of a fusion protein of HSA and hGBA and a ligand or protein (A), a pharmaceutical composition comprising, as an active ingredient, a conjugate of a fusion protein of HSA and hIL-10 and an antibody, or a conjugate of a fusion protein of HSA and hIL-10 and a ligand or protein (A), a pharmaceutical composition comprising, as an active ingredient, a conjugate of a fusion protein of HSA and hBDNF and an antibody, or a conjugate of a fusion protein of HSA and hBDNF and a ligand or protein (A), It may also be applied to pharmaceutical compositions containing as active ingredients a conjugate of a fusion protein of SA and hNGF and an antibody, or a
- the ratio of monomers to the total protein is preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, for example 95% or more.
- HSA-hGALC fusion proteins such as HSA-hGALC and hGALC-HSA, can be expressed as recombinant proteins to obtain homogeneous monomers. From the standpoint of production control, the method of producing hGALC as a fusion protein with HSA can be said to be an excellent method of producing hGALC.
- the conjugate of the antibody and the fusion protein of HSA and hGALC is expressed as a recombinant protein, most of it is obtained as a monomer.
- the ratio of monomer to total protein is preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more, for example 95% or more.
- the conjugate of the fusion protein of HSA and hGALC with an antibody is expressed as a recombinant protein, a homogeneous monomer can be obtained. Therefore, from the standpoint of production control, the method of producing hGALC as a fusion protein of HSA and an antibody can be said to be an excellent method of producing hGALC.
- a fusion protein between SA and a lysosomal enzyme, a fusion protein between SA and a cytokine, or a fusion protein between SA and a neurotrophic factor, as well as a nucleic acid molecule containing a gene encoding the conjugate can be used in gene therapy.
- a nucleic acid molecule containing a gene encoding a conjugate between any of these fusion proteins and an antibody, a nucleic acid molecule containing a gene encoding a conjugate between any of these fusion proteins and a ligand, and a nucleic acid molecule containing a gene encoding a conjugate between any of these fusion proteins and protein (A) can also be used in gene therapy.
- the fusion protein is, for example, a fusion protein of HSA and hGALC, a fusion protein of HSA and hGBA, a fusion protein of HSA and hIL-10, a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4.
- the antibody is, for example, an anti-transferrin receptor antibody.
- the ligand is, for example, transferrin or a fragment thereof.
- a nucleic acid molecule that can be used for gene therapy includes, for example, one having the following base sequence: (1) A base sequence including a first inverted terminal repeat (ITR) or a functional equivalent thereof, a base sequence including a gene expression control site downstream of the first inverted terminal repeat (ITR), a base sequence encoding a fusion protein further downstream, and a base sequence including a second inverted terminal repeat (ITR) or a functional equivalent thereof further downstream; (2) a nucleotide sequence containing a first long terminal repeat (LTR) or a functional equivalent thereof, a nucleotide sequence downstream thereof containing a gene expression control site, a nucleotide sequence further downstream thereof encoding a fusion protein, and a nucleotide sequence further downstream thereof containing a second long terminal repeat (LTR) or a functional equivalent thereof; (3) a base sequence containing a leader or a functional equivalent thereof, a base sequence downstream thereof containing a gene expression control site, a base
- nucleic acid molecule refers primarily to either DNA, which is a polymer of deoxyribonucleotides formed by phosphodiester bonds, or RNA, which is a polymer of ribonucleotides formed by phosphodiester bonds.
- the DNA may be single-stranded (single-stranded) or double-stranded with a complementary strand.
- the DNA may be a (+) strand or a (-) strand.
- the individual deoxyribonucleotides constituting the DNA may be of a naturally occurring type or may be modified from the natural type, so long as the gene encoding the protein contained in the DNA can be translated into mRNA in the cells of a mammal (particularly a human).
- the individual deoxyribonucleotides constituting the DNA may be of a naturally occurring type or may be modified from the natural type, so long as the gene encoding the protein contained in the DNA can be translated into mRNA in the cells of a mammal (particularly a human) and the whole or part of the DNA can be replicated.
- the RNA when the "nucleic acid molecule" is RNA, the RNA may be single-stranded (single-stranded) or double-stranded with a complementary strand. When the RNA is single-stranded, the RNA may be a (+) strand or a (-) strand.
- the individual ribonucleotides constituting the RNA may be of a naturally occurring type or may be modified, so long as the gene encoding the protein contained in the RNA can be reverse transcribed into DNA in a mammalian (particularly human) cell.
- the individual ribonucleotides constituting the RNA may be of a naturally occurring type or may be modified, so long as the gene encoding the protein contained in the RNA can be translated into a protein in a mammalian (particularly human) cell. Modification of ribonucleotides is performed, for example, to suppress degradation of RNA by RNase and to increase the stability of RNA in cells.
- the term "inverted terminal repeat (ITR)" refers to a base sequence that exists at the end of the viral genome and in which the same sequence is repeated.
- ITR those derived from adeno-associated virus and those derived from adenovirus can be preferably used.
- the ITR of the adeno-associated virus is a region of approximately 145 bases in length and functions as a replication origin, etc.
- there are two inverted terminal repeats (ITR) in the nucleic acid molecule which are called the first inverted terminal repeat (ITR) and the second inverted terminal repeat (ITR), respectively.
- the ITR located on the 5' side is called the first inverted terminal repeat (ITR), and the ITR located on the 3' side is called the second inverted terminal repeat (ITR).
- the inverted terminal repeat (ITR) may be derived from any virus as long as it has at least one of the functions of an original ITR, such as functioning as an origin of replication or inserting a gene into a host cell, and the ITR of the adeno-associated virus is one suitable one.
- the serotype of AAV is not particularly limited and may be any of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
- the inverted terminal repeat (ITR) of AAV serotype 2 has a first ITR that contains the base sequence shown in SEQ ID NO: 113 and a second ITR that contains the base sequence shown in SEQ ID NO: 114.
- the ITR is not limited to the wild-type ITR, and may be modified by substitution, deletion, addition, or the like, in the base sequence of the wild-type ITR.
- the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
- the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
- a mutation combining these base substitutions and deletions can also be added.
- a base When a base is added to the wild-type ITR, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added in the base sequence of the ITR or to the 5' end or 3' end. A mutation combining these base additions, substitutions, and deletions can also be added.
- the nucleotide sequence of the mutated ITR preferably exhibits 80% or more identity to the nucleotide sequence of the wild-type ITR, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.
- a functional equivalent of an ITR is something that can be used functionally in place of an ITR.
- an ITR artificially constructed based on an ITR is also a functional equivalent of an ITR as long as it can replace an ITR.
- a functional equivalent of an AAV ITR is one that can be used functionally in place of an AAV ITR.
- an ITR artificially constructed based on an AAV ITR is also a functional equivalent of an AAV ITR as long as it can replace an AAV ITR.
- first AAV-ITR An example of a functional equivalent of the artificially constructed first AAV inverted terminal repeat (first AAV-ITR) is one having the base sequence shown in SEQ ID NO: 115 (functional equivalent of the first AAV-ITR).
- This base sequence shown in SEQ ID NO: 115 to which substitutions, deletions, or mutations have been added is also included in the functional equivalent of the first AAV-ITR, so long as it can be used functionally in place of the ITR of the first AAV.
- the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3.
- the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3.
- an ITR to which mutations that combine the substitution and deletion of these bases have been added is also a functional equivalent of the first AAV-ITR.
- bases are added to the base sequence shown in SEQ ID NO:115, preferably 1 to 20 bases, more preferably 1 to 10 bases, even more preferably 1 to 5 bases, and even more preferably 1 to 3 bases are added to the base sequence or to the 5' end or 3' end.
- ITRs with mutations that combine the addition, substitution, and deletion of these bases are also included in the functional equivalent of the first AAV-ITR.
- the base sequence of the mutated ITR preferably shows 80% or more identity to the base sequence shown in SEQ ID NO:115, more preferably shows 85% or more identity, even more preferably shows 90% or more identity, even more preferably shows 95% or more identity, and even more preferably shows 98% or more identity.
- an example of a functional equivalent of an artificially constructed second inverted terminal repeat is one having the base sequence shown in SEQ ID NO: 116 (functional equivalent of the second AAV-ITR).
- This base sequence shown in SEQ ID NO: 116 to which substitutions, deletions, or mutations have been added is also included in the functional equivalent of the ITR of the second AAV, so long as it can be used functionally in place of the ITR of the second AAV.
- the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3.
- the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3.
- an ITR to which mutations have been added that combine the substitution and deletion of these bases is also a functional equivalent of the ITR of the second AAV.
- bases are added to the base sequence shown in SEQ ID NO:116, preferably 1 to 20 bases, more preferably 1 to 10 bases, even more preferably 1 to 5 bases, and even more preferably 1 to 3 bases are added to the base sequence or to the 5' end or 3' end.
- ITRs with mutations that combine the addition, substitution, and deletion of these bases are also included in the functional equivalent of the ITR of the second AAV.
- the base sequence of the mutated ITR preferably shows 85% or more identity with the base sequence shown in SEQ ID NO:116, more preferably shows 85% or more identity, even more preferably shows 90% or more identity, even more preferably shows 95% or more identity, and even more preferably shows 98% or more identity.
- the term "long terminal repeat (LTR)” refers to a base sequence in which the same sequence is repeated hundreds to thousands of times, for example, at the end of a retrotransposon of a eukaryote, or a retrovirus genome, lentivirus genome, etc.
- LTRs long terminal repeats
- the LTR located on the 5' side is called the first long terminal repeat (LTR), and the LTR located on the 3' side is called the second long terminal repeat (LTR).
- the long terminal repeat (LTR) may be derived from any virus as long as it has at least one of the functions of an original LTR, such as a function as a replication origin or gene insertion into a host cell, and preferred examples include retrovirus genomes and lentiviruses.
- the LTR is not limited to a wild-type LTR, and may be a wild-type LTR with a modified base sequence, such as a substitution, deletion, or addition.
- the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
- the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Mutations combining these base substitutions and deletions can also be added.
- bases are added to the wild-type LTR preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added in the LTR base sequence or at the 5' end or 3' end. Mutations combining these base additions, substitutions, and deletions can also be added.
- the mutated LTR base sequence preferably exhibits 80% or more identity with the wild-type LTR base sequence, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.
- a functional equivalent of an LTR is something that can be used functionally in place of an LTR.
- an artificially constructed LTR based on an LTR is also a functional equivalent of an LTR as long as it can replace an LTR.
- a functional equivalent of a lentiviral LTR is one that can be used functionally in place of the lentiviral LTR.
- an artificially constructed LTR based on a lentiviral LTR is also a functional equivalent of the lentiviral LTR as long as it can replace the lentiviral LTR.
- a functional equivalent of a retroviral LTR is one that can be used functionally in place of a retroviral LTR.
- an artificially constructed LTR based on a retroviral LTR is also a functional equivalent of a retroviral LTR as long as it can replace the retroviral LTR.
- An example of a functional equivalent of the first LTR is one having the base sequence shown in SEQ ID NO: 117.
- This base sequence shown in SEQ ID NO: 117 with substitutions, deletions, or mutations is also included in the functional equivalent of the first LTR, so long as it can be functionally used as the first LTR.
- the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 20, even more preferably 1 to 5, and even more preferably 1 to 3.
- the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3.
- an LTR with a mutation that combines the substitution and deletion of these bases is also a functional equivalent of the first LTR.
- bases are added to the base sequence shown in SEQ ID NO:117, preferably 1 to 20 bases, more preferably 1 to 10 bases, even more preferably 1 to 5 bases, and even more preferably 1 to 3 bases are added to the base sequence or to the 5' end or 3' end.
- LTRs with mutations that combine the addition, substitution, and deletion of these bases are also included in the functional equivalent of the first LTR.
- the base sequence of the mutated LTR preferably shows 80% or more identity with the base sequence shown in SEQ ID NO:117, more preferably shows 85% or more identity, even more preferably shows 90% or more identity, even more preferably shows 95% or more identity, and even more preferably shows 98% or more identity.
- an example of a functional equivalent of the second LTR is one having the base sequence shown in SEQ ID NO: 118.
- This base sequence shown in SEQ ID NO: 118 with substitutions, deletions, or mutations is also included in the functional equivalent of the second LTR as long as it can be functionally used as the second LTR.
- the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3.
- the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3.
- an LTR with a mutation that combines the substitution and deletion of these bases is also a functional equivalent of the second LTR.
- bases are added to the base sequence shown in SEQ ID NO:118, preferably 1 to 20 bases, more preferably 1 to 10 bases, even more preferably 1 to 5 bases, and even more preferably 1 to 3 bases are added to the base sequence or to the 5' or 3' end.
- LTRs with mutations that combine the addition, substitution, and deletion of these bases are also included in the functional equivalent of the second LTR.
- the base sequence of the mutated LTR preferably shows 80% or more identity with the base sequence shown in SEQ ID NO:118, more preferably shows 85% or more identity, even more preferably shows 90% or more identity, even more preferably shows 95% or more identity, and even more preferably shows 98% or more identity.
- leader and trailer refer to partially complementary base sequences present at the ends of the viral genome.
- Those derived from Sendai virus can be preferably used as leaders and trailers. Both the leader and trailer of Sendai virus are regions with a chain length of approximately 50 bases. Usually, the leader is located on the 5' side and the trailer is located on the 3' side.
- Examples that are preferably used in one embodiment of the present invention include a leader derived from Sendai virus having the base sequence shown in SEQ ID NO: 119 and a trailer derived from Sendai virus having the base sequence shown in SEQ ID NO: 120.
- Mutations in the leader and trailer derived from wild-type Sendai virus can also be suitably used in the present invention.
- the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
- the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3. Mutations combining these base substitutions and deletions can also be added.
- bases are added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added in the base sequence of the leader and/or trailer or to the 5' end or 3' end.
- the mutated base sequence preferably exhibits 80% or more identity to the wild-type base sequence, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.
- the base sequence that can be used as the base sequence containing the gene expression control site that controls the expression of the gene of the fusion protein is not particularly limited as long as it can express the fusion protein in the cells, tissues, or living body of a mammal (especially a human) into which the gene encoding the fusion protein is introduced.
- cytomegalovirus-derived promoter (optionally containing an enhancer), SV40 early promoter, human elongation factor-1 ⁇ (EF-1 ⁇ ) promoter, human ubiquitin C promoter, retroviral Rous sarcoma virus LTR promoter, dihydrofolate reductase promoter, and ⁇ -actin promoter, phosphoglycerate kinase (PGK) promoter, mouse albumin promoter, human albumin promoter, and human ⁇ -1 antitrypsin promoter.
- EF-1 ⁇ human elongation factor-1 ⁇
- ubiquitin C promoter human ubiquitin C promoter
- retroviral Rous sarcoma virus LTR promoter retroviral Rous sarcoma virus LTR promoter
- dihydrofolate reductase promoter and ⁇ -actin promoter
- PGK phosphoglycerate kinase
- a synthetic promoter having the base sequence shown in SEQ ID NO: 121 containing a mouse albumin promoter downstream of a mouse ⁇ -fetoprotein enhancer can be preferably used as the gene expression control site.
- a chicken ⁇ -actin/MVM chimeric intron having the base sequence shown in SEQ ID NO: 122 may be placed downstream of the mouse ⁇ -fetoprotein enhancer/mouse albumin promoter. By placing such an intron, the expression level of the protein controlled by the gene expression control site can be increased.
- the base sequence from 1 to 219 is the mouse ⁇ -fetoprotein enhancer
- the base sequence from 241 to 549 is the mouse albumin promoter.
- the gene regulatory site may be a promoter of a gene that is expressed in an organ-specific or cell type-specific manner.
- an organ-specific expression promoter or a cell type-specific expression promoter By using an organ-specific expression promoter or a cell type-specific expression promoter, the gene encoding the fusion protein incorporated into the nucleic acid molecule can be expressed specifically in a desired organ or cell.
- the nucleic acid molecule contains a gene encoding a conjugate of a fusion protein and an antibody between a first inverted terminal repeat (ITR) and a second inverted terminal repeat (ITR).
- the fusion protein is, for example, a fusion protein of HSA and hGALC, a fusion protein of HSA and hGBA, a fusion protein of HSA and hIL-10, a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4
- the antibody is, for example, an anti-transferrin receptor antibody.
- the following (1) to (4) are examples of the nucleic acid molecule: (1) A gene encoding a conjugate between a fusion protein and an antibody between a first inverted terminal repeat (ITR) and a second inverted terminal repeat (ITR), the gene including a base sequence encoding an internal ribosome binding site downstream of a gene encoding an antibody light chain, and a base sequence encoding a conjugate in which a fusion protein is bound directly or via a linker to the C-terminus or N-terminus of an antibody heavy chain downstream of the internal ribosome binding site; (2) A gene encoding a conjugate between a first inverted terminal repeat (ITR) and a second inverted terminal repeat (ITR), the gene including a base sequence encoding an internal ribosome binding site downstream of the gene encoding a conjugate in which the fusion protein is bound directly or via a linker to the C-terminus or N-terminus of the antibody heavy chain, and a base sequence encoding an antibody light chain downstream
- the nucleic acid molecule may further include a base sequence including a gene expression control site between the first inverted terminal repeat (ITR) and the gene encoding the conjugate of the fusion protein and the antibody.
- the base sequence encoding the internal ribosome binding site may be replaced with a base sequence including a gene expression control site.
- the first gene expression control site and the second gene expression control site are referred to in order from the first inverted terminal repeat (ITR).
- the base sequence encoding the internal ribosome binding site may be replaced with a base sequence encoding a 2A self-cleaving peptide.
- the 2A peptide derived from the porcine teschovirus is a suitable example of a 2A self-cleaving peptide.
- the nucleic acid molecule contains a gene encoding a conjugate of a fusion protein and an antibody between a first long terminal repeat (LTR) and a second long terminal repeat (LTR).
- the fusion protein is, for example, a fusion protein of HSA and hGALC, a fusion protein of HSA and hGBA, a fusion protein of HSA and hIL-10, a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4
- the antibody is, for example, an anti-transferrin receptor antibody.
- the following (1) to (4) are examples of the nucleic acid molecule: (1) A gene encoding a conjugate of a fusion protein and an antibody is contained between a first long terminal repeat (LTR) and a second long terminal repeat (LTR), and the gene contains a base sequence encoding an internal ribosome binding site downstream of a gene encoding the light chain of the antibody, and a base sequence encoding a conjugate in which the fusion protein is bound directly or via a linker to the C-terminus or N-terminus of the heavy chain of the antibody downstream of the internal ribosome binding site; (2) A gene encoding a conjugate of a fusion protein and an antibody between the first long terminal repeat (LTR) and the second long terminal repeat (LTR), the gene including a base sequence encoding an internal ribosome binding site downstream of the gene encoding a conjugate in which the fusion protein is bound directly or via a linker to the C-terminus or N-terminus of the antibody heavy chain, and a base
- the nucleic acid molecule may further include a base sequence including a gene expression control site between the first long terminal repeat (LTR) and the gene encoding the conjugate of the fusion protein and the antibody.
- the base sequence encoding the internal ribosome binding site may be replaced with a base sequence including a gene expression control site.
- the first gene expression control site and the second gene expression control site are referred to in order from the first inverted terminal repeat (ITR).
- the base sequence encoding the internal ribosome binding site may be replaced with a base sequence encoding a 2A self-cleaving peptide.
- the 2A peptide derived from the porcine teschovirus is a suitable example of the 2A self-cleaving peptide.
- the expression of one peptide chain constituting the fusion protein is controlled by the gene expression control site, while the expression of the other peptide chain is controlled by the base sequence encoding the internal ribosome binding site.
- the two peptide chains pair up within the cell to form a conjugate between the fusion protein and the antibody.
- the nucleic acid molecule in one embodiment of the present invention can be used in an AAV vector system.
- AAV inverted terminal repeats
- the genes of the viral genome integrated into the genome of the host cell are hardly expressed, but when the cell is infected with a helper virus, AAV is excised from the host genome and replication of the infectious virus begins.
- the helper virus is an adenovirus
- the genes responsible for the helper function are E1A, E1B, E2A, VA1, and E4.
- the host cell is a HEK293 cell, which is a human fetal kidney tissue-derived cell transformed with adenovirus E1A and E1B, the E1A and E1B genes are originally expressed in the host cell.
- the wild-type AAV genome contains two genes, rep and cap.
- the rep proteins (rep78, rep68, rep52, and rep40) produced by the rep gene are essential for capsid formation and mediate the integration of the viral genome into the chromosome.
- the cap gene is responsible for the production of three capsid proteins (VP1, VP2, and VP3).
- a recombinant AAV virion is a virion in which the region containing the rep gene and the cap gene has been replaced with a gene that codes for a foreign protein, and then packaged in a capsid.
- rAAV virions are used as vectors to deliver foreign genes to cells for therapeutic purposes.
- Plasmid 1 having a structure containing a base sequence including a first inverted terminal repeat (ITR) and a base sequence including a second inverted terminal repeat (ITR) derived from a virus such as AAV, and a gene encoding a desired protein arranged between these two ITRs;
- a plasmid (plasmid 2) containing an AAV Rep gene having the function required to integrate the base sequence of the region (including the ITR sequence) sandwiched between the ITR sequences into the genome of a host cell, and a gene encoding an AAV capsid protein;
- a plasmid (plasmid 3) containing the E2A region, E4 region, and VA1 RNA region of adenovirus.
- the desired protein is a conjugate of a fusion protein and an antibody.
- the fusion protein is, for example, a fusion protein of HSA and hGALC, a fusion protein of HSA and hGBA, a fusion protein of HSA and hIL-10, a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4, and the antibody is, for example, an anti-transferrin receptor antibody.
- these three types of plasmids are first introduced into host cells, such as HEK293 cells, in which the adenovirus E1a and E1b genes have been incorporated into the genome, by a general transfection method. Then, a region containing a base sequence containing a first inverted terminal repeat (ITR), a base sequence containing a second inverted terminal repeat (ITR), and a gene encoding a desired protein arranged between these two ITRs is replicated in the host cell, and the resulting single-stranded DNA is packaged into the capsid protein of AAV to form a recombinant AAV virion.
- ITR inverted terminal repeat
- ITR second inverted terminal repeat
- This recombinant AAV virion has infectivity and can be used to introduce a foreign gene into a cell, tissue, or living body.
- the foreign gene is a gene encoding a conjugate of a fusion protein and an antibody. In the cell or the like to which it is introduced, the conjugate is expressed from this gene.
- the fusion protein is, for example, a fusion protein of HSA and hGALC, a fusion protein of HSA and hGBA, a fusion protein of HSA and hIL-10, a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4, and the antibody is, for example, an anti-transferrin receptor antibody.
- the Rep protein of adeno-associated virus is encoded by the AAV rep gene.
- the Rep protein has the necessary function of integrating, for example, the AAV genome into the genome of a host cell via the ITRs present in the genome.
- Rep68 and Rep78 are the translation products of two types of mRNA transcribed by alternative splicing from the same gene.
- AAV Rep protein we mean one that includes at least the two types of proteins, Rep68 and Rep78.
- the base sequence encoding the Rep protein of an adeno-associated virus refers to a base sequence encoding at least Rep68 and Rep78, or a base sequence with a mutation added thereto.
- the Rep protein is preferably that of an AAV of serotype 2, but is not limited thereto, and may be any of serotypes 1, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
- a preferred example of a base sequence encoding the Rep protein of a wild-type AAV of serotype 2 is one having the base sequence shown in SEQ ID NO: 123.
- Rep68 may be a mutant in which the amino acid sequence of wild-type Rep68 of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 has been modified by substitution, deletion, addition, or other means, so long as it exerts its function. Rep68 with these mutations is also included in Rep68.
- the number of amino acids to be replaced is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
- the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
- Rep68 with mutations that combine these amino acid substitutions and deletions is also Rep68.
- amino acids are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or to the N-terminus or C-terminus of wild-type Rep68.
- Rep68 with mutations that combine these amino acid additions, substitutions, and deletions is also included in Rep68.
- the amino acid sequence of the mutated Rep68 preferably exhibits 80% or more identity to the amino acid sequence of wild-type Rep68, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.
- Rep78 may be a mutant in which the amino acid sequence of wild-type Rep78 of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 has been modified by substitution, deletion, addition, or other means, so long as it exerts its function. Rep78 with these mutations is also included in Rep78.
- the number of amino acids to be replaced is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
- the number of amino acids to be deleted is preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3.
- Rep78 with mutations that combine these amino acid substitutions and deletions is also Rep78.
- amino acids are added, preferably 1 to 10, more preferably 1 to 5, and even more preferably 1 to 3 amino acids are added to the amino acid sequence or to the N-terminus or C-terminus of wild-type Rep78.
- Rep78 with mutations that combine these amino acid additions, substitutions, and deletions is also included in Rep78.
- the amino acid sequence of the mutated Rep78 preferably exhibits 80% or more identity to the amino acid sequence of wild-type Rep78, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.
- the functional equivalent of the AAV Rep protein refers to a substance that can be used functionally in place of Rep68, and to a substance that can be used functionally in place of Rep78.
- the functional equivalent of the Rep protein may be a mutant of the wild-type Rep protein.
- the base sequence shown in SEQ ID NO:123 can be modified by substitution, deletion, addition, etc., so long as it encodes functional Rep68 and Rep78.
- the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
- the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
- a mutation that combines the substitution and deletion of these bases can also be used as a base sequence encoding a Rep protein.
- a base When a base is added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence shown in SEQ ID NO:123 or to the 5' end or 3' end.
- a mutation that combines the addition, substitution, and deletion of these bases can also be used as a nucleic acid molecule encoding a Rep protein.
- the mutated base sequence preferably exhibits 80% or more identity, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity to the base sequence shown in SEQ ID NO: 123.
- the start codon of the genes encoding the Rep proteins Rep68 and Rep78 is ACG.
- the base sequence encoding the Cap protein of an adeno-associated virus refers to a base sequence that encodes at least VP1, a protein that constitutes the capsid of AAV, or a base sequence that includes a mutated base sequence thereof.
- VP1 is preferably that of AAV serotype 8, but is not limited thereto, and may be any of serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.
- a suitable example of a base sequence of the Cap region of serotype 8 is one that includes the base sequence shown in SEQ ID NO: 124.
- VP1 may be modified by substitution, deletion, addition, or other alteration to the amino acid sequence of wild-type VP1 of any of AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, so long as it exerts its function. In one embodiment of the present invention, VP1 with these mutations is also included in VP1.
- the number of bases to be replaced is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
- the number of bases to be deleted is preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3.
- a mutation that combines the substitution and deletion of these bases can also be used as a base sequence encoding a Cap protein.
- bases are added, preferably 1 to 20, more preferably 1 to 10, and even more preferably 1 to 3 bases are added to the base sequence shown in SEQ ID NO: 124 or to the 5' end or 3' end.
- a mutation that combines the addition, substitution, and deletion of these bases can also be used as a nucleic acid molecule encoding a Cap protein.
- the mutated base sequence preferably exhibits 80% or more identity to the base sequence shown in SEQ ID NO:124, more preferably 85% or more identity, even more preferably 90% or more identity, even more preferably 95% or more identity, and even more preferably 98% or more identity.
- the AAV vector can produce a recombinant AAV virion in which a nucleic acid molecule containing a foreign gene is packaged between the first and second ITRs.
- the recombinant AAV virion has infectivity and can be used to introduce a foreign gene into cells, tissues, or living organisms.
- the foreign gene is a gene encoding a conjugate of a fusion protein and an antibody.
- the fusion protein is, for example, a fusion protein of HSA and hGALC, a fusion protein of HSA and hGBA, a fusion protein of HSA and hIL-10, a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4, and the antibody is, for example, an anti-transferrin receptor antibody.
- the fusion protein is expressed from this gene.
- an adenovirus vector system which is a recombinant virion in which a nucleic acid molecule containing a foreign gene is packaged between the first and second ITRs and can be used to introduce a gene into cells, etc.
- a recombinant adenovirus virion is obtained in which a nucleic acid molecule containing a foreign gene is packaged between the first and second ITRs.
- the recombinant adenovirus virion has infectivity and can be used to introduce a foreign gene into a cell, tissue, or living organism.
- Lentiviruses are viruses that belong to the Lentivirus genus in the Orthoretrovirinae subfamily of the Retroviridae family, and have a single-stranded (+) strand RNA genome (ssRNA).
- the genome of lentiviruses contains essential genes, gag (a region that codes for structural proteins including capsid proteins), pol (a region that codes for a group of enzymes including reverse transcriptase), and env (a region that codes for envelope proteins required for binding to host cells), which are sandwiched between two LTRs (5'LTR and 3'LTR).
- the genome of lentiviruses contains auxiliary genes, such as Rev (a region that codes for a protein that binds to the RRE (rev responsive element) present in the viral RNA and transports the viral RNA from the nucleus to the cytoplasm), tat (a region that codes for a protein that binds to the TAR in the 5'LTR and increases the promoter activity of the LTR), and others, such as vif, vpr, vpu, and nef.
- Rev a region that codes for a protein that binds to the RRE (rev responsive element) present in the viral RNA and transports the viral RNA from the nucleus to the cytoplasm
- tat a region that codes for a protein that binds to the TAR in the 5'LTR and increases the promoter activity of the LTR
- others such as vif, vpr, vpu, and nef.
- Lentiviruses are enveloped viruses that infect cells by fusing the envelope with the cell membrane. Lentiviruses are also RNA viruses, and reverse transcriptase is present in virions. After lentivirus infection, single-stranded plus-strand DNA is replicated from the (+) strand RNA genome by reverse transcriptase, and double-stranded DNA is then synthesized. Proteins that are components of virions are expressed from this double-stranded DNA, and the (+) strand RNA genome is packaged into this to cause virions to proliferate.
- the lentivirus vector has been developed based on the genome of HIV-1, a type of lentivirus, but is not limited to this.
- a first-generation lentiviral vector consists of three types of plasmids: a packaging plasmid, an Env plasmid, and a transfer plasmid.
- the packaging plasmid has the gag and pol genes under the control of a CMV promoter, etc.
- the Env plasmid has the env gene under the control of a CMV promoter.
- the transfer plasmid has a 5'LTR, an RRE, a gene encoding a desired protein under the control of a CMV promoter, and a 3'LTR.
- a recombinant virion is obtained in which a nucleic acid molecule containing a foreign gene between the first LTR and the second LTR is packaged in the capsid protein.
- the packaging plasmid of the first-generation lentiviral vector also contains the auxiliary genes rev, tat, vif, vpr, vpu, and nef derived from the virus.
- the desired protein in one embodiment of the present invention is a conjugate of a fusion protein and an antibody.
- the fusion protein is, for example, a fusion protein of HSA and hGALC, a fusion protein of HSA and hGBA, a fusion protein of HSA and hIL-10, a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4, and the antibody is, for example, an anti-transferrin receptor antibody.
- the promoter controlling the gene encoding the desired protein is preferably a promoter other than the CMV promoter, such as an SV40 early promoter, a human elongation factor-1 ⁇ (EF-1 ⁇ ) promoter, a human ubiquitin C promoter, a Rous sarcoma virus LTR promoter of a retrovirus, a dihydrofolate reductase promoter, a ⁇ -actin promoter, a phosphoglycerate kinase (PGK) promoter, a mouse albumin promoter, a human albumin promoter, and a human ⁇ -1 antitrypsin promoter.
- a promoter other than the CMV promoter such as an SV40 early promoter, a human elongation factor-1 ⁇ (EF-1 ⁇ ) promoter, a human ubiquitin C promoter, a Rous sarcoma virus LTR promoter of a retrovirus, a dihydrofolate reductase promoter, a ⁇ -
- it can be a synthetic promoter having the base sequence shown in SEQ ID NO: 121, which includes a mouse albumin promoter downstream of a mouse alpha-fetoprotein enhancer (mouse alpha-fetoprotein enhancer/mouse albumin promoter), etc.
- Second-generation lentiviral vectors like the first-generation, consist of three plasmids: a packaging plasmid, an Env plasmid (envelope plasmid), and a transfer plasmid.
- a packaging plasmid an Env plasmid (envelope plasmid)
- Env plasmid envelope plasmid
- transfer plasmid a transfer plasmid.
- the non-essential auxiliary genes vif, vpr, vpu, and nef have been deleted from the packaging plasmid.
- Third-generation lentiviral vectors consist of four types of plasmids: packaging plasmid, Env plasmid (envelope plasmid), Rev plasmid, and transfer plasmid.
- packaging plasmid envelope plasmid
- Env plasmid envelope plasmid
- Rev plasmid Rev plasmid
- transfer plasmid transfer plasmid.
- the rev that was in the packaging plasmid in the second generation has been separated to form the Rev plasmid.
- tat has been deleted from the packaging plasmid.
- the TAR in the 5'LTR of the transfer plasmid has been replaced with a CMV promoter.
- the foreign gene is a gene encoding a conjugate of a fusion protein and an antibody. In cells, etc., into which the gene is introduced, the conjugate is expressed from this gene.
- the fusion protein is, for example, a fusion protein of HSA and hGALC, a fusion protein of HSA and hGBA, a fusion protein of HSA and hIL-10, a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4, and the antibody is, for example, an anti-transferrin receptor antibody.
- Retroviruses have a single-stranded (+) strand RNA genome (ssRNA).
- the viral genome encodes the gag (encoding structural proteins including capsid protein), pol (encoding a group of enzymes including reverse transcriptase), env (encoding envelope proteins required for binding to host cells) and packaging signal ( ⁇ ) genes, which are flanked by two long terminal repeats (LTR, 5'LTR and 3'LTR).
- LTR long terminal repeats
- 5'LTR and 3'LTR packaging signal
- Retroviral vectors are mainly developed based on mouse leukemia viruses, and the viral genome is divided to lose the ability to self-replicate while maintaining its infectivity in order to eliminate pathogenicity and increase safety.
- the first generation retroviral vector consists of a packaging plasmid (the viral genome excluding the packaging signal) and an introduction plasmid (the packaging signal, a part of gag, and a foreign gene flanked by 5'LTR and 3'LTR at both ends). Therefore, when homologous recombination occurs in the gag sequence portion that is common to the packaging plasmid and the introduction plasmid, a self-replicating retrovirus, i.e., an RC (replication competent) virus, appears.
- RC replication competent
- the 3' LTR of the first generation packaging plasmid is replaced with a polyA addition signal.
- the third generation is composed of three plasmids, and the second generation packaging plasmid is further divided into a plasmid encoding gag/pol and a plasmid encoding env. This means that for an RC virus to emerge, homologous recombination must occur simultaneously at three sites, making the probability of this occurring extremely low, further increasing safety.
- these packaging plasmids and transfer plasmids are first transferred into host cells by a general transfection method. Then, the region containing the 5'LTR and 3'LTR and the gene encoding the desired protein located between these two LTRs is replicated in the host cell, and the resulting single-stranded (+) strand RNA is packaged in the retroviral capsid protein to form a recombinant retroviral virion.
- This recombinant retroviral virion has infectivity and can be used to transfer a foreign gene into a cell, tissue, or living body.
- the foreign gene is a gene encoding a conjugate of a fusion protein and an antibody.
- the conjugate is expressed from this gene.
- the fusion protein is, for example, a fusion protein of HSA and hGALC, a fusion protein of HSA and hGBA, a fusion protein of HSA and hIL-10, a fusion protein of HSA and hBDNF, a fusion protein of HSA and hNGF, a fusion protein of HSA and hNT-3, or a fusion protein of HSA and hNT-4
- the antibody is, for example, an anti-transferrin receptor antibody.
- the nucleic acid molecule may be encapsulated in a liposome, lipid nanoparticle (LNP), or the like.
- a liposome is a spherical vesicle with a lipid bilayer, and is composed mainly of phospholipids, particularly phosphatidylcholine.
- the present invention is not limited to this, and liposomes may contain other lipids, such as egg yolk phosphatidylethanolamine, as long as they form a lipid bilayer. Since cell membranes are mainly composed of phospholipid bilayers, liposomes have the advantage of being highly biocompatible.
- a lipid nanoparticle is a particle with a diameter of 10 nm to 1000 nm, typically less than about 200 nm, that is mainly composed of lipids, and can encapsulate hydrophobic (lipophilic) molecules and is mainly composed of biocompatible lipids, such as triglycerides, diglycerides, monoglycerides, fatty acids, and steroids. It is believed that when a gene encapsulated in liposomes or lipid nanoparticles is administered to a living body, it fuses directly with the cell membrane of the cell or is taken up by endocytosis, etc., and then migrates to the nucleus and is introduced into the cell.
- the gene introduction method using liposomes or lipid nanoparticles is superior to gene introduction using a viral vector in that there is no limit to the size of the gene to be introduced and that it is highly safe.
- the liposomes, lipid nanoparticles, etc. encapsulating the nucleic acid molecule of the present invention can be used to introduce a gene of a conjugate of a fusion protein and an antibody into a cell, tissue, or living body. In the cell, etc. into which the gene is introduced, the fusion protein is expressed from the gene.
- liposomes, lipid nanoparticles, etc. includes not only the above-mentioned liposomes and lipid nanoparticles, but also polymer nanoparticles, micelles, emulsions, nanoemulsions, microspheres, nanospheres, microcapsules, nanocapsules, dendrimers, nanogels, metal nanoparticles, and any other nano-microparticles that can be used as a drug delivery system (DDS).
- DDS drug delivery system
- the behavior of a nucleic acid molecule introduced into a cell, tissue, or living body in the form of a plasmid, encapsulated in a recombinant virus virion, or encapsulated in a liposome, lipid nanoparticle, or the like is exemplified below as (1) to (6).
- the behavior of the nucleic acid molecule is not limited to these.
- the nucleic acid molecule is a single-stranded (+) strand RNA, and when introduced into a cell, a gene encoding a conjugate between a fusion protein and an antibody contained in the nucleic acid molecule is translated, thereby expressing the conjugate.
- the nucleic acid molecule is a single-stranded (+) strand RNA, which, when introduced into a cell, is reverse transcribed to form a single-stranded (+) strand DNA, which is then transcribed and translated to express the conjugate.
- the nucleic acid molecule is a single-stranded (+) strand RNA or (-) strand RNA, and when introduced into a cell, the nucleic acid molecule is reverse transcribed to form double-stranded DNA, which is then transcribed and translated to express the conjugate.
- the nucleic acid molecule is a single-stranded (+) or (-) RNA, and when introduced into a cell, the nucleic acid molecule is reverse transcribed to form double-stranded DNA, which then undergoes random or homologous recombination with the genome of the host cell and is integrated into the genome, and the integrated DNA is transcribed and translated to express the conjugate.
- the nucleic acid molecule is a single-stranded (+) strand DNA, and when introduced into a cell, the nucleic acid molecule is transcribed and translated to express the conjugate.
- the nucleic acid molecule is double-stranded DNA, and when introduced into a cell, the nucleic acid molecule is transcribed and translated to express the conjugate.
- Nucleic acid molecules in the form of plasmids, encapsulated in recombinant viral virions, or encapsulated in liposomes, lipid nanoparticles, etc. can be introduced into cells, tissues, or organisms.
- the nucleic acid molecule When the nucleic acid molecule is to be introduced into a living body, the nucleic acid molecule is administered parenterally, such as subcutaneous injection, intramuscular injection, or intravenous injection, in the form of a plasmid, encapsulated in a recombinant virus virion, or encapsulated in a liposome or lipid nanoparticle.
- parenterally such as subcutaneous injection, intramuscular injection, or intravenous injection, in the form of a plasmid, encapsulated in a recombinant virus virion, or encapsulated in a liposome or lipid nanoparticle.
- nucleic acid molecule When the nucleic acid molecule is introduced into a cell, there is no particular limitation on the type of cell, but examples include mesenchymal stem cells, dental pulp-derived stem cells, hematopoietic stem cells, embryonic stem cells, endothelial stem cells, mammary stem cells, intestinal stem cells, hepatic stem cells, pancreatic stem cells, neural stem cells, and iPS cells.
- Cells that have been transfected with the nucleic acid molecule will express the fusion protein-antibody conjugate, which can then be transplanted into a patient for therapeutic purposes.
- Example 1 Preparation of wild-type hGALC expression plasmid, HSA-hGALC expression plasmid, and hGALC-HSA expression plasmid
- pCI-neo vector Promega Corp.
- restriction enzymes MluI and NotI were treated with restriction enzymes MluI and NotI, and gel extraction and purification were performed.
- Each DNA fragment treated with a restriction enzyme was mixed with each vector treated with a restriction enzyme, and a ligation reaction was performed at 16°C for 30 to 60 minutes using Ligation high Ver.2 (Toyobo Co., Ltd.), and the DNA fragment was inserted into the pCI vector.
- Escherichia coli E. coli DH5 ⁇ Competent Cells, Takara Bio
- Escherichia coli E. coli DH5 ⁇ Competent Cells, Takara Bio
- LB liquid medium LB Broth, Sigma-Aldrich
- plasmid DNA was purified from the cells using FastGene Plasmid Mini Kit (Nihon Genetics).
- the purified plasmid DNA was subjected to restriction enzyme treatment with MluI and NotI, and separated by agarose gel electrophoresis to confirm that the desired insert DNA had been inserted. Plasmids that were confirmed to contain wild-type hGALC, HSA-hGALC, and hGALC-HSA were purified using standard methods.
- Example 2 Transient expression of wild-type hGALC, HSA-hGALC, and hGALC-HSA
- plasmids were used in which the genes encoding wild-type hGALC, HSA-hGALC, and hGALC-HSA, respectively, were incorporated into the purified pCI-neo vector obtained in Example 1.
- ExpiCHO cells were transformed with plasmids incorporating genes encoding wild-type hGALC, HSA-hGALC, and hGALC-HSA, respectively. After transformation, the cells were cultured for 8 days, and wild-type hGALC, HSA-hGALC, and hGALC-HSA were expressed in the culture supernatant, respectively. The culture solutions 6, 7, and 8 days after the start of culture were centrifuged to collect the culture supernatant.
- Example 3 Confirmation of the expression levels of wild-type hGALC, HSA-hGALC, and hGALC-HSA by transient expression (SDS page electrophoresis) 10 ⁇ L of each culture supernatant obtained in Example 2 was mixed with 8 ⁇ L of 2 ⁇ Sample Buffer (Bio-Rad) and 2 ⁇ L of 2-Mercaptoethanol, and heated at 100° C. for 3 minutes to be heat-denatured under reducing conditions.
- SDS page electrophoresis SDS page electrophoresis
- Example 4 Confirmation of expression levels of wild-type hGALC, HSA-hGALC, and hGALC-HSA by transient expression (Western blotting) Electrophoresis was performed in the same manner as described in Example 3, and the nitrocellulose membrane and the gel after electrophoresis were sandwiched between blotting papers soaked in 25 mM Tris buffer/192 mM glycine buffer containing 20% methanol, and the proteins were transferred to the nitrocellulose membrane by applying electricity at 1.0 A and 25 V for 10 minutes in a blotting device.
- the nitrocellulose membrane after transfer was immersed in PBST containing 5% skim milk and shaken for 1 hour, and then immersed in Anti-GALC antibody (Rabbit polyclonal to GALC, Abcam) diluted to 0.4 ⁇ g/mL and shaken for 1 hour. After washing the membrane with PBST, it was immersed in Anti-mouse IgG (H+L), HRP Conjugate (Promega) solution diluted to 0.4 ⁇ g/mL and shaken for 30 minutes, and washed again with PBST.
- Anti-GALC antibody Rabbit polyclonal to GALC, Abcam
- Example 5 Confirmation of expression levels of wild-type hGALC, HSA-hGALC, and hGALC-HSA by transient expression (enzyme activity measurement)
- the culture supernatant obtained in Example 2 was diluted 100-fold with citrate buffer (pH 4.5) containing 0.5% Triton X-100 to prepare a sample solution.
- the sample solution was further diluted appropriately as necessary and used for measurement.
- a standard solution was prepared by serially diluting 4-MU (4-Methylumbelliferone, Sigma-Aldrich) from 75 to 4.39 ⁇ M with 100 mM citrate buffer (pH 4.2) containing 0.1% BSA.
- a substrate solution was prepared by diluting 4-Methylumbelliferyl- ⁇ -D-galactopyranoside (Sigma-Aldrich), an artificial substrate for hGALC, to 1 mmol/L with citrate buffer (pH 4.5) containing 0.5% Triton X-100. 25 ⁇ L/well of the sample solution or standard solution was added to a microplate, and 25 ⁇ L of substrate solution was added to each well and mixed by stirring with a plate shaker. After the plate was left at 37°C for 1 hour, 150 ⁇ L of 200 mmol/L glycine-NaOH buffer (pH 10.6) was added to each well to stop the reaction.
- the fluorescence intensity of each well was then measured using a fluorescence plate reader (Gemini XPS, Molecular Devices, Inc.) (excitation wavelength 365 nm, fluorescence wavelength 460 nm). This fluorescence intensity is proportional to the concentration of 4-MU (4-Methylumbelliferone) contained in the solution in each well.
- a calibration curve was created based on the measurement results of the standard solution, and the measured values of each sample solution were interpolated to determine the enzyme activity ( ⁇ M/hour). The unit of enzyme activity indicates the amount of substrate decomposed per hour.
- Electrophoretic images and Western blotting images obtained in Example 3 are shown in Figures 15(b) and (c), respectively. From the Western blotting images, bands corresponding to wild-type hGALC, HSA-hGALC, and hGALC-HSA on the electrophoretic images were identified. The expression levels (in terms of molecular number) of the parts of wild-type hGALC, HSA-hGALC corresponding to hGALC, and the parts of hGALC-HSA corresponding to hGALC, estimated from the density of the bands in the Western blotting images, were approximately correlated with the expression levels (in terms of enzyme activity) shown in Figure 15(a) and Table 1.
- the relative values of the expression levels shown as the expression levels (in terms of enzyme activity) in Table 1 can be said to be the expression levels (in terms of molecular number) of the parts of wild-type hGALC, HSA-hGALC corresponding to hGALC, and hGALC-HSA corresponding to hGALC.
- these results show that by making wild-type hGALC into a fusion protein with HSA, it is possible to express a larger number of molecules of hGALC, which is difficult to mass-produce as a recombinant in its wild form, without reducing its specific activity by making it into a fusion protein.
- the specific activity of hGALC in the fusion protein is calculated by multiplying the enzyme activity of hGALC per unit mass of the fusion protein ( ⁇ M/hour/mg protein) by (the molecular weight of the fusion protein/the molecular weight of the portion of the fusion protein corresponding to hGALC).
- Example 7 Cell viability, etc. in transient expression of wild-type hGALC, HSA-hGALC, and hGALC-HSA
- the viable cell density and cell viability in the culture medium 6, 7, and 8 days after the start of culture of transformed ExpiCHO cells were measured using an EVE Automated Cell Counter (Nano EnTek).
- Table 2 shows the viable cell density and cell viability in the culture medium 6, 7, and 8 days after the start of culture for each of the cells expressing wild-type hGALC, HSA-hGALC, and hGALC-HSA.
- the relative viability ratios were 1.1 and 1.1 for HSA-hGALC and hGALC-HSA expressing cells, respectively, 6 days after the start of culture, 1.3 and 1.2 for HSA-hGALC and hGALC-HSA expressing cells, respectively, 7 days after the start of culture, and 1.5 and 1.2 for HSA-hGALC and hGALC-HSA expressing cells, respectively, 8 days after the start of culture.
- Example 8 Summary The above results show that when producing hGALC as a recombinant protein, producing hGALC in the form of a fusion protein of HSA-hGALC or hGALC-HSA makes it possible to obtain about 1.5 to 3.6 times the number of molecules of hGALC without decreasing the specific activity of hGALC.
- the method of producing recombinant hGALC by expressing hGALC in the form of a fusion protein of HSA-hGALC or hGALC-HSA can be said to be an extremely effective means for producing a large amount of recombinant hGALC.
- hGALC in the form of a fusion protein of HSA-hGALC or hGALC-HSA, cell death during culture can be suppressed, making it easier to purify the expressed fusion protein. This also increases the purification efficiency during purification.
- Example 9 SE-HPLC analysis of wild-type hGALC, HSA-hGALC, and hGALC-HSA obtained by transient expression
- the culture supernatant of each protein was subjected to a purification process and then subjected to SE-HPLC analysis.
- the wild-type hGALC, HSA-hGALC, and hGALC-HSA adsorbed on the strong anion exchange column were then eluted with a salt concentration gradient using 25 mM MES buffer (pH 6.5) containing 500 mM NaCl.
- each eluate containing wild-type hGALC, HSA-hGALC, and hGALC-HSA obtained in the above purification process was loaded onto the column, and then 0.2 M sodium phosphate buffer solution containing 10% ethanol was passed through the column at a flow rate of 0.5 mL/min. During this time, the absorbance (measurement wavelength 215 nm) of the effluent from the column was measured to obtain an elution profile.
- the elution profile is shown in Figure 16.
- Wild-type hGALC has two main peaks, which are considered to correspond to the hGALC tetramer and the hGALC dimer, respectively, from the left side of the elution profile.
- HSA-hGALC and hGALC-HSA both have one main peak, which is considered to be the peak of the HSA-hGALC or hGALC-HSA monomer, respectively. This shows that, compared to the case where wild-type hGALC is expressed in cells, when the fusion protein of HSA and hGALC is expressed, a molecule having hGALC activity is stably obtained as a monomer.
- the mass ratio of the monomer to the total expression amount is 90% or more in each of HSA-hGALC and hGALC-HSA.
- HSA-hGALC and hGALC-HSA can be obtained as homogeneous monomers, so from the viewpoint of production control, the method of producing hGALC as a fusion protein of hGALC with HSA can be said to be superior.
- Example 10 Preparation of plasmids expressing conjugates of anti-hTfR antibody and fusion protein of HSA and hGALC Plasmids were prepared that express the four types of proteins shown below as conjugates of an anti-hTfR antibody (Fab) consisting of a heavy chain having the amino acid sequence shown in SEQ ID NO:25 and a light chain having the amino acid sequence shown in SEQ ID NO:23, and a fusion protein of HSA and hGALC.
- Fab anti-hTfR antibody
- a conjugate consisting of a fusion protein in which the N-terminus of HSA-hGALC is linked via a linker to the C-terminus of the heavy chain of an anti-hTfR antibody (Fab) and the light chain of an anti-hTfR antibody (Fab) (referred to as Fab-HSA-hGALC);
- a conjugate consisting of a fusion protein in which the C-terminus of HSA-hGALC is linked via a linker to the N-terminus of the heavy chain of an anti-hTfR antibody (Fab) and the light chain of an anti-hTfR antibody (Fab) (HSA-hGALC-Fab)
- a conjugate consisting of a fusion protein in which the N-terminus of hGALC-HSA is linked via a linker to the C-terminus of the heavy chain of an anti-hTfR antibody (Fab) and a light chain of an anti-hTfR antibody (Fab) (referred to as Fab-hGALC-HSA);
- the pCI vector (Promega) was digested with NheI and NotI (Takara Bio), and the above DNA fragment was inserted into it by ligation.
- the resulting plasmid was named pCI MCS-modified vector ( Figure 17).
- the pCI MCS-modified vector was digested with BamHI and BglII (Takara Bio), and a DNA fragment containing the CMV immediate-early enhancer/promoter region, multicloning site, and polyA sequence was excised.
- the pCI-neo vector (Promega) was digested with BamHI and treated with CIAP (Takara Bio), and the excised DNA fragment was inserted into it by ligation.
- the resulting plasmid was named Dual(+)pCI-neo vector ( Figure 18).
- the Dual(+)pCI-neo vector into which the gene encoding the anti-hTfR antibody Fab heavy chain-HSA-hGALC was inserted was digested with restriction enzymes AscI and AfeI (New England BioLabs), and a DNA fragment containing the base sequence shown in SEQ ID NO:36, including a gene encoding the light chain of an anti-hTfR antibody (Fab) having the amino acid sequence shown in SEQ ID NO:23, digested with AscI and AfeI, was inserted into the vector.
- the resulting plasmid was used as a Fab-HSA-hGALC expression plasmid.
- an HSA-hGALC-Fab expression plasmid was prepared using a DNA fragment containing the base sequence shown in SEQ ID NO: 31, which contains a gene encoding a conjugate in which the C-terminus of HSA-hGALC is linked via a linker to the N-terminus of the heavy chain of an anti-hTfR antibody (Fab) having the amino acid sequence shown in SEQ ID NO: 30, and a DNA fragment containing the base sequence shown in SEQ ID NO: 36, which contains a gene encoding the light chain of an anti-hTfR antibody (Fab) having the amino acid sequence shown in SEQ ID NO: 23.
- Fab anti-hTfR antibody
- a Fab-hGALC-HSA expression plasmid was prepared using a DNA fragment containing the base sequence shown in SEQ ID NO: 33, which contains a gene encoding a conjugate in which the N-terminus of hGALC-HSA is linked via a linker to the C-terminus of the heavy chain of an anti-hTfR antibody (Fab) having the amino acid sequence shown in SEQ ID NO: 32, and a DNA fragment containing the base sequence shown in SEQ ID NO: 36, which contains a gene encoding the light chain of an anti-hTfR antibody (Fab) having the amino acid sequence shown in SEQ ID NO: 23.
- Fab anti-hTfR antibody
- an hGALC-HSA-Fab expression plasmid was prepared using a DNA fragment containing the base sequence shown in SEQ ID NO: 35, which contains a gene encoding a conjugate in which the C-terminus of hGALC-HSA is linked via a linker to the N-terminus of the heavy chain of an anti-hTfR antibody (Fab) having the amino acid sequence shown in SEQ ID NO: 34, and a DNA fragment containing the base sequence shown in SEQ ID NO: 36, which contains a gene encoding the light chain of an anti-hTfR antibody (Fab) having the amino acid sequence shown in SEQ ID NO: 23.
- SEQ ID NO: 35 which contains a gene encoding a conjugate in which the C-terminus of hGALC-HSA is linked via a linker to the N-terminus of the heavy chain of an anti-hTfR antibody (Fab) having the amino acid sequence shown in SEQ ID NO: 34
- Fab anti-hTfR antibody
- each conjugate expression plasmid prepared in Example 10 was used to transform ExpiCHO cells. After transformation, the cells were cultured for 8 days, and Fab-HSA-hGALC, HSA-hGALC-Fab, Fab-hGALC-HSA, and hGALC-HSA-Fab were expressed in the culture supernatant. After 8 days from the start of culture, the culture solutions were centrifuged to collect the culture supernatants.
- Example 12 Confirmation of expression levels of Fab-HSA-hGALC, HSA-hGALC-Fab, Fab-hGALC-HSA, and hGALC-HSA-Fab by transient expression (enzyme activity measurement) Using the culture supernatant obtained in Example 11 as a sample solution, the expression levels of Fab-HSA-hGALC, HSA-hGALC-Fab, Fab-hGALC-HSA, and hGALC-HSA-Fab by transient expression were confirmed by enzyme activity measurement in the same manner as described in Example 5.
- FIG. 19 shows the results of enzyme activity measurement of wild-type hGALC, Fab-HSA-hGALC, HSA-hGALC-Fab, Fab-hGALC-HSA, and hGALC-HSA-Fab.
- the values obtained in Example 2 using the culture supernatant 8 days after the start of culturing cells expressing wild-type hGALC as the sample solution were used for the enzyme activity measurement results.
- Example 13 SE-HPLC analysis of Fab-HSA-hGALC, HSA-hGALC-Fab, Fab-hGALC-HSA, and hGALC-HSA-Fab obtained by transient expression
- the culture supernatant of each protein was subjected to a purification process, followed by SE-HPLC analysis.
- the Capture Select TM CH1-XL column is an affinity column in which a ligand having the property of specifically binding to the CH1 domain of an IgG antibody is immobilized on a carrier.
- the column was then washed by supplying the same buffer in a volume 5 times the column volume.
- the column was then further washed by supplying 25 mM MES buffer (pH 6.5) in a volume 3 times the column volume.
- the bound proteins were then eluted with 5 column volumes of 20 mM acetate buffer (pH 4.0) and immediately neutralized in a container that had previously contained 250 mM MES buffer (pH 6.0) and 2 M NaCl solution.
- each eluate containing Fab-HSA-hGALC, HSA-hGALC-Fab, Fab-hGALC-HSA, and hGALC-HSA-Fab obtained in the above purification process was loaded onto the column, and 0.2 M sodium phosphate buffer solution containing 10% ethanol was further passed through the column at a flow rate of 0.5 mL/min. During this time, the absorbance (measurement wavelength 215 nm) of the effluent from the column was measured to obtain an elution profile.
- these conjugates When expressed as recombinant proteins, these conjugates can be obtained as homogeneous monomers, so from the viewpoint of production control, the method of producing hGALC as a conjugate of HSA and an antibody (here, Fab) can be said to be excellent.
- Examples 14 to 20 show the experimental results of a fusion protein of HSA and hGBA.
- Example 14 Preparation of wild-type hGBA expression plasmid pE-neo7 vector and pCAGIPuro vector (Miyahara M. et.al., J. Biol. Chem. 275, 613-618(2000)) were treated with restriction enzymes BamHI and NotI (Takara Bio), respectively, and separated by agarose gel electrophoresis.
- the pE-neo7 vector was prepared by the method described in International Publication WO2012/101998. After EtBr staining, the band containing the target DNA fragment was excised under UV irradiation, and DNA was extracted from the gel using QIAEX II Gel Extraction Kit (QIAGEN).
- Ligation reaction was performed at 16°C for 60 minutes using Ligation high Ver.2 (Toyobo Co., Ltd.) to complete the pEI-puro vector.
- the DNA fragment was mixed with the pEI-puro vector that had been treated with the restriction enzymes MluI and NotI, and a ligation reaction was carried out at 16°C for 60 minutes using Ligation high Ver.2 to complete the pEI-puro-hGBA vector.
- the pEI-puro-hGBA vector and the pE-mIRES-GS vector were treated with restriction enzymes MluI and NotI, and then gel extracted and purified.
- the pE-mIRES-GS vector was prepared by the method described in International Publication WO2013/161958.
- the vectors treated with the restriction enzymes were mixed and ligated for 60 minutes at 16°C using Ligation high Ver.2 to complete the pEmIGS-hGBA vector ( Figure 21). ).
- the pEmIGS-hGBA vector and the pCIneo vector (Promega) were treated with restriction enzymes MluI and NotI, and gel extracted and purified. Then, the vectors treated with the restriction enzymes were mixed and ligated at 16°C for 60 minutes using Ligation high Ver.2 to complete the wild-type hGBA expression plasmid, the pCIneo-hGBA vector ( Figure 22).
- Example 15 Preparation of HSA-hGBA expression plasmid and hGBA-HSA expression plasmid
- a DNA fragment having a base sequence shown in SEQ ID NO: 40 was synthesized, including a gene encoding HSA-hGBA shown in SEQ ID NO: 39, which is a fusion protein in which the C-terminus of wild-type HSA (SEQ ID NO: 3) and the N-terminus of wild-type hGBA (SEQ ID NO: 37) are linked via a linker sequence shown in SEQ ID NO: 9.
- a ligation reaction was also carried out in the same manner with a DNA fragment having the base sequence shown in SEQ ID NO: 42, which contains a gene encoding hGBA-HSA shown in SEQ ID NO: 41, a fusion protein in which the C-terminus of wild-type hGBA (SEQ ID NO: 37) and the N-terminus of wild-type HSA (SEQ ID NO: 3) are linked via a linker sequence shown in SEQ ID NO: 9, to complete the pCIneo-hGBA-HSA vector, an hGBA-HSA expression plasmid.
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Abstract
Un mode de réalisation de la présente invention permet d'exprimer, sous la forme d'une protéine de fusion avec une sérum-albumine (SA), une protéine physiologiquement active exprimée en faible quantité et/ou moins active lorsqu'une cellule hôte telle qu'une cellule CHO est normalement utilisée pour exprimer la protéine physiologiquement active en tant que protéine recombinée, ce qui permet de produire efficacement la protéine physiologiquement active en tant que protéine recombinée hautement active. La protéine de fusion peut être obtenue par liaison de la protéine physiologiquement active au côté amino-terminal de la SA, ou peut être obtenue par liaison de la protéine physiologiquement active à une borne carboxyle. En outre, ladite protéine de fusion peut être un corps couplé à un ligand ou à un anticorps.
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JP2007522806A (ja) * | 2004-02-09 | 2007-08-16 | ヒューマン ジノーム サイエンシーズ, インコーポレイテッド | アルブミン融合蛋白質 |
WO2018124121A1 (fr) * | 2016-12-26 | 2018-07-05 | Jcrファーマ株式会社 | Nouvel anticorps anti-récepteur de la transferrine humaine capable de pénétrer dans la barrière hémato-encéphalique |
WO2023090409A1 (fr) * | 2021-11-19 | 2023-05-25 | Jcrファーマ株式会社 | Peptide ayant une affinité pour le récepteur de la transferrine humaine |
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JP2007522806A (ja) * | 2004-02-09 | 2007-08-16 | ヒューマン ジノーム サイエンシーズ, インコーポレイテッド | アルブミン融合蛋白質 |
WO2018124121A1 (fr) * | 2016-12-26 | 2018-07-05 | Jcrファーマ株式会社 | Nouvel anticorps anti-récepteur de la transferrine humaine capable de pénétrer dans la barrière hémato-encéphalique |
WO2023090409A1 (fr) * | 2021-11-19 | 2023-05-25 | Jcrファーマ株式会社 | Peptide ayant une affinité pour le récepteur de la transferrine humaine |
Non-Patent Citations (1)
Title |
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MATSUDA, JUNKO: "Significance of sphingolipid activator proteins-saposins and pro-saposin-in health and disease", JOURNAL OF JAPANESE BIOCHEMICAL SOCIETY, NIPPON SEIKAGAKKAI, TOKYO, JP, vol. 89, no. 6, 25 December 2017 (2017-12-25), JP , pages 808 - 819, XP009554110, ISSN: 0037-1017, DOI: 10.14952/SEIKAGAKU.2017.890808 * |
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