WO2022191253A1 - 融合タンパク質の製造方法 - Google Patents
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- WO2022191253A1 WO2022191253A1 PCT/JP2022/010398 JP2022010398W WO2022191253A1 WO 2022191253 A1 WO2022191253 A1 WO 2022191253A1 JP 2022010398 W JP2022010398 W JP 2022010398W WO 2022191253 A1 WO2022191253 A1 WO 2022191253A1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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- C12P21/00—Preparation of peptides or proteins
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- C07K2317/55—Fab or Fab'
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- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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- C12N2510/00—Genetically modified cells
- C12N2510/02—Cells for production
Definitions
- the present invention relates to a method for producing a fusion protein.
- Recombinant proteins are used in a wide range of fields. The recent growth of biopharmaceuticals has further increased their importance. Recombinant proteins are mainly produced using E. coli, yeast, insect cells, mammalian cells, etc. as host cells (for example, Japanese National Publication of International Patent Application No. 2007-524381 (Patent Document 1)). Large amounts of recombinant protein can be obtained in a short time using these host cells. On the other hand, the expressed recombinant protein does not perform correct folding or does not undergo post-translational modifications (e.g., addition of sugar chains), so that the original function of the recombinant protein is exhibited. Sometimes I could't.
- BDNF brain-derived neurotrophic factor
- DEP brain-derived neurotrophic factor
- TrkB high-affinity BDNF receptor
- TrkB tyrosine receptor kinase B, tropomyosin receptor kinase B, or tropomyosin-related kinase B
- BDNF can be used for neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, spinal cord degenerative diseases such as amyotrophic lateral sclerosis, diabetic neuropathy, cerebral ischemic disease, developmental disorders, and integration. It is expected to be developed as a therapeutic agent for various diseases such as ataxia, depression and Rett syndrome.
- BDNF was known to be difficult to produce in large amounts as a recombinant protein.
- a fusion protein containing BDNF and an anti-transferrin receptor antibody is known as a fusion protein capable of translocating into the brain because BDNF passes through the blood-brain barrier (Patent Documents 2 and 3). , there has been a demand for a method for mass production of this as a recombinant protein.
- the present invention has been made in view of the above circumstances, and the problem to be solved by the present invention is to provide a method for producing a fusion protein with improved production efficiency.
- the present inventors found that a gene encoding a fusion protein containing BDNF and an anti-transferrin receptor antibody or a fragment thereof and a predetermined The inventors have found that the production efficiency of the fusion protein is improved by co-expressing the gene encoding the chaperone protein, and have completed the present invention. That is, the present invention is as follows.
- the method for producing the fusion protein of the present invention comprises A method for producing a fusion protein comprising BDNF and an anti-transferrin receptor antibody or fragment thereof, comprising: culturing transformed mammalian cells containing the gene encoding the fusion protein and the gene encoding the exogenous chaperone protein in a protein production medium to produce the fusion protein; collecting the produced fusion protein; with
- the gene encoding the fusion protein includes the nucleotide sequence of the gene encoding BDNF and the nucleotide sequence of the gene encoding the anti-transferrin receptor antibody or fragment thereof
- the chaperone protein includes one or more selected from the group consisting of HSP90 ⁇ , HSP90 ⁇ , CDC37, HSP70, HSP40, HSP60, HSP10, HSP110 and HSP27.
- the method for producing the fusion protein of the present invention comprises A method for producing a fusion protein comprising the BDNF and the anti-transferrin receptor antibody or fragment thereof, providing a mammalian cell; transforming the mammalian cell with the gene encoding the fusion protein and the gene encoding the chaperone protein; culturing the transformed mammalian cell in a protein production medium to produce the fusion protein; collecting the produced fusion protein; with
- the gene encoding the fusion protein includes the nucleotide sequence of the gene encoding BDNF and the nucleotide sequence of the gene encoding the anti-transferrin receptor antibody or fragment thereof
- the chaperone protein includes one or more selected from the group consisting of HSP90 ⁇ , HSP90 ⁇ , CDC37, HSP70, HSP40, HSP60, HSP10, HSP110 and HSP27.
- the step of transforming the mammalian cell is preferably carried out using one or more recombinant protein expression vectors containing the gene encoding the fusion protein and the gene encoding the chaperone protein.
- the step of transforming the mammalian cell includes simultaneously transfecting one or more recombinant protein expression vectors containing the gene encoding the fusion protein and one or more expression enhancing vectors containing the gene encoding the chaperone protein. Alternatively, it is preferably carried out by separately contacting the mammalian cells.
- the method for producing the fusion protein of the present invention comprises A method for producing a fusion protein comprising the BDNF and the anti-transferrin receptor antibody or fragment thereof, providing mammalian cells containing one or more recombinant protein expression vectors containing genes encoding the fusion proteins; transforming the mammalian cell with at least one expression-enhancing vector containing a gene encoding the chaperone protein; culturing the transformed mammalian cell in a protein production medium to produce the fusion protein; collecting the produced fusion protein; with
- the gene encoding the fusion protein includes the nucleotide sequence of the gene encoding BDNF and the nucleotide sequence of the gene encoding the anti-transferrin receptor antibody or fragment thereof,
- the chaperone protein includes one or more selected from the group consisting of HSP90 ⁇ , HSP90 ⁇ , CDC37, HSP70, HSP40, HSP60, HSP10, HSP110 and HSP27.
- the expression-enhancing vector includes a first expression-enhancing vector containing a gene encoding a first chaperone protein and a second expression-enhancing vector containing a gene encoding a second chaperone protein, Preferably, said first chaperone protein is different from said second chaperone protein.
- the chaperone protein preferably contains either one or both of HSP90 ⁇ and CDC37.
- the BDNF is preferably bound directly or via a linker peptide to the anti-transferrin receptor antibody or fragment thereof.
- the linker peptide is Gly, Ser, Gly-Ser, Gly-Gly-Ser, Gly-Gly-Gly-Gly-Ser, Gly-Gly-Gly-Gly-Gly-Ser, Ser-Gly-Gly- It preferably contains an amino acid sequence selected from the group consisting of Gly-Gly and an amino acid sequence consisting of 1 to 10 of these amino acid sequences in succession.
- the BDNF preferably comprises an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO:32.
- the fragment of the anti-transferrin receptor antibody is preferably a Fab fragment, an F(ab') 2 fragment, or an F(ab') fragment.
- the mammalian cells preferably contain one or more selected from the group consisting of CHO cells, COS cells, BHK cells, HeLa cells, HEK293 cells, NS0 cells and Sp2/0 cells.
- the mammalian cell for recombinant protein production according to the present invention is A mammalian cell for recombinant protein production comprising one or more recombinant protein expression vectors containing a gene encoding a fusion protein, the fusion protein comprises BDNF and an anti-transferrin receptor antibody or fragment thereof;
- the gene encoding the fusion protein includes the nucleotide sequence of the gene encoding BDNF and the nucleotide sequence of the gene encoding the anti-transferrin receptor antibody or fragment thereof,
- the recombinant protein-producing mammalian cell further comprises one or more expression-enhancing vectors containing a gene encoding a chaperone protein,
- the chaperone protein includes one or more selected from the group consisting of HSP90 ⁇ , HSP90 ⁇ , CDC37, HSP70, HSP40, HSP60, HSP10, HSP110 and HSP27.
- the kit according to the present invention comprises A kit for enhancing production of a fusion protein comprising BDNF and an anti-transferrin receptor antibody or fragment thereof in mammalian cells, comprising: comprising one or more expression-enhancing vectors containing a gene encoding a chaperone protein;
- the chaperone protein includes at least one selected from the group consisting of HSP90 ⁇ , HSP90 ⁇ , CDC37, HSP70, HSP40, HSP60, HSP10, HSP110 and HSP27.
- FIG. 1 is a graph showing the production of human BDNF-human anti-transferrin receptor antibody Fab fragments in transformed mammalian cells.
- FIG. 2 is a graph showing the production of human BDNF-human anti-transferrin receptor antibody Fab fragments in transformed mammalian cells.
- FIG. 3 is a graph showing viable cell counts of transformed mammalian cells after production of human BDNF-human anti-transferrin receptor antibody Fab fragments in culture.
- this embodiment An embodiment of the present invention (hereinafter sometimes referred to as "this embodiment") will be described below. However, this embodiment is not limited to this.
- a designation of the form “A to Z” refers to the upper and lower limits of a range (ie, greater than or equal to A and less than or equal to Z). When no unit is described for A and only a unit is described for Z, the unit of A and the unit of Z are the same.
- the method for producing the fusion protein of this embodiment includes: A method for producing a fusion protein comprising BDNF and an anti-transferrin receptor antibody or fragment thereof, comprising: culturing transformed mammalian cells containing the gene encoding the fusion protein and the gene encoding the exogenous chaperone protein in a protein production medium to produce the fusion protein; collecting the produced fusion protein; with The gene encoding the fusion protein includes the nucleotide sequence of the gene encoding BDNF and the nucleotide sequence of the gene encoding the anti-transferrin receptor antibody or fragment thereof,
- the chaperone protein includes one or more selected from the group consisting of HSP90 ⁇ , HSP90 ⁇ , CDC37, HSP70, HSP40, HSP60, HSP10, HSP110 and HSP27.
- the transformed mammalian cell is providing mammalian cells containing one or more recombinant protein expression vectors containing genes encoding the fusion proteins; transforming the mammalian cell with at least one expression-enhancing vector containing a gene encoding a chaperone protein; can be obtained by a method comprising The details will be described in the "Method for Producing Fusion Protein (1)" below.
- the transformed mammalian cell is providing a mammalian cell; transforming the mammalian cell with the gene encoding the fusion protein and the gene encoding the chaperone protein; can be obtained by a method comprising The details will be described in the "Method for Producing Fusion Protein (2)" below.
- the method for producing the first fusion protein of this embodiment comprises A method for producing a fusion protein comprising BDNF and an anti-transferrin receptor antibody or fragment thereof, comprising: providing mammalian cells containing one or more recombinant protein expression vectors containing genes encoding the fusion proteins; transforming the mammalian cell with at least one expression-enhancing vector containing a gene encoding a chaperone protein; culturing the transformed mammalian cell in a protein production medium to produce the fusion protein; collecting the produced fusion protein; with
- the gene encoding the fusion protein comprises the nucleotide sequence of the gene encoding BDNF and the nucleotide sequence of the gene encoding the anti-transferrin receptor antibody or fragment thereof (hereinafter sometimes referred to as "additional protein").
- the chaperone protein includes one or more selected from the group consisting of HSP90 ⁇ , HSP90 ⁇ , CDC37, HSP70
- Step of preparing a mammalian cell containing a recombinant protein expression vector mammalian cells containing one or more recombinant protein expression vectors containing genes encoding fusion proteins are provided.
- the gene encoding the fusion protein includes the nucleotide sequence of the gene encoding the BDNF and the nucleotide sequence of the gene encoding the additional protein.
- the "fusion protein” in this embodiment includes BDNF and an anti-transferrin receptor antibody or fragment thereof (hereinafter sometimes referred to as "additional protein").
- the fusion protein may consist only of BDNF and an additional protein.
- the fusion protein may consist of BDNF, the additional protein, and a linker peptide connecting the BDNF and the additional protein. That is, the BDNF is preferably bound directly or via a linker peptide to the anti-transferrin receptor antibody or fragment thereof.
- the linker peptide is not particularly limited as long as it has a known amino acid sequence.
- Examples of the amino acid sequence of the linker peptide include an amino acid sequence (SEQ ID NO: 75) in which the amino acid sequence represented by the one-letter amino acid "GGGGS” (SEQ ID NO: 76) is repeated five times. Also included is a peptide linker in which the H4 linker (SEQ ID NO: 77) represented by "EAAAAK" is continuous two to four times.
- the linker peptide is Gly, Ser, Gly-Ser, Gly-Gly-Ser, Gly-Gly-Gly-Gly-Ser, Gly-Gly-Gly-Gly-Gly-Ser, Ser -Gly-Gly-Gly-Gly, and amino acid sequences selected from the group consisting of 1 to 10 consecutive identical or different amino acid sequences of these amino acid sequences.
- BDNF may be arranged on the N-terminal side and the additional protein may be arranged on the C-terminal side.
- the additional protein may be arranged on the N-terminal side, and BDNF may be arranged on the C-terminal side. That is, the BDNF may be bound directly or via a linker peptide to the C-terminal side or N-terminal side of the heavy chain and/or light chain of the anti-transferrin receptor antibody or fragment thereof.
- fusion protein specifically, the fusion protein described in International Publication: WO2016/208696 (Patent Document 2) or WO2018/124107 (Patent Document 3) (fusion protein of BDNF and anti-transferrin receptor antibody) is mentioned.
- BDNF BDNF
- BDNF proform BDNF precursor precursor having a signal peptide added to the N-terminus of the BDNF precursor
- BDNF is first produced as a BDNF prepro form from its gene transcription product, from which the signal peptide is cleaved to form a BDNF pro form. Thereafter, the N-terminal 110 amino acid residues are cleaved from the BDNF pro-form to give mature BDNF.
- the “additional protein” means an anti-transferrin receptor antibody or an anti-transferrin receptor antibody fragment that forms the fusion protein together with the BDNF.
- the additional protein may be a monomer, a dimer composed of two subunits, or a multimer composed of a plurality of subunits.
- Antibody fragments include, for example, Fab fragments, F(ab') 2 fragments, F(ab') fragments, antibody constant regions, which consist of an antibody heavy chain (H chain) fragment and an antibody light chain (L chain) fragment.
- recombinant protein expression vector means a DNA construct into which a gene encoding a recombinant protein of interest has been introduced so that it can be expressed in host cells.
- recombinant protein is meant a protein exogenous to the host cell.
- the recombinant protein of interest is the fusion protein. That is, the recombinant protein expression vector contains the gene encoding the fusion protein.
- the gene encoding the fusion protein includes the nucleotide sequence of the gene encoding the BDNF and the nucleotide sequence of the gene encoding the additional protein.
- the gene encoding the fusion protein comprises the nucleotide sequence of the gene encoding the BDNF and the nucleotide sequence of the gene encoding the first subunit constituting the additional protein. and a second gene containing the base sequence of the gene encoding the second subunit that constitutes the additional protein.
- the recombinant protein expression vector is composed of a first recombinant protein expression vector containing the first gene described above and a second recombinant protein expression vector containing the second gene described above. may have been The recombinant protein expression vector may also contain both the first gene and the second gene. Even when the additional protein is a multimer, a gene encoding the fusion protein and a recombinant protein expression vector can be designed in the same manner as in the case of the dimer described above.
- the nucleotide sequence of the gene encoding BDNF may be a wild-type nucleotide sequence, or a nucleotide sequence into which one or more mutations have been introduced with respect to the wild-type nucleotide sequence. good.
- the nucleotide sequence of the gene encoding the BDNF is (A) a nucleotide sequence having 90% or more and 100% or less sequence identity with the wild-type nucleotide sequence encoding the BDNF; (B) a nucleotide sequence in which one or several nucleotides are deleted, substituted, inserted or added to the wild-type nucleotide sequence encoding BDNF; (C) a nucleotide sequence that hybridizes under stringent conditions to an oligonucleotide having a nucleotide sequence complementary to the wild-type nucleotide sequence encoding BDNF; (D) a base sequence encoding an amino acid sequence having 90% or more and 100% or less sequence identity with the wild-type amino acid sequence of the BDNF; or (E) a base sequence encoding an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted or added to the wild-type amino acid sequence of the BDNF, and the
- “retaining the original function” means having a function equivalent to that of wild-type BDNF.
- “Homogeneous function” means, for example, physiologically or pharmacologically, that the property is qualitatively the same, and the degree of function (e.g., about 0.1 to about 10 times, preferably 0.5-2 fold), or quantitative factors such as the molecular weight of the protein may be different.
- BDNF functions possessed by wild-type BDNF, for example, (1) binding affinity to BDNF receptor (TrkB), (2) phosphorylation activity of BDNF receptor, (3) proliferation-promoting effect on nerve cells, (4) a survival-maintaining action on nerve cells, (5) a neurite-extending action on nerve cells, or (6) a protein that can be recognized by an antibody that specifically recognizes a protein consisting of the amino acid sequence of SEQ ID NO: 74.
- BDNF are considered "homogeneous proteins".
- the "intrinsic function” means, for example, the ability to produce a BDNF pro-form.
- “native function” means, for example, being able to produce mature BDNF or having binding affinity for the p75 receptor.
- sequence identity refers to the optimal alignment when two base sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm is introduction of gaps in one or both of the sequences)) means the ratio (%) of identical bases to all overlapping base sequences.
- sequence identity of base sequences can be easily confirmed by those skilled in the art. For example, NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) can be used. Sequence identity of amino acid sequences can also be confirmed by methods similar to those described above.
- the nucleotide sequence of the gene encoding the BDNF may have 95% or more and 100% or less sequence identity with the wild-type nucleotide sequence encoding the BDNF, or 98% or more and 100% or less. They may have sequence identity or they may have 100% sequence identity.
- nucleotide sequence in which one or several bases are deleted, substituted, inserted or added includes, for example, deleted, substituted, inserted or added by deletion, substitution, insertion or addition
- a base sequence having a sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more with respect to the base sequence of the previous sequence can be mentioned.
- the specific number of "one or several bases” includes the above-mentioned deletion, substitution, insertion or addition independently at 1, 2, 3, 4 or 5 positions. You can do it, or you can have a combination of multiple things.
- stringent conditions are 6 ⁇ SSC (composition of 1 ⁇ SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.5% SDS and 5 ⁇ Incubate at room temperature for 12 hours in a solution containing Denhardt, 100 ⁇ g/mL denatured salmon sperm DNA, and 50% (v/v) formamide, and then wash with 0.5 ⁇ SSC at a temperature of 50° C. or higher.
- more stringent conditions e.g., incubation at 45°C or 60°C for 12 hours, washing with 0.2 x SSC or 0.1 x SSC, washing at 60°C or 65°C It also includes more severe conditions such as washing under the above temperature conditions.
- the nucleotide sequence of the gene encoding BDNF is a nucleotide sequence in which codons have been optimized in consideration of codon usage in mammalian cells into which the gene is introduced. good too.
- the above codon optimization is performed, for example, as follows. That is, codon optimization can be performed using algorithms capable of optimizing transcription, translation effect, and folding formation, as typified by Codon W (for example, http://codonw.sourceforge.net/ index.html).
- the nucleotide sequence of the gene encoding BDNF includes the nucleotide sequence encoding the BDNF prepro form, the nucleotide sequence encoding the BDNF pro form, and the nucleotide sequence encoding mature BDNF.
- Examples of the nucleotide sequence encoding the BDNF prepro form include the nucleotide sequence of SEQ ID NO: 71 (GenBank No. NM_170735.6, human-derived wild nucleotide sequence).
- nucleotide sequence encoding the BDNF pro-body examples include, for example, the nucleotide sequence encoding the amino acid sequence of the BDNF pro-body lacking the signal peptide corresponding to the N-terminal 18 amino acid residues of the BDNF pre-pro body (e.g., SEQ ID NO: 31 ).
- the nucleotide sequence encoding the mature BDNF includes, for example, the nucleotide sequence encoding the mature BDNF lacking the N-terminal 110 amino acid residues of the BDNF pro-body (eg, SEQ ID NO: 73).
- the signal peptide in the BDNF prepro form may be a signal peptide possessed by a wild BDNF prepro form, or a signal peptide derived from another protein (for example, a signal peptide consisting of the amino acid sequence of SEQ ID NO: 36). may be
- the amino acid sequence of the BDNF may have a sequence identity of 95% or more and 100% or less, or a sequence identity of 98% or more and 100% or less, with the wild-type amino acid sequence of the BDNF. and may have 100% sequence identity.
- amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted or added includes, for example, deletion, substitution, insertion or addition by deletion, substitution, insertion or An amino acid sequence having a sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more with respect to the amino acid sequence before addition can be mentioned. can.
- Specific numbers of "one or several amino acid residues" include the above-mentioned deletions, substitutions, insertions or additions independently at 1, 2, 3, 4, or 5 positions. It may exist in each, or may occur in combination.
- the amino acid sequence of BDNF includes the amino acid sequence of the BDNF prepro-form, the amino acid sequence of the BDNF pro-form and the amino acid sequence of the mature BDNF described above.
- An example of the amino acid sequence of the BDNF prepro form is the amino acid sequence of SEQ ID NO: 72 (GenBank No. NP_733931).
- Examples of the amino acid sequence of the BDNF pro-body include an amino acid sequence lacking the N-terminal signal peptide of the BDNF pre-pro-body (eg, SEQ ID NO: 32).
- the amino acid sequence of the mature BDNF include an amino acid sequence lacking the N-terminal 110 amino acid residues of the pro-BDNF (eg, SEQ ID NO: 74).
- the nucleotide sequence of the gene encoding the additional protein may be a wild-type nucleotide sequence, or a nucleotide sequence into which one or more mutations have been introduced into the wild-type nucleotide sequence.
- the base sequence of the gene encoding the additional protein is (A) a nucleotide sequence having 90% or more and 100% or less sequence identity with the wild-type nucleotide sequence encoding the additional protein; (B) a base sequence in which one or several bases are deleted, substituted, inserted or added to the wild-type base sequence encoding the additional protein; (C) a nucleotide sequence that hybridizes under stringent conditions to an oligonucleotide having a nucleotide sequence complementary to the wild-type nucleotide sequence encoding the additional protein; (D) a base sequence encoding an amino acid sequence having 90% or more and 100% or less sequence identity with the wild-type amino acid sequence of the additional protein, or (E) a base sequence encoding an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted or added to the wild-type amino acid sequence of the additional protein, and It may be a nucleotide sequence that encodes a protein that retains
- the nucleotide sequence of the gene encoding the additional protein may have a sequence identity of 95% or more and 100% or less, or 98% or more and 100%, with the wild-type nucleotide sequence encoding the additional protein. It may have a sequence identity of less than or equal to 100% sequence identity.
- the base sequence of the gene encoding the additional protein is a base sequence whose codons have been optimized in consideration of the codon usage frequency in mammalian cells into which the gene is introduced.
- the above codon optimization is performed, for example, by the method described above.
- the nucleotide sequence of the gene encoding the heavy chain fragment (first subunit) of the antibody includes, for example, the nucleotide sequence of SEQ ID NO:37.
- the nucleotide sequence of the gene encoding the antibody light chain fragment (second subunit) include the nucleotide sequence of SEQ ID NO:41.
- the amino acid sequence of the additional protein is the amino acid sequence of the wild type of the additional protein (in this case, the Fab fragment containing the protein encoded by the nucleotide sequence of SEQ ID NO: 37 and the protein encoded by the nucleotide sequence of SEQ ID NO: 41). may have 95% or more and 100% or less sequence identity, may have 98% or more and 100% or less sequence identity, or may have 100% sequence identity may Said additional proteins are described in WO 2005/030003, Fab fragments of said added proteins comprising the heavy chain fragment of SEQ ID NO: 38 and the light chain fragment of SEQ ID NO: 42, and said added proteins having said sequence identity.
- the amino acid sequence of the heavy chain fragment (first subunit) of the antibody includes, for example, the amino acid sequence of SEQ ID NO:38.
- the amino acid sequence of the light chain fragment (second subunit) of the antibody includes, for example, the amino acid sequence of SEQ ID NO:42.
- the Fab fragment of the additional protein containing the heavy chain fragment of SEQ ID NO:38 and the light chain fragment of SEQ ID NO:42 is described in Patent Document 3.
- the recombinant protein expression vector contains a promoter sequence (e.g., cytomegalovirus (CMV) promoter, herpes simplex virus (HSV) thymidine kinase (TK) promoter, SV40 promoter, EF -1 promoter, actin promoter, ⁇ -globulin promoter and enhancer, etc.), Kozak sequences, terminator sequences, mRNA stabilization sequences.
- CMV cytomegalovirus
- HSV herpes simplex virus
- TK thymidine kinase
- SV40 promoter e.g., SV40 promoter
- EF -1 promoter e.g., actin promoter, ⁇ -globulin promoter and enhancer, etc.
- Kozak sequences e.g., cytomegalovirus (CMV) promoter, herpes simplex virus (HSV) thymidine kinase (TK) promoter, SV40 promoter
- the recombinant protein expression vector comprises an origin of replication, an enhancer sequence, a signal sequence, a drug resistance gene (e.g., ampicillin, tetracycline, kanamycin, chloramphenicol, neomycin, hygromycin, puromycin). , drug resistance genes such as zeocin), and genes encoding fluorescent proteins such as GFP.
- a drug resistance gene e.g., ampicillin, tetracycline, kanamycin, chloramphenicol, neomycin, hygromycin, puromycin.
- drug resistance genes such as zeocin
- genes encoding fluorescent proteins such as GFP.
- the recombinant protein expression vector is not particularly limited as long as the effect of the present invention is exhibited, and may be, for example, a plasmid vector or a virus vector. In one aspect of this embodiment, the recombinant protein expression vector is preferably a plasmid vector.
- the plasmid vectors include pcDNA3.1(+) vector, pEGF-BOS vector, pEF vector, pCDM8 vector, pCXN vector, pCI vector, episomal vector, transposon vector and the like.
- the plasmid vector is preferably a pcDNA3.1(+) vector.
- the viral vectors include lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, Sendai viral vectors, mammalian-expressing baculoviral vectors, and the like.
- Examples include pLenti4/V5-GW/lacZ, pLVSIN-CMV, pLVSIN-EF1 ⁇ , pAxcwit2, pAxEFwit2, pAAV-RCS, pSeV vector, pFastBacMam, pFastBacMam2.0 (VSV-G) and the like.
- mammalian cells refer to cells derived from mammals. Mammals include, for example, humans, hamsters (eg, Chinese hamsters), mice, rats, green monkeys, and the like. The mammalian cells may be immortalized cells.
- the mammalian cells are not particularly limited as long as they are used as host cells for expressing the recombinant protein.
- Examples of such mammalian cells include CHO cells (cell line derived from Chinese hamster ovary), COS cells (cell line derived from African green monkey kidney), and BHK cells (cells derived from baby hamster kidney). HeLa cells (cell line derived from human cervical cancer), HEK293 cells (cell line derived from human fetal kidney), NS0 cells (cell line derived from mouse myeloma) and Sp2/0 Cells (cell lines derived from mouse myeloma). That is, the mammalian cells preferably contain one or more selected from the group consisting of CHO cells, COS cells, BHK cells, HeLa cells, HEK293 cells, NS0 cells and Sp2/0 cells.
- Step of transforming mammalian cells with expression-enhancing vector the mammalian cells are transformed with at least one expression-enhancing vector containing a gene encoding a chaperone protein.
- the term "chaperone protein” means a protein that helps the fusion protein to fold correctly and acquire its original function.
- the “intrinsic function of the fusion protein” means the intrinsic function of each of the BDNF and the additional protein that constitute the fusion protein.
- the chaperone protein includes one or more selected from the group consisting of HSP90 ⁇ , HSP90 ⁇ , CDC37 (Cell Division Cycle 37, HSP90 cochaperone), HSP70, HSP40, HSP60, HSP10, HSP110 and HSP27.
- HSP is an abbreviation for heat shock protein.
- the chaperone protein preferably comprises any one of HSP90 ⁇ , HSP90 ⁇ , HSP40 and CDC37, or both HSP90 ⁇ , HSP90 ⁇ or HSP40 and CDC37. In another aspect of this embodiment, the chaperone protein preferably comprises either one or both of HSP90 ⁇ and CDC37.
- the animal species of origin of the chaperone protein may be the same as or different from the animal species of origin of the cysteine knot protein. In one aspect of this embodiment, the animal species of origin of the chaperone protein may be the same as or different from the animal species of origin of the host cells.
- the animal species of origin of the chaperone protein is preferably the same as either the animal species of origin of the cysteine knot protein or the animal species of origin of the host cell.
- the chaperone protein may be a human-derived chaperone protein or a Chinese hamster-derived chaperone protein. Preferably, it may be a human-derived chaperone protein.
- the chaperone protein when the fusion protein contains BDNF and a Fab fragment, preferably contains one or more selected from the group consisting of HSP90 ⁇ , HSP90 ⁇ , HSP60, HSP10, HSP70 and HSP27.
- expression-enhancing vector refers to a DNA construct into which a gene encoding the chaperone protein has been introduced so that it can be expressed in host cells.
- the nucleotide sequence of the gene encoding the chaperone protein may be a wild-type nucleotide sequence, or a nucleotide sequence into which one or more mutations have been introduced into the wild-type nucleotide sequence.
- the nucleotide sequence of the gene encoding the chaperone protein is (A) a nucleotide sequence having 90% or more and 100% or less sequence identity with the wild-type nucleotide sequence encoding the chaperone protein; (B) a base sequence in which one or several bases are deleted, substituted, inserted or added to the wild-type base sequence encoding the chaperone protein; (C) a nucleotide sequence that hybridizes under stringent conditions to an oligonucleotide having a nucleotide sequence complementary to the wild-type nucleotide sequence encoding the chaperone protein; (D) a base sequence encoding an amino acid sequence having 90% or more and 100% or less sequence identity with the wild-type amino acid sequence of the chaperone protein; or (E) a nucleotide sequence encoding an amino acid sequence in which one or several amino acid residues are deleted, substituted, inserted or added to the wild-type amino acid sequence of the chaperone protein, and
- the nucleotide sequence of the gene encoding the chaperone protein may have a sequence identity of 95% or more and 100% or less, or 98% or more and 100%, with the wild-type nucleotide sequence encoding the chaperone protein. It may have a sequence identity of less than or equal to 100% sequence identity.
- the nucleotide sequence of the gene encoding the chaperone protein is a nucleotide sequence whose codons have been optimized in consideration of the codon usage frequency in mammalian cells into which the gene is introduced.
- the above codon optimization is performed, for example, by the method described above.
- the nucleotide sequence of the gene encoding HSP90 ⁇ includes, for example, SEQ ID NO: 45 (GenBank No. NM_001017963, human-derived wild nucleotide sequence), SEQ ID NO: 47 (GenBank No. NM_005348, human-derived wild nucleotide sequence), SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 49 (GenBank No. NM — 001246821, Chinese hamster-derived wild nucleotide sequence), and SEQ ID NO: 5.
- SEQ ID NO: 45 GenBank No. NM_001017963, human-derived wild nucleotide sequence
- SEQ ID NO: 47 GenBank No. NM_005348, human-derived wild nucleotide sequence
- SEQ ID NO: 1 SEQ ID NO: 3
- SEQ ID NO: 49 GenBank No. NM — 001246821, Chinese hamster-derived wild nucleotide sequence
- SEQ ID NO: 5 examples of the
- Nucleotide sequences of genes encoding CDC37 include, for example, SEQ ID NO: 59 (GenBank No.
- NM_007065 human-derived wild nucleotide sequence
- SEQ ID NO: 15 SEQ ID NO: 61
- base sequence SEQ ID NO: 61
- base sequence SEQ ID NO: 61
- base sequence of SEQ ID NO: 17 The base sequences of genes encoding HSP60 include, for example, the base sequences of SEQ ID NO: 63 (GenBank No. NM_199440, human-derived wild base sequence) and SEQ ID NO: 19.
- the base sequences of genes encoding HSP40 include, for example, the base sequences of SEQ ID NO: 65 (GenBank No. NM_001539, human-derived wild base sequence) and SEQ ID NO: 21.
- nucleotide sequence of the gene encoding HSP10 examples include the nucleotide sequences of SEQ ID NO: 67 (GenBank No. NM_002157, human-derived wild nucleotide sequence) and SEQ ID NO: 23.
- nucleotide sequence of the gene encoding HSP110 examples include the nucleotide sequences of SEQ ID NO: 69 (GenBank No. NM — 006644, human-derived wild nucleotide sequence) and SEQ ID NO: 25.
- nucleotide sequence of the gene encoding HSP70 examples include the CHO-derived wild-type nucleotide sequence described in Journal of Biotechnology 143 (2009) 34-43 and the nucleotide sequence of SEQ ID NO:27.
- nucleotide sequence of the gene encoding HSP27 examples include the CHO-derived wild-type nucleotide sequence described in Journal of Biotechnology 143 (2009) 34-43 and the nucleotide sequence of SEQ ID NO:29.
- the amino acid sequence of the chaperone protein may have 95% or more and 100% or less sequence identity with the wild-type amino acid sequence of the chaperone protein, or 98% or more and 100% or less sequence identity. may have 100% sequence identity.
- the amino acid sequences of HSP90 ⁇ include, for example, the amino acid sequences of SEQ ID NO: 2 (GenBank No. NP_001017963), SEQ ID NO: 4 (GenBank No. NP_005339) and SEQ ID NO: 6 (GenBank No. NP_001233750).
- the amino acid sequences of HSP90 ⁇ include, for example, SEQ ID NO: 8 (GenBank No. NP_001258899), SEQ ID NO: 10 (GenBank No. NP_001258900), SEQ ID NO: 12 (GenBank No. NP_001258901) and SEQ ID NO: 14 (GenBank No. XP_003501716) sequence.
- the amino acid sequences of CDC37 include, for example, the amino acid sequences of SEQ ID NO: 16 (GenBank No. NP_008996) and SEQ ID NO: 18 (GenBank No. XP_003499785).
- the amino acid sequence of HSP60 includes, for example, the amino acid sequence of SEQ ID NO: 20 (GenBank No. NP_955472).
- the amino acid sequence of HSP40 includes, for example, the amino acid sequence of SEQ ID NO: 22 (GenBank No. NP — 001530).
- the amino acid sequence of HSP10 includes, for example, the amino acid sequence of SEQ ID NO: 24 (GenBank No. NP_002148).
- the amino acid sequence of HSP110 includes, for example, the amino acid sequence of SEQ ID NO: 26 (GenBank No. NP — 006635).
- the amino acid sequence of HSP70 includes, for example, the amino acid sequence of SEQ ID NO: 28 (described in Journal of Biotechnology 143 (2009) 34-43).
- the amino acid sequence of HSP27 includes, for example, the amino acid sequence of SEQ ID NO: 30 (described in Journal of Biotechnology 143 (2009) 34-43).
- the expression-enhancing vector contains, in addition to the gene encoding the chaperone protein, a promoter sequence (e.g., cytomegalovirus (CMV) promoter, herpes simplex virus (HSV) thymidine kinase (TK) promoter, SV40 promoter, EF-1 promoters, actin promoters, beta-globulin promoters and enhancers, etc.), Kozak sequences, terminator sequences, mRNA stabilization sequences.
- CMV cytomegalovirus
- HSV herpes simplex virus
- TK thymidine kinase
- SV40 promoter e.g., SV40 promoter
- EF-1 promoters e.g., actin promoters, beta-globulin promoters and enhancers, etc.
- Kozak sequences e.g., Kozak sequences, terminator sequences, mRNA stabilization sequences.
- the expression-enhancing vector comprises a replication origin, an enhancer sequence, a signal sequence, a drug resistance gene (e.g., ampicillin, tetracycline, kanamycin, chloramphenicol, neomycin, hygromycin, puromycin, zeocin It may further contain one or more selected from the group consisting of selectable marker genes such as drug resistance genes such as GFP, and genes encoding fluorescent proteins such as GFP.
- a drug resistance gene e.g., ampicillin, tetracycline, kanamycin, chloramphenicol, neomycin, hygromycin, puromycin, zeocin
- selectable marker genes such as drug resistance genes such as GFP, and genes encoding fluorescent proteins such as GFP.
- the expression-enhancing vector is not particularly limited as long as the effects of the present invention are exhibited, and may be, for example, a plasmid vector or a virus vector.
- the expression-enhancing vector is preferably a plasmid vector.
- the plasmid vectors include pcDNA3.1(+) vector, pEGF-BOS vector, pEF vector, pCDM8 vector, pCXN vector, pCI vector, episomal vector, transposon vector and the like.
- the plasmid vector is preferably a pcDNA3.1(+) vector.
- viral vectors examples include lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, Sendai viral vectors, mammalian-expressing baculoviral vectors, and the like.
- examples include pLenti4/V5-GW/lacZ, pLVSIN-CMV, pLVSIN-EF1 ⁇ , pAxcwit2, pAxEFwit2, pAAV-RCS, pSeV vector, pFastBacMam, pFastBacMam2.0 (VSV-G) and the like.
- designing the gene encoding the fusion protein, obtaining the gene fragment encoding the BDNF, obtaining the gene fragment encoding the additional protein, obtaining the gene fragment encoding the chaperone protein, and the plasmid Vectors can be constructed according to techniques commonly used in the fields of molecular biology, bioengineering, and genetic engineering (eg, Sambrook et al. "Molecular Cloning-A Laboratory Manual, second edition 1989").
- Host cells used for preparation of plasmid vectors include, for example, Escherichia coli commonly used in the art.
- the mammalian cell is a mammalian cell containing one or more recombinant protein expression vectors containing the gene encoding the fusion protein provided in the previous step.
- the expression-enhancing vector includes a first expression-enhancing vector containing a gene encoding a first chaperone protein and a second expression-enhancing vector containing a gene encoding a second chaperone protein. and the expression-enhancing vector of and the first chaperone protein is preferably different from the second chaperone protein. More preferably, the first chaperone protein is HSP90 ⁇ and the second chaperone protein is CDC37.
- mammalian cells may be transformed with expression-enhancing vectors containing genes encoding two or more chaperone proteins.
- the method of transformation using an expression-enhancing vector is not particularly limited as long as the effect of the present invention is exhibited, and known methods can be used (for example, Sambrook et al. "Molecular Cloning - A Laboratory Manual, second edition 1989").
- Known transformation methods include, for example, the lipofection method, calcium phosphate method, DEAE dextran method, electroporation method, polyethyleneimine method and polyethylene glycol method.
- the transformation described above may be performed using a commercially available kit.
- kits include, for example, ThermoFisher Scientific K.K. K. Gibco (trademark) Expi (trademark) Expression System (Cat. No.
- A29133 manufactured by Co., Ltd., and the like.
- 3 ⁇ g to 30 ⁇ g of expression vector is used per cell density (1 ⁇ 10 6 cells/mL to 9 ⁇ 10 6 cells/mL).
- a total of 20 ⁇ g of expression-enhancing vector is used for cells (6 ⁇ 10 6 cells/mL) in a 25 mL container.
- the transformed mammalian cells are cultured in a protein production medium to produce the fusion protein.
- a method for mass-producing a recombinant protein using E. coli as a host cell has been known.
- a method for mass-producing fusion proteins containing knot proteins as soluble fractions that retain their functional conformations has not been known.
- the production efficiency of the fusion protein is improved by co-expressing the gene encoding the fusion protein and the gene encoding the predetermined chaperone protein in the mammalian cell. .
- Cultivation of the transformed mammalian cells is carried out according to conditions such as medium composition, medium pH, glucose concentration, culture temperature, culture time, amount of expression inducer used, and time of use. It is adjusted appropriately so that the chaperone protein is efficiently expressed.
- the protein production medium used for culturing the transformed mammalian cells is not particularly limited as long as it is a known medium suitable for protein production, and may be a solid medium or a liquid medium.
- the protein production medium is preferably a liquid medium.
- Examples of the protein production medium include Dulbecco's Modified Eagle's Medium (DMEM), Eagle's Minimum Essential Medium (MEM), Roswell Park Memorial Institute Medium 1640 (RPMI 1640), Iscove's Modified Dulbecco's Medium (IMDM), F10 medium, and F12 medium. , DMEM/F12, FreeStyle293 expression medium, Freestyle CHO medium, and the like.
- the protein production medium may contain fetal calf serum (FCS).
- FCS fetal calf serum
- the medium for protein production may be a serum-free medium.
- Step of recovering the fusion protein the produced fusion protein is collected.
- This step includes recovering the produced fusion protein from the culture supernatant after completion of the culture. For example, after completion of the culture, the resulting culture supernatant can be treated by various purification methods to obtain a highly purified fusion protein.
- the purification method is, for example, heat treatment of the culture supernatant, salting out, and at least one selected from various chromatography such as anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, hydroxyapatite chromatography and affinity chromatography. may be
- the method for producing the second fusion protein of this embodiment comprises A method for producing a fusion protein comprising BDNF and an anti-transferrin receptor antibody or fragment thereof, comprising: providing a mammalian cell; transforming the mammalian cell with the gene encoding the fusion protein and the gene encoding the chaperone protein; culturing the transformed mammalian cell in a protein production medium to produce the fusion protein; collecting the produced fusion protein; with The gene encoding the fusion protein includes the nucleotide sequence of the gene encoding BDNF and the nucleotide sequence of the gene encoding the anti-transferrin receptor antibody or fragment thereof,
- the chaperone protein includes one or more selected from the group consisting of HSP90 ⁇ , HSP90 ⁇ , CDC37, HSP70, HSP40, HSP60, HSP10, HSP110 and HSP27.
- mammalian cells are prepared.
- the mammalian cells the mammalian cells exemplified in the above-mentioned "fusion protein production method (1)" can be used. That is, the mammalian cells preferably contain one or more selected from the group consisting of CHO cells, COS cells, BHK cells, HeLa cells, HEK293 cells, NS0 cells and Sp2/0 cells.
- a gene encoding a fusion protein and a gene encoding a chaperone protein are used to transform mammalian cells.
- the gene encoding the fusion protein includes the nucleotide sequence of the gene encoding BDNF and the nucleotide sequence of the gene encoding the anti-transferrin receptor antibody or fragment thereof.
- the fusion protein As the fusion protein, the fusion protein exemplified in the above-mentioned "fusion protein production method (1)" can be used.
- the fusion protein contains BDNF and an anti-transferrin receptor antibody or fragment thereof.
- Anti-transferrin receptor antibody fragments include, for example, a Fab fragment consisting of an antibody heavy chain (H chain) fragment and an antibody light chain (L chain) fragment, an Fc fragment in which the Fab fragment is added to the antibody constant region, Single chain antibodies (scFv) and bispecific antibodies (diabodies) are included.
- the chaperone protein As the chaperone protein, the chaperone protein exemplified in the above-mentioned "fusion protein production method (1)" can be used. That is, the chaperone protein includes one or more selected from the group consisting of HSP90 ⁇ , HSP90 ⁇ , CDC37, HSP70, HSP40, HSP60, HSP10, HSP110 and HSP27. In one aspect of this embodiment, the chaperone protein preferably comprises any one of HSP90 ⁇ , HSP90 ⁇ , HSP40 and CDC37, or both HSP90 ⁇ , HSP90 ⁇ or HSP40 and CDC37. In another aspect of this embodiment, the chaperone protein preferably comprises either one or both of HSP90 ⁇ and CDC37.
- the order in which the fusion protein-encoding gene and the chaperone protein-encoding gene are introduced into mammalian cells, which are host cells is not particularly limited.
- a gene encoding a fusion protein may be introduced into the mammalian cell, followed by introduction of a gene encoding a chaperone protein into the mammalian cell.
- a gene encoding a chaperone protein may be introduced into the mammalian cell, followed by introduction of a gene encoding a fusion protein into the mammalian cell.
- the fusion protein-encoding gene and the chaperone protein-encoding gene may be simultaneously introduced into the mammalian cell. For example, when both genes are introduced into a host cell, the ratio of the gene encoding the fusion protein and the gene encoding the chaperone protein is 1:1 to 10:1, more specifically 4:1. good too.
- transforming the mammalian cell is performed using one or more recombinant protein expression vectors containing the gene encoding the fusion protein and the gene encoding the chaperone protein. preferably. Since the recombinant protein expression vector contains the gene encoding the chaperone protein together with the gene encoding the fusion protein, it can also be understood as an expression-enhancing vector.
- the recombinant protein expression vector may have the same or different promoter sequences upstream of the gene encoding the fusion protein and the gene encoding the chaperone protein.
- the recombinant protein expression vector may have a promoter sequence, a gene encoding the fusion protein, and a gene encoding the chaperone protein in this order from the 5′ end, A promoter sequence, the gene encoding the chaperone protein, and the gene encoding the fusion protein may be arranged in order from the 5' end.
- the gene encoding the fusion protein is composed of the base sequence of the gene encoding BDNF and the first sub-protein constituting the additional protein. It may be composed of a first gene containing the base sequence of the gene encoding the unit, and a second gene containing the base sequence of the gene encoding the second subunit constituting the additional protein.
- the step of transforming the mammalian cell is performed using one or more recombinant protein expression vectors containing the first gene, the second gene, and the gene encoding the chaperone protein. preferably.
- transforming the mammalian cell comprises one or more recombinant protein expression vectors containing the gene encoding the fusion protein and the gene encoding the chaperone protein. It is preferably carried out by contacting said mammalian cells with one or more expression-enhancing vectors, either simultaneously or separately.
- transforming the mammalian cell comprises: a first recombinant protein expression vector containing the first gene described above; A second recombinant protein expression vector containing the second gene of and one or more expression enhancing vectors containing a gene encoding the chaperone protein are simultaneously or separately contacted with the mammalian cell. preferably.
- fusion protein production method (1) the method described in the above-mentioned “fusion protein production method (1)" can be used.
- Step of recovering the fusion protein In this step, the produced fusion protein is recovered.
- the method described in the above-mentioned "fusion protein production method (1)" can be used.
- the mammalian cell for recombinant protein production in this embodiment is A mammalian cell for recombinant protein production comprising one or more recombinant protein expression vectors containing a gene encoding a fusion protein, the fusion protein comprises BDNF and an anti-transferrin receptor antibody or fragment thereof;
- the gene encoding the fusion protein includes the nucleotide sequence of the gene encoding BDNF and the nucleotide sequence of the gene encoding the anti-transferrin receptor antibody or fragment thereof,
- the recombinant protein-producing mammalian cell further comprises one or more expression-enhancing vectors containing a gene encoding a chaperone protein,
- the chaperone protein includes one or more selected from the group consisting of HSP90 ⁇ , HSP90 ⁇ , CDC37, HSP70, HSP40, HSP60, HSP10, HSP110 and HSP27.
- kits in this embodiment are A kit for enhancing production of a fusion protein comprising BDNF and an anti-transferrin receptor antibody or fragment thereof in mammalian cells, comprising: comprising one or more expression-enhancing vectors containing a gene encoding a chaperone protein;
- the chaperone protein includes at least one selected from the group consisting of HSP90 ⁇ , HSP90 ⁇ , CDC37, HSP70, HSP40, HSP60, HSP10, HSP110 and HSP27.
- the kit comprises a buffer solution, a mammalian host cell, a recombinant protein expression vector, a medium for protein production, a sample tube, a microplate, an instruction manual for the user of the kit, and a transfection reagent. It may further contain one or more selected from the group consisting of:
- a fusion protein produced by the production method of the present invention can be used as a raw material for a pharmaceutical composition containing the fusion protein as an active ingredient.
- the present invention encompasses a method for producing the pharmaceutical composition comprising the step of contacting the fusion protein with an additive.
- the above additives are not particularly limited and can be appropriately selected as long as they are components generally known as additives contained in pharmaceutical compositions.
- NP — 008996 amino acid sequence: SEQ ID NO: 16) (base sequence after codon optimization: SEQ ID NO: 15), (3) human HSP60 gene (GenBank No. NP_955472, amino acid sequence: SEQ ID NO: 20) (base sequence after codon optimization: SEQ ID NO: 19), (4) human HSP10 gene (GenBank No. NP_002148, amino acid sequence: SEQ ID NO: 24) (base sequence after codon optimization: SEQ ID NO: 23), (5) human HSP110 gene (GenBank No. NP_006635, amino acid sequence: SEQ ID NO: 26) (base sequence after codon optimization: SEQ ID NO: 25), (6) human HSP40 gene (GenBank No.
- NP_NP_001530 amino acid sequence: SEQ ID NO: 22) (nucleotide sequence after codon optimization: SEQ ID NO: 21), (7) human HSJ1 gene (GenBank No. AAA09034, amino acid sequence: SEQ ID NO: 34) (nucleotide sequence after codon optimization: SEQ ID NO: 33), (8) Chinese hamster ovary-derived cell CHO HSP70 gene (J. Biotechnology 143 (2009) 34-43) (codon-optimized nucleotide sequence: SEQ ID NO: 27, amino acid sequence: SEQ ID NO: 28), (9) Chinese hamster ovary-derived cell CHO HSP27 gene (J. Biotechnology 143 (2009) 34-43) (base sequence after codon optimization: SEQ ID NO: 29, amino acid sequence: SEQ ID NO: 30).
- the optimal base sequences were determined in an expression system using CHO cells using Genscript's OptimumGene (codon optimization).
- a gene fragment was prepared by chemical synthesis by adding a Kozak sequence (ccacc) to the N-terminus and a stop codon (TGA) to the C-terminus of the determined optimal nucleotide sequence.
- ccacc Kozak sequence
- TGA stop codon
- Each gene fragment was inserted into the HindIII-EcoRI site of a mammalian expression vector pcDNA3.1(+) vector (Cat. No. V79020, Invitrogen) to prepare a plasmid vector (1 mg/mL) of an expression enhancer.
- pcDNA3.1(+) vector Cat. No. V79020, Invitrogen
- the Enhanced Green Fluorescent Protein (EGFP) gene (GenBank No. AAF62891.1) was used as a control for the above expression-enhancing factors.
- the optimal base sequence was determined in an expression system using CHO cells using Genscript's OptimumGene (codon optimization).
- a gene fragment having a Kozak sequence (ccacc) added to the N-terminus and a stop codon (TGA) added to the C-terminus of the determined optimal nucleotide sequence was prepared by chemical synthesis.
- the above gene fragment was inserted into the HindIII-EcoRI site of mammalian expression vector pcDNA3.1(+) vector (Cat. No. V79020, Invitrogen) to prepare a control plasmid vector (1 mg/mL).
- hBDNF-hFab(H) gene (nucleotide sequence after codon optimization: SEQ ID NO: 39, amino acid sequence: SEQ ID NO: 40, where hFab(H) is the Fab weight of the anti-transferrin receptor antibody chain)
- Second gene hFab(L) gene (nucleotide sequence after codon optimization: SEQ ID NO: 43, amino acid sequence: SEQ ID NO: 44, where hFab(L) is the Fab light chain of the anti-transferrin receptor antibody be).
- the hBDNF-hFab (H) gene is, in order from the N-terminal side, IgG signal sequence (GenBank No. 6SVL_B, base sequence after codon optimization: SEQ ID NO: 35, amino acid sequence: SEQ ID NO: 36), human BDNF (codon optimized Nucleotide sequence after modification: SEQ ID NO: 31, amino acid sequence: SEQ ID NO: 32), glycine linker (amino acid sequence: SEQ ID NO: 75) and the first subunit, human anti-transferrin receptor antibody Fab H chain fragment (codon-optimized It is a gene encoding a polypeptide consisting of the following nucleotide sequence: SEQ ID NO: 37, amino acid sequence: SEQ ID NO: 38).
- the hFab (L) gene is, in order from the N-terminal side, an IgG signal sequence (GenBank No. 6SVL_B, base sequence after codon optimization: SEQ ID NO: 35, amino acid sequence: SEQ ID NO: 36) and a second subunit. It is a gene encoding a polypeptide consisting of a human anti-transferrin receptor antibody Fab L chain fragment (nucleotide sequence after codon optimization: SEQ ID NO: 41, amino acid sequence: SEQ ID NO: 42).
- the optimal base sequences were determined in an expression system using CHO cells using Genscript's OptimumGene (codon optimization).
- a gene fragment was prepared by chemical synthesis by adding a Kozak sequence (ccacc) to the N-terminus and a stop codon (TAA) to the C-terminus of the determined optimal nucleotide sequence.
- ccacc Kozak sequence
- TAA stop codon
- Each gene fragment was inserted into the HindIII-EcoRI site of the mammalian expression vector pcDNA3.1(+) vector (Cat. No. V79020, Invitrogen) to prepare a plasmid vector (1 mg/mL) for each recombinant protein.
- Two types of recombinant protein expression vectors were obtained through the above steps.
- reagents (1 ml) containing the plasmid vectors shown in Table 1 below were prepared.
- Expifectamine Cat. No. A12129
- OptiPROTM SFM Cat. No. 12309050
- 920 ⁇ L were added to a tube different from the reagent containing the plasmid vector.
- the plasmid vector-containing reagent and the Expifectamine-containing reagent were each stirred and allowed to stand at room temperature for 5 minutes. Both reagents were then mixed gently to form ExpiFectamineTM CHO/plasmid DNA complexes and left at room temperature for 1 to 5 minutes.
- the complexes were added to a 125 mL Erlenmeyer flask containing Expi-CHO cells, and stirred overnight at 37° C., 8% CO 2 and 125 rpm.
- ExpiFectamine (trademark) CHO Enhancer (150 ⁇ L) and ExpiCHO (trademark) Feed (4 mL) were added to the culture medium, and the temperature was maintained at 32° C., 5% CO 2 , and 125 rpm. was cultured in Five days after the culture, ExpiCHO (trademark) Feed (4 mL) was further added to the culture solution, and culture was performed at 32°C, 5% CO 2 , and 125 rpm.
- BDNF-human anti-transferrin receptor antibody Fab fragment (hereinafter sometimes referred to as "fusion protein” or "BDNF-hFab").
- Productivity was calculated by ELISA.
- viability of cells was calculated by measuring the total cell number and viable cell number using a Countess II FL automatic cell counter (Cat. No. AMQAF1000, ThermoFisher Scientific K.K.).
- the concentration of the fusion protein in the culture supernatant was calculated using ELISA (Biosensis, Cat.No.BEK-2211-1P/2P). After culturing, the cells were collected on the 13th day, the number of viable cells was counted using a Countess II FL automatic cell counter, and the culture supernatant was collected after centrifugation at 10,000 xg for 5 minutes. The recovered culture supernatant was diluted (dilution ratio: 30,000-fold to 1,000,000-fold) to the range (7.8 to 500 pg/mL) that can be quantified by the standard.
- TMB reagent tetramethylbenzidine reagent
- HSP90 ⁇ (Sample 1-2) and CHO-derived HSP70 (Sample No. 1-3) enhanced the fusion protein production the most.
- CHO-derived HSP27 (sample No. 1-4), human CDC37 (sample No. 1-5), HSP40 (sample No. 1-6), HSP10 (sample No. 1-7), HSP110 (sample No. 1-8) was also found to enhance the production of the fusion protein.
- HSJ1 (Sample No. 1-14) showed no effect.
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Abstract
Description
また、BDNFと抗トランスフェリン受容体抗体を含む融合タンパク質は、BDNFが血液脳関門を通過するため、脳内に移行することができる融合タンパク質として知られているが(特許文献2及び特許文献3)、これを組換えタンパク質として大量に製造する方法が求められていた。
BDNF、及び抗トランスフェリン受容体抗体又はその断片を含む融合タンパク質の製造方法であって、
上記融合タンパク質をコードする遺伝子及び外因性のシャペロンタンパク質をコードする遺伝子を含有する形質転換された哺乳動物細胞をタンパク質生産用培地中で培養し、上記融合タンパク質を生産する工程と、
生産された上記融合タンパク質を回収する工程と、
を備え、
上記融合タンパク質をコードする遺伝子は、上記BDNFをコードする遺伝子の塩基配列と、上記抗トランスフェリン受容体抗体又はその断片をコードする遺伝子の塩基配列とを含み、
上記シャペロンタンパク質は、HSP90α、HSP90β、CDC37、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる1以上を含む。
上記BDNF、及び上記抗トランスフェリン受容体抗体又はその断片を含む融合タンパク質の製造方法であって、
哺乳動物細胞を準備する工程と、
上記融合タンパク質をコードする遺伝子及び上記シャペロンタンパク質をコードする遺伝子を用いて、上記哺乳動物細胞を形質転換する工程と、
形質転換された上記哺乳動物細胞をタンパク質生産用培地中で培養し、上記融合タンパク質を生産する工程と、
生産された上記融合タンパク質を回収する工程と、
を備え、
上記融合タンパク質をコードする遺伝子は、上記BDNFをコードする遺伝子の塩基配列と、上記抗トランスフェリン受容体抗体又はその断片をコードする遺伝子の塩基配列とを含み、
上記シャペロンタンパク質は、HSP90α、HSP90β、CDC37、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる1以上を含む。
上記哺乳動物細胞を形質転換する工程は、上記融合タンパク質をコードする遺伝子及び上記シャペロンタンパク質をコードする遺伝子を含有する1以上の組換えタンパク質発現ベクターを用いて実施されることが好ましい。
上記哺乳動物細胞を形質転換する工程は、上記融合タンパク質をコードする遺伝子を含有する1以上の組換えタンパク質発現ベクター、及び上記シャペロンタンパク質をコードする遺伝子を含有する1以上の発現増強ベクターを、同時又は別々に上記哺乳動物細胞に接触させることで実施されることが好ましい。
上記BDNF、及び上記抗トランスフェリン受容体抗体又はその断片を含む融合タンパク質の製造方法であって、
上記融合タンパク質をコードする遺伝子を含有する1以上の組換えタンパク質発現ベクターを含む哺乳動物細胞を準備する工程と、
上記シャペロンタンパク質をコードする遺伝子を含有する少なくとも1種の発現増強ベクターを用いて、上記哺乳動物細胞を形質転換する工程と、
形質転換された上記哺乳動物細胞をタンパク質生産用培地中で培養し、上記融合タンパク質を生産する工程と、
生産された上記融合タンパク質を回収する工程と、
を備え、
上記融合タンパク質をコードする遺伝子は、上記BDNFをコードする遺伝子の塩基配列と、上記抗トランスフェリン受容体抗体又はその断片をコードする遺伝子の塩基配列とを含み、
上記シャペロンタンパク質は、HSP90α、HSP90β、CDC37、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる1以上を含む。
上記発現増強ベクターは、第一のシャペロンタンパク質をコードする遺伝子を含有する第一の発現増強ベクターと、第二のシャペロンタンパク質をコードする遺伝子を含有する第二の発現増強ベクターとを含み、
上記第一のシャペロンタンパク質は、上記第二のシャペロンタンパク質と異なることが好ましい。
融合タンパク質をコードする遺伝子を含有する1以上の組換えタンパク質発現ベクターを含む、組換えタンパク質生産用哺乳動物細胞であって、
上記融合タンパク質は、BDNF、及び抗トランスフェリン受容体抗体又はその断片を含み、
上記融合タンパク質をコードする遺伝子は、上記BDNFをコードする遺伝子の塩基配列と、上記抗トランスフェリン受容体抗体又はその断片をコードする遺伝子の塩基配列とを含み、
上記組換えタンパク質生産用哺乳動物細胞は、シャペロンタンパク質をコードする遺伝子を含有する1種以上の発現増強ベクターを更に含み、
上記シャペロンタンパク質は、HSP90α、HSP90β、CDC37、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる1以上を含む。
哺乳動物細胞における、BDNF、及び抗トランスフェリン受容体抗体又はその断片を含む融合タンパク質の生産量を増強させるためのキットであって、
シャペロンタンパク質をコードする遺伝子を含有する1種以上の発現増強ベクターを含み、
上記シャペロンタンパク質は、HSP90α、HSP90β、CDC37、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる少なくとも1つを含む。
BDNF、及び抗トランスフェリン受容体抗体又はその断片を含む融合タンパク質の製造方法であって、
上記融合タンパク質をコードする遺伝子及び外因性のシャペロンタンパク質をコードする遺伝子を含有する形質転換された哺乳動物細胞をタンパク質生産用培地中で培養し、上記融合タンパク質を生産する工程と、
生産された上記融合タンパク質を回収する工程と、
を備え、
上記融合タンパク質をコードする遺伝子は、上記BDNFをコードする遺伝子の塩基配列と、上記抗トランスフェリン受容体抗体又はその断片をコードする遺伝子の塩基配列とを含み、
上記シャペロンタンパク質は、HSP90α、HSP90β、CDC37、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる1以上を含む。
上記融合タンパク質をコードする遺伝子を含有する1以上の組換えタンパク質発現ベクターを含む哺乳動物細胞を準備する工程と、
シャペロンタンパク質をコードする遺伝子を含有する少なくとも1種の発現増強ベクターを用いて、上記哺乳動物細胞を形質転換する工程と、
を備える方法によって、得ることができる。詳細は、後述する「融合タンパク質の製造方法(1)」において説明する。
哺乳動物細胞を準備する工程と、
上記融合タンパク質をコードする遺伝子及びシャペロンタンパク質をコードする遺伝子を用いて、上記哺乳動物細胞を形質転換する工程と、
を備える方法によって、得ることができる。詳細は、後述する「融合タンパク質の製造方法(2)」において説明する。
本実施形態の第一の融合タンパク質の製造方法は、
BDNF、及び抗トランスフェリン受容体抗体又はその断片を含む融合タンパク質の製造方法であって、
上記融合タンパク質をコードする遺伝子を含有する1以上の組換えタンパク質発現ベクターを含む哺乳動物細胞を準備する工程と、
シャペロンタンパク質をコードする遺伝子を含有する少なくとも1種の発現増強ベクターを用いて、上記哺乳動物細胞を形質転換する工程と、
形質転換された上記哺乳動物細胞をタンパク質生産用培地中で培養し、上記融合タンパク質を生産する工程と、
生産された上記融合タンパク質を回収する工程と、
を備え、
上記融合タンパク質をコードする遺伝子は、上記BDNFをコードする遺伝子の塩基配列と、上記抗トランスフェリン受容体抗体又はその断片(以下、「付加タンパク質」という場合がある)をコードする遺伝子の塩基配列とを含み、
上記シャペロンタンパク質は、HSP90α、HSP90β、CDC37、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる1以上を含む。以下詳細に説明する。
本工程では、融合タンパク質をコードする遺伝子を含有する1以上の組換えタンパク質発現ベクターを含む哺乳動物細胞を準備する。上記融合タンパク質をコードする遺伝子は、上記BDNFをコードする遺伝子の塩基配列と、上記付加タンパク質をコードする遺伝子の塩基配列とを含む。
本実施形態における「融合タンパク質」は、BDNF、及び抗トランスフェリン受容体抗体又はその断片(以下、「付加タンパク質」という場合がある。)を含む。本実施形態の一側面において、上記融合タンパク質は、BDNF及び付加タンパク質のみからなっていてもよい。本実施形態の他の側面において、上記融合タンパク質は、BDNF、上記付加タンパク質及びBDNFと上記付加タンパク質とを連結するリンカーペプチドとから構成されていてもよい。すなわち、上記BDNFは、上記抗トランスフェリン受容体抗体又はその断片に対して、直接又はリンカーペプチドを介して結合していることが好ましい。
上記融合タンパク質として、具体的には、国際公開公報:WO2016/208696(特許文献2)、又はWO2018/124107(特許文献3)に記載の融合タンパク質(BDNFと抗トランスフェリン受容体抗体との融合タンパク質)が挙げられる。
ここで、上述のBDNFは、1982年にBardeらによって発見され,1990年にJonesらによってクローニングされた公知のタンパク質である(EMBO J,(1982) 1: 549-553,Proc.Natl.Acad.Sci.USA(1990)87:8060-8064)。BDNFには、生体内でその機能を発揮する成熟BDNF、成熟前のBDNFプリカーサー(「BDNFプロ体」とも言う。)、及び上記BDNFプリカーサーのN末端にシグナルペプチドが付加したBDNFプリカーサーの前駆体(「BDNFプレプロ体」とも言う。)が包含される。すなわち、BDNFは、その遺伝子転写産物から、まずBDNFプレプロ体として生成され、そこからシグナルペプチドが切断されてBDNFプロ体となる。その後、上記BDNFプロ体からN末端の110アミノ酸残基が切断されて成熟BDNFとなる。
本実施形態において「付加タンパク質」とは、上記BDNFと共に上記融合タンパク質を構成する抗トランスフェリン受容体抗体又は抗トランスフェリン受容体抗体断片を意味する。上記付加タンパク質は、単量体であってもよいし、2つのサブユニットから構成される二量体であってもよいし、複数のサブユニットから構成される多量体であってもよい。抗体断片としては、例えば、抗体の重鎖(H鎖)フラグメントと抗体の軽鎖(L鎖)フラグメントとからなるFabフラグメント、F(ab’)2フラグメント、F(ab’)フラグメント、抗体定常領域に当該Fabフラグメントが付加したFcフラグメント、一本鎖抗体(scFv)及び二重特異性抗体(diabody)が挙げられる。
本実施形態において、「組換えタンパク質発現ベクター」とは、宿主細胞内において発現可能なように、目的の組換えタンパク質をコードする遺伝子が導入されているDNA構築物を意味する。「組換えタンパク質」とは、上記宿主細胞に対して外因性のタンパク質を意味する。本実施形態において上記目的の組換えタンパク質は、上記融合タンパク質である。すなわち、上記組換えタンパク質発現ベクターは、上記融合タンパク質をコードする遺伝子を含有する。上記融合タンパク質をコードする遺伝子は、上記BDNFをコードする遺伝子の塩基配列と、上記付加タンパク質をコードする遺伝子の塩基配列とを含む。
(A)上記BDNFをコードする野生型の塩基配列に対して、90%以上100%以下の配列同一性を有する塩基配列、
(B)BDNFをコードする野生型の塩基配列に対して、1若しくは数個の塩基が欠失、置換、挿入若しくは付加された塩基配列、
(C)BDNFをコードする野生型の塩基配列に相補的な塩基配列を有するオリゴヌクレオチドに対して、ストリンジェントな条件でハイブリダイズする塩基配列、
(D)上記BDNFの野生型のアミノ酸配列に対して、90%以上100%以下の配列同一性を有するアミノ酸配列をコードする塩基配列、又は、
(E)上記BDNFの野生型のアミノ酸配列に対して、1若しくは数個のアミノ酸残基が欠失、置換、挿入若しくは付加されたアミノ酸配列をコードする塩基配列、であり且つ
上記BDNFにおける本来の機能を保持するタンパク質をコードする塩基配列であってもよい。
また、BDNFがBDNFプレプロ体である場合、「本来の機能」とは、例えばBDNFプロ体を生成し得ることを意味する。BDNFがBDNFプロ体である場合、「本来の機能」とは、例えば、成熟BDNFを生成し得ること、又はp75受容体に対する結合親和性を有することを意味する。
(A)上記付加タンパク質をコードする野生型の塩基配列に対して、90%以上100%以下の配列同一性を有する塩基配列、
(B)上記付加タンパク質をコードする野生型の塩基配列に対して、1若しくは数個の塩基が欠失、置換、挿入若しくは付加された塩基配列、
(C)上記付加タンパク質をコードする野生型の塩基配列に相補的な塩基配列を有するオリゴヌクレオチドに対して、ストリンジェントな条件でハイブリダイズする塩基配列、
(D)上記付加タンパク質の野生型のアミノ酸配列に対して、90%以上100%以下の配列同一性を有するアミノ酸配列をコードする塩基配列、又は、
(E)上記付加タンパク質の野生型のアミノ酸配列に対して、1若しくは数個のアミノ酸残基が欠失、置換、挿入若しくは付加されたアミノ酸配列をコードする塩基配列、であり且つ
上記付加タンパク質における本来の機能を保持するタンパク質をコードする塩基配列であってもよい。
なお、上記付加タンパク質が二量体又は多量体である場合、上記付加タンパク質を構成するサブユニットをコードする遺伝子それぞれについて、上述の事項が適用される。
上記付加タンパク質は、配列番号38の重鎖フラグメント及び配列番号42の軽鎖フラグメントを含む付加タンパク質のFabフラグメント、及び上記配列同一性を有する付加タンパク質は、特許文献3に記載されている。
尚、配列番号38の重鎖フラグメント及び配列番号42の軽鎖フラグメントを含む付加タンパク質のFabフラグメントは、特許文献3に記載されている。
上記ウイルスベクターとしては、レンチウイルスベクター、アデノウイルスベクター、アデノ随伴ウイルスベクター、センダイウイルスベクター、哺乳類発現型バキュロウイルスベクター等が挙げられる。例えば、pLenti4/V5-GW/lacZ、pLVSIN-CMV、pLVSIN-EF1α、pAxcwit2、pAxEFwit2、pAAV-RCS、pSeVベクター、pFastBacMam、pFastBacMam2.0(VSV-G)等が挙げられる。
本実施形態において、「哺乳動物細胞」とは、哺乳動物に由来する細胞を意味する。哺乳動物としては、例えば、ヒト、ハムスター(例えば、チャイニーズハムスター)、マウス、ラット、ミドリザル等が挙げられる。上記哺乳動物細胞は、不死化細胞であってもよい。
本工程では、シャペロンタンパク質をコードする遺伝子を含有する少なくとも1種の発現増強ベクターを用いて、上記哺乳動物細胞を形質転換する。
本実施形態において「シャペロンタンパク質」とは、上記融合タンパク質が正しいフォールディングをして本来の機能を獲得することを補助するタンパク質を意味する。「融合タンパク質の本来の機能」とは、上記融合タンパク質を構成しているBDNF及び付加タンパク質それぞれが有する本来の機能を意味する。上記シャペロンタンパク質は、HSP90α、HSP90β、CDC37(Cell Division Cycle 37,HSP90 cochaperone)、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる1以上を含む。ここで、「HSP」は、熱ショックタンパク質の略称である。本実施形態の一側面において、上記シャペロンタンパク質は、HSP90α、HSP90β、HSP40及びCDC37のいずれか一つを含むこと、又は、HSP90α、HSP90βもしくはHSP40と、CDC37との両方を含むことが好ましい。
本実施形態の他の側面において、上記シャペロンタンパク質は、HSP90α及びCDC37のいずれか一方又は両方を含むことが好ましい。
本実施態様の一側面において、上記シャペロンタンパク質の由来動物種は、システインノットタンパク質の由来動物種と同じであっても、異なっていてもよい。
本実施態様の一側面において、上記シャペロンタンパク質の由来動物種は、宿主細胞の由来動物種と同一であっても異なっていてもよい。
本実施態様の一側面において、上記シャペロンタンパク質の由来動物種は、システインノットタンパク質の由来動物種又は宿主細胞の由来動物種のいずれかと同一であることが好ましい。
本実施形態の一側面において、上記シャペロンタンパク質は、ヒト由来のシャペロンタンパク質であってもよいし、チャイニーズハムスター由来のシャペロンタンパク質であってもよい。好ましくはヒト由来のシャペロンタンパク質であってもよい。
本実施形態において「発現増強ベクター」とは、宿主細胞内において発現可能なように、上記シャペロンタンパク質をコードする遺伝子が導入されているDNA構築物を意味する。
(A)上記シャペロンタンパク質をコードする野生型の塩基配列に対して、90%以上100%以下の配列同一性を有する塩基配列、
(B)上記シャペロンタンパク質をコードする野生型の塩基配列に対して、1若しくは数個の塩基が欠失、置換、挿入若しくは付加された塩基配列、
(C)上記シャペロンタンパク質をコードする野生型の塩基配列に相補的な塩基配列を有するオリゴヌクレオチドに対して、ストリンジェントな条件でハイブリダイズする塩基配列、
(D)上記シャペロンタンパク質の野生型のアミノ酸配列に対して、90%以上100%以下の配列同一性を有するアミノ酸配列をコードする塩基配列、又は、
(E)上記シャペロンタンパク質の野生型のアミノ酸配列に対して、1若しくは数個のアミノ酸残基が欠失、置換、挿入若しくは付加されたアミノ酸配列をコードする塩基配列、であり且つ
上記BDNFが正しいフォールディングをして本来の機能を獲得することを補助するタンパク質をコードする塩基配列であってもよい。
HSP90βをコードする遺伝子の塩基配列としては、例えば、配列番号53(GenBank No.NM_001271970、ヒト由来の野生の塩基配列)、配列番号55(GenBank No.NM_001271971、ヒト由来の野生の塩基配列)、配列番号51(GenBank No.NM_001271972、ヒト由来の野生の塩基配列)、配列番号7、配列番号9、配列番号11、配列番号57(GenBank No.XM_003501668.2、チャイニーズハムスター由来の野生の塩基配列)、及び配列番号13の塩基配列が挙げられる。
CDC37をコードする遺伝子の塩基配列としては、例えば、配列番号59(GenBank No.NM_007065、ヒト由来の野生の塩基配列)、配列番号15、配列番号61(GenBank No.XM_003499737、チャイニーズハムスター由来の野生の塩基配列)及び配列番号17の塩基配列が挙げられる。
HSP60をコードする遺伝子の塩基配列としては、例えば、配列番号63(GenBank No.NM_199440、ヒト由来の野生の塩基配列)、及び配列番号19の塩基配列が挙げられる。
HSP40をコードする遺伝子の塩基配列としては、例えば、配列番号65(GenBank No.NM_001539、ヒト由来の野生の塩基配列)、及び配列番号21の塩基配列が挙げられる。
HSP10をコードする遺伝子の塩基配列としては、例えば、配列番号67(GenBank No.NM_002157、ヒト由来の野生の塩基配列)、及び配列番号23の塩基配列が挙げられる。
HSP110をコードする遺伝子の塩基配列としては、例えば、配列番号69(GenBank No.NM_006644、ヒト由来の野生の塩基配列)、及び配列番号25の塩基配列が挙げられる。
HSP70をコードする遺伝子の塩基配列としては、例えば、Journal of Biotechnology 143 (2009) 34-43に記載のCHO由来の野生の塩基配列、及び配列番号27の塩基配列が挙げられる。
HSP27をコードする遺伝子の塩基配列としては、例えば、Journal of Biotechnology 143 (2009) 34-43に記載のCHO由来の野生の塩基配列、及び配列番号29の塩基配列が挙げられる。
HSP90βのアミノ酸配列としては、例えば、配列番号8(GenBank No.NP_001258899)、配列番号10(GenBank No.NP_001258900)、配列番号12(GenBank No.NP_001258901)及び配列番号14(GenBank No.XP_003501716)のアミノ酸配列が挙げられる。
CDC37のアミノ酸配列としては、例えば、配列番号16(Genbank No.NP_008996)及び配列番号18(GenBankNo.XP_003499785)のアミノ酸配列が挙げられる。
HSP60のアミノ酸配列としては、例えば、配列番号20のアミノ酸配列(GenBank No.NP_955472)のアミノ酸配列が挙げられる。
HSP40のアミノ酸配列としては、例えば、配列番号22のアミノ酸配列(GenBank No.NP_001530)のアミノ酸配列が挙げられる。
HSP10のアミノ酸配列としては、例えば、配列番号24(GenBank No.NP_002148)のアミノ酸配列が挙げられる。
HSP110のアミノ酸配列としては、例えば、配列番号26(GenBank No.NP_006635)のアミノ酸配列が挙げられる。
HSP70のアミノ酸配列としては、例えば、配列番号28(Journal of Biotechnology 143 (2009) 34-43記載)のアミノ酸配列が挙げられる。
HSP27のアミノ酸配列としては、例えば、配列番号30(Journal of Biotechnology 143 (2009) 34-43記載)のアミノ酸配列が挙げられる。
上記プラスミドベクターとしては、例えば、pcDNA3.1(+)ベクター、pEGF-BOSベクター、pEFベクター、pCDM8ベクター、pCXNベクター、pCIベクター、エピソーマルベクター、トランスポゾンベクター等が挙げられる。本実施形態の一側面において、上記プラスミドベクターは、pcDNA3.1(+)ベクターであることが好ましい。
上記ウイルスベクターとしては、レンチウイルスベクター、アデノウイルスベクター、アデノ随伴ウイルスベクター、センダイウイルスベクター、哺乳類発現型バキュロウイルスベクター等が挙げられる。例えば、pLenti4/V5-GW/lacZ、pLVSIN-CMV、pLVSIN-EF1α、pAxcwit2、pAxEFwit2、pAAV-RCS、pSeVベクター、pFastBacMam、pFastBacMam2.0(VSV-G)等が挙げられる。
または、2以上のシャペロンタンパク質をコードする遺伝子を含む発現増強ベクターで哺乳動物細胞を形質転換してもよい。
本実施形態において、発現増強ベクターを用いた形質転換の方法は、本発明の効果が奏される限りにおいて特に制限されず、公知の方法を用いることができる(例えば、Sambrook et al.”Molecular Cloning-A Laboratory Manual,second edition 1989”)。公知である形質転換の方法としては、例えば、リポフェクション法、リン酸カルシウム法、DEAEデキストラン法、エレクトロポレーション法、ポリエチレンイミン法及びポリエチレングリコール法等が挙げられる。また、市販されているキットを用いて上述の形質転換を行ってもよい。そのようなキットとしては、例えば、ThermoFisher Scientific K.K.社製のGibco(商標) Expi(商標) Expression System(Cat.No.A29133)等が挙げられる。
リポフェクション法の場合、例えば、細胞密度1×106cells/mL~9×106cells/mL)あたり、3μg~30μgの発現ベクターを用いる。例えば、25mL容器に入った細胞(6×106 cells/mL)に対して、トータル20μgの発現増強ベクターを用いる。
本工程では、形質転換された上記哺乳動物細胞をタンパク質生産用培地中で培養し、上記融合タンパク質を生産する。
本工程では、生産された上記融合タンパク質を回収する。本工程は、培養終了後の培養上清から、生産された上記融合タンパク質を回収することを含む。例えば、培養終了後、得られた培養上清を各種精製法により処理して、精製された高純度の融合タンパク質を得ることができる。
本実施形態の第二の融合タンパク質の製造方法は、
BDNF、及び抗トランスフェリン受容体抗体又はその断片を含む融合タンパク質の製造方法であって、
哺乳動物細胞を準備する工程と、
上記融合タンパク質をコードする遺伝子及びシャペロンタンパク質をコードする遺伝子を用いて、上記哺乳動物細胞を形質転換する工程と、
形質転換された上記哺乳動物細胞をタンパク質生産用培地中で培養し、上記融合タンパク質を生産する工程と、
生産された上記融合タンパク質を回収する工程と、
を備え、
上記融合タンパク質をコードする遺伝子は、上記BDNFをコードする遺伝子の塩基配列と、上記抗トランスフェリン受容体抗体又はその断片をコードする遺伝子の塩基配列とを含み、
上記シャペロンタンパク質は、HSP90α、HSP90β、CDC37、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる1以上を含む。
本工程では、哺乳動物細胞を準備する。上記哺乳動物細胞は、上述の「融合タンパク質の製造方法(1)」において、例示した哺乳動物細胞を用いることができる。すなわち、上記哺乳動物細胞は、CHO細胞、COS細胞、BHK細胞、HeLa細胞、HEK293細胞、NS0細胞及びSp2/0細胞からなる群より選ばれる1種以上を含むことが好ましい。
本工程では、融合タンパク質をコードする遺伝子及びシャペロンタンパク質をコードする遺伝子を用いて、哺乳動物細胞を形質転換する。上記融合タンパク質をコードする遺伝子は、上記BDNFをコードする遺伝子の塩基配列と、上記抗トランスフェリン受容体抗体又はその断片をコードする遺伝子の塩基配列とを含む。
例えば、両遺伝子を宿主細胞に導入する場合における、融合タンパク質をコードする遺伝子とシャペロンタンパク質をコードする遺伝子との比率は、1:1~10:1、より具体的には4:1であってもよい。
本工程では、形質転換された上記哺乳動物細胞をタンパク質生産用培地中で培養し、上記融合タンパク質を生産する。具体的な方法は、上述の「融合タンパク質の製造方法(1)」において、述べた方法を用いることができる。
本工程では、生産された上記融合タンパク質を回収する。具体的な方法は、上述の「融合タンパク質の製造方法(1)」において、述べた方法を用いることができる。
本実施形態における組換えタンパク質生産用哺乳動物細胞は、
融合タンパク質をコードする遺伝子を含有する1以上の組換えタンパク質発現ベクターを含む、組換えタンパク質生産用哺乳動物細胞であって、
上記融合タンパク質は、BDNF、及び抗トランスフェリン受容体抗体又はその断片を含み、
上記融合タンパク質をコードする遺伝子は、上記BDNFをコードする遺伝子の塩基配列と、上記抗トランスフェリン受容体抗体又はその断片をコードする遺伝子の塩基配列とを含み、
上記組換えタンパク質生産用哺乳動物細胞は、シャペロンタンパク質をコードする遺伝子を含有する1種以上の発現増強ベクターを更に含み、
上記シャペロンタンパク質は、HSP90α、HSP90β、CDC37、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる1以上を含む。
本実施形態におけるキットは、
哺乳動物細胞における、BDNF、及び抗トランスフェリン受容体抗体又はその断片を含む融合タンパク質の生産量を増強させるためのキットであって、
シャペロンタンパク質をコードする遺伝子を含有する1種以上の発現増強ベクターを含み、
上記シャペロンタンパク質は、HSP90α、HSP90β、CDC37、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる少なくとも1つを含む。
本発明の製造方法で製造される融合タンパク質は、当該融合タンパク質を有効成分として含有する医薬組成物の原料として用いることができる。本願発明は、当該融合タンパク質と添加物を接触させる工程を含む当該医薬組成物の製造方法を包含する。上記添加物は、医薬組成物に含まれる添加物として一般に知られている成分であれば、特に制限はなく適宜選択できる。
≪発現増強因子の哺乳類発現プラスミド(発現増強ベクター)の調製≫
発現増強因子(シャペロンタンパク質)として以下の9種類の遺伝子を検討に用いた。
(1)ヒト熱ショックタンパク質90α(HSP90α)遺伝子(HSP90AA1)(GenBank No.NP_001017963、アミノ酸配列:配列番号2)(コドン最適化後の塩基配列:配列番号1)、
(2)ヒトCell Division Cycle 37,HSP90 cochaperone(CDC37)遺伝子(Genbank No.NP_008996、アミノ酸配列:配列番号16)(コドン最適化後の塩基配列:配列番号15)、
(3)ヒトHSP60遺伝子(GenBank No.NP_955472、アミノ酸配列:配列番号20)(コドン最適化後の塩基配列:配列番号19)、
(4)ヒトHSP10遺伝子(GenBank No.NP_002148、アミノ酸配列:配列番号24)(コドン最適化後の塩基配列:配列番号23)、
(5)ヒトHSP110遺伝子(GenBank No.NP_006635、アミノ酸配列:配列番号26)(コドン最適化後の塩基配列:配列番号25)、
(6)ヒトHSP40遺伝子(GenBank No. NP_NP_001530、アミノ酸配列:配列番号22)(コドン最適化後の塩基配列:配列番号21)、
(7)ヒトHSJ1遺伝子(GenBank No. AAA09034、アミノ酸配列:配列番号34)(コドン最適化後の塩基配列:配列番号33)、
(8)チャイニーズハムスター卵巣由来細胞CHOのHSP70遺伝子(J.Biotechnology 143 (2009) 34-43)(コドン最適化後の塩基配列:配列番号27、アミノ酸配列:配列番号28)、
(9)チャイニーズハムスター卵巣由来細胞CHOのHSP27遺伝子(J.Biotechnology 143 (2009) 34-43)(コドン最適化後の塩基配列:配列番号29、アミノ酸配列:配列番号30)。
BDNF及び付加タンパク質からなる融合タンパク質をコードする遺伝子として以下の遺伝子を準備した。
(1)第一の遺伝子:hBDNF-hFab(H)遺伝子(コドン最適化後の塩基配列:配列番号39、アミノ酸配列:配列番号40、ここでhFab(H)は抗トランスフェリン受容体抗体のFab重鎖である)、
(2)第二の遺伝子:hFab(L)遺伝子(コドン最適化後の塩基配列:配列番号43、アミノ酸配列:配列番号44、ここでhFab(L)は抗トランスフェリン受容体抗体のFab軽鎖である)。
上記hFab(L)遺伝子は、N末端側から順に、IgG signal sequence(GenBank No. 6SVL_B、コドン最適化後の塩基配列:配列番号35、アミノ酸配列:配列番号36)及び第二のサブユニットであるヒト抗トランスフェリン受容体抗体Fab L鎖フラグメント(コドン最適化後の塩基配列:配列番号41、アミノ酸配列:配列番号42)からなるポリペプチドをコードする遺伝子である。
Gibco(商標) Expi(商標) Expression System(Cat.No.A29133,ThermoFisher Scientific K.K.)を利用し、Max Titerプロトコールに従い以下の操作を実施した。まず、培養したExpi-CHO細胞(6×106 cells/mL)を、ExpiCHO(商標) Expression Medium(Cat.No.A29100-01,ThermoFisher Scientific K.K.)(25mL)が入った125mL容量のErlenmeyer flask(Corning Inc.Cat.No.431143)に加えた。次に、以下の表1に示すプラスミドベクターが含まれる試薬(1ml)をそれぞれ調製した。また、上記プラスミドベクターが含まれる試薬とは異なるチューブにExpifectamine(Cat.No.A12129)(80μL)及び、OptiPRO(商標) SFM(Cat.No.12309050)(920μL)を加えた。上記プラスミドベクターが含まれる試薬及び上記Expifectamineが含まれる試薬をそれぞれ撹拌させ、室温で5分間放置した。その後、両試薬をゆっくりと混和し、ExpiFectamine(商標) CHO/plasmid DNA complexesとし、1から5分間、室温で放置した。そのcomplexesをExpi-CHO細胞の入った125mL容量のErlenmeyer flaskに加え、37℃、8%CO2、125rpm下で撹拌培養を一晩実施した。
Claims (14)
- BDNF、及び抗トランスフェリン受容体抗体又はその断片を含む融合タンパク質の製造方法であって、
前記融合タンパク質をコードする遺伝子及び外因性のシャペロンタンパク質をコードする遺伝子を含有する形質転換された哺乳動物細胞をタンパク質生産用培地中で培養し、前記融合タンパク質を生産する工程と、
生産された前記融合タンパク質を回収する工程と、
を備え、
前記融合タンパク質をコードする遺伝子は、前記BDNFをコードする遺伝子の塩基配列と、前記抗トランスフェリン受容体抗体又はその断片をコードする遺伝子の塩基配列とを含み、
前記シャペロンタンパク質は、HSP90α、HSP90β、CDC37、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる1以上を含む、融合タンパク質の製造方法。 - 前記BDNF、及び前記抗トランスフェリン受容体抗体又はその断片を含む融合タンパク質の製造方法であって、
哺乳動物細胞を準備する工程と、
前記融合タンパク質をコードする遺伝子及び前記シャペロンタンパク質をコードする遺伝子を用いて、前記哺乳動物細胞を形質転換する工程と、
形質転換された前記哺乳動物細胞をタンパク質生産用培地中で培養し、前記融合タンパク質を生産する工程と、
生産された前記融合タンパク質を回収する工程と、
を備え、
前記融合タンパク質をコードする遺伝子は、前記BDNFをコードする遺伝子の塩基配列と、前記抗トランスフェリン受容体抗体又はその断片をコードする遺伝子の塩基配列とを含み、
前記シャペロンタンパク質は、HSP90α、HSP90β、CDC37、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる1以上を含む、請求項1に記載の融合タンパク質の製造方法。 - 前記哺乳動物細胞を形質転換する工程は、前記融合タンパク質をコードする遺伝子及び前記シャペロンタンパク質をコードする遺伝子を含有する1以上の組換えタンパク質発現ベクターを用いて実施される、請求項2に記載の融合タンパク質の製造方法。
- 前記哺乳動物細胞を形質転換する工程は、前記融合タンパク質をコードする遺伝子を含有する1以上の組換えタンパク質発現ベクター、及び前記シャペロンタンパク質をコードする遺伝子を含有する1以上の発現増強ベクターを、同時又は別々に前記哺乳動物細胞に接触させることで実施される、請求項2に記載の融合タンパク質の製造方法。
- 前記BDNF、及び前記抗トランスフェリン受容体抗体又はその断片を含む融合タンパク質の製造方法であって、
前記融合タンパク質をコードする遺伝子を含有する1以上の組換えタンパク質発現ベクターを含む哺乳動物細胞を準備する工程と、
前記シャペロンタンパク質をコードする遺伝子を含有する少なくとも1種の発現増強ベクターを用いて、前記哺乳動物細胞を形質転換する工程と、
形質転換された前記哺乳動物細胞をタンパク質生産用培地中で培養し、前記融合タンパク質を生産する工程と、
生産された前記融合タンパク質を回収する工程と、
を備え、
前記融合タンパク質をコードする遺伝子は、前記BDNFをコードする遺伝子の塩基配列と、前記抗トランスフェリン受容体抗体又はその断片をコードする遺伝子の塩基配列とを含み、
前記シャペロンタンパク質は、HSP90α、HSP90β、CDC37、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる1以上を含む、請求項1に記載の融合タンパク質の製造方法。 - 前記発現増強ベクターは、第一のシャペロンタンパク質をコードする遺伝子を含有する第一の発現増強ベクターと、第二のシャペロンタンパク質をコードする遺伝子を含有する第二の発現増強ベクターとを含み、
前記第一のシャペロンタンパク質は、前記第二のシャペロンタンパク質と異なる、請求項5に記載の融合タンパク質の製造方法。 - 前記シャペロンタンパク質は、HSP90α及びCDC37のいずれか一方又は両方を含む、請求項1から請求項6のいずれか一項に記載の融合タンパク質の製造方法。
- 前記BDNFは、前記抗トランスフェリン受容体抗体又はその断片に対して、直接又はリンカーペプチドを介して結合している、請求項1から請求項7のいずれか一項に記載の融合タンパク質の製造方法。
- 前記リンカーペプチドは、Gly、Ser、Gly-Ser、Gly-Gly-Ser、Gly-Gly-Gly-Gly-Ser、Gly-Gly-Gly-Gly-Gly-Ser、Ser-Gly-Gly-Gly-Gly、及びこれらのアミノ酸配列が1~10個連続してなるアミノ酸配列からなる群から選択されるアミノ酸配列を含む、請求項8に記載の融合タンパク質の製造方法。
- 前記BDNFは、配列番号32のアミノ酸配列に対して少なくとも90%の配列同一性を有するアミノ酸配列を含む、請求項1から請求項9のいずれか一項に記載の融合タンパク質の製造方法。
- 前記抗トランスフェリン受容体抗体の断片は、Fabフラグメント、F(ab’)2フラグメント、又はF(ab’)フラグメントである、請求項1から請求項10のいずれか一項に記載の融合タンパク質の製造方法。
- 前記哺乳動物細胞は、CHO細胞、COS細胞、BHK細胞、HeLa細胞、HEK293細胞、NS0細胞及びSp2/0細胞からなる群より選ばれる1種以上を含む、請求項1から請求項11のいずれか一項に記載の融合タンパク質の製造方法。
- 融合タンパク質をコードする遺伝子を含有する1以上の組換えタンパク質発現ベクターを含む、組換えタンパク質生産用哺乳動物細胞であって、
前記融合タンパク質は、BDNF、及び抗トランスフェリン受容体抗体又はその断片を含み、
前記融合タンパク質をコードする遺伝子は、前記BDNFをコードする遺伝子の塩基配列と、前記抗トランスフェリン受容体抗体又はその断片をコードする遺伝子の塩基配列とを含み、
前記組換えタンパク質生産用哺乳動物細胞は、シャペロンタンパク質をコードする遺伝子を含有する1種以上の発現増強ベクターを更に含み、
前記シャペロンタンパク質は、HSP90α、HSP90β、CDC37、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる1以上を含む、組換えタンパク質生産用哺乳動物細胞。 - 哺乳動物細胞における、BDNF、及び抗トランスフェリン受容体抗体又はその断片を含む融合タンパク質の生産量を増強させるためのキットであって、
シャペロンタンパク質をコードする遺伝子を含有する1種以上の発現増強ベクターを含み、
前記シャペロンタンパク質は、HSP90α、HSP90β、CDC37、HSP70、HSP40、HSP60、HSP10、HSP110及びHSP27からなる群より選ばれる少なくとも1つを含む、キット。
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