WO2021039574A1 - Heavy-chain antibody in which o-linked sugar chain modification is suppressed - Google Patents

Heavy-chain antibody in which o-linked sugar chain modification is suppressed Download PDF

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WO2021039574A1
WO2021039574A1 PCT/JP2020/031425 JP2020031425W WO2021039574A1 WO 2021039574 A1 WO2021039574 A1 WO 2021039574A1 JP 2020031425 W JP2020031425 W JP 2020031425W WO 2021039574 A1 WO2021039574 A1 WO 2021039574A1
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amino acid
acid sequence
seq
heavy chain
chain antibody
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PCT/JP2020/031425
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French (fr)
Japanese (ja)
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将弘 荒武
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株式会社カネカ
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a heavy chain antibody that is not easily modified by an O-linked sugar chain.
  • the present invention also relates to a nucleic acid containing a base sequence encoding the amino acid sequence of the heavy chain antibody, a vector containing the nucleic acid, and a host cell.
  • the present invention also relates to a method for producing the heavy chain antibody.
  • the present invention also relates to a medicament for gene therapy, which comprises a nucleic acid containing a base sequence encoding the amino acid sequence of the heavy chain antibody.
  • Animal cells, Escherichia coli, yeast, etc. are used for the production of proteins such as antibodies for pharmaceutical use by gene recombination technology.
  • a host cell having a gene of an enzyme having an activity of adding an O-linked sugar chain to a serine or threonine residue of a protein such as a PMT (Protein O-Mannosyltransphase) gene
  • PMT Protein O-Mannosyltransphase
  • An O-linked sugar chain may be added to the serine or threonine residue of the foreign protein produced.
  • Patent Document 1 It has been recognized that a particular sugar chain structure on a protein can have a profound effect on the properties of the protein, including its pharmacokinetics, pharmacodynamics, receptor interactions, and tissue-specific targeting properties (non-).
  • Patent Document 1 it is known that the sugar chain structure on an antigen protein affects the binding property to an antibody, and in Patent Document 1, an immunogenic, hyperglycosylated IL-7 polypeptide is produced. Is described. However, it is difficult to predict which particular glycoform (s) contribute to the desired biological function, so it is desirable to produce a protein that has not undergone sugar chain modification.
  • Patent Document 2 a gene encoding an active HAC1 gene and / or an RRBP1 gene and a foreign protein under conditions of suppressing O-type sugar chain synthesis including addition of a PMT activity inhibitor to a medium and / or dysfunction of the PMT gene.
  • a method for producing a protein in which O-linked sugar chain modification is suppressed is described in which transformed cells into which the gene has been introduced is cultured in a medium and a foreign protein is collected from the culture.
  • VHH Very domain of the heavi-chain of heavi-chain antibody
  • a single domain heavy chain antibody composed of VHH has high chemical stability and is easy to manufacture, and is therefore suitable for application to pharmaceuticals and the like.
  • Patent Document 3 describes a method for generating an immunoglobulin sequence capable of binding to a cell-related antigen, which comprises gene vaccination of a non-human animal such as a camelid with a nucleic acid encoding a cell-related antigen. ing. And in Patent Document 3, a heavy chain variable domain sequence derived from a heavy chain antibody is described as the immunoglobulin sequence.
  • Sugar chain modification of an antibody may affect the antigen-binding activity and the immunogenicity of the antibody itself.
  • antigen-binding activity and immunogenicity may change depending on the position, length and number of sugar chains that modify the antibody. Therefore, when an antibody is produced by a gene recombination technique, it is considered that the quality of the antibody becomes non-uniform if the ratio of sugar chain modification is high. Therefore, one or more embodiments of the present invention provide a heavy chain antibody that is less susceptible to modification by O-linked sugar chains. Heavy chain antibodies that are not easily modified by O-linked sugar chains can be easily produced with uniform quality by transgenic cells.
  • FR1 framework regions 1
  • VH-CDR1 variable heavy chain complementarity determining regions 1
  • FR2 framework regions 2
  • VH-CDR3 variable heavy chain complementarity determining regions 3
  • FR4 framework regions 4
  • framework regions 1 variable heavy chain complementarity determining regions 1, framework regions 2, variable heavy chain complementarity determining regions 2, framework regions 3, variable heavy chain complementarity determining regions 3 and framework regions 4 Heavy chain antibodies containing heavy chain variable domains linked in this order.
  • Xaa7 is L or I
  • Xaa8 is A or T
  • Amino acid sequence shown in (1b) An amino acid sequence having 83% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.
  • the amino acid sequence of framework region 2 (2a) SEQ ID NO: 2: W-Xaa9-RQ-A-P-G-Xaa10-Xaa11-Xaa12-E-Xaa13-V-Xaa14 (Xaa9 is V, Y or F, Xaa10 is K or Q, Xaa11 is G or E, Xaa12 is L or R, Xaa13 is W, L, F or A, Xaa14 is S.
  • Amino acid sequence shown in (2b) An amino acid sequence having 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2.
  • Xaa21 is P or A
  • Xaa22 is E or D
  • Xaa23 is V or I
  • Xaa24 is T
  • a or G
  • Xaa25 is I, F, A.
  • R In the amino acid sequence shown in, at least one of the amino acids at positions 3, 5, 11, 12, 19 and 25 of SEQ ID NO: 3 is replaced with an amino acid other than serine or threonine.
  • Amino acid sequence in which is introduced or (3b) In the amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, the 3rd, 5th, 11th, 12th, 19th and 25th positions of SEQ ID NO: 3 An amino acid sequence in which substitution with an amino acid other than serine or threonine is introduced into at least one of the amino acids corresponding to the amino acid at the position.
  • the amino acid sequence of framework region 4 (4a) SEQ ID NO: 4: Xaa26-Xaa27-Xaa28-Xaa29-GT-Xaa30-VT-VS-S (Xaa26 is R, Y, N or S, Xaa27 is S, W or R, Xaa28 is S or G, Xaa29 is Q, L or R, Xaa30 is Q or L) Amino acid sequence shown in (4b) A heavy chain antibody characterized by having an amino acid sequence having 65% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4. [2] The amino acid sequence of framework region 1 is the amino acid sequence defined in (1a). The amino acid sequence of framework region 2 is the amino acid sequence defined in (2a).
  • the amino acid sequence of framework region 3 is the amino acid sequence defined in (3a)
  • the amino acid sequence of framework region 4 is the amino acid sequence defined in (4a).
  • the amino acid sequence defined in (3a) is at least the amino acids at positions 3, 5, 11, 12, 19, and 25 of SEQ ID NO: 3 in the amino acid sequence shown in SEQ ID NO: 3.
  • the second is an amino acid sequence in which substitution with an amino acid other than serine or threonine has been introduced.
  • [6] A vector containing the nucleic acid according to [5].
  • [7] A host cell containing the nucleic acid according to [5].
  • [8] The method for producing a heavy chain antibody according to any one of [1] to [3]. Culturing the host cell according to [7] and A method comprising recovering the heavy chain antibody according to any one of [1] to [3] from the culture.
  • [9] A drug for gene therapy containing the nucleic acid according to [5].
  • [11] The nucleic acid according to [5] for use in treating or ameliorating a disease treated or ameliorated by the heavy chain antibody according to any one of [1] to [3].
  • nucleic acid according to [5] for the production of a drug used for gene therapy.
  • nucleic acid according to [5] for producing a drug used for treating or ameliorating a disease treated or ameliorated by the heavy chain antibody according to any one of [1] to [3].
  • the disease which comprises administering the nucleic acid according to [5] to a patient in need of treatment or improvement of the disease treated or ameliorated by the heavy chain antibody according to any one of [1] to [3]. Treatment or improvement method.
  • a heavy chain antibody that is less susceptible to modification by O-linked sugar chains is provided.
  • FIG. 1 shows a known weight comprising a heavy chain variable domain having a structure of (FR1)-(VH-CDR1)-(FR2)-(VH-CDR2)-(FR3)-(VH-CDR3)-(FR4). It is a chain antibody, anti-Fc VHH containing the amino acid sequence shown in SEQ ID NO: 50, anti-HSA VHH containing the amino acid sequence shown in SEQ ID NO: 52, anti-IL6R VHH containing the amino acid sequence shown in SEQ ID NO: 57, SEQ ID NO: 58. Anti-vWF VHH containing the amino acid sequence shown in FIG.
  • anti-RANKL VHH containing the amino acid sequence shown in SEQ ID NO: 59
  • anti-TNF ⁇ VHH containing the amino acid sequence shown in SEQ ID NO: 60
  • anti-containing the amino acid sequence shown in SEQ ID NO: 61 anti-containing the amino acid sequence shown in SEQ ID NO: 61.
  • the outline of the results of aligning and comparing the amino acid sequences of cAbBcII10 VHH, anti-RSV VHH-1 containing the amino acid sequence shown in SEQ ID NO: 62, and anti-RSV VHH-2 containing the amino acid sequence shown in SEQ ID NO: 63 is shown.
  • amino acids and proteins are represented using the abbreviations adopted by the IUPAC-IUB Biochemical Nomenclature Committee (CBN) shown below. Unless otherwise specified, the sequence of amino acid residues of a protein is represented from the N-terminal to the C-terminal from the left end to the right end. Also, for ease of reference, the following commonly used nomenclature is applied.
  • One is a method described as "original amino acid; position; substituted amino acid”. For example, the substitution of threonine with aspartic acid at position 69 is expressed as "T69D".
  • sequence identity of a base sequence or an amino acid sequence can be obtained by using a method well known to those skilled in the art, sequence analysis software, or the like.
  • sequence analysis software or the like.
  • a BLASTn program or a Blastp program of the BLAST algorithm and a FASTA program of the FASTA algorithm can be mentioned.
  • sequence identity of a certain evaluation target base sequence with the base sequence X means that the base sequence X and the evaluation target base sequence are aligned (aligned) and a gap is introduced as necessary. It is a value indicating the frequency at which the same base appears at the same site in the base sequence including the gap portion when the degree of base matching between the two is set to be the highest.
  • sequence identity of a certain amino acid sequence to be evaluated with the amino acid sequence X means that the amino acid sequence X and the amino acid sequence to be evaluated are aligned and a gap is introduced as necessary to introduce both amino acids. It is a value indicating the frequency of appearance of the same base at the same site in the amino acid sequence including the gap portion when the degree of coincidence is set to be the highest in%.
  • the "nucleic acid” can also be called a polynucleotide, and refers to DNA or RNA, and typically refers to DNA.
  • the "nucleic acid” may exist in a form double-stranded with its complementary strand.
  • the DNA containing a predetermined base sequence exists in a double-stranded form with the DNA containing the complementary base sequence.
  • polypeptide refers to a peptide bond of two or more amino acids, and includes proteins, peptides and oligopeptides having a short chain length.
  • the "base sequence encoding" the amino acid sequence of a predetermined heavy chain antibody refers to the base sequence of a polynucleotide that produces a predetermined heavy chain antibody by transcription and translation, for example, the amino acid sequence of a heavy chain antibody. Refers to a base sequence designed based on the codon table.
  • the "host cell” refers to a cell into which nucleic acid is introduced and transformed, or a cell into which nucleic acid is introduced and transformed, and is also referred to as a "host”. Cells into which nucleic acids have been introduced and transformed may be particularly referred to as "transformants”.
  • “Expression” refers to the transcription and translation of a base sequence that results in the production of a heavy chain antibody polypeptide.
  • its expression may be in a substantially constant state without depending on external stimuli, growth conditions, etc., or may depend on it.
  • the promoter that drives the expression is not particularly limited as long as it is a promoter that drives the expression of the base sequence encoding the amino acid sequence of the heavy chain antibody.
  • yeast is preferable as the host organism.
  • the yeast may be a yeast having no methanol assimilation property such as Saccharomyces genus, Saccharomyces genus, Quiberomyces genus, Yarowina genus, or a methanol assimilation yeast, but may have methanol assimilation property.
  • Yeast is more preferred.
  • methanol-utilizing yeast is defined as yeast that can be cultivated using methanol as the only carbon source. Although it was originally methanol-utilizing yeast, it has the ability to assimilate methanol by artificial modification or mutation. Yeast that has lost the above is also included in the methanol-utilizing yeast in the present invention.
  • Examples of the methanol-utilizing yeast include yeasts belonging to the genus Pichia, Ogataea, Candida, Torulopsis, Komagataella and the like.
  • Pichia metanolica in the genus Ogataea, Ogataea angusta, Ogataea polymorpha, Ogataea polymorpha, Ogataea polymorpha, ), Candida boidini in the genus Candida, Pichia pastoris in the genus Komagataela, Pichia pastoris in the genus Komagataela, Pichia pastoris in the genus Komagataela, Pichia pastoris in the genus Komagataela, and the like.
  • Pichia yeast, Komagataera yeast, and Ogataea yeast are particularly preferable.
  • Komagataela pastoris As the yeast belonging to the genus Komagataella, Komagataela pastoris and Komagataela phaffii are preferable. Both Komagataera pastoris and Komagataera Fafi have another name for Pichia pastoris.
  • yeast strains that can be used as hosts include yeast strains such as Komagataera pastris ATCC76273 (Y-11430, CBS7435) and Komagataera pastris X-33. These yeast strains can be obtained from the American Type Culture Collection, Thermo Fisher Scientific, etc.
  • Ogataea As the yeast of the genus Ogataea, Ogataea angusta, Ogataea polymorpha, and Ogataea parapolymorpha are preferable. These three are closely related species, and all of them are also represented by another name, Hansenula polymorpha, or another name, Pichia angusta.
  • yeast strains that can be used include yeast strains such as Ogataea Angsta NCYC495 (ATCC14754), Ogataea polymorpha 8V (ATCC34438), and Ogataea parapolymorpha DL-1 (ATCC26012). These yeast strains can be obtained from the American Type Culture Collection and others.
  • yeast strains such as Pichia yeast, Komagataera yeast, and Ogataea yeast can also be used.
  • yeast strains such as Pichia yeast, Komagataera yeast, and Ogataea yeast
  • histidine is required
  • Komagataera Pastris GS115 strain available from Thermofisher Scientific
  • BY4329 derived from NCYC495, BY5242 derived from 8V, BY5243 derived from DL-1 (these can be sold from National BioResource Project) and the like can be mentioned.
  • derived strains and the like from these strains can also be used.
  • Heavy chain antibody> One or more embodiments of the present invention include framework regions 1 (FR1), variable heavy chain complementarity determining regions 1 (VH-CDR1), framework regions 2 (FR2), and variable heavy chain complementarity determining regions from the N-terminus.
  • the heavy chain antibody according to one or more embodiments of the present invention may be any one containing the heavy chain variable domain, and is a single composed of the heavy chain variable domain. It may be a domain antibody (VHH antibody), or it may be a heavy chain antibody containing the heavy chain variable domain and a heavy chain constant domain or a fragment thereof.
  • VHH antibody domain antibody
  • the heavy chain variable domain consists only of a polypeptide consisting of (FR1)-(VH-CDR1)-(FR2)-(VH-CDR2)-(FR3)-(VH-CDR3)-(FR4). It may be in the form of a fusion polypeptide in which another polypeptide is further linked to one or both of the N-terminal side and the C-terminal side of the polypeptide.
  • polypeptides include, but are not limited to, signal peptides, tag peptides, and the like. Specific examples of the signal peptide will be described later.
  • the tag peptide include a tag peptide (polyhistidine tag) composed of a plurality of (for example, 6 to 10) histidine residues and a FLAG tag peptide.
  • the heavy chain antibody according to one or more embodiments of the present invention is the heavy chain of the heavy chain antibody when expressed in a host cell transformed with a vector containing a nucleic acid containing a base sequence encoding the amino acid sequence. O-linked sugar chain modification at the serine or threonine residue of FR3 in the variable domain is suppressed.
  • the heavy chain antibody thus obtained is an antibody having high homogeneity and stable quality such as antigen binding.
  • the heavy chain antibody according to one or more embodiments of the present invention preferably has a low proportion of O-linked sugar chain modifiers, and specifically, the proportion of O-linked sugar chain modifiers to the total amount of the heavy chain antibody. It is 5% by mass or less, preferably 4% by mass or less, more preferably 3% by mass or less, particularly preferably 2% by mass or less, still more preferably 1% by mass or less, and most preferably 0% by mass.
  • the heavy chain antibody may be a multispecific antibody or a multivalent antibody in which a plurality of the antibodies are directly linked or linked via a linker.
  • a multispecific antibody is an antibody in which a plurality of the heavy chain antibodies having different antigens that specifically bind to each other are linked.
  • a multivalent antibody is an antibody in which a plurality of the heavy chain antibodies having the same specifically binding antigen are linked.
  • the present inventors are known to include a heavy chain variable domain having a structure of (FR1)-(VH-CDR1)-(FR2)-(VH-CDR2)-(FR3)-(VH-CDR3)-(FR4).
  • anti-Fc VHH containing the amino acid sequence shown in SEQ ID NO: 50
  • anti-HSA VHH containing the amino acid sequence shown in SEQ ID NO: 52
  • anti-IL6R VHH containing the amino acid sequence shown in SEQ ID NO: 57
  • Anti-vWF VHH containing the amino acid sequence shown in 58 anti-RANKL VHH containing the amino acid sequence shown in SEQ ID NO: 59
  • anti-TNF ⁇ VHH containing the amino acid sequence shown in SEQ ID NO: 60
  • antii containing the amino acid sequence shown in SEQ ID NO: 61.
  • amino acid sequences of -cAbBcII10 VHH, anti-RSV VHH-1 containing the amino acid sequence shown in SEQ ID NO: 62, and anti-RSV VHH-2 containing the amino acid sequence shown in SEQ ID NO: 63 were aligned and compared.
  • the outline of the comparison result is shown in FIG.
  • the amino acid sequences of FR1, FR2, FR3, and FR4 are highly conserved in the heavy chain variable domain of these heavy chain antibodies.
  • the amino acid sequence of FR1 can be represented by SEQ ID NO: 1.
  • SEQ ID NO: 1 of FR1 the partial amino acid sequence of positions 1 to 25 in the amino acid sequence of anti-Fc VHH shown in SEQ ID NO: 50, anti-HSA shown in SEQ ID NO: 52.
  • Examples thereof include a partial amino acid sequence at positions 1 to 25 in the amino acid sequence of VHH-1, and a partial amino acid sequence at positions 1 to 25 in the amino acid sequence of anti-RSV VHH-2 shown in SEQ ID NO: 63.
  • the amino acid sequence of FR2 can be represented by SEQ ID NO: 2.
  • SEQ ID NO: 2 of FR2 the partial amino acid sequence at positions 36 to 49 in the amino acid sequence of anti-Fc VHH shown in SEQ ID NO: 50, anti-HSA shown in SEQ ID NO: 52.
  • Examples thereof include the partial amino acid sequence at positions 36 to 49 in the amino acid sequence of VHH-1, and the partial amino acid sequence at positions 36 to 49 in the amino acid sequence of anti-RSV VHH-2 shown in SEQ ID NO: 63.
  • the amino acid sequence of FR3 can be represented by SEQ ID NO: 3.
  • SEQ ID NO: 3 of FR3 the partial amino acid sequence of positions 67 to 98 in the amino acid sequence of anti-Fc VHH shown in SEQ ID NO: 50, anti-HSA shown in SEQ ID NO: 52.
  • Partial amino acid sequence at positions 67 to 98 in the amino acid sequence of VHH partial amino acid sequence at positions 67 to 98 in the amino acid sequence of anti-RANKL VHH shown in SEQ ID NO: 59, anti-TNF ⁇ shown in SEQ ID NO: 60.
  • Partial amino acid sequence of positions 67 to 98 in the amino acid sequence of VHH, anti-cAbBcII10 shown in SEQ ID NO: 61 Partial amino acid sequence of positions 72 to 103 in the amino acid sequence of VHH, anti-RSV shown in SEQ ID NO: 62 Examples thereof include the partial amino acid sequence at positions 67 to 98 in the amino acid sequence of VHH-1, and the partial amino acid sequence at positions 67 to 98 in the amino acid sequence of anti-RSV VHH-2 shown in SEQ ID NO: 63.
  • the amino acid sequence of FR4 can be represented by SEQ ID NO: 4.
  • SEQ ID NO: 4 of FR4 the partial amino acid sequence of positions 114 to 125 in the amino acid sequence of anti-Fc VHH shown in SEQ ID NO: 50, anti-HSA shown in SEQ ID NO: 52.
  • Examples thereof include a partial amino acid sequence at positions 115 to 126 in the amino acid sequence of VHH-1, and a partial amino acid sequence at positions 115 to 126 in the amino acid sequence of anti-RSV VHH-2 shown in SEQ ID NO: 63.
  • the heavy chain antibody according to one or more embodiments of the present invention
  • the amino acid sequence of FR1 is (1a) The amino acid sequence shown in SEQ ID NO: 1 or (1b) An amino acid sequence having 83% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.
  • the amino acid sequence of FR2 is (2a) The amino acid sequence shown in SEQ ID NO: 2 or (2b) An amino acid sequence having 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2.
  • the amino acid sequence of FR3 is (3a) In the amino acid sequence shown in SEQ ID NO: 3, serine or threonine is added to at least one of the amino acids at positions 3, 5, 11, 12, 12, 19 and 25 of SEQ ID NO: 3.
  • amino acid sequence in which substitution with an amino acid other than is introduced or (3b) In the amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, the 3rd, 5th, 11th, 12th, 19th and 25th positions of SEQ ID NO: 3 An amino acid sequence in which substitution with an amino acid other than serine or threonine is introduced into at least one of the amino acids corresponding to the amino acid at the position.
  • the amino acid sequence of FR4 is (4a) The amino acid sequence shown in SEQ ID NO: 4 or (4b) An amino acid sequence having 65% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4.
  • amino acid sequences specified in (1a), (1b), (2a), (2b), (3a), (3b), (4a), and (4b) all contain the heavy chain variable domain as an antigen. Any amino acid sequence that can maintain the binding property is sufficient.
  • amino acid sequences specified in (1a), (1b), (2a), (2b), (3a), (3b), (4a), and (4b) are all variable heavy chains. It is an amino acid sequence in which the antigen binding property of the domain is equivalent to the antigen binding property of the heavy chain variable domain including FR1, FR2, FR3, and FR4 consisting of the above known amino acid sequences.
  • the amino acid sequences defined in (1a), (1b), (2a), (2b), (3a), (3b), (4a), and (4b) all contain heavy chains.
  • the antigen binding property of the variable domain is 70% or more and 150% or less, preferably 80% or more and 140% with respect to the antigen binding property of the heavy chain variable domain including FR1, FR2, FR3 and FR4 consisting of the above known amino acid sequences.
  • the amino acid sequence is particularly preferably 85% or more and 130% or less.
  • sequence identity is preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, still more preferably 98% or more. It is preferably 99% or more.
  • the amino acid sequence specified in (1b) is preferably the same as the amino acid sequence specified in (1a), and some amino acids are other amino acids in the amino acid sequence specified in (1a).
  • Amino acid sequence substituted with The number of substituted amino acid residues can be preferably 1 to several, preferably 1 to 3, more preferably 1 to 2, and particularly preferably 1.
  • the amino acid substitution is preferably a conservative amino acid substitution.
  • Constant amino acid substitution refers to a substitution between amino acids having similar properties such as charge, side chain, polarity, and aromaticity.
  • Amino acids with similar properties include, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagin, glutamine, serine, threonine, cysteine, tyrosine), non-polar amino acids. It can be classified into sex amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched amino acids (leucine, valine, isoleucine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine), etc. it can.
  • sequence identity is preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 93% or more, still more preferably 95% or more. It is more preferably 97%, more preferably 98% or more, and more preferably 99% or more.
  • the amino acid sequence specified in (2b) is preferably the same as the amino acid sequence specified in (2a), and some amino acids are other amino acids in the amino acid sequence specified in (2a).
  • the number of substituted amino acid residues can be preferably 1 to several, preferably 1 to 3, more preferably 1 to 2, and particularly preferably 1.
  • the amino acid substitution is preferably the above-mentioned conservative amino acid substitution.
  • sequence identity is preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, still more preferably 99% or more. ..
  • the amino acid sequence specified in (3b) is preferably the same as the amino acid sequence specified in (3a), and some amino acids are other amino acids in the amino acid sequence specified in (3a).
  • the number of substituted amino acid residues can be preferably 1 to several, preferably 1 to 3, more preferably 1 to 2, and particularly preferably 1.
  • the amino acid substitution is preferably the above-mentioned conservative amino acid substitution.
  • sequence identity is preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 93% or more. It is more preferably 95% or more, more preferably 97%, more preferably 98% or more, and even more preferably 99% or more.
  • the amino acid sequence specified in (4b) preferably has the same number of amino acid residues as the amino acid sequence specified in (4a), and some amino acids are other amino acids in the amino acid sequence specified in (4a).
  • the number of substituted amino acid residues can be preferably 1 to several, preferably 1 to 3, more preferably 1 to 2, and particularly preferably 1.
  • the amino acid substitution is preferably the above-mentioned conservative amino acid substitution.
  • the amino acid sequence of FR3 is (3a) In the amino acid sequence shown in SEQ ID NO: 3, serine or threonine is added to at least one of the amino acids at positions 3, 5, 11, 12, 12, 19 and 25 of SEQ ID NO: 3.
  • Amino acid sequence in which substitution with an amino acid other than is introduced or (3b) In the amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, the 3rd, 5th, 11th, 12th, 19th and 25th positions of SEQ ID NO: 3
  • substitution with an amino acid other than serine or threonine is introduced into at least one of the amino acids corresponding to the amino acid at the position. It is suppressed.
  • amino acid other than serine or threonine is not particularly limited, but alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, aspartic acid and glutamine are preferable, and alanine, aspartic acid or valine is particularly preferable.
  • amino acid corresponding to the amino acid at the 3rd, 5th, 11th, 12th, 19th and 25th positions of SEQ ID NO: 3 is defined in the above (3b). It refers to the amino acid corresponding to each position when the amino acid sequence is aligned so as to maximize the degree of coincidence with the amino acid sequence shown in SEQ ID NO: 3.
  • substitution with an amino acid other than serine or threonine is preferably introduced into at least one of the amino acids at positions 3, 5, 12, 19 and 25 of SEQ ID NO: 3.
  • at least one of the amino acids corresponding to the amino acids at positions 3, 5, 12, 19 and 25 of SEQ ID NO: 3 is an amino acid other than serine or threonine. Substitution has been introduced.
  • the amino acid sequence defined in (3a) is more preferably at least the amino acids at positions 3, 5, 12, 19 and 25 of SEQ ID NO: 3 in the amino acid sequence shown in SEQ ID NO: 3.
  • One is an amino acid sequence in which substitution with an amino acid other than serine or threonine has been introduced, and particularly preferably, in the amino acid sequence shown in SEQ ID NO: 3, the third, fifth, and eleventh positions of SEQ ID NO: 3 Serine is preferably at least two, more preferably at least three, more preferably at least four, more preferably at least five, and more preferably all of the amino acids at positions, 12, 19 and 25.
  • it is an amino acid sequence in which substitution with an amino acid other than threonine has been introduced.
  • the amino acid sequence defined in (3b) is more preferably at the 3rd, 5th, and 12th positions of SEQ ID NO: 3 in the amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3.
  • the amino acid corresponding to the amino acid at the 3rd, 5th, 11th, 12th, 19th and 25th positions of SEQ ID NO: 3 is preferable.
  • An amino acid sequence in which substitutions with amino acids other than serine or threonine have been introduced into at least two, more preferably at least three, more preferably at least four, more preferably at least five, and more preferably all.
  • nucleic acid comprising a base sequence encoding the amino acid sequence of the heavy chain antibody.
  • nucleic acids comprising a base sequence encoding the amino acid sequence of the heavy chain antibody.
  • vectors containing said nucleic acids relate to nucleic acids.
  • a vector is an artificially constructed nucleic acid molecule, and usually contains a base sequence derived from a heterologous organism in the nucleic acid molecule.
  • the vector according to one or more embodiments of the present invention can be introduced into a host cell and used to transform the host cell.
  • the vector according to one or more embodiments of the present invention can be a circular vector, a linear vector, an artificial chromosome, or the like.
  • the vector according to one or more embodiments of the present invention contains at least a nucleic acid containing a base sequence encoding the amino acid sequence of the heavy chain antibody, and the heavy chain antibody in a host cell is upstream and / or downstream of the base sequence. It may further contain a base sequence that controls the gene expression of.
  • the base sequence encoding the amino acid sequence of the heavy chain antibody can be contained in the vector in the form of being inserted into the expression cassette.
  • the "expression cassette” refers to an expression system that includes a base sequence encoding the amino acid sequence of the heavy chain antibody and can bring the heavy chain antibody into a state in which it can be expressed as a polypeptide.
  • the "expressible state” refers to a state in which the base sequence contained in the expression cassette is arranged under the control of an element necessary for gene expression so that it can be expressed in a host cell. Elements required for gene expression include promoters, terminators and the like.
  • the "promoter” is a base sequence region located upstream of the base sequence encoding the amino acid sequence of the heavy chain antibody, and in addition to RNA polymerase, various transcriptional regulators involved in promotion and suppression of transcription are mentioned.
  • Complementary RNA is synthesized (transcribed) by reading the base sequence encoding the amino acid sequence of the heavy chain antibody, which is a template by binding or acting on the region.
  • a promoter that expresses the heavy chain antibody a promoter that can be expressed by a selected carbon source may be appropriately used, and is not particularly limited.
  • the terminator is located downstream of the base sequence encoding the amino acid sequence of the heavy chain antibody.
  • the terminator can be appropriately selected depending on the promoter to be used and the host cell.
  • an In-Fusion cloning system of Clontech in order to further utilize a cloning site containing one or more restriction enzyme recognition sites, an In-Fusion cloning system of Clontech, a Gibson Assembly system of New England Biolabs, and the like. Can include the overlap region of the above, the base sequence of the selectable marker gene (nutrition-requiring complementary gene, drug resistance gene, etc.) and the like.
  • the vector according to one or more embodiments of the present invention can further include an Autonomous replication sequence (ARS), a centromere DNA sequence, and a telomere DNA sequence, depending on the host.
  • ARS Autonomous replication sequence
  • centromere DNA sequence centromere DNA sequence
  • telomere DNA sequence depending on the host.
  • the nucleic acid and vector according to one or more embodiments of the present invention further include, in addition to the base sequence encoding the amino acid sequence of the heavy chain antibody, one or both of the 5'end and 3'end of the base sequence. It may contain a base sequence encoding an amino acid sequence of a polypeptide.
  • Further polypeptides include a signal peptide that enables the secretion of the heavy chain antibody from a host cell, and a tag peptide. Such a further polypeptide is linked to one or both of the N-terminal side and the C-terminal side of the heavy chain antibody and expressed as a fusion peptide.
  • the signal peptide can be appropriately selected according to the host cell.
  • a Matting Factor ⁇ (MF ⁇ ) signal derived from Saccharomyces cerevisiae can be mentioned.
  • the signal sequence of Ogataea angusta acid phosphatase (PHO1), Komagataera pastris acid phosphatase (PHO1), Saccharomyces cerevisiae invertase (SUC2), or Saccharomyces cerevisiae PLB1 is also a peptide secretion from yeast. It is available as a signal peptide that enables.
  • Host cell> Another embodiment of the present invention relates to a host cell containing a nucleic acid containing a base sequence encoding the amino acid sequence of the heavy chain antibody.
  • the host cell according to this embodiment can contain the nucleic acid as part of the vector. Specific aspects of the host cell are as described above.
  • a known method can be appropriately used, and examples thereof include an electroporation method, a lithium acetate method, and a spheroplast method, but the method is not particularly limited thereto. Absent.
  • a yeast transformation method High efficiency transformation by electroporation of Pichia pastoris preserved with lithium acetate and dithiothreitol (Biotechnii) is described in (Biotechniq). Is the target.
  • the method for producing a heavy chain antibody includes a culturing step of culturing a host cell according to one or more embodiments of the present invention.
  • the target heavy chain antibody is recovered from the culture of the host cell obtained in the culture step.
  • the “culture” includes cultured cells or crushed cells, in addition to the supernatant of the culture solution.
  • the heavy chain antibody when expressed as a fusion protein with a signal peptide involved in secretion from the host cell to the outside of the cell, a fusion containing the heavy chain antibody. Since the protein is secreted and produced extracellularly, the culture solution supernatant is particularly preferable as the culture.
  • the culture conditions of the host cells according to one or more embodiments of the present invention are not particularly limited, and may be appropriately selected depending on the cells.
  • any medium containing a nutrient source capable of assimilating cells can be used.
  • the culture conditions of the host cells according to one or more embodiments of the present invention are not particularly limited, and may be appropriately selected depending on the cells.
  • any medium containing a nutrient source capable of assimilating cells can be used.
  • the nutrient source include lactose such as glucose, sucrose and maltose, organic acids such as acetic acid, citric acid and propionic acid, alcohols such as methanol, ethanol and glycerol, hydrocarbons such as paraffin, soybean oil and rapeseed oil.
  • Carbon sources of oils or mixtures thereof, ammonium sulfate, ammonium phosphate, urea, yeast extract, meat extract, peptone, nitrogen sources such as corn star chipliquor, and other nutrient sources such as inorganic salts and vitamins are appropriately used.
  • a mixed / blended normal medium can be used.
  • the culture can be either batch culture or continuous culture.
  • the carbon source when Pichia yeast or Ogataea yeast is used as the host cell, the carbon source may be one of glucose, glycerol, and methanol, or two or more. Further, these carbon sources may be present from the initial stage of culturing or may be added during culturing.
  • Culturing of host cells according to one or more embodiments of the present invention can usually be carried out under general conditions, for example, aerobically in a pH range of 2.5 to 10.0 and a temperature range of 10 ° C to 48 ° C. It can be carried out by culturing for 10 hours to 10 days.
  • the culture solution containing the host cells and the medium according to one or more embodiments of the present invention is centrifuged or filtered to remove the host cells from the liquid fraction, that is, the culture solution supernatant.
  • the obtained culture solution supernatant is subjected to salting (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fractionation precipitation method using acetone, ethanol, etc.), dialysis, gel filtration chromatography, ion exchange chromatography, hydrophobicity.
  • the heavy chain antibody can be recovered from the supernatant of the culture solution by using techniques such as chromatography, affinity chromatography, reverse phase chromatography, and ultrafiltration alone or in combination.
  • the heavy chain antibody produced by the production method according to the embodiment of the present invention has a low proportion of O-linked sugar chain modified product, and specifically, the O-linked sugar chain modified product with respect to the total amount of the heavy chain antibody. Is 5% by mass or less, preferably 4% by mass or less, more preferably 3% by mass or less, particularly preferably 2% by mass or less, still more preferably 1% by mass or less, and most preferably 0% by mass. That is, according to the production method according to one embodiment of the present invention, a heavy chain antibody having high uniformity and stable quality can be produced.
  • Another embodiment of the present invention relates to a medicament for gene therapy, which comprises a nucleic acid containing a base sequence encoding the amino acid sequence of the heavy chain antibody.
  • the medicament according to this embodiment can contain the nucleic acid as a part of the vector.
  • the medicament according to one or more embodiments of the present invention contains the nucleic acid in a form such that the heavy chain antibody can be expressed in the body of the target animal when administered to a target animal such as a human.
  • the heavy chain antibody can be expressed in a form in which the proportion of the O-linked sugar chain modifier is low in the body of the target animal.
  • the O-linked sugar chain modified product with respect to the total amount of the heavy chain antibody Is 5% by mass or less, preferably 4% by mass or less, more preferably 3% by mass or less, particularly preferably 2% by mass or less, still more preferably 1% by mass or less, and most preferably 0% by mass. It is expressed in the body of animals. That is, according to the drug according to the embodiment of the present invention, the heavy chain antibody having high uniformity and stable quality can be expressed in the body of the target animal.
  • the medicament according to one or more embodiments of the present invention preferably contains the nucleic acid in a form of being carried on a known gene therapy vector such as a retrovirus or adenovirus.
  • a known gene therapy vector such as a retrovirus or adenovirus.
  • the method of administering the drug according to one or more embodiments of the present invention is not particularly limited, but subcutaneous administration, intravenous administration, intramuscular administration, and intraarticular administration are all possible.
  • the plasmid used for yeast transformation is the constructed vector of Escherichia coli E. coli. It was prepared by introducing it into E. coli DH5 ⁇ competent cell (manufactured by Takara Bio Inc.) and culturing and amplifying the obtained transformant. The plasmid was prepared from the plasmid-carrying strain using a QIAprep spin miniprep kit (manufactured by QIAGEN).
  • the AOX1 promoter (SEQ ID NO: 5), AOX1 terminator (SEQ ID NO: 6), and HIS4 gene (SEQ ID NO: 7) used in the construction of the vector are the chromosomal DNA of the Komagataera Pastris ATCC76273 strain (the base sequence is EMBL (The European Molecular Biology Laboratory). ) ACCSESSION No. FR839628 to FR839631) Prepared by PCR using the mixture as a template.
  • the wild-type anti-Fc VHH gene is described in EP2170960B1 SEQ ID NO: 163, and the wild-type anti-HSA VHH gene is described in EP2069402A2 SEQ ID NO: 62, respectively.
  • a His tag sequence GGGGSHHHHHH
  • an anti-Fc VHH gene SEQ ID NO: 53
  • a His tag sequence addition to which a Matting Factor ⁇ signal sequence (MF sequence) (SEQ ID NO: 8) was added upstream.
  • Synthetic DNA of the anti-HSA VHH gene was prepared and used in the construction of the vector.
  • ⁇ Comparative Example 1 Construction of wild-type heavy chain antibody expression vector> A gene fragment (SEQ ID NO: 9) having a multi-cloning site of HindIII-BamHI-BglII-XbaI-EcoRI was totally synthesized and inserted between the HindIII-EcoRI sites of pUC19 (manufactured by Takara Bio) to pUC-1. Was built.
  • nucleic acid fragment having BamHI recognition sequences added to both sides of the AOX1 promoter was prepared by PCR using primers 1 (SEQ ID NO: 10) and 2 (SEQ ID NO: 11) using the chromosomal DNA mixture as a template, and treated with BamHI. It was later inserted into the BamHI site of pUC-1 to construct pUCPaox.
  • a nucleic acid fragment having XbaI recognition sequences added to both sides of the AOX1 terminator was prepared by PCR using the chromosomal DNA mixture as a template and primers 3 (SEQ ID NO: 12) and 4 (SEQ ID NO: 13), and after XbaI treatment.
  • a pUC-PaoxTaox was constructed by inserting it into the XbaI site of pUCPaox.
  • a nucleic acid fragment having EcoRI recognition sequences added to both sides of the HIS4 gene was prepared by PCR using the chromosomal DNA mixture as a template and primers 5 (SEQ ID NO: 14) and 6 (SEQ ID NO: 15), and after EcoRI treatment.
  • the pUC-PaoxTaoxHIS4 was constructed by inserting it into the EcoRI site of pUC-PaoxTaox.
  • BglII treatment was then inserted into the BglII site of pUC-PaoxTaoxHIS4 to construct pUC-Paoxanti-FcVHHTaxHIS4.
  • nucleic acid fragment in which the BglII recognition sequence is added to both sides of the anti-HSA VHH gene (heavy chain antibody gene) having the MF sequence added upstream and the His tag sequence added downstream, and the MF sequence (SEQ ID NO:) upstream Prepared by PCR using primers 7 (SEQ ID NO: 16) and 9 (SEQ ID NO: 18) using the synthetic DNA of the His-tag sequence-added anti-HSA VHH gene (SEQ ID NO: 55) to which 8) was added as a template.
  • BglII treatment was followed by insertion into the BglII site of pUC-PaoxTaoxHIS4 to construct pUC-Paoxanti-HSAVHHTaxHIS4.
  • pUC-Paoxanti-FcVHHTaxHIS4 and pUC-Paoxanti-HSAVHHTaxHIS4 are designed so that heavy chain antibodies are secreted and expressed under the control of the AOX1 promoter.
  • YPD medium 1% yeast extract bacto (manufactured by Difco), 2% polypeptone (manufactured by Nihon Pharmaceutical Co., Ltd.), 2% glucose
  • Preculture was obtained by shaking culture. 500 ⁇ L of the obtained preculture solution was inoculated into 50 mL of YPD medium, shake-cultured until the OD600 became 1 to 1.5, and then centrifuged (3000 ⁇ g, 10 minutes, 20 ° C.) to collect cells. Resuspended in 10 mL of 50 mM potassium phosphate buffer, pH 7.5, containing 250 ⁇ L of 1M 1,4-dithiothreitol (DTT) (final concentration 25 mM).
  • DTT 1,4-dithiothreitol
  • the cells were collected by centrifugation (3000 ⁇ g, 10 minutes, 20 ° C.), and pre-cooled 50 mL STM buffer (270 mM sucrose, 10 mM Tris-HCl, 1 mM). It was washed with magnesium chloride, pH 7.5). The washing solution is centrifuged (3000 ⁇ g, 10 minutes, 4 ° C.) to collect the cells, washed again with 25 mL of STM buffer, and then centrifuged (3000 ⁇ g, 10 minutes, 4 ° C.) to collect the cells. did. Finally, the cells were suspended in 250 ⁇ L ice-cold STM buffer, which was used as a competent cell suspension.
  • STM buffer 270 mM sucrose, 10 mM Tris-HCl, 1 mM
  • Escherichia coli was transformed with the heavy chain antibody expression vector pUC-Paoxanti-FcVHHTaxHIS4 or pUC-Paoxanti-HSAVHHTaoxHIS4 constructed in Comparative Example 1, and the obtained transformant was subjected to 2 mL of ampicillin-containing LB medium (1% Tryptone (1% Tryptone)). Cultivated in 0.5% Yeast extract (manufactured by Difco), 1% sodium chloride (manufactured by Difco)), and pUC using the QIAprep spin miniprep kit (manufactured by QIAGEN) from the obtained cells.
  • -Paoxanti-FcVHHTaxHIS4 or pUC-Paoxanti-HSAVHHTaxHIS4 was obtained.
  • This plasmid was treated with SalI to prepare a linear vector cleaved with the SalI recognition sequence in the HIS4 gene.
  • 60 ⁇ L of the above-mentioned competent cell suspension is mixed with 1 ⁇ L of a linear pUC-Paoxanti-FcVHHTaxHIS4 or pUC-Paoxanti-HSAVHHTaoxHIS4 solution, and a cuvette for electroporation (dispocubette electrode, electrode spacing 2 mm (manufactured by BM Instruments)).
  • a cuvette for electroporation dispenser for electroporation
  • the cells were subjected to 7.5 kV / cm, 25 ⁇ F, 200 ⁇ , and then the cells were suspended in 1 mL of YPD medium and allowed to stand at 30 ° C. for 1 hour.
  • the cells After standing for 1 hour, the cells are collected by centrifugation (3000 ⁇ g, 5 minutes, 20 ° C.) and suspended in 1 mL of YNB medium (0.67% yeast nitrogen base Without Amino acid (manufactured by Difco)). Then, the cells were collected by centrifugation (3000 ⁇ g, 5 minutes, 20 ° C.) again.
  • YNB medium 0.67% yeast nitrogen base Without Amino acid (manufactured by Difco)
  • the cells After resuspending the cells in an appropriate amount of YNB medium, the cells are applied to a YNB-selected agar plate (0.67% yeast nitrogen base Without Amino acid (manufactured by Difco), 2% agarose, 2% glucose) at 30 ° C., 3 Strains that grow in static culture for one day were selected to obtain wild-type anti-Fc VHH-expressing yeast or wild-type anti-HSA VHH-expressing yeast.
  • a YNB-selected agar plate 0.67% yeast nitrogen base Without Amino acid (manufactured by Difco), 2% agarose, 2% glucose
  • ⁇ Comparative Example 3 Culture of Transformed Yeast> Each wild-type heavy chain antibody-expressing yeast obtained in Comparative Example 2 was mixed with 3 mL of BMGMY medium (1% yeast extract bacto (manufactured by Difco), 2% polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 0.34% yeast nitrogen base with out. Amino Acid and Aminoium yeast, 1% ammonium sulfate, 0.4 mg / L biotin, 100 mM potassium phosphate (pH 7.0), 1% glycerol, 1% methanol) was inoculated, and this was shaken and cultured at 30 ° C. for 72 hours. The culture supernatant was recovered by centrifugation (12000 rpm, 5 minutes, 4 ° C.).
  • ⁇ Comparative Example 4 Purification of heavy chain antibody> The heavy chain antibody was purified by cation exchange chromatography using the culture solution supernatant obtained in Comparative Example 3. The solutions, materials and methods used are described below.
  • Load solution The culture solution supernatant was diluted 10-fold, and the pH of the diluted solution was adjusted to 4.0 to prepare a load solution.
  • Buffer A 20 mM sodium acetate pH 4.0
  • Buffer B 20 mM sodium acetate 0.5 M sodium chloride pH 4.0
  • a column packed with a cation exchange resin manufactured by Bio-Rad
  • Comparative Example 5 Sugar chain analysis of heavy chain antibody> The sample purified in Comparative Example 4 was subjected to LC / MS, and sugar chain analysis was performed. The equipment used and analysis conditions are described below.
  • a liquid chromatograph (Shimadzu UFLC Nexus X2, manufactured by Shimadzu Corporation) equipped with an analytical column (Waters Accuracy UPLC BEH300 C4 Volume 2.1 x 50 mm) was connected to a mass spectrometer (TripleTOF6600, manufactured by SCIEX), and a sample was connected to the column.
  • a mass spectrometer TripleTOF6600, manufactured by SCIEX
  • a sample was connected to the column.
  • For the separation of the heavy chain antibody 0.1% formic acid aqueous solution was used as the A solvent and 0.1% acetonitrile was used as the B solvent, and the A solvent was passed through the column in 0 to 0.5 minutes, and 0.5. Solvent A and solvent B were passed through the column so as to have a linear gradient of solvent B from 0% to 100% in 2 minutes.
  • the separated peptides were measured in a positive ion mode (applied voltage 5,500 V) by a quadrupole time-of-flight mass
  • a peak of a molecule having a theoretical mass based on the amino acid sequence of a heavy chain antibody and a peak of a plurality of isoforms having a mass increased by an integral multiple of 162 Da mass from the theoretical mass were observed.
  • the area ratio of each peak was calculated, where the peaks that coincided with the theoretical mass were Man0, and the peaks that increased by 162 Da were Man1, Man2, and so on, respectively.
  • the table below shows the peak area ratio of the results of sugar chain analysis of the wild-type anti-Fc VHH sample purified in Comparative Example 4. The results of sugar chain analysis of wild-type anti-HSA VHH are described in Example 6.
  • mutant heavy chain antibody expression vector Various mutant genes were prepared by PCR using the synthetic DNA of the wild-type anti-HSA VHH gene with the MF sequence added upstream and the His tag sequence added downstream as a template.
  • the synthetic DNA of the His-tag sequence-added wild-type anti-HSA VHH gene (SEQ ID NO: 55) to which the MF sequence (SEQ ID NO: 8) was added upstream was used as a template, and the 1st PCR-1 shown in Table 2 below was used.
  • PCR (1stPCR-1) using the combination of primers of 1stPCR-2 and PCR (1stPCR-2) using the combination of primers of 1stPCR-2 were performed.
  • PCR was performed with primers 7 and 9 using a mixture of the amplified fragment obtained by 1stPCR-1 and the amplified fragment obtained by 1stPCR-2 as a template, and the MF sequence was added upstream and His downstream.
  • DNA fragments in which BglII recognition sequences were added to both ends of various mutant heavy chain antibody genes to which tag sequences were added were prepared.
  • the mutation site indicates the position in the amino acid sequence shown in SEQ ID NO: 52 of anti-HSA VHH.
  • the DNA fragment containing the mutant heavy chain antibody gene prepared above is treated with BglII and inserted into the BglII site of pUC-PaoxTaoxHIS4 prepared in Comparative Example 1, and an MF sequence is added upstream and a His tag sequence is added downstream.
  • Various mutant heavy chain antibody gene expression vectors were constructed.
  • Example 2 Acquisition of transformed yeast> Using the mutant heavy chain antibody expression vector to which the MF sequence and His tag sequence constructed in Example 1 were added, Komagataera pastris was transformed in the same manner as in Comparative Example 2 to express the mutant heavy chain antibody. Obtained yeast.
  • Escherichia coli was transformed with the mutant heavy chain antibody expression vector constructed in Example 1, and the obtained transformant was cultured in 2 mL of ampicillin-containing LB medium, and a plasmid was obtained from the obtained bacterial cells. This plasmid was treated with SalI to make it linear.
  • Example 3 Culture of transformed yeast> The mutant heavy chain antibody-expressing yeast obtained in Example 2 was cultured by the method described in Comparative Example 3, and the culture broth supernatant was collected.
  • Example 4 Purification of heavy chain antibody> The heavy chain antibody was purified by cation exchange chromatography using the culture solution supernatant obtained in Example 3. The solutions, materials and methods used are described below.
  • Load solution The culture solution supernatant was diluted 10-fold, and the pH of the diluted solution was adjusted to 4.0 to prepare a load solution.
  • Buffer A 20 mM sodium acetate pH 4.0
  • Buffer B 20 mM sodium acetate 0.5 M sodium chloride pH 4.0
  • a column packed with a cation exchange resin manufactured by Bio-Rad
  • Example 5 Determination of sugar chain binding position of heavy chain antibody>
  • the anti-Fc VHH sample obtained in Comparative Example 4 was diluted to 100 ⁇ L with a 7M guanidine hydrochloric acid solution to denature the protein. Next, dithiothreitol was added, and the mixture was allowed to stand at 37 ° C. for 1 hour for reduction. Further, iodoacetamide was added, and the mixture was allowed to stand in a dark place for 30 minutes for alkylation. The solvent was replaced with 50 mM Tris-Cl (pH 7.5) by ultrafiltration. Trypsin was added and the mixture was allowed to stand overnight at 37 ° C.
  • peptides were separated by reverse phase high performance liquid chromatography (RP-HPLC).
  • the separated peptides were analyzed by a quadrupole time-of-flight mass spectrometer (QTOF-MS). From the obtained peaks, peaks of glycopeptides were extracted by BioPharmaView (manufactured by SCIEX) (Table 3). From Table 3, O-linked sugar chains were added to FR2 (positions 36 to 49 of SEQ ID NO: 54) and FR3 (positions 67 to 98 of SEQ ID NO: 54) of the His-tagged anti-Fc VHH (SEQ ID NO: 54). It became clear that they were combined.
  • Example 6 Sugar chain analysis of heavy chain antibody> Samples of wild-type anti-HSA VHH purified in Comparative Example 4 and samples of each mutant anti-HSA VHH obtained in Example 4 were subjected to LC / MS, and sugars of wild-type anti-HSA VHH and mutant anti-HSA VHH were subjected to LC / MS. Chain analysis was performed. The equipment used and analysis conditions are described below.
  • a liquid chromatograph (Shimadzu UFLC Nexus X2, manufactured by Shimadzu Corporation) equipped with an analytical column (Waters Accuracy UPLC BEH300 C4 Volume 2.1 x 50 mm) was connected to a mass spectrometer (TripleTOF6600, manufactured by SCIEX), and a sample was connected to the column. Was added to.
  • a mass spectrometer TripleTOF6600, manufactured by SCIEX
  • B solvent 0.1% formic acid aqueous solution as A solvent and 0.1% acetonitrile as B solvent, and pass A solvent through the column in 0 to 0.5 minutes, and from 0.5 minutes.
  • Solvent A and Solvent B were passed through the column so as to become a linear gradient from 0% of B solvent to 100% of B solvent in 2 minutes.
  • the separated peptides were measured in a positive ion mode (applied voltage 5,500 V) by a quadrupole time-of-flight mass spectrometer (QTOF-MS, manufactured by
  • a peak of a molecule having a theoretical mass based on the amino acid sequence of a heavy chain antibody and a peak of a plurality of isoforms having a mass increased by an integral multiple of 162 Da mass from the theoretical mass were observed.
  • the area ratio of each peak was calculated, where the peaks consistent with the theoretical mass were Man0 and the peaks increased by 162 Da were Man1, Man2, and so on, respectively (Table 4).
  • n. d. Indicates that no peak was detected.
  • the mutant anti-HSA VHH suppressed the O-binding sugar chain modification.
  • Example 7 Binding activity of heavy chain antibody> The binding activity of the mutant anti-HSA VHH obtained in Example 4 to the antigen was measured. The activity measurement method is described below.
  • HSA Human Serum Albumin
  • the contents of the plate were discarded and washed 3 times with the cleaning solution.
  • 100 ⁇ L of each sample solution was placed in each well (2 wells were used per sample), stirred with a plate mixer, and allowed to stand at room temperature for 1.5 hours. The contents of the plate were discarded and washed with the washing liquid four times.
  • 100 ⁇ L of a secondary antibody solution for detection (anti-HisTag antibody HRP conjugate (manufactured by Abcam) diluted 1000 times) was added to all the wells used, and the mixture was stirred with a plate mixer and allowed to stand at room temperature for 1 hour. The contents of the plate were discarded and washed with the washing liquid four times.

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Abstract

The present invention provides, in order to obtain a uniform quality for heavy-chain antibodies, a heavy-chain antibody that is less susceptible to modification by O-linked sugar chains. The heavy-chain antibody according to the present invention is characterized by including a heavy chain variable domain having a structure of (FR1)-(VH-CDR1)-(FR2)-(VH-CDR2)-(FR3)-(VH-CDR3)-(FR4), wherein, in the amino acid sequence of FR3, at least one serine or threonine residue is substituted with an amino acid other than serine or threonine. The present invention also pertains to: a nucleic acid having a base sequence encoding the amino acid sequence of the heavy-chain antibody; and a vector and a host cell including said nucleic acid.

Description

Ｏ結合型糖鎖修飾が抑制された重鎖抗体Heavy chain antibody with suppressed O-linked sugar chain modification
　本発明は、Ｏ結合型糖鎖による修飾を受けにくい重鎖抗体に関する。
　本発明はまた、前記重鎖抗体のアミノ酸配列をコードする塩基配列を含む核酸、それを含むベクター及び宿主細胞に関する。
　本発明はまた、前記重鎖抗体の製造方法に関する。
　本発明はまた、前記重鎖抗体のアミノ酸配列をコードする塩基配列を含む核酸を含む、遺伝子治療のための医薬に関する。 The present invention relates to a heavy chain antibody that is not easily modified by an O-linked sugar chain.
The present invention also relates to a nucleic acid containing a base sequence encoding the amino acid sequence of the heavy chain antibody, a vector containing the nucleic acid, and a host cell.
The present invention also relates to a method for producing the heavy chain antibody.
The present invention also relates to a medicament for gene therapy, which comprises a nucleic acid containing a base sequence encoding the amino acid sequence of the heavy chain antibody.
　医薬品用途の抗体等のタンパク質の遺伝子組み換え技術による生産のために、動物細胞、大腸菌、酵母等が用いられている。 Animal cells, Escherichia coli, yeast, etc. are used for the production of proteins such as antibodies for pharmaceutical use by gene recombination technology.
　タンパク質生産のための宿主細胞として、ＰＭＴ（Ｐｒｏｔｅｉｎ　Ｏ－Ｍａｎｎｏｓｙｌｔｒａｎｓｆｅｒａｓｅ）遺伝子等の、タンパク質のセリン又はスレオニン残基にＯ結合型糖鎖を付加する活性を有する酵素の遺伝子を有する宿主細胞を用いる場合、生産される外来タンパク質のセリン又はスレオニン残基にＯ結合型糖鎖が付加される場合がある。 When a host cell having a gene of an enzyme having an activity of adding an O-linked sugar chain to a serine or threonine residue of a protein, such as a PMT (Protein O-Mannosyltransphase) gene, is used as a host cell for protein production. An O-linked sugar chain may be added to the serine or threonine residue of the foreign protein produced.
　タンパク質上の特定の糖鎖構造が、タンパク質の、その薬物動態、薬力学、受容体相互作用、および組織特異的標的化特性を含む特性に、大いに影響を及ぼし得ることが認識されている（非特許文献１）。例えば、抗原タンパク質上の糖鎖構造が抗体との結合性に影響を与えることが知られており、特許文献１では、免疫原性を有する過剰グリコシル化されたＩＬ－７ポリペプチドを生産することが記載されている。しかしながら、どの特定のグライコフォーム（複数可）が所望の生物学的機能に寄与するかは予測が困難であるため、糖鎖修飾を受けていないタンパク質を製造することが望ましい。 It has been recognized that a particular sugar chain structure on a protein can have a profound effect on the properties of the protein, including its pharmacokinetics, pharmacodynamics, receptor interactions, and tissue-specific targeting properties (non-). Patent Document 1). For example, it is known that the sugar chain structure on an antigen protein affects the binding property to an antibody, and in Patent Document 1, an immunogenic, hyperglycosylated IL-7 polypeptide is produced. Is described. However, it is difficult to predict which particular glycoform (s) contribute to the desired biological function, so it is desirable to produce a protein that has not undergone sugar chain modification.
　特許文献２では、ＰＭＴ活性阻害剤の培地添加及び／又はＰＭＴ遺伝子の機能停止を含むＯ型糖鎖合成を抑制する条件下で、活性型ＨＡＣ１遺伝子及び／又はＲＲＢＰ１遺伝子と外来タンパク質をコードする遺伝子とを導入した形質転換細胞を培地に培養し、培養物から外来タンパク質を採取する、Ｏ結合型糖鎖修飾が抑制されたタンパク質の製造方法が記載されている。 In Patent Document 2, a gene encoding an active HAC1 gene and / or an RRBP1 gene and a foreign protein under conditions of suppressing O-type sugar chain synthesis including addition of a PMT activity inhibitor to a medium and / or dysfunction of the PMT gene. A method for producing a protein in which O-linked sugar chain modification is suppressed is described in which transformed cells into which the gene has been introduced is cultured in a medium and a foreign protein is collected from the culture.
　一方、偶蹄目、ラクダ科動物（フタコブラクダ、ヒトコブラクダ、ラマ、アルパカ）の血清中には軽鎖を持たず重鎖のみからなる特殊な抗体があることが知られている。重鎖２量体免疫グロブリンの重鎖可変ドメインはＶＨＨ（Ｖａｒｉａｂｌｅ ｄｏｍａｉｎ ｏｆ ｔｈｅ ｈｅａｖｙ－ｃｈａｉｎ ｏｆ ｈｅａｖｙ－ｃｈａｉｎ ａｎｔｉｂｏｄｙ（重鎖抗体の重鎖可変ドメイン））と呼ばれている。ＶＨＨは、それ単独で抗原への特異的結合能を有する単一ドメイン抗体としても用いられる。ＶＨＨからなる単一ドメイン重鎖抗体は、化学的な安定性が高く、製造が容易であることから、医薬等への応用に適している。 On the other hand, it is known that the sera of Artiodactyla and Camelids (Bactrian camel, Dromedary camel, Llama, Alpaca) have a special antibody that does not have a light chain and consists only of a heavy chain. The heavy chain variable domain of a heavy chain dimer immunoglobulin is called VHH (Variable domain of the heavi-chain of heavi-chain antibody) (heavy chain variable domain of a heavy chain antibody). VHH is also used as a single domain antibody, which by itself has the ability to specifically bind to an antigen. A single domain heavy chain antibody composed of VHH has high chemical stability and is easy to manufacture, and is therefore suitable for application to pharmaceuticals and the like.
　特許文献３では、ラクダ科動物等の非ヒト動物に、細胞関連抗原をコードする核酸を遺伝子ワクチン接種することを含む、細胞関連抗原と結合することのできる免疫グロブリン配列を生成する方法が記載されている。そして特許文献３では、前記免疫グロブリン配列として重鎖抗体に由来する重鎖可変ドメイン配列が記載されている。 Patent Document 3 describes a method for generating an immunoglobulin sequence capable of binding to a cell-related antigen, which comprises gene vaccination of a non-human animal such as a camelid with a nucleic acid encoding a cell-related antigen. ing. And in Patent Document 3, a heavy chain variable domain sequence derived from a heavy chain antibody is described as the immunoglobulin sequence.
特表２００９－５０１５４３号公報Special Table 2009-501543 ＷＯ２００７／１３２９４９WO2007 / 132949 特開２０１５－１５５４２９号公報JP-A-2015-155429
　抗体の糖鎖修飾は、抗原結合活性や、抗体自体の免疫原性に影響を与える可能性がある。また、抗体を修飾する糖鎖の位置、長さ及び数に応じて、抗原結合活性や免疫原性が変化する可能性がある。このため、遺伝子組み換え技術により抗体を生産する場合に、糖鎖修飾されている割合が高いと、抗体の品質が不均一になると考えられる。
　そこで本発明の一以上の実施形態は、Ｏ結合型糖鎖による修飾を受けにくい重鎖抗体を提供する。Ｏ結合型糖鎖による修飾を受けにくい重鎖抗体は、遺伝子組み換え細胞により、均一な品質で生産することが容易である。 Sugar chain modification of an antibody may affect the antigen-binding activity and the immunogenicity of the antibody itself. In addition, antigen-binding activity and immunogenicity may change depending on the position, length and number of sugar chains that modify the antibody. Therefore, when an antibody is produced by a gene recombination technique, it is considered that the quality of the antibody becomes non-uniform if the ratio of sugar chain modification is high.
Therefore, one or more embodiments of the present invention provide a heavy chain antibody that is less susceptible to modification by O-linked sugar chains. Heavy chain antibodies that are not easily modified by O-linked sugar chains can be easily produced with uniform quality by transgenic cells.
　本発明者らは、Ｎ末端からフレームワーク領域１（ＦＲ１）、可変重鎖相補性決定領域１（ＶＨ－ＣＤＲ１）、フレームワーク領域２（ＦＲ２）、可変重鎖相補性決定領域２（ＶＨ－ＣＤＲ２）、フレームワーク領域３（ＦＲ３）、可変重鎖相補性決定領域３（ＶＨ－ＣＤＲ３）及びフレームワーク領域４（ＦＲ４）がこの順で連結した重鎖可変ドメインを含む重鎖抗体を、宿主細胞中で発現させたところ、ＦＲ３にＯ結合型糖鎖が結合したこと、及び、ＦＲ３に存在するセリン又はスレオニン残基を他のアミノ酸残基に置換するとＯ結合型糖鎖修飾は抑制されたことを見出し、以下の本発明を完成するに至った。 From the N-terminus, the present inventors have framework regions 1 (FR1), variable heavy chain complementarity determining regions 1 (VH-CDR1), framework regions 2 (FR2), and variable heavy chain complementarity determining regions 2 (VH-). A heavy chain antibody containing a heavy chain variable domain in which CDR2), framework regions 3 (FR3), variable heavy chain complementarity determining regions 3 (VH-CDR3) and framework regions 4 (FR4) are linked in this order is used as a host. When expressed in cells, O-linked sugar chains were bound to FR3, and O-linked sugar chain modification was suppressed by substituting serine or threonine residues present in FR3 with other amino acid residues. This has led to the completion of the following inventions.
［１］
　Ｎ末端からフレームワーク領域１、可変重鎖相補性決定領域１、フレームワーク領域２、可変重鎖相補性決定領域２、フレームワーク領域３、可変重鎖相補性決定領域３及びフレームワーク領域４がこの順で連結した重鎖可変ドメインを含む重鎖抗体であって、
　フレームワーク領域１のアミノ酸配列が、
（１ａ）配列番号１：
Ｘａａ１－Ｖ－Ｑ－Ｌ－Ｘａａ２－Ｅ－Ｓ－Ｇ－Ｇ－Ｇ－Ｘａａ３－Ｖ－Ｑ－Ｘａａ４－Ｇ－Ｘａａ５－Ｓ－Ｌ－Ｘａａ６－Ｘａａ７－Ｓ－Ｃ－Ｘａａ８－Ａ－Ｓ
（Ｘａａ１はＥ、Ｑ又はＤであり、Ｘａａ２はＶ又はＱであり、Ｘａａ３はＬ又はＳであり、Ｘａａ４はＰ又はＡであり、Ｘａａ５はＧ又はＮであり、Ｘａａ６はＲ又はＳであり、Ｘａａ７はＬ又はＩであり、Ｘａａ８はＡ又はＴである）
に示すアミノ酸配列、又は、
（１ｂ）配列番号１に示すアミノ酸配列と８３％以上の配列同一性を有するアミノ酸配列であり、
　フレームワーク領域２のアミノ酸配列が、
（２ａ）配列番号２：
Ｗ－Ｘａａ９－Ｒ－Ｑ－Ａ－Ｐ－Ｇ－Ｘａａ１０－Ｘａａ１１－Ｘａａ１２－Ｅ－Ｘａａ１３－Ｖ－Ｘａａ１４
（Ｘａａ９はＶ、Ｙ又はＦであり、Ｘａａ１０はＫ又はＱであり、Ｘａａ１１はＧ又はＥであり、Ｘａａ１２はＬ又はＲであり、Ｘａａ１３はＷ、Ｌ、Ｆ又はＡであり、Ｘａａ１４はＳ又はＡである）
に示すアミノ酸配列、又は、
（２ｂ）配列番号２に示すアミノ酸配列と７０％以上の配列同一性を有するアミノ酸配列であり、
　フレームワーク領域３のアミノ酸配列が、
（３ａ）配列番号３：
Ｒ－Ｆ－Ｔ－Ｉ－Ｓ－Ｒ－Ｄ－Ｎ－Ａ－Ｋ－Ｘａａ１５－Ｘａａ１６－Ｘａａ１７－Ｘａａ１８－Ｌ－Ｑ－Ｍ－Ｎ－Ｘａａ１９－Ｌ－Ｘａａ２０－Ｘａａ２１－Ｘａａ２２－Ｄ－Ｔ－Ａ－Ｘａａ２３－Ｙ－Ｙ－Ｃ－Ｘａａ２４－Ｘａａ２５
（Ｘａａ１５はＴ、Ｎ又はＲであり、Ｘａａ１６はＴ又はＭであり、Ｘａａ１７はＬ、Ｖ又はＧであり、Ｘａａ１８はＹ、Ｔ又はＮであり、Ｘａａ１９はＮ又はＳであり、Ｘａａ２０はＲ、Ｋ、Ｅ又はＡであり、Ｘａａ２１はＰ又はＡであり、Ｘａａ２２はＥ又はＤであり、Ｘａａ２３はＶ又はＩであり、Ｘａａ２４はＴ、Ａ又はＧであり、Ｘａａ２５はＩ、Ｆ、Ａ又はＲである）
に示すアミノ酸配列において、配列番号３の第３位、第５位、第１１位、第１２位、第１９位及び第２５位のアミノ酸の少なくとも１つに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列、或いは、
（３ｂ）配列番号３に示すアミノ酸配列と８５％以上の配列同一性を有するアミノ酸配列において、配列番号３の第３位、第５位、第１１位、第１２位、第１９位及び第２５位のアミノ酸に対応するアミノ酸の少なくとも１つに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列であり、
　フレームワーク領域４のアミノ酸配列が、
（４ａ）配列番号４：
Ｘａａ２６－Ｘａａ２７－Ｘａａ２８－Ｘａａ２９－Ｇ－Ｔ－Ｘａａ３０－Ｖ－Ｔ－Ｖ－Ｓ－Ｓ
（Ｘａａ２６はＲ、Ｙ、Ｎ又はＳであり、Ｘａａ２７はＳ、Ｗ又はＲであり、Ｘａａ２８はＳ又はＧであり、Ｘａａ２９はＱ、Ｌ又はＲであり、Ｘａａ３０はＱ又はＬである）
に示すアミノ酸配列、又は、
（４ｂ）配列番号４に示すアミノ酸配列と６５％以上の配列同一性を有するアミノ酸配列である
ことを特徴とする、重鎖抗体。
［２］
　フレームワーク領域１のアミノ酸配列が、（１ａ）で規定するアミノ酸配列であり、
フレームワーク領域２のアミノ酸配列が、（２ａ）で規定するアミノ酸配列であり、
フレームワーク領域３のアミノ酸配列が、（３ａ）で規定するアミノ酸配列であり、且つ、
フレームワーク領域４のアミノ酸配列が、（４ａ）で規定するアミノ酸配列である、
［１］に記載の重鎖抗体。
［３］
　（３ａ）で規定するアミノ酸配列が、配列番号３に示すアミノ酸配列において、配列番号３の第３位、第５位、第１１位、第１２位、第１９位及び第２５位のアミノ酸の少なくとも２つに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列であり、
　（３ｂ）で規定するアミノ酸配列が、配列番号３に示すアミノ酸配列と８５％以上の配列同一性を有するアミノ酸配列において、配列番号３の第３位、第５位、第１１位、第１２位、第１９位及び第２５位のアミノ酸に対応するアミノ酸の少なくとも２つに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列である、
［１］又は［２］に記載の重鎖抗体。
［４］
　連結された２以上の［１］～［３］のいずれかに記載の重鎖抗体を含む多重特異性抗体又は多価抗体。
［５］
　［１］～［３］のいずれかに記載の重鎖抗体のアミノ酸配列をコードする塩基配列を含む核酸。
［６］
　［５］に記載の核酸を含むベクター。
［７］
　［５］に記載の核酸を含む宿主細胞。
［８］
　［１］～［３］のいずれかに記載の重鎖抗体の製造方法であって、
　［７］に記載の宿主細胞を培養すること、及び、
　培養物から［１］～［３］のいずれかに記載の重鎖抗体を回収すること
を含む方法。
［９］
　［５］に記載の核酸を含む、遺伝子治療のための医薬。
［１０］
　遺伝子治療に用いるための、［５］に記載の核酸。
［１１］
　［１］～［３］のいずれかに記載の重鎖抗体により治療又は改善される疾患の治療又は改善に用いるための、［５］に記載の核酸。
［１２］
　遺伝子治療に用いる医薬の製造のための、［５］に記載の核酸の使用。
［１３］
　［１］～［３］のいずれかに記載の重鎖抗体により治療又は改善される疾患の治療又は改善に用いる医薬の製造のための、［５］に記載の核酸の使用。
［１４］
　［５］に記載の核酸を、［１］～［３］のいずれかに記載の重鎖抗体により治療又は改善される疾患の治療又は改善を必要とする患者に投与することを含む、前記疾患の治療又は改善方法。 [1]
From the N-terminal, framework regions 1, variable heavy chain complementarity determining regions 1, framework regions 2, variable heavy chain complementarity determining regions 2, framework regions 3, variable heavy chain complementarity determining regions 3 and framework regions 4 Heavy chain antibodies containing heavy chain variable domains linked in this order.
The amino acid sequence of framework region 1
(1a) SEQ ID NO: 1:
Xaa1-VQL-Xaa2-ES-G-G-G-Xaa3-VQ-Xaa4-G-Xaa5-S-L-Xaa6-Xaa7-SC-Xaa8-AS
(Xaa1 is E, Q or D, Xaa2 is V or Q, Xaa3 is L or S, Xaa4 is P or A, Xaa5 is G or N, Xaa6 is R or S. , Xaa7 is L or I, Xaa8 is A or T)
Amino acid sequence shown in
(1b) An amino acid sequence having 83% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.
The amino acid sequence of framework region 2
(2a) SEQ ID NO: 2:
W-Xaa9-RQ-A-P-G-Xaa10-Xaa11-Xaa12-E-Xaa13-V-Xaa14
(Xaa9 is V, Y or F, Xaa10 is K or Q, Xaa11 is G or E, Xaa12 is L or R, Xaa13 is W, L, F or A, Xaa14 is S. Or A)
Amino acid sequence shown in
(2b) An amino acid sequence having 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2.
The amino acid sequence of framework region 3
(3a) SEQ ID NO: 3:
RFT I S R D-N-A-K-Xaa15-Xaa16-Xaa17-Xaa18-L-Q-M-N-Xaa19-L-Xaa20-Xaa21-Xaa22-DT- A-Xaa23-YYC-Xaa24-Xaa25
(Xaa15 is T, N or R, Xaa16 is T or M, Xaa17 is L, V or G, Xaa18 is Y, T or N, Xaa19 is N or S, Xaa20 is R. , K, E or A, Xaa21 is P or A, Xaa22 is E or D, Xaa23 is V or I, Xaa24 is T, A or G, and Xaa25 is I, F, A. Or R)
In the amino acid sequence shown in, at least one of the amino acids at positions 3, 5, 11, 12, 19 and 25 of SEQ ID NO: 3 is replaced with an amino acid other than serine or threonine. Amino acid sequence in which is introduced, or
(3b) In the amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, the 3rd, 5th, 11th, 12th, 19th and 25th positions of SEQ ID NO: 3 An amino acid sequence in which substitution with an amino acid other than serine or threonine is introduced into at least one of the amino acids corresponding to the amino acid at the position.
The amino acid sequence of framework region 4
(4a) SEQ ID NO: 4:
Xaa26-Xaa27-Xaa28-Xaa29-GT-Xaa30-VT-VS-S
(Xaa26 is R, Y, N or S, Xaa27 is S, W or R, Xaa28 is S or G, Xaa29 is Q, L or R, Xaa30 is Q or L)
Amino acid sequence shown in
(4b) A heavy chain antibody characterized by having an amino acid sequence having 65% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4.
[2]
The amino acid sequence of framework region 1 is the amino acid sequence defined in (1a).
The amino acid sequence of framework region 2 is the amino acid sequence defined in (2a).
The amino acid sequence of framework region 3 is the amino acid sequence defined in (3a), and
The amino acid sequence of framework region 4 is the amino acid sequence defined in (4a).
The heavy chain antibody according to [1].
[3]
The amino acid sequence defined in (3a) is at least the amino acids at positions 3, 5, 11, 12, 19, and 25 of SEQ ID NO: 3 in the amino acid sequence shown in SEQ ID NO: 3. The second is an amino acid sequence in which substitution with an amino acid other than serine or threonine has been introduced.
In the amino acid sequence defined in (3b), which has 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, the 3rd, 5th, 11th, and 12th positions of SEQ ID NO: 3 , An amino acid sequence in which substitutions with amino acids other than serine or threonine have been introduced into at least two of the amino acids corresponding to the amino acids at positions 19 and 25.
The heavy chain antibody according to [1] or [2].
[4]
A multispecific antibody or a multivalent antibody containing the heavy chain antibody according to any one of two or more linked [1] to [3].
[5]
A nucleic acid containing a base sequence encoding the amino acid sequence of the heavy chain antibody according to any one of [1] to [3].
[6]
A vector containing the nucleic acid according to [5].
[7]
A host cell containing the nucleic acid according to [5].
[8]
The method for producing a heavy chain antibody according to any one of [1] to [3].
Culturing the host cell according to [7] and
A method comprising recovering the heavy chain antibody according to any one of [1] to [3] from the culture.
[9]
A drug for gene therapy containing the nucleic acid according to [5].
[10]
The nucleic acid according to [5] for use in gene therapy.
[11]
The nucleic acid according to [5] for use in treating or ameliorating a disease treated or ameliorated by the heavy chain antibody according to any one of [1] to [3].
[12]
Use of the nucleic acid according to [5] for the production of a drug used for gene therapy.
[13]
Use of the nucleic acid according to [5] for producing a drug used for treating or ameliorating a disease treated or ameliorated by the heavy chain antibody according to any one of [1] to [3].
[14]
The disease, which comprises administering the nucleic acid according to [5] to a patient in need of treatment or improvement of the disease treated or ameliorated by the heavy chain antibody according to any one of [1] to [3]. Treatment or improvement method.
　本明細書は本願の優先権の基礎となる日本国特許出願番号２０１９－１５２８９６号の開示内容を包含する。 This specification includes the disclosure content of Japanese Patent Application No. 2019-152896, which is the basis of the priority of the present application.
　発明の一以上の実施形態によれば、Ｏ結合型糖鎖による修飾を受けにくい重鎖抗体が提供される。 According to one or more embodiments of the invention, a heavy chain antibody that is less susceptible to modification by O-linked sugar chains is provided.
図１は、（ＦＲ１）－（ＶＨ－ＣＤＲ１）－（ＦＲ２）－（ＶＨ－ＣＤＲ２）－（ＦＲ３）－（ＶＨ－ＣＤＲ３）－（ＦＲ４）の構造を有する重鎖可変ドメインを含む既知の重鎖抗体である、配列番号５０に示すアミノ酸配列を含むａｎｔｉ－Ｆｃ　ＶＨＨ、配列番号５２に示すアミノ酸配列を含むａｎｔｉ－ＨＳＡ　ＶＨＨ、配列番号５７に示すアミノ酸配列を含むａｎｔｉ－ＩＬ６Ｒ　ＶＨＨ、配列番号５８に示すアミノ酸配列を含むａｎｔｉ－ｖＷＦ ＶＨＨ、配列番号５９に示すアミノ酸配列を含むａｎｔｉ－ＲＡＮＫＬ ＶＨＨ、配列番号６０に示すアミノ酸配列を含むａｎｔｉ－ＴＮＦα ＶＨＨ、配列番号６１に示すアミノ酸配列を含むａｎｔｉ－ｃＡｂＢｃＩＩ１０　ＶＨＨ、配列番号６２に示すアミノ酸配列を含むａｎｔｉ－ＲＳＶ ＶＨＨ－１、配列番号６３に示すアミノ酸配列を含むａｎｔｉ－ＲＳＶ ＶＨＨ－２のアミノ酸配列をアライメントし比較した結果の概要を示す。FIG. 1 shows a known weight comprising a heavy chain variable domain having a structure of (FR1)-(VH-CDR1)-(FR2)-(VH-CDR2)-(FR3)-(VH-CDR3)-(FR4). It is a chain antibody, anti-Fc VHH containing the amino acid sequence shown in SEQ ID NO: 50, anti-HSA VHH containing the amino acid sequence shown in SEQ ID NO: 52, anti-IL6R VHH containing the amino acid sequence shown in SEQ ID NO: 57, SEQ ID NO: 58. Anti-vWF VHH containing the amino acid sequence shown in FIG. 59, anti-RANKL VHH containing the amino acid sequence shown in SEQ ID NO: 59, anti-TNFα VHH containing the amino acid sequence shown in SEQ ID NO: 60, anti-containing the amino acid sequence shown in SEQ ID NO: 61. The outline of the results of aligning and comparing the amino acid sequences of cAbBcII10 VHH, anti-RSV VHH-1 containing the amino acid sequence shown in SEQ ID NO: 62, and anti-RSV VHH-2 containing the amino acid sequence shown in SEQ ID NO: 63 is shown.
　以下、本発明を好ましい実施形態を用いて詳細に説明する。 Hereinafter, the present invention will be described in detail using preferred embodiments.
＜１．用語の定義＞
　本発明において、アミノ酸の表記は下記の通りである。
Ａ＝Ａｌａ＝アラニン、Ｃ＝Ｃｙｓ＝システイン、Ｄ＝Ａｓｐ＝アスパラギン酸、Ｅ＝Ｇｌｕ＝グルタミン酸、Ｆ＝Ｐｈｅ＝フェニルアラニン、Ｇ＝Ｇｌｙ＝グリシン、Ｈ＝Ｈｉｓ＝ヒスチジン、Ｉ＝Ｉｌｅ＝イソロイシン、Ｋ＝Ｌｙｓ＝リシン、Ｌ＝Ｌｅｕ＝ロイシン、Ｍ＝Ｍｅｔ＝メチオニン、Ｎ＝Ａｓｎ＝アスパラギン、Ｐ＝Ｐｒｏ＝プロリン、Ｑ＝Ｇｌｎ＝グルタミン、Ｒ＝Ａｒｇ＝アルギニン、Ｓ＝Ｓｅｒ＝セリン、Ｔ＝Ｔｈｒ＝スレオニン、Ｖ＝Ｖａｌ＝バリン、Ｗ＝Ｔｒｐ＝トリプトファン、Ｙ＝Ｔｙｒ＝チロシン <1. Definition of terms>
In the present invention, the notation of amino acids is as follows.
A = Ala = alanine, C = Cys = cysteine, D = Asp = aspartic acid, E = Glu = glutamic acid, F = Phe = phenylalanine, G = Gly = lysine, H = His = histidine, I = Ile = isoleucine, K = Lys = lysine, L = Leucine, M = Met = methionine, N = Asn = asparagine, P = Pro = proline, Q = Gln = glutamine, R = Arg = arginine, S = Ser = serine, T = Thr = Threonine, V = Val = Valin, W = Trp = Tryptophan, Y = Tyr = Tyrosine
　本発明において、アミノ酸、タンパク質は下記に示すＩＵＰＡＣ－ＩＵＢ生化学命名委員会（ＣＢＮ）で採用された略号を用いて表される。また、特に明示しない限り、タンパク質のアミノ酸残基の配列は、左端から右端にかけてＮ末端からＣ末端となるように表される。また、参照を容易にするため、一般的に用いられている下記の命名法を適用する。１つは、「もとのアミノ酸；位置；置換したアミノ酸」と記述する方法であり、例えば、第６９位におけるスレオニンのアスパラギン酸への置換は「Ｔ６９Ｄ」と表される。 In the present invention, amino acids and proteins are represented using the abbreviations adopted by the IUPAC-IUB Biochemical Nomenclature Committee (CBN) shown below. Unless otherwise specified, the sequence of amino acid residues of a protein is represented from the N-terminal to the C-terminal from the left end to the right end. Also, for ease of reference, the following commonly used nomenclature is applied. One is a method described as "original amino acid; position; substituted amino acid". For example, the substitution of threonine with aspartic acid at position 69 is expressed as "T69D".
　本発明において、塩基配列やアミノ酸配列の配列同一性は、当業者に周知の方法、配列解析ソフトウェア等を使用して求めることができる。例えば、ＢＬＡＳＴアルゴリズムのｂｌａｓｔｎプログラムやｂｌａｓｔｐプログラム、ＦＡＳＴＡアルゴリズムのｆａｓｔａプログラムが挙げられる。本発明において、ある評価対象塩基配列の、塩基配列Ｘとの「配列同一性」とは、塩基配列Ｘと評価対象塩基配列とを整列（アライメント）させ、必要に応じてギャップを導入して、両者の塩基一致度が最も高くなるようにしたときの、ギャップ部分も含んだ塩基配列において同一部位に同一の塩基が出現する頻度を％で表示した値である。なお、ＤＮＡの塩基配列とＲＮＡの塩基配列とを比較するとき、ＴとＵは同一の塩基配列とみなす。本発明において、ある評価対象アミノ酸配列の、アミノ酸配列Ｘとの「配列同一性」とは、アミノ酸配列Ｘと評価対象アミノ酸配列とを整列させ、必要に応じてギャップを導入して、両者のアミノ酸一致度が最も高くなるようにしたときの、ギャップ部分も含んだアミノ酸配列において同一部位に同一の塩基が出現する頻度を％で表示した値である。 In the present invention, the sequence identity of a base sequence or an amino acid sequence can be obtained by using a method well known to those skilled in the art, sequence analysis software, or the like. For example, a BLASTn program or a Blastp program of the BLAST algorithm, and a FASTA program of the FASTA algorithm can be mentioned. In the present invention, the "sequence identity" of a certain evaluation target base sequence with the base sequence X means that the base sequence X and the evaluation target base sequence are aligned (aligned) and a gap is introduced as necessary. It is a value indicating the frequency at which the same base appears at the same site in the base sequence including the gap portion when the degree of base matching between the two is set to be the highest. When comparing the base sequence of DNA and the base sequence of RNA, T and U are regarded as the same base sequence. In the present invention, the "sequence identity" of a certain amino acid sequence to be evaluated with the amino acid sequence X means that the amino acid sequence X and the amino acid sequence to be evaluated are aligned and a gap is introduced as necessary to introduce both amino acids. It is a value indicating the frequency of appearance of the same base at the same site in the amino acid sequence including the gap portion when the degree of coincidence is set to be the highest in%.
　本発明において「核酸」とはポリヌクレオチドと呼ぶこともでき、ＤＮＡ又はＲＮＡを指し、典型的にはＤＮＡを指す。本発明において「核酸」は、その相補鎖と二本鎖化された形態で存在していてもよい。特に「核酸」がＤＮＡである場合、所定の塩基配列を含むＤＮＡが、その相補塩基配列を含むＤＮＡと二本鎖化された形態で存在することが好ましい。 In the present invention, the "nucleic acid" can also be called a polynucleotide, and refers to DNA or RNA, and typically refers to DNA. In the present invention, the "nucleic acid" may exist in a form double-stranded with its complementary strand. In particular, when the "nucleic acid" is DNA, it is preferable that the DNA containing a predetermined base sequence exists in a double-stranded form with the DNA containing the complementary base sequence.
　本発明において、「ポリペプチド」とは、２個以上のアミノ酸がペプチド結合したものを指し、タンパク質の他、ペプチドやオリゴペプチドと呼ばれる鎖長の短いものが含まれる。 In the present invention, the "polypeptide" refers to a peptide bond of two or more amino acids, and includes proteins, peptides and oligopeptides having a short chain length.
　本発明において所定の重鎖抗体のアミノ酸配列を「コードする塩基配列」とは、転写及び翻訳によって所定の重鎖抗体の生成をもたらすポリヌクレオチドの塩基配列を指し、例えば、重鎖抗体のアミノ酸配列に対してコドン表に基づいて設計された塩基配列を指す。 In the present invention, the "base sequence encoding" the amino acid sequence of a predetermined heavy chain antibody refers to the base sequence of a polynucleotide that produces a predetermined heavy chain antibody by transcription and translation, for example, the amino acid sequence of a heavy chain antibody. Refers to a base sequence designed based on the codon table.
　本発明において、「宿主細胞」とは、核酸が導入され形質転換される細胞、又は、核酸が導入され形質転換された細胞をいい、「宿主」とも呼ばれる。核酸が導入され形質転換された細胞を特に「形質転換体」と称することがある。 In the present invention, the "host cell" refers to a cell into which nucleic acid is introduced and transformed, or a cell into which nucleic acid is introduced and transformed, and is also referred to as a "host". Cells into which nucleic acids have been introduced and transformed may be particularly referred to as "transformants".
　「発現」とは、重鎖抗体ポリペプチドの生成をもたらす塩基配列の転写及び翻訳を指す。また、その発現は外部刺激や生育条件等に依存せずにほぼ一定の状態でもよいし、依存してもよい。発現を駆動するプロモーターは、重鎖抗体のアミノ酸配列をコードする塩基配列の発現を駆動するプロモーターであれば特に限定されない。 "Expression" refers to the transcription and translation of a base sequence that results in the production of a heavy chain antibody polypeptide. In addition, its expression may be in a substantially constant state without depending on external stimuli, growth conditions, etc., or may depend on it. The promoter that drives the expression is not particularly limited as long as it is a promoter that drives the expression of the base sequence encoding the amino acid sequence of the heavy chain antibody.
　本発明において、宿主の生物種は酵母が好ましい。酵母としてはサッカロミセス属、シゾサッカロミセス属、クイベロマイセス属、ヤロウィナ属等のメタノール資化性を有さない酵母であってもよいし、メタノール資化性酵母であってもよいが、メタノール資化性酵母がより好ましい。一般に、メタノール資化性酵母は、唯一の炭素源としてメタノールを利用して培養可能な酵母と定義されるが、本来メタノール資化性酵母であったが人為的な改変あるいは変異によりメタノール資化能を喪失した酵母も、本発明におけるメタノール資化性酵母に包含される。 In the present invention, yeast is preferable as the host organism. The yeast may be a yeast having no methanol assimilation property such as Saccharomyces genus, Saccharomyces genus, Quiberomyces genus, Yarowina genus, or a methanol assimilation yeast, but may have methanol assimilation property. Yeast is more preferred. Generally, methanol-utilizing yeast is defined as yeast that can be cultivated using methanol as the only carbon source. Although it was originally methanol-utilizing yeast, it has the ability to assimilate methanol by artificial modification or mutation. Yeast that has lost the above is also included in the methanol-utilizing yeast in the present invention.
　メタノール資化性酵母としては、ピキア（Ｐｉｃｈｉａ）属、オガタエア（Ｏｇａｔａｅａ）属、キャンディダ（Ｃａｎｄｉｄａ）属、トルロプシス（Ｔｏｒｕｌｏｐｓｉｓ）属、コマガタエラ（Ｋｏｍａｇａｔａｅｌｌａ）属などに属する酵母が挙げられる。ピキア（Ｐｉｃｈｉａ）属ではピキア・メタノリカ（Ｐｉｃｈｉａ　ｍｅｔｈａｎｏｌｉｃａ）、オガタエア（Ｏｇａｔａｅａ）属ではオガタエア・アングスタ（Ｏｇａｔａｅａ　ａｎｇｕｓｔａ）、オガタエア・ポリモルファ（Ｏｇａｔａｅａ　ｐｏｌｙｍｏｒｐｈａ）、オガタエア・パラポリモルファ（Ｏｇａｔａｅａ　ｐａｒａｐｏｌｙｍｏｒｐｈａ）、オガタエア・ミヌータ（Ｏｇａｔａｅａ　ｍｉｎｕｔａ）、キャンディダ（Ｃａｎｄｉｄａ）属ではキャンディダ・ボイディニ（Ｃａｎｄｉｄａ　ｂｏｉｄｉｎｉｉ）、コマガタエラ（Ｋｏｍａｇａｔａｅｌｌａ）属ではコマガタエラ・パストリス（Ｋｏｍａｇａｔａｅｌｌａ　ｐａｓｔｏｒｉｓ）、コマガタエラ・ファフィ（Ｋｏｍａｇａｔａｅｌｌａ　ｐｈａｆｆｉｉ）などが好ましい例として挙げられる。
　上記のメタノール資化性酵母のなかでも、ピキア属酵母、コマガタエラ属酵母又はオガタエア属酵母が特に好ましい。 Examples of the methanol-utilizing yeast include yeasts belonging to the genus Pichia, Ogataea, Candida, Torulopsis, Komagataella and the like. In the genus Pichia, Pichia metanolica, in the genus Ogataea, Ogataea angusta, Ogataea polymorpha, Ogataea polymorpha, Ogataea polymorpha, ), Candida boidini in the genus Candida, Pichia pastoris in the genus Komagataela, Pichia pastoris in the genus Komagataela, Pichia pastoris in the genus Komagataela, and the like.
Among the above-mentioned methanol-utilizing yeasts, Pichia yeast, Komagataera yeast, and Ogataea yeast are particularly preferable.
　コマガタエラ（Ｋｏｍａｇａｔａｅｌｌａ）属酵母としては、コマガタエラ・パストリス（Ｋｏｍａｇａｔａｅｌｌａ　ｐａｓｔｏｒｉｓ）、コマガタエラ・ファフィ（Ｋｏｍａｇａｔａｅｌｌａ　ｐｈａｆｆｉｉ）が好ましい。コマガタエラ・パストリスおよびコマガタエラ・ファフィはどちらもピキア・パストリス（Ｐｉｃｈｉａ　ｐａｓｔｏｒｉｓ）の別名を有する。 As the yeast belonging to the genus Komagataella, Komagataela pastoris and Komagataela phaffii are preferable. Both Komagataera Pastoris and Komagataera Fafi have another name for Pichia pastoris.
　具体的に宿主として使用できる酵母株として、コマガタエラ・パストリスＡＴＣＣ７６２７３（Ｙ－１１４３０、ＣＢＳ７４３５）、コマガタエラ・パストリスＸ－３３などの酵母株が挙げられる。これらの酵母株は、アメリカン・タイプ・カルチャー・コレクションやサーモフィッシャーサイエンティフィック社等から入手することができる。 Specific examples of yeast strains that can be used as hosts include yeast strains such as Komagataera pastris ATCC76273 (Y-11430, CBS7435) and Komagataera pastris X-33. These yeast strains can be obtained from the American Type Culture Collection, Thermo Fisher Scientific, etc.
　オガタエア（Ｏｇａｔａｅａ）属酵母としては、オガタエア・アングスタ（Ｏｇａｔａｅａ　ａｎｇｕｓｔａ）、オガタエア・ポリモルファ（Ｏｇａｔａｅａ　ｐｏｌｙｍｏｒｐｈａ）、オガタエア・パラポリモルファ（Ｏｇａｔａｅａ　ｐａｒａｐｏｌｙｍｏｒｐｈａ）が好ましい。これら３つは近縁種であり、いずれも、別名ハンゼヌラ・ポリモルファ（Ｈａｎｓｅｎｕｌａ　ｐｏｌｙｍｏｒｐｈａ）、または別名ピキア・アングスタ（Ｐｉｃｈｉａ　ａｎｇｕｓｔａ）でも表される。 As the yeast of the genus Ogataea, Ogataea angusta, Ogataea polymorpha, and Ogataea parapolymorpha are preferable. These three are closely related species, and all of them are also represented by another name, Hansenula polymorpha, or another name, Pichia angusta.
　具体的に使用できる酵母株として、オガタエア・アングスタＮＣＹＣ４９５（ＡＴＣＣ１４７５４）、オガタエア・ポリモルファ８Ｖ（ＡＴＣＣ３４４３８）、オガタエア・パラポリモルファＤＬ－１（ＡＴＣＣ２６０１２）等の酵母株が挙げられる。これらの酵母株は、アメリカン・タイプ・カルチャー・コレクション等から入手できる。 Specific examples of yeast strains that can be used include yeast strains such as Ogataea Angsta NCYC495 (ATCC14754), Ogataea polymorpha 8V (ATCC34438), and Ogataea parapolymorpha DL-1 (ATCC26012). These yeast strains can be obtained from the American Type Culture Collection and others.
　また、宿主としてはピキア属酵母、コマガタエラ属酵母、オガタエア属酵母等の酵母株からの誘導株も使用でき、例えばヒスチジン要求性であればコマガタエラ・パストリスＧＳ１１５株（サーモフィッシャーサイエンティフィック社より入手可能）、ロイシン要求性株であれば、ＮＣＹＣ４９５由来のＢＹ４３２９、８Ｖ由来のＢＹ５２４２、ＤＬ－１由来のＢＹ５２４３（これらはＮａｔｉｏｎａｌ　ＢｉｏＲｅｓｏｕｒｃｅ　Ｐｒｏｊｅｃｔから分譲可能）等が挙げられる。本発明においては、これらの株からの誘導株等も使用できる。 In addition, as a host, derived strains from yeast strains such as Pichia yeast, Komagataera yeast, and Ogataea yeast can also be used. For example, if histidine is required, Komagataera Pastris GS115 strain (available from Thermofisher Scientific) ), If it is a leucine-requiring strain, BY4329 derived from NCYC495, BY5242 derived from 8V, BY5243 derived from DL-1 (these can be sold from National BioResource Project) and the like can be mentioned. In the present invention, derived strains and the like from these strains can also be used.
＜２．重鎖抗体＞
　本発明の一以上の実施形態は、Ｎ末端からフレームワーク領域１（ＦＲ１）、可変重鎖相補性決定領域１（ＶＨ－ＣＤＲ１）、フレームワーク領域２（ＦＲ２）、可変重鎖相補性決定領域２（ＶＨ－ＣＤＲ２）、フレームワーク領域３（ＦＲ３）、可変重鎖相補性決定領域３（ＶＨ－ＣＤＲ３）及びフレームワーク領域４（ＦＲ４）がこの順で連結した重鎖可変ドメインを含む重鎖抗体に関する。 <2. Heavy chain antibody>
One or more embodiments of the present invention include framework regions 1 (FR1), variable heavy chain complementarity determining regions 1 (VH-CDR1), framework regions 2 (FR2), and variable heavy chain complementarity determining regions from the N-terminus. A heavy chain containing a heavy chain variable domain in which 2 (VH-CDR2), framework region 3 (FR3), variable heavy chain complementarity determining region 3 (VH-CDR3) and framework region 4 (FR4) are linked in this order. Regarding antibodies.
　本発明の一以上の実施形態に係る重鎖抗体（以下「前記重鎖抗体」と称する場合がある）は、前記重鎖可変ドメインを含むものであればよく、前記重鎖可変ドメインからなるシングルドメイン抗体（ＶＨＨ抗体）あってもよいし、前記重鎖可変ドメインと重鎖定常ドメイン又はその断片とを含む重鎖抗体であってもよい。 The heavy chain antibody according to one or more embodiments of the present invention (hereinafter, may be referred to as “the heavy chain antibody”) may be any one containing the heavy chain variable domain, and is a single composed of the heavy chain variable domain. It may be a domain antibody (VHH antibody), or it may be a heavy chain antibody containing the heavy chain variable domain and a heavy chain constant domain or a fragment thereof.
　前記重鎖可変ドメインは、（ＦＲ１）－（ＶＨ－ＣＤＲ１）－（ＦＲ２）－（ＶＨ－ＣＤＲ２）－（ＦＲ３）－（ＶＨ－ＣＤＲ３）－（ＦＲ４）からなるポリペプチドのみからなるものであってもよいし、前記ポリペプチドのＮ末端側及びＣ末端側の一方又は両方に、更に他のポリペプチドが連結された融合ポリペプチドの形態であってもよい。他のポリペプチドとしては、シグナルペプチド、タグペプチド等が例示できるがこれらに限定されない。シグナルペプチドの具体例は後述する通りである。タグペプチドとしては複数（例えば６～１０個）のヒスチジン残基からなるタグペプチド（ポリヒスチジンタグ）や、ＦＬＡＧタグペプチドが例示できる。 The heavy chain variable domain consists only of a polypeptide consisting of (FR1)-(VH-CDR1)-(FR2)-(VH-CDR2)-(FR3)-(VH-CDR3)-(FR4). It may be in the form of a fusion polypeptide in which another polypeptide is further linked to one or both of the N-terminal side and the C-terminal side of the polypeptide. Examples of other polypeptides include, but are not limited to, signal peptides, tag peptides, and the like. Specific examples of the signal peptide will be described later. Examples of the tag peptide include a tag peptide (polyhistidine tag) composed of a plurality of (for example, 6 to 10) histidine residues and a FLAG tag peptide.
　本発明の一以上の実施形態に係る重鎖抗体は、そのアミノ酸配列をコードする塩基配列を含む核酸を含むベクターで形質転換した宿主細胞により発現させた場合に、前記重鎖抗体の前記重鎖可変ドメインのＦＲ３のセリン又はスレオニン残基でのＯ結合型糖鎖修飾が抑制される。こうして得られる前記重鎖抗体は、均一性が高く、抗原結合性等の品質が安定した抗体である。 The heavy chain antibody according to one or more embodiments of the present invention is the heavy chain of the heavy chain antibody when expressed in a host cell transformed with a vector containing a nucleic acid containing a base sequence encoding the amino acid sequence. O-linked sugar chain modification at the serine or threonine residue of FR3 in the variable domain is suppressed. The heavy chain antibody thus obtained is an antibody having high homogeneity and stable quality such as antigen binding.
　本発明の一以上の実施形態に係る重鎖抗体は、好ましくはＯ結合型糖鎖修飾体の割合が低く、具体的には、重鎖抗体の全量に対するＯ結合型糖鎖修飾体の割合が５質量％以下、好ましくは４質量％以下、より好ましくは３質量％以下、特に好ましくは２質量％以下、更に好ましくは１質量％以下、最も好ましくは０質量％である。 The heavy chain antibody according to one or more embodiments of the present invention preferably has a low proportion of O-linked sugar chain modifiers, and specifically, the proportion of O-linked sugar chain modifiers to the total amount of the heavy chain antibody. It is 5% by mass or less, preferably 4% by mass or less, more preferably 3% by mass or less, particularly preferably 2% by mass or less, still more preferably 1% by mass or less, and most preferably 0% by mass.
　前記重鎖抗体は、複数が直接又はリンカーを介して連結された多重特異性抗体又は多価抗体であってもよい。特異的に結合する抗原の異なる前記重鎖抗体が複数連結されている抗体が多重特異性抗体である。特異的に結合する抗原が同一の前記重鎖抗体が複数連結されている抗体が多価抗体である。 The heavy chain antibody may be a multispecific antibody or a multivalent antibody in which a plurality of the antibodies are directly linked or linked via a linker. A multispecific antibody is an antibody in which a plurality of the heavy chain antibodies having different antigens that specifically bind to each other are linked. A multivalent antibody is an antibody in which a plurality of the heavy chain antibodies having the same specifically binding antigen are linked.
　本発明者らは、（ＦＲ１）－（ＶＨ－ＣＤＲ１）－（ＦＲ２）－（ＶＨ－ＣＤＲ２）－（ＦＲ３）－（ＶＨ－ＣＤＲ３）－（ＦＲ４）の構造を有する重鎖可変ドメインを含む既知の重鎖抗体として、配列番号５０に示すアミノ酸配列を含むａｎｔｉ－Ｆｃ　ＶＨＨ、配列番号５２に示すアミノ酸配列を含むａｎｔｉ－ＨＳＡ　ＶＨＨ、配列番号５７に示すアミノ酸配列を含むａｎｔｉ－ＩＬ６Ｒ ＶＨＨ、配列番号５８に示すアミノ酸配列を含むａｎｔｉ－ｖＷＦ ＶＨＨ、配列番号５９に示すアミノ酸配列を含むａｎｔｉ－ＲＡＮＫＬ ＶＨＨ、配列番号６０に示すアミノ酸配列を含むａｎｔｉ－ＴＮＦα ＶＨＨ、配列番号６１に示すアミノ酸配列を含むａｎｔｉ－ｃＡｂＢｃＩＩ１０ ＶＨＨ、配列番号６２に示すアミノ酸配列を含むａｎｔｉ－ＲＳＶ ＶＨＨ－１、配列番号６３に示すアミノ酸配列を含むａｎｔｉ－ＲＳＶ ＶＨＨ－２のアミノ酸配列をアライメントし比較した。比較結果の概要を図１に示す。図１に示すように、これらの重鎖抗体の重鎖可変ドメインにおいてＦＲ１、ＦＲ２、ＦＲ３、ＦＲ４のアミノ酸配列は高度に保存されている。 The present inventors are known to include a heavy chain variable domain having a structure of (FR1)-(VH-CDR1)-(FR2)-(VH-CDR2)-(FR3)-(VH-CDR3)-(FR4). As the heavy chain antibody of, anti-Fc VHH containing the amino acid sequence shown in SEQ ID NO: 50, anti-HSA VHH containing the amino acid sequence shown in SEQ ID NO: 52, anti-IL6R VHH containing the amino acid sequence shown in SEQ ID NO: 57, SEQ ID NO: Anti-vWF VHH containing the amino acid sequence shown in 58, anti-RANKL VHH containing the amino acid sequence shown in SEQ ID NO: 59, anti-TNFα VHH containing the amino acid sequence shown in SEQ ID NO: 60, antii containing the amino acid sequence shown in SEQ ID NO: 61. The amino acid sequences of -cAbBcII10 VHH, anti-RSV VHH-1 containing the amino acid sequence shown in SEQ ID NO: 62, and anti-RSV VHH-2 containing the amino acid sequence shown in SEQ ID NO: 63 were aligned and compared. The outline of the comparison result is shown in FIG. As shown in FIG. 1, the amino acid sequences of FR1, FR2, FR3, and FR4 are highly conserved in the heavy chain variable domain of these heavy chain antibodies.
　ＦＲ１のアミノ酸配列は配列番号１で表すことができる。ＦＲ１の配列番号１に示すアミノ酸配列の既知の具体例としては、配列番号５０に示すａｎｔｉ－Ｆｃ　ＶＨＨのアミノ酸配列における第１位～第２５位の部分アミノ酸配列、配列番号５２に示すａｎｔｉ－ＨＳＡ　ＶＨＨのアミノ酸配列における第１位～第２５位の部分アミノ酸配列、配列番号５７に示すａｎｔｉ－ＩＬ６Ｒ ＶＨＨのアミノ酸配列における第１位～第２５位の部分アミノ酸配列、配列番号５８に示すａｎｔｉ－ｖＷＦ ＶＨＨのアミノ酸配列における第１位～第２５位の部分アミノ酸配列、配列番号５９に示すａｎｔｉ－ＲＡＮＫＬ ＶＨＨのアミノ酸配列における第１位～第２５位の部分アミノ酸配列、配列番号６０に示すａｎｔｉ－ＴＮＦα ＶＨＨのアミノ酸配列における第１位～第２５位の部分アミノ酸配列、配列番号６１に示すａｎｔｉ－ｃＡｂＢｃＩＩ１０ ＶＨＨのアミノ酸配列における第３位～第２７位の部分アミノ酸配列、配列番号６２に示すａｎｔｉ－ＲＳＶ ＶＨＨ－１のアミノ酸配列における第１位～第２５位の部分アミノ酸配列、配列番号６３に示すａｎｔｉ－ＲＳＶ ＶＨＨ－２のアミノ酸配列における第１位～第２５位の部分アミノ酸配列が例示できる。 The amino acid sequence of FR1 can be represented by SEQ ID NO: 1. As a known specific example of the amino acid sequence shown in SEQ ID NO: 1 of FR1, the partial amino acid sequence of positions 1 to 25 in the amino acid sequence of anti-Fc VHH shown in SEQ ID NO: 50, anti-HSA shown in SEQ ID NO: 52. Partial amino acid sequence of positions 1 to 25 in the amino acid sequence of VHH, partial amino acid sequence of positions 1 to 25 in the amino acid sequence of anti-IL6R VHH shown in SEQ ID NO: 57, anti-vWF shown in SEQ ID NO: 58. Partial amino acid sequence of positions 1 to 25 in the amino acid sequence of VHH, partial amino acid sequence of positions 1 to 25 in the amino acid sequence of anti-RANKL VHH shown in SEQ ID NO: 59, anti-TNFα shown in SEQ ID NO: 60. Partial amino acid sequence of positions 1 to 25 in the amino acid sequence of VHH, partial amino acid sequence of positions 3 to 27 in the amino acid sequence of anti-cAbBcII10 VHH shown in SEQ ID NO: 61, anti-RSV shown in SEQ ID NO: 62 Examples thereof include a partial amino acid sequence at positions 1 to 25 in the amino acid sequence of VHH-1, and a partial amino acid sequence at positions 1 to 25 in the amino acid sequence of anti-RSV VHH-2 shown in SEQ ID NO: 63.
　ＦＲ２のアミノ酸配列は配列番号２で表すことができる。ＦＲ２の配列番号２に示すアミノ酸配列の既知の具体例としては、配列番号５０に示すａｎｔｉ－Ｆｃ　ＶＨＨのアミノ酸配列における第３６位～第４９位の部分アミノ酸配列、配列番号５２に示すａｎｔｉ－ＨＳＡ　ＶＨＨのアミノ酸配列における第３６位～第４９位の部分アミノ酸配列、配列番号５７に示すａｎｔｉ－ＩＬ６Ｒ ＶＨＨのアミノ酸配列における第３６位～第４９位の部分アミノ酸配列、配列番号５８に示すａｎｔｉ－ｖＷＦ ＶＨＨのアミノ酸配列における第３６位～第４９位の部分アミノ酸配列、配列番号５９に示すａｎｔｉ－ＲＡＮＫＬ ＶＨＨのアミノ酸配列における第３６位～第４９位の部分アミノ酸配列、配列番号６０に示すａｎｔｉ－ＴＮＦα ＶＨＨのアミノ酸配列における第３６位～第４９位の部分アミノ酸配列、配列番号６１に示すａｎｔｉ－ｃＡｂＢｃＩＩ１０ ＶＨＨのアミノ酸配列における第４１位～第５４位の部分アミノ酸配列、配列番号６２に示すａｎｔｉ－ＲＳＶ ＶＨＨ－１のアミノ酸配列における第３６位～第４９位の部分アミノ酸配列、配列番号６３に示すａｎｔｉ－ＲＳＶ ＶＨＨ－２のアミノ酸配列における第３６位～第４９位の部分アミノ酸配列が例示できる。 The amino acid sequence of FR2 can be represented by SEQ ID NO: 2. As a known specific example of the amino acid sequence shown in SEQ ID NO: 2 of FR2, the partial amino acid sequence at positions 36 to 49 in the amino acid sequence of anti-Fc VHH shown in SEQ ID NO: 50, anti-HSA shown in SEQ ID NO: 52. Partial amino acid sequence at positions 36 to 49 in the amino acid sequence of VHH, partial amino acid sequence at positions 36 to 49 in the amino acid sequence of anti-IL6R VHH shown in SEQ ID NO: 57, anti-vWF shown in SEQ ID NO: 58. Partial amino acid sequence at positions 36 to 49 in the amino acid sequence of VHH, partial amino acid sequence at positions 36 to 49 in the amino acid sequence of anti-RANKL VHH shown in SEQ ID NO: 59, anti-TNFα shown in SEQ ID NO: 60. Partial amino acid sequence of positions 36 to 49 in the amino acid sequence of VHH, partial amino acid sequence of positions 41 to 54 in the amino acid sequence of anti-cAbBcII10 VHH shown in SEQ ID NO: 61, anti-RSV shown in SEQ ID NO: 62 Examples thereof include the partial amino acid sequence at positions 36 to 49 in the amino acid sequence of VHH-1, and the partial amino acid sequence at positions 36 to 49 in the amino acid sequence of anti-RSV VHH-2 shown in SEQ ID NO: 63.
　ＦＲ３のアミノ酸配列は配列番号３で表すことができる。ＦＲ３の配列番号３に示すアミノ酸配列の既知の具体例としては、配列番号５０に示すａｎｔｉ－Ｆｃ　ＶＨＨのアミノ酸配列における第６７位～第９８位の部分アミノ酸配列、配列番号５２に示すａｎｔｉ－ＨＳＡ　ＶＨＨのアミノ酸配列における第６７位～第９８位の部分アミノ酸配列、配列番号５７に示すａｎｔｉ－ＩＬ６Ｒ ＶＨＨのアミノ酸配列における第６６位～第９７位の部分アミノ酸配列、配列番号５８に示すａｎｔｉ－ｖＷＦ ＶＨＨのアミノ酸配列における第６７位～第９８位の部分アミノ酸配列、配列番号５９に示すａｎｔｉ－ＲＡＮＫＬ ＶＨＨのアミノ酸配列における第６７位～第９８位の部分アミノ酸配列、配列番号６０に示すａｎｔｉ－ＴＮＦα ＶＨＨのアミノ酸配列における第６７位～第９８位の部分アミノ酸配列、配列番号６１に示すａｎｔｉ－ｃＡｂＢｃＩＩ１０ ＶＨＨのアミノ酸配列における第７２位～第１０３位の部分アミノ酸配列、配列番号６２に示すａｎｔｉ－ＲＳＶ ＶＨＨ－１のアミノ酸配列における第６７位～第９８位の部分アミノ酸配列、配列番号６３に示すａｎｔｉ－ＲＳＶ ＶＨＨ－２のアミノ酸配列における第６７位～第９８位の部分アミノ酸配列が例示できる。 The amino acid sequence of FR3 can be represented by SEQ ID NO: 3. As a known specific example of the amino acid sequence shown in SEQ ID NO: 3 of FR3, the partial amino acid sequence of positions 67 to 98 in the amino acid sequence of anti-Fc VHH shown in SEQ ID NO: 50, anti-HSA shown in SEQ ID NO: 52. Partial amino acid sequences of positions 67 to 98 in the amino acid sequence of VHH, partial amino acid sequences of positions 66 to 97 in the amino acid sequence of anti-IL6R VHH shown in SEQ ID NO: 57, anti-vWF shown in SEQ ID NO: 58. Partial amino acid sequence at positions 67 to 98 in the amino acid sequence of VHH, partial amino acid sequence at positions 67 to 98 in the amino acid sequence of anti-RANKL VHH shown in SEQ ID NO: 59, anti-TNFα shown in SEQ ID NO: 60. Partial amino acid sequence of positions 67 to 98 in the amino acid sequence of VHH, anti-cAbBcII10 shown in SEQ ID NO: 61 Partial amino acid sequence of positions 72 to 103 in the amino acid sequence of VHH, anti-RSV shown in SEQ ID NO: 62 Examples thereof include the partial amino acid sequence at positions 67 to 98 in the amino acid sequence of VHH-1, and the partial amino acid sequence at positions 67 to 98 in the amino acid sequence of anti-RSV VHH-2 shown in SEQ ID NO: 63.
　ＦＲ４のアミノ酸配列は配列番号４で表すことができる。ＦＲ４の配列番号４に示すアミノ酸配列の既知の具体例としては、配列番号５０に示すａｎｔｉ－Ｆｃ　ＶＨＨのアミノ酸配列における第１１４位～第１２５位の部分アミノ酸配列、配列番号５２に示すａｎｔｉ－ＨＳＡ　ＶＨＨのアミノ酸配列における第１０４位～第１１５位の部分アミノ酸配列、配列番号５７に示すａｎｔｉ－ＩＬ６Ｒ ＶＨＨのアミノ酸配列における第１１０位～第１２１位の部分アミノ酸配列、配列番号５８に示すａｎｔｉ－ｖＷＦ ＶＨＨのアミノ酸配列における第１１７位～第１２８位の部分アミノ酸配列、配列番号５９に示すａｎｔｉ－ＲＡＮＫＬ ＶＨＨのアミノ酸配列における第１１５位～第１２５６位の部分アミノ酸配列、配列番号６０に示すａｎｔｉ－ＴＮＦα ＶＨＨのアミノ酸配列における第１０４位～第１１５位の部分アミノ酸配列、配列番号６１に示すａｎｔｉ－ｃＡｂＢｃＩＩ１０ ＶＨＨのアミノ酸配列における第１１９位～第１３０位の部分アミノ酸配列、配列番号６２に示すａｎｔｉ－ＲＳＶ ＶＨＨ－１のアミノ酸配列における第１１５位～第１２６位の部分アミノ酸配列、配列番号６３に示すａｎｔｉ－ＲＳＶ ＶＨＨ－２のアミノ酸配列における第１１５位～第１２６位の部分アミノ酸配列が例示できる。 The amino acid sequence of FR4 can be represented by SEQ ID NO: 4. As a known specific example of the amino acid sequence shown in SEQ ID NO: 4 of FR4, the partial amino acid sequence of positions 114 to 125 in the amino acid sequence of anti-Fc VHH shown in SEQ ID NO: 50, anti-HSA shown in SEQ ID NO: 52. Partial amino acid sequences of positions 104 to 115 in the amino acid sequence of VHH, partial amino acid sequences of positions 110 to 121 in the amino acid sequence of anti-IL6R VHH shown in SEQ ID NO: 57, anti-vWF shown in SEQ ID NO: 58. Partial amino acid sequence of positions 117 to 128 in the amino acid sequence of VHH, partial amino acid sequence of positions 115 to 1256 in the amino acid sequence of anti-RANKL VHH shown in SEQ ID NO: 59, anti-TNFα shown in SEQ ID NO: 60. Partial amino acid sequence of positions 104 to 115 in the amino acid sequence of VHH, partial amino acid sequence of positions 119 to 130 in the amino acid sequence of anti-cAbBcII10 VHH shown in SEQ ID NO: 61, anti-RSV shown in SEQ ID NO: 62 Examples thereof include a partial amino acid sequence at positions 115 to 126 in the amino acid sequence of VHH-1, and a partial amino acid sequence at positions 115 to 126 in the amino acid sequence of anti-RSV VHH-2 shown in SEQ ID NO: 63.
　本発明の一以上の実施形態に係る重鎖抗体は、
　ＦＲ１のアミノ酸配列が、
（１ａ）配列番号１に示すアミノ酸配列、又は、
（１ｂ）配列番号１に示すアミノ酸配列と８３％以上の配列同一性を有するアミノ酸配列であり、
　ＦＲ２のアミノ酸配列が、
（２ａ）配列番号２に示すアミノ酸配列、又は、
（２ｂ）配列番号２に示すアミノ酸配列と７０％以上の配列同一性を有するアミノ酸配列であり、
　ＦＲ３のアミノ酸配列が、
（３ａ）配列番号３に示すアミノ酸配列において、配列番号３の第３位、第５位、第１１位、第１２位、第１９位及び第２５位のアミノ酸の少なくとも１つに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列、或いは、
（３ｂ）配列番号３に示すアミノ酸配列と８５％以上の配列同一性を有するアミノ酸配列において、配列番号３の第３位、第５位、第１１位、第１２位、第１９位及び第２５位のアミノ酸に対応するアミノ酸の少なくとも１つに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列であり、
　ＦＲ４のアミノ酸配列が、
（４ａ）配列番号４に示すアミノ酸配列、又は、
（４ｂ）配列番号４に示すアミノ酸配列と６５％以上の配列同一性を有するアミノ酸配列である。 The heavy chain antibody according to one or more embodiments of the present invention
The amino acid sequence of FR1 is
(1a) The amino acid sequence shown in SEQ ID NO: 1 or
(1b) An amino acid sequence having 83% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.
The amino acid sequence of FR2 is
(2a) The amino acid sequence shown in SEQ ID NO: 2 or
(2b) An amino acid sequence having 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2.
The amino acid sequence of FR3 is
(3a) In the amino acid sequence shown in SEQ ID NO: 3, serine or threonine is added to at least one of the amino acids at positions 3, 5, 11, 12, 12, 19 and 25 of SEQ ID NO: 3. Amino acid sequence in which substitution with an amino acid other than is introduced, or
(3b) In the amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, the 3rd, 5th, 11th, 12th, 19th and 25th positions of SEQ ID NO: 3 An amino acid sequence in which substitution with an amino acid other than serine or threonine is introduced into at least one of the amino acids corresponding to the amino acid at the position.
The amino acid sequence of FR4 is
(4a) The amino acid sequence shown in SEQ ID NO: 4 or
(4b) An amino acid sequence having 65% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4.
　前記（１ａ）、（１ｂ）、（２ａ）、（２ｂ）、（３ａ）、（３ｂ）、（４ａ）、（４ｂ）で規定するアミノ酸配列はいずれも、それを含む重鎖可変ドメインが抗原結合性を保持できるアミノ酸配列であればよい。好ましくは、前記（１ａ）、（１ｂ）、（２ａ）、（２ｂ）、（３ａ）、（３ｂ）、（４ａ）、（４ｂ）で規定するアミノ酸配列はいずれも、それを含む重鎖可変ドメインの抗原結合性が、上記の既知のアミノ酸配列からなるＦＲ１、ＦＲ２、ＦＲ３、ＦＲ４を含む重鎖可変ドメインの抗原結合性と同等となるアミノ酸配列である。より好ましくは、前記（１ａ）、（１ｂ）、（２ａ）、（２ｂ）、（３ａ）、（３ｂ）、（４ａ）、（４ｂ）で規定するアミノ酸配列はいずれも、それを含む重鎖可変ドメインの抗原結合性が、上記の既知のアミノ酸配列からなるＦＲ１、ＦＲ２、ＦＲ３、ＦＲ４を含む重鎖可変ドメインの抗原結合性に対して７０％以上１５０％以下、好ましくは８０％以上１４０％以下、特に好ましくは８５％以上１３０％以下となるアミノ酸配列である。 The amino acid sequences specified in (1a), (1b), (2a), (2b), (3a), (3b), (4a), and (4b) all contain the heavy chain variable domain as an antigen. Any amino acid sequence that can maintain the binding property is sufficient. Preferably, the amino acid sequences specified in (1a), (1b), (2a), (2b), (3a), (3b), (4a), and (4b) are all variable heavy chains. It is an amino acid sequence in which the antigen binding property of the domain is equivalent to the antigen binding property of the heavy chain variable domain including FR1, FR2, FR3, and FR4 consisting of the above known amino acid sequences. More preferably, the amino acid sequences defined in (1a), (1b), (2a), (2b), (3a), (3b), (4a), and (4b) all contain heavy chains. The antigen binding property of the variable domain is 70% or more and 150% or less, preferably 80% or more and 140% with respect to the antigen binding property of the heavy chain variable domain including FR1, FR2, FR3 and FR4 consisting of the above known amino acid sequences. Hereinafter, the amino acid sequence is particularly preferably 85% or more and 130% or less.
　前記（１ｂ）において配列同一性は好ましくは８５％以上、より好ましくは９０％以上、より好ましくは９３％以上、より好ましくは９５％以上、より好ましくは９７％、より好ましくは９８％以上、より好ましくは９９％以上である。 In (1b), the sequence identity is preferably 85% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, still more preferably 98% or more. It is preferably 99% or more.
　前記（１ｂ）で規定するアミノ酸配列は、好ましくは、前記（１ａ）で規定するアミノ酸配列とアミノ酸残基数が同一で、前記（１ａ）で規定するアミノ酸配列において一部のアミノ酸が他のアミノ酸に置換されたアミノ酸配列である。置換されたアミノ酸残基の数は好ましくは１～数個、好ましくは１～３個、より好ましくは１～２個、特に好ましくは１個であることができる。ここでアミノ酸の置換は、保存的アミノ酸置換が好ましい。「保存的アミノ酸置換」とは、電荷、側鎖、極性、芳香族性等の性質の類似するアミノ酸間の置換をいう。性質の類似するアミノ酸は、例えば、塩基性アミノ酸（アルギニン、リジン、ヒスチジン）、酸性アミノ酸（アスパラギン酸、グルタミン酸）、無電荷極性アミノ酸（グリシン、アスパラギン、グルタミン、セリン、トレオニン、システイン、チロシン）、無極性アミノ酸（ロイシン、イソロイシン、アラニン、バリン、プロリン、フェニルアラニン、トリプトファン、メチオニン）、分枝鎖アミノ酸（ロイシン、バリン、イソロイシン）、芳香族アミノ酸（フェニルアラニン、チロシン、トリプトファン、ヒスチジン）等に分類することができる。 The amino acid sequence specified in (1b) is preferably the same as the amino acid sequence specified in (1a), and some amino acids are other amino acids in the amino acid sequence specified in (1a). Amino acid sequence substituted with. The number of substituted amino acid residues can be preferably 1 to several, preferably 1 to 3, more preferably 1 to 2, and particularly preferably 1. Here, the amino acid substitution is preferably a conservative amino acid substitution. “Conservative amino acid substitution” refers to a substitution between amino acids having similar properties such as charge, side chain, polarity, and aromaticity. Amino acids with similar properties include, for example, basic amino acids (arginine, lysine, histidine), acidic amino acids (aspartic acid, glutamic acid), uncharged polar amino acids (glycine, asparagin, glutamine, serine, threonine, cysteine, tyrosine), non-polar amino acids. It can be classified into sex amino acids (leucine, isoleucine, alanine, valine, proline, phenylalanine, tryptophan, methionine), branched amino acids (leucine, valine, isoleucine), aromatic amino acids (phenylalanine, tyrosine, tryptophan, histidine), etc. it can.
　前記（２ｂ）において配列同一性は好ましくは７５％以上、より好ましくは８０％以上、より好ましくは８５％以上、より好ましくは９０％以上、より好ましくは９３％以上、より好ましくは９５％以上、より好ましくは９７％、より好ましくは９８％以上、より好ましくは９９％以上である。 In (2b), the sequence identity is preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 93% or more, still more preferably 95% or more. It is more preferably 97%, more preferably 98% or more, and more preferably 99% or more.
　前記（２ｂ）で規定するアミノ酸配列は、好ましくは、前記（２ａ）で規定するアミノ酸配列とアミノ酸残基数が同一で、前記（２ａ）で規定するアミノ酸配列において一部のアミノ酸が他のアミノ酸に置換されたアミノ酸配列である。置換されたアミノ酸残基の数は好ましくは１～数個、好ましくは１～３個、より好ましくは１～２個、特に好ましくは１個であることができる。ここでアミノ酸の置換は、上記の保存的アミノ酸置換が好ましい。 The amino acid sequence specified in (2b) is preferably the same as the amino acid sequence specified in (2a), and some amino acids are other amino acids in the amino acid sequence specified in (2a). Amino acid sequence substituted with. The number of substituted amino acid residues can be preferably 1 to several, preferably 1 to 3, more preferably 1 to 2, and particularly preferably 1. Here, the amino acid substitution is preferably the above-mentioned conservative amino acid substitution.
　前記（３ｂ）において配列同一性は好ましくは９０％以上、より好ましくは９３％以上、より好ましくは９５％以上、より好ましくは９７％、より好ましくは９８％以上、より好ましくは９９％以上である。 In (3b), the sequence identity is preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, more preferably 98% or more, still more preferably 99% or more. ..
　前記（３ｂ）で規定するアミノ酸配列は、好ましくは、前記（３ａ）で規定するアミノ酸配列とアミノ酸残基数が同一で、前記（３ａ）で規定するアミノ酸配列において一部のアミノ酸が他のアミノ酸に置換されたアミノ酸配列である。置換されたアミノ酸残基の数は好ましくは１～数個、好ましくは１～３個、より好ましくは１～２個、特に好ましくは１個であることができる。ここでアミノ酸の置換は、上記の保存的アミノ酸置換が好ましい。 The amino acid sequence specified in (3b) is preferably the same as the amino acid sequence specified in (3a), and some amino acids are other amino acids in the amino acid sequence specified in (3a). Amino acid sequence substituted with. The number of substituted amino acid residues can be preferably 1 to several, preferably 1 to 3, more preferably 1 to 2, and particularly preferably 1. Here, the amino acid substitution is preferably the above-mentioned conservative amino acid substitution.
　前記（４ｂ）において配列同一性は好ましくは７０％以上、より好ましくは７５％以上、より好ましくは８０％以上、より好ましくは８５％以上、より好ましくは９０％以上、より好ましくは９３％以上、より好ましくは９５％以上、より好ましくは９７％、より好ましくは９８％以上、より好ましくは９９％以上である。 In (4b), the sequence identity is preferably 70% or more, more preferably 75% or more, more preferably 80% or more, more preferably 85% or more, more preferably 90% or more, more preferably 93% or more. It is more preferably 95% or more, more preferably 97%, more preferably 98% or more, and even more preferably 99% or more.
　前記（４ｂ）で規定するアミノ酸配列は、好ましくは、前記（４ａ）で規定するアミノ酸配列とアミノ酸残基数が同一で、前記（４ａ）で規定するアミノ酸配列において一部のアミノ酸が他のアミノ酸に置換されたアミノ酸配列である。置換されたアミノ酸残基の数は好ましくは１～数個、好ましくは１～３個、より好ましくは１～２個、特に好ましくは１個であることができる。ここでアミノ酸の置換は、上記の保存的アミノ酸置換が好ましい。 The amino acid sequence specified in (4b) preferably has the same number of amino acid residues as the amino acid sequence specified in (4a), and some amino acids are other amino acids in the amino acid sequence specified in (4a). Amino acid sequence substituted with. The number of substituted amino acid residues can be preferably 1 to several, preferably 1 to 3, more preferably 1 to 2, and particularly preferably 1. Here, the amino acid substitution is preferably the above-mentioned conservative amino acid substitution.
　本発明ではＦＲ３のアミノ酸配列が、
（３ａ）配列番号３に示すアミノ酸配列において、配列番号３の第３位、第５位、第１１位、第１２位、第１９位及び第２５位のアミノ酸の少なくとも１つに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列、或いは、
（３ｂ）配列番号３に示すアミノ酸配列と８５％以上の配列同一性を有するアミノ酸配列において、配列番号３の第３位、第５位、第１１位、第１２位、第１９位及び第２５位のアミノ酸に対応するアミノ酸の少なくとも１つに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列
であることにより、ＦＲ３のセリン又はスレオニン残基へのＯ結合型糖鎖修飾が抑制される。ここで「セリン又はスレオニン以外のアミノ酸」は特に限定されないが、アラニン、バリン、ロイシン、イソロイシン、アスパラギン酸、グルタミン酸、アスパラギン及びグルタミンが好ましく、アラニン、アスパラギン酸又はバリンが特に好ましい。 In the present invention, the amino acid sequence of FR3 is
(3a) In the amino acid sequence shown in SEQ ID NO: 3, serine or threonine is added to at least one of the amino acids at positions 3, 5, 11, 12, 12, 19 and 25 of SEQ ID NO: 3. Amino acid sequence in which substitution with an amino acid other than is introduced, or
(3b) In the amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, the 3rd, 5th, 11th, 12th, 19th and 25th positions of SEQ ID NO: 3 By using an amino acid sequence in which substitution with an amino acid other than serine or threonine is introduced into at least one of the amino acids corresponding to the amino acid at the position, the O-linked sugar chain modification to the serine or threonine residue of FR3 can be performed. It is suppressed. Here, "amino acid other than serine or threonine" is not particularly limited, but alanine, valine, leucine, isoleucine, aspartic acid, glutamic acid, aspartic acid and glutamine are preferable, and alanine, aspartic acid or valine is particularly preferable.
　前記（３ｂ）において「配列番号３の第３位、第５位、第１１位、第１２位、第１９位及び第２５位のアミノ酸に対応するアミノ酸」とは、前記（３ｂ）で規定するアミノ酸配列を、配列番号３に示すアミノ酸配列と一致度が最大になるようにアライメントしたときの、各位置に対応するアミノ酸を指す。 In the above (3b), "the amino acid corresponding to the amino acid at the 3rd, 5th, 11th, 12th, 19th and 25th positions of SEQ ID NO: 3" is defined in the above (3b). It refers to the amino acid corresponding to each position when the amino acid sequence is aligned so as to maximize the degree of coincidence with the amino acid sequence shown in SEQ ID NO: 3.
　前記（３ａ）において好ましくは、配列番号３の第３位、第５位、第１２位、第１９位及び第２５位のアミノ酸の少なくとも１つに、セリン又はスレオニン以外のアミノ酸への置換が導入されている。前記（３ｂ）において好ましくは、配列番号３の第３位、第５位、第１２位、第１９位及び第２５位のアミノ酸に対応するアミノ酸の少なくとも１つに、セリン又はスレオニン以外のアミノ酸への置換が導入されている。 In (3a), substitution with an amino acid other than serine or threonine is preferably introduced into at least one of the amino acids at positions 3, 5, 12, 19 and 25 of SEQ ID NO: 3. Has been done. In (3b) above, preferably, at least one of the amino acids corresponding to the amino acids at positions 3, 5, 12, 19 and 25 of SEQ ID NO: 3 is an amino acid other than serine or threonine. Substitution has been introduced.
　（３ａ）で規定するアミノ酸配列は、より好ましくは、配列番号３に示すアミノ酸配列において、配列番号３の第３位、第５位、第１２位、第１９位及び第２５位のアミノ酸の少なくとも１つに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列であり、特に好ましくは、配列番号３に示すアミノ酸配列において、配列番号３の第３位、第５位、第１１位、第１２位、第１９位及び第２５位のアミノ酸の好ましくは少なくとも２つ、より好ましくは少なくとも３つ、より好ましくは少なくとも４つ、より好ましくは少なくとも５つ、より好ましくは全てに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列である。 The amino acid sequence defined in (3a) is more preferably at least the amino acids at positions 3, 5, 12, 19 and 25 of SEQ ID NO: 3 in the amino acid sequence shown in SEQ ID NO: 3. One is an amino acid sequence in which substitution with an amino acid other than serine or threonine has been introduced, and particularly preferably, in the amino acid sequence shown in SEQ ID NO: 3, the third, fifth, and eleventh positions of SEQ ID NO: 3 Serine is preferably at least two, more preferably at least three, more preferably at least four, more preferably at least five, and more preferably all of the amino acids at positions, 12, 19 and 25. Alternatively, it is an amino acid sequence in which substitution with an amino acid other than threonine has been introduced.
　（３ｂ）で規定するアミノ酸配列は、より好ましくは、配列番号３に示すアミノ酸配列と８５％以上の配列同一性を有するアミノ酸配列において、配列番号３の第３位、第５位、第１２位、第１９位及び第２５位のアミノ酸に対応するアミノ酸の少なくとも１つに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列であり、特に好ましくは、配列番号３に示すアミノ酸配列と８５％以上の配列同一性を有するアミノ酸配列において、配列番号３の第３位、第５位、第１１位、第１２位、第１９位及び第２５位のアミノ酸に対応するアミノ酸の好ましくは少なくとも２つ、より好ましくは少なくとも３つ、より好ましくは少なくとも４つ、より好ましくは少なくとも５つ、より好ましくは全てに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列である。 The amino acid sequence defined in (3b) is more preferably at the 3rd, 5th, and 12th positions of SEQ ID NO: 3 in the amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3. , An amino acid sequence in which substitution with an amino acid other than serine or threonine is introduced into at least one of the amino acids corresponding to the amino acids at positions 19 and 25, and particularly preferably the amino acid sequence shown in SEQ ID NO: 3. Of the amino acid sequence having 85% or more sequence identity, the amino acid corresponding to the amino acid at the 3rd, 5th, 11th, 12th, 19th and 25th positions of SEQ ID NO: 3 is preferable. An amino acid sequence in which substitutions with amino acids other than serine or threonine have been introduced into at least two, more preferably at least three, more preferably at least four, more preferably at least five, and more preferably all.
＜３．核酸、ベクター＞
　本発明の一以上の実施形態は、前記重鎖抗体のアミノ酸配列をコードする塩基配列を含む核酸に関する。
　本発明の更に一以上の実施形態は、前記核酸を含むベクターに関する。 <3. Nucleic acid, vector>
One or more embodiments of the present invention relate to nucleic acids comprising a base sequence encoding the amino acid sequence of the heavy chain antibody.
One or more embodiments of the present invention relate to vectors containing said nucleic acids.
　ベクターとは人為的に構築された核酸分子であって、通常は、核酸分子内に異種生物に由来する塩基配列を含む。本発明の一以上の実施形態に係るベクターは宿主細胞に導入され宿主細胞を形質転換するために用いることができる。
　本発明の一以上の実施形態に係るベクターは環状ベクター、直鎖状ベクター、人工染色体等であることができる。 A vector is an artificially constructed nucleic acid molecule, and usually contains a base sequence derived from a heterologous organism in the nucleic acid molecule. The vector according to one or more embodiments of the present invention can be introduced into a host cell and used to transform the host cell.
The vector according to one or more embodiments of the present invention can be a circular vector, a linear vector, an artificial chromosome, or the like.
　本発明の一以上の実施形態に係るベクターは、前記重鎖抗体のアミノ酸配列をコードする塩基配列を含む核酸を少なくとも含み、前記塩基配列の上流及び／又は下流に、宿主細胞における前記重鎖抗体の遺伝子発現を制御する塩基配列を更に含んでいてよい。 The vector according to one or more embodiments of the present invention contains at least a nucleic acid containing a base sequence encoding the amino acid sequence of the heavy chain antibody, and the heavy chain antibody in a host cell is upstream and / or downstream of the base sequence. It may further contain a base sequence that controls the gene expression of.
　前記重鎖抗体のアミノ酸配列をコードする塩基配列は発現カセットに挿入された形態でベクター中に収めることができる。「発現カセット」とは、前記重鎖抗体のアミノ酸配列をコードする塩基配列を含み、前記重鎖抗体をポリペプチドとして発現可能な状態にすることのできる発現システムをいう。「発現可能な状態」とは、当該発現カセットに含まれる前記塩基配列が、宿主細胞において発現可能なように、遺伝子発現に必要なエレメントの制御下に配置されている状態をいう。遺伝子発現に必要なエレメントには、プロモーター、ターミネーター等が挙げられる。 The base sequence encoding the amino acid sequence of the heavy chain antibody can be contained in the vector in the form of being inserted into the expression cassette. The "expression cassette" refers to an expression system that includes a base sequence encoding the amino acid sequence of the heavy chain antibody and can bring the heavy chain antibody into a state in which it can be expressed as a polypeptide. The "expressible state" refers to a state in which the base sequence contained in the expression cassette is arranged under the control of an element necessary for gene expression so that it can be expressed in a host cell. Elements required for gene expression include promoters, terminators and the like.
　ここで、「プロモーター」は、前記重鎖抗体のアミノ酸配列をコードする塩基配列の上流に位置する塩基配列領域であり、ＲＮＡポリメラーゼのほか、転写の促進や抑制に関わる様々な転写調節因子が該領域に結合又は作用することによって鋳型である、前記重鎖抗体のアミノ酸配列をコードする塩基配列を読み取って相補的なＲＮＡを合成（転写）する。 Here, the "promoter" is a base sequence region located upstream of the base sequence encoding the amino acid sequence of the heavy chain antibody, and in addition to RNA polymerase, various transcriptional regulators involved in promotion and suppression of transcription are mentioned. Complementary RNA is synthesized (transcribed) by reading the base sequence encoding the amino acid sequence of the heavy chain antibody, which is a template by binding or acting on the region.
　前記重鎖抗体を発現させるプロモーターとしては、選択した炭素源にて発現が起こりうるプロモーターを適切に使用すればよく、特に限定されない。 As the promoter that expresses the heavy chain antibody, a promoter that can be expressed by a selected carbon source may be appropriately used, and is not particularly limited.
　本発明の一以上の実施形態に係るベクターの前記発現カセットにおいて、ターミネーターは、前記重鎖抗体のアミノ酸配列をコードする塩基配列の下流に位置する。ターミネーターは、使用するプロモーター及び宿主細胞に応じて適宜選択することができる。 In the expression cassette of the vector according to one or more embodiments of the present invention, the terminator is located downstream of the base sequence encoding the amino acid sequence of the heavy chain antibody. The terminator can be appropriately selected depending on the promoter to be used and the host cell.
　本発明の一以上の実施形態に係るベクターでは、更に、１つ以上の制限酵素認識部位を含むクローニングサイト、Ｃｌｏｎｔｅｃｈ社のＩｎ－ＦｕｓｉｏｎクローニングシステムやＮｅｗ　Ｅｎｇｌａｎｄ　Ｂｉｏｌａｂｓ社のＧｉｂｓｏｎ　Ａｓｓｅｍｂｌｙシステム等を利用するためのオーバーラップ領域、選択マーカー遺伝子（栄養要求性相補遺伝子、薬剤耐性遺伝子等）の塩基配列等を含むことができる。本発明の一以上の実施形態に係るベクターは更に、宿主に応じて、自律複製配列（Ａｕｔｏｎｏｍｏｕｓ　ｒｅｐｌｉｃａｔｉｏｎ　ｓｅｑｕｅｎｃｅ）（ＡＲＳ）、セントロメアＤＮＡ配列、テロメアＤＮＡ配列を含むことができる。 In the vector according to one or more embodiments of the present invention, in order to further utilize a cloning site containing one or more restriction enzyme recognition sites, an In-Fusion cloning system of Clontech, a Gibson Assembly system of New England Biolabs, and the like. Can include the overlap region of the above, the base sequence of the selectable marker gene (nutrition-requiring complementary gene, drug resistance gene, etc.) and the like. The vector according to one or more embodiments of the present invention can further include an Autonomous replication sequence (ARS), a centromere DNA sequence, and a telomere DNA sequence, depending on the host.
　本発明の一以上の実施形態に係る核酸及びベクターは、前記重鎖抗体のアミノ酸配列をコードする塩基配列に加えて、前記塩基配列の５’末端及び３’末端の一方又は両方に、更なるポリペプチドのアミノ酸配列をコードする塩基配列を含んでいてもよい。更なるポリペプチドとしては、前記重鎖抗体の宿主細胞からの分泌を可能にするシグナルペプチドや、タグペプチドが挙げられる。このような更なるポリペプチドは前記重鎖抗体のＮ末端側及びＣ末端側の一方又は両方に接続されて融合ペプチドとして発現する。 The nucleic acid and vector according to one or more embodiments of the present invention further include, in addition to the base sequence encoding the amino acid sequence of the heavy chain antibody, one or both of the 5'end and 3'end of the base sequence. It may contain a base sequence encoding an amino acid sequence of a polypeptide. Further polypeptides include a signal peptide that enables the secretion of the heavy chain antibody from a host cell, and a tag peptide. Such a further polypeptide is linked to one or both of the N-terminal side and the C-terminal side of the heavy chain antibody and expressed as a fusion peptide.
　シグナルペプチドは宿主細胞に応じて適宜選択することができる。例えば宿主細胞が酵母である場合のシグナルペプチドとしては、サッカロマイセス・セレビシエに由来するＭａｔｉｎｇ　Ｆａｃｔｏｒ　α（ＭＦα）シグナルが挙げられる。また、オガタエア・アングスタの酸性ホスファターゼ（ＰＨＯ１）、コマガタエラ・パストリスの酸性ホスファターゼ（ＰＨＯ１）、サッカロマイセス・セレビシエのインベルターゼ（ＳＵＣ２）、又はサッカロマイセス・セレビシエのＰＬＢ１のシグナル配列もまた、酵母からのポリペプチドの分泌を可能にするシグナルペプチドとして利用可能である。 The signal peptide can be appropriately selected according to the host cell. For example, as a signal peptide when the host cell is yeast, a Matting Factor α (MFα) signal derived from Saccharomyces cerevisiae can be mentioned. The signal sequence of Ogataea angusta acid phosphatase (PHO1), Komagataera pastris acid phosphatase (PHO1), Saccharomyces cerevisiae invertase (SUC2), or Saccharomyces cerevisiae PLB1 is also a peptide secretion from yeast. It is available as a signal peptide that enables.
＜４．宿主細胞＞
　本発明の別の一以上の実施形態は、前記重鎖抗体のアミノ酸配列をコードする塩基配列を含む核酸を含む宿主細胞に関する。この実施形態に係る宿主細胞は、前記核酸を、前記ベクターの一部として含むことができる。
　宿主細胞の具体的な態様は既述の通りである。 <4. Host cell>
Another embodiment of the present invention relates to a host cell containing a nucleic acid containing a base sequence encoding the amino acid sequence of the heavy chain antibody. The host cell according to this embodiment can contain the nucleic acid as part of the vector.
Specific aspects of the host cell are as described above.
　前記核酸を酵母へ導入する方法、すなわち形質転換法は公知の方法を適宜用いることができ、エレクトロポレーション法、酢酸リチウム法、スフェロプラスト法等が挙げられるが特にこれらに限定されるものではない。例えば、酵母の形質転換法としては、Ｈｉｇｈ　ｅｆｆｉｃｉｅｎｃｙ　ｔｒａｎｓｆｏｒｍａｔｉｏｎ　ｂｙ　ｅｌｅｃｔｒｏｐｏｒａｔｉｏｎ　ｏｆ　Ｐｉｃｈｉａ　ｐａｓｔｏｒｉｓ　ｐｒｅｔｒｅａｔｅｄ　ｗｉｔｈ　ｌｉｔｈｉｕｍａｃｅｔａｔｅ　ａｎｄ　ｄｉｔｈｉｏｔｈｒｅｉｔｏｌ（Ｂｉｏｔｅｃｈｎｉｑｕｅｓ．２００４　Ｊａｎ；３６（１）：１５２－４．）に記載されているエレクトロポレーション法が一般的である。 As a method for introducing the nucleic acid into yeast, that is, a transformation method, a known method can be appropriately used, and examples thereof include an electroporation method, a lithium acetate method, and a spheroplast method, but the method is not particularly limited thereto. Absent. For example, as a yeast transformation method, High efficiency transformation by electroporation of Pichia pastoris preserved with lithium acetate and dithiothreitol (Biotechnii) is described in (Biotechniq). Is the target.
＜５．重鎖抗体の製造方法＞
　前記重鎖抗体の製造方法は、本発明の一以上の実施形態に係る宿主細胞を培養する培養工程が含まれる。前記培養工程で得られた前記宿主細胞の培養物から目的とする前記重鎖抗体を回収する。ここで「培養物」とは、培養液上清のほか、培養細胞又は細胞の破砕物を包含する。本発明の一以上の実施形態に係る宿主細胞において、前記重鎖抗体が、宿主細胞から細胞外への分泌に関与するシグナルペプチドとの融合タンパク質として発現する場合は、前記重鎖抗体を含む融合タンパク質は細胞外に分泌生産されるため、培養物としては特に培養液上清が好ましい。 <5. Method for producing heavy chain antibody>
The method for producing a heavy chain antibody includes a culturing step of culturing a host cell according to one or more embodiments of the present invention. The target heavy chain antibody is recovered from the culture of the host cell obtained in the culture step. Here, the “culture” includes cultured cells or crushed cells, in addition to the supernatant of the culture solution. In the host cell according to one or more embodiments of the present invention, when the heavy chain antibody is expressed as a fusion protein with a signal peptide involved in secretion from the host cell to the outside of the cell, a fusion containing the heavy chain antibody. Since the protein is secreted and produced extracellularly, the culture solution supernatant is particularly preferable as the culture.
　本発明の一以上の実施形態に係る宿主細胞の培養条件は特に限定されず、細胞に応じて適宜選択すればよい。該培養においては、細胞が資化しうる栄養源を含む培地であれば何でも使用できる。 The culture conditions of the host cells according to one or more embodiments of the present invention are not particularly limited, and may be appropriately selected depending on the cells. In the culture, any medium containing a nutrient source capable of assimilating cells can be used.
　本発明の一以上の実施形態に係る宿主細胞の培養条件は特に限定されず、細胞に応じて適宜選択すればよい。該培養においては、細胞が資化しうる栄養源を含む培地であれば何でも使用できる。該栄養源としては、グルコース、スクロース、マルトース等の乳糖、酢酸、クエン酸、プロピオン酸等の有機酸類、メタノール、エタノール、グリセロール等のアルコール類、パラフィン等の炭化水素類、大豆油、菜種油等の油類、又はこれら混合物の炭素源、硫酸アンモニウム、リン酸アンモニウム、尿素、酵母エキス、肉エキス、ペプトン、コーンスターチープリカー等の窒素源、更に、その他の無機塩、ビタミン類等の栄養源を適宜混合・配合した通常の培地を用いることができる。また、培養はバッチ培養や連続培養のいずれでも可能である。 The culture conditions of the host cells according to one or more embodiments of the present invention are not particularly limited, and may be appropriately selected depending on the cells. In the culture, any medium containing a nutrient source capable of assimilating cells can be used. Examples of the nutrient source include lactose such as glucose, sucrose and maltose, organic acids such as acetic acid, citric acid and propionic acid, alcohols such as methanol, ethanol and glycerol, hydrocarbons such as paraffin, soybean oil and rapeseed oil. Carbon sources of oils or mixtures thereof, ammonium sulfate, ammonium phosphate, urea, yeast extract, meat extract, peptone, nitrogen sources such as corn star chipliquor, and other nutrient sources such as inorganic salts and vitamins are appropriately used. A mixed / blended normal medium can be used. In addition, the culture can be either batch culture or continuous culture.
　本発明の好ましい様態として、宿主細胞としてピキア属酵母又はオガタエア属酵母を用いた場合、上記炭素源は、グルコース、グリセロール、メタノールのうち１種でもよく、又は２種以上であってもよい。また、これらの炭素源は培養初期から存在していてもよいし、培養途中に添加してもよい。 As a preferable mode of the present invention, when Pichia yeast or Ogataea yeast is used as the host cell, the carbon source may be one of glucose, glycerol, and methanol, or two or more. Further, these carbon sources may be present from the initial stage of culturing or may be added during culturing.
　本発明の一以上の実施形態に係る宿主細胞の培養は通常一般の条件により行うことができ、例えば、ｐＨ２．５～１０．０、温度範囲１０℃～４８℃の範囲で、好気的に１０時間～１０日間培養することにより行うことができる。 Culturing of host cells according to one or more embodiments of the present invention can usually be carried out under general conditions, for example, aerobically in a pH range of 2.5 to 10.0 and a temperature range of 10 ° C to 48 ° C. It can be carried out by culturing for 10 hours to 10 days.
　前記培養物からの前記重鎖抗体の回収方法については、公知の精製法を適当に組み合わせて用いることができる。例えば、まず、本発明の一以上の実施形態に係る宿主細胞と培地とを含む培養液を遠心分離、或いは、ろ過処理して、液体画分、すなわち培養液上清から宿主細胞を除去する。得られた培養液上清を、塩析（硫酸アンモニウム沈澱、リン酸ナトリウム沈澱等）、溶媒沈澱（アセトン又はエタノール等によるタンパク質分画沈澱法）、透析、ゲルろ過クロマトグラフィー、イオン交換クロマトグラフィー、疎水クロマトグラフィー、アフィニティークロマトグラフィー、逆相クロマトグラフィー、限外ろ過等の手法を単独で、又は組み合わせて用いることにより、該培養液上清から前記重鎖抗体を回収することができる。 As a method for recovering the heavy chain antibody from the culture, a known purification method can be used in an appropriate combination. For example, first, the culture solution containing the host cells and the medium according to one or more embodiments of the present invention is centrifuged or filtered to remove the host cells from the liquid fraction, that is, the culture solution supernatant. The obtained culture solution supernatant is subjected to salting (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fractionation precipitation method using acetone, ethanol, etc.), dialysis, gel filtration chromatography, ion exchange chromatography, hydrophobicity. The heavy chain antibody can be recovered from the supernatant of the culture solution by using techniques such as chromatography, affinity chromatography, reverse phase chromatography, and ultrafiltration alone or in combination.
　本発明の一実施形態に係る製造方法で製造される前記重鎖抗体は、Ｏ結合型糖鎖修飾体の割合が低く、具体的には、重鎖抗体の全量に対するＯ結合型糖鎖修飾体の割合が５質量％以下、好ましくは４質量％以下、より好ましくは３質量％以下、特に好ましくは２質量％以下、更に好ましくは１質量％以下、最も好ましくは０質量％である。すなわち、本発明の一実施形態に係る製造方法によれば、均一性が高く品質の安定した重鎖抗体を製造することができる。 The heavy chain antibody produced by the production method according to the embodiment of the present invention has a low proportion of O-linked sugar chain modified product, and specifically, the O-linked sugar chain modified product with respect to the total amount of the heavy chain antibody. Is 5% by mass or less, preferably 4% by mass or less, more preferably 3% by mass or less, particularly preferably 2% by mass or less, still more preferably 1% by mass or less, and most preferably 0% by mass. That is, according to the production method according to one embodiment of the present invention, a heavy chain antibody having high uniformity and stable quality can be produced.
＜６．遺伝子治療のための医薬＞
　本発明の別の一以上の実施形態は、前記重鎖抗体のアミノ酸配列をコードする塩基配列を含む核酸を含む、遺伝子治療のための医薬に関する。この実施形態に係る医薬は、前記核酸を、前記ベクターの一部として含むことができる。 <6. Drugs for gene therapy>
Another embodiment of the present invention relates to a medicament for gene therapy, which comprises a nucleic acid containing a base sequence encoding the amino acid sequence of the heavy chain antibody. The medicament according to this embodiment can contain the nucleic acid as a part of the vector.
　本発明の一以上の実施形態に係る医薬は、ヒト等の対象動物に投与されたときに、前記重鎖抗体が対象動物の体内で発現できるような形態で、前記核酸を含有する。前記重鎖抗体は、対象動物の体内で、Ｏ結合型糖鎖修飾体の割合が低い形態で発現することができ、具体的には、前記重鎖抗体の全量に対するＯ結合型糖鎖修飾体の割合が５質量％以下、好ましくは４質量％以下、より好ましくは３質量％以下、特に好ましくは２質量％以下、更に好ましくは１質量％以下、最も好ましくは０質量％となるように対象動物の体内で発現する。すなわち、本発明の一実施形態に係る医薬によれば、均一性が高く品質の安定した前記重鎖抗体を対象動物の体内で発現することができる。 The medicament according to one or more embodiments of the present invention contains the nucleic acid in a form such that the heavy chain antibody can be expressed in the body of the target animal when administered to a target animal such as a human. The heavy chain antibody can be expressed in a form in which the proportion of the O-linked sugar chain modifier is low in the body of the target animal. Specifically, the O-linked sugar chain modified product with respect to the total amount of the heavy chain antibody. Is 5% by mass or less, preferably 4% by mass or less, more preferably 3% by mass or less, particularly preferably 2% by mass or less, still more preferably 1% by mass or less, and most preferably 0% by mass. It is expressed in the body of animals. That is, according to the drug according to the embodiment of the present invention, the heavy chain antibody having high uniformity and stable quality can be expressed in the body of the target animal.
　本発明の一以上の実施形態に係る医薬は、前記核酸を、レトロウィルス、アデノウィルスなどの公知の遺伝子治療用ベクターに担持させた形態で含むことが好ましい。
　本発明の一以上の実施形態に係る医薬の投与方法は特に限定されないが、皮下投与、静脈投与、筋肉内投与、関節内投与いずれも可能である。 The medicament according to one or more embodiments of the present invention preferably contains the nucleic acid in a form of being carried on a known gene therapy vector such as a retrovirus or adenovirus.
The method of administering the drug according to one or more embodiments of the present invention is not particularly limited, but subcutaneous administration, intravenous administration, intramuscular administration, and intraarticular administration are all possible.
　以下、実施例により本発明を詳細に説明するが、本発明はこれらにより限定されるものではない。なお、以下の実施例において用いた組換えＤＮＡ技術に関する詳細な方法などは、次の成書に記載されている：Ｍｏｌｅｃｕｌａｒ　Ｃｌｏｎｉｎｇ　２ｎｄ　Ｅｄｉｔｉｏｎ（Ｃｏｌｄ　Ｓｐｒｉｎｇ　Ｈａｒｂｏｒ　Ｌａｂｏｒａｔｏｒｙ　Ｐｒｅｓｓ，　１９８９）、Ｃｕｒｒｅｎｔ　Ｐｒｏｔｏｃｏｌｓ　ｉｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ（Ｇｒｅｅｎ　Ｐｕｂｌｉｓｈｉｎｇ　Ａｓｓｏｃｉａｔｅｓ　ａｎｄ　Ｗｉｌｌｅｙ－Ｉｎｔｅｒｓｃｉｅｎｃｅ）。 Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited thereto. Detailed methods related to the recombinant DNA technology used in the following examples are described in the following books: Molecular Cloning 2nd Edition (Cold Spring Harbor Biology Press, 1989), Current Protocols in Biology (Molecular Biology in Molecular Biology). Green Publishing Associates and Willy-Interscience).
　また、以下の実施例において、酵母の形質転換に用いるプラスミドは、構築したベクターを大腸菌Ｅ．Ｃｏｌｉ　ＤＨ５αコンピテントセル（タカラバイオ社製）に導入し、得られた形質転換体を培養して増幅することによって調製した。プラスミド保持株からのプラスミドの調製は、ＱＩＡｐｒｅｐ　ｓｐｉｎ　ｍｉｎｉｐｒｅｐ　ｋｉｔ（ＱＩＡＧＥＮ社製）を用いて行った。 Further, in the following examples, the plasmid used for yeast transformation is the constructed vector of Escherichia coli E. coli. It was prepared by introducing it into E. coli DH5α competent cell (manufactured by Takara Bio Inc.) and culturing and amplifying the obtained transformant. The plasmid was prepared from the plasmid-carrying strain using a QIAprep spin miniprep kit (manufactured by QIAGEN).
　ベクターの構築において利用した、ＡＯＸ１プロモーター（配列番号５）、ＡＯＸ１ターミネーター（配列番号６）、ＨＩＳ４遺伝子（配列番号７）はコマガタエラ・パストリスＡＴＣＣ７６２７３株の染色体ＤＮＡ（塩基配列はＥＭＢＬ（Ｔｈｅ　Ｅｕｒｏｐｅａｎ　Ｍｏｌｅｃｕｌａｒ　Ｂｉｏｌｏｇｙ　Ｌａｂｏｒａｔｏｒｙ）ＡＣＣＳＥＳＳＩＯＮ　Ｎｏ．ＦＲ８３９６２８～ＦＲ８３９６３１に記載）混合物をテンプレートにしてＰＣＲで調製した。 The AOX1 promoter (SEQ ID NO: 5), AOX1 terminator (SEQ ID NO: 6), and HIS4 gene (SEQ ID NO: 7) used in the construction of the vector are the chromosomal DNA of the Komagataera Pastris ATCC76273 strain (the base sequence is EMBL (The European Molecular Biology Laboratory). ) ACCSESSION No. FR839628 to FR839631) Prepared by PCR using the mixture as a template.
　野生型ａｎｔｉ－Ｆｃ　ＶＨＨ遺伝子はＥＰ２１７０９６０Ｂ１の配列番号：１６３に、野生型ａｎｔｉ－ＨＳＡ　ＶＨＨ遺伝子はＥＰ２０６９４０２Ａ２の配列番号：６２に、それぞれ記載されている。これらの公知情報に基づいて、上流にＭａｔｉｎｇ　Ｆａｃｔｏｒαシグナル配列（ＭＦ配列）（配列番号８）が付加された、Ｈｉｓタグ配列（ＧＧＧＧＳＨＨＨＨＨＨ）付加ａｎｔｉ－Ｆｃ　ＶＨＨ遺伝子（配列番号５３）又はＨｉｓタグ配列付加ａｎｔｉ－ＨＳＡ　ＶＨＨ遺伝子（配列番号５５）の合成ＤＮＡを調製し、ベクターの構築において利用した。 The wild-type anti-Fc VHH gene is described in EP2170960B1 SEQ ID NO: 163, and the wild-type anti-HSA VHH gene is described in EP2069402A2 SEQ ID NO: 62, respectively. Based on these known information, a His tag sequence (GGGGSHHHHHH) addition, an anti-Fc VHH gene (SEQ ID NO: 53) or a His tag sequence addition, to which a Matting Factor α signal sequence (MF sequence) (SEQ ID NO: 8) was added upstream. Synthetic DNA of the anti-HSA VHH gene (SEQ ID NO: 55) was prepared and used in the construction of the vector.
　ＰＣＲにはＰｒｉｍｅ　ＳＴＡＲ　Ｍａｘ　ＤＮＡ　Ｐｏｌｙｍｅｒａｓｅ（タカラバイオ社製）等を用い、反応条件は添付のマニュアルに記載の方法で行った。前記染色体ＤＮＡ混合物の調製は、コマガタエラ・パストリスＡＴＣＣ７６２７３株からＧｅｎとるくんＴＭ（タカラバイオ社製）等を用いて、これに記載の条件で実施した。 For PCR, Prime STAR Max DNA Polymerase (manufactured by Takara Bio Inc.) and the like were used, and the reaction conditions were carried out by the method described in the attached manual. The preparation of the chromosomal DNA mixture was carried out from the Komagataera pastris ATCC76273 strain using Gen Toru-kun TM (manufactured by Takara Bio Inc.) and the like under the conditions described therein.
＜比較例１：野生型重鎖抗体発現ベクター構築＞
　ＨｉｎｄＩＩＩ－ＢａｍＨＩ－ＢｇｌＩＩ－ＸｂａＩ－ＥｃｏＲＩのマルチクローニングサイトをもつ遺伝子断片（配列番号９）を全合成し、これをｐＵＣ１９（タカラバイオ社製）のＨｉｎｄＩＩＩ－ＥｃｏＲＩサイト間に挿入して、ｐＵＣ－１を構築した。 <Comparative Example 1: Construction of wild-type heavy chain antibody expression vector>
A gene fragment (SEQ ID NO: 9) having a multi-cloning site of HindIII-BamHI-BglII-XbaI-EcoRI was totally synthesized and inserted between the HindIII-EcoRI sites of pUC19 (manufactured by Takara Bio) to pUC-1. Was built.
　また、ＡＯＸ１プロモーターの両側にＢａｍＨＩ認識配列を付加した核酸断片を、前記染色体ＤＮＡ混合物を鋳型としたとしプライマー１（配列番号１０）及び２（配列番号１１）を用いたＰＣＲにより調製し、ＢａｍＨＩ処理後にｐＵＣ－１のＢａｍＨＩサイトに挿入して、ｐＵＣＰａｏｘを構築した。 In addition, a nucleic acid fragment having BamHI recognition sequences added to both sides of the AOX1 promoter was prepared by PCR using primers 1 (SEQ ID NO: 10) and 2 (SEQ ID NO: 11) using the chromosomal DNA mixture as a template, and treated with BamHI. It was later inserted into the BamHI site of pUC-1 to construct pUCPaox.
　次に、ＡＯＸ１ターミネーターの両側にＸｂａＩ認識配列を付加した核酸断片を、前記染色体ＤＮＡ混合物を鋳型としプライマー３（配列番号１２）及び４（配列番号１３）を用いたＰＣＲにより調製し、ＸｂａＩ処理後にｐＵＣＰａｏｘのＸｂａＩサイトに挿入して、ｐＵＣ－ＰａｏｘＴａｏｘを構築した。 Next, a nucleic acid fragment having XbaI recognition sequences added to both sides of the AOX1 terminator was prepared by PCR using the chromosomal DNA mixture as a template and primers 3 (SEQ ID NO: 12) and 4 (SEQ ID NO: 13), and after XbaI treatment. A pUC-PaoxTaox was constructed by inserting it into the XbaI site of pUCPaox.
　次に、ＨＩＳ４遺伝子の両側にＥｃｏＲＩ認識配列を付加した核酸断片を、前記染色体ＤＮＡ混合物を鋳型としプライマー５（配列番号１４）及び６（配列番号１５）を用いたＰＣＲにより調製し、ＥｃｏＲＩ処理後にｐＵＣ－ＰａｏｘＴａｏｘのＥｃｏＲＩサイトに挿入して、ｐＵＣ－ＰａｏｘＴａｏｘＨＩＳ４を構築した。 Next, a nucleic acid fragment having EcoRI recognition sequences added to both sides of the HIS4 gene was prepared by PCR using the chromosomal DNA mixture as a template and primers 5 (SEQ ID NO: 14) and 6 (SEQ ID NO: 15), and after EcoRI treatment. The pUC-PaoxTaoxHIS4 was constructed by inserting it into the EcoRI site of pUC-PaoxTaox.
　次に、上流にＭＦ配列が付加され下流にＨｉｓタグ配列が付加されたａｎｔｉ－Ｆｃ　ＶＨＨ遺伝子（重鎖抗体遺伝子）の両側にＢｇｌＩＩ認識配列を付加した核酸断片を、上流にＭＦ配列（配列番号８）が付加された、Ｈｉｓタグ配列付加ａｎｔｉ－Ｆｃ　ＶＨＨ遺伝子（配列番号５３）の前記合成ＤＮＡを鋳型とし、プライマー７（配列番号１６）及び８（配列番号１７）を用いたＰＣＲにより調製し、ＢｇｌＩＩ処理後にｐＵＣ－ＰａｏｘＴａｏｘＨＩＳ４のＢｇｌＩＩサイトに挿入して、ｐＵＣ－Ｐａｏｘａｎｔｉ－ＦｃＶＨＨＴａｏｘＨＩＳ４を構築した。 Next, a nucleic acid fragment in which the BglII recognition sequence was added to both sides of the anti-Fc VHH gene (heavy chain antibody gene) to which the MF sequence was added upstream and the His tag sequence was added downstream, and the MF sequence (SEQ ID NO:) was added upstream. Prepared by PCR using primers 7 (SEQ ID NO: 16) and 8 (SEQ ID NO: 17) using the synthetic DNA of the His-tagged sequence-added anti-Fc VHH gene (SEQ ID NO: 53) to which 8) was added as a template. , BglII treatment was then inserted into the BglII site of pUC-PaoxTaoxHIS4 to construct pUC-Paoxanti-FcVHHTaxHIS4.
　同様に、上流にＭＦ配列が付加され下流にＨｉｓタグ配列が付加されたａｎｔｉ－ＨＳＡ　ＶＨＨ遺伝子（重鎖抗体遺伝子）の両側にＢｇｌＩＩ認識配列を付加した核酸断片を、上流にＭＦ配列（配列番号８）が付加された、Ｈｉｓタグ配列付加ａｎｔｉ－ＨＳＡ　ＶＨＨ遺伝子（配列番号５５）の前記合成ＤＮＡを鋳型とし、プライマー７（配列番号１６）及び９（配列番号１８）を用いたＰＣＲにより調製し、ＢｇｌＩＩ処理後にｐＵＣ－ＰａｏｘＴａｏｘＨＩＳ４のＢｇｌＩＩサイトに挿入して、ｐＵＣ－Ｐａｏｘａｎｔｉ－ＨＳＡＶＨＨＴａｏｘＨＩＳ４を構築した。 Similarly, a nucleic acid fragment in which the BglII recognition sequence is added to both sides of the anti-HSA VHH gene (heavy chain antibody gene) having the MF sequence added upstream and the His tag sequence added downstream, and the MF sequence (SEQ ID NO:) upstream. Prepared by PCR using primers 7 (SEQ ID NO: 16) and 9 (SEQ ID NO: 18) using the synthetic DNA of the His-tag sequence-added anti-HSA VHH gene (SEQ ID NO: 55) to which 8) was added as a template. , BglII treatment was followed by insertion into the BglII site of pUC-PaoxTaoxHIS4 to construct pUC-Paoxanti-HSAVHHTaxHIS4.
　これらｐＵＣ－Ｐａｏｘａｎｔｉ－ＦｃＶＨＨＴａｏｘＨＩＳ４とｐＵＣ－Ｐａｏｘａｎｔｉ－ＨＳＡＶＨＨＴａｏｘＨＩＳ４は、重鎖抗体がＡＯＸ１プロモーター制御下で分泌発現するように設計されている。 These pUC-Paoxanti-FcVHHTaxHIS4 and pUC-Paoxanti-HSAVHHTaxHIS4 are designed so that heavy chain antibodies are secreted and expressed under the control of the AOX1 promoter.
＜比較例２：形質転換酵母の取得＞
　比較例１で構築した各重鎖抗体発現ベクターを用いて、以下のようにコマガタエラ・パストリスを形質転換した。 <Comparative Example 2: Acquisition of Transformed Yeast>
Using each heavy chain antibody expression vector constructed in Comparative Example 1, Komagataera pastris was transformed as follows.
　コマガタエラ・パストリスＡＴＣＣ７６２７３株由来ヒスチジン要求性株を３ｍＬのＹＰＤ培地（１％　ｙｅａｓｔ ｅｘｔｒａｃｔ　ｂａｃｔｏ（Ｄｉｆｃｏ社製）、２％　ｐｏｌｙｐｅｐｔｏｎｅ（日本製薬社製）、２％　ｇｌｕｃｏｓｅ）に接種し、３０℃で一晩振盪培養して前培養液を得た。得られた前培養液５００μＬを５０ｍＬのＹＰＤ培地に接種し、ＯＤ６００が１～１．５になるまで振盪培養後、遠心分離（３０００×ｇ、１０分、２０℃）して細胞を回収し、２５０μＬの１Ｍ　１，４－ジチオスレイトール（ＤＴＴ）（終濃度２５ｍＭ）を含む１０ｍＬの５０ｍＭリン酸カリウムバッファー、ｐＨ７．５に再懸濁した。 Inoculate a histidine-requiring strain derived from Komagataera pastris ATCC76273 strain into 3 mL of YPD medium (1% yeast extract bacto (manufactured by Difco), 2% polypeptone (manufactured by Nihon Pharmaceutical Co., Ltd.), 2% glucose) overnight at 30 ° C. Preculture was obtained by shaking culture. 500 μL of the obtained preculture solution was inoculated into 50 mL of YPD medium, shake-cultured until the OD600 became 1 to 1.5, and then centrifuged (3000 × g, 10 minutes, 20 ° C.) to collect cells. Resuspended in 10 mL of 50 mM potassium phosphate buffer, pH 7.5, containing 250 μL of 1M 1,4-dithiothreitol (DTT) (final concentration 25 mM).
　この懸濁液を３０℃で１５分インキュベート後、遠心分離（３０００×ｇ、１０分、２０℃）して細胞を回収し、予め予冷した５０ｍＬのＳＴＭバッファー（２７０ｍＭ　スクロース、１０ｍＭ　Ｔｒｉｓ－ＨＣｌ、１ｍＭ塩化マグネシウム、ｐＨ７．５）で洗浄した。洗浄液を遠心分離（３０００×ｇ、１０分、４℃）して細胞を回収し、２５ｍＬのＳＴＭバッファーで再度洗浄したのち、遠心分離（３０００×ｇ、１０分、４℃）して細胞を回収した。最終的に、２５０μＬの氷冷ＳＴＭバッファーに細胞を懸濁し、これをコンピテントセル懸濁液とした。 After incubating this suspension at 30 ° C. for 15 minutes, the cells were collected by centrifugation (3000 × g, 10 minutes, 20 ° C.), and pre-cooled 50 mL STM buffer (270 mM sucrose, 10 mM Tris-HCl, 1 mM). It was washed with magnesium chloride, pH 7.5). The washing solution is centrifuged (3000 × g, 10 minutes, 4 ° C.) to collect the cells, washed again with 25 mL of STM buffer, and then centrifuged (3000 × g, 10 minutes, 4 ° C.) to collect the cells. did. Finally, the cells were suspended in 250 μL ice-cold STM buffer, which was used as a competent cell suspension.
　比較例１で構築した重鎖抗体発現ベクターｐＵＣ－Ｐａｏｘａｎｔｉ－ＦｃＶＨＨＴａｏｘＨＩＳ４又はｐＵＣ－Ｐａｏｘａｎｔｉ－ＨＳＡＶＨＨＴａｏｘＨＩＳ４を用いて大腸菌を形質転換し、得られた形質転換体を２ｍＬのアンピシリン含有ＬＢ培地（１％　Ｔｒｙｐｔｏｎｅ（Ｄｉｆｃｏ社製）、０．５％　Ｙｅａｓｔ　ｅｘｔｒａｃｔ（Ｄｉｆｃｏ社製）、１％　塩化ナトリウム（Ｄｉｆｃｏ社製））で培養し、得られた菌体からＱＩＡｐｒｅｐ　ｓｐｉｎ　ｍｉｎｉｐｒｅｐ　ｋｉｔ（ＱＩＡＧＥＮ社製）を用いて、ｐＵＣ－Ｐａｏｘａｎｔｉ－ＦｃＶＨＨＴａｏｘＨＩＳ４又はｐＵＣ－Ｐａｏｘａｎｔｉ－ＨＳＡＶＨＨＴａｏｘＨＩＳ４を取得した。本プラスミドをＳａｌＩ処理し、ＨＩＳ４遺伝子内のＳａｌＩ認識配列で切断された直鎖状ベクターを調製した。 Escherichia coli was transformed with the heavy chain antibody expression vector pUC-Paoxanti-FcVHHTaxHIS4 or pUC-Paoxanti-HSAVHHTaoxHIS4 constructed in Comparative Example 1, and the obtained transformant was subjected to 2 mL of ampicillin-containing LB medium (1% Tryptone (1% Tryptone)). Cultivated in 0.5% Yeast extract (manufactured by Difco), 1% sodium chloride (manufactured by Difco)), and pUC using the QIAprep spin miniprep kit (manufactured by QIAGEN) from the obtained cells. -Paoxanti-FcVHHTaxHIS4 or pUC-Paoxanti-HSAVHHTaxHIS4 was obtained. This plasmid was treated with SalI to prepare a linear vector cleaved with the SalI recognition sequence in the HIS4 gene.
　上述のコンピテントセル懸濁液６０μＬと直鎖状のｐＵＣ－Ｐａｏｘａｎｔｉ－ＦｃＶＨＨＴａｏｘＨＩＳ４又はｐＵＣ－Ｐａｏｘａｎｔｉ－ＨＳＡＶＨＨＴａｏｘＨＩＳ４溶液１μＬを混合し、エレクトロポレーション用キュベット（ディスポキュベット電極、電極間隔２ｍｍ（ビーエム機器社製））に移し入れ、７．５ｋＶ/ｃｍ、２５μＦ、２００Ωに供した後、細胞を１ｍＬのＹＰＤ培地で懸濁し、３０℃で１時間静置した。１時間静置後、遠心分離（３０００×ｇ、５分、２０℃）して細胞を回収し、１ｍＬのＹＮＢ培地（０．６７％　ｙｅａｓｔ ｎｉｔｒｏｇｅｎ ｂａｓｅ Ｗｉｔｈｏｕｔ Ａｍｉｎｏ ａｃｉｄ（Ｄｉｆｃｏ社製））に懸濁後、再度遠心分離（３０００×ｇ、５分、２０℃）して細胞を回収した。細胞を適当量のＹＮＢ培地で再懸濁後、ＹＮＢ選択寒天プレート（０．６７％　ｙｅａｓｔ ｎｉｔｒｏｇｅｎ ｂａｓｅ Ｗｉｔｈｏｕｔ Ａｍｉｎｏ　ａｃｉｄ（Ｄｉｆｃｏ社製）、２％アガロース、２％グルコース）に塗布し、３０℃、３日間の静置培養で生育する株を選択し、野生型ａｎｔｉ－Ｆｃ　ＶＨＨ発現酵母又は野生型ａｎｔｉ－ＨＳＡ　ＶＨＨ発現酵母を取得した。 60 μL of the above-mentioned competent cell suspension is mixed with 1 μL of a linear pUC-Paoxanti-FcVHHTaxHIS4 or pUC-Paoxanti-HSAVHHTaoxHIS4 solution, and a cuvette for electroporation (dispocubette electrode, electrode spacing 2 mm (manufactured by BM Instruments)). ), And the cells were subjected to 7.5 kV / cm, 25 μF, 200 Ω, and then the cells were suspended in 1 mL of YPD medium and allowed to stand at 30 ° C. for 1 hour. After standing for 1 hour, the cells are collected by centrifugation (3000 × g, 5 minutes, 20 ° C.) and suspended in 1 mL of YNB medium (0.67% yeast nitrogen base Without Amino acid (manufactured by Difco)). Then, the cells were collected by centrifugation (3000 × g, 5 minutes, 20 ° C.) again. After resuspending the cells in an appropriate amount of YNB medium, the cells are applied to a YNB-selected agar plate (0.67% yeast nitrogen base Without Amino acid (manufactured by Difco), 2% agarose, 2% glucose) at 30 ° C., 3 Strains that grow in static culture for one day were selected to obtain wild-type anti-Fc VHH-expressing yeast or wild-type anti-HSA VHH-expressing yeast.
＜比較例３：形質転換酵母の培養＞
　比較例２で得られた各野生型重鎖抗体発現酵母を３ｍＬのＢＭＧＭＹ培地（１％　ｙｅａｓｔ　ｅｘｔｒａｃｔ　ｂａｃｔｏ（Ｄｉｆｃｏ社製）、２％　ｐｏｌｙｐｅｐｔｏｎｅ（日本製薬社製）、０．３４％ｙｅａｓｔ　ｎｉｔｒｏｇｅｎ　ｂａｓｅ　ｗｉｔｈｏｕｔ　Ａｍｉｎｏ　Ａｃｉｄ　ａｎｄ　Ａｍｍｏｎｉｕｍ　ｓｕｌｆａｔｅ、１％　硫酸アンモニウム、０．４ｍｇ／Ｌ　ビオチン、１００ｍＭ　リン酸カリウム（ｐＨ７．０）、１％　ｇｌｙｃｅｒｏｌ、１％　メタノール）に接種し、これを３０℃、７２時間振盪培養後、遠心分離（１２０００ｒｐｍ、５分、４℃）により培養液上清を回収した。 <Comparative Example 3: Culture of Transformed Yeast>
Each wild-type heavy chain antibody-expressing yeast obtained in Comparative Example 2 was mixed with 3 mL of BMGMY medium (1% yeast extract bacto (manufactured by Difco), 2% polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 0.34% yeast nitrogen base with out. Amino Acid and Aminoium yeast, 1% ammonium sulfate, 0.4 mg / L biotin, 100 mM potassium phosphate (pH 7.0), 1% glycerol, 1% methanol) was inoculated, and this was shaken and cultured at 30 ° C. for 72 hours. The culture supernatant was recovered by centrifugation (12000 rpm, 5 minutes, 4 ° C.).
＜比較例４：重鎖抗体の精製＞
　比較例３で取得した培養液上清を用いて、陽イオン交換クロマトグラフィーにて重鎖抗体を精製した。使用した溶液・資材・方法を以下に記す。
負荷液：培養液上清を１０倍希釈し、希釈液のｐＨを４．０に調整し負荷液とした。
緩衝液Ａ：２０ｍＭ酢酸ナトリウム　ｐＨ４．０
緩衝液Ｂ：２０ｍＭ酢酸ナトリウム　０．５Ｍ塩化ナトリウム　ｐＨ４．０
１）陽イオン交換樹脂（Ｂｉｏ－Ｒａｄ社製）を充填したカラムを、５カラム体積の緩衝液Ａで平衡化する。
２）平衡化した前記カラムに負荷液を添加する。
３）続いて前記カラムを５カラム体積の緩衝液Ａで洗浄する。
４）洗浄後の前記カラムに吸着した成分を５カラム体積の緩衝液Ｂで溶出する。
　溶出液を回収し、３ｋＤａカットの限外ろ過膜（メルク社製）を用いて、２５ｍＭ　酢酸アンモニウムに緩衝液を交換した。 <Comparative Example 4: Purification of heavy chain antibody>
The heavy chain antibody was purified by cation exchange chromatography using the culture solution supernatant obtained in Comparative Example 3. The solutions, materials and methods used are described below.
Load solution: The culture solution supernatant was diluted 10-fold, and the pH of the diluted solution was adjusted to 4.0 to prepare a load solution.
Buffer A: 20 mM sodium acetate pH 4.0
Buffer B: 20 mM sodium acetate 0.5 M sodium chloride pH 4.0
1) A column packed with a cation exchange resin (manufactured by Bio-Rad) is equilibrated with a buffer solution A having a volume of 5 columns.
2) Add the loading solution to the equilibrated column.
3) Subsequently, the column is washed with a buffer solution A having a volume of 5 columns.
4) The components adsorbed on the column after washing are eluted with a buffer solution B having a volume of 5 columns.
The eluate was collected and the buffer was replaced with 25 mM ammonium acetate using a 3 kDa cut ultrafiltration membrane (manufactured by Merck & Co., Inc.).
＜比較例５：重鎖抗体の糖鎖解析＞
　比較例４で精製したサンプルをＬＣ／ＭＳに供し、糖鎖解析を実施した。使用機器・分析条件を以下に記す。 <Comparative Example 5: Sugar chain analysis of heavy chain antibody>
The sample purified in Comparative Example 4 was subjected to LC / MS, and sugar chain analysis was performed. The equipment used and analysis conditions are described below.
　分析カラム（Ｗａｔｅｒｓ　Ａｃｑｕｉｔｙ　ＵＰＬＣ　ＢＥＨ３００　Ｃ４　Ｃｏｌｕｍｎ　２．１×５０ｍｍ）を装着した液体クロマトグラフ（Ｓｈｉｍａｄｚｕ　ＵＦＬＣ　Ｎｅｘｅｒａ　Ｘ２、島津製作所製）を質量分析計（ＴｒｉｐｌｅＴＯＦ６６００、ＳＣＩＥＸ社製）に接続し、サンプル５μＬをカラムに添加した。重鎖抗体の分離には、Ａ溶媒に０．１％ギ酸水溶液、Ｂ溶媒に０．１％アセトニトリルを用いて、０分から０．５分でＡ溶媒を前記カラムに通液し、０．５分から２分でＢ溶媒０％からＢ溶媒１００％のリニアグラジエントとなるようＡ溶媒及びＢ溶媒を前記カラムに通液した。分離したペプチドは、四重極飛行時間型質量分析計（ＱＴＯＦ－ＭＳ、ＳＣＩＥＸ社製）により、ポジティブイオンモード（印加電圧　５，５００Ｖ）で測定した。 A liquid chromatograph (Shimadzu UFLC Nexus X2, manufactured by Shimadzu Corporation) equipped with an analytical column (Waters Accuracy UPLC BEH300 C4 Volume 2.1 x 50 mm) was connected to a mass spectrometer (TripleTOF6600, manufactured by SCIEX), and a sample was connected to the column. Was added to. For the separation of the heavy chain antibody, 0.1% formic acid aqueous solution was used as the A solvent and 0.1% acetonitrile was used as the B solvent, and the A solvent was passed through the column in 0 to 0.5 minutes, and 0.5. Solvent A and solvent B were passed through the column so as to have a linear gradient of solvent B from 0% to 100% in 2 minutes. The separated peptides were measured in a positive ion mode (applied voltage 5,500 V) by a quadrupole time-of-flight mass spectrometer (QTOF-MS, manufactured by SCIEX).
　重鎖抗体のアミノ酸配列に基づく理論質量を有する分子のピークと、前記理論質量より１６２Ｄａ質量の整数倍が増加した質量を有する複数のアイソフォームのピークが観察された。理論質量と一致するピークをＭａｎ０として、１６２Ｄａずつ増加したピークをそれぞれＭａｎ１、Ｍａｎ２・・・として、各ピークの面積比を算出した。 A peak of a molecule having a theoretical mass based on the amino acid sequence of a heavy chain antibody and a peak of a plurality of isoforms having a mass increased by an integral multiple of 162 Da mass from the theoretical mass were observed. The area ratio of each peak was calculated, where the peaks that coincided with the theoretical mass were Man0, and the peaks that increased by 162 Da were Man1, Man2, and so on, respectively.
　比較例４で精製した野生型ａｎｔｉ－Ｆｃ　ＶＨＨのサンプルの糖鎖解析の結果のピーク面積比を下記表に示す。野生型ａｎｔｉ－ＨＳＡ　ＶＨＨの糖鎖解析の結果は実施例６に記載した。 The table below shows the peak area ratio of the results of sugar chain analysis of the wild-type anti-Fc VHH sample purified in Comparative Example 4. The results of sugar chain analysis of wild-type anti-HSA VHH are described in Example 6.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
＜実施例１：変異重鎖抗体発現ベクター構築＞
　上流にＭＦ配列が付加され下流にＨｉｓタグ配列が付加された野生型ａｎｔｉ－ＨＳＡ　ＶＨＨ遺伝子の合成ＤＮＡを鋳型として、各種変異体遺伝子をＰＣＲで調製した。 <Example 1: Construction of mutant heavy chain antibody expression vector>
Various mutant genes were prepared by PCR using the synthetic DNA of the wild-type anti-HSA VHH gene with the MF sequence added upstream and the His tag sequence added downstream as a template.
　まず、上流にＭＦ配列（配列番号８）が付加された、Ｈｉｓタグ配列付加野生型ａｎｔｉ－ＨＳＡ　ＶＨＨ遺伝子（配列番号５５）の前記合成ＤＮＡを鋳型とし、以下の表２に示した１ｓｔＰＣＲ－１のプライマーの組合せを用いたＰＣＲ（１ｓｔＰＣＲ－１）と、１ｓｔＰＣＲ－２のプライマーの組合せを用いたＰＣＲ（１ｓｔＰＣＲ－２）を行った。続いて、１ｓｔＰＣＲ－１で得られた増幅断片と１ｓｔＰＣＲ－２で得られた増幅断片とを混合したものを鋳型としプライマー７及びプライマー９でＰＣＲを行い、上流にＭＦ配列が付加され下流にＨｉｓタグ配列が付加された各種変異重鎖抗体遺伝子の両端にＢｇｌＩＩ認識配列が付加されたＤＮＡ断片を調製した。変異箇所はａｎｔｉ－ＨＳＡ　ＶＨＨの配列番号５２に示すアミノ酸配列での位置を示す。 First, the synthetic DNA of the His-tag sequence-added wild-type anti-HSA VHH gene (SEQ ID NO: 55) to which the MF sequence (SEQ ID NO: 8) was added upstream was used as a template, and the 1st PCR-1 shown in Table 2 below was used. PCR (1stPCR-1) using the combination of primers of 1stPCR-2 and PCR (1stPCR-2) using the combination of primers of 1stPCR-2 were performed. Subsequently, PCR was performed with primers 7 and 9 using a mixture of the amplified fragment obtained by 1stPCR-1 and the amplified fragment obtained by 1stPCR-2 as a template, and the MF sequence was added upstream and His downstream. DNA fragments in which BglII recognition sequences were added to both ends of various mutant heavy chain antibody genes to which tag sequences were added were prepared. The mutation site indicates the position in the amino acid sequence shown in SEQ ID NO: 52 of anti-HSA VHH.
　上記で調製した変異重鎖抗体遺伝子を含むＤＮＡ断片をＢｇｌＩＩ処理し、比較例１で調製したｐＵＣ－ＰａｏｘＴａｏｘＨＩＳ４のＢｇｌＩＩサイトに挿入して、上流にＭＦ配列が付加され下流にＨｉｓタグ配列が付加された各種変異重鎖抗体遺伝子発現ベクターを構築した。 The DNA fragment containing the mutant heavy chain antibody gene prepared above is treated with BglII and inserted into the BglII site of pUC-PaoxTaoxHIS4 prepared in Comparative Example 1, and an MF sequence is added upstream and a His tag sequence is added downstream. Various mutant heavy chain antibody gene expression vectors were constructed.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
＜実施例２：形質転換酵母の取得＞
　実施例１で構築したＭＦ配列及びＨｉｓタグ配列を付加した変異重鎖抗体発現ベクターを用いて、比較例２に示した方法と同様に、コマガタエラ・パストリスを形質転換して、変異重鎖抗体発現酵母を取得した。 <Example 2: Acquisition of transformed yeast>
Using the mutant heavy chain antibody expression vector to which the MF sequence and His tag sequence constructed in Example 1 were added, Komagataera pastris was transformed in the same manner as in Comparative Example 2 to express the mutant heavy chain antibody. Obtained yeast.
　実施例１で構築した変異重鎖抗体発現ベクターを用いて大腸菌を形質転換し、得られた形質転換体を２ｍＬのアンピシリン含有ＬＢ培地で培養し、得られた菌体からプラスミドを取得した。本プラスミドをＳａｌＩ処理し、直鎖状にした。 Escherichia coli was transformed with the mutant heavy chain antibody expression vector constructed in Example 1, and the obtained transformant was cultured in 2 mL of ampicillin-containing LB medium, and a plasmid was obtained from the obtained bacterial cells. This plasmid was treated with SalI to make it linear.
　コンピテントセルの調製、形質転換及び形質転換体の選択は比較例２に記載の方法で実施した。 Preparation of competent cells, transformation and selection of transformants were carried out by the method described in Comparative Example 2.
＜実施例３：形質転換酵母の培養＞
　実施例２で得られた変異重鎖抗体発現酵母を、比較例３に記載の方法で培養し、培養液上清を回収した。 <Example 3: Culture of transformed yeast>
The mutant heavy chain antibody-expressing yeast obtained in Example 2 was cultured by the method described in Comparative Example 3, and the culture broth supernatant was collected.
＜実施例４：重鎖抗体の精製＞
　実施例３で取得した培養液上清を用いて、陽イオン交換クロマトグラフィーにて重鎖抗体を精製した。使用した溶液・資材・方法を以下に記す。
負荷液：培養液上清を１０倍希釈し、希釈液のｐＨを４．０に調整し負荷液とした。
緩衝液Ａ：２０ｍＭ酢酸ナトリウム　ｐＨ４．０
緩衝液Ｂ：２０ｍＭ酢酸ナトリウム　０．５Ｍ塩化ナトリウム　ｐＨ４．０
１）陽イオン交換樹脂（Ｂｉｏ－Ｒａｄ社製）を充填したカラムを、５カラム体積の緩衝液Ａで平衡化する。
２）平衡化した前記カラムに負荷液を添加する。
３）続いて前記カラムを５カラム体積の緩衝液Ａで洗浄する。
４）洗浄後の前記カラムに吸着した成分を５カラム体積の緩衝液Ｂで溶出する。
　溶出液を回収し、３ｋＤａカットの限外ろ過膜（メルク社製）を用いて、２５ｍＭ　酢酸アンモニウムに緩衝液を交換した。 <Example 4: Purification of heavy chain antibody>
The heavy chain antibody was purified by cation exchange chromatography using the culture solution supernatant obtained in Example 3. The solutions, materials and methods used are described below.
Load solution: The culture solution supernatant was diluted 10-fold, and the pH of the diluted solution was adjusted to 4.0 to prepare a load solution.
Buffer A: 20 mM sodium acetate pH 4.0
Buffer B: 20 mM sodium acetate 0.5 M sodium chloride pH 4.0
1) A column packed with a cation exchange resin (manufactured by Bio-Rad) is equilibrated with a buffer solution A having a volume of 5 columns.
2) Add the loading solution to the equilibrated column.
3) Subsequently, the column is washed with a buffer solution A having a volume of 5 columns.
4) The components adsorbed on the column after washing are eluted with a buffer solution B having a volume of 5 columns.
The eluate was collected and the buffer was replaced with 25 mM ammonium acetate using a 3 kDa cut ultrafiltration membrane (manufactured by Merck & Co., Inc.).
＜実施例５：重鎖抗体の糖鎖結合位置決定＞
　比較例４で取得したａｎｔｉ－Ｆｃ　ＶＨＨ試料を７Ｍグアニジン塩酸溶液を用いて１００μＬに希釈し、タンパク質を変性させた。次に、ジチオスレイトールを添加し、３７℃で１時間静置することで還元した。さらに、ヨードアセトアミドを添加し、暗所で３０分間静置することでアルキル化した。限外ろ過により、溶媒を５０ｍＭ　Ｔｒｉｓ－Ｃｌ（ｐＨ７．５）に置換した。トリプシンを添加し３７℃で一晩静置した。結果として得られたペプチドを、逆相高速液体クロマトグラフィ（ＲＰ－ＨＰＬＣ）により分離した。分離したペプチドは、四重極飛行時間型質量分析計（ＱＴＯＦ－ＭＳ）により分析した。得られたピークから、ＢｉｏＰｈａｒｍａＶｉｅｗ（ＳＣＩＥＸ社製）により糖ペプチドのピークを抽出した（表３）。表３より、Ｈｉｓタグ付加ａｎｔｉ－Ｆｃ　ＶＨＨ（配列番号５４）のＦＲ２（配列番号５４の第３６～４９位）及びＦＲ３（配列番号５４の第６７位～９８位）にＯ－結合型糖鎖が結合している事が明らかとなった。 <Example 5: Determination of sugar chain binding position of heavy chain antibody>
The anti-Fc VHH sample obtained in Comparative Example 4 was diluted to 100 μL with a 7M guanidine hydrochloric acid solution to denature the protein. Next, dithiothreitol was added, and the mixture was allowed to stand at 37 ° C. for 1 hour for reduction. Further, iodoacetamide was added, and the mixture was allowed to stand in a dark place for 30 minutes for alkylation. The solvent was replaced with 50 mM Tris-Cl (pH 7.5) by ultrafiltration. Trypsin was added and the mixture was allowed to stand overnight at 37 ° C. The resulting peptides were separated by reverse phase high performance liquid chromatography (RP-HPLC). The separated peptides were analyzed by a quadrupole time-of-flight mass spectrometer (QTOF-MS). From the obtained peaks, peaks of glycopeptides were extracted by BioPharmaView (manufactured by SCIEX) (Table 3). From Table 3, O-linked sugar chains were added to FR2 (positions 36 to 49 of SEQ ID NO: 54) and FR3 (positions 67 to 98 of SEQ ID NO: 54) of the His-tagged anti-Fc VHH (SEQ ID NO: 54). It became clear that they were combined.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
＜実施例６：重鎖抗体の糖鎖解析＞
　比較例４で精製した野生型ａｎｔｉ－ＨＳＡ　ＶＨＨのサンプル及び実施例４で取得した各変異ａｎｔｉ－ＨＳＡ　ＶＨＨのサンプルをＬＣ／ＭＳに供し、野生型ａｎｔｉ－ＨＳＡ　ＶＨＨ及び変異ａｎｔｉ－ＨＳＡ　ＶＨＨの糖鎖解析を実施した。使用機器・分析条件を以下に記す。 <Example 6: Sugar chain analysis of heavy chain antibody>
Samples of wild-type anti-HSA VHH purified in Comparative Example 4 and samples of each mutant anti-HSA VHH obtained in Example 4 were subjected to LC / MS, and sugars of wild-type anti-HSA VHH and mutant anti-HSA VHH were subjected to LC / MS. Chain analysis was performed. The equipment used and analysis conditions are described below.
　分析カラム（Ｗａｔｅｒｓ　Ａｃｑｕｉｔｙ　ＵＰＬＣ　ＢＥＨ３００　Ｃ４　Ｃｏｌｕｍｎ　２．１×５０ｍｍ）を装着した液体クロマトグラフ（Ｓｈｉｍａｄｚｕ　ＵＦＬＣ　Ｎｅｘｅｒａ　Ｘ２、島津製作所製）を質量分析計（ＴｒｉｐｌｅＴＯＦ６６００、ＳＣＩＥＸ社製）に接続し、サンプル５μＬをカラムに添加した。試料の分離には、Ａ溶媒に０．１％ギ酸水溶液、Ｂ溶媒には０．１％アセトニトリルを用いて、０分から０．５分でＡ溶媒を前記カラムに通液し、０．５分から２分でＢ溶媒０％からＢ溶媒１００％のリニアグラジエントとなるようＡ溶媒及びＢ溶媒を前記カラムに通液した。分離したペプチドは、四重極飛行時間型質量分析計（ＱＴＯＦ－ＭＳ、ＳＣＩＥＸ社製）により、ポジティブイオンモード（印加電圧　５，５００Ｖ）で測定した。 A liquid chromatograph (Shimadzu UFLC Nexus X2, manufactured by Shimadzu Corporation) equipped with an analytical column (Waters Accuracy UPLC BEH300 C4 Volume 2.1 x 50 mm) was connected to a mass spectrometer (TripleTOF6600, manufactured by SCIEX), and a sample was connected to the column. Was added to. To separate the sample, use 0.1% formic acid aqueous solution as A solvent and 0.1% acetonitrile as B solvent, and pass A solvent through the column in 0 to 0.5 minutes, and from 0.5 minutes. Solvent A and Solvent B were passed through the column so as to become a linear gradient from 0% of B solvent to 100% of B solvent in 2 minutes. The separated peptides were measured in a positive ion mode (applied voltage 5,500 V) by a quadrupole time-of-flight mass spectrometer (QTOF-MS, manufactured by SCIEX).
　重鎖抗体のアミノ酸配列に基づく理論質量を有する分子のピークと、前記理論質量より１６２Ｄａ質量の整数倍が増加した質量を有する複数のアイソフォームのピークが観察された。理論質量と一致するピークをＭａｎ０として、１６２Ｄａずつ増加したピークをそれぞれＭａｎ１、Ｍａｎ２・・・として、各ピークの面積比を算出した（表４）。表４においてｎ．ｄ．はピークが検出されなかったことを示す。野生型ａｎｔｉ－ＨＳＡ　ＶＨＨと比較して、変異ａｎｔｉ－ＨＳＡ　ＶＨＨ（Ｍｕｔａｎｔ　１～１５）は、Ｏ結合型糖鎖修飾が抑制されていた。 A peak of a molecule having a theoretical mass based on the amino acid sequence of a heavy chain antibody and a peak of a plurality of isoforms having a mass increased by an integral multiple of 162 Da mass from the theoretical mass were observed. The area ratio of each peak was calculated, where the peaks consistent with the theoretical mass were Man0 and the peaks increased by 162 Da were Man1, Man2, and so on, respectively (Table 4). In Table 4, n. d. Indicates that no peak was detected. Compared with the wild-type anti-HSA VHH, the mutant anti-HSA VHH (Mutant 1 to 15) suppressed the O-binding sugar chain modification.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
＜実施例７：重鎖抗体の結合活性＞
　実施例４で取得した変異ａｎｔｉ－ＨＳＡ　ＶＨＨの抗原に対する結合活性を測定した。活性測定方法を以下に記す。 <Example 7: Binding activity of heavy chain antibody>
The binding activity of the mutant anti-HSA VHH obtained in Example 4 to the antigen was measured. The activity measurement method is described below.
　９６ウェルイムノプレートの各ウェルに１μｇ／ｍＬのＨｕｍａｎ　Ｓｅｒｕｍ　Ａｌｂｕｍｉｎ（ＨＳＡ）溶液を１００μＬ入れ、プレートミキサーで撹拌後、室温で１．５時間静置した。プレートのＨＳＡ溶液を捨て、洗浄液（ＰＢＳ－Ｔ：９．５７ｍＭリン酸ナトリウム、１３５ｍＭ塩化ナトリウム、２．７ｍＭ塩化カリウム、０．５％（ｖ／ｖ）Ｔｗｅｅｎ２０）で４回洗浄した。全てのウェルにブロッキング溶液（サーモフィッシャーサイエンティフィック社製）２００μＬを入れ、プレートミキサーで撹拌後、室温で１時間静置した。 100 μL of 1 μg / mL Human Serum Albumin (HSA) solution was added to each well of the 96-well immunoplate, stirred with a plate mixer, and allowed to stand at room temperature for 1.5 hours. The HSA solution on the plate was discarded and washed 4 times with a washing solution (PBS-T: 9.57 mM sodium phosphate, 135 mM sodium chloride, 2.7 mM potassium chloride, 0.5% (v / v) Tween 20). 200 μL of a blocking solution (manufactured by Thermo Fisher Scientific Co., Ltd.) was placed in all wells, stirred with a plate mixer, and allowed to stand at room temperature for 1 hour.
　プレートの内容液を捨て、洗浄液で３回洗浄した。各サンプル溶液をウェルに１００μＬずつ入れて（１サンプルにつき２ウェル使用する）、プレートミキサーで撹拌後、室温で１．５時間静置した。プレートの内容液を捨て、洗浄液で４回洗浄した。全ての使用ウェルに検出用二次抗体溶液（ａｎｔｉ－ＨｉｓＴａｇ抗体ＨＲＰ　ｃｏｎｊｕｇａｔｅ（アブカム社製）を１０００倍希釈したもの）を１００μＬずつ入れ、プレートミキサーで撹拌後、室温で１時間静置した。プレートの内容液を捨て、洗浄液で４回洗浄した。各ウェルに発色液を１００μＬずつ加え、プレートミキサーで撹拌後、室温で５～１０分間静置した。各ウェルに反応停止液（２Ｍリン酸）を１００μＬずつ加え、プレートミキサーで撹拌した。マイクロプレートリーダーにて４５０ｎｍの吸光度を測定した。同濃度の野生型ａｎｔｉ－ＨＳＡ　ＶＨＨの吸光度の相対値から相対結合活性を求めた（表５）。変異ａｎｔｉ－ＨＳＡ　ＶＨＨ（Ｍｕｔａｎｔ　１～１５）は、野生型ａｎｔｉ－ＨＳＡ　ＶＨＨと同程度の抗原結合活性を保持していることが確認された。 The contents of the plate were discarded and washed 3 times with the cleaning solution. 100 μL of each sample solution was placed in each well (2 wells were used per sample), stirred with a plate mixer, and allowed to stand at room temperature for 1.5 hours. The contents of the plate were discarded and washed with the washing liquid four times. 100 μL of a secondary antibody solution for detection (anti-HisTag antibody HRP conjugate (manufactured by Abcam) diluted 1000 times) was added to all the wells used, and the mixture was stirred with a plate mixer and allowed to stand at room temperature for 1 hour. The contents of the plate were discarded and washed with the washing liquid four times. 100 μL of the coloring liquid was added to each well, and the mixture was stirred with a plate mixer and allowed to stand at room temperature for 5 to 10 minutes. 100 μL of the reaction terminator (2M phosphoric acid) was added to each well, and the mixture was stirred with a plate mixer. The absorbance at 450 nm was measured with a microplate reader. The relative binding activity was determined from the relative value of the absorbance of the wild-type anti-HSA VHH at the same concentration (Table 5). It was confirmed that the mutant anti-HSA VHH (Mutant 1 to 15) retains the same level of antigen-binding activity as the wild-type anti-HSA VHH.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　本明細書で引用した全ての刊行物、特許及び特許出願はそのまま引用により本明細書に組み入れられるものとする。 All publications, patents and patent applications cited in this specification shall be incorporated herein by reference as is.

Claims (9)

  1. 　Ｎ末端からフレームワーク領域１、可変重鎖相補性決定領域１、フレームワーク領域２、可変重鎖相補性決定領域２、フレームワーク領域３、可変重鎖相補性決定領域３及びフレームワーク領域４がこの順で連結した重鎖可変ドメインを含む重鎖抗体であって、
    　フレームワーク領域１のアミノ酸配列が、
    （１ａ）配列番号１：
    Ｘａａ１－Ｖ－Ｑ－Ｌ－Ｘａａ２－Ｅ－Ｓ－Ｇ－Ｇ－Ｇ－Ｘａａ３－Ｖ－Ｑ－Ｘａａ４－Ｇ－Ｘａａ５－Ｓ－Ｌ－Ｘａａ６－Ｘａａ７－Ｓ－Ｃ－Ｘａａ８－Ａ－Ｓ
    （Ｘａａ１はＥ、Ｑ又はＤであり、Ｘａａ２はＶ又はＱであり、Ｘａａ３はＬ又はＳであり、Ｘａａ４はＰ又はＡであり、Ｘａａ５はＧ又はＮであり、Ｘａａ６はＲ又はＳであり、Ｘａａ７はＬ又はＩであり、Ｘａａ８はＡ又はＴである）
    に示すアミノ酸配列、又は、
    （１ｂ）配列番号１に示すアミノ酸配列と８３％以上の配列同一性を有するアミノ酸配列であり、
    　フレームワーク領域２のアミノ酸配列が、
    （２ａ）配列番号２：
    Ｗ－Ｘａａ９－Ｒ－Ｑ－Ａ－Ｐ－Ｇ－Ｘａａ１０－Ｘａａ１１－Ｘａａ１２－Ｅ－Ｘａａ１３－Ｖ－Ｘａａ１４
    （Ｘａａ９はＶ、Ｙ又はＦであり、Ｘａａ１０はＫ又はＱであり、Ｘａａ１１はＧ又はＥであり、Ｘａａ１２はＬ又はＲであり、Ｘａａ１３はＷ、Ｌ、Ｆ又はＡであり、Ｘａａ１４はＳ又はＡである）
    に示すアミノ酸配列、又は、
    （２ｂ）配列番号２に示すアミノ酸配列と７０％以上の配列同一性を有するアミノ酸配列であり、
    　フレームワーク領域３のアミノ酸配列が、
    （３ａ）配列番号３：
    Ｒ－Ｆ－Ｔ－Ｉ－Ｓ－Ｒ－Ｄ－Ｎ－Ａ－Ｋ－Ｘａａ１５－Ｘａａ１６－Ｘａａ１７－Ｘａａ１８－Ｌ－Ｑ－Ｍ－Ｎ－Ｘａａ１９－Ｌ－Ｘａａ２０－Ｘａａ２１－Ｘａａ２２－Ｄ－Ｔ－Ａ－Ｘａａ２３－Ｙ－Ｙ－Ｃ－Ｘａａ２４－Ｘａａ２５
    （Ｘａａ１５はＴ、Ｎ又はＲであり、Ｘａａ１６はＴ又はＭであり、Ｘａａ１７はＬ、Ｖ又はＧであり、Ｘａａ１８はＹ、Ｔ又はＮであり、Ｘａａ１９はＮ又はＳであり、Ｘａａ２０はＲ、Ｋ、Ｅ又はＡであり、Ｘａａ２１はＰ又はＡであり、Ｘａａ２２はＥ又はＤであり、Ｘａａ２３はＶ又はＩであり、Ｘａａ２４はＴ、Ａ又はＧであり、Ｘａａ２５はＩ、Ｆ、Ａ又はＲである）
    に示すアミノ酸配列において、配列番号３の第３位、第５位、第１１位、第１２位、第１９位及び第２５位のアミノ酸の少なくとも１つに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列、或いは、
    （３ｂ）配列番号３に示すアミノ酸配列と８５％以上の配列同一性を有するアミノ酸配列において、配列番号３の第３位、第５位、第１１位、第１２位、第１９位及び第２５位のアミノ酸に対応するアミノ酸の少なくとも１つに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列であり、
    　フレームワーク領域４のアミノ酸配列が、
    （４ａ）配列番号４：
    Ｘａａ２６－Ｘａａ２７－Ｘａａ２８－Ｘａａ２９－Ｇ－Ｔ－Ｘａａ３０－Ｖ－Ｔ－Ｖ－Ｓ－Ｓ
    （Ｘａａ２６はＲ、Ｙ、Ｎ又はＳであり、Ｘａａ２７はＳ、Ｗ又はＲであり、Ｘａａ２８はＳ又はＧであり、Ｘａａ２９はＱ、Ｌ又はＲであり、Ｘａａ３０はＱ又はＬである）
    に示すアミノ酸配列、又は、
    （４ｂ）配列番号４に示すアミノ酸配列と６５％以上の配列同一性を有するアミノ酸配列である
    ことを特徴とする、重鎖抗体。     From the N-terminal, framework regions 1, variable heavy chain complementarity determining regions 1, framework regions 2, variable heavy chain complementarity determining regions 2, framework regions 3, variable heavy chain complementarity determining regions 3 and framework regions 4 Heavy chain antibodies containing heavy chain variable domains linked in this order.
    The amino acid sequence of framework region 1
    (1a) SEQ ID NO: 1:
    Xaa1-VQL-Xaa2-ES-G-G-G-Xaa3-VQ-Xaa4-G-Xaa5-S-L-Xaa6-Xaa7-SC-Xaa8-AS
    (Xaa1 is E, Q or D, Xaa2 is V or Q, Xaa3 is L or S, Xaa4 is P or A, Xaa5 is G or N, Xaa6 is R or S. , Xaa7 is L or I, Xaa8 is A or T)
    Amino acid sequence shown in
    (1b) An amino acid sequence having 83% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.
    The amino acid sequence of framework region 2
    (2a) SEQ ID NO: 2:
    W-Xaa9-RQ-A-P-G-Xaa10-Xaa11-Xaa12-E-Xaa13-V-Xaa14
    (Xaa9 is V, Y or F, Xaa10 is K or Q, Xaa11 is G or E, Xaa12 is L or R, Xaa13 is W, L, F or A, Xaa14 is S. Or A)
    Amino acid sequence shown in
    (2b) An amino acid sequence having 70% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 2.
    The amino acid sequence of framework region 3
    (3a) SEQ ID NO: 3:
    RFT I S R D-N-A-K-Xaa15-Xaa16-Xaa17-Xaa18-L-Q-M-N-Xaa19-L-Xaa20-Xaa21-Xaa22-DT- A-Xaa23-YYC-Xaa24-Xaa25
    (Xaa15 is T, N or R, Xaa16 is T or M, Xaa17 is L, V or G, Xaa18 is Y, T or N, Xaa19 is N or S, Xaa20 is R. , K, E or A, Xaa21 is P or A, Xaa22 is E or D, Xaa23 is V or I, Xaa24 is T, A or G, and Xaa25 is I, F, A. Or R)
    In the amino acid sequence shown in, at least one of the amino acids at positions 3, 5, 11, 12, 19 and 25 of SEQ ID NO: 3 is replaced with an amino acid other than serine or threonine. Amino acid sequence in which is introduced, or
    (3b) In the amino acid sequence having 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, the 3rd, 5th, 11th, 12th, 19th and 25th positions of SEQ ID NO: 3 An amino acid sequence in which substitution with an amino acid other than serine or threonine is introduced into at least one of the amino acids corresponding to the amino acid at the position.
    The amino acid sequence of framework region 4
    (4a) SEQ ID NO: 4:
    Xaa26-Xaa27-Xaa28-Xaa29-GT-Xaa30-VT-VS-S
    (Xaa26 is R, Y, N or S, Xaa27 is S, W or R, Xaa28 is S or G, Xaa29 is Q, L or R, Xaa30 is Q or L)
    Amino acid sequence shown in
    (4b) A heavy chain antibody characterized by having an amino acid sequence having 65% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 4.
  2. 　フレームワーク領域１のアミノ酸配列が、（１ａ）で規定するアミノ酸配列であり、
    フレームワーク領域２のアミノ酸配列が、（２ａ）で規定するアミノ酸配列であり、
    フレームワーク領域３のアミノ酸配列が、（３ａ）で規定するアミノ酸配列であり、且つ、
    フレームワーク領域４のアミノ酸配列が、（４ａ）で規定するアミノ酸配列である、
    請求項１に記載の重鎖抗体。     The amino acid sequence of framework region 1 is the amino acid sequence defined in (1a).
    The amino acid sequence of framework region 2 is the amino acid sequence defined in (2a).
    The amino acid sequence of framework region 3 is the amino acid sequence defined in (3a), and
    The amino acid sequence of framework region 4 is the amino acid sequence defined in (4a).
    The heavy chain antibody according to claim 1.
  3. 　（３ａ）で規定するアミノ酸配列が、配列番号３に示すアミノ酸配列において、配列番号３の第３位、第５位、第１１位、第１２位、第１９位及び第２５位のアミノ酸の少なくとも２つに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列であり、
    　（３ｂ）で規定するアミノ酸配列が、配列番号３に示すアミノ酸配列と８５％以上の配列同一性を有するアミノ酸配列において、配列番号３の第３位、第５位、第１１位、第１２位、第１９位及び第２５位のアミノ酸に対応するアミノ酸の少なくとも２つに、セリン又はスレオニン以外のアミノ酸への置換が導入されているアミノ酸配列である、
    請求項１又は２に記載の重鎖抗体。     The amino acid sequence defined in (3a) is at least the amino acids at positions 3, 5, 11, 12, 19, and 25 of SEQ ID NO: 3 in the amino acid sequence shown in SEQ ID NO: 3. The second is an amino acid sequence in which substitution with an amino acid other than serine or threonine has been introduced.
    In the amino acid sequence defined in (3b), which has 85% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 3, the 3rd, 5th, 11th, and 12th positions of SEQ ID NO: 3 , An amino acid sequence in which substitutions with amino acids other than serine or threonine have been introduced into at least two of the amino acids corresponding to the amino acids at positions 19 and 25.
    The heavy chain antibody according to claim 1 or 2.
  4. 　連結された２以上の請求項１～３のいずれか１項に記載の重鎖抗体を含む多重特異性抗体又は多価抗体。 A multispecific antibody or a multivalent antibody containing the heavy chain antibody according to any one of claims 1 to 3 linked to each other.
  5. 　請求項１～３のいずれか１項に記載の重鎖抗体のアミノ酸配列をコードする塩基配列を含む核酸。 A nucleic acid containing a base sequence encoding the amino acid sequence of the heavy chain antibody according to any one of claims 1 to 3.
  6. 　請求項５に記載の核酸を含むベクター。 A vector containing the nucleic acid according to claim 5.
  7. 　請求項５に記載の核酸を含む宿主細胞。 A host cell containing the nucleic acid according to claim 5.
  8. 　請求項１～３のいずれか１項に記載の重鎖抗体の製造方法であって、
    　請求項７に記載の宿主細胞を培養すること、及び、
    　培養物から請求項１～３のいずれか１項に記載の重鎖抗体を回収すること
    を含む方法。     The method for producing a heavy chain antibody according to any one of claims 1 to 3.
    Culturing the host cell according to claim 7 and
    A method comprising recovering the heavy chain antibody according to any one of claims 1 to 3 from the culture.
  9. 　請求項５に記載の核酸を含む、遺伝子治療のための医薬。 A drug for gene therapy containing the nucleic acid according to claim 5.
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WO2005085433A1 (en) * 2004-03-03 2005-09-15 Meiji Dairies Corporation Method of regulating o-glycosylation in eucaryotic cell
JP2008539772A (en) * 2005-05-18 2008-11-20 アブリンクス エン．ヴェー． Improved Nanobody ™ for tumor necrosis factor-alpha
JP2008539775A (en) * 2005-05-20 2008-11-20 アブリンクス エン．ヴェー． Improved Nanobody ™ in the treatment of aggregation-mediated disorders
WO2008099968A1 (en) * 2007-02-15 2008-08-21 Tohoku University Zinc oxide-binding antibody and use thereof

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