WO2021039574A1 - Anticorps à chaîne lourde dans lequel une modification de chaîne de sucre liée à o est supprimée - Google Patents

Anticorps à chaîne lourde dans lequel une modification de chaîne de sucre liée à o est supprimée 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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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

La présente invention concerne, en vue d'obtenir une qualité uniforme pour des anticorps à chaîne lourde, un anticorps à chaîne lourde qui est moins susceptible de subir une modification par des chaînes de sucre liées à O. L'anticorps à chaîne lourde selon la présente invention est caractérisé en ce qu'il comprend un domaine variable de chaîne lourde ayant une structure de (FR1)-(VH-CDR1)-(FR2)-(VH-CDR2)-(FR3)-(VH-CDR3)-(FR4), dans laquelle, dans la séquence d'acides aminés de FR3, au moins un résidu de sérine ou de thréonine est substitué par un acide aminé autre que la sérine ou la thréonine. La présente invention concerne également : un acide nucléique ayant une séquence de base codant la séquence d'acides aminés de l'anticorps à chaîne lourde ; et un vecteur et une cellule hôte contenant ledit acide nucléique.
PCT/JP2020/031425 2019-08-23 2020-08-20 Anticorps à chaîne lourde dans lequel une modification de chaîne de sucre liée à o est supprimée WO2021039574A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023282162A1 (fr) * 2021-07-09 2023-01-12 株式会社カネカ Anticorps miniature

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WO2005085433A1 (fr) * 2004-03-03 2005-09-15 Meiji Dairies Corporation Methode de regulation de l'o-glycosylation dans une cellule eucaryote
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