WO2022045365A1 - Polypeptide contenant un échafaudage du domaine de la fibronectine de type iii - Google Patents

Polypeptide contenant un échafaudage du domaine de la fibronectine de type iii Download PDF

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WO2022045365A1
WO2022045365A1 PCT/JP2021/031961 JP2021031961W WO2022045365A1 WO 2022045365 A1 WO2022045365 A1 WO 2022045365A1 JP 2021031961 W JP2021031961 W JP 2021031961W WO 2022045365 A1 WO2022045365 A1 WO 2022045365A1
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amino acid
group
polypeptide
seq
formula
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裕 村上
成晃 都築
太志 近藤
剛介 林
公茂 藤野
真也 築地
達之 吉井
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国立大学法人東海国立大学機構
国立大学法人 名古屋工業大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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 is a polypeptide containing a fibronectin type III domain skeleton.
  • Proteins that specifically bind to small molecule ligands are important in biological research. The most common is the biotin (small molecule ligand) -avidin (protein) interaction. However, since avidin is a tetramer, it is difficult to bind it to a biotinylated target molecule on a one-to-one basis. In addition, biotin is a natural compound, and biotin and biotinylated proteins existing in the living body also bind to avidin, resulting in background noise. From this point of view, it is required to develop a protein that binds to unnatural small molecules in a one-to-one relationship and has high affinity and specificity. Although several lipocalin family proteins that bind to artificial small molecule ligands have been reported, they have the drawback of being difficult to express.
  • One of the problems of the present invention is to provide a polypeptide that binds to a specific artificial small molecule ligand.
  • the present inventors have been able to more easily express a polypeptide containing a fibronectin type III domain skeleton, and HPPU (1- (4-hydroxyphenyl) -3-phenylurea). Or, it specifically binds to an artificial small molecule ligand such as a derivative thereof, and the polypeptide-artificial small molecule ligand system can have a one-to-one bond as compared with a biotin-avidin system. It has been found that it has the advantage of reducing background noise and is useful for labeling assays. The present invention has been completed through repeated studies based on these findings.
  • the present invention includes the following aspects.
  • Item 1 A polypeptide containing a fibronectin type III domain skeleton,
  • the BC loop of the fibronectin type III domain skeleton has the following formula (1): YX 1a X 1b X 1c X 1d X 1e DX 1f X 1g D (1) (During the ceremony, X 1a is A, S, or R.
  • X 1b and X 1c are independent amino acids, respectively, and are arbitrary amino acids.
  • X 1d is N, H, W, or Y.
  • X 1e to X 1 g are independent amino acids, respectively)
  • Contains the amino acid sequence represented by The FG loop of the fibronectin type III domain skeleton has the following formula (2): X 2a X 2b X 2c X 2d X 2e KX 2f X 2g X 2h X 2i (2)
  • X 2a and X 2b are independent amino acids, respectively, and are arbitrary amino acids.
  • X 2c is G, M, N, Y, or W.
  • X 2d is an arbitrary amino acid and
  • X 2e is Y, F, P, W, V, or A.
  • X 2f is W, Y, V, F, or S.
  • X 2g is W, L, V, or M.
  • X 2h is V, C, G, or A.
  • X 2i is an arbitrary amino acid
  • X 1a is A or S
  • X 1b is L, R, or H
  • X 1c is L, A, R, or Y
  • X 1d is H, N, or W
  • X 1e is G, N, R, or M
  • X 1f is W, A, or H, Item 2.
  • the amino acid sequence represented by the above formula (1) is YALRHGDWED (SEQ ID NO: 2), YALRHGDHLD (SEQ ID NO: 3), YALRHGDAWD (SEQ ID NO: 4), YSHYWMDAWD (SEQ ID NO: 5), YSHYHRDWED (SEQ ID NO: 6), YSHYHGDHLD (SEQ ID NO: 7), Item 2.
  • the polypeptide according to Item 1 or 2 which is selected from the group consisting of YSHYHGDWED (SEQ ID NO: 8) and an amino acid sequence having 70% or more sequence identity with respect to these amino acid sequences.
  • X 2a is W or L
  • X 2b is W or Y
  • X 2c is G or Y
  • X 2d is Y
  • X 2e is Y
  • X 2f is W or Y
  • X 2g is W
  • X 2h is V
  • Item 6 The polypeptide according to any one of Items 1 to 3, wherein X 2i is P, A, or D. Item 5.
  • the amino acid sequence represented by the above formula (2) is WWGSYKWWVP (SEQ ID NO: 9), WWGSYKWWVD (SEQ ID NO: 10), LWYYYKWWVP (SEQ ID NO: 11), Item 6.
  • the polypeptide according to any one of Items 1 to 4 which is selected from the group consisting of LWYYYKWWVD (SEQ ID NO: 12) and an amino acid sequence having 70% or more sequence identity with respect to these amino acid sequences.
  • Item 6 The following formula (4): (In the formula, R 1 is an amino group which may have a substituent, R 2 is O or S, and R 3 is 1,4-phenylene which may have a substituent. Is the basis) Item 2.
  • Item 7. A polynucleotide comprising the coding sequence of the polypeptide according to any one of Items 1 to 6.
  • Item 9. The following formula (4): (In the formula, R 1 is an amino group which may have a substituent, R 2 is O or S, and R 3 is 1,4-phenylene which may have a substituent. Is the basis)
  • Item 10 A pharmaceutical composition comprising the polypeptide according to any one of Items 1 to 6, the polynucleotide according to Item 7, or the cell according to Item 8.
  • Item 11. Item 6.
  • Item 12. Item 11 and the following formula (4): (In the formula, R 1 is an amino group which may have a substituent, R 2 is O or S, and R 3 is 1,4-phenylene which may have a substituent. Is the basis) A kit containing a compound represented by.
  • the polypeptide-artificial small molecule ligand system has the advantage of being capable of one-to-one binding compared to the biotin-avidin system and further reducing background noise, and is used for labeling assays and the like. It is useful.
  • FIG. 1 is a diagram showing the recovery rate of cDNA bound to an artificial antibody bound to the compound represented by the formula (4) for each round.
  • FIG. 2 is a diagram showing the recovery rate of cDNA bound to the artificial antibody bound to the compound represented by the formula (4) in libraries A to D for each round.
  • the term "identity" of an amino acid sequence refers to the degree of coincidence of two or more comparable amino acid sequences with respect to each other. Therefore, the higher the match between two amino acid sequences, the higher the identity or similarity of those sequences.
  • the level of amino acid sequence identity is determined, for example, using FASTA, a tool for sequence analysis, with default parameters.
  • FASTA a tool for sequence analysis, with default parameters.
  • the algorithm BLAST by Karlin and Altschul Karlin S, Altschul SF. “Methods for assessment the statistical signature of molecular sequence features by using general scoring schemes” Proc Natl Acad Sci USA.
  • a program called BLASTX based on such a BLAST algorithm has been developed. Specific methods for these analysis methods are known, and the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov/) can be referred to.
  • NCBI National Center for Biotechnology Information
  • conservative substitution means that an amino acid residue is replaced with an amino acid residue having a similar side chain.
  • substitution between amino acid residues having basic side chains such as lysine (K), arginine (R), and histidine (H) is a conservative substitution.
  • Other amino acid residues with acidic side chains such as aspartic acid (D) and glutamic acid (E); glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y).
  • Amino acid residues with non-charged polar side chains such as cysteine (C); alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine Amino acid residues with non-polar side chains such as (M) and tryptophan (W); Amino acid residues with ⁇ -branched side chains such as treonine (T), valine (V) and isoleucine (I); Tyrosine (Y) , Phenylalanine (F), tryptophan (W), histidine (H) and other amino acid residues having aromatic side chains are also conservative substitutions.
  • nucleotides such as DNA and RNA may be subjected to known chemical modifications as exemplified below.
  • Substituting the phosphate residue (phosphate) of each nucleotide with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate to prevent degradation by hydrolases such as nucleases. Can be done.
  • the hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide is represented by -OR (R is, for example, CH 3 (2'-O-Me), CH 2 CH 2 OCH 3 (2'-O-MOE), CH 2 CH 2 NHC (NH) may be replaced with NH 2 , CH 2 CONHCH 3 , CH 2 CH 2 CN, etc.).
  • the base moiety pyrimidine, purine
  • examples thereof include those in which the phosphoric acid moiety and the hydroxyl moiety are modified with a biotin, an amino group, a lower alkylamine group, an acetyl group and the like, but the present invention is not limited thereto.
  • BNA LNA
  • BNA LNA or the like in which the formation of the sugar portion is fixed to N-type by cross-linking the 2'oxygen and 4'carbon of the sugar part of the nucleotide can also be preferably used.
  • amino acid mutations are specifically deletions, substitutions, insertions, or additions of amino acids.
  • a polypeptide containing a fibronectin type III domain skeleton A fibronectin type III domain (hereinafter referred to as “Fn3 domain”) is one of the structural units (or modules) present in fibronectin, or the same entity present in a protein other than fibronectin. Or a similar structural unit.
  • the polypeptide of the present invention can contain an Fn3 domain skeleton derived from any organism, and more preferably contains a Fn3 domain skeleton derived from a mammal, and more preferably contains a Fn3 domain skeleton derived from a human.
  • the polypeptide of the present invention may contain a skeleton derived from any Fn3 domain in fibronectin, but preferably contains a skeleton derived from the 10th Fn3 domain from the N-terminal side.
  • the polypeptide of the present invention has two or more types of Fn3 domain skeletons (for example, a skeleton derived from the 9th Fn3 domain from the N-terminal side and a skeleton derived from the 10th Fn3 domain from the N-terminal side). You may be.
  • the Fn3 domain has a plurality of ⁇ chains and a loop connecting each ⁇ chain, and typically 7 ⁇ chains (“A chain”, “B chain”, “C” from the N-terminal side. "Chain”, “D chain”, “E chain”, “F chain”, “G chain”) and 6 loops (AB loop connecting A chain and B chain, BC connecting B chain and C chain) It has a loop, a CD loop connecting the C chain and the D chain, a DE loop connecting the D chain and the E chain, an EF loop connecting the E chain and the F chain, and an FG loop connecting the F chain and the G chain). Multiple ⁇ -chains in the Fn3 domain typically form two antiparallel ⁇ -sheets.
  • the loops in the Fn3 domain are typically exposed on the surface, the BC loops, DE loops, and FG loops are located at the top, and the AB loops, CD loops, and EF loops are located at the bottom.
  • the Fn3 domain has a structure similar to immunoglobulin, and the BC loop, DE loop, and FG loop correspond to CDR1, CDR2, and CDR3 of the heavy chain variable region of immunoglobulin, respectively, and thus Fn3 of the present invention.
  • the polypeptide containing a domain skeleton can be referred to as an antibody mimic, an artificial antibody, a monobody, or the like.
  • the polypeptide of the present invention has SEQ ID NO: 1: VSDVPRDLEVVAATPTSLLISWDA PAVT VRYYRITYGETGGNSPVQEFTVPG SKS TATISGLKPGVDYTITVYAVT GRGDSPASSK PISINYRT It is preferable to include an Fn3 domain skeleton derived from the amino acid sequence shown in.
  • SEQ ID NO: 1 corresponds to the amino acid sequence of the 10th Fn3 domain present in wild-type human fibronectin.
  • the 25th to 28th regions correspond to the BC loop
  • the 53rd to 55th regions correspond to the DE loop
  • the 77th to 86th regions correspond to the FG loop.
  • the Fn3 domain skeleton of the polypeptide of the present invention is not particularly limited as long as it has the same or similar structure as the above Fn3 domain structure.
  • the amino acid sequence of the Fn3 domain skeleton of the polypeptide of the present invention contains one or more amino acid mutations in the amino acid sequence of the wild-type Fn3 domain (SEQ ID NO: 1 and the like), and the amino acid sequence of the wild-type Fn3 domain (SEQ ID NO: 1). Etc.), preferably 70% or more, more preferably 75% or more of sequence identity.
  • the BC loop of the Fn3 domain skeleton of the polypeptide of the present invention has the following formula (1): YX 1a X 1b X 1c X 1d X 1e DX 1f X 1g D (1)
  • X 1a is A, S, or R.
  • X 1b and X 1c are independent amino acids, respectively, and are arbitrary amino acids.
  • X 1d is N, H, W, or Y.
  • X 1e to X 1 g are independent amino acids, respectively) It is not particularly limited as long as it includes the amino acid sequence represented by.
  • the BC loop may be one in which the amino acid sequence represented by the formula (1) is inserted at an arbitrary position (including the terminal) of the BC loop of the wild-type Fn3 domain, and the BC loop of the wild-type Fn3 domain. A part or all of the amino acids in the above may be replaced with the amino acid sequence represented by the formula (1).
  • the polypeptide of the present invention has an amino acid sequence in which the amino acid sequences from the 25th to the 28th of SEQ ID NO: 1 are replaced with the amino acid sequence represented by the formula (1) as a BC loop of the Fn3 domain skeleton. Is preferable.
  • X 1a is preferably A or S.
  • X 1b is preferably L, R, T, K, V, S, I, A, H, Y, N, M, E, or Q, and more preferably L, R, T, K, V, S, I, A, or H, more preferably L, R, or H.
  • X 1c is preferably L, A, R, K, Q, T, M, I, V, E, H, S, or Y, and more preferably L, A, R, or Y.
  • X 1d is preferably H, N, or W.
  • X 1e is preferably G, N, H, R, E, S, M, Q, A, C, L, K, or D, and more preferably G, N, H, R, E, S, Or M, more preferably G, N, H, R, or M, and particularly preferably G, N, R, or M.
  • X 1f is preferably W, A, S, H, Y, C, F, V, L, Q, R, E, I, or D, and more preferably W, A, S, or H. , More preferably W, A, or H.
  • X 1 g is preferably E, L, K, R, W, Y, F, C, V, I, A, M, D, or S, and more preferably E, L, K, R, or W. , And more preferably E, L, K, or W.
  • X 1a , X 1b , X 1c , X 1d , and X 1e is preferably selected from the group consisting of (1-1) to (1-6) in Table 1.
  • X 1d , X 1e , X 1f , and X 1g is preferably selected from the group consisting of (1-7) to (1-12) in Table 2.
  • the amino acid sequence represented by the formula (1) is YALRHGDWED (SEQ ID NO: 2), YALRHGDHLD (SEQ ID NO: 3), YALRHGDAWD (SEQ ID NO: 4), YSHYWMDAWD (SEQ ID NO: 5), YSHYHRDWED (SEQ ID NO: 6), YSHYHGDHLD (SEQ ID NO: 7), YSHYHGDWED (SEQ ID NO: 8), and an amino acid sequence having 70% or more (preferably 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more) sequence identity with respect to these amino acid sequences. It is preferably selected from the group consisting of.
  • the FG loop of the Fn3 domain skeleton of the polypeptide of the present invention has the following formula (2): X 2a X 2b X 2c X 2d X 2e KX 2f X 2g X 2h X 2i (2)
  • X 2a and X 2b are independent amino acids, respectively, and are arbitrary amino acids.
  • X 2c is G, M, N, Y, or W.
  • X 2d is an arbitrary amino acid and X 2e is Y, F, P, W, V, or A.
  • X 2f is W, Y, V, F, or S.
  • X 2g is W, L, V, or M.
  • X 2h is V, C, G, or A.
  • X 2i is an arbitrary amino acid
  • the FG loop may be one in which the amino acid sequence represented by the formula (2) is inserted at an arbitrary position (including the terminal) of the FG loop of the wild-type Fn3 domain, and the FG loop of the wild-type Fn3 domain. A part or all of the amino acids in the above may be replaced with the amino acid sequence represented by the formula (2).
  • the polypeptide of the present invention has an amino acid sequence in which the amino acid sequence from position 77 to 86 of SEQ ID NO: 1 is replaced with the amino acid sequence represented by the formula (2) as an FG loop of the Fn3 domain skeleton. Is preferable.
  • X 2a is preferably W, Y, F, H, I, L, V, or C, more preferably W, Y, F, H, I, or L, and even more preferably W or L. be.
  • X 2b is preferably W, Y, G, A, S, F, H, Q, or M, and more preferably W or Y.
  • X 2c is preferably G, M, N, Y, or W, more preferably G, M, N, or Y, and even more preferably G or Y.
  • X 2d is preferably Y, S, C, W, T, V, F, D, A, I, H, L, or G, and more preferably Y, S, C, W, or T. , More preferably Y, S, or T.
  • X 2e is preferably Y, F, P, W, V, H, or A, more preferably Y, F, P, or W, still more preferably Y or F, and particularly preferably Y. Is.
  • X 2f is preferably W, Y, V, or F, and more preferably W or Y.
  • X 2g is preferably W or L, and more preferably W.
  • X 2h is preferably V, C, or G, and more preferably V.
  • X 2i is preferably P, A, R, E, D, S, L, N, or M, more preferably P, A, R, E, or D, and even more preferably P, A, Or D.
  • X 2a , X 2b , X 2c , X 2d , and X 2e is preferably selected from the group consisting of (2-1) to (2-4) in Table 3.
  • X 2e , X 2f , X 2g , X 2h , and X 2i is preferably selected from the group consisting of (2-5) to (2-8) in Table 4.
  • the amino acid sequence represented by the formula (2) is preferably the following formula (2a) :. X 2a X 2b X 2c X 2d YKX 2f WVX 2i (2a) (In the formula, X 2a to X 2d , X 2f , and X 2i are the same as described above). It is an amino acid sequence represented by, and more preferably the following formula (2b) :. X 2a WX 2c X 2d YKWWVX 2i (2b) (In the equation, X 2a , X 2c , X 2d , and X 2i are the same as described above). It is an amino acid sequence represented by.
  • the amino acid sequence represented by the formula (2), (2a), or (2b) is WWGSYKWWVP (SEQ ID NO: 9), WWGSYKWWVD (SEQ ID NO: 10), LWYYYKWWVP (SEQ ID NO: 11), LWYYKWWVD (SEQ ID NO: 12), and an amino acid sequence having 70% or more (preferably 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more) sequence identity with respect to these amino acid sequences. It is preferably selected from the group consisting of.
  • the DE loop of the Fn3 domain skeleton of the polypeptide of the present invention may have the same amino acid sequence as the DE loop of the wild-type Fn3 domain (for example, SEQ ID NO: 1), and one or more amino acid mutations (for example) in the amino acid sequence.
  • it may have an amino acid sequence containing (conservative substitution).
  • the DE loop is 70% or more (preferably 75% or more, 80% or more, 85% or more, 90%) of the amino acid sequence from the 53rd to 55th sequence of SEQ ID NO: 1, that is, SKS, or this amino acid sequence. It is preferable to have an amino acid sequence having the above, or 95% or more) sequence identity.
  • the amino acid sequences of the regions other than the BC loop and the FG loop correspond to the wild-type Fn3 domain (for example, SEQ ID NO: 1). It may be the same as the amino acid sequence of the region to be subjected to, or may contain one or more amino acid mutations (for example, conservative substitution), and 70% or more, 75% or more, 80% or more, etc. with respect to the amino acid sequence. It is preferable to have 85% or more, 90% or more, or 95% or more sequence identity.
  • the number of the amino acid mutations is preferably 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.
  • the Fn3 domain skeleton of the polypeptide of the present invention may have an amino acid sequence from the 1st to the 7th of SEQ ID NO: 1 at the N-terminal, and has an amino acid sequence different from the amino acid sequence. It may or may not have the amino acid sequence.
  • the Fn3 domain skeleton of the polypeptide of the invention is located at or near the N-terminus (eg, at positions 1-7 amino acids away from the N-terminus) from amino acids 8 to 20 of SEQ ID NO: 1. It is preferable to have a sequence or an amino acid sequence having 70% or more, 75% or more, or 80% or more sequence identity with respect to the amino acid sequence, and in order to improve solubility, the amino acid shown in SEQ ID NO: 13: LEVVEASPTSIQI It is preferable to have a sequence.
  • the Fn3 domain skeleton of the polypeptide of the present invention is, in a preferred embodiment, the following formula (3): LEVVEASPTSIQISWDA YX 1a X 1b X 1c X 1d X 1e DX 1f X 1g D VRYYRITYGETGGNSPVQEFTVPG SKS TATISGLKPGVDYTITVYAVT X 2a X 2b X 2c X 2d X 2e KX 2f X 2g X 2h X 2i PISINYRT And X 2a to X 2i are the same as above) An amino acid sequence represented by, or an amino acid sequence having 70% or more (preferably 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more) sequence identity with respect to this amino acid sequence. Have.
  • the polypeptide of the present invention has the following formula (4): (In the formula, R 1 is an amino group which may have a substituent, R 2 is O or S, and R 3 is 1,4-phenylene which may have a substituent. Is the basis) It is preferable to specifically bind to the compound represented by, and the binding dissociation constant Kd for the compound represented by the formula (4) is, for example, 50 nM or less, preferably 45 nM or less, and more preferably 40 nM or less. It is also preferable that the polypeptide of the present invention specifically binds to a compound having a terminal SH instead of the terminal OH of the formula (4), and the binding dissociation constant Kd for such a compound is in the same range as described above. Can be.
  • the binding dissociation constant Kd can be measured according to the binding assay method described in Examples below.
  • the substituent of the "amino group which may have a substituent" represented by R1 is not particularly limited, and any substituent which can be introduced into the amino group can be used. ..
  • the substituent include an organic group.
  • the organic group include an alkyl group, a cycloalkyl group, an aryl group, an aliphatic heterocyclic group, an aromatic heterocyclic group (heteroaryl group) and the like.
  • an aryl group for example, a C6-12 aryl group such as a phenyl group or a naphthyl group is preferable.
  • These groups may further have a substituent, and the substituent may be, for example, an alkyl group (for example, a C 1-6 alkyl group such as a methyl group, an ethyl group, a propyl group or a butyl group).
  • an alkyl group for example, a C 1-6 alkyl group such as a methyl group, an ethyl group, a propyl group or a butyl group.
  • the substituent may be a group derived from a biotin-labeled object (for example, sugar, nucleic acid, amino acid, protein, fluorescent dye, polymer beads) in the biotin-avidin system.
  • R 1 is preferably a monophenylamino group which may have a substituent.
  • R 2 is preferably O.
  • R 3 is not particularly limited, and examples thereof include the same substituents exemplified by R 1 .
  • R 3 is preferably a 1,4-phenylene group.
  • the compound represented by the formula (4) is preferably HPU (1- (4-hydroxyphenyl) urea).
  • the compound represented by the formula (4) is the following formula (4a) :.
  • R 1a is a phenyl group which may have a substituent). It is preferably a compound represented by.
  • Examples of the compound represented by the formula (4a) include the following compounds.
  • the phenyl group substituted with a hydroxyl group may further have a substituent, and the following formula (4b): (In the formula, R 1a is the same as described above, and R 3a , R 3a , R 3a , and R 3a are independently hydrogen atoms or substituents, respectively, except that R 3a , R 3a , and R 3a . , And at least one of R 3a is a substituent) It may be a compound represented by. Such a compound is also included in the compound represented by the formula (4).
  • R 3a , R 3a , R 3a , and R 3a are preferably hydrogen atoms, halogen atoms, or alkyl groups.
  • Examples of the compound represented by the formula (4b) include the following compounds.
  • R 1a may be a phenyl group having a plurality of substituents, and the plurality of substituents may be two substituents having an ortho positional relationship.
  • the two substituents may be bonded to each other to form a ring (for example, a benzene ring), or may be, for example, the following compound.
  • a compound is also included in the compound represented by the formula (4).
  • the compound represented by the formula (4a) or (4b) has two or more of the following formulas: (In the formula, each benzene ring portion may have a substituent)
  • the group represented by may be a compound linked via a linker (for example, a linker having a polyethylene glycol chain, a linker having a polyglycine chain, or the like represented by L), and as an example, the following may be used.
  • Linker for example, a linker having a polyethylene glycol chain, a linker having a polyglycine chain, or the like represented by L
  • Compounds include.
  • the compound represented by the formula (4) can be synthesized by combining known reactions.
  • the compound represented by the formula (4) includes a compound represented by the formula: R 1 H (in the formula, R 1 is the same as described above) and a compound represented by the formula: H 2 N-R 3 -OH (formula).
  • R3 can be synthesized by a method including a step of reacting the compound represented by ( the same as above) with triphosgene.
  • the polypeptide of the present invention has a structure similar to that of the compound represented by the formula (4), and has the following formula (5): (In the formula, R 4 is an amino group which may have a substituent, R 5 is O or S, and R 6 is 1,4-phenylene which may have a substituent. Is the basis) It is preferable not to bind to the compound represented by.
  • R 4 to R 6 correspond to R 1 to R 3 , respectively, and the same groups as R 1 to R 3 can be adopted.
  • the polypeptide of the present invention recognizes, for example, the difference between the terminal group (hydroxyphenyl group) of the compound represented by the formula (4) and the terminal group (benzyloxyphenyl group) of the compound represented by the formula (5). It is possible to specifically bind to the compound represented by the formula (4).
  • the polypeptide of the present invention may contain other amino acid sequences in addition to the Fn3 domain skeleton as long as the binding property to the compound represented by the formula (4) is not significantly impaired.
  • the polypeptide of the present invention may be a signal sequence such as a protein tag, a fluorescent protein, a luminescent protein, a secretory signal sequence (Ig ⁇ signal sequence, etc.), a protease recognition sequence (TEV protease recognition sequence, etc.), an expression-enhancing sequence, and / or possible. It may be one to which a protein or peptide such as a lysed sequence is added (or fused).
  • the protein tag include biotin, His tag, FLAG tag, Halo tag, MBP tag, HA tag, Myc tag, V5 tag, PA tag and the like.
  • the polypeptide of the invention may contain the amino acid sequence of all or part of the antibody.
  • the antibody is not particularly limited, and examples thereof include IgA, IgD, IgE, IgG, IgM, and further subclasses thereof.
  • the origin of the antibody is also not particularly limited, and may be, for example, a human-derived antibody, a mouse-derived antibody, a rat-derived antibody, a rabbit-derived antibody, a monkey-derived antibody, a chimpanzee-derived antibody, or the like.
  • the amino acid sequence preferably comprises an Fc region. This makes it possible to exert the effector function.
  • the polypeptide of the present invention can be linked via the Fc region to form a multimerization.
  • the polypeptide of the present invention may be added with a multimerization domain and / or a linker (or a hinge region), and is a multimerized multimer via the domain or linker.
  • the multimer may be a homomultimer or a heteromultimer.
  • the domains are domains that can bind to each other to form multimers, and are not particularly limited as long as they are. Examples of the domain include cartilage oligomer matrix protein domain, leucine zipper domain, collagen-like domain, cholera toxin B subunit domain, tetrabrachyon coiled core domain, leovirus ⁇ 1 protein domain, hepatitis delta antigen domain and the like.
  • the domain may be part of a human dimer protein, such as the Liprin-b2 Coil2, RP_P2 domain.
  • the linker is also not particularly limited, and for example, the following formula: (In the formula, X is an n-valent organic group, n is an integer of 2 or more (for example, 2 to 8), and Z is a binding site with a polypeptide). It may be a linker represented by, or may be a peptide linker. Examples of the peptide linker include a glycine-rich linker and a serine-rich linker [for example, a linker represented by -T- (SNSS) a -EL- (a is an integer, for example, 1, 2, 3, or 4)]. , Proline-rich linker [eg, a linker represented by-(PSTPPTPS) b- (b is an integer, eg 1, 2, 3, or 4)] and the like.
  • polypeptide of the present invention may be added (or fused) with another protein or peptide such as toxin (eg, diphtheria toxin), albumin (eg, serum albumin).
  • toxin eg, diphtheria toxin
  • albumin eg, serum albumin
  • the polypeptide of the present invention may be in the form of a salt with an acid or a base.
  • the salt is not particularly limited, and either an acidic salt or a basic salt can be adopted.
  • acidic salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; acetate, propionate, tartrate, fumarate, maleate, and apple.
  • Organic acid salts such as acid salts, citrates, methane sulfonates and paratoluene sulfonates; amino acid salts such as asparaginates and glutamates can be mentioned.
  • basic salts include alkali metal salts such as sodium salt and potassium salt; alkaline earth metal salts such as calcium salt and magnesium salt.
  • the polypeptide of the present invention may be in the form of a solvate.
  • the solvent is not particularly limited, and examples thereof include water, ethanol, glycerol, acetic acid and the like. Further, the polypeptide of the present invention may be freeze-dried.
  • polypeptide of the present invention may be chemically modified or may be in the form of a complex as long as the binding property to the compound represented by the formula (4) is not significantly impaired.
  • the polypeptide of the present invention may have a C-terminal of any of a carboxyl group (-COOH), a carboxylate (-COO- ) , an amide group (-CONH 2 ), and an ester group (-COOQ).
  • the Q in the ester group is, for example, a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl; for example, a C 3-8 cycloalkyl group such as cyclopentyl, cyclohexyl; for example.
  • C 6-12 aryl groups such as phenyl, ⁇ -naphthyl; phenyl-C 1-2 alkyl groups such as benzyl, phenethyl; C 7 such as ⁇ -naphthyl-C 1-2 alkyl groups such as ⁇ -naphthylmethyl -14 Aralkyl group; Pivaloyloxymethyl group and the like are used.
  • a carboxyl group (or carboxylate) other than the C-terminal may be amidated or esterified.
  • ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
  • the amino group of the N-terminal amino acid residue is protected by a protective group (for example, a C 1-6 acyl group such as C 1-6 alkanoyl such as a formyl group or an acetyl group).
  • a protective group for example, a C 1-6 acyl group such as C 1-6 alkanoyl such as a formyl group or an acetyl group.
  • N-terminal glutamine residue that can be cleaved and produced in vivo is pyroglutamine oxidized, substituents on the side chain of amino acids in the molecule (eg -OH, -SH, amino group, imidazole group, indole group) , A guanidino group, etc.) is protected by a suitable protective group (eg, a C 1-6 acyl group such as a C 1-6 alkanoyl group such as a formyl group or an acetyl group), or a so-called sugar chain attached.
  • a suitable protective group eg, a C 1-6 acyl group such as a C 1-6 alkanoyl group such as a formyl group or an acetyl group
  • Complex proteins such as glycoproteins are also included.
  • the polypeptide of the present invention may be modified with polyalkylene glycol.
  • the polyalkylene glycol include polyethylene glycol and polypropylene glycol.
  • the method for modifying the polyalkylene glycol is, for example, a method of reacting the polypeptide with a polyalkylene glycol having a reactive group at the terminal.
  • the reactive group is not particularly limited as long as it is a group capable of reacting with the polypeptide, and examples thereof include a reactive group for an amino group, a carboxyl group, and a thiol group.
  • Examples of the reactive group for the amino group include an isothiocyano group, a carboxyl group, an active ester group and the like, and examples of the active ester group include the following formula: Examples thereof include an N-hydroxysuccinimide ester group which may have a substituent, such as a group represented by.
  • Examples of the reactive group for the carboxyl group include an amino group.
  • the amino group before the reaction may be protected by a protecting group such as a Boc group (t-butoxycarbonyl group) and an Fmoc group (9-fluorenylmethyloxycarbonyl group).
  • Examples of the reactive group for the thiol group include the following formula: Examples thereof include a maleimide group represented by.
  • the polypeptide of the present invention may be one to which a drug is bound via a linker.
  • the linker is not particularly limited as long as the polypeptide and the drug can be linked, and for example, all the linkers used in the antibody drug complex (ADC) can be applied.
  • the linker include a linker having an alkylene chain (here, at least one methylene of the alkylene chain may be replaced with O, S, or NH) as a main skeleton.
  • the linker includes a linker having a polyalkylene glycol as a main skeleton and the like.
  • the drug is not particularly limited, and may be a small molecule drug or a high molecular weight drug (for example, nucleic acid, antibody).
  • ADC antibody drug complex
  • Typical agents are, for example, anti-cancer agents.
  • the anticancer agent include cytotoxic anticancer agents, and examples thereof include alkylating agents, antimetabolites, platinum compounds, topoisomerase inhibitors, antibiotics, microtubule agents and the like.
  • alkylating agent examples include cyclophosphamide, iphosphamide, melphalan, thiotepa, busulfan, dacarbazine, nimustine, ranimustine, carmustine, lomustine, chlorampsyl, temozolomid and the like.
  • Antimetabolites include, for example, fluorouracil, capecitabine, gemcitabine, enocitabine, tegafur, carmofur, doxifluridine, cytarabine, mercaptopurine, fludarabine, cladribine, methotrexate, pemetrexed, hydroxyurea and the like.
  • Examples of the platinum compound include cisplatin, carboplatin, nedaplatin, oxaliplatin and the like.
  • Examples of the topoisomerase inhibitor include irinotecan, nogitecan, etoposide and the like.
  • Examples of the antibiotic include actinomycin, doxorubicin, daunorubicin, bleomycin, peplomycin, mitomycin, aclarubicin, pirarubicin, epirubicin, idarubicin, amrubicin, dinostatin stimalamar and the like.
  • Examples of the microtubule agonist include vincristine, vinblastine, vindesine, vinorelbine, paclitaxel, docetaxel and the like.
  • the method of binding the drug may be, for example, a method of reacting the polypeptide with the drug and a cross-linking agent capable of forming a linker.
  • the cross-linking agent is not particularly limited as long as it can form a linker between the polypeptide and the drug, and is, for example, protein A, carbodiimide, 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), S-.
  • SATA N-succinimidyl
  • oPDM o-phenylenedimaleimide
  • SPDP 3-maleimide propionic acid N-succinimid
  • the polypeptide of the present invention can be easily produced according to a known genetic engineering method. For example, it can be produced by using PCR, restriction enzyme cleavage, DNA ligation technology, in vitro transcription / translation technology, recombinant protein production technology, and the like.
  • the polynucleotides of the invention contain the coding sequences of the polypeptides of the invention.
  • the coding sequence is not particularly limited as long as it is a polynucleotide consisting of a base sequence encoding the polypeptide of the present invention.
  • the polynucleotide of the present invention in one embodiment, comprises an expression cassette of the polypeptide of the present invention.
  • the expression cassette is not particularly limited as long as it is a polynucleotide capable of expressing the polypeptide of the present invention in cells.
  • Typical examples of the expression cassette include a promoter and a polynucleotide containing the coding sequence of the polypeptide of the present invention arranged under the control of the promoter.
  • the promoter is not particularly limited and can be appropriately selected according to the target cells.
  • various pol II promoters can be used.
  • the polII promoter is not particularly limited, and examples thereof include a CMV promoter, an EF1 promoter, an SV40 promoter, and an MSCV promoter.
  • Other promoters include, for example, tryptophan promoters such as trc and tac, lac promoters, T7 promoters, T5 promoters, T3 promoters, SP6 promoters, arabinose-inducible promoters, cold shock promoters, tetracycline-inducible promoters and the like.
  • the expression cassette may contain other elements, if necessary.
  • Other elements include, for example, multicloning sites (MCS), drug resistance genes, origins of replication, enhancer sequences, repressor sequences, insulator sequences, reporter protein (eg, fluorescent proteins, etc.) coding sequences, drug resistance gene coding sequences. And so on.
  • MCS multicloning sites
  • drug resistance genes include, for example, drug resistance genes, origins of replication, enhancer sequences, repressor sequences, insulator sequences, reporter protein (eg, fluorescent proteins, etc.) coding sequences, drug resistance gene coding sequences. And so on.
  • Examples of the drug resistance gene include a chloramphenicol resistance gene, a tetracycline resistance gene, a neomycin resistance gene, an erythromycin resistance gene, a spectinomycin resistance gene, a canamycin resistance gene, a hyglomycin resistance gene, a puromycin resistance gene, and the like.
  • the reporter protein is not particularly limited as long as it is a luminescent (color-developing) protein that emits light (color-developing) in response to a specific substrate or a fluorescent protein that emits fluorescence by excitation light.
  • luminescent (color-developing) protein include luciferase, ⁇ -galactosidase, chloramphenicol acetyltransferase, ⁇ -glucuronidase, and the like
  • examples of the fluorescent protein include GFP, Azami-Green, ZsGreen, GFP2, HyPer, Sirius, and BFP.
  • Examples include CFP, Turquoise, Cyan, TFP1, YFP, Venus, ZsYellow, Banana, KusabiraOrange, RFP, DsRed, AsRed, Strawberry, Jred, KillerRed, Cherry, HcRed, mPlum and the like.
  • the polynucleotide of the present invention can be in the form of a vector.
  • An appropriate vector is selected according to the purpose of use (cloning, protein expression) and also considering the type of host cell.
  • Vectors hosting Escherichia coli include M13 phage or variants thereof, ⁇ phage or variants thereof, pBR322 or variants thereof (pB325, pAT153, pUC8, etc.), and vectors hosting yeast include pYepSec1, pMFa, pYES2. , PPIC3.5K, etc., pAc, pVL, etc. can be exemplified as a vector having an insect cell as a host, and pcDNA, pCDM8, pMT2PC, etc. can be exemplified as a vector having a mammalian cell as a host.
  • Cell The cell of the present invention is not particularly limited as long as it contains the polynucleotide of the present invention.
  • Examples of cells include Escherichia coli K12 and other Escherichia coli, Bacillus subtilis MI114 and other Bacillus bacteria, Saccharomyces cerevisiae AH22 and other yeast, Spodoptera frugiperda-derived Sf cell lineage or Trichoplusia ni-derived High Five cell lineage, and olfactory nerve cells.
  • Examples include animal cells such as insect cells and COS7 cells.
  • animal cells are preferably cultured cells derived from mammals, specifically, COS7 cells, CHO cells, HEK293 cells, HEK293FT cells, Hela cells, PC12 cells, N1E-115 cells, SH-SY5Y cells and the like. Be done.
  • the cell of the present invention expresses the polypeptide of the present invention in one embodiment.
  • the cell of the invention in one embodiment, secretes the polypeptide of the invention or has the polypeptide of the invention on the cell surface.
  • the ligand binder that binds to the ligand represented by the formula (4) of the present invention is not particularly limited as long as it contains the polypeptide of the present invention.
  • the Kd of the ligand binder with respect to the ligand represented by the formula (4) is, for example, 50 nM or less, preferably 45 nM or less, and more preferably 40 nM or less. It is preferable that the ligand binder does not bind to the compound represented by the formula (5).
  • compositions of the present invention are not particularly limited as long as it contains the polypeptide of the present invention, the polynucleotide of the present invention, or the cells of the present invention.
  • the content of the polypeptide contained in the pharmaceutical composition is not particularly limited and may be, for example, 0.001 to 90% by mass.
  • the pharmaceutical composition of the present invention may further contain other drugs.
  • the other drug may be a low molecular weight drug or a high molecular weight drug (eg, nucleic acid, antibody), for example, an anticancer drug, an antihypertensive drug, an antidiabetic drug, an anticardiac disease drug, an anti.
  • examples include, but are not limited to, neuropsychiatric drugs, anti-immune disease drugs, anti-allergic disease drugs, anti-infectious disease drugs, and the like.
  • the form of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include, but are not limited to, tablets, granules, capsules, liquids, gels, ointments, and creams.
  • the pharmaceutical composition of the present invention usually contains a pharmaceutically acceptable excipient or carrier.
  • the excipient or carrier include starch, lactose, crystalline cellulose, sorbitol, calcium hydrogen phosphate, water, ethanol, (poly) ethylene glycol, (poly) propylene glycol, glycerol, vegetable oil and the like.
  • the pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable additive.
  • the additive can be appropriately selected depending on the dosage form and the like, and for example, a binder, a disintegrant, a lubricant, a flavoring agent, a flavoring agent, a preservative, a buffering agent, an emulsifier, a surfactant, and a thickening agent. Examples include agents, dispersants, isotonic agents, antioxidants and the like. These additives may be used alone or in combination of two or more.
  • the pharmaceutical composition of the present invention can be used to treat or prevent various diseases.
  • diseases include cancer, hypertension, diabetes, heart disease, neuropsychiatric disease, immune disease, allergic disease, infectious disease and the like.
  • the route of administration of the pharmaceutical composition of the present invention is not particularly limited, and is, for example, oral, intraretinal, intranasal, subcutaneous, intramuscular, intravenous, intrabronchial, intraosseous, intraarticular, intraperitoneal, intrarectal, and colon. Inside, inside the spinal cord, etc.
  • the administration target of the pharmaceutical composition of the present invention is not particularly limited, and can be, for example, human or non-human animals (for example, dogs, cats, cows, horses, sheep, mice, guinea pigs, rabbits).
  • the administration frequency of the pharmaceutical composition of the present invention is not particularly limited, and for example, it can be administered once, twice, or three times a day, once every two days, or once a week.
  • the reagents of the invention are not particularly limited as long as they include the polypeptides of the invention, the polynucleotides of the invention, or the cells of the invention.
  • the reagent of the present invention can further contain the excipient or carrier exemplified in the pharmaceutical composition of the present invention, and / or an additive or the like.
  • the reagent of the present invention may be dissolved or dispersed in a solvent, or may be freeze-dried.
  • kit of the present invention is not particularly limited as long as it contains the reagent of the present invention and the compound represented by the formula (4).
  • the compound represented by the formula (4) may be dissolved or dispersed in a solvent, and in addition to the compound represented by the formula (4), a solvent for dissolving or dispersing the compound is included in the kit of the present invention. It may be.
  • kits of the invention include other reagents (eg, polypeptides of the invention, polynucleotides of the invention, or reagents for detecting cells of the invention), instruments, and instruments necessary for the use of the reagents of the invention, as needed. Instructions for use and the like may be included as appropriate.
  • Fmoc deprotection was performed with 20% piperidine in DMF for 15 minutes at room temperature.
  • the amino acid coupling reaction was carried out in DMF, Fmoc-protected amino acid (3.0 eq), 1- [bis (dimethylamino) methylene] -1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU, 2.9 eq), 1 -A mixture of hydroxybenzotriazole (HOBt, 2.9 eq) and N, N-diisopropylethylamine (DIPEA, 6.0 eq) was used at room temperature. All Fmoc deprotection and coupling steps were also monitored by the Kaiser test. In addition, all cleaning procedures were performed using DMF.
  • the Sieber amide resin (0.79 mmol / g) (126 mg, 100 ⁇ mol) was deprotected from Fmoc and washed. Subsequently, Fmoc-Cys (Trt) -OH was coupled to the resin and washed with DMF.
  • Trt deprotection, Boc deprotection and cleavage from the resin were performed with dichloromethane with 5% triisopropylsilane (TIS) and 30% TFA. Subsequently, the solvent was removed under reduced pressure and the crude product was purified on a semi-prepared C18 column by reverse phase HPLC using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA to make compound 4 white. Obtained as a solid.
  • the premix is reacted with the premix (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2% (v / v) DMSO, 2 mM DDL4, 0.2 mM each dNTP, 0.375 ⁇ M.
  • Amplified A-fragment DNA and B-fragment DNA were purified by phenol / chloroform extraction and isopropanol precipitation.
  • One end of each DNA product was digested with BsaI (New England Biolabs), and the DNA product was purified by phenol / chloroform extraction and isopropanol precipitation.
  • the products were ligated to each other (1 ⁇ M, 200 ⁇ L) to synthesize a full-length DNA product and amplified using T7SD8M2.F44, G5S-4Gan21-3.R42, and Pfu-S DNA polymerase (60 mL total, 4 cycles of PCR). ).
  • the product was purified by phenol / chloroform extraction and isopropanol precipitation.
  • the DNA template was transcribed by in vitro run-off transcription, the mRNA was purified by isopropanol precipitation, and PAGE purification was performed. mRNA / HEX-mPuL was prepared by the same method. The resulting complex was used in the first selection.
  • the reaction was carried out with stirring for 30 minutes.
  • the immobilization rate and concentration were measured by the fluorescence of tryptophan in the reaction solution.
  • Add 20 ⁇ L of 1-thioglycerin solution 500 mM 1-thioglycerin, 100 mM HEPES-K pH8.0, 600 mM NaCl, 50% DMF
  • suspend and stir at 37 ° C for 2 hours to carry out the reaction.
  • rice field After washing with DMF and 10 mM Tris-HCl pH 8.0, a bead storage solution (10 mM AcONa pH 5, 1 mM DTT) was added and stored at 4 ° C.
  • HMLV (27.5 ⁇ L) was added and the resulting solution was incubated at 42 ° C. for 15 minutes.
  • the buffer was replaced with HBST buffer using a Zebra TM spin desalting column.
  • the obtained solution was mixed with Dynabeads M270 Amine on which compound 4 was immobilized at 25 ° C. for 20 minutes.
  • the obtained beads were washed twice with HBST buffer and PCR premix (690 ⁇ L) was added. The beads were heated at 95 ° C. for 5 minutes and the amount of eluted cDNA was quantified by SYBR green-based quantitative PCR using T7SD8M2.F44 and FN3Lip.R20 as primers.
  • the eluted cDNA was PCR amplified using T7SD8M2.F44, G5S-4Gan21-3.R42, and Pfu-S DNA polymerase, and purified by phenol / chloroform extraction and isopropanol precipitation. Transcription was performed on the DNA purified using T7 RNA Polymerase, and the DNA was purified by phenol / chloroform extraction and isopropanol precipitation.
  • the buffer was replaced with HBST buffer using a Zebra TM spin desalting column.
  • the obtained solution was mixed with compound 5-Dynabeads M270 Amine at 25 ° C for 5 minutes, and then the solution was recovered. Mixing with compound 5-Dynabeads M270 Amine was repeated 8 times in total. The solution was recovered and mixed with compound 4-Dynabeads M270 Amine at 25 ° C for 20 minutes. After recovering the target protein using the beads, the obtained beads were washed twice with HBST buffer, and PCR premix (50 ⁇ L) was added. Complementary DNA quantification, DNA amplification and purification (no transcription) was performed in the same manner as in the first round selection.
  • the obtained DNA (final concentration of about 5 nM) was added to the TRAP system and the reaction mixture (5 ⁇ L) was incubated at 37 ° C for 30 minutes. After the reaction, 1 ⁇ L of 100 mM EDTA (pH 8.0) was added to the translation mixture.
  • the reverse transfer mixture (3 ⁇ L; 150 mM Tris-HCl pH 8.4, 225 mM KCl, 75 mM MgCl2, 16 mM DTT, 1.5 mM dNTP, 7.5 ⁇ M FN3S.R29 (primer), 3.4 ⁇ M HMLV) was added to the translation mixture, and the obtained solution was added. Incubated at 42 ° C for 15 minutes. The buffer was replaced with HBST buffer using a Zebra TM spin desalting column. Mixing with beads, washing of beads, quantification of cDNA, amplification and purification of DNA were carried out in the same manner as in the second round selection.
  • the 4th to 6th round selections were performed in the same way as the 3rd round selection.
  • the concentration of compound 4 in the system was changed from 500 nM to 50 nM.
  • Other operations were performed in the same way as the 3rd round selection.
  • cDNA recovery rate (%) amount of recovered cDNA x 100 / amount of puromycin linker
  • Figure 1 shows the recovery rate of cDNA in each round. As is clear from FIG. 1, the cDNA recovery rate increased as the round progressed. In addition, the low recovery rate for compound 5 suggests that an artificial antibody with high specificity for compound 4 has been recovered.
  • the DNA sequence recovered in the 10th round was analyzed by the next-generation sequencer.
  • SEQ ID NO: 14 MQANSGSLEVVEASPTSIQISWDA YSHYWMDAWD VRYYRITYGETGGNSPVQEFTVPG SKS TATISGLKPGVDYTITVYAVT LWYYYKWWVD PISINYRT It was found that the sequence shown in (Monobody-1) showed a very high ratio to the total number of reads (about 70%). Monobody-1 expression in E. coli, Ni-NTA purification, and affinity for compounds 4 and 5 immobilized on streptavidin biosensor (ForteBio) using Octet system (ForteBio, CA, USA) Sex measurement was performed. The binding assay was performed at 30 ° C.
  • Mutant Saturation Library In order to further improve the affinity and specificity of artificial antibodies, four new mutant saturation libraries were constructed using Monobody-1 as the parent clone and the sequence as a template. FN3F0.F83 (1 ⁇ M), FN3F1-2.F29 (P) (1 ⁇ M), FN3FF1NNK6L.F79 (1 ⁇ M, corresponding to library A), or FN3FF1NNK6R to prepare A-fragment DNA for monobody libraries.
  • Fn3an1.R20 (2 ⁇ M) and Fn3an2-1.R20 (3NH2) (2 ⁇ M) with .F79 (1 ⁇ M, corresponding to library B) or FN3FF1temp.F79 (1 ⁇ M, used for libraries C and D) ) was used as an auxiliary agent, and each was ligated with T4 DNA ligase (5 ⁇ L in total). After ligation, the premix is reacted with the premix (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2% (v / v) DMSO, 2 mM DDL4, 0.2 mM each dNTP, 0.375 ⁇ M.
  • FN3FF2co.F72 P
  • FN3F3NNK6L.F70 compatible with library C
  • FN3F3NNK6R.F70 compatible with library D
  • FN3F3temp.F70 used for libraries A and B
  • Fn3an3.R20 3NH2 for ligation.
  • FN3BsaI.F33 and FN3Pri2.R44 for amplification were used to prepare B-fragment DNA in the same manner.
  • Amplified A-fragment DNA and B-fragment DNA were purified by phenol / chloroform extraction and isopropanol precipitation.
  • One end of each DNA product was digested with BsaI (New England Biolabs), and the DNA product was purified by phenol / chloroform extraction and isopropanol precipitation.
  • the corresponding products were ligated (0.15 ⁇ M, 10 ⁇ L), respectively (0.15 ⁇ M, 10 ⁇ L) to synthesize full-length DNA products, which were amplified using T7SD8M2.F44, G5S-4Gan21-3.R42, and Pfu-S DNA polymerase (total 400 ⁇ L, 6-8 cycles of PCR).
  • the product was purified by phenol / chloroform extraction and isopropanol precipitation.
  • the DNA template was transcribed by in vitro run-off transcription and the mRNA was purified by isopropanol precipitation.
  • mRNA / HEX-mPuL was prepared by the same method. The resulting complex was used in the first selection.
  • the obtained beads were washed twice with HBST buffer and PCR premix (50 ⁇ L) was added. Complementary DNA quantification, DNA amplification and purification (no transcription) was performed in the same manner as in the first round selection.
  • the obtained DNA (final concentration of about 5 nM) was added to the TRAP system and the reaction mixture (5 ⁇ L) was incubated at 37 ° C for 30 minutes. The following was carried out in the same way as the second round selection.

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Abstract

L'invention concerne un polypeptide qui se lie spécifiquement à un ligand de faible poids moléculaire artificiel. Le polypeptide, dans lequel une boucle BC d'un échafaudage de domaine de la fibronectine de type III contient une séquence d'acides aminés représentée par la formule suivante (1) : X1aX1bX1cX1dX1eDX1fX1gD (où X1a est A, S ou R, X1b et X1c sont chacun indépendamment un acide aminé arbitraire, X1d est N, H, W ou Y, et X1e à X1g sont chacun indépendamment un acide aminé arbitraire), et une boucle FG contient une séquence d'acides aminés représentée par la formule (2) suivante : X2aX2bX2cX2dX2eKX2fX2gX2hX2i (dans laquelle X2a et X2b sont chacun indépendamment un acide aminé arbitraire, X2c est G, M, N, Y, ou W, X2d est un acide aminé arbitraire, X2e est Y, F, P, W, V, ou A, X2f est W, Y, V, F, ou S, X2g est W, L, V, ou M, X2g est V, C, G, ou A, et X2i est un acide aminé arbitraire), se lie spécifiquement à un ligand artificiel de faible poids moléculaire tel que la 1-(4-hydroxyphényl)-3-phénylurée (HPPU) ou un dérivé de celle-ci.
PCT/JP2021/031961 2020-08-31 2021-08-31 Polypeptide contenant un échafaudage du domaine de la fibronectine de type iii WO2022045365A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001500531A (ja) * 1997-06-12 2001-01-16 リサーチ コーポレイション テクノロジーズ，インコーポレイティド 人工抗体ポリペプチド
JP2005132733A (ja) * 2003-10-28 2005-05-26 Mitsubishi Gas Chem Co Inc レジスト用化合物および感放射線性組成物

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001500531A (ja) * 1997-06-12 2001-01-16 リサーチ コーポレイション テクノロジーズ，インコーポレイティド 人工抗体ポリペプチド
JP2005132733A (ja) * 2003-10-28 2005-05-26 Mitsubishi Gas Chem Co Inc レジスト用化合物および感放射線性組成物

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Novel Ligand-Protein System : HPPU-Monobody ", BIONAUTS.JP, 8 February 2021 (2021-02-08), XP055903092, Retrieved from the Internet <URL:https://bionauts.jp/?p=1678> *

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