Polypeptides containing a fibronectin type III domain scaffold

Abstract

Provided is a polypeptide that binds specifically to an artificial low molecular weight ligand. This polypeptide, in which a BC loop of a fibronectin type III domain scaffold contains an amino acid sequence represented by the following formula (1): X1aX1bX1cX1dX1eDX1fX1gD (wherein X1a is A, S, or R, X1b and X1c are each independently an arbitrary amino acid, X1d is N, H, W, or Y, and X1e to X1g are each independently an arbitrary amino acid), and an FG loop contains an amino acid sequence represented by the following formula (2): X2aX2bX2cX2dX2eKX2fX2gX2hX2i (wherein X2a and X2b are each independently an arbitrary amino acid, X2c is G, M, N, Y, or W, X2d is an arbitrary amino acid, X2e is Y, F, P, W, V, or A, X2f is W, Y, V, F, or S, X2g is W, L, V, or M, X2g if V, C, G, or A, and X2i is an arbitrary amino acid), binds specifically to an artificial low molecular weight ligand such as 1-(4-hydroxyphenyl)-3-phenylurea (HPPU) or a derivative thereof.

Description

[Technical Field]

[0001] The present invention relates to a polypeptide comprising a fibronectin type III domain scaffold. [Background technology]

[0002] Proteins that specifically bind to small molecule ligands are important in biological research. The most common example is the biotin (small molecule ligand)-avidin (protein) interaction. However, because avidin is a tetramer, it is difficult to achieve one-to-one binding with a biotinylated target molecule. Furthermore, biotin is a natural compound, and biotin and biotinylated proteins present in living organisms also bind to avidin, resulting in background noise. For these reasons, there is a demand for the development of proteins that bind to non-natural small molecules in a one-to-one relationship and have high affinity and specificity. Several lipocalin family proteins that bind to artificial small molecule ligands have been reported, but they have the disadvantage of being difficult to express. Summary of the Invention [Problem to be solved by the invention]

[0003] An object of the present invention is to provide a polypeptide that binds to a specific artificial small molecule ligand. [Means for solving the problem]

[0004] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that a polypeptide containing a fibronectin type III domain scaffold can be more easily expressed and specifically binds to an artificial small molecule ligand such as HPPU (1-(4-hydroxyphenyl)-3-phenylurea) or its derivatives, and that this polypeptide-artificial small molecule ligand system allows one-to-one binding compared to a biotin-avidin system and further has the advantage of reducing background noise, making it useful for labeling assays, etc. The present invention was completed through extensive research based on these findings.

[0005] The present invention includes the following aspects. Section 1. A polypeptide comprising a fibronectin type III domain scaffold, The BC loop of the fibronectin type III domain scaffold has the following formula (1): YX 1a X 1b X 1c X 1d X 1e DX 1f X 1g D (1) (In the formula, X 1a is A, S, or R, X 1b and X 1c are each independently any amino acid, X 1d is N, H, W, or Y; X 1e ~X 1g are each independently any amino acid) The amino acid sequence represented by The FG loop of the fibronectin type III domain scaffold has the following formula (2): X 2a X 2b X 2c X 2d X 2e KX 2f X 2g X 2h X 2i (2) (In the formula, X 2a and X 2b are each independently any amino acid, X 2c is G, M, N, Y, or W, X 2d is any amino acid, X 2e is Y, F, P, W, V, or A; X 2f is W, Y, V, F, or S, X2g is W, L, V, or M, X 2h is V, C, G, or A, X 2i is any amino acid) The polypeptide comprising the amino acid sequence represented by: Section 2. In the formula (1), 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, X 1g is E, L, K, or W. Section 3. The amino acid sequence represented by the formula (1) 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 Amino acid sequences with 70% or more sequence identity to these amino acid sequences Item 3. The polypeptide according to Item 1 or 2, selected from the group consisting of: Section 4. In the formula (2), X 2a is W or L, X 2b is W or Y, X 2c is G or Y, X 2d is Y, S, or T; X 2e is Y, X 2f is W or Y, X 2g But W, X 2h But V, X 2i is P, A, or D. Section 5. The amino acid sequence represented by the formula (2) WWGSYKWWVP (SEQ ID NO: 9), WWGSYKWWVD (SEQ ID NO: 10), LWYYYKWWVP (SEQ ID NO: 11), LWYYYKWWVD (SEQ ID NO: 12), and Amino acid sequences with 70% or more sequence identity to these amino acid sequences Item 5. The polypeptide according to any one of Items 1 to 4, selected from the group consisting of: Section 6. The following formula (4): [ka] (In the formula, R 1 is an amino group which may have a substituent, and R 2 is O or S, and R 3 is an optionally substituted 1,4-phenylene group. Item 6. The polypeptide according to any one of Items 1 to 5, which has a binding dissociation constant Kd of 50 nM or less for a compound represented by the formula: Section 7. A polynucleotide comprising a coding sequence for the polypeptide according to any one of Items 1 to 6. Section 8. A cell comprising the polynucleotide according to item 7. Section 9. The following formula (4): [ka] (In the formula, R1 is an amino group which may have a substituent, and R 2 is O or S, and R 3 is an optionally substituted 1,4-phenylene group. A ligand-binding agent that binds to a ligand represented by the formula (I): wherein the ligand-binding agent comprises the polypeptide according to any one of Items 1 to 6. Section 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. Section 11. A reagent 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. Section 12. Item 11 and the reagent according to the following formula (4): [ka] (In the formula, R 1 is an amino group which may have a substituent, and R 2 is O or S, and R 3 is an optionally substituted 1,4-phenylene group. and a compound represented by the formula: [Effects of the Invention]

[0006] The present invention provides a polypeptide that specifically binds to an artificial small molecule ligand such as HPPU or its derivative. Furthermore, compared with the biotin-avidin system, this polypeptide-artificial small molecule ligand system allows for one-to-one binding and has the advantage of reducing background noise, making it useful for labeling assays. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 is a diagram showing the recovery rate of cDNA bound to an artificial antibody that binds to the compound represented by formula (4) for each round. [Figure 2]FIG. 2 is a diagram showing the recovery rates of cDNA bound to an artificial antibody that binds to the compound represented by formula (4) for each round in libraries A to D. DETAILED DESCRIPTION OF THE INVENTION

[0008] 1.Definitions etc. In this specification, the expression "comprise" includes the concepts of "consist essentially of" and "consist only of."

[0009] As used herein, the "identity" of an amino acid sequence refers to the degree of correspondence between the amino acid sequences of two or more comparable amino acid sequences. Therefore, the greater the identity between two amino acid sequences, the greater the identity or similarity between those sequences. The level of identity between amino acid sequences can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul S F. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes," Proc. Natl. Acad. Sci. USA. 87:2264-2268 (1990); Karlin S, Altschul S F. "Applications and statistics for multiple high-scoring segments in molecular sequences," Proc. Natl. Acad. Sci. USA. 90:5873-7 (1993)). A program called BLASTX, based on the BLAST algorithm, has been developed. Specific techniques for these analysis methods are known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). The "identity" of nucleotide sequences is also defined in the same manner as above.

[0010] As used herein, the term "conservative substitution" refers to substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, a conservative substitution is substitution of amino acid residues having basic side chains, such as lysine (K), arginine (R), and histidine (H). Conservative substitutions also include substitutions between amino acid residues with acidic side chains, such as aspartic acid (D) and glutamic acid (E); uncharged polar side chains, such as glycine (G), asparagine (N), glutamine (Q), serine (S), threonine (T), tyrosine (Y), and cysteine ​​(C); nonpolar side chains, such as alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), and tryptophan (W); beta-branched side chains, such as threonine (T), valine (V), and isoleucine (I); and aromatic side chains, such as tyrosine (Y), phenylalanine (F), tryptophan (W), and histidine (H).

[0011] As used herein, nucleotides such as DNA and RNA may be chemically modified as described below. To prevent degradation by hydrolases such as nucleases, the phosphate residue of each nucleotide may be substituted with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may be substituted with -OR (where R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base moiety (pyrimidine or purine) may be chemically modified, for example by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Further examples include, but are not limited to, those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc. Also preferably used are BNA (LNA), in which the conformation of the sugar moiety of the nucleotide is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety.

[0012] As used herein, amino acid mutation specifically refers to amino acid deletion, substitution, insertion, or addition.

[0013] 2. Polypeptides containing fibronectin type III domain scaffolds A fibronectin type III domain (hereinafter referred to as "Fn3 domain") refers to one of the structural units (or modules) present in fibronectin, or an identical or similar structural unit present in a protein other than fibronectin.

[0014] Fibronectin derived from various organisms is known. The polypeptide of the present invention can contain an Fn3 domain backbone derived from any organism, preferably a mammalian Fn3 domain backbone, and more preferably a human Fn3 domain backbone.

[0015] Fibronectin contains at least 15 Fn3 domains. The polypeptides of the present invention may contain a backbone derived from any of the Fn3 domains in fibronectin, but preferably contain a backbone derived from the 10th Fn3 domain from the N-terminus. The polypeptides of the present invention may contain two or more types of Fn3 domain backbones (for example, a backbone derived from the 9th Fn3 domain from the N-terminus and a backbone derived from the 10th Fn3 domain from the N-terminus).

[0016] The Fn3 domain has multiple β strands and loops connecting the β strands, and typically has seven β strands (referred to from the N-terminus as "A strand," "B strand," "C strand," "D strand," "E strand," "F strand," and "G strand") and six loops (the AB loop connecting the A strand and the B strand, the BC loop connecting the B strand and the C strand, the CD loop connecting the C strand and the D strand, the DE loop connecting the D strand and the E strand, the EF loop connecting the E strand and the F strand, and the FG loop connecting the F strand and the G strand). The multiple β strands in the Fn3 domain typically form two antiparallel β sheets. The loops of the Fn3 domain are typically exposed to the surface, with the BC loop, the DE loop, and the FG loop located at the top and the AB loop, the CD loop, and the EF loop located at the bottom. The Fn3 domain has a structure similar to that of an immunoglobulin, and the BC loop, DE loop, and FG loop correspond to CDR1, CDR2, and CDR3 of the heavy chain variable region of an immunoglobulin, respectively. Therefore, a polypeptide comprising the Fn3 domain scaffold of the present invention can be referred to as an antibody mimetic, an artificial antibody, a monobody, or the like.

[0017] The polypeptide of the present invention is SEQ ID NO:1: VSDVPRDLEVVAATPTSLLISWDA PAVT VRYYRITYGETGGNSPVQEFTVPG SKS TATISGLKPGVDYTITVYAVT GRGDSPASSK PISINYRT Preferably, the Fn3 domain comprises a scaffold derived from the amino acid sequence shown in

[0018] SEQ ID NO: 1 corresponds to the amino acid sequence of the 10th Fn3 domain present in wild-type human fibronectin. In SEQ ID NO: 1, the region from positions 25 to 28 corresponds to the BC loop, the region from positions 53 to 55 corresponds to the DE loop, and the region from positions 77 to 86 corresponds to the FG loop.

[0019] The Fn3 domain backbone of the polypeptide of the present invention is not particularly limited, as long as it has a structure identical to or similar to the above-mentioned Fn3 domain structure. The amino acid sequence of the Fn3 domain backbone 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 (e.g., SEQ ID NO: 1), and has a sequence identity of preferably 70% or more, more preferably 75% or more, to the amino acid sequence of the wild-type Fn3 domain (e.g., SEQ ID NO: 1).

[0020] The BC loop of the Fn3 domain scaffold 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) (In the formula, X 1a is A, S, or R, X 1b and X 1c are each independently any amino acid, X 1d is N, H, W, or Y; X 1e ~X 1g are each independently any amino acid) The BC loop is not particularly limited as long as it contains the amino acid sequence represented by formula (1). The BC loop may be one in which the amino acid sequence represented by formula (1) has been inserted at any position (including the terminus) of the BC loop of a wild-type Fn3 domain, or one in which some or all of the amino acids in the BC loop of a wild-type Fn3 domain have been replaced with the amino acid sequence represented by formula (1). In one aspect, the polypeptide of the present invention preferably has, as the BC loop of the Fn3 domain scaffold, an amino acid sequence in which the amino acid sequence from positions 25 to 28 of SEQ ID NO: 1 has been replaced with the amino acid sequence represented by formula (1).

[0021] X 1a is preferably A or S.

[0022] X 1b is preferably L, R, T, K, V, S, I, A, H, Y, N, M, E, or Q, more preferably L, R, T, K, V, S, I, A, or H, and even more preferably L, R, or H.

[0023] 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.

[0024] X 1d is preferably H, N, or W.

[0025] X 1e is preferably G, N, H, R, E, S, M, Q, A, C, L, K, or D, more preferably G, N, H, R, E, S, or M, even more preferably G, N, H, R, or M, and particularly preferably G, N, R, or M.

[0026] X 1f is preferably W, A, S, H, Y, C, F, V, L, Q, R, E, I, or D, more preferably W, A, S, or H, and even more preferably W, A, or H.

[0027] X 1gis preferably E, L, K, R, W, Y, F, C, V, I, A, M, D, or S, more preferably E, L, K, R, or W, and even more preferably E, L, K, or W.

[0028] X 1a , X 1b , X 1c , X 1d , and X 1e The combination is preferably selected from the group consisting of (1-1) to (1-6) in Table 1. [Table 1]

[0029] X 1d , X 1e , X 1f , and X 1g The combination is preferably selected from the group consisting of (1-7) to (1-12) in Table 2. [Table 2]

[0030] The amino acid sequence represented by 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 Amino acid sequences that have a sequence identity of 70% or more (preferably 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more) to these amino acid sequences It is preferably selected from the group consisting of:

[0031] The FG loop of the Fn3 domain scaffold 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) (In the formula, X 2a and X 2b are each independently any amino acid, X 2c is G, M, N, Y, or W, X 2d is any amino acid, 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 any amino acid) The FG loop is not particularly limited as long as it contains the amino acid sequence represented by formula (2). The FG loop may be one in which the amino acid sequence represented by formula (2) has been inserted at any position (including the terminus) of the FG loop of a wild-type Fn3 domain, or one in which some or all of the amino acids in the FG loop of a wild-type Fn3 domain have been replaced with the amino acid sequence represented by formula (2). In one aspect, the polypeptide of the present invention preferably has, as the FG loop of the Fn3 domain scaffold, an amino acid sequence in which the amino acid sequence from positions 77 to 86 of SEQ ID NO: 1 has been replaced with the amino acid sequence represented by formula (2).

[0032] 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.

[0033] X 2bis preferably W, Y, G, A, S, F, H, Q, or M, and more preferably W or Y.

[0034] X 2c is preferably G, M, N, Y, or W, more preferably G, M, N, or Y, and even more preferably G or Y.

[0035] X 2d is preferably Y, S, C, W, T, V, F, D, A, I, H, L, or G, more preferably Y, S, C, W, or T, and even more preferably Y, S, or T.

[0036] X 2e is preferably Y, F, P, W, V, H, or A, more preferably Y, F, P, or W, even more preferably Y or F, and particularly preferably Y.

[0037] X 2f is preferably W, Y, V, or F, and more preferably W or Y.

[0038] X 2g is preferably W or L, and more preferably W.

[0039] X 2h is preferably V, C, or G, and more preferably V.

[0040] 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.

[0041] X 2a , X 2b , X 2c , X 2d , and X 2e The combination is preferably selected from the group consisting of (2-1) to (2-4) in Table 3. [Table 3]

[0042] X 2e , X 2f , X 2g , X 2h , and X 2i The combination is preferably selected from the group consisting of (2-5) to (2-8) in Table 4. [Table 4]

[0043] The amino acid sequence represented by formula (2) is preferably represented by the following formula (2a): X 2a X 2b X 2c X 2d YKX 2f WVX 2i (2a) (In the formula, X 2a ~X 2d , X 2f , and X 2i is the same as above) and more preferably an amino acid sequence represented by the following formula (2b): X 2a WX 2c X 2d YKWWVX 2i (2b) (In the formula, X 2a , X 2c , X 2d , and X 2i is the same as above) It is an amino acid sequence represented by the following formula:

[0044] The amino acid sequence represented by formula (2), (2a), or (2b) is WWGSYKWWVP (SEQ ID NO: 9), WWGSYKWWVD (SEQ ID NO: 10), LWYYYKWWVP (SEQ ID NO: 11), LWYYYKWWVD (SEQ ID NO: 12), and Amino acid sequences that have a sequence identity of 70% or more (preferably 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more) to these amino acid sequences It is preferably selected from the group consisting of:

[0045] The DE loop of the Fn3 domain scaffold of the polypeptides of the invention may have the same amino acid sequence as the DE loop of a wild-type Fn3 domain (e.g., SEQ ID NO: 1), or may have an amino acid sequence that contains one or more amino acid mutations (e.g., conservative substitutions) in the amino acid sequence.

[0046] The DE loop preferably has the amino acid sequence from positions 53 to 55 of SEQ ID NO: 1, i.e., SKS, 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 to this amino acid sequence.

[0047] In the Fn3 domain backbone of the polypeptide of the present invention, the amino acid sequence of a region other than the BC loop and the FG loop (particularly other than the BC loop, the DE loop, and the FG loop) may be identical to the amino acid sequence of the corresponding region of the wild-type Fn3 domain (e.g., SEQ ID NO: 1) or may contain one or more amino acid mutations (e.g., conservative substitutions), and preferably has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more sequence identity to the amino acid sequence. The number of 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.

[0048] In one embodiment, the Fn3 domain backbone of the polypeptide of the present invention may have, at its N-terminus, the amino acid sequence from positions 1 to 7 of SEQ ID NO: 1, or may have an amino acid sequence different from said amino acid sequence, or may not have said amino acid sequence.

[0049] In one aspect, the Fn3 domain scaffold of the polypeptide of the present invention preferably has, at or near the N-terminus (e.g., at a position 1 to 7 amino acids away from the N-terminus), the amino acid sequence from positions 8 to 20 of SEQ ID NO: 1, or an amino acid sequence having 70% or more, 75% or more, or 80% or more sequence identity to said amino acid sequence, and preferably has the amino acid sequence shown in SEQ ID NO: 13: LEVVEASPTSIQI to improve solubility.

[0050] In one preferred embodiment, the Fn3 domain backbone of the polypeptide of the present invention has 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 (3) (where X 1a ~X 1g and X 2a ~X 2i is the same as above) 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 to this amino acid sequence.

[0051] The polypeptide of the present invention has the following formula (4): [ka] (In the formula, R 1 is an amino group which may have a substituent, and R 2 is O or S, and R 3 is an optionally substituted 1,4-phenylene group. The polypeptide of the present invention preferably specifically binds to a compound represented by formula (4), and the binding dissociation constant Kd for the compound represented by 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 formula (4), and the binding dissociation constant Kd for such a compound can be in the same range as described above. The binding dissociation constant Kd can be measured according to the binding assay method described in the Examples below.

[0052] R 1 The substituent of the "amino group which may have a substituent" represented by the formula (I) is not particularly limited, and any substituent which can be introduced into the amino group can be used. Examples of the substituent include organic groups. Examples of the organic group include alkyl groups, cycloalkyl groups, aryl groups, aliphatic heterocyclic groups, and aromatic heterocyclic groups (heteroaryl groups). Among these groups, aryl groups (e.g., C groups such as phenyl groups and naphthyl groups) are preferred. 6-12 These groups may further have a substituent, and examples of the substituent include alkyl groups (e.g., C groups such as methyl, ethyl, propyl, and butyl groups). 1-6alkyl group), cycloalkyl group, aryl group, aliphatic heterocyclic group, aromatic heterocyclic group, alkoxy group, alkylthio group, monoalkylamino group, dialkylamino group, amido group (—CONH2), nitro group, halogen atom (for example, fluorine atom, chlorine atom), group combining these groups (for example, haloalkyl group such as trifluoromethyl group, -L-Ph-NH-C(═R2)-NH-R3-OH (wherein Ph is a 1,4-phenylene group which may have a substituent, L is a linker, for example, a linker having an alkylene chain, the alkylene chain may have a substituent such as an oxo group (═O) or a thioxo group (═S), and at least one methylene of the alkylene chain may be replaced by O, S, or NH), groups represented by the following formula: [ka] The substituent may be a group derived from the object to be biotin-labeled in the biotin-avidin system (for example, sugar, nucleic acid, amino acid, protein, fluorescent dye, polymer bead). In one embodiment, R 1 is preferably a monophenylamino group which may have a substituent.

[0053] In one embodiment, R 2 is preferably O.

[0054] R 3 The substituent of the "optionally substituted 1,4-phenylene group" represented by the following formula is not particularly limited, and examples thereof include R 1 In one embodiment, R 3 is preferably a 1,4-phenylene group.

[0055] In one embodiment, the compound represented by formula (4) is preferably HPU (1-(4-hydroxyphenyl)urea).

[0056] In one embodiment, the compound represented by formula (4) has the following formula (4a): [ka] (In the formula, R 1a is an optionally substituted phenyl group) It is preferable that the compound is represented by the following formula:

[0057] In one embodiment, R 1a is a phenyl group, a halogen atom, a monoalkylaminocarbonylamino group (for example, —CH—C(═O)—NH—CH—CH, etc.), or a group represented by the following formula: [ka] Preferably, it is a phenyl group substituted with .

[0058] Examples of the compound represented by formula (4a) include the following compounds. [ka]

[0059] In the compound represented by formula (4a), the phenyl group substituted with a hydroxyl group may further have a substituent, and may be represented by the following formula (4b): [ka] (In the formula, R 1a is the same as above, and R 3a , R 3a , R 3a , and R 3a are each independently a hydrogen atom or a substituent. 3a , R 3a , R 3a , and R 3a at least one of which is a substituent Such compounds are also included in the compounds represented by formula (4).

[0060] In one embodiment, R 3a , R 3a , R 3a, and R 3a is preferably a hydrogen atom, a halogen atom, or an alkyl group.

[0061] Examples of the compound represented by formula (4b) include the following compounds. [ka]

[0062] In the compound represented by formula (4a) or (4b), R 1a may be a phenyl group having multiple substituents, and when the multiple substituents include two substituents in an ortho-positional relationship, the two substituents may be bonded to each other to form a ring (e.g., a benzene ring), such as the compound shown below. Such compounds are also included in the compounds represented by formula (4). [ka]

[0063] The compound represented by formula (4a) or (4b) is a compound represented by two or more of the following formula: [ka] (wherein each benzene ring moiety may have a substituent) and a group represented by the formula (I) may be linked via a linker (for example, a linker represented by L, such as a linker having a polyethylene glycol chain or a linker having a polyglycine chain), and examples thereof include the following compounds: [ka]

[0064] The compound represented by formula (4) can be synthesized by combining known reactions. For example, the compound represented by formula (4) can be synthesized by combining a compound represented by formula: R 1 H (wherein, R 1 is the same as above) and a compound represented by the formula: HN-R 3-OH(in the formula, R 3 The compound can be synthesized by a method including a step of reacting a compound represented by the formula (wherein R is the same as above) with triphosgene.

[0065] The polypeptide of the present invention has a structure similar to that of the compound represented by formula (4), which is represented by the following formula (5): [ka] (In the formula, R 4 is an amino group which may have a substituent, and R 5 is O or S, and R 6 is an optionally substituted 1,4-phenylene group. It is preferred that the compound does not bind to the compound represented by the formula:

[0066] R 4 ~R 6 are R 1 ~R 3 corresponds to R 1 ~R 3 The same groups as those shown below can be used.

[0067] The polypeptide of the present invention can recognize the difference between the terminal group (hydroxyphenyl group) of the compound represented by formula (4) and the terminal group (benzyloxyphenyl group) of the compound represented by formula (5), for example, and can specifically bind to the compound represented by formula (4).

[0068] The polypeptides of the present invention may further comprise other amino acid sequences in addition to the Fn3 domain backbone, as long as the binding ability to the compound represented by formula (4) is not significantly impaired. The polypeptides of the present invention may be those to which proteins or peptides, such as protein tags, fluorescent proteins, luminescent proteins, signal sequences such as secretory signal sequences (e.g., Igκ signal sequence), protease recognition sequences (e.g., TEV protease recognition sequence), expression-enhancing sequences, and / or solubilizing sequences, have been added (or fused). Examples of protein tags include biotin, His tags, FLAG tags, Halo tags, MBP tags, HA tags, Myc tags, V5 tags, and PA tags.

[0069] The polypeptides of the present invention may comprise the entire or partial amino acid sequence of an antibody. Examples of antibodies include, but are not limited to, IgA, IgD, IgE, IgG, and IgM, as well as 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, or a chimpanzee-derived antibody. When using a partial amino acid sequence of an antibody, the amino acid sequence preferably contains an Fc region, which allows the antibody to exert its effector function. Furthermore, the polypeptides of the present invention can be linked via the Fc region to form multimers. Thus, the polypeptides of the present invention may have a multimerization domain and / or a linker (or hinge region) added thereto, or may form multimers formed via the domain or linker. The multimers may be homomultimers or heteromultimers. The domains are not particularly limited, as long as they are capable of binding to each other to form multimers. Examples of such domains include cartilage oligomeric matrix protein domain, leucine zipper domain, collagen-like domain, cholera toxin B subunit domain, tetrabrachion coiled core domain, reovirus σ1 protein domain, Hepatitis delta antigen domain, etc. The domain may be a part of a human dimeric protein, such as Liprin-b2 Coil2 or RP_P2 domain. The linker is not particularly limited, and may be, for example, a linker represented by the following formula: [ka] (wherein X is an n-valent organic group, n is an integer of 2 or more (e.g., 2 to 8), and Z is a binding site to a polypeptide). The peptide linker may be, for example, a glycine-rich linker, a serine-rich linker [for example, -T-(SNSS)] a -EL- (wherein a is an integer, for example, 1, 2, 3, or 4))], proline-rich linkers [for example, -(PSTPPTPS)b -(b is an integer, for example, 1, 2, 3, or 4)].

[0070] The polypeptides of the present invention may be attached (or fused) with other proteins or peptides such as toxins (eg, diphtheria toxin), albumins (eg, serum albumin), etc.

[0071] The polypeptides 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 acid salt or a basic salt can be used. Examples of acid salts include inorganic acid salts such as hydrochloride, hydrobromide, sulfate, nitrate, and phosphate; organic acid salts such as acetate, propionate, tartrate, fumarate, maleate, malate, citrate, methanesulfonate, and paratoluenesulfonate; and amino acid salts such as aspartate and glutamate. Examples of basic salts include alkali metal salts such as sodium salt and potassium salt; and alkaline earth metal salts such as calcium salt and magnesium salt.

[0072] 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, etc. The polypeptide of the present invention may also be lyophilized.

[0073] The polypeptide of the present invention may be chemically modified or may be in the form of a complex, as long as its binding ability to the compound represented by formula (4) is not significantly impaired.

[0074] The polypeptide of the present invention has a C-terminus containing a carboxyl group (-COOH), a carboxylate group (-COO - ), an amide group (-CONH), or an ester group (-COOQ). Here, Q in the ester group may be, for example, a C group such as methyl, ethyl, n-propyl, isopropyl, or n-butyl. 1-6 Alkyl groups; for example, C groups such as cyclopentyl and cyclohexyl 3-8Cycloalkyl groups such as phenyl and α-naphthyl 6-12 Aryl groups; for example, phenyl-C such as benzyl and phenethyl 1-2 Alkyl groups; α-naphthyl-C such as α-naphthylmethyl 1-2 C such as alkyl group 7-14 Aralkyl groups, pivaloyloxymethyl groups, etc. are used.

[0075] In the polypeptide of the present invention, a carboxyl group (or carboxylate) other than that at the C-terminus may be amidated or esterified. In this case, the ester used may be, for example, the C-terminal ester described above.

[0076] The polypeptide of the present invention is a polypeptide in which the amino group of the N-terminal amino acid residue is protected by a protecting group (e.g., a C group such as a formyl group or an acetyl group). 1-6 C such as alkanoyl 1-6 those in which the N-terminal glutamine residue that can be generated by cleavage in vivo is pyroglutamated; those in which the substituents on the side chains of amino acids in the molecule (e.g., -OH, -SH, amino group, imidazole group, indole group, guanidino group, etc.) are protected by an appropriate protecting group (e.g., C group such as formyl group, acetyl group, etc.); 1-6 C such as alkanoyl group 1-6 Also included are those protected by an acyl group or other suitable glycan, or conjugated proteins such as glycoproteins to which sugar chains are attached.

[0077] The polypeptide of the present invention may be modified with polyalkylene glycol. Examples of polyalkylene glycol include polyethylene glycol and polypropylene glycol. A method for modifying with polyalkylene glycol is, for example, reacting the polypeptide with polyalkylene glycol having a reactive group at the end. The reactive group is not particularly limited as long as it is a group that can react with the polypeptide, and examples thereof include groups reactive with amino groups, carboxyl groups, or thiol groups.

[0078] Examples of the reactive group with an amino group include an isothiocyano group, a carboxyl group, and an active ester group. Examples of the active ester group include a group represented by the following formula: [ka] and an N-hydroxysuccinimide ester group which may have a substituent, such as a group represented by the following formula:

[0079] Examples of the reactive group with a carboxyl group include an amino group, etc. The amino group before the reaction may be protected with a protecting group such as a Boc group (t-butoxycarbonyl group) or an Fmoc group (9-fluorenylmethyloxycarbonyl group).

[0080] Examples of the reactive group for a thiol group include a group represented by the following formula: [ka] Examples thereof include a maleimide group represented by the following formula:

[0081] The polypeptide of the present invention may be linked to a drug via a linker.

[0082] The linker is not particularly limited as long as it can link the polypeptide and the drug, and for example, all linkers used in antibody-drug conjugates (ADCs) are applicable. Examples of such linkers include linkers with an alkylene chain as the main skeleton (wherein at least one methylene of the alkylene chain may be replaced with O, S, or NH). Examples of such linkers include linkers with a polyalkylene glycol as the main skeleton.

[0083] The drug is not particularly limited and may be a low molecular weight drug or a high molecular weight drug (e.g., nucleic acid, antibody). For example, all drugs used in antibody-drug conjugates (ADCs) are applicable. A representative drug is, for example, an anticancer drug. Examples of anticancer drugs include cytotoxic anticancer drugs, such as alkylating agents, antimetabolites, platinum compounds, topoisomerase inhibitors, antibiotics, and microtubule-active agents. Examples of alkylating agents include cyclophosphamide, ifosfamide, melphalan, thiotepa, busulfan, dacarbazine, nimustine, ranimustine, carmustine, lomustine, chlorampucil, and temozolomide. Examples of antimetabolites include fluorouracil, capecitabine, gemcitabine, enocitabine, tegafur, carmofur, doxifluridine, cytarabine, mercaptopurine, fludarabine, cladribine, methotrexate, pemetrexed, and hydroxycarbamide. Examples of platinum compounds include cisplatin, carboplatin, nedaplatin, and oxaliplatin. Examples of topoisomerase inhibitors include irinotecan, nogitecan, and etoposide. Examples of antibiotics include actinomycin, doxorubicin, daunorubicin, bleomycin, peplomycin, mitomycin, aclarubicin, pirarubicin, epirubicin, idarubicin, amrubicin, and zinostatin stimalamer. Examples of microtubule-interfering agents include vincristine, vinblastine, vindesine, vinorelbine, paclitaxel, and docetaxel.

[0084] The method for binding the drug may be, for example, a method of reacting the polypeptide, the drug, and a cross-linking agent capable of forming a linker. The crosslinking agent is not particularly limited as long as it can form a linker between the polypeptide and the drug, and examples thereof include protein A, carbodiimide, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), N-succinimidyl S-acetyl-thioacetate (SATA), o-phenylenedimaleimide (oPDM), N-succinimidyl 3-maleimidopropionate, N-succinimidyl 4-maleimidobutyrate, N-succinimidyl 6-maleimidohexanoate, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexanecarboxylate (SMCC), sulfo-SMCC, and N-succinimidyl 4,7,10,13,16-pentaoxanonadecanedioate.

[0085] The polypeptides of the present invention can be easily produced by known genetic engineering techniques, such as PCR, restriction enzyme digestion, DNA ligation, in vitro transcription / translation, and recombinant protein production techniques.

[0086] 3. Polynucleotides The polynucleotide of the present invention comprises a coding sequence for the polypeptide of the present invention. The coding sequence is not particularly limited as long as it is a polynucleotide consisting of a nucleotide sequence that encodes the polypeptide of the present invention.

[0087] In one aspect, the polynucleotide of the present invention comprises an expression cassette for the polypeptide of the present invention. The expression cassette is not particularly limited as long as it is a polynucleotide that is capable of expressing the polypeptide of the present invention in cells. Typical examples of such expression cassettes include a polynucleotide comprising a promoter and a coding sequence for the polypeptide of the present invention placed under the control of the promoter.

[0088] The promoter is not particularly limited and can be selected appropriately depending on the target cell. For example, various Pol II promoters can be used. Pol II promoters are not particularly limited, but examples include the CMV promoter, EF1 promoter, SV40 promoter, and MSCV promoter. Other examples of promoters include tryptophan promoters such as trc and tac, lac promoters, T7 promoters, T5 promoters, T3 promoters, SP6 promoters, arabinose-inducible promoters, cold-shock promoters, and tetracycline-inducible promoters.

[0089] The expression cassette may contain other elements as necessary, such as a multiple cloning site (MCS), a drug resistance gene, a replication origin, an enhancer sequence, a repressor sequence, an insulator sequence, a reporter protein (e.g., a fluorescent protein) coding sequence, a drug resistance gene coding sequence, etc.

[0090] Examples of drug resistance genes include the chloramphenicol resistance gene, tetracycline resistance gene, neomycin resistance gene, erythromycin resistance gene, spectinomycin resistance gene, kanamycin resistance gene, hygromycin resistance gene, and puromycin resistance gene.

[0091] The reporter protein is not particularly limited as long as it is a luminescent (color-producing) protein that emits light (color) upon reaction with a specific substrate, or a fluorescent protein that emits fluorescence upon excitation light. Examples of luminescent (color-producing) proteins include luciferase, β-galactosidase, chloramphenicol acetyltransferase, and β-glucuronidase. Examples of fluorescent proteins include GFP, Azami-Green, ZsGreen, GFP2, HyperPer, Sirius, BFP, CFP, Turquoise, Cyan, TFP1, YFP, Venus, ZsYellow, Banana, KusabiraOrange, RFP, DsRed, AsRed, Strawberry, JRed, KillerRed, Cherry, HcRed, and mPlum.

[0092] The polynucleotide of the present invention may be in the form of a vector. An appropriate vector is selected depending on the intended use (cloning, protein expression) and the type of host cell. Examples of vectors using E. coli as a host include M13 phage or modified versions thereof, λ phage or modified versions thereof, and pBR322 or modified versions thereof (pB325, pAT153, pUC8, etc.); examples of vectors using yeast as a host include pYepSec1, pMFa, pYES2, and pPIC3.5K; examples of vectors using insect cells as a host include pAc and pVL; and examples of vectors using mammalian cells as a host include pcDNA, pCDM8, and pMT2PC.

[0093] 4.Cells The cells of the present invention are not particularly limited as long as they contain the polynucleotide of the present invention. Examples of cells include Escherichia coli such as Escherichia coli K12, Bacillus bacteria such as Bacillus subtilis MI114, yeast such as Saccharomyces cerevisiae AH22, the Sf cell line derived from Spodoptera frugiperda or the HighFive cell line derived from Trichoplusia ni, insect cells such as olfactory nerve cells, and animal cells such as COS7 cells. Preferred animal cells include cultured cells derived from mammals, specifically COS7 cells, CHO cells, HEK293 cells, HEK293FT cells, Hela cells, PC12 cells, N1E-115 cells, SH-SY5Y cells, etc.

[0094] In one embodiment, the cell of the present invention expresses the polypeptide of the present invention. For example, in one embodiment, the cell of the present invention secretes the polypeptide of the present invention or has the polypeptide of the present invention on the cell surface.

[0095] 5. Ligand-binding agents The ligand binding agent of the present invention that binds to the ligand represented by formula (4) is not particularly limited as long as it contains the polypeptide of the present invention. The Kd of the ligand binding agent for the ligand represented by 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 binding agent does not bind to the compound represented by formula (5).

[0096] 6. Pharmaceutical Compositions The pharmaceutical composition of the present invention is not particularly limited as long as it contains the polypeptide of the present invention, the polynucleotide of the present invention, or the cell 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.

[0097] The pharmaceutical composition of the present invention may further contain other drugs. The other drugs may be low molecular weight drugs or high molecular weight drugs (e.g., nucleic acids, antibodies), and examples thereof include, but are not limited to, anticancer drugs, antihypertensive drugs, antidiabetic drugs, anticardiac drugs, antipsychotic drugs, antiimmune disease drugs, antiallergic disease drugs, and antiinfective drugs.

[0098] 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.

[0099] The pharmaceutical composition of the present invention typically contains a pharmaceutically acceptable excipient or carrier, such as starch, lactose, crystalline cellulose, sorbitol, calcium hydrogen phosphate, water, ethanol, (poly)ethylene glycol, (poly)propylene glycol, glycerol, vegetable oil, etc.

[0100] 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, etc., and examples thereof include binders, disintegrants, lubricants, flavoring agents, odorants, preservatives, buffers, emulsifiers, surfactants, thickeners, dispersants, isotonicity agents, antioxidants, etc. These additives may be used alone or in combination of two or more.

[0101] The pharmaceutical composition of the present invention can be used to treat or prevent various diseases, such as cancer, hypertension, diabetes, heart disease, neuropsychiatric disorders, immune disorders, allergic diseases, and infectious diseases.

[0102] The route of administration of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include oral, intraretinal, intranasal, subcutaneous, intramuscular, intravenous, intrabronchial, intraosseous, intraarticular, intraperitoneal, intrarectal, intracolonic, and intraspinal routes.

[0103] The subjects to which the pharmaceutical composition of the present invention is administered are not particularly limited, and can be, for example, humans or non-human animals (for example, dogs, cats, cows, horses, sheep, mice, guinea pigs, and rabbits).

[0104] The frequency of administration of the pharmaceutical composition of the present invention is not particularly limited, and it can be administered, for example, once, twice, or three times a day, once every two days, or once a week.

[0105] 7. Reagents The reagent of the present invention is not particularly limited as long as it contains the polypeptide of the present invention, the polynucleotide of the present invention, or the cell of the present invention. The reagent of the present invention may further contain an excipient or carrier and / or an additive, etc., as exemplified for the pharmaceutical composition of the present invention. The reagent of the present invention may be dissolved or dispersed in a solvent, or may be lyophilized.

[0106] 8. Kit The kit of the present invention is not particularly limited as long as it contains the reagent of the present invention and a compound represented by formula (4). The compound represented by formula (4) may be dissolved or dispersed in a solvent, and in addition to the compound represented by formula (4), the kit of the present invention may also contain a solvent for dissolving or dispersing the compound.

[0107] The kit of the present invention may also contain other reagents (e.g., reagents for detecting the polypeptide of the present invention, the polynucleotide of the present invention, or the cell of the present invention), instruments, instructions for use, etc., as needed. [Example]

[0108] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0109] [Synthesis Examples of Compounds Represented by Formulas (4) and (5)] As examples of the compounds represented by formulas (4) and (5), compounds 4 and 5 were synthesized according to the following scheme. [ka]

[0110] Synthesis of compound 2 Compound 1 (400 mg, 2.0 mmol, 1.0 eq.) and triethylamine (420 μL, 3.0 mmol, 1.5 eq.) were dissolved in dichloromethane (20 mL) and stirred at 0 °C under an argon atmosphere. Triphosgene (654 mg, 2.2 mmol, 1.1 eq.) was added and stirred at 0 °C for 1 h. The solvent was removed under reduced pressure, and the residue was dissolved in N,N-dimethylformamide (DMF) (10 mL). p-amino-phenylacetic acid (303 mg, 2.0 mmol, 1.0 eq.) and triethylamine (1.05 mL, 7.6 mmol, 3.8 eq.) were added under an argon atmosphere and stirred at room temperature for 24 h. After removing the solvent under reduced pressure, the residue was washed with ethyl acetate. The resulting solid was dissolved in 20 mL of water and 2 mL of 1 M NaOH. 1 M hydrochloric acid was added until the pH reached 3, and the precipitated solid was collected by filtration and dried under reduced pressure to give target compound 2 (570 mg, 75%) as a white solid. 1 H-NMR(DMSO-d6): δ 12.22 (brs, 1H), 8.70 (brs, 1H), 8.60 (brs, 1H), 7.45-7.32 (m, 9H), 7.14 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 9.2 Hz, 2H), 5.06 (s, 2H), 3.48 (s, 2H).

[0111] Synthesis of compound 3 Compound 2 (200 mg, 0.53 mmol, 1.0 eq.) and 10% Pd / C (20 mg) were dissolved in methanol (50 mL) and stirred under a hydrogen atmosphere at room temperature for 19 hours. The Pd / C was removed by filtration through Celite. The solvent was removed under reduced pressure to give the desired compound 3 (146 mg, 96%) as a white solid. 1H-NMR(CD3OD): δ 7.34 (d, J = 8.4 Hz, 2H), 7.21-7.17 (m, 4H), 6.72 (d, J = 8.8 Hz, 2H), 3.53 (s, 2H).

[0112] Synthesis of compound 4 Compound 4 was synthesized on Sieber amide resin following standard solid phase peptide synthesis protocols utilizing the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group.

[0113] Fmoc deprotection was carried out using 20% ​​piperidine in DMF for 15 minutes at room temperature. Amino acid coupling reactions were carried out using a mixture of Fmoc-protected amino acid (3.0 equiv.), 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU, 2.9 equiv.), 1-hydroxybenzotriazole (HOBt, 2.9 equiv.), and N,N-diisopropylethylamine (DIPEA, 6.0 equiv.) in DMF at room temperature. All Fmoc deprotection and coupling steps were monitored by Kaiser assay. All washing procedures were carried out using DMF.

[0114] First, Sieber amide resin (0.79 mmol / g) (126 mg, 100 μmol) was Fmoc-deprotected and washed, followed by coupling of Fmoc-Cys(Trt)-OH to the resin and washing with DMF.

[0115] Subsequently, Fmoc-Trp(Boc)-OH, Fmoc-Adox-OH, and compound 3 were used as building blocks, and Fmoc deprotection and coupling reactions were repeated.

[0116] Trt deprotection, Boc deprotection, and cleavage from the resin were carried out using 5% triisopropylsilane (TIS) and 30% TFA in dichloromethane. The solvent was then removed under reduced pressure, and the crude product was purified by reverse-phase HPLC on a semi-preparative C18 column using a linear gradient of acetonitrile containing 0.1% TFA and 0.1% hydrated TFA to give compound 4 as a white solid. 1 H-NMR (400MHz, CD3OD): δ 7.60 (d, J = Hz, 1H), 7.43-7.29 (m, 9H), 7.22 (d, J = 8.4 Hz, 2H), 7.16 (s, 1H), 7.12-7.07 (m, 1H), 7.04-7.00 (m, 1H), 6.94 (d, J = 8.8 Hz, 2H), 5.05 (s, 2H), 4.75-4.71 (m, 1H), 4.48-4.44 (m, 1H), 4.00-3.86 (m, 6H), 3.64-3.33 (m, 28H), 2.85-2.72 (m, 2H).

[0117] Synthesis of compound 5 The same reaction as for Compound 4 in the above scheme was carried out, except that Compound 2 was used instead of Compound 3. The product was then purified by reverse-phase HPLC to obtain Compound 5 as a white solid. 1 H-NMR (400 MHz, CD3OD): δ 7.60 (d, J = 8.0 Hz, 1H), 7.36-7.32 (m, 3H), 7.22-7.17 (m, 4H), 7.15 (s,1H), 7.11-7.07 (m, 1H), 7.04-7.00 (m, 1H), 6.73 (d, J = 8.8 Hz, 2H), 4.75-4.70 (m, 1H), 4.48-4.29 (m, 1H), 4.00-3.86 (m, 6H), 3.64-3.33 (m, 28H), 2.84-2.71 (m, 2H).

[0118] [Example of polypeptide (artificial antibody, monobody) with Fn3 domain framework] material Oligonucleotides (manufactured by Fasmac Co., Ltd. or Nippon Bio Service) were used, except that for NNK (N: any base selected from A, G, C, and T; K: any base selected from G and T), trinucleotide synthesis was performed for SEQ ID NOs: 26, 27, 31, and 32. TIFF0007788106000023.tif220149

[0119] Preparation of monobody mRNA library for selection against compound 4 To prepare A-fragment DNA for the monobody library, FN3F0.F83 (1 μM), FN3F1-2.F29(P) (1 μM), FN3FF1coR8.F73(P) (0.5 μM), or FN3FF1coR10.F79(P) (0.5 μM) were ligated with T4 DNA ligase (75 pmol of each oligonucleotide, 75 μL total volume) in the presence of Fn3an1.R20(3NH2) (2 μM) and Fn3an2-1.R20(3NH2) (2 μM). The codon mixture for randomized residues had the following proportions: 20% Tyr, 10% Ser, 15% Gly, 10% Trp, and 3% Cys for all amino acids except for Cys. After ligation, the premix was added to the reaction mixture (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2% (v / v) DMSO, 2 mM MgSO, 0.2 mM each dNTP, 0.375 μM T7SD8M2.F44, 0.375 μM FN3BsaI.R40, and 2 nM PFu-S DNA polymerase) and amplified by PCR (total 15 mL, 7 cycles). B-fragment DNA was prepared similarly using FN3FF2co.F72(P), FN3F3coR10.F70(P), FN3F3coR12.F76(P), and Fn3an3.R20(3NH2) for ligation and FN3BsaI.F33 and FN3Pri2.R44 for amplification.

[0120] The 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 products were purified by phenol / chloroform extraction and isopropanol precipitation. The products were ligated together (1 μM, 200 μL) to synthesize full-length DNA products, which were then amplified using T7SD8M2.F44, G5S-4Gan21-3.R42, and Pfu-S DNA polymerase (total volume: 60 mL, 4 cycles of PCR). The products were 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 and then PAGE-purified. mRNA / HEX-mPuL was prepared in a similar manner. The resulting complex was used in the first round of selection.

[0121] Immobilization of small molecule ligands on magnetic beads Unmodified Dynabeads M270 Amine (20 μL) was suspended and separated. The magnetic beads were collected with a magnet and the supernatant was removed. After washing five times with DMF, 10 μL of 2 M DIC in DMF and 10 μL of 2 M bromoacetic acid in DMF were added sequentially and suspended. The reaction was carried out by mixing at 25°C for 10 minutes. After washing five times with DMF, 4 μL of the prepared ligand solution (0.1 mM Compound 4 or 5, 100 mM HEPES-K pH 8.0, 600 mM NaCl, 50% DMF) was added and suspended. The reaction was carried out by stirring at 37°C for 30 minutes. The immobilization rate and concentration were measured by tryptophan fluorescence in the reaction solution. The beads were suspended in 20 μL of 1-thioglycerol solution (500 mM 1-thioglycerol, 100 mM HEPES-K pH 8.0, 600 mM NaCl, 50% DMF), and the reaction was carried out with stirring at 37 °C for 2 hours. After washing with DMF and 10 mM Tris-HCl pH 8.0, beads were added with bead storage solution (10 mM AcONa pH 5, 1 mM DTT) and stored at 4 °C.

[0122] In vitro selection In the first round of selection, 1 μM mRNA / Pu-OMe-linker was added to the reconstituted translation system, and the reaction mixture (500 μL) was incubated at 37°C for 30 minutes. After the reaction, 41.7 μL of 200 mM EDTA (pH 8.0) was added to the translation mixture. Reverse transcription buffer (41.1 μL of 0.78 M Tris-HCl pH 8.4, 1.16 M KCl, 0.37 M MgCl, 0.08 M DTT), 5 mM dNTPs (66.7 μL), 100 μM FN3S.R29 (10 μL), and 28.7 μM HMLV (27.5 μL) were added to the translation mixture, and the resulting solution was incubated at 42°C for 15 minutes. The buffer was exchanged into HBST buffer using a Zebra™ spin desalting column. The resulting solution was mixed with Dynabeads M270 Amine loaded with compound 4 at 25°C for 20 minutes. After target protein recovery using the beads, the resulting beads were washed twice with HBST buffer and PCR premix (690 μL) 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 amplified by PCR using T7SD8M2.F44, G5S-4Gan21-3.R42, and Pfu-S DNA polymerase, and purified by phenol / chloroform extraction and isopropanol precipitation. The purified DNA was transcribed using T7 RNA polymerase and purified by phenol / chloroform extraction and isopropanol precipitation.

[0123] In the second round of selection, 1 μM mRNA / Pu-OMe-linker was added to the reconstituted translation system, and the reaction mixture (10 μL) was incubated at 37°C for 30 minutes. After the reaction, 1.8 μL of 100 mM EDTA (pH 8.0) was added to 9 μL of the translation mixture. Reverse transcription mixture (5.4 μL; 150 mM Tris-HCl pH 8.4, 225 mM KCl, 75 mM MgCl2, 16 mM DTT, 1.5 mM dNTPs, 7.5 μM FN3S.R29 (primer), 3.4 μM HMLV) was added to the translation mixture, and the resulting solution was incubated at 42°C for 15 minutes. The buffer was exchanged into HBST buffer using a Zebra™ spin desalting column. The resulting solution was mixed with compound 5 - Dynabeads M270 Amine at 25°C for 5 minutes, and then the solution was collected. The mixture with compound 5 and Dynabeads M270 Amine was repeated a total of eight times. The solution was collected and mixed with compound 4 and Dynabeads M270 Amine at 25°C for 20 minutes. After target protein collection using the beads, the resulting beads were washed twice with HBST buffer and PCR premix (50 μL) was added. cDNA quantification, DNA amplification, and purification (no transcription) were performed in the same manner as in the first round of selection.

[0124] In the third round of selection, the resulting DNA (approximately 5 nM final concentration) was added to the TRAP system, and the reaction mixture (5 μL) was incubated at 37°C for 30 minutes. After the reaction, 1 μL of 100 mM EDTA (pH 8.0) was added to the translation mixture. The reverse transcription mixture (3 μL; 150 mM Tris-HCl pH 8.4, 225 mM KCl, 75 mM MgCl, 16 mM DTT, 1.5 mM dNTPs, 7.5 μM FN3S.R29 (primer), 3.4 μM HMLV) was added to the translation mixture, and the resulting solution was incubated at 42°C for 15 minutes. The buffer was exchanged for HBST buffer using a Zebra™ spin desalting column. Mixing with beads, bead washing, cDNA quantification, DNA amplification, and purification were performed in the same manner as in the second round of selection.

[0125] The fourth to sixth rounds of selection were carried out in the same manner as the third round of selection.

[0126] In the seventh to tenth rounds of selection, the concentration of compound 4 in the system was changed from 500 nM to 50 nM. Other operations were the same as in the third round of selection.

[0127] The cDNA recovery rate for each round was calculated based on the following formula: cDNA recovery rate (%) = amount of recovered cDNA × 100 / amount of puromycin linker. The cDNA recovery rate for each round is shown in Figure 1. As is clear from Figure 1, the cDNA recovery rate increased as the rounds progressed. Furthermore, the low recovery rate for compound 5 suggested that an artificial antibody with high specificity for compound 4 had been recovered. The sequence of the DNA recovered in the 10th round was analyzed using a next-generation sequencer. As a result, SEQ ID NO: 14: MQANSGSLEVVEASPTSIQISWDA YSHYWMDAWD VRYYRITYGETGGNSPVQEFTVPG SKS TATISGLKPGVDYTITVYAVT LWYYYKWWVD PISINYRT The sequence shown in Figure 1 (Monobody-1) was found to account for a very high proportion of the total reads (approximately 70%). Monobody-1 was expressed in E. coli, purified by Ni-NTA, and affinity measurements were performed on compounds 4 and 5 immobilized on a streptavidin biosensor (ForteBio) using the Octet system (ForteBio, CA, USA). Binding assays were performed at 30°C using assay buffer (50 mM Hepes-KOH pH 7.5, 300 mM NaCl, 0.1% (v / v) Tween 20, and 1% (w / v) PEG 6000). Each step of the binding assay consisted of 300 seconds of equilibration, 1000 seconds of association, and 1000 seconds of dissociation. Monobody-1 showed an affinity of 33.5 nM for compound 4. It did not bind to compound 5, demonstrating high specificity.

[0128] Mutation saturation library construction To further improve the affinity and specificity of the artificial antibodies, we constructed four new mutation saturation libraries using Monobody-1 as the parent clone and its sequence as a template. To prepare the A-fragment DNA for the monobody libraries, FN3F0.F83 (1 μM), FN3F1-2.F29(P) (1 μM), FN3FF1NNK6L.F79 (1 μM, corresponding to library A), FN3FF1NNK6R.F79 (1 μM, corresponding to library B), or FN3FF1temp.F79 (1 μM, used for libraries C and D) were ligated with T4 DNA ligase (5 μL total) in the presence of Fn3an1.R20(3NH2) (2 μM) and Fn3an2-1.R20(3NH2) (2 μM). After ligation, the premix was added to the reaction mixture (10 mM Tris-HCl pH 8.4, 100 mM KCl, 0.1% (v / v) Triton X-100, 2% (v / v) DMSO, 2 mM MgSO, 0.2 mM each dNTP, 0.375 μM T7SD8M2.F44, 0.375 μM FN3BsaI.R40, and 2 nM PFu-S DNA polymerase) and amplified by PCR (total 400 μL, 6 cycles). B-fragment DNA was prepared similarly using FN3FF2co.F72(P), FN3F3NNK6L.F70 (for library C), FN3F3NNK6R.F70 (for library D), or FN3F3temp.F70 (used for libraries A and B), and Fn3an3.R20(3NH2) for ligation, and FN3BsaI.F33 and FN3Pri2.R44 for amplification.

[0129] The 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 products were purified by phenol / chloroform extraction and isopropanol precipitation. The corresponding products (0.15 μM, 10 μL) were ligated to synthesize full-length DNA products, which were then amplified using T7SD8M2.F44, G5S-4Gan21-3.R42, and Pfu-S DNA polymerase (400 μL total volume, 6–8 cycles of PCR). The products were 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 in a similar manner. The resulting complexes were used in the first round of selection.

[0130] Reselection of artificial antibodies In the second first-round selection, 1 μM mRNA / Pu-OMe-linker was added to the reconstituted translation system, and the reaction mixture (10 μL) was incubated at 37°C for 30 minutes. After the reaction, 1 μL of 100 mM EDTA (pH 8.0) was added to 9 μL of the translation mixture. 3 μL of reverse transcription mixture (150 mM Tris-HCl pH 8.4, 225 mM KCl, 75 mM MgCl, 16 mM DTT, 1.5 mM dNTPs, 7.5 μM FN3S.R29 (primer), 3.4 μM HMLV) was added to the translation mixture, and the resulting solution was incubated at 42°C for 15 minutes. The buffer was exchanged into HBST buffer using a Zebra™ spin desalting column. The resulting solution was mixed with Dynabeads M270 Amine immobilized with compound 5 at 25°C for 10 minutes, and then the solution was recovered (the concentration of compound 5 in the system was 2.5 μM). This mixing with Dynabeads M270 Amine immobilized with compound 5 was repeated four times. The solution was then recovered and mixed with Dynabeads M270 Amine immobilized with compound 4 at 25°C for 10 minutes (the concentration of compound 4 in the system was 50 nM). After the target protein was recovered using the beads, the resulting beads were washed twice with HBST buffer and PCR premix (50 μL) was added. cDNA quantification, DNA amplification, and purification (transcription was not performed) were performed in the same manner as in the first round of selection.

[0131] In the second to fifth rounds of selection, the obtained DNA (final concentration: approximately 5 nM) was added to the TRAP system, and the reaction mixture (5 μL) was incubated at 37°C for 30 minutes. The following procedures were carried out in the same manner as in the first round of selection.

[0132] Artificial antibody selection was performed for four rounds for libraries A and C, and for five rounds for libraries B and D. The results are shown in Figure 2. As is clear from Figure 2, the recovery rate of cDNA increased with each round. The sequence of the DNA recovered in the final round was determined using a next-generation sequencer. The amino acid occurrence rate in the randomized region of the BC loop was calculated from libraries A and B, and the amino acid occurrence rate in the randomized region of the FG loop was calculated from libraries C and D. The results, normalized to a percentage, are shown in Tables 5 to 8. In Tables 5 to 8, the symbol O indicates a read-through stop codon.

[0133] [Table 5]

[0134] [Table 6]

[0135] [Table 7]

[0136] [Table 8]

[0137] As shown in Table 9, a total of 11 types of Monobodies (Monobodies-2 to 12) were produced, including the sequences that appeared most frequently in each library and sequences that were combinations of these. [Table 9]

[0138] The affinity of Monobodies 1 to 12 for Compound 4 was analyzed using Octet in the same manner as described above. The results are shown in Table 10. [Table 10]

Claims

1. A polypeptide comprising a fibronectin type III domain scaffold, The BC loop of the fibronectin type III domain scaffold has the following formula (1): YX １ａ X １ｂ X １ｃ X １ｄ X １ｅ DX １ｆ X １ｇ D (1) (In the formula, X １ａ is A, S, or R; X １ｂ and X １ｃ are each independently any amino acid, X １ｄ is N, H, W, or Y; X １ｅ ~X １ｇ are each independently any amino acid) The amino acid sequence represented by The FG loop of the fibronectin type III domain scaffold has the following formula (2): X ２ａ X ２ｂ X ２ｃ X ２ｄ X ２ｅ KX ２ｆ X ２ｇ X ２ｈ X ２ｉ (2) (In the formula, X ２ａ and X ２ｂ are each independently any amino acid, X ２ｃ is G, M, N, Y, or W; X ２ｄ is any amino acid, 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 any amino acid) The polypeptide, which is capable of specifically binding to 1-(4-hydroxyphenyl)-3-phenylurea or a derivative thereof, comprises an amino acid sequence represented by the following formula:

2. In the formula (1), 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; X １ｇ is E, L, K, or W.

3. 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 Amino acid sequences that have 70% or more sequence identity with these amino acid sequences 3. The polypeptide of claim 1 or 2, selected from the group consisting of:

4. In the formula (2), X ２ａ is W or L, X ２ｂ is W or Y; X ２ｃ is G or Y, X ２ｄ is Y, S, or T; X ２ｅ is Y, X ２ｆ is W or Y; X ２ｇ But W, X ２ｈ is V, X ２ｉ The polypeptide according to any one of claims 1 to 3, wherein is P, A, or D.

5. The amino acid sequence represented by the formula (2) is WWGSYKWWVP (SEQ ID NO: 9), WWGSYKWWVD (SEQ ID NO: 10), LWYYYKWWVP (SEQ ID NO: 11), LWYYYKWWVD (SEQ ID NO: 12), and Amino acid sequences that have 70% or more sequence identity with these amino acid sequences The polypeptide according to any one of claims 1 to 4, selected from the group consisting of:

6. The following formula (4): 【Chemistry 1】 (In the formula, R １ is an amino group which may have a substituent, and R ２ is O or S, and R ３ represents a 1,4-phenylene group which may have a substituent. The polypeptide according to any one of claims 1 to 5, wherein the binding dissociation constant Kd for a compound represented by the formula (I) is 50 nM or less.

7. A polynucleotide comprising a coding sequence for the polypeptide of any one of claims 1 to 6.

8. A cell comprising the polynucleotide of claim 7.

9. The following formula (4): 【Chemistry 2】 (In the formula, R １ is an amino group which may have a substituent, and R ２ is O or S, and R ３ represents a 1,4-phenylene group which may have a substituent. A ligand binding agent that binds to a ligand represented by the formula:

10. A reagent comprising the polypeptide according to any one of claims 1 to 6, the polynucleotide according to claim 7, or the cell according to claim 8.

11. The reagent according to claim 10 and a compound of the following formula (4): 【Transformation 3】 (In the formula, R １ is an amino group which may have a substituent, and R ２ is O or S, and R ３ represents a 1,4-phenylene group which may have a substituent. and a compound represented by the formula:

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