HSA-binding protein

Abstract

To provide HSA-binding protein that is considered to increase retentivity of a medicine.SOLUTION: Protein in which protein chain B made up of 9 to 11 pieces of amino acid having an amino acid sequence shown by sequence number 3 is peptide-bonded between C-terminal of protein chain A shown by sequence number 1 and N-terminal of protein chain C shown by sequence number 2. Note that all of amino acid X in sequence number 1,2,3 are arbitrary L-amino acid normally found out in protein present in nature, amino acid X of N-terminal in sequence number 3 is G or deleted, 6th amino acid X from N-terminal is any of V,A,R, 7th amino acid X from N-terminal is any of E,V,S, 10th amino acid X from N-terminal is R or V, and 11th amino acid X from N-terminal is G or deleted.SELECTED DRAWING: Figure 6

Description

[Technical Field]

[0001] The present invention relates to human serum albumin binding proteins. [Background technology]

[0002] Human serum albumin (HSA) accounts for approximately 60% of serum proteins and transports fatty acids, bilirubin, metal ions, and other molecules through the blood. While most proteins are degraded by lysosomes after entry into cells, HSA binds to the cell surface receptor FcRn, allowing it to escape lysosomal degradation and be released back into the extracellular space (HSA recycling function). Therefore, by conjugating various drugs to HSA-binding proteins, they can be taken up into cells along with HSA and then released back into the extracellular space, potentially extending the drug's blood half-life. For example, insulin regulates blood glucose levels. Diabetic patients require insulin (insulin preparations) for diabetes control. The recycling function of HSA could extend the blood half-life of insulin, reducing the frequency of administration and reducing the dose. From this perspective, for example, Patent Documents 1 and 2 propose HSA-binding ligands containing specific amino acid sequences as HSA-binding proteins.

[0003] The present inventors have proposed a protein (hereinafter referred to as "HLH protein") with a helix-loop-helix structure that is relatively low in molecular weight and maintains a stable three-dimensional structure in blood (see, for example, Patent Document 1). This HLH protein has A chains and C chains with α-helices, and B chain, also called the loop chain, that connects these A chains and C chains. The A chain and C chain each consist of 14 amino acids, and the seven alanine residues in the outward-facing positions of the A chain and C chain maintain a stable structure even if substituted with any amino acid. HLH proteins with various functions have been proposed (see, for example, Patent Documents 1 to 3), but no HSA-binding HLH protein has been discovered to date. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 2003-518075 [Patent Document 2] Special Publication No. 2004-535373 [Patent Document 3] JP 2014-1189 A [Patent Document 4] Japanese Patent Application Laid-Open No. 2011-231085 [Patent Document 5] Japanese Patent Application Laid-Open No. 2008-214254 Summary of the Invention [Problem to be solved by the invention]

[0005] The present inventors focused on the recycling function of HSA and believed that if HLH proteins that have a stable structure in blood could be bound to HSA, it would be possible to extend the half-life of various drugs in blood. An object of the present invention is to provide proteins having various HSA-binding ligands, preferably proteins that can adopt an HLH protein structure. [Means for solving the problem]

[0006] The HSA-binding protein of the present invention has a ligand having the amino acid sequence shown in SEQ ID NO:3, and typically has the amino acid sequence shown in SEQ ID NO:11. [Effects of the Invention]

[0007] According to the present invention, an HSA-binding protein that can contribute to the blood half-life is provided. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows a vector map of the phagemid vector used in the phage surface-displayed protein library. [Figure 2] FIG. 2 shows a vector map of the yeast transformation vector used in error-prone PCR. [Figure 3] FIG. 3 is a schematic diagram showing the procedure for yeast transformation by homologous recombination. [Figure 4] FIG. 4 (A) to (E) are diagrams showing the affinity for HSA of yeast presenting HSA-binding proteins, which are examples of the present invention. [Figure 5] Figure 5 is a schematic diagram showing a method for synthesizing a conjugate of HSA-binding protein and insulin, in which (A) shows the steps for synthesizing an alkyne derivative of insulin, (B) shows the steps for synthesizing an azide derivative of HSA-binding protein, and (C) shows the steps for synthesizing the conjugate. [Figure 6] FIG. 6 shows the effect of the HSA-binding protein and insulin complex on insulin retention. DETAILED DESCRIPTION OF THE INVENTION

[0009] The HSA-binding ligand of the present invention has the amino acid sequence shown in SEQ ID NO: 3 (wherein the amino acid X constituting SEQ ID NO: 3 is any L-amino acid commonly found in naturally occurring proteins, the N-terminal amino acid X is G or is deleted, the sixth amino acid X from the N-terminus is V, A, or R, the seventh amino acid X from the N-terminus is E, V, or S, the tenth amino acid X from the N-terminus is R or V, or the eleventh amino acid X from the N-terminus is G or is deleted), such as an amino acid sequence shown in SEQ ID NO: 4 to 8. An HSA-binding protein having this ligand may be, for example, a protein having a helix-loop-helix structure comprising an A chain and a C chain each having an α-helical structure and a B chain (also called a loop chain) connecting the A chain and the C chain. The protein having a helix-loop-helix structure is preferably a protein consisting of 41 to 43 amino acids, for example, the amino acid sequence shown in SEQ ID NO: 9. A protein having this helix-loop-helix structure has, for example, the amino acid sequence shown in SEQ ID NO: 1 in chain A, the amino acid sequence shown in SEQ ID NO: 2 in chain C, and the amino acid sequence shown in SEQ ID NO: 3 in chain B. In the present invention, the amino acids constituting the HSA-binding protein preferably refer to amino acids that are normally found in naturally occurring proteins.

[0010] The HSA-binding protein of the present invention is characterized by having the amino acid sequence of SEQ ID NO: 3 in the loop region, and the ligand consisting of the amino acid sequence of SEQ ID NO: 3 is a sequence commonly found in converged HSA-binding proteins in a population of proteins that bind relatively strongly to HSA, as shown in the Examples. The loop region may lack one or both Gs at either end of the amino acid sequence of SEQ ID NO: 3, and the Gs at both ends may be replaced by other amino acids. This is because, even if both or either end of the C chain, which constitutes the loop region, is deleted, the distance between the A chain and C chain, which have two α-helices (the distance between the surfaces facing inward), does not change significantly, and it can be said that the protein binds stably to HSA.

[0011] In general, HLH proteins maintain a stable three-dimensional structure due to hydrophobic interactions between the leucine side chains located inside the two α-helices, and are further stabilized by disulfide bonding between the cysteine at the N-terminus of the A chain and the cysteine at the C-terminus of the C chain to form a cyclized structure. Therefore, regardless of whether cyclization occurs, as long as this stable three-dimensional structure is maintained, the amino acids that make up the two α-helices (the A chain and the C chain) and the amino acids facing outward from the two α-helices can be substituted as desired. In particular, while the HSA-binding HLH protein of a preferred embodiment of the present invention has the amino acid sequence shown in SEQ ID NO: 11 (protein AY01), some of the alanines that make up the two α-helices, for example, those at positions 3, 13, 30, 31, 34, 37, 38, and 41 from the N-terminus of the HLH protein (hereinafter, the amino acid positions refer to the positions from the N-terminus of the HLH protein), can be substituted with other amino acids, as shown in SEQ ID NO: 10. Furthermore, because HSA binding is maintained as long as the HLH protein structure is stabilized, all of the A amino acids facing outward from the two α-helices, i.e., the A's at positions 2, 3, 6, 7, 10, 13, 14, 30, 31, 34, 37, 38, 41, and 42 from the N-terminus of the HLH protein, can be substituted with any other amino acid, while still maintaining a stable structure (protein having the amino acid sequence of SEQ ID NO: 9). Even when amino acids other than the A's constituting the two α-helices are substituted, it is sufficient that the loop portion of the B chain contains the aforementioned characteristic seven amino acid sequence, i.e., the amino acid sequence of SEQ ID NOs: 3 to 8 minus the G's at both ends, and that the loop portion is composed of 9 to 11 amino acids sufficient to maintain good binding between the two α-helices. Of course, the HLH proteins of the present invention do not necessarily need to maintain a stable three-dimensional structure.

[0012] Amino acids that can be substituted for the alanine that constitutes the α-helix include, in chain A, the third A from the N-terminus of the HLH protein may be T or S, the sixth A may be S, and the thirteenth A may be S; in chain C, the 30th A from the N-terminus of the HLH protein may be D or N, the 31st A may be F, the 34th A may be Q, G, or S, the 37th A may be E, P, or H, the 38th A may be V or F, and the 41st A may be D or N; and these substitutions may occur at one or more positions.

[0013] In the HLH protein of the present invention, amino acids other than alanine that constitute the α-helix can also be substituted. In the two α-helices, the following amino acids can be substituted: in the A chain, the fourth amino acid E from the N-terminus of the HLH protein can be substituted with D; the fifth amino acid L can be substituted with P or Q; the ninth amino acid E can be substituted with K; and the twelfth amino acid L can be substituted with R or Q. In the C chain, the 28th amino acid K from the N-terminus of the HLH protein can be substituted with E; the 29th amino acid L can be substituted with R; the 33rd amino acid K can be substituted with R; the 40th amino acid K can be substituted with N or R; and the 43rd amino acid C can be substituted with S. One or more of these amino acids can be substituted. Of these, the substitution of leucine (L), which is considered important for maintaining the three-dimensional structure of the HLH protein, is preferably one or two, and more preferably one, in each of the A chain and C chain.

[0014] The HLH protein of the present invention exhibits HSA binding even when one to three amino acids from the N-terminus of the B chain (loop chain) of the amino acid sequence shown in SEQ ID NO: 4 are substituted, particularly V at position 22, E at position 23, and R at position 26. For example, the HLH protein has V at position 22 substituted with A or R, E at position 23 substituted with V or S, and R at position 26 substituted with V. Specifically, the HLH protein has the B chain amino acid sequences shown in SEQ ID NOs: 5 to 8. In proteins with amino acid substitutions in the B chain, the α-helix A and C chains may have the amino acid sequences shown in SEQ ID NOs: 1 and 2. Preferably, in the A chain, E at position 4 from the N-terminus of SEQ ID NO: 1 is substituted with D, and L at position 5 is substituted with Q. Furthermore, the C chain preferably has K at position 28 substituted with E, and L at position 29 is substituted with R. These substitutions may occur at one or more positions. The amino acids in the α-helix are as follows: the 6th amino acid X is A or S, the 30th amino acid X is A or N, the 31st amino acid X is A or F, the 34th amino acid X is A or S, the 37th amino acid X is A or H, the 38th amino acid X is A or F, and the 41st amino acid X can also be A or N.

[0015] More specifically, an HSA-binding protein is a protein having the amino acid sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10. In the amino acid sequence, amino acid X is preferably A, but the third amino acid A may be substituted with T or S, the sixth amino acid A with S, the thirteenth amino acid A with S, the thirtyth amino acid A with D or N, the thirty-first amino acid A with F, the thirty-fourth amino acid A with Q, G, or S, the thirty-seventh amino acid A with E, P, or H, the thirty-eighth amino acid A with V or F, or the forty-first amino acid A with D or N. Furthermore, the 28th amino acid K from the N-terminus may be substituted with E, the fifth amino acid L with P or Q, the ninth amino acid E with K, the twelfth amino acid L with Q or P, the thirty-third amino acid K with R, the fortieth amino acid K with N or Q, and the fortieth amino acid C with S. These substitutions of amino acids A and amino acids other than A may occur at one or more positions.

[0016] A specific example of an HSA-binding protein is a protein having the amino acid sequence shown in SEQ ID NO: 10, in which the amino acid K at position 28 is substituted with E. In this case, too, the amino acid X in the amino acid sequence is preferably A, but the amino acid A at position 3 can also be T or S, and the amino acid A at position 13 can also be S. Furthermore, the amino acid E at position 9 can be substituted with K, the L at position 29 with R, the K at position 33 with R, the K at position 40 with N, and the C at position 43 with S.

[0017] Other examples of HSA-binding proteins include a protein having the amino acid sequence set forth in SEQ ID NO: 10 in which the amino acid L at the fifth position from the N-terminus is substituted with P, a protein in which the amino acid E at the fourth position from the N-terminus is substituted with D, and a protein in which the L at the twelfth position from the N-terminus is substituted with Q or R. In these proteins, the amino acid X is preferably A, but particularly in the protein in which the amino acid L at the fifth position from the N-terminus is substituted with P, the amino acid X at the 30th position from the N-terminus may be A, D, or N, the amino acid X at the 34th position may be A, Q, G, or S, the amino acid X at the 37th position may be A, E, P, or H, the amino acid at the 38th position may be A or V, and the amino acid X at the 41st position may be A, D, or N.

[0018] When the HLH protein of the present invention has a sulfide group at the N-terminus of the A chain and the C-terminus of the C chain, it is preferable that the N-terminus of the A chain and the C-terminus of the C chain form a disulfide bond to form a cyclic structure.

[0019] The HLH protein of the present invention has the ability to bind to HSA and can extend the blood circulation in animals, particularly humans. Examples of drugs that can be used include, but are not limited to, insulin, glucagon-like peptides, human growth hormone, interleukin 2, and G-CSF (granulocyte-colony stimulating factor), which have previously been considered to have short blood circulation.

[0020] The method for producing the HSA-binding protein of the present invention comprises the steps of: substituting at least one of the amino acids constituting the protein shown in SEQ ID NO: 11 (however, the 17th and / or 27th G from the N-terminus may be deleted) with any L-amino acid normally found in other naturally occurring proteins; contacting the protein having the substituted amino acid sequence with HSA; determining the amino acid sequence of the HSA-binding protein; and synthesizing the protein from the determined amino acid sequence.

[0021] The protein before substitution is an HLH protein that has been shown to bind to HSA, and this method involves searching for modified proteins that bind to HSA. In particular, HLH proteins that retain the amino acid sequence of the loop region are thought to exhibit HSA binding.

[0022] The amino acids to be substituted are at least one of any amino acids in the protein consisting of chains A, B, and C, and are preferably the 2nd, 3rd, 6th, 7th, 10th, 13th, 14th, 30th, 31st, 34th, 37th, 38th, 41st, and 42nd amino acids from the N-terminus, i.e., at least one amino acid A facing outward from the α-helices of chains A and C. The amino acids may also be amino acids constituting chain B, which is the loop portion, but substitution is preferably made so as to retain the amino acid sequence of chain B, which is the loop portion.

[0023] The contact of HLH proteins with HSA is a step for confirming their binding to HSA. The contacting method is not particularly limited, and HLH proteins that have been confirmed to bind to HSA are then separated from other HLH proteins. The determination of binding to HSA can be appropriately determined by, for example, the concentration of HSA used for contact. HSA-binding proteins that have been confirmed to bind are then separated using a magnet, for example, by binding to an anti-biotin antibody bound to magnetic beads. The amino acid sequences of the separated HSA-binding proteins can then be determined by various methods.

[0024] The HLH protein may be produced by various known chemical methods, such as solid-phase synthesis, or by transforming yeast or phage with a vector such as a plasmid that is configured to express the substituted protein.

[0025] The amino acid substitution method may be either a method in which the target amino acid or the base corresponding to that amino acid is specifically substituted, or a method in which the amino acid is randomly substituted. In the former method, the corresponding amino acid or base can be used during the chemical synthesis of the protein. In the latter method, proteins with diverse amino acid sequences can be simultaneously produced (expressed) by using a plasmid into which a base sequence for randomization has been introduced so that any amino acid can be introduced. The latter method produces a library of so-called HSA-binding proteins. In this case, a plasmid that displays the protein on the surface of yeast or phage is preferably used. This is because it allows for the simple synthesis of a wide variety of proteins and the easy confirmation of their binding to HSA.

[0026] Alternatively, proteins with substituted amino acids may be produced by error-prone PCR using the base sequence determining the protein before substitution as a template. Error-prone PCR is also known, and is a method of amplifying genes encoding HSA proteins having the amino acid sequences of SEQ ID NOS: 4 to 8, such as proteins having HSA-binding amino acid sequences shown in SEQ ID NOS: 11 and 34, by PCR (polymerase chain reaction) with reduced replication strictness. HSA-binding proteins can be synthesized using genes into which mutations have been introduced using this method, or by transforming yeast or other organisms with vectors containing such genes, in a manner similar to that described above. In this manner, HSA-binding proteins with various amino acid sequences can be produced.

[0027] The present invention will now be described with reference to the following examples. [Example]

[0028] [HSA-binding protein screening 1] Based on the report by Ramanayake et al. (Ramanayake Mudiyanselage TMR et al. An Immune-Stimulatory Helix-Loop-Helix Peptide: Selective Inhibition of CTLA-4-B7 Interaction. ACS Chem. Biol. 15, 360-368 (2020)), we created a protein library in which HLH proteins were expressed on the yeast cell surface using agglutinin present on the yeast cell surface. The HLH proteins constituting the library have the amino acid sequence shown in SEQ ID NO: 38 (CAAELAALEAELAALEGXXXXXXXXXGKLAALKAKLAALKAAC), with nine amino acids in the loop randomized. In addition, a vector was constructed to introduce the HLH proteins into yeast so that they could express a FLAG tag via a linker at the C-terminus.

[0029] The library was expressed by culturing yeast cells, and magnetic beads bearing biotinylated HSA and anti-biotin antibodies were added to the cell suspension, allowing the library to react. After the reaction, yeast expressing proteins that bound to HSA were separated from the cell suspension using a column equipped with a magnet. The isolated yeast were then further cultured and subjected to similar screening several times to enrich for yeast expressing HSA-binding proteins.

[0030] The enriched yeast was incubated with anti-FLAG tag mouse antibody and biotinylated HSA, followed by the addition of streptavidin-conjugated fluorescent label APC (Allophycocyanin) and anti-mouse IgG goat antibody-conjugated fluorescent label Alexa488 to obtain a labeled HSA-binding protein library. The fluorescence intensity of this library was monitored using a flow cytometer. Since APC fluorescence intensity indicates the amount of HSA bound to HLH proteins, and Alexa488 fluorescence indicates the expression level of HLH proteins on the yeast surface, yeast populations with high fluorescence intensity for both were sorted. The sorted yeast were then re-cultured and subjected to the same procedure. Eight clones were selected from the cell populations that showed high fluorescence intensity, and their DNA sequences were analyzed. These clones demonstrated the expression of a protein (AY01) with the amino acid sequence (CAAELAALEAELAALEGVNFGVELFRGKLAALKAKLAALKAAC) shown in SEQ ID NO: 11.

[0031] Furthermore, when a single yeast strain displaying the protein was used to evaluate its binding to HSA and MSA (mouse serum albumin) using flow cytometry, it was confirmed that the protein (AY01) had binding affinity to both HSA and MSA.

[0032] Furthermore, when the protein AY01 was synthesized using a peptide synthesizer by the Fmoc solid-phase synthesis method, it was confirmed that this protein also had binding affinity to both HSA and MSA, and that it retained its three-dimensional structure by CD spectroscopy. [Example]

[0033] [HSA-binding protein screening 2] Next, we constructed a phage-displayed protein library using the phage expression plasmid (pComb3d) based on the paper by Fujii et al. (Fujii, I. et al. Evolving catalytic antibodies in a phage-displayed combinatorial library. Nat. Biotechnol. 16, 463 (1998)) (see Figure 1). The proteins constituting this library have the amino acid sequence shown in SEQ ID NO: 39 (CAAELAALEAELAALEGXXXXXXXXXGKLXXLKXKLXXLKXAC). In addition to the amino acids in the loop region, the amino acids constituting the C-terminal helix region were also randomized. Furthermore, the helix region was designed to avoid proline residues, which destabilize the three-dimensional structure, as well as alanine and threonine residues. The vector map for the library is shown in Figure 1.

[0034] The resulting library was subjected to repeated biopanning to screen for phages expressing proteins that bind to HSA. The amino acid sequence of the resulting phage protein was determined, and a protein (AY03) was obtained having the amino acid sequence (CAAELAALEAELAALEGVNFGRSLFVGKLNFLKSKLHFKANAC) shown in SEQ ID NO: 34, which is similar to the AY01 protein obtained in Example 1. [Example]

[0035] [Modified from AY01 and AY03] Using the genes encoding AY01 and AY03 as templates, error-prone PCR was performed three times with primers NcoI forward and new lib (ss) reverse to introduce mutations into the DNA base sequence. PCR was then performed again on the mutated DNA using primers HR forward and HR reverse to generate inserts with sequences homologous to the pYD11-BxXN vector (see Figure 2) at their termini. These inserts were mixed with the vector digested with restriction enzymes NcoI-HF and XhoI and introduced into yeast EBY100 by electroporation. A secondary library was generated, displaying the protein library on the yeast surface (see Figure 3).

[0036] The library was screened using magnetic beads in the same manner as in Example 1 to enrich the yeast. Flow cytometry was performed on the enriched yeast in the same manner as in Example 1, and several clones were selected from the yeast population with strong binding to HSA, resulting in proteins having the amino acid sequences shown in Table 1 (AY01-1 to AY01-13, AY03-2 to AY03-9: SEQ ID NOS: 12 to 32). Table 1 shows a list of the amino acid sequences mentioned in the present invention. In Table 1, "-" indicates an amino acid that is the same as the amino acid in the amino acid sequence of SEQ ID NOS: 11 (AY01).

[0037] [Table 1]

[0038] Because glutamine frequently appears at the 28th amino acid from the N-terminus in proteins AY01-1 to AY01-8, we investigated the affinity for HSA of five proteins: three proteins in which glutamine appears, AY01-1 (SEQ ID NO: 12), AY01-12 (SEQ ID NO: 31), and AY01-13 (SEQ ID NO: 32), and two proteins in which glutamine does not appear, AY01 (SEQ ID NO: 11) and AY01-11 (SEQ ID NO: 30). As a result, as shown in Figure 4, proteins with glutamine at the 28th amino acid from the N-terminus were found to have a higher affinity for HSA than proteins AY01 and AY01-11 that do not have glutamine.

[0039] In addition, a protein (AY-all: sequence number 33) was synthesized that contained all of the mutations found in the three proteins (AY01-11 to 13) that had mutations in the loop region, and it was found that this protein also had the ability to bind to HSA.

[0040] These five proteins, which were found to bind to HSA, were also found to bind to HSA, just like AY01. [Example]

[0041] The improvement of insulin retention was investigated using the AY01 protein obtained in Example 1. As shown in Figure 5, an alkyne derivative of insulin was first synthesized (Figure 5(A)), and the C-terminus of the cyclized AY01 was azidotated (Figure 5(B)). The synthesized alkyne derivative of insulin and the azidotated AY01-1 were then reacted at room temperature for 1 hour in the presence of copper sulfate and ascorbic acid to produce an AY01-1 insulin conjugate (insulin-peptide conjugate) (Figure 5(C)).

[0042] The conjugate was administered subcutaneously (38 μg / kg insulin equivalent) to ddy mice (male, 6 weeks old) that had been fasted for 15 hours, and the time course of plasma glucose concentrations was examined. The results are shown in Figure 6. As shown in Figure 6, in the initial phase, approximately 1 hour after administration of the insulin conjugate (AY-insulin), a decrease in plasma glucose concentration was observed to the same extent as human insulin. However, for approximately 8 hours after administration, no significant increase in plasma glucose concentration was observed, and plasma glucose concentrations remained almost constant at a lower level than with human insulin. These results suggest that by conjugating the HSA-binding protein with a helix-loop-helix structure of the present invention, a new type of insulin conjugate can be obtained that combines the rapid action of free insulin with a prolonged insulin retention time. [Industrial Applicability]

[0043] According to the present invention, there is provided an HSA-binding protein that increases the blood retention of various drugs. The present disclosure provides, for example: [Section 1] A ligand that exhibits binding to HSA and has the amino acid sequence (XVNFGXXLFXX) shown in SEQ ID NO: 3. provided that amino acid X in SEQ ID NO: 3 is any L-amino acid normally found in naturally occurring proteins, the N-terminal amino acid X is G or is deleted, the sixth amino acid X from the N-terminus is V, A, or R, the seventh amino acid X from the N-terminus is E, V, or S, the tenth amino acid X from the N-terminus is R or V, and the eleventh amino acid X from the N-terminus is G or is deleted. [Section 2] A ligand that exhibits binding affinity to HSA, having an amino acid sequence set forth in any of SEQ ID NO: 4 (GVNFGVELFRG), SEQ ID NO: 5 (GVNFGVVLFRG), SEQ ID NO: 6 (GVNFGAELFRG), SEQ ID NO: 7 (GVNFGAVLFRG), and SEQ ID NO: 8 (GVNFGRSLFVG). However, the amino acid G at the N-terminus and / or the amino acid X at the 11th position from the N-terminus of the amino acid sequences shown in SEQ ID NOs: 4 to 8 may be deleted. [Section 3] HSA-binding protein with a helix-loop-helix structure. [Section 4] Item 3. An HSA-binding protein consisting of 41 to 43 amino acids, comprising the ligand according to Item 1 or 2. [Section 5] A protein in which protein chain B having the amino acid sequence shown in SEQ ID NO: 3 is peptide-bonded between the C-terminus of protein chain A shown in SEQ ID NO: 1 and the N-terminus of protein chain C shown in SEQ ID NO: 2. SEQ ID NO: 1: CXXELXXLEXELXXLE, where all of the amino acids X are any L-amino acid commonly found in naturally occurring proteins. SEQ ID NO: 2: KLXXLKXKLXXLKXXC, where all of the amino acids X are any L-amino acid commonly found in naturally occurring proteins. SEQ ID NO: 3: XVNFGXXLFXX, wherein the N-terminal amino acid X is G or is deleted, the sixth amino acid X from the N-terminus is V, A, or R, the seventh amino acid X from the N-terminus is E, V, or S, the tenth amino acid X from the N-terminus is R or V, and the eleventh amino acid X from the N-terminus is G or is deleted. [Section 6] Item 6. The protein according to Item 5, wherein the protein has one or more of the following substitutions: amino acid E at the fourth position from the N-terminus is replaced with D; amino acid L at the fifth position from the N-terminus is replaced with P or Q; amino acid E at the ninth position from the N-terminus is replaced with K; and amino acid L at the twelfth position from the N-terminus is replaced with Q or R. [Section 7] Item 6. The protein according to Item 5, wherein the protein has one or more amino acid substitutions: the amino acid K at the 28th position from the N-terminus is replaced with E; the amino acid L at the 29th position from the N-terminus is replaced with R; the amino acid K at the 33rd position from the N-terminus is replaced with R; the amino acid K at the 40th position from the N-terminus is replaced with N or R; and the amino acid C at the 43rd position from the N-terminus is replaced with S. [Section 8] Item 8. The protein according to Item 7, further comprising one or more substitutions of the following: amino acid E at the fourth position from the N-terminus of the protein is replaced with D; amino acid L at the fifth position from the N-terminus is replaced with P or Q; amino acid E at the ninth position from the N-terminus is replaced with K; and amino acid L at the twelfth position from the N-terminus is replaced with Q or R. [Section 9] Item 9. The protein according to any one of Items 5 to 8, wherein either or both of the amino acid X at the N-terminus and the amino acid X at the C-terminus of the protein chain B is G or is missing. [Section 10] Item 9. The protein according to any one of Items 5 to 8, wherein the amino acid sequence of the protein chain B is any one of SEQ ID NO: 4 (GVNFGVELFRG), SEQ ID NO: 5 (GVNFGVVLFRG), SEQ ID NO: 6 (GVNFGAELFRG), SEQ ID NO: 7 (GVNFGAVLFRG), and SEQ ID NO: 8 (GVNFGRSLFVG). [Section 11] Item 11. The protein according to Item 10, wherein the amino acid sequence of the protein chain B lacks G at the N-terminus and / or C-terminus. [Section 12] 12. A protein according to any one of items 5 to 11, wherein in the amino acid sequence shown in SEQ ID NO: 1, all amino acids X are A, and in the amino acid sequence shown in SEQ ID NO: 2, all amino acids X are A. [Section 13] 12. A protein according to any one of items 5 to 11, wherein in the amino acid sequence shown in SEQ ID NO: 1, the third amino acid X from the N-terminus is any one of A, T, or S, the sixth amino acid X from the N-terminus is A or S, and the thirteenth amino acid X from the N-terminus is A or S; and in the amino acid sequence shown in SEQ ID NO: 2, the third amino acid X from the N-terminus is any one of A, D, or N, the fourth amino acid X from the N-terminus is A or F, the seventh amino acid X is any one of A, Q, G, or S, the tenth amino acid X is any one of A, E, P, or H, the eleventh amino acid X is any one of A, V, or F, and the fourteenth amino acid X is any one of A, D, or N. [Section 14] Item 14. The protein according to Item 13, wherein all amino acids X other than the amino acid X that can be substituted are A. [Section 15] A protein consisting of the amino acid sequence set forth in SEQ ID NO: 10, wherein all of the amino acids X in said amino acid sequence are any L-amino acid commonly found in naturally occurring proteins. [Section 16] A protein consisting of the amino acid sequence set forth in SEQ ID NO: 10, in which the amino acid K at the 28th position from the N-terminus in the amino acid sequence set forth in SEQ ID NO: 10 is substituted with E, and in which all of the amino acid Xs in the amino acid sequence are any L-amino acid normally found in naturally occurring proteins. [Section 17] Item 17. The protein according to Item 16, wherein in the amino acid sequence of SEQ ID NO: 10, the third amino acid X from the N-terminus is A, T, or S, and the 13th amino acid X from the N-terminus is A or S. [Section 18] 18. The protein according to any one of items 15 to 17, wherein, in the amino acid sequence shown in SEQ ID NO: 10, the amino acid E at the 9th position from the N-terminus is substituted with K, the amino acid L at the 29th position from the N-terminus is substituted with R, the amino acid K at the 33rd position from the N-terminus is substituted with R, the amino acid K at the 40th position from the N-terminus is substituted with N, or the amino acid C at the 43rd position from the N-terminus is substituted with S. [Section 19] 19. The protein according to any one of items 15 to 18, wherein all amino acids X in the amino acid sequence shown in SEQ ID NO: 10 are A except for the case where X is an amino acid other than A. [Section 20] A protein consisting of the amino acid sequence set forth in SEQ ID NO: 10, in which the amino acid L at the fifth position from the N-terminus is substituted with P, the amino acid E at the fourth position from the N-terminus is substituted with D, or the amino acid L at the twelfth position from the N-terminus is substituted with Q or R, and the amino acid X is any L-amino acid normally found in naturally occurring proteins. [Section 21] 21. The protein according to item 20, wherein K at the 40th position from the N-terminus in the amino acid sequence shown in SEQ ID NO: 10 is substituted with R. [Section 22] 22. The protein according to item 20 or 21, wherein all amino acids X in the amino acid sequence shown in SEQ ID NO: 10 are A. [Section 23] 23. The protein according to any one of Items 20 to 22, wherein, in the amino acid sequence shown in SEQ ID NO: 10, the amino acid X at the 30th position from the N-terminus is any one of A, D, and N, the amino acid X at the 31st position from the N-terminus is A or F, the amino acid X at the 34th position is any one of A, Q, G, and S, the amino acid X at the 37th position is any one of A, E, P, and H, the amino acid X at the 38th position is any one of A, V, and F, and the amino acid X at the 41st position is any one of A, D, and N. [Section 24] Item 24. The protein according to Item 22 or 23, wherein the other amino acid X is A. [Section 25] A protein consisting of an amino acid sequence shown in any one of SEQ ID NOs: 11 to 35. [Section 26] 25. The protein according to any one of items 15 to 24, wherein the amino acid G at the 17th position and / or the amino acid G at the 27th position from the N-terminus is deleted. [Section 27] A protein according to Item 3 or 4, wherein the N-terminus and C-terminus are cysteines, or a protein according to any one of Items 5 to 26, wherein a ring is formed between the N-terminal cysteine and the C-terminal cysteine. [Section 28] Item 28. A drug conjugate in which the protein according to any one of Items 3 to 27 is bound to a drug. [Section 29] A method for producing a protein in which a protein chain B having the amino acid sequence of SEQ ID NO: 4 (wherein G at the N-terminus and / or C-terminus of protein chain B may be deleted) is peptide-bonded between the C-terminus of protein chain A shown in SEQ ID NO: 36 and the N-terminus of protein chain C shown in SEQ ID NO: 37, or a protein according to any one of Items 3 to 27, with any L-amino acid normally found in other naturally occurring proteins; contacting the protein having the substituted amino acid sequence with HSA; determining the amino acid sequence of the protein bound to HSA; and synthesizing a protein from the determined amino acid sequence. [Section 30] Item 30. The method according to Item 29, wherein the step of contacting the protein having the substituted amino acid sequence with HSA is a step of contacting the protein with a protein expressed on the surface of a cell or phage into which a gene having a nucleotide sequence corresponding to the amino acid sequence after the substitution has been introduced. [Section 31] Item 31. The method according to Item 29 or 30, wherein the substituted amino acid is alanine in the amino acid sequence shown in SEQ ID NO: 36 and / or the amino acid sequence shown in SEQ ID NO: 37.

Claims

1. An HSA-binding protein comprising a ligand that exhibits binding affinity to HSA, the HSA-binding protein consisting of 41 to 43 amino acids, wherein the ligand has the amino acid sequence (XVNFGXXLFXX) shown in SEQ ID NO:

3. However, amino acid X in SEQ ID NO: 3 is any L-amino acid normally found in naturally occurring proteins, the N-terminal amino acid X is G or is deleted, the 6th amino acid X from the N-terminus is any of V, A, or R, the 7th amino acid X from the N-terminus is any of E, V, or S, the 10th amino acid X from the N-terminus is R or V, and the 11th amino acid X from the N-terminus is G or is deleted.

2. An HSA-binding protein comprising a ligand that exhibits binding affinity to HSA, the HSA-binding protein consisting of 41 to 43 amino acids, wherein the ligand has an amino acid sequence set forth in any one of SEQ ID NOs: 4 (GVNFGVELFRG), 5 (GVNFGVVLFRG), 6 (GVNFGAELFRG), 7 (GVNFGAVLFRG), and 8 (GVNFGRSLFVG). However, the amino acid G at the N-terminus of the amino acid sequences shown in SEQ ID NOs: 4 to 8 may be deleted.

3. A protein in which a protein chain B having the amino acid sequence shown in SEQ ID NO: 3 is peptide-bonded between the C-terminus of a protein chain A shown in SEQ ID NO: 1 and the N-terminus of a protein chain C shown in SEQ ID NO:

2. SEQ ID NO: 1: CXXELXXLEXELXXLE, where all of the amino acids X are any L-amino acid commonly found in naturally occurring proteins. SEQ ID NO: 2: KLXXLKXKLXXLKXXC, where all of the amino acids X are any L-amino acid commonly found in naturally occurring proteins. SEQ ID NO: 3: XVNFGXXLFXX wherein the N-terminal amino acid X is G or is deleted, the sixth amino acid X from the N-terminus is V, A, or R, the seventh amino acid X from the N-terminus is E, V, or S, the tenth amino acid X from the N-terminus is R or V, and the eleventh amino acid X from the N-terminus is G or is deleted.

4. The protein according to claim 3, wherein one or more of the following substitutions have been made: the fourth amino acid from the N-terminus of the protein, E, to D; the fifth amino acid from the N-terminus, L, to P or Q; the ninth amino acid from the N-terminus, E, to K; and the twelfth amino acid from the N-terminus, L, to Q or R.

5. The protein according to claim 3, wherein the protein has one or more of the following amino acid substitutions: the amino acid K at the 28th position from the N-terminus is replaced with E; the amino acid L at the 29th position from the N-terminus is replaced with R; the amino acid K at the 33rd position from the N-terminus is replaced with R; the amino acid K at the 40th position from the N-terminus is replaced with N or R; and the amino acid C at the 43rd position from the N-terminus is replaced with S.

6. The protein of claim 5, further comprising one or more substitutions of the following: the fourth amino acid from the N-terminus of the protein, E, with D; the fifth amino acid from the N-terminus, L, with P or Q; the ninth amino acid from the N-terminus, E, with K; and the twelfth amino acid from the N-terminus, L, with Q or R.

7. 7. The protein according to claim 3, wherein either or both of the amino acid X at the N-terminus and the amino acid X at the C-terminus of the protein chain B is G or is deleted.

8. The protein according to any one of claims 3 to 6, wherein the amino acid sequence of the protein chain B is any one of SEQ ID NO: 4 (GVNFGVELFRG), SEQ ID NO: 5 (GVNFGVVLFRG), SEQ ID NO: 6 (GVNFGAELFRG), SEQ ID NO: 7 (GVNFGAVLFRG), and SEQ ID NO: 8 (GVNFGRSLFVG).

9. The protein according to claim 8, wherein the N-terminal and / or C-terminal G of the amino acid sequence of the protein chain B is deleted.

10. 10. The protein according to any one of claims 3 to 9, wherein all amino acids X in the amino acid sequence shown in SEQ ID NO: 1 are A, and all amino acids X in the amino acid sequence shown in SEQ ID NO: 2 are A.

11. 10. The protein according to any one of claims 3 to 9, wherein in the amino acid sequence shown in SEQ ID NO: 1, the third amino acid X from the N-terminus is A, T, or S, the sixth amino acid X from the N-terminus is A or S, and the thirteenth amino acid X from the N-terminus is A or S; and in the amino acid sequence shown in SEQ ID NO: 2, the third amino acid X from the N-terminus is A, D, or N, the fourth amino acid X from the N-terminus is A or F, the seventh amino acid X is A, Q, G, or S, the tenth amino acid X is A, E, P, or H, the eleventh amino acid X is A, V, or F, and the fourteenth amino acid X is A, D, or N.

12. The protein according to claim 11, wherein all amino acids X other than the amino acid X that can be substituted are A.

13. A protein consisting of the amino acid sequence shown in SEQ ID NO: 10, wherein all of the amino acids X in said amino acid sequence are any L-amino acids commonly found in naturally occurring proteins.

14. A protein consisting of the amino acid sequence shown in SEQ ID NO: 10, in which the amino acid K at the 28th position from the N-terminus in the amino acid sequence shown in SEQ ID NO: 10 is substituted with E, and in which all of the amino acids X in the amino acid sequence are any L-amino acids normally found in naturally occurring proteins.

15. 15. The protein according to claim 14, wherein in the amino acid sequence shown in SEQ ID NO: 10, the third amino acid X from the N-terminus is A, T, or S, and the 13th amino acid X from the N-terminus is A or S.

16. The protein according to any one of claims 13 to 15, wherein, in the amino acid sequence shown in SEQ ID NO: 10, the amino acid E at the 9th position from the N-terminus is substituted with K, the amino acid L at the 29th position from the N-terminus is substituted with R, the amino acid K at the 33rd position from the N-terminus is substituted with R, the amino acid K at the 40th position from the N-terminus is substituted with N, or the amino acid C at the 43rd position from the N-terminus is substituted with S.

17. 17. The protein according to any one of claims 13 to 16, wherein all amino acids X in the amino acid sequence shown in SEQ ID NO: 10 are A, except for the case where the amino acid X is an amino acid other than A.

18. A protein consisting of the amino acid sequence shown in SEQ ID NO: 10, in which the amino acid L at the fifth position from the N-terminus is substituted with P, the amino acid E at the fourth position from the N-terminus is substituted with D, or the amino acid L at the twelfth position from the N-terminus is substituted with Q or R, and the amino acid X is any L-amino acid normally found in naturally occurring proteins.

19. The protein according to claim 18, wherein K at the 40th position from the N-terminus in the amino acid sequence shown in SEQ ID NO: 10 is replaced with R.

20. 20. The protein according to claim 18 or 19, wherein all amino acids X in the amino acid sequence shown in SEQ ID NO: 10 are A.

21. 21. The protein according to claim 18, wherein, in the amino acid sequence shown in SEQ ID NO: 10, the amino acid X at the 30th position from the N-terminus is any one of A, D, and N, the amino acid X at the 31st position from the N-terminus is A or F, the amino acid X at the 34th position is any one of A, Q, G, and S, the amino acid X at the 37th position is any one of A, E, P, and H, the amino acid X at the 38th position is any one of A, V, and F, and the amino acid X at the 41st position is any one of A, D, and N.

22. 22. The protein according to claim 20 or 21, wherein the other amino acid X is A.

23. A protein consisting of an amino acid sequence shown in any of SEQ ID NOs: 11 to 35.

24. The protein according to any one of claims 13 to 22, wherein the 17th amino acid G and / or the 27th amino acid G from the N-terminus is deleted.

25. A protein in which the N-terminus and C-terminus of the protein according to claim 1 or 2 are cysteines, or a protein in which a ring is formed between the N-terminal cysteine and the C-terminal cysteine of the protein according to any one of claims 3 to 24.

26. A drug conjugate comprising the protein according to any one of claims 1 to 25 and a drug bound thereto.

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