Peptide screening methods

Abstract

To provide a method for efficiently selecting peptides that have high affinity for target molecules and that also possess functional properties. [Solution] A method for screening peptides, comprising the following steps. (1) A step of reacting a functional crosslinking agent with a peptide to form a peptide complex having a structure in which the peptide and the functional crosslinking agent are crosslinked intermolecularly. (2) A step of mixing the peptide complex obtained in step (1) with the target molecule and selecting the peptide complex bound to the target molecule.

Description

[Technical Field]

[0001] The present invention relates to a method for screening peptides. More specifically, the present invention relates to a method for screening peptides that have high affinity for a target molecule and that have been conferred with functionality. [Background technology]

[0002] In the manufacturing of pharmaceuticals and other substances, peptides with high affinity to target molecules are selected. Furthermore, after selecting peptides with high affinity to target molecules, they are modified to impart functionality. For example, when using peptides as therapeutic agents, peptides are modified based on rational design to impart desired chemical and pharmacological properties, and various modified peptides such as stapled peptides, cell-permeable peptides, retro-inverso peptides, and cyclic peptides are designed (Non-Patent Literature 1). However, when peptides are modified to impart functionality, their physical properties change, requiring further optimization after modification. For example, it has been reported that to stabilize the secondary structure of short-chain peptides, stapled peptides with cross-linked side chains are further optimized for properties such as solubility and binding affinity through sequence exploration (Non-Patent Literature 2).

[0003] On the other hand, Non-Patent Document 3 discloses designing a bi-cycle peptide consisting of a cyclic peptide that binds to a target molecule and a cyclic peptide that has membrane permeability, and then performing screening. However, membrane permeability is conferred by incorporating a membrane permeability sequence into the peptide backbone, and not by a crosslinking agent. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Future Med Chem.2010 Dec;2(12):1813-22. [Non-Patent Document 2] “Design-Rules for Stapled Peptides with in vivo Activity and their Application to Mdm2 / X antagonists”, published in Nature Communications, 2024 Jan 12;15(1):489.doi:10.1038 / s41467-023-43346-4. [Non-Patent Document 3] ACS Comb.Sci.2016,18.75-85 [Overview of the project] [Problems that the invention aims to solve]

[0005] The present invention aims to provide a method for efficiently selecting peptides that have high affinity for a target molecule and that possess added functionality. [Means for solving the problem]

[0006] As a result of diligent research to solve the above problems, the present inventors have discovered that, in selecting a peptide with high affinity to a target molecule, it is possible to easily and reliably select a peptide that has high affinity to a target molecule and is also functionally conferred to it by first reacting the candidate peptide with a functional crosslinking agent to form a peptide complex before contacting the target molecule, then mixing the peptide complex with the target molecule, and selecting the peptide complex bound to the target molecule. This has led to the completion of the present invention.

[0007] In other words, the present invention encompasses the following: [1] A method for screening peptides, comprising the following steps. (1) A step of reacting a functional crosslinking agent with a peptide to form a peptide complex having a structure in which the peptide and the functional crosslinking agent are crosslinked intermolecularly. (2) A step of mixing the peptide complex obtained in step (1) with the target molecule and selecting the peptide complex bound to the target molecule. [2] The screening method according to [1], wherein the peptide complex is a peptide library. [3] The screening method according to [2], wherein the peptide library is a ribosome display peptide library. [4] The screening method according to any one of [1] to [3], wherein the peptide comprises an amino acid residue having a nucleophilic side chain, and the functional crosslinking agent comprises a leaving group. [5] The screening method according to any one of [1] to [3], wherein the functional crosslinking agent comprises at least one of a cell membrane permeability-conferring group and a cyclizing group for cyclizing peptides. [Effects of the Invention]

[0008] According to the present invention, peptides possessing both functionality and high affinity for target molecules can be efficiently screened. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1 is a schematic diagram of the mRNA structure contained in the RNA library prepared in Production Example 1. [Figure 2] Figure 2 is a schematic diagram showing the design of the sample used for NGS analysis. [Figure 3A] Figure 3A shows the results of evaluating the binding ability of samples obtained by reacting 1,3-dibromo-2-propanone with sequences obtained from selection experiments using a peptide library modified with 1,3-dibromo-2-propanone. [Figure 3B] Figure 3B shows the results of evaluating the binding ability of samples obtained by reacting the functional crosslinking agent prepared in Production Example 4 with sequences obtained from selection experiments using a peptide library modified with 1,3-dibromo-2-propanone. [Figure 4A]Figure 4A shows the results of evaluating the binding ability of a sample obtained by reacting 1,3-dibromo-2-propanone with the sequence obtained from the selection experiment using the peptide library modified with the functional crosslinking agent obtained in Production Example 4. [Figure 4B] Figure 4B shows the results of evaluating the binding ability of a sample obtained by reacting the functional crosslinking agent prepared in Production Example 4 with the sequence obtained from the selection experiment using the peptide library modified with the functional crosslinking agent obtained in Production Example 4.

Mode for Carrying Out the Invention

[0010] The method for screening a peptide having high affinity for the target molecule of the present invention comprises the following steps: (1) A step of reacting a functional crosslinking agent with a peptide to form a peptide complex having a structure in which the peptide and the functional crosslinking agent are crosslinked intermolecularly, (2) A step of mixing the peptide complex obtained in step (1) with the target molecule and selecting the peptide complex bound to the target molecule. It includes.

[0011] Hereinafter, each step and term will be described. Process (1) : In step (1), a functional crosslinking agent is reacted with a peptide to form a peptide complex having a structure in which the peptide and the functional substance are crosslinked intermolecularly.

[0012] <Functional crosslinking agent> The functional crosslinking agent is a modifier for imparting functionality to a peptide by crosslinking a functional substance to the peptide intermolecularly, and preferably does not bind to the target molecule. Further, the functional crosslinking agent preferably has a leaving group.

[0013] Functional crosslinking agents have functional groups that are imparted to peptides. The functional groups can be appropriately selected according to the intended use and are not particularly limited, but examples include cell membrane permeability imparting groups, linker compounds that cyclize polypeptides, luminescent substances such as fluorescent substances, dyes, radioactive substances, drugs, toxins, nucleic acids, amino acids, peptides, sugars, lipids, and various polymers, as well as combinations thereof. Examples of fluorescent substances include fluorescent dyes such as fluorescein, rhodamine, coumarin, pyrene, and cyanine.

[0014] A preferred embodiment of the functional crosslinking agent in the present invention has at least one of a cell membrane permeability-conferring group and a cyclizing group that cyclizes peptides. The functional crosslinking agent may also have a linking group.

[0015] The cell membrane permeability-conferring group is a group that confers cell membrane permeability to the peptide, and it is preferable that it has a basic functional group.

[0016] There are no particular restrictions on the form of the cell membrane permeability-conducting group, and it can be appropriately selected depending on the purpose, but a form having a dendritic structure is preferred. There are no particular restrictions on the number of dendritic structural units in the cell membrane permeability-conducting group, and it can be appropriately selected depending on the purpose, and it may be one or two or more. There are no particular restrictions on the number of branches per dendritic structural unit, and it can be appropriately selected depending on the purpose, but three or more is preferred.

[0017] There are no particular restrictions on the branch structure of a dendritic structure; it can be appropriately selected according to the purpose.

[0018] There are no particular restrictions on the basic functional group; for example, guanidino groups, amino groups, and imidazole groups are examples, but guanidino groups are preferred. The basic functional group may be used alone or in combination of two or more.

[0019] There are no particular restrictions on the number of basic functional groups, and they can be appropriately selected depending on the purpose, but two or more are preferred. The basic functional group is preferably one having two or more guanidino groups.

[0020] There are no particular restrictions on the position of the basic functional group in the functional crosslinking agent, and it can be appropriately selected depending on the purpose, but it is preferable that it be located at the end of the branch of the dendritic structure.

[0021] Specific examples of cell membrane permeability-conferring groups having a dendritic structure include, for example, those represented by the following general formula (I).

[0022] [ka]

[0023] The general formula (I) above represents a cell membrane permeability-conducting group having a dendritic structure with 3 branches, where "Y" represents a basic functional group. The basic functional group may be formed at the end of all branches or at the end of some branches. Furthermore, the cell membrane permeability-conducting group may have multiple dendritic structures represented by general formula (I), in which case the number of branches may be, for example, 6 or 9.

[0024] Cyclizing groups contribute to the cyclization of peptides by reacting with reactive amino acid residues in the peptide. Reactive amino acid residues are amino acid residues that react with the cyclizing group, and may be amino acid residues that react directly with the cyclizing group, or amino acid residues that have been modified to react with the cyclizing group. There are no particular restrictions on the cyclizing group, and it can be appropriately selected depending on the purpose, but electron-withdrawing groups are preferred.

[0025] There are no particular restrictions on the electron-withdrawing group, and it can be appropriately selected depending on the purpose, but it is preferable that it contains a halogen.

[0026] There are no particular restrictions on the type of halogen; it can be selected appropriately depending on the purpose. There are no particular restrictions on the number of halogens; it can be selected appropriately depending on the purpose, but two or more are preferred.

[0027] The electron-withdrawing group preferably has two or more chlorine atoms, more preferably a benzyl chloride which may have substituents, and particularly preferably a 3,5-bis(chloromethyl)benzyl group.

[0028] Cyclization of peptides using cycloforming groups is preferably carried out by a reaction between the cycloforming group and at least one group selected from the group consisting of thiol groups, amino groups, and hydroxyl groups contained in the peptide.

[0029] The linking group is a group that connects the cell membrane permeability-conferring group with the cyclizing group, and its structure is not particularly limited and can be appropriately selected depending on the purpose.

[0030] Specific examples of functional crosslinking agents include 1,3-dibromo-2-propanone, 1,3,5-tris(bromomethyl)benzene, and compounds represented by the following structural formula. The functional crosslinking agent represented by the following structural formula has a 3,5-bis(chloromethyl)benzyl group as a cyclizing group and three guanidino groups at the end of the dendritic structure as a cell membrane permeability-conferring group, and the cyclizing group and the cell membrane permeability-conferring group are linked via a linking group.

[0031] The functional crosslinking agent represented by the following structural formula can be obtained by the method described in International Publication No. 2020 / 195302.

[0032] [ka]

[0033] <Peptide complex> The peptide complex of the present invention is obtained by reacting a peptide with a functional crosslinking agent, and has a structure in which the peptide and the functional substance are crosslinked intermolecularly.

[0034] The peptide in the peptide complex may have a structural site capable of binding to the target molecule, as well as other structural sites. The binding is not limited as long as the peptide can bind to the target molecule, and may be chemical or physical. Examples of chemical bonds include covalent bonds, ionic bonds, and metallic bonds. Examples of physical bonds include hydrogen bonds, polar attractive forces, and intermolecular forces such as van der Waals forces. Furthermore, the binding may be by adsorption.

[0035] There are no particular restrictions on the lower limit of the number of amino acid residues of the peptide (or polypeptide) that binds to the target molecule, and it can be appropriately selected depending on the purpose, but it is preferably 2 or more, more preferably 3 or more, even more preferably 5 or more, even more preferably 10 or more, especially preferably 15 or more, and most preferably 20 or more. Similarly, there are no particular restrictions on the upper limit of the number of amino acid residues of the peptide (or polypeptide), and it can be appropriately selected depending on the purpose, but it is preferably 1000 or less, more preferably 500 or less, and even more preferably 200 or less.

[0036] The peptide may be a peptide-containing substance, a peptide-containing substance containing one type of peptide, or a peptide-containing substance containing two or more types of peptides. Among these, a peptide-containing substance containing two or more peptides is preferred, a peptide library is more preferred, and a ribosome display peptide library, which is a peptide library of ribosome display complexes (RD complexes), is even more preferred.

[0037] In the present invention, the "structural site capable of binding to a target molecule" preferably has a (poly)peptide structure. The structural site capable of binding to a target molecule preferably contains random sequences at specific positions so that it is useful as a (poly)peptide library. From such random sequences, useful amino acid sequences can be identified according to a predetermined purpose.

[0038] The lower limit of the number of amino acid residues in the structural site capable of binding to the target molecule is preferably 2 or more, more preferably 3 or more, even more preferably 5 or more, and particularly preferably 10 or more. The upper limit of the number of amino acid residues in the structural site capable of binding to the target molecule is preferably 1000 or less, more preferably 500 or less, even more preferably 200 or less, even more preferably 100 or less, particularly preferably 50 or less, and most preferably 20 or less.

[0039] The RNA encoding a structural site capable of binding to the target molecule is preferably one that does not have a stop codon.

[0040] The structural site capable of binding to the target molecule preferably contains a reactive amino acid residue, and more preferably contains an amino acid residue having a nucleophilic side chain. The peptide complex can be obtained by reacting a reactive amino acid residue or an amino acid residue having a nucleophilic side chain with a functional crosslinking agent having a leaving group.

[0041] There are no particular restrictions on the structure of the structural site capable of binding to the target molecule, and it can be appropriately selected depending on the purpose, but a cyclic structure is preferred. In particular, forming a cyclic structure through modification restricts the conformation of the structure. This is expected to improve affinity with the target molecule and chemical and biological stability.

[0042] Examples of reactive amino acid residues include cysteine ​​residues, lysine residues, serine residues, and threonine residues. Reactive amino acid residues may be used individually or in combination of two or more.

[0043] The number of reactive amino acid residues in the peptide is preferably two or more, in terms of cyclizing the peptide. The upper limit for the number of reactive amino acid residues in the peptide is preferably 10 or less, because if the number of reaction sites increases, the number and position of the functional crosslinking agent bound to the peptide may become unstable, making it difficult to compare the properties of the peptide derived from the amino acid sequence.

[0044] Furthermore, if, for example, cysteine ​​residues in the peptide are involved in stabilizing the peptide's higher-order structure via disulfide bonds, it is preferable to introduce reactive amino acid residues into the peptide separately.

[0045] There are no particular restrictions on the position of reactive amino acid residues in a peptide; they can be appropriately selected depending on the purpose.

[0046] For example, when using a ribosome display complex (hereinafter sometimes referred to as the "RD complex") containing an mRNA molecule, its translated peptide chain (hereinafter sometimes referred to as the "polypeptide chain"), and ribosomes as the peptide (or polypeptide), it is preferable to use the portion that exits the ribosome's exit tunnel, specifically between the 2nd position from the N-terminus and the 30th position from the C-terminus (including the 2nd position from the N-terminus and the 30th position from the C-terminus), in that the modification reaction by the functional crosslinking agent may be less likely to be sterically inhibited by the ribosome.

[0047] The position of the reactive amino acid residue from the C-terminus is preferably the 50th position from the C-terminus, and more preferably the 100th position. Furthermore, when counting the positions of the reactive amino acid residues from the N-terminus, the position can be appropriately set according to the peptide chain length, but for example, it is between the 2nd and 1000th positions from the N-terminus, preferably between the 2nd and 100th positions from the N-terminus, and more preferably between the 2nd and 50th positions from the N-terminus.

[0048] There are no particular restrictions on the method for manufacturing the RD complex; known methods can be appropriately selected, such as the method described in International Publication No. 2017 / 213158. It can also be manufactured using commercially available kits.

[0049] There are no particular restrictions on the position of the random sequence in the peptide; it can be appropriately selected depending on the purpose. For example, similar to the position of reactive amino acid residues, when using an RD complex, it is preferable to select a position between the 2nd position from the N-terminus and the 30th position from the C-terminus (including the 2nd position from the N-terminus and the 30th position from the C-terminus). In other words, it is preferable that the reactive amino acid residues are included within the random sequence. Therefore, the preferred position of the random sequence can be set from the same range as the preferred position of the reactive amino acid residues.

[0050] The number of random sequences in a peptide may be one or two or more. There is no particular upper limit to the number of random sequences, and it can be appropriately selected depending on the purpose, but it is preferable to have 10 or fewer.

[0051] There are no particular restrictions on the number of amino acid residues per random sequence; it can be appropriately selected depending on the purpose, for example, it can be between 1 and 30.

[0052] The longer a single random sequence is, and the greater the number of random sequences, the greater the diversity of the peptide library.

[0053] Other structures are not particularly limited and can be appropriately selected depending on the purpose, for example, a protease recognition structure site and a linker structure site. The protease recognition structure site is not particularly limited and can be appropriately selected depending on the purpose, for example, a TEV protease recognition structure site.

[0054] Process (2) : Step (2) involves mixing the peptide complex obtained in step (1) with the target molecule and selecting the peptide complex bound to the target molecule.

[0055] As for the mixing method, for example, a method of mixing the peptide complex and the target molecule and stirring by inversion is preferred. Furthermore, it is preferable that the mixing be carried out in a buffer solution.

[0056] The mixing temperature can be set appropriately depending on the type of peptide complex and target molecule, and is not limited, but its upper limit is preferably 50°C or lower, more preferably 40°C or lower, even more preferably 20°C or lower, particularly preferably 10°C or lower, and most preferably 5°C or lower. Its lower limit is preferably 0°C or higher, and more preferably 4°C or higher.

[0057] The pH of the buffer solution can be set appropriately depending on the type of peptide complex and target molecule, and is not limited, but its upper limit is preferably pH 12 or lower, more preferably pH 10 or lower, and even more preferably pH 8 or lower. Its lower limit is preferably pH 2 or higher, more preferably pH 4 or higher, and even more preferably pH 6 or higher.

[0058] <Target molecule> In the present invention, "target molecule" refers to a specific molecule for which a peptide with high affinity is selected for the purpose of controlling its function. Specific examples of target molecules include biomolecules such as antigens, receptors, enzymes, ion channels, and transporters.

[0059] The target molecule is preferably a target molecule bound to a substrate (support). The binding method is not particularly limited as long as the target molecule and the substrate (support) can be bound, and can be appropriately selected depending on the purpose, but streptavidin-biotin binding is preferred. Specifically, the target molecule can be biotin-labeled, streptavidin can be chemically bonded to the substrate (support), and the target molecule can be bound to the substrate (support) by streptavidin-biotin binding.

[0060] The substrate (support) to which the target molecule is bound is preferably spherical beads, and although the type is not particularly limited, magnetic beads are preferred.

[0061] The lower limit of the volume-average diameter of the beads is preferably 10 nm or more, more preferably 100 nm or more, even more preferably 200 nm or more, even more preferably 300 nm or more, particularly preferably 500 nm or more, and most preferably 800 nm or more. The upper limit of the volume-average diameter of the beads is preferably 1000 μm or less, more preferably 100 μm or less, even more preferably 10 μm or less, even more preferably 5 μm or less, particularly preferably 2 μm or less, and most preferably 1200 nm or less. The volume-average diameter can be measured using Partica LA-960 (HORIBA).

[0062] In step (2), after mixing, the peptide complex bound to the target molecule is a modified peptide that retains its binding ability to the target molecule even after modification of the peptide to impart functionality, and therefore does not require an optimization step. The peptide complex bound to the target molecule can be recovered, for example, by utilizing the magnetism of the beads bound to the target substance.

[0063] Other steps are not particularly limited and can be selected as appropriate depending on the purpose, but examples include introducing mutations into the peptide complex recovered through the above steps. Methods for introducing mutations include site-directed mutagenesis using mixed bases and random mutations using error-prone PCR. [Examples]

[0064] The following describes embodiments of the present invention, but the present invention is not limited in any way to these embodiments.

[0065] (Manufacturing Example 1) RNA Production Using the NNK method, similar to the method described in International Publication No. 2017 / 213158, (NNK) 10 RNA having a sequence containing the sequence (wherein N represents A, U, G, or C, K represents G or U, and NNK corresponds to all codons) is 10 12 RNA library including the above (10 12The above types of RNA are composed of approximately one molecule each, totaling 10 molecules. 12 We created (more than 1) libraries.

[0066] First, an RNA library (nucleotide sequence: SEQ ID NO: 2) was prepared in preparation for constructing a polypeptide library (amino acid sequence: SEQ ID NO: 1) capable of binding to both the target molecule and the selection auxiliary, as shown in Figure 1. The RNA library was prepared by a transcription reaction using the DNA library (nucleotide sequence: SEQ ID NO: 3) as a template, and the DNA library was prepared by a PCR reaction using plasmid DNA (nucleotide sequence: SEQ ID NO: 4) as a template. The plasmid DNA was constructed using pUC19 (Takara) and the DNA library (DNA shown in SEQ ID NO: 3) by homologous recombination (In Fusion HD Cloning Kit (Takara)).

[0067] Specifically, a reaction solution having the composition shown in Table 1 was used, and PCR was performed using the plasmid DNA as template DNA in the PCR cycle shown in Table 2 to prepare the 5' fragment. KOD FX Neo (TOYOBO) was used as the PCR enzyme. The 2× buffer and 2mM dNTPs were those provided with the KOD FX Neo (TOYOBO).

[0068] In Table 1, 5FFnew_150409 is a forward primer (SEQ ID NO: 5), and Ma5FragR_withoutHis_150409 is a reverse primer (SEQ ID NO: 6). Both primers were purchased from Eurofin Genomics. The same applies to the following primers.

[0069] [Table 1]

[0070] [Table 2]

[0071] Next, using a reaction solution having the composition shown in Table 3, the 3'-fragment of the template DNA was prepared with the PCR cycle shown in Table 4. The PCR enzyme used was KOD FX Neo (TOYOBO). The 2× buffer and 2 mM dNTPs used were those attached to the KOD FX Neo (TOYOBO).

[0072] In Table 3, MaNNK5ProNNK5_F150310 is a forward primer (SEQ ID NO: 7), and 3F-Rnew_150409 is a reverse primer (SEQ ID NO: 8). The sequence of MaNNK5ProNNK5_F150310 contains a random nucleotide sequence, and its diversity is up to 20 10 ways (about 1×10 13 ways). In this reaction solution, since 4×10 13 primers are used, about 4 of each sequence are included.

[0073]

Table 3

[0074]

Table 4

[0075] Next, using a reaction solution having the composition shown in Table 5, overlapping PCR was performed with the PCR cycle shown in Table ⑥ to ligate the above 5'-fragment and 3'-fragment. The PCR enzyme used was KOD FX Neo (TOYOBO). The 2× buffer and 2 mM dNTPs used were those attached to the KOD FX Neo (TOYOBO).

[0076] In Table 5, X~Z indicate that the total volume was adjusted to 100 μL by adding H2O to the reaction solution using 1×10 12 of the 5'-fragment and 3'-fragment. Specifically, 10 μL of the 5'-fragment at 1×10 12 / μL, 1×10 1210 μL of 3' fragments at a concentration of 3' particles / μL and 8 μL of H2O were used.

[0077] [Table 5]

[0078] [Table 6]

[0079] Furthermore, using the obtained DNA strand (overlapping PCR product) as a template, PCR was performed using a reaction mixture with the composition shown in Table 7 and the PCR cycle shown in Table 8 to amplify the full-length DNA and obtain template DNA. KOD FX Neo (TOYOBO) was used as the PCR enzyme. The 2× buffer and 2 mM dNTPs were those provided with the KOD FX Neo (TOYOBO).

[0080] In Table 7, 5FFnew_150409 is a forward primer (SEQ ID NO: 5), and 3F-Rnew_150409 is a reverse primer (SEQ ID NO: 8). In Table 7, X to Y are 1 × 10⁻⁶. 12 This shows that 5 × 10 overlapping PCR products were used, and the total volume was adjusted to 200 μL by adding H2O to the reaction mixture. Specifically, 5 × 10 10 20 μL of overlapping PCR product with a concentration of cells / μL and 24 μL of H2O were used.

[0081] [Table 7]

[0082] [Table 8]

[0083] Using the obtained DNA as a template, and reacting it with a reaction solution having the composition shown in Table 9 at 37°C for 2 hours, a 1×10¹⁶ DNA having the base sequence of Sequence ID No. 3 is obtained. 12 An RNA library containing the above mRNAs was obtained.

[0084] The transcriptase used was T7 RNA polymerase (TAKARA). The 10× buffer, 50 mM DTT, was the one provided with the T7 RNA polymerase (TAKARA).

[0085] In Table 9, X to Y are 1 × 10 12 This indicates that more than 1 × 10¹⁶ template DNA molecules were used, and H₂O was added to the reaction mixture to adjust the total volume to 100 μL. Specifically, 1 × 10¹⁶ 12 10 μL of template DNA (with a particle / μL concentration) and 45 μL of H2O were used.

[0086] As shown in Figure 1, the mRNA contained in this library has, in order from the 5' end, a FLAG® sequence, a random sequence, a TEV protease recognition sequence, and a spacer sequence (linker sequence), and does not contain a stop codon.

[0087] [Table 9]

[0088] (Manufacturing Example 2) Fabrication of Ribosome Display Complex Ribosome display (RD) complexes were prepared from the RNA library created in Production Example 1 using a reconstituted cell-free protein synthesis kit (Gene Frontier "PURE frexRD®").

[0089] The above RD complex reaction solution was mixed with agarose gel beads, Anti-FLAG M2 Affinity Gel (Merck), and stirred at 4°C for 1 hour. Through this process, agarose gel beads to which RD complexes containing the FLAG sequence were selectively bound were recovered.

[0090] (Manufacturing Example 3) Preparation of peptide complexes using crosslinking reaction with functional crosslinking agents In the agarose gel to which the RD complex recovered in Production Example 2 was selectively bound, the RD complex was still trapped. Tris(2-carboxyethyl)phosphine hydrochloride (Nacalai tesque) was added to this suspension to a final concentration of 0.5 mM, and 1,3-dibromo-2-propanone (Fujifilm Wako Pure Chemical Industries) was added as a functional crosslinking agent to a final concentration of 2 mM. The modification reaction was carried out by inversion and stirring at 4°C overnight. After washing and removing the functional crosslinking agent, FLAG peptide (Merck) was added to dissociate the RD complex containing the peptide complex from the agarose gel beads.

[0091] (Manufacturing Example 4) Preparation of Functional Crosslinking Agents A functional crosslinking agent represented by the following structural formula was obtained, similar to the method described in International Publication No. 2020 / 195302.

[0092] [ka]

[0093] (Example 1) Selection of RD complexes that bind to antigens (1) Preparation of beads (antigen-immobilized magnetic beads) on which the antigen (corresponding to the target molecule) is immobilized. 50 μg of Galectin-3 (R&D Systems) was mixed with 20 μg of NHS-PEG4-Biotin (ThermoFisher Scientific), and the mixture was reacted on ice for 2 hours. Unreacted reagents were then removed by dialysis to obtain biotinylated antigen.

[0094] To Nanolink Sterptavidin Magnetic Beads (Solulink, volume average diameter 1.0 μm), five times the binding capacity of the magnetic beads, the biotinylated antigen (35 ng) was added, and the beads were washed by inversion and agitation at 4°C for 1 hour to obtain beads to which the antigen had bound and immobilized (antigen-immobilized magnetic beads).

[0095] (2) Recovery of RD complex bound to antigen The RD complex containing the peptide complex obtained in Production Example 3 was mixed with the antigen-immobilized magnetic beads and reacted with the antigen by inversion and stirring at 4°C for 1 hour. Subsequently, the mixture was washed with a buffer containing 15 mM magnesium ions to remove the RD complex that was not bound to the antigen, and only the RD complex bound to the antigen was recovered.

[0096] (Test Example 1) Amplification of recovered RNA (1) Preparation of cDNA The RD complex bound to the antigen obtained in Example 1 was destabilized by adding 50 mM EDTA, and the RNA from the RD complex was recovered. Since EDTA affects subsequent operations, this RNA was purified using an RNA enrichment and purification kit (QIAGEN's "RNeasy MinElute Cleanup Kit"). Using the obtained purified RNA as a template, the reaction was carried out at 65°C for 5 minutes with a primer (SEQ ID NO: 9) at the composition shown in Table 10. Then, the reaction mixture (reaction product) was reverse transcribed by reacting it at 50°C for 1 hour and then at 70°C for 15 minutes at the composition shown in Table 11 to obtain cDNA.

[0097] 2 mM dNTPs were used, as provided with the KOD FX Neo (TOYOBO). Superscript III Reverse Transcriptase (ThermoFisher Scientific) was used as the reverse transcriptase. 5 × buffer and 0.1 M DTT were used, as provided with the Superscript III Reverse Transcriptase (ThermoFisher Scientific).

[0098] In Table 10, X to Y indicate that an appropriate amount of template RNA was used, and H2O was added to the reaction mixture to adjust the total volume to 13 μL. Specifically, 7 μL of the 15 μL total volume of the purified RNA solution and 0.8 μL of H2O were used.

[0099] [Table 10]

[0100] [Table 11]

[0101] (2) Preparation of full-length DNA Using the obtained cDNA as a template, PCR was performed using a reaction solution with the composition shown in Table 12 and the PCR cycle shown in Table 13 to amplify the full-length template DNA.

[0102] The PCR enzyme used was 2×KAPA HiFi HS Ready Mix (Roche). In Table 12, the forward primer is the sequence of sequence number 10, and the reverse primer is the sequence of sequence number 11.

[0103] In Table 12, X to Y indicate that an appropriate amount of cDNA was used, and H2O was added to the reaction mixture to adjust the total volume to 200 μL. Specifically, 7 μL of the 15 μL total volume of purified cDNA solution was used, and 1.8 μL of H2O was added.

[0104] [Table 12]

[0105] [Table 13]

[0106] (3) RNA preparation Using the obtained DNA as a template, RNA was obtained by reacting it at 37°C for 2 hours using a reaction solution having the composition shown in Table 9.

[0107] The transcriptase used was T7 RNA polymerase (TAKARA). The 10× buffer, 50 mM DTT, was the one provided with the T7 RNA polymerase (TAKARA).

[0108] In Table 9 above, X to Y are 1 × 10 12 This indicates that more than 1 × 10¹⁶ template DNA molecules were used, and H₂O was added to the reaction mixture to adjust the total volume to 100 μL. Specifically, 1 × 10¹⁶ 12 10 μL of template DNA (with a particle / μL concentration) and 45 μL of H2O were used.

[0109] After the reaction, the RNA was purified using an RNA enrichment and purification kit (QIAGEN's "RNeasy Mini Kit"). The purified samples were then subjected to concentration quantification using Nanodrop (ThermoFisher Scientific) and amplification confirmation by agarose gel electrophoresis to confirm that amplification was successful.

[0110] Using RNA that was confirmed to have been amplified without problems, the procedures described in Production Example 2, Production Example 3, Example 1, and Test Example 1 were performed until it was confirmed that the amount of RD complex recovered from the antigen-immobilized beads had increased sufficiently. The recovered samples obtained in this way were then used for the NGS analysis described in the next section.

[0111] (Example 2) Analysis of enriched sequences (1) Sample preparation for NGS analysis NGS analysis was performed using Miseq (Illumina). The recovered RNA was used as a template and reacted with primer (SEQ ID NO: 9) at the composition shown in Table 10 at 65°C for 5 minutes. The reaction mixture (reaction product) was then reverse transcribed by reacting it at the composition shown in Table 11 at 50°C for 1 hour, and then at 70°C for 15 minutes.

[0112] 2 mM dNTPs were used, as provided with the KOD FX Neo (TOYOBO). Superscript III Reverse Transcriptase (ThermoFisher Scientific) was used as the reverse transcriptase. 5 × buffer and 0.1 M DTT were used, as provided with the Superscript III Reverse Transcriptase (ThermoFisher Scientific).

[0113] In Table 10 above, X to Y indicate that an appropriate amount of template RNA was used, and H2O was added to the reaction mixture to adjust the total volume to 13 μL. Specifically, 1 × 10 6 We used 1 μL of template RNA with a concentration of cells / μL and 6.8 μL of H2O.

[0114] Furthermore, using the cDNA obtained in this reaction as a template, tailed PCR was performed using a reaction solution with the composition shown in Table 12 and the PCR cycle shown in Table 13 to produce fragments of the design shown in Figure 2, to which the sequences necessary for analysis were added.

[0115] After analysis, in order to distinguish the source sample, samples were prepared using reverse primers with different index sequences for each sample: truseq1R(709) (SEQ ID NO: 12) for samples recovered from magnetic beads with antigen, truseq1R(710) (SEQ ID NO: 13) for samples recovered from magnetic beads without antigen, and truseq1R(711) (SEQ ID NO: 14) for samples recovered from beads after off-rate selection. Note that in truseq1R(709) (SEQ ID NO: 12), truseq1R(710) (SEQ ID NO: 13), and truseq1R(711) (SEQ ID NO: 14), the index sequence is from the 25th to the 30th base.

[0116] The PCR enzyme used was 2×KAPA HiFi HS Ready Mix (Roche). In Table 12 above, X to Y indicate that an appropriate amount of cDNA was used, and H2O was added to the reaction mixture to adjust the total volume to 20 μL. Specifically, 1×10 5 We used 1 μL of cDNA with a concentration of cells / μL and 7.8 μL of H2O.

[0117] After denaturation with 0.2N NaOH, the sample solution for analysis was prepared by diluting it with HT1 buffer (Illumina) while cooling it on ice, and then mixing it with equimolar PhiX Control (Illumina).

[0118] (2)NGS analysis The sample prepared using the above procedure was read using Paired-End sequencing. As a result of the sequence analysis, multiple enriched sequences were obtained.

[0119] (Test Example 3) Peptide synthesis of enriched sequences (1) Synthesis of linear peptides Based on the information in International Publication No. 2020 / 195302, a peptide having the following sequence, which is one of the concentrated sequences obtained in Test Example 2, was synthesized on Rink Amide resin (0.2 mmol / g) using a microwave solid-phase synthesis method. Ac-Cys-Pro-Leu-Phe-Pro-Trp-Pro-Ser-Leu-Trp-His-Arg-Cys-NH2 (SEQ ID NO: 15)

[0120] The resin on which the peptide was formed was immersed in TFA / water / triisopropylsilane / 3,6-dioxa-1,8-octanedithiol (92.5 / 2.5 / 2.5 / 2.5 (volume ratio)) for 3 hours, and the peptide was cleaved from the resin. The obtained peptide was purified by reverse-phase HPLC and freeze-dried to obtain a peptide having the above sequence.

[0121] (2) Preparation of peptide complexes using functional crosslinking agents To confirm the desirability of modification before the selection experiment, in addition to the 1,3-dibromo-2-propanone modified peptide used in the selection experiment, peptides modified with the functional crosslinking agent prepared in Production Example 4 were also prepared. 3.0 μmol of peptide was dissolved in 1.0 mL of 50 mM ammonium bicarbonate buffer, and 3.3 μmol of TCEP was added and the mixture was stirred at 25°C for 1 hour. A solution consisting of 4.5 μmol of G3-DCX and 500 μL of water was added, and the mixture was stirred at 25°C for 20 hours. The reaction mixture was purified by preparative HPLC to obtain the cyclic peptide (G3-DCX-P).

[0122] (Test Example 4) Evaluation of the effect of changing the functional crosslinking agent on peptides obtained from a library modified with 1,3-dibromo-2-propanone. The binding ability of peptides modified with 1,3-dibromo-2-propanone, obtained in Test Example 3, and peptides modified with the functional crosslinking agent prepared in Production Example 4 was evaluated. The evaluation was performed using a Biacore S200 (cytiva). A Series S Sensor Chip SA was used as the sensor chip, and biotinylated antigens were immobilized as ligands by contacting them at a flow rate of 10 μL / min for 600 seconds. A lane with no immobilization was set up as a reference. The binding behavior to the antigen was confirmed by flowing peptides (conc. = 62.5 nM ~ 500 nM) at a flow rate of 30 μL / min for 60 seconds, and then the dissociation behavior was confirmed by flowing only buffer for 120 seconds.

[0123] The results of the binding behavior confirmation are shown in Figure 3A (peptide modified with 1,3-dibromo-2-propanone) and Figure 3B (peptide modified with the functional crosslinking agent prepared in Production Example 4). Compared with the synthetic peptide obtained by modifying with the same functional crosslinking agent as in the selection experiment, a decrease in binding response was observed in the peptide obtained by modifying with a different functional crosslinking agent. As described in Example 1, in this selection experiment, peptides that strongly bind to the target molecule were selected from a variety of peptides (affinity screening). When peptides modified with a functional crosslinking agent are used in the selection experiment, the binding ability to the target molecule changes depending on the physical properties of the functional crosslinking agent and the change in the overall three-dimensional structure of the peptide due to the modification. Therefore, after the selection experiment, sequences of peptides that have or have improved binding ability to the target molecule due to modification with a functional crosslinking agent are obtained, or sequences of peptides that are not significantly affected by modification with a functional crosslinking agent. In other words, even if the peptide obtained in the above selection experiment does not show strong binding ability to the target molecule as a peptide alone, it shows strong binding ability to the target molecule when modified with a functional crosslinking agent.

[0124] On the other hand, the results of the binding ability evaluation above confirmed that modifying the peptide with a functional crosslinking agent after the selection experiment significantly altered the peptide's physical properties and structure compared to the selection experiment, resulting in a decrease in binding ability.

[0125] In peptide pharmaceuticals, when a peptide alone may not exhibit the desired function from the standpoint of functionality or chemical and biological stability, it is conceivable to modify the peptide with a modifying agent. Therefore, by modifying the peptide beforehand before conducting selection experiments, it becomes possible to efficiently obtain a peptide that has been given function by the modifying agent and also possesses the ability to bind to the target molecule, all in one go.

[0126] (Test Example 5) Evaluation of the effect of changing the functional crosslinking agent on peptides obtained from a library modified with the functional crosslinking agent obtained in Manufacturing Example 4. In Production Example 1, an RNA library was constructed in the same manner as in Production Example 1, except that the primer for SEQ ID NO: 7 was changed to MaNNK15_F150310 (SEQ ID NO: 16). A ribosome display complex was prepared in the same manner as in Production Example 2. In Production Example 3, a peptide complex was prepared in the same manner as in Production Example 3, except that 1,3-dibromo-2-propanone was changed to the functional crosslinking agent prepared in Production Example 4. RD complexes were selected in the same manner as in Example 1, and enriched sequences were obtained from the obtained RD complexes using the method described in Test Example 1. Based on the information obtained from the sequence analysis, a peptide designed as Ac-Cys-Leu-Val-His-Ile-Ala-Pro-Pro-Leu-Arg-Met-Ala-Leu-Ile-Thr-Gly-NH2 (SEQ ID NO: 17) was prepared using the same method as in Test Example 3. This was also prepared with modifications using 1,3-dibromo-2-propanone and with the functional crosslinking agent prepared in Production Example 4. The binding ability of these peptides was evaluated using the same method as in Test Example 4. The results of the binding ability evaluation are shown in Figure 4A (peptides modified with 1,3-dibromo-2-propanone) and Figure 4B (peptides modified with the functional crosslinking agent prepared in Production Example 4). Similar to Figures 3A and 3B, the peptides modified with the same functional crosslinking agent as in the selection experiment showed stronger binding ability. This confirmed the reproducibility of the phenomenon observed in Test Example 4.

Claims

1. A method for screening peptides, comprising the following steps. (1) A step of reacting a functional crosslinking agent with a peptide to form a peptide complex having a structure in which the peptide and the functional crosslinking agent are crosslinked intermolecularly. (2) A step of mixing the peptide complex obtained in step (1) with the target molecule and selecting the peptide complex bound to the target molecule.

2. The screening method according to claim 1, wherein the peptide complex is a peptide library.

3. The screening method according to claim 2, wherein the peptide library is a ribosome display peptide library.

4. The peptide comprises an amino acid residue having a nucleophilic side chain, and the functional crosslinking agent comprises a leaving group. The screening method according to any one of claims 1 to 3.

5. The screening method according to any one of claims 1 to 3, wherein the functional crosslinking agent has at least one of a cell membrane permeability-conferring group and a cyclizing group for cyclizing peptides.

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