Peptide screening methods and peptides

Abstract

To provide methods for screening peptides that specifically bind to a target site of a living organism and such peptides.SOLUTION: Disclosed herein is a method for screening peptides that specifically bind to a target site of a living organism, comprising the steps of: (a) administering to a non-human living organism a phage population displaying a peptide library; (b) separately recovering a population of phages that bound to the target site and a population of phages that did not bind to the target site; (c) reading the base sequences encoding peptides displayed by the target site binding phage population and the base sequences encoding peptides displayed by the phage population not bound to the target site using a next generation sequencer, to obtain a lead population of base sequences encoding peptides presented by the target site-binding phage population (target site lead population) and a non-target site lead population, where the peptides encoded by leads in the target site lead population but not contained in the non target site lead population are the peptides that specifically bind to the target site of the living organism.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for screening peptides and peptides.

Background Art

[0002] Techniques for targeting specific organs are expected to be applied to, for example, drug delivery systems (DDS) for drug delivery. For example, Patent Document 1 describes a peptide specific to neovascularization. Patent Document 2 also describes a peptide specific to the heart.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] An object of the present invention is to provide a method for screening a peptide that specifically binds to a target site in a living body and the peptide.

Means for Solving the Problems

[0005] The present invention includes the following aspects. [1] A method for screening a peptide that specifically binds to a target site of a living body, comprising: a step (a) of administering a phage population presenting a peptide library to a non-human living body; a step (b) of recovering, respectively, the phage population bound to the target site of the non-human living body (target-site-bound phage population) and the phage population bound to a non-target site (non-target-site-bound phage population); and a step (c) of decoding, using a next-generation sequencer, the nucleotide sequences encoding the peptides presented by the target-site-bound phage population and the non-target-site-bound phage population recovered in step (b), to obtain a population of reads of the nucleotide sequences encoding the peptides presented by the target-site-bound phage population (target-site read population) and a population of reads of the nucleotide sequences encoding the peptides presented by the non-target-site-bound phage population (non-target-site read population), wherein a peptide encoded by a read included in the target-site read population obtained in step (c) and not included in the non-target-site read population is a peptide that specifically binds to the target site of the living body. [2] The method according to [1], wherein steps (a) and (b) are repeated two or more times. [3] The method according to [1] or [2], wherein the peptide is a peptide consisting of 7 amino acids. [4] The method according to any one of [1] to [3], wherein the living body is an animal or a plant. [5] The method according to any one of [1] to [4], wherein the target site is an organ, a tissue, or a single cell. [6] A brain-specific binding agent comprising a peptide consisting of an amino acid sequence set forth in any of SEQ ID NOs: 57 to 66. [7] A kidney-specific binding agent comprising a peptide consisting of an amino acid sequence set forth in any of SEQ ID NOs: 67 to 75. [8] A liver-specific binding agent comprising a peptide consisting of an amino acid sequence set forth in any of SEQ ID NOs: 76 to 84. [9] A bone marrow-specific binding agent comprising a peptide consisting of an amino acid sequence set forth in any of SEQ ID NOs: 85 to 94.

Advantages of the Invention

[0006] According to the present invention, a method for screening a peptide that specifically binds to a target site of a living body and a peptide can be provided.

Mode for Carrying Out the Invention

[0007] [Screening Method] In one embodiment, the present invention provides a method for screening a peptide that specifically binds to a target site of a living body, the method comprising: step (a) of administering a phage population presenting a peptide library to a non-human living body; step (b) of recovering a phage population bound to the target site of the non-human living body (target site-bound phage population) and a phage population bound to a non-target site (non-target site-bound phage population); and step (c) of decoding, using a next-generation sequencer, the nucleotide sequences encoding the peptides presented by the target site-bound phage population and the non-target site-bound phage population recovered in step (b), to obtain a population of reads of the nucleotide sequences encoding the peptides presented by the target site-bound phage population (target site read population) and a population of reads of the nucleotide sequences encoding the peptides presented by the non-target site-bound phage population (non-target site read population), wherein a peptide encoded by a read included in the target site read population obtained in step (c) and not included in the non-target site read population is a peptide that specifically binds to the target site of the living body.

[0008] The living body is not particularly limited, and examples include animals and plants. Examples of animals include vertebrates and invertebrates. Examples of vertebrates include mammals, birds, reptiles, amphibians, and fish. Examples of mammals include rodents such as mice, rats, hamsters, and guinea pigs; ungulates such as pigs, cows, goats, horses, and sheep; carnivores such as dogs and cats; and primates such as rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, and humans. Examples of invertebrates include arthropods and mollusks.

[0009] Examples of plants include edible crops, non-edible plants, etc. Examples of edible crops include fruit trees, vegetables, mushrooms, grains, forage crops, etc. Examples of non-edible plants include medicinal crops, flowers / foliage plants, trees, etc.

[0010] In step (a), a phage population presenting a peptide library is administered to a non-human living body. The phage is not particularly limited as long as it is usually used in a phage display library. For example, M13 phage can be used. As the phage population presenting the peptide library, a phage display library in which a gene encoding the peptide library is inserted into the gene encoding the pIII coat protein of M13 phage can be used. As the peptide library, a library of random peptides consisting of 7 amino acids is preferred.

[0011] The administration of the phage population to the non-human living body may be performed by a method according to the non-human living body. For example, when the non-human living body is a mouse, phage in an amount of about 1×10 11 colony forming units (cfu) may be administered via the tail vein. For example, when the non-human living body is a monkey, phage in an amount of about 1×10 11 cfu may be administered via the vein.

[0012] Subsequently, in step (b), the phage population adsorbed (bound) to the target site of the non-human living body (hereinafter sometimes referred to as "target site-bound phage population") and the phage population bound to the non-target site (hereinafter sometimes referred to as "non-target site-bound phage population") are each recovered. Examples of the target site include organs, tissues, single cells, etc. Examples of the non-target site include organs, tissues, single cells, etc. other than the target site. The non-target site preferably includes as many organs, tissues, single cells, etc. as possible other than the target site.

[0013] The organs are not particularly limited, and examples include the brain, kidney, liver, bone marrow, eyeball, tongue, salivary gland, thyroid gland, lung, organ, thymus, mammary gland, aorta, heart, liver, gallbladder, spleen, pancreas, bladder, esophagus, stomach, small intestine, large intestine, spinal cord, testis, prostate gland, uterus, ovary, etc.

[0014] The tissues are not particularly limited, and examples include the cerebrum, cerebellum, pituitary gland, adrenal gland, skin, skeletal muscle, sciatic nerve, dorsal root ganglion of the spinal cord, etc.

[0015] The single cells are not particularly limited, and examples include cortical neurons, striatal neurons, hippocampal neurons, hypothalamic neurons, PVN neurons, substantia nigra neurons, etc. in the brain. Also, retinal neurons, retinal pigment epithelial cells, lens epithelial cells, etc. in the eyeball. Also, taste bud cells, filiform papilla epithelial cells, lingual muscle cells, Ebner gland cells, etc. in the tongue. Also, atrial muscle cells, ventricular muscle cells, coronary artery vascular smooth muscle cells, etc. in the heart. Also, hepatocytes, liver capsule cells, bile duct epithelial cells, Kupffer cells, stellate cells, etc. in the liver. Also, Langerhans cells, exocrine gland cells, duct epithelial cells, etc. in the pancreas. Also, glomerular epithelial cells, mesangial cells, podocytes, renal tubular epithelial cells, etc. in the kidney. Also, mucosal epithelial cells, smooth muscle cells, etc. in the stomach.

[0016] The collection of the phage population adsorbed to the target site can be carried out by collecting the target site from a non-human living body, homogenizing the collected target site, culturing it, etc. Similarly, the collection of the phage population bound to the non-target site can be carried out by collecting the non-target site from a non-human living body, homogenizing the collected non-target site, culturing it, etc.

[0017] When the target site or non-target site is an organ, the organ can be collected by visually cutting the organ using scissors or forceps. When the target site or non-target site is a tissue, the tissue may be collected by visually cutting the tissue using scissors or forceps, or the tissue may be collected by a method such as laser capture microdissection (LCM). When the target site or non-target site is a single cell, the cell can be collected by a method such as laser capture microdissection (LCM).

[0018] The operations in step (a) and step (b) may be referred to as panning, in vivo panning, bio-panning, etc.

[0019] Subsequently, in step (c), the nucleotide sequences encoding the peptides presented by the target site-binding phage population and the non-target site-binding phage population recovered in step (b) are decoded using a next-generation sequencer, and a population of reads of the nucleotide sequences encoding the peptides presented by the target site-binding phage population (target site read population) and a population of reads of the nucleotide sequences encoding the peptides presented by the non-target site-binding phage population (non-target site read population) are obtained. Among the reads included in the target site read population obtained in step (c), the peptides encoded by the reads not included in the non-target site read population are peptides that specifically bind to the target site of the living body.

[0020] As will be described later in the examples, a peptide having binding affinity for the target site may also have binding affinity for the non-target site. The sequence information of the peptides obtained by decoding with a next-generation sequencer can be compared in silico (on the data), and peptides having binding affinity for the non-target site can be removed. In this specification, the operation of removing peptides having binding affinity for the non-target site from peptides having binding affinity for the target site (the operation of identifying peptides encoded by reads not included in the non-target site read population among the reads included in the target site read population) may be referred to as digital subtraction.

[0021] As will be described later in the examples, by performing digital subtraction, the sensitivity and specificity of the peptide specifically binding to the target site can be significantly improved. In addition, by including all possible organs, tissues, single cells, etc. other than the target site in the non-target site, the non-target site lead population can be enlarged, and a peptide with higher specificity for the target site can be obtained by digital subtraction.

[0022] In the screening method of the present embodiment, it is preferable to repeat steps (a) and (b) two or more times, and more preferably three times. By repeating steps (a) and (b) two or more times, a peptide with higher specificity for the target site can be obtained.

[0023] When steps (a) and (b) are repeated two or more times, the non-human biological entity used in the first round of panning may be dead. In this case, the non-human biological entity used in the second and subsequent rounds of panning may be a different individual from the non-human biological entity used in the first round of panning.

[0024] Also, in the second and subsequent rounds of panning, the phage population administered to the non-human biological entity may be a phage population recovered from a specific target site, or a phage population obtained by mixing phage populations recovered from a plurality of target sites respectively.

[0025] For example, when screening for a peptide that specifically binds to a target site using monkeys, instead of using monkeys from the first round of panning, for example, mice can be used for three rounds of panning, and the phage population recovered in the third round of panning can be administered to monkeys. Since the difference between mice and monkeys is slight in terms of the base sequence of the genome, such a screening method can also obtain results close to those obtained by performing three rounds of panning using monkeys. Thereby, the number of monkeys required for screening can be reduced. Since monkeys are precious, this method is advantageous.

[0026] [Brain-specific binder] In one embodiment, the present invention provides a brain-specific binder comprising a peptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 57 to 66.

[0027] As will be described later in the examples, the peptide of the present embodiment is obtained by excluding peptides that bind to non-specific organs by digital subtraction, and thus binds specifically to the brain and has high sensitivity and specificity. The peptide of the present embodiment can be used, for example, in a drug delivery system for specifically delivering a drug to the brain.

[0028] [Kidney-specific binder] In one embodiment, the present invention provides a kidney-specific binder comprising a peptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 67 to 75.

[0029] As will be described later in the examples, the peptide of the present embodiment is obtained by excluding peptides that bind to non-specific organs by digital subtraction, and thus binds specifically to the kidney and has high sensitivity and specificity. The peptide of the present embodiment can be used, for example, in a drug delivery system for specifically delivering a drug to the kidney.

[0030] [Liver-specific binder] In one embodiment, the present invention provides a liver-specific binder comprising a peptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 76 to 84.

[0031] As will be described later in the examples, the peptide of the present embodiment is obtained by excluding peptides that bind to non-specific organs by digital subtraction, and thus binds specifically to the liver and has high sensitivity and specificity. The peptide of the present embodiment can be used, for example, in a drug delivery system for specifically delivering a drug to the liver.

[0032] [Bone marrow-specific binder] In one embodiment, the present invention provides a bone marrow-specific binding agent comprising a peptide consisting of the amino acid sequence set forth in any of SEQ ID NOs: 85 to 94.

[0033] As will be described later in the examples, the peptide of the present embodiment is obtained by excluding peptides that bind to non-specific organs by digital subtraction, and thus binds specifically to bone marrow and has high sensitivity and specificity. The peptide of the present embodiment can be used, for example, in a drug delivery system for specifically delivering a drug to bone marrow.

Examples

[0034] Next, examples are shown to explain the present invention in more detail, but the present invention is not limited to the following examples.

[0035] [Experimental Example 1] (In vivo panning using mice) A phage library of 1×10 11 cfu was administered to mice via the tail vein. As the phage library, a commercially available kit (product name "Ph.D TM -7 Phage Display Peptide Library Kit", New England Biolabs) was used. This phage library expressed peptides consisting of random 7 amino acids on the phage surface.

[0036] Five minutes after administering the phage library, the mice were anesthetized, blood was collected from the heart, and then perfusion and bloodletting were performed using phosphate-buffered saline (PBS). Subsequently, the target sites were collected from the mice. As the target sites, 37 organs and 152 types of cells shown in Table 9 below, which will be described later, were collected. The cells were collected by the laser capture microdissection (LCM) method.

[0037] Subsequently, the collected target sites were each homogenized to obtain phage solutions. The phage solutions were infected with Escherichia coli, added to top agar, and seeded on LB plates. They were cultured overnight at 37°C. After confirming the phage plaques, the phages were recovered and propagated using SM buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 8 mM MgSO4, 0.01% Gelatin). PCR amplification was performed using the SM buffer as a sample, the PCR products were electrophoresed, gel-extracted and purified, and samples for next-generation sequencing were prepared for each target site.

[0038] Subsequently, the phages collected for each target site were mixed, and again administered to mice via the tail vein at 1×10 11 cfu, and the second in vivo panning was performed in the same manner as above, and samples for next-generation sequencing were prepared for each target site.

[0039] Subsequently, the phages collected for each target site in the second in vivo panning were mixed, and again administered to mice via the tail vein at 1×10 11 cfu, and the third in vivo panning was performed in the same manner as above, and samples for next-generation sequencing were prepared.

[0040] Subsequently, using a next-generation sequencer, the nucleotide sequences of the prepared samples for next-generation sequencing were analyzed.

[0041] [Experimental Example 2] (Analysis Result 1 by Next-Generation Sequencing) Focusing on the brain, liver, kidney, and bone marrow, based on the results of next-generation sequencing after the third panning, the amino acid sequences of peptides having organ-specific binding properties were analyzed.

[0042] Table 1 below shows the results of showing four or five amino acid sequences of the peptides expressed on the phage surface that adsorbed to each of the brain, liver, kidney, and bone marrow organs in descending order of the detected read counts. As a result, it became clear that peptides with the same amino acid sequence bound to different organs.

[0043]

Table 1

[0044] [Experimental Example 3] (Analysis Results 2 by Next-Generation Sequencing) Focusing on the brain, liver, kidney, and bone marrow, based on the results of next-generation sequencing after the third panning, the amino acid sequences of the peptides with organ-specific binding were analyzed after removing the peptides with confirmed binding to other organs from the data. Hereinafter, the operation of removing the peptides with confirmed binding to sites other than the target site from the data may be referred to as "digital subtraction".

[0045] The following Table 2 shows the amino acid sequences of the peptides with binding specific to each of the brain, liver, kidney, and bone marrow organs, in groups of 9 or 10 in descending order of the number of reads.

[0046]

Table 2

[0047] [Experimental Example 4] (Analysis Results 3 by Next-Generation Sequencing) Regarding the specific organs (target sites) and non-specific organs, attention was paid to the organs shown in Table 3 below.

[0048]

Table 3

[0049] Based on the results of next-generation sequencing after the third panning, after removing (digital subtraction) the peptides with confirmed binding to the non-specific organs shown in Table 3 above from the data, the amino acid sequences of the peptides with binding specific to the brain, liver, kidney, and bone marrow were analyzed respectively.

[0050] Table 4 below shows the amino acid sequences of peptides having binding specificity for each of the organs of the brain, liver, kidney, and bone marrow, with 9 or 10 sequences listed in descending order of the number of leads.

[0051] [Table 4]

[0052] [Experimental Example 5] (Analysis Results 4 by Next-Generation Sequencing) For the 29 types of organs shown in Tables 5 and 6 below, sensitivity and specificity were calculated based on the results of next-generation sequencing after the first and third pannings. Digital subtraction was not performed. Tables 5 and 6 below show true positive, false positive, true negative, false negative, sensitivity, specificity, positive predictive value, and negative predictive value for each organ. Table 5 below shows the results of the first panning, and Table 6 below shows the results of the third panning. Tables 5 and 6 show the results for all peptides for which one or more leads were detected.

[0053] Based on the results of next-generation sequencing, peptides detected in specific organs and peptides detected in serum were noted. The number of leads of peptides detected only in specific organs was regarded as true positives. Also, for peptides detected in both specific organs and serum, the number of leads detected in serum was regarded as false negatives. Also, for peptides detected in both specific organs and serum, the number of leads detected in specific organs was regarded as false positives. Also, the number of leads of peptides detected only in serum was regarded as true negatives.

[0054] [Table 5]

[0055] [Table 6]

[0056] [Experimental Example 6] [Analysis Result 5 by Next-Generation Sequencing] For 29 types of organs shown in Tables 7 and 8 below, based on the results of next-generation sequencing after the third panning, sensitivity and specificity were calculated for the cases where digital subtraction was not performed and where digital subtraction was performed. Tables 7 and 8 below show true positives, false positives, true negatives, false negatives, sensitivity, specificity, positive predictive value, and negative predictive value for each organ. Table 7 below shows the results without performing digital subtraction, and Table 8 below shows the results with digital subtraction performed. Tables 7 and 8 show the results of all peptides for which 5 or more leads were detected.

[0057] [Table 7]

[0058] [Table 8]

[0059] As a result, it became clear that both sensitivity and specificity were significantly increased by performing digital subtraction.

[0060] [Experimental Example 7] [Analysis Result 6 by Next-Generation Sequencing] Tables 9-1 and 9-2 below show the number of peptides with specific binding properties identified for 37 organs and 152 types of cells in mice. In Tables 9-1 and 9-2, the numerical values in parentheses indicate the number of peptides with specific binding properties.

[0061] [Table 9-1]

[0062] [Table 9-2]

[0063] [Experimental Example 8] (In Vivo Panning Using Monkeys) In Experimental Example 1, the phages collected for each target site in the third in vivo panning using mice were mixed, and 1×10 11 cfu was administered via the vein.

[0064] Five minutes after administering the phage library, the monkeys were anesthetized, blood was collected from the heart, and then perfusion and exsanguination were performed using phosphate-buffered saline (PBS). Subsequently, the target sites were collected from the monkeys. As the target sites, 37 organs and 162 types of cells shown in Table 10 below were collected. The cells were collected by the laser capture microdissection (LCM) method.

[0065] Subsequently, the collected target sites were each homogenized to obtain a phage solution. The phage solution was infected with Escherichia coli, added to top agar, and seeded on an LB plate. It was cultured overnight at 37°C. After confirming the phage plaques, the phages were recovered and propagated using SM buffer. PCR amplification was performed using SM buffer as a sample, the PCR products were electrophoresed, gel extraction and purification were performed, and samples for next-generation sequencing were prepared for each target site.

[0066] Tables 10-1 and 10-2 below show the number of peptides having specific binding properties identified for 37 organs and 162 types of cells of monkeys. In Tables 10-1 and 10-2, the numerical values in parentheses indicate the number of peptides having specific binding properties.

[0067] [Table 10-1]

[0068] [Table 10-2] [Industrial Applicability]

[0069] According to the present invention, it is possible to provide a method for screening a peptide that specifically binds to a target site of a living body and the peptide.

Claims

1. A method for screening a peptide that specifically binds to a target site of a living body, comprising: administering a phage population presenting a peptide library to a non-human living body (step (a)); recovering the phage population bound to the target site (target site-bound phage population) and the phage population bound to the non-target site (non-target site-bound phage population) of the non-human living body, respectively (step (b)); decoding the nucleotide sequences encoding the peptides presented by the target site-bound phage population and the non-target site-bound phage population recovered in step (b) using a next-generation sequencer, and obtaining a population of reads of the nucleotide sequences encoding the peptides presented by the target site-bound phage population (target site read population) and a population of reads of the nucleotide sequences encoding the peptides presented by the non-target site-bound phage population (non-target site read population) (step (c)); A method, wherein among the reads included in the target site read population obtained in step (c), the peptides encoded by the reads not included in the non-target site read population are peptides that specifically bind to the target site of the living body.

2. The method according to claim 1, wherein steps (a) and (b) are repeated two or more times.

3. The method according to claim 1 or 2, wherein the peptide is a peptide consisting of 7 amino acids.

4. The method according to claim 1 or 2, wherein the living body is an animal or a plant.

5. The method according to claim 1 or 2, wherein the target site is an organ, a tissue, or a single cell.

6. A brain-specific binding agent comprising a peptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 57 to 66.

7. A kidney-specific binding agent comprising a peptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 67 to 75.

8. A liver-specific binding agent comprising a peptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 76 to 84.

9. A bone marrow-specific binding agent comprising a peptide consisting of the amino acid sequence set forth in any one of SEQ ID NOs: 85 to 94.

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