WO2019098291A1 - Synthesis method of positron-emitting nuclide-labeled protein - Google Patents

Abstract

This method uses a positron-emitting nuclide-labeled amino acid or a derivative thereof to synthesize a positron-emitting nuclide-labeled protein by means of a cell-free protein synthesis system. Said cell-free protein synthesis system is a system reconfigured by means of a factor involved in protein synthesis, and said method uses a positron-emitting nuclide-labeled unnatural amino acid as the positron-emitting nuclide-labeled amino acid or derivative thereof; the method further uses a template nucleic acid into which a stop codon has been introduced, tRNA which recognizes the introduced stop codon and binds to the positron-emitting nuclide-labeled unnatural amino acid, and an aminoacyl-tRNA synthetic enzyme which causes bonding between the tRNA and the positron-emitting nuclide-labeled unnatural amino acid.

Description

Method for synthesizing positron-emitting radionuclide labeled protein

[Cross-reference to related applications]
The present application claims priority based on Japanese Patent Application No. 2017-219798, filed on Nov. 15, 2017, the entire disclosure of which is incorporated herein by reference.
The present invention relates to a method of protein labeling with positron emitting nuclides.

Positron-emitting nuclides are extremely useful factors for molecular imaging, and researches are being conducted on their use in diagnosis and drug conductors using Positron Emission Tomography (PET). Here, a peptide, a protein, etc. attract attention as a target to be labeled with a positron-emitting nuclide. Under such circumstances, the present inventors used a positron-emitting radionuclide-labeled amino acid or a derivative thereof to positron-emitting radionuclide labeling by a cell-free protein synthesis system reconstituted with factors involved in protein synthesis extracted and purified from cells. A method has been developed for protein synthesis, and a patent for the method has also been obtained (Patent Document 1). The method according to Patent Document 1 relates to a method of synthesizing a positron-emitting radionuclide labeled protein by a cell-free protein synthesis system by adding one positron labeled amino acid instead of one kind of natural amino acid from 20 types of natural amino acids Figure 1). However, since this method substitutes a specific amino acid contained in the amino acid sequence of the target protein, depending on the type of positron-emitting nuclide labeled amino acid, the type of amino acid sequence of the protein can be introduced. Since a labeled amino acid such as ¹⁸ F-labeled amino acid 1 is introduced using a natural aminoacyl synthetase / tRNA pair, the introduction efficiency is not necessarily high.

On the other hand, in order to use a positron-emitting radionuclide labeled amino acid as a raw material for cell-free labeling synthesis of a protein, it is necessary to obtain the positron-emitting radionuclide labeled amino acid dissolved in a trace amount of aqueous solution (buffer). In Non-Patent Document 4, in order to use ¹⁸ F-fluorine ion produced by using a cyclotron for labeling reaction, ¹⁸ F-fluorine ion is added to Kryptofix 2.2.2 (K. 222) (registered trademark) and potassium. A method is disclosed in which an ion is dissolved in an anhydrous organic solvent together with a complex (cryptand, K. 222 / K +) and activated, and this is concentrated as it is to 20 μL or less. In this method, K. The concentration of 222 / K + becomes higher than necessary and the labeling reaction is inhibited. Further, in the conventional method, it takes time and labor to repeat the azeotropic drying operation with acetonitrile for ¹⁸ F-fluorine ion obtained in the aqueous solution to be dehydrated repeatedly.

Japanese Patent No. 5590540

An object of the present invention is to provide a new positron-emitting nuclide labeled protein synthesis method different from the conventional method such as a method of replacing one of 20 kinds of natural amino acids with corresponding positron labeled amino acids. .

An object of the present invention is to provide a new method for solving the problems in the synthesis of positron-emitting radionuclide-labeled amino acids which are raw materials for the synthesis of such positron-emitting radionuclide-labeled proteins.

Under these circumstances, as a result of intensive studies under the circumstances, the present inventors have found that a target encoding a protein of interest instead of using a corresponding positron labeled amino acid instead of one of 20 natural amino acids. It was found that a positron-emitting nuclide labeled protein can be synthesized by inserting a stop codon into the template nucleic acid and introducing a positron-emitting nuclide-labeled amino acid at the position of the stop codon. Furthermore, in the synthesis of positron-emitting radionuclide labeled amino acids, the present invention adsorbs the aqueous solution of positron-emitting radionuclide ion obtained by cyclotron with an anion exchange column, desorbs it with a solution containing cryptand and then removes most of cryptand by cation exchange column. Is removed as a solution of positron-emitting nuclide ions and trace amounts of cryptands, and mixed with reaction precursors containing unnatural amino acids to avoid labeling reaction inhibition due to excessive concentration of cryptands, time or process It was found that the number could be reduced and synthesized. The present invention is based on such novel findings. Accordingly, the present invention provides the invention according to the following items:
Item 1. A method of synthesizing a positron emitting nuclide labeling protein by a cell-free protein synthesis system using a positron emitting nuclide labeling amino acid or a derivative thereof,
The cell-free protein synthesis system is a system reconstituted with factors involved in protein synthesis,
The method uses a positron-emitting radionuclide-labeled non-natural amino acid as a positron-emitting radionuclide-labeled amino acid or a derivative thereof,
The method further comprises a template nucleic acid into which a stop codon has been introduced,
A method using a tRNA that recognizes the introduced stop codon and binds to the positron-emitting radionuclide-labeled non-natural amino acid, and an aminoacyl-tRNA synthetase that combines the tRNA with the positron-emitting radionuclide-labeled non-natural amino acid.

Item 2. The method according to Item 1, wherein the positron-emitting radionuclide used for labeling is ¹¹ C, ¹⁸ F, or ¹²⁴ I.

Item 3. Item 1 wherein the positron-emitting radionuclide labeled non-natural amino acid is O-[( ¹⁸ F) fluoromethyl] tyrosine, O-[( ¹⁸ F) fluoroethyl] tyrosine, or 4-borono-2- ( ¹⁸ F) fluorophenylalanine Or the method as described in 2.

Item 4. The template nucleic acid has a stop codon inserted at any position not causing a frame shift of the open reading frame of the nucleic acid sequence encoding the native protein, or specifies a tyrosine residue or phenylalanine residue on the open reading frame The method according to any one of Items 1 to 3, comprising a nucleic acid sequence in which the codon and the stop codon are substituted.

Item 5. 5. The method according to any one of Items 1 to 4, wherein the stop codon is an amber codon.

Item 6. A kit for synthesizing positron-emitting radionuclide labeled proteins in a cell-free protein synthesis system, comprising the following components:
1) Positron emitting nuclide labeled unnatural amino acid 2) Template nucleic acid with introduced stop codon 3) tRNA which recognizes the introduced stop codon and binds to the positron emitting nuclide labeled unnatural amino acid
4) An aminoacyl-tRNA synthetase that combines the tRNA with the positron-emitting radionuclide-labeled unnatural amino acid.

Item 7. Positron-emitting radionuclide-labeled non-natural amino acid, template DNA into which a stop codon has been introduced, tRNA which recognizes the introduced stop-codon and binds to the positron-emitting radionuclide-labeled non-natural amino acid, the tRNA and the positron-emitting nuclide-labeled non-naturally-occurring A positron-emitting radionuclide-labeled protein synthesizer, comprising means for synthesizing a positron-emitting radionuclide-labeled protein by a cell-free protein synthesis system using an aminoacyl-tRNA synthetase that binds to an amino acid.

Item 8. Item 7. The kit according to Item 6 or the apparatus according to Item 7, wherein the positron-emitting radionuclide used for labeling is ¹¹ C, ¹⁸ F, or ¹²⁴ I.

Item 9. Item 6 wherein the positron-emitting radionuclide labeled unnatural amino acid is O-[( ¹⁸ F) fluoromethyl] tyrosine, O-[( ¹⁸ F) fluoroethyl] tyrosine, or 4-borono-2- ( ¹⁸ F) fluorophenylalanine Item 8. The kit according to Item 7, the device according to Item 7, or the kit or device according to Item 8.

Item 10. A method for producing a PET diagnostic agent or test agent using the method according to any one of Items 1 to 5.

Item 11. Positron-emitting radionuclide labeled protein comprising one or more positron-emitting radionuclide-labeled non-natural amino acids.

Item 12. The term wherein a positron-emitting nuclide-labeled non-natural amino acid is introduced by insertion at any position in the amino acid sequence of a naturally occurring protein, or substitution at the position of a tyrosine residue or phenylalanine residue in the amino acid sequence of a naturally occurring protein 11. The positron-emitting radionuclide labeled protein according to 11.

Item 13. Item 11 wherein the positron-emitting radionuclide-labeled non-natural amino acid is O-[( ¹⁸ F) fluoromethyl] tyrosine, O-[( ¹⁸ F) fluoroethyl] tyrosine, or 4-borono-2- ( ¹⁸ F) fluorophenylalanine Or the positron emitting nuclide labeled protein according to 12.

Item 14. A method for producing a positron-emitting radionuclide-labeled non-natural amino acid, comprising
A method comprising: obtaining a mixture for performing a labeling reaction between a positron-emitting nuclide ion and a reaction precursor derived from an amino acid by the following steps:
(1) applying an aqueous solution containing positron-emitting nuclide ions to an anion exchange column, and adsorbing positron-emitting nuclide ions onto the anion exchange column;
(2) applying a solution containing cryptand to the anion exchange column to desorb positron-emitting radionuclide ions and recovering a mixed solution containing positron-emitting radionuclide ions and cryptand;
(3) applying the mixed solution to a cation exchange column to adsorb the cryptand onto the cation exchange column, and recovering a concentrated mixed solution in which positron-emitting nuclide ions are concentrated;
(4) adding the reaction precursor to the concentrated mixed solution or the residue after evaporation thereof to obtain a mixture containing positron-emitting nuclide ions, cryptand and the reaction precursor.

Item 15. A solvent having a boiling point higher than that of the concentrated mixed solution is added as a high boiling point solvent to the concentrated mixed solution recovered in the step (3), and the residue after evaporation of the concentrated mixed solution to which the high boiling point solvent is added is Item 15. The method according to Item 14, which is a residue after evaporation described in the step (4).

Item 16. 16. The method according to item 14 or 15, wherein the positron-emitting radionuclide used for labeling is ¹¹ C, ¹⁸ F, or ¹²⁴ I.

Item 17. The method according to any one of items 14 to 16, wherein the cryptand is complexed with potassium ion.

According to the present invention, it is possible to provide a new positron emitting nuclide labeled protein synthesis method different from the conventional method such as a method of replacing one of 20 kinds of natural amino acids with the corresponding positron labeled amino acids. . Furthermore, according to the present invention, it is possible to provide a new synthesis method capable of improving the recovery rate by reducing the time and the number of steps as compared with the conventional synthesis method of positron-emitting nuclide-labeled amino acid.

FIG. 1 shows a method of synthesizing a positron-emitting radionuclide labeled protein using a conventional cell-free protein synthesis reagent and a positron-emitting radionuclide-labeled amino acid (Japanese Patent No. 5590540). A: 19 kinds of natural amino acids. B: Normal tRNA and its synthetic enzyme (proline). C: Normal cell-free protein reagent and ribosome. D: Positron labeled protein. 1. ¹⁸ F-labeled amino acid ( ¹⁸ F-fluoroproline) 1 shows a method of the invention. A: 20 kinds of natural amino acids. B: Normal cell-free protein reagents and ribosomes. C: Positron labeled protein. 1. ¹⁸ F-labeled amino acid ( ¹⁸ F-fluoromethyltyrosine). 2. Fluorotyrosine-tRNA synthetase. Improved aminoacyl-tRNA synthetase capable of introducing fluorotyrosine into tRNA. 3. tRNA. A tRNA that binds to fluorothiocin by a fluorotyrosine tRNA synthetase. 4. A gene into which a stop codon has been introduced (ATG-TAG- ...). DNA in which a stop codon is introduced at the position where you want to introduce a nonnatural amino acid. ⁷ shows the amino acid sequence of human IL-8, including ¹⁸ F-FET. In gel autoradiography, the synthesis result of ¹⁸ F-FET-IL-8 is shown. In gel autoradiography, the synthesis results of ¹⁸ F-FET-IL-8 with different concentrations of mutant pCNF RS and tRNA CUA _opt are shown. The amino acid sequence of HE-tag-Z _{HER2: 342} including ¹⁸ F-FET is shown. In gel autoradiography, the synthesis result of ¹⁸ F-FET-HE-tag-Z _{HER2: 342} is shown. (A) 8.9 MBq and (B) 492 MBq for the ¹⁸ F-FET In gel autoradiography, the radiochemical purity of ¹⁸ F-FET-HE-tag-Z _{HER2: 342} is shown. The result of the cell binding experiment of HE-tag-Z _{HER2: 342} is shown. ¹⁸ F-FET-HE-tag-Z _{HER2: 342} PET images in HER2-positive SKOV-3 transplanted nude mice. The right side shows Blocking image by unlabeled Z _{HER2: 342} administration. An example of a microscale labeling synthesis procedure of ¹⁸ F-FET is shown. The relationship between the synthesis yield of ¹⁸ F-FET and the amount of reaction solvent is shown. ((A) When the reaction is carried out by adding a precursor solution after distilling off methanol containing ¹⁸ F-fluorine ions equivalent to the amount of reaction solvent (B) Distilling methanol containing 300 μL of ¹⁸ F-fluorine ions (C) Add 300% of ¹⁸ F-Fluoride ion in methanol to react, then add DMSO to distill off the precursor solution, and then add the precursor solution to react (Method 1) (D) In the case of synthesizing by adding a precursor solution to methanol containing 300 μL of ¹⁸ F-fluorine ion and distilling off and reacting (method 2). An example of rapid purification of ¹⁸ F-FET by analytical HPLC column is shown. The result of the binding test in Example 7 is shown. The amino acid sequence of _ZPD-L1 including ¹⁸ F-FET is shown. The result of gel autoradiography of O- [ ¹⁸ F] fluoroethyl-L-tyrosine labeled Affibody (HE-tag-Z _{PD-L1_1} ) synthesized in Example 8 is shown. The result of the binding test in Example 9 is shown. The result of PET imaging in Example 10 is shown.

The present invention is a method for synthesizing a positron emitting nuclide labeled protein by a cell-free protein synthesis system using a positron emitting nuclide labeled amino acid or a derivative thereof,
The cell-free protein synthesis system is a system reconstituted with factors involved in protein synthesis,
The method uses a positron-emitting radionuclide-labeled non-natural amino acid as a positron-emitting radionuclide-labeled amino acid or a derivative thereof,
The method further comprises a template nucleic acid into which a stop codon has been introduced,
Provided is a method using a tRNA that recognizes the introduced stop codon and binds to the positron-emitting radionuclide-labeled non-natural amino acid, and an aminoacyl-tRNA synthetase that combines the tRNA with the positron-emitting radionuclide-labeled non-natural amino acid Do.

As used herein, "nucleic acid" includes both DNA and RNA. The terms "nucleotide", "oligonucleotide" and "polynucleotide" are all encompassed in "nucleic acid". In addition, they may be double-stranded or single-stranded, and when referred to as "nucleotide" (or "oligonucleotide", "polynucleotide") having a certain sequence, they are complementary unless otherwise stated. "Nucleotide" (or "oligonucleotide" and "polynucleotide") having the following sequence is also meant to be comprehensive. Furthermore, when "nucleotide" (or "oligonucleotide" and "polynucleotide") is RNA, the base symbol "T" shown in the sequence listing shall be read as "U".

In the present invention, "protein" and "peptide" are used to include oligopeptides and polypeptides. Moreover, in the present specification, “protein” and “peptide” include both proteins modified with sugar chains and the like and unmodified proteins, unless otherwise stated. The same is true for proteins that are not specified to be proteins.

The method of the present invention uses, as a positron-emitting nuclide-labeled amino acid or a derivative thereof, a positron-emitting nuclide-labeled non-natural amino acid as a raw material. In the present invention, “non-naturally occurring amino acids” are amino acids or derivatives thereof, and 20 kinds of natural amino acids (glycine, alanine, valine, leucine, isoleucine, serine, threonine, aspartic acid, glutamic acid, asparagine, glutamine, lysine And arginine, cysteine, methionine, phenylalanine, tyrosine, tryptophan, histidine, proline), typically, which do not bind to the tRNA corresponding to the 20 natural amino acids. In the present specification, a non-natural amino acid labeled with a positron-emitting radionuclide may be simply referred to as simply an acid as a positron-emitting radionuclide-labeled non-natural amino acid. Here, positron (positron) indicates an electron having a positive charge. In addition, a positron-emitting nuclide means an element (radioactive isotope) having the ability to emit positron. Examples of positron-emitting nuclides include ¹¹ C, ¹³ N, ¹⁵ O, ¹⁸ F, ⁶² Cu, ⁶⁸ Ga, ⁸² Rb, ¹²⁴ I and the like, with ¹¹ C, ¹⁸ F, ¹²⁴ I and the like being preferable.

Examples of positron-emitting radionuclide-labeled non-natural amino acids include, but are not limited to, for example, positron-emitting nuclide-labeled methyltyrosine, ethyltyrosine, phenylalanine and the like. More specifically, for example, O-[( ¹⁸ F) fluoromethyl] tyrosine, O-[( ¹⁸ F) fluoroethyl] tyrosine, or 4-borono-2- ( ¹⁸ F) fluorophenylalanine can be mentioned.

These positron-emitting nuclide-labeled non-natural amino acids can be produced, for example, according to the methods described in the following examples. Also, positron-emitting nuclide-labeled non-natural amino acids other than those specifically shown in the following examples can be produced by the same method based on the description of the following examples. Due to the short half-life of positron-emitting radionuclides, it is desirable that the positron-emitting radionuclide labeled unnatural amino acids be prepared for use at the site of use.

The method of the present invention further uses a template nucleic acid into which a stop codon has been introduced. As a method for introducing a stop codon into a template nucleic acid, it can be appropriately carried out according to a method known in this field (for example, the method described in Non-Patent Document 5). The position of introduction of the stop codon into the template nucleic acid is not particularly limited, but typically, for example, the stop codon is inserted at any position which does not cause a frame shift of the open reading frame of the nucleic acid sequence encoding a natural protein. be able to. In addition, codons specifying tyrosine residues on the open reading frame can be replaced with stop codons. Such a method is preferable when using positron emitting nuclide-labeled methyltyrosine, ethyltyrosine or the like as the positron-emitting nuclide-labeled non-natural amino acid. In addition, the codon specifying the phenylalanine residue on the open reading frame can be replaced with the stop codon. Such a method is preferable when using positron-emitting radionuclide-labeled phenylalanine or the like as the positron-emitting radionuclide-labeled non-natural amino acid. The stop codon includes, but is not limited to, amber codon (UAG), opal codon (UGA), ochre codon (UAA), and amber codon is preferable. Although the number of stop codons introduced into the template nucleic acid is not particularly limited, for example, it is preferable to introduce 1 to 4 and preferably 3 stop codons to one nucleic acid sequence of the template nucleic acid. The labeled protein obtained by the method of the present invention is useful because it can be detected by PET or the like even if the number of positron-emitting nuclide labeled amino acids introduced to one protein molecule is relatively small.

The method of the present invention further comprises a tRNA that recognizes the stop codon introduced into the template nucleic acid and binds to the positron-emitting radionuclide labeled non-natural amino acid, and combines the tRNA with the positron-emitting radionuclide labeled non-natural amino acid Use aminoacyl-tRNA synthetase. Examples of the tRNA that recognizes the stop codon introduced into the template nucleic acid and binds to the positron-emitting radionuclide-labeled non-natural amino acid include the above-mentioned stop codon (amber codon (UAG), opal codon (UGA), Those which recognize ochre (ochre) codon (UAA) and the like, preferably amber codon) can be mentioned.

An aminoacyl-tRNA synthetase that combines the tRNA with the positron-emitting radionuclide-labeled non-natural amino acid can be produced according to a method known in the art, for example, the method described in Non-Patent Document 1. In addition, a tRNA that recognizes the stop codon introduced into the template nucleic acid and binds to the positron-emitting radionuclide-labeled non-natural amino acid is also produced according to a method known in the art, for example, based on the method described in Non-Patent Document 2. Can. The method of the present invention uses the tRNA that recognizes the stop codon introduced into the template nucleic acid and binds to the positron-emitting radionuclide-labeled non-natural amino acid and the corresponding aminoacyl-tRNA synthetase to obtain the target protein Positron emitting radionuclide-labeled non-natural amino acids can be inserted into the desired position. The amount of tRNA that recognizes the stop codon introduced into the template nucleic acid and binds to the positron-emitting radionuclide-labeled non-natural amino acid in the method of the present invention is not particularly limited. The aminoacyl-tRNA synthetase (11 to 33 μM) to which is bound is preferably formulated so that the tRNA binding to the positron-emitting nuclide labeled unnatural amino acid is 5 μM to 15 μM, More preferably, 5 μM of tRNA is added to 22 μM).

In the present invention, the tRNA and the aminoacyl-tRNA synthetase are preferably orthogonal tRNA and orthogonal aminoacyl-tRNA synthetase. In the present invention, “orthogonal system” with respect to a certain tRNA and aminoacyl-tRNA synthetase means that the tRNA does not receive an amino acid from an enzyme other than the aminoacyl-tRNA synthetase.

According to the method of the present invention, it is possible to obtain a positron-emitting nuclide-labeled protein at a desired position by synthesizing a protein by a cell-free protein synthesis system using the above-mentioned raw material.

The cell-free protein synthesis system is a synthetic system that carries out a series of protein synthesis flows of transcription and translation in a test tube without directly using cells such as E. coli. Cell-free protein synthesis systems have the advantage of being able to produce proteins that are toxic to cells.

As the cell-free protein synthesis system, those known in the art can be used appropriately. More specifically, as a cell-free protein synthesis system, for example, one using cell extract obtained by disrupting cells such as cell E. coli, wheat germ, rabbit reticulocytes and removing membrane components by centrifugation may be mentioned. . As a cell-free protein synthesis system using a cell extract, RTS 100 E. coli. E. coli HY Kit (biotech rabbit), RYTS Kit (Protein Express), cell-free kun (solar sun acid) and the like.

The cell-free protein synthesis system includes, as factors constituting the system, unlabeled amino acids (typically, natural amino acids and the like) necessary for protein synthesis. Moreover, factors that constitute a cell-free protein synthesis system include, for example, factors for transcription / translation, enzymes, enzymes for regenerating energy in a reaction system, and degradation of inorganic pyrophosphate produced in transcription and translation. Examples include protein components such as enzymes. Each of these factors is preferably tagged (labeled) and prepared separately. And, these protein components are preferably labeled with one of the substances adhering to each other. In such an embodiment, it is preferable to use the other substance of the substances adhering to each other as an adsorbent, since the protein component can be captured after completion of translation. Thus, after synthesis of the target positron-emitting radionuclide labeled protein, the protein component that is a factor constituting the cell-free protein synthesis system can be removed by affinity chromatography or the like (see Japanese Patent Application Laid-Open No. 2003-102495).

Examples of substances that are in a mutually attached relationship include a combination of a protein and a metal ion, a combination of an antigen and an antibody, a combination of a protein or a peptide fragment, a protein and a specific amino acid, DNA, a dye, a vitamin, a lectin And combinations of proteins and sugars, and combinations of proteins and ion exchange resins. As a combination of a protein and a metal ion, for example, a histidine tag and a nickel complex or a cobalt complex can be mentioned. Moreover, the substance in the mutually adhering relationship may be a substance which adheres magnetically.

Examples of factors for transcription / translation and enzymes include initiation factors, elongation factors, termination factors, aminoacyl-tRNA synthetase, methionyl-tRNA transformase, RNA polymerase and the like. Moreover, as an enzyme for regenerating energy in the reaction system, for example, creatinine kinase, myokinase and nucleoside diphosphate kinase can be mentioned. Examples of enzymes for degradation of inorganic pyrophosphate produced in transcription and translation include, for example, inorganic pyrophosphatase.

In the present invention, the cell-free protein synthesis system may further contain known reagents and the like in the field of protein synthesis, such as ribosomes, ATP, GTP, unnatural amino acids, etc., acids and bases constituting buffers, and the like.

In the present invention, as a cell-free protein synthesis system, a system reconstructed with factors involved in protein synthesis (such as those produced by an enzyme possessed by cells etc.) may be used.

As the "reconstituted cell-free protein synthesis system", for example, PURESYSTEM (registered trademark) can be mentioned. PURESYSTEM® is reconstituted from extracted and purified ribosomes of E. coli, amino acids, NTPs, factors for transcription / translation, enzymes. In PURESYSTEM®, all protein components except ribosomal proteins are tagged separately with a histidine tag. After completion of synthesis, ribosomal proteins are removed by ultrafiltration and other protein components are removed by affinity chromatography using a histidine tag. The reconstituted cell-free protein synthesis system can be constructed in the same manner as PURESYSTEM (registered trademark) using cells other than E. coli known in the art (for example, insect cells, wheat germ, rabbit reticulocytes) as raw materials. It may be

In the method of the present invention, in the above-mentioned cell-free protein synthesis system, a positron-emitting radionuclide-labeled non-natural amino acid, a template nucleic acid into which a stop codon has been introduced, the introduced stop codon is recognized and the positron-emitting radionuclide-labeled non-natural amino acid It can be carried out by mixing a tRNA to be bound and an aminoacyl-tRNA synthetase to bind the tRNA and the positron-emitting radionuclide-labeled non-natural amino acid.

Although the temperature in the reaction of protein synthesis in the present invention is not particularly limited, it is preferably 30 ° C. The reaction time for protein synthesis in the present invention is not particularly limited, but preferably 30 minutes to 120 minutes. Since positron has a relatively short half life, the method of the present invention, which requires a short time and short synthesis time, is very useful as a method for synthesizing positron-emitting radionuclide labeled proteins.

According to the present invention, since the tRNA recognizing the stop codon is previously present in the template nucleic acid in the reaction system, the protein synthesis reaction is not completed at the position of the introduced stop codon, and the tRNA is bound to the tRNA at the position. The positron-emitting radionuclide labeled unnatural amino acid is introduced, and protein synthesis proceeds further. Therefore, it is possible to obtain a target protein in which a positron-emitting nuclide-labeled non-natural amino acid is introduced at a desired position. FIG. 2 outlines an exemplary embodiment of the invention using fluorotyrosine as the positron emitting nuclide labeled unnatural amino acid.

Various proteins can be synthesized by using the method of the present invention. Although the molecular weight of the target protein is not particularly limited, for example, a protein having a molecular weight of 10 kDa to 100 kDa can be produced. In the present invention, relatively small peptides (eg, 7 kDa to 10 kDa in molecular weight) can also be produced.

The present invention inserts a stop codon into a target template nucleic acid encoding a target protein, and introduces a positron-emitting radionuclide labeled amino acid at the position of the stop codon, regardless of the amino acid sequence of the target protein. Positron emitting radionuclide labeled amino acids can be introduced into the protein of the amino acid sequence. Further, according to the present invention, it is useful because it is possible to introduce the positron emitting nuclide labeled amino acid and detect the obtained positron emitting nuclide labeled protein even if only a very small amount of positron nuclide releasing labeled amino acid is used. In the present invention, the concentration of the positron nuclide-releasing labeled amino acid in the cell-free protein synthesis system described above is preferably 0.5 to 7 nanomoles / L, and more preferably 1 to 5 nanomoles / L. .

Positron-emitting nuclide labeled protein synthesis kit The present invention is a kit for synthesizing a positron-emitting nuclide-labeled protein in a cell-free protein synthesis system, which comprises the following components:
1) Positron emitting nuclide labeled unnatural amino acid 2) Template nucleic acid with introduced stop codon 3) tRNA which recognizes the introduced stop codon and binds to the positron emitting nuclide labeled unnatural amino acid
4) To provide an aminoacyl-tRNA synthetase that combines the tRNA with the positron-emitting radionuclide-labeled non-natural amino acid.

A positron-emitting radionuclide-labeled non-natural amino acid; a template DNA into which a stop codon has been introduced; a tRNA that recognizes the introduced stop-codon and binds to the positron-emitting radionuclide-labeled non-natural amino acid; The aminoacyl-tRNA synthetase that binds the positron-emitting radionuclide-labeled non-natural amino acid is as described above.

The kit of the present invention may further contain, for example, liposomes, ATP, GTP, unnatural amino acids, etc., acids and bases constituting a buffer, reagents known in the field of protein synthesis, etc. In addition, the kit of the present invention may contain a document in which the procedure for carrying out the above-mentioned protein synthesis method is written.

The present invention relates to a positron-emitting radionuclide-labeled non-natural amino acid, a template DNA into which a stop codon has been introduced, a tRNA that recognizes the introduced stop codon and binds to the positron-emitting nuclide-labeled non-natural amino acid And a means for synthesizing a positron-emitting radionuclide labeled protein by a cell-free protein synthesis system using an aminoacyl-tRNA synthetase that combines the tRNA with the positron-emitting radionuclide-labeled unnatural amino acid, provide. In addition, the device of the present invention may also contain a document in which the procedure for carrying out the above-mentioned protein synthesis method is written.

The present invention also provides a method for producing a PET diagnostic agent or test agent using the above-mentioned method of the present invention. The present invention also provides a positron-emitting radionuclide labeled protein comprising one or more positron-emitting radionuclide-labeled non-natural amino acids. Such positron-emitting radionuclide-labeled non-naturally occurring amino acid-containing positron-emitting radionuclide-labeled proteins can only be obtained by the method of the present invention described above.

Methods of Producing Positron-Emitting Nuclide-Labeled Unnatural Amino Acids The present invention also provides a new method of synthesizing positron-emitting nuclide-labeled amino acids. More specifically, the present invention relates to a method for producing a positron-emitting radionuclide-labeled non-natural amino acid as a non-limiting preferred embodiment,
The following steps provide a method characterized by obtaining a mixture for performing a labeling reaction between a positron-emitting nuclide ion and a reaction precursor derived from an unnatural amino acid:
(1) applying an aqueous solution containing positron-emitting nuclide ions to an anion exchange column, and adsorbing positron-emitting nuclide ions onto the anion exchange column;
(2) applying a solution containing cryptand to the anion exchange column to desorb positron-emitting radionuclide ions and recovering a mixed solution containing positron-emitting radionuclide ions and cryptand;
(3) applying the mixed solution to a cation exchange column to adsorb the cryptand onto the anion exchange column, and recovering a concentrated mixed solution in which positron-emitting nuclide ions are concentrated;
(4) The reaction precursor is added to the concentrated mixed solution or the residue after evaporation thereof to obtain an anhydrous organic solvent capable of dissolving positron-emitting nuclide ion, cryptand and the reaction precursor as a mixture for performing the labeling reaction. Process.

According to the method of the present invention, the labeling reaction for synthesizing positron-emitting radionuclide-labeled amino acid, which is a raw material for synthesis of positron-emitting radionuclide-labeled protein, can be performed with a minute solution of 20 μL or less. Further, in the present invention, it is possible to avoid the unnecessary concentration of cryptand and the inhibition of the labeling reaction as a result thereof, and it is possible to improve the labeling efficiency (the acquisition rate and the amount of the labeled form). The present invention makes it possible to reduce the number of distillation steps, especially to the repetition of evaporation and to the decrease in the labeling efficiency (acquisition rate and amount of labeled substance) as a result of the repetition, and to improve the labeling efficiency as well as the synthesis. The time required can be shortened.

In a preferred embodiment, the method of the present invention comprises first providing an aqueous solution of positron emitting nuclide ions to an anion exchange column. By adsorption onto the column and desorption in a later step, it is possible to concentrate positron-emitting nuclide ions, whereby the volume of the aqueous solution (buffer solution) containing the ions can be reduced to a small amount.

Next, in a preferred embodiment of the present invention, a solution containing cryptand is applied to the anion exchange column to desorb positron-emitting radionuclide ions and recover a mixed solution containing positron-emitting radionuclide ions and cryptand. May be In the present invention, “cryptand” is a generic name of a complex of a polydentate ligand consisting of two or more rings, and a polydentate ligand and an ion, unless otherwise specified. As the cryptand, for example, one obtained by complexing Cryptofix 2.2.2 (registered trademark) with potassium ion can be mentioned as an example of use. Since the solution containing cryptand is applied to the same column for desorption of positron-emitting nuclide ions adsorbed on the anion exchange column, the concentration increase of cryptand more than necessary can be suppressed.

In a preferred embodiment of the present invention, the step of subjecting the mixed solution to a cation exchange column to adsorb the cryptand onto the cation exchange column and recovering a concentrated mixed solution in which positron-emitting nuclide ions are concentrated is performed. May be The concentrated mixed solution contains positron-emitting nuclide ions, and cryptands not adsorbed to the cation exchange column in the above step. The recovered product from the anion exchange column as a fraction containing positron-emitting radionuclide ions and cryptands is further subjected to a cation exchange column to remove most cryptands, and a mixed solution of the positron-emitting radionuclide-enriched cryptands To prevent mode reaction inhibition due to the presence of excess cryptand. Further, according to the present invention, after the step of applying the mixed solution to a cation exchange column, the solvent and / or the concentrated mixed solution is added to the concentrated mixed solution before the step of adding the reaction precursor to the concentrated mixed solution obtained in the step. Alternatively, it may include the step of adding a cryptand. In the present invention, the concentration of cryptand can be adjusted to one suitable for the labeling reaction by appropriately adding a solvent and / or cryptand as appropriate.

The present invention includes the step of adding a reaction precursor to the concentrated mixed solution or the residue after evaporation thereof. In the present invention, an ion exchange column is used to recover fractions containing positron-emitting nuclide ions and cryptands. By adding a reaction precursor derived from an unnatural amino acid to this and making it in a state of being dissolved in an anhydrous organic solvent, a mixture to be subjected to a labeling reaction is obtained. The reaction precursor is preferably prepared as a solution dissolved in an anhydrous organic solvent represented by acetonitrile. As the reaction precursor, at least one selected from the group consisting of tosyl group, trityl group, tert-butyl group with respect to amino acids such as methyltyrosine, ethyltyrosine and phenylalanine (preferably ethyltyrosine etc.) (preferably And the like. The reaction precursor may be added to any of the fraction containing positron-emitting nuclide ion and cryptand, or the residue obtained by distilling off the solvent. According to the present invention, by adding a reaction precursor to the above concentrated mixed solution or the residue after evaporation, a mixture containing a positron-emitting nuclide ion, cryptand and a reaction precursor in an anhydrous organic solvent can be labeled It can be obtained as a raw material for performing In the mixture containing positron-emitting nuclide ion, cryptand and reaction precursor obtained in the above step, the cryptand content is preferably 10 to 80 mmol / L, more preferably 20 to 40 mmol / L. . The content ratio of the cryptand to the reaction precursor in the mixture obtained in the above step is not particularly limited, but for example, the reaction precursor is preferably 0.1 to 1.5 moles relative to 1 mole of cryptand, More preferably, it is 0.2 to 1 mole.

In the present invention, a solvent having a boiling point higher than that of the solvent of the positron-emitting nuclide ion and the cryptand prior to the addition of the reaction precursor (this is referred to as a high-boiling point solvent in the present invention) Is added and the fraction to which the high boiling point solvent is added is distilled off to obtain a residue, to which a reaction precursor may be added. A high yield positron-emitting radionuclide-labeled amino acid can be obtained by adding a distillation step in which a high boiling point solvent is added. In the present invention, the addition amount of the high boiling point solvent is preferably equivalent to that of the fraction. In the present invention, the high boiling point solvent is preferably selected from aprotic polar solvents represented by dimethyl sulfoxide (DMSO) and N, N dimethylformamide (DMF).

In the present invention, a positron-emitting radionuclide ion is caused to react with a reaction precursor in the presence of cryptand, using as a raw material a mixture containing the positron-emitting radionuclide ion, cryptand and the reaction precursor obtained in the above steps. Release nuclide labeled unnatural amino acids can be produced. According to the present invention, the steps after obtaining an anhydrous organic solvent capable of dissolving positron-emitting nuclide ion, cryptand and a reaction precursor as a mixture for carrying out a labeling reaction are the same as the contents described in Non-Patent Document 2 as an example. It is sufficient to carry out a treatment usually performed by a person skilled in the art as a labeling reaction to finally obtain a positron-emitting nuclide-labeled amino acid.

Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited to these examples.

Example 1 Synthesis of O- [ ¹⁸ F-Fluoroethyl] -L-Tyrosine Labeled IL-8 The protein synthesized in this example is human IL-8. In order to synthesize with cell-free protein synthesis reagent from E. coli, the signal sequence was removed and methionine at the start codon was added. This is used in the method for synthesizing positron-emitting radionuclide labeled proteins (Japanese Patent No. 5590,540). A DNA in which an amber codon (TAG) was introduced after the initiation codon (ATG) of this original DNA template pET-28a IL-8 (Met + 28-99) was prepared, and a template DNA of this example (pET-28a) was prepared. Used as IL-8 TAG). The amino acid sequence to be synthesized is shown in FIG. Therefore, IL-8 is selectively labeled by incorporating O- [ ¹⁸ F-fluoroethyl] -L-tyrosine ( ¹⁸ F-FET) only at this amber codon.

1. Methods and Materials for Synthesis of O- [ ¹⁸ F-Fluoroethyl] -L-Tyrosine Labeled IL-8 For cell-free protein synthesis reagents RTS 100 E. coli HY Kit (biotech rabbit) was used. The template DNA used was pET-28a IL-8 TAG containing the amber codon described above. Archaea M. A variant aminoacyl-tRNA synthetase (N-His-pCNF RS) derived from Jannaschii was expressed by E. coli and prepared by His-tag purification (Non-patent Document 1). The orthogonal pair of tRNA CUA _opt is GeneDesign Inc. Obtained by consignment RNA synthesis according to (Non-patent Document 2). In this example, the above reagents were mixed in a 1.5 mL tube as shown in Table 1 below, and reacted at 30 ° C. for 30 to 120 minutes. As a negative control, a sample not containing pCNF RS, tRNA CUA _opt and pET-28a IL-8 TAG was similarly prepared. The reaction solution 10μL mixed with ^NuPAGE TM LDS sample buffer ^(including NuPAGE TM Reducing ^Agent), subjected to electrophoresis using NuPAGE TM 12% Bis-Tris Protein gel (200 V, 30 minutes), gel imaging plate ( The synthesis of ¹⁸ F-FET labeled IL-8 was confirmed by contacting BAS-IP MS (GE Healthcare) overnight and obtaining an autoradiographic image with FLA-9500.

Result 1. Synthesis of O- [ ¹⁸ F-Fluoroethyl] -L-Tyrosine Labeled IL-8 As shown in FIG. 4, time-dependent O- [ ¹⁸ F-fluoroethyl] in lanes 1, 2, 3, 4 An increase in the band of -L-tyrosine labeled IL-8 was observed. On the other hand, lane 5 (reaction solution in which negative control lane 5 (pCNF RS, tRNA CUA _opt was removed and pET-28a IL-8 was added instead of pET-28a IL-8 TAG), lane 6 (pCNF RS removed) ), Lane 7 (reaction solution from which tRNA CUA _opt was removed), and lane 8 (reaction solution from which pCNF RS, tRNA CUA _opt were removed), no band was confirmed at the same position. From the above, it was shown that a protein to which ¹⁸ F-FET is selectively introduced can be synthesized by using a template DNA containing pCNF RS, tRNA CUA o _opt and TAG (amber codon).

2. Examination of optimum concentrations of mutant pCNF RS and tRNA CUA _opt Methods and Materials The reagents in Table 1 were mixed at a concentration of 0-30 μM pCNF RS and 0-15 μM tRNA CUA _opt and reacted at 30 ° C. for 120 minutes. Mixed with the reaction solution 10μL ^NuPAGE TM LDS sample buffer ^(including NuPAGE TM Reducing ^Agent), subjected to electrophoresis using NuPAGE TM 4-12% Bis-Tris Protein gel (200 V, 30 minutes), imaging plate gel Was contacted overnight, and an autoradiographic image was obtained at FLA-9500.

Results As shown in FIG. 5, it was shown that when the concentration of pCNF RS is 22 μM (0.8 mg / mL) and the tRNA CUAopt is reacted at 5 μM, the synthesis amount is maximized.

Example 2 Synthesis of O- [ ¹⁸ F- Fluoroethyl ] -L-Tyrosine-Labeled Affibody (HE-tag-Z _{HER2: 342} ) Affibody is a small molecule protein with a molecular weight of 6-7 kDa and produced using Protein G as a mother nucleus The ligand, Z _{HER2: 342} is an Affibody created targeting HER2 highly expressed in breast cancer (FIG. 6), and small animal PET imaging has been reported (Non-patent Document 3). In order to simplify the purification, HE-tag-Z _{HER2: 342} into which HE-tag in which accumulation in the liver was reduced was introduced was synthesized in this example.

Methods and Materials. Consigned DNA synthesis with Genescript yielded pET-21a HE-tag-Z _{HER2: 342} . PET-21a HE-tag-Z _{HER2: 342} TAG, in which an amber codon TAG was introduced after the start codon (ATG) of this template DNA, was used as a template DNA of Example 2. At the concentration optimized in Example 1, the reagents were mixed with the compositions shown in Table 2 or Table 3 below, reacted at 30 ° C. for 30 to 120 minutes, and gel autoradiography was performed.

Result 1. Synthesis of O- [ ¹⁸ F-Fluoroethyl] -L-Tyrosine-Labeled Affibody (HE-tag-Z _{HER2: 342} ) As shown in FIG. 7, time-dependent ¹⁸ F in lanes 1, 2, 3 and 4 An increase in the band of _-FET tag HE-tag-Z _{HER2: 342} was observed. Also, high activity (492 MBq) ¹⁸ F-FET is dissolved in Reconstitution Buffer (RTS kit), and it is used to dissolve a solid sample (E. coli Lysate, Reaction mix, Amino acids (RTS kit)) When it was made to react and it was made to react, it succeeded in obtaining an ¹⁸ F-FET labeled HE-tag-Z _{HER2: 342} in high yield even for 30 minutes.

Example 3 Purification of O- [ ¹⁸ F-Fluoroethyl] -L-Tyrosine-Labeled Affibody (HE-tag-Z _{HER2: 342} ) Since each of the positron-emitting compounds has a short half-life and a short half-life. A rapid purification method is required. ^{Since 18} F-FET labeled HE-tag-Z _{HER2: 342} contains HE-tag, it can be purified using a spin column, a cartridge column or the like on the principle of commercially available His-tag purification. In addition, although pCNF RS has a His-tag, it is possible to separate it from ¹⁸ F-FET labeled HE-tag-Z _{HER2: 342} by using a low concentration of imidazole. Finally, since it is necessary to make the solvent an injectable solution that can be administered to animals etc., desalting using NAP-5 (GE Healthcare) equilibrated with phosphate saline (PBS) Pure ¹⁸ F-FET labeled HE-tag-Z _{HER2: 342} can be obtained.

Methods and Materials The reaction solution was reacted at 60 ° C. for 5 minutes, centrifuged (20,000 g, 10 minutes), and the supernatant was recovered. The supernatant is diluted to 0.6 mL with binding buffer (PBS, pH 7.4), added to a pre-equilibrated His SpinTrap column (GE Healthcare) and eluted, and after washing the column with binding buffer, Elution Elute with buffer (Binding buffer containing 50 mM imidazole). The eluted sample was desalted with NAP-5 previously equilibrated with PBS to obtain 1 mL of ¹⁸ F-FET labeled HE-tag-Z _{HER2: 342} . The amount of radioactivity of the obtained sample was measured by a Curie meter, and the radiochemical yield was calculated. In addition, radiochemical purity was calculated by analyzing the obtained sample by NuPAGE (GE Healthcare) -autoradiography (ARG).

Results As a result of NuPAGE-ARG, a radioactive band was detected as a single band, and the radiochemical purity was calculated to be> 99% (FIG. 8).

Example 4: Binding test of O- [ ¹⁸ F-fluoroethyl] -L-tyrosine labeled Affibody (HE-tag-Z _{HER2: 342} ) to HER2-positive cells (SKOV-3) ¹⁸ synthesized by the present invention The avidity of the F-FET tag HE-tag-Z _{HER2: 342} was evaluated.

Methods and Materials ¹⁸ F-FET labeled HE-tag-Z _{HER2: 342} prepared to a concentration of 0.296 MBq / mL was added to SKOV-3 cells cultured in a 12-well plate, and allowed to react at 37 ° C. for 1 hour. The amount of nonspecific binding was calculated by measuring the amount of binding in the presence of unlabeled HE-tag-Z _{HER2: 342} (15 μg / mL). After the reaction, the drug solution was removed and the medium was washed twice. Finally, 1 mL of 0.1 M NaOH was added to lyse the cells, the cells were collected in an Eppendorf tube, and the amount of radioactivity was measured with a γ counter (Aloka) to determine the bound amount. HEK293 was used as a control cell.

Results As shown in FIG. 9, higher binding was observed in SKOV-3 of HER2-positive cells as compared to control HEK 293 cells, and this binding was specific because the binding was completely blocked with unlabeled form. It was shown to be a selective bond.

Example 5
O- [ ¹⁸ F-Fluoroethyl] -L-Tyrosine-labeled Affibody (HE-tag-Z _{HER2: 342} ) was synthesized according to the present invention by PET imaging in nude mice transplanted with HER2-positive cell line (SKOV-3) The in vivo binding of ¹⁸ F-FET labeled HE-tag-Z _{HER2: 342} was evaluated.

Methods and Materials 1.1 x 10 ⁸ cells / 0.2 mL of HER2-positive cell line (SKOV-3) cells were transplanted to the left axilla of nude mice (BALB / cAJc1-nu / nu, CLEA Japan, 8 weeks old) Used one to two months later. Intravenous administration of ¹⁸ F-FET labeled HE-tag-Z _{HER2: 342} formulated in PBS at 3.3 ± 0.2 MBq and dynamic for 120 minutes using small animal PET (Clarvivo PET / CT, Shimadzu Corporation) I took a picture. In addition, in order to evaluate specific binding in vivo, unlabeled HE-tag-Z _{HER2: 342} (250 μg) was intravenously administered first, followed by ¹⁸ F-FET labeled HE-tag-Z _{HER2: 342} After 2 hours, I took a 30 minute static shot. Tumor accumulation of ¹⁸ F-FET labeled HE-tag-Z _{HER2: 342} was analyzed using AMIDE.

Results As shown in FIG. 10, significant accumulation of ¹⁸ F-FET labeled HE-tag-Z _{HER2: 342} was observed at the site where the HER2-positive cell line (SKOV-3) was transplanted. Furthermore, unlabelled _{HE-tag-Z HER2: 342} and ¹⁸ F-FET labeled _{HE-tag-Z HER2: 342} administered treatment to before ¹⁸ F-FET labeled _{HE-tag-Z HER2: 342} integrated significant Decreased to Therefore, accumulation of the tracer in vivo is shown to be specific binding, and the O- [ ¹⁸ F-fluoroethyl] -L-tyrosine labeled Affibody (HE-tag-Z _{HER2: 342} ) In synthesized according to the present invention It has been shown to function in vivo.

Example 6: Labeling method of O- [ ¹⁸ F] fluoroethyl-L-tyrosine ( ¹⁸ F-FET) (1)
As shown in FIG. 11, ¹⁸ F-FET was synthesized according to the procedure of 20 μL scale shown below using ¹⁸ F-fluorine ion aqueous solution (1.5-2 mL) manufactured by cyclotron.
1. ¹⁸ F-Fluoride ion water (1.5-2 mL) was passed through the coupled Oasis MAX + MCX cartridge, then the cartridge was washed with MeOH (2 mL).
2. K. The ¹⁸ F-Fluoride ion was collected in a reaction vial (300 μL) with 222 / KHCO ₃ -MeOH (20 mM, 200 μL) and MeOH (100 μL).
3. In the recovered MeOH solution K.I. An equal volume of DMSO was added with 222 / KHCO ₃ -MeOH (20 mM, 5, 10, 20 μL), placed in a 85 ° C. block heater and flushed with He (200 mL / min) for 10 minutes to evaporate MeOH.
4. Add an equal volume of the precursor (L-tyrosine, O- (2-tosyl-oxyethyl) -N-trityl, tert-butyl ester, ABX) in acetonitrile solution (12 mg / mL, 18 mM) to the reaction vial and He was flushed for a minute to evaporate MeCN.
5. It was then sealed completely with a cap and heated (85 ° C., 5 minutes).
6. To the reaction solution was added HCl (2 M, 30 μL), sealed with a cap to carry out deprotection reaction (120 ° C., 10 minutes).
7. After the vial was immersed in ice water and cooled, KF (1 M, 30 μL) and Meilon (40 μL) were added to the reaction solution.
8. The reaction solution was injected onto an ODS column for HPLC analysis, and fractions containing ¹⁸ F-FET were collected (eg, InertSustain, 150 × 4.6 mm, eluent: ethanol / 50 mM acetic acid, 2.0 mL / min).
The eluate was distilled off with a rotary evaporator, and the purified ¹⁸ F-FET was dissolved in a suitable solvent for cell-free synthesis.

Method (2)
In step 3 of the above method (1), the equal amount of precursor solution is used instead of the equal amount of DMSO, and the equal amount of acetonitrile is used instead of the precursor solution in the step 4. Other operations are the same as method (1).

1. Preparation of methanol solution of ¹⁸ F-fluorine ion A commercially available cation exchange cartridge that captures 222 / K ⁺ was used. Two Oasis MAX cartridges (10 mg, 30 mg) of different polymer based capacities and a silica based Sep-Pak CM Light (130 mg) were added to K.S. A comparison was made in terms of the rate of 222 / K ⁺ leakage and the recovery of ¹⁸ F-fluoride. As apparent from the results shown in Table 4, the combination of Oasis MAX (10 mg) and MCX (30 mg) was optimal.

2. Microscale Labeling Synthesis of ¹⁸ F-FET As shown in FIG. 12, the newly developed method (1) and (F) as compared to the yield (yield shown in (B)) obtained by ordinary evaporation of methanol The synthesis yield of ¹⁸ F-FET was improved in any of 2), and a substantial difference was hardly observed between the synthesis yields obtained by the two methods. From this result, it was shown that it is a practical method giving a sufficient yield up to the 10 μL scale. These microscale synthesis methods are applicable to many other ¹⁸ F-labeled probe syntheses as well as ¹⁸ F-FETs. Table 5 is an example of a representative ¹⁸ F-probe synthesis performed on a 20 μL scale. Method (2) is not suitable because the precursor is unstable and decomposes during methanol evaporation and the yield is greatly reduced, but method (1) is not influenced by the stability of the precursor and gives a high yield. It is a general purpose method.

3. Rapid purification by analytical column As shown in Figure 13, ¹⁸ F-FET synthesized on a 10 μL scale can be separated and purified with sufficiently high chemical and radiochemical purity even with analytical columns, and the time required for elution is 6 It was within minutes. In addition, the volume of the separated solution was as small as about 1 mL, and the subsequent drying operation with a rotary evaporator was also quick. Radiochemical yield of ¹⁸ F-FET based on starting the ¹⁸ F- fluoride ion is 40-50% (decay corrected value), the time required for the synthesis, including HPLC purification is 40 minutes, the micro It shows the practicability of scale synthesis.

Example 7: Comparative binding test with [ ¹⁸ F] SFB labeled Z _{HER2: 342}
¹⁸ using a protein are well utilized as a labeling technique ^[18 F] SFB of F _{Z HER2: 342} were labeled, ¹⁸ were synthesized binding to HER2 by the method of the present invention F-FET labeled HE-tag-Z Compared to _{HER2: 342} .

Methods and Materials Z _HER2: 0.12 mL (2 mg / mL), 0.1 mL of [ ¹⁸ F] SFB (348 MBq) ethanol solution, 0.5 mL of 40 mM boric acid buffer (pH 8.5) mixed, at 50 ° C. It was allowed to react for 10 minutes. After the reaction, purification was performed with HisSpinTrap as in Example 3, and the eluted sample was desalted with NAP-5 previously equilibrated with PBS, and 1 mL of ¹⁸ F-FSB labeled HE-tag-Z _HER2: Obtained ₃₄₂ (3 MBq). Existing SFB-labeled _{Z HER2: 342} and _Z labeled with the labeling present invention _{HER2: 342} to compare, were binding test to similarly HER2-positive cells Example 4 (SKOV-3) .

As a result, it is impossible to calculate the exact specific activity because it is difficult to distinguish the unlabeled Z _{HER2: 342} and the labeled Z _{HER2: 342.} However, calculating the specific activity from the starting material and the amount of activity recovered is> 0. 25 It was estimated to be GBq / μmol. The present invention suggests that an extremely high specific activity of> 18.5 GBq / μmol can be realized in comparison with this. The results of the cell binding test, Figure 14 ¹⁸ F-FSB labeled as shown in _{HE-tag-Z HER2: 342} as compared to the specific binding ^of, 18 F-FET labeled _{HE-tag-Z HER2: 342} of The amount of binding is shown to be extremely high.

Example 8: Synthesis of O- [ ¹⁸ F] fluoroethyl-L-tyrosine labeled Affibody (HE-tag-Z _{PD-L1_1} )
Affibody is a small molecule protein ligand with a molecular weight of 6-7 kDa and made with Protein G as a mother nucleus, and is currently made for various targets. In this example, Z _PD-L1 (WO2015072280A1) targeted to Programmed cell Death ligand 1 (PD-L1) at the immune checkpoint was synthesized. In addition, two [ ¹⁸ F] FET-Z _PD-L1 with different labeling sites are designed by designing an N-terminal methionine followed by an FET (M1_H3insFET) and Y12 replaced with a FET (Y12FET). Were examined (Figure 15).

Methods and materials
Template DNA was obtained by commissioned DNA synthesis by Genescript. Using pET-21a HE-tag-Z _PD-L1 into which TAG was introduced at different sites, the reagents were mixed at the concentration optimized in Example 2 and reacted at 30 ° C. for 30 minutes. Purification was performed as in Example 2 and analyzed by gel autoradiography.

Results As a result of NuPAGE-ARG, a band of [ ¹⁸ F] FET-Z _PD-L 1 is detected at any FET introduction site after purification (FIG. 16), and [ ¹⁸ F] FET is introduced at any site. Was shown to be possible.

Example ^9: Binding Test invention to PD-L1-positive cells (MDA-MB-231) _of: ^O-[18 F- fluoroethyl] -L- tyrosine labeled _{Affibody (342 HE-tag-Z} HER2) The binding properties of the synthesized ¹⁸ F-FET labeled HE-tag-Z _{HER2: 342} were evaluated.

Methods and Materials ¹⁸ F-FET labeled HE-tag-Z _{PD-L1_1} M1_H3InsFET or ¹⁸ F-FET labeled HE-tag prepared at a concentration of 0.250 MBq / mL in MDA-MB-231 cells cultured in a 24-well plate -Z _{PD-L1_1 Y12} YET was added and allowed to react at 37 ° C. for 1 hour. The amount of nonspecific binding was calculated by measuring the amount of binding in the presence of unlabeled HE-tag-Z _{HER2: 342} (20 μg / mL). After the reaction, the drug solution was removed and the medium was washed twice. Finally, 1 mL of 0.1 M NaOH was added to lyse the cells, the cells were collected in an Eppendorf tube, and the amount of radioactivity was measured with a γ counter (Aloka) to determine the bound amount.

Results As shown in FIG. 17, ¹⁸ F-FET labeled HE-tag-Z _{PD-L1_1 in} which FET was introduced at any site also _showed specific binding to MDA-MB-231 cells, and the difference is It was not.

Example 10
O- by ^[18 F- fluoroethyl] -L- tyrosine labeled _{Affibody (HE-tag-Z PD} -L1_1) MDA-MB-231 cells PET imaging in transplanted nude mice of, ¹⁸ F synthesized by the present invention The in vivo binding of _-FET -labeled HE-tag-Z _{PD-L1_1} was evaluated.

Methods and Materials: 1.0 x 10 ⁶ cells / 0.2 mL of MDA-MB-231 cells were transplanted into the left axilla of nude mice (BALB / cAJc1-nu / nu, CLEA Japan, 8 weeks old), and 1 month I used it later. 1.8 MBq of ¹⁸ F-FET labeled HE-tag-Z _{PD-L1_1} formulated in PBS was intravenously administered and dynamic imaging was performed for 120 minutes using small animal PET (Clarvivo PET / CT, Shimadzu Corporation) . Tumor accumulation of ¹⁸ F-FET labeled HE-tag-Z _{PD-L1_1} was analyzed using AMIDE.

Results As shown in FIG. 18, accumulation of prominent ¹⁸ F-FET labeled HE-tag-Z _{PD-L1_1} was observed at the site where MDA-MB-231 was implanted. Therefore, it is shown that O- [ ¹⁸ F-fluoroethyl] -L-tyrosine labeled Affibody (HE-tag-Z _{PD-L1_1} ) synthesized in the present invention also functions in vivo as HE-tag-Z _{HER2: 342} It was done.

Claims (17)

1. A method of synthesizing a positron emitting nuclide labeling protein by a cell-free protein synthesis system using a positron emitting nuclide labeling amino acid or a derivative thereof,
   The cell-free protein synthesis system is a system reconstituted with factors involved in protein synthesis,
   The method uses a positron-emitting radionuclide-labeled non-natural amino acid as a positron-emitting radionuclide-labeled amino acid or a derivative thereof,
   The method further comprises a template nucleic acid into which a stop codon has been introduced,
   A method using a tRNA that recognizes the introduced stop codon and binds to the positron-emitting radionuclide-labeled non-natural amino acid, and an aminoacyl-tRNA synthetase that combines the tRNA with the positron-emitting radionuclide-labeled non-natural amino acid.
2. Positron-emitting nuclide used for labeling is ^{11 C, 18 F,} or ¹²⁴ I, The method of claim 1.
3. The positron-emitting radionuclide labeled unnatural amino acid is O-[( ¹⁸ F) fluoromethyl] tyrosine, O-[( ¹⁸ F) fluoroethyl] tyrosine, or 4-borono-2- ( ¹⁸ F) fluorophenylalanine The method described in 1.
4. The template nucleic acid has a stop codon inserted at any position not causing a frame shift of the open reading frame of the nucleic acid sequence encoding the native protein, or specifies a tyrosine residue or phenylalanine residue on the open reading frame The method according to claim 1, comprising a nucleic acid sequence in which the codon and the stop codon are substituted.
5. The method according to claim 1, wherein the stop codon is an amber codon.
6. A kit for synthesizing positron-emitting radionuclide labeled proteins in a cell-free protein synthesis system, comprising the following components:
   1) Positron emitting nuclide labeled unnatural amino acid 2) Template nucleic acid with introduced stop codon 3) tRNA which recognizes the introduced stop codon and binds to the positron emitting nuclide labeled unnatural amino acid
   4) An aminoacyl-tRNA synthetase that combines the tRNA with the positron-emitting radionuclide-labeled unnatural amino acid.
7. Positron-emitting radionuclide-labeled non-natural amino acid, template DNA into which a stop codon has been introduced, tRNA which recognizes the introduced stop-codon and binds to the positron-emitting radionuclide-labeled non-natural amino acid, the tRNA and the positron-emitting nuclide-labeled non-naturally-occurring A positron-emitting radionuclide-labeled protein synthesizer, comprising means for synthesizing a positron-emitting radionuclide-labeled protein by a cell-free protein synthesis system using an aminoacyl-tRNA synthetase that binds to an amino acid.
8. Positron-emitting nuclide used for labeling is ^{11 C, 18} F, or ¹²⁴ I, kit of claim 6, apparatus according to the kit or claim 7 according to claim 6.
9. The positron-emitting radionuclide labeled unnatural amino acid is O-[( ¹⁸ F) fluoromethyl] tyrosine, O-[( ¹⁸ F) fluoroethyl] tyrosine, or 4-borono-2- ( ¹⁸ F) fluorophenylalanine A kit according to claim 6, a device according to claim 7, or a kit or device according to claim 8.
10. A method for producing a PET diagnostic agent or test agent using the method according to claim 1.
11. Positron-emitting radionuclide labeled protein comprising one or more positron-emitting radionuclide-labeled non-natural amino acids.
12. The positron-emitting nuclide-labeled non-naturally occurring amino acid is introduced by insertion at any position in the amino acid sequence of a naturally occurring protein, or substitution at the position of a tyrosine residue or phenylalanine residue in the amino acid sequence of a naturally occurring protein Item 11. The positron-emitting radionuclide labeled protein according to Item 11.
13. The positron-emitting radionuclide-labeled non-natural amino acid is O-[( ¹⁸ F) fluoromethyl] tyrosine, O-[( ¹⁸ F) fluoroethyl] tyrosine, or 4-borono-2- ( ¹⁸ F) fluorophenylalanine 11. The positron-emitting radionuclide labeled protein according to 11.
14. A method for producing a positron-emitting radionuclide-labeled non-natural amino acid, comprising
    A method comprising: obtaining a mixture for performing a labeling reaction between a positron-emitting nuclide ion and a reaction precursor derived from an amino acid by the following steps:
    (1) applying an aqueous solution containing positron-emitting nuclide ions to an anion exchange column, and adsorbing positron-emitting nuclide ions onto the anion exchange column;
    (2) applying a solution containing cryptand to the anion exchange column to desorb positron-emitting radionuclide ions and recovering a mixed solution containing positron-emitting radionuclide ions and cryptand;
    (3) applying the mixed solution to a cation exchange column to adsorb the cryptand onto the cation exchange column, and recovering a concentrated mixed solution in which positron-emitting nuclide ions are concentrated;
    (4) adding the reaction precursor to the concentrated mixed solution or the residue after evaporation thereof to obtain a mixture containing positron-emitting nuclide ions, cryptand and the reaction precursor.
15. A solvent having a boiling point higher than that of the concentrated mixed solution is added as a high boiling point solvent to the concentrated mixed solution recovered in the step (3), and the residue after evaporation of the concentrated mixed solution to which the high boiling point solvent is added is The method according to claim 14, wherein the residue after distillation according to the step (4) is used.
16. 15. The method according to claim 14, wherein the positron emitting nuclide used for labeling is ¹¹ C, ¹⁸ F or ¹²⁴ I.
17. 15. The method of claim 14, wherein the cryptand is complexed with potassium ions.

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