JP2019041588A - Process for producing labeled proteins, process for producing labeled peptides and kits - Google Patents

Abstract

To provide labeled protein-producing methods for easy production of labeled proteins and labeled peptides labeled withF orF in a short period of time as well as kits, PET reagents, labeled trastuzumab, pharmaceutical compositions, kits for detecting HER2 protein.SOLUTION: Disclosed herein is a labeled protein-producing process comprising synthesizing a protein labeled withF orF in a cell-free protein synthesis system using a labeled indole labeled withF orF. Also disclosed is a labeled peptide-producing process comprising synthesizing a polypeptide labeled withF orF in a cell-free protein synthesis system using a labeled indole labeled withF orF. Further disclosed is a kit for synthesizing a protein or peptide labeled withF orF comprising a cell extract for acellular protein synthesis.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing a labeled protein, a method for producing a labeled peptide, and a kit.

  Since proteins have various functions, they are used in various industrial applications such as pharmaceuticals, diagnostic agents, and food processing enzymes, and labeled proteins labeled with specific elements are particularly useful. For example, since antibodies can specifically bind to a wide variety of molecules, labeled antibodies are used in various applications such as in vivo and in vivo diagnosis and treatment. Specifically, positron-labeled antibodies can be powerful imaging probes for positron emission tomography (hereinafter referred to as "PET imaging"). In addition, labeled proteins labeled with elements that can be useful probes are also used in NMR analysis of peptides and proteins.

As a method of labeling a protein such as an antibody or a peptide, for example, a method of labeling with a radioactive isotope has been proposed. As a method of labeling a protein or peptide with a radioactive isotope, for example, a method of binding ⁶⁸ Ga or ⁶⁴ Cu chelated to a peptide or protein has been proposed (Patent Document 1). In this method, various peptides and proteins can be labeled with ⁶⁸ Ga or ⁶⁴ Cu and used for PET imaging, but the large molecule of the chelating agent means that the nature of the labeled peptide or protein is the natural type. May be different. In particular, when a small molecular weight peptide is labeled, the derivatization moiety of the large chelating agent has a large influence on the localization and kinetics.

In addition, a method has been proposed in which a labeling reagent having a maleimide group, a polyethylene glycol (PEG) chain and ¹⁸ F is allowed to act on the thiol group of a cysteine residue of a peptide or protein to label with ¹⁸ F (Patent Document 2) ). The labeling reagent used in this method is excellent in hydrophilicity and in vivo stability due to having a PEG region, but the structure of the peptide or protein largely changes from the natural form by labeling, so localization particularly in low molecular weight peptides The influence on the behavior is large. In addition, in peptides and proteins, in many cases, a cysteine residue in the molecule plays an important role in their functions, and if the cysteine residue is modified by labeling, an adverse effect is likely to occur.

In addition, a protein is synthesized in a cell-free protein synthesis system using [ ¹¹ C] methionine, [ ¹¹ C] methionine derivative, [ ¹⁸ F] proline, [ ¹⁸ F] fluoroethionine, or [ ¹⁸ F] proline derivative, ¹¹ C Alternatively, a method for obtaining a labeled protein labeled with ¹⁸ F has been proposed (Patent Document 3, Non-patent Document 1). In this method, the structural change of the peptide or protein by labeling is smaller than that of the method using the above-mentioned chelating agent or labeling reagent, and the influence on the localization and the dynamics thereof is small.

JP 2014-65725 A JP 2012-519176 gazette JP, 2010-263809, A

Matsuda T, et al. Bioorg. Med. Chem. 2012. 20: 6579-6582.

However, since the half life of ¹¹ C is as short as 20 minutes, the method using [ ¹¹ C] methionine or [ ¹¹ C] methionine derivative in Patent Document 3 is unsuitable for practical use considering the time required for protein synthesis and purification It is. On the other hand, ¹⁸ F has a relatively long half life, and labeling with ¹⁸ F is useful for disease diagnosis, drug discovery research and the like. However, while [ ¹¹ C] methionine and [ ¹¹ C] methionine derivatives are easily incorporated into proteins, [ ¹⁸ F] proline and [ ¹⁸ F] proline derivatives have a low efficiency of incorporation into proteins. Therefore, if it is attempted to obtain a sufficient amount of labeled protein using [ ¹⁸ F] proline or [ ¹⁸ F] proline derivative, an excessive synthesis reaction is required, which is disadvantageous in terms of economics and radiation exposure.

The present invention relates to a method for producing labeled protein and labeled peptide which can easily produce labeled protein or labeled peptide labeled with ¹⁸ F or ¹⁹ F in a short time, a method for producing labeled peptide, reagent for PET and method for producing the same, labeled protein or label It is an object of the present invention to provide a kit capable of efficiently producing a peptide, and a labeled trastuzumab, a pharmaceutical composition and a kit for detecting HER2 protein.

The present invention has the following configuration.
[1] In a cell-free protein synthesis system, labeled with ¹⁸ F or ¹⁹ F labeled indole with tryptophanase or tryptophan synthase and L-serine, or pyruvate and ammonium ion labeled with ¹⁸ F or ¹⁹ F A method for producing a labeled protein, comprising: synthesizing the labeled tryptophan described above; and further synthesizing a labeled protein labeled with ¹⁸ F or ¹⁹ F using the labeled tryptophan in the cell-free protein synthesis system.
[2] The method for producing a labeled protein according to [1], wherein the labeled protein is labeled trastuzumab in which the indole ring of the side chain of tryptophan residue in trastuzumab is labeled with ¹⁸ F.
[3] A reagent for PET, which produces a reagent for PET comprising a labeled protein in which the indole ring of the side chain of the tryptophan residue is labeled with ¹⁸ F by the method for producing a labeled protein according to [3] [1] or [2] Manufacturing method.
In [4] a cell-free protein synthesis system, by tryptophanase or tryptophan ^{synthase, 18} F or a labeled indole labeled with ¹⁹ F, L-serine, or a label and a pyruvic acid and ammonium ions at ¹⁸ F or ¹⁹ F A method for producing a labeled peptide, which comprises synthesizing the labeled tryptophan described above, and further synthesizing a labeled peptide labeled with ¹⁸ F or ¹⁹ F using the labeled tryptophan in the cell-free protein synthesis system.
[5] A kit for synthesizing a labeled protein or a labeled peptide labeled with ¹⁸ F or ¹⁹ F, which comprises a cell-free protein synthesis system.
[6] A reagent for PET, which comprises a labeled protein in which the indole ring of the side chain of a tryptophan residue is labeled with ¹⁸ F.
[7] Labeled trastuzumab in which the indole ring of the side chain of tryptophan residue is labeled with ¹⁸ F.
[8] A pharmaceutical composition comprising trastuzumab according to [7].
A HER2 protein detection kit comprising the kit according to [9] [5].

According to the present invention, a labeled protein labeled with ¹⁸ F or ¹⁹ F, a labeled peptide, a reagent for PET, a labeled trastuzumab, a pharmaceutical composition and a HER2 protein detection kit can be easily produced in a short time.

It is the figure which showed the quantity of the labeled protein (CAT protein) in Examples 1-5. FIG. 10 is a diagram showing the amount of labeled protein (CAT protein) in Example 6. FIG. 10 is a diagram showing the amount of labeled protein (CAT protein) in Example 7. FIG. 16 is a view showing an imaging result of a PET imaging test of a normal model mouse in Example 9. FIG. 16 is a view showing an imaging result of a PET imaging test of a tumor model mouse in Example 9. FIG. 16 shows the results of observation of tumor tissue sections by in vitro autoradiography in Example 10.

[Method for producing labeled protein]
The method for producing a labeled protein of the present invention is a method for producing a labeled protein labeled with ¹⁸ F or ¹⁹ F.
In the present invention, “protein” is defined as a compound in which 25 or more amino acids are linked by peptide bond. In addition, " ¹⁸ F or ¹⁹ F-labeled protein" is defined as a protein having ¹⁸ F or ¹⁹ F in the molecule.

In the method for producing a labeled protein of the present invention, a cell-free protein synthesis system is used to synthesize a labeled protein labeled with ¹⁸ F or ¹⁹ F.
“Cell-free protein synthesis system” refers to a cell-free transcription / translation system including a cell-free transcription system that synthesizes RNA using DNA as a template, and a cell-free translation system that reads mRNA information and synthesizes a protein on ribosomes. As well as both cell free translation systems.

  In the cell-free protein synthesis system, cell extract solution for cell-free protein synthesis (hereinafter simply referred to as "cell extract") extracted from cells containing components necessary for cell-free protein synthesis system (hereinafter simply referred to as "cell extract") It is possible to use a solution used for reconstituted cell-free protein synthesis method in which factors are separately purified and mixed.

Extracted from plant cells, animal cells, fungal cells and bacterial cells containing components necessary for translation system involved in protein synthesis such as ribosome and tRNA, or components necessary for transcription system and translation system as cell extract Cell extract is mentioned. For example, cell extracts from E. coli, wheat germ, rabbit reticulocytes, mouse L-cells, Ehrlich ascites tumor cells, HeLa cells, CHO cells, budding yeast and the like can be mentioned.
Specific examples of the cell extract include cell extracts from E. coli S30 cells. As the S30 cell extract, one prepared by a known method may be used, or a commercially available product may be used.

  A known method can be employed as a method for preparing a cell extract, for example, cells are disrupted with a French press or glass beads, and a buffer containing several kinds of salts for solubilizing protein components and ribosomes is added. The method includes homogenizing, centrifuging and precipitating insoluble components for separation.

In the method for producing a labeled protein of the present invention, for example, ¹⁸ F or ¹⁹ F labeled indole, 19 amino acids other than tryptophan, energy source, the amino acid sequence of the labeled protein of interest are encoded in cell extract. Nucleic acid etc. are added. As a result, in the reaction solution of the cell-free protein synthesis system, tryptophanase or tryptophan synthase synthesizes labeled tryptophan in which the side chain indole ring is labeled with ¹⁸ F or ¹⁹ F from labeled indole and L-serine. Ru. Moreover, protein synthesis using the labeled tryptophan proceeds further in a cell-free protein synthesis system, and the labeled tryptophan is incorporated, whereby a labeled protein in which the indole ring of the side chain of the tryptophan residue is labeled with ¹⁸ F or ¹⁹ F Is obtained.

In the method for producing a labeled protein of the present invention, pyruvate and ammonium ion may be added to the cell extract. In a cell-free protein synthesis system, tryptophanase or tryptophan synthase synthesizes labeled tryptophan whose side chain indole ring is labeled with ¹⁸ F or ¹⁹ F even from labeled indole, pyruvate and ammonium ion (Nakazawa, Chem. 1972. 36: 2523-2528 and Watanabe, T. Snell, EE Proc. Natl. Acad. Sci. USA 1972. 69: 1086-H. Enei, H. Okamura, S. Yamada, H. Agr. 1090). For example, ammonium ions are added to the reaction solution by adding ammonium chloride.

  The content of the cell extract in the reaction solution is preferably 10 to 40% by volume with respect to the total mass of the reaction solution.

^As the labeled indole labeled with ¹⁸ F or ¹⁹ F, indole in which the hydrogen atom at 4th, 5th or 6th position is substituted with ¹⁸ F or ¹⁹ F is preferable, and the hydrogen atom at 5th position is ¹⁸ F or ¹⁹ F More preferred is 5-fluoroindole substituted by As labeled indole, one type may be used alone, or two or more types may be used in combination.
Labeled indole can be produced by known methods. For example, treatment of unprotected indoleboronic acid with K ¹⁸ F gives a labeled indole in which the hydrogen atom at position 5 is substituted with ¹⁸ F.

  The content of the labeled indole in the reaction solution is preferably 0.001 to 20 mM, more preferably 0.01 to 3.0 mM, and still more preferably 0.04 to 1.5 mM relative to the total amount of the reaction solution. If the content of the labeled indole is at least the lower limit value of the above range, the labeled protein is easily synthesized. If the content of labeled indole is below the upper limit value of the above range, labeled protein can be synthesized with higher yield.

  In the cell-free protein synthesis system of the present invention, tryptophanase or tryptophan synthase is used for the synthesis of labeled tryptophan from labeled indole, L-serine, or pyruvate and ammonium ions. Since cell extracts of cell-free protein synthesis systems usually contain tryptophanase or tryptophan synthase, it is not always necessary to add tryptophanase or tryptophan synthase. However, tryptophanase or tryptophan synthase may be further added to the reaction solution for the purpose of enhancing the synthesis efficiency of labeled tryptophan and producing the labeled protein more efficiently.

  The addition amount of tryptophanase or tryptophan synthase to the reaction solution is preferably 0,001 to 1.0 mg / mL, more preferably 0.05 to 0.4 mg / mL, relative to the total amount of the reaction solution. If the addition amount of tryptophanase or tryptophan synthase is within the above range, compared to before addition, the labeled protein can be synthesized in higher yield.

Amino acids other than tryptophan are 19 types other than tryptophan among 20 types of amino acids which comprise a protein. Specifically, L-alanine, L-arginine, L-aspartic acid, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L- Proline, L-serine, L-threonine, L-tyrosine, L-valine, L-cysteine, L-asparagine, L-glutamine may be mentioned.
In the present invention, L-tryptophan may be used as long as the effects of the present invention are not impaired.

  0.01-20 mM is preferable and, as for content of each 18 types of amino acids other than tryptophan and L-serine in a reaction liquid, 1.0-5.0 mM is more preferable. If the content of each amino acid is equal to or more than the lower limit value of the above range, a protein having labeled tryptophan incorporated therein is synthesized. If the content of each amino acid is below the upper limit value of the above range, a protein in which labeled tryptophan is incorporated can be synthesized with higher yield.

Since L-serine is used for the synthesis of labeled tryptophan, it is preferable that L-serine in the reaction solution is larger than other amino acids.
0.001-20 mM is preferable and, as for content of L- serine in a reaction liquid, 0.01-5.0 mM is more preferable. If content of L-serine is more than the lower limit of the said range, it will be easy to manufacture labeled protein highly efficiently. If content of L-serine is below the upper limit of the said range, it will be easy to manufacture labeled protein with high efficiency.

The energy source is not particularly limited as long as it is a substance used as an energy source in vivo, and ATP (adenosine 5'-triphosphate), GTP (guanosine 5'-triphosphate), CTP (cytidine 5'-) Substances having a high energy phosphate bond such as triphosphate), UTP (uridine 5'-triphosphate), creatine phosphate, phosphoenolpyruvate and the like are preferable. As an energy source, 1 type may be used independently and 2 or more types may be used together.
The content of the energy source in the reaction solution can be appropriately set depending on the protein synthesis activity of the cell extract, the type of labeled protein to be synthesized, and the like.

As a nucleic acid encoding an amino acid sequence of interest, a DNA of an appropriate sequence encoding the amino acid sequence of interest, which can be transcribed and translated, or an RNA of an appropriate sequence encoding an amino acid sequence of interest, which can be translated it can. In the present invention, the DNA encoding the amino acid sequence of the labeled protein of interest may be added to the cell-free transcription translation system to synthesize the labeled protein, and the RNA encoding the amino acid sequence of the labeled protein of interest to the cell-free translation system In addition, labeled proteins may be synthesized.
For example, by using a nucleic acid encoding the amino acid sequence of trastuzumab, it is possible to produce labeled trastuzumab in which the indole ring of the side chain of tryptophan residue in trastuzumab is labeled with ¹⁸ F.

  The content of the nucleic acid in the reaction solution can be appropriately set depending on the protein synthesis activity of the cell extract, the type of labeled protein to be synthesized, and the like, and can be, for example, about 0.5 to 10 μg / mL.

  In the reaction solution, if necessary, an enzyme involved in ATP regeneration (for example, a combination of phosphoenolpyruvate and pyruvate kinase, or a combination of creatine phosphate and creatine kinase), various RNA polymerases (T7, T3, and SP6 RNA polymerase etc., chaperone proteins (for example, DnaJ, DnaK, GroE, GroEL, GroES and HSP70 etc.) which have a function to help the protein form a three-dimensional structure may be added.

  In addition, the reaction solution can be supplemented with non-proteinaceous components, if necessary. The non-proteinaceous component is a component originally contained in the cell extract, but is a component capable of improving the ability to synthesize proteins by adding it separately, and examples include tRNA.

Furthermore, the reaction solution may contain various additives for protection and / or stabilization of protein and RNA, as necessary. As additives, for example, ribonuclease (RNase) inhibitor (RNase inhibitor etc.), reducing agent (dithiothreitol etc.); RNA stabilizer (spermidine etc.), protease inhibitor (phenylmethane sulfonyl fluoride (PMSF) Etc., salts (magnesium acetate, magnesium chloride, potassium acetate, calcium chloride etc.), antibacterial agents (sodium azide, ampicillin etc.) and the like.
The content of the additive in the reaction solution may be appropriately set in accordance with the protein synthesis activity of the cell extract to be used, the type of the target labeled protein, and the like.

For the synthesis of the labeled protein in the present invention, a conventionally known batch method may be used, or a dialysis method may be used.
When the batch method is used, for example, a nucleic acid encoding an amino acid sequence of a labeled protein of interest, a labeled indole labeled with ¹⁸ F or ¹⁹ F, an amino acid composition containing 19 amino acids other than tryptophan, in a cell extract In addition to pyruvate and ammonium ions, ATP, GTP, CTP, UTP, buffers, salts, RNase inhibitors, antibacterial agents, if necessary, RNA polymerase such as T7 RNA polymerase (when DNA is used as a template), tRNA etc. The reaction mixture

The reaction conditions may be appropriately set depending on the cell extract to be used, the labeled protein to be synthesized, and the like.
The reaction temperature is preferably 20 to 40 ° C., and more preferably 23 to 37 ° C. in both the batch method and the dialysis method.
In the case of a batch method, reaction time is preferably within 90 minutes, more preferably 5 to 90 minutes. In the case of the dialysis method, 1 to 24 hours are preferable, and 5 to 16 hours are more preferable.

  When the labeled protein is produced using the dialysis method, the reaction solution is used as a dialysis inner fluid, dialyzed against the dialysis solution of 5 to 10 times the volume of the reaction solution, and the generated labeled protein is recovered from the dialysis inner fluid . The dialysis is preferably carried out with stirring. In addition, dialysis may be performed while periodically replenishing the reaction solution with a nucleic acid encoding the amino acid sequence of the target labeled protein. Dialysis can be carried out by using a dialysis device capable of shaking or stirring (rotational stirring or the like) in a state where the inner fluid and the outer fluid are separated by a dialysis membrane.

  As the dialysis external fluid, for example, one obtained by removing cell extract, RNase inhibitor, nucleic acid, RNA polymerase, pyruvate kinase or creatine kinase etc. from the composition of dialysis internal fluid can be used. Specifically, for example, a solution containing a buffer, ATP, GTP, CTP, UTP, salts, amino acids and the like can be used as a dialysis outer solution.

3,500-100,000 are preferable and, as for the molecular weight cut off of the dialysis membrane which isolate | separates a dialysis internal fluid and a dialysis external fluid, 10,000-50,000 are more preferable.
20-40 degreeC is preferable and, as for the temperature of dialysis, 23-37 degreeC is more preferable.
The shaking speed or the stirring speed can be, for example, 100 to 300 rpm.
The reaction time in the case of dialysis can be appropriately selected while monitoring the production of the labeled protein of interest.

After synthesizing the labeled protein, it is preferable to purify the labeled protein. As a purification method of the labeled protein, a known purification method of protein can be adopted. For example, ammonium sulfate or acetone precipitation, acid extraction, anion or cation exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, gel filtration chromatography Hydroxyapatite, isoelectric point chromatography, chromatofocusing and the like. These purification methods may be performed alone or in combination of two or more. In addition, it is possible to use an affinity purification method in which a tag (a histidine tag, a GST tag, a maltose binding tag, etc.) is added to a labeled protein at the time of synthesis and adsorption specifically recognizing the tag is used.
Identification and quantification of the obtained labeled protein can be performed by activity measurement, immunological measurement, spectroscopic measurement, amino acid analysis, etc., while comparing with a standard sample as necessary.

As described above, in the method for producing a labeled protein of the present invention, the labeled protein is synthesized because a labeled protein is synthesized by incorporating a labeled tryptophan synthesized in the system using a labeled indole in a cell-free protein synthesis system. It can be easily manufactured.
Specifically, in the case of synthesizing a labeled amino acid, a plurality of protecting groups are required, and there is a problem that the purification of the labeled amino acid takes a long time. On the other hand, labeled indole labeled with ¹⁸ F or ¹⁹ F can be synthesized without protection and can be easily purified with a simple column such as silica gel or ODS because it is highly hydrophobic. In the present invention, since labeled indole can be used as a raw material, and labeled protein can be synthesized at one time via labeled tryptophan, there is no need to synthesize labeled tryptophan using a plurality of protecting groups, and there is no need to purify labeled tryptophan once. . In this manner, ¹⁸ F or ¹⁹ F-labeled protein can be easily produced in a short time without complicated synthesis and purification.
In addition, since labeled indole can be produced at low cost compared to labeled tryptophan labeled with ¹⁸ F or ¹⁹ F, labeled protein can be produced at low cost.

  In addition, the labeled protein obtained by the production method of the present invention is smaller in structural change of the protein due to labeling compared to the method using a chelating agent or a labeling reagent as described in Patent Documents 1 and 2, and the label is localized to the protein And the influence on the behavior is small. In addition, since a cell-free protein synthesis system is adopted, various types of proteins can be labeled, including proteins that are difficult to obtain. Therefore, it can contribute to image diagnosis of various diseases by, for example, PET imaging etc. and antibody drug development.

[Method for producing peptide]
The method for producing a labeled peptide of the present invention is a method for producing a labeled peptide labeled with ¹⁸ F or ¹⁹ F.
In the present invention, “peptide” is defined as a compound in which 2 or more and less than 25 amino acids are linked by peptide bond. In addition, “ ¹⁸ F or ¹⁹ F-labeled peptide” is defined as a peptide having ¹⁸ F or ¹⁹ F in the molecule.

In the method for producing a labeled peptide of the present invention, a cell-free protein synthesis system is used to synthesize a labeled peptide labeled with ¹⁸ F or ¹⁹ F. Similar to the method for producing a labeled protein of the present invention, the method for producing a labeled peptide of the present invention uses labeled indole labeled with ¹⁸ F or ¹⁹ F.

  The method for producing a labeled peptide of the present invention can be carried out in the same manner as the method for producing a labeled protein except that the number of amino acids bound by peptide bond in the target labeled compound is 2 or more and less than 25. Specifically, in the method for producing a labeled peptide of the present invention, the label of the present invention is used except that a nucleic acid encoding the amino acid sequence of the labeled peptide of interest is used instead of the nucleic acid encoding the amino acid sequence of the labeled protein of interest. It can be carried out in the same manner as the method for producing proteins.

  In the method for producing the labeled peptide of the present invention, as in the method for producing the labeled protein of the present invention, the labeled indole can be used as a raw material, and the labeled protein can be synthesized at one time via the labeled tryptophan. Can be manufactured. In addition, the structural change of the peptide by labeling is small, and the influence of the labeling on the localization and kinetics of the peptide is small.

[kit]
The kit of the present invention is a kit for synthesizing labeled protein or labeled peptide labeled with ¹⁸ F or ¹⁹ F, and comprises a cell-free protein synthesis system. As a cell-free protein synthesis system, the above-mentioned cell extract may be provided, and instead of the cell extract, a solution used in the above-described reconstituted cell-free protein synthesis method may be provided.
The kit of the present invention preferably contains, among the components described above, a component other than a labeled tryptophan or a labeled indole, and a nucleic acid encoding an amino acid sequence of a labeled protein or a peptide of interest. Specifically, the kit of the present invention preferably contains 19 amino acids other than tryptophan, an energy source and the like.

In the kit of the present invention, at least one of tryptophan labeled with ¹⁸ F or ¹⁹ F at the side chain indole ring and labeled indole labeled with ¹⁸ F or ¹⁹ F, and an amino acid of the labeled protein or peptide of interest By adding a nucleic acid encoding a sequence, a labeled protein or a labeled peptide of interest can be conveniently produced.

  The content of tryptophanase or tryptophan synthase in the kit is preferably 0.001 to 1.0 mg / mL, more preferably 0.05 to 0.4 mg / mL. If the addition amount of tryptophanase or tryptophan synthase is at least the lower limit value of the above range, a labeled protein can be efficiently produced as compared with before addition. When the addition amount of tryptophanase or tryptophan synthase is not more than the upper limit value of the above range, the labeled protein can be produced more efficiently.

  0.001-20 mM is preferable and, as for content of L- serine in a kit, 0.01-3.0 mM is more preferable. If content of L-serine is more than the lower limit of the said range, it will be easy to manufacture labeled protein highly efficiently. If content of L-serine is below the upper limit of the said range, it will be easy to manufacture labeled protein with high efficiency.

[Reagent for PET]
The reagent for PET of the present invention is a reagent for PET, which comprises a labeled protein in which the indole ring of the side chain of the tryptophan residue is labeled with ¹⁸ F. The reagent for PET is a reagent used for PET imaging, that is, a positron-labeled antibody which is an imaging probe for PET imaging.
The reagent for PET of the present invention can be produced by the method for producing a labeled protein of the present invention described above.

The labeled protein used in the PET reagent is not particularly limited, for example, labeled trastuzumab indole ring of the side chain labeled with ¹⁸ F tryptophan residues ^{([18 F] tra-scFv} ) and the like.
Since trastuzumab specifically binds to HER2 protein which is a gene product of human oncogene HER2 / neu (c-erbB-2), labeled trastuzumab is useful as a reagent for PET.

[Pharmaceutical composition, HER2 protein detection kit]
The pharmaceutical composition of the present invention comprises the labeled trastuzumab. The pharmaceutical composition of the present invention may contain other components other than labeled trastuzumab. Examples of other components contained in the pharmaceutical composition include stabilizers, solubilizers, buffers for affinity column purification, and the like.

The HER2 protein detection kit of the present invention includes the kit described above, and includes at least a cell-free protein synthesis system. In addition, the HER2 protein detection kit of the present invention may further contain trastuzumab template DNA, tryptophanase, labeled indole labeled with ¹⁸ F or ¹⁹ F, and L-serine or pyruvate and ammonium ion.

EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but the present invention is not limited by the following description.
[Quantification of labeled protein (CAT protein)]
For quantification of the labeled protein (CAT protein) produced in each example, W. V. It carried out according to the method of Shaw et al. (See Methods Enzymol. 735-755, 1975). That is, acetyl coenzyme A and chloramphenicol are used as a substrate for acetylation reaction of chloramphenicol with CAT protein, and the resulting reduced coenzyme A is obtained as 5,5'-dithiobis-2-nitrobenzoic acid (DNTB The color was quantified using The activity of CAT protein was quantified from the amount of increase per unit time of absorbance at 412 nm at 30 ° C., and the amount of CAT protein was determined using this as an index.

[Example 1 (Example)]
A reaction solution having a composition shown in Table 1 was prepared, reacted at 37 ° C. for 1 hour, and used in a cell-free protein synthesis system containing labeled indole (5- ¹⁹ F-indole) in which the hydrogen atom at position 5 was substituted with ¹⁹ F. , Chloramphenicol acetyltransferase (CAT protein) was labeled with ¹⁹ F to produce a labeled protein. As a cell extract, the method of Kikawa et al. (WO 2003/053910, Patent No. 3317983, Patent No. 3145431, Tugarinov, V., et. Al., J. Am. Chem. Soc., (2003) vol. 125, pp. 13868-13878.), The S30 extract extracted from E. coli was used. The S30 extract contains tryptophanase or tryptophan synthase. PUC-CAT (see Kim et al., Eur. J. Biochem. 239, 881-886, 1996) was used as a template DNA.

[Example 2 (Example)]
A labeled protein was produced in the same manner as in Example 1 except that the concentration of labeled indole (5- ¹⁹ F-indole) in the reaction solution was changed to 3.0 mM.

[Example 3 (Example)]
A labeled protein was produced in the same manner as in Example 1 except that the concentration of labeled indole (5- ¹⁹ F-indole) in the reaction solution was changed to 7.5 mM.

[Example 4 (Reference Example)]
A labeled protein was produced in the same manner as in Example 1 except that L-tryptophan was added to the reaction solution to a concentration of 1.5 mM instead of labeled indole (5- ¹⁹ F-indole).

[Example 5 (Reference Example)]
A CAT protein was produced in the same manner as in Example 1 except that indole was used instead of the labeled indole.

The results of Examples 1 to 3 and Examples 4 and 5 are shown in FIG. As shown in FIG. 1, in Examples 1 to 3 in which indole labeled with ¹⁹ F was used, labeled protein labeled with ¹⁹ F was obtained with high efficiency. The production efficiency of the labeled proteins of Examples 1 to 3 was also sufficient compared to Examples 4 and 5 where natural L-tryptophan and indole were used. Moreover, in the comparison of Examples 1 to 3, the amount of synthesized labeled protein was the highest in Example 1 in which the concentration of 5- ¹⁹ F-indole was 1.5 mM.
Thus, it is confirmed that labeled tryptophan is synthesized from 5- ¹⁹ F-indole by the action of tryptophanase or tryptophan synthase inherent in the cell-free protein synthesis system and further incorporated into protein synthesis to obtain a labeled protein. It was done.

[Example 6]
The labeled protein was produced in the same manner as in Example 1 except that tryptophanase was added to the reaction solution so as to be 0.05, 0.1, and 0.4 mg / mL, respectively, and reacted at 37 ° C. for 1 hour. . Also, the CAT protein was produced in the same manner as in Example 1 and Example 5. The results are shown in FIG.

  As shown in FIG. 2, by adding 50 μg / mL or more of tryptophanase to the reaction solution using the S30 extract, the synthesis amount of the labeled protein was improved.

[Example 7]
Instead of labeled indole (5- ¹⁹ F-indole), 2-methylindole, 4-nitriindole, 5-amino-2-methylindole, 5-aminoindole, 5-bromoindole, 5-cyanoindole, 5- A labeled protein was prepared in the same manner as in Example 1 except that reaction was carried out at 37 ° C. for 1 hour using fluoroindole (5- ¹⁸ F-indole), 5-hydroxyindole, 5-methoxyindole, 6-cyanoindole or indole. (CAT protein) was produced. In addition, 0.05 mg / mL of tryptophanase was added to the reaction solution to similarly produce a labeled protein (CAT protein). The results are shown in FIG.

  As shown in FIG. 3, the CAT protein was not synthesized with modified indoles other than 5-fluoroindole. On the other hand, when 5-fluoroindole is used, labeled tryptophan is synthesized in a cell-free protein synthesis system, the labeled tryptophan is incorporated into CAT protein, and the labeled protein is efficiently synthesized. Was confirmed.

[Example 8 (Example)]
A labeled trastuzumab in which the hydrogen atom at the 5-position of the indole ring of the tryptophan residue is substituted with ¹⁸ F by preparing a reaction solution (500 μl in total) having the composition shown in Table 2 below and heating at 37 ° C. for 30 minutes ([ ¹⁸ F] tra-scFv) was produced. The obtained [ ¹⁸ F] tra-scFv was purified using the affinity of the SBP tag bound to [ ¹⁸ F] tra-scFv and the affinity column.

[Example 9]
Trastuzumab is an anticancer agent that exerts an antitumor effect by specifically binding to HER2 protein that is involved in malignant transformation of cells, and HER2 protein is present in SK-OV-3.
In order to confirm the accumulation of [ ¹⁸ F] tra-scFv produced in Example 8 at the tumor site, ICR mice (female, 13) which were artificially tumored by implanting normal mice and SK-OV-3 cells. Age, tumor model: BALB / c Slc-nu / nu, 30 days after SK-OV-3 transplantation, 200 μL (8.6 to 9.2 MBq) of [ ¹⁸ F] tra-scFv solution injected from tail vein The PET imaging test was performed. PET imaging tests were performed under isoflurane anesthesia (flow rate: 1.0 L / min, concentration: 1.0%), and mice were imaged for 90 minutes after administration of the [ ¹⁸ F] tra-scFv solution. Clairvivo PET was used as an imaging device, and the display image was a maximum value projection display (MIP). The results are shown in FIG.
Also, for normal ICR mice (male, 6 weeks old, 30 to 32 g) not treated with tumor, 200 μL (3.0 to 5.3 MBq) of [ ¹⁸ F] tra-scFv solution is similarly added to the tail vein. The PET imaging test was performed. PET imaging studies were performed under isoflurane anesthesia (flow rate: 1.5 L / min, concentration: 1.5%) and tumors were generated except for imaging the mouse for 60 minutes after administration of the [ ¹⁸ F] tra-scFv solution The procedure was the same as for the control mice. The results are shown in FIG.

As shown in FIG. 5, in normal model mice, accumulation of [ ¹⁸ F] tra-scFv only in urine, kidney, liver and blood was confirmed. On the other hand, as shown in FIG. 4, in the tumor model mouse, accumulation of [ ¹⁸ F] tra-scFv in SK-OV-3 cells was confirmed, and the availability as a tumor imaging agent was shown.

[Example 10]
HER2 protein is also present in MCF7. [ ¹⁸ F] tra-scFv (100 μL) prepared in Example 8 was used as a tumor tissue section of a tumor model prepared using each of SK-OV-3 (HER2 high expression) and MCF7 (HER2 low expression) tumor cell lines. 0.2 MBq) was added, and the binding state after 30 minutes by in vitro autoradiography was observed. The results of tumor tissue sections using a tumor cell line of SK-OV-3 are shown in FIG. 6A, and the results of tumor tissue sections using a tumor cell line of MCF7 are shown in FIG. 6B.
In addition, the binding state in each tumor tissue section was observed in the same manner except that unlabeled trastuzumab was added to block the HER2 protein 60 minutes before adding [ ¹⁸ F] tra-scFv. The results of tumor tissue sections (HER2 block) using a tumor cell line of SK-OV-3 are shown in FIG. 6C, and the results of tumor tissue sections (HER2 block) using a tumor cell line of MCF7 are shown in FIG. 6D.

As shown in FIGS. 6A and 6B, in tumor tissue fragments not blocked with HRR2 protein, HRR2 of [ ¹⁸ F] tra-scFv was obtained using either SK-OV-3 or MCF7 tumor cell line. Binding to the protein was confirmed. On the other hand, as shown in FIG. 6C and FIG. 6D, autoradiography derived from [ ¹⁸ F] tra-scFv was not obtained in the tumor tissue fragment in which the HRR2 protein was blocked. These results indicate that [ ¹⁸ F] tra-scFv recognizes the same HER2 protein as unlabeled trastuzumab, and that ¹⁸ F labeling does not alter the structure or function of the antigen binding site. Indicated.

Claims (9)

In a cell-free protein synthesis system, labeled with ¹⁸ F or ¹⁹ F labeled indole with tryptophanase or tryptophan synthase and L-serine or with pyruvate and ammonium ion labeled with ¹⁸ F or ¹⁹ F Let tryptophan be synthesized,
Furthermore, a method for producing a labeled protein, wherein a labeled protein labeled with ¹⁸ F or ¹⁹ F is synthesized using the labeled tryptophan in the cell-free protein synthesis system. The method for producing a labeled protein according to claim 1, wherein the labeled protein is labeled trastuzumab in which the indole ring of the side chain of tryptophan residue in trastuzumab is labeled with ¹⁸ F. The manufacturing method of the reagent for PET which manufactures the reagent for PET which the indole ring of the side chain of a tryptophan residue manufactures the labeled protein which consists of a ¹⁸ F label by the manufacturing method of the labeled protein of Claim 1 or 2. In a cell-free protein synthesis system, labeled with ¹⁸ F or ¹⁹ F labeled indole with tryptophanase or tryptophan synthase and L-serine or with pyruvate and ammonium ion labeled with ¹⁸ F or ¹⁹ F Let tryptophan be synthesized,
Furthermore, a method for producing a labeled peptide, wherein a labeled peptide labeled with ¹⁸ F or ¹⁹ F is synthesized using the labeled tryptophan in the cell-free protein synthesis system. A kit for synthesizing a labeled protein or a labeled peptide labeled with ¹⁸ F or ¹⁹ F, which comprises a cell-free protein synthesis system. A reagent for PET comprising a labeled protein in which an indole ring of a side chain of a tryptophan residue is labeled with ¹⁸ F. Labeled trastuzumab in which the indole ring of the side chain of tryptophan residue is labeled with ¹⁸ F.   A pharmaceutical composition comprising the labeled trastuzumab according to claim 7.   A HER2 protein detection kit comprising the kit according to claim 5.