JP2002322196A - Method for labeling protein or peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for labeling a protein and a peptide having disulfide bond with radioactive technetium or radioactive rhenium and completing all steps from reducing the disulfide bond to radioactively labeling without any complex operation for purification. SOLUTION: The method comprises reacting the protein or the peptide having disulfide bond with a reducing agent in a solution of pH 8 to 11 in a molar ratio of 4 to 30 folds based on the protein and the peptide to cleave the disulfide bond and form a sulfhydryl group, adding the radioactive technetium or the radioactive rhenium in the reaction solution, and reducing the radioactive technetium or the radioactive rhenium to react with the sulfhydryl group.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method and a kit for radiolabeling a protein or peptide having at least one disulfide bond with a radioisotope.

[0002]

2. Description of the Related Art Proteins radioactively labeled with radioisotopes are used in the diagnosis and treatment of various pathological conditions in living organisms. For example, antibodies labeled with radioisotopes of technetium are used in vivo. The antibody recognizes the antigen at the lesion site, and by tracking the radioactivity of the labeled antibody, it can be used to confirm the presence or absence of the lesion site from outside the body and its distribution. Isotopically labeled antibodies can be used as internal radiotherapeutic agents.

[0003] In recent years, directional preparations containing monoclonal antibodies and the like have been used for treatment, and therapeutic results have been achieved. Diagnosis of the distribution and expression level of a monoclonal antibody in a living body with respect to the target molecule of the monoclonal antibody can provide very useful information for treatment planning or treatment effect judgment.

[0004] Proteins, especially antibodies, are radioactive isotopes of technetium or rhenium, such as technetium-99.
Many methods for radiolabeling with m have been reported to date and can be broadly classified into two types: indirect labeling and direct labeling.

[0005] Indirect labeling is a method in which an antibody molecule is bound to a chelating agent such as diethylenetriaminepentaacetic acid (DTPA), and the chelating agent is chelated with a radioactive metal to label. This method has a problem that a chelating agent must be introduced into a protein, and the functional group of the amino acid introduced by the chelating agent used is limited. Further, there is a problem that an unreacted chelating agent present in excess must be removed, and if this operation is neglected, the labeling rate will be significantly reduced. At the same time, this operation is complicated and requires a large amount of antibodies.

[0006] On the other hand, in the direct labeling method, an antibody molecule is reduced with an appropriate reducing agent to cleave disulfide (SS) bonds present in the antibody molecule, thereby generating a sulfhydryl (SH) group, and then forming the sulfhydryl (SH) group. In this method, technetium-99m reduced via the sulfhydryl group is directly bound to the antibody molecule.

As a reducing agent for cleaving the disulfide bond, 2-mercaptoethanol (2-ME), dithiothreitol (DTT) and the like have been disclosed (Japanese Patent Application Laid-Open No. 2-5044387, Japanese Patent Publication No. 7-6203
No. 2, JP-B-7-23326, JP-A-6-1
No. 00469, Japanese Patent No. 2550481, Japanese Patent No. 252168, Japanese Patent No. 2769244), since these compounds are relatively toxic substances, when these reducing agents reduce the antibody, After the reduction, the reducing agent used must be removed.

Other reducing agents include tin (II) and ascorbic acid. However, the reducing power of tin (II) and ascorbic acid is
Since it is weaker than 2-ME and DTT, it requires an excess of tin (II) or ascorbic acid to cleave the disulfide bond of the antibody molecule, and the excess tin (I
I) and ascorbic acid are reduced technetium-9
Excess reducing agent must be removed prior to labeling, or by-products removed after labeling, as it binds with 9m to produce by-products.

For example, Japanese Patent No. 3070763 and Japanese Patent Application Laid-Open No. 2000-53590 disclose a method in which a protein is reduced by tin (II) as a metal reducing agent, and then the protein is radiolabeled with technetium or rhenium. It is described that a step of reducing excess tin (II) after reducing the protein with tin (II) in a molar ratio of 100 times or more is essential. On the other hand, the excessively added tin (II) reduces the disulfide bond of the antibody and forms a complex with the sulfhydryl group generated by the reduction to protect the sulfhydryl group from returning to the non-reactive disulfide. It can be said that this is a characteristic of this labeling method. However, tin (II) present in excess not only causes a reduction in the labeling rate due to the production of by-products, but also presents a danger of denaturation or inactivation of the antibody due to excessive reduction of the antibody. I do.

In Japanese Patent Application Laid-Open No. 6-505990, a protein is reduced by a molar ratio of 30 to 50 equivalents of tin (II), and then technetium-99m
However, there is a risk that excessively existing tin (II) reduces the antibody more than necessary and denatures the antibody. In addition, it is described that tin (II) used here is preferably used as a sucrose salt or a glucaric acid salt.
It has been pointed out that when used alone, a radioactive colloid unsuitable for use is formed. Further, it is described that this labeling method requires the addition of a chelating agent after labeling. The addition of this chelating agent
It is for reacting with weak or unbound technetium-99m, thereby terminating the reaction and binding and removing free or weakly bound technetium-99m, and this step cannot be excluded.

As described above, in the prior method, in a method of directly labeling a protein such as an antibody with technetium-99m or the like, a step of removing an excessive reducing agent after reducing the antibody, A step of removing technetium-99m having weak binding or unbound after labeling with technetium-99m or treating with technetium-99m in advance is required, and a purification step from reduction to labeling of the protein is not required. Could not be prepared.

[0012]

DISCLOSURE OF THE INVENTION In view of the above problems, the present invention did not participate in the reduction reaction when radioactively labeling a protein or peptide having a disulfide bond with radioactive technetium or radioactive rhenium. Disulfides contained in proteins or peptides without the need for complicated purification operations such as the step of removing excess reducing agents and impurities generated thereby, or the step of adding a chelating agent before and after radiolabeling. An object of the present invention is to provide a labeling method in which all steps from reduction of binding to radiolabeling are completed.

[0013]

Means for Solving the Problems In order to solve the above problems, the present inventors have conducted intensive studies. As a result, when a protein or peptide having a disulfide bond is radioactively labeled with radioactive technetium or radioactive rhenium, the protein or the peptide is labeled. Reduction of the disulfide bond of the peptide is labile to the disulfide bond and tin (I
When carried out in a solution having a high pH, which is an unfavorable condition such that the reducing agent such as I) tends to be in a colloidal state, the disulfide bond can be reduced with a surprisingly small amount of the reducing agent. In a series of steps from reduction of binding to radiolabeling, all purification steps such as removing excess reducing agent and impurities generated by it, or adding a chelating agent before and after radiolabeling are required. It has been found that labeling can be performed at a high labeling rate of 90% or more without performing the method, and the present invention has been completed.

That is, the present invention relates to a method of labeling a protein or peptide having a disulfide bond with radioactive technetium or radioactive rhenium, wherein the protein or peptide having a disulfide bond and a reducing agent are dissolved in a solution of pH 8-11. The reaction is carried out in a molar ratio of the reducing agent to the protein or peptide of 4 to 30 times to cleave the disulfide bond of the protein or peptide to generate a sulfhydryl group, and the reaction solution containing the protein or peptide and the reducing agent Radioactive technetium or radioactive rhenium is added to the mixture to reduce radioactive technetium or radioactive rhenium, and the sulfhydryl group of the protein or peptide is combined with the reduced radioactive technetium or radioactive rhenium to form a protein. A protein or peptide labeling method which comprises labeling the quality or peptide.

Further, the present invention relates to a method for manufacturing a container, comprising:
A reducing agent that cleaves disulfide bonds and reduces radioactive technetium or rhenium, and (b) a protein or peptide having at least one disulfide bond, wherein the molar ratio of the reducing agent to the protein or peptide is 4 to 4. 30 times the range,
Further, the pH is in the range of 8 to 11, and the disulfide bond of the protein or peptide is cleaved into a sulfhydryl group by the reducing agent,
A kit for labeling a protein or peptide with radioactive technetium or radioactive rhenium.

[0016]

DETAILED DESCRIPTION OF THE INVENTION In the present invention, the pH of a solution containing a protein or peptide to be radioactively labeled is determined.
Is adjusted to a range of about 8 to 11, preferably about 8.5 to 10, and reacting with a reducing agent such as tin (II) in the solution to cleave the disulfide bond of the protein or peptide to obtain sulfhydryl. Generate a group. The pH of the solution may be adjusted using a caustic soda solution, a hydrochloric acid solution, or the like, or may be adjusted using a buffer solution. Examples of the buffer include a carbonate buffer and a phosphate buffer. At this time, if the pH of the solution is lower than 8, a decrease in the labeling rate is recognized, and if the pH of the solution exceeds 11, it is not preferable because it is not suitable for proteins and peptides.

In the present invention, the reducing agent is required in a sufficient amount to reduce the disulfide bond of the protein or peptide and the radioactive technetium or rhenium added in the next step. Is mixed in a molar ratio of 4 to 30 times, preferably 4 to 10 times, more preferably 6 to 8 times.

If the amount of the reducing agent is less than 4 times the molar ratio of the protein or peptide, the disulfide bond of the protein or peptide will not be sufficiently cleaved, resulting in a decrease in the labeling rate. On the other hand, if the amount of the reducing agent exceeds 30 times the molar ratio to the protein or peptide, precipitation may be observed when the protein or peptide is mixed with the reducing agent depending on the concentration of the protein or peptide. Therefore, it is not preferable.

As the reducing agent, those containing at least one compound selected from tin (II), copper (I), iron (II), titanium (III) and the like can be used. Can be used as chlorides, and can also be used as sucrose salts, tartrate salts, and the like. Stannous chloride is preferred because it is easily available, has a suitable reducing power, and is easy to handle.

As the protein or peptide used in the present invention, any kind of protein or peptide having at least one disulfide bond can be used, and any protein or peptide used as a diagnostic or therapeutic agent can be used. Can be used. For example,
Selected from antibodies, antibody fragments, hormones, cytokines, other signaling substances, etc., and the antibodies and antibody fragments include polyclonal antibodies, monoclonal antibodies, humanized antibodies, chimeric antibodies, hybrid antibodies, and all of these antibody fragments And, in particular, polyclonal and monoclonal immunoglobulins can be used. In addition, immunoglobulin is IgG, IgM, IgA,
Any immunoglobulin class such as IgD and IgE can be used.

As the radioactive technetium which is a radioisotope, for example, technetium-99m can be used, and as the radioactive rhenium, for example, rhenium-1
86 or rhenium-188 can be used, technetium-99m labeled protein or peptide can be used as a diagnostic agent, and rhenium-186 or rhenium-188 labeled protein or peptide can be used for internal radiation. It can be used as a therapeutic.

In a preferred embodiment of the labeling method of the present invention, for example, the antibody and the reducing agent are mixed in a vessel whose inside has been sufficiently replaced with an inert gas, for example, helium, nitrogen, argon, or the like. For 12 hours or more, preferably 18 to 24 hours. All operations are performed under an inert gas atmosphere. At the time of incubation, appropriate heating can also be performed in consideration of the properties of the target compound. Further, the container can be shaken during the incubation. The reaction solution after the incubation does not require purification such as removal of a reducing agent, and the reaction solution after the incubation can be stored frozen or lyophilized until labeling.

After the above incubation, for example, a sodium pertechnetate solution is added to the reaction solution of the antibody and the reducing agent without removing the reducing agent in the reaction solution,
Further, the incubation is usually performed for 10 minutes or more, preferably 15 minutes or more, for example, 15 to 30 minutes. Thereby, sodium pertechnetate is reduced and technetium (I
II) From -99m to technetium (V) -99m, and then the sulfhydryl group of the antibody reacts with the reduced technetium-99m to bind and complete the labeling of the antibody. The amount of the radioisotope added, such as sodium pertechnetate, varies depending on the type of antibody in the case of an antibody, for example, but is usually preferably up to 30 mCi per mg of antibody. The solution after the labeling can be used after being subjected to a filter treatment if necessary.

Furthermore, the present invention provides a kit used for labeling a protein or peptide having a disulfide bond with radioactive technetium or radioactive rhenium, which is prepared by a method comprising the above-mentioned steps.

The kit of the present invention comprises, in a single container,
(A) a reducing agent that cleaves a disulfide bond and reduces radioactive technetium or radioactive rhenium; and (b)
A protein or peptide having at least one disulfide bond, so that the protein or peptide is contained in a state where the disulfide bond is cleaved into a sulfhydryl group by a coexisting reducing agent.

As the reducing agent, those containing at least one compound selected from tin (II), copper (I), iron (II), titanium (III) and the like can be used, and disulfide of protein or peptide can be used. Sufficient amounts are required to reduce the bond to produce sulfhydryl groups and to reduce the radioactive technetium or rhenium added later. After reducing the disulfide bond of the protein or peptide in the kit, there remains a reducing agent that has not been used to reduce the disulfide bond, and the remaining reducing agent reduces the radioactive technetium or rhenium that is added later.

This kit has a pH of about 8 to 1 for a solution containing a protein or peptide to be radiolabeled.
1, preferably in the range of about 8.5 to 10, wherein the reducing agent in the solution has a molar ratio of reducing agent to protein or peptide of 4 to 30 times, preferably 4 to 10 times, more preferably 6 to 10 times. It is obtained by, for example, transferring the reaction solution to an appropriate container, sealing, and freezing, without removing the reducing agent in the reaction solution, by allowing the reaction to proceed in a range of up to 8 times.
Alternatively, the reaction solution transferred to the container can be freeze-dried and sealed. In the case of lyophilization, if the pH fluctuates significantly when redissolved, use a predetermined pH.
The buffer for adjusting the pH may be enclosed in another container and attached. As the buffer, a carbonate buffer, a phosphate buffer, and the like can be used as described above.

The reaction solution may contain additives which do not interfere with the labeling of the protein or peptide and which have high physiological acceptability, such as buffers and stabilizers. For example, as a buffer, carbonate, phosphate,
Acetate and the like, and as a stabilizer,
It may include ascorbate, citrate, tartrate, reducing sugars and the like.

The kit is prepared, for example, by adding 1 to 10 mg of an antibody and 7.2 to stannous chloride (anhydrous) in a container such as a vial.
96 μg in a solution having a pH of 8 to 9 and reacted for 12 hours or more, preferably for 18 to 24 hours, and then, without removing the reducing agent in the reaction solution, freeze in a solution state and freeze-preserve, or It is obtained by lyophilizing and storing the solution. For example, in the case of a monoclonal antibody, it can be usually stored in a freezer at minus 80 degrees.

When the kit is used, for example, when stored frozen, the solution is dissolved and then added with a sodium pertechnetate solution for at least 10 minutes, preferably 15 minutes.
It can be used by reacting for more than 1 minute, and when it is stored by freeze-drying, after the container is brought to room temperature, a sodium pertechnetate solution is added and dissolved to react for 10 minutes or more, preferably 15 minutes or more. When a buffer solution is attached, the buffer solution can be added before and after the sodium pertechnetate solution is added. The amount of the radioisotope added, such as sodium pertechnetate, varies depending on the type of antibody in the case of an antibody, for example, but is usually preferably up to 30 mCi per mg of antibody. This completes the labeling of the protein or peptide. Since the labeled solution is labeled at a high labeling rate without requiring purification, it can be used as it is.

As described above, the present invention provides a method for labeling a protein or a peptide having a disulfide bond and a radioisotope with a reducing agent using the same reducing agent. By performing the reduction reaction in a solution, a large amount of a reducing agent is not required unlike the prior art, and specifically, about 1/5 or less (molar) of the amount of the reducing agent used in the prior method. Ratio), the disulfide bond of the protein or peptide can be efficiently reduced, and as a result, the production of by-products is suppressed. A protein or peptide can be labeled at a high labeling rate without any complicated purification operation such as a step of adding a chelating agent before and after labeling. It is.

[0032]

Example 1 Influence of the amount of reducing agent (pH 8) A tin solution was prepared by dissolving 5 mg of stannous chloride in 10 mL of 0.01N hydrochloric acid solution which had been sufficiently bubbled with argon gas in advance, and then subjected to a filter treatment. Prepared.

Under an atmosphere of argon gas, 100 μL of a human immunoglobulin preparation, Venoglobulin-IH (trade name, manufactured by Welfide) and 900 μL of 0.1 M phosphate buffer (pH = 8) were added to the vial, and further added. 12 to 72 μL of a tin solution was added, and the mixture was sufficiently stirred by a stream of argon gas, and finally stoppered and sealed with something like a flip-off cap.

The vial was incubated overnight (about 21 hours) at room temperature to cleave the disulfide bonds of the antibody. At this time, if necessary, the solution may be heated to such an extent that the antibody is not denatured, or may be shaken slowly so that the liquid level of the vial does not collapse.

To this vial was added 1 mL of a sodium pertechnetate solution (46 mCi / mL), and the mixture was inverted and left standing for 30 minutes to prepare technetium-labeled human immunoglobulin ( ^99m Tc-HIG). Sodium pertechnetate solutions are generally commercially available
⁹⁹ Mo- ^99m Tc generator in which Mejitteku (Japan Medi-Physics Co., Ltd., trade name) was used which was eluted from.

The labeling rate was confirmed by an electrophoresis method using a cellulose acetate membrane (constant voltage: 210 V, 20 mi).
n).

Labeling is performed when the molar ratio of the amount of the antibody to the amount of stannous chloride is in the range of antibody: tin = 1: 1 to 1: 6, and the labeling rate is measured 1 hour and 3 hours after the labeling. I asked. Table 1 shows the results.

[0038]

[Table 1]

As a result, when the pH of the solution is 8 and the amount of tin is 4 to 6 times that of the antibody, removal of excess reducing agent and impurities, which was conventionally required, and chelation after radiolabeling, A high labeling rate could be achieved without adding an agent or the like.

Example 2: Influence of the pH of the solution (molar ratio of antibody: reducing agent = 1: 6) The pH of the solution was adjusted to 5 to 10, and the other conditions were the amount of antibody (5 mg) and the amount of radioactivity (31 -42 mCi), and labeling was performed in the same manner as in Example 1 except that the amount of stannous chloride (36 μg) was used. The incubation of the reaction solution containing the antibody and stannous chloride is 24 when the pH is 5-8.
When the pH is 8.5 to 10, the reaction is performed for 22 hours. When the pH of the solution is 5 to 8, the phosphate buffer and the p
When H was 8.5 to 10, the reaction was performed using a carbonate buffer.
After the labeling, the labeling rate was measured 1 hour and 6 hours later to determine the labeling rate. Table 2 shows the results.

[0041]

[Table 2]

The pH values in parentheses are actually measured values one week after labeling.

As can be seen from these results, when the pH of the solution was on the acidic side, the amount of the reducing agent was too small, so that a decrease in the labeling rate was observed. On the other hand, when the pH of the solution was labeled on the alkaline side, the disulfide bond of the antibody was sufficiently cleaved, resulting in a high labeling rate. In particular, when the pH of the solution was 8.5 to 10, a high labeling rate of 93% or more could be obtained.

Example 3 Influence of Amount of Reducing Agent (pH 9) When the pH of the solution was 9, the amount of antibody (5 mg), and the amount of radioactivity (35)
Labeling was performed in the same manner as in Example 1 except that mCi) and 0.1 M carbonate buffer (pH = 9) were used. Incubation of the reaction solution containing the antibody and stannous chloride was carried out for 22 hours.
Time went. After the labeling, the labeling rate was measured at 1 hour and 3 hours after the labeling. Table 3 shows the results.

[0045]

[Table 3]

As a result, when the pH of the solution was 9, a high labeling ratio could be obtained when the amount of tin was in the range of 6 to 8 times that of the antibody.

Example 4: Influence of the amount of radioactivity The amount of radioactivity was in the range of 50 to 200 mCi, and the amount of antibody (5
mg), 0.1 M sodium hydrogen carbonate solution (pH = 8 to
8.5), except that the amount of stannous chloride (36 μg) was used.
Labeling was performed in the same manner as in Example 1. The reaction solution containing the antibody and stannous chloride was incubated for 22 hours. After the labeling, the labeling rates were measured at 0.5 hours, 2.5 hours, and 21 hours after the labeling. Table 4 shows the results.

[0048]

[Table 4]

From this result, in this embodiment, 50 to 15
It was found that at a radioactivity of 0 mCi, a high labeling rate was achieved.

Example 5: Confirmation of labeling stability Antibody amount (10 mg), radioactivity amount (200 mCi),
Example 1 except that the amount of stannous chloride (72 μg) and the 0.1 M sodium hydrogen carbonate solution (pH = 8 to 8.5) were used.
Labeling was performed in the same manner as described above. The reaction solution containing the antibody and stannous chloride was incubated for 22 hours. After the labeling, the labeling rate was measured at 1 hour, 3 hours and 20 hours after the labeling. The labeling rate was shown as an average of three times.

Table 5 shows the results.

[0052]

[Table 5]

From these results, it was found that when the amount of antibody was 10 mg, a high labeling rate was achieved at a radioactivity of 200 mCi, and the stability was excellent.

[0054]

According to the present invention, when a protein or peptide having a disulfide bond is radioactively labeled with radioactive technetium or radioactive rhenium, a step of removing excess reducing agents or impurities, or before or after radioactive labeling, It is possible to provide a labeling method and a kit in which all steps from reduction of a protein or peptide to radiolabeling are completed without requiring a complicated purification operation such as a step of adding a chelating agent. In addition, the antibody can be labeled at a high labeling rate even at a high radioactivity of 200 mCi, and is excellent in stability. This means that the protein or peptide can be labeled the day before and delivered as a pre-prepared injection.

Claims (19)

[Claims] 1. A method for labeling a protein or peptide having a disulfide bond with radioactive technetium or radiorhenium, comprising: dissolving a protein or peptide having a disulfide bond and a reducing agent in a solution having a pH of 8 to 11; To reduce the disulfide bond of the protein or peptide to generate a sulfhydryl group, and to react with the radioactive technetium or the radioactive technetium in the reaction solution containing the protein or peptide and the reducing agent. A radioactive rhenium is added to reduce radioactive technetium or radioactive rhenium, and a protein or peptide is formed by binding the sulfhydryl group of the protein or peptide to the reduced radioactive technetium or radioactive rhenium. Protein or peptide labeling method which comprises labeling. 2. The method for labeling a protein or peptide according to claim 1, wherein the pH is in the range of 8.5 to 10. 3. The method according to claim 1, wherein the molar ratio of the reducing agent to the protein or the peptide is in the range of 4 to 10 times. 4. The method for labeling a protein or peptide according to claim 1, wherein the molar ratio of the reducing agent to the protein or peptide is in the range of 6 to 8 times. 5. The method of claim 1, wherein the reducing agent comprises at least one compound selected from the group consisting of tin (II), copper (I), iron (II), and titanium (III). . 6. The method according to claim 1, wherein the protein or peptide is selected from an antibody, an antibody fragment, a hormone, and a cytokine. 7. The radioactive technetium is technetium-9.
The method according to claim 1, which is 9 m. 8. The method according to claim 1, wherein the radioactive rhenium is rhenium-186 or rhenium-188. 9. A single container comprising: (a) a reducing agent that cleaves a disulfide bond and reduces radioactive technetium or rhenium, and (b) a protein or peptide having at least one disulfide bond. The molar ratio of the reducing agent to the protein or peptide is in the range of 4 to 30 times, and the pH is 8 to 11;
Wherein the disulfide bond of the protein or peptide is cleaved to a sulfhydryl group by the reducing agent. A kit for labeling a protein or peptide with radioactive technetium or radioactive rhenium. 10. The kit according to claim 9, wherein the pH ranges from 8.5 to 10. 11. The kit according to claim 9, wherein the molar ratio of the reducing agent to the protein or peptide is in the range of 4 to 10 times. 12. The kit according to claim 9, wherein the molar ratio of the reducing agent to the protein or peptide ranges from 6 to 8 times. 13. The method according to claim 12, wherein the reducing agent is tin (II), copper (I),
The kit according to claim 9, comprising at least one compound selected from the group consisting of iron (II) and titanium (III). 14. The protein or peptide is an antibody,
The kit according to claim 9, which is selected from an antibody fragment, a hormone, and a cytokine. 15. The radioactive technetium is technetium-
The kit according to claim 9, which is 99 m. 16. The kit according to claim 9, wherein the radioactive rhenium is rhenium-186 or rhenium-188. 17. The kit according to claim 9, wherein a solution containing the protein or peptide and the reducing agent is frozen. 18. The kit according to claim 9, wherein the solution containing the protein or peptide and the reducing agent is freeze-dried. 19. The kit according to claim 18, further comprising another container containing a buffer for reconstitution.