JP5376130B2 - Separation and purification of carrier-free 177Lu capable of antibody labeling - Google Patents

Description

The present invention relates to a method for separating and purifying carrier-free ¹⁷⁷ Lu capable of antibody labeling.

  Cancer treatment using RI (radioisotope) is performed by administering a RI-labeled drug into the body and accumulating it specifically in cancer cells, and irradiating the lesion tissue or cells with a short range of radiation emitted from the RI. This is a method of treating the disease by destroying the tissue and cells of the target lesion, and can reduce the effects of radiation on normal body tissues and cells, and there are few side effects. This is a method that can be expected to improve QOL (quality of life). However, there are many problems to be solved such as improvement in cancer accumulation of RI-labeled drugs and reduction in accumulation in non-target organs, and practical application is still limited.

Currently, there are three types of RI for cancer treatment currently used in Japan, ¹³¹ I, ⁸⁹ Sr and ⁹⁰ Y, all of which are β-ray emitting RIs. The range of these β-rays in the tissue is about several to several tens of millimeters, and is an appropriate length for irradiation of small to medium-sized tumors. In addition, many RIs that emit β rays useful for cancer treatment have an appropriate half-life in the range of several hours to several days, and have various chemical properties. In addition, when gamma rays suitable for imaging from outside the body are emitted, the biodistribution can be confirmed at a low dose prior to the administration at a high dose at the therapeutic level. Can contribute to the determination of quantity. Furthermore, by monitoring, it is possible to accurately grasp the treatment status of cancer and to evaluate the absorbed dose of each tissue simultaneously with the treatment.

In order to use RI for cancer treatment, in addition to its physical properties (half-life, β-ray energy), the specific activity of RI (ratio of radioisotope to stable isotope) is one of the important factors. . That is, in order to enhance the therapeutic effect, it is necessary to irradiate the target cancer tissue with more β-ray energy, and therefore RI having a high specific activity is advantageous. ¹⁷⁷ Lu emits 113 keV and 208 keV γ-rays suitable for imaging, with a half-life of 6.73 days, the maximum energy of β-rays is 498 keV, and the range of β-rays in tissues is as short as 1.8 mm. Moreover, lutetium, yttrium clinical application was observed ^{(90 Y)} and a related elements, for chemical properties are similar, it is possible to effectively utilize the accumulated knowledge in the development of ^{90 Y-labeled} agent it can.

There are two types of methods for producing ¹⁷⁷ Lu, which are considered promising as radioisotopes for cancer treatment, a direct method using a nuclear reactor and an indirect method.

The direct method is a method of producing ¹⁷⁷ Lu by a ¹⁷⁶ Lu (n, γ) ¹⁷⁷ Lu reaction, and Lu is used as a target. ¹⁷⁷ Lu produced by the direct method includes a Lu carrier that is a stable isotope.

The indirect method is a method of manufacturing by ¹⁷⁶ Yb (n, γ) ¹⁷⁷ Yb (half-life 1.91 hours) → ¹⁷⁷ Lu reaction, and uses Yb as a target. ¹⁷⁷ Lu produced by the indirect method does not contain Lu carrier, which is a stable isotope. That is, carrier-free ¹⁷⁷ Lu can be obtained.

The ^{177 Lu} as a method for use in cancer therapy, the ^{177 Lu} specifically to antibodies capable of binding to antigens expressed in cancer cells to produce a labeled ^{177 Lu-antibody,} a method of performing cancer therapy is administered to the body . In the case of this therapeutic method, if a Lu carrier that is a stable isotope is contained, the labeling rate of the ¹⁷⁷ Lu-antibody is lowered, and in the body, the stable Lu-antibody is bound to the antigen in addition to the ¹⁷⁷ Lu-antibody. Reducing the therapeutic effect of ¹⁷⁷ Lu. Therefore, it is necessary to use carrier-free ¹⁷⁷ Lu produced by the indirect method for such cancer therapy.

^{177 Lu-antibodies} used in cancer therapy, and no carrier ^{177 Lu, 1,4,7,10-tetraazacyclododecan-N} , N ', N'',N''' - tetraacetic acid and (DOTA) is bound to the antibody It can be obtained by reacting with DOTA-antibody.

The production, separation and purification method of carrier-free ¹⁷⁷ Lu produced by the indirect method is described in “Production of No-carrier-added ¹⁷⁷ Lu via the ¹⁷⁶ Lu (n, γ) ¹⁷⁷ Yb → ¹⁷⁷ Lu process”, K. Hashimoto , H. Matsuoka, S. Uchida, Journal of Radioanalytical and Nuclear Chemistry, Vol. 255, No. 3 (2003) 575-579.

  The separation and purification method described in the paper by Hashimoto et al. Is a method using a reverse phase silica gel column, and the procedure is as follows.

Irradiation with ¹⁷⁶ Yb ₂ O ₃ is performed in a nuclear reactor to obtain irradiated ¹⁷⁶ Yb ₂ O ₃ . Next, the irradiated ¹⁷⁶ Yb ₂ O ₃ is dissolved in hydrochloric acid and hydrogen peroxide solution, evaporated to dryness, and made into a 0.01M HCl solution. This solution was charged into HPLC (reverse phase silica gel column: Waters Resolve C ₁₈ Radial-Pack 8 mmφ × 300 mm), and 0.25M 2-hydroxyisobutyric acid (2-HIBA) /0.1M 1-octanesulfonic acid was used as an eluent. Using sodium (1-OS), ¹⁷⁷ Lu and target Yb are separated at a flow rate of 2 ml / min. The separated Lu fraction was passed through a cation exchange column (BioRad AG50WX, 8 mmφ × 20 mm), and Lu was adsorbed to the resin, and 0.1 M HCl was flowed to completely remove 2-HIBA / 1-OS. Then, ¹⁷⁷ Lu is eluted with 6M HCl and evaporated to dryness for labeling experiments.

However, in the conventional method of Hashimoto et al., ¹⁷⁷ Lu after separation and purification contains a large amount of Ca, Fe, Zn, and DOTA-antibody and Ca, Fe, Zn are preferentially bound to form Ca-antibody, Fe - antibody, to create a Zn- antibody, no longer ¹⁷⁷ Lu is will not be able to bind to the DOTA- ^antibody, can not be a sign of 177 Lu-DOTA- antibody, there is a problem in that.

"Production of No-carrier-added 177Lu via the 176Lu (n, γ) 177Yb → 177Lu process", K. Hashimoto, H. Matsuoka, S. Uchida, Journal of Radioanalytical and Nuclear Chemistry, Vol. 255, No. 3 ( 2003) 575-579

An object of the present invention is to provide a ¹⁷⁷ Lu antibody having a high labeling rate by separating and purifying carrier-free ¹⁷⁷ Lu free of Ca, Fe, and Zn.

As a result of diligent research, the present inventors have determined that the cause of contamination of Ca, Fe, and Zn that inhibits the binding of ¹⁷⁷ Lu to the antibody is mainly impurities contained in the eluent, and purifies the eluent in advance. It has been found that the above problem can be solved by adding a step of removing impurities at the end of the separation step.

That is, according to the present ^invention, 176 _Yb and 2 _{O 3} was irradiated in a nuclear reactor, obtaining a irradiated ¹⁷⁶ Yb ₂ O _3, the irradiated ¹⁷⁶ Yb ₂ O ₃ through the HCl solution in hydrochloric acid HPLC A method for separating and purifying ¹⁷⁷ Lu, comprising: separating ¹⁷⁷ Lu from target Yb; and separating the ¹⁷⁷ Lu by passing the separated Lu fraction through a cation exchange column. In the separation step to be used, an eluent purified in advance by cation exchange and chelate exchange is used, and after the separation step of ¹⁷⁷ Lu using cation exchange, a solution containing ¹⁷⁷ Lu is further passed through an anion exchange column to form Fe. A separation / purification method characterized by comprising a step of removing

  As HPLC, a reverse phase silica gel column using silica gel as a filler can be used.

  As an eluent used in HPLC, 0.25M 2-hydroxyisobutyric acid (2-HIBA) /0.1M sodium 1-octanesulfonate (1-OS) can be used as in the conventional method. However, in the present invention, it is necessary to purify the eluent by cation exchange and chelate exchange before passing through the HPLC column. The cation exchange resin that can be used for purification of the eluent is preferably a sulfonic acid type ion exchange resin, and specifically includes AG560Wx8 (manufactured by Bio Rad). As the chelate resin, an iminodiacetic acid type chelate resin is preferable, and specifically, Chelex-100 (manufactured by Bio Rad) can be mentioned.

As the cation exchange resin packed in the cation exchange column that can be used in the step of separating ¹⁷⁷ Lu from the Lu fraction, a sulfonic acid type ion exchange resin is preferable. Specifically, AG560Wx8 (manufactured by Bio Rad) is used. Can be mentioned.

An anion exchange resin that can be packed in an anion exchange column that can be used in the step of removing Fe from a solution containing ¹⁷⁷ Lu after separation includes a trimethylammonium type, specifically AG1 × 8 (Bio Rad). Manufactured). Further, this resin can remove not only Fe but also various elements such as Zn, Ga, and Mo.

In addition, according to the present invention, the ¹⁷⁷ Lu final solution separated and purified by the above-described method is evaporated to dryness to obtain an acetic acid solution, and then the antibody solution is added and reacted to synthesize a ¹⁷⁷ Lu antibody. A method for producing a ¹⁷⁷ Lu-antibody is provided.

^In order to bind the antibody to ¹⁷⁷ Lu, the chelator is first bound to the antibody and then the chelator-antibody is bound to ¹⁷⁷ Lu. As the chelating agent that can be used at this time, polyazapolycarboxylic acid derivatives and polyaminopolycarboxylic acid derivatives can be preferably used. As the polyazapolycarboxylic acid derivative, in particular, 1,4,7,10-tetraazacyclododecane-N, N ′, N ″, N ′ ″-tetraacetic acid (1,4,7,10-tetraazacyclododecan- N, N ′, N ″, N ′ ″-tetraacetic acid (DOTA)), 1,4,8,11-tetraazacyclotetradecane-N, N, N ″, N ″ ′-tetraacetic acid (TETA) Examples of polyaminopolycarboxylic acid derivatives include diethylenetriamine-N, N, N ′, N ″, N ″ -pentaacetic acid. (DTPA)), ethylenediamine-N, N, N ′, N′-tetraacetic acid (EDTA).

  Examples of antibodies to be bound to the chelating agent include NuB2 that recognizes CD20 antigen that is highly expressed in malignant lymphoma, anti-Erb-B2 antibody (trastuzumab) for the treatment of breast cancer and lung cancer, and anti-CD22 for the treatment of malignant lymphoma. Antibody (epratuzumab), anti-CEA antibody (cT84.66) for the treatment of metastatic colorectal cancer, anti-PMSA antibody (J591) for the treatment of prostate cancer, for the treatment of metastatic renal cell carcinoma And anti-CA-IXG250 / MN antibody (cG250).

According to the present invention, since the purification purity of the carrier-free ¹⁷⁷ Lu can be improved, the labeling rate of ¹⁷⁷ Lu-chelating agent-antibody can be greatly improved. ¹⁷⁷ Lu is a promising nuclide for cancer treatment because it has a half-life of 6.73 days and is suitable for cancer treatment and emits β-rays with a maximum energy of 498 keV. Advances in research on cancer treatment using ¹⁷⁷ Lu-antibodies that specifically bind to antigens expressed in cancer cells can be expected using ¹⁷⁷ Lu isolated and purified in the present invention.

FIG. 1 is a separation and purification flow of ¹⁷⁷ Lu according to the present invention.

  Hereinafter, the present invention will be specifically described with reference to examples.

FIG. 1 shows the separation and purification flow of ¹⁷⁷ Lu of the present invention. ^In order to obtain ¹⁷⁷ Lu, ¹⁷⁶ Yb ₂ O ₃ as a target is irradiated in a nuclear reactor to obtain irradiated ¹⁷⁶ Yb ₂ O ₃ (3). An HCl solution in which irradiated ¹⁷⁶ Yb ₂ O ₃ is dissolved in hydrochloric acid is passed through an HPLC reverse phase silica gel column to separate ¹⁷⁷ Lu and Yb as a target (4). At this time, 2-HIBA is passed through a cation exchange column to remove impurities (1), 1-OS is passed through a chelate exchange column to remove impurities (2), and both are mixed to prepare an eluent. And introduced into the HPLC column. The separated Lu fraction is passed through a cation exchange column to adsorb ¹⁷⁷ Lu, and the HPLC eluent is removed to separate ¹⁷⁷ Lu (5). The separated solution containing ¹⁷⁷ Lu is passed through an anion exchange column to remove Fe (6). A solution containing purified ¹⁷⁷ Lu is obtained (7).

(1) Preparation of Eluent Cation exchange resin AG50Wx8 (manufactured by Bio Rad) was packed in a 15 mmφ × 113 mm column to prepare a cation exchange column. A 0.5M 2-HIBA (2-hydroxyisobutyric acid) solution was passed through the cation exchange column to purify 2-HIBA.

  A chelate exchange column was prepared by packing chelate resin Chelex-100 in a 15 mmφ × 113 mm column. A 0.2M 1-OS (sodium 1-octanesulfonate) solution was passed through the chelate exchange column to purify 1-OS.

An equivalent amount of 0.5M 2-HIBA solution and 0.2M 1-OS solution was mixed to prepare a 0.25M 2-HIBA / 0.1M 1-OS solution.
(2) Production of carrier-free ¹⁷⁷ Lu
¹⁷⁶ Yb ₂ O ₃ was irradiated for 6 hours using a nuclear reactor (JRR3HR-2 hole (1 × 10 ¹⁴ n.cm ^−2.s-1 .) Of Japan Atomic Energy Agency).

Irradiated ¹⁷⁶ Yb ₂ O ₃ is dissolved in 3 mL of 6M hydrochloric acid and 2 mL of 30% hydrogen peroxide solution, evaporated to dryness, and subjected to HPLC as a 0.01M HCl solution (column: Waters Resolve C ₁₈ Radial-Pack 8 mmφ × 300 mm). Prepared. A pre-purified eluent (0.25 M 2-HIBA / 0.1 M 1-OS solution) was passed through an HPLC column to separate ¹⁷⁷ Lu from the target Yb.

The separated ¹⁷⁷ Lu fraction was passed through a cation exchange column (Bio Rad AG50WX8, 8 mmφ × 20 mm), and Lu was adsorbed on the resin, and 0.1 M HCl was flowed to completely remove 2-HIBA / 1-OS. did. The cation exchange column was then flushed with 6M HCl to elute ¹⁷⁷ Lu.

Finally, the eluate containing the eluted ¹⁷⁷ Lu was passed through an anion exchange column (anion exchange resin AG1x8 manufactured by Bio Rad was packed in an 8 mmφ × 20 mm column) to obtain a ¹⁷⁷ Lu final solution.
(3) Impurity content in ¹⁷⁷ Lu final solution For comparison, Control 1 (HPLC eluate was not purified and HPLC eluate was not purified by anion exchange. Same as conventional method) and Control 2 (The HPLC eluent was purified, but the HPLC eluent was not purified by anion exchange).
(4) Antibody labeling
^{The 177} Lu final solution was evaporated to dryness and dissolved by adding 35 μl of 0.1 M acetic acid. To this, 7 μl of 3M acetate buffer (pH = 6) and 26 μl of 5 mg / ml DOTA-NuB2 antibody solution were added and reacted at 40 ° C. for 1.5 hours to synthesize ¹⁷⁷ Lu-DOTA-NuB2. The DOTA-NuB2 antibody solution was prepared as follows.

  NuB2 in 500 μL borate buffer (0.1 M, pH = 8) was added to 1,4,7,10-tetraazacyclododecane-N, N ′, N ″, N in 5 μL dimethyl sulfoxide (DMSO). '"-Tetraacetic acid mono (N-hydroxysuccinimidyl ester) (1,4,7,10-tetraazacyclodocecane-N, N', N '', N '' '-tetracetic acid mono (N-hydroxysuccinimidyl ester)) mDOTA) was added, and the mixture was reacted at room temperature for 15 hours, and then purified by a gel filtration column through 0.5 M acetate buffer (pH = 6).

Next, 100 mM of EDTA (ethylenediaminetetraacetic acid) was added to the synthesized solution containing ¹⁷⁷ Lu-DOTA-NuB2, and unreacted ¹⁷⁷ Lu was changed to ¹⁷⁷ Lu-EDTA.

The labeling rate was determined by thin layer chromatography (TLC). Specifically, a solution containing the synthesized ¹⁷⁷ Lu-DOTA-NuB2 was spotted on a TLC paper (ITLC SG of Gelman Science Inc.) and developed with physiological saline to obtain ¹⁷⁷ Lu-DOTA-NuB2 and ¹⁷⁷ Lu-EDTA. Separately, each radioactivity was measured. The amount of radioactivity was determined by measuring ¹⁷⁷ Lu 208 keV gamma rays with a Ge detector. In this experiment, radioactivity of about 20 MBq is used for antibody labeling, but 100-200 MBq is required for animal treatment.

  The labeling rate was determined by the following formula.

Labeling rate (%) = ¹⁷⁷ Lu -DOTA-NuB2 radioactivity / ¹⁷⁷ Lu radioactivity used for labeling x 100

The “radioactivity of ¹⁷⁷ Lu used for labeling” is divided into ¹⁷⁷ Lu-DOTA-NuB2 and ¹⁷⁷ Lu-EDTA, and the ratio of the generated ¹⁷⁷ Lu-DOTA-NuB2 is the labeling rate.

  Table 1 shows the measurement results of the residual amounts of Ca, Fe, and Zn and the labeling rates of the examples according to the present invention and the controls 1 and 2.

  From Table 1, it can be seen that in the examples in which the separation / purification method of the present invention was performed, the residual amounts of Ca, Fe, and Zn were greatly reduced and the labeling rate was greatly improved as compared with the conventional method.

Claims (6)

Irradiating ¹⁷⁶ Yb ₂ O ₃ in a nuclear reactor to obtain irradiated ¹⁷⁶ Yb ₂ O ₃ ;
Passing an HCl solution of irradiated ¹⁷⁶ Yb ₂ O ₃ in hydrochloric acid through HPLC to separate ¹⁷⁷ Lu and target Yb;
Separating the ¹⁷⁷ Lu by passing the separated Lu fraction through a cation exchange column, and a method for separating and purifying ¹⁷⁷ Lu,
In the separation step using HPLC, an eluent purified in advance by cation exchange and chelate exchange is used ^. After the separation step of ¹⁷⁷ Lu using cation exchange, a solution containing ¹⁷⁷ Lu is further passed through an anion exchange column. Including a step of removing Fe
A method for separating and purifying ¹⁷⁷ Lu, The ¹⁷⁷ Lu final solution separated and purified by the method according to claim 1 is evaporated to dryness and dissolved in an acid solution, and then a chelating agent-antibody solution is added and reacted. ¹⁷⁷ Lu-chelating agent-antibody A method for producing a ¹⁷⁷ Lu-chelating agent-antibody, comprising the step of synthesizing. The chelator-antibody solution is obtained by reacting the chelator with the antibody in borate buffer and then purifying with a gel filtration column through acetate buffer,
The ¹⁷⁷ Lu-chelator - antibody obtained the chelator - ¹⁷⁷ Lu-chelating agent according to claim 2, characterized in that it is obtained by reacting the antibody solution was added to the ¹⁷⁷ Lu acid solution - Antibody production method.   The method according to claim 2 or 3, wherein the chelating agent is selected from a polyazapolycarboxylic acid derivative and a polyaminopolycarboxylic acid derivative. Polyazapolycarboxylic acid derivatives are 1,4,7,10-tetraazacyclododecane-N, N ′, N ″, N ′ ″-tetraacetic acid (DOTA) and 1,4,8,11-tetraaza Selected from cyclotetradecane-N, N, N ″, N ′ ″-tetraacetic acid (TETA);
The polyaminopolycarboxylic acid derivative is selected from diethylenetriamine-N, N, N ′, N ″, N ″ -pentaacetic acid ((DTPA) and ethylenediamine-N, N, N ′, N′-tetraacetic acid (EDTA) The manufacturing method according to claim 4. The antibody is selected from NuB2, Erb-B2, CD22, CEA, PMSA, CA-IXG250 / MN;
The method for producing a ¹⁷⁷ Lu-chelating agent-antibody according to any one of claims 2 to 5.

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