KR100762359B1 - Vascular-specific contrast media for magnetic resonance imaging and process for preparing the same - Google Patents

Abstract

Gd-DTPA, which is mainly used as a conventional T1 MR imaging contrast agent, is limited in angiography due to its short molecular time due to its small molecular weight. To overcome this problem, a contrast agent that has been increased by using various types of carbohydrates has been tried. However, this also does not provide clear vascular cell specific images.

The present invention provides a novel Gd-DTPA-anti-VEGFR2 antibody conjugate and a method for preparing the same that can solve the problems of the existing MR imaging contrast agent.

The Gd-DTPA-anti-VEGFR2 antibody conjugate provided in the present invention can be used to specifically diagnose neoplastic tissues with increased neovascularization, and the amount of the Gd-DTPA-anti-VEGFR2 antibody conjugate is lower than that of the conventional contrast agent Gd-DTPA. MR imaging can be used to obtain neovascularization of tumors and other tissues for a long residence time.

Gd-DTPA-anti-VEGFR2 Antibody, Angiogenic T1 Contrast, MRI Contrast, VEGFR2

Description

Neovascular-specific MR contrast agent and method for preparing the same {Vascular-specific contrast media for magnetic resonance imaging and process for preparing the same}

1 is a MALDI-TOF analysis data of the anti-VEGFR2 antibody and the resulting Gd-DTPA-anti-VEGFR2 antibody conjugate used in the synthesis in the preparation of the contrast agent of the present invention.

Figure 2 is a T1 MR image of tumor-causing mice using Gd-DTPA, a conventional contrast agent.

Figure 3 is a vascular specific T1 MR image in tumor-causing mice using the contrast agent Gd-DTPA-anti-VEGFR2 antibody conjugate of the present invention.

4 is a graph of the results of FIGS. 2 and 3.

The present invention relates to a contrast agent and a method for producing the same, and more particularly to a neovascular specific MR contrast agent and a method for producing the same.

Paramagnetic metal-chelate compounds and compounds combined with macromolecules are widely used as T1 contrast agents for magnetic resonance imaging (MRI), of which gd (gadolinium) is the most representative contrast material.

Gd-DTPA (gadolinium-diethylenetriaminepentaacetic acid) compound is stable in vivo and does not have toxicity, but due to its small molecular weight, it is easily lost from vascular tissues and thus its use range is limited.

In order to overcome this, contrast agents have been tried to increase the size by using various types of carbohydrates, but they also do not provide clear vascular cell-specific images.

All commercially available contrast agents are passive imaging steps that aid in diagnosis by providing contrast effects between tissues and space because the contrast agent stays in the spaces of blood vessels or gastrointestinal tracts.

In the present invention, by contrast contrast agent directly to the neovascularization of cancer, to implement an active contrast agent to enable the diagnosis of cancer by implementing cancer vascular specific contrast.

Specifically, angiogenesis is mostly activated in lesion tissues such as tumors, and since vascular endothelial growth factor receptor2 (VEGFR2) is expressed in vascular endothelial cells of neovascular vessels, specific angiogenesis is possible. By combining the anti-VEGFR2 antibody with Gd-DTPA, it was confirmed that the neovascularization can be specifically ported and the present invention was completed.

Accordingly, an object of the present invention is to provide an neovascular specific molecular contrast agent and method for preparing the neovascularized tumor and other tissues specifically for MR imaging for an effective diagnosis of diseases involving angiogenesis. To provide.

The present invention, in order to achieve the above object, Gd-DTPA represented by the following formula (3) prepared by reacting a diethylenetriaminepentaacetic acid bisanhydride (DTPABA) represented by the formula (1), an anti-VEGFR2 antibody represented by the formula (2) and Gd Provided is a contrast agent comprising an anti-VEGFR2 antibody conjugate.

Unlike the conventional contrast agent, the contrast agent of the present invention may be referred to as an active contrast agent that enables diagnosis of a disease by implementing a disease-specific contrast by directly binding the contrast agent to the lesion tissue.

In detail, angiogenesis is mostly activated in lesion tissues such as tumors, and since angiogenesis factors (VEGFR2) are expressed in vascular endothelial cells of neovascularization, the present invention provides anti-VEGFR2 which can specifically bind thereto. By binding the antibody to Gd-DTPA, the neovascularization can be specifically characterized.

The Gd-DTPA-anti-VEGFR2 antibody conjugate according to the present invention can be effectively used for diagnosis by specifically contrasting lesion tissue with angiogenesis in MR images. In addition, as the molecular weight is increased by binding an antibody which is a macromolecule, it is possible to effectively realize a vascular specific MR image by prolonging the residence time in the blood vessel.

Specifically, the contrast agent according to the present invention by specifically binding to neovascularization, MR imaging of tissues with neovascularization, differential diagnosis of local lesions, determination of extent of tumor lesions and determination of disease progression, tumors Can be used for early diagnosis of lesions.

The anti-VEGFR2 antibody used in the present invention can be a human or animal-derived antibody, and in one embodiment of the present invention, a mouse hybridoma against VEGFR2 of a mouse as an anti-VEGFR2 antibody is ATCC (No. CRL-1878). The product obtained from the company was incubated in Dulbecco's modification of Eagle's medium (DMEM) medium, and then purified from the culture medium by protein-G affinity chromatography.

In addition, the present invention comprises the steps of (a) adding the DTPABA represented by the formula (1) in the solvent and then added to the purified anti-VEGFR2 antibody solution represented by the formula (2); (b) preparing a DTPA-anti-VEGFR2 antibody conjugate by stirring and reacting the mixed solution; (c) adding a Gd solution to the prepared DTPA-anti-VEGFR2 antibody solution; And (d) preparing a Gd-DTPA-anti-VEGFR2 antibody conjugate represented by Chemical Formula 3 by stirring and reacting the mixed solution, thereby providing a method for preparing a contrast agent comprising a Gd-DTPA-anti-VEGFR2 antibody conjugate. do.

As the solvent for DTPABA in step (a), DMF (dimethylformamide), anhydrous DMSO (dimethylsulfoxide), or the like may be used.

Both solvents are available, and DMF is significantly lower in solubility in DTPABA than DMSO and suspending for addition in small amounts. DMSO has high solubility in DTPABA but is toxic and has limited use. Both solvents must be anhydrous, since the reactivity is reduced if the anhydrous form of DTPABA is converted to carboxylic acid (-CO ₂ H) by hydrolysis prior to reaction with the antibody.

In the steps (b) and (d), the mixed solution may be reacted by stirring at room temperature for about 1 hour, or the mixed solution may be reacted by stirring at about 4 ° C. for about 24 hours.

Both reaction conditions are possible and do not significantly affect the yield or product properties but only affect the reaction rate. At room temperature, one hour is sufficient because the reaction is faster than the reaction at low temperature. However, if more time is required, since the antibody is a protein, physical property change is expected.

The contrast agent according to the present invention is specifically prepared by the following method.

① Dissolve DTPABA 40eq (equivalent) in a small amount of DMF or anhydrous DMSO and add it to 1eq anti-VEGFR2 antibody solution in PBS (phosphate buffered saline, pH 7.4) buffer.

② The reaction is stirred at room temperature for 1 hour or the mixture is stirred at 4 ° C. for 24 hours. At this time, the pH of the reaction solution is maintained at 8.5 using 1M NaOH solution. This reaction results in the formation of a DTPA-anti-VEGFR2 antibody conjugate.

③ The reaction solution is treated with 0.5M sodium acetate (pH 5.5) solution using PD-10 column (sephadex G 25M) to separate DTPA-anti-VEGFR2 antibody product from unreacted and side reactions.

④ Dissolve GdCl ₃ 40eq in deionized distilled water.

⑤ Add GdCl ₃ (gadolinium chloride) solution to the separated 1eq DTPA-anti-VEGFR2 antibody solution.

⑥ The reaction is stirred at room temperature for 1 hour or the mixture is stirred at 4 ° C. for 24 hours. At this time, the pH of the reaction solution is maintained at 5.0 to 5.5. This reaction results in the formation of a Gd-DTPA-anti-VEGFR2 antibody conjugate.

⑦ Gd-DTPA-anti-VEGFR2 antibody product is separated from unreacted and side reactions by treating the reaction solution with 0.15M sodium chloride (pH 5.5) solution using PD-10 column (sephadex G 25M). .

⑧ Concentrate the separated solution.

⑨ Check the product using MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time Of Flight) spectrometry and ICP-MS (Inductively Coupled Plasma Mass Spectrometry).

In short, the contrast agent according to the present invention is synthesized according to the following Scheme 1. Where Ab is an antibody. DTPA and anti-VEGFR2 antibodies are bound by amide bonds (-CONH-). Gd-DTPA is a metal-chelate compound in which Gd is coordinated to DTPA.

The present invention will be described in detail with reference to the following Examples.

Example 1

(1) DTPABA (40eq) was suspended in a small amount of DMF, and then slowly added to an anti-VEGFR2 antibody solution (1eq) contained in PBS (pH 7.4) buffer. ② stirred at room temperature for 1 hour. At this time, the pH of the reaction solution was maintained at 8.5 using 1M NaOH solution. ③ The reaction solution was treated with 0.5 M sodium acetate (pH 5.5) solution using PD-10 column (sephadex G 25M) to separate DTPA-anti-VEGFR2 antibody product from unreacted and side reactions. ④ GdCl ₃ (40eq) was dissolved in deionized distilled water. ⑤ GdCl ₃ solution was slowly added to the separated DTPA-anti-VEGFR2 antibody solution (1eq). ⑥ Stir at room temperature for 1 hour. At this time, the pH of the reaction solution was maintained at 5.0 to 5.5. ⑦ The reaction solution was treated with 0.15 M sodium chloride (pH 5.5) solution using PD-10 column (sephadex G 25M) to separate Gd-DTPA-anti-VEGFR2 antibody product from unreacted and side reactions. ⑧ The separated solution was concentrated. ⑨ The product was confirmed using MALDI-TOF and ICP-MS.

Example 2

(1) DTPABA (40eq) was suspended in a small amount of DMF, and then slowly added to an anti-VEGFR2 antibody solution (1eq) contained in PBS (pH 7.4) buffer. ② stirred at 4 ° C. for 24 hours. At this time, the pH of the reaction solution was maintained at 8.5 using 1M NaOH solution. ③ The reaction solution was treated with 0.5 M sodium acetate (pH 5.5) solution using PD-10 column (sephadex G 25M) to separate DTPA-anti-VEGFR2 antibody product from unreacted and side reactions. ④ GdCl ₃ (40eq) was dissolved in deionized distilled water. ⑤ GdCl ₃ solution was slowly added to the separated DTPA-anti-VEGFR2 antibody solution (1eq). ⑥ Stir at 4 ° C. for 24 hours. At this time, the pH of the reaction solution was maintained at 5.0 to 5.5. ⑦ The reaction solution was treated with 0.15 M sodium chloride (pH 5.5) solution using PD-10 column (sephadex G 25M) to separate Gd-DTPA-anti-VEGFR2 antibody product from unreacted and side reactions. ⑧ The separated solution was concentrated. ⑨ The product was confirmed using MALDI-TOF and ICP-MS.

Example 3

① DTPABA (40eq) was dissolved in a small amount of anhydrous DMSO, and then slowly added to the anti-VEGFR2 antibody solution (1eq) contained in PBS (pH 7.4) buffer solution. ② stirred at room temperature for 1 hour. At this time, the pH of the reaction solution was maintained at 8.5 using 1M NaOH solution. ③ The reaction solution was treated with 0.5 M sodium acetate (pH 5.5) solution using PD-10 column (sephadex G 25M) to separate DTPA-anti-VEGFR2 antibody product from unreacted and side reactions. ④ GdCl ₃ (40eq) was dissolved in deionized distilled water. ⑤ GdCl ₃ solution was slowly added to the separated DTPA-anti-VEGFR2 antibody solution (1eq). ⑥ Stir at room temperature for 1 hour. At this time, the pH of the reaction solution was maintained at 5.0 to 5.5. ⑦ The reaction solution was treated with 0.15 M sodium chloride (pH 5.5) solution using PD-10 column (sephadex G 25M) to separate Gd-DTPA-anti-VEGFR2 antibody product from unreacted and side reactions. ⑧ The separated solution was concentrated. ⑨ The product was confirmed using MALDI-TOF and ICP-MS.

Example 4

(1) DTPABA (40eq) was suspended in a small amount of anhydrous DMSO and slowly added to the anti-VEGFR2 antibody solution (1eq) contained in PBS (pH 7.4) buffer. ② stirred at 4 ° C. for 24 hours. At this time, the pH of the reaction solution was maintained at 8.5 using 1M NaOH solution. ③ The reaction solution was treated with 0.5 M sodium acetate (pH 5.5) solution using PD-10 column (sephadex G 25M) to separate DTPA-anti-VEGFR2 antibody product from unreacted and side reactions. ④ GdCl ₃ (40eq) was dissolved in deionized distilled water. ⑤ GdCl ₃ solution was slowly added to the separated DTPA-anti-VEGFR2 antibody solution (1eq). ⑥ Stir at 4 ° C. for 24 hours. At this time, the pH of the reaction solution was maintained at 5.0 to 5.5. ⑦ The reaction solution was treated with 0.15 M sodium chloride (pH 5.5) solution using PD-10 column (sephadex G 25M) to separate Gd-DTPA-anti-VEGFR2 antibody product from unreacted and side reactions. ⑧ The separated solution was concentrated. ⑨ The product was confirmed using MALDI-TOF spectrometry (Voyager-DE STR 4349) and ICP-MS (Agilent 7500A).

1 is a MALDI-TOF analysis data of the anti-VEGFR2 antibody and the resulting Gd-DTPA-anti-VEGFR2 antibody conjugate used in the synthesis in the preparation of the contrast agent of the present invention.

147464, 153367 in FIG. The molecular weight of the anti-VEGFR2 antibody as a starting material and the Gd-DTPA-anti-VEGFR2 antibody produced after the reaction, respectively, wherein the unit is Dalton, which means the molecular weight of the material. The difference in molecular weight means that new material has been produced from the starting material, and it can be calculated how much Gd-DTPA is bound to the anti-VEGFR2 antibody.

Table 1 shows the results of quantitative analysis of DTPA-anti-VEGFR2 antibody conjugate and Gd-DTPA-anti-VEGFR2 antibody conjugate using ICP-MS.

Conjugated product (protein quantification results) yield(%) DTPA-anti-VEGFR2 antibody conjugate 95 to 98 Gd-DTPA-Anti-VEGFR2 Antibody Conjugates 75 to 85 Analysis of Gd Using ICP-MS Molar ratio Anti VEGFR2 Antibody (mAb): Gd 1:20

The yield of DTPA-anti-VEGFR2 antibody conjugate and Gd-DTPA-anti-VEGFR2 antibody conjugate is a quantitative result of antibody protein using BCA (Bicinchoninic acid) analysis method.

The molar ratio of mAb (monoclonal antibody): Gd was determined by pretreatment of the sample with nitric acid, Gd concentration of Gd-DTPA-anti-VEGFR2 antibody conjugate, and Gd-DTPA-anti-VEGFR2 obtained by ICP-MS instrumental analysis. After obtaining the concentration of only the anti-VEGFR2 antibody contained in the antibody, it is a result of comparing the total number of moles. That is, it means that 20 Gds are bound to one anti-VEGFR2 antibody in the Gd-DTPA-anti-VEGFR2 antibody conjugate.

Figure 2 is a T1 MR image taken in tumor-causing mice using a conventional contrast agent Gd-DTPA, the conventional MR contrast agent Gd-DTPA (2.5μmole Gd) in tumor-causing mice made by injecting 1 million E. coli into the muscle After scanning, T1-weighted spin echo (TR / TE = 500/15 ms) imaging using a 47 mm surface coil was performed on a 1.5T magnetic resonance imaging system (Gyroscan Integra, Philips Med.System). It only shows the overall contrast increase over time.

Figure 3 is a vascular specific T1 MR image in tumor-induced mice using the contrast agent Gd-DTPA-anti-VEGFR2 antibody conjugate of the present invention, taken as shown in Figure 2, Gd-DTPA-anti-VEGFR2 antibody Injecting the conjugate (250 nmole Gd), unlike the case of injecting Gd-DTPA of Figure 2 shows a tumor tissue specific contrast increase for 24 hours. In addition, the Gd-DTPA-anti-VEGFR2 antibody conjugate can effectively observe the difference between tumor tissue and normal tissue even in a small amount of 1/10 of the conventional contrast agent.

FIG. 4 is a graphical representation of MR signals of FIGS. 2 and 3 as contrast-to-noise ratios (CNRs) over time. One minute in the lesion tissue after injection with the Gd-DTPA contrast agent. It can be seen that it shows the highest increase in the time zone and decreases rapidly from the body within 1 hour. On the other hand, in the case of Gd-DTPA-anti-VEGFR2 antibody conjugates, the VEGFR2 produced in the tumor site and the anti-VEGFR2 antibody specifically bind to each other, and the video signal gradually increases to last for 24 hours.

By these results, it was confirmed that the Gd-DTPA-anti-VEGFR2 antibody conjugate according to the present invention can specifically image neoplastic blood vessels with longer residence time than conventional Gd-DTPA. It was.

Unlike conventional contrast agents, the Gd-DTPA-anti-VEGFR2 antibody conjugate according to the present invention is an active contrast agent capable of diagnosing a disease by implementing a disease-specific contrast by directly binding the contrast agent to lesion tissue. By increasing the molecular weight, the vascular-specific MR image can be effectively realized by prolonging the residence time in the blood vessel, and the MR image can be obtained by using less than the amount of Gd of the conventional contrast agent Gd-DTPA. It is possible to easily diagnose diseases involving angiogenesis.

Contrast agent according to the present invention by specifically binding to neovascularization, MR imaging of tissues with neovascularization, differential diagnosis of local lesions, determination of extent of tumor lesions and determination of disease progression, early onset of tumor lesions Specific diagnosis of lesion tissues involving neovascularization, such as diagnosis, can be effectively used for diagnosis.

Claims (5)

It is prepared by reacting diethyltriaminepentaacetic acid bisanhydride (DTPABA) represented by Formula 1, an anti-VJFR2 (VEGFR2) antibody and gadolinium (Gd). A MR imaging contrast comprising a gadolinium-diTP-anti-VGFR2 (Gd-DTPA (diethylenetriaminepentaacetic acid) -anti-VEGFR2) antibody conjugate represented by Formula 3 below. [Formula 1]

[Formula 2]

[Formula 3]

The MR imaging contrast of claim 1, wherein the anti-VGF2 antibody is a human or animal-derived antibody. The MR imaging contrast medium of claim 1 or 2, wherein the contrast agent specifically binds to angiogenesis. The method of claim 3, wherein the contrast agent is magnetic resonance (MR) images of tissues with neovascularization, differential diagnosis of local lesions, determination of extent of tumor lesions and determination of the extent of disease progression, initial stage of tumor lesions MR imaging contrast agent used for diagnosis. (a) An anti-VGFR2 (VEGFR2) vascular endothelial growth factor receptor2 (VEGFR2) antibody, which is purified after the diethyltriaminepentaacetic acid bisanhydride (DTPABA) represented by the following Chemical Formula 1 is suspended in a solvent. Adding to the solution; (b) preparing a DTP-anti-VEGFR2 (diethylenetriaminepentaacetic acid) -anti-VEGFR2 antibody conjugate by stirring the mixed solution; (c) adding a gadolinium (Gd) solution to the prepared DTP-anti-VGF2 antibody solution; And (d) preparing a gadolinium-dTPA-anti-VEGFR2 antibody conjugate represented by the following Chemical Formula 3 by stirring the mixed solution: A method for preparing an MR imaging contrast agent comprising a gadolinium-DTP-anti-VGF2 antibody conjugate. [Formula 1]

[Formula 2]

[Formula 3]

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