KR101041058B1 - Kit for Evaluating Enduronidase Activity Using Lymphocyte Line - Google Patents

Abstract

The present invention provides a transgenic lymphatic cell line in which immunized lymphocytes collected from the body of the recipient are immortalized with EB (Epstein-Bar) virus; Ii) α-L-eduronidase enzyme specimens with a fluorescent label attached thereto; And iii) a kit for evaluating α-L-eduronidase activity comprising a fluorescent label recognition light absorbing device for quantitatively detecting the uptake or secretion of the α-L-eduronidase enzyme sample in lymphatic cell lines via fluorescent labeling. to provide.

Lymphocyte, EB (Epstein-Bar) virus, fluorescent label, α-L-eduronidase enzyme, Herr syndrome, kit

Description

Kit for evaluating iduronidase activity using lymphocyte cell line}

The present invention relates to a kit for assessing eduronidase activity using lymph cell lines. More specifically, to prepare an immortal lymph cell line infected with transformed EB (Epstein-Bar) virus after harvesting the lymphocytes or leukocytes of the patient as a subject administered, and through the immortal lymph cell line Hurr syndrome or It is most suitable for the treatment of individual patients by measuring the uptake or secretion of lymphoid cell lines of various species of iduronidase enzymes or their homologues for the treatment of Schiey syndrome through fluorescent labels conjugated to the iduronidase enzyme. The present invention relates to a kit for evaluating the eduronidase activity capable of selectively evaluating a first enzyme homologue.

Mucopolysaccharide deposition is a serious genetic disease caused by abnormal accumulation of mucopolysaccharides. The mucopolysaccharide accumulates partially in cells and tissues due to the lack of enzymes necessary for metabolic processes in the human body.

These mucopolysaccharide depositions can be classified into seven types, of which the type 1 mucopolysaccharide deposition, Hurler syndrome or Hurler-Scheie syndrome, two types of heparin sulfate and dermatan sulfate. It is a hereditary disease caused by a deficiency of α-L-eduronidase, an enzyme that breaks down glycosaminoglycans.

These or Herr-Scheier syndromes develop several disorders throughout the body within the years of life, resulting in delayed growth and development, frequent upper respiratory tract infections, heart disease, liver and splenomegaly, and hardening of the joints, which are also difficult to achieve basic behavior. It causes hernias, spinal malformations, abnormal facial growth due to the accumulation of glycosaminoglycans in bone and connective tissue, language developmental disorders with a large tongue, and intelligence.

In the case of patients with Herr syndrome or Herr-Scheier syndrome, the growth of normal one year is normal, but bone marrow transplantation (BMT) has been developed for the treatment, but the risk is high. It could not be used as.

Recently, Genzyme Co., Ltd. developed a technology for mass production of α-L-eduronidase by genetic engineering method using CHO cells for the treatment of Herr syndrome or Herr-Scheie syndrome. Aldurazyme is commercialized under the US FDA approval in 2003 and is used in over 20 countries as an enzymatic drug for the treatment or alleviation of symptoms of Herr syndrome or Herr-Scheie syndrome.

The α-L-eduronidase is an enzyme consisting of 653 amino acids and has six N-linked oligosaccharide sites, which are expressed by transforming CHO cells, and 50% of the overexpressed enzyme is directly lysosome. The α-L-eduronidase is prepared by migrating and the remainder secreted into the medium and recovered via purification. Mannose 6-phosphate chains are bonded to the 336th and 451th amino acid sites among the six N-linked oligosaccharide sites, and such mannose 6-phosphate chains and M6PR (mannose 6-phosphate) in the cells. Uptake into the cell from outside the cell through the affinity of the receptor).

However, even in the case of the α-L-iduronidase brand name Aldurazim enzyme, which is produced by such genetic recombination method, the enzyme is not sufficiently absorbed into the cell from outside the cell when administered directly to the patient, such as the enzyme produced by the general genetic recombination method. There is a problem that does not show its utility in the body. In other words, the commercially available Aldurazime enzyme is also absorbed into cells through M6PR (Mannose 6-Phosphate Receptor) or carrier, but the loss of Aldurazyme Enzyme occurs when the drug is delivered to the cell membrane. It was difficult to obtain sufficient effects of treatment or symptomatic treatment of Herr syndrome or Herr-Scheie syndrome.

Meanwhile, a method for treating Herr syndrome or Herr-Scheie syndrome may include direct enzyme replacement of α-L-eduronidase or a homologue thereof, α-L-eduronidase or a homologue thereof. Source cell expression, cell transplantation, and vector-mediated gene delivery methods have been developed, but not all of them have yet been developed to produce sufficient therapeutic effects, as well as determining which treatment is most appropriate for the individual patient. The method for doing this has not been developed yet.

Accordingly, the present invention has been completed by developing a kit for evaluating the endurodinase activity required to determine a method for treating such Herr syndrome or Herr-Scheie syndrome.

The problem to be solved by the present invention is to develop an activity evaluation kit for the selection of an optimal enduronidase enzyme for the treatment of Herr syndrome or Herr-Scheie syndrome by enzyme replacement method. To develop a kit for quantitative activity evaluation of the absorption or secretion of the first enzyme.

The object of the present invention is iii) transfected lymph cell lines immortalized with EB (Epstein-Bar) virus lymphocytes collected in the body of the recipient; Ii) α-L-eduronidase enzyme specimens with a fluorescent label attached thereto; Iii) provides a kit for the evaluation of α-L-eduronidase activity consisting of a fluorescent label recognition light absorbing device for quantitatively detecting the absorption or secretion of the α-L-eduronidase enzyme sample in lymphatic cell lines through fluorescent labeling It is.

At this time, the transformed lymph cell line was cultured by adding an equivalent amount of EB (Epstein-Bar) virus soup to 0.5-1.5 × 10 ⁶ cells / ml of lymphocytes centrifuged in the blood of the recipient, followed by addition of cyclosporin A. It is characterized by being an immortalized transformed cell line.

In addition, the α-L-eduronidase sample is characterized in that the α-L-eduronidase enzyme or a homologue thereof for the treatment of Herr syndrome or Herr-Scheie syndrome by direct enzyme replacement.

The effect of the present invention is to provide a kit for activity evaluation for the selection of an optimal enduronidase enzyme for the treatment of Herr syndrome or Herr-Scheie syndrome by enzyme replacement. Or to provide a kit for quantitative activity assessment of secretion.

The present invention is described in more detail below.

Infected lymphocytes, which have been difficult to develop into normal cell lines, are infected with EB (Epstein-Bar) virus and transformed into immortalized cell lines, which can be specifically receptive to the α-L-eduronidase sample. It is possible to obtain a suitable cell line for evaluation with M6PR).

At this time, the transformed lymph cell line was cultured by adding an equivalent amount of EB (Epstein-Bar) virus soup to 0.5-1.5 × 10 ⁶ cells / ml of lymphocytes centrifuged in the blood of the recipient, followed by addition of cyclosporin A. Cell line transformed by immortalization.

In this case, the lymphocytes are preferably lymphocytes collected from the patient's blood to remove immune rejection reactions such as hemolysis from the patient during administration. Only the patient's own lymphocytes should be used as a raw material for the transformed cell line to accurately and quantitatively measure the enzyme uptake and secretion in the cell line of the α-L-eduronidase enzyme sample through elimination of the immune rejection response.

On the other hand, in order to quantitatively measure the α-L-eduronidase enzyme sample, a fluorescent label should be attached to the α-L-eduronidase enzyme sample. By recognizing, the uptake secretion of the α-L-eduronidase enzyme sample in the lymph cell line can be quantitatively measured.

In addition, the present invention is a method for the treatment of patients with Herr syndrome or Herr-Scheer syndrome, the α-L-eduronidase enzyme or its homologues are selected from the enzyme specimens by direct enzyme replacement method, Herr syndrome or Herr-Shay To provide a method for treating the syndrome.

The present invention will be described in more detail with reference to the following examples. However, these examples do not limit the scope of the present invention.

Preparation Example 1 Lymphocyte Line Prepared Using Lymphocytes Collected from Patient A

Primary culture

After adding whole blood cells extracted from Hero syndrome patient A to Histopaque, centrifuged at 3,000 rpm X for 15 minutes, the buffy coat layer was extracted, washed with phosphate buffer, and 15 at 1,500 rpm X. Centrifuge again for a minute. After removing the supernatant, washed with phosphate buffer solution and centrifuged again at 1,500rpm X for 15 minutes.

After 5 ml of phosphate buffer is removed, 5 ml of the residual solution is mixed well and the final cell count in the medium is calculated. At this time, the final cell number of the medium is 1 × 10 ⁶ cells / ml. Place in 1/4 of 10% medium cell pellet of final culture and add equal amount of EBvirus soup. After 2 hours incubation at 37 ℃, add the same amount of 10% medium and add 4 to the final concentration of cyclosporin A 0.5 ug / ml. After 2 weeks of incubation, observe the transformation under a microscope. At this time, if the badge color is yellow, replace the badge about 1/2.

Cell line formation

One week after the medium replacement, the transformed lymphocytes are cultured as cell lines are formed.

freezing

Freeze when cell number is 25 × 10 ⁶ cells / ml or more (based on 5 vials).

Preparation Example 2 Lymphocyte Line Prepared Using Lymphocytes Collected from Patient B

Except for the collection of blood cells from the patient B, the freezing was performed in the same manner as in Preparation Example 1, after forming the primary culture and the cell line, when the number of cells reached 25 × 10 ⁶ cells / ml or more (based on five vials). To prepare a cell line.

Preparation Example 3 Lymphocyte Line Prepared Using Lymphocytes Collected from Patient C 4

Except for taking blood cells from the patient C, the cells were formed in the same manner as in Production Example 1, and then frozen when the cell number reached 25 × 10 ⁶ cells / ml or more (based on 5 vials). Prepare cell lines.

Preparation Example 4 Preparation of α-L-Eduronidase Enzyme

A commercially available brand name Aldurazime enzyme is used as α-L-eduurodase.

Preparation Example 5 Preparation of α-L-eduronidase Enzyme Homolog

The α-L-eduronidase homolog in which the oligosaccharide site is replaced with polyethylene glycol at amino acid residue No. 110 in the commercially available brand name Aldurazime enzyme is used.

Preparation Example 6 Preparation of α-L-eduronidase Enzyme Homolog

An α-L-eduronidase homologue is used which removes the mannose-6-phosphate moiety attached to amino acid residue 336 in the commercially available trade name Aldurazyme enzyme.

Example 1 Selection of Optimal α-L-Eduronidase Enzyme Required for Enzyme Replacement Method

The α-L-eduronidase enzymes prepared in Preparation Examples 4 to 6 were applied to the respective lymph cell lines using the lymphocyte lines prepared in Preparation Examples 1 to 3 through blood cells collected from patients A, B, and C. Are added to select the α-L-eduronidase enzyme homolog that exhibits optimal uptake within the cell line.

In this case, the fluorescent label trade name Alexa Fluor 488 having a wavelength of 488 nm was conjugated to the ends of the α-L-eduronidase enzyme homologs prepared in Preparation Examples 4 to 6 for smooth quantitative measurement of the α-L-eduronidase enzyme homolog. Gating

Table 1 shows the uptake results of the α-L-eduronidase enzyme homologs prepared in Preparation Examples 4 to 6 for the lymph cell lines prepared in Preparation Examples 1-3.

     (Unit nmol / hr / mg enzyme) Enzyme 1
(Production Example 4) Enzyme 2
(Production Example 5) Enzyme 3
(Production Example 6) Lymphatic cell line 1
(Production Example 1) 2216 1840 1750 Lymphatic cell line 2
(Production Example 2) 1845 1910 2040 Lymphatic cell line 3
(Production Example 3) 1940 2110 1650

Based on the enzyme uptake results of the lymph cell line shown in Table 1, the lymph cell line prepared from Patient A (Herbo Syndrome) A (Production Example 1) showed the highest absorption rate of Enzyme 1 prepared in Preparation Example 4, The prepared lymph cell line (Preparation Example 2) showed the highest absorption rate with enzyme 3 prepared in Preparation Example 6, and the lymphatic cell line (Preparation Example 3) prepared from the Herer syndrome patient C was prepared in Preparation Example 5. Enzyme 2 had the highest absorption rate.

Through this test, the enzyme showing the optimum absorption rate in patient A was enzyme 1, the enzyme showing the optimum absorption rate in patient B was enzyme 3, and the enzyme showing the optimum absorption rate in patient C was enzyme 2.

Example 2 Treatment of Patients Using Enzyme Replacement

Through the results shown in Example 1, the patient A was treated with enzyme 1, the patient B with enzyme 3, and the patient C with enzyme 2 to treat the Herr syndrome or Herr-Scheier syndrome by enzyme replacement.

Claims (3)

delete Iii) transformed lymph cell lines in which lymphocytes harvested in the body of the administered patient are immortalized with Epstein-Bar (EB) virus; Ii) α-L-eduronidase enzyme therapeutics with a fluorescent label attached thereto; And iii) a fluorescent label-recognized light absorbing device for quantitatively detecting the absorption or secretion of the α-L-eduronidase enzyme therapeutic agent in the lymphatic cell line through fluorescent labeling. In the kit, the transformed lymph cell line was cultured by adding an equivalent amount of EB (Epstein-Bar) virus soup to 0.5-1.5 × 10 ⁶ cells / ml of lymphocytes centrifuged in the blood of the administered patient, followed by cyclosporin A. A kit for evaluating the activity of α-L-eduronidase enzyme therapeutic agent for the treatment of patients with Herr syndrome or Herr-Scheier syndrome, characterized in that the cell line was transformed into immortalized by the addition of delete