JPH024800A - Module for removing interleukin ii receptor and method for removing interleukin ii receptor using said module - Google Patents

Abstract

PURPOSE:To obtain the subject module useful for the therapy of adult T-cell leukemia, etc., composed of a column packed with a carrier supporting a monoclonal antibody having especially highly specific reacting with a human interleukin II receptor. CONSTITUTION:A column placed with 2000 hollow fiber carriers made of a high polymer such as polypropylene is washed with ethanol by pumping thereinto from one open end, rewashed with purified water adequately. Then a solution dissolving, a monoclonal antibody having especially highly specific reactivity with a human interleukin II receptor [example; monoclonal antibody H-48 (FERM No.1913)] is passed through the column by a pump to deposit the monoclonal antibody onto an inner surface of the hollow fiber with physical adsorption to afford the objective module.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to the human interleukin 2 receptor (a high molecular protein that has an affinity for human interleukin 2) in human blood. The present invention relates to a module for removing leukin-2 (which is thought to regulate the biological effects of leukin-2 (hereinafter sometimes abbreviated as IL-2R) and a method for removing IL-2R using the module. It is.

[Description of prior art]

Human interleukin 2 (hereinafter sometimes abbreviated as IL-2) has a molecular weight of about 15,000 and is a type of lymphokine synthesized and secreted by antigen-activated T cells. IL-2 is thought to exhibit biological effects such as stimulation of proliferation of activated T cells through interaction with IL-2R present on the surface of antigen-activated T cells.

By the way, the onset and progression of adult T-cell leukemia (hereinafter sometimes abbreviated as ATL), which has recently been attracting attention due to its frequent occurrence in western Japan, especially in the Kyushu region, is due to the origin of leukemic cells. The involvement of the rL-2 system is suggested by the fact that these are mature helper Tt, t[l cells, and a large number of L-2Rs are constitutively abnormally expressed on their surfaces.

In other words, the explanation for the onset and progression of ATL is as follows.
The first step is when the helper TiJIl cells are infected with the HTLV-1 virus and constitutively abnormally express a large number of IL-2Rs on the surface of the T cell.
RJ and its HTLV-1 viral gene-derived product (p40X) and IL- abnormally produced based on antigen stimulation.
The second stage consists of abnormal proliferation of TjII cells due to reaction with 2J! There is a two-stage activation model for the L-2 system [Maruyama M, et al.
, cel148.343+ (1987)].

Therefore, if IL-2R can be removed from the blood of ATL patients or those infected with viruses such as HTLV-I or Hl■, it will be possible to treat those patients or prevent the onset of symptoms in those infected.

In addition, it has been reported that T cells with IL-2R play an important role in conditions where local GVHR (graft-versus-host reaction) rapidly increases (H, D, Volk, e.g.
t atSEur, J. Immunol, 16s 13
09-1312 (1986)], and IL
It is also considered possible to suppress rejection reactions that occur during organ transplantation by removing cells containing -2R.

However, until now, I
There is no known method for removing L-2R [Problem to be solved by the invention] The purpose of the present invention is to remove IL-2R from IL-2R-containing substances.
The present invention provides a module comprising a carrier carrying a monoclonal antibody that can be used to easily and effectively remove R, and a method for removing IL-2R using the module.

[Means for solving problems]

As a result of intensive research to solve the above problems, the present inventors have found that IL-2R can be removed very easily and effectively from IL-2R-containing substances using the module of the present invention. They discovered that it is possible to do this, and completed the present invention.

Particularly, the present invention relates to an IL-2 removal module characterized by comprising a column having a carrier carrying a monoclonal antibody having extremely high specific reactivity against IL-2H. be.

Furthermore, the present invention provides IL-2R using the module.
The present invention relates to a method for removing IL-2R from contained substances.

The present invention will be explained in more detail below.

The module (hereinafter abbreviated as "Module 1") that can be used to remove rL-2R from IL-2R-containing substances of the present invention essentially includes a monoclonal antibody, its carrier, and a column. .

Examples of the II, -2R-containing substance used to remove IL-2R include human blood. When using human blood, whole blood can be used as it is, but plasma obtained by separating it in advance with a plasma separator or the like can also be used, or it can also be used as serum. When the IL-2R-containing substance is not in a solution state, it is necessary to make it into a solution state in advance using an appropriate solvent (eg, water, buffer, etc.).

The monoclonal antibody used in the r module j of the present invention may be obtained by immunizing a mouse (for example, BALB/?mouse) with IL-2R (for example, cells expressing IL-2R1IL-2R). IL-2R produced by a hybridoma strain obtained by cell fusion of a mouse lymphocyte and a mouse myeloma cell (for example, a cell that does not secrete or produce antibodies such as SP210-Ag14) using polyethylene glycol. Any monoclonal antibody may be used as long as it has a very high specific reactivity against the monoclonal antibody, but monoclonal antibody H-48 (Feikoken Joyori No. 1913) is preferable.
, HA-26 (Feikoken Joyori No. 1914) is preferably used, and more preferably H-48 is used.

The invention? The carrier supporting the monoclonal antibody used in module 1 includes synthetic polymeric substances such as polyethylene, polypropylene, polymethyl methacrylate, polyvinyl alcohol, 4-methylpentene, polycarbonate, polystyrene, and AS resin (acrylonitrile-styrene resin). , inorganic substances such as glass and silica gel, or insoluble biopolymer substances derived from living organisms such as proteins and polysaccharides. Considering these points, it is preferable to use polyethylene or polypropylene.

The shape of such a carrier may be any shape such as a bead, a plate, a rod, or a tube as long as it is compatible with the purpose of the present invention, but it is preferable to use a hollow fiber that is a tubular carrier. It's better to be there.

Columns used in the “r module of the present invention” include:
Examples include those using synthetic polymer substances such as polycarbonate, polystyrene, As resin (acrylonitrile-styrene resin), glass, and the like. The shape of the column may include, for example, one having at least one inlet portion at one end that can be easily connected to a solution circuit for the IL-2R-containing substance, and at least one outlet portion at the other end. However, the shape is not particularly limited as long as it can provide a place where the carrier carrying the monoclonal antibody and the solution of the IL-2R-containing substance can come into effective contact.

As for the size of the hollow fibers that are the tubular carriers, the inner diameter is preferably 100 to 500 μm and the membrane thickness is 10 to 200 μm, but the inner diameter is preferably 300 to 400 μm, and the total area based on the inner diameter of the hollow fibers is 0.3 to 1.0 m" is preferable.

Surface (inner surface and/or outer surface) of the carrier in the present invention
The method for supporting the monoclonal antibody and the monoclonal antibody is such that the supporting is relatively strong and the monoclonal antibody and the IL-2R
Any method may be used as long as the reactivity with the glutaraldehyde is not impaired. can.

The “removal of IL-2R using module j” of the present invention
IL-2R-containing substances (such substances include, for example, solutions of blood, plasma, serum, etc. containing IL-2R. If the substance is not in a solution state, add water, buffer solution, etc. It is necessary to make it into a solution state with a suitable solvent such as.

) can be carried out by passing it through the module j of the present invention.

In the present invention, the concentration and flow rate of the IL-2R-containing substance passed through module j are not particularly limited, but IL-2R
If the 2R concentration is high, the object of the present invention can be achieved by reducing the flow rate and/or by additionally connecting the r module j of the present invention.

In this way, IL-
The r module used to remove 2R is
By increasing the salt concentration and/or pH and removing IL-2R that has been affinity bound to the monoclonal antibody of module 1, IL-2R in the IL-2R-containing substance can be released.
-Can be reused for removal of 2R.

〔Example〕

Hereinafter, the present invention will be specifically explained with reference to Examples. Note that these Examples do not limit the scope of the present invention.

Example 1 [Production of "module"] Polypropylene hollow system made of polypropylene as a carrier (membrane area 0.35 m'', inner diameter 320 μm, outer diameter 430 μm)
A peristaltic pump was inserted from one end of the opening of a column (made of polycarbonate, inner diameter 3 cm, length 20 cm, with one opening at each end) containing 2,000 columns (film thickness 55 μm, length 18 cm). (manufactured by Iuchi Seieido), the flow rate is 500mj! /hr for about 30 minutes, and then thoroughly washed with purified water.

Next, a solution containing a monoclonal antibody (H-48) that has highly specific reactivity to IL-2R (dissolved in a phosphate buffer solution of pH 7.4, 200 μg/m
This monoclonal antibody was supported by physical adsorption on the inner surface of the hollow fiber by flowing 400 mf of the sample (referred to as J2) through the ethanol-washed column at a flow rate of about 25 ml/hr using a peristaltic pump (manufactured by Iuchi Seieido). .

Next, add 55% of 1% human T-globulin solution to this column.
0 mj2 was injected and left to stand at room temperature for 3 hours to coat the hollow fiber surface not coated with this monoclonal antibody with human γ-globulin. This was thoroughly washed with 0°1Ml-Lis-HCl buffer (pH 8, 0, 0, 15M sodium chloride). It was stored at 4°C until use.

Incidentally, even when the r module j prepared in this way was stored as it was, and even when it was used in ordinary affinity chromatography, almost no protein detachment from the V module j was observed.

Example 2 [Removal of IL-2R in buffer] 200 mj! 0. I M I-Lis-HCl buffer (
pH 8,0,0,15M sodium chloride) at a concentration of 40 ng of IL-2R prepared from human T cell culture supernatant.
/mf! This IL-2R-containing substance was poured into module j prepared in Example 1 at a flow rate of 500 mf/hr using a peristaltic pump (manufactured by Iuchi Seieido), and the effluent from the outlet of r module J was was fractionated into 10ml portions using a fraction collector.

The IL-2H concentration in each fraction and the IL-2H concentration before flowing into the r module were measured according to the conventional Sandwich enzyme-linked immunosorbent assay (ELISA) as shown below.

Standard IL-
Add 100μ aliquots of 2R or effluent and allow to react for 1 hour. Wash each well and add 100μ of HA-26 solution of monoclonal antibody labeled with peroxidase.
Add 100μ2 each of a substrate solution (pH 5,0) consisting of orthophenylenediamine, H20□ and react for 30 minutes at room temperature, and then add 2N sulfuric acid solution. After adding 50 μ2 each, absorbance at 492 nm was measured.

As a result, the average removal rate of IL-2R was 92.3%.
Met.

Examples 3 to 4 [Removal of IL-2R in normal human serum] IL-2R was added to 10 m2 of Nescor X (manufactured by Kaketsuken), which is a commercially controlled human serum.
IL-2R was added so that the concentration of -2R was 39.5 ng/ml. This IL-2R-containing substance was applied to an affinity ram with a membrane area of 0.008 m'' (adsorbed monoclonal antibody amount was approximately 10 mg) prepared in the same manner as in Example 1 using a peristaltic pump (manufactured by Iuchi Seieido) at a flow rate. Target 20ml/hr or 60mj2/
hr, and the concentration of IL-2R in the serum flowing out from the outlet of r module J and the serum before flowing into r module J was measured by the same ELISA method as in Example 2.

As a result, the average removal rate of IL-2R was approximately 2
It was 97.5% or more at 0 ml/hr, and 97.5% at 60 mf/hr.

Examples 5 to 7 [Removal of IL-2R in human whole blood] 15 mj of human whole blood to which heparin was added at 1 unit/m1! IL-2R was added to the solution at a concentration of 40.0 ng/ml. This rL-2'R-containing substance was prepared in the same manner as in Example 1, with a membrane area of 0.008 m.
” using a peristaltic pump (manufactured by Iuchi Seieido) through an affinity ram (absorbed monoclonal antibody amount is about 10 mg) at a flow rate of 20 m1/hr, 40 mI!, /hr or 80 mI!, /hr, and r module J. The whole blood that flowed out from the outlet and the whole blood before flowing into module J were subjected to plasma separation at 2000 rpm for 15 minutes), and the concentration of IR-2R in each plasma was measured by the same ELISA method as in Example 2.

As a result, the average value of the removal rate of IL-2H was approximately 2
0m/! /hr, it is 79.7%, and 40mj
! /hr, it is 78.3%, and 80ml/hr.
When it was hr, it was 68.8%.

Examples 8 to 9 (Removal of IL-2R in ATL patient serum) 10 mf of ATL patient serum, which is an IL-2R-containing substance, was prepared in the same manner as in Example 1 with an affinity force of 0.008 m'' membrane area. Lamb (the amount of monoclonal antibody adsorbed is approximately 10 m
Using a peristaltic pump (manufactured by Iuchi Seieido) for g), the flow rate is 20 m! / hr r or 40mj2/hr, r
The concentration of IR-2R in the serum flowing out from the outlet of the module and the serum before flowing into r module j was measured by the same ELISA method as in Example 2.

As a result, the average removal rate of IL-2R was approximately 2
At 0mA/hr, it is 92.9%, and 40mj2
/hr, it was 90.2%. Furthermore, "the concentration of IR-2R in serum before flowing through module 1 is 460
It was ng/ml.

〔Effect of the invention〕

By using the r module 1 of the present invention, TL can be easily and effectively removed from IL-2R-containing substances.

Claims (2)

[Claims] (1) A human interleukin 2 receptor removal module comprising a column having a support carrying a monoclonal antibody having extremely high specific reactivity with human interleukin 2 receptor. (2) A method for removing human interleukin 2 receptor from a substance containing human interleukin 2 receptor, which comprises using the module according to claim 1.