JPH07106219B2 - Interleukin 2 receptor removal device and extracorporeal blood circulation device including the device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to interleukin 2 receptor (hereinafter, IL-2R).
Or a device for removing IL-2R-expressing cells from the blood and an extracorporeal blood circulation device equipped with the device for removing IL-2R, in particular, by removing IL-2R from the blood of an adult T-cell leukemia patient. The present invention relates to a device for removing interleukin 2 receptor for preventing deterioration or treatment, and an extracorporeal blood circulation device including the device.

[Prior Art] Interleukin 2 is a kind of lymphokine synthesized and secreted by antigen-activated T cells. IL-2 is considered to exhibit biological effects such as stimulation of proliferation of activated T cells by interacting with specific high affinity receptor IL-2R existing on the surface of antigen-activated T cells. ing.

Recently, adult T-cell leukemia (hereinafter abbreviated as ATL), which has been attracting attention because it frequently occurs mainly in western Japan, especially in Kyushu region, is caused by the helper T-cells derived from mature leukemia cells. And a large number of IL-
The involvement of the IL-2 system has been suggested from the fact that 2R is constitutively abnormally expressed. As such an explanation, a two-step activation model of the IL-2 system in the development of ATL has been proposed, which removes IL-2R from the blood of ATL patients and patients infected with viruses such as HTLV-I or HIV. If possible, it is considered possible to treat patients or prevent the onset of infected persons [Maruyama M. et al, Cel1 48 , 343, (198
7)].

Conventionally, as a method for removing IL-2R from blood, there are a method using various separation membranes, a method using an ion exchange resin column or an affinity column using a gel, and the like.

[Problems to be Solved by the Invention] However, IL-2R cannot be specifically removed by conventional methods using various separation membranes, and an ion exchange resin column, or cellulose, In the method that uses an affinity column that uses a gel such as Sepharose, the ion exchange resin and the affinity gel itself are expensive, and there is a problem in their performance (sample processing amount, sample processing time, sample processing efficiency, etc.) .

[Means for Solving the Problems] Therefore, the present inventors have solved the above-mentioned problems, and
The present invention has been completed as a result of earnest research to find a device that can remove IL-2R effectively at low cost.

That is, the present invention relates to a device for removing interleukin 2 receptor, which comprises adsorbing and immobilizing a monoclonal antibody of interleukin 2 receptor on a carrier, and contacting blood and / or plasma with the carrier, wherein the carrier comprises a plurality of carriers. Hollow fiber membranes with a diameter of 100-1000 μm
And the total membrane area of the plurality of hollow fiber membranes is 0.01 to 1.
An apparatus for removing an interleukin 2 receptor having a size of 0 m ² (first invention) is provided.

In the above-described interleukin 2 receptor removing apparatus, the plurality of hollow fiber membranes are bundled inside a tubular container, and both ends of the hollow fiber membranes are embedded in partition walls in an open state,
It is preferable that both ends of the hollow fiber membrane are hermetically sealed in the tubular container by partition walls, and the hollow fiber membrane is made of a polymer resin.

The present invention also provides an extracorporeal blood circulation device (second invention) comprising the above-mentioned interleukin 2 receptor removal device.
Is provided.

Here, as a typical example of the extracorporeal blood circulation device, there is a plasmapheresis treatment device, and in this case, the blood processing device is a plasma separator.

As the material of the hollow fiber membrane used in the present invention, synthetic polymers, inorganic substances such as glass and silica gel, or insoluble biopolymer substances derived from organisms such as proteins and polysaccharides can be used.

[Examples] Hereinafter, the present invention will be described in more detail based on the illustrated examples, but it will be apparent that the present invention is not limited to these examples.

FIG. 1 shows the present invention from blood and / or plasma
1 shows an example of an IL-2R removal device.

Inside the cylindrical container 1, a plurality of polymer resin hollow fiber membranes 2 on which IL-2R monoclonal antibody is adsorbed and fixed are arranged. Both ends of the container 1 and the hollow fiber membrane 2 are supported in a fluid-tight manner by partition walls 3 made of potting material such as polyurethane resin, and both ends of the hollow fiber membrane 2 are open at both ends of the tubular container 1. .

In the above configuration, blood and / or plasma enter the hollow portion of the hollow fiber membrane 2 to which the monoclonal antibody of IL-2R is adsorbed and fixed from the inlet 4 of the cylindrical container 1, and contact with the monoclonal antibody, IL-2R in blood and / or plasma
The blood and / or plasma that has been removed and then purified flows out of the cylindrical container 1 via the outlet 5.

The hollow fiber membrane, which is a carrier for adsorbing and immobilizing the monoclonal antibody against IL-2R in the above examples, may be made of various high molecular weight polymers, and the type thereof is not particularly limited, and examples thereof include polyethylene, Polypropylene, polymethylmethacrylate, polyvinyl alcohol, 4-methylpentene, polycarbonate, polyvinylidene fluoride, silicone rubber and the like and modified resins thereof can be usually used, but from the viewpoint of the binding method with a monoclonal antibody and the binding force. Considering these, polypropylene and polyethylene are preferable.

The size of the hollow fiber membrane is not particularly limited, but the inner diameter is 100 to 100.
It is desired that the thickness is 0 μm, preferably 200 to 400 μm, and the film thickness is 10 to 200 μm, preferably 20 to 60 μm.

Further, the membrane area of the hollow fiber membrane is generally preferably about 0.01 to 1.0 m ² .

As a method for adsorbing / fixing the monoclonal antibody on the hollow fiber membrane, a well-known method such as a physical adsorption method or a chemical binding method can be applied, but it can be fixed relatively firmly and does not impair the antibody activity. Must be the way.

As the monoclonal antibody adsorbed and immobilized on the carrier in the present invention, any monoclonal antibody may be used as long as it has a very high specific reactivity with IL-2R, but is preferably Monoclonal antibody H-48 is used.

As the material of the cylindrical container used in the present invention, polycarbonate, polystyrene, ABS resin, glass and the like can be used.

The apparatus for removing IL-2R from blood and / or plasma of the present invention can flow whole blood as it is, but can also flow only plasma previously separated by a plasma separator.

FIG. 2 shows an example of an extracorporeal blood circulation device for removing IL-2R in blood. The blood of the patient is guided to the interleukin 2 receptor removing device 6 from the blood collecting unit 8 by the blood pump 7, and the blood from which IL-2R has been removed is returned from the blood returning unit 9 into the body of the patient.

FIG. 3 shows an example of an extracorporeal blood circulation device for removing IL-2R from plasma.

Reference numeral 10 denotes a blood collecting unit, which guides the blood of the patient from the body to the plasma separator 12 by the blood pump 11. The blood that has entered the plasma separator 12 is separated into plasma components, is guided by the plasma pump 13 to the interleukin 2 receptor removal device 14, and the plasma from which IL-2R has been removed is discharged from the plasma separator 12 at point A. The mixed blood is mixed with the concentrated blood and returned to the patient's body from the blood returning section 15.

Example 1 Production of Interleukin 2 Receptor Removal Device 2000 polypropylene hollow fiber membranes having an inner diameter of 320 μm, a film thickness of 55 μm, and a length of 210 mm were bundled and inserted into a polycarbonate cylindrical container to form a polyurethane resin cylindrical container. Both ends of the hollow fiber membrane were fixed to prepare a module having a membrane area of 0.35 m ² .

Ethanol was passed through this module at 500 ml / hr for 30 minutes for washing, and then ethanol was thoroughly washed off with purified water.

Next, a monoclonal antibody (H-48) having a very high specific reactivity to IL-2R was added at a concentration of 200 μg.
solution was dissolved in phosphate-buffered saline so that the concentration of the monoclonal antibody was adjusted to 100 ml / ml, and 400 ml of this solution was flowed through the ethanol-cleaned module at a flow rate of 25 ml / hr to bind the monoclonal antibody to the inner surface of the hollow fiber by physical adsorption. did.

Next, 50 ml of a 1% human γ-globulin solution was injected into this module, and the mixture was allowed to stand at room temperature for 3 hours to coat the surface of the hollow fiber not coated with the monoclonal antibody with human γ-glopurin. This is a 0.1 M Tris-HCl buffer (pH 8.0,
It was thoroughly washed with 0.15 M sodium chloride) and stored at 4 ° C. until use as a module used for removing IL-2R from blood.

In addition, even when the module thus produced was used for ordinary affinity chromatography or stored, the protein was hardly released from the module.

(Example 2) Removal of IL-2R from human clean serum 10 of commercially available controlled human serum Nescor X (manufactured by KEIKAKEN)
IL-2R was added to ml so that the concentration of IL-2R was about 40 ng / ml.

A membrane area of 0.01 m ² prepared in the same manner as in Example 1 was obtained.
Module (The amount of adsorbed monoclonal antibody is about 10 m
In g), a blood pump (perista pump, manufactured by Inei SEIEIDO) 9 shown in the apparatus of FIG. 2 is used, and the flow rate is about 20 ml / hr or 60 m.
Serum flown out at 1 / hr and the IL-2R concentration before flowing out into the module outlet and the module were measured by the sandwich oxygen immunoassay (ELISA) shown below.

The results are shown in Table-1.

(Example 3) Removal of IL-2R from human whole blood 15 ml of whole blood to which heparin was added at 1 unit / ml
In addition, IL-2R was added at a concentration of about 40 ng / ml. A module having a membrane area of 0.01 m ² prepared in the same manner as in Example 1 (the amount of adsorbed monoclonal antibody was about 10 mg)
Using a peristaltic pump (made by Inei Seieidou) for a flow rate of about 20 ml
/ hr, 40 ml / hr or 80 ml / hr, at each flow rate, whole blood flowing out from the module outlet and whole blood before flowing to the module are separated by centrifugation at 2000 rpm for 15 minutes to separate the plasma. The concentration of IL-2R in the same ELISA as in Example 2
Was measured by the method.

The results are shown in Table-2.

(Example 4) Removal of IL-2R from serum of ATL patient 10 ml of serum of ATL patient was placed in a module having a membrane area of 0.01 m ² prepared in the same manner as in Example 1 (the amount of adsorbed monoclonal antibody was about 10 mg). Using a peristaltic pump (manufactured by Inei Seieidou) at a flow rate of about 20 ml / hr or 40 ml / hr, the concentrations of IL-2R in the serum flowing out from the outlet of the module and the serum before flowing to the module were measured by the ELISA method. .

The results are shown in Table-3.

[Effects of the Invention] As described above, according to the interleukin-2 receptor removing apparatus of the present invention, a hollow fiber membrane having a monoclonal antibody adsorbed thereon is used, and blood and / or plasma is brought into contact with the hollow fiber membrane. It is possible to efficiently and inexpensively remove IL-2R in blood and / or plasma. Especially adult T
By removing IL-2R from the blood of a cell patient, the disease state can be prevented from worsening or treated, and the onset of infection can be prevented.

Further, according to the extracorporeal blood circulating apparatus of the present invention, since the interleukin 2 receptor removing apparatus using the hollow fiber membrane adsorbing the monoclonal antibody is used as the blood treating apparatus, the blood and / or plasma There is an effect that IL-2R can be efficiently removed.

[Brief description of drawings]

1 is a schematic diagram showing an example of an IL-2R removing apparatus according to the present invention, FIG. 2 is an explanatory view showing an example of an extracorporeal blood circulating apparatus for removing IL-2R in blood, and FIG. 3 is plasma It is explanatory drawing which shows an example of the extracorporeal blood circulation apparatus which removes IL-2R in blood. 1 ... Cylindrical container, 2 ... Hollow fiber membrane, 3 ... Support member, 4
…… Inlet, 5 …… Outlet, 6 …… Interleukin 2 receptor removal device (module), 7 …… Blood pump, 8
…… Blood collection unit, 9 …… Blood return unit, 10 …… Blood collection unit, 11 …… Blood pump, 12 …… Plasma separator, 13 …… Plasma pump, 14 ……
Interleukin-2 receptor removal device (module), 15 ... Blood return part.

Claims (3)

[Claims] 1. A device for removing interleukin 2 receptor, which comprises adsorbing and immobilizing a monoclonal antibody for interleukin 2 receptor on a carrier, and contacting blood and / or plasma with the carrier, the carrier comprising a plurality of carriers. Hollow fiber membrane, wherein the diameter of each hollow fiber membrane is 100 to 100 μm, and the total membrane area of the plurality of hollow fiber membranes is 0.01 to 1.0 m ² , removal of interleukin 2 receptor apparatus. 2. A plurality of hollow fiber membranes are bundled inside a tubular container, and both end portions of the hollow fiber membranes are embedded in partition walls in an open state,
The device for removing interleukin 2 receptor according to claim 1, wherein both ends of the hollow fiber membrane are hermetically sealed in the tubular container by the partition wall, and the hollow fiber membrane is made of a polymer resin. 3. An extracorporeal blood circulation device comprising at least a blood pump and a blood processing device, for sending blood to the blood processing device and returning blood from the blood processing device, wherein the blood processing device is claimed. An external blood circulation apparatus comprising the interleukin 2 receptor removing apparatus according to item 1.