JPS6219533A - Expression-inducing substance b for interleukin 2 receptor - Google Patents

Abstract

PURPOSE:To produce the titled substance which is a simple substance separated from T-leukemia cell strain, inducing the expression of interleukin 2 receptor and useful for the remedy of immunoinsufficiency, cancer and autoimmune infectious diseases. CONSTITUTION:The expression-inducing substance (abbreviated as ADF) for interleukin 2 receptor has the following characteristics. Molecular weight, 20-10Kd (Sephacryl-S200 gel-filtration); isoelectric point, pI 5.5-4.5; amino acid sequence (from N-terminal), shown in the formula. ADF has been found in a supernatant liquid of the cultured T-leukemia cell strain (ATL cell) established from a human adult-type T-leukemia patient. Although known ADF fraction is a mixture of several proteins, the ADF of the present invention corresponds to the simple substance having lower molecular weight in two ADF activity peaks obtained by the fractionation of a concentrated ATL cell liquid using a Sephacryl S-200 column. The ADF can be used as a remedy singly or in combination with IL2 or other lymphokine.

Description

[Detailed Description of the Invention] [Industrial Application Field] This invention relates to interleukin 2 (hereinafter referred to as rlL2) receptor (interleukin 2 receptor).
The present invention relates to a substance (hereinafter referred to as rADFJ) that induces the expression of cep-tor (hereinafter referred to as rlL2RJ).

[Conventional technology]

ADF is derived from Tac antigen (N
ture, 300, 267-269 (1982),
Pro, N, A, S, 80°6957-6951 (19
83), Nature, 311.635-638 (1
984)) was found to exist in the culture supernatant of T leukemia cell lines (rATL cells) established from human adult T leukemia patients (rATL cells).
J, Immunol, 134.1623-1630. (
(1985), J. Mol.

Ce11. Immunol, 2.17-26. (i9
85)) Therefore, by inducing the expression of IL2R, ADF causes the expression of IL2 activities such as T cell differentiation and proliferation in vivo, or allows IL2 to function effectively in vivo. Due to its unique biological activity, it can be expected to be widely applied as a treatment for immunodeficiency, tumors, etc.

According to research by the present inventors, interleukin 1 (inter-I) is a soluble substance that induces Tac antigen.
eukin 1), but the chemical properties such as the amino acid sequence of interleukin 1 (Pro, N, A, S, 8
1.7907-7911, (1984)) and that of the ADF of the present invention are completely different.

In addition, the present inventors previously subjected the culture supernatant of ATL cells to gel filtration chromatography, and found that the molecular weight ranged from 40 to 35k.
d and 20 to 15 kd with ADF activity (J, Mo1.Ce11.In+muno1.2+
17-26, (1985)), and subsequent studies revealed that the ADF fraction obtained here was not pure but a mixture of several proteins.

Therefore, this ADF protein fraction and the purified AD of the present invention
This substance is different from the F protein preparation, and it was only after such purification that it was revealed that a single substance was involved in inducing IL2R expression. In other words, the conventional ADF fraction is a mixture of several proteinaceous substances.
It had L2R expression inducing activity. In addition, various problems can be expected when using such mixtures as medicines.

[Problem that the invention seeks to solve]

Therefore, an object of the present invention is to obtain ADF purified to only the proteins necessary for its activity.

[Means for solving problems]

In order to solve the problem of ridges, the present inventors conducted inoculation research and finally succeeded in isolating ADF as a single substance having the following properties. That is (1) Molecular weight: 2
0 to 10 kd (Sephacryl S-200 gel filtration method) (2) Isoelectric point: I) I5.5 to 4.5 (3) Amino acid sequence (from N-terminus): Pro-Met-Phe-
X-Val-Asn-Thr-Asn-Va 1-Pr
o-Arg-Val-Ser-Val-Pro-ATL
There are, for example, the following two methods for obtaining cells.

The first method is to establish it from peripheral blood of ATL patients. That is, peripheral blood was collected from a patient diagnosed with ATL, and white blood cells were collected using RP containing (Fe2) using the Ficoll method.
Culture is started in MI-1640 medium. After stable growth was shown, crude IL2 was gradually removed from the medium and culture started. After several injections, RPMI-164 containing 10% FC3 was added.
Obtain a cell line that can be grown and passaged only in 0 medium. To confirm that the obtained cell line is an ATL cell, search for surface traits such as E rosette OKT series, s4g, ATLA, and Tac. It should be noted that these antigen markers are all well-known and widely used clinically.
It is used.

E rosette is a search for receptors for sheep red blood cells, which are mainly expressed on T lymphocytes.

OKT series includes 0KT-3, 4i 6.8. T-3 is a T-cell antigen receptor-related antigen and is used to identify human peripheral blood T lymphocytes, T-4 is used to identify helper T lymphocyte subsets, and T-6 is used to identify β-2 microcells. Among globulin-related antigens, T-8 is used to identify common thymus-derived T lymphocytes and suppressor/killer T lymphocyte subsets. s-Ig is a cell surface immunoglobulin and is specifically expressed in B lymphocytes. ATLA is a human adult T leukemia antigen, and the antigen is a virus-derived protein. Matata Tac
is said to be IL2R. Since monoclonal antibodies against these antigens are commercially available or can be easily obtained, cell surface antigens can be easily identified by binding each monoclonal antibody to FITC and reacting with sample cells.

The second method uses human adult T leukemia virus (ATLV)
Peripheral blood transformation using
itro). (Science, 217, 73
7 (1982)).

That is, ATLV-producing ATL cells, such as MT-2, whose proliferation ability has been completely stopped by irradiation with γ-rays (12,000 rad)
Cells and white blood cells obtained by Ficoll treatment of peripheral blood from healthy individuals are cultured in RPMI-1640. After injury, a cell line that can be passaged can be obtained, but if proliferation is unstable, human Add IL2 derived from peripheral blood. To confirm that the obtained cell line is an ATL cell,
Rosette, OKT series + 5-1g + ATLA
Search for surface traits such as ITac is performed in the same manner as in the first method.

Next, an example of a method for producing ADF using ATL cells will be shown. Conditions suitable for growing ATL cells include, for example:
ATL cells are cultured in a medium containing fetal bovine serum (Fe2) to increase the number of ATL cells, and then the cells are separated and washed under the optimal conditions for ADF production, for example, completely free of proteins such as Fe2. By transferring the cells to a synthetic medium and further culturing, ADF with less contaminant proteins can be obtained.

What is the main component of the medium used to culture ATL cells? may be a commercially available medium. For example, RfMI-1640 medium,
Modified Eagle's medium (MEM), Dulbecco's modified Eagle's medium (DMEM), or Click's medium may be used. As an additive to these media i) 1+4! Hit 20-2
50 units, ideally III! Approximately 100 units of penicillin, 1i) 1++j! lμg~100μg per
, ideally 1+aj! 10 μg gentamicin per 1 ii) 20-250 #g per 1111, ideally 100 μg streptomycin, iV) about 100-1000 μg per 1 m42, ideally about 3
00 μg fresh L-glutamine, v) 1-3 g/l ideally 2 g/It NaHCO*, vi)
5 x 10-' to 5' x 10-6 M%, ideally 5 x 10-'M of 2-mercaptoethanol, etc. can be used as needed. As an optimal medium for increasing the number of ATL cells, for example, add 1 ml to the above-mentioned medium.
A medium supplemented with ~30%, preferably 10% FC3 is used.

When producing lymphokines from T cells, it is usually necessary to add proteins such as Fe2 or mitogens to the culture medium.
When producing ADF using TL cells, it is not necessary to add serum components such as Fe2 or other serum components to the culture medium, and also add the commonly used mitogens for T cells or B cells. There's no need.

The above method of producing ADF using ATL cells is performed under a variety of environmental conditions. That is, preferably the ATL cell culture is about 5%
It should be kept in humidified air containing ~10% carbon dioxide. As an incubator, I use Fasion Loveware Division.

Becton Disokinson Endo Corporation (Falcon Labware+ Div, Bec
Tissue culture flasks such as the Falcon 11H3013 or 3025 commercially available from John Dickinson and Co.) can be used. It is also possible to use a roller bottle, such as Bottle Na3027, commercially available from Fashion Labware, mentioned above, or a spinner jar flask.

The cell density at the start of culturing ATL cells to increase the number of cells is lXl0'/mj! When ATL cells are cultured under the above-mentioned conditions, it is usually 4-6X10'/mj in 4-5 days! Since the cell density increases, new medium is added again to lower the cell density to 1×1oS/IIIt, and culture is continued again. After culturing ATL cells in this manner until the desired cell number is reached, the cells are centrifuged, washed with a protein-free complete synthetic medium, and then inoculated into a new complete synthetic medium. The initial cell density at this time is preferably 1X10' to 1X10', ideally 5X10'/ml. ATL
The amount of ADF produced by culturing cells changes over time. For example, ATL cells were cultured in RPMI-1640 medium (penicillin 100 units/It, streptomycin 100 #g) at an initial cell density of 5X10S/mJ.
/mj! and NaHCO (containing 2 g/l), ADF activity reaches its maximum after 3 to 5 days. In this way, AD from ATL cells in RPMI-1640 medium.
The optimal culture time to produce F is about 3 to 5 days.

From the culture supernatant of ATL cells obtained in this way,
ADF is salting out, vacuum dialysis, and ultrafiltration.

It can be concentrated and purified from the above-mentioned culture supernatant by various methods such as gel filtration chromatography, ion exchange chromatography, affinity chromatography, chromatofocusing, reversed phase chromatography, and gel electrophoresis. For example, Sephacryl S-200 (Ph
armacia Fine Chemi-cals, Sweden) column chromatography.

Sperose HR/12 column chromatography (Su
perose ) IR/12+ Pharmacia
Fine Chemicals).

Hydroxyapatite chromatography.

Octa Dodesyle
It is possible to purify ADF by a combination of reverse phase high performance liquid chromatography (HPLC") using a 5ilane (ODS Cl8) column, etc.

In addition, when the concentrated solution of ATL cells is fractionated using a Sephacryl S-200 column, the molecular weight is 40 to 30 kd and 2O
Two ADF activity peaks corresponding to -10kd are observed, but the ADF of the present invention has a low molecular weight, that is, the molecular weight is 2O-
This corresponds to the substance found at the 10 kd position.

The physicochemical properties of the obtained ADF are as follows.

(11 molecular weight) ADF produced from ATL-2 by the method described above has the following properties.

When the S-condensed ATL-2 culture supernatant is added to a Sephacryl S-200 column previously equilibrated with PBS and eluted with PBS, ADF is eluted at a position corresponding to a molecular weight of 40-30.2O-10 kd. In addition, FPLC system (P
harn+acia Fine Chemicals
.

In the Sperose HR/12 column chromatography using
ADF was eluted at a position corresponding to 5-13 kd.

(2) Isoelectric point The culture solution containing ADF obtained from ATL-2 by the method described above was concentrated by ultrafiltration, and the crude ADF was fractionated by Sephacryl S-200 gel filtration chromatography, that is,
Samples containing high-molecular and low-molecular ADF at pH 6~
When chromatofocusing is performed using a Flumacia monop column in the range of pH 5.4, ADF has a pH of 5.5 to 4.4.
It was revealed that the protein was eluted between 5.5 and 4.5, and was an acidic protein with a pl of 5.5 to 4.5. Furthermore, Sephacryl S-
Using a sample containing low-molecular-weight ADF that has undergone several purification steps after 200 gel chromatography, chromatography is performed in the same manner as described above, and ADF has a pH of 5, 2, 4, 7, 4, 4. The low molecular weight ADF eluted at the position has a pl of 5.2. It was shown to be an acidic protein exhibiting microheterogeneity of 4.7.4.4. This is thought to reflect the difference in sugar chain structure.

(3) Amino acid sequence of N-terminal part Low molecular weight A that became a single peak in the final purification step, reversed-phase high performance liquid chromatography using an ODS column.
When the amino acid secondary structure of the N-terminal portion of DF was determined using an amino acid sequencer, it was revealed that the molecule consists of the following N-terminal structure.

Pro-Met-Phe-X-Val-Asn-Thr
-Asn-Val-Pro-Arg-Val-Ser-
Val-Pro-The ADF thus obtained is useful as a therapeutic agent for immunodeficiency, cancer, autoimmune infections, etc.
ADF alone or in combination with IL2, other lymphokines, and immune active substances can be used in a wide range of applications as a therapeutic agent. At the same time, the present protein and its constituent peptides can be used by themselves or in combination with antibodies against them to diagnose various diseases. Furthermore, by using ADF and IL2 together, it is possible to enhance the action of IL2. Mengi ADF not only enhances IL2R expression, but also exhibits many other immunoactivities and has usefulness as an immunoactive substance well known in the art.

ADF activity is measured by the following method.

YT cells, an NK-like tumor line in which expression of IL2R (Tac antigen) is regulated (J, Immunol, 134
.

1623-1630. (1985)), the activity of ADF was measured using the induction of Tac antigen expression as an indicator. That is, 3 x 10' YT cells are cultured in the presence of the sample for 24 hours under 5% CO□ aeration. 1%FC5 and 0.1%N
After washing twice with Hank's balanced solution containing aN3, the fluorescent substance FIT C (fluorecein 1) was washed twice.
Stain by reacting with anti-Tac antibody conjugated with sothiocyanate for 30 minutes at 40°C. FITC-goat anti-mouse Ig antibody is used as a negative control. The fluorescence intensity of the stained cells was determined by flow cytometry (Spectrum m-0rtho Pharma).
Ceutical Co., NJ) to determine the positive rate.

Example 1 (1) Establishment of ADF-producing ATL cells Examples of ADF-producing ATL cells include the ATL-2 cell line. The ATL-2 cell line is an adult T-cell leukemia (AT
It was established from the peripheral blood of a 61-year-old boy diagnosed with L). The patient's peripheral blood at the start of culture was determined to be leukemic cells based on atypical cell nuclei, and the surface characteristics of leukemic cells were Eroseft (+) and 0KT-3 (+).
), 4 (+), 6 (-),
8 (-), Ia (+) and AT in serum
The L^ antibody was positive. The medium used was RPMI-1640 medium (containing 10% FC3) supplemented with human peripheral blood or lung cell-derived crude IL2.

Three months after the start of culture, stable growth began to occur, and 3-
It became possible to subculture every 4 days. 10 more times after the start of culture
After about 2 months, the IL2 dependence was gradually released, and it became possible to grow and subculture using only RPMI-1640 medium containing 10% Fe2 without adding IL2. It continues. The cell traits of ATL-2 are ED Z) (+), 0KT-3 (-), 4
(+).

8 (), Tac (+), SIg (), and A
In TLA(+), many type C retrovirus particles (ATLV) are observed by electron microscopy.

ATL cell lines with various surface characteristics are established from ATL patients, but most of the ADF-producing cell lines are of the 0KT-4 (+), ie helper phenotype, like ATL-2 cells.

(2) Production of ADF using ATL-2 21 capacity plastic crawler incubator (Falcon 3027) (
11 10% FC3-containing R in (hereinafter referred to as roller)
PMI-1640 medium (2mM glutamine, 100 units/ml penicillin.

100μg/INaH, 2g/INaH
(contains CO=) to lXl0'/mj! AT at the cell density of
L-2 was inoculated and cultured at 37° C. for 5 days while rotating at 20 rpa+. After culturing, the cells were collected by centrifuging the culture, and after washing the cells once with RPMI-1640 medium, the cells were suspended in 11 volumes of RPMI-1640 medium in a 21-volume roller to a concentration of 5 × 10S/IIIt cells. did. The cells were cultured at 37° C. for 4 days while rotating the roller at 2 Orpm. After culturing, the culture was centrifuged to obtain a culture supernatant.

When assaying the ADF activity of the main culture supernatant, it was confirmed that ATL-
2 cells were shown to produce a substance that induces the expression of IL2R on YT cells.

(3) Purification of ADF ADF was purified from the ATL-2 culture supernatant as follows.

Sephacryl S-200 gel Chromatography 111 ATL-2 serum-free culture supernatant (RPMI-164
0) is Amicon's DC-2 hollow fiber system (HI PIO-8 hollow fiber).

molecular cut at 10kd Am1
con, corp, , lexington.

After concentrating to 10 ml using LA), gel filtration chromatography was performed using a Sephacryl S-200 column equilibrated with PBS for fractionation.

ADF activity has a molecular weight of 4O-30kd and 2O-10kd.
It was eluted in both fractions corresponding to . As standard proteins, bovine serum albumin (66,000), chymotrypsinogen (24,000), and cytochrome C (130,000) were used. Both fractions corresponding to a molecular weight of 10 kd to 50 kd were collected and concentrated using an Amicon DiafloY M-5 membrane filter (Amicon, molecular
arcut at 5000) Hydroxyapatite chromatography followed by concentration A
DF samples were diluted with 10 mM Na1PO4 (pH 6,8
) equilibrated with a hydroxyapatite column (10X
1.5 cm) and 20 ml of 10°40.11
0,400. Proteins were sequentially eluted with mM NazPO, pH 6 and 8, respectively. Mainly 110mM Na1P
ADF activity was eluted at O.

The ADF active fraction (110mM NazPO, eluate) obtained from the hydroxyapatite column was further equilibrated with PBS using a Spherose HR10/3012 column.
uid Chromatography system,
Pharmacia, 1lppsala, Sweden) system for separation and purification at a flow rate of 0.5 m//min. Similar to Sephacryl S-200 chromatography, ADF was eluted into two components, high molecular weight and low molecular weight, corresponding to molecular weights of 30-25 kd and 15-13 kd.

Sperose IIR1 was added to a hydroxyapatite column equilibrated with 10mM Na1PO* (pH 6,8).
The low-molecular ADF active fraction eluted from the 0/3012 column, that is, both fractions corresponding to a molecular weight of 15-13 kd, were added and separated by HPLC. Protein elution was performed using a linear concentration gradient (10mM/Tll1n) of 101 to 400Mn+ Na1POa (pH 6,8), and the elution rate was 0.
5 tal/win. The ADF activity was divided into two activity peaks, and both peaks eluted with a high concentration of NaLPOn, which accounted for more than 90% of the total activity, were pooled and transferred to the next purification step.

The ADF active fraction obtained from the reverse hydroxyapatite column containing the ODS column (C18) was further subjected to Synchropak RP-C.
I B (Sychrom, Inc, +Linden
, Indiana) column and separated by a reverse phase HPLC system. The column was prefilled with 0.1% TF.
Equilibrate with A, and elute the protein at a flow rate of 1. Os 17m1
A linear concentration gradient of 0-100% a7tonitrile with 0.1% TFA in n was performed. ADF activity is CH
3CN eluted at 58-62% and protein absorption (OD28
0) was completely consistent.

(4) Properties of ADF purified protein As mentioned above, ADF gives molecular weights of 40-30 and 20-10 kd in Sephacryl S-200 chromatography, and 30-20 kd and 15 kd in Sperose HR/12 column chromatography. Gives a molecular weight of ~13 kd.

Furthermore, low molecular weight ADF is 10-
Gives a molecular weight of 13 kd.

! 25mM bis-Tris-HCl
The ADF active fraction was passed through a monop column equilibrated with (pH 6,3). Elution of protein is at 10m at pH 6-4.0.
The column was operated using an M polybuffer 74 and an FPLC system. Sephacryl S-200 eluted ADF fraction, that is, a sample containing a mixture of high-molecular and low-molecular ADF, gave a pH of 5.5-4.5, and the hydroxyapatite column (HPLC system) eluted fraction of low-molecular ADF pH 5, 2, 4 for samples containing only
,7,4,4 was given.

In order to determine the amino acid sequence of the low molecular weight ADF protein, the purified ADF was run on a protein sequencer (Appli).
ed Biosystems Co., Ltd.). The method for determining the amino acid sequence is J, Biol, Chen+, 1
93.265-275 (1951).

It has been found that low molecular weight ADF consists of molecules having the N-terminal structure shown below.

Pro-Met-Phe-X-Val-Asn-Thr
-^sn-Val-Pro-Arg-Val-Ser-
Val-Pro-(5) Immune activity of purified ADF A single purified ADF sample was used to confirm the expression of IL2R1, ie, Tac antigen, in YT cells by HPLC. 3X10' YT cells and purified ADF samples were cultured in RPMI medium containing 10% FC3 (100 μg/ml streptomycin, 100 units/ml penicillin, 2 g/l NaHCOs).
(containing) for 24 hours.

The cells were washed twice with Hank's balanced solution containing 1% FC3 and 0.1% Na)4+, and reacted with FITC-conjugated anti-Tac antibody at 4°C for 30 minutes. After the reaction, the cells were washed and the amount of FITC-anti-Tac antibody bound to the cells was analyzed using flow cytometry (Figure 1).

To explain Figure 1, a is the control experiment and the solid line is the ADF.
YT cells to which no sample was added, and the broken line is a negative control, which is a fluorescence pattern using goat anti-mouse immunoglobulin antibody instead of FITC-anti-Tac antibody. b is a fluorescence pattern of YT cells to which purified ADF was added.

The horizontal axis shows the fluorescence intensity, and the vertical axis shows the number of cells for each fluorescence intensity. That is, the more the curve shifts to the left on the horizontal axis, the greater the amount of FITC on the cell surface of the sample.

As shown in Figure 1, clearly purified ADF is YT
It induces cellular expression of Tac antigen.

Note that under these conditions, not only the expression of Tac antigen but also its purification and enhanced binding of combination IL2 were confirmed.

Furthermore, it was confirmed that when ADF and rL2 coexisted, Tac antigen expression and IL2 binding were further enhanced than when ADP was used alone, demonstrating the usefulness of the combination of ADF and IL2.

[Brief explanation of the drawing]

FIG. 1 shows the results of examining the Tac antigen inducing activity of the interleukin 2 receptor expression inducer of the present invention.

Claims (1)

[Scope of Claims] Interleukin-2 receptor expression inducer (1) having the following properties: Molecular weight: 20 to 10 kd (cephacryl-52
00 gel filtration method) (2) Isoelectric point: pI 5.5 to 4.5 (3) Amino acid sequence (from N-terminus): Pro-Met-Phe-X-Val-Asn-Thr
-Asn-Val-Pro-Arg-Val-Ser-
Val-Pro-