JPS6216430A - Interleukin receptor expression inducing substance a - Google Patents

Abstract

NEW MATERIAL:An interleukin receptor expression inducing substance ADF [Molecular weight; 2-10kd (Sephacryl-S200 gel filtration method); Isoelectric point; pI5.5-4.5. Amino acid sequence; Sequence expressed by the formula (from the N-terminal)]. USE:A remedy for immunological insufficiency, cancer and autoimmune infectious diseases, etc. PREPARATION:A cultivation supernatant liquid of ATL cells (T leukemic cell strain established from human adult type T leukemic patients) is concentrated and purified by salting out, vacuum dialysis, ultrafiltration, gel filtration and ion exchange chromatography, etc., to give an ADF protein specimen. The resultant specimen is applicable to a wide range as a remedy alone or by use together with interleukin 2 (IL2), another lymphokine or immunologically active substance, and the specimen or peptides constituting the specimen are usable for diagnosis of various diseases individually or by combination with their antibody.

Description

[Detailed Description of the Invention] [Industrial Application Field] This invention relates to interleukin 2 (hereinafter referred to as rlL2) receptor (interleukin 2rec).
The present invention relates to a substance (hereinafter referred to as rADFJ) that induces the expression of ep-tor+ (hereinafter referred to as rIL2RJ).

[Conventional technology]

ADF is derived from Tac antigen (N
ture, 300, 267-269 (1982),
Pro, N, A, S, 80°6957-6951 (19
83), Nature, 31). 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.

Ce1). Immunol, 2.17-26. (19
85)) Therefore, by inducing the expression of IL2R, ADF causes IL2 to express 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-1) 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
1.7907-791), (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 of ADF activity were obtained (J, Mo1.Ce1). Immunol, 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 the induction of 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 problems of soft soil, the present inventors conducted various researches and finally succeeded in isolating ADF as a single substance having the following properties. Namely (1) Molecular weight: 20 to 10 kd (Sephacryl S-200 gel filtration method) (2) Isoelectric point: pIs, s to 4.5 (3) Amino acid sequence (from N-terminus): Val-Lys
-Glu-1)e-Glu-Ser-Lys-Thr-
Ala-Phe-Gln-Glu-Ala-Leu-A
There are, for example, the following two methods for obtaining sp-Ala-AlaATL cells.

The first method is to establish it from peripheral blood of ATL patients. Specifically, peripheral blood was collected from a patient diagnosed with ATL, white blood cells were obtained using the Ficoll method, and 10% l fetal serum (10% l) supplemented with crude IL-2 prepared from human peripheral blood or human tile cells was added. Fe2)
Culture is started in RP old-1640 medium containing. After stable growth was shown, crude IL2 was gradually removed from the medium and culture was started. After several injections, RPMI containing 10% FC3 was added.
Obtain a cell line that can be grown and passaged only in -1640 medium.

To confirm that the obtained cell line is an ATL cell, E rosette OKT series, 5-1g, ATLA,
Search for surface traits such as Tac. All of these antigen markers are well known and widely used in clinical practice.

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

The OKT series includes 0KT-3, 4, 6, 8, etc., but T-3 is a T cell antigen receptor-related antigen;
For the identification of human peripheral blood T lymphocytes, T-4 is also used as T helper.
For the identification of lymphocyte subsets, T-6 is a β-2 microglobulin-related antigen, which is used to identify common thymus-derived T lymphocytes, and T-8 is used to identify suppressor/killer T lymphocyte subsets. s4g 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. Also, Tac is I
It is said to be L2R. 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
tro). (Science, 217,737
(1982)).

That is, by irradiating with γ-rays (12,000 rad), ATLV-producing ATLIII cells, such as MT
-2 cells and white blood cells obtained by Ficoll treatment of peripheral blood from healthy individuals were combined in RP old-1640 medium - after injury.
A cell line that can be passaged is obtained, but if proliferation is unstable, add IL2 derived from human peripheral blood or the like.

To confirm that the obtained cell line is an ATL cell, E rosette, OKT series + s4g, 8TLA,
Search for surface traits such as Tac 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 (FCs:+) to increase the number of ATL cells, and then the cells are separated and washed under the optimal conditions for ADF production, such as containing proteins such as Fe2. A-DF with less contaminant proteins can be obtained by transferring the cells to a complete synthetic medium and culturing them further.

What is the main component of the medium used to culture ATL cells? may be a commercially available medium. For example, R [MI-1640 medium,
Modified Eagle's medium (MEM), Dulbecco's modified Eagle's medium (DMEM), or Click's medium may be used. As additives to these media i) 20-25 per mJ;
0 units of penicillin, ideally about 100 units per ml, 1i) lnj! gentamicin at 1 μg to 100 μg per square meter, ideally 10 μg per m42, iii) 1
20-250 μg per mj2, ideally 100 μg
of streptomycin, 1v) approx. 100 per In+42
~1000μg, ideally about 300μg/m42
of fresh L-glutamine, V) 1-3 g/l ideally 2
g/l NaHCO3, vi) 5 x 10-' ~
5 x 10-'M, ideally 2- of 5 x 10-'M
Mercaptoethanol and the like can be used as needed. As the optimal medium for increasing the number of ATL cells, for example, a medium obtained by adding 1 to 30%, preferably 10% FC3 to the above-mentioned medium 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 Dickinson End Corporation (Falcon Labware, Div, Bect
Tissue culture flasks such as the Fasion Nf13Q13 or 3025 commercially available from Dickinson and Co., Ltd., can be used. Also, bottle N1 commercially available from Fasion Loveware mentioned above.
) 3027 or spinner jar flasks can also be used. A.T.
In order to increase the cell number by culturing L cells, the cell density at the start of culture is preferably I x 10'/ml. When ATL cells are cultured under the above conditions, the cell density usually increases to 4 to 6 x 10'/ml in 4 to 5 days, so add new medium again, lower the cell density to lXl0'/m#, and culture again. Continue. 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 density of the cells at this time is preferably 1×10 4 to 1×10′, ideally 5×10′/mβ. A.T.
The amount of ADF produced by culturing L cells changes over time. For example, ATL cells were cultured in RPMI-1640 medium (7 ml of 100 units of penicillin, 1 ml of streptomycin) at an initial cell density of 5 x 10'/nu.
r/mz and NaHCOz containing 2 g/l), ADF activity reaches its maximum after 3 to 5 days. As described above, the optimal culture time for producing ADF from ATL cells in RPMI-1640 medium is about 3 to 5 days.

From the culture supernatant of ATL cells obtained in this way,
ADF can be prepared as described above by various methods such as salting out, vacuum dialysis, ultrafiltration, two-gel filtration chromatography, ion exchange chromatography, affinity chromatography, chromatofocusing, reversed phase chromatography, and gel electrophoresis. It can be concentrated and purified from culture and culture supernatants. For example, Sephacryl S-200 (Pharmacyl
a Pine Chen+i-cals, Sweden) column chromatography.

Sperose HR/12 column chromatography (Su
perose HR/12+ Pharmacia F
ine 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 Sephacryl S-200 column, the molecular weight is 40-3°Okd 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 10kd position.

The physicochemical properties of the obtained ADF are as follows.

(1) Molecular Weight ADF produced from ATL-2 by the method described above has the following properties.

When the concentrated 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 the molecule 1t40-30.2O-10kd. In addition, FPLC system (
Pharmacia Fine Chemicals
ADF was eluted at positions corresponding to molecular weights of 30-25 kd and 15-13 kd in Speros HR/12 column chromatography using a 30-25 kd column (Sweden).

(2) Isoelectric point - Crude ADF obtained by concentrating the ADF-containing culture solution obtained from ATL-2 by the method described above by ultrafiltration and fractionating it by Sephacryl S-200 gel filtration chromatography, that is,
Samples containing high-molecular and low-molecular ADF at pH 6~
When performing chromatofocusing using a Flumacia monop column in the range of 4, the ADF is pos, s ~ 4.
．． It was revealed that the protein was eluted between 5 and 5 and was an acidic protein with a pI 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.
is eluted at pH 5,2, 4,7, 4,4, and low molecular weight ADF has a pI of 5.2.4.7. It was shown to be an acidic protein with a microheterogeneity of 4.4. This is II! This is thought to reflect the difference in 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.

Val-Lys-Glu-1)e-Glu-Ser-L
ys-Thr-Ala-Phe-Gln-Glu-Al
a-Leu-Asp-Ala-Ala The ADF thus obtained is useful as a therapeutic agent for immunodeficiency, cancer, autoimmune infections, etc., and can be used alone or in combination with IL2, other lymphokines, or immune active substances. , it has 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. Also AD
By using F and IL2 together, it is also 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.

N in which the expression of IL2R (Tac antigen) is regulated
YT cells, a K-like tumor line (J, lnmunol,
134 +1623-1630. (1985)),
ADF activity was measured using Tac antigen expression induction as an index. That is, 3 x 10' YT cells are cultured in the presence of the sample for 24 hours under 5% CO2 aeration. After washing twice with Hank's balanced solution containing 1% FC3 and 0.1% NaN3, the fluorescent substance FIT C (fluorec
The cells are reacted with anti-Tac antibody conjugated with ein 1 sothiocyanate for 30 minutes at 40°C and stained. FITC-goat anti-mouse Ig as negative control
Use antibodies. The fluorescence intensity of the stained cells was measured by flow cytometry (Spectrum m-0rtho P
Pharmaceutical 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 hypocellular 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 E rosette (+) and 0KT-3 (+).
, 4 (+), 6 (-), 8 (
-), Ia (+), and the ATL^ antibody in the serum was positive. The medium is RPMI-1640 medium (10%
FC3-containing) and human peripheral blood or lung cell-derived crude I
The one to which L2 was added was used.

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 subculture was possible without adding IL2 using only RPMI-1640 medium containing 10% Fe2, and subculture has been continued for 3 years and 8 months up to the present. ing. ATL-2 cell traits are E rosette (+), 0KT-3 (-), 4
(+).

8 (), Tac (+), SIg (-)
In ATLA(+), 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.

(1) 10% FC3-containing RPM in a 21 volume plastic crawler incubator (Fashion 3027) (hereinafter referred to as roller)
I-1640 medium (2mM glutamine, 100 units)
m1l penicillin.

1004g/mf streptomycin, 2g/ItNa
) containing 1 cO3) to a cell density of lXIO3/mJ
TL-2 was inoculated and cultured at 37° C. for 5 days while rotating at 2 Orpm. After culturing, the cells were collected by centrifuging the culture, and after washing the cells once with RPMI-1640 medium, the cells were placed in a 21 volume roller IN (7) RPMI-164.
The cells were suspended in 0 medium to a concentration of 5 x IQ'/me cells. 4 at 37℃ while rotating the roller at 2Orpm.
Cultured for 1 day. 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 filtration chromatography 1) ATL-2 serum-free culture supernatant of 1 (RPMI-164
0) is Amicon's DC-2 hollow fiber system (HI PIO-8 hollow fiber).

molecular cut at' 10kd Am
1con Corp., Lexington.

After concentrating to 10 mf 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 molecular weights of 10 kd to 50 kd were collected and concentrated using an Amicon DiafloY M-5 membrane filter. (Amicon, molecular
rcut at 5000) Hydroxyapatite chromatography followed by concentration A
DF sample 101) IMNaiP0101) I, 8
) equilibrated with a hydroxyapatite column (10X
1.5cm) and 20mj+ 10°40.1)
0,400. Proteins were sequentially eluted with mM Na4PO4 (pH 6, 8). Mainly 1) 0mM Na
ADF activity was eluted at a P Oa.

The ADF active fraction (1) 0mM Na1PO4 eluate obtained from the hydroxyapatite column was further analyzed using F PLC (Fast Protein Liq) using a Sperose HR10/3012 column equilibrated with PBS.
uid Chromatography system,
Pharmacia, Uppsala, Sweden) system for separation and purification at a flow rate of 0.5 ml/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 HR1 was applied to a hydroxyapatite column equilibrated with 10 mM Na, POa (pH 6, 8).
A low molecular weight ADF active fraction eluted from the 0/3012 column, ie a fraction corresponding to a molecular weight of 15-13 kd, was added and separated by HPLC. Protein elution was performed using a linear concentration gradient (10mM/ml) of Na1POa (pH 6,8) from 10mM to 400Mm, and the elution rate was 0.5
ml/min. ADF activity is divided into two activity peaks, and high concentration of N accounts for more than 90% of the total activity.
a1PO.

Both fractions eluted were pooled and transferred to the next purification step.

The ADF active fraction obtained from the reversed-phase frozen hydroxyapatite column using an ODS column (C18) was further subjected to Synchroρak 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 1.0 ml/m.
A linear concentration gradient of 0-100% acetonitrile containing 0.1% TFA in in. ADF activity is C
H3CN eluted at 58-62% and protein absorption (OD2
80) was in complete agreement.

(4) Properties of ADF purified protein (1) As mentioned above, ADF gives molecular weights of 40 to 30 and 20 to 10 kd in Sephacryl S-200 chromatography, and 30 to 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.

Isoelectric point 25mM bis-Tris-HCl
The ADF active fraction was passed through a monorecolumn equilibrated with (pH 6,3). Elution of protein is at 10m at pH 6-4.0.
The column was operated using M polybuffer 74 and an FPLC system was used. The Sephacryl S-200 eluted ADF fraction, i.e., a sample containing a mixture of high-molecular and low-molecular ADF, gave a pxs of 5-4.5, and the low-molecular-weight fraction eluted from the hydroxyapatite column (HPLC system) gave a pxs of 5-4.5. In the sample containing only sexual ADF, p15.
2. 4.7. 4.4 was given.

In order to determine the amino acid sequence of the low-molecular-weight ADF protein, purified ADF was processed using a protein sequencer (8 ppli).
ed Biosystem Co.). The method for determining the amino acid sequence is described in J. Biol. Chem, 193.
．． 265-275 (1951).

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

Val-Lys-Glu-1)e-'Glu-3er-
Lys-Thr-Ala-Phe-Gln-Glu-A
la-Leu-Asp-Ala-Ala (5) Immunoreactivity of Purified ADF Expression of IL2R1, ie, Tac antigen, in YT cells was confirmed using a single purified ADF sample by HPLC. 3X10' YT cells and purified ADF samples were grown in RPMI medium containing 10% FC3 (100 #g/nl 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% NaN3, and reacted with FITC-conjugated anti-Tac antibody at 4°C for 30 minutes. After repulsion, 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
The expression of Tac antigen in i cells is induced.

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 IL2 was present in addition to ADF, Tac antigen expression and IL2 binding were further enhanced than when ADF was used alone, demonstrating the usefulness of the combination of ADF and IL2.

[Brief explanation of drawings]

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-S2
00 gel filtration method) (2) Isoelectric point: pI 5.5 to 4.5 (3) Amino acid sequence (from N-terminus): Val-Lys-Glu-Ile-Glu-Ser-L
ys-Thr-Ala-Phe-Gln-Glu-Al
a-Leu-Asp-Ala-Ala