JPH0657720B2 - B cell differentiation factor - Google Patents

Description

TECHNICAL FIELD The present invention relates to a B cell differentiation factor (hereinafter referred to as “BCDF”) protein preparation having human activity.

[Conventional technology]

Antigen-stimulated and activated mature B cells divide and proliferate with the help of T cells, and in order for B cells to finally differentiate into antibody-producing cells, one or more T cells may be generated.
It is known that a cell-derived differentiation-inducing substance is essential. The existence of this substance is RW Dutton et al., Transplant.R.
ev. 23 66 (1975), A.Schimpl and E.Wecker et al, Na
ture N. Biol. 237 , 15 (1972). They found that the substance present in the culture supernatant of a mouse lymphocyte mixture culture or in the lymphocyte catalytic supernatant of a mouse stimulated by an antigen or mitogen
It was found that the primary immune response to sheep red blood cells (SRBC) of lymphocyte cell populations from which cells have been removed or lymphocytes derived from nude mice is amplified, and an active substance having such an action has a T lymphocyte replacement factor, namely, The name TRF was given. Since then, TRF acts on B cells in a non-antigen-specific manner that does not require concordance of major histocompatibility complex (MHC).
It is defined as a humoral factor that does not induce mitotic proliferation of cells but induces differentiation of B cells into antibody-producing cells.

Thereafter, evidence showing the existence of such a B cell differentiation factor has been accumulated, and the existence of a differentiation factor similar to that of the mouse has been suggested in humans. At present, the factors for differentiating B cells defined as described above into antibody-producing cells have been generically called BCDF.

As described above, BCDF plays an important role in the antibody producing function of B cells in the human body. There are three major clinical applications of BCDF. First is BCDF by BCDF
Make antibody and BCDF by BCDF and anti-BCDF antibody
Can be used for analysis of immunological pathological conditions. The second application is to treat various diseases. For example, in patients with immunodeficiency due to a decrease in B cell antibody production due to a decrease in T cell helper function, BCD
It is considered that administration of F alone or with other lymphokines restores the antibody production function to normal.

Furthermore, the following are possible applications of BCDF. B
Cell growth factor (BCGF) (K. Yoshizaki et al., J. of Immu
nol. 130, 1241 (1983) ), including the other lymphokines T
It has been reported that normal B cells can be cultured for a long period of time by adding a cell factor to the medium (B, Sredni et al., J. Exp. Me.
d., 154 , 1500 (1981)). Antibodies can be produced in vitro by allowing BCDF to act on these cultured normal B cells or B cells transformed with EB virus at an appropriate time. B cells producing specific antibodies, for example, antibodies that recognize specific antigens on the surface of pathogenic bacteria, pathogenic viruses, pathogenic protozoa, cancer cells, etc. are monocloned and transformed with cloned normal B cells or EB virus. Cells can be cultured in combination with BCDF and other lymphokines to produce useful monoclonal antibodies. These antibodies can be used for treatment and diagnosis of infectious diseases and eyes.

Conventionally, in order to obtain BCDF, a method of producing BCDF by stimulating normal human T cells separated from human peripheral blood or the like with mitogen has been adopted. In this method, it is difficult to obtain T cells sufficiently, and since mitogen is used, harmful mitogen is mixed into BCDF and it is difficult to remove it, and fetal bovine serum is used for T cell culture. It is necessary to add serum components to the medium, and these added proteins and BCDF cannot be separated sufficiently,
There are many problems in using BCDF for medical treatment, such as the fact that purified BCDF cannot be obtained, and it is industrially
It was not possible to produce CDF. In addition, a method has been reported in which human T cells are fused with human cancer cells to obtain human T fused cells and thereby BCDF is produced (Okada).
Et al., J. Exp. Mod., 157 , 583 (1983)). However, human fused cells often lose their lymphokine-producing ability during passage, and there is no practical BCDF-producing human fused cell yet.

[Problems to be solved by the invention]

Therefore, an object of the present invention is to provide a protein preparation containing BCDF.

[Means for solving problems]

The present inventors have found that human T-cell leukemia virus (hereinafter referred to as “HTL
We have found that human T cells transformed with V.) produce BCDF with high efficiency. That is, the present invention is a B cell differentiation factor having the following properties, which is produced from human T cells transformed with HTLV and obtained by purification by gel filtration chromatography, chromatofocusing and reverse phase chromatography.

(1) Molecular weight 3.5 ± 0.5 × 10 ⁴ Dalton (gel filtration method) 2.2 ± 0.2 × 10 ⁴ Dalton (SDS / polyacrylamide gel electrophoresis method) (2) N-terminal amino acid sequence Pro Val Pro Pro Glu Asp Ser The Lys Asp Val Ala Ala human BCDF producing human T cell line can be prepared as follows. Lymphocytes were isolated from human peripheral blood, tonsils, umbilical cord blood, etc. by density gradient centrifugation using a film call pack, etc., and N. Yamamoto, science 217 , 737 (19).
According to the method of 82), human T cells are transformed (transformed) using HTLV. For example, the following method can be used. Virus-producing cell line MT
-2 and cell 1 × ¹⁰ 7 / m inactivated by X-ray irradiation (12,000 to 14,000 rads) and human lymphocytes 1 × ¹⁰ 7 / m with 20% FCS obtained as described above, 100 microns
g / m kanamycin, 2 μg / m NaHCO ₃ , 25 mM
N-2-hydroxyethyl-piperazine-N'-2-ethansu
Plastic petri dish (Falcon # 300 containing RPMI1640 medium containing lfonic acid (HEPES))
Contact with 8) and culture at 37 ° C. in the presence of 5% CO ₂ .
2 times a week, replacing half of the medium with fresh medium
After culturing for 3 months, the strain is established by the limiting dilution method. The BCDF activity of the culture supernatant of the established cells is measured to obtain a strain having BCDF activity.

As the cells established by this method, for example, a human T cell line labeled with VT-1 can be used. VT-1
There is no special condition for growing the strain, and generally used culture conditions may be appropriately adopted. Also, B
Although CDF may be produced by a general method, it should preferably be performed using a protein-free medium.

The following is one of the methods for producing BCDF using VT-1.
Here is an example:

VT-1 is cultured in a medium suitable for growing VT-1, for example, a medium containing fetal calf serum (FCS), and V
After increasing the number of T-1 cells, the cells were separated and washed and BC
BCDF containing less contaminating proteins can be obtained by transferring the cells to the optimal conditions for DF production, for example, a completely synthetic medium containing no protein such as FCS and further culturing.

The main component of the medium used for culturing VT-1 may be a commercially available medium. For example, RPMI1640 medium, modified Eagle medium (MEM), Dulbecco modified eagle medium (DME
M) or click medium. As an additive to these media i) about 20-250 units per m, ideally about 100 units penicillin per m, ii) 1 m
1 μg to 100 μg, ideally 10 per 1 m
μg geltamycin, iii) 20-250 per m
μg, ideally 100 μg streptomycin, i
v) About 100 to 1000 μg per 1m, ideally 1
About 300 μg of fresh L-glutamine per m, v) 10
~ 60 mM, ideally 25 mM Hepes buffer, vi)
8-20 mM, ideally 16 mM NaHCO ₃ , vii) 5
× 10 ^{−4 to} 5 × 10 ⁻⁶ M, ideally 5 × 10 ⁻⁵
M 2-mercaptoethanol or the like can be used as necessary.

The optimal medium for increasing the number of VT-1 cells is, for example, 1% to 30%, preferably 20% in the above-mentioned medium.
The medium to which FCS is added is used. The optimal medium for BCDF production may be the above-mentioned medium without the addition of FCS. The viability of VT-1 is maintained at 70% or more even after culturing for 48 hours in a completely synthetic medium without the addition of FCS.

When producing BCDF from T cells, FC was conventionally used in the medium.
It was essential to add proteins such as S and mitogens (T. Teranishi et al., J. of
Immunol. 128 , 1903 (1982), A. Muraguchi
J. of Immunol. 127 , 412 (1981)).
On the other hand, when VT-1 is used to produce BCDF, it is not necessary to add serum such as FCS, protein components in blood, and other protein components to the medium, and the T cells or B It is worth noting that there is no need to add mitogens to the cells either. Therefore, not only can BCDF be produced inexpensively without using expensive FCS, but also a safe BCDF that does not contain a heterologous protein or mitogen harmful to the human body can be easily obtained.

The above method of producing BCDF using VT-1 is performed under various environmental conditions. However, preferably VT-1
The culture is about 5-10 in the temperature range of about 35-38 ° C.
It should be kept in humidity-controlled air containing% carbon dioxide.
Also ideally, the pH of the medium should be maintained under slightly alkaline conditions of about 7.0-7.4. VT-1 is inoculated in various volumes such as 100 μ units on various types of incubators such as flat bottom microplates. Falcon Labware, Div.Becton, Dic, Becton Dickinson End Corporation
Flask No. 301 commercially available from Kinson and Co.)
Tissue culture flasks such as 3 or 3025 can also be used. Alternatively, a rotating bottle such as bottle No. 3027 commercially available from Falcon Labware can also be used as the culture container.

As an optimum condition for culturing VT-1 to increase the number of cells, the initial density of cells is 1 × 10 ⁴ to 5 × 10 ⁵ cells, preferably 2 × 10 ⁵ cells, per 1 m of the medium. When VT-1 is cultured under the above-mentioned conditions, the cell density usually increases from 5 × 10 ⁵ cells to 2 × 10 ⁶ cells per 1 m of the medium in 2 to 7 days. The cell density is reduced to × 10 ^{4 to} 5 × 10 ⁵ cells, and compensation is continued again. In this way, after culturing VT-1 until the target number of cells is reached, the cells are separated by centrifugation, washed with a protein-free complete synthetic medium, and then inoculated into a new complete synthetic medium. To do. The initial density of cells at this time is about 1 × per 1 m of medium.
It is preferably 10 ⁴ to 1 × 10 ⁷ cells, and ideally 1 × 10 ⁶ cells per 1 m of the medium.

The amount of BCDF produced by culturing VT-1 changes with time. For example, VT-1 at an initial cell density of 1 × 10 ⁶ cells per 1 m was added to RPMI1640 medium (100 units of penicillin, 100 μm of matretomycin per 1 m).
g, containing 10 μg of gentamicin and 16 μM of NaHCO ₃ ), BCDF activity reaches a peak level after 48 hours. Furthermore, BC existing in the next 24 hours
DF activity is slightly reduced. In this way RPMI164
The optimal culture time for producing BCDF with VT-1 in 0 medium is about 24-78 hours.

Purification of BCDF BCDF can be purified by various methods such as salting out, vacuum dialysis, ultrafiltration, gel filtration chromatography, ion exchange chromatography, affinity chromatography, chromatofocusing, reverse phase chromatography, focus electrophoresis and gel electrophoresis. It can be concentrated and purified from the above-mentioned culture supernatant (see Example 1).

Physicochemical Properties of BCDF BCDF produced from VT-1 by the above method has the following properties.

(1) Molecular weight From AcA 34 equilibrated with PBC in advance (LKB, B
(Romma, Swed.), the concentrated VT-1 culture supernatant containing BCDF was run and eluted with PBS to obtain a molecular weight of 3.5 ± 0.5 ×
BCDF is eluted at a position corresponding to 10 ⁴ daltons.
The BCDF purified by the above method was subjected to HPLC by the above method.
When gel filtration is performed using a TSK-2000SEG (Toyo Soda), BCDF is eluted at a position corresponding to a molecular weight of 3.5 ± 0.5 × 10 ⁴ daltons. Also SDS-PAGE
When electrophoresed by (SDS / polyacrylamide gel electrophoresis), BC is located at a position corresponding to 2.2 ± 0.2 × 10 ⁴ daltons.
DF is eluted.

(2) Isoelectric point The culture medium containing BCDF obtained from VT-1 by the above-mentioned method was concentrated by ultrafiltration, and BCDF separated and purified by an AcA 34 column was added to Pharmashima Mo in the range of pH 7 to 4.
When chromatofocusing is performed using a no P column, BCDF is eluted at the position of pH 4.9 to 5.1. From this, the isoelectric point of BCDF is estimated to be pH 4.9-5.1.

(3) N-terminal amino acid sequence Pro Val Pro Pro Gly Glu Asp Ser Lys Asp Val Ala Ala BCDF activity assay method Human B cell line CES that produces IgG in response to human BCDF
S (K. Yoshizaki et al., J. of Immunology, 132 , 294).
BC (8 (1984)) for measuring BCDF activity
Test solution for measuring DF activity and 6 × 10 ³ CESS
RPMI 1640 medium containing 00 μ of 10% FCS (100 units of punicillin per 1 m, streptomycin 100 μg, gentamicin 10 μg and NaHCO ₃ 1).
6 mM). This mixture is cultured in a 96-well microplate for 3 days in the presence of 5% CO ₂ at 37 ° C., and the amount of 1 gG of the culture supernatant is measured by enzyme immunoassay. Under this condition, the activity of BCDF showing 50% of the maximum 1 gG production (the highest CESS reaction) was 1 U.
/ M.

[Action]

On the other hand, the transformed human T cell line obtained by infecting T cells with HTLV according to the present invention is the above-mentioned BC.
Compared to the DF production method, a large amount of BCDF is produced in the medium, and this cell line can be subcultured and BCDF can be subcultured during passage.
In that the production capacity does not decrease, and this cell line
BCDF in protein-free synthetic medium without any stimulant such as mitogen
The present invention enables the industrial production of human BCDF for the first time by the present invention. It is characterized in that it can obtain BCDF containing a small amount of contaminating proteins and can obtain purified BCDF with a relatively easy purification method.

Example 1 (1) Production of BCDF by VT-1 2 volume plastic roller incubator (Falcon # 30
27) 1 20% FC in (hereinafter referred to as roller)
RPMI 1640 medium containing S (2 mM glutamine, 5 ×
10 ⁻⁵ M 2ME, 100 units / m penicillin, 1
2 × 10 ^{5 in} 00 μg / m streptomycin, 20 μg / m gentamicin, 16 mM NaHCO ₂ )
/ M cells were inoculated with VT-1 and cultured at 37 ° C. for 3 days while rotating at 8 rpm. After culturing, the culture was centrifuged to collect the cells, and the cells were washed twice with RPMI1640 medium, and then the cells were mixed with 1 RPMI164 in a 2-volume roller.
The cells were suspended in 0 medium at a cell concentration of 1 × 10 ⁶ / m. Incubate at 37 ° C. for 2 days with the roller rotating at 8 rpm.
After the culture, the culture was centrifuged to obtain a culture supernatant.

As described above, BCDF was purified from the culture supernatant containing BCDF obtained by culturing VT-1 by the following method. The cell-free supernatant 10 is equipped with an ultrafiltration membrane (Amicon YM-10, Amicon Corporation, Massachusetts, USA) and equipped with an ultrafiltration device (Amicon mass treatment cell 200).
Type 0, Amicon Corporation, Massachusetts, USA) and filtration with nitrogen gas at a pressure of 4 kg / cm ² . The concentration of 100 m remaining on the upper part of the filtration membrane was further applied with an ultrafiltration device (Amicon, Standard Cell 52 type) equipped with an ultrafiltration membrane (Amicon, Standard Cell 52 type) to apply a pressure of 4 kg / cm ² with nitrogen gas. Filtered. The 5 m concentrated liquid remaining on the upper part of the filtration membrane was collected.

The concentrated supernatant described above was transferred to an AcA-34 gel filtration column (LK
B Produker.Sweden, 2.6 × 90 cm). In addition,
The gel filtration column was previously equilibrated with PBC (phosphate buffer saline, 0.01 M phosphate buffer containing 0.15 M sodium chloride, pH 7.0). The concentrated supernatant was eluted with PBC, the eluate was fractionated in 5 m increments, and the BCDF activity of the fractionated fraction was measured. The fraction having BCDF activity was found to contain BCDF in the fraction corresponding to the molecular weight (3.5 ± 0.5 × 10 ⁴ Daltons. The gel filtration column was assayed with the following molecular weight markers: Blue Dextran 2000 (Pharmacia Fine Chemicals, Sweden) 2 × 10 ⁶ , ferritin 4.5 × 10
⁵ , aldolase 1.58 × 10 ⁵ , ovalbumin 4.5 ×
10 ⁴ , chymotrypsinogen 2.5 × 10 ⁴ , cytochrome C1.17 × 10 ⁴ Fractions containing BCDF were collected, and 25 mM piperazine-hydrochloric acid was prepared using an ultrafiltration device equipped with an ultrafiltration membrane (Amicon YM-10). It was replaced with a buffer solution (pH 6.3).

Chromatofocusing B fractionated by AcA-34 column chromatography
The CDF fraction was passed through a MonoP column (Pharmacia Fine Chemicals, Sweden) which had been equilibrated with 25 mM piperazine-hydrochloric acid buffer (pH 6.3) in advance. The column was washed with 25 mM piperazine-hydrochloric acid buffer solution and then eluted with 40 m of 1/10 diluted polybuffer-74 (Pharmacia Fine Chemicals, Sweden) adjusted to pH 4.5 with hydrochloric acid. The column operation was performed using Fast Protein Liquid Chromatography and FPLC (Pharmacia Fine Chemicals, Sweden) at a flow rate of 0.5 m / min. The eluate was collected by 1 m and the BCDF activity and pH were measured. BCDF activity was eluted at pH 4.9-5.1.

The BCDF active fraction obtained from the Mono P column was added to 0.1% T
Reversed-phase chromatography column P ro RPCHR 5/10 buffered with FA (trifluoroacetic acid aqueous solution)
(Pharmacia Fine Chemicals), eluate, 0.1% TFA in acetonitrile concentration 0 to 6
The BCDF was eluted with a linear increase to 0%. Elute with 50-55% acetonitrile. O. D. The peak at ₂₈₀ is different from that of other O.I. D. It was completely separated from the ₂₈₀ peak, and BCDF activity was detected corresponding to this peak. This peak was freeze-dried to obtain purified BCDF.

B purified from culture supernatant 1.8 of FCS-free VT-1
As shown in Table 1, the recovery rate of activity of CF was 18%, and the activity per protein amount increased about 1,000 times.

(2) Properties of BCDF purified protein (i) Molecular weight AcA 34 column (LKB previously equilibrated with PBC)
Concentrated VT-1 containing BCDF in Bromma, Swed.)
When the culture supernatant was poured and eluted with PBC, the molecular weight was 3.5
BCDF was eluted at a position corresponding to ± 0.5 × 10 ⁴ daltons. When the BCDF purified by the above method was subjected to gel filtration using the TSK-2000SWG for HPLC (Toyo Soda) by the above method, BCDF was eluted at a position corresponding to a molecular weight of 3.5 ± 0.5 × 10 ⁴ daltons. Also SDS
When it was electrophoresed by PAGE (SDS / polyacrylamide gel electrophoresis), BCDF was eluted at the position corresponding to a molecular weight of 2.2 ± 0.2 × 10 ⁴ daltons.

(Ii) Isoelectric point The culture medium containing BCDF obtained from VT-1 by the method described above was concentrated by ultrafiltration, and BCDF separated and purified by an AcA34 column was used to obtain Pharmacia Mono in a pH range of 7 to 4.
When chromatofocusing was performed using the P column, BCDF was eluted at the position of pH 4.9 to 5.1. From this, the isoelectric point of BCDF is estimated to be pH 4.9-5.1.

(Iii) Amino acid sequence To determine the amino acid sequence of the BCDF protein, 6 μg of purified BCDF was added to a protein sequencer (Applied Bio
system Co., Calf, Model 470A). The amino acid sequence was determined by the method described in J. Biol. Chem., 193 , 265-275 (1951).

The amino acid sequence from the N-terminus was as follows.

(3) Immunological properties of BCDF (i) B cells were prepared from human peripheral blood, and blasted B cells were isolated by this (K. Yoshizaki et al., J. of Immunolo
gy 132 , 2948 (1984)). That is, human B cells were centrifuged (4 ° C, 400G, 15 minutes) in a Percoll gradient (50% to 70%) to separate the cell layer existing in Percoll PBS solution 50% to 55%, Plastified low density B cells were collected.

Low specific gravity B cells were added to 2 × 10 ⁵ cells / m in 200 μ RPMI in a 96-well plastic microplate.
Suspended in 1640 medium. 1 unit / m BC of medium
VT-1 culture supernatant containing DF, 10% FCS, 0.0025%
SAC, 100 units of penicillin per 1m, 1m
100 μg / streptomycin, 10 / m
μg Gentai Machine, 16 mM NaHCO ₃ was included.

Culture in a humidity controlled environment containing 5% carbon dioxide in the air 37
The temperature was kept at 0 ° C, and after 5 days, B cells were collected. The antibody-producing ability of the cells was assayed as described in the BCDF activity assay section.
As shown in Table 2, plaques due to antibody-producing B cells corresponding to the added amount of BCDF were detected.

(Ii) B cells were prepared from human peripheral blood, and a medium containing SAC 0.0025% (10% FCS, 0.0025% SAC, 100 units penicillin per m, 100 μg streptomycin per m, 10 μg gentamicin per m, 16 mM) RPMI 1 containing NaHCO ₃
After culturing in 640 medium) for 3 days, blasted B cells were separated by the same Percoll gradient treatment as in (1).

The antibody production ability of B cells was assayed by the same method as in (i) below. The results are shown in Table 3.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location C12P 21/08 8214-4B (C12P 21/02 C12R 1:91) Patent Act Article 30 Paragraph 1 Application for application 43rd General Report of the Japanese Cancer Society (August 25, 1984) Published on pages 24 and 104 of the Japanese Cancer Society Application for application of Article 30, Paragraph 1 of the Patent Act October 4, 1984 Presentation at the 43rd Annual Meeting of the Japanese Cancer Society held in Fukuoka City on the 5th

Claims (1)

[Claims] 1. A B cell differentiation factor having the following properties, which is produced by human T cells transformed with human T cell leukemia virus and obtained by purification by gel filtration chromatography, chromatofocusing and reverse phase chromatography. (1) Molecular weight 3.5 ± 0.5 × 10 ⁴ Dalton (gel filtration method) 2.2 ± 0.2 × 10 ⁴ Dalton (SDS / polyacrylamide gel electrophoresis method) (2) N-terminal amino acid sequence Pro Val Pro Pro Gly Glu Asp Ser Lys Asp Val Ala Al
a