JPH01161000A - Neutrophil-activating factor, its production and antitumor agent and antibacterial agent containing said factor - Google Patents

Abstract

NEW MATERIAL:A neutrophil-activating factor having a molecular weight of about 25,000 and containing a fragment having amino acid sequences of formula I-IV. USE:An antitumor agent and antibacterial agent. PREPARATION:An MT-2 cell which is an antigen-producing cell strain originated from human umbilical leukocyte is established by the mixed culture with leukemia cell collected from the peripheral blood of an adult T-cell leukemia patient. The MT-2 cell is cultured in a medium containing bovine fetus serum, etc., and the cultured liquid is centrifuged to precipitate and separate the cells and obtain the supernatant liquid. The supernatant liquid of the cultured product is passed through a molecular sieve membrane, concentrated, dialyzed, subjected to ion exchange column chromatography and eluted by the concentration gradient of sodium chloride. The eluted active fraction is purified by gel filtration and reversed phase high-performance liquid chromatography, etc., to obtain the objective neutrophil activating factor.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a neutrophil activating factor, a method for producing the same, and an antitumor agent and an antibacterial agent containing the same.

Lymphocytes act on various other white blood cells to cause them to proliferate and produce various protein-based microactive substances that activate the functions of these cells.

These active substances are collectively called lymphokines.

The present invention relates to a new lymphokine that activates neutrophils, a method for producing the same, and antitumor and antibacterial agents containing the same.

[Conventional technology]

Neutrophils are not only known as important cells involved in biological defense against bacterial infections by sterilizing pathogenic bacteria, but also have been recently reported to have antitumor effects against cancer. (Katano M, a
nd Torisu M,, Cancer+50,
62-68 (1982), Korec S, *Il
erberman R, B, + Dean J, H, an
d Cannon G, B, Ce11. Immuno
l.

54, 104-115 (1980), Gerrard
T.L., Cohen D.

J, and Xaplan A, M, + J,
Natl, Cancer In5t, +66,
483-488 (1981), 5endo F,,
5eiji S,.

Watabe Sot Fuyan+a S, a
nd Arai Sol Transplan・t
ation Proceedfngs, 12. 1
60-163 (1980), Chee O, 0,
, Townsend cJ, l Galbraith
M.A.

B11ber F, D, and Morton
D.L., Cancer Res.

Kawa, 4534-4539 (197B)).

In particular, neutrophils activated by lymphokines have strong antibacterial and antitumor effects. As a lymphokine that activates neutrophils, one whose amino acid sequence is already known is granulocyte-macrophage colony stimulating factor (GM-CSF) [Weisbart
R, B9. Golde D, WlClark S,C
0t Wong G, G, and Ga55on J
,C,,Nature, 314 361 363(
1985)), tumor necrosis factor (TNF) (Shal
aby M, R,, Aggarwal B, B,, R
ink-erknech E, +5redersk
yL, P., Finkle B.S., and PaLla.
dtno M, A,, J, Immunol,,
Y Shaiko 2069 2073 (1985), Kleban
off S, J,, Vadas M, A,, H
arlanJ, M,, 5parks L, tl,,
Gamble J, R, + Agosti J,
M.

and Waltersdorf A, M, t, r
, Immunol, +tae.

4220-4225 (1986)), α-interferon (Yukiko Miyake, Arata Abi, Akemi Araki, Fujibe Sendo.

Japanese Society of Immunology General Meeting/Academic Meeting Record, Volume 16, Page 39゜, 1986), β-interferon (Yukiko Miyake, Arata Ani, Akemi Araki, Fuji Sendo 9 Japanese Society of Immunology General Meeting/
Academic Conference Record, Volume 16, Page 390.

1986), γ-interferon (Sharaby
M.

R, + Aggarwal B, B,, Rinkerk
nech U,, 5rederskyL, P,, F
inkle B, S, and Pa1ladtno
M, A,, J.

Immunol,, 135.2069 2073 (
(1985), Basham T.

Y, + Sm1th W, and Meriga
n r, c, l Ce11. Imm-unol,,8
8.393 (1984)) is known.

When normal lymphocytes are cultured in vitro in the presence of plant lectins such as concanavalin A, the lymphocytes are stimulated and
Produces lymphokines in the culture supernatant. Some leukemia cells proliferate while producing lymphokines without receiving such stimulation.

[Structure of the invention]

Due to resource constraints, it is difficult to use normal lymphocytes for the production of human-derived neutrophil activating factor. If lymphoid cells derived from leukemia patients (hereinafter abbreviated as leukemia cells) that can proliferate indefinitely produce a high amount of neutrophil activating factor, it is advantageous in terms of resources to use these leukemia cells. be. The present inventors screened a large number of leukemia cells, found human-derived leukemia cells that produce neutrophil-activating lymphokines in high units, and detected a novel neutrophil-activating factor in the culture supernatant of these cells. This finding led to the completion of the present invention.

That is, the present invention relates to a neutrophil activating factor containing fragments A, B, C, and D having the following amino acid sequences and having a molecular weight of about 25,000.

Fragment A Lys-Asp-His-Pro-
Lys-Tyr fragment B Pro-11e-S
er-Asp-Val-Leu-Lys fragment CTyr-Glu-Tyr-11e-As
p-Gly-Arg-Ser fragment D Leu-Glu-Gly-＾rg-
Glu-5 to Thr-Tyr-8la-Asp-Tyr
er-Val-Asn-Glu-Met The above-mentioned neutrophil activating factor (hereinafter abbreviated as NAS) according to the present invention was established by mixed culture with leukemia cells collected from the peripheral blood of adult T-cell leukemia patients. It is produced by extraction and purification from the culture supernatant of MT-2 cells, which are an antigen-producing cell line derived from human p-band leukocytes.

Proliferation culture of MT-2 cells was carried out using R containing 10% tallow serum.
It is carried out using PMI-1640 medium (manufactured by Hinaga Pharmaceutical Co., Ltd.). The doubling time of this cell is 3 days,
The saturation density is I x 10h pieces/@7.

A serum-free culture supernatant is required as a starting material for purification, and therefore cells suitable for serum-free culture were cloned to obtain strain A-6. This strain is serum-free RPMI-164
NAS was continued to be produced in 0 medium for 2 weeks, and the NAS activity of the obtained supernatant was i, oo.

Units/m7, cell viability after 2 weeks is 50%.

Since the A-6 strain produces adult T-cell leukemia virus, it is passed through an ultrafiltration membrane with 111 million molecules to remove the virus, then concentrated, and used as a starting material for purification.

The neutrophil activating substance produced by the A-6 cell line includes DEA.
Experiments by the present inventors have confirmed that there are three types of isoelectric points that can be separated by BSepharose ion exchange chromatography, but considering their physical properties and antitumor activity, we decided to set the isoelectric point on the most acidic side. Proceed with purification of the active fraction.

Purification is performed by the steps shown below.

That is, after dialysis of the A-6 cell line culture supernatant, three types of neutrophil activating substance peaks 1, 2.
3 is fractionated, both desired fractions (beak 3, hereinafter referred to as NAS) are collected and purified using a total of 7 steps of purification.

It is thought that NAS interacts with contaminant proteins from the beginning of purification, so purification is performed in a system in which a dissociating agent is added. As a result, the second step was Q-3epha-rose ion exchange chromatography, and the third step was Protein W.
In gel filtration HPLC using S2003 & Zorbax GF250, chromatography is performed in the presence of urea, and in reverse phase HPLC using the old Trapore RPSC in the 6th stage, chromatography is performed in the presence of SDS, and good results can be obtained. can.

The physical properties of the obtained NAS are as follows.

(1) Molecular weight As a result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on purified NAS, it is thought that the molecular weight of NAS is about 25,000.

(2) Thermal stability NAS retains its activity even after heat treatment at 56°C for 130 minutes and at 85°C for 130 minutes and is stable to heat.

(3) Stability NAS towards SO5 is 1% at 25°C for 3 hours in the presence of o, i% SOS.
It is stable under incubation.

(4) Protease-sensitive NA
Trypsin, chymotrypsin, and pronase (1μ9/−) were each added to S, and enzymatic digestion was performed at 37°C for 4 hours. As a result, NAS was inactivated by each protease. As a result, NAS is considered to be a protein.

(5) Amino acid analysis The purified NAS was analyzed for amino acid composition. First, as shown in FIG. 7, the main active fraction was divided into two parts and their compositions were analyzed. As shown in fraction 3.4 in Table 1, the compositions were the same. Furthermore, the amino acid composition of both fractions other than the active main fraction was different. Table 2 shows the amino acid composition values of NAS. From this result, NAS is As
x (Asp, Asn), Glx (Glu, Gln)
It was shown to contain characteristically large amounts of Leu, Val, Ala, and Gly.

Table 1: Amino acid composition around the active fraction in the final purification process Table 2: Amino acid composition of purified NAS The structure of NAS was analyzed by the method shown below.

(1) Using N-terminal amino acid sequence purification NAS 5μs, gas phase sequencer (A
The N-terminal amino acid sequence was analyzed using Biosystems, Inc.). Analysis was performed on the N-terminal IO residue, but no significant amino acid sequence was detected, suggesting the possibility of blocking the N-terminal amino acid (modification of the Ni1. group).

Therefore, next, purified NAS 1.8 ttH was added to CNBr.
After being subjected to degradation and fragmenting specifically at the Met position, N-terminal analysis was performed again under the same conditions as before. As a result, multiple amino acid sequences consisting of at least four components were clearly identified for the N-terminal 10 residues.

From the above results, it was considered that the N-terminal amino acid of NAS was blocked in some way. In order to determine whether this substance has a new structure, we further analyzed the internal structure.

internal amino acid sequence The internal amino acid sequence was analyzed by the steps shown below.

Purified NAS 16μ9 ↓ Enzyme degradation with Lysyl Endopeptidase ↓ Amino acid sequence analysis (Applied Biosystem
ms 470^5equenator) That is, using purified NAS 16py, CNBr decomposition and reductive carboxymethylation were performed to cleave and fragment NAS at the Met position. The reactants were then subjected to reverse phase II P
Although the fragments were separated and purified by LC, the fragments were not separated into distinct peaks.

This suggests that the peptide fragments are extremely similar in terms of molecular weight or hydrophobic properties. As a preliminary test, we performed amino acid sequence analysis on some of the fragments to check their purity, and found that there were multiple fragments mixed together.
It became clear that the separation was still insufficient.

Therefore, we put the CNBr fragments together and added them to LysylEnd.
Enzymatic decomposition using opeptidase was performed, and Lys
When the peptide fragment was specifically cleaved at the position of
The peaks derived from the two peptide fragments were well separated.

As a result of analyzing each amino acid sequence for the main peaks,
The partial amino acid sequences of fragments A-D shown below were identified.

Fragment A Lys-Asp-His-Pro-
Lys-Tyr fragment B Pro-11e-5
er-Asp-Val-Leu-Lys fragment CTyr-Glu-Tyr-11e-As
p-Gly-Arg-Ser fragment D Leu-Glu-Gly-Arg-
Thr-Tyr-Ala-Asp-Tyr-Glu-S
For er-Val-Asn-Glu-Met fragment D, 15 residues were identified. Ne
Since it contains t, it is considered to be one of the best synthetic probes for future gene sequence analysis.

(3) Novelty of structure Regarding the identified sequence, NBRF Protein D
We searched for differences with known structures using ataBase and CAS on Line, but found no matches or relatively highly homologous structures. We also searched for recent literature, but found nothing relevant.

In addition, interferon α, interferon β, and interferon T are lymphokines that activate neutrophils.
, TNF, lymphotoxin (TNF-β), GM-C
3F has been reported, but NAS differs from these lymphokines because it has a different amino acid sequence.

From the above results, it was concluded that the NAS isolated and purified this time is a novel active protein.

NAS obtained in the present invention is a lymphokine that activates neutrophils, and activated neutrophils exhibit anticancer and antibacterial effects. Therefore, the present invention also provides antitumor agents and antibacterial agents containing NAS as an active ingredient.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 (Culture of MT-2 cell line) For culturing MT-2 cells, RP old-1640 medium (Hinaga Pharmaceutical RPMI-1640-■) was supplemented with 10% calf fat serum,
A medium supplemented with 100 units/ml of penicillin G, LOOug/ml of streptomycin, 0.2% NaHCO3, and 0.03% glutamine was used as a medium. MT-2 cells were inoculated into this medium 141 at a cell concentration of 5 x 10' cells/d, placed in a spinner flask, and kept at 37°C.
It was stirred with The dissolved oxygen concentration of the medium is 95% oxygen, 5%
Carbon dioxide gas was passed through the culture medium and a dissolved oxygen meter was used to measure 0. was adjusted to 0.4 to 0.5 ppm.

MT-2 cells reached 1 x 10 cells/cell after 3-4 days of culture, half of the culture 71 was removed and fresh medium 7. After 3 to 4 days, culture medium 71 is removed and fresh medium 71 is added. By repeating this operation and continuing culturing, one spinner flask can produce 1X10"/
d elbow-2 cell culture every 3-4 days 71! , produced.

Example 2 (Collection of serum-free culture supernatant of MT-2 cells) The MT-2 cell culture solution obtained in Example 1 was centrifuged at 4°C to precipitate cells. Cells receive 100 units of penicillin G/
ml, streptomycin 100 μ3/mf, N
Contains aHCO30.2% and glutamine 0.03%! ?
PMI-1640 medium (Hinaga Pharmaceutical RPMI-1640-
■) (hereinafter this medium is referred to as serum-free RPMI-1640 medium) after centrifugal washing three times,
After adjusting the 0 medium to a cell concentration of 1×106 cells/m7, it was cultured for 5 days at 37°C in a spinner flask with an oxygen concentration of 4 to 4.5 ppm using a dissolved oxygen meter for 4 to 5 days, and the centrifuged supernatant was was used as serum-free culture supernatant.

Example 3 (Measurement of antibacterial activity based on neutrophil activation of NAS) Human peripheral blood was collected at a final concentration of heparin of 10 u/-, and physiological saline containing 6% dextran was added to 10 ffi7 of whole blood.
Add at a ratio of 3-, leave at room temperature for 30 minutes to precipitate red blood cells, take white blood cell fraction in a centrifuge tube, cool, and add 1 + 0.
The white blood cells were precipitated by centrifugation at 00 rpm+ for 10 minutes. These white blood cells were collected using Percoll (Pharmacia) with a specific gravity of 1
．． 040.1, 080.1.100, centrifuged at 1900 rpm for 20 minutes, and collected cells with specific gravity between 1.080 and 1.100. Neutrophils with a purity of over 97% were obtained. Obtained. Neutrophils were 10% calf fat serum, H
RPMI-1 containing EPES 15mM, Penicillin G100 units, Streptomycin 100μ3/l117
640 medium (hereinafter simply referred to as ASSAY medium) 1×1
The number was adjusted to 0b pieces/-.

On the other hand, in a medium containing 2% grape #M, 0.3% yeast extract, and 0.5% peptone, Candida
parapsilosios), and after culturing for several hours, the cells were washed with physiological saline and adjusted to 2.5 x 10' cells/-.

This is 1/10 for lXl0' of neutrophils/- solution.
0 volume was added and placed at 4°C.

501J1 A for each person in 96 large flat bottom microplates
Add SSAY medium and 25 tons of test solution containing NAS in holes 1 and 7! A 3-fold dilution series was prepared by adding 25 IJ1 from wells 1 to 6 and from wells 7 to 12. In this way, the number of dilution terms will be from 3 times to 729 times, and the measurement will be dup-licated for each dilution. A mixture of neutrophils and yeast 504 was added thereto, and cultured in a 5% carbon dioxide incubator for about 16 hours.

When neutrophils are activated by NAS, they phagocytize and sterilize Candida bacteria, and even after 16 hours, the cells remain transparent with no growth of Candida bacteria. As the amount of NAS decreased, the growth of Candida increased, and the amount of NASO was measured by measuring the turbidity of each person at 0.0660 nm using a microplate reader. O, D of Candida in ASSAY medium only 50% when 660nm is 100
The dilution rate at which O and D were obtained was determined as the unit of the analyte.

Example 4 (Manufacture of NAS) The serum-free culture supernatant obtained in Example 2 was passed through a molecular sieve membrane with a molecular weight cutoff of 1 million using a Pellicon cassette (Millibore Corporation). This filtrate,
It was concentrated 100 times using a sieve membrane with a molecular weight cutoff of 10,000 molecules. DEAE-3epharose 4B, in which 1°C of 100-fold concentrated serum-free culture soil was placed in a cellophane tube, dialyzed against 20mM trisaminomethane-hydrochloric acid buffer (pH 7,4), and the content was equilibrated with the same buffer.
(Pharmacia) column and eluted with a salt concentration gradient. The elution pattern is shown in Figure 1.

NAS was measured by the method shown in Example 3.

The active fraction eluted at a salt concentration of 0.11 M was collected and
Q-5ephar using 50 InM trisaminomethane-HCl buffer (pH 7,5) in the presence of M urea.
ose ion exchange chromatography (Figure 2), Protein WS2003 & Zorbax G using 50mM lisaminomethane-hydrochloric acid buffer (pl! 7.5) in the presence of 6M urea and 0.5M NaCl
Gel filtration with F250 + 1 PLC (Figure 3), 0.
IJltrapore in the presence of 1% TFA/CH3CN
Reverse phase HPLC using RPSC (Figure 4) was performed. At this stage, NAS activity was divided into two groups, A and B. Collect active fraction B and make 5111M NH4HCO3/CI1
．． Reversed phase) IPLc using Phenyl 5PW-RP in the presence of CN (Figure 5), 0.1% SOS/CH3
Reversed phase 11PLC using Ultrapore RPSC in the presence of CN (Figure 6), 200mM NH40
1(/Phenyl 5PW- in the presence of C113CN
Reverse-phase HPLC using RP was performed in sequence (Figure 7), and N
I was able to get AS.

Example 5 (Measurement of anticancer activity of NAS based on neutrophil activation) NA
The anticancer activity of S based on neutrophil activation was investigated using antibody-dependent cytotoxic activity.

After labeling P815 mastocytoma cells with 5ICr, anti-H-2
The 0' antibody was reacted for 2 hours. After the reaction, P815 mastocytoma cells 1X10'/@Z 100m were dispensed into a 96-well U-shaped plate, and 50I of neutrophils and NAS50 of each concentration were added.
After incubation for 4 hours, supernatant 1001
The amount of % I Cr in i1 was measured with a gamma counter.

A: Count of experimental group B: Count of spontaneous release from P815 mastocytoma C: Total label count of P815 mastocytoma As shown in Table 3, in the group without neutrophils, total release from P815 mastocytoma was (While no cytotoxic activity was observed, in the neutrophil-added group, the cytotoxic activity increased with the concentration of NAS at any E/T ratio.

Table 3: ADCC activity of NAS note) Book 1: Ratio of neutrophils to P815 mastocytoma cells $2: Neutrophil-free group

[Brief explanation of the drawing]

1 to 7 are diagrams showing the results of each purification step of NAS carried out in Example 4, and FIG. 1 shows DEAE-5eph-ar
ose ion exchange chromatography, Figure 2 is Q-5
Epharose ion exchange chromatography, Figure 3
is Protein WS2003 & Zorbax
Gel filtration with GF250 + 1PLc, Figure 4 is tll
Reverse phase 11PLc using trapore RPSC, Figure 5 shows reverse phase 11PL using Phenyl 5PW-RP.
C, FIG. 6 shows the results of reversed phase 11PLC using 1ltrapore RPSC, and FIG. 7 shows the results of reversed phase HPLC using Phenyl 5PW-RP. Applicant's agent: Kaoru Furuya (Volunteer to write the amendment) February 3, 1986 Neutrophil activating factor and its manufacturing method, anti-ulcer agents and antibacterial agents containing it 3, relationship with the person making the amendment case Patent applicant (021) Eisai Co., Ltd. 4, Agent 5 Nakai Building, 1-3 Nihonbashi Yokoyama-cho, Chuo-ku, Tokyo Column 7 for detailed explanation of the invention in the specification subject to amendment Contents of the amendment (1) Description Page 15, line 5 and line 8 from the bottom, “carboxymethylation” was corrected to “carboxamitomethylation”.

Claims (1)

[Claims] 1. Fragments A and B having the following amino acid sequences,
A neutrophil activating factor containing C and D and having a molecular weight of approximately 25,000. Fragment A [There is a gene sequence. ] Fragment B [There is a gene sequence. ] Fragment C [There is a gene sequence. ] Fragment D [There is a gene sequence. 2. Fragment A having the following amino acid sequence, which is extracted and purified from MT-2 cell culture supernatant,
A method for producing a neutrophil activating factor containing B, C, and D and having a molecular weight of about 25,000. Fragment A [There is a gene sequence. ] Fragment B [There is a gene sequence. ] Fragment C [There is a gene sequence. ] Fragment D [There is a gene sequence. 3. The manufacturing method according to claim 2, wherein urea is used for extraction and purification. 4. Fragments A and B having the following amino acid sequences,
An antitumor agent and an antibacterial agent containing C and D and a neutrophil activating factor with a molecular weight of about 25,000 as an active ingredient. Fragment A [There is a gene sequence. ] Fragment B [There is a gene sequence. ] Fragment C [There is a gene sequence. ] Fragment D [There is a gene sequence. ]