JPS6229598A - Anti-epidermal growth factor monoclonal antibody - Google Patents

Abstract

NEW MATERIAL:A monoclonal antibody reacting specifically with both human Epidermal Growth Factor (h-EGF for short) and mouse Epidermal Growth Factor (m-EGF for short), produced by using a highly purified h-EGF as an antigen. USE:For purifying h-EGF or m-EGF or for detecting biochemical or clinical h-EGF or m-EGF, etc. Easily producible at low cost. PREPARATION:Firstly, an animal such as mouse, etc., is immunized against highly purified h-EGF having preferably >=99% purity as an antigen, the splenocyte of the animal is extracted to prepare an antibody-forming cell. Then, the splenocyte is fused with a tumor cell to give a hybridoma and a hybridoma producing a desired antibody is selected. Finally, the hybridoma, for example, is cultivated in a growth medium and the aimed antibody is collected from the culture supernatant liquid according to a conventional procedure.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a monoclonal antibody that specifically reacts with both human epidermal growth factor and mouse epidermal growth factor.

[Conventional technology]

Cell fusion technology [Nature 25
6, 495 (1975)), various monoclonal antibodies have been created using this technique, and human epidermal growth factor (human epidermal growth factor)
mal Growth Fac-tor/h-EG
F) and mouse epidermal growth factor (mouse
Hptdern+al Grosvth Factor
There is a report that a monoclonal antibody that specifically reacts with m-HGF (hereinafter referred to as m-HGF) has been created [Collection of Abstracts of the 105th Annual Meeting of the Pharmaceutical Society of Japan (1985)]. h-EGF and m-EGF that have been created so far
A monoclonal antibody that reacts with either of these can be obtained by obtaining a hybridoma by fusion of animal B lymphoid cells prepared using h-EGF extracted from human urine as an antigen and tumor cells, and produced by this hybridoma. This is the antibody that was tested.

-a) Since the h-EGF content in human urine is low and contains many impurities, it is extremely difficult to isolate and purify h-EGF from human urine, and it is necessary to obtain a sufficient amount of highly pure h-EGF. I couldn't. Therefore, h-E derived from human urine
It is difficult to efficiently immunize animals with GF, and therefore it has not always been easy to obtain anti-h-EGF antibody-producing B lymphocytes. Therefore, screening methods for obtaining hybridomas having desired specificity are complicated, and monoclonal antibodies produced using such hybridomas are expensive.

[Problem that the invention seeks to solve]

Therefore, the present invention provides an anti-EGF monoclonal antibody that reacts with (that is, crosses) both h-EGF and m-EGF, which is obtained by a method that enables highly specific anti-EGF monoclonal antibodies to be produced efficiently and at low cost. This is what we are trying to provide.

[Means for solving problems]

As mentioned above, it has not been possible to produce highly pure h-EGF, and it has therefore been impossible to attempt to produce a monoclonal antibody against highly purified h-EGF. The present inventors and other co-inventors have previously purified h-EGF to an extremely high purity of over 99% using genetic engineering techniques.
(Japanese Patent Application No. 60-22630) discovered that a highly specific anti-EGF monoclonal antibody could be easily produced by using this highly purified h-EGF as an antigen. Completed the invention.

Therefore, the present invention relates to an anti-epidermal growth factor monoclonal antibody that is produced against highly purified human epidermal growth factor and reacts with both human epidermal growth factor and mouse epidermal growth factor. Monoclonal antibodies are produced by culturing hybridomas obtained by cell fusion of B lymphoid cells of mammals immunized with highly purified human epidermal growth factor and tumor cells;
It is produced by collecting a monoclonal antibody having the above-mentioned specificity from a culture solution.

[Detailed explanation] ■-Anti-Journey In the present invention, highly purified h-EGF and m-EGF are used for preparing antibody-producing cells and detecting a desired monoclonal antibody. .

Here, "refined to a high degree of purity" means that it is substantially single, for example, about 90% or more pure, preferably about 95% or more, and more preferably about 99% or more pure. . h-EGF purified to high purity in this way
In order to produce h-EGF in large quantities and at a lower cost than before, there is a method of producing h-EGF using genetic engineering techniques and then purifying it, particularly the method proposed by the present inventors and co-researchers [Patent Application No. 1983]. -22630]. This method is a method for producing purified h-EGF characterized by comprising the following steps A to C.

That is, (A) (B) A gene encoding a signal peptide, and h-EGF is present immediately after the downstream end of the gene.
The gene encoding h-EGF is inserted into a vector that can be linked with the structural gene of ``h-EGF'' and can be propagated in the intended host cell, and a Durham-negative microorganism is transformed with this recombinant. λ) The resulting transformed microorganism is cultured in a medium containing an amount of inorganic phosphorus necessary to induce protein synthesis ability from the late logarithmic growth phase to the early arrest phase during the growth process of the microorganism. After that, the microorganisms are collected, and (d) the microorganisms are then treated by the osmotic shock method to recover both components containing h-EGF; (B) the collected h-
Both fractions containing EGF were subjected to ion exchange chromatography, and then the h-EGF fraction was collected; and (C) both fractions containing h-EGF collected above were further subjected to high performance liquid chromatography. , collect the h-EGF fraction.

A three-cycle preparation of m-EGF was published in The Journal on Biological Chemistry (J, Biol, Chem.
,).

237, 1555-1562 (1962), Recent Progress - Hormone Research (Recent
Prog, HormoneRes, ), 30,
551-574 (1974), JP-A-59-20412
This can be carried out according to Publication No. 3, etc., but since there is a commercially available product (from Toyobo Co., Ltd.), it is convenient to use that product.

(2) Monoclonal! The monoclonal antibody according to the present invention is produced as follows. First, (1) prepare antibody-producing cells by immunizing animals with highly purified h-EGF, (2) obtain hybridomas by fusing these cells with tumor cells, and (2) obtain anti-h-EGF having the above characteristics. Select hybridomas that produce monoclonal antibodies. Then, (1) this hybridoma is cultured, and (2) the desired anti-h-EGF monoclonal antibody is obtained from the culture. Note that the above general method itself is publicly known, for example,
-45407, JP-A-59-128397, and Journal on Immunological Men's (J.

Immunol, Methods), 39285-3
08 (1980), etc.

■ Preparation of antibody-producing cells Antibody-producing cells are prepared by immunizing an animal with an antigen, extracting lung cells, and using a known method [for example, Methods in ENZ-YMOLO].
GY), 73.14 (1981) Academic Press].

It can also be prepared by sensitizing B lymphoid cells with antigens in vitro [Japanese Patent Application No. 59-212578 "
(See “Preparation of Antibody-Producing Cells”).

In the present invention, for example, h-EGF (created by genetic engineering techniques, isolated and purified) is administered as an antigen into the abdominal cavity of a mouse (primary immunization), and 10 days later, immunization is performed (booster immunization). Three days after the final immunization, lung cells are removed from the animal and antibody-producing cells are prepared.

-■ Cell fusion This is a step in which the antibody-producing cells prepared above and tumor cells are fused in the presence of a fusion agent to obtain a subcultureable hybridoma. Such cell fusion operation is carried out by Nature (Na
ture) (supra), Journal on Immunological Men (J.

Is-munol, Methods) (above), monoclonal antibodies - hybridomas and ELISA-j
This can be done with reference to p50-60 published by Kodansha Scientific. In the case of the present invention, for example, the above antibody-producing cells and tumor cells p3-X63-AgaU, (P,
Ul) (/Method
ds in ll! NZ-YMOLOGY), mutually,
3-46 (1981)) into RPMI-1640.
The test is carried out in the presence of about 40% polyethylene glycol (PEG) in the medium.

■ Selection of hybridomas The desired hybridomas are first selected using P2, which was used in the present invention.
For tumor cells such as O3, HAT medium (R containing hypoxanthine, aminopterin and thymidine)
PMI-1640 medium) [Science
e), 145, 709 (1964)) [Methods in EN
ZYMOLOGY), Mutual, 16-18 (1981))
.

Next, the hybridomas obtained by the above procedure are further cultured, and the culture supernatant is analyzed (confirming the presence of antibodies) to select hybridomas producing the desired antibody.

Culture supernatants can be analyzed using the plaque method, agglutination reaction method, radioimmunoassay method, etc., but the simple method is usually the ELISA method [Methods in Enzymology (Met)].
Hods in II! NZYMOLOGY), 70,
419-439 (1980) etc.].

■ Cultivate hybridomas in a growth medium [e.g. RPM] according to the conventional method.
I-1640 medium (10-20%), fetal bovine serum ((F
e2)))] or [Methods in ENZYMOLO
GY).

42-43 (1981)), or by intraperitoneally administering the hybridoma to an experimental animal (mice, rat, etc.) in which the cells can proliferate and culturing it in vivo [
Methods in enzymology
ENZYMQLOGY), 73.43-44 (198
1)).

■ Recovery of antibodies Antibodies can be collected from culture supernatants when hybridomas are grown in growth medium according to conventional methods, or from ascites of animals when hybridomas are grown in vivo in experimental animals. For example, it can be recovered according to the ammonium sulfate fractionation method). Furthermore, purified samples with high antibody titers can be obtained by gel filtration, ion exchange chromatography, affinity column chromatography, or an appropriate combination of these methods [monoclonal
Antibodies (MONOCLONAL ANTIB)
ODIES) J 405 (1980), Plenum
Published by Press].

Confirmation of the species specificity of the monoclonal antibody created by
This can be carried out according to the BLISA method described above using a system in which EGF or m-EGF is immobilized. Also, h-E
Antigen-antibody reaction between GF or m-EGF and the constructed anti-EGF monoclonal antibody, and the ability to promote epithelial cell proliferation, which is a biological activity of EGF [Proceedings on the National Academy of Sciences on・The United Stake on America (Pr.
oc, Natl, Acad.

Sci, USA, ), 72, 1317 132
1 (1975)), the specificity of the antibody can also be confirmed. For example, h-EGF or m-EGF is added to a system in which the monoclonal antibody of the present invention is immobilized (
Here, h-EGF or m-EG is added to the system by antigen-antibody reaction.
After F is immobilized), the supernatant of the system is collected.

This supernatant is then used to inject epithelial cells (e.g. mouse 3T3 cells).
to measure the proliferation ability of cells. The specificity of the antibody was confirmed by comparing this result with the results of a control experiment in which the above procedure was performed without using the antibody and a control experiment in which h-EGF or m-EGF was simply applied to epithelial cells. do.

Furthermore, the so-called susceptor assay method [e.g., radioreceptor assay] combines the binding of EGF to the EGF receptor present on epithelial cells and the antigen-antibody reaction between EGF and an anti-EGF monoclonal antibody.
(RRA) Law Journal on Biological Chemistry (J.

Biol, Chew, ), 257.3053 3
060 (1982)) to h-EGF or m-EG.
The specificity of the antibody can also be confirmed by measuring the biological activity of F. Furthermore, the same antigen (h-EG
By utilizing the competitive reaction between antibodies against F), the specificity of the antigenic site between the antibodies can be confirmed.

(4) Monoclonal - Eight types of hybridomas, namely 5-3-3.6-4-6.7-2-3.9-6-11.
8-4-7.24-5-5.6A-4 and 6F-9 were obtained, and monoclonal antibodies were obtained from each. Each of these monoclonal antibodies is designated by the same name as the hybridoma that produces it. When the specificity of these eight hybridomas was examined as described above, all of the above monoclonal antibodies reacted with both h-EGF and m-EGF.

The subclass of these monoclonal antibodies was IgG + or IgGzm, and their light chains were IgG.

〔Effect of the invention〕

In the method for producing monoclonal antibodies of the present invention, highly purified h-EGF is used as the antigen;
Compared to the conventional method of using low-purity h-EGF derived from human urine, it is easier to immunize experimental animals, and it is easier to screen hybridomas, thus producing monoclonal antibodies with the desired specificity. The resulting hybridomas can be obtained efficiently and monoclonal antibodies can be produced at low cost.

The anti-EGF monoclonal antibody of this invention reacts with both h-EGF and m-EGF. Therefore h-EGF
Or purification of m-EGF [affinity cam using the antibody (for practical methods, see, for example, published patent publication No. 59-500720)], as well as biochemical and clinical h-EGF Alternatively, it may also be used for detection of m-EGF [detection in biological components (human urine, blood, etc.)]. Furthermore, h-EGF and m-
A monoclonal antibody that reacts with any of EGF,
By combining it with an anti-h-EGF monoclonal antibody or an anti-m-EGF monoclonal antibody, it can be widely used in biochemical experiments.

On a blood spot (1) Preparation of cells h-EGF created and isolated by genetic engineering method in Bal b/c mice (Patent Application No. 60-226 mentioned above)
Immunization (initial immunization) was performed by intraperitoneally administering 30 gg (see No. 30), and subsequent booster immunizations were performed once at 10-day intervals. Three days after the final immunization, lung cells were removed from the mice aseptically, the cells were loosened, and RPMI-
After suspending in 1640 medium, the mouse tile cell suspension (2 x 106 cells/
ml) was prepared.

On the other hand, mouse tumor cells P and U1 to be fused with the above cells
(Flow Corporation) suspension (RPMI-1640 medium) was prepared according to a conventional method.

(2) Cell fusion The mouse tile cell suspension prepared above and the P,01 cell suspension were mixed so that the ratio of the number of lung cells to P,Ul cells was 10=
After mixing at a ratio of 1:1, the mixture was centrifuged and the supernatant was removed. Then, add about 40% PE G4 to the cells at the bottom of the centrifuge tube.
1 m/ml of PBS (-) solution containing 000 was slowly added dropwise. After leaving this at 37℃ for 4 minutes, RP old-1
By adding 640 (containing 1.0% FC5), PE
G was diluted. After centrifugation and removing the supernatant, the final concentration of P301 cells was 1.0 x 10' cells/m
After diluting the solution with RP Old-1640 (containing 10% FC3) to a concentration of 1, the solution was dispensed into a 96-well plate at a rate of 100 μl/well. In addition, thymocytes of Bal b/c mice within 3 weeks of age were previously dispensed into this plate as feeder cells at a ratio of 5 x 10' cells/well in an amount of 100 μl/well.

(3) Selection of hybridomas Starting from the day after the above fusion operation, replace half of the medium (100 μl) in each well with HAT medium every day for 4 days, and
1 while replacing the medium with HAT medium every other day.
Culture was performed for 0 days. In addition, the above HAT medium contains 100 μM hypoxanthine and 0.1 g in RP old-1640 medium.
M aminopterin, 1.6 μM thymidine, 10% FC3
, 5x 10-'M2-mercaptoethanol, 2mM
Glutamine, 100 units/ml Penicillin,
and 0.1 mg/m 1 streptomycin.

Moreover, PBS (-) is sodium chloride 8.0g.

Potassium dihydrogen phosphate (anhydrous) 0.2 g, sodium hydrogen phosphate (anhydrous) 1.15 g, potassium chloride 0,
Mix 2g for 1000m! ! (p
H7,4).

The culture supernatant was then analyzed using the following procedure.

(1) Preparation of analysis plate Coating buffer (100mM NaHCOi,
50mM NazCOt, (pH 9,7-10
h-EGF (0,5 μg/m7 and m-EGF (0,5 p g/m I-) dissolved in ))
Dispense 50 μβ of each, leave at room temperature for 2 hours, then add PB
The plate was washed three times with S(-)-Tween (Tween 20 (0.5 mA/β) added to the above PBS(-)).

Then PBS (-) (1% bovine serum albumin (B
After adding P
Analytical plates were prepared by washing with B S (-)-Tween (described above).

(ii) Analysis of supernatant Take the supernatant of the well where colonies appeared and transfer it to the above two types of analysis plates. / well was added. After leaving it for 2 hours, it was washed 3 times with PBS ()-Tween and peroxidase-labeled goat anti-mouse immunoglobulin (
Capel) 50 μl/well was added and incubated for 2 hours (37”, 5% carbon dioxide gas).PBS
After washing 5 times with (-)-Tween, 0.1 M citrate buffer (pH 4,
2) was prepared by dissolving 2.5mM of ABTS (2,2'-azinovir (3-ethylbenzothiazoline)-6-sulfonic acid) and 5mM of hydrogen peroxide immediately before use, and added 100μl/well of this solution. 5 to 5 at room temperature after injection at a rate of
The reaction was carried out for 15 minutes. After stopping the reaction (adding 2mM sodium azide at 100μl/well), O
D4° was measured.

Eight types of wells showing a positive reaction were selected by this reaction,
Cells were then taken out from the colonies in these eight wells and cloned by the limiting dilution method to establish eight types of hybridomas.

(4) Culture of hybridomas and recovery of antibodies
The cells were cultured in a medium (containing 0% FC3) at 37°C in the presence of 5% carbon dioxide in a carbon dioxide gas incubator, and then antibodies were recovered from the culture supernatant by ammonium sulfate fractionation. On the other hand, the same cells were intraperitoneally administered to a mouse to obtain the antibody as ascites fluid, which was crudely fractionated by ammonium sulfate fractionation and purified antibody was recovered by DEAR-cellulose column chromatography. When these antibodies were characterized, the results shown in Table 1 were obtained. In the table, the expression 1gG+/ means that the subclass is IgG.
, indicates that the light chain is . IgGzm / indicates that the subclass is IgGza and the light chain is .

Table 1 (5) Confirmation of antibody specificity The specificity of the above monoclonal antibody was determined by ELISA method! Approved.

A 96-well plate (Falcon) in which h-EGF and m-EGF#meba=廿 were each immobilized using a coating buffer, and a well in which no antigen was immobilized was also present in the same system as a control. prepared.

Using this plate, the specificity of the antibody obtained according to the present invention was confirmed according to the above-mentioned ELISA method. In addition, the antibodies at that time (hybridoma strain 5-3-3, 6-4-6
Strains, 7-2-3 stocks, 9-6-11 stocks, 8-4-7 stocks, 2
4-5-5 strain, 6A-4 strain, and 6F-9 strain) were each used after adjusting the concentration to 1.15 μg/ml with PBS (-). Absorbance OD of the supernatant of each well at that time. ,
are shown in Table 2. The antibody used in this experiment was collected from mouse ascites, subjected to ammonium sulfate fractionation, and then purified using a DEAE-cellulose column.
Except for 6-11, which is IgGga/, all others are IgG□/, and the antibody concentrations are also the same. All values of 0D4os in Table 2 are obtained by subtracting the control value.

The remainder is 0 below.The common amino acid sequence of h-EGF and m-EGF is about 70% of the total amino acid sequence width. However, it is possible that the binding and dissociation constant between the antigen h-EGF or m-EGF and each antibody of the present invention may differ due to differences in the antigen recognition site of the obtained antibodies or differences in the antibodies themselves. Therefore, if the binding and dissociation constants of each antibody are all close values, although it cannot be determined simply from the crossover rate in Table 2, the antibodies of the present invention can be classified into five groups based on the crossover rate. That is, 7-
2-3 and 9-6-11, 5-3-3 and 6-4-6
, 24-5-5, 8-4-7 and 6F-9 and 6A-4
There are 5 groups.

Tsunako Charcoal Preparation of High Purity h-EGF High purity h-EC;F used in the present invention was manufactured by the following method.

Transformed microorganism E. coli (E, coli) K
12YK537 (pTA 1522) was cultured, the cultured cells were collected, an osmotic supernatant was prepared, and DEAE-
Purified by ion exchange chromatography using TOYOPEARL 650M and high performance liquid chromatography using HCL 803D system.

The purity of the h-EGF preparation thus purified was determined by conventional methods of polyacrylamide gel electrophoresis and reversed phase high performance liquid chromatography.

From these results, it was found that the purity was 99% or more. The results of electrophoresis and high performance liquid chromatography are shown in FIG. 1 and FIG. 2, respectively. In addition, (a) in Figure 1 is a reproduction of an electrophoresis gel, and the arrow (O=) indicates the h-EGF band.
Also (mouth) is densitometry.

Next, the above specimen was analyzed using one HLC-825A machine for amino acid analysis.
A (Toyo Soda) was used, and the amino acid composition completely matched the literature value. In addition, the determination of the -order structure was carried out in the Journal of Biological Chemistry (J, B
iol, Chem,)2!16.7990-7997
(1981), and the secondary structure was further determined. As a result of the above, h-EGF used in the present invention
The primary structure and secondary structure were proposed by Gregory in 1975 [Nature, 257.325
(1975)), it was completely consistent with that of the previous study.

[Brief explanation of the drawing]

Figure 1 shows the results of determining the purity of the antigen h-EGF used in this invention by electrophoresis, in which (a) is a reproduction of the electrophoresis gel, and (b) is its densitometry. It is. FIG. 2 shows the results of high performance liquid chromatography of the antigen h-EGF used in this invention.

Claims (1)

[Claims] 1. Anti-epithelial cell growth that specifically reacts with both human epidermal growth factor and mouse epidermal growth factor, which is produced against highly purified human epidermal growth factor. Factor monoclonal antibody. 2. The monoclonal antibody according to claim 1, wherein the highly purified human epidermal cell growth factor is produced by genetic engineering techniques and then purified to a purity of 95% or more.