JPH0780918B2 - Monoclonal anti-CEA antibody 3 - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention relates to carcinoembryonic antigen (hereinafter referred to as C
EA)).

[0002]

BACKGROUND OF THE INVENTION CEA is a well-known cancer-associated fetal antigen, which has a molecular weight of about 200,000 ± 80,000 and a ratio of sugar to protein of about 1: 1.
Is a kind of glycoprotein. The presence of the cancer antigen CEA in human digestive adenocarcinoma was reported by Gold and Freedman [J. Exp. Med., 121 ,.
439 (1965); ibid., 122 , 467 (1965)]. CEA is used for the diagnosis and treatment of cancer and various basic medical studies clinically as a marker showing the presence and the growth of cancer tissue, and its usefulness and importance. Sex is well known. However, there are certain CEA-related normal antigens, and these are CE
Since it has immunological cross-reactivity with A, the cancer specificity of CEA is unclear.

Examples of CEA-related antigens of this type are non-specific cross-reactive antigens (hereinafter NCA) and normal fecal antigens (hereinafter NFA). NCA has a molecular weight of about 80,000 ±
It is a glycoprotein of 30,000 with a sugar content of about 40-60%, which is present in human lungs and spleen, for example [Proc. Natl. Acad.
Sci. USA., 69 , 2492 (1972)]. Next, NFA is further classified into NFA-1, NFA-2 and normal fecal cross-reactive antigen (hereinafter referred to as NFCA). NFA-2 is a kind of glycoprotein having a molecular weight of 200,000 ± 50,000 and a sugar-protein ratio of about 1: 1 and its antigenicity and physicochemical properties are very similar to those of CEA. NFA-1 and NFCA are NFA-2
It seems to be a decomposition product of. NFA-1 has a molecular weight of about 2
~ 30,000, a small molecule antigen of about 13% sugar content, NFCA
Is a glycoprotein having a molecular weight of about 80,000 ± 30,000. other,
Fecal non-specific cross-reactive antigen (hereinafter referred to as f
-NCA) is present. f-NC
The antigenicity of A is substantially the same as that of NCA described above, and CEA,
It shows cross-reactivity with NFCA and NFA-2. Therefore,
There are four CEA-related antigens relevant to the purpose of the present invention in normal human feces.

In order to improve the usefulness of CEA as a cancer marker, CEA-related antigens and CEA must be accurately distinguished. For this reason, it has been attempted to provide an anti-CEA antibody having clear specificity for the antigenic determinant of CEA. However, various known polyclonal antibodies have a common drawback that the reaction specificity is unclear. That is, these polyclonal CEA antibodies are
Various antibody mixtures are reactive with almost all of the many antigenic determinants on the CEA molecule. In order to eliminate this drawback, various monoclonal CEA antibodies are emphasized. The reason is as follows.

(1) Since the monoclonal CEA antibody obtained by the conventional method of cell fusion has specificity for only one antigenic determinant, it will have uniform reactivity with the antigen. (2) Monoclonal expansion will yield large amounts of antibody with the desired homogeneity. (3) It is possible to obtain many kinds of monoclones. Overall, they will have a wide range of specificities, similar to known polyclonal anti-CEA antibodies. In this way, the production of monoclonal anti-CEA antibody
For example, it is attempted as in the following document. Accolla,
RS et al., Proc. Natl. Acad. Sci. USA., 77 , 56
3 (1980); Mitchell, KF, Cancer Immunol, Immunot
her., 10 , 1 (1980); Rogers, GTet al., Br. J. Can
cer, 43 , 1 (1981); Kupchik, HZ et al., Cancer R
es., 41 , 3306 (1981).

The reaction specificity of these previously reported monoclonal antibodies is summarized as follows. (1) Accolla et al. Antibodies obtained from the two hybridomas reacted weakly with NGP (probably equivalent to NCA) and strongly with CEA. No competitive inhibition was found in the reaction of these two antibodies with CEA. Each antibody appears to react with another antigenic determinant on the CEA molecule. (2) Mitchell. One anti-CEA antibody was obtained.
It reacted with CEA but not NCA. C
The reaction with EA could not be blocked by the polyclonal goat anti-CEA antibody. (3) Rogers et al. One monoclonal anti-CEA antibody was obtained. It reacted weakly with CEA preparations from tumor tissue but strongly with CEA in the serum of patients. (4) Kupchik. One of 9 clones was examined for monoclonal anti-CEA antibody. When compared with its reaction specificity and the polyclonal goat anti-CEA antibody, the monoclonal anti-CEA antibody reacted only with part of the CEA molecules that reacted with the polyclonal anti-CEA antibody. No more detailed search has been done on the specificity of the reaction of these known monoclonal anti-CEA antibodies.

In the meantime, we found that some CEA-related antigens, namely the above-mentioned NFA-1, NFA-2 and NFCA are present in normal human feces, and succeeded in their isolation [JP-A-56- 46819, Cancer Res.,
41 , 713-720 (1981)]. Furthermore, as a result of examining the antigen structure of the CEA molecule using these CEA-related normal antigens, C
It was proposed that many antigenic determinants on the EA molecule could be classified, for example as follows.

(1) Individual-specific antigenic determinant A specific antigenic determinant found only in individual CEA preparations used as immunogens, and CEA obtained from other individuals.
Things not found in the standard. (2) CEA-specific determinant An antigenic determinant commonly found in CEA preparations obtained from cancer tissues, but not found in CEA-related normal antigens such as NFA and NCA. It is the most cancer-specific antigenic determinant. (3) NFA-1 common determinant An antigenic determinant commonly found among CEA, NFA-2 and NFA-1, which is one of the major antigenic determinants on the CEA molecule. (4) NFCA common determinant An antigenic determinant commonly found among CEA, NFA-2 and NFCA, which is also one of the major antigenic determinants on the CEA molecule. (5) NCA common determinant It is an antigenic determinant commonly found in CEA, NFA-2, NFCA and NCA, and is the most commonly recognized antigenic determinant in CEA and related antigens. The present invention provides the above-mentioned C isolated from normal human feces.
By using EA-related antigen, monoclonal anti-C
It is based on the finding that a clone having EA antibody-producing ability can be selected from the viewpoint of reactivity with an antigen.

[0009]

DISCLOSURE OF THE INVENTION The object of the present invention is to provide a monoclonal antibody specific for carcinoembryonic antigen.

[0010]

The present invention provides a specific monoclonal antibody obtained by the following method. According to the present invention, by immunizing a first mammal with a carcinoembryonic antigen of the first individual (hereinafter referred to as a first carcinoembryonic antigen), cells having the ability to produce antibodies against the antigen are produced, and the resulting cells Was collected from this mammal, the collected cells were fused with a myeloma cell line derived from a second mammal, the fused cells thus obtained were subjected to cloning, and the obtained monoclonal hybridoma was cultured. The desired monoclonal antibody is recovered from the obtained culture medium, and (a) the first carcinoembryonic antigen is used as the first marker antigen, and the monoclonal hybridoma is used as the first monoclonal antibody.
(B) Carcinoembryonic antigen (hereinafter referred to as second carcinoembryonic antigen) of individuals other than the first immunized individual in the recovery step, normal feces, selected based on the ability to produce antibodies that react with marker antigens. Two or more kinds of antigens selected from the group consisting of antigen 1, normal fecal antigen 2 and non-specific cross-reactive antigen are used as marker antigens for selection, and the monoclonal hybridoma is used to detect reactivity with marker antigens for selection. It is selected as a standard, and at that time (c) the normal fecal antigen 2 is used as the second
A monoclonal hybridoma is selected using as a marker antigen, a monoclonal antibody capable of producing an antibody (antibody B) that reacts with normal fecal antigen 2 is separated, and then normal fecal antigen 1 is used as a third marker antigen. Specificity for carcinoembryonic antigen, which comprises the steps of separating a monoclonal hybridoma capable of producing an antibody that reacts with normal fecal antigen 1 and culturing the selected monoclonal hybridoma to obtain the desired antibody By the method for producing a monoclonal antibody having
Reactive with non-specific part of carcinoembryonic antigen, normal fecal antigen 1 and normal fecal antigen 2, but with individual-specific part of carcinoembryonic antigen and non-specific cross-reactive antigen A monoclonal antibody (antibody 3) having no

In a practical recovery step, two or more kinds selected from the group consisting of a first carcinoembryonic antigen, a second carcinoembryonic antigen, a normal fecal antigen 1, a normal fecal antigen 2 and a non-specific cross-reactive antigen. The monoclonal antibody produced by the hybridoma is selected based on its reactivity with the marker antigen for selection by using the antigen of 1. Preferably, the selection is performed by an immunoassay method using a marker labeled with various labeling substances.

The monoclonal antibody obtained from the clone according to the present invention has a uniform and clear reaction with the corresponding antigenic determinant on the CEA molecule. It can be advantageously used for basic medical research.

The clone of the present invention can be obtained by a conventional method, for example, as follows. The mammal to be immunized may be a small animal such as mouse, rat or guinea pig, or a large animal such as rabbit, goat, cow or horse.
Animals are immunized in a conventional manner. For example, a mouse is given C
Immunizations are carried out by subcutaneous injection of 0.2 ml of emulsion with EA standard, ie Freund's complete adjuvant containing 20 μg of first CEA, and 5 weeks later, by intravenous injection of the same amount of CEA in 0.2 ml of saline. After 3 days, the spleen and lymph node cells of the animal are collected by a conventional method. Collected antibody-producing cells and appropriate tumor cells [eg P3-X63-A
g8, 6, 5, 3, (for example, P3-X63-Ag8-u
1), Sp2 / 0-Ag14, 210, RCY3, Ag
1, 2, 3, etc.] and 1 × 10 ⁸ antibody-producing cells / ml to 1
Mix at a ratio of −2 × 10 ⁷ tumor cells / ml, ie at a ratio of 5: 1 to 10: 1 and fuse by standard methods, eg using HVJ (Sendai virus) or PEG (polyethylene glycol). When the cells are selected using HAT medium, normal cells die and fused cells remain. The fused cells are selected in terms of the ability to produce antibodies that react with the first CEA.
The selected fused cells are fused cells having an anti-CEA antibody-producing ability. This was cloned by a conventional method, and the obtained clone was cloned with the antibody derived from the clone and CEA and C.
Selection is performed from the viewpoint of reactivity with the EA-related antigen. It can be practically selected by an immunoassay method known per se. The selection method used in the examples below is the ammonium sulfate precipitation method of Farr (Farr, RS, J. Inf. Dis., 103 , 239 (1958)).
Is. Practical examples of CEA-related antigens are NFA-1, NFA-2 and f-NCA derived from normal adult feces, and their properties are as described above. The production of these CEA-related antigens is described in Reference Example below.

The results of the screening performed in Example 1 below are shown in Table 1.

[0015]

[Table 1]

The monoclonal antibodies obtained by the method of the present invention can be classified as shown in Table 1. Antibody 1 has reactivity with the first carcinoembryonic antigen, but
It is a monoclonal antibody having no reactivity with any of carcinoembryonic antigens other than carcinoembryonic antigen, normal fecal antigen 1, normal fecal antigen 2 and non-specific cross-reactive antigens. Antibody 2 has reactivity with two or more carcinoembryonic antigens, but does not have reactivity with any of normal fecal antigen 1, normal fecal antigen 2 and nonspecific cross-reactive antigens. It is a clonal antibody. Antibody 3 has reactivity with two or more carcinoembryonic antigens, has reactivity with normal fecal antigen 1 and normal fecal antigen 2, but does not have reactivity with non-specific cross-reactive antigens It is a monoclonal antibody. Antibody 4 is 2
It is a monoclonal antibody that has reactivity with one or more carcinoembryonic antigens and normal fecal antigen 2, but has no reactivity with normal fecal antigen 1 and nonspecific cross-reactive antigens. Antibody 5
Is a monoclonal antigen having reactivity with two or more carcinoembryonic antigens, normal fecal antigen 2 and non-specific cross-reactive antigen, but having no reactivity with normal fecal antigen 1. Practically, these monoclonal antibodies are in the form of antisera.

In Table 1, CEA derived from normal adult feces
The relevant antigen NFA-2 is radioiodinated and then added to the monoclonal culture supernatant and radioimmunoassay is performed. As a result, a single clone (clone A, about 10 strains in Example 1) and a single clone (clone B, Example 1)
Then about 200 strains) are selected. Antibody A produced by clone A does not react with NFA-2. Antibody B produced by clone B reacts with NFA-2. Antibody A is human CE
Reacts with A and does not react with CEA-related antigens from normal adult feces.

One or more second CEAs (four in the example)
When a radioimmunoassay was carried out in the same manner as above, clone A to clone A1 (8 strains in Example 1)
Antibody 1 produced by Escherichia coli reacts with the first CEA, but the second CE
Does not react with A. Antibody 2 produced by clone A2 is first
And second CEA (2 strains in Example 1).

In a similar manner, NFA-1 was used to
Clone B, which produces an antibody that reacts with -2, yields clones B1 and B2. Clone B1
The antibody 3 produced by (about 70 strains in Example 1) is NFA.
-1 and NFA-2. The antibody produced by clone B2 (about 100 strains in Example 1) is B2 is NFA-
Reacts with 2, not with NFA-1. Since NFA-1 has a small molecular weight, but has a strong antigenic activity, about 70 strains of clone B1 were obtained in Example 1.

Clone B2-1 (about 60 strains in the example) and B2-2 (about 40 strain in the example 1) are selected from clone B2 by the same method using f-NCA. The antibody 4 produced by clone B2-1 does not react with f-NCA, but the antibody 5 produced by clone B2-2 is f-NCA.
Reacts with.

If desired, other antigens besides NFA-2 can be used for the initial selection of the clones obtained by cloning. For example, by using NFA-1 first, it is possible to select a single clone from the viewpoint of the reactivity of the produced antibody with NFA-1.

The monoclonal anti-CEA antibody according to the present invention can be advantageously used for various clinical applications and basic medical research. For example, antibody 2 is considered to be most advantageous for clinical use, and antibody 3 is considered to be valuable for measuring blood CEA concentration. The clone according to the present invention can be propagated on an industrial scale by a conventional method.

The usage of the monoclonal anti-CEA antibody according to the present invention varies depending on the reaction specificity and use of the antibody, but typical examples of usage are shown below.

I. Histological search method For example, a cell suspension such as a cell suspension, a smear, or a tissue section was treated with the monoclonal anti-CEA antibody of the present invention.
After treatment at 7 ℃ for 30 minutes and washing thoroughly with saline, fluorescent dye (tetra methylrhodamine isothiocyanate or fluo
The cells are treated with an anti-mouse immunoglobulin antibody solution labeled with rescein isothiocyanate) at 37 ° C. for 30 minutes, and fluorescence positive cells are searched for using a fluorescence microscope. If desired, a search by an enzyme antibody method using peroxidase bound to an anti-mouse immunoglobulin antibody instead of a fluorescent dye can also be performed.

II. Blood CEA Concentration Measurement Method Accurate measurement of blood CEA concentration by the monoclonal anti-CEA antibody of the present invention can be carried out, for example, by the Farr method by ammonium sulfate precipitation, the two-antibody method using anti-mouse immunoglobulin, as exemplified below. , Z-gel method using zirconium gel, solid-phase antibody sandwich method and the like.

(1) Example of double antibody method using anti-mouse immunoglobulin antibody. Add 50 μl of human serum to a plastic tube (eg
Eiken tube No. 1: manufactured by Eiken Chemical Co., Ltd .; diameter 9.8 mm, length 8 cm), and diluted by adding 200 μl of 0.1 M acetic acid buffer solution (pH 6.0) containing 0.5% BSA. 0.1
Using normal mouse serum diluted 50-fold with M acetate buffer (pH 6.0), 50 μl of the appropriately diluted monoclonal anti-CEA antibody solution of the present invention was taken and placed in the above tube at 37 ° C. React for minutes. An appropriate CEA preparation (1.5 ng) in an acetate buffer solution (50 μl) prepared by the method described in Reference Example 1 below and labeled with ¹²⁵ I is added to the tube and reacted at 37 ° C. for 1 hour. Then, 100 μl of rabbit or goat anti-mouse immunoglobulin serum containing an amount of anti-mouse immunoglobulin antibody capable of sufficiently precipitating immunoglobulin in normal mouse serum (× 50; 50 μl) is added to the reaction mixture and reacted at 4 ° C. overnight. . The resulting precipitate was removed by centrifugation (3000 rpm / 30 minutes) to give 0.9%
After washing the precipitate with a saline solution, the radioactivity of the precipitate is measured with a gamma counter. CEA from 1 ng / ml to 50
0.1 M acetate buffer solution (containing calf gamma globulin 1.2% and BSA 0.5%, pH 6.0) containing each concentration up to ng / ml was treated in the same manner as the sample serum, and standard calibration Make a line. The blood CEA concentration is calculated from the measured value of CEA in the sample serum.

Since the measured value (normal value) of normal human serum generally differs depending on the type of monoclonal anti-CEA antibody used, it cannot be easily specified, but it is twice the standard deviation (SD) of the average normal value. It is practical to use a value equal to or higher than the value obtained by adding the value as a pathologically high value for the determination of malignant tumor detection.

(2) Example of solid-phase antibody sandwich method. This method is the most sensitive. In principle, one of the monoclonal anti-CEA antibodies obtained in the present invention is adsorbed and bound to a plastic bead or tube as a solid-phase primary antibody. After reacting this solid-phase primary antibody with CEA in the sample, another monoclonal anti-CEA antibody having a different corresponding antigenic determinant from the primary antibody was reacted as a labeled secondary antibody iodinated with ¹²⁵ I. Then, CEA in the sample
The labeled secondary antibody binds depending on the amount. The CEA amount in the sample is calculated from a calibration curve obtained by measuring a known amount of CEA by the same operation.

(A) Preparation of solid-phase primary antibody The characteristics of a monoclonal anti-CEA antibody with which the primary antibody is selected may vary depending on the purpose of the assay system, but the most general purpose is to use CEA. An antibody 3 directed to the NFA-1 common part, which is considered to be the most predominant antigenic site on the molecule, may be chosen, so an example of antibody 3 is described below. Among the monoclonal anti-CEA antibodies classified as antibody 3, those having a strong reaction affinity (Ka) with CEA (at least Ka ≧ 1 × 10 ⁹ M ⁻¹ ) are selected.

The Ka is measured as follows. CEA [0.01M] labeled with ¹²⁵ I of various amounts up to 200 μg was added to a fixed amount (eg 10 μg) of the antibody 3 preparation.
Borate buffered saline (BBS) pH 8.0; containing normal rabbit serum (1%) and NaN3 (0.05%)]. After incubating each mixed solution at 37 ° C for 18 hours, 75% saturated ammonium sulfate solution (400 µl) was added to each mixture and kept at 4 ° C for 1 hour, and then the supernatant was removed by centrifugation (1800 xg / 30 minutes). . The precipitate is washed with 50% saturated ammonium sulfate solution. The radioactivity of the supernatant (200 μl) and the precipitate is measured using a gamma counter. Repeat the same method,
The ratio of bound CEA and free CEA was determined, and from this, the standard method [Steward MW & Petty RE, Immunol., 22 , 7
47 (1972)], the reaction affinity (Ka) is calculated. The thus-obtained high-affinity antibody 3 preparation was treated with a 0.001 M phosphate buffer (pH 6.0) containing sodium lauryl sulfate (0.01%) and EDTA (0.05%), for example, to give 5
It is diluted to 00 times (about 5 to 10 μg / ml as the antibody concentration) and used for coating plastic beads or tubes.

Various types of plastic tubes and beads can be used as the solid phase matrix, for example, a diameter of about 6
mm polystyrene beads (eg Precision Plas, USA)
ticBall) was thoroughly washed, then immersed in the above-mentioned monoclonal anti-CEA antibody (antibody 3) solution, and allowed to stand at room temperature overnight. The beads were washed twice with distilled water, treated with 0.001 M glycine-hydrochloric acid buffer (pH 2.3), and further washed twice with distilled water, then with 0.5 M sodium chloride and 0.5% bovine serum albumin. Soak in 0.1 M phosphate buffer containing (BSA) for 3 hours, and finally vacuum dry and store in a cool place.

(B) Preparation of labeled secondary antibody The results obtained are significantly different depending on what is used as the labeled secondary antibody. As can be seen from Table 2 below, for example, CE obtained by using antibody 2 as a secondary antibody
The A measurement system reacts only with CEA in cancer tissue, but NF
Although it is a system with the highest specificity that does not react with A or NCA, the assay system obtained by using antibody 5 as a secondary antibody also measures all related antigens other than NFA-1 together with CEA. It is a system to obtain. Further, among various antibodies identified as the antibody 3, those having an antigenic determinant that reacts with the same antibody as the antigenic determinant of the primary antibody cannot be used for the secondary antibody, but among the antibodies belonging to the antibody 3, the primary Any antibody that reacts with an antigenic determinant other than the corresponding antigenic determinant of the antibody can be used as the secondary antibody. The following example is an assay system with the highest cancer specificity using antibody 2.

Of the various antibodies identified as antibody 2, the highest reaction affinity constant with CEA (at least K
a ≧ 1 × 10 ⁹ M ⁻¹ ) is selected. C in which CEA and Sepharose 4B are bound (10 mg / 1.0 g dry gel)
The above monoclonal anti-CEA antibody was specifically purified using an EA adsorbent (see Reference Example 2), and the obtained antibody preparation was used as a known chloramine T method [Hunter & Greenwood, 196 2, N.
ature (London) 194 , 495] and labeled with ¹²⁵ I to give a labeled antibody having a radioactivity intensity of about 5 nCi / ng antibody. If desired, a secondary antibody labeled with an enzyme such as peroxidase can be used instead of ¹²⁵ I.

(C) Radioimmunoassay 50 μl of human serum (or plasma) obtained as a sample was placed in a plastic test tube (for example, Eiken tube 9.8 × 80 mm).
Then, 200 μl of 0.1 M acetate buffer (pH 6.0) containing BSA (0.5%) was added, and the above anti-CE was added thereto.
One bead to which the A antibody is bound is added and reacted at room temperature for 4 hours while rotating the test tube. After the reaction, the reaction solution was removed by suction and washed once with 1 ml of 0.9% saline solution to iodize antibody 2 to obtain a labeled secondary antibody solution (containing 1% BSA of 0.1%).
Add 200 μl of labeled antibody diluted to about 600 nCi / ml with 05M Tris-HCl buffer, and rotate at room temperature for 2
Allow to react for 4 hours. After completion of the reaction, the beads are washed twice with 0.9% saline, and the radioactivity of the beads is measured with a gamma counter.

To prepare a calibration curve, purified CEA was added at 1 ng /
ml, 3ng / ml, 10ng / ml, 30ng / ml and 100ng
1.2% bovine gamma globulin and 0.
Each standard solution prepared with 0.1 M acetate buffer (pH 6.0) containing 5% BSA is used. 50 μl of each standard solution is taken and reacted with the antibody-coated beads and the radioiodine-labeled secondary antibody by the same procedure as the treatment of the above-mentioned specimen. In this way, the radioactivity (B cpm) of each standard solution is obtained. CB 'is calculated by subtracting the control value B'without CEA from B cpm, the corresponding CEA concentration is plotted to draw a calibration curve, and this is used to calculate the CEA concentration in the sample.

In this example using antibody 2 as the labeled secondary antibody,
The reaction with normal human serum or plasma is weak. C of normal person
The average value of the EA concentration significantly differs depending on the characteristics of the anti-CEA antibody preparation used, for example, its affinity constant Ka, and a specific numerical value cannot be easily specified. However, practically, a value equal to or larger than a value obtained by adding a value twice the standard deviation (SD) to the average value of normal persons can be used as a pathologically high value for the determination of detection of malignant tumor.

By using the monoclonal anti-CEA antibody according to the present invention, as compared with the conventional polyclonal and monoclonal anti-CEA antibodies used for the same purpose,
The desired antigen concentration can be measured more accurately.

In addition to the diagnostic treatment of cancer, the monoclonal anti-CEA antibody according to the present invention is used as a ancestral return phenomenon to the fetal stage of gene discovery associated with malignant changes of cells.
It can be used to elucidate the biological activity and molecular structure of A production and CEA.

In the examples of the invention described below, the Farr radioimmunoassay procedure was performed as follows. CEA preparations labeled with ¹²⁵ I (5-10 ng; 50 μl) and culture supernatant of monoclones (50 μl) were placed in each well of a 96-well microtiter plate and mixed well.
After standing at 37 ° C for 1 hour, saturated ammonium sulfate solution (100 µl)
Was added to each well, and the plate was allowed to stand at 37 ° C. for 1 hour. Next, the reaction mixture is centrifuged (2000 rpm / 30 minutes).
Then, the supernatant was separated, and 100 μl of the radioactivity was measured with a gamma counter. Radioactive C precipitated with antibody
From the amount of EA, fused cells capable of producing a monoclonal anti-CEA antibody were identified.

[0040]

[Example 1] Two BALB / c mice (SPF mouse: obtained from Shizuoka Experimental Animal Agricultural Cooperative Association) of about 5 weeks old were C
Immunization was performed with EA (prepared by the method of Reference Example 1). That is, for each animal, 20 μg of CEA for the first time was added to 0.2 ml of an emulsion using Freund's complete adjuvant (manufactured by DIFCO).
Then, 5 weeks later, the same amount of CEA (20 μg of CEA in 0.2 ml of saline) was intravenously administered to each mouse. Three days after that, the animals were killed, and antibody-producing cells (lymphocytes) were collected from the spleen and lymph nodes. About 1 x 10 ⁸
Lymphocytes and 1 × 10 ⁷ azaguanine-resistant mouse myeloma-derived cell line P3-X63-Ag8-ul [Y
elton, DE et al., Curr. Top. Microbiol. Immuno
l., 81 , 1 (1978)]. 1 ml of 45% (v / v) polyethylene glycol 4000 (manufactured by Sigma, USA) was added dropwise to the mixture and kept at 37 ° C for 7 minutes, and then D-ME.
Polyethylene glycol was diluted by dropping 15 ml of M (Nissui Pharmaceutical) into the mixture.

The fused cells were washed with D-MEM (30 ml),
10% fetal calf serum (FCS) (manufactured by Gibco, USA), penicillin (100 units / ml) and gentamicin (50 μg)
/ ml) and was suspended in D-MEM (100 ml). Put the cell suspension (1 ml each) into 96 wells (2 each)
4-well microtiter plate was used), left overnight at 37 ° C. in the presence of carbon dioxide, and then HAT medium (4 × 10 ⁻⁷ M aminopterin, 1.6 × 10 ⁻⁵ M thymidine and 1 ×)
Medium containing 10 ^-4 M hypoxanthine, pH 7) 1 ml each
Was dispensed into each well, then half of the medium was replaced with fresh HAT medium every two days. On the 11th day, half of the medium is
Replace with T medium (the above HAT medium minus aminopterin), then half of the medium is replaced with 10% F every 2 days.
It was replaced with D-MEM containing CS. Almost all (9
Growth of fused cells was observed in the medium (8% or more). The supernatant of the medium was collected from each well, and the fused cells (hybridomas) producing the anti-CEA antibody were selected by the Farr's ammonium sulfate precipitation method using the first CEA. Then anti-CEA
The fused cells recognized to have antibody-producing ability were cloned by the limiting dilution method as follows.

D containing 10% FCS (fetal calf serum)
-Dilute the fused cells to 3 cells / ml in MEM,
0.2 ml of each was placed in each well of the microtiter plate. 3 using D-MEM containing thymocytes (about 5 × 10 ⁶ cells / ml) of X-ray-irradiated young BALB / c mice
After culturing at 7 ° C for 2 weeks, cells proliferating in the wells were identified as monoclonal fused cells, and the limiting dilution method was repeated to ensure monoclonality. Thus, a monoclonal fused cell having an anti-CEA antibody-producing ability of about 10 ng / ml or more was obtained in the culture supernatant.

The first CEA used in Example 1 was obtained by the method described in Reference Example 1 below, but other suitable CEA preparations can be used for the same purpose, if desired. Usually, 20 to 30 monoclonal anti-CEA antibody-producing fused cells are obtained from two mice. The culture supernatant of about 300 strains of anti-CEA antibody-producing fused cells obtained by repeating the above operation a dozen times and CEA labeled with radioactive iodine,
The reactivity with NFA-2, NFA-1 and f-NCA was determined by Fa
It was searched by radioimmunoassay by ammonium sulfate precipitation method of rr. The results are shown in Table 1 above.

[0044]

Example 2 Antibodies 1 to 5 (see Table 1) obtained by the method of Example 1 were separately analyzed for CEA-related antigens derived from the first CEA, second CEA and normal human feces, namely NFA-1, N.
It was investigated by Farr's radioimmunoassay method using FA-2 and f-NCA, and the reactivity between all antibodies 1 to 5 and the above all antigens was elucidated. In Table 2 showing the results, “+” and “−” represent the presence or absence of reactivity.

[0045]

[Table 2] Reactivity of monoclonal anti-CEA antibody with CEA and CEA-related antigens 1 2 3 4 5 Human CEA 1 * ++++++++ 2 ***-+++++ Normal associated antigen NFA-1 − − + − − NFA-2 − − + + + f-NCA − − − − + (Note) * CEA for animal immunization ** CEA other than 1st CEA

From Table 2, it can be seen that the monoclonal anti-CEA antibody of the present invention has the following reaction specificity. (1) All antibodies 1-5 react with the first CEA, ie CEA used for immunization of animals. (2) Antibody 1 is an individual-specific antibody that reacts only with the first CEA. This antibody is considered to be useful for diagnostic treatment of cancer in each individual. (3) Antibody 2 reacts with the first CEA and the second CEA, but with all CEA-related normal fecal antigens or NFA-
1, does not react with NFA-2 and f-NCA. In addition, in Example 1, four CEA preparations obtained from different patients were used as the second CEA. Since this antibody is an anti-CEA-specific antibody and has the highest cancer specificity, it has various clinical uses (eg, confirmation of cancer diagnosis, carriers for delivering radioactive substances and anticancer agents to cancer tissues) and basic medical research. It can be used for universal purposes such as (4) Since antibody 3 reacts with the first CEA and the second CEA, and also with NFA-1 and NFA-2, it is an antibody of practical value. In addition, a single clone having antibody 3 producing ability can be selected by a simpler method than other clones having antibody producing ability. (5) The other antibodies, that is, the antibodies 4 and 5, also have more clear reaction specificity and homogeneity as compared with the conventional polyclonal and monoclonal anti-CEA antibodies.

[0047]

[Example 3] Anti-CEA antibody-producing fused cells were mixed with BALB / c
Example in which a monoclonal anti-CEA antibody was produced by transplantation into the abdominal cavity of a mouse. Commercially available (obtained from Shizuoka Prefecture Experimental Animal Agricultural Cooperative Association) BALB / c mice (5-6 weeks old, female or male) were used as test animals. Each mouse was intraperitoneally administered with 0.5 ml of pristane, and after 7 to 10 days, D-containing 5 × 10 ⁶ to 10 ⁷ cells / ml of the monoclonal fused cells (obtained by the method of Example 1). MEM (0.5 ml) was also injected intraperitoneally. After 7 to 10 days, ascites was collected from the mouse in which ascites was sufficiently accumulated by abdominal puncture, and thereafter, ascites was collected 5 times every 2 days, and then the mouse was killed to collect whole blood. The amount of ascites that can be obtained depends on the properties of the fused cells.
Although it was 0 ml, an average of about 10 ml was obtained. The concentration of the monoclonal anti-CEA antibody contained in the ascites and serum of each animal also varies depending on the properties of the fused cells, from about 1 mg / ml to about 1
It was 0 mg / ml. The amount of antibody obtained is also 2-3 mg to 1
A considerable difference was observed at more than 00 mg.

[0048]

Reference Example In the following Reference Example, the concentration of the solution by the pressure air drying method was performed by the following method (hereinafter referred to as the air drying method). The solution to be air dried was placed in several cellophane tubes. For example, the solution (3 l) was dispensed into 8 to 9 tubes (manufactured by Visking, USA, 18/32, length 150 cm), and the upper part of the tube was connected to a pressurizing machine, for example, through a suitable conduit. Support the tube vertically, and
Dip 5 in a container filled with demineralized water or a suitable buffer,
The rest of the room was blown by a fan.

[0049]

Reference Example 1 CEA preparations described in this specification were prepared by the following method unless otherwise specified [Data. JP-A-56
-47762]. Human colon cancer liver metastases 120g
To the mixture was added 700 ml of physiological saline, and the mixture was ground and centrifuged to obtain a supernatant (9000 rpm / 40 minutes). 80 ml of 60% perchloric acid solution (perchloric acid concentration 0.6M) was added to 710 ml of the supernatant, and the resulting precipitate was centrifuged (9000r.
pm / 40 minutes) and the resulting supernatant was dialyzed against running water overnight to remove perchloric acid and dialyzable impurities. The dialysis solution is a cellophane tube (made by Visking, 18/3
(2, length 150 cm) was dispensed into 3 tubes and concentrated to a total of 8 ml by the air-drying method described above. Centrifuge the concentrated solution (15000 r.
pm / 30 minutes) to obtain a CEA crude extract.

Sepharose 4B (Pharmacia Fine Chemicals, Sweden) column (2.6 ×
171 cm) to 0.05 M sodium phosphate buffer (pH
It was washed with 5.2), loaded with 7.5 ml of the above CEA crude extract, and eluted with the same buffer as above. Fraction 4B-1 that elutes at the same position as the solvent volume of the column, as well as fraction 4B that elutes at 2.1, 2.5, and 2.7-fold positions, respectively.
-2 to 4B-4 were obtained. Fraction 4B-2 containing most of CEA was collected and concentrated (3.5 ml) by the air-drying method described above. The Sepharose 6B (Pharmacia Fine Chemicals, Sweden) column (1.9 x 145 cm) was washed with the same buffer as above,
When 3.5 ml of the above concentrated solution was loaded and eluted with the same buffer, an asymmetrical peak appeared at a position 1.6 times the solvent capacity of the column. Fraction 6B-1 obtained early in elution
(About 20 ml) contained high molecular weight impurities and the final fraction 6B-3 (about 1/3) contained NCA (non-specific cross-reactive antigen). The middle part (about 60 ml) corresponding to about 2/3 of the whole was collected and collected, and fractionated by air-drying method 6
B-2 (3 ml) was obtained. Sephadex G-200 (Pharmacia Fine Chemicals, Sweden)
Column (1.9 x 145 cm) was washed with the same sodium phosphate buffer as above, then loaded with Fraction 6B-2 (3 ml) and eluted with the same buffer. A symmetrical peak appeared at a position of about 1.2 times. Fraction located in the middle 3/4 of this peak (approx. 45 m
l) was collected, collected and concentrated by air-drying method. Purified CEA (50 mg) was recovered from this concentrate. Its high purity was confirmed by, for example, immunoelectrophoresis (using a rabbit antibody). Purified CEA is a kind of glycoprotein, the molecular weight is about 200,000 ± 80,000, the ratio of sugar to protein is about 1: 1, the maximum absorption in the UV absorption spectrum is 277 nm, and the small shoulder is 283 nm. Was given.

[0051]

[Reference Example 2] CEA-related normal antigens described in the present specification, namely NFA-1, NFA-2 and f-NCA were obtained by the following method (Reference: JP-A-56-46819). 250 g of normal adult feces was added to 2500 ml of a 0.6 M perchloric acid solution, stirred well and filtered with gauze to remove insoluble substances such as food residues. Centrifuge this (700
The supernatant obtained at 0 rpm./30 minutes) was dialyzed against running water overnight to remove perchloric acid and dialyzable impurities. 9 dialysis solution
Book of cellophane tubes (Bisking, 18/32,
It was dispensed to a length of 150 cm) and concentrated by the air-drying method to a total of 30 ml. By repeating the same operation four times, a total of 120 ml of crude extract was obtained. This is about 10mg in total
Contained the desired CEA-related normal antigen of

An anti-CEA antibody adsorbent bound with a specific anti-CEA antibody (10 mg, produced by the method described below) was added to the above crude extract and reacted at 4 ° C. for 24 hours. After removing the reaction solution, a cold borate buffer solution (pH = 8.0; 0.01M
Boric acid buffer solution; washed with 0.15 M NaCl), glycine hydrochloric acid buffer solution (0.175 M; pH 2.3; 200 m)
l) and then extracted with glycine NaOH buffer (1.0 M; p
By neutralizing with H = 9), a solution containing crude CEA-related antigen (dry weight 9 mg) was obtained. This solution was concentrated to 1.2 ml by the air-drying method described above and loaded on a column (1.3 × 80 cm) of Sepharose 6B (manufactured by Pharmacia Fine Chemicals, Sweden). Phosphate buffer (0.01 M; pH 5.0; containing 0.15 M NaCl)
Elution was carried out and each 1 ml fraction was collected in a test tube. Test tube numbers 60 to 75 and 81 to 95 were collected and designated as fraction 1 and fraction 2, respectively.

Fraction 1 was concentrated to about 1 ml, loaded onto a Sephadex G-200 (100 ml) column and eluted with the same phosphate buffer as above, and 1 ml fractions were collected in a test tube. did. Test tube numbers 30-40 containing purified NFA-2 were collected and concentrated. This concentrated liquid itself can be used as it is as a preparation of purified NFA-2. The NFA-2 thus obtained is a kind of glycoprotein and has a molecular weight of 200,000 ±
30,000, the ratio of sugar to protein is about 1: 1, and the maximum absorption is 277 nm in the ultraviolet absorption spectrum. The amino acid composition of this product is very similar to that of CEA, and NHA-2 NH2
The terminal amino acid sequence is identical to that of CEA up to at least position 11.

The brown dye was removed from Fraction 2 by diethylaminoethyl cellulose chromatography [see Cancer Res., 41 , 713 (1981)], and 3.5 ml of the anti-NCA antibody adsorbent described below was added to the remaining solution. added. The mixture was reacted at 4 ° C for 2 days. The adsorbent is collected, washed well, and then glycine hydrochloride buffer (0.175M; pH =
2.3; 20 ml) and then neutralized with glycine NaOH buffer (1M; pH = 9) before B.I. B. S. The solution was dialyzed against and the dialysis solution was concentrated by the air-drying method. This concentrate contains purified NCA (1 mg dry weight) and can be used for the purposes of the present invention without further purification. The f-NCA thus obtained is a kind of glycoprotein having a molecular weight of 80,000 ± 30,000,
The sugar content is about 20%. At least the 20th position in the NH2-terminal amino acid sequence of this product is identical to CEA. f
-The physicochemical properties of NCA are, for example, N existing in lung and spleen.
It is substantially the same as that of CA.

A solution containing a substance which was not adsorbed by the above-mentioned anti-NCA antibody adsorbent was concentrated to 0.5 ml, and a column of Sephadex G-100 Super Fine (manufactured by Pharmacia Fine Ke 1 Cals, Sweden) was used. 1.3 x
43.4 cm) and the same phosphate buffer as above (0.01 M; pH 5.0; containing 0.15 M NaCl)
Elution was carried out at 1 ml, and 1 ml each was collected in a test tube. Test tube number 4
5-55 (fraction a) and 65-75 (fraction b) were each collected and collected. Fractions a and b were each rechromatographed using the same column of Sephadex G-100 Superfine (1.3 x 43.4 cm). In this way, from fraction a to purified NFCA, fraction b
From this, purified NFA-1 (0.5 mg dry weight each) was obtained. These could be used for the purposes of the present invention without further purification. The NFA-1 thus obtained is a kind of glycoprotein having a molecular weight of 20,000 to 30,000 and a sugar content of about 13
%. The amino acid composition of NFA-1 is similar to that of CEA, but some but significant differences were found with some aminos such as phenylalanine, lysine and proline. However, NH ₂ terminal amino acid sequence of NFA-1 is CEA, is quite different from that of NFA-2 or NCA.

The anti-CEA antibody adsorbent and the anti-NCA antibody adsorbent described above were obtained by the following method. The CEA preparation (10 mg dry weight) obtained by the method of Reference Example 1 was combined with CNBr Sepharose 4B (1 g dry gel; manufactured by Pharmacia Fine Chemicals, Sweden) by a conventional method to obtain a CEA adsorbent. Anti-CEA serum (5
0 ml) at 4 ° C for 3 days. Any CEA can be used to make this antiserum. Anti-CE
The CEA adsorbent bound with the A antibody was recovered and cooled until the appearance of protein was not observed. B. S. Washed with glycine-HCl buffer (10 ml each; 0.175M; pH = 2.
It was eluted 5 times in 3). Collect the eluted liquid to collect glycine Na
Neutralize with OH buffer (1M; pH = 9), then B.I. B.
S. Against 4 ° C overnight. The dialyzed solution was concentrated to 10 ml. This solution contained 50 mg of the specific polyclonal anti-CEA antibody. 20 ml of the concentrated solution obtained by repeating the above-mentioned method twice contained 100 mg of the specifically purified polyclonal anti-CEA antibody. 100 mg of the thus obtained polyclonal anti-CEA antibody was added to CNB.
R Sepharose 4B (10 g; manufactured by Pharmacia Fine Chemicals, Sweden) was combined by a conventional method. Thereby, the specifically purified anti-CEA antibody 1
The desired anti-CEA antibody adsorbent combined with 00 mg was obtained. By repeating the above method using an anti-NCA antibody instead of the anti-CEA antibody, an anti-NCA antibody adsorbent bound to Sepharose 4B can be obtained.

[0057]

INDUSTRIAL APPLICABILITY The monoclonal antibody obtained by the present invention is not only an antibody that specifically reacts with carcinoembryonic antigen, but also any of antibodies 1, 2, 3, 4, 5 is different from other antibodies It shows different reactivity. For example, antibody 1 exhibits individual-specific reactivity, and antibody 2 is carcinoembryonic antigen-specific. Antibody 3,
Since each of 4 and 5 shows a characteristic reactivity with respect to the normal fecal antigen 1 and the non-specific cross-reacting antigen, there is a characteristic use corresponding to each antibody. That is, the antibody 2 is particularly useful as an antibody having high cancer specificity in quantifying and detecting carcinoembryonic antigen as a tumor marker. Antibodies 3, 4, and 5 are also very useful for analysis of the molecular structure and tissue distribution of each antigen molecule.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location G01N 33/577 B (C12P 21/08 C12R 1:91)

Claims (1)

[Claims] By 1. A immunizing a first mammal in the first individual carcinoembryonic antigen, it was produced cells having antibody-producing ability to the antigen, were taken resulting cells from the mammal are harvested Fused cells with the myeloma cell line derived from the second mammal, the fused cells thus obtained are subjected to cloning, the obtained monoclonal hybridomas are cultured, and the desired culture medium is cultured. A clonal antibody is recovered, wherein (a) the carcinoembryonic antigen of the first individual is used as a first marker antigen, and the monoclonal hybridoma is used as a reference for the ability to produce an antibody that reacts with the first marker antigen. (B) In the collecting step, (b) consists of carcinoembryonic antigen, normal fecal antigen 1, normal fecal antigen 2 and non-specific cross-reactive antigen of individuals other than the first immunized individual Using two or more antigens selected from the group as selection marker antigens, the sorted Monoclonal high <br/> Buridoma based on the reactivity with the screening marker antigens, and at that time (c) It was selected monoclonal hybridoma using normal fecal antigen 2 as a second marker antigen, separating the monoclonal antibodies with the antibody (antibody B) producing ability of reacting with the normal fecal antigen 2, then the normal fecal antigen 1
Carcinoembryonic fetus comprising a step of isolating a monoclonal hybridoma capable of producing an antibody that reacts with normal fecal antigen 1 by using as a third marker antigen, and culturing the selected monoclonal hybridoma to obtain a desired antibody obtained by preparation of monoclonal antibodies with specificity for sexual antigen, individual non-specific portion of an oncofetal antigen, although reactive with normal stool antigen 1 and normal faecal antigen 2, cancer Fetus
A monoclonal antibody (antibody 3) having no reactivity with the individual-specific portion of the sex antigen and the non-specific cross-reactive antigen.