JPH06312942A - Carcinostatic agent for human hepatoma cell producing alpha-fetoprotein - Google Patents

Abstract

PURPOSE:To provide a powerful carcinostatic agent giving specific recognition for ATP on the surface of human hepatoma cell membrane, and to provide a method for purifying the agent. CONSTITUTION:The carcinostatic agent, an immunotoxin prepared by binding a type 1 liposome-deactivated protein to a monoclonal antibody (or its fragment) capable of specifically binding to alpha-fetoprotein(AFP) on the surface of human hepatoma cell membrane. And, the method for purifying the agent, comprising a treatment of the immunotoxin by an affinity chromatography using, as substrate, a cross-linked agarose gel with a chlorotriazine pigment covalently bonded thereto.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anticancer agent which is an immunotoxin for human hepatoma cells producing α-fetoprotein (hereinafter abbreviated as AFP), and a method for purifying such immunotoxin.

[0002]

2. Description of the Related Art Liver cancer is a cancer that frequently occurs in Japanese, but there is no effective chemotherapeutic agent until now, and the development of new anticancer agents is earnestly desired. About 80% of liver cancers produce AFP, which is present on the surface of the cancer cell membrane. Therefore, anti-AFP monoclonal antibody (hereinafter, anti-AFPMo
A targeting therapy of an anticancer agent using an anti-AFPMoAb as a carrier targeting liver cancer is expected, which utilizes the accumulation property of Ab for short) to AFP-producing cells.

Since AFP is a secretory protein, it is known that it exists in high concentration in the serum of liver cancer patients.
Therefore, as the above-mentioned carrier targeting liver cancer cells, it is considered desirable to use an antibody whose binding to AFP existing on the surface of a cancer cell membrane is less likely to be inhibited by free AFP. , There is no report using such an appropriate antibody.

Conventionally, as delivery drugs by targeting therapy, adriamycin, daunomycin, and erliptinum acetate (elli) have been used.
ptinumacetate), mitomycin C, etc. are used, but the cytocidal effect of these drugs is weaker than that of toxins including ribosome inactivating protein and protein toxins such as diphtheria toxin. In order to obtain a therapeutic effect, it is necessary to devise a method in which a drug having a larger number of molecules is bound to one molecule of the antibody as a carrier, or a large amount of antibody is used. Problems such as decreased, increased antigenicity, and increased costs are occurring.

The above-mentioned toxins having a high cell killing effect may be used in a smaller amount than the above-mentioned drugs and do not have mutagenicity like general anticancer agents, and further, the binding between the antibody and the above-mentioned toxins. In the production of the body, that is, the immunotoxin, there are various advantages that conditions do not need to depend on complicated chemical reactions with low yields and that genetic engineering techniques can be applied.

[0006] As an example of using an immunotoxin having anti-AFPMoAb as a targeting carrier, a type 2 ribosome inactivating protein (hereinafter abbreviated as type 2 RIP) such as lysine is reduced and separated from the A chain and anti-AFP.
The conjugate with MoAb is disclosed in JP-A-60-67434, Cancer Re
s., 45 , 1834 (1985) and Cancer Res., 49 , 361 (1989).
However, the expected effect was not obtained,
No specific pharmacological data are given.

[0007] Conventionally, Lamb was used for purification of crude immunotoxin.
ert et al. (J. Biol. Chem., 260 , 12035,
(1985)) ion exchange chromatography is used, but a more efficient method is desired.

[0008]

DISCLOSURE OF THE INVENTION The present invention is to solve the above-mentioned conventional problems, and an object thereof is a potent immunotoxin which specifically recognizes AFP present on the surface of human hepatoma cell membrane. And an effective method for purifying such immunotoxins.

[0009]

[Means for Solving the Problems] In view of the above points, the present inventors have diligently studied and existed on the surface of a cancer cell membrane as a monoclonal antibody (hereinafter abbreviated as MoAb) suitable as a targeting carrier for human hepatoma cells. A type 1 ribosome-inactivating protein (hereinafter referred to as a type 1 RI) was used as a toxin by using a substance that specifically binds to AFP.
The target immunotoxin was produced by binding (abbreviated as P) to produce the anticancer agent of the present invention.

The anticancer agent of the present invention comprises an immunotoxin in which a type 1 RIP binds to MoAb or a fragment thereof which specifically binds to AFP existing on the surface of human hepatoma cell membrane.

The present inventors have further established a useful purification method for these immunotoxins as an alternative to conventional ion exchange chromatography. The present invention provides MoA that specifically binds to AFP existing on the surface of human hepatoma cell membrane.
A method for purifying an immunotoxin in which a type 1 ribosome-inactivating protein is bound to b or a fragment thereof, which is obtained by affinity chromatography using a crosslinked agarose gel to which a chlorotriazine dye is covalently bound as a support. Including processing steps.

The present invention will be described in detail below.

The MoAb that can be used as a targeting carrier in the present invention is an antibody that specifically binds to AFP present on the surface of a human hepatoma cell membrane, and is preferably an antibody that is less susceptible to inhibition even in the presence of free AFP. Is. Therefore, the antibody and Mo as defined herein
Ab includes not only the original immunoglobulin molecule such as IgG and IgM but also a fragment derived from the above having an AFP binding ability. A good example of such a fragment is F (ab ') ₂ obtained by digesting IgG with pepsin.
Is.

An example of preferred antibodies for use in the present invention is provided by the present inventors in Microbiol. Immunol., 36 , 873 (199
80 which is a MoAb prepared by the method reported in 2)
G and 67D. These are produced by hybridomas of spleen cells derived from mice immunized with AFP and mouse myeloma cells. Most preferred example is MoAb
It is 80G.

MoAb80G is an IgG subclass I
It belongs to gG1 and has the preferable property of suppressing the growth of AFP-producing cancer cells by antibody-dependent cellular cytotoxicity.
Furthermore, although 80G forms an immune complex with serum AFP, unlike general anti-AFP antibodies, the immune complex does not disappear rapidly from the blood, and it binds to cell superficial AFP. It also has the feature of staying in the blood as an immune complex for a long time while maintaining the activity. These facts indicate that 80G is an antibody that is less susceptible to free AFP inhibition.

Examples of the type 1 RIP used in the present invention include gelonin, saporin and momordin. Type 1 RI
P, like the A chain of type 2 RIP, is known to have an enzymatic action to inactivate the ribosome and inhibit protein synthesis. Also, like the A chain, it is not taken into the cell by itself and has low toxicity. Furthermore, the type 1 RIP does not have a subunit corresponding to the B chain of the type 2 RIP, and is biochemically and physically compared with the A chain of the type 2 RIP which originally exists as a dimer with the B chain. Being stable, it is more suitable for immunotoxin production.

The above-mentioned MoAb and type 1 RIP are combined using a suitable crosslinking agent to provide the immunotoxin of the present invention.

As the crosslinking method, a conventional method in the art can be used. For example, N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP) or 2-iminothiolane (IT) is used to introduce an SH group into Type 1 RIP, and then N-succinimidyl 4- (N-maleimidomethyl) cyclohexane- 1-carboxylate (SMCC) or S
Using a PDP, a maleimide group or a dithiopyridyl group was introduced into MoAb, and then a type 1 RIP having an SH group introduced therein and an MoAb having a maleimide group or a dithiopyridyl group introduced therein were oxidized to form a thioether conjugate, respectively. Or a disulfide bond is created.

To purify the crude immunotoxin obtained as described above, affinity chromatography using a crosslinked agarose gel to which a chlorotriazine dye is covalently bound as a support is used. In this purification, a combination of gel filtration and the above affinity chromatography is desirable.

Chlorotriazine dyes have affinities for various proteins and easily react with polysaccharides such as agarose under alkaline conditions, and thus are suitable for use as a support for affinity chromatography. The immunotoxin can be effectively purified. The preferred chlorotriazine dye is Cibacron Blue F3G
A (manufactured by Ciba-Geigy). The support used in the immunotoxin purification method of the present invention is prepared by binding the above dye by the triazine coupling method or the like (J. Chro.
matogr., 69 , 209, (1972)), or a commercially available support may be used. A suitable commercial support is Blue Sepharose (Pharmacia).

After gel filtration of the crude immunotoxin, the fraction containing the immunotoxin having a molecular weight of about 200,000 is treated by affinity chromatography using the above-mentioned support to give the unreacted MoAb and the above-mentioned conjugate. Can be effectively separated. In the present purification method, a purification step such as dialysis and various chromatographies may be further added depending on the use of the anticancer agent comprising the immunotoxin.

Conventionally, purification of crude immunotoxin has been carried out by Lamber
(J. Biol. Chem., 260, 12035.
(1985)), the MoA specifically binds to the above AFP.
The conjugate of b or a fragment thereof with type 1 RIP is not effectively purified by the above-mentioned treatment by ion exchange chromatography.

The anticancer agent composed of the immunotoxin prepared as described above can be delivered to AFP-producing hepatocarcinoma cells by various clinical methods, generally by intravenous injection. The antitumor effect of the above anticancer agent on human liver cancer cells,
In vitro well known to those skilled in the art (eg trypan blue method)
Alternatively, it can be evaluated by any of in vivo methods (eg, nude mouse method).

The present invention will be described in more detail with reference to the following examples, which, however, do not limit the present invention in any way.

[0025]

EXAMPLES Example 1 (Method for producing and purifying immunotoxin having IgG1 as a constituent) (1) Test material 80G prepared as a MoAb by the method reported in Microbiol. Immunol., 36, 873 (1992). , And gelonin as a type 1 RIP (hereinafter abbreviated as GL; Pierce
(Made by the company) was used.

(2) Preparation and Purification of Immunotoxin An immunotoxin which is a disulfide bond type and an immunotoxin which is a thioether bond type were prepared as follows.

Introduction of SH group into GL is carried out by the method of Thorpe et al. (Eur. J. Biochem., 116 , 447 (198) using SPDP.
1)) or using IT according to the method of Lambert et al. (J. Biol. Chem., 260, 12035 (1985)),
About 2 moles of SH groups were introduced per mole of GL. MoA
The introduction of the dithiopyridyl group into b80G is carried out by the method of Lambert et al. using SPDP, and the introduction of the maleimide group is carried out by the method of Ishikawa et al. (Immunoassa) using SMCC.
y, 4 , 209 (1983)), MoAb80
About 2 mol of dithiopyridyl group or maleimide group was introduced per mol of G.

In order to prepare an immunotoxin which is a disulfide bond, the SH group-introduced GL and the dithiopyridyl group-introduced MoAb80G are mixed at an equimolar ratio, and the pH of the mixed solution (0.1 M phosphate buffer) is mixed. Was adjusted to 7.5. Next, Centriprep-10 (manufactured by Amicon) was used to concentrate each concentration of GL and MoAb80G in the mixed solution to 50 to 100 µM, and then the mixture was reacted at room temperature for 20 hours. After the reaction was completed, the remaining SH group was blocked with 2 mM iodoacetamide. The immunotoxin preparation, which is a thioether conjugate of SH group-introduced GL and maleimide group-introduced MoAb80G, is carried out in the same manner as the disulfide conjugate preparation step except that the above reaction was carried out at pH 7 and 4 ° C. It was

Purification of the above conjugate was carried out by a combination of gel filtration and affinity chromatography. First, in order to remove unreacted GL in the reaction solution, gel filtration was performed with Sephacryl S-200 (manufactured by Pharmacia) to obtain a fraction having a molecular weight of about 200,000. Next, this fraction was treated by affinity chromatography using Blue Sepharose CL-6B (Pharmacia) to remove unreacted MoAb80G with a phosphate buffer containing 0.15 M NaCl, and then 1
The conjugate was eluted with phosphate buffer containing M NaCl.

Table 1 shows the four kinds of conjugates obtained in the above steps. In Table 1, (a) and (b) are disulfide type conjugates, and (c) and (d) are thioether type conjugates.

[0031]

[Table 1]

SDS-PAGE method (Nature (Lond.), 227 , 680 (19
70)), the above four types of conjugates are respectively MoAb80
G: GL = 1: 1 (mol / mol) molecular weight about 190 kDa
It was confirmed that this is an immunotoxin. In addition, ELIS
By method A (Microbiol. Immunol., 36 , 873 (1992)), it was confirmed that the anti-AFP activity of these conjugates was maintained at the same level as that of unmodified MoAb80G.

Example 2 (Method for producing and purifying immunotoxin having F (ab ') ₂ as a constituent component) The above 80G was digested with pepsin by a conventional method, and then Ultrogel Ac was used.
F (ab ') ₂ was fractionated and collected by gel filtration using A44 (manufactured by IBF Biotechnics). Then, according to Example 1, an immunotoxin, which is a thioether conjugate in which the 80G portion of the conjugate shown in Table 1 (d) was substituted with F (ab ′) ₂ , was prepared and purified. The obtained conjugate was treated with SDS- in the same manner as in Example 1.
Tested by PAGE method and ELISA method, this conjugate showed F (ab ') ₂ : G
It is an immunotoxin with a molecular weight of about 130 kDa consisting of L = 1: 1 (mol / mol), and its anti-AFP activity is unmodified F (ab ').
It was confirmed that it was maintained at the same level as ₂ .

Example 3 (In Vitro Antitumor Effect Evaluation Test of Immunotoxin Constituting IgG1) (1) Materials and Methods (i) Cell Line AFP-producing Human Hepatocellular Carcinoma Culture HuH-7 (Okayama University,
(Obtained from Dr. Sato) and AFP non-producing human hepatocellular carcinoma cell line HuH-13 (obtained from Tumor Laboratory, Dr. Hirai) were used. (ii) Culturing method RPMI1640 medium containing 15% fetal bovine serum at 37 ° C, 5% CO ₂ , 95%
Cultured under air conditions. (iii) Specimens Test group: Immunotoxin that is a conjugate of four types ((a) to (d) in Table 1) prepared in Example 1 Control group: 80G alone and GL alone (iv) Test method (trypan blue Method) HuH-7 and HuH-13 in the logarithmic growth phase were seeded on a 24-well culture plate (10 ⁵ / well), and after preculture for 2 days, each conjugate (10 ^-13 to 10 ^-7 M) was ^added. The cells were cultured for 2 days in the medium containing them, and the viable cells were counted by the trypan blue method. In each group, the concentration at which cell growth was suppressed to 50% (IC ₅₀ value) was calculated as compared to the group without addition, and the antitumor effect was compared.

(2) Test results For all immunotoxins at concentrations lower than 10 ^-7 M, Hu
Inhibition of H-7 proliferation by 50% or more, whereas HuH-1
No growth inhibition for 3 was observed even at 10 ⁻⁷ M. On the other hand, with GL alone and 80G alone,
The concentration of ^-5 M and 10 ^-6 M did not suppress the growth of both cells.
The results are shown in Table 2.

[0036]

[Table 2]

Example 4 (In vitro antitumor effect evaluation test of immunotoxin having F (ab ') ₂ as a constituent) (1) Material and method (i) Cell line The same as in Example 3. (ii) Culture method The same as in Example 3. (iii) Sample Test group: immunotoxin prepared in Example 2 Control group: mixture of 80 (G) -derived F (ab ′) ₂ and GL (iv) Test method (trypan blue method) The same as in Example 3.

[0038] (2) immunotoxin tried test results today, Hu at a concentration below 10 ^{over 10} M
Inhibition of H-7 proliferation by 50% or more, whereas HuH-1
No growth inhibition was observed for 3 even at 10 ⁻⁷ M. On the other hand, in the case of the mixture of F (ab ′) ₂ and GL in the control group,
Even at a concentration of 0 ^{over 7} M did not inhibit proliferation of HuH-7. The results are shown in Table 3.

[0039]

[Table 3]

Example 5 (In Vivo Antitumor Effect Evaluation Test of Immunotoxin Constituting IgG1) (1) Materials and Methods (i) Cell Line AFP Producing Human Hepatoma Cancer Culture HuH-7, AFP Non-Producing Human hepatocellular carcinoma cell lines HuH-13 transplanted into nude mice, HuH-7N and HuH-13N, respectively
Was used. (ii) Culturing method Male nude mice were subcutaneously subcultured. The experiment includes
A subculture of 10 or more generations was used. (iii) Samples Test group: immunotoxin of two kinds of conjugates produced in Example 1, that is, 80G-SS-GL (IT) and 80G-CS-GL (IT) Control group: phosphate buffered saline ( PBS), and a mixture of 80 G and GL in an amount corresponding to the tested immunotoxin (iv) Test method (nude mouse method) HuH-7N and HuH-13N were separately transplanted to nude mice, and the estimated tumor weight was 100 to Treatment was started when the dose reached 300 mg (after 8 and 14 days, respectively). Estimated tumor weight was determined by the method of Ovejera et al. (Annals of Clinical an
d Laboratory Science, 8 , 50, (1978)) (W ² xL)
Calculated from / 2 (mg) {W, minor axis (mm); L, major axis (mm)}.
Administration of 4 mg / kg of immunotoxin {80G-SS-GL (IT) or 80G-CS-GL (IT)} in the tail vein for 4 consecutive days from the start of treatment, and estimated tumor weight and body weight over time Measured.

(2) Test results The results are shown in FIGS. In each figure, the symbol ○ is a control group administered with PBS only, ● is a control group administered with a mixture of 80G and GL, △ is a test group administered with 80G-SS-GL (IT) and □ is 80G-CS. -GL (IT) represents a test group administered, and the mean value of the relative tumor weights after the start of each treatment ± standard error (Fig. 1
N in 8 to 9 and n in Fig. 2 indicate changes in 8). 80G-CS-
Both the GL (IT) and 80G-SS-GL (IT) test groups significantly suppressed the growth of AFP-producing HuH-7N as compared with the control group (T / C = 35-45%). This action is due to AFP non-producing HuH-
Not observed for 13N.

80G-SS-GL (IT) and 80G used in this test
-CS-GL (IT) was administered to normal mice by the same method, and then a conventional serodiagnosis was performed.As a result, hepatotoxicity was observed at 16 mg / kg in the 80G-SS-GL (IT) treatment group. , 80G-CS-GL (IT)
In the administration group, no toxicity was observed even at 32 mg / kg. The above results indicate that the immunotoxin selectively acts on AFP-producing liver cancer cells.

Example 6 (In Vivo Antitumor Effect Evaluation Test of Immunotoxin Constituting F (ab ′) ₂ as Component) (1) Materials and Methods (i) Cell Line The same as in Example 5. (ii) Culture method The same as in Example 5. (iii) Specimens Test group: immunotoxin prepared in Example 2 Control group: PBS, and the amount of F corresponding to the immunotoxin tested
Mixture of (ab ′) ₂ and GL (iv) Test method (nude mouse method) The same as in Example 5. However, the dose is 2.7 and 5.5 mg / k
g

(2) Test results The results are shown in FIGS. The symbol ○ in each figure represents a control group administered with PBS only, and the symbol ● represents a mixture of F (ab ′) ₂ and GL (5.5
(mg / kg) control group, △ indicates F (ab ') ₂ -CS-GL (IT) (2.
7 mg / kg) administered in the test group and □ are F (ab ') ₂ -CS-GL (I
T) (5.5 mg / kg) was administered to the test group, and the mean value of the relative values of tumor weights after the start of each treatment ± standard error (n in FIG. 3 is 4
6 and n in FIG. 4 indicate the transition of 6). Compared to the control group, the tested immunotoxin test group had AFP-producing HuH
The growth of -7N was significantly suppressed (T / C = 30-50%). This effect was not observed for AFP non-produced HuH-13N.

[0045]

INDUSTRIAL APPLICABILITY The anti-cancer agent of the present invention, which is an immunotoxin, selectively delivers type 1 RIP to AFP-producing human hepatoma cells, so that a high hepatoma therapeutic effect can be obtained. Furthermore, the immunotoxin purification method of the present invention provides high production efficiency of the above anticancer agent.

[Brief description of drawings]

FIG. 1 shows the in vivo antitumor action of immunotoxin, which is an anticancer agent of the present invention, which comprises 80G (IgG1) as a constituent
It is the result of examining the nude mouse transplant strain HuH-7N of the P-producing human hepatocellular carcinoma culture strain HuH-7. The vertical axis is
The relative value of the tumor weight, the horizontal axis represents the number of days after the start of treatment.

FIG. 2 shows the in vivo antitumor action of immunotoxin, which is an anticancer agent of the present invention, which comprises 80G (IgG1) as a constituent,
It is the result of examining the nude mouse transplant strain HuH-13N of the P non-producing human hepatocellular carcinoma culture strain HuH-13. The vertical axis represents the relative value of tumor weight, and the horizontal axis represents the number of days after the start of treatment.

FIG. 3 shows the in vivo antitumor effect of immunotoxin, which is an anticancer agent of the present invention, which comprises F (ab ′) ₂ derived from 80G as a constituent, and shows the AFP-producing human hepatocellular carcinoma culture HuH-7 in a nude mouse transplant. It is the result of examining HuH-7N.
The vertical axis represents the relative value of tumor weight, and the horizontal axis represents the number of days after the start of treatment.

FIG. 4 shows the in vivo antitumor effect of the immunotoxin, which is an anticancer agent of the present invention, which comprises F (ab ′) ₂ derived from 80G as a component, and transplants the AFP non-producing human hepatocellular carcinoma culture HuH-13 into nude mice. It is the result of examining the strain HuH-13N. The vertical axis represents the relative value of tumor weight, and the horizontal axis represents the number of days after the start of treatment.

Claims (6)

[Claims] 1. An anti-cancer agent, which is an immunotoxin comprising a type 1 ribosome-inactivating protein bound to a monoclonal antibody or a fragment thereof which specifically binds to α-fetoprotein present on the surface of a human hepatoma cell membrane. 2. The anticancer agent according to claim 1, wherein the monoclonal antibody or a fragment thereof is 80G or a fragment thereof. 3. The anticancer agent according to claim 1 or 2, wherein the fragment is F (ab ′) ₂ . 4. The anticancer agent according to claim 1, wherein the type 1 ribosome inactivating protein is gelonin. 5. A method for purifying an immunotoxin in which a type 1 ribosome inactivating protein is bound to a monoclonal antibody or a fragment thereof which specifically binds to α-fetoprotein present on the surface of a human liver cancer cell membrane, the method comprising: Treating the immunotoxin by affinity chromatography using a crosslinked agarose gel to which a chlorotriazine dye is covalently bound, as a support.
Method. 6. The method according to claim 5, wherein the support is blue sepharose.