JPH0655680B2 - Human monoclonal antibody conjugate for malignant tumor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to a class and / or amount of malignant tumor-binding human monoclonal antibody that is difficult to exert a practical killing effect on malignant cells when used alone. The present invention relates to a human monoclonal antibody composite agent for treating malignant tumor, which contains at least two kinds and exhibits an excellent additive effect which can selectively exert a highly unexpected and surprisingly high killing effect on malignant tumor cells.

More specifically, the present invention provides a human monoclonal antibody (α) produced by a human / human hybridoma capable of producing an antibody capable of binding to a malignant tumor, and another antibody capable of producing an antibody capable of binding to a malignant tumor of the same species. The present invention relates to a human monoclonal antibody composite agent for treating malignant tumor, which comprises at least one human monoclonal antibody (β) produced by human / human hybridoma.

Conventionally, a human monoclonal antibody produced by a human / human hybridoma having a malignant tumor-binding antibody-producing ability has been included, and a pathogenic antigen-specific human immunoglobulin-containing substance or a method for producing the human immunoglobulin, and Examples of the malignant tumor-binding human monoclonal antibody obtained by the method described in JP-A-58-201994 and 59-59.
It is disclosed and known in JP-A-135898 and JP-A-59-137497. However, these proposals do not describe or suggest any findings regarding the combined use of a plurality of human monoclonal antibodies produced by human / human hybridomas.

On the other hand, a mouse that produces antibodies that bind to human malignant melanoma /
Regarding the test using mouse monoclonal antibody produced by mouse hybridoma and mouse monoclonal antibody produced by other mouse / mouse hybridoma producing antibody that binds to the tumor of the same species, The Journal of Imm
unology, vol, 127, NO.1, July, 1981, p. 1
57-160 reports are known. According to this report, it is reported that the above-mentioned combined use in the coexistence of complement invented the phase-addition effect. However, this report does not describe or suggest the combined use of multiple human monoclonal antibodies produced by human / human hybridomas.

The present inventors have found that human / human hybridoma-produced human monochrome obtainable by the method disclosed in JP-A-58-201994, JP-A-59-135898 and JP-A-59-137497. Studies have been conducted on the treatment of malignant tumors using null antibodies, particularly human monoclonal antibodies that bind to malignant tumors.

As a result, when used alone, it is necessary to use at least two types of human monoclonal antibodies capable of exerting a practically satisfactory killing effect on malignant tumor cells and / or malignant tumor-binding human monoclonal antibodies. It was discovered that it is possible to selectively exert a highly unexpected and surprisingly high killing effect on the malignant tumor cells.

According to the study by the present inventors, as shown in the test example below,
Of human monoclonal antibody (α) produced by a human / human hybridoma capable of producing a malignant tumor-binding antibody,
The amount of human monoclonal antibody (β) produced by another human / human hybridoma having the ability to produce an antibody that binds to a malignant tumor of the same species by itself does not have a substantial killing effect on the tumor. It has been discovered that, by using the same amount in combination, an excellent reductive killing effect can be selectively exerted on the malignant tumor cells under the preservation of complement.

Furthermore, the Journal of Immunology, vol. 127, N.
O.1, july, 1981, p. 157-160 report that the two mouse monoclonal antibodies have two binding sites p9 of the same antigen in one malignant melanoma cell.
7 ^a and be coupled to p97 ^b, competitive binding analysis (co
mpetition binding assays). According to the study by the present inventors, the human monoclonal antibody (α) and the human monoclonal antibody (β) in the combination drug of the present invention are associated with one malignant tumor cell, for example, one brain tumor cell U-373MG (ATCC HTB17). It was confirmed by the affinity chromatography and the fluorescent antibody method that they each bind to one binding site of different antigens in (1). This is completely different from the known mechanism of action in the case of the above two mouse monoclonal antibodies,
It may be meant that the inventive combination has a mechanism of action with a much increased chance of attack on malignant tumor cells. And, although the details of the mechanism of action are not yet clear, as shown in the test examples below, similar to human monoclonal antibody (α), which does not show a substantial killing effect on malignant tumor by itself, as shown in the following test examples. It has been found that a combination of different amounts of human monoclonal antibody (β) achieves a high killing effect of about 80% or more. On the other hand, according to the above report, regarding the combined use of the two mouse monoclonal antibodies, in addition to the different mechanism of action,
It has only been shown that at most about 70% of the killing effect is achieved by the combined use in amounts which do not show a substantial killing effect on their own.

Therefore, an object of the present invention is to provide a new type of human monoclonal antibody conjugate for malignant tumor.

These and many other objects and advantages of the present invention will become more apparent from the description below.

According to the present invention, a human / human hybridoma (α) produced by a human / human hybridoma capable of producing an antibody capable of binding to a malignant tumor, and another human / human capable of producing an antibody capable of binding to a malignant tumor of the same species. It is possible to provide a human monoclonal antibody complex for malignant tumor, which comprises at least one kind of human monoclonal antibody (β) produced by a hybridoma.

Examples of the human monoclonal antibody produced by a human / human hybridoma capable of producing a malignant tumor-binding antibody used in the complex of the present invention include those described in JP-A-58-2.
01994, JP-A-59-135898, JP-A-5
Human / human hybridomas having malignant tumor-binding antibody-producing ability, which can be obtained by a method known per se disclosed in 9-137497, etc., are produced by the method known in the proposal, and collected. Human malignant tumor-binding human monoclonal antibody can be used.

In the present invention, for example, a human monoclonal antibody (α) produced by a human / human hybridoma capable of producing a malignant tumor-binding antibody that can be obtained as described above and a homologous antibody that can be formed in the same manner as There are two types of human monoclonal antibodies (β) produced by other human / human hybridomas having the ability to produce antibodies that bind to malignant tumors, or still other human / human hybridomas having the same ability to produce antibodies. At least two human / human hybridomas each having the ability to produce the same type of malignant tumor-binding antibody, such as three types of the produced human monoclonal antibodies (γ), or four or more types, were produced respectively. Species of human monoclonal antibodies are used in combination.

Examples of such human monoclonal antibodies include those described in JP-A-58-201994 and JP-A-59-135898.
No. and human / human hybridoma CLN / SUZ H5 disclosed in detail in JP-A-59-137497.
Human I produced by a strain (ATCCHB8206; Notification of Refusal of Deposit of Micro Engineering Laboratory, Notification No. 57: Micro Contribution No. 637)
gM, human / human hybridoma CLN / SUZ H
Human IgG produced by the 11 strains (ATCC HB8307; Notification of Refusal of Deposit of Microtechnology Research Institute, Notification No. 57: Microcontribution No. 638), and further, JP-A-60 relating to the application of the same applicant.
-210310 (filed September 24, 1985), a subclone of human B-cell of a human gastric cancer patient and a human lymphoblastoid cell line, for example, UC729-6 strain (ATCC CRL8061; refused to deposit by IIT) Notification, Human IgG produced by human / human hybridoma SLNF10 (Notification No. 57 slightly contributed 664) (Notification of refusal of deposit accepted by Institute of Mechanical Engineering, Notification No. 60 slightly contributed 1197), and other special features mentioned above. Human monoclonal antibodies produced by various malignant tumor-binding human / human hybridomas, which can be similarly formed, can be mentioned in accordance with the method known in Japanese Patent Laid-Open No. 58-201994.

Human / human hybridoma CLN / S exemplified above
Human IgM produced by the UZ H5 strain includes, for example, cell lines Hela and CaSki derived from cervical cancer, cell lines Calu-1, SK-MES-1 and T293H derived from lung cancer, Ln-Cop derived from prostate cancer, Human I produced by the human / human hybridoma CLN / SUZ H11 strain, which is capable of binding to malignant tumors such as cell line AGS derived from gastric cancer and cell line G361 derived from malignant melanoma
gG is, for example, a cell line Hal derived from cervical cancer.
a, Hela229 and CaSki, lung cancer-derived cell line A549, prostate cancer-derived cell line PC-3, gastric cancer-derived cell line AGS, malignant melanoma-derived cell line G3
61, a colon adenocarcinoma-derived cell line T84, a brain tumor-derived cell line U-373MG, and the like, which are binding to malignant tumors, and human / human hybridoma SLN F10
The human IgG produced by the strain is, for example, cell line Hela229 derived from cervical cancer, cell line A54 derived from lung cancer.
9, cell line AGS derived from gastric cancer, cell line U-373MG derived from brain tumor, cell line G361 derived from malignant melanoma,
Since it is capable of binding to malignant tumors such as cell line PC-3 derived from prostate cancer and to at least one common malignant tumor, they can be appropriately selected and used in combination.

As described above, the human monoclonal antibody complex for malignant tumor of the present invention comprises at least two human / human hybridomas having the same malignant tumor-binding antibody-producing ability,
Each of them may contain at least two kinds of human monoclonal antibodies produced, and each human / human hybridoma is separately cultured, and the human monoclonal antibody isolated from each culture is prepared. It is not necessary to obtain at least two types of human / human obtained by culturing both human / human hybridomas together and separating them from a culture containing both human monoclonal antibodies in a mixed state. -The hybrid agent may be a composite agent containing at least two human monoclonal antibodies produced by each hybridoma. However, it is usually preferable to adopt the former mode.

The human monoclonal antibody complex for malignant tumors of the present invention contains at least two types of human monoclonal antibodies (α) and (β) capable of producing an antibody that binds to malignant tumors of the same species. Various known biopharmaceutical additives and other additives for preparations can be contained.

Examples of such additives include, for example, polymerization inhibitors such as human serum albumin; sugars such as mannitol, glucose, hexose, fructose, sucrose, etc .; for example, glycine, glutamine, itleucine, leucine, methionine. , Amino acids such as valine, arginine, triplophane, threonine, etc .; for example, sodium chloride, potassium chloride, calcium chloride,
Inorganic salts such as phosphates; Excipients such as lactose, soluble starch; Lubricants such as magnesium stearate; Dispersed income such as light anhydrous silicic acid; Surfactants such as hydroxycholic acid; binders such as hydroxypropyl cellulose, carboxymethyl cellulose, methyl cellulose; bases such as polyethylene glycol, glycerin, petrolatum, paraffin, wax, glycerogelatin, etc .; For example, oils and fats such as olive oil, soybean oil, corn oil, peanut oil, sesame oil, camellia oil, pork oil, cacao butter, and purified lanolin; for example, cresol, chlorobutanol, benzyl alcohol, paraoxybenzoates, thimerosal, Benzalco chloride Um, and the like can be exemplified such preservatives such as benzethonium chloride.

The human monoclonal antibody complex for malignant tumors of the present invention can be applied in various dosage forms depending on the type of tumor and the site of development. Such dosage forms include various injection drug forms, orally administered drug forms, drip drug forms, suppository drug forms,
It may be a dosage form such as an eye drop dosage form or a patch dosage form. Dispensing techniques per se for such dosage forms are well known and can be used in the present invention.

The human monoclonal antibody complex for malignant tumors of the present invention can be applied by various administration methods suitable for the type and site of malignant tumor. For example, injection methods such as local injection, subcutaneous injection, intramuscular injection, intravenous injection, arterial injection, drip application, topical external application, eye drop application, endoscopic spraying and injection application, inhalation application, etc. You can

The human monoclonal antibody complex for malignant tumor of the present invention exerts an excellent effect in the presence of complement, but since complement exists in the body, it is not necessary to mix it with the agent of the present invention. The complex agent of the present invention is useful for diagnosis, prevention, treatment, prevention of recurrence and metastasis of various malignant tumors. Examples of such malignant tumor include, for example, brain tumor, tongue cancer, laryngeal cancer, lung cancer, esophageal cancer, gastric cancer, intestinal cancer, liver cancer, pancreatic cancer, breast cancer, uterine cancer, renal cancer, prostate cancer, blood cancer, skin. Examples include cancer osteosarcoma.

The dose of the complex agent of the present invention can be appropriately selected and changed depending on the combination of human monoclonal antibodies, the tumor to be administered, the symptoms, etc.
About 20 μg to 2 mg / kg / day for daily administration by injection, about 1 mg to about 1 per 1 g of base material for topical administration such as ointment for external use
0 mg, in the case of suppository administration about 1 mg to about 20 per 1 g of base material
A dose such as mg can be exemplified.

The pharmacological effects of the human monoclonal antibody complex of the present invention are shown below.

(Test Example 1) Glioma cells (CU-373), which is one type of brain tumor cells
MG) was suspended in DF medium (DME: F-12 = 1: 1) containing 10% fetal bovine serum (FBS). This cell suspension was added to a microplate with 96 holes to obtain 10 cells.
^The cells were seeded at ⁵ / well to adsorb the cells to the plate. The medium was removed, and RDF medium containing 10% fetal bovine serum (RPMI1640: DME: F-12 = 2: 1:
Human x human hybridoma (LN / SU) cultured in 1)
Human x human hybridoma SLNF10 cultured in RDF medium containing 50 ml of culture supernatant containing IgG produced by ZH11 (derived concentration of 10 ⁶ / ml) and 10% fetal bovine serum.
50 μl of culture supernatant containing (produced IgG at a cell concentration of 10 ⁶ / ml) and 50 μl of a rabbit-containing complement-containing solution
Was added and the cells were cultured for 4 hours in an incubator under the conditions of 37 ° C. and 5% CO ₂ . After culturing, remove the medium and add 0.25
% Cells were treated with a trypsin solution and then killed with 0.2% trypan blue staining solution, and the number of unstained live cells was counted by a hemocytometer. The results are shown in Table 1 below (Rwm NO.8).

For comparison, a control example in which U-373MG cells were cultured in an RDF medium containing 10% fetal bovine serum without complement (Ru
n NO. 1), the above-mentioned two types of IgG-containing culture supernatants and the above-mentioned complement-containing liquid, one or two of which were omitted, and comparative examples (Run NO. 2 to NO. The results of 7) are shown together in Table 1.

From the results shown in Table 1 above, the amounts by which human monoclonal antibodies alone have no substantial killing effect (Run NO. 2 and N
O.3), by using these two human monoclonal antibodies in combination in the presence of complement, an excellent synergistic killing effect can be achieved as shown in Run NO.8. Recognize.

(Test Example 2) Glioma cells (U-373MG), which is one type of brain tumor
1 × 10 ⁴ cells are inoculated into a medium containing 10% FBS in a basal medium of DME: F-12 = 1: 1 and cultured for 5 days. Remove the old medium and run each Run N shown in Table 2 below.
200μ so that the concentration of constituents is from O.1 to NO.8
The reaction solution of was prepared. Specifically, in 200 μ, CLN-IgG (CL
N / SUZ H11-produced IgG) was used in an amount of 5 µg or 10 µg, and SLN-IgG (IiG produced by SLN F10) was used in an amount of 5 µg or 10 µg. The above U-373MG cells were cultured at 37 ° C. in a 5% CO ₂ incubator for 9 hours in a culture medium composed of Run Nos. 1 to 8 respectively. After culturing, remove the medium and treat the cells with a 0.25% trypsin solution.
Dead cells were stained with a 2% trypan blue staining solution, and the number of unstained live cells was counted by a hemocytometer. The results are shown in Table 2 below.

From the results shown in Table 2, it was found that by using two human monoclonal antibodies in combination in the presence of complement in an amount such that each human monoclonal antibody alone has no killing effect, excellent synergistic effect was obtained as shown in Run NO.8. It can be seen that a great killing effect is achieved.

Hereinafter, some examples of the composite agent of the present invention will be shown.

Taking the above, it is manufactured by the Japanese Pharmacopoeia injection method.

Taking the above, it is manufactured by the Japanese Pharmacopoeia injection method.

Formulation Example 3 (pasta) CLN-IgG 50 mg SIN-IgG 50 mg Zinc oxide 25 g Starch 25 g White petrolatum 48 g Manitol 2 g Take the above ingredients and prepare according to the Japanese Pharmacopoeia method and suppository method.

Claims (1)

[Claims] 1. A human monoclonal antibody (α) produced by a human / human hybridoma capable of producing an antibody capable of binding to a malignant tumor, and another antibody capable of producing an antibody capable of binding to a different antigen of the same type of malignant tumor. A human monoclonal antibody composite agent for malignant tumor, comprising at least one kind of human monoclonal antibody (β) produced by human / human hybridoma.