JPH0355450B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a new form of anticancer formulation. More specifically, in order to selectively transport anticancer substances to cancer tissues and maintain high local drug concentrations,
This is a cancerous drug that uses liposomes with antibodies against cancer bound to the membrane. Anticancer drugs currently on the market are mainly chemotherapeutic agents consisting of cytotoxic substances against cancer cells, and immunotherapeutic agents that have an indirect anticancer effect by enhancing the immune capacity of cancer-bearing patients. In immunotherapy as well, concomitant use of other chemotherapeutic agents is required to enhance the anticancer effect. However, chemotherapeutic agents generally do not have selectivity between cancer cells and normal cells, and severe side effects are unavoidable. In view of these circumstances, the present inventor has completed the present invention as a result of research in order to selectively exert the strong cytotoxicity of chemotherapeutic agents on cancer cells. That is, the present invention provides actinomycin D in the membrane.
This is an IgM monoclonal antibody-modified liposome containing. In carrying out the present invention, the anticancer substance is mixed with a solution of lipids constituting liposomes, and the lipid-soluble anticancer substance is mixed with a solution of lipids constituting liposomes, and the liposomes are formed into unilamellar liposomes by adding operations such as ultrasonication using conventional methods. The water-soluble anticancer substance can be encapsulated in the lipid vesicles and present in the form of microcapsules. On the other hand, in order to bind a monoclonal antibody onto a membrane, an antibody fragment having an SH group is prepared and used. In the case of IgM, the IgM antibody is reduced under mild conditions, for example with cysteine, to reduce only the J chain and prepare IgM subunits (IgMs) having two mercapto groups. In the case of an IgG antibody, it is digested with pepsin and then reduced with mercaptoethanol to isolate and purify Fab' having one mercapto group for use. On the other hand, when forming liposomes, phospholipids having amino groups, which are one of the constituent components, such as dipalmitoylphosphatidylethanolamine (DPPEA) and m-maleimidobenzoyl-N-
m-maleimidobenzoyl-N-(dipalmitoylphosphatidyl)ethanolamine (MBPEA) obtained by reaction with hydroxysuccinimide ester is mixed with other lipids such as dimyristoyl lecithin, dipalmitoyl lecithin, distearoyl lecithin, etc. One or more of lecithins, phosphatidylinositol, phosphatidylserine, phosphatidic acid and cholesterol are mixed in an appropriate ratio and dissolved in a fat-soluble organic solvent such as chloroform, followed by forming liposomes by a conventional method. . The liposomes obtained here are placed on the membrane.
There are many maleimide groups derived from MBPEA. IgMs prepared with light was added to the phosphate buffered saline (PBS) solution of this liposome and incubated at 37°C for about 1 hour to perform the SH addition reaction. Add cysteine. In this way, multiple IgMs can be bound to the liposome membrane using the Fc portion without any effect on the Fab portion, which is important for antigen recognition. The liposome of the present invention, which contains an anticancer substance and has a monoclonal antibody against a cancer antigen on its membrane, which is obtained in this way, acts specifically on cancer tissues and transfers the anticancer substance into cancer cells. Therefore, it is used as an anticancer drug with few side effects. Production example (1) Preparation of monoclonal antibody Hyperimmunized with MM-46 cells (ascitic breast cancer cells naturally occurring in C3H mice),
(BALB/c×C3H/He) _F1 mouse splenocytes and 8-azaguanine-resistant myeloma cells NS producing cytotoxic antibodies (antibody titer 1/640)
Hybridoma 2-11-G clone obtained by fusion of
1×10 ⁶ cells were inoculated into 20 to 25 ml of MEM-10% FCS medium and cultured for 4 to 7 days at 37° C. in a 5% CO ₂ incubator. Collect the supernatant, perform 50% ammonium sulfate salting out twice, and apply it to a Sephacryl S-300 column (90 x 3.2 cm).
The purified IgM fraction was eluted with 0.2M Tris-HCl buffer (PH8.6) and concentrated by ultrafiltration. (2) Adjustment of IgMs Cysteine was added to the IgM obtained in (1) to a final concentration of 0.05M, and Miller &
Following Metzger's method, after reacting and reducing at 24°C for 8 minutes, a Sepharose CL6B column (30
x 1.8 cm), PBS containing 2 m MEDTA (PH
7.0) for elution and purification. The yield from IgM was approximately 50% in terms of protein content. (3) Synthesis of MBPEA 25μM m-maleimidobenzoyl-N-hydroxysuccinimide ester and
Dissolve 20 μM of DPPEA in 5 ml of chloroform-methanol (9:1) and add triethylamine.
Add 30 μM and stir at room temperature. After the reaction is complete,
Add 3.5 ml of methanol and 2 ml of water to the reaction solution, collect the lower chloroform layer, add a small amount of benzene to this, distill off the solvent, add 2 ml of chloroform to the residue, and purify by applying to a Unisil column. The resulting MBPEA 1M contains 1M phosphate ion, which was determined by colorimetric determination by turning an aqueous molybdenum solution blue in the presence of a reducing agent, and also contained 1M phosphate ion, which was determined by colorimetric determination by turning an aqueous molybdenum solution blue in the presence of a reducing agent. Quantitative tests confirmed the presence of 1M maleimide groups. (4) Preparation of liposome Dipalmitoyl lecithin 50μM MBPEA 5μM Cholesterol 35μM was dissolved in chloroform, further 500μg of actinomycin D was added thereto, the mixture was made homogeneous and the solvent was distilled off.
After stirring for 10 minutes, the mixture was subjected to ultrasonic treatment while sufficiently cooling, and centrifuged at 10,000 rpm for 30 minutes to obtain a supernatant containing small-sized liposomes. (5) Add the IgMs obtained in (2) to 4 ml of the supernatant obtained in (4).
Add 2 ml of the fraction and incubate at 37°C for 1 hour, then add 5 mg of cysteine to remove unreacted maleimide groups to prevent reaction with SH groups such as serum albumin, and incubate at 37°C for 30 minutes to incubate. Activated. Liposomes were then isolated using a concentration gradient (0-20%) of dextran to remove unencapsulated actinomycin D and excess cysteine. Test example [anticancer effect] C3H/He mice (male, 6 weeks old) 5×10 ⁴ in the peritoneal cavity
MM-46 cancer cells were transplanted. After 24 hours, 50μ of the liposomes of the present invention prepared with varying amounts of actinomycin D were administered to examine the anticancer effect. For comparison, actinomycin D alone (AD),
AD-free antibody-conjugated liposomes (2-11-
GsLip), AD-containing liposomes without antibody (Lip-AD) AD with BSA conjugated instead of antibody
The table shows the results of administering each of the containing liposomes (BSA-Lip-AD) and examining the effects on cancer. In addition, each liposome for comparison was prepared according to the above-mentioned production example.

[Table]
As is clear from the table, the liposomes of the present invention have excellent anticancer effects. Although this example shows the case of AD, the liposome of the present invention containing other chemical therapeutic agents in a much smaller amount than the usual dose can also bring about significant anticancer effects. .

Claims (1)

[Claims] 1. IgM containing actinomycin D in the membrane
Monoclonal antibody-modified liposomes. 2. A step of reducing only the J chain of a cancer-specific IgM monoclonal antibody to prepare an IgM subunit having a mercapto group in the Fc portion, using a reagent having a reactive group that reacts with a mercapto group and a liposome-forming material. When liposomes are formed by a conventional method, actinomycin D, which is a fat-soluble anticancer drug, is added to the liposomes to produce liposomes containing actinomycin D in the liposome membrane and having a large number of mercapto groups on the liposome membrane. A method for producing an IgM monoclonal antibody-modified liposome containing actinomycin D in its membrane, comprising the steps of: reacting the above liposome with the above IgM subunit. 3. The method for producing an IgM monoclonal antibody-modified liposome according to claim 2, wherein the reagent having a reactive group that reacts with a mercapto group is m-maleimidobenzoyl-N-(dipalmitoylphosphatidyl)ethanolamine.