JPH0390037A - Liposome pharmaceutical - Google Patents

Abstract

PURPOSE:To obtain an antitumor agent, composed of a conjugant of a liposome containing a lymphotoxin to a monoclonal antibody and capable of exhibiting powerful tumor proliferation inhibitory effects as compound with those in single use of the lymphotoxin. CONSTITUTION:A liposome pharmaceutical obtained by containing a lymphotoxin(LT) in a liposome and then linking the resultant LT-containing liposome to a monoclonal antibody(e.g. monoclonal antibody against an antigen held by cancer cells). Methods for linking the antibody to a membranous lipid of the liposome, modifying the antibody, introducing hydrophobic groups and thereby integrating the antibody onto liposome membranes; integrating a polymer, having hydrophobic groups and rich in reactivity onto the liposome membranes and linking the aforementioned polymer to the antibody, etc., are cited as a method for linking both.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention contains at least lymphotoxin (
The present invention relates to an antitumor agent comprising a conjugate of a monoclonal antibody and a liposome containing LT (hereinafter sometimes abbreviated as LT).

[Conventional technology]

LT receives antigen stimulation from lymphocytes and lymphoid cell lines,
Alternatively, it is a type of lymphokine produced by mitogen stimulation and is an antitumor agent (Evans C) because it has toxicity against various cancer cells.

■, et Cancer Res, 37, p 8
98 (1977)), antiviral agents (G, H, W, Wo
ng & D., V., Goeddel et al. Nature32
3. p819. (1986)) and is expected to be applied to medicine as a macrophage activator [JP-A-64-42441]. However, if a large amount of LT is administered in order to produce these effects, side effects are observed, so it is desired to provide a device that is effective with a smaller amount or a technical means with fewer side effects.

On the other hand, as a conventional technique, there has been an attempt to encapsulate LT in liposomes, but there is no data and no effect has been shown (Japanese Patent Laid-Open No. 61-56197).

[Problem to be solved by the invention]

With the aim of obtaining a preparation that exhibits the above-mentioned effects when used in a small amount, the present inventor has conducted extensive studies and found that after incorporating LT into liposomes, the resulting LT-containing liposomes are combined with a monoclonal antibody. The present invention was completed by discovering that it is sufficient to do so.

[Means to solve the problem]

That is, the present invention provides an antitumor agent comprising a conjugate of a lymphotoxin-containing liposome and a monoclonal antibody.

The present invention will be explained in detail below.

The LT used in this invention has an amino acid sequence substantially equivalent to the amino acid sequence represented by the following general formula: There is one. Such LT may be either a natural type or a genetically recombinant type, but preferably a genetically recombinant type. Specific examples include JP-A-64-6298, JP-A-63-
Examples include proteins disclosed in JP-A No. 8698, JP-A-63-8399, and JP-A-62-151182.

Furthermore, examples of monoclonal antibodies include those directed against antigens possessed by target sites, lesions, and cells. If you want to accumulate it against cancer, you should use monoclonal antibodies against antigens possessed by the cancer cells, such as proteins such as CEA, transferrin receptor (Tf-R), AFP, etc., or cancer-specific m chains. Antibodies are listed. If you want to accumulate it in the tissue of a lesion, a monoclonal antibody against an antigen specific to the tissue cell is a good example of application, and if you want to have it act on immunocompetent cells, you can use a marker antigen possessed by the target immunocompetent cell. Examples include monoclonal antibodies. Actual CE
It has been confirmed that monoclonal antibodies accumulate in cancer tissues when monoclonal antibodies are administered in vivo to cancer cells containing A, Tf-R, etc., and are used in the examples of the present invention. Human melanoma cell line A-37
Mouse monoclonal antibody against 5 (AM-4E3)
It has also been confirmed that antibodies accumulate at tumor sites in experiments using nude mice (Watanabe, et al., J. Biol., Re5ponse nod, 6).
(5), 556° (1987)). From the above, it can be inferred that monoclonal antibody-modified liposomes in which monoclonal antibodies and liposomes are bound also accumulate at the target site. Further, these monoclonal antibodies may be human antibodies, human-mouse chimeric antibodies, mouse antibodies, etc., and may be F(ab')g or F(ab'), which are parts of antibodies, in addition to the antibodies themselves.

In the present invention, liposomes are closed vesicles with a lipid multilayer structure obtained by dispersing phospholipids in a saline solution.
The formation of liposomes can be carried out by known methods such as a thin film method, a solution injection method, a surfactant treatment method, an ultrasonic method, a freeze-thaw method, and a reverse phase evaporation method.

Materials include phospholipids, such as phosphatidylcholine,
Phosphatidylserine, phosphatidylethanolamine, lysolecithin, sphingomylin, dicetyl phosphate, stearylamine, phosphatidylglycerol,
Examples include, but are not limited to, phosphatidic acid, phosphatidylinositol, modified versions thereof, and mixtures thereof. Further, as a reinforcing agent for the liposome bilayer membrane, a neutral lipid such as cholesterol may be used as a stabilizer, and glycolipids, glycoproteins, etc. may be added. In short, any phospholipid that can form liposomes may be used, and phosphatidylcholine such as lecithin is a preferable material for the phospholipid that is the basic constituent of liposomes.

The monoclonal antibody/lipid molar ratio is between 1 and 10-10. In addition, methods for binding monoclonal antibodies to liposomes include: ■ adsorbing the antibody onto the surface of the liposome; ■ incorporating the antibody onto the liposome membrane;
■ A method of binding the antibody to the liposome membrane lipid via a spacer such as 5PDP, ■ A method of incorporating the antibody onto the liposome membrane by modifying the antibody and introducing a hydrophobic group, ■
A method of incorporating a highly reactive polymer with hydrophobic groups onto the liposome membrane and binding the polymer to an antibody (Tor
chilin V.

P,, CRCCr1tical Reviews i
n Therapeutic Drug Carrier
Systems vol, 2+ l5sue l
), etc., but methods ①, ②, and ③ are particularly preferred because they actively bind the antibody to the membrane.

An example of this will be given below, and a method of coupling via a spacer such as 5PDP will be explained. monoclonal antibody
-Succinimidyl 3 (2-pyrt
dyidHhio ) propinate (S
After conversion into 5PDP using PDP), dithiothreitol (DTT) is added and SH conversion is performed to obtain an SH-antibody. On the other hand, phosphatidylethanolamine (PE) and N-(3-
(2-pyridyldithio) propyn
yl) phosphotidyl ethanol a
Bottom out a+ine (P D P - P E ) (
Martin, F. J. et al., Bioche
s+1try Dori, 4229. (1981)). Next, a monoclonal antibody-modified LT-containing liposome can be obtained by preparing an LT-containing liposome incorporating FDP-PE and reacting it with an SH-antibody.

Monoclonal antibody-modified LT-containing liposomes can be used as an injection as a suspension, as an inhaler as an aerosol, or as a powder after freeze-drying, but depending on the antibody selected, it can be accumulated at a high concentration at the desired target site. Therefore, compared to direct administration of LT, the effect can be expected with a smaller amount, and since LT is less administered to unnecessary areas, side effects can also be reduced.

〔Example〕

EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the following examples.

The monoclonal antibody producing strain is disclosed in JP-A-62-11193.
As shown in 4, Ba1b/c mice (5 weeks old, female)
The human melanoma cell line A-375 was immunized with the human melanoma cell line A-375, and the lung cells were fused with the mouse myeloma m cell line 5p-2, and the binding ability to fixed A-375 was used as an indicator. A monoclonal antibody (AM-423) against the human melanoma cell line A-375 produced by this strain was obtained in large quantities from mouse ascites and purified using ammonium sulfate salting out, gel filtration, and ion exchange chromatography. This antibody was used as N-SucN-5u.
ccint 3 (2pyridyldithio
) propionate (SP D
) was added and converted into SH to obtain an SH-antibody. On the other hand, SH
Phosphatidyl ethanol agone (PE) and N-(3(2pyrid
yldithio)propynyl)phosp
liatidyl ethanol amine (P
D P -P E ) was synthesized. Go or Mart
The method of in et al. (Martin, F.

J, et al., Biochemistry20
, 4229.

(1981)), 5PDP25 was dissolved in methanol 3 and 18.4 μl of triethylamine, PE100
d was added and reacted for 5 hours at room temperature in the dark, and the unreacted 5
It was obtained by removing PDP with a silica gel column.

Egg yolk lecithin, FDP-PE, cholesterol total 10■
were mixed in a molar ratio of 9:l:to, dissolved in 300μl of ether, and mixed with the recombinant LT solution (5.5X 10'μ/
d) LOOμl was added and emulsified by sonication (2 minutes).

The ether was removed under reduced pressure and dissolved in PBS (sodium chloride 7.
5% containing 5mM phosphate buffer, pH 7.2) 4.7
Add wit, mix, and centrifuge (50,0OOG, 1
The supernatant (LT?V solution that was not encapsulated) was removed, and 500 μl of SH-antibody solution (1 μ/v) was added to the precipitate, mixed, and reacted overnight at 4'C. After the reaction, wash twice with PBS using a centrifuge, and add the precipitate to 500 μA of PBS.
The suspension was suspended in water and sterilized by passing it through a 0.4 μm polycarbonate membrane (manufactured by UCRIBOAR). When the LT uptake rate was examined using 3H-labeled fucose, the yield was 8.4%. Therefore, the monoclonal antibody-modified LT-containing liposome prepared was 2.5X103μ/-
It was estimated that

Four-week-old male Ba1b/c nu/nu mice were used to examine the tumor growth suppressive effects of monoclonal antibody-modified LT-containing liposomes and warm LT solutions against human melanoma A-375 in vivo. A-375 subcutaneously in the right inguinal region of Ba1b/c nu/nu mice! [1 x l0' cells were transplanted, and from the date of transplantation 2.5.9.12.16.19 and 2
Once on the third day/total of 7 times a day, monoclonal antibody-modified LT-containing liposomes prepared in Example 1, monoclonal antibody-modified LT-free liposomes (Comparative Example 1), monoclonal antibody-modified LT-incompatible liposomes, and LT-containing liposomes were tested. Mixed solution (Comparative Example 2), LT warm solution Comparative Example 3) at 200 μl/
administered to animals.

In order to prevent monoclonal antibody-modified LT-containing liposomes from being ingested by the reticuloendothelial system, 2 ml of liposomes containing phosphatidylserine, phosphatidylcholine, and cholesterol were administered to each animal one hour before administration of each sample.

The tumor area (breadth axis x long axis) on the 25th day was examined, and the growth inhibition rate (%) was calculated by the following formula by comparing with the physiological saline administration group.

Growth inhibition rate (%) = Results are shown in Table 1. The tumor area in the physiological saline administration group was 39 m''.

Surprisingly, the group administered with the monoclonal antibody-modified LT-containing posome showed a significant tumor growth suppressive effect despite the dose at which LT alone had little effect.

〔Effect of the invention〕

Monoclonal antibody-modified LT-containing liposomes showed a stronger tumor growth suppressive effect than when LT was used alone. From the above, it is possible to reduce the amount of LTO required to expect the same effect, and also to reduce side effects, so it can be expected to be used as an excellent antitumor agent.

Special permission Out wish Man Denki Kagaku Kogyo Co., Ltd. hand Continued Supplementary Positive book Flat $, October 1 year 4 days

Claims (1)

[Claims] An antitumor agent consisting of a conjugate of a liposome containing lymphotoxin and a monoclonal antibody.