JPH09110722A - Immunoliposome for transducing tumorous cell of antitumor active substance and its preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To produce an immunoliposome, capable of effectively and selectively transducing an antitumor active substance into a tumorous cell by bonding a cationic liposome membrane containing the antitumor active substance to a specific antibody selectively reactive with the tumorous cell. SOLUTION: This immunoliposome for transducing an antitumor active substance into a tumorous cell is obtained by bonding a cationic liposome membrane containing the antitumor active substance to a monoclonal antibody G-22 (antiCD 44 antibody) selectively reactive with the tumorous cell. A ceramide or an antiFas antibody capable of efficiently inducing apoptosis, a magnetite capable of manifesting hyperthermic effects and a gene effective in antitumor effects are preferred as the antitumor active substance. Dimethyldioctadecylammonium bromide and dioleylphosphatidylethanolamine are preferred as a lipid constituting the cationic liposome membrane. A cell of a glioma, a cell of melanoma, etc., which are cells excessively expressing CD44 are preferred as the tumorous cell to be an object of treatment. Furthermore, the daily dose of the immunoliposome for an adult is preferably 10-1000nmol expressed in terms of the amount of the lipid.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an immunoliposome capable of efficiently inducing apoptosis by selectively introducing ceramide or anti-Fas antibody into tumor cells, and hyperthermia by selectively introducing magnetite or gene into tumor cells. Alternatively, it relates to an immunoliposome having characteristics as a vector for gene therapy, and a method for preparing them.

[0002]

[Prior art]

1. Ceramide or anti-Fas antibody Apoptosis is a typical mode of death of programmed cell death (normal development, physiological cell death occurring at a specific site at a specific time as an essential condition for differentiation). It is characterized by chromatin condensation, cytoplasmic enrichment, nuclear enrichment and their fragmentation, membrane-encapsulated spheroids, phagocytosis and digestion of spheroids by adjacent macrophages and epithelial cells. In conventional cancer treatments (chemotherapy, radiation therapy, etc.), most of the tumor cells were caused to die by necrosis. Necrosis, unlike apoptosis, induces an inflammatory reaction in the surrounding tissues, which is accompanied by strong adverse effects such as fever, nausea, and vomiting. On the other hand, apoptosis is expected to have very few adverse effects because it does not involve an inflammatory reaction in the surrounding tissues. Therefore, it is considered that the induction of cell death by apoptosis of tumor cells is effective in reducing the harmful effects and leading to an efficient therapeutic method in view of clinical application.

The mechanism of apoptosis is diverse and there are many points that have not yet been elucidated, but the sphingomyelin metabolic pathway in which ceramide is involved is one of the cytokines, TNF-.
It is thought to constitute an early event in the apoptotic cascade of programmed cell death induced by α (tumor necrosis factor) [Jarvis WD, Kolesnick RN, Fornari FA, Tra
ylor RS, Gewirtz DA, Grant S, Induction of apopto
tic DNA damage and cell death by activation of the
sphingomyelin pathway, Proc. Natl. Acad. Sci. USA,
91, 73-77 (1994)]. Ceramide, which is formed by hydrolysis of sphingomyelin, is considered to be the second messenger that mediates the effects of TNF-α on cell proliferation and differentiation as well as apoptosis [Kolesn
ick R, Golde DW, The sphingomyelin pathway in tumo
r necrosis factor and interleukin-1 signaling, Cel
l, 77, 325-328 (1994)]. In some types of cells,
Apoptosis is induced by stimulation with TNF-α. In some glioma cells, induction of apoptosis was observed only at high doses, but rather it was exceptional and apoptosis was not induced in many glioma cells. On the other hand, there is a paper showing a method of inducing apoptosis by dissolving C2-ceramide, a synthetic ceramide analog, in ethanol to efficiently permeate cell membranes [Obleid LM, Lin
ardic CM, Karolak LA, Hannun YA, Programmed cell d
eath induced by ceramide, Science, 259, 1769-1771
(1993)], but no significant induction of apoptosis was observed in this method by glioma cells. This fact can be said when anti-Fas antibody that induces apoptosis in various cells is added to glioma cells. Anti-Fas antibody has been characterized as a typical antibody capable of inducing apoptosis in various cells, but in glioma cells, its sensitivity is slightly observed only at a high dose. Therefore, in the conventional method, another approach is desired in order to efficiently induce apoptosis in glioma cells using ceramide or anti-Fas antibody.

[0004] 2. Magnetite or Gene Magnetite, which is a magnetic fine particle, is attracting attention as a new player in local hyperthermia. The heat generation mechanism and its characteristics of magnetite as magnetic particles have already been studied in detail, and its status as a thermal derivative is being established [Shinkai M, Suzuki M, Iijima S, Kobayashi T, A
ntibody-conjugated magnetoliposomesfor targeting c
ancer cells and their application in hyperthermia,
Biotechnol. Appl. Biochem, 21, 125-137 (1994)].
Therefore, the success of local hyperthermia depends on how efficiently magnetite can be introduced into tumor cells.

On the other hand, many human gene therapies have been started mainly in the United States, and studies have been conducted using liposomes, which are lipid capsules having high safety and high introduction efficiency, as gene carriers. For example, Felgner et al. Provide that cationic liposomes are an efficient and convenient means of delivering nucleic acids and proteins to various cells.
It proved to have many advantages in in vivo gene transfer [Felgner PL, et al., Proc. Nat
l. Acad. Sci. USA, 84, 7413-7414 (1987), Felgner P
L, et al., Nature, 337, 387-388 (1989)]. Yagi et al. Conducted research on liposome gene transfer in terms of the rate of plasmid embedding in liposomes (referred to as embedding a substance in liposomes), gene transfer into cells, and expression efficiency. N- (α-trimethylammonioacetyl) -didodecyl-D-glutamate chloride (TMAG)
, Which was combined with dilauroylphosphatidylcholine (DLPC) and dioleoylphosphatidylethanolamine (DOPE) in fixed ratios, showed high DNA uptake.
zaka T, Hayashi Y, YagiK, Novel liposomes for effi
cient transfection of β-galactosidase gene into C
OS-1 cells. J. Clin. Biochem. Nutr. 7, 185-192 (19
89)], and that the multilamellar liposome prepared only by shaking is simple and easy to prepare [Yagi K, Noda H, Kurono M, Ohishi N, Effect.
ive gene transfer with less cytotoxicity by means
of combined multilamellar liposomes. Biochem. Biop
hys. Res. Commun 3, 1042-1048 (1993)]. Furthermore, another advantage of the cationic liposome is that it binds a monoclonal antibody or a ligand to attack a specific cell on the liposome. The present inventors, in collaboration with Yagi et al., Targeting human glioma using the above-mentioned cationic liposome (targetin
g) 10 studies of cells studied for therapy and transgenic
˜20% expression of the gene in vitro after liposome-mediated gene transfer, and binding of the anti-human glioma cell monoclonal antibody G-22 to the liposome improves the transfer efficiency and tumor cell specificity. Proved [Mizuno M, Yoshida J, Sugita K, Inoue I,
Seo H, Hayashi Y, Koshizaka T, Yagi K, Growth inhib
ition of glioma cells transfected with the human
β-interferon gene by liposomes coupled with a mo
noclonal antibody. Cancer Res, 50, 7826-7829 (199
0)]. In addition, the binding of these monoclonal antibodies to liposomes is performed by chemical reactions such as Leserman et al. [Leserman et al.
al., Nature, 288,602-604 (1980)], cross-linking agent, N
What was done by using -hydroxysuccinimidyl-3- (2-pyridyldithio) propionate (SPDP), the present inventor uniquely only shakes without performing a chemical reaction. By the method, immunoliposomes having excellent embedding rate in liposomes, gene transfer into tumor cells and expression efficiency were prepared.

Based on the above findings, ceramide or anti-Fas
The use of immunoliposomes as described above is highly expected for the introduction of antibodies or magnetite or genes into tumor cells.

[0007]

An object of the present invention is to provide an immunoliposome capable of efficiently inducing apoptosis by selectively introducing ceramide or anti-Fas antibody into tumor cells, and selectively selecting magnetite or gene into tumor cells. It is intended to provide an immunoliposome having a characteristic as a vector for hyperthermia or gene therapy, and a method for preparing them.

[0008]

As a result of repeated studies to solve the above problems, the inventors of the present invention have found that a monoclonal antibody G-22 which selectively reacts ceramide, anti-Fas antibody, magnetite or gene with tumor cells. By encapsulating or embedding in a cationic liposome bound with (anti-CD44 antibody), it was proved that these can be efficiently and selectively introduced into tumor cells. Furthermore, in the former two cases, they found that the introduction can efficiently induce apoptosis in glioma cells, and completed the present invention.

That is, the present invention is a cationic liposome in which a monoclonal antibody (anti-CD44 antibody) which selectively reacts with an antitumor active substance selected from the group consisting of ceramide, anti-Fas antibody, magnetite and gene is reacted with tumor cells. BEST MODE FOR CARRYING OUT THE INVENTION The present invention relating to an immunoliposome characterized by being encapsulated in or embedded in and a method for preparing the same will be specifically described below.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Liposome Membrane The liposome membrane on which the present invention is based comprises two types of lipids, dimethyldioctadecyl ammonium bromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE).
It is composed of a monoclonal antibody (anti-CD44 antibody) that selectively reacts with tumor cells (glioma cells, melanoma cells, lung cancer cells, etc.) that overexpress CD44. A choline lipid such as dilauroylphosphatidylcholine (DLPC) may be added to stabilize the shape of the liposome. DDAB, one of the constituent lipids, is a lipid that imparts a positive charge to the liposome. By using such a lipid, both the inside and outside of the liposome are positively charged, and as a result, additives to tumor cells having a negative charge, that is, various antitumor active substances (ceramide or anti-Fas antibody or magnetite) according to the present invention are used. Or the efficiency of gene introduction is improved. Furthermore, since the gene itself has a negative charge, it also leads to an improvement in the encapsulation or embedding efficiency during liposome preparation. The constituent molar ratio of lipid is 2: 1 for DDAB and DOPE.
Alternatively, 1: 2 is exemplified, and an appropriate amount of additives may be added.

Antitumor active substance introduced into tumor cells The antitumor active substance introduced into tumor cells in the present invention is
The mechanism is not limited as long as it can be introduced into tumor cells and exert an antitumor effect. Specifically, those that can be selectively introduced into tumor cells and efficiently induce apoptosis,
For example, ceramide (C ₂ or C ₆ ceramide or other ceramide analog capable of inducing apoptosis), anti-Fas
It means an antibody, or magnetite that can be selectively introduced into a tumor cell to exert a thermotherapeutic effect, and a gene effective for antitumor. The gene effective for antitumor is preferably a suicide gene, specifically a gene encoding herpes simplex thymidine kinase, or a cytokine gene, specifically interferon (IFN) -α, β, γ, granulocyte macrophage. Colony stimulating factor (GM-CSF), interleukin (IL) -1α, IL-1β, IL-1α, IL-1β, IL-2,
IL-3, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL
-15, Tumor necrosis factor (TNF) -α, lymphotoxin (LT)-
β, granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), macrophage migration inhibitory factor (MIF), leukemia inhibitory factor (LIF), T cell activating costimulator B7 (CD80) and Examples include genes encoding various cytokines such as B7-2 (CD86), kit ligand, and oncostatin M. The above gene used in the present invention is cD obtained by isolation from cells using known techniques.
Although it may be NA, it may be chemically synthesized according to a method such as a polymerase chain reaction (PCR) based on information disclosed in publicly known documents, etc., in order to minimize immunological rejection. In addition, in order to improve the therapeutic effect, those of human origin are desirable.

G-22 monoclonal antibody (anti-CD44
Antibody ) According to the research conducted by the present inventors, some of human glioma cells, melanoma cells, or lung cancer cells are homing recep of lymphocytes.
The fact that CD44, identified as tor, is overexpressed has been found. Based on this fact, a specific antibody against human glioma cells (anti-CD44 antibody) was prepared [Wakabayashi T, Yoshida J, Seo H, et al.,
Characterization of neuroectodermal antigen by am
onoclonal antibody and its application in CSF diag
nosis of human glioma. J Neurosurg, 68, 449-455 (1
988), Okada H, Yoshida J, Seo H, et al., Anti- (g
lioma surface antigen) monoclonal antibody G-22 re
cognized overexpressedCD44 in glioma cells. Cancer
Immunol. Immunother. 39, 313-317 (1994)]. The production can be carried out by a conventional means, that is, by fusing mouse splenocytes immunized with an antigen expressed on the cells and myeloma cells, and culturing the obtained hybridoma. The monoclonal antibody may be whole IgG or a part thereof, for example, F (ab ') 2.

Tumor cells The tumor cells to be treated with the immunoliposome in the present invention are glioma cells which are CD44 overexpressing cells,
Examples include melanoma cells and lung cancer cells.

Preparation of Immunoliposomes The immunoliposomes of the present invention can be prepared by a slight modification of the shaking method which is a conventional means for preparing multilamellar liposomes. Specifically, the above lipids or mixed lipids dissolved in chloroform (in the case of ceramide, it is dissolved in chloroform and then mixed with liposome lipids at this point) are added to Spitz, and the solvent is evaporated by a rotary evaporator to form a liposome membrane. Prepare. A phosphate buffer containing anti-Fas antibody, magnetite, or gene was added to this, and 2 ng to 2 μg of G-22 monoclonal antibody (anti-CD44 antibody) was added to 1 μmol.
And shake for 1 to 5 minutes. Mixing ratio of lipid and other additives (including monoclonal antibody) is 1μm lipid.
For example, the amount of monoclonal antibody is 2 ng to 2 μg, the amount of magnetite is 5 to 30 μg, and the amount of gene is 5 to 30 μg per mol.

Formulation The above-mentioned lipids or mixed lipids dissolved in chloroform (in the case of ceramide, it is dissolved in chloroform and then mixed with liposome lipids at this point) are added to Spitz, and the solvent is evaporated by a rotary evaporator to form a liposome membrane. To prepare. According to this method, the liposome membrane is made into a vial and sterilized and stored. Cold storage is desirable to prevent lipid oxidation. On the other hand, other additives such as antibody, magnetite, or gene are stored in separate vials. In this case as well, storage in a cool place is desirable. A vial (two kinds in some cases) of this additive and a vial of liposome membrane are mixed immediately before use and shaken before use.

The dosage form of the immunoliposome of the present invention includes local injection at the expected dissemination or metastatic site corresponding to the tumor lesion or tumor, and in some cases, usual systemic administration such as intravenous and intraarterial administration. To be The dose of the immunoliposome of the present invention includes age, sex, symptoms, administration route,
Although it depends on the number of administrations, it is appropriate to adjust the amount of lipid per day for an adult to the range of 10 to 1000 nmol.

[0017]

The present invention will be described in more detail with reference to the following examples. These examples are illustrative and do not limit the scope of the invention. Example 1 Preparation of Ceramide-Containing Immunoliposomes (1) Ceramide and Lipid for Liposomes C2-ceramide was used as MATREYA INC., Pleasant Gap, PA, USA.
More; dimethyldioctadecyl ammonium bromide (D
DAB), dilauroylphosphatidylcholine (DLPC) Sigm
a Chemical Co., St., Louis, MO; and dioleoylphosphatidyl-ethanolamine (DOPE) from Avanti
It was purchased from Polar Lipids, Birmingham, AL and used.

(2) Preparation of G-22 monoclonal antibody G-22 monoclonal antibody specific to human glioma cell surface antigen (CD44) (mouse IgG monoclonal antibody)
, Wakabayashi, T. et al., J. Neurosurg., 68, 449.
-455, (1988). First, the spleen cells of a mouse immunized with the human glioma cell line SK-MG-4 were mixed with mouse myeloma cell line NS-1 and Dippold WG, Lloyd KO, Li L.
YC et al., Proc. Natl. Acad. Sci. USA 77, 6114-611
8 (1980). While monitoring the antibody activity in the culture supernatant on a panel of cultured cell lines derived from glioma cells, neuroblastoma, and other tumor cells, hybridomas were selected and cloned by the limiting dilution method. BALB / c mice were inoculated with the selected hybridoma,
The desired monoclonal antibody was obtained from ascites.

(3) Preparation of Liposomes The preparation of liposomes was carried out by a simple method with some improvements made to the shaking method which is a conventional means for preparing multilamellar liposomes. First, add DDAB, DOPE and C2-ceramide 1: 2: 2.
Alternatively, it was dissolved and mixed in chloroform at a ratio of 1: 2: 4 (total amount 2 μmol), and the solvent was removed using a rotary evaporator at the bottom of the siliconized test tube. In this lipid film,
500 μl of Dulbecco's phosphate buffer containing 2 μg of G-22 monoclonal antibody prepared in (2) was added. The target ceramide-immunoliposome was obtained by shaking the test tube containing the lipid film and the phosphate buffer solution for 1 to 5 minutes until the mixture became clean.

(4) Introduction of ceramide into tumor cells Human glioma cells U-251SP, SK-MG-1, and T98G were 10%
Fetal calf serum (FCS), streptomycin (100 μg / ml)
, Penicillin (100 U / ml), 4 mM L-glutamic acid and Dulbecco's minimum essential supplemented with non-essential amino acids
Maintained in medium (DMEM).

Culture medium suspension of the above glioma cells (7.
5 x 10 ⁴ cells / ml) 2 ml to Falcon plate (#
3046) was seeded in each well and incubated at 37 ° C. for 24 hours in a humid atmosphere of 5% CO ₂ and 95% air. Liposome solution
30 μl (60 nmol lipid) with a molar ratio of 1: 2: 2 (= DDAB: DOP
E: C2-ceramide) so that the final concentration of ceramide is 12 μM, or when the molar ratio is 1: 2: 4 (= DDAB: DOPE: C2-ceramide), the final concentration of ceramide is 24 μM. Added to the culture medium. After culturing at 37 ° C for 16 hours, the medium was exchanged and the cells were further incubated at 37 ° C for a certain period of time.
Control empty liposomes with DDAB, DLPC, and DO
Using PE, the molar ratio was adjusted to 1: 2: 2, respectively.

[Test Example 1] (1) Analysis of DNA fragmentation (Method) The analysis of DNA fragmentation was performed using [Jarvis WD, Kolesnic
k RN, Fornari FA, Traylor RS, Gewirtz DA, Grant S,
Induction of apoptotic DNA damage and cell deathb
y activation of the sphingomyelin pathway, Proc. N
Atl. Acad. Sci., USA, 91, 73-77 (1994)] with a slight modification to 2% agarose gel electrophoresis. Two days after treatment with ceramide-immunoliposomes (24 μM), floating cells or adherent cells were collected and 0.5% SDS / 0.1
It was dissolved in M NaCl / 1 mM EDTA, pH 7.5, and the solution was treated with proteinase K (100 μg / ml) at 50 ° C. for 12 hours. Proteinase K was inactivated by heat denaturation at 94 ° C for 5 minutes and 1/10 volume of 3M sodium acetate and 2.5 volumes of cold ethanol were added to precipitate the DNA. The lysate was kept at -70 ° C for 15 minutes and centrifuged at 30,000 xg for 10 minutes. Finally, the pellet was dissolved in 20 μl of TE and electrophoresed on a 2% agarose gel containing 0.4 μg / ml ethidium bromide. The resolution pattern was visualized under UV light.

(Results) DNA extracted from cultured cells treated with ceramide-immunoliposome showed a ladder-type electrophoresis profile of oligonucleosome DNA fragment which is a characteristic of apoptosis (FIG. 1).

(2) Antitumor effect of ceramide-liposomes on cultured cells (Method) Ceramide-immunoliposomes or ceramide liposomes prepared in the same manner without binding antibody (molar ratio 1: 2: 2 (= DDAB: DOPE: C2-ceramide); final concentration of ceramide in the medium 12 μM, or molar ratio 1: 2: 4 (= DDAB: DOPE: C2-
Ceramide); final concentration of ceramide in the medium is 24 μM],
The growth inhibitory effect of ceramide introduced into cultured glioma cells was examined. 48 cultured liposomes on glioma cells
After hours, it was assessed by counting the number of viable cells under a hemocytometer without trypan-blue staining. As a control, treatment with empty liposomes or ethanol-dissolved ceramide (4 mM in ethanol) was performed.

(Results) In Table 1, cytotoxicity (%),
That is, the reduction rate of the number of viable cells relative to untreated cells was shown for each liposome type and cell line. No significant cytotoxicity was observed with the control empty liposomes, whereas ceramide liposomes 12 μM showed cytotoxicity in all three types of cells, and ceramide-immunoliposomes 12 μM showed higher cytotoxicity. It was found that the effect is further increased by the binding of. Student-t t compared to ethanol-dissolved liposomes
Statistical differences due to est (p <0.01) were found in U-251SP and SK-MG-1 treated with ceramide-liposomes and all cells treated with ceramide-immunoliposomes.

Further, 24 μM of ceramide liposome showed higher cytotoxicity than 12 μM of ceramide liposome, and it was found that the effect was dose-dependent, but the dose-dependence was also found in ceramide-immunoliposome. It was In addition, a statistical difference was found between SK-MG-1 and T98G.

[0027]

[Table 1] ──────────────────────────────────── Cell line treated liposome U-251SP SK-MG1 T98G ──────────────────────────────────── Empty liposome 2.0 ± 0 5.8 ± 2 8.1 ± 1.2 Ethanol − Dissolved ceramide (12 μM) 7.5 ± 1 35.4 ± 2 50.0 ± 2.4 Ceramide liposomes (12 μM) 34.7 ± 2 ^* 54.6 ± 1.5 ^* 60.6 ± 1.4 Ceramide-immunoliposomes (12 μM) 56.3 ± 2.5 ^* 63.8 ± 1.5 ^* 80.3 ± 0.7 ^* Ethanol -Dissolved ceramide (24 μM) 33.1 ± 1.5 39.2 ± 3 56.1 ± 2.4 Ceramide liposomes (24 μM) 44.2 ± 2.5 69.1 ± 0.35 ^* 81.8 ± 0.3 ^* Ceramide-immunoliposomes (24 μM) 61.8 ± 2 72.4 ± 1.7 ^* 86.3 ± 0.6 ^* ─ ─────────────────────────────────── Cytotoxicity (%) ^* There is a statistical difference student-t test ( p <0.01)

Example 2 Preparation of Anti-Fas Antibody-Containing Immunoliposomes (1) Anti-Fas Antibodies and Lipids for Liposomes Anti-Fas antibodies are available in Medical and biological laboratories (MB
Purchased from L), Co., Ltd., Nagoya, Japan. The liposome lipid is as described above.

(2) G-22 monoclonal antibody (anti-CD
44 antibody) As described above.

(3) Preparation of anti-Fas antibody-containing immunoliposome The anti-Fas antibody-containing immunoliposome is prepared by a method which is a modification of the shaking method which is a conventional means for preparing multilamellar liposomes. It was First, a choline lipid such as dilauroylphosphatidylcholine (DLPC) was added for the purpose of stabilizing the shape of the liposome. Ie DDA
B, DOPE and choline lipid were dissolved and mixed in chloroform at a molar ratio of 1: 2: 2 or 1: 3: 2, respectively, and the solvent was removed using a rotary evaporator at the bottom of the siliconized spitz. To this lipid film (liposome membrane), anti-Fas antibody dissolved in phosphate buffer was added at a ratio of 2 ng to 2 μg per 1 μmol of total lipid, and G-22 monoclonal antibody (anti-CD44 antibody) was added to 1 μmol of total lipid. 2 ng to 2 μg,
Shake for 1 to 5 minutes. After that, phosphate buffer was added to adjust the total volume to 500 μl. By this method, the desired immunoliposomes containing anti-Fas antibody were obtained.

[Test Example 2] Antitumor effect of anti-Fas antibody-containing immunoliposome on cultured cells (1) Antitumor effect of anti-Fas antibody-containing immunoliposome on cultured cells (Method) 15 to 30 nmol of anti-Fas antibody-containing immunoliposome Lipids / ml were added to cultured glioma cells to examine the growth inhibitory effect of glioma cells. The same amount of free anti-Fas antibody was added as a control.

(Results) In Table 2, cytotoxicity (%),
That is, the reduction rate of the number of viable cells relative to untreated cells was shown for each liposome type and cell line. Anti-Fas antibody and anti-Fas
Cytotoxicity was observed in all of the cultured cell lines with any of the antibody-containing immunoliposomes. The cytotoxicity of the anti-Fas antibody-containing immunoliposomes was stronger with a statistically significant difference than when the free anti-Fas antibody was added. Moreover, the cytotoxic form was proved to be apoptosis by the TUNEL method.

[0033]

[Table 2] ──────────────────────────────────── Cell line U-251 SP SK-MG- 1 T98G ──────────────────────────────────── Empty liposome 1.2 ± 0.3 4.2 ± 1.8 5.0 ± 1.1 Anti-Fas antibody 0.1 μg 4.3 ± 0.7 2.5 ± 0.4 3.2 ± 1.5 1.0 μg 8.4 ± 2.5 10.1 ± 2.1 12.7 ± 3.0 100 μg 56.7 ± 4.4 60.3 ± 3.8 58.9 ± 5.5 Liposome antibody amount 0.1 μg 52.2 ± 3.8 48.3 ± 5.4 50.1 ± 6.3 1.0 μg 78.4 ± 4.5 67.5 ± 4.7 67.4 ± 3.8 Immunoliposome antibody amount 0.1 μg 69.3 ± 4.1 64.5 ± 3.4 66.6 ± 4.2 1.0 μg 88.7 ± 6.3 80.5 ± 7.1 85.3 ± 3.9 ───────────── ───────────────────────

Example 3 Preparation of Immunoliposomes Containing Magnetite (1) Lipids for Magnetite and Liposomes Magnetite is Sinkai M, Honda H, Kabayashi T, Pr.
eparation of fine magnetic particles and applicati
on for enzyme immobilization Biocatalysis, 5, 61-69
It was prepared according to the method of (1991).

The lipid for liposome is as described above.

(2) G-22 monoclonal antibody (anti-CD
44 antibody) As described above.

(3) Preparation of Magnetite-Containing Immunoliposomes The magnetite-containing immunoliposomes were prepared by a slight modification of the shaking method, which is a conventional means for preparing multilamellar liposomes. First, for the purpose of stabilizing the shape, dilauroylphosphatidylcholine (DLP
C) and other choline lipids were added. DDAB, DOPE, and choline lipid were dissolved and mixed in chloroform at a molar ratio of 1: 2: 2, and the solvent was removed using a rotary evaporator at the bottom of the siliconized spitz. Magnetite dissolved in a phosphate buffer was added to this lipid film (liposome membrane) at a ratio of 20 μg to 1 μmol of total lipid, and further G-22 monoclonal antibody (anti-CD) was added.
44 antibody) was added at a ratio of 2 ng to 2 μg to 1 μmol of total lipid and shaken for 1 to 5 minutes. After that, phosphate buffer was added to adjust the total volume to 500 μl. By this method, a target magnetite-containing immunoliposome was obtained.

[Test Example 3] Incorporation of iron into cultured cells by magnetite-containing immunoliposomes (1) Incorporation of magnetite (method) Magnetite-containing immunoliposomes were added to cultured glioma cells at a ratio of 15 to 30 nmol lipid / ml, and glioma cells were added. The amount of magnetite uptake in cells was examined. As an object, the same amount of free magnetite was added.
The amount of incorporated magnetite was calculated by dissolving the cells with concentrated hydrochloric acid, removing the cell lysate with TCA, adding StCl, and measuring the concentration of magnetite by atomic absorption spectrometry to calculate the amount of magnetite.

(Results) In Table 3, the amount of magnetite incorporated into cultured glioma cells is shown for each liposome type. When the same amount of free magnetite was added to cultured glioma cells, intracellular uptake of magnetite was hardly observed. On the other hand, a large amount of magnetite uptake was observed in the magnetite-containing immunoliposomes. The uptake amount was 10 times or more that of a liposome prepared with a conventional lipid such as phosphatidylcholine, phosphatidylserine or cholesterol. The amount of iron uptake was significantly higher in the immunoliposomes conjugated with anti-CD44 antibody than in normal liposomes not conjugated with anti-CD44 antibody.

[0040]

[Table 3] ──────────────────────────── Cell line U-251 SP SK-MG-1 T98G ────── ────────────────────── Liposomes 17.0 18.0 19.0 Immunoliposomes 42.0 36.0 37.0 ──────────────────── ───────── The numerical value shows the uptake of magnetite per cell (pg-Fe3O4 / cell) 16 hours after administration of magnetite-containing liposome or immunoliposome.

[Example 4] Preparation of gene-containing immunoliposome (1) Gene and lipid for liposome Gene pCH110 (purchased from Pharmacia), pSV2IFN-β
Eukaryotic cell expression vectors such as those provided by Toray Industries, Inc. were used. The liposome lipid is as described above.

(2) G-22 monoclonal antibody (anti-CD
44 antibody) As described above.

(3) Preparation of Gene-Containing Immunoliposomes The preparation of gene-containing immunoliposomes was carried out by a slight modification of the shaking method which is a conventional means for preparing multilamellar liposomes. First, a choline lipid such as dilauroylphosphatidylcholine (DLPC) was added for the purpose of stabilizing the shape. DDAB, DOPE, and choline lipid were dissolved and mixed in chloroform at a molar ratio of 1: 2: 2, and the solvent was removed using a rotary evaporator at the bottom of the siliconized spitz. Genes dissolved in phosphate buffer were added to this lipid film (liposome membrane) at a ratio of 20 μg to 1 μmol of total lipid, and G-22 monoclonal antibody (anti-CD44 antibody) was added from 2 ng to 1 μmol of total lipid. Add at a rate of 2 μg,
Shake for 1 to 5 minutes. After that, phosphate buffer was added to adjust the total volume to 500 μl. By this method, a target gene-containing immunoliposome was obtained.

[Test Example 4] Gene expression in cultured cells by gene-containing immunoliposome (1) Gene expression (method) 15 to 30 gene-containing immunoliposomes
The nmol lipid / ml was added to the cultured glioma cells, and the expression of the transgene in the glioma cells was examined. The same amount of free gene was added as a control.

(Results) In Table 4, the expression of the gene introduced into the cultured glioma cells is shown for each liposome type. No expression was observed when the same amount of free gene was added to cultured glioma cells. On the other hand, its expression was observed in gene-containing immunoliposomes. In addition, the expression of the transgene was significantly enhanced in the immunoliposomes bound with anti-CD44 antibody as compared with the normal liposomes not bound with anti-CD44 antibody.

[0046]

[Table 4] ──────────────────────────────────── Cell line U-251 SP SK-MG- 1 T98G ──────────────────────────────────── Empty liposome 2.5> 2.5> 2.5> Free gene 2.5> 2.5> 2.5> Liposome 7.5 nmol 23.8 ± 4.0 30.7 ± 4.8 39.8 ± 8.8 15.0 nmol 52.0 ± 7.8 60.8 ± 5.6 77.8 ± 12.7 30.0 nmol 126.3 ± 12.1 112.1 ± 10.7 168.3 ± 17.5 Immunoliposomes 7.5 nmol 85.0 ± 7.6 88.9 ± 8.5 63.3 ± 9.7 15.0 nmol 175.8 ± 10.8 170.5 ± 11.6 140.8 ± 15.3 30.0 nmol 256.3 ± 15.7 240.4 ± 14.7 263.3 ± 19.5 ────────────────────────── ─────────── The numerical value indicates the β-interferon concentration (IU / ml) in the culture medium on the 4th day after gene transfer.

[0047]

EFFECT OF THE INVENTION The ceramide-containing immunoliposome or anti-Fas antibody-containing immunoliposome of the present invention can efficiently and selectively introduce ceramide or anti-Fas antibody into tumor cells to induce apoptosis. Effective in treatment. It is also considered that they are useful for elucidating the mechanism of apoptosis.

According to the magnetite-containing immunoliposome of the present invention, it is possible to introduce magnetite into tumor cells much more efficiently than that of freely added magnetite, and it is effective for local hyperthermia treatment for malignant tumors. According to the gene-containing immunoliposome of the present invention, a gene can be efficiently introduced into a tumor cell and expressed, and therefore, it is effective in gene therapy for malignant tumor.

[Brief description of the drawings]

FIG. 1 shows an agarol gel electrophoresis photograph of DNA extracted from glioma cells by treatment with ceramide-immunoliposomes. Lane 1: Molecular weight marker Lane 2: Extracted DNA

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI Technical display area A61K 33/26 A61K 33/26 31/715 39/395 T 39/395 48/00 48/00 A61N 1/40 // A61N 1/40 A61K 37/20 (72) Inventor Hideho Okada 4-26 Kawanamachi, Showa-ku, Aichi Prefecture Nagoya No. 301 Mirakawana

Claims (9)

[Claims] 1. An immunoliposome in which a monoclonal antibody G-22 (anti-CD44 antibody) that selectively reacts with tumor cells is bound to a cationic liposome membrane containing an antitumor active substance. 2. The immunoliposome according to claim 1, wherein the antitumor active substance is selected from the group consisting of ceramide, anti-Fas antibody, magnetite, and gene. 3. The immunoliposome according to claim 2, wherein the gene is a suicide gene or a cytokine gene. 4. The cytokine is interferon (IF
N) -α, β, γ, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin (IL) -1α, IL-1β, IL-1
α, IL-1β, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10,
IL-12, IL-13, IL-15, tumor necrosis factor (TNF) -α, lymphotoxin (LT) -β, granulocyte colony stimulating factor (G-CS
F), macrophage colony stimulating factor (M-CSF), macrophage migration inhibitory factor (MIF), leukemia inhibitory factor (LIF)
, T cell activating co-stimulators B7 (CD80) and B7-2 (CD86),
The immunoliposome according to claim 3, which is selected from the group consisting of kit ligand and oncostatin M. 5. The lipid constituting the cationic liposome membrane is dimethyldioctadecyl ammonium bromide (DD
AB) and dioleoylphosphatidylethanolamine (DOPE). 6. The immunoliposome according to claim 1, wherein the tumor cell is a CD44 overexpressing cell. 7. The CD44 overexpressing cell is a glioma cell,
The immunoliposome according to claim 6, which is a melanoma cell or a lung cancer cell. 8. An immunological method characterized in that a monoclonal antibody G-22 (anti-CD44 antibody) which selectively reacts with tumor cells is added to a cationic liposome membrane containing an anti-tumor active substance, mixed and shaken. Method for preparing liposome. 9. The method for preparing an immunoliposome according to claim 8, wherein the antitumor active substance is selected from the group consisting of ceramide, anti-Fas antibody, magnetite, and gene.