WO1992004887A1

WO1992004887A1 - Induction of cytotoxic t cell

Info

Publication number: WO1992004887A1
Application number: PCT/JP1990/001231
Authority: WO
Inventors: Junzo Sunamoto; Hiroshi Shiku
Original assignee: Kyowa Hakko Kogyo Co., Ltd.
Priority date: 1990-09-25
Filing date: 1990-09-25
Publication date: 1992-04-02

Abstract

A complex of a cancer cell antigen or a viral antigen with liposome covered with a polysaccharide including mannose, and a method of inducing a cytotoxic T cell specific for a cancer cell or a virus antigen by using said complex.

Description

Specification

Methods for inducing cytotoxic τ cells

Technical field

The present invention relates to a cancer cell antigen or a virus antigen-ribosome complex coated with a polysaccharide containing mannose and a cancer cell or virus antigen using the complex. The present invention relates to a method for inducing a novel cytotoxic T cell (hereinafter abbreviated as “CT”), and a method for treating or preventing cancer or a virus disease using the CTL.

Background technology

It is known that CTL specifically recognizes and impairs cancer cells or virus- and virus-sensitized cells by their antigen receptors, and CTLs are used to detect in vivo cancer or virus. It is also known to be important in host defense mechanisms against Luss.

[Dai Iey, M.0., Et al., Proceeding 'ob' National ♦ Academic 'ob' Science 'USA (Pro Natl. Acad. Sci. USA) 79, 5384-5387 (1984); Nakayama, Ε., Et al., Journal of ォ ob 'experimental, Medicine (J. Exp. Med,) 161, 345-355. (1985); Greenberg, P.0., J. Immunol. 136, 1917-1922 (1986) J; Klarnet, J. Ρ, et al., Journal 'ob' Immunology U. Immunol.) 138, 4012-4017 (1987); Von der Hoorn et al., Journal of ブ Bec-Experimental 'Medicine (J. Exp. Med.) 162, 123-144 (1985)) _c Furthermore, recent studies have shown that the antigenic protein of cancer cells or virus and virus-sensitized cells does not induce CTLs when presented alone, and that MHC It was found that CTL induction was possible only when co-expressed with class I [Morrison, et al., Et al. J. Bxpjed., 163, 903-921 (1986); Garmain, RJ., Nature, 322, 687-689. (1986)].

Therefore, to date, it has been necessary to use cancer cells themselves, or virus or virus-sensitized cells themselves, for CTL induction. Was restricted. On the other hand, with the development of genetic recombination technology, it is possible to produce cancer-specific antigens and virus-specific antigens safely and in large quantities, but they used antigen polypeptides obtained by genetic recombination technology. No efficient CT guidance method has been found yet.

Disclosure of the invention

The present invention provides a cancer cell antigen or a virus antigen-liposome complex, which is coated with a polysaccharide containing mannose.

The phrase "antigen" refers to substances that elicit an immune response, such as polypeptides, polypeptide complexes, glycoproteins, nucleic acids, and the like. It may be part of the pathological target itself, or may be an antigen expressed by a diseased tissue such as a neoplastic tissue or a virus-infected cell. As the antigen in the present invention, an antigen protein is preferably used. As the antigen protein, any origin and purity can be used as long as it includes an antigen determination site, but those prepared by a gene recombination method are preferable. The molecular weight of the antigen protein is not particularly limited as long as it includes at least one antigen-determining site, but is generally 500 to 1,000,000, preferably 200 to 100,000. is there.

Specific examples include the gag protein and env protein of ATLV, the env protein of HIV, and partial proteins thereof.

[Gipy Sipno, 'Knod U. Schupbach', ed., Current Topics 'In', My Chromology 'And' ♦ Immunology Topics in Microbiology and Immunology), 142 (1989), Springer-Verlag; Ed. P. Vogt, Current Topics in Mic. Ronoolo's 'and' imiuno. G (Current Topics in Microbiology and Immunology), 115 (1985), Springer-Verlag) _c

Further, as an example having a plurality of antigen-determining sites, a gag-env fusion protein of ATLV described in the following literature can be mentioned. [JP-A-62-48699; Kuga, T. et al., Japanese 'Journal of Cancer Research., Jpn. J. Cancer Res., 79, 1168-1173 (1988) )].

The method of preparing the liposome, the method of encapsulating the antigen in the liposome, and the method of coating the liposome with the Mannos polysaccharide are prepared by the method developed by the present inventors. However, it is also possible to use a general liposome preparation method. [M's Jy's Best Mouth (MJ Ostro), Liposomes, MARCEL DEKER, INC (1987, New York).

The mannose-containing polysaccharide may be a chemically synthesized product, a natural extract, or a chemically modified product thereof, as long as mannose is contained as a constituent sugar. Preferably, readily available mannans and mannan derivatives are used.Particularly, cholesterol residues are partially introduced into mannans for the purpose of producing stable coated polysaccharide ribosomes. It is preferred to do so. [JP-A-61-268628; Sunamoto et al., Chem. Lett. 1781 (1988); Takada, A. et al., Biohi Mika. Biochim. Biophys. Acta 802, 237 (1984); Sunamoto, Pathophysiology, 771 (1987)].

The liposome can be coated with the mannose-containing polysaccharide by incubating the polysaccharide.ice solution and the liposome at an appropriate temperature.

An example of a method for preparing an antigen-liposome complex coated with a mannan derivative is performed by the following procedure. (1) Preparation of liposomes, (2) Modification of liposomes to antigens, (3) Coating of antigen-modified ribosomes with mannan derivatives

① Creation of reposome

In the mid-1960s, liposomes formed a bilayer membrane with the same basic structure as cell plasma membrane when Bangham in the United Kingdom redispersed phospholipids derived from natural products in water. Since it was found that it has a Balta structure and closed vesicle morphology, it has been used as a membrane model or as a microcapsule, and it has been reported that the membrane fluidity ゃ barrier function (membrane permeation barrier) etc. It has been widely used in research on cell structural characteristics and also on research on animal functions of cells such as aggregation and fusion.

In carrying out the present invention, it is preferable to use a liposome having excellent stability, and the partial structure of sphingomyelin, which has been developed by the present inventors and is considered to be a kind of boundary lipid in nature, is considered. As an analog, it is preferable to use 1.2-dimyristylamide 1.2-dexoxyphosphatidylcholine (hereinafter referred to as “DDPC j”) having an amide bond in a polysyl group. More specifically, the above-mentioned liposome is prepared by using various liposome-forming phospholipids and the above-mentioned DDPC, and various methods known per se. For example, typically performed according to Vortex (V 0 rte X 1 ng method or Hydrat ion method) (Bangham, AD, Standish, MM & Watkins, JCJ Mol. Biol., 13, 238 (1965)) The above lipids for ribosome formation are More specifically, for example, lipids having an amide group capable of forming a hydrogen bond zone in a membrane of lecithin, sphingomyelin, etc. extracted and purified from soybean, egg yolk, etc. And preferably yolk lecithin, dimyristylphosphatidylzircholine (DMPC), etc. In the above, a particularly preferred liposome is a combination of yolk lecithin and DDPC It is usually prepared by combining DMPC and DDPC in a ratio of about 4: 6 or 2: 8 (weight ratio).

Modification of antigen to liposome

The modification of the antigen to the ribosome is basically performed by simply mixing the two, that is, adding a predetermined amount of the antigen suspension to the ribosome suspension. It can be performed by mixing.

The amount of antigen added to the liposome in the above is very important in relation to the ability to express the activity of the obtained complex, and is generally from 10 to L000, preferably about 200 to 10 mg of lecithin in 1 Use the antigen mass.

③ Coating of antigen-modified ribosome with mannan derivative

The coating with a mannan derivative is basically performed by simply mixing the two, that is, adding a predetermined amount of a cholesteryl-modified mannan permanent solution to the antigen-modified liposome suspension and mixing. <Generally, the mass of mannan derivative is 0.1 to 10 mg, preferably about 2 mg per 10 mg of lecithin in 1 mi.

The thus obtained antigen-ribosome complex coated with mannose monosaccharide polysaccharide can be used in the present invention as it is, but of course-if necessary, sterilization or the like may be carried out according to a conventional method. Operations can also be performed.

The liposome complex of the present invention can be immunized by administering a predetermined value to the animal in the same manner as other conventionally known pectins. The animal to which the vaccine can be applied is not particularly limited, and may be any of various mammals including humans and other animals. Immunization of these animals by administration of the vaccine of the present invention can also be performed according to a general method. The route of administration of the vaccine of the present invention is generally subcutaneous injection, but is not particularly limited thereto. The dose of the pectin of the present invention for the above immunization can be determined as appropriate, but usually the dose is about 50 to: L 50 #g Z ratton times as the antigen. It is appropriate to administer about 1 to 4 times.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of a comparison of the effect of a mannan-coated antigen-liposome complex on TARS-1 cells.

A is a physiological saline, B is a mannan-coated antigen-ribosome complex (the present invention), C is a non-mannan-coated antigen-liposome complex, D is And the results obtained when the liposomes coated with mannan were respectively administered.

FIG. 2 shows a comparison result of induction of CTL activity of the antigen-liposome complex coated with mannan.

A is a physiological saline, B is a mannan-coated antigen-ribosome complex (the present invention), C is an unmannan-coated antigen-ribosome complex, and D is a man-ribosome complex. The results were obtained when each of the ribosomes coated with nannan was administered. '

The best form for carrying out KIKI

Hereinafter, examples for describing the present invention in more detail will be described.

Example 1 Preparation of antigen-liposome complex coated with mannan derivative

1) Preparation of liposome

Egg yolk phosphatid zirconine (EggPC) according to the method described in the literature (WS Singleton, MS Gray, Μ · Sh. Brown, JL White, J. Am. Oil Chem. Soc,, 42, 53 (1965)) ) Was extracted and purified using alumina.The purity was confirmed by TLC. Further, DDPC was obtained from 2,3-diaminopropionic acid by the method described in JP-A-61-267509 (Junzo Sunamoto et al., Journal of the Chemical Society of Japan, 1987 (3), P569-574). Was synthesized.

10.6 mg of the above-mentioned EggPC and 7.0 mg of DDPC were mixed, and dissolved in 2 mg of clog-mouthed form in an eggplant-shaped flask. The crotch form was distilled off under reduced pressure using a rotary evaporator, and the mixture was allowed to stand overnight in a reduced pressure desiccator.

The resulting solution was then swollen on a vortex mixer into 1 of buffer A (10 mM carbonate buffer, 0.35 urea, 0.07 mM EDTA, 0.035¾2-mercaptoethanol, ΡΗ? .4), I was crowded. Then, using a UR200P probe ultrasonic breaker (manufactured by Tomi Corporation), irradiate ultrasonic waves at 25 ° at 0 ° for 5 minutes under a nitrogen gas flow to obtain the desired liposome Suspension) Repair of antigen to ribosome

The gag-env fusion protein aqueous solution 1 rafi (200 as protein) prepared by the method described in JP-A-62-48699 and the liposome suspension 1 m2 prepared in 1) above were used. The mixture was mixed and incubated at 371: 6 for 6 hours to obtain an antigen-encapsulated liposome suspension 2.

) Preparation of antigen-liposome complex coated with mannan derivative Two ml of the liposome suspension obtained in '2) above was prepared by the method described in the following literature. 0.5 mg of a 5 mg aqueous solution of resterol-modified mannan was added, and the mixture was incubated at room temperature for 30 minutes to obtain the desired product (T, Sato, .ojima, T.I hda, and J. Sunatnoto, Macrophage Activation by Poly (maleicacid-alt-zc clohe y 1-1, 3-di oxap-5-ene) Encapsulated in Pol saccharide- Coated Liposomes, J.

Bioactive and Compatible Polym., _1, 448-460 (1986): _c

Example 2 Effect of Antigen-Liposome Complex Coated with Mannan Derivative I [In Vivo (i_vo) Effect]

WA / H female rats per group, 4 animals, were subcutaneously administered twice a week with the liposome complex suspension lOOmg prepared in Example 1 at 1-week intervals to induce CTL, and 1 week after the last administration Later, in the skin of the test animal, the literature [Noguchi, Ϊ., Et al., Jannarele ob., I. Immunol., 143, 3737-3742 (1989) 1) 1 × 10 ⁷ TARS-1 cells prepared by the method described above were transplanted.

Thereafter, the diameter of the colony of the transplanted TAR S-1 cells was measured by the method described above to determine the effect. (B in Fig. 1)

As a comparison and a control, instead of the ribosome complex, physiological saline (A in FIG. 1) and a non-mannan-coated antigen-ribosome complex (C in FIG. 1) were used. A group was also administered with mannan-coated ribosome alone (D in FIG. 1).

As a result, in the group administered with the antigen-liposome complex of the present invention, almost no tumor growth was observed, and it was found that a significant tumor growth inhibitory effect was exhibited.

Example 3 Effect of antigen-ribosome complex coated with mannan derivative [In vitro effect]

Rats were immunized as in Example 2. 1 week after the last dose Later, spleen cells of the test animals were collected. 4 × 10 ⁷ spleen cells and mitomycin in RPM1 1640 medium 20 ^ containing 20% fetal calf serum. 4 x 10 ^{s of the} treated TARS-1 cells were co-cultured at 37: for 1 week in a carbon dioxide incubator. The cytotoxicity after culturing was determined by the ⁵¹ Cr release method [Nakayama, E. et al., Proc. Of the National University of California, California, Science, USA (Proc. Natl. Acad. Sci. USA) 76, 3486 (1979)] (Fig. 2, B). As a comparison and a control, instead of the liposomal complex, physiological saline (A in FIG. 2) and an antigen-ribosomal complex not coated with a mannan derivative (FIG. 2) were used. C) and ribosome alone coated with a mannan derivative (D in FIG. 2) were similarly administered.

As is clear from FIG. 2, high CTL activity was induced only in the case of the antigen-ribosome complex coated with the mannan derivative of the present invention (FIG. 2B).

Claims

The scope of the claims

1. A cancer cell antigen or virus antigen-liposome complex coated with a polysaccharide that contains mannose.

2. The complex according to claim 1, wherein the antigen is gag protein, env protein, or a part thereof of adult T-cell leukemia virus (hereinafter abbreviated as "ATLV").

3. The complex according to claim 1, wherein the antigen is an ATLV gag-env fusion protein.

4. Administer a cancer cell or virus antigen-liposome complex coated with a polysaccharide containing mannose to a human or animal, and obtain the cancer in the human or animal. A method for inducing cytotoxic T cells specific for cellular or viral antigens.

5. The method according to claim 4, wherein the antigen is the gag protein, the env protein or a part thereof of ATLV.

6. The method according to claim 4, wherein the antigen is an ATLV gag-env fusion protein.