JPH0665100A - Pharmaceutical composition for AIDS - Google Patents

Abstract

(57) [Summary] [Purpose] To provide therapeutic and diagnostic agents for AIDS. [Structure] Mucin-type glycolipid Galβ1 → 4GlcNAcβ
AIDS is treated and diagnosed using an antibody that recognizes 1 → 6GalNAcα1 → 1Cer and the like.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a pharmaceutical composition for AIDS containing an antibody that recognizes glycolipid.

[0002]

2. Description of the Related Art AIDS is an HIV (human
It is a disease that causes immunodeficiency due to infection with unodeficity virus, and frequently causes carinii pneumonia and Kaposi's sarcoma. Due to the infectivity of this virus and the high mortality rate of this disease, it has become a social problem all over the world, and early diagnosis of HIV infection and development of anti-HIV drugs have become urgent issues.

HIV gradually changes its gene,
Since the constituent proteins and core proteins are changed, it is very difficult to develop antibodies useful for diagnosis and vaccines for treatment and prevention. Furthermore, in recent years, a virus species (Chermann) having an infectious pathway that seems not to be mediated by CD4
Et al., Human Retroviruses (eds.
Stehelin, D.M. ) 14, 1988) was also isolated, so that the viral coat protein gp120
There is an urgent need to search for other viral markers.

It has been reported that Lewis Y antigen, which is a kind of sugar chain antigen, is abnormally expressed in cells infected with HIV (Adachi et al., J. Exp. Med., 1).
67, p323, 1988). Also recently, HIV
It has been reported that an antibody that recognizes the sugar chain of gp120, which is a coat protein of Escherichia coli, inhibited HIV infection (H
ansen et al. Virology, 64, p283
3, 1990). Thus, there is a deep link between HIV infection and sugar chains.

[0005]

[Problems to be Solved by the Invention] However, what kind of sugar chain is induced in T lymphocytes by HIV infection, and which sugar chain recognizing antibody is effective as a diagnostic or therapeutic agent for AIDS? Not well known about. The reason is that it is difficult to prepare an antibody that recognizes a sugar chain whose structure is clearly defined.

[0006]

In order to solve these problems, the present inventors have already synthesized a glycolipid in which a mucin-type sugar chain is bound to ceramide, and filed a patent application (JP-A-3- 251594, Japanese Patent Application Laid-Open No. 275694, Japanese Patent Application No. 3
-48627, JP-A-3-275694, Japanese Patent Application No. 3-1
67500). In addition, immunize mice with these,
As a result of selecting a large number of clones, a monoclonal antibody that recognizes these sugar chains was prepared, and a patent application was filed (JP-A-3-280894, July 28, 1992).
Date application “monoclonal antibody and its production method”, application “July 28, 1992 application“ monoclonal antibody and its production method ”, July 28, 1992 application“ monoclonal antibody and its production method ”).

This time, the present inventors have used the above antibody to
The inventors reached the present invention by finding the expression of sugar chains on the cell surface due to HIV infection and the HIV infection inhibitory effect of these antibodies, and finding that an antibody that recognizes a certain mucin-type glycolipid can be a drug for AIDS. did. That is, the present invention uses the formulas (F1), (F3), (F4), or (F3).
6) Galβ1 → 4GlcNAcβ1 → 6GalNAcα1 → 1Cer (F1) Galβ1 → 4GlcNAcβ1 → 3GalNAcα1 → 1Cer (F3) Galβ1 → 4GlcNAcβ1 → 3GalNAcβ1 → 6G4NAcβ1 → 6GalNAcα1 → 4GlcNAcβ1 → 6GalNAcβ1 → 6GalNAcβ1 → 6GalNAcβ1 → 6GalNAcβ1 → 4GlcNAcβ1 → 6GalNAcβ1 → 6GalNAcβ1 → 4 A pharmaceutical composition for AIDS, comprising as an active ingredient an antibody that recognizes a mucin-type glycolipid represented by GalNAcα1 → 1Cer (F36). Hereinafter, the present invention will be described in detail.

The pharmaceutical composition as used in the present invention includes, for example, AIDS diagnostic agents and therapeutic agents. Examples of the AIDS diagnostic agent of the present invention include a test for examining the presence or absence of HIV infection, a test for examining the presence or absence of AIDS in HIV-infected persons, and a test for examining the degree thereof. These may or may not apply to health insurance. The test method can be applied to any method using an antibody, for example, an enzyme immunoassay method (ELISA) or a radioimmunoassay method (RIA) by a sandwich assay method or a competitive assay method, and further cytofluorometric analysis (FACS). ) Can be exemplified.

The therapeutic agent for AIDS of the present invention includes, for example, a therapeutic agent for preventing further infection from HIV infected persons, H
It includes all the therapeutic agents for preventing the onset of AIDS in IV infected persons, the therapeutic agents for delaying the progression of the disease in AIDS patients, and the like. Specifically, a method of administering the antibody as it is or an antibody to which a toxin or the like is bound as a therapeutic agent into the body,
Examples include a method of passing the patient's blood through a carrier to which an antibody is bound in advance.

The antibody used in the present invention has the formula (F
1), (F3), (F4), or (F36), which recognizes the glycolipid represented by the formula (F1), for example, a monoclonal antibody F1α7 that recognizes the glycolipid represented by the formula (F1).
5, monoclonal antibody F3α25A3, F3α328 that recognizes the glycolipid represented by formula (F3), and monoclonal antibody F4α3 that recognizes the glycolipid represented by formula (F4)
D8, F4α3F10, F4α3E5, F4α2F1
1, and a monoclonal antibody F36α2G4 that recognizes the glycolipid represented by the formula (F36).

Examples of the method for preparing the antibody of the present invention include conventional methods and methods using genetic engineering. In the conventional method, a distinction is made between a monoclonal antibody and a polyclonal antibody. Monoclonal antibodies have the formula (F1), (F3),
It is produced by a hybridoma obtained by immunizing a mouse with the glycolipid represented by (F4) or (F36), extracting spleen cells, and fusing the splenocytes with established myeloma cells. As the animal for immunization, most animals such as humans, rabbits, mice and rats can be used, but mice are preferable, and BALB / c mice are more preferable. Rearing mice and collecting spleen cells follow conventional methods. On the other hand, as the myeloma cells, myeloma cells of most animals such as humans, rabbits, mice, and rats can be used, but preferably BALB / c mouse-derived S
P2 / 0-Ag8 is used. Polyethylene glycol or the like can be used as a fusing agent used in cell fusion. Fusion cells (hybridomas) are selected by screening cells that produce a culture supernatant that reacts with the glycolipid used for immunization.
Just train. At this time, 1 for a 96-well plate
It is preferable to screen 0 plate or more. Also, care must be taken not to miss clones that produce only small amounts of antibody. Hybridomas are cloned by the limiting dilution method, the methyl cellulose method, the soft agarose method, or the like. When the hybridoma is cultured in the same manner as in normal animal cell culture, the antibody used in the present invention is produced in the medium. Alternatively, the hybridoma may be transplanted to a mouse or the like to produce a monoclonal antibody in ascites and purified. Also for polyclonal antibodies, the above-mentioned animals may be immunized with the glycolipids represented by the formula (F1), (F3), (F4), or (F36) by a conventional method, and the antiserum thereof may be obtained.

In the method using genetic engineering, the formula (F
1), (F3), (F4), or (F36) is immunized with a glycolipid represented by (F36), spleen cells are taken out, and RN
A is extracted, a cDNA library is prepared, and the target gene is isolated using the antigen-binding property as an index. Then, the microorganisms or cells into which the antibody expression vector has been introduced may be cultured and purified by a usual method. For purification, ordinary methods such as ammonium sulfate precipitation and salting out may be used.

As described above, the antibody used in the present invention may be not only a natural type antibody but also an artificial type antibody produced by a genetic engineering method or a protein chemical method. As an example of an artificial antibody produced by using a genetic engineering method, a variable region necessary for binding to an antigen is derived from a mouse, and an invariant region not necessary to bind to an antigen is a human-derived chimeric antibody. To be done. Such an antibody can be produced by a method well known in the art per se. Further, as an example of an artificial antibody produced by using a protein chemical method, a fragmented antibody such as Fab which can be prepared by cleaving an antibody molecule with papain can be mentioned.

[0014]

EXAMPLES Examples will be described below in order to explain the present invention in more detail, but the present invention is not limited to these examples.

The antibodies used in the examples are control mouse antibodies, monoclonal antibody F1α75 obtained by immunizing the glycolipid represented by the formula (F1), and glycolipid represented by the formula (F3). Obtained monoclonal antibody F3
α25A3, F3α328, monoclonal antibodies F4α3D8, F obtained by immunization with the glycolipid represented by the formula (F4)
The monoclonal antibody F36α2G4 obtained by immunizing 4α3F10, F4α3E5, F4α2F11 and the glycolipid represented by the formula (F36) was used. These antibodies are described in Japanese Patent Application Laid-Open No. 3-280894, 1992, by the present inventors.
Manufactured as described in the application “Monoclonal antibody and its manufacturing method” dated March 28, the application “Monoclonal antibody and its manufacturing method” dated July 28, 1992, and the application “Monoclonal antibody and its manufacturing method” dated July 28, 1992 did.

Example 1 Recognition of HIV-Infected Cells by Monoclonal Antibodies Recognizing Glycolipids The reactivity of various monoclonal antibodies recognizing glycolipids with HIV-infected cells was examined.

The antibody is a control mouse antibody or antibody F.
1α75, F3α25A3, F4α3D8, F4α3F
10, F4α3E5, and F4α2F11 were used.
For HIV, type 1 Bru (Barre-Sinou)
ssi et al., Science, 220, p868, 198.
3rd year) and type 2 Rod (Clavel et al., Sc
Ience, 233, p343, 1986). Infected cells include T cell line H9 and lymphoma cell U93
7. PHA blast of lymphocytes and PHA blast of macrophages were used. Experiments using cytofluorometric analysis (FACS) are described in HONDA et al.
Immunology, 145, p4131, 1990.
Followed the method.

The results are shown in Tables 1 and 2. Tables 1 and 2 are HI
The percentage (%) of various V-infected cells stained with various antibodies is shown. As is clear from the table, Br is added to H9.
Infection of u reduced the percentage of F1α75 cell staining. In addition, U1937 was infected with Rod, F1α75
The percentage of cell staining of F4α3E5 and F4α2F11 increased, while the percentage of cell staining of F4α2E11 increased. Furthermore U
Infection of 937 with Bru increased the rate of cell staining for all antibodies.

On the other hand, when PHA-blast (macrophage) was infected with Rod or Bru, the ratio of cell staining increased in almost all the antibodies.

From the above results, it is understood that this method provides a method for detecting AIDS-infected cells with an antibody that recognizes a mucin-type sugar chain, and that this antibody is useful as a diagnostic agent for AIDS.

Example 2 Inhibition of HIV Infection of Various Monoclonal Antibodies Recognizing Glycolipids The inhibitory effect on HIV infection of cells by various monoclonal antibodies recognizing glycolipids was examined. The antibodies are control mouse antibodies, antibodies F1α75, F3α25A3, F
3α328, F4α3D8, F4α3F10, F4α2
F11 and F36α2G4 were used. HIV has
CD4 antigen-free type NDK (Cher
mann et al., Human Retroviruses
(Eds. Stehelin, D.) 14, 1988), and type 1 Bru and type 2 Rod. U937 lymphoma cells were used as infected cells. The degree of infection depends on the amount of p24 antigen that is the HIV core protein
It was measured by solid phase enzyme immunoassay (ELISA) using 24 antibodies. The measurement was carried out on the day, 3, 7 and 7 of the day when each cell was infected with HIV, and the amount of p24 in the infected cell (p
g / ml) was measured.

The results are shown in Tables 3 and 4. As is clear from the table, it can be seen that HIV infection of cells is inhibited by various antibodies. For example, NDK infection is F1α7
5, F4α3D8, F4α3F10, and F4α2F
It was completely inhibited by 11. See also Bru and Rod
Some inhibition was observed with all the antibodies for H. pylori infection.

From the above results, the present method provides a detailed method for analyzing the inhibitory effect on HIV infection of cells by an antibody that recognizes a mucin-type sugar chain, and the present antibody is useful as a therapeutic agent for AIDS. It is understood that there is.

Table 1 Unit (%) Cell H9 U937 ───────────── ──────────── Antibody Virus (−) Bru (−) Bru Rod ─── ──────────────────────────────── Control 0.7 1.0 1.0 0.7 0.7 0.9 F1α75 48 0.5 19.6 19.5 12.5 4.9 F3α25A3 0.6 0.6 0.6 2.7 1.4 F4α3D8 5.7 4.8 0.5 2.4 2.4 F4α3F10 5.7 4.3 0.3 1.5 0.7 F4α3E5 3.7 4.6 0.3 2.4 1.0 F4α2F11 5.0 4.7 4.7 0.2 1.7 1.3 Table 2 Unit (%) PHA-blast PHA-blast cells (lymphocytes) (macrophages) ─────────────────── ────────── Antibody virus (−) Bru Rod (−) Bru Rod ────────────────────────────── ────── Control 1.0 1.0 1.0 1.0 0.9 0.8 1.0 F1α75 24.3 24.9 19.7 65.9 89.7 82.2 F3α25A3 1.7 1. 0 2.2 0.9 0.6 2.6 F4α3D8 5.4 7.1 9.4 2.1 11.3 23.3 F4α3F10 5.5 6.0 8.8 1.5 10.2 26. 8 F4α3E5 5.6 5.2 9.3 1.0 4.2 17.9 F4α2F11 4.9 6.5 8.7 1.2 NT 12.3

Table 3 Unit (pg / ml) NDK Bru ────────── ─────────────── Antibody days 0 3 7 0 3 7 7 ───── ─────────────────────────────── Control ND ND 140.4 ND 127.3 531.0 F1α75 ND ND ND ND ND 157.9 F3α25A3 ND ND 132.9 ND 110.5 157.3 F3α328 ND ND 146.6 ND 98.0 128.6 F4α3D8 ND ND ND ND 10D. 9 F4α2F11 ND ND ND ND ND 144.2 F36α2G4 ND ND 424.3 ND 128.6 266.5

[0026]

Industrial Applicability The application of the antibody provided by the present invention, which recognizes an antibody recognizing a mucin-type sugar chain, to a diagnostic agent or therapeutic agent for AIDS is very useful for the diagnosis and treatment of AIDS, which is expected to increase in the future. It is expected. In addition, it is expected that studies using these antibodies will advance basic research on HIV infection and the onset of AIDS, and elucidate a more detailed mechanism.

Claims (1)

[Claims] 1. Formula (F1), (F3), (F4), or (F36) Galβ1 → 4GlcNAcβ1 → 6GalNAcα1 → 1Cer (F1) Galβ1 → 4GlcNAcβ1 → 3GalNAcα1 → 1Cer (F3) Galβ1 → 1G6NA1β4 → 1G6NAcβ1 → 4GlcNA 3) A pharmaceutical composition for AIDS containing an antibody recognizing a mucin-type glycolipid represented by GalNAcα1 → 1Cer (F4) Galβ1 → 4GlcNAcβ1 → 6 (Galβ1 → 4GlcNAcβ1 → 3) GalNAcα1 → 1Cer (F36) as an active ingredient.