CN1339319A - Medicine for treating AIDS and its preparing method - Google Patents

Abstract

The invention discloses a medicine for treating AIDS and a preparation method thereof. The medicine for treating AIDS of the invention basically includes at least one monoclonal antibody, and the antigen of the antibody is a neutralizing antibody on the membrane protein gp120 of human immunodeficiency virus. The epitope GPGRAFY epitope and its variant epitope, the variant epitope is GPGQTFY or GPGQAWY. The preparation method of the medicine of the present invention is (1) artificially synthesizing at least one neutralizing epitope GPGRAFY or/and its mutated epitope respectively on the human immunodeficiency virus membrane protein gp120; (2) coupling the above polypeptides to the carrier respectively (3) immunize animals with the above-mentioned conjugate; (4) prepare hybridoma; (5) mix the obtained antibody with a pharmaceutical adjuvant to prepare a drug.

Description

A kind of medicine for treating AIDS and preparation method thereof

The invention relates to a medicine for treating AIDS prepared by bioengineering technology and a preparation method thereof, in particular to a medicine for treating AIDS prepared by using a monoclonal antibody of HIV membrane protein and a preparation method thereof.

AIDS, also known as acquired immunodeficiency syndrome, is an immune disease caused by the human immunodeficiency virus (HIV). Due to its rapid spread, high case fatality rate, and currently no specific treatment, and no vaccine to prevent it, it is known as the "plague of the century".

The existing clinically used anti-AIDS drugs can only delay the onset and prolong the patient's life, and are expensive and highly toxic. The results of recent clinical studies of foreign anti-AIDS drugs have proved that the combined use of neutralizing antibodies (monoclonal antibodies) targeting several specific neutralizing epitopes on the HIV-1 membrane protein gp120 in passive immunotherapy can inhibit the growth of HIV-1 virus. Mucosal infection and mother-to-child transmission, and can clear the HIV-1 virus in the blood (Nature Medicine 1999, 5: 204; Nature Medicine 2000, 6: 200; Nature Medicine 1999, 5: 211); the main on HIV-1 gp120 The neutralizing epitope GPGRAFY and the antibody recognizing the epitope can inhibit HIV-1 from infecting target cells in vitro, and can prevent HIV-1 from infecting chimpanzees (Nature 1990, 345: 622; Science 1992, 256: 1687; Nature 1992, 355: 728-730; J. Biol. Chem. 1995, 271:8236).

The purpose of the present invention is to provide an effective medicine for treating AIDS, which can deal with the variation of HIV.

Another object of the present invention is to provide a method for preparing the above-mentioned medicine for treating AIDS.

To achieve the above object, the present invention adopts the following design scheme: a medicine for treating AIDS, which basically includes at least one monoclonal antibody, the antigen of which is the neutralizing epitope GPGRAFY on the human immunodeficiency virus membrane protein gp120 Epitopes and their variant epitopes.

The variant epitope is GPGQTFY or GPGQAWY.

The medicine for treating AIDS of the present invention preferably consists of the following components:

    GPGRAFY-epitope-specific monoclonal antibody

      GPGQTFY-epitope-specific monoclonal antibody

GPGQAWY-epitope-specific monoclonal antibody

A kind of method for preparing above-mentioned medicine for treating AIDS, comprises the following steps basically:

(1), artificially synthesize at least one epitope polypeptide respectively containing the neutralizing epitope on the human immunodeficiency virus membrane protein gp120, and the neutralizing epitope is selected from the GPGRAFY epitope or/and its variant epitope or at least once Repeated GPGRAFY epitopes or/and its variant epitopes;

(2) Coupling the above-mentioned polypeptides to carrier proteins or carrier polypeptides to form conjugates;

(3) Immunizing animals with the above-mentioned conjugates respectively with acceptable adjuvants;

(4), using conventional cell fusion technology to prepare hybridomas respectively;

(5) Purify the antibodies obtained from the above-mentioned different hybridoma cell lines, and mix them to make a drug for treating AIDS.

Wherein, the at least one artificially synthesized polypeptide preferably contains at least one repeated GPGRAFY epitope or its variant epitope on human immunodeficiency virus membrane protein gp120. The variant epitope of the GPGRAFY epitope is GPGQTFY or GPGQAWY.

Studies have shown that antibodies against neutralizing epitopes on the HIV-1 virus membrane protein can reduce the viral load and delay the progress of the disease in the treatment of AIDS. The medicine for the treatment of AIDS of the present invention is the medicine that contains the GPGRAFY epitope on the human immunodeficiency virus membrane protein gp120 and its variant epitope (such as GPGQTFY, GPGQAWY) the medicine of multiple antibodies, even under the situation that AIDS virus produces mutation, injection People who have taken the drug will also have corresponding antibodies to kill the mutated virus.

The medicine of the invention is not only non-toxic, but also can reduce the cost of AIDS treatment while improving the immunotherapy effect of AIDS.

The present invention adopts the method of artificially synthesizing epitope polypeptides to induce and prepare monoclonal antibodies with predetermined epitope specificity, which overcomes the need to use natural protein or recombinant protein for immunization, and then a large number of screening and identification can obtain predetermined epitope specificity. The complex work of monoclonal antibodies.

According to the present invention, according to the variation of AIDS virus, the corresponding type of medicine can be produced quickly without long-term testing, thereby reducing the production cost. This technology will have a major impact on the world's preventive medicine research, and will bring huge economic and social benefits.

The present invention will be further described below in conjunction with non-limiting specific examples.

Embodiment 1: The drug for treating AIDS made by the antibody of the neutralizing epitope GPGRAFY on the anti-HIV-1 gp120 as the main active ingredient is produced by the following steps:

(1), artificially synthesize an epitope polypeptide C-(GPGRAFY)4 containing 4 times of repeated GPGRAFY neutralizing epitopes on the human immunodeficiency virus membrane protein gp120;

(2), using MBS (m-maleimidobenzoyl-N-hydroxy succinimide ester) to couple the above-mentioned epitope polypeptide to the carrier protein BSA;;

(3) Mix the above-mentioned conjugate with Freund's adjuvant (the weight ratio of the two substances is: conjugate:Freund's adjuvant=1:1) to immunize Balb/c mice. Complete Freund's adjuvant for the first time, then immunize once every two weeks, and use incomplete Freund's adjuvant. The antigen dose for each immunization is: conjugates containing 10 micrograms of epitope polypeptides/time/only, co-immunization 3 times;

(4) Fusion of immunized mouse splenocytes and mouse myeloma cell lines using conventional cell fusion techniques to prepare hybridomas;

(5) Screening clones capable of producing monoclonal antibodies with predefined epitope specificity from the above hybridoma cell lines, and making the purified antibodies into drugs for treating AIDS.

The dosage of the drug used in actual application is determined according to the patient's condition.

Embodiment two: the drug for the treatment of AIDS made of anti-HIV-1 gp120 neutralizing epitope GPGRAFY and its variant epitope GPGQTFY, GPGQAWY antibody as the main active ingredient, produced by the following steps:

(1), 4 artificially synthesized epitope polypeptides containing the neutralizing epitope GPGRAFY and its variant epitopes GPGQTFY and GPGQAWY respectively on the human immunodeficiency virus membrane protein gp120:

CGPGRAFYGPGRAFYGPGRAFYGPGRAFY

CGPGQTFYGPGQTFYGPGQTFYGPGQTFY

CGPGQAWYGPGQAWYGPGQAWYGPGQAWY

(2) Using glutaraldehyde or MBS to couple the above polypeptides to the carrier protein bovine serum albumin to form a conjugate;

(3) Mix the above-mentioned conjugate with Freund's adjuvant (the volume ratio of the two substances is: conjugate:Freund's adjuvant=1:1) to immunize Balb/c mice. Complete Freund's adjuvant was used for the first time, and then every two weeks, incomplete Freund's adjuvant was used for immunization. The dose of antigen for each immunization is: Conjugates containing 10 micrograms of epitope polypeptides/time/piece, immunized 3 times in total;

(4) Fusion of immunized mouse splenocytes and mouse myeloma cell lines using conventional cell fusion techniques to prepare hybridomas;

(5) Screening clones capable of producing monoclonal antibodies with predefined epitope specificity from the above hybridoma cell lines.

(6) Three kinds of antibodies against the neutralizing epitope GPGRAFY on HIV-1 gp120 and its variant epitopes GPGQTFY and GPGQAWY were respectively obtained from the above-mentioned hybridoma cell lines, and the above-mentioned antibodies were made into a drug for treating AIDS, wherein each The amount of each antibody added is adjusted by the occurrence probabilities of various epitopes obtained from the statistical processing of the applicable population.

Using the medicine of the invention can obviously resist the variation of HIV, reduce the load of HIV, delay the progress of the disease, and finally achieve the purpose of cure.

Claims (6)

1, a kind of medicine for the treatment of acquired immune deficiency syndrome (AIDS), it consists essentially of at least a monoclonal antibody, and the antigen of described antibody is neutralizing epitope GPGRAFY epi-position and the variant epitope thereof on human immunodeficiency virus's memebrane protein gp120.

2, a kind of medicine for the treatment of acquired immune deficiency syndrome (AIDS) according to claim 1, it is characterized in that: described variant epitope is GPGQTFY or GPGQAWY.

3, a kind of medicine for the treatment of acquired immune deficiency syndrome (AIDS) according to claim 1 and 2, it is characterized in that: it is composed of the following components: the monoclonal antibody of GPGRAFY-epitope specificity

The monoclonal antibody of GPGQTFY-epitope specificity

The monoclonal antibody of GPGQAWY-epitope specificity

4, a kind of method for preparing the medicine of the described treatment acquired immune deficiency syndrome (AIDS) of claim 1-3 consists essentially of following steps:

(1), synthetic at least one contains the epitope polypeptide of the neutralizing epitope on human immunodeficiency virus's memebrane protein gp120 respectively, described neutralizing epitope be selected from the GPGRAFY epi-position or/and its variant epitope or at least once multiple GPGRAFY epi-position or/and its variant epitope;

(2), aforementioned polypeptides is coupled to respectively on carrier protein or the carrier polypeptide forms coupling matter;

(3), be equipped with acceptable adjuvant immunity animal respectively with above-mentioned coupling matter;

(4), adopt conventional cell-fusion techniques to prepare hybridoma respectively;

(5), the antibody purification that will obtain from above-mentioned different hybridoma cell lines, be mixed and made into the medicine of treatment acquired immune deficiency syndrome (AIDS).

5, the method for the medicine of preparation treatment acquired immune deficiency syndrome (AIDS) according to claim 4, it is characterized in that: at least one polypeptide of described synthetic, contain at least once multiple GPGRAFY epi-position or its variant epitope on human immunodeficiency virus's memebrane protein gp120 respectively.

6, according to the method for the medicine of claim 4 or 5 described preparations treatment acquired immune deficiency syndrome (AIDS), it is characterized in that: the variant epitope of described GPGRAF epi-position is GPGQTF or GPGQAWF.