WO2002032452A1 - Composition for aids and method producing it - Google Patents
Composition for aids and method producing it Download PDFInfo
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- WO2002032452A1 WO2002032452A1 PCT/CN2001/001192 CN0101192W WO0232452A1 WO 2002032452 A1 WO2002032452 A1 WO 2002032452A1 CN 0101192 W CN0101192 W CN 0101192W WO 0232452 A1 WO0232452 A1 WO 0232452A1
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- 238000000034 method Methods 0.000 title claims abstract description 13
- 239000000203 mixture Substances 0.000 title abstract description 6
- 208000030507 AIDS Diseases 0.000 claims abstract description 30
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 18
- 230000003472 neutralizing effect Effects 0.000 claims abstract description 17
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 17
- 229920001184 polypeptide Polymers 0.000 claims abstract description 16
- 101800001690 Transmembrane protein gp41 Proteins 0.000 claims abstract description 11
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 10
- 239000000427 antigen Substances 0.000 claims abstract description 5
- 102000036639 antigens Human genes 0.000 claims abstract description 5
- 108091007433 antigens Proteins 0.000 claims abstract description 5
- 230000008878 coupling Effects 0.000 claims abstract description 5
- 238000010168 coupling process Methods 0.000 claims abstract description 5
- 238000005859 coupling reaction Methods 0.000 claims abstract description 5
- 102000014914 Carrier Proteins Human genes 0.000 claims abstract description 4
- 108010078791 Carrier Proteins Proteins 0.000 claims abstract description 4
- 241001465754 Metazoa Species 0.000 claims abstract description 3
- 239000003814 drug Substances 0.000 claims description 31
- 241000725303 Human immunodeficiency virus Species 0.000 claims description 24
- 229940079593 drug Drugs 0.000 claims description 11
- 239000002671 adjuvant Substances 0.000 claims description 10
- 230000007910 cell fusion Effects 0.000 claims description 4
- 108091005703 transmembrane proteins Proteins 0.000 claims description 3
- 102000035160 transmembrane proteins Human genes 0.000 claims description 3
- 238000011282 treatment Methods 0.000 abstract description 7
- 230000003053 immunization Effects 0.000 abstract description 5
- 241000700605 Viruses Species 0.000 abstract description 4
- 230000015572 biosynthetic process Effects 0.000 abstract description 2
- 238000003786 synthesis reaction Methods 0.000 abstract description 2
- 239000003795 chemical substances by application Substances 0.000 abstract 2
- 230000007812 deficiency Effects 0.000 abstract 2
- 230000001225 therapeutic effect Effects 0.000 abstract 1
- 108010052285 Membrane Proteins Proteins 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
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- 239000000126 substance Substances 0.000 description 3
- 241000699670 Mus sp. Species 0.000 description 2
- 206010035226 Plasma cell myeloma Diseases 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 239000002259 anti human immunodeficiency virus agent Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 201000000050 myeloid neoplasm Diseases 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 206010061818 Disease progression Diseases 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
- C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- the present invention relates to a medicament for treating AIDS prepared by bioengineering technology and a method for preparing the same, in particular to a medicament for treating AIDS prepared by using a monoclonal antibody against HIV membrane protein and a preparation method thereof.
- the disease also known as acquired immunodeficiency syndrome, is an immune disease caused by the human immunodeficiency virus (HIV). Because of its rapid spread and extremely high mortality, there are no specific treatments, and no vaccine can prevent it.
- HIV human immunodeficiency virus
- the object of the present invention is to provide an effective medicine for treating AIDS, which medicine can deal with the mutation of the HIV virus.
- Another object of the present invention is to provide a method for preparing the above medicament for treating AIDS.
- the present invention adopts the following design scheme: A drug for treating AIDS, which basically includes at least one monoclonal antibody whose antigen is the ELDKWA epitope on human immunodeficiency virus transmembrane protein gp41 and Its variant epitope.
- the variant epitope is ELEKWA, ELNKWA or ELDEWA.
- the medicine for treating AIDS of the present invention preferably consists of the following components:
- ELDKWA epidermal-specific monoclonal antibody
- ELEKWA epidermal-specific monoclonal antibody
- a method for preparing the above medicament for treating AIDS basically includes the following steps: (1) artificially synthesizing at least one epitope polypeptide containing a neutralizing epitope on human immunodeficiency virus transmembrane protein gp41, and the neutralizing The epitope is selected from the ELDKWA epitope or / and its variant epitope or at least one repeating ELDKWA epitope or / and its variant epitope;
- the at least one artificially synthesized polypeptide is preferably: at least one repeated neutralizing epitope ELDKWA or its variant epitope on human immunodeficiency virus transmembrane protein gp41, respectively.
- the variant epitope of the ELDKWA epitope is ELEKWA, ELNKWA or ELDEWA.
- the medicine for treating AIDS of the present invention is a medicine containing various antibodies against the main neutralizing epitope ELDKWA epitope on human immunodeficiency virus transmembrane protein gp41 and its variant epitopes (such as ELEKWA, ELNKWA, ELDEWA), even in AIDS
- ELDKWA epitope on human immunodeficiency virus transmembrane protein gp41
- its variant epitopes such as ELEKWA, ELNKWA, ELDEWA
- the medicament of the invention is not only non-toxic, but also can reduce the cost of AIDS treatment while improving the effect of AIDS immunotherapy.
- the invention adopts a method of artificially synthesizing epitope polypeptide to induce and prepare a predetermined monoclonal antibody specific for an epitope, which overcomes the need to immunize with a natural protein or a recombinant protein, and then performs a large number of screening and identification to obtain the predetermined epitope specificity.
- Sexual monoclonal antibodies perform many complex tasks.
- the corresponding type of medicine can be produced quickly according to the mutation of the HIV virus, without the need for long-term tests, and the production cost can be reduced.
- This technology will have a significant impact on the world's preventive medicine research and will bring huge economic and social benefits.
- Example 1 A medicament for treating AIDS made from a neutralized epitope ELDKWA on HIV-1 gp41, a variant epitope ELNKWA-epitope-specific antibody as a main active ingredient, produced by the following steps:
- the dosage of the drug in actual application is determined according to the patient's condition.
- Example 2 From neutralizing epitope ELDKWA and its variant epitope ELNKWA on anti-HIV-1 g p4l,
- Antibodies to ELEKWA and ELDEWA are the main active ingredients for the treatment of AIDS. They are produced by the following steps-(1), artificial synthesis of 4 human immunodeficiency virus transmembrane protein gp4l neutralizing epitopes ELDKWA and its variants.
- Epitope ELNKWA, ELEKWA or ELDEWA polypeptide are produced by the following steps-(1), artificial synthesis of 4 human immunodeficiency virus transmembrane protein gp4l neutralizing epitopes ELDKWA and its variants.
- the mutation of HIV can be obviously resisted, the load of HIV can be reduced, the progress of the disease can be delayed, and finally the purpose of cure is achieved.
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Abstract
Therapeutic composition for treatment of AIDS is disclosed, and the method producing the agent is also disclosed. The composition for treatment of AIDS of the invention substantially comprise at least a monoclonal antibody to which the antigen is ELDKWA neutralizing epitope on the human immune deficiency associated virus transmembrane protein gp41 or the variants thereof, and the said variant epitopes are ELEKWA,ELNKWA, ELDEWA. The method producing the agent comprise: (1) artificially synthesis of at least a strand of peptide containing neutralizing epitope ELDKWA on the human immue deficiency associated virus transmembrane protein gp41 and/or the variants thereof; (2) coupling the said peptides to a carrier protein or a carrier polypeptide individually; (3) immunizing an animal with the conjugate; (4) producing hybridoma; (5) mixing the four neutralizing antibodies to obtain the composition.
Description
治疗艾滋病的药物及其制备方法 Medicine for treating AIDS and preparation method thereof
技术领域 Technical field
本发明涉及用生物工程技术制备的治疗艾滋病的药物及其制各方 法， 特别是涉及用艾滋病病毒膜蛋白的单克隆抗体制备的治疗艾滋病的 药物及其制备方法。 The present invention relates to a medicament for treating AIDS prepared by bioengineering technology and a method for preparing the same, in particular to a medicament for treating AIDS prepared by using a monoclonal antibody against HIV membrane protein and a preparation method thereof.
背景技术 Background technique
文滋病又称获得性免疫缺陷综合征， 是由人类免疫缺陷病毒 （HIV) 引 起的一种免疫性疾病。 由于其传播迅速、 病死率极高， 且目前尚无特效治疗 方法， 更无疫苗可以预防。 The disease, also known as acquired immunodeficiency syndrome, is an immune disease caused by the human immunodeficiency virus (HIV). Because of its rapid spread and extremely high mortality, there are no specific treatments, and no vaccine can prevent it.
现有的用于临床的抗艾滋病药物仅能推迟发病期和延长病人的生命， 并且价格昂贵，毒性大。最近国外抗艾滋病药物临床研究结果证明，针对 HIV - 1 膜蛋白 gpl60 (膜蛋白 gP120及跨腠蛋白 gp41的前体蛋白） 上的几个特定中和 表位的中和抗体（其中包括 ELDKWA—表位特异性的单克隆抗体 2F5)在被动免 疫治疗中的组合使用能抑制 HIV— 1病毒的粘膜传染以及母婴传播，并能清除 血液中的 HIV— 病毒（Nature Medicine 1999， 5 : 204; Nature Medicine 2000, 6 : 200 ; Nature Medicine 1999, 5 : 211)； 人免疫缺陷病毒（HIV- 1)跨膜蛋 白 gp41上的主要中和表位 ELDKWA的表位特异性单克隆抗体能够体外抻制多种 HIV— 1病毒株感染靶细胞 (J. Virology 1993, 67 : 6642 ; AIDS Res. Human Retroviruses 1994, 10 : 1651 ; AIDS 1996， 10 : 587 )。 Existing anti-AIDS drugs for clinical use can only delay the onset and prolong the life of patients, and are expensive and highly toxic. Recent clinical studies of anti-AIDS drugs abroad have shown that several specific neutralizing epitope neutralizing antibodies (including ELDKWA) against HIV-1 membrane protein gpl60 (precursor protein of membrane protein g P 120 and streptavidin gp41) —The combination of epitope-specific monoclonal antibody 2F5) in passive immunotherapy can inhibit mucosal transmission of HIV-1 virus and mother-to-child transmission, and can clear HIV-virus from the blood (Nature Medicine 1999, 5: 204 ; Nature Medicine 2000, 6: 200; Nature Medicine 1999, 5: 211); Human immunodeficiency virus (HIV-1) epitope-specific monoclonal antibody on the transmembrane protein gp41, ELDKWA, is able to lyse in vitro A variety of HIV-1 strains were produced to infect target cells (J. Virology 1993, 67: 6642; AIDS Res. Human Retroviruses 1994, 10: 1651; AIDS 1996, 10: 587).
发明公开 Invention Disclosure
本发明的目的是提供一种有效的治疗艾滋病的药物， 该药物可对付艾滋 病病毒的变异。 The object of the present invention is to provide an effective medicine for treating AIDS, which medicine can deal with the mutation of the HIV virus.
本发明的另一个目的是提供一种制备上述治疗艾滋病的药物的方法。 为实现上述目的， 本发明采取以下设计方案： 一种治疗艾滋病的药物， 它基本上包括至少一种单克隆抗体， 所述抗体的抗原是人免疫缺陷病毒跨膜 蛋白 gp41上的 ELDKWA表位及其变异表位。 Another object of the present invention is to provide a method for preparing the above medicament for treating AIDS. In order to achieve the above object, the present invention adopts the following design scheme: A drug for treating AIDS, which basically includes at least one monoclonal antibody whose antigen is the ELDKWA epitope on human immunodeficiency virus transmembrane protein gp41 and Its variant epitope.
所述变异表位为 ELEKWA、 ELNKWA或 ELDEWA。 The variant epitope is ELEKWA, ELNKWA or ELDEWA.
本发明治疗艾滋病的药物优选由以下组分组成： The medicine for treating AIDS of the present invention preferably consists of the following components:
ELDKWA—表位特异性的单克隆抗体
ELEKWA—表位特异性的单克隆抗体 ELDKWA—epitope-specific monoclonal antibody ELEKWA—epitope-specific monoclonal antibody
ELNKWA―表位特异性的单克隆抗体 ELNKWA-epitope-specific monoclonal antibody
ELDE —表位特异性的单克隆抗体 ELDE — epitope-specific monoclonal antibody
一种制备上述治疗艾滋病的药物的方法， 基本上包括以下步骤： ( 1 )、 人工合成至少一条分别含有人免疫缺陷病毒跨膜蛋白 gp41上的中 和表位的表位多肽， 所述中和表位选自 ELDKWA表位或 /和它的变异表位或至 少一次重复的 ELDKWA表位或 /和它的变异表位； A method for preparing the above medicament for treating AIDS basically includes the following steps: (1) artificially synthesizing at least one epitope polypeptide containing a neutralizing epitope on human immunodeficiency virus transmembrane protein gp41, and the neutralizing The epitope is selected from the ELDKWA epitope or / and its variant epitope or at least one repeating ELDKWA epitope or / and its variant epitope;
( 2 )、 将上述表位多肽分别耦联到载体蛋白或载体多肽上形成耦联物； (2) coupling the epitope polypeptide to a carrier protein or a carrier polypeptide to form a conjugate;
( 3 )、 用上述耦联物分别配以可接受的佐剂免疫动物； (3) Immunize the animals with the above-mentioned conjugates and accept an acceptable adjuvant, respectively;
( 4)、 采用常规的细胞融合技术分别制备杂交瘤； (4) preparing conventional hybridomas using conventional cell fusion techniques;
( 5)、 将自上述不同杂交瘤细胞系（克隆)产生得到的抗体纯化， 混合制 成治疗艾滋病的药物。 (5) Purify the antibodies produced from the above-mentioned different hybridoma cell lines (clones) and mix them to produce a drug for treating AIDS.
其中， 所述人工合成的至少一条多肽优选为： 分别含有人免疫缺陷病毒 跨膜蛋白 gp41上的至少一次重复的中和表位 ELDKWA或它的变异表位。 Wherein, the at least one artificially synthesized polypeptide is preferably: at least one repeated neutralizing epitope ELDKWA or its variant epitope on human immunodeficiency virus transmembrane protein gp41, respectively.
所述 ELDKWA表位的变异表位为 ELEKWA、 ELNKWA或 ELDEWA。 The variant epitope of the ELDKWA epitope is ELEKWA, ELNKWA or ELDEWA.
研究表明， 针对 HIV— 1膜蛋白上中和表位的抗体在艾滋病治疗中能降低 病毒的载量， 延缓疾病的进行。 本发明治疗艾滋病的药物是含有抗人免疫缺 陷病毒跨膜蛋白 gp41上的主要中和表位 ELDKWA表位及其变异表位 （如 ELEKWA, ELNKWA, ELDEWA ) 的多种抗体的药物， 即使在艾滋病病毒产生变异 的情况下， 注射了该药物的人体内也会有相应的抗体来杀灭变异的病毒。 Studies have shown that antibodies against neutralizing epitopes on HIV-1 membrane proteins can reduce viral load and delay disease progression in AIDS treatment. The medicine for treating AIDS of the present invention is a medicine containing various antibodies against the main neutralizing epitope ELDKWA epitope on human immunodeficiency virus transmembrane protein gp41 and its variant epitopes (such as ELEKWA, ELNKWA, ELDEWA), even in AIDS When the virus is mutated, the person who has injected the drug will also have corresponding antibodies to kill the mutated virus.
本发明的药物不但无毒性， 而且在提高艾滋病的免疫治疗效果的同时， 还可降低艾滋病治疗成本。 The medicament of the invention is not only non-toxic, but also can reduce the cost of AIDS treatment while improving the effect of AIDS immunotherapy.
本发明采用人工合成表位多肽的方法来诱导、 制备预先确定的表位特异 性的单克隆抗体， 克服了需要利用天然蛋白或重组蛋白免疫， 然后大量筛选 和鉴定才能得到预先确定的表位特异性的单克隆抗体的诸多复杂工作。 The invention adopts a method of artificially synthesizing epitope polypeptide to induce and prepare a predetermined monoclonal antibody specific for an epitope, which overcomes the need to immunize with a natural protein or a recombinant protein, and then performs a large number of screening and identification to obtain the predetermined epitope specificity. Sexual monoclonal antibodies perform many complex tasks.
根据本发明， 可以按照艾滋病病毒的变异情况， 很快生产出其相应类型 的药物， 不需进行长时间的试验， 降低生产成本。 该技术将对世界预防医学 研究产生重大影响， 并将带来巨大的经济效益和社会效益。 According to the present invention, the corresponding type of medicine can be produced quickly according to the mutation of the HIV virus, without the need for long-term tests, and the production cost can be reduced. This technology will have a significant impact on the world's preventive medicine research and will bring huge economic and social benefits.
下而结合非限制性具体实施例对本发明作进一步说明。
实施发明的最佳方式 The present invention will be further described in combination with non-limiting specific examples. The best way to implement the invention
实施例 1 : 由抗 HIV— 1 gp41上的中和表位 ELDKWA的变异表位 ELNKWA—表 位特异性抗体为主要活性成分制成的治疗艾滋病的药物， 由以下步骤生产： Example 1: A medicament for treating AIDS made from a neutralized epitope ELDKWA on HIV-1 gp41, a variant epitope ELNKWA-epitope-specific antibody as a main active ingredient, produced by the following steps:
( 1 )、 人工合成一条含有人免疫缺陷病毒跨膜蛋白 gp41上的 4次重复的 ELNKWA中和表位的表位多肽 (1) An epitope polypeptide containing 4 repetitive ELNKWA neutralizing epitopes on human immunodeficiency virus transmembrane protein gp41 was artificially synthesized
CELNKWAGELNKWAGELNKWAGELNKWA； CELNKWAGELNKWAGELNKWAGELNKWA;
( 2 )、 禾 [J用 MBS (nrmaleimidobenzoyl- - hydroxy succinimide ester)将上 述表位多肽与载体蛋白 BSA耦联； (2) He (J used MBS (nrmaleimidobenzoyl--hydroxy succinimide ester) to couple the epitope polypeptide with the carrier protein BSA;
( 3 )、 将上述耦联物与福氏佐剂混合 （两种物质的体积比为， 耦联物： 福氏佐剂 = 1: 1 ) 免疫 Balb/c小鼠。 第一次用完全福氏佐剂， 以后每两周免 疫一次， 用不完全福氏佐剂。 每次免疫的抗原剂量为： 含 10微克表位多肽的 耦联物 /次 /只， 共免疫 3次； (3) Mix the above conjugate with Freund's adjuvant (the volume ratio of the two substances is: conjugate: Freund's adjuvant = 1: 1). Immunize Balb / c mice. Full Freund's adjuvant was used for the first time and immunized every two weeks thereafter, and incomplete Freund's adjuvant was used. The antigen dose for each immunization is: conjugates containing 10 micrograms of epitope polypeptide / times / only, a total of 3 immunizations;
(4)、 将免疫后的小鼠脾细胞与小鼠骨髓瘤细胞系采用常规的细胞融合 技术融合， 并制备杂交瘤； (4) Fusion of the immunized mouse spleen cells and the mouse myeloma cell line using conventional cell fusion technology, and preparing a hybridoma;
( 5 )、 从上述杂交瘤细胞系中筛选出能产生预先定义的表位特异性的单 克隆抗体的克隆， 将提纯的抗体制成治疗艾滋病的药物。 (5) Screening clones capable of producing a pre-defined epitope-specific single-clonal antibody from the above-mentioned hybridoma cell lines, and using the purified antibodies to prepare drugs for the treatment of AIDS.
该药物在实际应用中的使用剂量根据患者的病情来确定。 The dosage of the drug in actual application is determined according to the patient's condition.
实施例 2 : 由抗 HIV- 1 gp4l上的中和表位 ELDKWA及其变异表位 ELNKWA、Example 2: From neutralizing epitope ELDKWA and its variant epitope ELNKWA on anti-HIV-1 g p4l,
ELEKWA和 ELDEWA的抗体为主要活性成分制成的治疗艾滋病的药物， 由以下步 骤生产- ( 1 )、 人工合成 4条分别含有人免疫缺陷病毒跨膜蛋白 gp4l上的中和表 位 ELDKWA及其变异表位 ELNKWA、 ELEKWA或 ELDEWA的多肽： Antibodies to ELEKWA and ELDEWA are the main active ingredients for the treatment of AIDS. They are produced by the following steps-(1), artificial synthesis of 4 human immunodeficiency virus transmembrane protein gp4l neutralizing epitopes ELDKWA and its variants. Epitope ELNKWA, ELEKWA or ELDEWA polypeptide:
C ELDKWA G ELDKWA G ELDKWA G ELDKWA C ELDKWA G ELDKWA G ELDKWA G ELDKWA
C ELNKWA G ELNKWA G ELNKWA G ELNKWAC ELNKWA G ELNKWA G ELNKWA G ELNKWA
C ELEKWA G ELEKWA G ELEKWA G ELEKWA C ELDEWA G ELDEWA G ELDEWA G ELDEWA C ELEKWA G ELEKWA G ELEKWA G ELEKWA C ELDEWA G ELDEWA G ELDEWA G ELDEWA
(2 )、 利用戊二醛或 MBS将上述多肽分别耦联到载体蛋白牛血清白蛋白 上形成耦联物； (2) using glutaraldehyde or MBS to respectively couple the above polypeptides to the carrier protein bovine serum albumin to form a conjugate;
( 3 )、 将上述耦联物分别与福氏佐剂混合 （两种物质的体积比为， 耦联
物： 福氏佐剂 = 1 : 1 ) 后， 分别免疫 Balb/c小鼠。 第一次用完全福氏佐剂， 以后每两周免疫一次， 用不完全福氏佐剂。 每次免疫的抗原剂量为： 含 10微 克表位多肽的耦联物 /次 /只， 共免疫 3次； (3) Mixing the above couplings with Freund's adjuvant respectively (the volume ratio of the two substances is, coupling Substance: Freund's adjuvant = 1: 1), Balb / c mice were immunized separately. Full Freund's adjuvant was used for the first time and immunized every two weeks thereafter, and incomplete Freund's adjuvant was used. The antigen dose for each immunization is: conjugates containing 10 micrograms of epitope polypeptide / times / only for a total of 3 immunizations;
(4)、 将免疫后的小鼠脾细胞与小鼠骨髓瘤细胞系采用常规的细胞融合 技术融合， 并制备杂交瘤； (4) Fusion of the immunized mouse spleen cells and the mouse myeloma cell line using conventional cell fusion technology, and preparing a hybridoma;
( 5 )、 从上述杂交瘤细胞系中筛选出能产生预先定义的 4种表位特异性 的单克隆抗体的克隆。 (5) From the above hybridoma cell lines, select clones capable of producing monoclonal antibodies specific for the four epitopes defined in advance.
( 6 )、 自上述杂交瘤细胞系 （克隆） 的产物中分别得到抗 HIV— 1 gP41 上的中和表位 ELDKWA及其变异表位 ELNKWA、 ELEKWA、 ELDEWA的 4种单克隆抗 体， 将上述抗体混合， 制成治疗艾滋病的药物， 其中， 各种抗体的加入量由 适用人群的统计学处理得出的各种表位的出现概率进行调整。 (6) Obtaining four monoclonal antibodies against the neutralizing epitope ELDKWA and its variant epitopes ELNKWA, ELEKWA, and ELDEWA on HIV-1 g P 41 from the products of the above hybridoma cell lines (clones), respectively. The above antibodies are mixed to make a drug for treating AIDS, wherein the amount of various antibodies added is adjusted by the appearance probability of various epitopes obtained by statistical processing of the applicable population.
工业应用 Industrial applications
使用本发明的药物， 可以明显抵抗艾滋病病毒的变异， 降低艾滋病病毒 的载量， 延缓疾病的进行， 最后达到治愈的目的。
By using the medicine of the present invention, the mutation of HIV can be obviously resisted, the load of HIV can be reduced, the progress of the disease can be delayed, and finally the purpose of cure is achieved.
Claims
1、 一种治疗艾滋病的药物， 它基本上包括至少一种单克隆抗体， 所述 抗体的抗原是人免疫缺陷病毒跨膜蛋白 gp41上的 ELDKWA表位及其变异表位。 1. A medicament for treating AIDS, which basically comprises at least one monoclonal antibody whose antigen is the ELDKWA epitope and its variant epitope on the human immunodeficiency virus transmembrane protein gp41.
2、 根据权利要求 1所述的一种治疗艾滋病的药物， 其特征在于： 所述变 异表位为 ELEKWA、 ELNKWA或 ELDEWA。 2. A medicament for treating AIDS according to claim 1, characterized in that: the variant epitope is ELEKWA, ELNKWA or ELDEWA.
3、 根据权利要求 1或 2所述的一种治疗艾滋病的药物， 其特征在于： 它 是由以下组分组成的： ELDKWA—表位特异性的单克隆抗体 3. A medicine for treating AIDS according to claim 1 or 2, characterized in that it is composed of the following components: ELDKWA—epitope-specific monoclonal antibody
ELEKWA—表位特异性的单克隆抗体 ELEKWA—epitope-specific monoclonal antibody
ELNKWA—表位特异性的单克隆抗体 ELNKWA—epitope-specific monoclonal antibody
ELDEWA—表位特异性的单克隆抗体 ELDEWA—epitope-specific monoclonal antibody
4、 一种制备权利要求 1一 3所述的治疗艾滋病的药物的方法， 基本上包 括以下步骤- 4. A method for preparing a medicament for treating AIDS according to claims 1 to 3, basically comprising the following steps −
( 1 )、 人工合成至少一条分别含有人免疫缺陷病毒跨膜蛋白 gP41上的中 和表位的表位多肽， 所述中和表位选自 ELDKWA表位或 /和它的变异表位或至 少一次重复的 ELDKWA表位或 /和它的变异表位； (1) At least one epitope polypeptide containing a neutralizing epitope on human immunodeficiency virus transmembrane protein g P 41 is artificially synthesized, and the neutralizing epitope is selected from the ELDKWA epitope or / and its variant epitope Or at least one repeated ELDKWA epitope or / and its variant epitope;
( 2 )、 将上述表位多肽分别耦联到载体蛋白或载体多肽上形成耦联物； (2) coupling the epitope polypeptide to a carrier protein or a carrier polypeptide to form a conjugate;
( 3 )、 用上述耦联物分别配以可接受的佐剂免疫动物； (3) Immunize the animals with the above-mentioned conjugates and accept an acceptable adjuvant, respectively;
( 4 )、 采用常规的细胞融合技术分别制备杂交瘤； (4) preparing conventional hybridomas using conventional cell fusion techniques;
( 5 )、 将自上述不同杂交瘤细胞系得到的抗体纯化， 混合制成治疗艾滋 病的药物。 (5) Purify the antibodies obtained from the above-mentioned different hybridoma cell lines and mix them to prepare a drug for treating AIDS.
5、 根据权利要求 4所述的制备治疗艾滋病的药物的方法， 其特征在于： 所述人工合成的至少一条多肽分别含有人免疫缺陷病毒跨膜蛋白 gp4l上的至 少一次重复的中和表位 ELDKWA或它的变异表位。 5. The method for preparing a medicament for treating AIDS according to claim 4, characterized in that: said at least one artificially synthesized polypeptide contains at least one repeated neutralizing epitope ELDKWA on human immunodeficiency virus transmembrane protein gp4l, respectively. Or its variant epitope.
6、 根据权利要求 4或 5所述的制备治疗艾滋病的药物的方法， 其特征在 于： 所述 ELDKWA表位的变异表位为 ELEKWA、 ELNKWA或 ELDEWA。
6. The method for preparing a medicament for treating AIDS according to claim 4 or 5, characterized in that: the variant epitope of the ELDKWA epitope is ELEKWA, ELNKWA or ELDEWA.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2002214920A AU2002214920A1 (en) | 2000-08-18 | 2001-07-20 | Composition for aids and method producing it |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB001234838A CN1172717C (en) | 2000-08-18 | 2000-08-18 | A kind of medicine for treating AIDS and preparation method thereof |
| CN00123483.8 | 2000-08-18 |
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| WO2002032452A1 true WO2002032452A1 (en) | 2002-04-25 |
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| PCT/CN2001/001192 WO2002032452A1 (en) | 2000-08-18 | 2001-07-20 | Composition for aids and method producing it |
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| CN (1) | CN1172717C (en) |
| AU (1) | AU2002214920A1 (en) |
| WO (1) | WO2002032452A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017093985A1 (en) | 2015-12-05 | 2017-06-08 | Centre Hospitalier Universitaire Vaudois | Hiv binding agents |
| WO2020012435A1 (en) | 2018-07-13 | 2020-01-16 | Lausanne University Hospital | Hiv binding agents |
| WO2021009697A2 (en) | 2019-07-15 | 2021-01-21 | Lausanne University Hospital | Hiv binding agents |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2500248C (en) * | 2002-09-24 | 2013-03-19 | Frontier Biotechnologies Co., Ltd. | Peptide derivative fusion inhibitors of hiv infection |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0492560A2 (en) * | 1990-12-26 | 1992-07-01 | Joseph P. Cotropia | Human monoclonal antibodies directed against the transmembrane glycoprotein (gp41) of HIV-1, and related peptides |
| WO1994012533A1 (en) * | 1992-11-23 | 1994-06-09 | President And Fellows Of Harvard College | Gp41 mutants and their use as hiv therapeutics |
| WO1994029339A1 (en) * | 1993-06-09 | 1994-12-22 | Connaught Laboratories Limited | Tandem synthetic hiv-1 peptides |
| WO1995001993A1 (en) * | 1993-07-06 | 1995-01-19 | University Of Utah Research Foundation | COMPOUNDS HAVING THE ANTIGENICITY OF hCG |
| WO1995007354A1 (en) * | 1993-09-11 | 1995-03-16 | Polymun Scientific Immunobiologische Forschung Gmbh | Peptides that elicit neutralizing antibodies against genetically divergent hiv-1 strains |
-
2000
- 2000-08-18 CN CNB001234838A patent/CN1172717C/en not_active Expired - Fee Related
-
2001
- 2001-07-20 AU AU2002214920A patent/AU2002214920A1/en not_active Abandoned
- 2001-07-20 WO PCT/CN2001/001192 patent/WO2002032452A1/en active Application Filing
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0492560A2 (en) * | 1990-12-26 | 1992-07-01 | Joseph P. Cotropia | Human monoclonal antibodies directed against the transmembrane glycoprotein (gp41) of HIV-1, and related peptides |
| WO1994012533A1 (en) * | 1992-11-23 | 1994-06-09 | President And Fellows Of Harvard College | Gp41 mutants and their use as hiv therapeutics |
| WO1994029339A1 (en) * | 1993-06-09 | 1994-12-22 | Connaught Laboratories Limited | Tandem synthetic hiv-1 peptides |
| WO1995001993A1 (en) * | 1993-07-06 | 1995-01-19 | University Of Utah Research Foundation | COMPOUNDS HAVING THE ANTIGENICITY OF hCG |
| WO1995007354A1 (en) * | 1993-09-11 | 1995-03-16 | Polymun Scientific Immunobiologische Forschung Gmbh | Peptides that elicit neutralizing antibodies against genetically divergent hiv-1 strains |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017093985A1 (en) | 2015-12-05 | 2017-06-08 | Centre Hospitalier Universitaire Vaudois | Hiv binding agents |
| US10730933B2 (en) | 2015-12-05 | 2020-08-04 | Centre Hospitalier Universitaire Vaudois | HIV binding agents |
| WO2020012435A1 (en) | 2018-07-13 | 2020-01-16 | Lausanne University Hospital | Hiv binding agents |
| WO2021009697A2 (en) | 2019-07-15 | 2021-01-21 | Lausanne University Hospital | Hiv binding agents |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1172717C (en) | 2004-10-27 |
| AU2002214920A1 (en) | 2002-04-29 |
| CN1339318A (en) | 2002-03-13 |
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