WO2004003021A1 - HIV感染細胞にアポトーシスを誘導するヒトIgM抗体及びHIV感染症治療剤 - Google Patents
HIV感染細胞にアポトーシスを誘導するヒトIgM抗体及びHIV感染症治療剤Info
- Publication number
- WO2004003021A1 WO2004003021A1 PCT/JP2003/008305 JP0308305W WO2004003021A1 WO 2004003021 A1 WO2004003021 A1 WO 2004003021A1 JP 0308305 W JP0308305 W JP 0308305W WO 2004003021 A1 WO2004003021 A1 WO 2004003021A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antibody
- hiv
- infected cells
- cells
- human igm
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
Definitions
- the present invention relates to a human IgM monoclonal antibody that specifically reacts with HIV-infected cells and induces apoptosis in HIV-infected cells and an HIV infection therapeutic agent containing such an antibody as an active ingredient.
- Multi-drug combination therapy combining 3 or 4 types of these drugs is effective in HIV-infected patients, resulting in a drastic reduction of HIV level in blood and CD4 lymphocyte level. It has become possible to bring about improvements and the like.
- latently infected cells can not be eliminated even by combination therapy, and it is difficult to completely cure HIV-infected patients, and cells that have been HIV-infected latently will not receive HIV if they stop drug administration. If it proliferated again, there was a problem with the problem.
- Human (type) monoclonal antibodies that specifically react with HIV-infected cells have been engineered to produce humanized antibodies, which are IgG type antibodies. They are neutralizing antibodies that block HIV infection, but can not damage infected cells. Forces such as species-specific receptor regulatory membrane factors (DAF, Decay accelerating factor; MCP, Membrane cofactor Dratein; HRF20, 20 kDa Homologous restriction factor or the like) exist on the human cell membrane In order to prevent the reaction, there is no cell lysis reaction via the capture reaction.
- DAF species-specific receptor regulatory membrane factors
- MCP Decay accelerating factor
- MCP Membrane cofactor Dratein
- HRF20 20 kDa Homologous restriction factor or the like
- IgM antibodies in human serum that respond to HIV-infected cells can overcome HIV-infected cells over complement regulatory membrane factors to cause human complement-mediated cytolytic reaction. It was found that IgM antibody exerts such an effect on gangliosides such as GM2 and Gg4 whose expression is increased by HIV infection (Japanese Patent Laid-Open Publication No. 0 9 ”)).
- L55 produced by human B lymphoblastoid cells immortalized with EB virus has been reported as a human IgM monoclonal antibody against GM2 of ganglioside. It has been found that HIV-infected cells treated with this human IgM monoclonal antibody cause cell lysis via the reaction of human complement. However, since the L55 antibody is not specific for HIV-infected cells, it may also respond to normal cells other than HIV-infected cells. Disclosure of the invention
- An object of the present invention is to provide an agent for treating HIV-infected patients, etc., which comprises a human IgM antibody as an active ingredient, which specifically reacts with HIV-infected cells and induces apoptosis in infected cells.
- a monoclonal antibody is provided, which belongs to human IgM that specifically recognizes HIV-infected cells and induces apoptosis.
- the second solution of the present invention provides a therapeutic agent for HIV infection characterized by specifically recognizing HIV-infected cells and inducing apoptosis in HIV-infected cells as a human IgM antibody as an active ingredient. Do.
- the third solution of the present invention provides the therapeutic agent according to claim 2, which is for preventing the onset of AIDS.
- a fourth solution of the present invention is characterized in that the human IgM monoclonal antibody reactive to HIV-infected cells is a 2G9 antibody in which the nucleic acid sequence of the variable region of the H chain has the nucleic acid sequence of SEQ ID NO: 1.
- a human IgM monoclonal antibody according to any one of 3 to 7 is provided.
- the fifth solution according to the present invention is characterized in that the nucleic acid sequence of the variable region of the light chain of human IgM monoclonal antibody reactive to HIV-infected cells is the 2G9 antibody having the nucleic acid sequence of SEQ ID NO: 2.
- a human IgM monoclonal antibody according to any one of claims 1 to 4. Brief description of the drawings
- FIG. 1 is a drawing showing the specificity of the 2G9 antibody.
- PBMC peripheral blood lymphocytes
- FIG. 2 is a drawing showing that 2G9 antibody also responds to HIV latently infected cells.
- the latently infected OM10.1 cells show that the antibody against the HIV membrane protein antigen gpl20 does not react (0.5 ⁇ ) but the 2G9 antibody does.
- FIG. 3 is a drawing showing apoptosis of HIV-infected cells by 2G9 antibody.
- FIG. 4 is a drawing showing apoptosis of HIV latently infected cells by 2G9 antibody.
- FIG. 5 shows a schematic diagram of 2G9 ⁇ chain expression plasmid construction. MODE FOR CARRYING OUT THE INVENTION
- the present inventors immunize HIV-infected cells with a Kirin beer mouse (TC mouse: trans-chromosome mouse) into which a chromosome containing a gene related to human immunoglobulin has been introduced.
- TC mouse trans-chromosome mouse
- mice that produced human antibodies that specifically react with HIV-infected cells.
- the spleen cells of this immunized mouse were fused with a mouse myeloma cell line to prepare a hybridoma according to a standard method. From the obtained hybridomas, a clone producing a monoclonal antibody that reacts to HIV-infected cells was selected, and the hyply doma clone was designated as 2G9 cell line.
- the 2G9 antibody a monoclonal antibody produced by the 2G9 cell line, is a human IgM monoclonal antibody consisting of human ⁇ chain and human ⁇ chain.
- the present invention 2G9 antibody-producing cell line 2G9 is manufactured by the National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, Institute of Advanced Industrial Science and Technology (Ibaraki Pref. Tsukuba, East 1- 1-1-Central 6th), May 8, 2003 It has been deposited internationally and has been assigned the deposit number FE RM BP-837.
- the antigen (2G9 antigen) that reacts with the 2G9 antibody is considered to lose reactivity with the 2G9 antibody by SDS treatment.
- the antibody according to claim 1, for example 2G9 antibody has a feature to induce apoptosis against HIV-infected cells, including OM10.1 cells. That is, this is a human IgM monoclonal antibody that can specifically bring HIV-infected cells into apoptosis, and can also induce apoptosis against HIV latently infected cells such as OM10.1 cells. Chemotherapeutic agents can not exert effects It can also be used as a therapeutic agent to eliminate HIV latent infection in the body of HIV-infected patients.
- a therapeutic agent for use in a therapeutic agent for HIV-infected patients, etc. comprising a human IgM antibody as an active ingredient, which reacts specifically with HIV-infected cells and induces apoptosis in HIV-infected cells.
- Physiologically acceptable carriers are well known in the art and are physiologically buffered saline or other buffered aqueous solutions, or solvents, or glycols, glycerol, oils (eg, olive oil), Or contain solvents such as injectable organic esters.
- Physiologically acceptable carriers also include the ability to stabilize IgM antibodies, compounds that increase absorption.
- physiologically acceptable compounds may be, for example, sugars such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents or proteins such as albumin or other stabilizers. Contains agents and excipients. Furthermore, it is also possible to add other physiologically active substances such as reverse transcriptase inhibitors, protease inhibitors and the like.
- physiologically acceptable carrier can be combined with each other depending on the administration route and target disease.
- the reactivity of the 2G9 antibody to HIV-infected cells was analyzed by flow cytometry.
- Cultured cell lines U937 cells, MOLT-4 cells and CEM cells were used as test cells.
- U937 cells were infected with the virus strain of HIV-1 U937 / mB, and primary isolates were infected with momo strain U937 / primary isolate, MN strains were infected.
- U937 / MN and MOLT-4 / ⁇ infected with MOLT-4 were used. After washing each cell with 2G9 antibody and washing, fluorescent dye labeled anti-human IgM antibody PT / JP2003 / 008305
- OM10.1 cells are considered to be HIV latently infected cell lines and HIV antigens such as gpl20 are not normally expressed.
- the 2G9 antibody reacted to the OM10.1 cells, it was revealed that the 2G9 antibody can also react to the latently infected cells (FIG. 2).
- HIV-1 infected MOLT-4 / ⁇ cells are prepared at 5 x 10 s / ml in RPMIIMO medium supplemented with 20% inactive human serum, to which an equal volume of 100 ⁇ g / ml of 2G9 antibody is added.
- the cells were cultured in a 5% CO 2 incubator (37 ° C.) for 2 days. After culture, DNA fragmentation, which is an indicator of apoptosis, was stained using the TUNEL method, then cells were fixed with 1% paraformaldehyde and analyzed by flow cytometry. As shown in FIG.
- OM10.1 cells an HIV-1 latently infected cell line, were prepared in cell culture medium (RPMI 1640 medium) containing 20% inactivated human serum in lxlOV ml, and added with an equal volume of 12.5 ⁇ g / ml of 2G9 antibody.
- the cells were cultured at 37 degrees for 2 days. After incubation, the cells were stained with Annexin V, an apoptosis detection reagent, and then cells were fixed with 1% paraformaldehyde and analyzed by flow cytometry. As a result, as shown in FIG.
- the method of reconstructing the 2G9 antibody based on the nucleotide sequence of the 2G9 antibody variable region shown in Table 1 is the shot-gun ligation method (. Grundstrom, T. et al. Nucleic Acid Res. 13, 3305- A method for establishing a cell line producing 2G9 antibody using genetic engineering techniques such as 3316 (1985)) is exemplified.
- the indicated nucleotide sequence is translated to obtain the amino acid sequence of the variable region of 2G9 antibody.
- the nucleotide sequence encoding the amino acid sequence of the variable region of the 2G9 antibody is polymorphic as shown in Table 2 in addition to the base sequence of the original 2G9 antibody variable region and further changing the codons used. Among them, those having certain restriction enzyme recognition fragments were selected for each appropriate length that can be chemically synthesized as oligonucleotides (Table 2).
- the synthesized oligonucleotides were sequentially digested with the respective restriction enzymes, and ligated to obtain the full-length base sequence encoding the amino acid sequence of the 2G9 antibody variable region.
- the cDNA fragments of the 2G9 antibody variable region (L v / z 2G 9 and rV 2 2 G 9 respectively) obtained in the same manner as in the chimeric antibody preparation method are the same as in the chimeric antibody preparation method.
- the recombinant 2G9 ⁇ chain, ⁇ chain expression plasmid was obtained by integration into a vector having the sequence (CM, C ic) (FIG. 5).
- the gene was introduced using ribofectamine reagent according to the protocol of CO company. Thereafter, the culture was continued for 2 days under normal culture conditions, and the culture supernatant of the transfected cells was recovered. The obtained culture supernatant was used to identify recombinant 2 G9 antibody present in the culture supernatant by an assay system using a sandwich ELISA method using an anti-human antibody and an anti-human kappa antibody. In addition, using this culture supernatant, FACS analysis is performed using U937 cells, MOLT-4 cells and U937 cells infected with HIV-1 strains by using ⁇ 937 / ⁇ , MOLT-4 / ⁇ , etc. It was confirmed that the antibody had the above specificity.
- the activity of the recombinant 2G9 antibody was confirmed by a competitive inhibition test in which the fluorescently labeled original 2G9 antibody and this culture supernatant were simultaneously allowed to act on ⁇ 937 / ⁇ , MOLT-4 / ⁇ .
- the 2G9 antibody obtained by the present invention was also found to have an action of inducing apoptosis on OM10.1 cells of latently infected cells.
- the ability to induce apoptosis in infected cells was found to be 2G9 antibody.
- such non-infectious cells did not show such damaging effects on U937 and MO LT-4 cells.
- the ability to induce apoptosis in latently infected cells, etc. is also used as a therapeutic agent for eliminating latent infections in the body of infected patients whose chemotherapy agents can not exert their effect. sell.
- the present invention also provides a nucleotide sequence of a ⁇ chain variable region and a gene encoding a nucleotide sequence of a ⁇ chain variable region, which are extremely useful for producing a recombinant anti-HIV antibody.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Virology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Oncology (AREA)
- Communicable Diseases (AREA)
- Tropical Medicine & Parasitology (AREA)
- AIDS & HIV (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2003244158A AU2003244158A1 (en) | 2002-07-01 | 2003-06-30 | HUMAN IgM ANTIBODY INDUCING APOPTOSIS IN HIV-INFECTED CELLS AND REMEDY FOR HIV-INFECTION |
CA002491859A CA2491859A1 (en) | 2002-07-01 | 2003-06-30 | Human igm antibody inducing apoptosis in hiv-infected cells and remedy for hiv-infection |
US10/519,855 US7595050B2 (en) | 2002-07-01 | 2003-06-30 | Human IGM monoclonal antibody capable of inducing apoptosis in HIV-infected cells |
CN038155982A CN1665842B (zh) | 2002-07-01 | 2003-06-30 | 诱导HIV感染细胞凋亡的人IgM抗体和用于HIV感染的药物 |
EP03761838A EP1533320B1 (en) | 2002-07-01 | 2003-06-30 | HUMAN IgM ANTIBODY INDUCING APOPTOSIS IN HIV-INFECTED CELLS AND REMEDY FOR HIV-INFECTION |
JP2004517333A JP4310498B2 (ja) | 2002-07-01 | 2003-06-30 | HIV感染細胞にアポトーシスを誘導するヒトIgM抗体及びHIV感染症治療剤 |
DE60326243T DE60326243D1 (de) | 2002-07-01 | 2003-06-30 | HUMANER IgM-ANTIKÖRPER, DER IN HIV-INFIZIERTEN ZELLEN APOPTOSE INDUZIERT, UND MITTEL GEGEN HIV-INFEKTION |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2002227953 | 2002-07-01 | ||
JP2002-227953 | 2002-07-01 | ||
JP2003-74316 | 2003-03-18 | ||
JP2003074316 | 2003-03-18 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2004003021A1 true WO2004003021A1 (ja) | 2004-01-08 |
Family
ID=30002402
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2003/008305 WO2004003021A1 (ja) | 2002-07-01 | 2003-06-30 | HIV感染細胞にアポトーシスを誘導するヒトIgM抗体及びHIV感染症治療剤 |
Country Status (9)
Country | Link |
---|---|
US (1) | US7595050B2 (ja) |
EP (1) | EP1533320B1 (ja) |
JP (1) | JP4310498B2 (ja) |
CN (1) | CN1665842B (ja) |
AT (1) | ATE423141T1 (ja) |
AU (1) | AU2003244158A1 (ja) |
CA (1) | CA2491859A1 (ja) |
DE (1) | DE60326243D1 (ja) |
WO (1) | WO2004003021A1 (ja) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2018512863A (ja) * | 2015-04-17 | 2018-05-24 | アイジーエム バイオサイエンシズ エー/エス | 多価ヒト免疫不全ウイルス抗原結合分子およびその使用方法 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112094352B (zh) * | 2020-09-27 | 2021-03-16 | 南京妙迪生物科技有限公司 | 一种抗IgM单克隆抗体 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0510691A1 (en) * | 1991-04-26 | 1992-10-28 | Osaka Bioscience Institute | DNA coding for human cell surface antigen |
WO1997022361A1 (fr) * | 1995-12-18 | 1997-06-26 | Otsuka Pharmaceutical Co., Ltd. | Anticorps reconnaissant la chaine du glucose et medicament contre l'infection par vih |
WO2000018426A1 (fr) * | 1998-09-30 | 2000-04-06 | The Institute Of Physical And Chemical Research | Inducteurs d'apoptose |
-
2003
- 2003-06-30 DE DE60326243T patent/DE60326243D1/de not_active Expired - Fee Related
- 2003-06-30 EP EP03761838A patent/EP1533320B1/en not_active Expired - Lifetime
- 2003-06-30 AT AT03761838T patent/ATE423141T1/de not_active IP Right Cessation
- 2003-06-30 WO PCT/JP2003/008305 patent/WO2004003021A1/ja active Application Filing
- 2003-06-30 CA CA002491859A patent/CA2491859A1/en not_active Abandoned
- 2003-06-30 JP JP2004517333A patent/JP4310498B2/ja not_active Expired - Fee Related
- 2003-06-30 AU AU2003244158A patent/AU2003244158A1/en not_active Abandoned
- 2003-06-30 US US10/519,855 patent/US7595050B2/en not_active Expired - Fee Related
- 2003-06-30 CN CN038155982A patent/CN1665842B/zh not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0510691A1 (en) * | 1991-04-26 | 1992-10-28 | Osaka Bioscience Institute | DNA coding for human cell surface antigen |
WO1997022361A1 (fr) * | 1995-12-18 | 1997-06-26 | Otsuka Pharmaceutical Co., Ltd. | Anticorps reconnaissant la chaine du glucose et medicament contre l'infection par vih |
WO2000018426A1 (fr) * | 1998-09-30 | 2000-04-06 | The Institute Of Physical And Chemical Research | Inducteurs d'apoptose |
Non-Patent Citations (5)
Title |
---|
"TIA-1 Cytotoxic cell marker", BECKMAN COULTER KABUSHIKI KAISHA, 1999, XP002973375, Retrieved from the Internet <URL:http://www.bc-cytometry-com/reagent/TIA-1.html> [retrieved on 20030801] * |
ITOH N. ET AL.: "The polypeptide encoded by the cDNA for human cell surface antigen fas can mediate apoptosis", CELL, vol. 66, no. 2, 1991, pages 233 - 243, XP000579691 * |
KLEIN R. ET AL.: "Expressed human immunoglobulin kappa genes and their hypermutation", EUR. J. IMMUNOL., vol. 23, 1993, pages 3248 - 3271, XP002971863 * |
WANG X., STOLLAR B.D.: "Immunoglobulin VH gene expression in human aging", CLINICAL IMMUNOLOGY, vol. 93, no. 2, 1999, pages 132 - 142, XP002971864 * |
YOSHIHIDE TSUJIMOTO: "Experimental Medicine", 25 March 1997, BIO MANUAL UP SERIES, article "Saishin apoptosis jikkenho", pages: 112 - 117, XP002973374 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2018512863A (ja) * | 2015-04-17 | 2018-05-24 | アイジーエム バイオサイエンシズ エー/エス | 多価ヒト免疫不全ウイルス抗原結合分子およびその使用方法 |
US11192941B2 (en) | 2015-04-17 | 2021-12-07 | Igm Biosciences, Inc. | Multi-valent human immunodeficiency virus antigen binding molecules and uses thereof |
Also Published As
Publication number | Publication date |
---|---|
EP1533320A4 (en) | 2006-05-03 |
US20060292160A1 (en) | 2006-12-28 |
US7595050B2 (en) | 2009-09-29 |
DE60326243D1 (de) | 2009-04-02 |
AU2003244158A1 (en) | 2004-01-19 |
ATE423141T1 (de) | 2009-03-15 |
JP4310498B2 (ja) | 2009-08-12 |
EP1533320A1 (en) | 2005-05-25 |
CN1665842A (zh) | 2005-09-07 |
CA2491859A1 (en) | 2004-01-08 |
EP1533320B1 (en) | 2009-02-18 |
CN1665842B (zh) | 2011-09-28 |
JPWO2004003021A1 (ja) | 2005-10-27 |
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