Polypeptide inducing apoptosis

A technology of apoptosis and blood cells, applied in the direction of peptides, specific peptides, peptide/protein components, etc., can solve problems such as side effects and agglutination reactions

Inactive Publication Date: 2003-06-04
CHUGAI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It shows that the administration of large doses of monoclonal antibodies th...

Method used

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  • Polypeptide inducing apoptosis
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Examples

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Embodiment 1

[0068] To illustrate the preparation method of the reconstituted polypeptide of the present invention, an example of single chain Fv is prepared as shown below. Mouse anti-human IAP antibodies, MABL-1 and MABL-2, were used in the example of preparing the reconstructed polypeptide. On September 11, 1997, the hybridomas MABL-1 and MABL-2 that produced these two antibodies were released internationally respectively. Deposited as FERM BP-6100 and FERM BP-6101 at the Microorganism Depository - National Institute of BioScience and human Technology, Agency of Industrial Science and Technology, Minister of International Trade and Industry Industry) (1-3 Higasi 1-Chome, Tsukuba-shi, Ibaraki-Ken, Japan). Example 1 (cloning the DNA encoding the V region of the mouse anti-human IAP monoclonal antibody)

[0069] The DNAs encoding the variable regions of the mouse anti-human IAP monoclonal antibodies MABL-1 and MABL-2 were cloned as follows. 1.1 Preparation of messenger RNA (mRNA)

[00...

Embodiment 2

[0088] A plasmid containing the gene encoding the mouse H chain V region derived from hybridoma MABL-2 was prepared from the purified DNA fragment and named pGEM-M2H. Embodiment 2 (DNA sequencing)

[0089] The nucleotide sequence of the cDNA coding region in the above plasmid was determined using Auto DNA Sequencer (Applied Biosystem) and ABI PRISM DyeTerminator Cycle Sequencing Ready Reaction Kit (Applied Biosystem) according to the manufacturer's protocol.

[0090]The plasmid pGEM-M1L contains the gene encoding the V region of the L chain of the mouse antibody MABL-1, and its nucleotide sequence is shown in SEQ ID No.5.

[0091] The plasmid pGEM-M1H contains the gene encoding the H chain V region of the mouse antibody MABL-1, and its nucleotide sequence is shown in SEQ ID No.6.

[0092] The plasmid pGEM-M2L contains the gene encoding the L chain V region of the mouse antibody MABL-2, and its nucleotide sequence is shown in SEQ ID No.7.

[0093] The plasmid pGEM-M2H contain...

Embodiment 3

[0093] The plasmid pGEM-M2H contains the gene encoding the H chain V region of the mouse antibody MABL-2, and its nucleotide sequence is shown in SEQ ID No.8. Embodiment 3 (determining CDR)

[0094] The V regions of the L chain and the H chain are generally similar in structure, and every four framework regions are connected by three hypervariable regions, ie, complementarity determining regions (CDRs). The amino acid sequences of the framework are relatively conserved, whereas those of the CDRs are extremely highly variable (Kabat E.A. et al., "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services, 1983).

[0095] Based on these facts, the amino acid sequence of the variable region of the mouse anti-human IAP monoclonal antibody was entered into the antibody amino acid sequence database established by Kabat et al., and its homology was searched. Table 1 shows the CDR regions determined by homology.

[0096] Table 1

[0097] Plasmid SEQ ID...

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Abstract

A reconstituted polypeptide characterized by inducing apoptosis in nuclear blood cells having integrin associated protein (IAP) without causing the agglutination of erythrocytes. This reconstituted polypeptide contains at least two H chain V domains and at least two L chain V domains of a monoclonal antibody which induces apoptosis in nuclear blood cells having IAP. This reconstituted polypeptide is useful as a remedy for blood diseases such as leukemia.

Description

technical field [0001] The present invention relates to a reconstructed polypeptide, which is characterized in that it can induce the apoptosis of nucleated blood cells with integrin-associated protein (Integrin Associated Protein, IAP), without causing hemagglutination reaction. The present invention particularly relates to a reconstructed polypeptide comprising two or more H chain V regions and two or more L chain V regions of a monoclonal antibody capable of inducing apoptosis of nucleated blood cells with IAP. The reconstituted polypeptide can be used as a therapeutic agent for blood diseases, such as leukemia. Background technique [0002] Japanese patent application 9-67499 discloses the preparation of specific monoclonal antibodies using splenic stromal cell lines as sensitizing antigens. The purpose is to develop specific antibodies that can recognize the above-mentioned splenic stromal cells. Production of novel monoclonal antibodies recognizing the protein (mouse ...

Claims

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Application Information

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IPC IPC(8): A61K38/00A61P35/02C07K16/28C07K16/30C12N15/12
CPCC07K2317/622C07K16/3061C07K16/28A61K38/00A61K2039/505C07K2317/73C07K2317/56A61P35/02C07K16/18
Inventor 福岛直土屋政幸大枝匡义宇野慎介菊地康文
Owner CHUGAI PHARMA CO LTD
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