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Degraded agonist antibody

An agonist, antibody technology, applied in the direction of antibodies, anti-inflammatory agents, anti-tumor drugs, etc.

Inactive Publication Date: 2006-01-18
CHUGAI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are no reports of altered antibodies that reduce molecular size

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1 (cloning the DNA encoding the V region of the mouse anti-human IAP monoclonal antibody)

[0114] The DNAs encoding the variable regions of the mouse anti-human IAP monoclonal antibodies MABL-1 and MABL-2 were cloned as follows.

[0115] 1.1 Preparation of messenger RNA (mRNA)

[0116] The mRNAs of hybridomas MABL-1 and MABL-2 were obtained using mRNA Purification Kit (Pharmacia Biotech).

[0117] 1.2 Synthesis of double-stranded cDNA

[0118] Double-stranded cDNA was synthesized from approximately 1 μg of mRNA using Marathon cDNA Amplification Kit (CLONTECH), and ligated with an adapter.

[0119] 1.3 PCR amplification of the coding group of the antibody variable region

[0120] PCR was performed using a Thermal Cycler (PERKIN ELMER).

[0121] (1) Amplify the gene encoding MABL-1 L chain V region

[0122] The primer that is used for PCR method is Adapter primer-1 shown in SEQ ID No.1, (CLONTECH), and it hybridizes with the partial sequence of linker,...

Embodiment 2

[0143] Embodiment 2 (DNA sequencing)

[0144] The nucleotide sequence of the cDNA coding region in the above plasmid was determined using Auto DNA Sequencer (Applied Biosystem) and ABI PRISMDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystem) according to the manufacturer's protocol.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] 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

[0149] Embodiment 3 (determining CDR)

[0150] The V regions of the L chain and the H chain are generally similar in structure, and each of the four framework regions is 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).

[0151] Based on these facts, the amino acid sequence of the variable region of the mouse anti-human IAP monoclonal antibody was applied to the antibody amino acid sequence database established by Kabat et al. to search for its homology. Table 1 shows the CDR regions determined by homology.

[0152] Table 1

[0153] plasmid SEQ ID No. CDR(1) CDR(2) CDR(3)

[0154] pGEM-M1L 5 43-58 74-80 113-121

[0155] pGEM-M1H 6 50-54 69-85 118-125

[0156] pGEM-M2L7 43-58 74-80 113-121 ...

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Abstract

The present invention relates to a modified antibody which contains two or more H chain V regions and two or more L chain V regions of monoclonal antibody and can transduce a signal into cells by crosslinking TPO receptor to thereby exert TPO agonist action. The modified antibody can be used as a TPO signal transduction agonist and, therefore, useful as a preventive and / or remedy for various diseases such as platelet-reduction-related blood diseases, thrombopenia following chemotherapy for cancer or leukemia, etc.

Description

[0001] This application is a divisional application of the Chinese invention patent application "low molecular weight agonist antibody" with application number 01817431.0 filed on October 22, 2001. technical field [0002] The present invention relates to altered antibodies comprising two or more H chain V regions and two or more L chain V regions of monoclonal antibodies that exhibit agonism by cross-linking cell surface molecules or intracellular molecules agent activity. The altered antibodies have agonist activity that transduces signals into cells by cross-linking cell surface molecules, and are useful as drugs for a variety of purposes. Background technique [0003] JP-A 9-295999 discloses the preparation of specific monoclonal antibodies using spleen stromal cell lines as sensitizing antigens, the purpose of which is to develop specific antibodies that can recognize the above-mentioned spleen stromal cells, and discloses that mouse integrin Production of novel monocl...

Claims

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Application Information

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IPC IPC(8): A61K39/395C07K16/18C07K16/28A61P35/00A61P29/00A61P7/00A61P5/00
Inventor 福岛直土屋政幸宇野慎介大友俊彦薮田尚弘角田浩行
Owner CHUGAI PHARMA CO LTD
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