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Antibody modification method for purifying bispecific antibody

A bispecific antibody, multispecific antibody technology, applied in chemical instruments and methods, antibodies, cells modified by introducing foreign genetic material, etc., can solve problems such as difficulties

Inactive Publication Date: 2009-06-17
CHUGAI PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When the two antibodies constituting the bispecific antibody have the same constant region sequence, the A chain and B chain hybrid dimer must be separated only based on the difference in the variable region sequence, but the amino acid sequence of the variable region of the antibody is between the antibodies. The homology is very high (non-patent literature 9), and it is difficult to purify the A-chain B-chain hybrid dimer to a pharmaceutically acceptable high purity only based on the difference in the sequence of the variable region

Method used

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  • Antibody modification method for purifying bispecific antibody
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  • Antibody modification method for purifying bispecific antibody

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preparation example Construction

[0105] First, the present invention provides antibody modification methods for preparing multispecific antibodies. The preferred version of the preparation method of the present invention comprises modifying both or one of the nucleic acid encoding the amino acid residues of the first polypeptide and the nucleic acid encoding the amino acid residues of the second polypeptide, so that the isoelectric points of the first polypeptide and the second polypeptide method of making a difference. That is, by changing the charge of the amino acid residues of the first polypeptide and the second polypeptide, a difference in isoelectric point (pI) can be introduced into the polypeptides, and a multispecific antibody can be prepared using the difference in isoelectric point. Specifically, it is a preparation method comprising the steps of (a)-(c) below.

[0106] (a) modifying both or one of the nucleic acid encoding the amino acid residues of the first polypeptide and the nucleic acid enc...

Embodiment 1

[0237] [Example 1] Humanization of bispecific antibody with hybrid L chain

[0238] In Japanese Patent Application No. 2005-112514, a bispecific antibody containing a combination of anti-FactorIXa antibody A69-VH, anti-FactorX antibody B26-VH, and hybrid L chain (BBA) with the highest coagulation time shortening effect was humanized as follows .

[0239] 1-1 Homology search of human antibody

[0240] Human antibody amino acid sequence data were obtained from the generally published Kabat database (ftp: / / ftp.ebi.ac.uk / pub / databases / kabat / ) and the IMGT database (http: / / imgt.cines.fr / ), using the construct The database is divided into mouse A69-H chain variable region (amino acid sequence: SEQ ID NO.19), mouse B26-H chain variable region (amino acid sequence: SEQ ID NO.20), mouse BBA-L chain The variable region (amino acid sequence: SEQ ID NO.21) was searched for homology. As a result, it was confirmed that it has a high homology with the human antibody sequence shown below, ...

Embodiment 2

[0273] [Example 2] Determination of amino acid modification positions in the variable region for the isolation of bispecific antibodies In the expression of bispecific antibodies, two types of H chains and one type of L chain were used to humanize the A69-H chain and humanized BBA-L chain homodimer, humanized B26-H chain and humanized BBA-L chain homodimer, humanized A69-H chain and humanized B26-H chain Three antibodies were expressed as heterodimers with humanized BBA-L chains. These three antibodies were separated, and the isoelectric point of the humanized A69H chain variable region was lowered, and the isoelectric point of the humanized B26H chain variable region was raised, and amino acid modifications were performed in order to purify only the bispecific antibody.

[0274] First, in order to confirm the amino acid residues exposed on the surface of the variable regions of the humanized A69 antibody and the humanized B26 antibody, the humanized A69 antibody was prepared ...

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Abstract

The present inventors devised methods for efficiently purifying bispecific antibodies using a chromatography column based on the difference in isoelectric points between the H chains of two types of antibodies, wherein the difference is introduced by modifying the amino acids present on the surface of the antibody variable regions of two types of antibodies that constitute a bispecific antibody. Furthermore, the inventors devised methods for efficiently purifying bispecific antibodies using a chromatography column by linking respective antigen binding sites (heavy chain variable regions) to the antibody constant regions having different isoelectric points, and then coexpressing these antibodies.

Description

technical field [0001] The present invention relates to an antibody modification method for purifying a bispecific antibody, a method for isolating the bispecific antibody, a pharmaceutical composition containing the bispecific antibody as an active ingredient, and the like. Background technique [0002] Antibodies have high stability in blood and few side effects, so they are attracting attention as drugs. Among them, there is a bispecific antibody capable of simultaneously recognizing two kinds of antigens (antigen A and antigen B) (Non-Patent Document 1). MDX-210, which is currently undergoing clinical trials, is an IgG-type bispecific antibody reconstituted from monocytes or the like expressing FcγRI to cancer cells expressing HER-2 / neu (Non-Patent Document 2). The preparation of antibodies usually mostly adopts gene recombination technology. Specifically, DNA encoding an antibody protein is cloned from antibody-producing cells such as hybridomas, sensitized lymphocyte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K39/395A61P43/00C07K16/00C07K19/00C12N1/15C12N1/19C12N1/21C12N5/10C12P21/02
CPCC07K16/46
Inventor 井川智之角田浩行
Owner CHUGAI PHARMA CO LTD
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