Fragment of humanized anti-egfr antibody substituted-lysine variable fragment and use thereof

Abstract

The purpose of the present invention is to provide fragments of humanized anti-EGFR antibody substituted-lysine light-chain or heavy-chain variable regions, and single-chain antibodies, etc, comprising the same, having sufficient binding activity (affinity) with target cells, and having the ability to undergo various site-specific and uniform chemical modifications. The present invention pertains to a humanized variable region on the light-chain or heavy-chain of antibody 528 against human epidermal cell growth factor receptor 1 (Her-1), said variable region comprising the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, wherein fragments of humanized anti-EGFR antibody lysine-substituted light-chain variable regions are formed by substituting a different amino acid for all of the lysine residues, or all of the lysine residues except for the lysine residue(s) of one specified moiety.

What is claimed: 
     
         1 . A lysine-substituted humanized variable region fragment of a light-chain of an anti-human epithelial cell growth factor (EGF) receptor 1 (Her1) antibody 528 (5L) that consists of the amino acid sequence of SEQ ID NO:2, wherein all of the lysine residues, or all of the lysine residues except for one lysine residue at a particular site in the humanized variable region of the light chain is replaced by other amino acids. 
     
     
         2 . The lysine-substituted humanized variable region fragment of the light-chain according to  claim 1 , wherein all of the lysine residues except for lysine (Lys) 108 in the 5L is replaced by other amino acids. 
     
     
         3 . The lysine-substituted humanized variable region fragment of the light-chain according to  claim 1 , which contains at least one of the following replacements in the 5L:
 Lys 44 is replaced by Arg;   Lys 55 is replaced by Gln;   Lys 79 is replaced by Thr;   Lys 108 is replaced by Glu; or   Lys 112 is replaced by Glu.   
     
     
         4 . The lysine-substituted humanized variable region fragment of the light-chain according to  claim 1 , which contains the following replacements in the 5L:
 Lys 44 is replaced by Arg;   Lys 55 is replaced by Gln;   Lys 79 is replaced by Thr;   Lys 108 is replaced by Glu; and   Lys 112 is replaced by Glu.   
     
     
         5 . An antibody molecule comprising as a constituent the lysine-substituted humanized variable region fragment of the light-chain according to  claim 1 , or a lysine-substituted humanized variable region fragment of a heavy-chain of an anti-human epithelial cell growth factor (EGF) receptor 1 (Her1) antibody 528 (5H) that consists of an amino acid sequence of SEQ ID NO:4, wherein all of the lysine residues, or all of the lysine residues except for one lysine residue at a particular site in the humanized variable region of the heavy chain (5H) is replaced by other amino acids,
 said antibody molecule is selected from the group consisting of IgG-type antibody molecule, single-chain antibody (scFv), dimer of scFv, bispecific antibody, diabody-type bispecific antibody, highly functional bispecific antibody, and polymerized low-molecular antibody, and   wherein the humanized variable region (OL) of a light chain of an anti-CD3 antibody OKT3 and humanized variable region (OH) of a heavy chain of the anti-CD3 antibody OKT3 consist of an amino acid sequence represented by SEQ ID NO: 6 and SEQ ID NO: 8, respectively.   
     
     
         6 . A nucleic acid molecule encoding a lysine-substituted humanized variable region fragment of a light-chain of an anti-human epithelial cell growth factor (EGF) receptor 1 (Her1) antibody 528 (5L) that consists of an amino acid sequence of SEQ ID NO:2, wherein all of the lysine residues, or all of the lysine residues except for one lysine residue at a particular site in the humanized variable region of the light chain is replaced by other amino acids, or a single-chain polypeptide constituting an antibody molecule,
 wherein said antibody molecule comprising as a constituent the lysine-substituted humanized variable region fragment of the light-chain according to  claim 1 , or a lysine-substituted humanized variable region fragment of a heavy-chain of an anti-human epithelial cell growth factor (EGF) receptor 1 (Her1) antibody 528 (5H) that consists of an amino acid sequence of SEQ ID NO:4, wherein all of the lysine residues, or all of the lysine residues except for one lysine residue at a particular site in the humanized variable region of the heavy chain (5H) is replaced by other amino acids.   
     
     
         7 . A nucleic acid molecule encoding both of the two kinds of the single-chain polypeptides constituting an antibody molecule,
 wherein said antibody molecule comprising as a constituent the lysine-substituted humanized variable region fragment of the light-chain according to  claim 1 , or a lysine-substituted humanized variable region fragment of a heavy-chain of an anti-human epithelial cell growth factor (EGF) receptor 1 (Her1) antibody 528 (5H) that consists of an amino acid sequence of SEQ ID NO:4, wherein all of the lysine residues, or all of the lysine residues except for one lysine residue at a particular site in the humanized variable region of the heavy chain (5H) is replaced by other amino acids.   
     
     
         8 . A replicable cloning vector or an expression vector containing the nucleic acid molecule of  claim 6  or  7 . 
     
     
         9 . The vector of  claim 8 , which is a co-expression vector. 
     
     
         10 . The vector of  claim 8 , which is a plasmid vector. 
     
     
         11 . A host cell transformed with the vector of  claim 10 .