Fragment of humanized anti-EGFR antibody substituted-lysine variable fragment and use thereof
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.
1. A lysine-substituted humanized variable region fragment of a heavy-chain of an anti-human epithelial cell growth factor (EGF) receptor 1 (Her 1) antibody 528 (5H) that consists of the amino acid sequence of SEQ ID NO:4, wherein all of the lysine residues except for lysine (Lys) 19 in the 5H is replaced by other amino acids.
2. The lysine-substituted humanized variable region fragment of the heavy chain according to claim 1 , which contains at least one of the following replacements in the 5H:
Lys 12 is replaced by Ala;
Lys 13 is replaced by Glu;
Lys 23 is replaced by Ala;
Lys 63 is replaced by Glu; or
Lys 65 is replaced by Gln.
3. The lysine-substituted humanized variable region fragment of the heavy chain according to claim 1 , which contains the following replacements in the 5H:
Lys 12 is replaced by Ala;
Lys 13 is replaced by Glu;
Lys 23 is replaced by Ala;
Lys 63 is replaced by Glu; and
Lys 65 is replaced by Gln.
4. The lysine-substituted humanized variable region fragment of the heavy chain according to claim 1 , wherein the one lysine residue or N-terminal amino acid is site-specifically modified.
5. The lysine-substituted humanized variable region fragment of the heavy-chain according to claim 4 , wherein the one lysine residue or N-terminal amino acid is site-specifically modified with polyethylene glycol.
6. An antibody molecule comprising as a constituent the lysine-substituted humanized variable region fragment of the heavy chain according to claim 1 .
7. The antibody molecule according to claim 6 , which 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.
8. The dimer according to claim 7 , wherein the one lysine residue contained in the lysine-substituted variable region fragment of a heavy chain is cross-linked.
9. A single-chain polypeptide constituting the antibody molecule of claim 6 .
10. A method for the production of the antibody molecule of claim 6 , comprising
culturing a host cell transformed with a vector, which is a co-expression vector containing 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 and
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 except for lysine (Lys) 19 in the 5H is replaced by other amino acids
to express the two kinds of the single-chain polypeptides constituting said antibody molecule,
collecting and purifying said single-chain polypeptides,
assembling the two kinds of the single-chain polypeptides,
and separating and collecting the antibody molecule thus formed.
11. The method of claim 10 wherein the host cell is E. coli , and the two kinds of the single-chain polypeptides are collected from supernatant of a culture medium, periplasm fraction, intracellular soluble fraction or intracellular insoluble fraction.
12. A pharmaceutical composition comprising the antibody molecule of claim 6 as an active ingredient.
13. The pharmaceutical composition of claim 12 suitable for use in eliminating, hurting, damaging and/or reducing tumor cells.