LH-type bispecific antibody
Provided are a novel diabody type bispecific antibody, the function of which as a bispecific antibody is improved to provide a higher additional value, such as cost saving caused by a reduction in dose, to a drug; and a method for producing the same. A humanized diabody type bispecific antibody (LH-diabody type bispecific antibody) characterized in that an L-chain is located in the N-terminal side in each polypeptide (LH type); a humanized high-functional bispecific antibody which contains said LH diabody type bispecific antibody; a nucleic acid molecule encoding both of two kinds of single-stranded polypeptides constituting said bispecific antibody; and a method for producing said antibody which comprises culturing a host cell having been transformed by an expression vector containing said nucleic acid molecule.
1. A single-chain polypeptide which is either a first or a second polypeptide of a diabody-type bispecific antibody, said diabody-type bispecific antibody consisting of said first polypeptide comprising a humanized variable region of the light chain (5L) of an anti-human EGF receptor 1 antibody 528 and a humanized variable region of the heavy chain (OH) of an anti-CD3 antibody OKT in this order from its N-end to C-end and said second polypeptide comprising a humanized variable region of the light chain (OL) of an anti-CD3 antibody OKT and a humanized variable region of the heavy chain (5H) of an anti-human EGF receptor 1 antibody 528 in this order from its N-end to C-end.
2. A single-chain polypeptide which has the structure of (5LOH) or (OL5H) bonded to Fc region of a human antibody via hinge region of a humanized highly functional bispecific antibody comprising humanized variable regions of the heavy chain (5H) and the light chain (5L) of an anti-human EGF receptor 1 antibody 528, and humanized variable regions of the heavy chain (OH) and the light chain (OL) of an anti-CD3 antibody OKT3 having the following structure: a humanized diabody-type bispecific antibody consisting of two kinds of the single-chain polypeptides of (5LOH) and (OL5H) is bonded to two Fc regions of a human antibody via each hinge region through either of the two single-chain polypeptides.
3. A diabody-type bispecific antibody consisting of a first polypeptide comprising a humanized variable region of the light chain (5L) of an anti-human EGF receptor 1 antibody 528 and a humanized variable region of the heavy chain (OH) of an anti-CD3 antibody OKT in this order from its N-end to C-end; and a second polypeptide comprising a humanized variable region of the light chain (OL) of an anti-CD3 antibody OKT and a humanized variable region of the heavy chain (5H) of an anti-human EGF receptor 1 antibody 528 in this order from its N-end to C-end.
4. A bispecific antibody of claim 3 wherein the 5L, 5H, OL and OH have an amino acid sequence of SEQ ID NO: 2, 4, 6 and 8, respectively.
5. A pharmaceutical composition comprising the bispecific antibody of claim 3 as an active ingredient, and a pharmaceutically acceptable carrier.
6. The diabody-type bispecific antibody of claim 3 , wherein the first polypeptide and the second polypeptide are separate polypeptide chains, and wherein the first polypeptide and second polypeptide are expressed in E. coli at their final length.
7. A humanized highly functional bispecific antibody comprising humanized variable regions of the heavy chain (5H) and the light chain (5L) of an anti-human EGF receptor 1 antibody 528, and humanized variable regions of the heavy chain (OH) and the light chain (OL) of an anti-CD3 antibody OKT3; and having the following structure:
a humanized diabody-type bispecific antibody consisting of two kinds of the single-chain polypeptides of (5LOH) and (OL5H) is bonded to two Fc regions of a human antibody via each hinge region through either of the two single-chain polypeptides.
8. The humanized highly functional bispecific antibody of claim 7 , wherein the humanized diabody-type bispecific antibody is bonded to the hinge regions via a protease cleavage site.
9. A bispecific antibody of claim 7 wherein the 5L, 5H, OL and OH have an amino acid sequence of SEQ ID NO: 2, 4, 6 and 8, respectively.
10. The diabody-type bispecific antibody of claim 3 or 7 wherein the humanized variable region of the light chain and the humanized variable region of the heavy chain are linked via a peptide linker in the single-chain polypeptide.
11. A method for inhibiting or reducing tumor cells in a subject, comprising:
administering to the subject composition of claim 5 in an effective amount to inhibit or reduce tumor cells.