Lh-type bispecific antibody

Abstract

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.

What is claimed: 
     
         1 . A nucleic acid molecule encoding the single-chain polypeptide constituting the 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. 
     
     
         2 . A nucleic acid molecule encoding both of the two kinds of the single-chain polypeptides constituting the bispecific antibody of  claim 1 . 
     
     
         3 . A replicable cloning vector or an expression vector containing the nucleic acid molecule of  claim 1 . 
     
     
         4 . The vector of  claim 3 , which is a co-expression vector. 
     
     
         5 . The vector of  claim 3 , which is a plasmid vector. 
     
     
         6 . A host cell transformed with the vector of  claim 3 . 
     
     
         7 . The host cell of  claim 6 , which is a prokaryotic cell. 
     
     
         8 . The host cell of  claim 7 , which is  E. coli.    
     
     
         9 . A method for the production of the bispecific antibody, comprising culturing a host cell according to  claim 6  to express the two kinds of the single-chain polypeptides constituting said bispecific antibody, collecting and purifying said single-chain polypeptides, assembling the two kinds of the single-chain polypeptides, and separating and collecting the bispecific antibody thus formed. 
     
     
         10 . The method of  claim 9  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. 
     
     
         11 . A method for the production of the bispecific antibody, comprising culturing a host cell transformed with the co-expression vector of  claim 4  to express the two kinds of the single-chain polypeptides constituting said bispecific antibody, allowing the transformed cell to form the diabody-type bispecific antibody in said cell, and separating and collecting the bispecific antibody thus formed. 
     
     
         12 . A method for the production of a humanized diabody-type bispecific antibody consisting of two kinds of the single-chain polypeptides of (5L OH) and (OL5H) or 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 structure:
 (5LOH)-(a peptide linker)-(OL5H) or (OL5H)-(a peptide linker)-(5LOH),   the method comprising:   digesting with a protease to cleave the Fc region and the hinge region 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 structure of an antibody wherein 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 wherein the humanized diabody-type bispecific antibody is bonded to the hinge regions via a protease cleavage site.   
     
     
         13 . A method for the production of a humanized diabody-type bispecific antibody consisting of two kinds of the single-chain polypeptides of (5L OH) and (OL5H) or 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 structure:
 (5LOH)-(a peptide linker)-(OL5H) or (OL5H)-(a peptide linker)-(5LOH),   the method comprising:   digesting with a protease to cleave the Fc region and the hinge region 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 structure of an antibody wherein the single-chain polypeptide of (5LOH)-(a peptide linker)-(OL5H) or (OL5H)-(a peptide linker)-(5LOH) is bonded to two Fc regions of a human antibody via each hinge region wherein the single-chain polypeptide is bonded to the hinge regions via a protease cleavage site.