Process for producing humanized chimera antibody

Abstract

A humanized chimera antibody, a pharmaceutical composition comprising a humanized chimera antibody and a pharmaceutically acceptable carrier, and a method of treating cancer which comprises administering to a patient a pharmaceutically acceptable amount of the humanized chimera antibody, are disclosed.

1 . A process for producing human chimeric antibody which comprises the steps of 
 (1) constructing a cassette vector by inserting a cDNA coding for a heavy chain constant region (Cγ1) of human antibody into an expression vector for animal cell use and establishing a cloning site in the upstream region of the heavy chain constant region (Cγ1) of said cassette vector for inserting a cDNA which encodes a heavy chain variable region of non human animal antibody;    (2) digesting a cDNA coding for the heavy chain variable region of non human animal antibody with restriction enzymes;    (3) inserting said cDNA coding for the heavy chain variable region of non human animal antibody into the cassette vector, using a synthetic DNA which comprises a base sequence corresponding to the 5′-end side of said heavy chain constant region of human antibody as defined by the 31st to 43rd base in SEQ ID NO:13 and a base sequence corresponding to the 3′-end side of said heavy chain variable region of non human animal antibody and is possessed of the restriction enzyme recognition sites on both of its ends, thereby constructing a human chimeric antibody heavy chain expression vector in which said cDNA coding for the heavy chain constant region of human antibody and said cDNA coding for the heavy chain variable region of non human animal antibody are linked together through said synthetic DNA;    (4) constructing a cassette vector by inserting a cDNA coding for a light chain constant region (Cκ) of human antibody into an expression vector for animal cell use and establishing a cloning site in the upstream region of the light chain constant region (Cκ) of said cassette vector for inserting a cDNA which encodes a light chain variable region of non human animal antibody;    (5) digesting a cDNA coding for the light chain variable region of non human animal antibody with restriction enzymes;    (6) inserting said cDNA coding for a light chain variable region of non human animal antibody into the cassette vector, using a synthetic DNA which comprises a base sequence corresponding to the 5′-end side of said light chain constant region of human antibody as defined by the 48th to 61st base in SEQ ID NO:15 and a base sequence corresponding to the 3′-end side of said light chain variable region of non human animal antibody and is possessed of the restriction enzyme recognition sites on both of its ends, thereby constructing a humanized chimera antibody light chain expression vector in which said cDNA coding for the light chain constant region of human antibody and said cDNA coding for the light chain variable region of non human animal antibody are linked together through said synthetic DNA;    (7) introducing these expression vectors into host cells to obtain a transformant; and    (8) culturing said transformant in an appropriate culture medium, thereby allowing the transformant to produce and accumulate a human chimeric antibody, and collecting said human chimeric antibody from the resulting culture broth.    
     
     
         2 . The process according to  claim 1 , wherein said non human animal is mouse.  
     
     
         3 . The process according to  claim 1 , wherein said non human animal antibody binds to ganglioside.  
     
     
         4 . The process according to  claim 1 , wherein said non human animal antibody binds to ganglioside GD 3 .  
     
     
         5 . The process according to  claim 1 , wherein said human chimeric antibody binds to ganglioside GD 3 .  
     
     
         6 . The process according to  claim 1 , wherein said heavy chain variable region of antibody comprises the polypeptide having the amino acid sequence as defined by the 11st to 129th residue in SEQ ID NO: 18 and said light chain variable region of antibody comprises the polypeptide having the amino acid sequence as defined by the 21st to 127th residue in SEQ ID NO: 19.  
     
     
         7 . The process according to  claim 1 , wherein said human chimeric antibody is an antibody KM-871 binds to ganglioside GD 3 .  
     
     
         8 . The process according to  claim 1 , wherein said human chimeric antibody binds to ganglioside GD 3  and recognizes the epitope which is recognized by the human chimeric antibody KM-871 produced by a transformant KM-871 (FERM BP-3512).  
     
     
         9 . The process according to  claim 1 , wherein the synthetic DNA used for the heavy chain has the DNA sequence showing SEQ ID NO: 13.  
     
     
         10 . The process according to  claim 1 , wherein the synthetic DNA used for the light chain has the DNA sequence showing SEQ ID NO: 15.