The tyrosine residue at position 97 in the VH CDR3 region of a mouse/human chimeric anti-colorectal carcinoma antibody contributes hydrogen bonding to the TAG72 antigen.
One amino acid, tyrosine at position 96 and 97 in the VH CDR3 region of a mouse/human chimeric anti-TAG72 antibody cB72.3m4 was substituted by the phenylalanine residue and by a number of amino acids from different amino acid groups by the site-directed mutagenesis technique. The expression vector mpSV2neo-EP1-Vm11-16C1 containing mutant VH region fragments (Vm11-16) as well as the immunoglobulin enhances (E), promoter (P1) and the human genomic C1 region fragments, were transfected into a heavy-chain-loss mutant cell line B72.3Mut(K), respectively. Mutant chimeric cB72.3m11-16 antibodies were purified from the transfectant supernates and compared based upon their binding affinity for the TAG72 antigen relative to that of the original cB72.3m4 antibody. The data showed that a single amino acid substitution of tyrosine by phenylalanine and a number of amino acids including serine, asparagine, histidine and arginine at position 97 in the VH CDR3 region all resulted in approximate 18-fold lower binding affinity, whereas the substitution of tyrosine by phenylalanine at position 96 in the VH CDR3 region did not affect the binding affinity of the cB72.3m4 antibody. This suggests that the tyrosine residue at position 97 in the VH CDR3 region is in a contact position in the B72.3/TAG72 antibody/antigen interaction, and that the terminal hydroxyl group of the position 97 tyrosine side-chain contributes hydrogen bonding to the TAG72 antigen, whereas the position 96 tyrosine side-chain does not.
Xiang
J
J
Saskatoon Cancer Center, Department of Microbiology, University of Saskatchewan, Canada.
Liu
E
E
Delbaere
L T
LT
Chen
Z
Z
Luo
X
X
Qi
Y
Y
Rathgeber
C
C
D000818:Animals; D000912:Antibodies, Neoplasm / Q000378:metabolism*; D000915:Antibody Affinity; D000937:Antigen-Antibody Reactions; D000951:Antigens, Neoplasm / Q000378:metabolism*; D001483:Base Sequence; D015179:Colorectal Neoplasms / Q000276:immunology*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D006860:Hydrogen Bonding; D007143:Immunoglobulin Heavy Chains / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism
D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D006023:Glycoproteins; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; C047052:tumor-associated antigen 72; D014443:Tyrosine