Genetic engineering of high affinity anti-human colorectal tumour mouse/human chimeric antibody

Two amino acids, tyrosine at position 96 and histidine at position 99 in the variable heavy chain (VH) CDR3 region of a mouse/human chimeric anti-TAG72 antibody cB72.3-1-3 were substituted with phenylalanine and asparagine respectively by site-directed mutagenesis technique. The expression vector mpSV2neo-EP1-Vm1-3-C gamma 1 containing mutant VH region fragments (Vm1-3) as well as the immunoglobulin enhancer (E), promoter (P1) and human genomic C gamma 1 region fragments, was transfected into a heavy-chain loss mutant cell line B72.3Mut(k). Mutant chimeric cB72.3m1-3 antibodies were purified from the transfectant supernates and compared based upon their binding affinity for the TAG72 antigen relative to that of the original cB72.3-1-3 antibody. The data show that a single amino acid substitution of histidine with asparagine at position 99 in VH CDR3 region contributes to four times increase in binding affinity for the TAG72 antigen. This suggests that the residue at position 99 in VH CDR3 region may play some role in antibody/antigen (B72.3/TAG72) interaction.