Complementarity determining region residues aspartic acid at H55, serine at H95 and tyrosines at H97 and L96 play important roles in the B72.3 antibody-TAG72 antigen interaction |
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Authors: | Xiang Jim; Sha Yuhua; Prasad Lata; Delbaere Louis TJ |
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Affiliation: | Saskatoon Cancer Center, Departments of Microbiology Saskatoon, Saskatchewan S7N 0W0, Canada
2Department of Biochemistry, University of Saskatchewan Saskatoon, Saskatchewan S7N 0W0, Canada |
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Abstract: | Structural analysis derived from the crystallographic studyof the chimeric B72.3 antibody illustrated some major atomicinteractions between complementarity determining region (CDR)residues. For example, hydrogen bonds are formed between H35/H95,L50/H97, H53/H55 and H96/L96 respectively. These CDR residuesmay play important roles in the B72.3TAG72 (antibody-antigen)interaction either by direct interaction with the TAG72 antigenor by maintaining a CDR loop conformation through atomic interactionsbetween CDR residues. In order to confirm these assumptions,we altered these CDR residues by site-directed mutagenesis anddetermined binding affinities of these mutant chimeric antibodiesfor the TAG72 antigen in a solid-phase radioimmunoassay. Wefound that H55, H95, H97 and L96 are important CDR residuesfor the B72.3TAG72 interaction. Single amino acid substitutionsof aspartic acid and serine by alanine at H55 of CDR2 and atH95 of CDR3 respectively and of tyrosine by phenylalanine atH97 and L96 of CDR3, significantly reduced the binding affinityfor the TAG72 antigen by 20-, 8-, 16- and 45-fold respectively.Therefore, this study reveals some of the requirements for maintainingthe integrity of the B72.3 antibody combining sites. |
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Keywords: | antigen-binding affinity// CDR residues// site-directed mutagenesis// TAG72 antigen |
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