Modification in framework region I results in a decreased affinity of chimeric anti-TAG72 antibody.
A mouse/human chimeric B72.3P-1-6 antibody was produced by construction of a novel expression vector mpSV2neo-EP2-V-Crl containing the same gene fragments as the expression vector mpSV2neo-EP1-V-Crl (Xiang J., Roder J. and Hozunni N., submitted to Molec. Immun., 1991) except the promoter (P2) fragment in which the translation start codon ATG is retained. The expression vector was transfected into a heavy chain loss mutant cell line, B72.3M1. The translation of the chimeric heavy chain may start at the exogenous start codon ATG within the P2 fragment, which is 27 base pairs upstream of the endogenous start codon ATG in B72.3 heavy chain V region cDNA fragment, leading to an alteration in leader sequence cleavage sites and the formation of chimeric heavy chain with an elongation in the FR1 region. Chimeric B72.3P-1-6 antibody retained binding specificity to TAG72 antigen, but showed an eight-fold decrease in binding affinity to TAG72 compared with chimeric B72.3-1-3 antibody. This suggests that residues in FRI contribute to the correct folding of the antibody binding region of the B72.3 antibody.
Xiang
J H
JH
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Department of Immunology and Medical Genetics, University of Toronto, Ontario, Canada.
Roder
J
J
Pan
Z G
ZG
Roifman
C
C
Hozumi
N
N
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000912:Antibodies, Neoplasm / Q000737:chemistry* / Q000235:genetics; D000915:Antibody Affinity; D000951:Antigens, Neoplasm / Q000276:immunology*; D001483:Base Sequence; D003001:Cloning, Molecular; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D005822:Genetic Vectors; D006023:Glycoproteins / Q000276:immunology*; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship
D000596:Amino Acids; D000912:Antibodies, Neoplasm; D000951:Antigens, Neoplasm; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins; C047052:tumor-associated antigen 72