Influences of amino acid sequences in FR1 region on binding activity of the scFv and Fab of an antibody to human gastric cancer cells.
Murine anti-gastric cancer mAb 3H11 has promising clinical applications. In this work, engineering of 3H11 scFv and Fab was conducted to increase its usefulness. 3H11 scFv and Fab were constructed by PCR amplification of the V-domains with degenerate primers for FR1. The bacterially expressed 3H11 Ab fragments showed no antigen binding activity. Then the N-terminal sequences of V regions were mutated to the 3H11 original sequence. The expressed scFv and Fab in bacterial culture supernatant showed binding activity to gastric cancer cells. Comparing the expression of unmutated and mutated 3H11 Fab, we found that the sequence changes of the V region N terminus introduced by PCR may seriously affect antigen binding but not the expression of antibody. Correction of either VL or VH N-terminal sequences can partially restore the antigen binding activity (61% for VL and 73% for VH).
Li
J
J
The School of Oncology, Beijing Medical University, Beijing, PR China. jli01@emerald.tufts.edu
Wang
Y
Y
Wang
Z
Z
Dong
Z
Z
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology; D000912:Antibodies, Neoplasm / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017931:DNA Primers; D015870:Gene Expression; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000276:immunology*; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012995:Solubility; D013274:Stomach Neoplasms / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D017931:DNA Primers; D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region