Aim: To prepare humanized Fab antibody by guided selection and chain shuffling technique.
Methods: Human Fd and C(L) repertoire genes were amplified by RT-PCR from PBMCs of patients with hepatocellular carcinoma. Human-murine chimeric C(L) genes were paired with the human Fd repertoire genes to construct phage antibody library. After four rounds of panning against HAb18GE, chimeric antibodies containing humanized Fd gene and binding to HAb18GE were obtained. Then the selected Fd genes were paired with the human C(L) repertoire genes to construct a humanized Fab antibody gene library. After four rounds of panning, completely humanized Fab antibodies were obtained.
Results: After six rounds of panning, 7 chimeric Fab genes were obtained from the Fab gene library with 2 x 10 (7) pfu. Using the selected seven Fd genes, the humanized Fab gene library of 0.8 x 10 (7) pfu was constructed. After four rounds of panning, 2 humanized Fab genes with the strongest reactivity to HAb18G were obtained. DNA sequencing showed that Fds of the two humanized Fabs had the same DNA sequence, which belonged to IgG2, just the same as the parent antibody. And the C(L) belonged to kappa type, and V kappa 3 family.
Conclusion: By guided-selection and chain shuffling technique, the humanized Fab antibody against HAb18G is obtained, which lays the foundation for further research.