Objective: With the pComb3X-displaying Fab antibody libraries, to achieve the humanization of murine HAb18 against HCC by guided selection.
Methods: With the optimized primers, the human Fd and C(L) repertoire genes were amplified by RT-PCR from PBMC of HCC patients. The Fd repertoire genes were paired with murine HAb18 C(L) gene to construct pComb3X-displaying hybrid Fab library. The recombinant HAb18GE was used as antigens to select the target antibodies and got the Fd fragments. Then the human C(L) genes were paired with the selected human Fds to construct human Fab library. After the panning, the complete human Fab antibodies were got and analyzed.
Results: With the murine HAb18 C(L) gene as template, the heavy chain Fd shuffling was achieved by panning the hybrid Fab library. Then with the selected Fds as template, the human Fabs were obtained through the light chain shuffling. Two of the resulting human Fabs (HuFab2 and HuFab11), with same Fd and different light chains, bound to HAb18G/CD147 specifically. The competitive ELISA, Western blotting, FCM, fluorescent cell staining and so on demonstrated that the human Fabs resembled its parental murine Fab in that they both perhaps recognized the same epitope. K(D) indicated (HuFab2=210 nm and HuFab11=280 nm) the selected Fabs had available affinity.
Conclusion: Through guided-selection, we got the available human Fab antibodies for the subsequent research. These results suggest that guided selection is a promising strategy in murine mAb humanization.