Humanized heavy variable region of anti human fibrin ScFv 8E5
Objective To humanize the V H of anti human fibrin ScFv 8E5 for minimizing the human anti mouse antibody response. Methods We have constructed a “humanized ” antibody by combining the complementarity determining regions (CDRs) of V H in the anti human fibrin ScFv 8E5 with human framework. The human framework regions were chosen to maximize homology (71 8%) with the anti human fibrin ScFv 8E5 sequence. A few mouse amino acids were also retained in the humanized antibody. Humanized V H 8E5 fragments were synthesized and combined into an intact heavy variable region. An expression vector pOPE101 V H 8E5 was constructed by substituting the V H 8E5 with humanized V H 8E5.The transformed E.coli JM109 cells were propagated and induced by IPTG. Results Expression product was found in the total cell extracts and periplastic fractions by SDS PAGE and ELISA. It was a 30kD single chain fragment with human fibrin binding specificity. The humanized ScFv 8E5 has an affinity for human fibrin about 4/5 that of murine ScFv 8E5. The yield of the humanized ScFv was 11 2% of the total bacterial proteins. Conclusion The humanized ScFv 8E5 showed antigen binding capacity of the parental ScFv. It should be successful to humanize the V H of ScFv 8E5.