Using A Combinatorial Library Optimize Strategy For Humanization Of A Single Chain Variable Fragment Against Hepatocellular Carcinoma

OBJECTIVE With the fundamental work of obtaining the scFv DM with high specificity and affinity against hepatocellular carcinoma (HCC) from panning, we combined homology modeling, CDR-grafting with phage display techniqs to simultaneously humanize and optimize the murine scFv DM against hepatocellular carcinoma and get humanized scFvs with high specificity and affinity against HCC and low immuogenicity and provide a new method and means for the early diagnosis and therapy of HCC.METHODS With the aid of computer, the three-dimensional structure of scFv DM against HCC was built by means of homology modeling. Human germ-line VH and VL sequence with the highest degree of homologh with the framework regions of the scFv DM were identified as templates from GenBank by using BLAST. The amino acid sequence and the model structure of scFv DM were investigated to identify murine framework residues that most likely contribute to the integrity of binding site. All the residues that were difficult to decide were included as human and murine alternatives in the combinatorial library. The full-length humanized scFv gene fragment were synthesized with splicing overlapping extension-PCR. Phage display techniq was used to construct a humanized combinatorial library. Hepatocarcinoma cell-specific scFvs were selected by panning with the hepatocellular line.And the specificity for HCC of those selected scFvs was detected by ELISA. The relative affinities of phage antibodies of the positive clones and the primitive clone DM were measured by ELISA using thiocyanate elution.RESULTS The modeled structure of scFv DM manifested the common features of a classical antibody.Six CDRs located on the top of the Fv molecules and form a cavity for the antigen contact.The depth of cavity is 16.1A. The result of the ProStat is that 79.8%?-?angles of the model lie in the core region of the Ramachandran plot and that of the template 1NQB is 83.2%.The selection for the heavy chain template was AAW67378 with 76.1% homology to the framework of DM, and that of light chain was BAB 18257, having 71.6% homology of the framdwork of DM. The framework sequences of heavy chain and light chain and the corresponding selected human framework sequences were aligned, and 20 different residues in the heavy chain and 23 different residues in the light chain were detected and were divided into three kinds residues:28 humanized residues,8 backmutated residues and 7 difficult decided residues. A humanized combinatorial library was constructd successfully and the resulting library contained 25 different heavy chains and 22 different light chains for a total of 128 variant humanized DM. Three selected positive clones HDM1?HDM2?HDM3 were obtained after two rounds of panning and ELISA detection. The relative affinity of the positive clone HDM1 was 1.6mol/L?HDM2 was 1.2mol/L?HDM3 was 2.2mol/L.CONCLUSION The 3D structure of scFv DM against hepatocellular carcinoma was successfully constructed and our calculated result implies that the modeling structure is reasonable. Combinatorial library strategy was an effective and simple method for humanization of antibodies. All of the selected positive clones HDM1?HDM2?HDM3 had high specificity and affinity against HCC as the primitive clone DM. Which help to make a fundamental work for exploring its utilization on the early diagnosis and treatment of hepatocellular carcinoma.