| Primary liver cancer was endowed with high malignance and being liable to invade and metastasize,and it was inherently resistant to drug. Therefore, anti-liver cancer therapy is still in the predicament that hardly effective drugs and treatment technic can be used. It has an annual incidence rate of 564,000 cases, 55% of which are in China. HAb18G/CD147 was a novel cancer target defined by our laboratory and can be used as a significant independent predictor of poor prognosis in HCC patients after surgery. Using it as a drug target, a radioimmunotherapeutic agent LICARTIN, a national class I new drug, generated by labeling HAb18-F(ab')2 with 131I, developed in our lab, has showed a significant suppressing efficacy on tumor progression in HCC patients, and can significantly decrease the tumor recurrence/metastasis rate and prolong the patients'survival in post-liver transplantation patients with advanced HCC. However, alternative generation of the HAb18-based antibody drugs should be developed for higher production cost and HAMA response detected in the patients who used Licartin. Basing on mAb HAb18, its single chain fragment of variable region was humanized, and was then fabricated with human IgG1 Fc fragment. It was demonstrated that the resulting antibody fragment (HAb18-huScFv)2-Fc has binding activity to HAb18G/CD147, anti-tumor activity, including cytotoxicity and inhibition of MMPs secretion and cell invasion, and decreased immunogenicity additionally.Part I: construction and expression of (HAb18-huScFv)2-FcObjective: obtaining (HAb18-huScFv)2-FcMethod: first, analysis of mAb HAb18 variable region genes and BlastP with sequences in non-redundant human immunoglobulin VL and VH sequences of Genbank were performed. All the chosen sequences were selected from two species (Mus musculus and Homosapiens) since each containing light and heavy chain types. Then, we selected 100 sequences from Mus musculus and Homosapiens banks, respectively, for analysis patterns of surface exposed residues that most closely matched the patterns found on VL and VH of HAb18 according to the scores of similarity. Preternatural residues and differential residues of HAb18 were obtained. After that, with the 3-D structure from modeling of HAb18 variable region, we obtained intermolecular and intramolecular hydrogen bonds between HAb18 VH and VL. Besides, we considered relative solvent accessibility and relative conservatism, and eventually, identified the mutated amino acid residues for humanization. With HAb18-ScFv as a template, The humanized VH and VL gene sequences of (HAb18-huScFv)2-Fc were synthesized by overlapping polymerase chain reaction after site-specific mutagenesis. The genetic fragment of HAb18-huScFv was subcloned to construct pCDNA5/FRT-(HAb18-huScFv)2-Fc. The expression vector was transfected into Flp-In CHO cells using lipofectin, and the cells with stable expression were generated by selecting and cloning. The supernatant was purified by HiTrap MabSelect SuRe and HiTrap chelating HP column successively to obtain (HAb18-huScFv)2-Fc protein. It was detected by indirect ELISA, SDS-PAGE and Western blot. Meanwhile, its murine counterpart, (HAb18-ScFv)2-Fc, was constructed following the same process except the introduction of site-directed mutation.Results: The different CDR and FR of HAb18 sequences was marked according to Kabat, Abm, Chothia and Contact rules, after alignment and screening antibody sequences, the surface exposed positions in a set of heavy and light chain variable region framework were determined and the most closely identical to the set of Mus musculus or homo sapiens surface residues was also labeled. Preternatural residues and differential residues were obtained after alignment. After that we determined the candidate mutation sites were that H19K, H90A and L10F were replaced by H19A, H90T and L10S, respectively, for humanization, according to relative solvent accessibility, relative conservatism and intermolecular and intramolecular hydrogen bonds between HAb18 VH, VL. After overlapping PCR after site-directed mutation, identified gene fragment was subcloned into expression vector. The vector was transfected into expression cells, and screening under pressure, cloning were performed for production. It was showed by SDS-PAGE and Western blot that the protein molecular weight was about 110 kDa under non-reduction and that was about 55 kDa under reduction. Its murine counterpart was of identical molecular weight and right gene sequence thereof.Part II: characterization of (HAb18-huScFv)2-FcObjective: characterizing binding activity, anti-tumor activity and immunogenicity of in vitro Method: Indirect competitive ELISA was performed to analyze (HAb18-huScFv)2-Fc binding activity to HAb18G/CD147, (HAb18-huScFv)2-Fc of various concentration was used to compete with its parental antibody HAb18 to bind HAb18G/CD147 and affinity and specificity were observed. Surface plasmon resonance was used to detect association constant (Kon) and dissociation constant (Koff) of (HAb18-huScFv)2-Fc and (HAb18-ScFv)2-Fc, respectively, then affinity constant (KD) was calculated. Cell immunofluorescence and IHC assay were used to compare binding activity of (HAb18-huScFv)2-Fc and (HAb18-ScFv)2-Fc to antigen HAb18G/CD147 of liver cancer cells FHCC-98 and HCC tissue. In gelatin zymograph assay, those two antibodies were used to treat HPF-1 cells and FHCC-98 cells co-culture, respectively, and blank control was set. In invasion assay, mixture of FHCC-98 cells and HPF-1 cells in equal volume was added to chamber, invasion inhibition of antibodies was observed after the cells were treated with antibodies. ADCC and CDC assays were performed to observe mediation function of (HAb18-huScFv)2-Fc to cytotoxicity of PBMC and rabbit serum. To analyze the immunogenicity of antibodies, sera samples from patients with HAMA response after treated with HAb18 were processed by 5A5, which recognize epitopes distinct from that of HAb18, through pre-absorption, to deplete free CD147, anti-isotypic antibodies and anti-allotypic antibodies in the serum. Respectively, those tow antibodies of various concentrations were used to compete with HAb18-F(ab')2 to bind anti-idiotypic antibodies in the serum. By indirected competitive ELISA, we obtained 50% inhibition concentration (IC50) of those two antibodies in each sample. The difference of antibodies'immunogenicity were reflected by their inhibition activity to the binding of HAb18-F(ab')2 to anti-idiotypic antibody in the serum. Results: (HAb18-huScFv)2-Fc bind the same epitope of HAb18G/CD147 to HAb18, and (HAb18-huScFv)2-Fc and (HAb18-ScFv)2-Fc have similar binding activity to liver cancer cells and tissues (the former has affinity constant (KD) of 1.5×10-9 M, and the latter has affinity constant (KD) of 1.2×10-9 M). Compared with the blank control, those two antibodies can inhibit MMPs secretion and invasion of HCC cells FHCC-98 (p < 0.05), and difference can hardly observed between them. (HAb18-huScFv)2-Fc was an efficient mediator of ADCC and CDC (p < 0.05) and the effect was dose-dependent. The immunogenicity of those two antibodies was decreased. In all four serum samples, IC50 values (HAb18-huScFv)2-Fc were significantly higher than those of (HAb18-ScFv)2-Fc, and P values of them were p = 0.0440, p < 0.0001, p = 0.0338, p < 0.0001, respectively.Conclusion: humanized antibody fragment (HAb18-huScFv)2-Fc could be a more efficient antibody fragment with anti-tumor activity, less immunogenicity and additional cytotoxicity function. |
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