Cloning And Expression Of An Anti-human Melanoma Single-chain Fv Gene And Construction Of ScFv-TNF Bifunctional Antibody

Malignant melanomas, seen mostly on the skin, originate mainly from epithelial melanocytes and some originate from chromatophore nevus and dermis. Early metastasis may occur through lymph and blood stream. Effects of conventional treatment such as surgical removal, radiotherapy, and chemotherapy are far from satisfactory. As some melanomas have no pigment, it is very hard to distinguish the metastatic tumors from other kind of tumors.Recent developments in genetic antibodies have made it possible to use engineered antibodies in immunotherapy. Antibody directed therapy, first put into use in 80s, has used many murine antibodies to tumor-associated antigens in conjunction with chemicals, toxins, radioisotopes and prodrugs. But the immunogenicity of these antibodies greatly limited their clinical applications. Inrecent years, several kinds of engineered antibodies, especially the single chain antibodies, were constructed in order to reduce the immunogenicity and to facilitate clinical applications. Here we report a study on the cloning and expression of an anti-human melanoma single-chain antibody gene and the construction of ScFv-TNF bifunctional antibody.The VH and VL gene were amplified from a mouse anti -human melanoma hybridoma cell line HB8760 by RT-PCR. The two genes were sequenced and compared with those published genes in EMBL genbank. The results showed that they were homologous with the published mouse antibody variable region gene sequences. The VH and VL genes were 363bp and 327bp respectively and both of them were capable of encoding 121 and 109 amino acids. The deduced amino acid...