Genetic engineering of a recombinant fusion possessing anti-tumor F(ab')2 and tumor necrosis factor

The construction, synthesis and expression of a genetically engineered bifunctional antibody/cytokine fusion protein is described. In order to target alpha-tumor necrosis factor (TNF) to tumor cells, recombinant antibody techniques were used to construct an RM4/TNF fusion protein containing the chimeric anti-tumor F(ab')2 (RM4) as well as the TNF moiety. The recombinant cDNA of human TNF was linked to the 3' end of the chimeric heavy-chain gene fragment (M4) containing the VH, the CH1 and the hinge region to form the fused heavy-chain gene fragment M4-TNF. Transfection of the M4-TNF gene fragment into a VKCK cell line producing the chimeric light-chain of the same antibody allowed the transfectant secreting the bifunctional fusion protein RM4/TNF. The RM4/TNF was purified by affinity chromatography. Our data showed that RM4/TNF retained the TAG72 antigen-binding reactivity as well as TNF activity as measured by ELISA, Western blotting, flow cytometry analysis, immunohistochemistry and cytotoxicity assays using the human colon cancer cell line LS174T. Therefore, the bifunctional fusion protein RM4/TNF may prove useful in targeting the biological effects of TNF to tumor cells, and in this way stimulate the immune destruction of tumor cells.