Construction And Biological Activity Of Anti-CD3/Anti-Osteosarcoma Bispecific Single-Chain Antibody

Objective: To construct bispecific single chain antibody fragment anti-CD3 × anti-TP-3 diabody by genetic engineering with dual specificity for both the human osteosarcoma cell antigen TP-3 and CD3e-chain of CD3/TCR. The diabody was incubated with human T cells and osteosarcoma cells to investigate its activity to stimulate T cells to kill target cells compared with its parental monoclonal antibody(McAb) and single-chain variable fragment(scFv).Methods:1 Vector construction Plasmids pHOG-aTP-3 and pHOG-dmOKT3 encoding the scFv fragments derived from hybridoma HP7 specific for human TP-3 and OKT3 specific for human CD3. A PCR fragment of the V_h domain of anti-TP-3 preceded by a BglII site and followed by a segment coding for a LysLeuGlyGly linker was generated using two relative primers. The PCR fragment was digested with EcoRI and EcoRV and ligated with the EcoRI/EcoRV linearized plasmid pHOG-dmOKT3, generating the vector pHOGTP-3-CD3 encoding the first hybrid scFv (V_h of anti-TP-3 and V_L of anti-CD3). The other PCR fragment of the V_L domain of anti-TP-3 followed by a segment coding for a c-myc epitope and a hexahistidinyl tail was generated using two relative primers. The PCR fragment of the V_l domain of anti-TP-3 contained a BglII site near the 3 □ end of the coding strand. It was digested with HindIII and XbaI and ligated with the HindIII/XbaI linearized plasmid pHOGdmOKT3, generating the vectors pHOGCD3-TP-3 encoding the second hybrid scFv(V_L of anti-TP-3 and V_H of anti-CD3). The expression plasmid pKIDCD 3 ×...