Construction, Expression, And Activity Of A Genetic Engineered Anti-HER2/neu×Anti-CD16 Bispecific Antibody

Bispecific antibodies (BsAbs) have significant potential for cancer therapy because they can be used to retarget cytotoxic effector cells against tumor cells. BsAbs are artificially created antibodies with antigen-binding sites for two different epitopes, that can make a bridge between the tumor cell and the immune effector cell, followed by triggering the cytotoxic responses that result in a 'lethal hit' to the tumor cell. In this study, a genetic engineered anti-HER2/neu anti-CD16 BsAb was constructed. The binding characteristics and ability in vitro killing of HER2/neu over-expressing tumor cells of the BsAb were also analyzed.1 Cloning, expression and characterization of the variable region genes of the monoclonal antibody against human CD16The 5'-RACE (rapid amplification of cDNAend, RACE)method was used to clone the immunoglobulin (Ig) genes encoding variable regions of heavy and light chains (VH and VL) from the hybridoma cell line B88-9, which secrets the monoclonal antibody against human CD 16 . A Linker Peptide sequence was inserted between the VH and VL by overlap extension PCR. The assembled scFv gene and the Fc gene of human IgGl were cloned into expression vector pCI-neo and expressed in CHO cells. The scFv-Fc fusion protein was purified from serum-free conditioned supernatant using a Protein A affinity chromatography column. Two-color flow cytometry studies revealed that the scFv-Fc fusion protein recognizes the subset of CD56+ NK cells which express CD 16.2 Expression and characterization of scFv-Fc fusion protein against human HER2/neu proteinThe anti-HER2/neu scFv gene and the Fc gene of human IgGl were cloned into expression vector pCI-neo and expressed in CHO cells. The high producing clone was selected with G418 and the scFv-Fc fusion protein was purified from serum-free conditioned supernatant using a Protein A column. The scFv-Fc fusion protein could specially immunoprecipitate 185 kD HER2/neu protein from SK-BR-3 cell lysates. Flow cytometric analysis indicated the fusion protein bound to extracellular domain of the HER2/neu protein. The affinity of the scFv-Fc fusion protein, determined by ELISA,was K=1.33X10"9mol/L.3 Construction of an anti-HER2/neu X anti-CD 16 bispecific antibody by 'knobs-into-holes' engineering of antibody CH3 domainsA 'knobs-into-holes' strategy was adopted to engineer the CH3 domains of human IgGl for heterodimerization. A konb variant T366Y was obtained by replacement of a small amino acid with a larger one in the CH3 domain of anti-HER2/neu scFv-Fc chain. A hole mutation Y407T was constructed by replacement of a large residue with a smaller one in the CH3 domain of anti-CD 16 scFv-Fc chain. The two chains were functionally expressed in CHO cells and assembled into heterodimers with dual antigen-binding specificity. The highly producing clone F7, which had a yield of approximate 13.4 ug/ml for the BsAb, was selected with MTX and G418. The clone F7 was adapted to serum-free medium and grew in suspension by decreasing serum concentration in medium gradually. In vitro experiments demonstrated that the BsAb was able to recruit human PBMC for killing SK-BR-3 cells more effectively than anti-HER2/neu antibody Herceptin? P< 0.001.4 Design and construction of a new format bispecific antibody with three binding valancesA new format BsAb with three binding valances (trivalent BsAb) was designed using the first constant region of human IgGl (CHI) and the constant of human K chain (CL) as heterodimerization domains. In the trivalent BsAb, one binding valance was designed for effector cells, that would reduce or avoid side effects caused by systemic activation of effector cells; two binding valances were designed for target cells, so the BsAb would have higher affinity for the target and localize at the tumor site quickly in vivo. As a example, a trivalent anti-HER2/neu X anti-CD 16 BsAb was constructed and expressed in E. coli.