RESUMO
Transferrin receptor (TfR) has been explored as a target for antibody-based therapy of cancer. In the previous study, we reported a murine anti-TfR monoclonal antibody (mAb) 7579 had good anti-tumor activities in vitro. In an attempt to reduce its immunogenicity and enhance its ability to recruit immune effector mechanism in vivo, we herein developed its chimera in the baculovirus/insect cell expression system based on the mating-assisted genetically integrated cloning (MAGIC) strategy. The chimeric light and heavy chains, containing human IgG1 constant regions, were correctly processed and assembled in insect cells, and then secreted into the mediums as heterodimeric H(2)L(2) immunoglobulins. Furthermore, analyses of antigen-binding assay and competitive binding assay indicated that the chimeric antibody possessed specificity and affinity similar to that of its parental murine antibody. Results of the antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assay verified that the chimeric antibody could efficiently mediate ADCC and CDC against TfR-overexpressing tumor cells. These results suggested that this baculovirus-expressed chimeric anti-TfR IgG1 might have the potential to be used for cancer immunotherapy. Meanwhile, the MAGIC strategy, facilitating the rapid generation of chimeric mAbs, could be one of the efficient strategies for antibody engineering.
Assuntos
Anticorpos/química , Baculoviridae/genética , Engenharia de Proteínas , Receptores da Transferrina/imunologia , Proteínas Recombinantes de Fusão/química , Animais , Anticorpos/genética , Anticorpos/imunologia , Citotoxicidade Celular Dependente de Anticorpos , Ligação Competitiva , Linhagem Celular , Humanos , Hibridomas , Camundongos , Reação em Cadeia da Polimerase , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
BACKGROUND: RNA interference (RNAi) has become a powerful means for silencing target gene expression in mammalian cells and is envisioned to be useful in therapeutic approaches to human disease. In recent years, high-throughput, genome-wide screening of siRNA/miRNA libraries has emerged as a desirable approach. Current methods for constructing siRNA/miRNA expression vectors require the synthesis of long oligonucleotides, which is costly and suffers from mutation problems. RESULTS: Here we report an ingenious method to solve traditional problems associated with construction of siRNA/miRNA expression vectors. We synthesized shorter primers (< 50 nucleotides) to generate a linear expression structure by PCR. The PCR products were directly transformed into chemically competent E. coli and converted to functional vectors in vivo via homologous recombination. The positive clones could be easily screened under UV light. Using this method we successfully constructed over 500 functional siRNA/miRNA expression vectors. Sequencing of the vectors confirmed a high accuracy rate. CONCLUSION: This novel, convenient, low-cost and highly efficient approach may be useful for high-throughput assays of RNAi libraries.
