Targeting c-Met on gastric cancer cells through a fully human fab antibody isolated from a large naive phage antibody library.

PURPOSE: The aberrant Hepatocyte growth factor (HGF)/ mesenchymal-epithelial transition factor (c-Met) signaling pathway in various malignancies and its correlation with tumor invasion and poor prognosis has validated c-Met as a compelling therapeutic target. Up to now, several monoclonal antibodies and small molecule inhibitors targeting c-Met have been introduced with different outcomes, none are yet clinically approved. Toward the generation of novel fully human anti-c-Met molecules, we generated a large naïve Fab antibody library using phage display technology, which subsequently screened for novel Fabs against c-Met. METHODS: A phage library, with a functional size of 5.5 × 1010 individual antibody clones, was prepared using standard protocols and screened for c-Met-specific Fabs by successive rounds of panning. A panel of Fabs targeting c-Met were isolated, from which four clones were selected and further characterized by DNA sequencing. The c-Met binding ability of our selected Fabs was evaluated by c-Met ELISA assay and flow cytometry techniques. RESULTS: Among the confirmed anti-c-Met Fabs, clone C16, showed the highest affinity (Kaff: 0.3 × 109 M-1), and 63% binding to MKN45 cells (a human gastric adenocarcinoma cell-line) as compared to c-Met negative T47D cell-line (9.03%). CONCLUSION: Together, our study presents a single-pot antibody library, as a valuable source for finding a range of antigen-specific Fab antibodies, and also, a fully human, high affinity and specific anti c-Met Fab antibody, C16, which has the potential of developing as a therapeutic or chemotherapeutic delivery agent for killing c-Met-positive tumor cells.

Quantitative Proteomic Analysis of Cellular Responses to a Designed Amino Acid Feed in a Monoclonal Antibody Producing Chinese Hamster Ovary Cell Line

Background: Chinese hamster ovary (CHO) cell line is considered as the most common cell line in the biopharmaceutical industry because of its capability in performing efficient post-translational modifications and producing the recombinant proteins, which are similar to natural human proteins. The optimization of the upstream process via different feed strategies has a great impact on the target molecule expression and yield. Methods: To determine and understand the molecular events beneath the feed effects on the CHO cell, a label-free quantitative proteomic analysis was applied. The proteome changes followed by the addition of a designed amino acid feed to the monoclonal antibody producing CHO cell line culture medium were investigated. Results: The glutathione synthesis, the negative regulation of the programmed cell death, proteasomal catabolic process, and the endosomal transport pathway were up-regulated in the group fed with a designed amino acid feed compared to the control group. Conclusion: Our findings could be helpful to identify new targets for metabolic engineering.

Biomolecular Corona Dictates Aß Fibrillation Process.

Amyloid beta (Aß), which forms toxic oligomers and fibrils in brain tissues of patients with Alzheimer's disease, is broadly used as a model protein to probe the effect of nanoparticles (NPs) on oligomerization and fibrillation processes. However, the majority of the reports in the field have ignored the effect of the biomolecular corona on the fibrillogenesis of the Aß proteins. The biomolecular corona, which is a layer composed of various types of biomolecules that covers the surface of NPs upon their interaction with biological fluids, determines the biological fates of NPs. Therefore, during in vivo interaction of NPs with Aß protein, what the Aß actually "sees" is the human plasma and/or cerebrospinal fluid (CSF) biomolecular-coated NPs rather than the pristine surface of NPs. Here, to mimic the in vivo effects of therapeutic NPs as antifibrillation agents, we probed the effects of a biomolecular corona derived from human CSF and/or plasma on Aß fibrillation. The results demonstrated that the type of biomolecular corona can dictate the inhibitory or acceleratory effect of NPs on Aß1-42 and Aß25-35 fibrillation processes. More specifically, we found that the plasma biomolecular-corona-coated gold NPs, with sphere and rod shapes, has less inhibitory effect on Aß1-42 fibrillation kinetics compared with CSF biomolecular-corona-coated and pristine NPs. Opposite results were obtained for Aß25-35 peptide, where the pristine NPs accelerated the Aß25-35 fibrillation process, whereas corona-coated ones demonstrated an inhibitory effect. In addition, the CSF biomolecular corona had less inhibitory effect than those obtained from plasma.