Generation of Potent Anti-Vascular Endothelial Growth Factor Neutralizing Antibodies from Mouse Phage Display Library for Cancer Therapy

Abstract

Vascular endothelial growth factor (VEGF) is an important stimulator for angiogenesis in solid tumors. Blocking VEGF activity is an effective therapeutic strategy to inhibit tumor growth and metastasis. Avastin, a humanized monoclonal antibody recognizes VEGF, has been approved by the US Food and Drug Administration. To generate potential VEGF-recognizing antibodies with better tumor regression ability than that of Avastin, we have designed a systematic antibody selection plan. From mice immunized with recombinant human VEGF, we generated three phage display libraries, scFv-M13KO7, Fab-M13KO7, and scFv-Hyperphage, in single-chain Fv (scFv) or Fab format, displayed using either M13KO7 helper phage or Hyperphage. Solid-phase and solution-phase selection strategies were then applied to each library, generating six panning combinations. A total of sixty-four antibodies recognizing VEGF were obtained. Based on the results of epitope mapping, binding affinity, and biological functions in tumor inhibition, eight antibodies were chosen to examine their abilities in tumor regression in a mouse xenograft model using human COLO 205 cancer cells. Three of them showed improvement in the inhibition of tumor growth (328%–347% tumor growth ratio (% of Day 0 tumor volume) on Day 21 vs. 435% with Avastin). This finding suggests a potential use of these three antibodies for VEGF-targeted therapy.

Continuous microfluidic assortment of interactive ligands (CMAIL)

“…Typically magnet beads may provide increased surface area for binding, and thoroughness of washing, and hence better efficiency than plate biopanning363943. Compared with the initial capture efficiency of bead biopanning, the recovery rate of CMAIL is more than an order of magnitude higher.…”

“…However, it only confirms positive binding interactions, since its signal depends not only on the binding affinity, but also the heterogeneity of phage preparation as well as the level and avidity of scFv display. Further evaluation of selected clones involves DNA sequencing, scFv synthesis, as well as competitive ELISA, surface plasmon resonance or other comparable techniques for measuring the binding affinity and kinetics 36 . This approach is costly, time-consuming, and restricts the number of clones that can be evaluated.…”

“…Biopanning has been implemented using both plate and magnetic bead formats with different performance benchmarks, cost, and time required 39 40 41 42 . Typically magnet beads may provide increased surface area for binding, and thoroughness of washing, and hence better efficiency than plate biopanning 36 39 43 . Compared with the initial capture efficiency of bead biopanning, the recovery rate of CMAIL is more than an order of magnitude higher.…”

The Dual Role of the Liver in Nanomedicine as an Actor in the Elimination of Nanostructures or a Therapeutic Target

“…A number of studies have been carried out to isolate scFvs capable of specifically targeting signature proteins of a particular cancer cell, mainly exploiting the phage display technology [124]. Many scFvs have been generated against known proteins overexpressed by a number of different kinds of cancer cells, such as HER [125] and EpCAM [126], or against target proteins that are essential for cancer growth and spreading, such as VEGF [127] (involved in neo-angiogenesis). Recently, a novel scFv able to bind CD24, a target overexpressed by hepatocellular carcinoma (HCC), has been developed, showing a higher accumulation in the hepatocellular carcinoma xenograft mouse model and proving its potential as a therapeutic and diagnostic agent [128].…”

Production of Antigen-Binding Fragment against O,O-Diethyl Organophosphorus Pesticides and Molecular Dynamics Simulations of Antibody Recognition

“…A combination of Fab expression and display has revolutionized the isolation and engineering of antibodies [ 21 ]. Some of the most successful applications of this technology include cancer therapy [ 22 ], poisoning treatment [ 23 ], and immunoassay [ 24 ]. However, a limiting factor on the wide use of recombinant Fab, whether in bacteria like Escherichia coli , fungi, or higher eukaryotes in cell culture, has been the low yield of Fabs, with on average only a few milligrams of Fab produced per liter of cell culture [ 25 ].…”