Parallel evaluation of cell‑based phage display panning strategies: Optimized selection and depletion steps result in...

Phage display technology (PD) is a powerful technique for the generation of tumor‑targeting antibodies. However, there are a number of different selection methods established in different laboratories around the world. Cell‑based PD panning methods using primary tumor cells are particularly heterogeneous between laboratories, which can lead to inconsistent results. Therefore, the present study evaluated different cell‑based PD selection methods regarding their potential to generate acute myeloid leukemia (AML) blast‑binding antibodies. In addition to this evaluation, the present study improved the PD procedure by optimizing selection as well as depletion strategies. To the best of our knowledge, the current study demonstrated for the first time that antigen diversity during the depletion step is of importance for the enrichment of tumor‑targeting phage antibodies. It is demonstrated that medium levels of depletion antigen diversity led to the most promising antibody candidates. In addition, it was determined that purification of blast cells from patients with AML by immunomagnetic separation ameliorated the selection of AML‑binding phages during panning. Furthermore, suggesting a common design‑related mechanism using a 'single‑pot' PD library, such as the well‑known Tomlinson single‑chain fragment variable (scFv) library, the present study identified specific binding consensus phage particles in independent panning procedures. By means of these optimized strategies, four promising AML blast‑binding phage particles were isolated and soluble scFv‑Fc (scFv cloned to a fragment crystallizable of an IgG2a mouse antibody) fusion proteins were produced. These scFv‑Fc antibodies bound the surface of AML blasts and were successfully internalized into their cytoplasm, indicating that they are potential immunoconjugate candidates for AML immunotherapy.