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.

中文翻译：

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基于细胞的噬菌体展示淘选策略的平行评估：优化的选择和消耗步骤导致 AML 爆炸结合共有抗体。

噬菌体展示技术 (PD) 是一种用于产生肿瘤靶向抗体的强大技术。然而，世界各地的不同实验室建立了许多不同的选择方法。使用原发性肿瘤细胞的基于细胞的 PD 淘选方法在实验室之间特别异质，这可能导致结果不一致。因此，本研究评估了不同的基于细胞的 PD 选择方法关于它们产生急性髓性白血病 (AML) 爆炸结合抗体的潜力。除此评估外，本研究还通过优化选择和消耗策略改进了 PD 程序。据我们所知，目前的研究首次证明，耗尽步骤中的抗原多样性对于富集肿瘤靶向噬菌体抗体很重要。已证明中等水平的耗尽抗原多样性导致最有希望的抗体候选者。此外，确定通过免疫磁性分离纯化 AML 患者的胚细胞改善了淘选过程中 AML 结合噬菌体的选择。此外，本研究提出了使用“单罐”PD 文库（例如著名的 Tomlinson 单链片段变量 (scFv) 文库）的常见设计相关机制，在独立的淘选程序中确定了特异性结合的共有噬菌体颗粒。通过这些优化策略，分离了四种有希望的 AML 胚细胞结合噬菌体颗粒，并产生了可溶性 scFv-Fc（scFv 克隆到可结晶的 IgG2a 小鼠抗体片段）融合蛋白。这些 scFv-Fc 抗体结合 AML 原始细胞的表面并成功内化到它们的细胞质中，表明它们是 AML 免疫治疗的潜在免疫偶联物候选物。