Toward selection of internalizing antibodies from phage libraries
Toward selection of internalizing antibodies from phage libraries
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DOI：
10.1006/bbrc.1999.0177
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发表时间：
1999-02-16
影响因子：
3.1
通讯作者：
Marks, JD
中科院分区：
文献类型：
--
作者：
Becerril, B;Poul, MA;Marks, JD
以使其被内吞的方式结合细胞表面受体的抗体是用于将药物、毒素或DNA递送到哺乳动物细胞的胞质溶胶中以用于治疗应用的有用分子。传统上，通过筛选杂交瘤来鉴定内化抗体。为了这项工作，我们研究了结合ErbB2的人scFv（C6.5），以确定从噬菌体文库中直接选择内化抗体的可行性，并鉴定最有效的展示形式。使用野生型C6.5单链抗体展示噬菌粒上的单链抗体，我们证明了抗ErbB2噬菌体抗体可以进行受体介导的内吞作用。使用亲和突变体和二聚体的C6.5双抗体显示为噬菌粒上的单拷贝或噬菌体上的多个拷贝，我们定义了亲和性，效价和展示形式对噬菌体内吞作用的作用，并确定了导致最大富集内化的因素。展示二价双抗体或多拷贝scFv的噬菌体比展示单体scFv的噬菌体更有效地内吞，并且通过用氯喹预孵育细胞来增加感染性噬菌体的回收。作为应用的噬菌体滴度的函数的从胞质溶胶内的噬菌体回收的测量表明，即使在10（9）的文库中单个噬菌体抗体成员存在的低浓度下，也可以选择可内吞的抗体。(C)北京：科学出版社.
Antibodies which bind cell surface receptors in a manner whereby they are endocytosed are useful molecules for the delivery of drugs, toxins, or DNA into the cytosol of mammalian cells for therapeutic applications. Traditionally, internalizing antibodies have been identified by screening hybridomas. For this work, we studied a human scFv (C6.5) which binds ErbB2 to determine the feasibility of directly selecting internalizing antibodies from phage libraries and to identify the most efficient display format. Using wild-type C6.5 scFv displayed monovalently on a phagemid, we demonstrate that anti-ErbB2 phage antibodies can undergo receptor-mediated endocytosis. Using affinity mutants and dimeric diabodies of C6.5 displayed as either single copies on a phagemid or multiple copies on phage, we define the role of affinity, valency, and display format on phage endocytosis and identify the factors that lead to the greatest enrichment for internalization. Phage displaying bivalent diabodies or multiple copies of scFv were more efficiently endocytosed than phage displaying monomeric scFv and recovery of infectious phage was increased by preincubation of cells with chloroquine. Measurement of phage recovery from within the cytosol as a function of applied phage titer indicates that it is possible to select for endocytosable antibodies, even at the low concentrations that would exist for a single phage antibody member in a library of 10(9). (C) 1999 Academic Press.