Toward selection of internalizing antibodies from phage libraries.
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).
Becerril
B
B
Department of Pharmaceutical Chemistry, University of California, San Francisco 94110, USA.
Poul
M A
MA
Marks
J D
JD
D018033:Antibodies, Bispecific / Q000378:metabolism; D000915:Antibody Affinity / Q000235:genetics; D001943:Breast Neoplasms; D004705:Endocytosis / Q000235:genetics; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D017912:Inovirus / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014407:Tumor Cells, Cultured; D010948:Viral Plaque Assay
D018033:Antibodies, Bispecific; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D018719:Erb-b2 Receptor Tyrosine Kinases; D011994:Recombinant Proteins; C055966:immunoglobulin Fv