Phage Display Technology for Human Monoclonal Antibodies

Abstract

During the last 15 years in vitro technologies opened powerful routes to combine the generation of large libraries together with fast selection procedures to identify lead candidates. One of the commonest methods is based on the use filamentous phages. Antibodies (Abs) can be displayed successfully on the surface of phage by fusing the coding sequence of the antibody variable (V) regions to the phage minor coat protein pIII. By creating large libraries, antibodies with affinities comparable to those obtained using traditional hybridomas technology can be selected by a series of cycles of selection on antigen. As in this system antibody genes are cloned simultaneously with selection they can be easily further engineered for example by increasing their affinity (to levels unobtainable in the immune system), modulating their specificity or their effector function (by recloning into a full-length immunoglobulin scaffold). This chapter describes the basic protocols for antibody library construction, handling, and selection.

Mammalian Cell Surface Display as a Novel Method for Developing Engineered Lectins with Novel Characteristics

“…Previously, we generated several mutated lectins with altered sugar-binding specificities using phage-display [ 30 ] and ribosome-display systems [ 31 , 32 ]. Although phage-display and ribosome-display are convenient screening methods for the selection and evolution of peptides or single-chain antibodies [ 33 , 34 , 35 ], they have some disadvantages when used for the selection of mutated lectins. First, high molecular weight proteins are not suitable for expression in these systems because the correct folding of recombinant proteins does not occur efficiently in E. coli cells or in vitro .…”

Identification of novel proteins binding the AU-rich element of α-prothymosin mRNA through the selection of open reading frames (RIDome)

“…The library selection was performed as reported 31,32 with minor changes of solutions composition to safeguard the RNA bait (see the Materials and Methods section). After two cycles of selection, phagemid DNAs were recovered, and ORF inserts were amplified and sequenced according to a 454 protocol.…”

“…32 For biopanning experiments, phage particles were suspended in PBS buffer at a concentration of 10 11 cfu/ml, and for each selection, 10 12 phages were used. The bait was a 3 0 biotinylated RNA oligonucleotide corresponding to the AU-rich element of a-prothymosin (ARE PTMA , 5 0 -GGAAAUUUGUUUGUAUUUUUAGCU-biotin).…”

“…The amplified phages were used for a second cycle of selection as reported. 32,56 Deep Sequencing of cDNA Inserts After the second round of selection, colonies growing on agar plates were harvested, and the phagemid DNAs were isolated using a standard miniprep procedure. The cDNA inserts were processed for 454 sequencing according to the Titanium Rapid Library preparation method manual (Roche, Milano, Italy).…”

EPOR-Based Purification and Analysis of Erythropoietin Mimetic Peptides from Human Urine by Cys-Specific Cleavage and LC/MS/MS

“…Implementing the sophisticated Phage Display Technology (PDT) into pharmaceutical proceedings entails a fast and customized in vitro method to screen for highly specific active peptide regions that interact with physiological structures of interest [4]. The acquired peptide libraries establish the fund of the modern medicinal drug treasury and are utilized for e.g., full human antibody modeling and manufacturing [5,6]. Fast evolving classes of bioactive substances pose unforeseen complications for analytical procedures and fast adaptable methods are desirable in sports drug testing.…”