A method for the generation of combinatorial antibody libraries using pIX phage display

Abstract

For more than a decade, phage displayed combinatorial antibody libraries have been used to generate and select a wide variety of antibodies. We previously reported that the phage coat proteins pVII and pIX could be used to display the heterodimeric structure of the antibody Fv region. Herein, aspects of this technology were invoked and extended to construct a large, human single-chain Fv (scFv) library of 4.5 ؋ 10 9 members displayed on pIX of filamentous bacteriophage. Furthermore, the diversity, quality, and utility of the library were demonstrated by the selection of scFv clones against six different protein antigens. Notably, more than 90% of the selected clones showed positive binding for their respective antigens after as few as three rounds of panning. Analyzed scFvs were also found to be of high affinity. For example, kinetic analysis (BIAcore) revealed that scFvs against staphylococcal enterotoxin B and cholera toxin B subunit had a nanomolar and subnanomolar dissociation constant, respectively, affording affinities comparable to, or exceeding that, of mAbs obtained from immunization. High specificity was also attained, not only between very distinct proteins, but also in the case of the Ricinus communis (''ricin'') agglutinins (RCA60 and RCA120), despite >80% sequence homology between the two. The results suggested that the performance of pIX-display libraries can potentially exceed that of the pIII-display format and make it ideally suited for panning a wide variety of target antigens.

Selection and characterization of human antibodies neutralizing Bacillus anthracis toxin

“…In brief, PA was added to cultured RAW264.7 cells and incubated for a period of 5,15,25,30,45 and 60 min at 37 °C. PA was removed and the cells were washed by fresh culture medium, followed by the addition of LF and incubation at 37 °C for 4 hr before conducting the MTT assay.…”

The effect of pH dependence of antibody-antigen interactions on subcellular trafficking dynamics

“…1 Antibodies with high affinity and specificity for an almost unlimited number of targets can be isolated using different display technologies. [2][3][4] More recently, considerable effort has been directed toward producing next generation antibodies that are improved over their parent molecules in one or more respects. For example, Fc engineering can be used to enhance ADCC 5,6 or increase in vivo persistence [7][8][9] by modifying binding to FcγRs or FcRn, respectively.…”

Phage escape libraries for checkmate analysis

“…Fortunately, the most widely used phagemid vectors have been constructed in such a fashion that the inserted gene is located 5Ј to the pIII coat protein with an amber suppressor codon between the two proteins ( Fig. 2A) to allow for rapid conversion from a selection vector to an expression vector (3,6). This vector design perfectly fulfills our requirements for a checkmate analysis.…”

“…The methods for generation of combinatorial small-molecule or antibody libraries (3)(4)(5)(6) are well established. Many libraries are currently available or easily constructed.…”