Selection of full‐length IgGs by tandem display on filamentous phage particles and Escherichia coli fluorescence‐activated cell sorting screening

Phage display of antibody libraries is a powerful tool for antibody discovery and evolution. Recombinant antibodies have been displayed on phage particles as scFvs or Fabs, and more recently as bivalent F(ab′)2. We recently developed a technology (E‐clonal) for screening of combinatorial IgG libraries using bacterial periplasmic display and selection by fluorescence‐activated cell sorting (FACS) [Mazor Y et al. (2007) Nat Biotechnol25, 563–565]. Although, as a single‐cell analysis technique, FACS is very powerful, especially for the isolation of high‐affinity binders, even with state of the art instrumentation the screening of libraries with diversity > 108 is technically challenging. We report here a system that takes advantage of display of full‐length IgGs on filamentous phage particles as a prescreening step to reduce library size and enable subsequent rounds of FACS screening in Escherichia coli. For the establishment of an IgG phage display system, we utilized phagemid‐encoded IgG with the fUSE5–ZZ phage as a helper phage. These phage particles display the Fc‐binding ZZ protein on all copies of the phage p3 coat protein, and are exploited as both helper phages and anchoring surfaces for the soluble IgG. We demonstrate that tandem phage selection followed by FACS allows the selection of a highly diversified profile of binders from antibody libraries without undersampling, and at the same time capitalizes on the advantages of FACS for real‐time monitoring and optimization of the screening process.

[1]  G. Georgiou,et al.  E-clonal antibodies: selection of full-length IgG antibodies using bacterial periplasmic display , 2008, Nature Protocols.

[2]  R. Scheller,et al.  Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs. , 2008, Journal of immunological methods.

[3]  A. Honegger,et al.  The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies. , 2008, Journal of molecular biology.

[4]  D. Kirchhofer,et al.  Structural insight into distinct mechanisms of protease inhibition by antibodies , 2007, Proceedings of the National Academy of Sciences.

[5]  Frederic A. Fellouse,et al.  High-throughput generation of synthetic antibodies from highly functional minimalist phage-displayed libraries. , 2007, Journal of molecular biology.

[6]  P. Daugherty Protein engineering with bacterial display. , 2007, Current opinion in structural biology.

[7]  G. Georgiou,et al.  A scFv antibody mutant isolated in a genetic screen for improved export via the twin arginine transporter pathway exhibits faster folding. , 2007, Journal of molecular biology.

[8]  George Georgiou,et al.  Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli , 2007, Nature Biotechnology.

[9]  M. Tessier-Lavigne,et al.  Function blocking antibodies to neuropilin-1 generated from a designed human synthetic antibody phage library. , 2007, Journal of molecular biology.

[10]  A. Takaoka,et al.  Comparing antibody and small-molecule therapies for cancer , 2006, Nature Reviews Cancer.

[11]  K Dane Wittrup,et al.  Isolating and engineering human antibodies using yeast surface display , 2006, Nature Protocols.

[12]  Gary Walsh,et al.  Biopharmaceutical benchmarks 2006 , 2006, Nature Biotechnology.

[13]  Iftach Yacoby,et al.  Targeting Antibacterial Agents by Using Drug-Carrying Filamentous Bacteriophages , 2006, Antimicrobial Agents and Chemotherapy.

[14]  H. Hoogenboom,et al.  Selecting and screening recombinant antibody libraries , 2005, Nature Biotechnology.

[15]  Janice M Reichert,et al.  Monoclonal antibody successes in the clinic , 2005, Nature Biotechnology.

[16]  C. Rupprecht,et al.  The human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries , 2005, European journal of immunology.

[17]  J. Osbourn,et al.  From rodent reagents to human therapeutics using antibody guided selection. , 2005, Methods.

[18]  E. H. Cohen,et al.  Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity , 2005, Nature Biotechnology.

[19]  A. Bradbury,et al.  Antibodies from phage antibody libraries. , 2004, Journal of immunological methods.

[20]  Frederic A. Fellouse,et al.  Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions. , 2004, Journal of molecular biology.

[21]  Peter Bohlen,et al.  Tailoring in Vitro Selection for a Picomolar Affinity Human Antibody Directed against Vascular Endothelial Growth Factor Receptor 2 for Enhanced Neutralizing Activity* , 2003, Journal of Biological Chemistry.

[22]  Rivka Adar,et al.  Human Combinatorial Fab Library Yielding Specific and Functional Antibodies against the Human Fibroblast Growth Factor Receptor 3* , 2003, Journal of Biological Chemistry.

[23]  George Georgiou,et al.  Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis. , 2003, Journal of immunological methods.

[24]  P. Hudson,et al.  Recombinant antibodies for cancer diagnosis and therapy , 2003, Expert opinion on biological therapy.

[25]  J. Marks,et al.  Phage versus phagemid libraries for generation of human monoclonal antibodies. , 2002, Journal of molecular biology.

[26]  R. Jefferis,et al.  Interaction sites on human IgG-Fc for FcγR: current models , 2002 .

[27]  A. Plückthun,et al.  Stability engineering of antibody single-chain Fv fragments. , 2001, Journal of molecular biology.

[28]  A. Pini,et al.  Phage display of antibody fragments. , 2000, Current protein & peptide science.

[29]  A. Plückthun,et al.  Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. , 2000, Journal of molecular biology.

[30]  A. D. de Vos,et al.  Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen. , 1999, Journal of molecular biology.

[31]  Hennie R. Hoogenboom,et al.  A Large Non-immunized Human Fab Fragment Phage Library That Permits Rapid Isolation and Kinetic Analysis of High Affinity Antibodies* , 1999, The Journal of Biological Chemistry.

[32]  J. Marks,et al.  Toward selection of internalizing antibodies from phage libraries. , 1999, Biochemical and biophysical research communications.

[33]  J. Gerhart,et al.  Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[34]  L. Presta,et al.  Antibody Humanization Using Monovalent Phage Display* , 1997, The Journal of Biological Chemistry.

[35]  R. Webster,et al.  Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero-residue linker a trimer. , 1997, Protein engineering.

[36]  G. Adams,et al.  Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site. , 1996, Journal of molecular biology.

[37]  Tristan J. Vaughan,et al.  Human Antibodies with Sub-nanomolar Affinities Isolated from a Large Non-immunized Phage Display Library , 1996, Nature Biotechnology.

[38]  J S Tung,et al.  Affinity maturation of a high-affinity human monoclonal antibody against the third hypervariable loop of human immunodeficiency virus: use of phage display to improve affinity and broaden strain reactivity. , 1996, Journal of molecular biology.

[39]  J. Bye,et al.  Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection. , 1996, Journal of molecular biology.

[40]  D R Burton,et al.  CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range. , 1995, Journal of molecular biology.

[41]  T. Logtenberg,et al.  Selection and application of human single chain Fv antibody fragments from a semi-synthetic phage antibody display library with designed CDR3 regions. , 1995, Journal of molecular biology.

[42]  Hennie R. Hoogenboom,et al.  Guiding the Selection of Human Antibodies from Phage Display Repertoires to a Single Epitope of an Antigen , 1994, Bio/Technology.

[43]  P. T. Jones,et al.  Isolation of high affinity human antibodies directly from large synthetic repertoires. , 1994, The EMBO journal.

[44]  I. Tomlinson,et al.  Antibody fragments from a ‘single pot’ phage display library as immunochemical reagents. , 1994, The EMBO journal.

[45]  T Prospero,et al.  "Diabodies": small bivalent and bispecific antibody fragments. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[46]  E. Voss,et al.  Molecular stabilization effects of interactions between anti-metatype antibodies and liganded antibody. , 1992, The Journal of biological chemistry.

[47]  T. Yokota,et al.  Construction, binding properties, metabolism, and tumor targeting of a single-chain Fv derived from the pancarcinoma monoclonal antibody CC49. , 1991, Cancer research.

[48]  H R Hoogenboom,et al.  By-passing immunization. Human antibodies from V-gene libraries displayed on phage. , 1991, Journal of molecular biology.

[49]  C. Barbas,et al.  Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[50]  T. Clackson,et al.  Making antibody fragments using phage display libraries , 1991, Nature.

[51]  H R Hoogenboom,et al.  Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. , 1991, Nucleic acids research.

[52]  G. Winter,et al.  Phage antibodies: filamentous phage displaying antibody variable domains , 1990, Nature.

[53]  R. R. Robinson,et al.  Secretion of functional antibody and Fab fragment from yeast cells. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[54]  A. Plückthun,et al.  Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. , 1988, Science.

[55]  R. R. Robinson,et al.  Escherichia coli secretion of an active chimeric antibody fragment. , 1988, Science.

[56]  M. Uhlén,et al.  A synthetic IgG-binding domain based on staphylococcal protein A. , 1987, Protein engineering.

[57]  C. Broder,et al.  UvA-DARE ( Digital Academic Repository ) Neutralizing antibodies to the HIV-1 envelope glycoproteins , 2009 .

[58]  S. Sidhu,et al.  Bivalent antibody phage display mimics natural immunoglobulin. , 2004, Journal of immunological methods.

[59]  H. Hoogenboom,et al.  Selection of antibodies against biotinylated antigens. , 2002, Methods in molecular biology.

[60]  R. Jefferis,et al.  Interaction sites on human IgG-Fc for FcgammaR: current models. , 2002, Immunology letters.

[61]  G. Georgiou Analysis of large libraries of protein mutants using flow cytometry. , 2000, Advances in protein chemistry.

[62]  Gary Walsh,et al.  Biopharmaceutical benchmarks , 2000, Nature Biotechnology.

[63]  G. P. Smith,et al.  Libraries of peptides and proteins displayed on filamentous phage. , 1993, Methods in enzymology.