During the past decade several display methods and other library screening techniques have been developed for isolating monoclonal antibodies (mAbs) from large collections of recombinant antibody fragments. These technologies are now widely exploited to build human antibodies with high affinity and specificity. Clever antibody library designs and selection concepts are now able to identify mAb leads with virtually any specificity. Innovative strategies enable directed evolution of binding sites with ultra-high affinity, high stability and increased potency, sometimes to a level that cannot be achieved by immunization. Automation of the technology is making it possible to identify hundreds of different antibody leads to a single therapeutic target. With the first antibody of this new generation, adalimumab (Humira, a human IgG1 specific for human tumor necrosis factor (TNF)), already approved for therapy and with many more in clinical trials, these recombinant antibody technologies will provide a solid basis for the discovery of antibody-based biopharmaceuticals, diagnostics and research reagents for decades to come.
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Winter, G. & Milstein, C. Man-made antibodies. Nature 349, 293–299 (1991).
Orlandi, R., Gussow, D.H., Jones, P.T. & Winter, G. Cloning immunoglobulin variable domains for expression by the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86, 3833–3837 (1989).
Ward, E.S., Gussow, D., Griffiths, A.D., Jones, P.T. & Winter, G. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341, 544–546 (1989).
Huse, W.D. et al. Generation of a large combinatorial library of the immunoglobulin repertoire in phage lambda. Science 246, 1275–1281 (1989).
McCafferty, J., Griffiths, A.D., Winter, G. & Chiswell, D.J. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552–554 (1990).
Clackson, T., Hoogenboom, H.R., Griffiths, A.D. & Winter, G. Making antibody fragments using phage display libraries. Nature 352, 624–628 (1991).
Marks, J.D. et al. By-passing immunization: human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222, 581–597 (1991).
Burton, D.R. et al. A large array of human monoclonal antibodies to type 1 human immunodeficiency virus from combinatorial libraries of asymptomatic individuals. Proc. Natl. Acad. Sci. USA 88, 10134–10137 (1991).
Winter, G., Griffiths, A.D., Hawkins, R.E. & Hoogenboom, H.R. Making antibody by phage display technology. Annu. Rev. Immunol. 12, 433–455 (1994).
Hoogenboom, H.R. Overview of antibody phage-display technology and its applications. Methods Mol. Biol. 178, 1–37 (2002).
Griffiths, A.D. et al. Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 13, 3245–3260 (1994).
Vaughan, T.J. et al. Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nat. Biotechnol. 14, 309–314 (1996).
de Haard, H.J. et al. A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies. J. Biol. Chem. 274, 18218–18230 (1999).
Knappik, A. et al. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J. Mol. Biol. 296, 57–86 (2000).
Hoet, R.M. et al. Generation of high-affinity human antibodies by combining donor-derived and synthetic complementarity-determining-region diversity. Nat. Biotechnol. 23, 344–348 (2005).
Yang, W.P. et al. CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range. J. Mol. Biol. 254, 392–403 (1995).
Schier, R. 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. J. Mol. Biol. 263, 551–567 (1996).
Lu, D. et al. Tailoring in vitro selection for a picomolar affinity human antibody directed against vascular endothelial growth factor receptor 2 for enhanced neutralizing activity. J. Biol. Chem. 278, 43496–43507 (2003).
Chames, P., Hufton, S.E., Coulie, P.G., Uchanska-Ziegler, B. & Hoogenboom, H.R. Direct selection of a human antibody fragment directed against the tumor T-cell epitope HLA-A1-MAGE-A1 from a nonimmunized phage-Fab library. Proc. Natl. Acad. Sci. USA 97, 7969–7974 (2000).
Huie, M.A. et al. Antibodies to human fetal erythroid cells from a nonimmune phage antibody library. Proc. Natl. Acad. Sci. USA 98, 2682–2687 (2001).
Moulard, M. et al. Broadly cross-reactive HIV-1-neutralizing human monoclonal Fab selected for binding to gp120–CD4-CCR5 complexes. Proc. Natl. Acad. Sci. USA 99, 6913–6918 (2002).
Kramer, R.A. et al. The human antibody repertoire specific for rabies virus glycoprotein as selected from immune libraries. Eur. J. Immunol. 35, 2131–2145 (2005).
Lipovsek, D. & Pluckthun, A. In-vitro protein evolution by ribosome display and mRNA display. J. Immunol. Methods 290, 51–67 (2004).
Hanes, J., Schaffitzel, C., Knappik, A. & Pluckthun, A. Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat. Biotechnol. 18, 1287–1292 (2000).
Schaffitzel, C. et al. In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc. Natl. Acad. Sci. USA 98, 8572–8577 (2001).
Jermutus, L., Honegger, A., Schwesinger, F., Hanes, J. & Pluckthun, A. Tailoring in vitro evolution for protein affinity or stability. Proc. Natl. Acad. Sci. USA 98, 75–80 (2001).
Hanes, J., Jermutus, L., Weber-Bornhauser, S., Bosshard, H.R. & Pluckthun, A. Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries. Proc. Natl. Acad. Sci. USA 95, 14130–14135 (1998).
Zahnd, C. et al. Directed in vitro evolution and crystallographic analysis of a peptide-binding single chain antibody fragment (scFv) with low picomolar affinity. J. Biol. Chem. 279, 18870–18877 (2004).
Boder, E.T. & Wittrup, K.D. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15, 553–557 (1997).
Boder, E.T., Midelfort, K.S. & Wittrup, K.D. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc. Natl. Acad. Sci. USA 97, 10701–10705 (2000).
Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. Development of an optimized expression system for the screening of antibody libraries displayed on the Escherichia coli surface. Protein Eng. 12, 613–621 (1999).
Chen, G. et al. Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS). Nat. Biotechnol. 19, 537–542 (2001).
Harvey, B.R. et al. Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries. Proc. Natl. Acad. Sci. USA 101, 9193–9198 (2004).
Urban, J.H. et al. Selection of functional human antibodies from retroviral display libraries. Nucleic Acids Res. 33, e35 (2005).
Odegrip, R. et al. CIS display: In vitro selection of peptides from libraries of protein-DNA complexes. Proc. Natl. Acad. Sci. USA 101, 2806–2810 (2004).
Reiersen, H. et al. Covalent antibody display–an in vitro antibody-DNA library selection system. Nucleic Acids Res. 33, e10 (2005).
Sepp, A., Tawfik, D.S. & Griffiths, A.D. Microbead display by in vitro compartmentalisation: selection for binding using flow cytometry. FEBS Lett. 532, 455–458 (2002).
Mossner, E., Koch, H. & Pluckthun, A. Fast selection of antibodies without antigen purification: adaptation of the protein fragment complementation assay to select antigen-antibody pairs. J. Mol. Biol. 308, 115–122 (2001).
Urech, D.M., Lichtlen, P. & Barberis, A. Cell growth selection system to detect extracellular and transmembrane protein interactions. Biochim. Biophys. Acta 1622, 117–127 (2003).
Jermutus, L. et al. Ligand binding of a ribosome-displayed protein detected in solution at the single molecule level by fluorescence correlation spectroscopy. Eur. Biophys. J. 31, 179–184 (2002).
Binz, H.K., Amstutz, P. & Pluckthun, A. The matrix reloaded: specific binding proteins based on nonimmunoglobulin domains. Nat. Biotechnol. 23, in the press (2005).
Zauderer, M. & Smith, E.S. In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells. US patent application 20,020,123,057A1 (2002).
Cumbers, S.J. et al. Generation and iterative affinity maturation of antibodies in vitro using hypermutating B-cell lines. Nat. Biotechnol. 20, 1129–1134 (2002).
Weller, S. et al. Hypermutation in human B cells in vivo and in vitro. Ann. NY Acad. Sci. 987, 158–165 (2003).
Nicolaides, N.C., Grasso, L. & Sass, P.M. Methods for generating genetically altered antibody producing cell lines with improved antibody characteristics. US patent 6,808,894 (2004).
Bradbury, A.R. & Marks, J.D. Antibodies from phage antibody libraries. J. Immunol. Methods 290, 29–49 (2004).
Mutuberria, R., Hoogenboom, H.R., van der Linden, E., de Bruïne, A.P. & Roovers, R.C. Model systems to study the parameters determining the success of phage antibody selections on complex antigens. J. Immunol. Methods 231, 65–81 (1999).
VanAntwerp, J.J. & Wittrup, K.D. Fine affinity discrimination by yeast surface display and flow cytometry. Biotechnol. Prog. 16, 31–37 (2000).
Zaccolo, M., Griffiths, A.P., Prospero, T.D., Winter, G. & Gherardi, E. Dimerization of Fab fragments enables ready screening of phage antibodies that affect hepatocyte growth factor/scatter factor activity on target cells. Eur. J. Immunol. 27, 618–623 (1997).
Larocca, D. et al. Evolving phage vectors for cell targeted gene delivery. Curr. Pharm. Biotechnol. 3, 45–57 (2002).
Janda, K.D. et al. Direct selection for a catalytic mechanism from combinatorial antibody libraries. Proc. Natl. Acad. Sci. USA 91, 2532–2536 (1994).
Becerril, B., Poul, M.A. & Marks, J.D. Toward selection of internalizing antibodies from phage libraries. Biochem. Biophys. Res. Commun. 255, 386–393 (1999).
Feldhaus, M.J. & Siegel, R.W. Yeast display of antibody fragments: a discovery and characterization platform. J. Immunol. Methods 290, 69–80 (2004).
Bradbury, A. et al. Antibodies in proteomics II: screening, high-throughput characterization and downstream applications. Trends Biotechnol. 21, 312–317 (2003).
Chambers, R.S. High-throughput antibody production. Curr. Opin. Chem. Biol. 9, 46–50 (2005).
Lou, J. et al. Antibodies in haystacks: how selection strategy influences the outcome of selection from molecular diversity libraries. J. Immunol. Methods 253, 233–242 (2001).
Liu, B., Huang, L., Sihlbom, C., Burlingame, A. & Marks, J.D. Towards proteome-wide production of monoclonal antibody by phage display. J. Mol. Biol. 315, 1063–1073 (2002).
Walter, G., Konthur, Z. & Lehrach, H. High-throughput screening of surface displayed gene products. Comb. Chem. High Throughput Screen. 4, 193–205 (2001).
Pavlik, P. et al. Predicting antigenic peptides suitable for the selection of phage antibodies. Hum. Antibodies 12, 99–112 (2003).
Van Beijnum, J.R. et al. Target validation for genomics using peptide-specific phage antibodies: a study of five gene products overexpressed in colorectal cancer. Int. J. Cancer 101, 118–127 (2002).
Frisch, C. et al. From EST to IHC: human antibody pipeline for target research. J. Immunol. Methods 275, 203–212 (2003).
Hoet, R. et al. Method and apparatus for washing magnetically responsive particles. US patent application 20,030,170,686A1 (2003).
Edwards, B.M. et al. The remarkable flexibility of the human antibody repertoire; isolation of over one thousand different antibodies to a single protein, BLyS. J. Mol. Biol. 334, 103–118 (2003).
Hallborn, J. & Carlsson, R. Automated screening procedure for high-throughput generation of antibody fragments. Biotechniques Suppl., 30–37 (2002).
Angenendt, P. et al. Seeing better through a MIST: evaluation of monoclonal recombinant antibody fragments on microarrays. Anal. Chem. 76, 2916–2921 (2004).
Vanhercke, T., Ampe, C., Tirry, L. & Denolf, P. Rescue and in situ selection and evaluation (RISE): a method for high-throughput panning of phage display libraries. J. Biomol. Screen. 10, 108–117 (2005).
Rungpragayphan, S. et al. High-throughput, cloning-independent protein library construction by combining single-molecule DNA amplification with in vitro expression. J. Mol. Biol. 318, 395–405 (2002).
Holt, L.J., Bussow, K., Walter, G. & Tomlinson, I.M. By-passing selection: direct screening for antibody-antigen interactions using protein arrays. Nucleic Acids Res. 28, e72 (2000).
Watkins, J.D. et al. Discovery of human antibodies to cell surface antigens by capture lift screening of phage-expressed antibody libraries. Anal. Biochem. 256, 169–177 (1998).
Pini, A., Ricci, C. & Bracci, L. Phage display and colony filter screening for high-throughput selection of antibody libraries. Comb. Chem. High Throughput Screen. 5, 503–510 (2002).
Michaud, G.A. et al. Analyzing antibody specificity with whole proteome microarrays. Nat. Biotechnol. 21, 1509–1512 (2003).
Poetz, O. et al. Protein microarrays for antibody profiling: specificity and affinity determination on a chip. Proteomics 5, 2402–2411 (2005).
Jostock, T. et al. Rapid generation of functional human IgG antibodies derived from Fab-on-phage display libraries. J. Immunol. Methods 289, 65–80 and corrigendum in 294, 209 (2004).
Sarantopoulos, S., Kao, C.Y., Den, W. & Sharon, J. A method for linking VL and VH region genes that allows bulk transfer between vectors for use in generating polyclonal IgG libraries. J. Immunol. 152, 5344–5351 (1994).
Persic, L. et al. An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene 187, 9–18 (1997).
Schoonbroodt, S. et al. Oligonucleotide-assisted cleavage and ligation: a novel directional DNA cloning technology to capture cDNAs. Application in the construction of a human immune antibody phage-display library. Nucleic Acids Res. 33, e81 (2005).
Marzari, R. et al. Molecular dissection of the tissue transglutaminase autoantibody response in celiac disease. J. Immunol. 166, 4170–4176 (2001).
Roovers, R.C. et al. Evidence for a bias toward intracellular antigens in the local humoral anti-tumor immune response of a colorectal cancer patient revealed by phage display. Int. J. Cancer 93, 832–840 (2001).
Haurum, J. & Bregenholt, S. Recombinant polyclonal antibodies: therapeutic antibody technologies come full circle. IDrugs 8, 404–409 (2005).
Hoogenboom, H.R. & Winter, G. By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro. J. Mol. Biol. 227, 381–388 (1992).
Barbas, C.F., Bain, J.D., Hoekstra, D.M. & Lerner, R. Semisynthetic combinatorial libraries: a chemical solution to the diversity problem. Proc. Natl. Acad. Sci. USA 89, 4457–4461 (1992).
Sidhu, S.S. et al. Phage-displayed antibody libraries of synthetic heavy chain complementarity determining regions. J. Mol. Biol. 338, 299–310 (2004).
Silacci, M. et al. Design, construction, and characterization of a large synthetic human antibody phage display library. Proteomics 5, 2340–2350 (2005).
de Wildt, R.M., Mundy, C.R., Gorick, B.D. & Tomlinson, I.M. Antibody arrays for high-throughput screening of antibody-antigen interactions. Nat. Biotechnol. 18, 989–994 (2000).
Loset, G.A. et al. Construction, evaluation and refinement of a large human antibody phage library based on the IgD and IgM variable gene repertoire. J. Immunol. Methods 299, 47–62 (2005).
Soderlind, E. et al. Recombining germline-derived CDR sequences for creating diverse single-framework antibody libraries. Nat. Biotechnol. 18, 852–856 (2000).
Hust, M. & Dubel, S. Mating antibody phage display with proteomics. Trends Biotechnol. 22, 8–14 (2004).
Hoogenboom, H.R. & Chames, P. Natural and designer binding sites made by phage display technology. Immunol. Today 21, 371–378 (2000).
Xu, J.L. & Davis, M.M. Diversity in the CDR3 region of V(H) is sufficient for most antibody specificities. Immunity 13, 37–45 (2000).
Senn, B.M. et al. Combinatorial immunoglobulin light chain variability creates sufficient B cell diversity to mount protective antibody responses against pathogen infections. Eur. J. Immunol. 33, 950–961 (2003).
Lee, M.S. et al. Selection of scFvs specific for HBV DNA polymerase using ribosome display. J. Immunol. Methods 284, 147–157 (2004).
He, M. et al. Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display. J. Immunol. Methods 231, 105–117 (1999).
Bakker, A. et al. Novel human antibody combination effectively neutralizing natural rabies virus variants and individual in vitro escape mutants. J. Virol. 79, 9062–9068 (2005).
Wu, H. et al. Ultra-potent antibodies against Respiratory Syncytial Virus: effects of binding kinetics and binding valency on viral neutralization. J. Mol. Biol. 350, 126–144 (2005).
Weiner, L.M. & Carter, P. Tunable antibodies. Nat. Biotechnol. 23, 556–557 (2005).
Wu, H. et al. Stepwise in vitro affinity maturation of Vitaxin, an alphav beta3-specific humanized mAb. Proc. Natl. Acad. Sci. USA 95, 6037–6042 (1998).
Ho, M., Kreitman, R.J., Onda, M. & Pastan, I. In vitro antibody evolution targeting germline hot spots to increase activity of an anti-CD22 immunotoxin. J. Biol. Chem. 280, 607–617 (2005).
Foote, J. & Eisen, H.N. Kinetic and affinity limits on antibodies produced during immune responses. Proc. Natl. Acad. Sci. USA 92, 1254–1256 (1995).
Pini, A. et al. Design and use of a phage display library. Human antibodies with subnanomolar affinity against a marker of angiogenesis eluted from a two-dimensional gel. J. Biol. Chem. 273, 21769–21776 (1998).
Rauchenberger, R. et al. Human combinatorial Fab library yielding specific and functional antibodies against the human fibroblast growth factor receptor 3. J. Biol. Chem. 278, 38194–38205 (2003).
Midelfort, K.S. et al. Substantial energetic improvement with minimal structural perturbation in a high affinity mutant antibody. J. Mol. Biol. 343, 685–701 (2004).
Baca, M. & Presta, L.G. SJ, O'Connor, S.J. & Wells, J.A. Antibody humanization using monovalent phage display. J. Biol. Chem. 272, 10678–10684 (1997).
Wu, H., Nie, Y., Huse, W.D. & Watkins, J.D. Humanization of a murine monoclonal antibody by simultaneous optimization of framework and CDR residues. J. Mol. Biol. 294, 151–162 (1999).
Dall'acqua, W.F. et al. Antibody humanization by framework shuffling. Methods 36, 43–60 (2005).
Beiboer, S.H. et al. Guided selection of a pan carcinoma specific antibody reveals similar binding characteristics yet structural divergence between the original murine antibody and its human equivalent. J. Mol. Biol. 296, 833–849 (2000).
Salfeld, J. et al. Human antibodies that bind human TNFα. US patent 6,090,382 (2000).
Naundorf, S. et al. In vitro and in vivo activity of MT201, a fully human monoclonal antibody for pancarcinoma treatment. Int. J. Cancer 100, 101–110 (2002).
Jespers, L.S., Roberts, A., Mahler, S.M., Winter, G. & Hoogenboom, H.R. Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen. Bio/Technology 12, 899–903 (1994).
Rader, C., Cheresh, D.A. & Barbas, C.F., III. A phage display approach for rapid antibody humanization: designed combinatorial V gene libraries. Proc. Natl. Acad. Sci. USA 95, 8910–8915 (1998).
Jung, S., Honegger, A. & Pluckthun, A. Selection for improved protein stability by phage display. J. Mol. Biol. 294, 163–180 (1999).
Kristensen, P. & Winter, G. Proteolytic selection for protein folding using filamentous bacteriophages. Fold. Des. 3, 321–328 (1998).
Brockmann, E.C., Cooper, M., Stromsten, N., Vehniainen, M. & Saviranta, P. Selecting for antibody scFv fragments with improved stability using phage display with denaturation under reducing conditions. J. Immunol. Methods 296, 159–170 (2005).
Jespers, L., Schon, O., Famm, K. & Winter, G. Aggregation-resistant domain antibodies selected on phage by heat denaturation. Nat. Biotechnol. 22, 1161–1165 (2004).
Shusta, E.V., Raines, R.T., Pluckthun, A. & Wittrup, K.D. Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments. Nat. Biotechnol. 16, 773–777 (1998).
Shusta, E.V., Holler, P.D., Kieke, M.C., Kranz, D.M. & Wittrup, K.D. Directed evolution of a stable scaffold for T-cell receptor engineering. Nat. Biotechnol. 18, 754–759 (2000).
Graff, C.P., Chester, K., Begent, R. & Wittrup, K.D. Directed evolution of an anti-carcinoembryonic antigen scFv with a 4-day monovalent dissociation half-time at 37 degrees C. Protein Eng. Des. Sel. 17, 293–304 (2004).
Roodveldt, C., Aharoni, A. & Tawfik, D.S. Directed evolution of proteins for heterologous expression and stability. Curr. Opin. Struct. Biol. 15, 50–56 (2005).
Topping, K.P., Hough, V.C., Monson, J.R. & Greenman, J. Isolation of human colorectal tumour reactive antibodies using phage display technology. Int. J. Oncol. 16, 187–195 (2000).
Roovers, R.C., van der Linden, E., de Bruïne, A.P., Arends, J.W. & Hoogenboom, H.R. Identification of colon tumour-associated antigens by phage antibody selections on primary colorectal carcinoma. Eur. J. Cancer 37, 542–549 (2001).
Tur, M.K. et al. A novel approach for immunization, screening and characterization of selected scFv libraries using membrane fractions of tumor cells. Int. J. Mol. Med. 11, 523–527 (2003).
Mutuberria, R. et al. Isolation of human antibodies to tumor-associated endothelial cell markers by in vitro human endothelial cell selection with phage display libraries. J. Immunol. Methods 287, 31–47 (2004).
Liu, B., Conrad, F., Cooperberg, M.R., Kirpotin, D.B. & Marks, J.D. Mapping tumor epitope space by direct selection of single-chain Fv antibody libraries on prostate cancer cells. Cancer Res. 64, 704–710 (2004).
Nizak, C. et al. Recombinant antibodies against subcellular fractions used to track endogenous Golgi protein dynamics in vivo. Traffic 4, 739–753 (2003).
Ridgway, J.B. et al. Identification of a human anti-CD55 single-chain Fv by subtractive panning of a phage library using tumor and nontumor cell lines. Cancer Res. 59, 2718–2723 (1999).
Geuijen, C.A. et al. A proteomic approach to tumour target identification using phage display, affinity purification and mass spectrometry. Eur. J. Cancer 41, 178–187 (2005).
Bakker, A.B. et al. C-type lectin-like molecule-1: a novel myeloid cell surface marker associated with acute myeloid leukemia. Cancer Res. 64, 8443–8450 (2004).
Visintin, M., Meli, G.A., Cannistraci, I. & Cattaneo, A. Intracellular antibodies for proteomics. J. Immunol. Methods 290, 135–153 (2004).
Martineau, P., Jones, P. & Winter, G. Expression of an antibody fragment at high levels in the bacterial cytoplasm. J. Mol. Biol. 280, 117–127 (1998).
Gargano, N. & Cattaneo, A. Rescue of a neutralizing anti-viral antibody fragment from an intracellular polyclonal repertoire expressed in mammalian cells. FEBS Lett. 414, 537–540 (1997).
Gennari, F. et al. Direct phage to intrabody screening (DPIS): demonstration by isolation of cytosolic intrabodies against the TES1 site of Epstein Barr virus latent membrane protein 1 (LMP1) that block NF-kappaB transactivation. J. Mol. Biol. 335, 193–207 (2004).
Visintin, M., Tse, E., Axelson, H., Rabbitts, T.H. & Cattaneo, A. Selection of antibodies for intracellular function using a two-hybrid in vivo system. Proc. Natl. Acad. Sci. USA 96, 11723–11728 (1999).
auf der Maur, A. et al. Direct in vivo screening of intrabody libraries constructed on a highly stable single-chain framework. J. Biol. Chem. 277, 45075–45085 (2002).
Tanaka, T., Lobato, M.N. & Rabbitts, T.H. Single domain intracellular antibodies: a minimal fragment for direct in vivo selection of antigen-specific intrabodies. J. Mol. Biol. 331, 1109–1120 (2003).
Amstutz, P. et al. Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins. J. Biol. Chem. 280, 24715–24722 (2005).
Pendley, C., Schantz, A. & Wagner, C. Immunogenicity of therapeutic monoclonal antibodies. Curr. Opin. Mol. Ther. 5, 172–179 (2003).
Zemlin, M. et al. Expressed murine and human CDR-H3 intervals of equal length exhibit distinct repertoires that differ in their amino acid composition and predicted range of structures. J. Mol. Biol. 334, 733–749 (2003).
Harris, R.J., Shire, S.J. & Winter, C. Commercial manufacturing scale formulation and analytical characterization of therapeutic recombinant antibodies. Drug Dev. Res. 61, 137–154 (2004).
Fellouse, F.A., Wiesmann, C. & Sidhu, S.S. Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition. Proc. Natl. Acad. Sci. USA 101, 12467–12472 (2004).
Fellouse, F.A. et al. Molecular recognition by a binary code. J. Mol. Biol. 348, 1153–1162 (2005).
Rao, B.M., Lauffenburger, D.A. & Wittrup, K.D. Integrating cell-level kinetic modeling into the design of engineered protein therapeutics. Nat. Biotechnol. 23, 191–194 (2005).
De Genst, E., Areskoug, D., Decanniere, K., Muyldermans, S. & Andersson, K. Kinetic and affinity predictions of a protein-protein interaction using multivariate experimental design. J. Biol. Chem. 277, 29897–29907 (2002).
Bond, C.J., Wiesmann, C., Marsters, J.C. Jr. & Sidhu, S.S. A structure-based database of antibody variable domain diversity. J. Mol. Biol. 348, 699–709 (2005).
Almagro, J.C. Identification of differences in the specificity-determining residues of antibodies that recognize antigens of different size: implications for the rational design of antibody repertoires. J. Mol. Recognit. 17, 132–143 (2004).
Kirkham, P.M., Neri, D. & Winter, G. Towards the design of an antibody that recognises a given protein epitope. J. Mol. Biol. 285, 909–915 (1999).
Calarese, D.A. et al. Antibody domain exchange is an immunological solution to carbohydrate cluster recognition. Science 300, 2065–2071 (2003).
Darbha, R. et al. Crystal structure of the broadly cross-reactive HIV-1-neutralizing Fab X5 and fine mapping of its epitope. Biochemistry 43, 1410–1417 (2004).
Jespers, L., Bonnert, T.P. & Winter, G. Selection of optical biosensors from chemisynthetic antibody libraries. Protein Eng. Des. Sel. 17, 709–713 (2004).
Ehrlich, P. On autoimmunity with special references to cell life. Proc. R. Soc. 66, 424–448 (1900).
Salvatore, G., Beers, R., Margulies, I., Kreitman, R.J. & Pastan, I. Improved cytotoxic activity toward cell lines and fresh leukemia cells of a mutant anti-CD22 immunotoxin obtained by antibody phage display. Clin. Cancer Res. 8, 995–1002 (2002).
Chames, P. et al. TCR-like human antibodies expressed on human CTLs mediate antibody affinity-dependent cytolytic activity. J. Immunol. 169, 1110–1118 (2002).
Chen, Y. et al. Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen. J. Mol. Biol. 293, 865–881 (1999).
Colby, D.W. et al. Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody. Proc. Natl. Acad. Sci. USA 101, 17616–17621 (2004).
Desiderio, A. et al. A semi-synthetic repertoire of intrinsically stable antibody fragments derived from a single-framework scaffold. J. Mol. Biol. 310, 603–615 (2001).
Azriel-Rosenfeld, R., Valensi, M. & Benhar, I. A human synthetic combinatorial library of arrayable single-chain antibodies based on shuffling in vivo formed CDRs into general framework regions. J. Mol. Biol. 335, 177–192 (2004).
Yau, K.Y. et al. Affinity maturation of a V(H)H by mutational hotspot randomization. J. Immunol. Methods 297, 213–224 (2005).
De Pascalis, R. et al. In vitro affinity maturation of a specificity-determining region-grafted humanized anticarcinoma antibody: isolation and characterization of minimally immunogenic high-affinity variants. Clin. Cancer Res. 9, 5521–5531 (2003).
Maynard, J.A. et al. Protection against anthrax toxin by recombinant antibody fragments correlates with antigen affinity. Nat. Biotechnol. 20, 597–601 (2002).
Hoogenboom, H.R.J.M. & Somers, V. Hybridization control of sequence variation. US patent application 20,040,005,709A1 (2004).
Sblattero, D. & Bradbury, A. Exploiting recombination in single bacteria to make large phage antibody libraries. Nat. Biotechnol. 18, 75–80 (2000).
Lutz, R. & Bujard, H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1–I2 regulatory elements. Nucleic Acids Res. 25, 1203–1210 (1997).
Hoet, R., Ladner, R.C. & Frans, N. Methods and compositions for controlling valency of phage display. US patent application 20,040,180,422A1 (2004).
McGuinness, B.T. et al. Phage diabody repertoires for selection of large numbers of bispecific antibody fragments. Nat. Biotechnol. 14, 1149–1154 (1996).
Lee, C.V., Sidhu, S.S. & Fuh, G. Bivalent antibody phage display mimics natural immunoglobulin. J. Immunol. Methods 284, 119–132 (2004).
Poul, M.A. & Marks, J.D. Targeted gene delivery to mammalian cells by filamentous bacteriophage. J. Mol. Biol. 288, 203–211 (1999).
O'Connell, D., Becerril, B., Roy-Burman, A., Daws, M. & Marks, J.D. Phage versus phagemid libraries for generation of human monoclonal antibodies. J. Mol. Biol. 321, 49–56 (2002).
de Wildt, R.M., Tomlinson, I.M., Ong, J.L. & Holliger, P. Isolation of receptor-ligand pairs by capture of long-lived multivalent interaction complexes. Proc. Natl. Acad. Sci. USA 99, 8530–8535 (2002).
Mattheakis, L.C., Bhatt, R.R. & Dower, W.J. An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc. Natl. Acad. Sci. USA 91, 9022–9026 (1994).
He, M. & Taussig, M.J. Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites. Nucleic Acids Res. 25, 5132–5134 (1997).
Hanes, J. & Pluckthun, A. In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. USA 94, 4937–4942 (1997).
Wilson, D.S., Keefe, A.D. & Szostak, J.W. The use of mRNA display to select high-affinity protein-binding peptides. Proc. Natl. Acad. Sci. USA 98, 3750–3755 (2001).
Xu, L. et al. Directed evolution of high-affinity antibody mimics using mRNA display. Chem. Biol. 9, 933–942 (2002).
Feldhaus, M.J. et al. Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat. Biotechnol. 21, 163–170 (2003).
Yeung, Y.A. & Wittrup, K.D. Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture. Biotechnol. Prog. 18, 212–220 (2002).
van den Beucken, T. et al. Affinity maturation of Fab antibody fragments by fluorescent-activated cell sorting of yeast-displayed libraries. FEBS Lett. 546, 288–294 (2003).
Hufton, S.E. & Hoogenboom, H.R.J.M. Multi-chain eukaryotic display vectors and uses thereof. US patent application 20,030,186,374A1 (2003).
Blaise, L. et al. Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to the affinity maturation of Fab antibody fragments. Gene 342, 211–218 (2004).
Swers, J.S., Kellogg, B.A. & Wittrup, K.D. Shuffled antibody libraries created by in vivo homologous recombination and yeast surface display. Nucleic Acids Res. 32, e36 (2004).
I thank many colleagues including Jane Osbourn and Lutz Jermutus, Clive Wood, Zhenping Zhu, Patrick Bauerle, Herren Wu, David Chen and Lex Bakker for sharing unpublished results and am grateful to Mark Alfenito for reviewing the manuscript.
The author declares no competing financial interests.
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Hoogenboom, H. Selecting and screening recombinant antibody libraries. Nat Biotechnol 23, 1105–1116 (2005). https://doi.org/10.1038/nbt1126
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