Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity.

Single-chain antibody mutants have been evolved in vitro with antigen-binding equilibrium dissociation constant K(d) = 48 fM and slower dissociation kinetics (half-time > 5 days) than those for the streptavidin-biotin complex. These mutants possess the highest monovalent ligand-binding affinity yet reported for an engineered protein by over two orders of magnitude. Optimal kinetic screening of randomly mutagenized libraries of 10(5)-10(7) yeast surface-displayed antibodies enabled a >1,000-fold decrease in the rate of dissociation after four cycles of affinity mutagenesis and screening. The consensus mutations are generally nonconservative by comparison with naturally occurring mouse Fv sequences and with residues that do not contact the fluorescein antigen in the wild-type complex. The existence of these mutants demonstrates that the antibody Fv architecture is not intrinsically responsible for an antigen-binding affinity ceiling during in vivo affinity maturation.

[1]  D. Kranz,et al.  Thermodynamic properties of ligand binding by monoclonal anti-fluorescyl antibodies. , 1986, Biochemistry.

[2]  D. Leckband,et al.  4-4-20 anti-fluorescyl IgG Fab' recognition of membrane bound hapten: direct evidence for the role of protein and interfacial structure. , 1995, Biochemistry.

[3]  S. Batra,et al.  Pharmacokinetics and biodistribution of genetically engineered antibodies. , 2002, Current opinion in biotechnology.

[4]  G. Georgiou,et al.  In vitro scanning saturation mutagenesis of an antibody binding pocket. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[5]  M. Neuberger,et al.  Affinity dependence of the B cell response to antigen: a threshold, a ceiling, and the importance of off-rate. , 1998, Immunity.

[6]  J. Foote,et al.  Kinetic and affinity limits on antibodies produced during immune responses. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[7]  D. Kranz,et al.  Mechanisms of ligand binding by monoclonal anti-fluorescyl antibodies. , 1982, The Journal of biological chemistry.

[8]  G Schreiber,et al.  Interaction of barnase with its polypeptide inhibitor barstar studied by protein engineering. , 1993, Biochemistry.

[9]  P. Talbot,et al.  Protection from lethal coronavirus infection by immunoglobulin fragments. , 1995, Journal of immunology.

[10]  A. Plückthun,et al.  Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting. , 1997, Protein engineering.

[11]  G. Adams,et al.  Increased affinity leads to improved selective tumor delivery of single-chain Fv antibodies. , 1998, Cancer research.

[12]  J. Herron,et al.  Molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment. 1. Computer simulations. , 1995, Biochemistry.

[13]  K. D. Hardman,et al.  1.85 A structure of anti-fluorescein 4-4-20 Fab. , 1995, Protein engineering.

[14]  M. Hensler,et al.  Stepwise in vitro affinity maturation of Vitaxin, an αvβ3-specific humanized mAb , 1998 .

[15]  K D Wittrup,et al.  Optimal Screening of Surface‐Displayed Polypeptide Libraries , 1998, Biotechnology progress.

[16]  W. Stemmer.  Rapid evolution of a protein in vitro by DNA shuffling , 1994, Nature.

[17]  T. Lybrand,et al.  A structural snapshot of an intermediate on the streptavidin-biotin dissociation pathway. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[18]  G. Winter,et al.  Antibody framework residues affecting the conformation of the hypervariable loops. , 1992, Journal of molecular biology.

[19]  E. Goldman,et al.  Anatomy of an antibody molecule: structure, kinetics, thermodynamics and mutational studies of the antilysozyme antibody D1.3 , 1998, Immunological reviews.

[20]  K. Meek,et al.  Assembly of IgH CDR3: mechanism, regulation, and influence on antibody diversity. , 1992, International reviews of immunology.

[21]  L. Nieba,et al.  Disrupting the hydrophobic patches at the antibody variable/constant domain interface: improved in vivo folding and physical characterization of an engineered scFv fragment. , 1997, Protein engineering.

[22]  U. Piran,et al.  Dissociation rate constant of the biotin-streptavidin complex. , 1990, Journal of immunological methods.

[23]  Allen H. Terry,et al.  High resolution structures of the 4-4-20 Fab-fluorescein complex in two solvent systems: effects of solvent on structure and antigen-binding affinity. , 1994, Biophysical journal.

[24]  Y. Chien,et al.  T cell receptor biochemistry, repertoire selection and general features of TCR and Ig structure. , 1997, Ciba Foundation symposium.

[25]  K D Wittrup,et al.  Isolation of anti-T cell receptor scFv mutants by yeast surface display. , 1997, Protein engineering.

[26]  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.

[27]  E. Kabat,et al.  Identical V region amino acid sequences and segments of sequences in antibodies of different specificities. Relative contributions of VH and VL genes, minigenes, and complementarity-determining regions to binding of antibody-combining sites. , 1991, Journal of immunology.

[28]  M. Arkin,et al.  Probing the importance of second sphere residues in an esterolytic antibody by phage display. , 1998, Journal of molecular biology.

[29]  G. Georgiou,et al.  Antibody affinity maturation using bacterial surface display. , 1998, Protein engineering.

[30]  Eric T. Boder,et al.  Yeast surface display for screening combinatorial polypeptide libraries , 1997, Nature Biotechnology.

[31]  J. Arends,et al.  High-affinity recombinant phage antibodies to the pan-carcinoma marker epithelial glycoprotein-2 for tumour targeting. , 1998, British Journal of Cancer.

[32]  C. Barbas,et al.  Passive immunization with a human monoclonal antibody protects hu-PBL-SCID mice against challenge by primary isolates of HIV-1 , 1997, Nature Medicine.

[33]  E. Voss,et al.  Identification of the binding site of two monoclonal antibodies to human protamine , 1993 .

[34]  D. Allen,et al.  Antigen specificity and tumour targeting efficiency of a human carcinoembryonic antigen-specific scFv and affinity-matured derivatives. , 1998, British Journal of Cancer.

[35]  T. Lehner,et al.  Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans , 1998, Nature Medicine.

[36]  A. Ward.  Single-step purification of shuttle vectors from yeast for high frequency back-transformation into E. coli. , 1990, Nucleic acids research.

[37]  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.

[38]  J. Herron,et al.  Molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment. 2. Time-resolved fluorescence spectroscopy. , 1995, Biochemistry.

[39]  W. Jiskoot,et al.  Role of electrostatic interactions in the binding of fluorescein by anti-fluorescein antibody 4-4-20. , 1993, Biochemistry.

[40]  H. Fritz,et al.  Biochemistry and applications of aprotinin, the kallikrein inhibitor from bovine organs. , 1983, Arzneimittel-Forschung.

[41]  Ronald T. Raines,et al.  Increasing the secretory capacity of Saccharomyces cerevisiae for production of single-chain antibody fragments , 1998, Nature Biotechnology.

[42]  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.

[43]  R C Stevens,et al.  Structural insights into the evolution of an antibody combining site. , 1997, Science.

[44]  M Aguet,et al.  The role of antibody concentration and avidity in antiviral protection. , 1997, Science.

[45]  K. Wittrup,et al.  Fine Affinity Discrimination by Yeast Surface Display and Flow Cytometry , 2000, Biotechnology progress.

[46]  A. Plückthun,et al.  Selection for improved protein stability by phage display. , 1999, Journal of molecular biology.