Selection by phage display of llama conventional V(H) fragments with heavy chain antibody V(H)H properties.

A llama single domain antibody (dAb) library designed and constructed to contain only heavy chain antibody variable domains (V(H)Hs) also contained a substantial number of typical conventional antibody heavy chain variable sequences (V(H)s). Panning the library against two carbohydrate-specific antibodies yielded anti-idiotypic dAbs and enriched solely for sequences from the V(H) subpopulation of the library. The conventional antibody origin of these V(H)s was confirmed by using oligonucleotide probes, specific for the enriched V(H)s, to identify the parental sequences in the message employed in library construction. Surprisingly, these V(H) dAbs, which are produced in high yield in Escherichia coli, are highly soluble, have excellent temperature stability profiles and do not display any aggregation tendencies. The very close similarity of these molecules to human V(H)s makes them potentially very useful as therapeutic dAbs.

[1]  L. Wyns,et al.  Selection and identification of single domain antibody fragments from camel heavy‐chain antibodies , 1997, FEBS letters.

[2]  A. Lesk,et al.  Elbow motion in the immunoglobulins involves a molecular ball-and-socket joint , 1988, Nature.

[3]  R. Karlsson,et al.  Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology. , 1991, BioTechniques.

[4]  L. Wyns,et al.  Camel heavy‐chain antibodies: diverse germline VHH and specific mechanisms enlarge the antigen‐binding repertoire , 2000, The EMBO journal.

[5]  L. Wyns,et al.  A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops. , 1999, Structure.

[6]  P. Kirkham,et al.  Immunoglobulin VH clan and family identity predicts variable domain structure and may influence antigen binding. , 1992, The EMBO journal.

[7]  P. T. Jones,et al.  Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli , 1989, Nature.

[8]  Lode Wyns,et al.  Potent enzyme inhibitors derived from dromedary heavy‐chain antibodies , 1998, The EMBO journal.

[9]  L. Riechmann,et al.  ‘Camelising’ human antibody fragments: NMR studies on VH domains , 1994, FEBS letters.

[10]  R. MacKenzie,et al.  The role of valency in the selection of anti-carbohydrate single-chain Fvs from phage display libraries. , 1998, Journal of immunological methods.

[11]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[12]  S. Muyldermans,et al.  Unique single‐domain antigen binding fragments derived from naturally occurring camel heavy‐chain antibodies , 1999, Journal of molecular recognition : JMR.

[13]  K. Sharp,et al.  Linkage of thioredoxin stability to titration of ionizable groups with perturbed pKa. , 1991, Biochemistry.

[14]  D. Rose,et al.  Exploring the mimicry of polysaccharide antigens by anti-idiotypic antibodies. The crystallization, molecular replacement, and refinement to 2.8 A resolution of an idiotope-anti-idiotope Fab complex and of the unliganded anti-idiotope Fab. , 1994, Journal of molecular biology.

[15]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[16]  C. MacKenzie,et al.  Optimal Design Features of Camelized Human Single-domain Antibody Libraries* , 2001, The Journal of Biological Chemistry.

[17]  E. Padlan,et al.  Anatomy of the antibody molecule. , 1994, Molecular immunology.

[18]  B. de Geus,et al.  Llama heavy-chain V regions consist of at least four distinct subfamilies revealing novel sequence features. , 2000, Molecular immunology.

[19]  S. Muyldermans,et al.  Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. , 1994, Protein engineering.

[20]  R. Aitken,et al.  Bovine IgG repertoire is dominated by a single diversified VH gene family. , 1997, Journal of immunology.

[21]  A. Lesk,et al.  Canonical structures for the hypervariable regions of immunoglobulins. , 1987, Journal of molecular biology.

[22]  A. Plückthun,et al.  Engineered turns of a recombinant antibody improve its in vivo folding. , 1995, Protein engineering.

[23]  L. Wyns,et al.  Comparison of llama VH sequences from conventional and heavy chain antibodies. , 1997, Molecular immunology.

[24]  G. Winter,et al.  Screening of phage antibody libraries. , 1996, Methods in enzymology.

[25]  Dominique Bourgeois,et al.  The crystal structure of a llama heavy chain variable domain , 1996, Nature Structural Biology.

[26]  C. MacKenzie,et al.  Molecular basis for the lack of mimicry of Brucella polysaccharide antigens by Ab2gamma antibodies. , 1999, Molecular immunology.

[27]  C Cambillau,et al.  Thermal unfolding of a llama antibody fragment: a two-state reversible process. , 2001, Biochemistry.

[28]  R. Cortese,et al.  Affinity selection of a camelized V(H) domain antibody inhibitor of hepatitis C virus NS3 protease. , 1997, Protein engineering.

[29]  L. Riechmann,et al.  Antibody VH Domains as Small Recognition Units , 1995, Bio/Technology.

[30]  M. Little,et al.  Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity. , 1997, Protein engineering.

[31]  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.

[32]  L. Frenken,et al.  The structure of the llama heavy chain constant genes reveals a mechanism for heavy-chain antibody formation , 1999, Immunogenetics.

[33]  C. Pace,et al.  How to measure and predict the molar absorption coefficient of a protein , 1995, Protein science : a publication of the Protein Society.

[34]  R. Poljak,et al.  Structural patterns at residue positions 9, 18, 67 and 82 in the VH framework regions of human and murine immunoglobulins. , 1993, Journal of molecular biology.

[35]  S. Muyldermans,et al.  Naturally occurring antibodies devoid of light chains , 1993, Nature.

[36]  S. Berens,et al.  Use of a single VH family and long CDR3s in the variable region of cattle Ig heavy chains. , 1997, International immunology.

[37]  L. Riechmann,et al.  Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability. , 1996, Protein engineering.

[38]  K. Constantine,et al.  Redefining the minimal antigen-binding fragment , 1996, Nature Structural Biology.

[39]  P. Schuck,et al.  An antibody single-domain phage display library of a native heavy chain variable region: isolation of functional single-domain VH molecules with a unique interface. , 1999, Journal of molecular biology.

[40]  A. Plückthun,et al.  The nature of antibody heavy chain residue H6 strongly influences the stability of a VH domain lacking the disulfide bridge. , 1998, Journal of molecular biology.

[41]  B. de Geus,et al.  Induction of immune responses and molecular cloning of the heavy chain antibody repertoire of Lama glama. , 2000, Journal of immunological methods.

[42]  S V Evans,et al.  SETOR: hardware-lighted three-dimensional solid model representations of macromolecules. , 1993, Journal of molecular graphics.

[43]  H. Jennings,et al.  Conformational epitope of the type III group B Streptococcus capsular polysaccharide. , 1999, Journal of immunology.

[44]  Lode Wyns,et al.  Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme , 1996, Nature Structural Biology.

[45]  M. Perry,et al.  Antigenic S-type lipopolysaccharide of Brucella abortus 1119-3 , 1984, Infection and immunity.

[46]  L. Wyns,et al.  Camel single‐domain antibody inhibits enzyme by mimicking carbohydrate substrate , 1998, Proteins.

[47]  E. Kabat,et al.  Sequences of proteins of immunological interest , 1991 .

[48]  R. Oomen,et al.  Crystal structure to 2.45 Å resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen , 1993, Protein science : a publication of the Protein Society.