Cambridge Antibody Technology Limited

Medical Research Council

Other

Medimmune Limited

Glaxo Wellcome Inc.

Kevin Stuart Johnson

Gregory Paul Winter

Andrew David Griffiths

John Gerald McCafferty

Ronald Henry Jackson

Anthony Richard Pope

James David Marks

David John Chiswell

Timothy Peter Bonnert

Timothy Piers Clackson

Kaspar Philipp Holliger

Henricus Renerus Jacobus Mattheus Hoogenboom

Tristan J Vaughan

Andrew John Smith

Peter Michael Waterhouse

Elaine Joy Derbyshire

Jane Katharine Osbourn

Andrew John Hammond Smith

Steven Neville Chatfield

Ian George Charles

Andrew James Williams

Carlos Estenio Hormaeche

Gordan Dougan

Ahuva Nissim

Samuel Cameron Williams

Alison Jane Wilton

Philip Ronald Tempest

Hendricus Renerus Jacobus Mattheus Hoogenboom

Julia Elizabeth Thompson

Kevin Pritchard

Henricus Renerus Hoogenboom

Steven Paul Ruddock

Raymond Paul Field

Louise Bacon

Jonathan Alexander Green

Hendricus Reneus Jacobus Mattheus Hoogenboom

Kasper Philipp Holliger

A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a filamentous bacteriophage particle containing DNA encoding the sbp member, wherein the sbp member has a binding domain that consists of a dAb fragment. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected filamentous bacteriophage particles for expression of the selected sbp members.

Methods for producing members of specific binding pairs

A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof, by virtue of the sbp member or a polypeptide component thereof being expressed as a fusion with a capsid component of the rgdp. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected rgdps for expression of the selected sbp members. Antibody sbp members may be thus obtained, with the different chains thereof expressed, one fused to the capsid component and the other in free form for association with the fusion partner polypeptide. A phagemid may be used as an expression vector, with said capsid fusion helping to package the phagemid DNA. Using this method libraries of DNA encoding respective chains of such multimeric sbp members may be combined, thereby obtaining a much greater genetic diversity in the sbp members than could easily be obtained by conventional methods.

A member of a specific binding pair (sbp) is identified by expressing DNA encoding a genetically diverse population of such sbp members in recombinant host cells in which the sbp members are displayed in functional form at the surface of a secreted recombinant genetic display package (rgdp) containing DNA encoding the sbp member or a polypeptide component thereof, by virtue of the sbp member or a polypeptide component thereof being expressed as a fusion with a capsid component of the rgdp. The displayed sbps may be selected by affinity with a complementary sbp member, and the DNA recovered from selected rgdps for expression of the selected sbp members. A phagemid may be used as an expression vector, with said capsid fusion helping to package the phagemid DNA.

Specific binding members comprising human antibody antigen binding domains specific for human transforming growth factor beta (TGF&#x3b2;) bind specifically isoforms TGF&#x3b2;2 and TGF&#x3b2;1 or both, preferentially compared with TGF&#x3b2;3. Specific binding members may be isolated and utilized in the treatment of disease, particularly fibrotic disease and also immune/inflammatory diseases. Therapeutic utility is demonstrated using in vitro and in vivo models. Full sequence and binding information is provided, including epitope sequence information for particularly advantageous specific binding member which binds the active form of TGF&#x3b2;2, neutralizing its activity, but does not bind the latent member.

Human antibodies specific for TGF&#x3b2;2

Methods, recombinant host cells and kits are disclosed-for the production of members of specific binding pairs (sbp), e.g. antibodies, using display on the surface of secreted replicable genetic display packages (rgdps), e.g. filamentous phage. To produce a library of great diversity recombination occurs between first and second vectors comprising nucleic acid encoding first and second polypeptide chains of sbp members respectively, thereby producing recombinant vectors each encoding both a first and a second polypeptide chain component of a sbp member. The recombination may take place in vitro or intracellularly and may be site-specific, e.g. involving use of the loxP sequence and mutants thereof. Recombination may take place after prior screening or selecting for rgdps displaying sbp members which bind complementary sbp member of interest.

A method of labelling molecules which includes providing in a common medium a label molecule, a marker ligand able to bind a member of a specific binding pair, such as an antigen, a sbp member, an enzyme able to catalyze binding of the label molecule to other molecules, the enzyme being associated with the marker ligand; causing or allowing binding of the marker ligand to the sbp member; and causing or allowing binding of the label molecule to other molecules in the vicinity of the marker ligand bound to the sbp member. The marker ligand may be an antibody or any specific binding molecule, such as a chemokine or cytokine. A complementary member of the specific binding pair may be included, e.g. an antibody, or a diverse population of such sbp members, e.g. antibodies, may be included within which those which bind the counterpart sbp member, e.g. antigen, may be labelled and subsequently isolated for manipulation and/or use. Suitable labels include biotin-tyramine with signal transfer being catalysed by hydrogen peroxidase. Cells, virus particles and other moieties may be labelled, for identification or obtention of proteins which interact or are in close proximity with a particular sbp member, or of cells of interest, or for enhancement of labelling, e.g. for cell sorting.

Labelling and selection of molecules

A method of labelling molecules which includes providing in a common medium a label molecule, a marker ligand able to bind a member of a specific binding pair, such as an antigen, a sbp member, an enzyme able to catalyse binding of the label molecule to other molecules, the enzyme being associated with the marker ligand; causing or allowing binding of the marker ligand to the sbp member; and causing or allowing binding of the label molecule to other molecules in the vicinity of the marker ligand bound to the sbp member. The marker ligand may be an antibody or any specific binding molecule, such as a chemokine or cytokine. A complementary member of the specific binding pair may be included, e.g. an antibody, or a diverse population of such sbp members, e.g. antibodies, may be included within which those which bind the counterpart sbp member, e.g. antigen, may be labelled and subsequently isolated for manipulation and/or use. Suitable labels include biotin-tyramine with signal transfer being catalysed by hydrogen peroxidase. Cells, virus particles and other moieties may be labelled, for identification or obtention of proteins which interact or are in close proximity with a particular sbp member, or of cells of interest, or for enhancement of labelling, e.g. for cell sorting.

The invention provides methods and kits for producing specific binding pairs (sbp) members. Populations of polypeptide chain components of sbp members are combined to form libraries of sbps displayed by secreted replicable genetic display packages (rgdp). At least one of the polypeptide chains is expressed as a fusion with a component of an rgdp which thereby displays that polypeptide chain at the surface of rgdp. At least one population of polypeptide chains is expressed from nucleic acid which is capable of being packaged using a component of an rgdp, whereby the genetic material of rgdps produced encodes a polypeptide chain. The methods enable production of libraries of multimeric sbp members from a very large number of possible combinations. In one embodiment of the invention a method employs &ldquo;chain shuffling&rdquo; in the production of sbp members of desired specificity for a counterpart sbp member. Selection procedures are also described.

Methods, recombinant host cells and kits are disclosed for the production of members of specific binding pairs (sbp), e.g. antibodies, using display on the surface of secreted replicable genetic display packages (rgdps), e.g. filamentous phage. To produce a library of great diversity, recombination occurs between first and second vectors comprising nucleic acid encoding first and second polypeptide chains of sbp members respectively, thereby producing recombinant vectors each encoding both a fist and a second polypeptide chain component of an sbp member. The recombination may take place in vitro or intracellularly and may be site-specific, e.g. involving use of the loxP sequence and mutants thereof. Recombination may take place after prior screening or selecting for rgdps displaying sbp members which bind complementary sbp member of interest.

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Cambridge, United Kingdom

Kevin Stuart Johnson