GB2408332A - Phage display assay - Google Patents

Abstract

A method of producing specific binding members for a cell surface antigen, wherein cells with the antigen at their surface and control cells without the antigen at their surface are immobilised on a solid surface, without fixation, and are contacted with a phage display library with blocking of non-specific binding, and a population of phage displaying specific binding members for the cell surface antigen is identified, the method further comprising obtaining nucleic acid which has a nucleotide sequence encoding specific binding members displayed on phage of the population and producing one or more specific binding members for the cell surface antigen by recombinant expression from nucleic acid with the nucleotide sequence or a derivative nucleotide sequence to produce a product specific binding member.

Description

H I GH THROUGHPUT AS SAY

The present invention relates to phage display assays to identify specific binding members for a desired target. It especially relates to phage display employing antibody molecules (so-called "phage antibodies") and is primarily concerned with identifying antibody molecules displayed on phage that bind to and preferably inhibit the function of a cell surface expressed antigen.

A high-throughput initial screen provided in accordance with the present invention can enable prioritization of panels of candidates for analysis by a functional high-throughput screen.

In various embodiments of the present invention, the process involves phage antibodies binding to cells and may utilise IS Europium labelled anti-M13 conjugate and the DELFIA_ reagent system.

The application of phage display in the area of receptor-ligand interactions has great potential (Hartley 2002). However, there are two problems that need to be considered in the use of phage display for identifying phage antibody binders to cell surface expressed antigens, such as receptors.

The first relates to the selection and enrichment of phage antibody binders that recognise the native/natural antigen, i.e., the target expressed in the correct conformation, rather than phage antibody binders that recognise the recombinant antigen.

Recombinant receptor antigen is typically available in the extracellular domain form, which precludes any tertiary structure involving other parts of the receptor or other factors involved in the cell surface architecture.

The second issue is that phage particles themselves are inherently "sticky", meaning that previous studies assessing enrichment of phage antibody binders to cell surface antigens have suffered from high nonspecific background. Such studies have used a hydrogen peroxidase (HRP) conjugated anti-M13 secondary reagent, which will also detect endogenously expressed HRP, also accounting for the high non-specific background. A conventional phage ELISA on recombinant antigen will use either anti-M13 HRP conjugate, or an anti-fc antibody with an alkaline phosphatase conjugated secondary antibody for detection of phage antibody binding.

The alternatives to using anti-M13 HRP conjugate in a conventional phage ELISA have been either: to sort the population by FACS (Van der Vuurst de Vries and Logtenberg 1999; Hegmans et al 2002; Pereira et al 1997); to express soluble scFv and detect the soluble scFv binding to cells by FACS (Peipp et al 2001); to specifically detect scFv binding to human blood group antigens by agglutination (Marks et al 1993); or to detect myctagged scFv binding using the anti-myc antibody 9E10 (Hoogenboom et al 1999).

Differential staining in immunohistochemical analysis of tissue sections (Palmer et al 1997) has been investigated, as well as assessing pools of phage clones by polyclonal phage ELISA (fur et al 2003). All of these are useful tools, but are not suited to the rapid high throughput assessment of multiple selection strategies.

This invention in various embodiments provides a high-throughput initial screen, which has been used on a number of targets for assessing selection outputs in order to prioritise the most successful strategies for the main screening assay. The invention in preferred embodiments, e.g. employing a combination of an anti-M13 Europium (Eu3+) conjugate and the DELFIA_ reagent system (Perkin Elmer), allows for a much greater sensitivity in detection than previously observed with the anti-M13 HRP conjugate and its associated detection system.

Cells have previously been used in phage antibody selections using established protocols (i.e., blocking with 2%(w/v) Marvel_/PBS, washing with PBS/Tween 20 and PBS) in combination with standard panning and soluble selection approaches (Osbourn et al 1998a; Edwards et al 2000). However, before the present invention, there was no rapid method to assess the enrichment for cell surface expressed antigen specific phage antibodies.

Previous attempts to perform phage ELISAs on cells and as described in the literature generally utilized 2%(w/v) Marvel_/PBS as block, with PBS/Tween 20 and PBS as washes and detection with anti-M13 HRP conjugate (Edwards et al 2000).

Alternatively, media alone has been used for block and washes and detection with anti-M13 HRP conjugate (Spear et al 2001).

Generally, whole cells were either fixed or plasma membranes prepared and fixed, using cross-linking fixatives such as gluteraldehyde or paraldebyde, which could feasibly modify the presentation of the receptor (Cal and Garen 1995; Figini et al 1998; Osbourn et al 1998a; Mutaberia et al 1999; Topping et al 2000; Williams and Sharon 2002; Williams et al 2002). When unfixed cells were used, results were variable with specific signal being only twice that of background (Spear et al 2001); thus the procedure was not robust. In addition, non-standard reagents, such as media, have been reported to interfere with TMB conversion and reading (Spear et al 2001).

In accordance with aspects and embodiments of the present invention there is provided subject-matter as defined in the claims. Further aspects and embodiments are disclosed in the

description herein.

The present invention provides methods of producing specific binding members for cell surface antigens. The invention especially provides for the use of methods as disclosed herein in a screening or assessment process to identify specific binding members, including antibody molecules that may be used therapeutically.

The cell surface antigen may be any antigen with a surface accessible region. A cell surface antigen may be a protein, carbohydrate, lipid, and/or glycolipid. The cell surface antigen is preferably a protein. Examples of cell surface antigens include G-protein coupled receptors, cytokine receptors, hormone receptors, adhesion molecules and ion channels.

Blocking in a method according to preferred embodiments of the invention may employ 4-5% (w/v) milk powder (e.g. Tradename "Marvel"; Ingredients 99.5% Dried Skimmed Milk, Vitamins A&D; UK Supplier is Premier International Foods (UK) Ltd., Bridge Road, Long Sutton, Spalding Lincs PEl2 9EQ) or other blocking agent.

Where a 96-well plate is employed in embodiments of the invention, preferably this conforms to the internationally recommended standard, wherein a 96 well plate is multireaction vessel that has 96 separate wells allowing multiple experimental conditions to be tested in one vessel; each plate is approximately 127mm long, approximately 85 mm wide and approximately 14mm high. A 384 well plate is a similar multireaction vessel, of similar size, that has 384 separate wells.

Preferred embodiments within the present invention are directed to producing specific binding members that are antibody molecules, whether whole antibody (e.g. IgG, such as IgG4) or antibody fragments (e.g. scFv, Fab, dab). An antibody molecule comprises an antibody antigen binding and this may consist of or comprise a VH domain and/or a VL domain. Within VH and VL domains are provided complementarily determining regions, CDR's, which may be provided within different framework regions, Fit's, to form VH or VL domains as the case may be.

An antigen binding site may be provided by means of arrangement of CDR's on non-antibody protein scaffolds such as fibronectin or cytochrome B etc. (Koide et al (1998) Journal of Molecular Biology, Vol 284:1141-1151; Nygren et al (1997) Current Opinion in Structural Biology, Vol 7:463-469). Scaffolds for engineering novel binding sites in proteins have been reviewed in detail by Nygren et al. supra. Protein scaffolds for antibody mimics are disclosed in WO/0034784 in which the inventors describe proteins (antibody mimics) which include a fibronectin type III domain having at least one randomised loop.

A specific binding member produced by a method as disclosed herein, and optionally comprising an antibody antigen-binding domain comprising a VH and/or VL domain, is also provided by the present invention.

Techniques useful for making substitutions within amino acid sequences of CDR's, antibody VH or VL domains and specific binding members generally are available in the art. Derivative or variant sequences may be made, with substitutions that may or may not be predicted to have a minimal or beneficial effect on activity, and tested for ability to bind target antigen S (generally a cell surface antigen) and/or for any other desired property.

Any one or more CDR's of a specific binding member may be provided in, e. g. by way of grafting, a VH domain that is used as a specific binding member alone or in combination with a VL domain. A VH domain may be provided with a set of HCDR's, and if such a VH domain is paired with a VL domain, then the VL domain may be provided with a set of LCDR's. The framework regions of the VH and/or VL domains may be germline frameworks.

One or more CDRs may be taken from a VH or VL domain of a specific binding member identified in a product population in accordance with the invention, and incorporated into a suitable framework.

An antibody VH domain, an antibody VL domain, a set of HCDR's, a set of LCDR's, a set of CDR's, one or more HCDR's e.g. an HCDR3, and/or one or more LCR's e.g. an LCDR3, of a specific binding member that is identified using the invention as binding the target antigen, may be employed for instance in methods of mutation and selection of antigen binding sites with improved affinity and/or potency.

Variants or derivatives of selected VH and VL domains and CDRs which can be employed in specific binding members for a target antigen can be obtained by means of methods of sequence alteration or mutation and screening. Such methods are encompassed within methods of the present invention.

Particular variants or derivaties may include one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue), may be less than about 20 alterations, less than about 15 alterations, less than about 10 alterations or less than about 5 alterations, 4, 3, 2 or 1. Alterations may be made in one or more framework regions and/or one or more CDR's.

A product specific binding member according to the present invention may comprise amino acids other than antibody sequences, e.g. forming a peptide or polypeptide, such as a folded domain, or imparting to the molecule another functional characteristic in addition to ability to bind antigen. Specific binding members of the invention may carry a detectable label, or may be conjugated to a toxin or a targeting moiety or enzyme (e. g. via a peptidyl bond or linker).

A further aspect of the present invention provides nucleic acid, generally isolated, encoding an specific binding member of a product population as identified herein.

Such nucleic acid is useful in subsequent production of the specific binding member, any fragment or component thereof (e.g. a VH domain of an scFv molecule), and may be used as a template in production of multiple nucleic acid copies. A derivative nucleic acid may be provided by alteration of the sequence, whether by substitution, addition, insertion or removal of one or more nucleotides, or any combination thereof. For instance, additional nucleotides may be added encoding a peptide or protein domain, imparting an additional functionality on the encoded product. Nucleic acid encoding a VH domain of a scFv antibody molecule, for example, may have added to it nucleic acid encoding an antibody CH domain, providing thereby an antibody heavy chain.

This may be used in combination with antibody light chains to produce a whole antibody, e.g. an IgG antibody. VH and/or VL domains of initially selected specific binding members, e.g. scFv or Fab antibody molecules, may be produced by expression from derivative nucleic acid in which the nucleotide sequence or nucleotide sequences encoding the VH and/or VL domains are joined with nucleic acid encoding other peptide or polypeptide components, such as marker peptides and/or antibody constant regions. Derivative nucleic acid may comprise one or more mutations, e.g. within the region of the nucleotide sequence encoding a VH CDR3 or other CDR, thereby potentially altering a CDR sequence, possibly affecting a property of the encoded product.

A further aspect provides a host cell transformed with nucleic acid of the invention.

"Transformation" extends to any suitable means or mechanism for introducing nucleic acid into a cell. A cell containing nucleic acid as a result of introduction using recombinant or genetic engineering techniques is said to be "transgenic".

A yet further aspect provides a method of production of a specific binding member, the method including causing expression from encoding nucleic acid. Such a method may comprise culturing host cells under conditions for production of said specific binding member.

A method of production may comprise a step of isolation and/or purification of the product.

Following production of a product specific binding member by recombinant expression, it may be recovered from the expression system and may be isolated. It may be formulated into a composition comprising at least one additional component. It may be modified.

A method of production may comprise formulating the product into a composition including at least one additional component, such as a pharmaceutically acceptable excipient.

Specific binding members produced according to the invention may be used in a method of treatment or diagnosis of the human or animal body, such as a method of treatment (which may include prophylactic treatment) of a disease or disorder in a human patient which comprises administering to said patient an effective amount of a specific binding member of the invention.

Conditions treatable in accordance with the present invention include any in which the target antigen plays a role.

TERMINOLOGY

Specific binding member This describes a member of a pair of molecules which have binding specificity for one another. The members of a specific binding pair may be naturally derived or wholly or partially synthetically produced. One member of the pair of molecules has an area on its surface, or a cavity, which specifically binds to and is therefore complementary to a particular spatial and polar organization of the other member of the pair of molecules. Thus the members of the pair have the property of binding specifically to each other. Examples of types of specific binding pairs are antigen-antibody, biotin-avidin, hormone- hormone receptor, receptor-ligand, enzyme-substrate. The present invention is primarily concerned with antigen-antibody type reactions.

Antibody molecule This describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein comprising an antibody binding domain.

Antibody fragments which comprise an antigen binding domain are molecules such as Fab, scFv, Fv, dab, Ed; and diabodies.

It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarily determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP-A-184187, GB 2188638A or EP-A-239400, and a large body of subsequent literature. A hybridoma or other cell producing an antibody may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.

As antibodies can be modified in a number of ways, the term "antibody molecule" should be construed as covering any specific binding member or substance having an antibody antigen-binding domain with the required specificity. Thus, this term covers antibody fragments and derivatives, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A- 0125023; and a large body of subsequent literature.

Synthetic antibody molecules may be created by expression from genes generated by means of oligonucleotides synthesized and assembled within suitable expression vectors, for example as described by Knappik et al. J. Mol. Biol. (2000) 296, 57-86 or Krebs et al. Journal of Immunological Methods 254 2001 67-84.

It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments are (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Ed fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward, E.S.

et al., Nature 341, 544-546 (1989), McCafferty et al (1990) Nature, 348, 552-554) which consists of a VH domain; (v) isolated CUR regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two linked Fab fragments (vii) single chain Fv molecules (scFv) , wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, Science, 242, 423-426, 1988; Huston et al, PNAS USA, 85, 5879-5883, 1988); (viii) bispecific single chain Fv diners (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; P. Holliger et al, Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993). Fv, scFv or diabody molecules may be stabilised by the incorporation of disulphide bridges linking the VH and VL domains (Y. Reiter et al, Nature Biotech, 14, 1239-1245, 1996). Minibodies comprising a scFv joined to a CH3 domain may also be made (S. Ho et al, Cancer Res., 56, 3055-3061, 1996).

Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger, P. and Winter G. Current Opinion Biotechnol. 4, 446-449 (1993)) , e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. Examples of bispecific antibodies include those of the BITE_ technology in which the binding domains of two antibodies with different specificity can be used and directly linked via short flexible peptides. This combines two antibodies on a short single polypeptide chain. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the effects of anti- idiotypic reaction.

Bispecific diabodies, as opposed to bispecific whole antibodies, may also be particularly useful because they can be readily constructed and expressed in E. cold. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be readily selected using phage display (WO94/13804) from libraries. If one arm of the diabody is to be kept constant, for instance, with a specificity directed against a particular antigen, say Receptor C, then a library can be made where the other arm is varied and an antibody of appropriate specificity selected. Bispecific whole antibodies may be made by knobs-into-holes engineering (J. B. B. Ridgeway et al, Protein Eng., 9, 616-621, 1996).

An tiger -binding Coma in This describes the part of an antibody molecule which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding domain may be provided by one or more antibody variable domains (e.g. a so-called Ed antibody fragment consisting of a VH domain). Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

Speci fi c This may be used to refer to the situation in which one member of a specific binding pair will not show any significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen binding domain is specific for a particular epitope which is carried by a number of antigens, in which case the specific binding member carrying the antigen binding domain will be able to bind to the various antigens carrying the epitope.

Compri s e This is generally used in the sense of include, that is to say permitting the presence of one or more features or components.

Isola ted This refers to the state in which specific binding members of the invention, or nucleic acid encoding such binding members, will generally be in accordance with the present invention. Isolated members and isolated nucleic acid will be free or substantially free of material with which they are naturally associated such as other polypeptides or nucleic acids with which they are found in their natural environment, or the environment in which they are prepared (e.g. cell culture) when such preparation is by recombinant DNA technology practiced in vitro or in viva.

Members and nucleic acid may be formulated with diluents or adjuvants and still for practical purposes be isolated - for example the members will normally be mixed with gelatin or other carriers if used to coat microtitre plates for use in immunoassays, or will be mixed with pharmaceutically acceptable carriers or diluents when used in diagnosis or therapy. Specific binding members may be glycosylated, either naturally or by systems of heterologous eukaryotic cells (e.g. CHO or NSO (ECACC 85110503) cells, or they may be (for example if produced by expression in a prokaryotic cell) unglycosylated.

The structure for carrying a CDR or a set of CDR's in a specific binding member of the invention will generally be of an antibody heavy or light chain sequence or substantial portion thereof in which the CDR or set of CDR's is located at a location corresponding to the CDR or set of CDR's of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. The structures and locations of immunoglobulin variable domains may be determined by reference to (Kabat, E.A. et al, Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof, now available on the Internet (http://immuno.bme.nwu.edu or find "Kabat" using any search engine). CDRs are defined according to Kabat et al. (1991).

As noted, CDR's can also be carried by other scaffolds such as fibronectin or cytochrome B. although preferably CDR amino acid sequences are carried as a CDR in a human variable domain or a substantial portion thereof.

Variable domains employed in the invention may be obtained from any germline or rearranged human variable domain, or may be a synthetic variable domain based on consensus sequences of known human variable domains. A CDR sequence (e.g. CDR3) may be introduced into a repertoire of variable domains lacking a CDR (e.g. CDR3), using recombinant DNA technology.

For example, Marks et al (Bio/Technology, 1992, 10:779-783) describe methods of producing repertoires of antibody variable domains in which consensus primers directed at or adjacent to the 5' end of the variable domain area are used in conjunction with consensus primers to the third framework region of human VH genes to provide a repertoire of VH variable domains lacking a CDR3.

Marks et al further describe how this repertoire may be combined with a CDR3 of a particular antibody. Using analogous techniques, CDR3-derived sequences may be shuffled with repertoires of VH or VL domains lacking a CDR3, and the shuffled complete VH or VL domains combined with a cognate VL or VH domain to provide specific binding members invention.

Information on phage display systems useful in methods of the invention can be found in WO92/01047 and any of a subsequent large body of literature, including Kay, B.K., Winter, J., and McCafferty, J. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, San Diego: Academic Press, so that suitable specific binding members may be selected.

Novel specific binding members, e.g. antibody molecules, VH and/or VL regions carrying CDR-derived sequences, may be provided using random mutagenesis of one or more genes, e.g. selected VH and/or VL genes, to generate mutations within the entire polypeptide, e.g. variable domain. Such a technique is described by Gram et al (1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580), who used error-prone PCR. In preferred embodiments one or two amino acid substitutions are made within a set of HCDR's and/or LCDR's.

Another method which may be used is to direct mutagenesis to CDR regions of VH or VL genes. Such techniques are disclosed by Barbas et al, (1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813) and Schier et al (1996, J. Mol. Biol. 263:551-567).

All the above described techniques are known as such in the art and in themselves do not form part of the present invention. The skilled person will be able to use such techniques to provide specific binding members in the context of the invention using routine methodology in the art.

A substantial portion of an immunoglobulin variable domain will comprise at least the three CDR regions, together with their intervening framework regions. Preferably, the portion will also include at least about 50% of either or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Additional residues at the N-terminal or Cterminal end of the substantial part of the variable domain may be those not normally associated with naturally occurring variable domain regions. For example, construction of specific binding members of the present invention made by recombinant DNA techniques may result in the introduction of N- or C-terminal residues encoded by linkers introduced to facilitate cloning or other manipulation steps. Other manipulation steps include the introduction of linkers to join variable domains of the invention to further protein sequences including immunoglobulin heavy chains, other variable domains (for example in the production of diabodies) or protein labels as discussed in more detail elsewhere herein.

Although in a preferred aspect of the invention specific binding members comprising a pair of VH and VL domains are preferred, single binding domains based on either VH or VL domain sequences form further aspects of the invention. It is known that single immunoglobulin domains, especially VH domains, are capable of binding target antigens in a specific manner.

Specific binding members of the present invention may further comprise antibody constant regions or parts thereof. For example, a VL domain may be attached at its C-terminal end to antibody light chain constant domains including human CK or Ck chains. Similarly, a specific binding member based on a VH domain may be attached at its C-terminal end to all or part (e.g. a CH1 domain) of an immunoglobulin heavy chain derived from any antibody isotype, e.g. IgG, IgA, IgE and IgM and any of the isotype sub-classes, particularly IgG1 and IgG4. IgG4 is preferred. IgG4 may be preferred for certain therapeutic indications because it does not bind complement and does not create effecter functions. Any synthetic or other constant region variant that has these properties and stabilizes variable regions is also preferred for use in embodiments of the present invention.

Specific binding members of the invention may be labelled with a detectable or functional label. Detectable labels include radiolabels such as 13lI or 99Tc, which may be attached to antibodies of the invention using conventional chemistry known in the art of antibody imaging. Labelsalso include enzyme labels such as horseradish peroxidase. Labels further include chemical moieties such as biotic which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin.

Anti-phage antibodies may be labelled with Europium chelate using a commercially available kit (Perkin Elmer, BELGIAN Eu-Labelling Kit, Catalogue Number 1244-302). All antibodies used in this work were labelled using the protocol supplied by the manufacturer with this kit, the only modification being that the "Labelling Buffer" was at pH 8.5 rather than the recommended pH of 9.3.

DELFIA detection technology is reported to be a highly sensitive detection technology with a wide dynamic range e.g. Suonpaa et al (1992) J Immunol Methods 149: 247. Comparative studies have shown that DELFIA detection offers increased sensitivity and a broader dynamic range in a wide range of systems compared to certain other non-fluorescent ELISA detection methods e.g. Smith et al (2001) Clin Diagn Lab Immunol 8: 1070; Butcher et al (2003) J Immunol Methods 272: 247; Allicotti et al (2003) J Immunoassay Immunochem 24: 345; Schoket et al (1993) Cancer Epidemiol Biomarkers Prev 2: 349; Peruski et al (2002) J Immunol Methods 263: 35.

Specific binding members of the present invention may be used in methods of diagnosis or treatment in human or animal subjects, preferably human.

Accordingly, further aspects of the invention provide methods of treatment comprising administration of a specific binding member as provided, pharmaceutical compositions comprising such a specific binding member, and use of such a specific binding member in the manufacture of a medicament for administration, for example in a method of making a medicament or pharmaceutical composition comprising formulating the specific binding member with a pharmaceutically acceptable excipient.

Treatment with a specific binding member may be given orally, by injection (for example, subcutaneously, intravenously, intraperitoneal or intramuscularly), by inhalation, or topically (for example intraocular, intranasal, rectal, into wounds, on skin). The route of administration can be determined by the physicochemical characteristics of the treatment, by special considerations for the disease or by the requirement to optimise efficacy or to minimise side- effects.

Combination treatments may be used to provide significant synergistic effects, particularly the combination of a specific binding member with one or more other drugs.

In accordance with the present invention, compositions provided may be administered to individuals. Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to a patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated.

Prescription of treatment, e.g. decisions on dosage etc. is within the responsibility of general practitioners and other medical doctors. Appropriate doses of antibody are well known in the art; see Ledermann J. A. et al. (1991) Int. J. Cancer 47: 659 664; Bagshawe K.D. et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922.

The precise dose will depend upon a number of factors, including whether the antibody is for diagnosis or for treatment, the size and location of the area to be treated, the precise nature of the antibody (e.g. whole antibody, fragment or diabody), and the nature of any detectable label or other molecule attached to the antibody. A typical antibody dose will be in the range loopy to 1 gm for systemic applications, and log to lmg for topical applications. Typically, the antibody will be a whole antibody, preferably the IgG4 isotype. This is a dose for a single treatment of an adult patient, which may be proportionally adjusted for children and infants, and also adjusted for other antibody formats in proportion to molecular weight. Treatments may be repeated at daily, twice-weekly, weekly or monthly intervals, at the discretion of the physician. In preferred embodiments of the present invention, treatment is periodic, and the period between administrations is about two weeks or more, preferably about three weeks or more, more preferably about four weeks or more, or about once a month.

Specific binding members of the present invention will usually be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the specific binding member.

Thus pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may comprise, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. intravenous.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharine solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.

A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

Specific binding members of the present invention may be formulated in liquid or solid forms depending on the physicochemical properties of the molecule and the route of delivery. Formulations may include excipients, or combinations of excipients, for example: sugars, amino acids and surfactants.

Liquid formulations may include a wide range of antibody concentrations and pH. Solid formulations may be produced by lyophilisation, spray drying, or drying by supercritical fluid technology, for example. Formulations of specific binding member will depend upon the intended route of delivery.

The present invention provides a method comprising causing or allowing binding of a specific binding member as provided herein to its antigen. As noted, such binding may take place in viva, e.g. following administration of a specific binding member, or nucleic acid encoding a specific binding member, or it may take place in vitro, for example in ELISA, Western blotting, immunocytochemistry, immuno-precipitation, affinity chromatography, or cell based assays.

The amount of binding of specific binding member to antigen may be determined. Quantitation may be related to the amount of the antigen in a test sample, which may be of diagnostic interest.

A kit comprising a specific binding member or antibody molecule according to any aspect or embodiment of the present invention is also provided as an aspect of the present invention. In a kit of the invention, the specific binding member or antibody molecule may be labelled to allow its reactivity in a sample to be determined, e.g. as described further below. Components of a kit are generally sterile and in sealed vials or other containers.

Kits may be employed in diagnostic analysis or other methods for which antibody molecules are useful. A kit may contain instructions for use of the components in a method, e.g. a method in accordance with the present invention. Ancillary materials to assist in or to enable performing such a method may be included within a kit of the invention.

The reactivities of antibodies in a sample may be determined by any appropriate means. Radioimmunoassay (RIA) is one possibility. Radioactive labelled antigen is mixed with unlabelled antigen (the test sample) and allowed to bind to the antibody. Bound antigen is physically separated from unbound antigen and the amount of radioactive antigen bound to the antibody determined. The more antigen there is in the test sample the less radioactive antigen will bind to the antibody. A competitive binding assay may also be used with non-radioactive antigen, using antigen or an analogue linked to a reporter molecule. The reporter molecule may be a fluorochrome, phosphor or laser dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin, Texas Red, and Lanthanide chelates or cryptates. Suitable chromogenic dyes include diaminobenzidine.

Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded. These molecules may be enzymes which catalyse reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed.

The signals generated by individual antibody-reporter conjugates may be used to derive quantifiable absolute or relative data of the relevant antibody binding in samples (normal and test).

The present invention also provides the use of a specific binding member as above for measuring antigen levels in a competition assay, that is to say a method of measuring the level of antigen in a sample by employing a specific binding member as provided by the present invention in a competition assay. This may be where the physical separation of bound from unbound antigen is not required. Linking a reporter molecule to the specific binding member so that a physical or optical change occurs on binding is one possibility. The reporter molecule may directly or indirectly generate detectable, and preferably measurable, signals. The linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non- covalently. Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding antibody and reporter molecule.

The present invention also provides for measuring levels of antigen directly, by employing a specific binding member according to the invention for example in a biosensor system.

The mode of determining binding is not a feature of these aspects of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, yeast and baculovirus systems and transgenic plants and animals.

The expression of antibodies and antibody fragments in prokaryotic cells such as E. cold is well established in the art.

For a review, see for example Pluckthun, A. Bio/Technology 9: 545-551 (1991). A common, preferred bacterial host is E. coli.

Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of a specific binding member for example Chadd HE and Chamow SM (2001) 110 Current Opinion in Biotechnology 12: 188-194, Andersen DC and Krummen L (2002) Current Opinion in Biotechnology 13: 117, Larrick JW and Thomas DW (2001) Current opinion in Biotechnology 12:411-418. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells, human embryonic retina cells and many others.

Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.

Vectors may be plasmids, viral e.g. 'phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook and Russell, 2001, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Second Edition, Ausubel et al. ads., John Wiley & Sons, 1988, Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons, 4th edition 1999. The disclosures of Sambrook et al. and Ausubel et al. (both) are incorporated herein by reference.

Thus, a further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. Such a host cell may be in vitro and may be in culture. Such a host cell may be in viva.

A still further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction may employ any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE Dextran, electroporation, liposome-mediated transfection and transduction using retrovirus or other virus, e.g. vaccinia or, for insect cells, baculovirus. Introducing nucleic acid in the host cell, in particular a eukaryotic cell may use a viral or a plasmid based system. The plasmid system may be maintained episomally or may incorporated into the host cell or into an artificial chromosome (Csonka E et al (2000) Journal of Cell Science, 113: 3207-3216; Vanderbyl S et al (2002) Molecular Therapy, 5(5): 10). Incorporation may be either by random or targeted integration of one or more copies at single or multiple loci. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation and transfection using bacteriophage.

The introduction may be followed by causing or allowing expression from the nucleic acid, e.g. by culturing host cells under conditions for expression of the gene. The nucleic acid may become integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.

It is convenient to point out here that "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B. just as if each is set out individually herein.

Brief Description of the Figures

Figure 1 shows a schematic representation of a typical lead isolation process for the identification of inhibitory scFv binders Figure 2 shows a schematic representation of identification of inhibitory scFv binders incorporating DELISA as an initial, in accordance with an embodiment of the present invention.

Figure 3 shows a schematic representation of issues addressed for optimization of the high throughput initial screening assay. . Figure 4 shows binding of reference mAbs to recombinant and native Receptor A in media and in bacterial supernatant using TMB detection. MAbl and mAb2 were blocked in Marvel_/media or Marvel_/bacterial supernatant (sin) and corresponding cells or wells were blocked. Detection was with an antimouse HRP conjugate using the TMB detection system. ECD is the extracellular domain of Receptor A (recombinant antigen).

Figure 4a shows results for mAbl.

Figure 4b shows results for mAb2.

Figure 5 shows binding of reference mAbs to recombinant and native Receptor A in media and in bacterial supernatant using DELFIA detection mAbl and mAb2 were blocked in Marvel_/media or Marvel_/bacterial supernatant (sin) and corresponding cells or wells were blocked similarly. Detection was with an anti-mouse Europium conjugate using the DELFIA_ reagent system. A higher specific binding signal: noise ratio was observed with the Europium/DELFIA_ detection system than with the HRP/TMB detection system. In addition, no detectable receptor internalization (and therefore depression of specific binding signal) was observed. This was also seen in the screening assay set-up. Note that the specific binding signal of mAbl on ECD (recombinant antigen) is significantly lower than the specific binding signal of mAb2 on ECD, however the specific binding signals of both mAbs are comparable on cells (native receptor).

This provides indication that the epitope availability for mAbl on immobilized ECD has been significantly reduced.

Figure 5a shows results for mAbl.

Figure 5b shows results for mAb2.

Figure 6 illustrates optimization of phage titre. Optimal phage titre and growth conditions were established by PEG precipitation of 25ml cultures of the dominant clone lineage and titrating this crude preparation in tenfold step-wise dilutions. These were compared with signal obtained with phage supernatant rescued in 25ml cultures. All phage, controls and cells were blocked in 4% (w/v) Marvel_/media final concentration. A comparison of specific binding with non-specific binding (on non-transfected cells, irrelevant clones, M13 helper phage and anti-M13 Europium only) was made. Background is approx. 2000 counts, whereas specific signal for a lOell phage/ml tithe was 5-fold higher for clone 1 (phage supernatant was 8-fold higher), 6- fold higher for clone 2 (phage supernatant was 4-fold higher) and 7-fold higher for clone 3 phage supernatant in this example.

Figure 7 shows variability using deep well conditions. Multiple wells containing the phage antibodies representing the functional hit scFv lineage show variable specific binding with phage supernatant derived from lml cultures (96-well deep well plate, Beckman). All phage, controls and cells were blocked in 4% (w/v) Marvel_/media final concentration. A comparison of specific binding with non-specific binding (on nontransfected cells, irrelevant clone, M13 helper phage and anti-M13 Europium only) was made. Background is approx. 2000 counts. An irrelevant clone and a phage clone representing the functional hit were plated in duplicate (wells a and b) in replicate experiments and screened using the anti-Ml3 Europium/DELFIA_ reagent system on non transfected and transfected Receptor A NIH3T3 cell lines. The positive control does consistently screen positive, however it ranges from approx. 5000 to approx. 13000 counts. This is probably due to the phage titre being lower than that obtained from 25ml cultures, as the lml cultures in 96-well format will not be as well aerated and therefore the phage titre will be reduced.

Figure 8 illustrates optimization of growth conditions. All phage, controls and cells were blocked in 4% (w/v) Marvel_/media final concentration. A comparison of specific binding with non specific binding using shallow well and deep well growth conditions for phage rescue was made. Multiple wells containing the phage antibodies representing the functional hit scFv lineage had shown variable specific binding with phage supernatant derived from lml cultures (Figure 7), therefore a second comparison was made between shallow 96-well plate growth/phage rescue (Costar) and deep 96-well plate growth/phage rescue (Beckman). Shallow well growth and rescue uses a volume of 100- 200, e.g. 150, Ill per well in 96 well plates, or 501 or less if 384 well plates are used, and produces higher specific signals presumably due to less higher phage titres. presumably due to less variable phage titres. Better aeration of the culture and a higher concentration of phage particles in a smaller volume achieve this. These conditions were then applied to a high throughput format pre-screen of 88 phage ELISA (ECD) positive unique sequence clones (see Figure 9). The remaining 8 wells on the 96-well plate are used for controls.

Figure 9 shows optimization on high-throughput application. 96- well DELISA on Receptor A transfected NIH3T3 cells high throughput analysis of phage ELISA positive clones Plate 1. Cells were immobilized by seeding the prior day into collagen-coated 96-well plates. Phage antibodies representing the functional hit scFv lineage were internal positive controls for this plate.

Cells and phage were blocked in 4% (w/v) Marvel_/media final concentration, blocked phage was bound to blocked cells for lh at rt. Cells were washed three times with lx PBS and then bound phage detected with anti-M13 Eu3+ conjugate diluted to lOOng/ml in DELFIA_ reagent buffer (lh at rt). Unbound conjugate was removed by washing seven times in DELFIA_ wash buffer prior to the addition of DELFIA_ enhancement solution. Plates were left to incubate at rt for 5 min prior to detection of phage bound to cells by fluorometric measurement using the Wallac Victor 2 (Perkin Elmer) with excitation at 340nm and emission at 615nm.

Phage clones were rescued and grown overnight in shallow well format in duplicate (Plates A and B), as well as deep well format (Plate C).

Hits on transfected cells are compared to the corresponding well position for non-transfected cells to ensure the robustness of this method. Only the controls and clones demonstrating a specific binding signal have been shown. The results not only show that the shallow well format (A and B) provides consistent results compared with deep well format (C), but also reveal that 11% of phage ELISA positives on recombinant antigen were phage binders in the cell DELISA, i.e., were positive for binding to recombinant antigen and transfected cells. This can be described as the overlap population.

Figure 10a shows results of cell DELISA on transfected NIH3T3 cell line expressing Receptor A. Data showing functional hits that are phage antibody binders for Receptor A transfected NIH3T3 cells (adherent) are shown. Phage antibodies were prepared as follows. A colony from a freshly prepared bacterial streak of a scFv lineage that was a functional hit (derived from the preliminary screening campaign) was used to inoculate 2ml of 2TYAG and incubated for 4h at 37 C shaking. M13KO7 helper phage was added according to the MOI and cells infected for 30 minutes stationary at 37 C, followed by 30 minutes at 180rpm 37 C. The culture was centrifuged at 3000rpm for 10 minutes and resuspended in 20ml 2TYAK prior to incubating overnight at 30 C 300rpm.

Figure lOb shows results of cell DELISA on transfected HEK293 cell line expressing Receptor B. Cell DELISA optimization using functional hit (phage antibody binder) from screen in initial lead isolation campaign for Receptor B transfected HEK293 cells (adherent). Shallow well growth conditions were used for phage rescue.

Figure lOc illustrates cell DELISA optimization on non transfected IM9 and HepG2 cell lines expressing Receptor C using reference mAbs. Reference mAbs for Receptor C expressing IM9 and HepG2 cells (non transfected suspension cell lines) were used initially to investigate optimal conditions for the cell DELISA prior to pre-screening selection output clones.

Figure lOd shows cell DELISA on non transfected IM9 and HepG2 cell lines expressing Receptor C: examples of phage antibody binders. Examples of phage antibody binders classed as hits in the pre-screen are shown. Shallow well growth conditions were used for phage rescue.

Poly-D-lysine coated 96-well plates were chosen as immobilization surfaces for HEK293, IM9 and HepG2 cells and collagen-coated plates for NIH3T3 cells, as these surfaces not only maintain the cells in a confluent monolayer but also provide optimal signal:noise ratio. Detection of bound phage was performed using anti-M13 Europium conjugate and the DELFIA_ reagent system as previously described.

Signals expected for specific binding can be in the region of 10 20x background for transfected cell lines (Figure lOa and Figure lob). This is usually lower for non-transfected cell lines (Figure led), where a lower limit of 3-4x background is applied as a cut- off, which reflects the relative expression level of the receptor of interest.

A typical lead isolation strategy consists of several distinct stages in the process used to identify inhibitory scFv clones (see for example Figure 1). The first part is the selection of phage libraries on antigen to isolate a population of phage antibody binders using various parallel methodologies (for example, panning selection, soluble selection, etc.). The resulting populations of phage antibody binders are referred to as selection outputs.

A representation (typically, 48 scFv clones) of each selection output is assessed for diversity by sequence analysis. The same phage antibodies are also assessed for recognition of antigen by phage ELISA. Selection outputs containing unique sequence phage ELISA positive binders from preliminary assessments are then targeted for functional screening.

Targeted selection outputs are subjected to a screening campaign, employing a functional high-throughput assay, of periplasmic extracts, phage or scFv supernatants in order to identify a population of functionally active scFvs. The expected hit rate for a functional screening campaign is 2%, as observed in previous lead isolation programmed. Finally, the hits from the screening campaign are profiled as scFv by IC50 analysis which assesses the potency of their method of action.

A key element for success in identifying lead candidate phage antibodies lies with the format of antigen for selection (normally recombinant human antigen) and the format of antigen used for the screen (normally cell surface expressed antigen). A low hit rate from the functional assay screening campaign could be indicative of a disparity between the receptor used for selection (recombinant format of receptor) and that used for the screen (native format of receptor). Therefore, in terms of available inhibiting epitopes, it is possible that confirmational differences between the purified recombinant forms of antigen and cell surface expressed native receptor would be responsible for a low hit rate. In practice, phage ELISAs are performed on recombinant antigen so although a certain percentage of phage antibodies may be identified as recombinant antigen binders, this will not necessarily equate to the same phage antibodies being inhibitors of the native receptor.

For example, in terms of Receptor A (see Example 1), the preliminary lead isolation campaign utilized recombinant human antigen which generated populations of phage antibodies (i.e., selection outputs). The initial screening campaign employed native Receptor A in a functional cell-based assay. Despite the inclusion of control mAbs to confirm the availability of epitopes for binding on the recombinant human antigen (and therefore available for selection), only a 0.02% hit rate was achieved which was unusually low.

Using ELISA, it had been demonstrated that there was binding of the known reference mAb (mAbl) to an epitope on both the recombinant ECD and cell (native) Receptor A. However, mAbl was found to be a partial inhibitor ofligand binding when recombinant ECD was coated on a plastic surface in a biochemical assay; it was a full inhibitor in the cell-based functional screen. Another reference mAb, mAb2, that recognises a different epitope, was a full inhibitor in both assays. A confirmational difference between the different formats of receptor is also suggested by the data when comparing the specific binding signal on recombinant ECD of mAbl with that of mAb2. It is reasonable to deduce this as there is equivalence in affinities of the reference mAbs 1 and 2. Thus, it was hypothesized that a confirmational difference between the recombinant form of Receptor A used for selection and the native form of Receptor A used for screening was the reason for the unusually low hit rate.

The establishment of a cell-based phage antibody binding assay (DELISA) in accordance with embodiments of the present invention allows for a large sample of selection outputs to be tested in a pre-screen to the scFv-binding cell-based functional screen (see e.g. Figure 2), based on our observations.

When an embodiment of the present invention was used for the rapid identification of phage selection outputs demonstrating a high percentage of phage antibodies that were native Receptor A binders, the hit rate was restored to 2% as selection outputs were successfully targeted and prioritised for the functional screening campaign.

Further aspects and embodiments of the invention will be apparent to those skilled in the art given the disclosure herein. All documents mentioned anywhere in the text are incorporated by reference.

EXAMPLE 1

DELISA optimism tion for Receptor A Selections and screening strategies are iterative, so not only will the screening assay provide feedback on the direction of the selections, but it has also been utilised to establish optimal conditions for the cells for the duration of the cell DELISA method (see e.g. Figure 3).

1. Cell growth conditions It is preferred in embodiments of the invention to bind phage antibodies to a confluent monolayer of cells. This was achieved by seeding in 96-well plate format the previous day at an appropriate concentration of cells related to the growth rate, for example, a confluent monolayer of NIH3T3 Receptor A transfected cells was achieved by seeding at 2.5 x 109 cells/well the previous day. No fixation of cells was involved and therefore the native conformation of the receptor was presented.

In accordance with embodiments of the invention, cells were immobilized on a suitable surface with no further modification (such as fixation), for example, adherent NIH3T3 cells were immobilized on collagen-coated plates, and suspension IM-9 or HepG2 cells were immobilized on poly-D-lysine. Other surfaces that may be employed, taking into account optimal signal:noise ratio obtained using for example reference mAbs or known phage antibody binders, as well as optimal immobilization by using microscopic observation for verification of an intact monolayer of cells at the end of the procedure, include poly-L- lysine, fibronectin, laminin, gelatin, polyornithine, CellTak_ prepared surfaces. The optimum surface is cell-line dependent and therefore optimization is to be on a case-by-case basis.

2. Blocking conditions A final concentration of 4%(w/v) Marvel_/appropriate media plus FCS, which was used in the cell-based functional assay, was employed as the blocking agent in this initial screen for phage antibody binders. Other blocking agents may be used in cell-based phage antibody binding assays (DELISAs).

3. Reference cabs Reference mAbs and a scFv lineage with known inhibitory properties were utilised to probe the nature of neutralizing epitopes on cell expressed Receptor A in order to further develop the screen and to set up the cell DELISA procedure. It is noteworthy that the NIH3T3 transfected cell line demonstrated no detectable receptor internalization (Figure 4 and Figure 5) as determined with the reference mAbs. It was also demonstrated during assay conditions that there was no receptor internalization of iodinated ligand. The cell line was tolerant of extended exposure to bacterial and phage supernatants (Figure 4, Figure 5 and Figure 6) and periplasmic extracts (determined by screen).

4. Sensitivity of detection Phage titre The tithe of phage in the phage supernatant rescued for the ELISA is crucial to providing a specific signal, as determined by titrating crude preparations of PEG precipitated phage. The phage antibodies used for this were from the scFv lineage that produced functional hits in the preliminary screening campaign (Figure 6).

The specific binding signal:noise ratio dramatically decreased as the phage titre was reduced, with 101 phage particles/ml to 10i phage particles/ml being the lower limit of phage titre able to produce a high specific binding signal.

Analysis of multiple wells containing the known scFv lineage revealed variable specific binding with phage supernatant derived from lml cultures (Figure 7). The culture conditions for generation of phage particles were investigated; shallow well conditions were compared with deep well conditions. The former provided less variability in specific binding signal, greater culture aeration and greater phage concentration in a smaller volume. Hence, shallow well overnight growth conditions were determined as optimal (Figure 8 and Figure 9).

b) Detection system Detection of bound phage was achieved using an antiM13Eu3+ conjugate (lOOng/ml with a labelling efficiency of 6.6 Eu3+ /molecule), which gave a specific signal that could be 10-20 times greater than background with transfected cell lines (Figure lea). However, the signal was lower for non-transfected cell lines, for example, 3-4 times greater than background reflecting the relative level of receptor expression. Anti-M13Eu3+ gave a much greater sensitivity of detection than anti-M13 HRP.

It is preferable for a transfected cell line to be employed in this method, thereby biasing the target towards being an abundant cell surface antigen, i.e., where there is a moderate to high level of expression. However, some non-transfected cell lines may

also be suitable.

5. Controls A non-transfected cell line (i.e., the parental cell line) should be used for comparison to distinguish cell binders from putative receptor binders (Figure 9), as employed for Receptor A. A null cell line (i.e. no expressed receptor) could be used in instances where the cell line in question is a primary cell line/not transfected. Positive controls should be included, such as a known phage antibody binder that is a functional hit in the screen or reference mAbs. All samples are assessed in duplicate with known controls (see e.g. Figure 9).

6. High throughput application An optimised DELISA was used to test two plates of unique sequence phage ELISA positive clones from the initial selection strategy, which had been performed using standard methods on the recombinant purified forms of antigen only. Each plate was screened in duplicate on Receptor A transfected NIH3T3 cells and the parental cell line (non-transfected). This yielded 11% and 13% putative native receptor binders for Plate 1 and Plate 2, respectively. Data for Plate 1 is shown in Figure 9. The results not only show that the shallow well format (A and B) provide consistent results compared with deep well format (C), but also reveal that 11% of phage ELISA positives on recombinant antigen were phage antibody binders in the cell DELISA, i.e., were positive for binding to recombinant antigen and transfected cells. This can be described as the overlap population. Outputs are prioritized for high throughput screening in the functional assay based on the extent of the sequence diversity and a high percentage of cell binders (and therefore native receptor binders). Particularly preferred outputs have a high percentage of overlap population.

EXAMPLE 2

Cell DELISA on adherent transfected cell line expressing Receptor

A

1. Introduction

As described above, confirmational differences between the purified recombinant forms of antigen and cell-expressed native receptor may have been responsible for the low hit rate observed in the preliminary lead isolation campaign. Experimental observations using control mAbs reinforced this hypothesis.

The lead isolation process outlined in Figure 2 was then followed all the way through to identify inhibitors of Receptor A, to validate the use of the pre-screen in the overall process and to increase the hit rate achieved in the functional assay. As part of the output assessment, the pre-screen indicates which selection strategies to prioritise for the high throughput functional screen.

2. Selection Ensuring optimal presentation of the recombinant form of antigen is critical to the successful isolation of phage antibodies that inhibit ligand binding to the native form of Receptor A. Complicating factors can include modifications such as biotinylation as used in soluble selection strategies. A novel plate based presentation, which involves covalent coupling (for example, Exiqon Protein Immobiliser_) to ensure optimal presentation of recombinant antigen, was employed.

Two rounds of panning selection were carried out on a naive phage antibody library in order to provide a sub-population of phage antibody binders, which were known to recognise the recombinant ECU of Receptor A. A proportion of this sub-population recognizes the receptor in its native conformation.

_ Assessment and pre-screen Selection outputs were simultaneously assessed by phage ELISA on recombinant ECD of Receptor A, cell DELISA and sequence diversity (see Figure 2). Outputs demonstrating a good hit rate in the cell DELISA (for example, 40% cell binders) were then targeted for the high throughput cell-based functional screen and further diverse hits were isolated.

Approximately 10,500 clones were screened as scFv periplasmic extracts giving 201 hits, restoring the expected hit rate to 2%.

Assessment of selection outputs by pre-screening with the cell DELISA prior to the functional screening assay was the key factor to rapid identification of inhibitory scFv binders, as evidenced by this strategy providing 8 unique clones with sub-50nM IC50 values and 4 unique clones with sub-lOOnM ICED values.

Additional benefits of this invention could be to use the conditions established for the cell DELISA to modify a cell selection strategy and to use the cell DELISA as a simple measure of validation for a cell-based selection strategy.

EXAMPLE 3

Cell DELISA on adherent, transfected cell line expressing Receptor B The cell DELISA was used to along with a Proximol_ technique (Osbourn et al 1998b; W098/01757; US5994519, US6180336, US6342588, US6489123), which was employed on HEK293 cells transfected with Receptor B. The transfected and non-transfected cells were immobilized on poly-D- lysine coated plates using a cell seeding concentration of 2 x 109 cells/well the previous day. Conditions were optimised using a phage antibody binder that was a functional hit in a previous lead isolation campaign.

Although this functional hit was clearly shown to be positive in the cell DELISA (Figure lOb), providing a specific binding signal times greater than background, no novel phage DELISA hits on cells (or phage ELISA hits on recombinant antigen) were observed and therefore alternative selection techniques were pursued.

EXAMPLE 4

Cell DELISA on suspension, non-transfected cell lines expressing Receptor C The cell DELISA was used to assess selection strategies after two rounds of selection on recombinant antigen representing the ECD of Receptor C. DELISA conditions for IM9 and HepG2 cells were validated using reference mAbs (Figure lOc). Whereas the level of specific signal for phage antibodies binding to transfected cells can be 10-20 times background, this is usually lower for non transfected cell lines (Figure led), where a lower limit of 3-4x background is applied as a cut-off. This reflects the relative expression level of the receptor of interest, but is sufficient for an effective "pre-screen".

EXPERIMENTAL PROCEDURES

1. Selection of phagemid library using Exiqon Protein Immobiliser_ plates Several wells of an Exigon Protein Immobiliser_ plate were coated with 300ul per well of recombinant antigen ECD Receptor A at lOug/ml in carbonate buffer (O.lM pH 9.6) and incubated overnight at 4 C. The following day the antigen was removed from the wells and the wells were washed three times with PBS/Tween.

Prior to this, a 50ul naive phage antibody library aliquot containing 1012 - 1013 of phage particles was blocked in 5% (w/v) Marvel_/PBS/Tween in a total volume of 200ul for the first round of selection. For subsequent rounds of selection, a 10-20ul volume of rescued phage was used. Phage were incubated in block for one hour at room temperature, after which the blocked phage were added to the washed selection wells. Note that the Exiqon Protein Immobiliser_ surface does not require blocking. The selection was incubated for 1.5 hours at 1050rpm (Titramax 100) at room temperature.

After this step, the plate wells were washed in the following manner at room temperature using 300ul buffer per well each time: one PBS/Tween wash for 30 seconds static, three PBS/Tween washes for 5 minutes at 750rpm (Titramax 100). Both wash steps were then repeated using PBS only in order to remove any unbound or weak phage antibody binders. Bound phage antibodies were eluted by pH elusion. Phage input and output tithes were determined.

2. Rescue of phagemid particles from selection output Bacterial lawn scrapes from each Bioassay plate were decanted into separate 50 ml Falcon tubes using 10ml of 2xTY broth per selection output and placed on a rotating mixer for 10 minutes at room temperature. 25ml of 2xTY broth containing 100ug/ml ampicillin and 2% (v/v) glucose (2TYAG) in a 250ml flask was inoculated with 100ul of the output (to give an OD600 of 0.3 to 0.5). The volume of cells should be sufficient to represent the output by at least 10-fold; if the output was less than 10 then 25ml would be sufficient. Each culture was grown for at least 1 hour at 37 C, 300rpm to mid-log (OD600 = 0.5 to 1.0). 6.25ul of M13K07 helper phage was added to each flask and rescues performed as described in Marks et al 1991.

For subsequent rounds of selection, lml of rescued phage culture was centrifuged in a microfuge for 10 minutes at 13000rpm to pellet the cells. The phage supernatant was transferred to a fresh Eppendorf microfuge tube and placed on ice. This was the phage input for the next round of selection (10-20ul).

3. Phage ELISA screening and sequencing of clones.

Individual colonies from the output tithe plates were picked and transfered into 100ul 2TYAG per well in 96-well format.

Microtitre plates were incubated at 37 C, 120rpm overnight. 50ul per well of 50% (v/v) sterile glycerol was added and the plates store at -70 C. These were the master plates used for subaulturing replica plates to be used for making sequencing template or for use in phage ELISA or cell DELISA. Phage ELISAs were carried out as previously described (Marks et al 1991) and ELISA plates were coated with recombinant antigen at a concentration of 1ug/ml in a similar manner to that used for coating selection surfaces. DNA sequencing was carried out on the same phage antibodies (Vaughan et al 1996) 4. Reference mAb ELISA mAbl and mAb2 were blocked in Marvel_/media or Marvel_/bacterial supernatant and corresponding cells or wells coated with recombinant antigen were blocked similarly. Washes were performed as for phage ELISAs and detection of bound reference mAbs either used an anti-mouse HRP conjugate with the TMB detection system or an anti-mouse Europium conjugate with the DELFIA_ reagent system.

5. Phage antibody cell-binding DELFIA assay (DELISA) If cells are not adherent, for this assay it is highly preferred to immobilise a confluent monolayer of cells and maintain this throughout the procedure in order to obtain meaningful data.

This protocol should be applied to determining the optimal surfaces for immobilization (e.g., poly-D-lysine, poly-L-lysine, collagen, fibronectin, gelatin, laminin, polyornithine coated 96 well plates) and checking that the media used for dissolving Marvel_ does not interfere with the system. This should be performed prior to any high throughput pre-screening campaign.

Note that MEM/1% NEAA/FCS, the media used for growth of HepG2 cells, has an inhibitory effect on the performance of this procedure, however RPMI/FCS is a suitable substitute for the duration of the pre-screen. A confluent monolayer of cells is required so seeding concentrations the prior day will be cell line dependent. Marvel_ is dissolved in media supplemented with FCS to a final concentration of 5%, which is adjusted to pH7.4 with 1M Hepes pH8.0 immediately prior to use.

If there is no parental cell line available for direct comparison, then putative receptor binders will be defined as a positive on duplicate plates, otherwise a hit is positive on duplicate transfected cell plates and negative on parental cells.

Positive controls and hits should give at least 3x to 4x background. As transfected cell lines give a greater specific signal, the cut-off can be higher. An irrelevant phage clone should be included as a negative control. Again, this may be cell line dependent. Should a positive phage control not be available, then use reference mAbs - these are usually mouse mAbs and therefore should be detected with anti-mouse Eu3+ conjugate (stock used is 1:200 dilution in DELFIA_ assay buffer). Other anti-host conjugates can be readily made as commercial labelling kits are available from Perkin Elmer and the anti-host antibody preparation to be conjugated should be BSA and azide free.

DELFIA_ assay wash was made in the following manner: lox wash stock 444g Trizma hydrochloride 265g Trizma base 806g sodium chloride 20g potassium chloride made to 10 litres with MilliQ water Working concentration DELFIA_ wash 1 litre 10x DELFIA_ wash stock 10ml Tween 20 (Sigma) made to 10 litres with MilliQ water DAY1: Shallow plate grow-up and phage rescue 2 x 96 well cultures containing 150,ul/well 2TYAG were set up by replica inoculation of a 96-well plate from the frozen master glycerol stock plate. 4 plates per sample were required (2 for transfected cells and 2 for parental cell line). Plates were incubated at 37 C, 120rpm for 5 hours. Stock helper phage M13 KO7 (103 titre) was diluted by taking 10,ul stock into 10ml 2TY and 15,ul of this dilution added to each well. Plates were incubated for 30 minutes at 37 C static and then for 30 minutes at 37 C, 120rpm, after which they were centrifuged at 2000rpm for 10 minutes and the supernatant decanted.

Pellets were resuspended in 150 ul/well 2TYAK (lOOug/ml ampicillin and 50ug/ml kanamycin) and phagemid particles were rescued in shallow well plates overnight at 30 C in 2TYAK, 120 rpm DAY2: Cell-binding assay (DELISA) Plates were centrifuged at 2000rpm for 10 minutes and the phage containing supernatants immediately transfered to a fresh plate, combining duplicate wells.

Media was gently removed off the monolayer of immobilized cells and the cells blocked with 4%(w/v) Marvel_/media (final concentration) 200 ul/well for 1 hour at room temperature.

Phage were blocked for 1 hour at room temperature with 4%(w/v) MarvelTM/media (final concentration). The block was gently decanted from the cells and the cells were washed once in xl PBS.

The plates were gently blotted dry and then the blocked phage were added, 50 ul/well, incubating for 1 hour at room temperature.

Plates were washed three times each in lx PBS and then anti- M13Eu3+ conjugate diluted to lOOng/ml in DELFIA_ assay buffer added (warm DELFIA_ assay buffer up to rt prior to this step).

50 ul/well was used and the plates were incubated for 1 hour at room temperature. The plates were then washed seven times in DELFIA_ assay wash and 100 ul/well enhancement solution added.

Plates were left at room temperature for 5 minutes prior to reading on Wallac Victor (excitation at 340nm and emission at 615nm). Positives on duplicate plates of transfected cells were compared with each other and the duplicate plates of parental cells to define a hit.

REFERENCES

Cal and Garen. Proc Natl Acad Sci USA 92 1995 pp6537-6541 Edwards et al. J Immunological Methods 245 2000 pp67-78 Figini et al. (1998). Cancer Research. 58, 991-996.

Hartley. J Receptor and Signal Transduction 22 (1-4) 2002 pp373 Hegmans et al. J Immunological Methods 262 2002 ppl91-204 Hoogenboom et al. Eur J Biochem 260 1999 pp774-784 Marks et al. J Mol Biol 222 1991 581-597 Marks et al. Bio/Technology 11 1993 ppll45-1149 Mutaberia et al. J Immunological Methods 231 1999 pp65-81 Osbourn et al. Immunotechnology 3 1998b 293-302 Osbourn et al. Nature Biotechnology 16 1998a pp778-781 Palmer et al. Immunology 91 1997 pp473-478 Peipp et al. J Immunological Methods 251 2001 ppl61-176 Pereira et al. J Immunological Methods 203 1997 ppll-24 Spear et al. Cancer Gene Therapy 8 (7) 2001 pp506-511 Topping et al. (2000) International Journal of Oncology. 16, 187-195.

Tur et al. Int J Molecular Medicine 11 2003 pp523-527 Van der Vuurst de Vries and Logtenberg. Immunology 98 1999 pp55 Vaughan et al. Nature Biotechnology 14 1996 309-314 Williams and Sharon (2002) Immunology Letters. 81, 141-148.

Williams et al. (2002) Combinatorial Chemistry and High Throughput Screening. 5, 489-499.

Project Receptor A Receptor B Receptor C Cell line NIH3T3 HEK293 IM-9 transfected transfected HepG2 Parental Parental HL60* no null Level of expn 10e5/cell HEK293 Moderate moderate (FACS) Type Adherent Adherent Suspension Immobilisation Collagen Poly-D- lysine Poly-D-lysine Cell seeding 2.5x10e4 2 x 10e4 1 x 10e5 (cells/well) Val idatio n mAb 1 Functional hit mAb3 mAb2 in original Ll mAb4 functional Ll hit App I ication Hybrid Val idation of Hybrid selections selection selections Pre-screen to technique Pre-screen to prioritise prioritise outputs for outputs for

HTS HTS

Overlap 11% No hits on rec 7% Ag or cells Functional hits Yes (cell) N/a Yes in HT screen (biochemical)

Claims (30)

1. I

   CLAIMS: 1. A method of producing specific binding members for a cell surface antigen, the method comprising: (i) providing cells containing nucleic acid encoding the cell surface antigen so the cells produce the cell surface antigen and incorporate it into the outer surface of the cells; (ii) immobilizing the cells on a solid surface, without fixation of the cells; (iii) contacting or incubating the cells on the solid surface with a phage display library, in which phage particles collectively display a diversity of specific binding members from which specific binding members for the cell surface antigen may

   be selectable;

   (iv) contacting or incubating control cells with the phage display library, which control cells do not produce the cell surface antigen and incorporate it into their outer surface; wherein non-specific binding in (iii) and (iv) of specific binding members displayed in the phage display library is blocked; (v) identifying phage displaying a specific binding member for the cell surface antigen by: (a) determining binding of an anti-phage binding molecule to phage bound to cells in (iii); (b) determining binding of the anti-phage binding molecule to phage bound to cells in (iv); (c) comparing binding of the anti-phage binding molecule in (a) and (b), to identify a first product population of phage displaying specific binding members for the cell surface antigen; (vi) obtaining nucleic acid which has a nucleotide sequence encoding the specific binding members displayed on phage of the first product population and producing one or more specific binding members for the cell surface antigen by recombinant expression from nucleic acid with the nucleotide sequence or a derivative nucleotide sequence to produce a product specific binding member.
2. 2. A method according to claim 1 comprising providing the phage display library contacted with the cells on the solid surface in step (iii) as a fraction of an initial phage display library, which fraction is provided by performing a selection to identify the fraction of phage in the initial phage display library that display specific binding members that bind isolated cell surface antigen.
3. 3. A method according to claim 2 wherein the isolated cell surface antigen is recombinantly produced.
4. 4. A method according to any one of claims 1 to 3 comprising performing a further selection with the first product population to identify phage displaying specific binding members that bind isolated cell surface antigen, to identify a subpopulation of the first product population.
5. 5. A method according to claim 4 wherein the isolated cell surface antigen is recombinantly produced.
6. 6. A method according to any one of claims 1 to 5 further comprising performing in parallel a selection with the same phage display library as employed in (iii) on isolated cell surface antigen to obtain a second product population of phage displaying specific binding members for the cell surface antigen, and comparing the first product population or said subpopulation of the first product population and the second product population to identify phage displaying specific binding members common to both the first product population or said subpopulation of the first product population and the second product population.
7. 7. A method according to claim 6 wherein the isolated cell surface antigen is recombinantly produced.
8. 8. A method according to any one of claims 1 to 7 wherein the cells provided in (i) are transgenic for nucleic acid encoding the cell surface antigen so that the cells produce the cell surface antigen recombinantly and incorporate it into the outer surface of the cells.
9. 9. A method according to any one of claims 1 to 8 wherein the cells are immobilized on a collagen-coated surface.
10. 10. A method according to any one of claims 1 to 8 wherein the cells are immobilised on a poly-D-lysine-coated surface.
11. 11. A method according to any one of claims 1 to 8 wherein the cells are immobilised on a surface coated with a material selected from the group consisting of poly-L-lysine, fibronectin, laminin and polyornithine.
12. 12. A method according to any one of claims 1 to 11 wherein contacting the cells on the solid surface with a phage display library is in multiple wells with the phage at a titre of at least 101 pfu per well.
13. 13. A method according to any one of claims 1 to 12 wherein the phage display library is provided by growth and rescue in 100 200ul culture volume in 96 well plates or SOpl culture volume or less in 384 well plates.
14. 14. A method according to any one of claims 1 to 13 wherein non specific binding in (iii) and (iv) is blocked with 4-5%(w/v) milk powder.
15. 15. A method according to any one of claims 1 to 14 wherein in (a) and (b) the anti-phage binding molecule is labelled with a label.
16. 16. A method according to claim 15 wherein the label is Europium.
17. 17. A method according to any one of claims 1 to 16 wherein contacting of cells with a phage display in step (iii) and/or (iv) is performed in duplicate experiments.
18. 18. A method according to any one of claims 1 to 17 wherein binding of the anti-phage binding molecule in (a) and (b) is compared in (c) in duplicate experiments.
19. 19. A method according to any one of claims 1 to 18 wherein a derivative nucleotide sequence is provided by mutation of nucleic acid with said nucleotide sequence.
20. 20. A method according to any one of claims 1 to 19 wherein a derivative nucleotide sequence is provided by fusion of additional nucleotides to nucleic acid with said nucleotide sequence.
21. 21. A method according to any one of claims 1 to 20 wherein a derivative nucleotide sequence is provided by removal of nucleotides from nucleic acid with said nucleotide sequence.
22. 22. A method according to any one of claims 1 to 21 wherein the displayed specific binding members each comprise an antibody VH domain.
23. 23. A method according to claim 22 wherein the displayed specific binding members each comprise an antibody VL domain.
24. 24. A method according to claim 23 wherein the displayed specific binding members are scFv antibody molecules.
25. 25. A method according to any one of claims 22 to 24 wherein a derivative nucleotide sequence is provided comprising a nucleotide sequence encoding an antibody VH domain.
26. 26. A method according to claim 25 wherein a product specific binding member which is an antibody IgG molecule is produced by recombinant expression from nucleic acid comprising a derivative nucleotide sequence.
27. 27. A method according to any one of claims 1 to 26 wherein a product specific binding member is produced by recombinant expression and formulated into a composition comprising at least one additional component.
28. 28. A method according to claim 27 wherein the composition comprises a pharmaceutically acceptable excipient, vehicle or caroler.
29. 29. A method according to any one of the preceding claims further comprising bringing into contact the product specific binding member and an antigen.
30. 30. A method according to claim 29 wherein said contact is in vitro. :