WO2009089998A1 - Binding members for tenascin-c domain a2 - Google Patents

Abstract

Binding members, e.g. antibody molecules, for domain A2 of human tenascin-C, capable of recognising tenascin-C isoforms containing domain A2 in human tissue. Binding members that bind a loop region of domain A2 comprising sequence Thr-Ala-Pro-Glu-Gly-Ala-Tyr-Glu-Tyr-Phe. Use of the binding members for diagnosis and/or treatment of conditions in which tenascin-C isoforms containing domain A2 are expressed, e.g. proliferative disorders such as cancer.

Description

Binding Members for Tenascin-C Domain A2

This invention relates to binding members directed to tenascin-C, in particular human antibodies against human tenascin-C, and their use in medicine, for example in the diagnosis and/or treatment of cell proliferation, such as cancer, tumourigenesis and/or angiogenesis .

A promising avenue towards the development of selective anti-cancer therapies is the targeted delivery of bioactive molecules to the tumour site by means of ligands specific to tumour associated antigens. Proteins that are preferentially expressed in the modified tumour extracellular matrix are ideal antigens for tumour targeting applications [1, 2].

Tenascin-C is a large hexameric glycoprotein of the extracellular matrix which modulates cellular adhesion. It is involved in processes such as cell proliferation and cell migration and is associated with changes in tissue architecture as occurring during morphogenesis and embryogenesis as well as under tumorigenesis or angiogenesis.

The schematic domain structure of tenascin-C is depicted in Figure 1. It comprises several fibronectin type-III repeats that can be either included or omitted in the primary transcript by alternative splicing, leading to small and large isoforms. Several isoforms of tenascin-C can be generated as a result of alternative splicing which may lead to the inclusion of (multiple) domains in the central part of this protein, ranging from domain Al to domain D [3, 4]. The large isoform of tenascin-C, containing Al-D alternatively spliced domains, is virtually undetectable in healthy adult tissues but is expressed during embryogenesis and tissue remodelling including neoplasia [3, 4] .

The alternatively spliced domains can be independently omitted or included in the molecule leading to several different tenascin-C splicing variants with different expression patterns. For example, the spliced repeat C is barely detectable in tumours positive for Al and D (breast and lung carcinomas) but it is extremely abundant in high-grade astrocytoma (grade III and glioblastoma) [5, 6, 7].

An additional level of complexity is provided by the presence or absence of post-translational modifications (e.g. glycosylation) , which may modify certain epitopes on the surface of individual protein domains and make them unavailable to a specific molecular recognition in vitro or in vivo to specific monoclonal antibodies.

Several monoclonal antibodies have been generated against the alternatively spliced domains of tenascin-C. Radiolabeled antibodies specific to domains Al and D have been successfully used in the clinic for the treatment of glioma and lymphoma [8, 9] and efficient tumor targeting by anti-tenascin antibodies has been shown by the avidin/biotin-based pre-targeting approach [10].

Even though the rapid isolation of antibodies specific to virtually any protein of interest can be accomplished with existing methodologies in vitro, such antibodies do not necessarily recognise the epitope in biological specimens or in animal models of disease. Possible reasons for lack of binding in vivo include post- translational modifications of the epitope, masking of the epitope and insufficient antibody specificity or stability. It is therefore difficult to assess the suitability of monoclonal antibodies for practical applications based solely on their reactivity with recombinant antigens (or antigen fragments) in typical solid-phase assays, such as enzyme-linked immunosorbent assays (ELISA) , which are routinely used for the screening of monoclonal antibodies. Monoclonal antibodies to the individual domains of the tenascin-C large isoforms therefore need to be analysed individually, in order to evaluate their suitability for diagnostic and therapeutic applications .

Zardi et al. [11] isolated and characterised monoclonal antibodies specific for domains of human tenascin-C, including one antibody to domain A2. Some of the antibodies were able to react strongly in immunoblotting but showed only weak interactions in immunofluorescence or immunohistochemistry . These data indicate that some epitopes are masked in the tissue, which may be due to the conformation of the native tenascin-C molecule, to the interactions of tenascin with other proteins and/or to the glycosylation state of the molecule [H].

Binding to different epitopes of the alternatively spliced tenascin-C domains may be required in order to achieve recognition of the antigen in the native environment.

This invention relates to binding members for domain A2 of human tenascin-C and their use in therapeutic and/or diagnositic applications .

We expressed recombinant A2 domain of human tenascin-C and generated a human monoclonal antibody against this domain. The antibody, designated C12, was able to recognise the antigen not only in ELISA, but also on freshly frozen glioblastoma sections where it showed an impressive peri-vascular staining.

Furthermore we were able to identify the epitope of the anti-A2 antibody C12 using mass-spectrometry analysis.

Disclosed herein are binding members for domain A2 of human tenascin- C, wherein the binding members bind human tenascin-C comprising domain A2 in human tissue, for example in biological specimens, such as in glioblastoma tissue (e.g. in a U87 human glioblastoma cell xenograft) . Binding of binding members to the antigen in human tissue may be demonstrated for example by immunofluorescence or immunohistochemistry, as illustrated in Example 3.

The ability of binding members of the invention to recognise domain A2 of tenascin-C in human tissue indicates that they bind an epitope in domain A2 that is not masked in the tissue. We investigated the region of domain A2 that is bound by monoclonal antibody molecule C12, which binds human tenascin-C comprising domain A2 in human tissue as demonstrated herein.

Epitope mapping experiments indicated that C12 recognised residues located on a loop between two β-strands of domain A2.

Our data indicated that the epitope comprised one or more residues within the sequence Thr-Ala-Pro-Glu-Gly-Ala-Tyr-Glu-Tyr-Phe (SEQ ID NO: 19, TAPEGAYEYF) at positions 26 to 35 of domain A2. Further investigation of the epitope indicated that C12 binds one or more residues within the sequence Gly-Ala-Tyr-Glu (SEQ ID NO: 38, GAYE) at positions 30 to 33 of domain A2. The sequence of domain A2 is shown in SEQ ID NO: 18.

We have thus identified an epitope in domain A2 that appears to be present in the native conformation of the tenascin-C molecule comprising domain A2 and is not masked by interaction of tenascin-C with other proteins and/or by glycosylation . The invention relates to binding members for this region of domain A2.

The invention provides a binding member that binds at least one residue of Thr-Ala-Pro-Glu-Gly-Ala-Tyr-Glu-Tyr-Phe (SEQ ID NO: 19) at positions 26 to 35 of domain A2 (SEQ ID NO: 18) of human tenascin-C. The binding member may bind at least one residue of Gly-Ala-Tyr-Glu (SEQ ID NO: 38) at positions 30 to 33 of domain A2, and optionally may also bind one or more other residues of Thr-Ala-Pro-Glu-Gly-Ala- Tyr-Glu-Tyr-Phe (SEQ ID NO: 19) at positions 26 to 35 of domain A2 of human tenascin-C.

A binding member may bind one, two, three, four or all residues of SEQ ID NO: 19 and/or SEQ ID NO: 38. Optionally a binding member may bind flanking residues or structurally neighbouring residues in the tenascin-C molecule, e.g. in domain A2, in addition to binding one or more residues in SEQ ID NO: 19 and/or SEQ ID NO: 38. Any suitable method may be used to determine the residues bound by a binding member, e.g. mass spectrometry, hydrogen-deuterium exchange, site-directed mutagenesis, NMR and X-ray crystallography.

For example, as described in Example 4, a method of identifying residues of domain A2 bound by a binding member may comprise incubating domain A2 of human tenascin-C with the binding member and a protease, under conditions for digestion of domain A2 by the protease, and analysing the resulting fragments. Typically, a protease with a broad cleavage site specificity, such as proteinase K, is used. The presence of a fragment containing a cleavage site for the protease within its sequence indicates that the binding member protected that cleavage site from digestion by the protease, and thus indicates that the binding member binds one or more residues in that fragment. A binding member of the invention may mask a protease cleavage site in the amino acid sequence SEQ ID NO: 19, identifiable by the presence of SEQ ID NO: 19 or a fragment thereof, e.g. SEQ ID NO: 38, containing the cleavage site. A binding member of the invention may mask a protease cleavage site in SEQ ID NO: 19 or SEQ ID NO: 38. Where proteinase K is used, this may be identifiable by the detection of SEQ ID NO: 19 or SEQ ID NO: 38, respectively. Mass spectrometry may be used to analyse the fragments obtained by protease digestion.

The method may optionally further comprise contacting the fragments of domain A2, which were obtained from the protease digestion, with the binding member immobilised on a support, and determining whether the fragment containing a cleavage site for the protease within its sequence is bound by the binding member. For example, the binding member may be bound to a column, the fragments of domain A2 may be run through the column, and the flow-through may be analysed for the presence of the fragments. Mass spectrometry may be used to analyse the fragments. The absence of one or more fragments from the flow- through indicates that the fragment or fragment was bound by the binding member on the column. Where a fragment containing a cleavage site for the protease is absent from the flow-through, this provides further evidence that the binding member binds one or more residues of that fragment, indicating that the fragment contains at least part of the epitope of domain A2 recognised by the binding member.

Binding members which bind a particular peptide may be isolated for example from a phage display library by panning with the peptide with or a polypeptides comprising the peptide.

Isolated peptides consisting of SEQ ID NO: 19 or fragments thereof, e.g. fragments comprising SEQ ID NO: 38, or polypeptides comprising SEQ ID NO: 19 or fragments thereof e.g. fragments comprising SEQ ID NO: 38, may be utilised in methods of generating, isolating and/or testing further binding members for domain A2 of human tenascin-C according to the present invention.

Accordingly, further aspects of the invention relate to isolated fragments of domain A2 of human tenascin-C, comprising or consisting of amino acid sequence SEQ ID NO: 19 or fragments thereof, e.g. fragments comprising or consisting of SEQ ID NO: 38. Fragments of SEQ ID NO: 19 may for example be up to 4, 5, 6, 7, 8 or 9 amino acids long. One or more fragments may be contained within a longer isolated peptide or polypeptide sequence which is not a domain A2 amino acid sequence, e.g. which is not SEQ ID NO: 18. Peptides or polypeptides comprising an isolated fragment or fragments of the domain A2 amino acid sequence may comprise additional amino acid residues, wherein the additional residues are non-contiguous with a domain A2 amino acid sequence. For example, SEQ ID NO: 19 or SEQ ID NO: 38 may be followed and/or or preceded by one or more residues non-contiguous with SEQ ID NO: 18.

Binding members according to the invention represent good candidates for use in binding tenascin-C comprising domain A2 in human tissue, such as in human therapeutic and/or diagnostic applications where it is desirable to specifically target isoforms of tenascin-C comprising domain A2. As noted above, human tenascin-C isoforms comprising domain A2 are generally not found in healthy adult tissue. Isoforms comprising domain A2 are alternatively spliced variants that are predominantly found in neoplastic tissue such as cancers, tumours and sites of angiogenesis e.g. pathological angiogenesis . Thus, binding members according to the invention may be tumour specific, binding preferentially to tumour tissue relative to normal tissue. Binding members may, for example, bind to stroma and/or neo- and perivascular structures of tumour tissue preferentially to normal tissue.

A binding member of the invention may bind human tenascin-C comprising domain A2 , and optionally comprising domain Al, A3, A4, B, AD, C and/or D. A binding member of the invention may not bind human tenascin-C that does not contain domain A2, or may bind it with lower affinity, e.g. at least 10-fold or at least 100-fold lower affinity. Thus, a binding member may bind preferentially to tenascin-C large isoform relative to tenascin-C small isoform.

Binding members of the invention are useful for treatment or diagnosis of individuals. For example, a binding member of the invention may be used in a method of treatment of the human body by surgery or therapy, or in a diagnostic method practised on the human body or a tissue sample.

For example, a binding member of the invention may be used for treatment and/or diagnosis of a proliferative disorder in which a tenascin-C isoform containing domain A2 is expressed, as discussed in detail elsewhere herein.

A binding member of the invention may have an affinity for domain A2 of human tenascin-C wherein K_D of interaction of the binding member with domain A2 is less than 100 μM, e.g. K_D may be less than 50 μM, less than 10 μM, less than 5 μM, less than 2 μM or less than 1.5 μM. Methods of affinity optimisation are known, and may be used to obtain binding members of even higher affinity.

Binding kinetics and affinity of binding members for domain A2 of human tenascin-C may be determined using surface plasmon resonance e.g. BIAcore, as described in Example 2. Surface plasmon resonance involves passing an analyte in fluid phase over a ligand attached to a support, and determining binding between analyte and ligand. Surface plasmon resonance may for example be performed whereby a binding member is passed in fluid phase over domain A2 attached to a support. The isolated A2 domain of tenascin-C, SEQ ID NO: 18, may be recombinantly produced by expression in E. coli for use in this method. An affinity constant K_D may be calculated from the ratio of rate constants kdl/kal as determined by surface plasmon resonance. K_D of interaction of antibody C12 with domain A2 was estimated at approximately 1.18 x 10^~6 M using surface plasmon resonance - see Example 2.

A binding member of the invention may comprise an antibody molecule, e.g. a human antibody molecule. The binding member normally comprises an antibody VH and/or VL domain. VH and VL domains of binding members are also provided as part of the invention. Within each of the VH and VL domains are complementarity determining regions, ("CDRs") , and framework regions, ("FRs") . A VH domain comprises a set of HCDRs, and a VL domain comprises a set of LCDRs. An antibody molecule may comprise an antibody VH domain comprising a VH CDRl, CDR2 and CDR3 and a framework. It may alternatively or also comprise an antibody VL domain comprising a VL CDRl, CDR2 and CDR3 and a framework. A VH or VL domain framework comprises four framework regions, FRl, FR2, FR3 and FR4 , interspersed with CDRs in the following structure:

FRl - CDRl - FR2 - CDR2 - FR3 - CDR3 - FR4.

Examples of antibody VH and VL domains, FRs and CDRs according to the present invention are as listed in the appended sequence listing that forms part of the present disclosure. The sequence listing shows the VH domain, VL domain, HCDRs, LCDRs and FRs for antibody C12. The VH and VL sequences, CDR sequences, sets of CDRs and sets of HCDRs and sets of LCDRs disclosed herein represent aspects and embodiments of the invention. As described herein, a "set of CDRs" comprises CDRl, CDR2 and CDR3. Thus, a set of HCDRs refers to HCDRl, HCDR2 and HCDR3, and a set of LCDRs refers to LCDRl, LCDR2 and LCDR3. Unless otherwise stated, a "set of CDRs" includes HCDRs and LCDRs. Typically binding members of the invention are monoclonal antibodies.

A binding member of the invention may comprise an antigen-binding site within a non-antibody molecule, normally provided by one or more CDRs e.g. a set of CDRs in a non-antibody protein scaffold, as discussed further below.

The C12 antibody molecule described herein has a set of CDRs in which : the amino acid sequence of HCDRl is SEQ ID NO: 3, the amino acid sequence of HCDR2 is SEQ ID NO: 4, the amino acid sequence of HCDR3 is SEQ ID NO: 5, the amino acid sequence of LCDRl is SEQ ID NO: 8, the amino acid sequence of LCDR2 is SEQ ID NO: 9, and the amino acid sequence of LCDR3 is SEQ ID NO: 10.

Thus, the C12 set of HCDRs consists of HCDRl SEQ ID NO: 3, HCDR2 SEQ ID NO: 4 and HCDR3 SEQ ID NO: 5, and the C12 set of LCDRs consists of LCDRl SEQ ID NO: 8, LCDR2 SEQ ID NO: 9 and LCDR3 SEQ ID NO: 10.

As described in the Examples, the C12 antibody was isolated from the ETH-2 GOLD library [66], in which the antibody sequence diversity is provided by randomisation of residues in HCDR3 and LCDR3.

A binding member of the invention may comprise: an HCDR3 amino acid sequence SEQ ID NO: 5 or an amino acid sequence having one or two amino acid substitutions in SEQ ID NO: 5, and/or an LCDR3 amino acid sequence SEQ ID NO: 10 or an amino acid sequence having one or two amino acid substitutions in SEQ ID NO: 10. A binding member of the invention may comprise an HCDRl amino acid sequence SEQ ID NO: 3 or an amino acid sequence having one or two amino acid substitutions in SEQ ID NO: 3.

A binding member of the invention may comprise an HCDR2 amino acid sequence SEQ ID NO: 4 or an amino acid sequence having one or two amino acid substitutions in SEQ ID NO: 4.

A binding member of the invention may comprise an LCDRl amino acid sequence SEQ ID NO: 8 or an amino acid sequence having one or two amino acid substitutions in SEQ ID NO: 8.

A binding member of the invention may comprise an LCDR2 amino acid sequence SEQ ID NO: 9 or an amino acid sequence having one or two amino acid substitutions in SEQ ID NO: 9.

A binding member of the invention may comprise an HCDRl, HCDR2, HCDR3, LCDRl, LCDR2, and/or LCDR3 from the C12 set of HCDRs.

A binding member of the invention may comprise a set of HCDRs:

HCDRl, HCDR2 and HCDR3, wherein the set of HCDRs has no more than 10 amino acid substitutions compared with the C12 set of HCDRs, in which : the amino acid sequence of HCDRl is SEQ ID NO: 3, the amino acid sequence of HCDR2 is SEQ ID NO: 4, and the amino acid sequence of HCDR3 is SEQ ID NO: 5.

A binding member of the invention may comprise a set of LCDRs: LCDRl, LCDR2 and LCDR3, wherein the set of LCDRs has no more than 10 amino acid substitutions compared with the C12 set of LCDRs in which: the amino acid sequence of LCDRl is SEQ ID NO: 8, the amino acid sequence of LCDR2 is SEQ ID NO: 9, and the amino acid sequence of LCDR3 is SEQ ID NO: 10. A binding member of the invention may comprise a set of CDRs: HCDRl, HCDR2, HCDR3, LCDRl, LCDR2 and LCDR3, wherein the set of CDRs has no more than 10 amino acid substitutions compared with the C12 set of CDRs in which: the amino acid sequence of HCDRl is SEQ ID NO: 3, the amino acid sequence of HCDR2 is SEQ ID NO: 4, the amino acid sequence of HCDR3 is SEQ ID NO: 5, the amino acid sequence of LCDRl is SEQ ID NO: 8, the amino acid sequence of LCDR2 is SEQ ID NO: 9, and the amino acid sequence of LCDR3 is SEQ ID NO: 10.

There may be less than 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid substitutions in the set of CDRs, HCDRs or LCDRs. For example, there may be one or two substitutions.

A binding member of the invention may comprise an HCDRl, HCDR2,

HCDR3, LCDRl, LCDR2 and/or LCDR3, wherein: the amino acid sequence of HCDRl is SEQ ID NO: 3, the amino acid sequence of HCDR2 is SEQ ID NO: 4, the amino acid sequence of HCDR3 is SEQ ID NO: 5, the amino acid sequence of LCDRl is SEQ ID NO: 8, the amino acid sequence of LCDR2 is SEQ ID NO: 9, and the amino acid sequence of LCDR3 is SEQ ID NO: 10.

For example, the binding member may comprise a set of HCDRs wherein: the amino acid sequence of HCDRl is SEQ ID NO: 3, the amino acid sequence of HCDR2 is SEQ ID NO: 4, and the amino acid sequence of HCDR3 is SEQ ID NO: 5 and/or may comprise a set of LCDRs wherein the amino acid sequence of LCDRl is SEQ ID NO: 8, the amino acid sequence of LCDR2 is SEQ ID NO: 9, and the amino acid sequence of LCDR3 is SEQ ID NO: 10.

Typically, an antibody VH domain is paired with an antibody VL domain to provide an antibody antigen-binding site, although as discussed further below a VH or VL domain alone may be used to bind antigen. A VH domain comprising the C12 set of HCDRs, e.g. the C12 VH domain SEQ ID NO: 2, may be paired with a VL domain comprising the C12 set of LCDRs, e.g. the C12 VL domain SEQ ID NO: 7. Thus, the C12 VH domain may be paired with the C12 VL domain so that an antibody antigen- binding site is formed comprising both the antibody C12 VH and VL domains. Alternatively, the a VH domain comprising the C12 set of HCDRs may be paired with a VL domain other than the a VL domain comprising the C12 set of LCDRs. Light-chain promiscuity is well established in the art.

One aspect of the invention is an isolated antibody molecule comprising a VH domain with the VH domain amino acid sequence shown in SEQ ID NO: 2 and a VL domain with the VL domain amino acid sequence shown in SEQ ID NO: 7.

A binding member may comprise an antibody molecule having one or more CDRs, e.g. a set of CDRs, within an antibody framework. For example, one or more CDRs or a set of CDRs of an antibody may be grafted into a framework (e.g. human framework) to provide an antibody molecule. The framework regions may be of human germline gene segment sequences. Thus, the framework may be germlined, whereby one or more residues within the framework are changed to match the residues at the equivalent position in the most similar human germline framework. The skilled person can select a germline segment that is closest in sequence to the framework sequence of the antibody before germlining and test the affinity or activity of the antibodies to confirm that germlining does not significantly reduce antigen binding or potency in assays described herein. Human germline gene segment sequences are known to those skilled in the art and can be accessed for example from the VBase compilation.

A binding member of the invention may be an isolated human antibody molecule having a VH domain comprising a set of HCDRs in a human germline framework, e.g. DP47. Thus, the VH domain framework regions FRl, FR2 and/or FR3 may comprise framework regions of human germline gene segment DP47 and/or may be germlined by mutating framework residues to match the framework residues of this human germline gene segment. FR4 may comprise a framework region of a human germline j segment. The amino acid sequence of VH FRl may be SEQ ID NO: 30. The amino acid sequence of VH FR2 may be SEQ ID NO: 31. The amino acid sequence of VH FR3 may be SEQ ID NO: 32. The amino acid sequence of VH FR4 may be SEQ ID NO: 33. The binding member may also have a VL domain comprising a set of LCDRs, e.g. in a human germline framework, e.g. DPLl 6. Thus, the VL domain framework regions may comprise framework regions FRl, FR2 and/or FR3 of human germline gene segment DPLl 6 and/or may be germlined by mutating framework residues to match the framework residues of this human germline gene segment. FR4 may comprise a framework region of a human germline j segment. The amino acid sequence of VL FRl may be SEQ ID NO: 34. The amino acid sequence of VL FR2 may be SEQ ID NO: 35. The amino acid sequence of VL FR3 may be SEQ ID NO: 36. The amino acid sequence of VL FR4 may be SEQ ID NO: 37. A germlined VH or VL domain may or may not be germlined at one or more Vernier residues.

An antibody molecule or a VH domain of the invention may comprise the following set of heavy chain framework regions: FRl SEQ ID NO: 30;

FR2 SEQ ID NO: 31;

FR3 SEQ ID NO: 32;

FR4 SEQ ID NO: 33; or may comprise the said set of heavy chain framework regions with one, two, three, four or five amino acid alterations, e.g. substitutions .

An antibody molecule or a VL domain of the invention may comprise the following set of light chain framework regions: FRl SEQ ID NO: 34;

FR2 SEQ ID NO: 35;

FR3 SEQ ID NO: 36;

FR4 SEQ ID NO: 37; or may comprise the said set of light chain framework regions with one, two, three, four or five amino acid alterations, e.g. substitutions . For example, an antibody molecule of the invention may comprise a set of heavy and light chain framework regions, wherein heavy chain FRl is SEQ ID NO: 30; heavy chain FR2 is SEQ ID NO: 31; heavy chain FR3 is SEQ ID NO: 32; heavy chain FR4 is SEQ ID NO: 33; light chain FRl is SEQ ID NO: 34; light chain FR2 is SEQ ID NO: 35; light chain FR3 is SEQ ID NO: 36; light chain FR4 is SEQ ID NO: 37; or may comprise the said set of heavy and light chain framework regions with 10 or fewer, e.g. five or fewer, amino acid alterations, e.g. substitutions. For example there may be one or two amino acid substitutions in the said set of heavy and light chain framework regions .

A binding member of the invention may be one which competes for binding to domain A2 of human tenascin-C with an antibody molecule comprising the C12 VH domain SEQ ID NO: 2 and the C12 VL domain SEQ

ID NO: 7. Thus, a further aspect of the present invention provides a binding member comprising a human antibody antigen-binding site that competes with an antibody molecule comprising the VH and VL domain of antibody C12 for binding to domain A2 of human tenascin-C, e.g. competes with scFv C12 as shown in SEQ ID NO: 12.

Competition between binding members may be assayed easily in vitro, for example using ELISA and/or by tagging a specific reporter molecule to one binding member which can be detected in the presence of one or more other untagged binding members, to enable identification of binding members which bind the same epitope or an overlapping epitope. Competition may be conveniently determined using the isolated A2 domain, rather than the whole tenascin-C molecule. The A2 domain sequence is shown in SEQ ID NO: 18. In addition to antibody sequences, a binding member according to the present invention may comprise other amino acids, e.g. forming a peptide or polypeptide, such as a folded domain, or to impart to the molecule another functional characteristic in addition to ability to bind antigen.

Binding members of the invention may carry a detectable label, for example an agent which facilitates tumour detection such as a radionuclide or fluorophore, or may be conjugated to an agent capable of triggering a biocidal event, such as a radionuclide, photosensitiser, drug, cytokine, pro-coagulant factor, toxin or enzyme (e.g. via a peptidyl bond or linker), for use in a method of therapy.

In further aspects, the invention provides an isolated nucleic acid which comprises a sequence encoding a binding member, VH domain and/or VL domain according to the present invention, and methods of preparing a binding member, a VH domain and/or a VL domain of the invention, which comprise expressing said nucleic acid under conditions to bring about production of said binding member, VH domain and/or VL domain, and recovering it.

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

Further aspects of the present invention provide for compositions containing binding members of the invention, and their use in methods of binding tenascin-C comprising domain A2, including methods of treatment and diagnosis.

Binding members according to the invention may be used in a method of treatment or diagnosis, such as a method of treatment (which may include prophylactic treatment) of a disease or disorder in the human or animal body (e.g. in a human patient), which comprises administering to said patient an effective amount of a binding member of the invention. Conditions treatable in accordance with the present invention include any in which tenascin-C containing domain A2 plays a role, as discussed in detail elsewhere herein.

These and other aspects of the invention are described in further detail below.

Terminology

Binding member

This describes one member of a pair of molecules that bind one another. The members of a 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 binds to and is therefore complementary to a particular spatial and polar organization of the other member of the pair of molecules. Examples of types of binding pairs are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The present invention is concerned with antigen-antibody type reactions.

A binding member normally comprises a molecule having an antigen- binding site. For example, a binding member may be an antibody molecule or a non-antibody protein that comprises an antigen-binding site .

An antigen binding site may be provided by means of arrangement of CDRs on non-antibody protein scaffolds, such as fibronectin or cytochrome B etc. [12, 13, 14], or by randomising or mutating amino acid residues of a loop within a protein scaffold to confer binding specificity for a desired target. Scaffolds for engineering novel binding sites in proteins have been reviewed in detail by Nygren et al. [14]. Protein scaffolds for antibody mimics are disclosed in WO/0034784, which is herein incorporated by reference in its entirety, in which the inventors describe proteins (antibody mimics) that include a fibronectin type III domain having at least one randomised loop. A suitable scaffold into which to graft one or more CDRs, e.g. a set of HCDRs or an HCDR3 and/or LCDR3, may be provided by any domain member of the immunoglobulin gene superfamily. The scaffold may be a human or non-human protein. An advantage of a non- antibody protein scaffold is that it may provide an antigen-binding site in a scaffold molecule that is smaller and/or easier to manufacture than at least some antibody molecules. Small size of a binding member may confer useful physiological properties, such as an ability to enter cells, penetrate deep into tissues or reach targets within other structures, or to bind within protein cavities of the target antigen. Use of antigen binding sites in non-antibody protein scaffolds is reviewed in Wess, 2004 [15]. Typical are proteins having a stable backbone and one or more variable loops, in which the amino acid sequence of the loop or loops is specifically or randomly mutated to create an antigen-binding site that binds the target antigen. Such proteins include the IgG-binding domains of protein A from S. aureus, transferrin, tetranectin, fibronectin (e.g. 10th fibronectin type III domain) , lipocalins as well as gamma-crystalline and other Affilin™ scaffolds (Scil Proteins) . Examples of other approaches include synthetic "Microbodies" based on cyclotides - small proteins having intra-molecular disulphide bonds, Microproteins (Versabodies™, Amunix) and ankyrin repeat proteins (DARPins, Molecular Partners) .

In addition to antibody sequences and/or an antigen-binding site, a binding member according to the present invention may comprise other amino acids, e.g. forming a peptide or polypeptide, such as a folded domain, or to impart to the molecule another functional characteristic in addition to ability to bind antigen. 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) . For example, a binding member may comprise a catalytic site (e.g. in an enzyme domain) as well as an antigen binding site, wherein the antigen binding site binds to the antigen and thus targets the catalytic site to the antigen. The catalytic site may inhibit biological function of the antigen, e.g. by cleavage . Although, as noted, CDRs can be carried by non-antibody scaffolds, the structure for carrying a CDR, e.g. CDR3, or a set of CDRs of the invention will generally be an antibody heavy or light chain sequence or substantial portion thereof in which the CDR or set of CDRs is located at a location corresponding to the CDR or set of CDRs 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, et al . , 1987 [16] and updates thereof. A number of academic and commercial on-line resources are available to query this database. For example, see ref. [17] and the associated on-line resource, currently at the web address of http: //www. bioinf.org.uk/abs/simkab.html .

By CDR region or CDR, it is intended to indicate the hypervariable regions of the heavy and light chains of the immunoglobulin as defined by Kabat et al. 1991 [18], and later editions. An antibody typically contains 3 heavy chain CDRs and 3 light chain CDRs. The term CDR or CDRs is used here in order to indicate, according to the case, one of these regions or several, or even the whole, of these regions which contain the majority of the amino acid residues responsible for the binding by affinity of the antibody for the antigen or the epitope which it recognises.

Among the six short CDR sequences, the third CDR of the heavy chain (HCDR3) has a greater size variability (greater diversity essentially due to the mechanisms of arrangement of the genes which give rise to it) . It may be as short as 2 amino acids although the longest size known is 26. CDR length may also vary according to the length that can be accommodated by the particular underlying framework. Functionally, HCDR3 plays a role in part in the determination of the specificity of the antibody [19, 20, 21, 22, 23, 24, 25, 26].

In binding members according to the invention: HCDRl may be 5 amino acids long. HCDR2 may be 17 amino acids long.

HCDR3 may be 5 amino acids long.

LCDRl may be 11 amino acids long.

LCDR2 may be 7 amino acids long.

LCDR3 may be 9 amino acids long.

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 antigen-binding site. It must be understood here that the invention does not relate to the antibodies in natural form, that is to say they are not in their natural environment but that they have been able to be isolated or obtained by purification from natural sources, or else obtained by genetic recombination, or by chemical synthesis, and that they can then contain unnatural amino acids as will be described later. Antibody fragments that comprise an antibody antigen-binding site include, but are not limited to, molecules such as Fab, Fab', Fab' -SH, scFv, Fv, dAb and Fd. Various other antibody molecules including one or more antibody antigen-binding sites have been engineered, including for example Fab₂, Fab₃, diabodies, triabodies, tetrabodies and minibodies. Antibody molecules and methods for their construction and use are described in [27] .

It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules that bind the target antigen. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the 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 binding member or substance having an antibody antigen-binding site with the required specificity and/or binding to antigen. Thus, this term covers antibody fragments and derivatives, including any polypeptide comprising an antibody antigen-binding site, whether natural or wholly or partially synthetic. Chimeric molecules comprising an antibody antigen-binding site, or equivalent, fused to another polypeptide (e.g. derived from another species or belonging to another antibody class or subclass) 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.

Further techniques available in the art of antibody engineering have made it possible to isolate human and humanised antibodies. For example, human hybridomas can be made as described by Kontermann & Dubel [28]. Phage display, another established technique for generating binding members has been described in detail in many publications, such as Kontermann & Dubel [28] and WO92/01047 (discussed further below), and US patents US5969108, US5565332,

US5733743, US5858657, US5871907, US5872215, US5885793, US5962255, US6140471, US6172197, US6225447, US6291650, US6492160, US6521404.

Transgenic mice in which the mouse antibody genes are inactivated and functionally replaced with human antibody genes while leaving intact other components of the mouse immune system, can be used for isolating human antibodies [29]. Humanised antibodies can be produced using techniques known in the art such as those disclosed in for example WO91/09967, US 5,585,089, EP592106, US 565,332 and WO93/17105. Further, WO2004/006955 describes methods for humanising antibodies, based on selecting variable region framework sequences from human antibody genes by comparing canonical CDR structure types for CDR sequences of the variable region of a non-human antibody to canonical CDR structure types for corresponding CDRs from a library of human antibody sequences, e.g. germline antibody gene segments. Human antibody variable regions having similar canonical CDR structure types to the non-human CDRs form a subset of member human antibody sequences from which to select human framework sequences. The subset members may be further ranked by amino acid similarity between the human and the non-human CDR sequences. In the method of WO2004/006955, top ranking human sequences are selected to provide the framework sequences for constructing a chimeric antibody that functionally replaces human CDR sequences with the non-human CDR counterparts using the selected subset member human frameworks, thereby providing a humanized antibody of high affinity and low immunogenicity without need for comparing framework sequences between the non-human and human antibodies. Chimeric antibodies made according to the method are also disclosed.

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. [30] or Krebs et al. [31].

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 CHl domains; (ii) the Fd fragment consisting of the VH and CHl domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment [32, 33, 34], which consists of a VH or a VL domain; (v) isolated CDR 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 [35, 36]; (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94/13804; [37]). Fv, scFv or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains [38]. Minibodies comprising a scFv joined to a CH3 domain may also be made [39]. Other examples of binding fragments are Fab', which differs from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CHl domain, including one or more cysteines from the antibody hinge region, and Fab' -SH, which is a Fab' fragment in which the cysteine residue (s) of the constant domains bear a free thiol group.

Qui et al. [40] described antibody molecules containing just two CDRs linked by a framework region. CDR3 from the VH or VL domain was linked to the CDRl or CDR2 loop of the other domain. Linkage was through the C terminus of the selected CDRl or CDR2 to the N terminus of the CDR3, via a FR region. Qui et al. selected the FR region having the fewest hydrophobic patches. The best combination for the antibody tested was found to be VL CDRl linked by VH FR2 to VH CDR3 (VHCDR1-VHFR2-VLCDR3) . At a molecular weight of around 3 kDa, these antibody molecules offer advantages in terms of improved tissue penetration as compared with full immunoglobulins (approx. 150 kDa) or scFv (approx. 28 kDa) .

Antibody fragments of the invention can be obtained starting from a parent antibody molecule such as antibody C12 by methods such as digestion by enzymes e.g. pepsin or papain and/or by cleavage of the disulfide bridges by chemical reduction. In another manner, the antibody fragments comprised in the present invention can be obtained by techniques of genetic recombination likewise well known to the person skilled in the art or else by peptide synthesis by means of, for example, automatic peptide synthesisers, such as those supplied by the company Applied Biosystems, etc., or by nucleic acid synthesis and expression.

Functional antibody fragments according to the present invention include any functional fragment whose half-life is increased by a chemical modification, especially by PEGylation, or by incorporation in a liposome.

A dAb (domain antibody) is a small monomeric antigen-binding fragment of an antibody, namely the variable region of an antibody heavy or light chain [34]. VH dAbs occur naturally in camelids (e.g. camel, llama) and may be produced by immunizing a camelid with a target antigen, isolating antigen-specific B cells and directly cloning dAb genes from individual B cells. dAbs are also producible in cell culture. Their small size, good solubility and temperature stability makes them particularly physiologically useful and suitable for selection and affinity maturation. Camelid VH dAbs are being developed for therapeutic use under the name "nanobodies™" . A binding member of the present invention may be a dAb comprising a VH or VL domain substantially as set out herein, or a VH or VL domain comprising a set of CDRs substantially as set out herein.

As used herein, the phrase "substantially as set out" refers to the characteristic (s) of the relevant CDRs of the VH or VL domain of binding members described herein will be either identical or highly similar to the specified regions of which the sequence is set out herein. As described herein, the phrase "highly similar" with respect to specified region (s) of one or more variable domains, it is contemplated that from 1 to about 5, e.g. from 1 to 4, including 1 to 3, or 1 or 2, or 3 or 4, amino acid substitutions may be made in the CDR and/or VH or VL domain.

Bispecific or bifunctional antibodies form a second generation of monoclonal antibodies in which two different variable regions are combined in the same molecule [41]. Their use has been demonstrated both in the diagnostic field and in the therapy field from their capacity to recruit new effector functions or to target several molecules on the surface of tumour cells. Where bispecific antibodies are to be used, these may be conventional bispecific antibodies, which can be manufactured in a variety of ways [42], e.g. prepared chemically or from hybrid hybridomas, or may be any of the bispecific antibody fragments mentioned above. These antibodies can be obtained by chemical methods [43, 44] or somatic methods [45, 46] but likewise and preferentially by genetic engineering techniques which allow the heterodimerization to be forced and thus facilitate the process of purification of the antibody sought [47]. 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. coli. 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 domain A2 of tenascin-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 alternative engineering methods as described in Ridgeway et al., 1996 [48] .

Various methods are available in the art for obtaining antibodies against domain A2 of human tenascin-C. The antibodies may be monoclonal antibodies, especially of human, murine, chimeric or humanized origin, which can be obtained according to the standard methods well known to the person skilled in the art.

Antigen-binding site This describes the part of a molecule that binds to and is complementary to all or part of the target antigen. In an antibody molecule it is referred to as the antibody antigen-binding site, and comprises the part of the antibody that binds to and is complementary to all or part of the target 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 antibody antigen-binding site may be provided by one or more antibody variable domains. An antibody antigen-binding site may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH) . Isola ted

This refers to the state in which binding members of the invention, or nucleic acid encoding such binding members, will generally be in accordance with the present invention. Thus, binding members, VH and/or VL domains, and encoding nucleic acid molecules and vectors according to the present invention may be provided isolated and/or purified, e.g. from their natural environment, in substantially pure or homogeneous form, or, in the case of nucleic acid, free or substantially free of nucleic acid or genes of origin other than the sequence encoding a polypeptide with the required function. 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 practised in vitro or in vivo. 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. 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.

Brief Description of the Drawings

Figure 1. Schematic representation of the small (A) and large (B) tenascin-C isoform. Several fibronectin type III like domains are subject to alternative splicing, either being included (B) or omitted (A) in the molecule.

Figure 2. Cloned recombinant domain A2 of tenascin-C. Residues from the pQE12 vector are underlined. Sequence of residues identified in epitope mapping for antibody molecule C12 is boxed. Figure 3. Specificity ELISA of anti-A2 domain on different fibronectin type-III respeats Al, A2, A4, B, C and D, respectively, of human tenascin-C, and on plastic and on bovine serum albumin.

Figure 4. Amino acid sequence (A) and nucleotide sequence (B) of scFv C12 including C terminal myc tag.

Figure 5. BIAcore analysis of C12 antibody molecule.

Figure 6. Immunohistochemistry of C12 antibody molecule on U87 human glioblastoma section.

Figure 7. Structure of human tenascin-C domain A2 modelled using SWISS-MODEL-REPOSITORY software based on the template entry lfnf.

The mapped epitope is depicted in dark grey indicated by the arrow.

Detailed Description

Variable domains employed in the invention may be obtained or derived from any germline or rearranged human variable domain, or may be a synthetic variable domain based on consensus or actual sequences of known human variable domains . A variable domain can be derived from a non-human antibody. A CDR sequence of the invention (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. [49] 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, the CDR3-derived sequences of the present invention 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 binding members of the invention. The repertoire may then be displayed in a suitable host system, such as the phage display system of WO92/01047, which is herein incorporated by reference in its entirety, or any of a subsequent large body of literature, including Kay, Winter & McCafferty [50], so that suitable binding members may be selected. A repertoire may consist of from anything from 10⁴ individual members upwards, for example at least 10⁵, at least 10⁶, at least 10⁷, at least 10⁸, at least 10⁹ or at least 10¹⁰ members or more. Other suitable host systems include, but are not limited to yeast display, bacterial display, T7 display, viral display, cell display, ribosome display and covalent display.

Analogous shuffling or combinatorial techniques are also disclosed by Stemmer [51], who describes the technique in relation to a β- lactamase gene but observes that the approach may be used for the generation of antibodies.

A further alternative is to generate novel VH or VL regions carrying CDR-derived sequences of the invention using random mutagenesis of one or more selected VH and/or VL genes to generate mutations within the entire variable domain. Such a technique is described by Gram et al. [52], who used error-prone PCR. Another method that may be used is to direct mutagenesis to CDR regions of VH or VL genes. Such techniques are disclosed by Barbas et al. [53] and Schier et al. [54]. 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 binding members of the invention using routine methodology in the art.

A further aspect of the invention provides a method for obtaining an antibody antigen binding domain specific for domain A2 of tenascin-C, the method comprising providing, by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a VH domain set out herein, a VH domain which is an amino acid sequence variant of the VH domain, optionally combining the VH domain thus provided with one or more VL domains, and testing the VH domain or VH/VL combination or combinations for ability to bind domain A2 of tenascin-C. Said VL domain may have an amino acid sequence which is substantially as set out herein. In some embodiments, the one or more amino acids may added, deleted, substituted or inserted into one or more CDRs of the VH domain, for example CDRl, CDR2 and/or CDR3.

An analogous method may be employed comprising providing, by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a VL domain set out herein, a VL domain which is an amino acid sequence variant of the VL domain, combining the VL domain thus provided with one or more VH domains, and testing the VH/VL combination or combinations for ability to bind domain A2 of tenascin-C. Said VH domain may have an amino acid sequence which is substantially as set out herein or may be an amino acid sequence variant of a VH domain which is substantially as set out herein obtained as described above. In some embodiments, the one or more amino acids may added, deleted, substituted or inserted into one or more CDRs of the VL domain.

A further aspect of the invention provides a method of preparing a binding member for domain A2 of human tenascin-C, which method comprises :

(a) providing a starting repertoire of nucleic acids encoding a VH domain which either include a CDR to be replaced or lack a CDR encoding region;

(b) combining said repertoire with a donor nucleic acid encoding an amino acid sequence substantially as set out herein for a VH CDR such that said donor nucleic acid is inserted into the CDR region in the repertoire, so as to provide a product repertoire of nucleic acids encoding a VH domain;

(c) expressing the nucleic acids of said product repertoire;

(d) selecting a binding member for domain A2 of tenascin-C; and

(e) recovering the binding member or nucleic acid encoding it. The CDR may be a VH CDRl, CDR2 or CDR3. Again, an analogous method may be employed in which a VL CDR of the invention is combined with a repertoire of nucleic acids encoding a VL domain which either include a CDR to be replaced or lack a CDR encoding region. The CDR may be a VL CDRl, CDR2 or CDR3.

Similarly, one or more, or all three CDRs may be grafted into a repertoire of VH or VL domains that are then screened for a binding member or binding members for tenascin-C containing domain A2.

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 C-terminal 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 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 detectable/functional labels as discussed in more detail elsewhere herein.

Although in some aspects of the invention binding members comprise a pair of VH and VL domains, 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. For example, see the discussion of dAbs above. In the case of either of the single chain binding domains, these domains may be used to screen for complementary domains capable of forming a two-domain binding member able to bind domain A2 of tenascin-C. This may be achieved by phage display screening methods using the so-called hierarchical dual combinatorial approach as disclosed in WO92/01047 in which an individual colony containing either an H or L chain clone is used to infect a complete library of clones encoding the other chain (L or H) and the resulting two-chain binding member is selected in accordance with phage display techniques such as those described in that reference. This technique is also disclosed in Marks et al. [49] .

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 Cλ chains, preferably Cλ chains. Similarly, a binding member based on a VH domain may be attached at its C-terminal end to all or part 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 IgGl and IgG4.

Binding members of the invention may be labelled with a detectable or functional label. Detectable labels may include radionuclides, such as iodine-131, yttrium-90, indium-Ill and technicium-99, which may be attached to antibodies of the invention using conventional chemistry known in the art of antibody imaging. A binding member labelled with a radioactive isotope may be used to selectively deliver radiation to a specific target, such as a tumour. This may be useful in imaging the tumour or in delivering a cytoxic dose of radiation, as described below.

Other detectable labels may include enzyme labels such as horseradish peroxidase, chemical moieties such as biotin which may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin, fluorochromes such as fluorescein, rhodamine, phycoerythrin and Texas Red and near infrared fluorophores, including cyanine dye derivatives such as Cy7 (Amersham Pharmacia) and Alexa750 (Molecular probes) .

A detectable label may comprise a microbubble derivative, which is detectable by ultrasound [55], or a magnetic particle [56] .

A functional label may include an agent which is capable of triggering a biocidal event or has an anti-cancer effect. Suitable labels include radionuclides, photosensitisers, toxin polypeptides, toxic small molecules and other drugs, cytokines (e.g. IL-2, IL-12, TNF), chemokines, pro-coagulant factors (e.g. tissue factor), enzymes, liposomes, and immune response factors [57].

Radionuclides include iodine-131, yttrium-90, indium-Ill and technicium-99 and are described in more detail above.

A toxin polypeptide or peptide has cytotoxic or apoptotic activity and may be derived from a microbial, plant, animal or human source. In some embodiments, a toxin polypeptide may be inserted directly into the constant regions of a binding member. Examples of toxin polypeptides include Pseudomonas exotoxin, ricin α-chain and angiogenin .

Toxic small molecules include chemical compounds with cytotoxic activity, including, for example, DNA-complexing agents or cell cycle inhibitors. In some embodiments, the toxic molecule may be liberated in the vicinity of the target cell by cleavage of a pH- or enzyme- sensitive linker (e.g. linkers containing imine bonds) . Examples of toxic small molecules include maytansine, calicheamicin, epothilone and tubulysin and derivatives thereof.

Immune response factors may include binding members which bind to immune effector cells. The binding of the binding member may invoke a cell-mediated immune response against the target cell. A binding member of the invention may be conjugated with a cytokine. A fusion protein comprising the binding member or a polypeptide component thereof (e.g. a heavy chain or a light chain of an antibody or multi-chain antibody fragment, such as a Fab) and the cytokine may be produced. Thus, for example, a VH domain or VL domain of a binding member of the invention may be fused to the cytokine. Typically the binding member, or component thereof, and cytokine are joined via a peptide linker, e.g. a peptide of about 5-25 residues, e.g. 10-20 residues, preferably about 15 residues. The cytokine is may be IL-2, e.g. human IL2. The cytokine may be fused upstream (N- terminal) or downstream (C-terminal) of the binding member or polypeptide component thereof. For example, a fusion protein may comprise the binding member (especially an antibody molecule, e.g. scFv molecule) of the invention and IL-2. Amino acid sequences of such fusion proteins, and nucleic acids comprising nucleotide sequences encoding them, form part of the invention.

Binding members of the invention may be useful in methods of diagnosis, such as tumour imaging, or in the treatment in human or animal subjects, for example for cancer conditions.

Accordingly, further aspects of the invention provide methods of treatment comprising administration of a binding member as provided, pharmaceutical compositions comprising such a binding member, and use of such a 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 binding member with a pharmaceutically acceptable excipient.

A binding member for use in a method of treatment may be conjugated with or linked to a functional label which elicits an anti-tumour effect. In preferred embodiments, as noted above, the binding member is conjugated with or linked to a cytokine e.g. IL-2.

Clinical indications in which a binding member as described herein may be used to provide therapeutic benefit include proliferative disorders such as pre-malignant and malignant neoplasms and tumours, (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g., lung cancer, small cell lung cancer, gastrointestinal cancer, bowel cancer, colon cancer, breast carcinoma, ovarian carcinoma, prostate cancer, testicular cancer, liver cancer, kidney cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukaemias and angiogenic diseases.

A pre-malignant or malignant condition may occur in any cell-type, including but not limited to, lung, colon, breast, ovarian, prostate, liver, pancreas, brain, and skin.

A proliferative disorder suitable for treatment as described herein may be characterised by the presence of cells or tissue expressing a tenascin-C large isoform comprising the A2 domain, or in which expression of such an isoform is increased above normal levels.

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, eg 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 [58, 59] . 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 0.5 mg to 100 g for systemic applications, and 10 μg to 1 mg for local applications. Typically, the antibody will be a whole antibody, preferably the IgGl or 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.

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 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 saccharide 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, stabilisers, 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. Other treatments may include the administration of suitable doses of pain relief drugs such as nonsteroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or anti- emetics.

A further aspect of the invention provides a method of detecting and/or imaging tumour cells comprising administering an antibody as described herein to an individual and detecting the binding of said antibody to tumour cells in said individual.

Preferred antibodies for use in such methods may be conjugated or linked to a detectable label such as a radionuclide or flurophor.

A method of the invention may comprise causing or allowing binding of a binding member as provided herein to domain A2 of tenascin-C. As noted, such binding may take place in vivo, e.g. following administration of a binding member, or nucleic acid encoding a binding member.

The amount of binding of binding member to human tenascin-C comprising domain A2 may be determined. In some embodiments, the binding of the binding member to a sample obtained from an individual may be determined. In other embodiments, binding of the binding member to an antigen may be determined in in vivo, for example in imaging or detecting tumours in the body of an individual. Quantitation may be related to the amount of the antigen, which may be of diagnostic interest.

The binding of antibodies may be determined by any appropriate means. For example, the antibody may be linked or conjugated to a reporter molecule or detectable label and the presence, amount or localisation of the label or reporter on the sample determined.

Binding of an antibody in vivo, for example in a method of molecular imaging, may be determined by radioactive detection (e.g. PET, SPECT), near infrared fluorescence imaging (e.g. diffuse optical tomography, endoscopy), ultrasound (e.g. with targeted microbubble derivatives) and MRI (with targeted magnetic particles) .

In other embodiments, binding of the antibody may take place in vitro, for example in ELISA, Western blotting, immunocytochemistry, immuno-precipitation or affinity chromatography.

A method of detecting and/or imaging tumour cells may thus comprise contacting an antibody as described herein with a sample obtained from an individual and detecting the binding of said antibody to tumour cells in said sample.

Preferred antibodies for use in such in vitro methods may be conjugated or linked to a reporter molecule. The reporter molecule may be a radionuclide, fluorochrome, phosphor or laser dye with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red. Suitable chromogenic dyes include diaminobenzidine . For in vivo imaging, radionuclides or flurophors are preferred.

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

The present invention further provides an isolated nucleic acid encoding a binding member of the present invention. Nucleic acid includes DNA and RNA. In a preferred aspect, the invention provides a nucleic acid which codes for a CDR or VH or VL domain of the invention as defined above.

The present invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as above.

The present invention also provides a recombinant host cell which comprises one or more constructs as above. A nucleic acid encoding any CDR, VH or VL domain, or binding member as provided itself forms an aspect of the present invention, as does a method of production of the encoded product, which method comprises expression from encoding nucleic acid therefor. Expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the nucleic acid. Following production by expression a VH or VL domain, or binding member may be isolated and/or purified using any suitable technique, then used as appropriate. A method of production may comprise formulating the product into a composition including at least one additional component, such as a pharmaceutically acceptable excipient.

Nucleic acid according to the present invention may comprise DNA or RNA and may be wholly or partially synthetic. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

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, yeast and baculovirus systems. The expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. For a review, see for example Pluckthun [60]. A common 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 binding member [61, 62, 63] . 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, NSO mouse melanoma 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 e.g. phagemid, or viral e.g. 'phage, as appropriate [64]. 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 Ausubel et al. [65] .

A further aspect of the present invention provides a host cell containing nucleic acid as disclosed herein. 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. 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.

In one embodiment, the nucleic acid of the invention is 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.

The present invention also provides a method which comprises using a construct as stated above in an expression system in order to express a binding member or polypeptide as above.

Other methods of the invention comprise conjugating or linking a binding member as described herein with a detectable label or anticancer agent. Suitable labels and agents are described above.

Examples

Example 1: Cloning of human A2 domain and isolation of human anti- tenascin A2 antibodies

Human tenascin-C A2 DNA sequence was amplified from genomic clones (RZPDB737F032145D) and cloned into a bacterial expression vector (pQE12-Qiagen) which appends a His-TAG at the C-terminus of the recombinant proteins (Figure 2) . The antigen was then purified by affinity chromatography with Ni-NTA resin and analysed by SDS-PAGE and size exclusion chromatography.

One anti-tenascin A2 monoclonal antibody, designated C12, was isolated from a large synthetic human antibody library (ETH2-Gold,

[66]) using published procedures [67]. The isolated clone was able to recognise the recombinant antigen in ELISA and its binding specificity was demonstrated by ELISA on other fibronectin type-III repeats (Al, A2, A4, B, C, D of human tenascin-C) , BSA and plastic (Figure 3) .

The anti-tenascin antibody clone C12 was sequenced and purified by protein-A affinity chromatography for further analysis. C12 comprises a DP47 heavy chain and a DPL16 light chain (Figure 4) .

Example 2: BIAcore analysis

The affinity-purified C12 anti-A2 antibody was used in real time interaction analysis using surface plasmon resonance technology

(BIAcore 3000) . C12 showed an apparent dissociation constant in the micromolar range (Figure 5) . K_D was estimated to be approximately 1.18 x 10^~6 M.

Example 3: Immunohistochemistry

Immunohistochemical analysis was performed on sections of human U87 glioblastoma xenografted in nude mice. Clone C12 anti-A2 domain showed an impressive peri-vascular staining (Figure 6) indicated by the darker shades around blood vessels, thereby demonstrating that its epitope is not masked neither by other extracellular matrix proteins nor by glycosylation events. These results demonstrate the ability of the anti-tenascin antibody to recognise the antigen in the tissue .

Example 4 : Epitope mapping

The location of the epitope recognized by C12 antibody was determined using mass-spectrometry (MS) . Recombinant antigen A2 was incubated with the C12 antibody clone, treated with a protease (proteinase K) with a very broad cleavage site specificity (it cuts at C-terminus of Ale, Phe, Tyr, Trp, Leu, lie and VaI) . The fragments obtained were analysed by MS. The antibody-antigen interaction was able to mask the cleavage site contained in the epitope, leading to the identification of a 1147.43 Da fragment whose sequence is TAPEGAYEYF (SEQ ID NO: 19). In the proteinase K-digested A2 domain alone no 1147.43 fragment was detectable.

To demonstrate the reliability of this epitope-mapping detection, proteolysed A2 antigen was loaded on to an A2-antibody functionalized CnBr-resin (C12-resin) [68], and peptides contained in the flow through were analysed by MS.

Trypsinised or GIuC cleaved A2-domain (Trypsin cuts at C-terminus of Lys and Arg, while GIuC cuts at C-terminus of GIu and Asp in phosphate buffer) were incubated for about 1 hour at RT with the C12- resin and no fragments containing the amino acid sequence GAYE (SEQ ID NO: 38) were observed in the flow through.

In a 3D-structure model of domain A2 of human tenascin-C we observed that the tetrapeptide recognized by C12 antibody maps on a loop between two β-strands (Figure 7) . The position of the epitope on the antigen structure and the type of amino acid within and surrounding this tetrapeptide suggests that this loop is probably exposed to the external aqueous environment.

Materials and Methods for Examples 1 to 4

Cloning, expression and purification of recombinant antigens

Human tenascin-C A2, A4 and B domains DNA sequences were amplified from the genomic clones RZPDB737F032145D using primers: TnC-A2BamBA (5' CGGGTACCTCCACAGGGGAAACTCCCAATTTG 3' - SEQ ID NO: 20) and TnC- A2BglFO (5' GAAGATCTCTCCTCTGTAAAGACTTCAAC 3' - SEQ ID NO: 21) for domain A2; TnC-A4BamBA (5' CGGGATCCGTCACAGAGGATCTCCCACAG 3' - SEQ ID NO: 22) and TnC-A4BglFO (5' GAAGATCTGGCTGTGGAGGCCTCAGCAGA 3' - SEQ ID NO: 23) for domain A4 ; TnC-BBamBA (5' CGGGATCCTCCACAGCCAAAGAACCTGAA 3' - SEQ ID NO: 24) and TnC-BBgIFO (5' GAAGATCTCTCTGTCGTGGCTGTGGCACTGAT 3' - SEQ ID NO: 25) for domain B.

The resulting PCR fragments were cloned into BamHI and Ncol digested and dephosphorylated pQE12 vector (Qiagen) . These domains were expressed in E.coli TG-I whose cultures were grown at 37°C in 2xTY/100 μg/ml ampicillin and 0.1% glucose. Once reached an OD₆₀O= 0.5, 1 mM of isopropyl-thio-galactopiranoside (IPTG) was added to induce the protein expression. Cultures were incubated at 30⁰C overnight on orbital shaking. Spinned bacterials were lysed by sonication and proteins were purified from the cleared lysate using Ni-NTA column on Akta FPLC system (Amersham Biosciences) . Proteins were analysed by SDS-PAGE and size-exclusion chromatography using a superdex 75 10/30 column .

ELISA

Bacterial supernatant containing scFv fragments were used in ELISA assay essentially as described [69]. Individual colonies were inoculated in 180 μl 2xTY/ 100 μg/ml ampicillin/ 0.1% glucose in 96- well plates (NunclonTM Surface, Nunc) . The plates were incubated 3 hrs at 37 ⁰C in a shaker incubator. Expression was induced by addition of ImM IPTG and cultures were grown overnight at 30⁰C. Biotinylated antigen (EZ-link sulfo-NHS-SS-biotin, Pierce) was added to streptavidin 96-well plate (Roche) at a concentration of 10^"6 M for 30' at 37°C.

Binding of 80 microliters of myc-tagged scFv to antigen was detected with anti-myc mouse monoclonal antibody 9E10 (0.5 micrograms / ml), followed by anti-mouse IgG-horseradish peroxidase conjugate (Sigma) with a 1:1000 dilution. The colorimetric reaction was developed with BM-Blue POD soluble substrate (Roche), stopped by addition of 333 mM H₂SO₄ and the absorbance was measured at 450 nm using a microtiter plate reader. Sequencing of recombinant tenascin-C domains and scFv antibody genes

Antibodies were sequenced using Big Dye® Terminator vl .1 Cycle Sequencing kit (Applied Biosystems) on an ABI PRISM 3130 Genetic analyzer. Termination reactions were performed either on miniprep DNA or on PCR products using primers pQEseq.ba (5'

ATAGATTCAATTGTGAGCGGATAA 3' - SEQ ID NO: 26) and pQEseq.fo (5' GTCATTACTGGATCTATCAACAGG 3' - SEQ ID NO: 27) for tenascin domains and LMB31ong (5' CAGGAAACAGCTATGACCATGATTAC 3' - SEQ ID NO: 28) and fdseqlong (5' GACGTTAGTAAATGAATTTTCTGTATGAGG 3' - SEQ ID NO: 29) for scFv.

BIAcore

Purified anti-A2 C12-scFv at a concentration of 0.1 mg/ml and 0.05 mg/ml was analyzed by surface plasmon resonance (BIAcore 3000 system) using a low-density coated streptavidin chip. Chips coated with the recombinant A2 domain of tenascin-C were prepared by coupling biotinylated antigen to a SA-sensor chip (BIAcore) . 20 μl of monomeric antibody were injected using the kinject command at a flow of 20 μl/min. The binding curves were analysed with the BIAevaluation 3.2 software.

Immunohistochemistry

U87 human glioblastoma (cell line HBT-14. ATCC) xenograft tumors were obtained by injecting 3 x 10⁶ U87 cells into 6 to 8-week-old female BaIb-C nu/nu mice. Immunohistochemistry with scFv fragments was performed essentially as previously described [6]. Ten μm thick sections were treated with ice-cold acetone, rehydrated in TBS (5OmM Tris, 10OmM NaCl, pH 7.4), blocked with fetal bovine serum (Invitrogen) and then incubated with 20 μg/ml of purified scFv (myc- tagged) together with the biotinylated 9E10 anti-myc antibody (5μg/ml) . Bound antibody was detected using streptavidin :biotinylated alkaline phosphatase complex (Biospa, Milano, Italy) and subsequent staining reaction with Fast-Red TR (Sigma) (in the presence of 1 mM levamisole to inhibit endogenous alkaline phosphatase activity) . Hematoxylin solution (Sigma) was used for counterstaining . Epitope mapping

Twenty-five picomoles of recombinant antigen were pre-incubated with 50 picomoles of scFv fragment for 1 hour at RT. Proteinase K (Promega) was then added at the concentration of 0.001 mg/ml and incubated at 37°C for 2 hours. Single antigen digestions were performed on 25 picomoles of recombinant protein using either Proteinase K (1 μg/ml) for 2 hours at 37°C, or trypsin (0.01 μg/ml) for 2 hours at 37°C, or GluC-phosphate buffer (1-0.1 μg/ml) for 2 hours at 25°C. CnBr resin (GE Healthcare) was prepared essentially as described in ref . [68] . After resin resuspension and washing with 1 mM HCl, 9E10 antibody (in coupling buffer: NaHCO3 0. IM and NaCl 0.5M) was added at a final concentration of 30 μg/ml for 2 hours at RT on orbital-shacking (400 rpm) . The coupling reaction was blocked with 0.1 M Tris-HCl pH 8.00. The resin was then washed according to manufacturer manual, incubated with 0.1 mg/ml scFv in phospate buffer pH 7.4, BSA 10% and washed again several time with phosphate buffer. Amicon Ultrafree-MC 5.0 μm centrifugal filter devices were used for resin handling (washing, buffer changing and collecting flow through) .

Sequence listing

SEQ ID NO: 1

C12 VH domain

GAG GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTA CAG CCT GGG GGG TCC CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC ACC TTT AGC AGC TAT GCC ATG

AGC TGG GTC CGC CAG GCT CCA GGG AAG GGG CTG GAG TGG GTC TCA GCT ATT

AGT GGT AGT GGT GGT AGC ACA TAC TAC GCA GAC TCC GTG AAG GGC CGG TTC

ACC ATC TCC AGA GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC AGC

CTG AGA GCC GAG GAC ACG GCC GTA TAT TAC TGT GCG AAA CAG GCT CGT AGG TCG TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCG AGT SEQ ID NO: 2 Cl2 VH domain

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCAKQARRSFDYWGQGTLVTVSS

SEQ ID NO: 3 C12 VH CDRl SYAMS

SEQ ID NO: 4 C12 VH CDR2 AISGSGGSTYYADSVKG

SEQ ID NO: 5 C12 VH CDR3 QARRS

SEQ ID NO: 6 C12 VL domain TCG TCT GAG CTG ACT CAG GAC CCT GCT GTG TCT GTG GCC TTG GGA CAG ACA

GTC AGG ATC ACA TGC CAA GGA GAC AGC CTC AGA AGC TAT TAT GCA AGC TGG

TAC CAG CAG AAG CCA GGA CAG GCC CCT GTA CTT GTC ATC TAT GGT AAA AAC

AAC CGG CCC TCA GGG ATC CCA GAC CGA TTC TCT GGC TCC AGC TCA GGA AAC

ACA GCT TCC TTG ACC ATC ACT GGG GCT CAG GCG GAA GAT GAG GCT GAC TAT TAC TGT AAC TCC TCT GGT ATT TCT ATG AGT CCC GTG GTA TTC GGC GGA GGG

ACC AAG CTG ACC GTC CTA GGC

SEQ ID NO: 7 C12 VL domain SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNT ASLTITGAQAEDEADYYCNSSGISMSPVVFGGGTKLTVLG

SEQ ID NO: 8 C12 VL CDRl QGDSLRSYYAS SEQ ID NO: 9 C12 VL CDR2 GKNNRPS

SEQ ID NO: 10 C12 VL CDR3 NSSGISMSP

SEQ ID NO: 11 Tagged C12 scFv

GAG GTG CAG CTG TTG GAG TCT GGG GGA GGC TTG GTA CAG CCT GGG GGG TCC

CTG AGA CTC TCC TGT GCA GCC TCT GGA TTC ACC TTT AGC AGC TAT GCC ATG

AGC TGG GTC CGC CAG GCT CCA GGG AAG GGG CTG GAG TGG GTC TCA GCT ATT

AGT GGT AGT GGT GGT AGC ACA TAC TAC GCA GAC TCC GTG AAG GGC CGG TTC ACC ATC TCC AGA GAC AAT TCC AAG AAC ACG CTG TAT CTG CAA ATG AAC AGC

CTG AGA GCC GAG GAC ACG GCC GTA TAT TAC TGT GCG AAA CAG GCT CGT AGG

TCG TTT GAC TAC TGG GGC CAG GGA ACC CTG GTC ACC GTC TCG AGT GGT GGA

GGC GGT TCA GGC GGA GGT GGC TCT GGC GGT GGC GGA TCG TCT GAG CTG ACT

CAG GAC CCT GCT GTG TCT GTG GCC TTG GGA CAG ACA GTC AGG ATC ACA TGC CAA GGA GAC AGC CTC AGA AGC TAT TAT GCA AGC TGG TAC CAG CAG AAG CCA

GGA CAG GCC CCT GTA CTT GTC ATC TAT GGT AAA AAC AAC CGG CCC TCA GGG

ATC CCA GAC CGA TTC TCT GGC TCC AGC TCA GGA AAC ACA GCT TCC TTG ACC

ATC ACT GGG GCT CAG GCG GAA GAT GAG GCT GAC TAT TAC TGT AAC TCC TCT

GGT ATT TCT ATG AGT CCC GTG GTA TTC GGC GGA GGG ACC AAG CTG ACC GTC CTA GGC gcg gcc gca gaa caa aaa etc ate tea gaa gag gat ctg aat ggg gcc gca tag

SEQ ID NO: 12 Tagged C12 scFv EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAVYYCAKQARRSFDYWGQGTLVTVSSGGGGSGGGGSGGGGSSELTQD PAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTITG AQAEDEADYYCNSSGISMSPWFGGGTKLTVLGAAAEQKLISEEDLNGAA

SEQ ID NO: 13 Linker GGT GGA GGC GGT TCA GGC GGA GGT GGC TCT GGC GGT GGC GGA

SEQ ID NO: 14 Linker

GGGGSGGGGSGGGG SEQ ID NO: 15

Myc tag gcg gcc gca gaa caa aaa etc ate tea gaa gag gat ctg aat ggg gcc gca

SEQ ID NO: 16 Myc tag AAAEQKLISEEDLNGAA

SEQ ID NO: 17 Cloned recombinant A2 domain expressed from pQE12 vector

MRGSSTGETPNLGEVVVAEVGWDALKLNWTAPEGAYEYFFIQVQEADTVEAAQNLTVPGGLRSTDLPGL KAATHYTITIRGVTQDFSTTPLSVEVLTEERSHHHHHH

SEQ ID NO: 18 A2 domain

STGETPNLGEVVVAEVGWDALKLNWTAPEGAYEYFFIQVQEADTVEAAQNLTVPGGLRSTDLPGLKAAT HYTITIRGVTQDFSTTPLSVEVLTEE

SEQ ID NO: 19 A2 domain mapped epitope TAPEGAYEYF

SEQ ID NO: 20 Primer TnC-A2BamBA CGGGTACCTCCACAGGGGAAACTCCCAATTTG

SEQ ID NO: 21 Primer TnC-A2BglFO GAAGATCTCTCCTCTGTAAAGACTTCAAC

SEQ ID NO: 22

Primer TnC-A4BamBA

CGGGATCCGTCACAGAGGATCTCCCACAG SEQ ID NO : 23

Primer TnC-A4BglFO

GAAGATCTGGCTGTGGAGGCCTCAGCAGA

SEQ ID NO: 24

Primer TnC-BBamBA CGGGATCCTCCACAGCCAAAGAACCTGAA

SEQ ID NO: 25 Primer TnC-BBgIFO

GAAGATCTCTCTGTCGTGGCTGTGGCACTGAT

SEQ ID NO: 26 Primer pQEseq.ba ATAGATTCAATTGTGAGCGGATAA

SEQ ID NO: 27 Primer pQEseq.fo GTCATTACTGGATCTATCAACAGG

SEQ ID NO: 28

Primer LMB31ong

CAGGAAACAGCTATGACCATGATTAC

SEQ ID NO: 29

Primer fdseqlong GACGTTAGTAAATGAATTTTCTGTATGAGG

SEQ ID NO: 30 C12 VH FRl

EVQLLESGGGLVQPGGSLRLSCAASGFTFS

SEQ ID NO: 31 C12 VH FR2 WVRQAPGKGLEWVS SEQ ID NO: 32

C12 VH FR3

RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK

SEQ ID NO: 33 C12 VH FR4 FDYWGQGTLVTVSS

SEQ ID NO: 34 C12 VL FRl

SSELTQDPAVSVALGQTVRITC

SEQ ID NO: 35 C12 VL FR2 WYQQKPGQAPVLVIY

SEQ ID NO: 36 C12 VL FR3

GIPDRFSGSSSGNTASLTITGAQAEDEADYYC

SEQ ID NO: 37 C12 VL FR4 VVFGGGTKLTVLG

SEQ ID NO: 38

A2 domain epitope mapped tetrapeptide GAYE

References

1 Adams, G. P. and L. M. Weiner (2005). Nat Biotechnol 23(9): 1147-57

2 Neri, D. and R. Bicknell (2005). Nat Rev Cancer 5(6): 436-46

3 Borsi L et al Int J Cancer 1992; 52:688-692

4 Carnemolla B et al. Eur J Biochem 1992; 205:561-567

5 Carnemolla, B., et al. (1999). Am J Pathol 154(5): 1345-52

6 Brack, S. et al . (2006). Clin Cancer Res 12(10): 3200-8

7 Silacci, M., et al. (2006). Protein Enq Pes SeI 19(10): 471-8

8 Riva, P., et al . (1999). Acta Oncol 38(3): 351-9

9 Akabani, G., et al. (2005). J Nucl Med 46(6): 1042-51

10 Paganelli, G., et al . (1994). Eur J Nucl Med 21(4): 314-21

11 Balza, E., et al . (1993). FEBS Lett 332(1-2): 39-43

12 Haan & Maggos (2004) BioCentury, 12(5): A1-A6

13 Koide et al . (1998) Journal of Molecular Biology, 284: 1141-1151.

14 Nygren et al . (1997) Current Opinion in Structural Biology, 7: 463-469

15 Wess, L. In: BioCentury, The Bernstein Report on BioBusiness, 12(42), A1-A7, 2004

16 Kabat, E.A. et al, Sequences of Proteins of Immunological Interest. 4^th Edition. US Department of Health and Human Services. 1987

17 Martin, A. C. R. Accessing the Kabat Antibody Sequence Database by Computer PROTEINS: Structure, Function and Genetics, 25 (1996), 130- 133

18 Kabat, E. A. et al . (1991) Sequences of Proteins of Immunological Interest, 5th Edition. US Department of Health and Human Services, Public Service, NIH, Washington

19 Segal et al . , PNAS, 71:4298-4302, 1974

20 Amit et al., Science, 233:747-753, 1986

21 Chothia et al., J. MoI. Biol., 196:901-917, 1987

22 Chothia et al . , Nature, 342:877- 883, 1989

23 Caton et al . , J. Immunol., 144:1965-1968, 1990

24 Sharon et al., PNAS, 87:4814-4817, 1990

25 Sharon et al . , J. Immunol., 144:4863-4869, 1990 26 Kabat et al., J. Immunol., 147:1709-1719, 1991

27 Holliger & Hudson, Nature Biotechnology 23 (9) : 1126-1136 2005

28 Kontermann, R & Dubel, S, Antibody Engineering, Springer-Verlag New York, LLC; 2001, ISBN: 3540413545

29 Mendez, M. et al . (1997) Nature Genet, 15(2): 146-156

30 Knappik et al . J. MoI. Biol. (2000) 296, 57-86

31 Krebs et al. Journal of Immunological Methods 254 2001 67-84

32 Ward, E. S. et al., Nature 341, 544-546 (1989)

33 McCafferty et al (1990) Nature, 348, 552-554

34 Holt et al (2003) Trends in Biotechnology 21, 484-490

35 Bird et al, Science, 242, 423-426, 1988

36 Huston et al, PNAS USA, 85, 5879-5883, 1988

37 Holliger, P. et al, Proc. Natl. Acad. Sci. USA 90 6444-6448, 1993

38 Reiter, Y. et al, Nature Biotech, 14, 1239-1245, 1996

39 Hu, S. et al, Cancer Res., 56, 3055-3061, 1996

40 Qui et al., Nat. Biotechnol. 25:921-929 2007

41 Holliger and Bohlen 1999 Cancer and metastasis rev. 18: 411-419

42 Holliger, P. and Winter G. Current Opinion Biotechnol 4, 446-449 1993

43 Glennie M J et al . , 1987 J. Immunol. 139, 2367-2375

44 Repp R. et al . , 1995 J. Hemat . 377-382

45 Staerz U. D. and Bevan M. J. 1986 PNAS 83

46 Suresh M. R. et al . , 1986 Method Enzymol. 121: 210-228

47 Merchand et al., 1998 Nature Biotech. 16:677-681

48 Ridgeway, J. B. B. et al, Protein Eng., 9, 616-621, 1996

49 Marks et al Bio/Technology, 1992, K): 779-783

50 Kay, B. K., Winter, J., and McCafferty, J. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, San Diego: Academic Press

51 Stemmer, Nature, 1994, 370:389-391

52 Gram et al., 1992, Proc. Natl. Acad. Sci., USA, 89:3576-3580

53 Barbas et al., 1994, Proc. Natl. Acad. Sci., USA, 91:3809-3813

54 Schier et al., 1996, J. MoI. Biol. 263:551-567

55 Joseph S et al Pharm Res. 2004 Jun; 21 (6) : 920-6

56 Schellenberger EA et al . Bioconjug. Chem. 2004 Sep-Oct; 15(5): 1062-7

57 Neri (2004) CHIMIA "Tumor Targeting" vol. 58, pages 723-726 58 Ledermann J. A. et al . (1991) Int. J. Cancer 47: 659-664

59 Bagshawe K. D. et al . (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922

60 Plϋckthun, A. Bio/Technology 9: 545-551 (1991)

61 Chadd HE and Chamow SM (2001) Current Opinion in Biotechnology 12: 188-194

62 Andersen DC and Krummen L (2002) Current Opinion in Biotechnology 13: 117

63 Larrick JW and Thomas DW (2001) Current Opinion in Biotechnology 12:411-418

64 Sambrook and Russell, Molecular Cloning: a Laboratory Manual: 3rd edition, 2001, Cold Spring Harbor Laboratory Press

65 Ausubel et al . eds . , Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, John Wiley & Sons, 4^th edition 1999

66 Silacci, M., et al . (2005). Proteomics 5(9): 2340-50

67 Giovannoni, L., et al . (2001). Nucleic Acids Res 29(5): E27

68 Peter, J. F. and K. B. Tomer (2001). Anal Chem 73(16): 4012-9

69 Viti et al. 2000 Methods Enzymol., 326, 480-505

References

The references mentioned herein are all expressly incorporated by reference .

Claims

Claims

1. An isolated binding member for domain A2 of human tenascin C, wherein the binding member binds at least one residue of Gly-Ala-Tyr- GIu at positions 30 to 33 of domain A2 (SEQ ID NO: 18) of human tenascin C.

2. An isolated binding member for domain A2 of human tenascin C, wherein the binding member binds human tenascin C comprising domain A2 in human tissue, and wherein K_D of interaction of the binding member with domain A2 of human tenascin C is less than 100 μM as determined by surface plasmon resonance.

3. A binding member according to claim 1 or claim 2, comprising a set of complementarity determining regions (CDRs): HCDRl, HCDR2, HCDR3, LCDRl, LCDR2 and LCDR3, wherein the amino acid sequence of HCDR3 is SEQ ID NO: 5 or an amino acid sequence having one or two amino acid substitutions in SEQ ID NO: 5.

4. A binding member according to any of claims 1 to 3, comprising a set of CDRs: HCDRl, HCDR2, HCDR3, LCDRl, LCDR2 and LCDR3, wherein the amino acid sequence of LCDR3 is SEQ ID NO: 10 or an amino acid sequence having one or two amino acid substitutions in SEQ ID NO: 10.

5. A binding member according to any of claims 1 to 4, comprising a set of CDRs: HCDRl, HCDR2, HCDR3, LCDRl, LCDR2 and LCDR3, wherein the set of CDRs has no more than 10 amino acid substitutions compared with a set of CDRs in which: the amino acid sequence of HCDRl is SEQ ID NO: 3, the amino acid sequence of HCDR2 is SEQ ID NO: 4, the amino acid sequence of HCDR3 is SEQ ID NO: 5, the amino acid sequence of LCDRl is SEQ ID NO: 8, the amino acid sequence of LCDR2 is SEQ ID NO: 9, and the amino acid sequence of LCDR3 is SEQ ID NO: 10.

6. A binding member according to any of claims 3 to 5, wherein the amino acid sequence of HCDR3 is SEQ ID NO: 5.

7. A binding member according to any of claims 4 to 6, wherein the amino acid sequence of LCDR3 is SEQ ID NO: 10.

8. A binding member according to any of claims 4 to 7, comprising a set of CDRs wherein the amino acid sequence of HCDRl is SEQ ID NO: 3, the amino acid sequence of HCDR2 is SEQ ID NO: 4, the amino acid sequence of HCDR3 is SEQ ID NO: 5, the amino acid sequence of LCDRl is SEQ ID NO: 8, the amino acid sequence of LCDR2 is SEQ ID NO: 9, and the amino acid sequence of LCDR3 is SEQ ID NO: 10.

9. A binding member according to any of the preceding claims, wherein the binding member comprises an antibody molecule comprising an antibody VH domain and an antibody VL domain, wherein the VH domain comprises HCDRl, HCDR2, HCDR3 and heavy chain framework regions FRl, FR2, FR3 and FR4, and the VL domain comprises LCDRl, LCDR2, LCDR3 and light chain framework regions FRl, FR2, FR3 and FR4.

10. A binding member according to claim 9, wherein the antibody molecule is an scFv.

11. A binding member according to claim 9 or claim 10, wherein heavy chain FRl is SEQ ID NO: 30, heavy chain FR2 is SEQ ID NO: 31, heavy chain FR3 is SEQ ID NO: 32, heavy chain FR4 is SEQ ID NO: 33, light chain FRl is SEQ ID NO: 34, light chain FR2 is SEQ ID NO: 35, light chain FR3 is SEQ ID NO: 36 and light chain FR4 is SEQ ID NO: 37.

13. A binding member according to claim 12, wherein the VH domain amino acid sequence is SEQ ID NO: 2 and the VL domain amino acid sequence is SEQ ID NO: 7.

14. An isolated antibody molecule comprising a heavy chain comprising amino acid sequence SEQ ID NO: 2 and a light chain comprising amino acid sequence SEQ ID NO: 7.

15. An isolated antibody molecule for domain A2 of human tenascin C, wherein the antibody molecule comprises a VH domain amino acid sequence at least 90 % identical to SEQ ID NO: 2 and a VL domain amino acid sequence at least 90 % identical to SEQ ID NO: 7.

16. An antibody molecule according to claim 14 or claim 15, wherein the antibody molecule is an scFv.

17. An antibody molecule according to claim 16, comprising SEQ ID NO: 2 connected at its C terminus by a linker amino acid sequence SEQ ID NO: 14 to the N terminus of SEQ ID NO: 7.

18. An isolated VH domain of an antibody molecule according to any of claims 9 to 15.

19. An isolated VL domain of an antibody molecule according to any of claims 9 to 15.

20. An isolated polypeptide comprising SEQ ID NO: 19, wherein the polypeptide does not comprise domain A2 of human tenascin C.

21. A composition comprising an isolated binding member according to any of claims 1 to 13 or antibody molecule according to any of claims 14 to 17, and a pharmaceutically acceptable excipient.

22. A composition comprising an isolated binding member for domain A2 of human tenascin-C, for use in a method for treatment of the human body by surgery or therapy or for use in a diagnostic method practised on the human body.

23. A composition comprising an isolated binding member according to any of claims 1 to 13 or antibody molecule according to any of claims 14 to 17, for use in a method for treatment of the human body by surgery or therapy or for use in a diagnostic method practised on the human body.

24. A composition comprising a binding member for domain A2 of human tenascin-C, for use in treating or diagnosing a proliferative disorder .

25. A composition according to claim 24, wherein the binding member is a binding member according to any of claims 1 to 13 or an antibody molecule according to any of claims 14 to 17.

26. Use of a binding member for domain A2 of human tenascin-C for the manufacture of a medicament for use in treatment or in vivo diagnosis of a proliferative disorder.

27. Use according to claim 26, wherein the binding member is a binding member according to any of claims 1 to 13 or antibody molecule according to any of claims 14 to 17.

28. A method of treating a proliferative disorder in an individual comprising administering a binding member for domain A2 of human tenascin-C to the individual.

29. A method according to claim 28, wherein the binding member is a binding member according to any of claims 1 to 13 or an antibody molecule according to any of claims 14 to 17.

30. A method of detecting and/or imaging tumour cells in an individual comprising; administering a binding member for domain A2 of human tenascin-C to the individual; and detecting binding of the binding member or antibody molecule to tumour cells in the individual.

31. A method according to claim 30, wherein the binding member is a binding member according to any one of claims 1 to 13 or an antibody molecule according to any of claims 14 to 17.

32. A method according to claim 30 or claim 31 wherein the binding member is conjugated with a detectable label.

33. An isolated nucleic acid molecule comprising a nucleotide sequence encoding a binding member according to any of claims 1 to 13, an antibody molecule according to any of claims 14 to 17, a VH domain according to claim 18 or a VL domain according to claim 19.

34. A host cell in vitro transformed with nucleic acid according to claim 33.

35. A method of producing a binding member, antibody molecule, VH domain or VL domain, comprising culturing host cells according to claim 34 under conditions for production of the binding member, antibody molecule, VH domain or VL domain.

36. A method according to claim 35, further comprising isolating and/or purifying the binding member, antibody molecule, VH domain or VL domain.

37. A method according to claim 35 or claim 36, further comprising formulating the binding member, antibody molecule, VH domain or VL domain into a composition comprising at least one additional component .

38. A method for producing an antibody antigen-binding domain for domain A2 of human tenascin C, the method comprising providing, by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VH domain comprising HCDRl SEQ ID NO: 3, HCDR2 SEQ ID NO: 4 and HCDR3 SEQ ID NO: 5, a VH domain which is an amino acid sequence variant of the parent VH domain, and optionally combining the VH domain thus provided with one or more VL domains to provide one or more VH/VL combinations; and testing said VH domain which is an amino acid sequence variant of the parent VH domain or the VH/VL combination or combinations to identify an antibody antigen binding domain for domain A2 of human tenascin C.

39. A method according to claim 38, wherein the parent VH domain comprises amino acid sequence SEQ ID NO: 2.

40. A method according to claim 38 or claim 39 wherein said one or more VL domains is provided by way of addition, deletion, substitution or insertion of one or more amino acids in the amino acid sequence of a parent VL domain comprising LCDRl SEQ ID NO: 8, LCDR2 SEQ ID NO: 9 and LCDR3 SEQ ID NO: 10, producing one or more VL domains each of which is an amino acid sequence variant of the parent VL domain .

41. A method according to claim 40 wherein the parent VL domain comprises amino acid sequence SEQ ID NO: 7.

42. A method according to any one of claims 38 to 41, wherein the VH domain which is an amino acid sequence variant of the parent VH domain is provided by CDR mutagenesis.

43. A method according to any one of claims 38 to 41 further comprising producing the antibody antigen-binding domain as a component of an IgG, scFv or Fab antibody molecule.

44. A method for producing a binding member that binds domain A2 of human tenascin C, the method comprising: providing starting nucleic acid encoding a VH domain or a starting repertoire of nucleic acids each encoding a VH domain, wherein the VH domain or VH domains either comprise a HCDRl, HCDR2 and/or HCDR3 to be replaced or lack a HCDRl, HCDR2 and/or HCDR3 encoding region; combining said starting nucleic acid or starting repertoire with donor nucleic acid or donor nucleic acids encoding or produced by mutation of the amino acid sequence of an HCDRl SEQ ID NO: 3, HCDR2 SEQ ID NO: 4, and/or HCDR3 SEQ ID NO: 5, such that the donor nucleic acid is or donor nucleic acids are inserted into the CDRl, CDR2 and/or CDR3 region in the starting nucleic acid or starting repertoire, so as to provide a product repertoire of nucleic acids encoding VH domains; expressing the nucleic acids of the product repertoire to produce product VH domains; optionally combining said product VH domains with one or more VL domains; selecting a binding member for domain A2 of human tenascin C, wherein the binding member comprises a product VH domain and optionally a VL domain; and recovering the binding member or nucleic acid encoding it.

45. A method according to claim 44 wherein the donor nucleic acids are produced by mutation of HCDRl and/or HCDR2.

46. A method according to claim 44 wherein the donor nucleic acid is produced by mutation of HCDR3.

47. A method according to claim 44, comprising providing the donor nucleic acid by random mutation of nucleic acid.

48. A method according to any of claims 44 to 47 comprising providing an IgG, scFv or Fab antibody molecule comprising the product VH domain and a VL domain.

49. A method according to any of claims 38 to 48, further comprising testing the antibody antigen-binding domain or binding member for ability to bind human tenascin C comprising domain A2 in human tissue and/or to bind domain A2 of human tenascin C with a K_D of less than 100 μM as determined by surface plasmon resonance.

50. A method according to claim 49, wherein a binding member is obtained that comprises an antibody molecule that binds human tenascin C comprising domain A2 in human tissue and/or binds domain A2 of human tenascin C with a K_D of less than 100 μM as determined by surface plasmon resonance.

51. A method according to claim 50, wherein the antibody molecule is an scFv.

52. A method for producing an antibody molecule composition, comprising obtaining an antibody molecule using a method according to any of claims 38 to 51, and formulating the antibody molecule into a composition comprising at least one additional component.