Antibodies for treatment and diagnosis of inflammatory bowel disease

Abstract

The invention relates to the diagnosis and treatment of diseases, including inflammatory disorders, proliferative disorders and autoimmune diseases. The invention provides, and involves the use of, antibody molecules that bind: i) lysozyme, ii) neutrophil elastase, iii) tissue inhibitor of metalloproteinase-1 (TIMP-1), or iv) the D domain of Tenascin-C. The invention also relates to the use of antibody molecules that bind v) the IIICS isoform of fibronectin, vi) the Extra Domain-B (ED-B) of fibronectin, vii) matrix-metalloproteinase 3 (MMP3), or viii) the A1 domain of tenascin-C in the diagnosis and treatment of inflammatory disorders such as inflammatory bowel disease.

Claims

1. An antibody molecule that binds domain D of Tenascin-C, wherein the antibody molecule comprises a VH domain comprising a framework and a set of complementarity determining regions HCDR1, HCDR2 and HCDR3, and a VL domain comprising a framework and a set of complementarity determining regions LCDR1, LCDR2 and LCDR3, wherein: HCDR1 has the amino acid sequence of SEQ ID NO: 27; HCDR2 has the amino acid sequence of SEQ ID NO: 91; HCDR3 has the amino acid sequence of SEQ ID NO: 29; LCDR1 has the amino acid sequence of SEQ ID NO: 30; LCDR2 has the amino acid sequence of SEQ ID NO: 31; and LCDR3 has the amino acid sequence of SEQ ID NO: 92.

2. The antibody molecule according to claim 1, wherein the VH domain comprises or consist of the amino acid sequence of SEQ ID NO: 89, and/or the VL domain comprises or consist of the amino acid sequence of SEQ ID NO: 90.

3. The antibody molecule according to claim 1 wherein the antibody molecule is or comprises a single chain Fv (scFv), is a small immunoprotein (SIP), is a diabody, or is an IgG molecule.

4. A conjugate comprising an antibody molecule according to claim 1 and an immunosuppressive or anti-inflammatory agent.

5. The conjugate according to claim 4, wherein the anti-inflammatory agent is a cytokine.

6. The conjugate according to claim 4 wherein the conjugate is a fusion protein comprising the antibody molecule and an immunosuppressive agent, an anti-inflammatory agent, or a cytokine.

7. A conjugate comprising an antibody molecule according to claim 1 and a detectable label.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1A shows the Biacore characterization of the antibody CT01 and confirms its binding to human lysozyme. FIG. 1B shows the Biacore characterization of the antibody 2PC10 and confirms its binding to TIMP-1. FIG. 1C shows the Biacore characterization of the antibody CPR01 and confirms its binding to the domain D of human Tenascin-C. FIG. 1D shows the Biacore characterization of the antibody CPR01 and confirms its binding to the domain BCD of murine Tenascin-C. FIG. 1 E shows the Biacore characterization of the antibody CPR01.1 and confirms its binding to the domain D of human Tenascin-C. FIG. 1F shows the Biacore characterization of the antibody CPR01.1 and confirms its binding to the domain BCD of murine Tenascin-C.

(2) FIG. 2 shows immunofluorescence experiments performed in sections of ulcerative colitis patients using different antibodies. FIG. 2A shows strong positive staining of neovascular structures with the anti-IIICS antibody SW01. FIG. 2B shows positive staining of neovascular structures with the anti-EDB antibody L19, although less strong than the staining observed with the anti-IIICS antibody SW01. FIG. 2C shows positive staining of neovascular structures with the anti-Al TNC antibody F16. The staining observed with the F16 antibody is similar to that observed with antibody L19, and less strong than the staining observed with the anti-IIICS antibody SW01. FIG. 2D shows that the anti-CTNC antibody G11 did not stain neovascular structures. FIG. 2E shows positive staining of neovascular structures with the anti-MMP3 antibody CH01, although the observed staining was again less strong than that observed with the anti-IIICS antibody SW01. FIG. 2F shows that the anti-hens egg lysozyme (HEL) antibody KSF does not stain neovascular structures. The KSF antibody was used as a negative control, as hen egg lysozyme is not expressed in mammalian tissues.

(3) FIG. 3 shows the results of immunofluorescence experiments performed in cultured HT29 cells and HeLA cells using the anti-human lysozyme antibody CT01. FIG. 3A demonstrates that the anti-human lysozyme antibody CT01 clearly stains HT29 cells, while FIG. 3C shows that the negative control antibody anti-egg hen lysozyme KSF does not. FIG. 3B shows the DAPI staining of the nuclei of the cells shown in FIG. 3A, while FIG. 3D shows the staining of the nuclei of the cells shown in FIG. 3C.

(4) FIG. 4 shows the results immunofluorescence experiments performed on colitis/Crohn's disease biopsy samples from two patients with colitis and two patients with Crohn's disease. When the sections were probed with the anti-human lysozyme antibody CT01 antibody in SIP format, fluorescent staining of epithelia is clearly visible but not when probed with the negative control antibody anti-egg hen lysozyme KSF in the same SIP format. An anti-CD31 antibody (specific for an endothelial marker) was used as a positive control. DAPI staining of the nuclei was also performed. FIGS. 4A and 4B are for patient with colitis n 1; FIG. 4C and 4D are for patient with colitis n 2. FIGS. 4E and 4F are for patient with Crohn's disease n 1 and FIGS. 4G and 4H are for patient with Crohn's disease n 2.

(5) FIG. 5 shows the results of immunofluorescence experiments performed on samples taken from the intestine of a mouse with colitis. The experiments were run to confirm whether or not the anti-human lysozyme antibody CT01 cross-reacts with murine lysozyme. When the sections were probed with the CT01 antibody in SIP format, fluorescence staining is clearly visible but not when probed with the negative control antibody anti-egg hen lysozyme KSF in the same SIP format. An anti-CD31 antibody (specific for an endothelial marker) was used as positive control. DAPI staining of the nuclei was also performed.

(6) FIG. 6 shows the ELISA characterization of the FF01 and FF02 antibodies and confirms their binding to human neutrophil elastase. FIG. 6A and B show the binding of the FF01 antibody to human neutrophil elastase. FIG. 6C and D show the binding of the FF02 antibody to human neutrophil elastase. ug/ml refers to g/ml. The y-axis in FIG. 6A-D shows the optical density (OD) as measured at 450 nM.

(7) FIG. 7 shows the results of immunofluorescence experiments performed with the FF02 antibody on sections taken from patients with Crohn's disease and ulcerative colitis.

(8) FIG. 8 shows the results of immunofluorescence experiments performed with the CPR01.1 antibody on sections taken from the intestine of a mouse with colitis. A stronger intensity signal can be detected in the colon of animals stained with CPR01.1 (FIG. 8A) when compared to the signal observed with an irrelevant antibody (KSF) (FIG. 8B). An anti-CD31 antibody (specific for an endothelial marker) was used as a positive control. DAPI staining of the nuclei was also performed.

(9) FIG. 9 shows the in vivo near-infrared fluorescence imaging CPR01.1 in mouse bearing PC-3 xenograft. A mouse bearing subcutaneous PC3 tumors was injected intravenously with IRDye 800CW (Licor) labeled CPR01.1 (200 L). After 72 hours fluorescence images were acquired on an IVIS Spectrum imaging system. The mouse was then sacrificed by cervical dislocation and images of isolated organs were acquired using the same settings. The images show a preferential and selective accumulation CPR01.1 in the tumor. The healthy organs are virtually negative for any uptake of CPR01.1. The signal recorded in the liver is consistent with the expected hepatic excretion route of an IgG labelled with a new-infrared dye.

(10) FIG. 10 shows the results of immunofluorescence experiments with the CPR01.1 antibody against the D domain of Tenascin-C on sections from the human xenograft melanoma A375 (FIG. 10A), from the human xenograft glioblastoma U87 (FIG. 10B) and from the murine xenograft teratocarcinoma F9 (FIG. 10C). An anti-CD31 antibody (specific for an endothelial marker) was used as positive control. DAPI staining of the nuclei was also performed. The anti-hen egg Lysozyme antibody ScFv(KSF) was used as an isotype-negative control for the staining and the anti-fibronectin EDA domain antibody SIP(F8) was used as a positive control. The antibody anti-D domain of Tenascin-C SIP (CPR01.1) was able to stain xenograft tumours derived from both murine and human tumour cell lines.

DETAILED DESCRIPTION

(11) The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

(12) In one aspect, the present invention relates to antibodies which bind (i) Human Lysozyme, (ii) Neutrophil Elastase, iii) Tissue Inhibitor of Metalloproteinase-1 (TIMP1), or (iv) the D domain of Tenascin-C.

(13) Antibody Molecule

(14) The term antibody molecule describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is substantially homologous to, an antibody binding domain.

(15) Examples of antibodies are the immunoglobulin isotypes and their isotypic subclasses; fragments which comprise an antigen binding domain such single chain diabodies. The antibody molecule or fragment thereof may be human or humanised. It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the 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. 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.

(16) As antibodies can be modified in a number of ways, the term antibody molecule should be construed as covering antibody fragments, derivatives, functional equivalents and homologues of antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023.

(17) The term specific may be used to refer to the situation in which the antibody molecule will not show any significant binding to molecules other than its specific binding partner(s). The term is also applicable where e.g. an antigen-binding site of an antibody molecule is specific for a particular epitope that is carried by a number of antigens, in which case the antibody molecule carrying the antigen-binding site will be able to bind to the various antigens carrying the epitope.

(18) The antibody molecule may be monovalent or bivalent i.e. may have two antigen binding sites. Where the antibody molecule is bivalent, the two antigen binding sites may be identical or different. An antigen binding site describes the part of an antibody which comprises the area which specifically binds to and is complementary to part or all of an antigen. Where an antigen is large, an antibody molecule may only bind to a particular part of the antigen, which part is termed an epitope. An antigen binding site may be provided by one or more antibody variable domains (e.g. a so-called Fd antibody fragment consisting of a VH domain). Preferably, an antigen binding site comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

(19) The VH and VL can be further subdivided into regions of hyper-variability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Herein, the three CDRs of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3 and the three CDRs of the light chain are referred to as LCDR1, LCDR2 and LCDR3. The CDRs contain most of the residues which form specific interactions with the antigen.

(20) Various systems are used to define the CDR residues within the antibody variable domains. The most commonly used systems are the Kabat and Chothia systems. The Kabat CDR definition (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991)) is based upon antibody sequence variability. The Chothia CDR definition (Chothia et al., Canonical structures for the hypervariable regions of immunoglobulins, Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., Standard conformations for the canonical structures of immunoglobulins, Journal of Molecular Biology, 273, 927-948 (1997)) is based on three-dimensional structures of antibodies and topologies of the CDR loops. The CDRs defined by the Chothia system are identical to the CDRs defined by the Kabat system with the exception of HCDR1 and HCDR2.

(21) In the present disclosure, the Kabat definition is used for HCDR2.

(22) In the present disclosure, HCDR1 is defined using the Kabat system for the L19 and of the F16 antibody (L19: SEQ ID NO: 51, F16 SEQ ID NO: 59) in order to be consistent with previous patent disclosures. For other antibodies, HCDR1 is defined using the Chothia system. According to the Kabat definition, HCDR1 starts eight residues after the first cysteine of the heavy chain, whereas the Chothia definition of HCDR1 starts three residues after this cysteine. HCDR1 ends at the same residue in both systems. For example, the HCDR1 of CT01, FF01, FF02, SW01, CPR01 and CPR01.1 according to the Chothia system is GFTFSSYAMS and according to the Kabat system is SSYAMS; the HCDR1 of CH01 according to the Chothia system is GFTFSPYAMS and according to the Kabat system is SPYAMS; and the HCDR1 of 2PC10 according to the Chothia system is GFTFSSAAMS and according to the Kabat system is SSAAMS. Although the Chothia definition of HCDR1 is preferred for these antibodies, the present disclosure also encompasses these antibodies with HCDR1 defined according to the Kabat system.

(23) An antibody molecule of the invention preferably comprises the HCDR3 of antibody CT01, antibody FF01, antibody FF02, antibody 2PC10, or antibody CPR01/CPR01.1. The HCDR3 is known to play a role in determining the specificity of an antibody molecule (Segal et al., (1974), PNAS, 71:4298-4302; Amit et al., (1986), Science, 233:747-753; Chothia et al., (1987), J. Mol. Biol., 196:901-917; Chothia et al., (1989), Nature, 342:877-883; Caton et al., (1990), J. Immunol., 144:1965-1968; Sharon et al., (1990a), PNAS, 87:4814-4817; Sharon et al., (1990b), J. Immunol., 144:4863-4869; Kabat et al., (1991b), J. Immunol., 147:1709-1719).

(24) The antibody molecule may further comprise the HCDR1, HCDR2, LCDR1, LCDR2 and/or LCDR3 of antibody CT01, antibody FF01, antibody FF02, antibody 2PC10, antibody CPR01, or antibody CPR01.1.

(25) The antibody may also comprise the VH and/or VL domain of antibody CT01, antibody FF01, antibody FF02, antibody 2PC10, antibody CPR01, or antibody CPR01.1.

(26) An antibody molecule of the invention may comprise a VH domain having at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the VH domain of antibody CT01, antibody FF01, antibody FF02, antibody 2PC10, antibody CPR01, or antibody CPR01.1.

(27) An antibody molecule of the invention may comprise a VL domain having at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity to the VL domain of antibody CT01, antibody FF01, antibody FF02, antibody 2PC10, antibody CPR01, or antibody CPR01.1.

(28) Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty=12 and gap extension penalty=4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used.

(29) Variants of these VH and VL domains and CDRs may also be employed in antibody molecules for use in as described herein. Suitable variants can be obtained by means of methods of sequence alteration, or mutation, and screening.

(30) Particular variants for use as described herein may include one or more amino acid sequence alterations (addition, deletion, substitution and/or insertion of an amino acid residue), maybe less than about 20 alterations, less than about 15 alterations, less than about 10 alterations or less than about 5 alterations, 4, 3, 2 or 1.

(31) Alterations may be made in one or more framework regions and/or one or more CDRs. In particular, alterations may be made in HCDR1, HCDR2 and/or HCDR3.

(32) The antibody molecule may be a whole antibody or a fragment thereof, in particular an antigen-binding fragment thereof.

(33) Whole antibodies include IgA, IgD, IgE, IgG or IgM. Preferably, the whole antibody is IgG.

(34) Antigen-binding fragments of whole antibodies include (i) the Fab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward et al. (1989) Nature 341, 544-546; McCafferty et al., (1990) Nature, 348, 552-554; Holt et al. (2003) Trends in Biotechnology 21, 484-490), 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 (Bird et al. (1988) Science, 242, 423-426; Huston et al. (1988) PNAS USA, 85, 5879-5883); (viii) bispecific single chain Fv dimers (PCT/US92/09965) and (ix) diabodies, multivalent or multispecific fragments constructed by gene fusion (WO2013/014149; WO94/13804; Holliger et al. (1993a), Proc. Natl. Acad. Sci. USA 90 6444-6448). Fv, scFv or diabody molecules may be stabilized by the incorporation of disulphide bridges linking the VH and VL domains (Reiter et al. (1996), Nature Biotech, 14, 1239-1245). Minibodies comprising a scFv joined to a CH3 domain may also be made (Hu et al. (1996), Cancer Res., 56(13):3055-61). 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 CH1 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.

(35) A single chain Fv (scFv) may be comprised within a mini-immunoglobulin or small immunoprotein (SIP), e.g. as described in (Li et al., (1997), Protein Engineering, 10: 731-736). An SIP may comprise an scFv molecule fused to the CH4 domain of the human IgE secretory isoform IgE-S2 (.sub.S2-CH4; Batista et al., (1996), J. Exp. Med., 184: 2197-205) forming an homo-dimeric mini-immunoglobulin antibody molecule

(36) Where the antibody molecule is a diabody, the VH and VL domains are preferably linked by a 5 to 12 amino acid linker. A diabody comprises two VH-VL molecules which associate to form a dimer. The VH and VL domains of each VH-VL molecule are preferably linked by a 5 to 12 amino acid linker. For example, the VH and VL domains may be linked by an amino acid linker which is 5, 6, 7, 8, 9, 10, 11, or 12 amino acid in length. Preferably, the amino acid linker is 5 amino acids in length. Suitable linker sequences are known in the art and include the linker sequence set forth in SEQ ID NO: 68.

(37) Where the antibody molecule is an scFv, the VH and VL domains of the antibody are preferably linked by a 14 to 20 amino acid linker. For example, the VH and VL domains may be linked by an amino acid linker which is 14, 15, 16, 17, 18, 19, or 20 amino acid in length. Suitable linker sequences are known in the art and include the linker sequence set forth in SEQ ID NO: 67.

(38) Where the antibody is a small immunoprotein (SIP), the VL domain of the scFv antibody is preferably linked to the CH4 domain of human IgE via a 5 to 20 amino acid linker, more preferably a 5 to 10 amino acid linker. Suitable linker sequences are known in the art and include the linker sequence set forth in SEQ ID NO: 83. The CH4 domain of human IgE preferably has the sequence set forth in SEQ ID NO: 84, or a sequence which has at least 70%, more preferably one of at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO: 84.

(39) Preferably the antibody molecule comprises or consists of a single chain Fv, a small immunoprotein, a diabody, or a (whole) IgG molecule.

(40) Conjugates

(41) Conjugates of the invention comprise an antibody molecule of the invention and a therapeutic or diagnostic agent. The therapeutic agent may be an immunosuppressive or anti-inflammatory agent, such as a cytokine.

(42) The diagnostic agent may be a detectable label, such as a radioisotope, e.g. a non-therapeutic radioisotope.

(43) Radioisotopes which may be conjugated to a binding member of the invention include isotopes such as .sup.94mTc, .sup.99mTc, .sup.186Re, .sup.188Re, .sup.203Pb, .sup.67Ga, .sup.68Ga, .sup.47Sc, .sup.111In, .sup.97Ru, .sup.62Cu, .sup.64Cu, .sup.86Y, .sup.88Y, .sup.90Y, .sup.121Sn, .sup.161Tb, .sup.153Sm, .sup.166Ho, .sup.105Rh, .sup.177Lu, .sup.123I, .sup.124I, .sup.125I, .sup.131I, .sup.18F, .sup.211At and .sup.225Ac. Preferably, positron emitters, such as .sup.18F and .sup.124I or gamma emitters, such as .sup.99mTc, .sup.111In and .sup.123I, are used for diagnostic applications (e.g. for PET), while beta-emitters, such as .sup.131I, .sup.90Y and .sup.177Lu, are preferably used for therapeutic applications. Alpha-emitters, such as .sup.211At and .sup.225Ac may also be used for therapy. In one example, the specific binding member may be conjugated to .sup.177Lu or .sup.90Y.

(44) The specific binding member may be conjugated to the immunosuppressive or anti-inflammatory agent, or detectable label, by means of a peptide bond or linker, i.e. within a fusion polypeptide comprising said molecule and the specific binding member or a polypeptide chain component thereof. Other means for conjugation include chemical conjugation, especially cross-linking using a bifunctional reagent (e.g. employing DOUBLE-REAGENTS' Cross-linking Reagents Selection Guide, Pierce).

(45) Linkers

(46) The antibody molecule and the therapeutic or diagnostic agent may be connected to each other directly, for example through any suitable chemical bond or through a linker, for example a peptide linker.

(47) The peptide linker may be a short (2-20, preferably 2-15, residue stretch of amino acids). Suitable examples of peptide linker sequences are known in the art. One or more different linkers may be used. The linker may be about 5 amino acids in length.

(48) The chemical bond may be, for example, a covalent or ionic bond. Examples of covalent bonds include peptide bonds (amide bonds) and disulphide bonds. For example the antibody molecule and therapeutic or diagnostic agent may be covalently linked. For example by peptide bonds (amide bonds). Thus, the antibody molecule and therapeutic or diagnostic agent may be produced (secreted) as a single chain polypeptide. The individual components that form the antibody molecule or the therapeutic or diagnostic agent may also be connected directly, for example through any suitable chemical bond, or through a linker, for example a peptide linker. Examples of individual components which may be linked within the antibody molecule are CDRs or VH or VL sequences.

(49) For example, an immunosuppressive or anti-inflammatory agent, such as a cytokine, or a detectable label, may be conjugated, either through an amino acid linker, or directly, to the N-terminus or C-terminus of the antibody molecule. For example, where the antibody molecule is or comprises an scFv, the immunosuppressive or anti-inflammatory agent, or the detectable label, may be conjugated, either through an amino acid linker, or directly to the N-terminus of the VH domain of the scFv, or to the C-terminus of the VL domain of the scFv. Where the antibody molecule is a diabody (which comprises two scFv molecules), the immunosuppressive or anti-inflammatory agent, or the detectable label, may be conjugated, either through an amino acid linker, or directly, to the N-terminus of one or both of the VH domains, or to the C-terminus of one or both of the VL domains, of the two scFvs making up the diabody.

(50) Methods of Treatment and Diagnosis

(51) An antibody molecule or conjugate of the invention may be used in a method of treatment of the human or animal body, such as a method of treatment (which may include prophylactic treatment) of a disease or disorder in a patient (typically a human patient) comprising administering the antibody molecule or conjugate to the patient.

(52) Accordingly, such aspects of the invention provide methods of treatment comprising administering an antibody molecule or conjugate of the invention, pharmaceutical compositions comprising such an antibody molecule or conjugate for the treatment of a condition or disease, and a method of making a medicament or pharmaceutical composition comprising formulating the antibody molecule or conjugate of the present invention with a physiologically acceptable carrier or excipient.

(53) An antibody molecule or conjugate as herein described may be used in a method of treating an inflammatory disorder, preferably an inflammatory bowel disorder, or treating an autoimmune disease in a patient. The method may comprise targeting a therapeutic agent to the neovasculature in vivo. The agent may be any therapeutic agent discussed herein, which is suitable for treatment of the disease or disorder in question.

(54) Also contemplated is a method of treating an inflammatory disorder, preferably an inflammatory bowel disorder, or treating an autoimmune disease in a patient by targeting a therapeutic agent to the neovasculature in a patient, the method comprising administering a therapeutically effective amount of an antibody molecule or conjugate as herein described to the patient.

(55) An antibody molecule or conjugate as herein described may also be used in a method of imaging, detecting, or diagnosing a disease or disorder in a patient. A method of imaging, detecting, or diagnosing a disease or disorder comprising administering an antibody or conjugate as described herein to a patient is similarly contemplated. The disease or disorder may be an inflammatory disorder, preferably an inflammatory bowel disorder, or an autoimmune disease. The method may comprise targeting a diagnostic agent, such as a detectable label, to the neovasculature in vivo.

(56) Inflammatory disease and/or disorder refers to diseases and/or disorders which are accompanied and/or characterised by inflammation. An inflammatory disease and/or disorder is preferably associated with and/or characterised by angiogenesis. An inflammatory disease and/or disorder may be an inflammatory disease and/or disorder characterised by angiogenesis, wherein the neovasculature expresses the ED-A isoform of fibronectin, the ED-B isoform of fibronectin and/or alternatively spliced Tenascin-C.

(57) An autoimmune disease is preferably associated with and/or characterised by angiogenesis. An autoimmune disease may be an autoimmune disease characterised by angiogenesis, wherein the neovasculature expresses the ED-A isoform of fibronectin, the ED-B isoform of fibronectin and/or alternatively spliced Tenascin-C. The autoimmune disease may be an inflammatory autoimmune disease, i.e. an autoimmune disease associated with and/or characterised by inflammation. The inflammatory autoimmune disease may be any inflammatory autoimmune disease which is characterised by expression of the ED-A isoform of fibronectin, the ED-B isoform of fibronectin, and/or alternatively spliced tenascin C, in particular at sites of inflammation in the patient. The autoimmune disease may be selected from the group consisting of: inflammatory bowel disease (IBD), atherosclerosis, rheumatoid arthritis (RA), multiple sclerosis (MS), endometriosis, autoimmune diabetes (such as diabetes mellitus type 1), psoriasis, psoriatic arthritis, and periodontitis. Preferably, the autoimmune disease is IBD.

(58) Inflammatory Bowel Disease is a group of inflammatory conditions that affect the colon and small intestine. The major types of IBD are Crohn's disease and ulcerative colitis, while other types of IBD include collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behet's disease and indeterminate colitis. Crohn's disease can affect any part of the gastrointestinal tract, whereas ulcerative colitis is typically restricted to the colon and rectum.

(59) IBD, as referred to herein, may be Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behet's disease or indeterminate colitis. In particular, the terms Crohn's disease, ulcerative colitis, collagenous colitis, lymphocytic colitis, ischaemic colitis, diversion colitis, Behet's disease and indeterminate colitis, as used herein, may refer to active Crohn's disease, active ulcerative colitis, active collagenous colitis, active lymphocytic colitis, active ischaemic colitis, active diversion colitis, and active indeterminate colitis, respectively.

(60) The inflammatory disorder or autoimmune disease is preferably inflammatory bowel disease, such as ulcerative colitis or Crohn's disease.

(61) Another aspect of the invention provides an antibody molecule which binds to the D domain of Tenascin-C described herein for use in treating a proliferative disorder, such as cancer.

(62) Another aspect of the invention provides a method of treating a proliferative disorder, such as cancer, comprising administering an antibody molecule which binds to the D domain of Tenascin-C described herein to an individual in need thereof. Preferably the individual is human.

(63) Another aspect of the invention provides a method of diagnosing a proliferative disorder, such as cancer, in an individual, wherein the method comprises administering the antibody molecule which binds to the D domain of Tenascin-C described herein to the individual and detecting binding of the antibody molecule in the individual.

(64) Other aspects of invention provide an antibody molecule which binds to the D domain of Tenascin-C described herein for use in a method of treating or diagnosing a proliferative disorder, such as cancer, and the use of an antibody molecule described herein in the manufacture of a medicament for use in a method of treating or diagnosing a proliferative disorder, such as cancer.

(65) Preferred antibody molecules which bind to the D domain of Tenascin-C are described above and include CPR01.1.

(66) An individual suitable for treatment with an antibody molecule which binds to D domain of Tenascin-C may have a proliferative disorder, preferably a cancer.

(67) Proliferative disorders are caused or characterized by increased cell growth and proliferation and may include a pre-malignant or malignant neoplasm or tumour, (e.g. histocytoma, glioma, astrocyoma, osteoma), cancer (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, merkel cell carcinoma, pancreas cancer, brain cancer such as glioma, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), a disease characterized by neovasculature or an angiogenic disease. Noncancerous tumours of any of these tissues may also be treated. Cancers may be familial or sporadic.

(68) Treatment of a tumour or cancer in an individual may comprise eradication of the tumour. However, for many forms of tumours, especially malignant cancers and aggressive forms such as glioblastoma, complete cure may not be possible. Treatment may comprise retarding tumour growth and/or reducing tumour volume. Treatment may comprise lengthening the overall survival or progression free survival of the individual. Treatment may comprise improving quality of life of the individual, e.g. by reducing one or more symptoms caused by the tumour. Treatment may comprise inhibiting regrowth of the tumour following tumour regression. Treatment according to the present invention may be used to achieve any or all of these therapeutic effects.

(69) The antibody molecule which binds to the D domain of Tenascin-C as described herein may be administered alone or in combination with other treatments, concurrently or sequentially or as a combined preparation with another therapeutic agent or agents, for the treatment of a disease, including a proliferative disorder, such as cancer. For example, an antibody molecule which binds to the D domain of Tenascin-C as described herein may be used in combination with an existing therapeutic agent for the treatment of a proliferative disorder, such as cancer. Therapeutic agents may include anti-cancer compounds which are well-known in the art such as: alkylating agents, antimetabolites, plant alkaloids and terpenoids, topoisomerase inhibitors, anti-tumour antibiotics, monoclonal antibodies, and corticosteroids.

(70) Pharmaceutical Compositions

(71) A further aspect of the present invention relates to a pharmaceutical composition comprising at least one antibody molecule or conjugate of the invention and optionally a pharmaceutically acceptable excipient.

(72) Pharmaceutical compositions of the present invention typically comprise a therapeutically effective amount of an antibody molecule or conjugate according to the invention and optionally auxiliary substances such as pharmaceutically acceptable excipient(s). Said pharmaceutical compositions are prepared in a manner well known in the pharmaceutical art. A carrier or excipient may be a liquid material which can serve as a vehicle or medium for the active ingredient. Suitable carriers or excipients are well known in the art and include, for example, stabilisers, antioxidants, pH-regulating substances, controlled-release excipients. The pharmaceutical composition of the invention may be adapted, for example, for parenteral use and may be administered to the patient in the form of solutions or the like.

(73) Pharmaceutical compositions comprising the antibody molecule or conjugate of the present invention may be administered to a patient. Administration is preferably in a therapeutically effective amount, this being sufficient to show benefit to the patient. Such benefit may be amelioration of at least one symptom. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc., is within the responsibility of general practitioners and other medical doctors. Treatments may be repeated at daily, twice-weekly, weekly, or monthly intervals at the discretion of the physician.

(74) A pharmaceutical composition of the invention may be administered to a patient in need of treatment via any suitable route, usually by injection into the bloodstream and/or directly into the site to be treated. The precise dose and its frequency of administration will depend upon a number of factors, the route of treatment, the size and location of the area to be treated.

(75) 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

(76) For intravenous injection, or injection at the site of affliction, the pharmaceutical composition 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.

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

(78) Kits

(79) Another aspect of the invention provides a therapeutic kit for use in the treatment of a disease or disorder comprising an antibody molecule or conjugate of the invention. The components of a kit are preferably sterile and in sealed vials or other containers.

(80) A kit may further comprise instructions for use of the components in a method described herein. The components of the kit may be comprised or packaged in a container, for example a bag, box, jar, tin or blister pack.

(81) Further aspects and embodiments of the invention will be apparent to those skilled in the art given the present disclosure including the following experimental exemplification.

(82) All documents mentioned in this specification are incorporated herein by reference in their entirety for all purposes.

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

(84) Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

(85) Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described above.

EXAMPLES

Example 1

Preparation and Characterisation of the Novel Antibodies CT01 against Lysozyme, FF01 and FF02 against Neutrophil Elastase, 2PC10 against TIMP-1, CPR01 and CPR01.1 against Domain D of Tenascin C

(86) The antibodies against human lysozyme, human neutrophil elastase, human TIMP-1 and domain D of human tenascin C, antibodies CT01, FF01 and FF02, 2PC10, CPR01 and CPR01.1, were isolated in single-chain Fv (scFv) configuration from phage display libraries, which included the libraries described in PCT/EP2009/006487, Weber et al. (PLoS One, 2014, 9 (6) doi: 10/1361) and Silacci et al. (Protein Engineering Design & Selection, 2006, 19, 471-478), according to the screening technique described by Silacci et al. (Protein Engineering Design & Selection, 2006, 19, 471-478) using lysozyme, neutrophil elastase, TIMP-1, and domain D of tenascin C, respectively, as the screening antigen.

(87) Antibodies in SIP format were prepared by fusing the VL domain sequence of the antibody in scFv format to the CH4 domain of human IgE via using overlap extension PCR. The SIP construct was then fused to a mammalian leader sequence using overlap extension PCR and subcloned into the pcDNA3.1 vector using the restriction enzymes HindIII and NotI. The protein was expressed in CHO.S cells using PEI-mediated transient gene expression, and purified from the culture supernatant by affinity chromatography using protein A resin (Sino Biological Inc.). The sequence of the linker linking the VL domain to the CH4 domain of human IgE is shown in SEQ ID NO: 83. The sequence of the CH4 domain of human IgE is shown in SEQ ID NO: 84. The sequence of the FF02 antibody in SIP format is shown in SEQ ID NO: 82.

(88) Results:

(89) The binding of antibodies CT01 to lysozyme, 2PC10 to TIMP-1, CPR01 and CPR01.1 to domain D of tenascin-C was further confirmed by Biacore analysis as described below. The corresponding results are shown in FIG. 1. The binding of antibodies FF01 and FF02 to human neutrophil elastase was confirmed by ELISA. The corresponding results are shown in FIG. 6.

(90) The affinity measurements for the binding of CT01 to the lysozyme are shown in the table 1. The Biacore analysis demonstrates that scFv(CT01) had an affinity (KD) for the lysozyme of 8.910.sup.7 M.

(91) TABLE-US-00001 TABLE 1 Ka (1/Ms) Kd (1/s) KD (M) 8.72E03 7.7E03 8.9E07

(92) The affinity measurements for the binding of CPR01 to the D domain of human Tenascin-C are shown in the table 2.

(93) TABLE-US-00002 TABLE 2 Ka (1/Ms) Kd (1/s) KD (M) 151000 0.011 7.0E08

(94) The affinity measurements for binding of CPR01 to the BCD domains of murine tenascin-C are shown in table 3.

(95) TABLE-US-00003 TABLE 3 Ka (1/Ms) Kd (1/s) KD (M) 240000 7.0E3 2.9E08

(96) The Biacore analysis demonstrates that scFv(CPR01) had an affinity (KD) for the D domain of human Tenascin-C of 7.010.sup.8 M and a KD for the BCD domains of murine tenascin-C of 2.910.sup.8 M and thus the species cross-reactivity of this antibody.

(97) The affinity measurements for the binding of CPR01.1 to the D domain of human tenascin-C are shown in the table 4.

(98) TABLE-US-00004 TABLE 4 Ka (1/Ms) Kd (1/s) KD (M) 78300 0.067 8.5E07

(99) The affinity measurements for binding of CPR01.1 to the BCD domains of murine tenascin-C are shown in table 5.

(100) TABLE-US-00005 TABLE 5 Ka (1/Ms) Kd (1/s) KD (M) 150000 0.064 4.3E07

(101) The Biacore analysis demonstrates that scFv(CPR01.1) had an affinity (K.sub.D) for the D domain of human Tenascin-C of 8.510.sup.7 M and a KD for the BCD domains of murine tenascin-C of 4.310.sup.7 M and thus the species cross-reactivity of this antibody.

(102) The affinity measurements for the binding of CH01 to human MMP3 is shown in the table 6.

(103) TABLE-US-00006 TABLE 6 Ka (1/Ms) Kd (1/s) KD (M) 1.7E05 4.7E03 2.8E08

(104) The affinity measurements for the binding of CH01 to murine MMP3 is shown in the table 7.

(105) TABLE-US-00007 TABLE 7 Ka (1/Ms) Kd (1/s) KD (M) 8.0E04 2.5E03 2.9E08

(106) The Biacore analysis demonstrates that scFv(CH01) had an affinity (K.sub.D) for the human MMP3 of 2.810.sup.8 M and an affinity (K.sub.D) for the murine MMP3 of 2.910.sup.8 M.

(107) The affinity measurements for the binding of SW01 to IIICS isoform of fibronectin is shown in the table 8. The Biacore analysis demonstrates that scFv(SW01) had an affinity (KD) for the IIICS isoform of fibronectin of 2.6210.sup.7 M

(108) TABLE-US-00008 TABLE 8 Ka (1/Ms) Kd (1/s) KD (M) 1.84E05 0.048 2.62E07
Biacore Analysis:

(109) CT01, CPR01 and 2PC10 were expressed in CHO.S cells as ScFv antibody fragments and purified from the culture supernatant by affinity chromatography using protein A resin (Sino Biological Inc.).

(110) CT01 was analysed for antigen binding by Biacore using a CM5 chip coated with commercial native human lysozyme (from human neutrophils; Cat-No. L8402; SIGMA) to a final coating density of 2000 resonance units (RU). The flow rate for the Biacore analysis was set at 20 L/min.

(111) CPR01 was analysed for antigen binding by Biacore using a CM5 chip coated with the human tenascin-C D domain. The flow rate for the Biacore analysis was set at 10 L/min.

(112) ScFv (CPR01.1) antibody fragments were expressed in CHO.S cells and purified from the culture supernatant by affinity chromatography using protein A resin (Sino Biological Inc.)

(113) The eluted fractions containing ScFv antibody fractions were analysed for antigen binding by BIAcore using a CM5 chip coated with the D domain of human Tenascin-C. Binding to the BCD domain of murine Tenascin-C coated on a CM5 chip was also assessed to determine species cross-reactivity of the antibody. Analysis was run in phosphate buffer (100 mM sodium phosphate pH 7.4, 100 mM NaCl) at a flow rate of 10 L/min and after each injection the chip was regenerated with 5 L of 10 mM HCl.

(114) 2PC10 was analysed for antigen binding by Biacore using a CM5 chip coated with rHsTIMP1 (a recombinant polypeptide which includes the hs-TIMP1 sequence, aa24-207, and 6His at the C-terminus of the protein) to achieve a final coating density of 3500 resonance units (RU). The flow rate for the Biacore analysis was set at 10 L/min.

(115) ELISA Analysis

(116) Elastase (ELANE) protein from human neutrophils was purchased from MyBiosource (MBS173384). Human Neutrophil Elastase (MyBiosource) was biotinylated according to the standard protocol. The degree of biotinylation was 0.3-0.6 biotin/molecule.

(117) ELISA with the FF01 and FF02 Antibodies in scFv Format

(118) MAXIsorp strips (Nunc) were coated with HNE at 10.sup.6 M. Coated plates were incubated with myc-tagged FF01, FF02 scFv fragments or with a negative control anti-hen egg lysozyme scFv at different concentrations (100 g/ml, 50 g/ml, 25 g/ml or 12.5 g/ml) for 1 hour. Bound antibody was detected with the anti-Myc antibody 9E10 (1:500 dilution) and an HRP-conjugated anti-Murine F.sub.c antibody (Sigma). (1:1000 dilution). Colorimetric detection of antibody-antigen binding was performed using BM-Blue POD soluble substrate (Roche). The optical density was measured at 450 nM.

(119) ELISA with FF01 and FF02 Antibodies in SIP Format

(120) Streptawell High Bind strips (Roche) were coated with biotinylated HNE at 3.510.sup.6 M. Coated plates were incubated with the FF01 or with the FF02 antibody in SIP format (50 pg/ml) for 1 hour. Bound antibody was detected with a Rabbit anti-human IgE (Sigma) and an HRP-conjugated anti-Rabbit IgG antibody (Sigma). The plate coating density was assessed by detecting the immobilized HNE using an HNE-specific Rabbit IgG (Abcam 68672, 1:150 dilution) and detection with an HRP-conjugated anti-Rabbit IgG antibody (1:1000 dilution, Sigma).

(121) Colorimetric detection of antibody-antigen binding was performed using BM-Blue POD soluble substrate (Roche). The optical density was measured at 450 nM.

(122) Results:

(123) The results are shown in FIG. 6 and confirm that the FF01 and FF02 antibodies bind to human neutrophil elastase both in scFv and in SIP format.

Example 2

Immunofluorescence Staining of Sections from Ulcerative Colitis Patients

(124) Freshly frozen biopsy samples of Ulcerative Colitis were stained according to published methods (S. Pfaffen. Eur J Nucl Med Mol Imaging 2010). In brief, purified biotinylated antibodies in SIP (KSF) or IgG (SW01, L19, F16, CH01, G11) format were added at the final concentration of 2pg/ml to the sections. Detection of the primary antibody was performed with streptavidine-Alexa-488 antibody (Invitrogen).

(125) Positive control was performed by staining blood vessels with an antibody (eBioscience, 1:100) specific for CD31, an endothelial cell marker, the signal revealed with goat anti-mouse Alexa-594 (Invitrogen). Further positive control was performed by counterstaining for cell nuclei was performed with DAPI (eBioscience). Sections were mounted with fluorescent mounting medium (DAKO) followed by analysis using an Axioskop2 microscope with a 10 objective (Carl Zeiss A G, Jena, Germany). The results are shown in FIG. 2.

Example 3

Immunofluorescence Staining of HT29 Cells with the CT01 Antibody

(126) Coverslips were placed into culture dishes (100 mm20 mm), and HT29 cells (both human adenocarcinoma cell lines) were then seeded at 100,000 cells/mL in the culture dishes respectively. The culture was incubated until proper confluency was reached. Cells were fixed and permeabilized by ice-cold methanol incubation at 20 C. Cells were then blocked with 10% FBS/2% BSA. Affinity-purified antibody CT01, in SIP format (final concentration 5 g/ml) was first incubated with the cells, followed by incubation with a rabbit anti-human IgE antibody (1:500 dilution; DAKO). The antibody was then detected with goat anti-rabbit IgG Alexa 488 (1:1000 dilution; Invitrogen). DAPI was used for nuclei staining. The anti-hen egg Lysozyme SIP antibody (KSF) was used as an isotype-negative control for the staining. The results demonstrate that the anti-human lysozyme antibody CT01 is capable of staining lysozyme in biological samples (FIG. 3).

Example 4

Immunofluorescence Staining of Biopsies taken from Ulcerative Colitis and Crohn's Disease Patients with CT01 Antibody

(127) Staining for lysozyme and CD31 (as an endothelial marker) was performed on human colitis and Crohn's disease biopsy samples. 10 m thickness frozen specimens were defrosted at room temperate and treated with ice-cold methanol, rehydrated in PBS and blocked with 20% FCS. Affinity-purified SIPs (final concentration 5 g/mL) were first incubated with the tissue samples, followed by rabbit anti-human IgE antibody (1:500 dilution; DAKO) and the mouse anti-human CD31 antibody (1:200 dilution; eBioscience). Bound SIPs were detected with goat anti-rabbit IgG Alexa 488 (1:200 dilution; Invitrogen), while the anti-CD31 antibody was detected using goat anti-mouse IgG Alexa 594 (1:200 dilution; Invitrogen). DAPI was used for nuclei staining. The anti-hen egg lysozyme antibody ScFv(KSF) was used as an isotype-negative control for the staining. The results demonstrate that the anti-human lysozyme antibody CT01 antibody is capable of staining epithelia in human biopsy samples of colitis and Crohn's disease (FIG. 4).

Example 5

Immunofluorescence Staining of Samples taken from Mice with Colitis with the CT01 Antibody

(128) 10 m thick frozen biopsy specimens from a colitis mouse model were defrosted at room temperate and treated with ice-cold methanol, rehydrated in PBS and blocked with 20% FCS. Affinity-purified SIPs (final concentration 5 g/mL) were first incubated with the tissue sample, followed by rabbit anti-human IgE antibody (1:500 dilution; DAKO) and the rat anti-mouse CD31 antibody (1:200 dilution; BD Biosciences). Bound SIPs were detected with goat anti-rabbit IgG Alexa 488 (1:200 dilution; Invitrogen), while the anti-CD31 antibody was detected using donkey anti-rat IgG Alexa 594 (1:200 dilution; Invitrogen). DAPI was used for nuclei staining. The anti-hen egg lysozyme antibody ScFv(KSF) was used as an isotype-negative control for the staining. The results demonstrate that the anti-human lysozyme antibody CT01 is capable of binding to mouse lysozyme, i.e. cross-reacts with mouse lysozyme, which is useful for testing the CT01 antibody, and conjugates thereof, in mouse models of IBD (FIG. 5).

Example 6

Immunofluorescence Staining of Samples taken from Patients with Ulcerative Colitis or Crohn's Disease with the FF02 Antibody

(129) Frozen human colitis and Crohn's disease patient tissue specimens (10 m sections) were defrosted at room temperate and fixed with ice-cold acetone, and thereafter blocked with 20% FCS in PBS. The slides were first incubated for two hours either with FF02 SIP or with KSF SIP used as negative control, both at a concentration of 5 pg/mL and thereafter revealed with a Rabbit anti-human IgE (1:1000 dilution, Sigma). As positive control a mouse anti-human CD31 antibody (1:200 dilution, eBioscience) was used. The SIP's were detected with and an Alexa488-conjugated goat anti-Rabbit IgG antibody (1:200 dilution, Sigma), and the anti-CD31 antibody was detected with an Alexa 594-conjugated goat anti-mouse IgG (1:200 dilution, Invitrogen). Cells were also counterstained with DAPI (Sigma), and slides were mounted with fluorescent mounting medium (Dako) and analyzed with an Axioskope2 mot plus microscope (Zeiss), using the 20 objective. The results are shown in FIG. 7, and demonstrate that both in ulcerative colitis and in Crohn's disease there was a good correspondence between the staining of the FF02 antibody and the nuclei of the cells as well as the anti-CD31 antibody. No staining was visible for the negative control antibody.

Example 7

Immunofluorescence Staining of Samples from Mice having Colitis with the CPR01.1 Antibody

(130) After 2 weeks of acclimatization, 8-week-old specific pathogen-free female C57BL/6 mice (Janvier Labs) received 3.0% (wt/vol) DSS (40,000 g/mol, TdB Consultancy) in drinking water ad libitum. Five days later, DSS water was replaced by water supplemented with 5% glucose and 0.25% NaHCO.sub.3 for 7 days, followed by nonsupplemented (i.e., normal) water. Body weight and disease score were assessed daily. On day 10 after colitis induction, mice were sacrificed and colon harvested and embedded in O.C.T. compound. Eight micrometers thick sections were cut and stained following a standard immunofluorescence protocol. Briefly, purified antibodies in full IgG format (CPR01.1 and KSF) were added to a final concentration of 2 g/ml onto the sections. Detection of the primary antibody was performed with a rabbit anti human-IgG (DAKO). Signals were revealed with a goat anti-rabbit Alexa 488. Blood vessels were revealed with a rat anti mouse-CD31 antibody (BD Pharmingen), followed by donkey anti-rat Alexa 594. Counterstaining for cell nuclei was performed with DAPI (eBioscience).

(131) Results:

(132) The results are shown in FIG. 8. A stronger intensity signal can be detected in the colon of animals stained with CPR01.1 when compared to the signal observed with an irrelevant antibody (KSF).

Example 8

In Vivo Targeting Properties of the CPR01.1 Antibody by IVIS Imaging in Mouse Bearing PC3 Xenograph

(133) PC-3 cells were purchased from American Type Culture Collection (ATCC) and were grown to 80% confluence and detached with Trypsin-EDTA 0.05% (Life Technologies).

(134) The PC-3 model of human prostatic cancer has been described in Kaighn et al. (1983) Invest. Urol. 17: 16-23. Cells were washed with HBSS medium (Gibco) twice, counted and resuspended in HBSS medium to a final concentration of 100 mio cells ml-1. Aliquots of 5 mio cells (50 l of a suspension) were mixed 1:1 with Matrigel (Corning Matrigel, 734-1101, VWR) injected subcutaneously in the lower back of Balb/c nude mice (7 weeks of age, Charles River).

(135) In vivo targeting properties of CPR01.1 in IgG format was investigated using near-infrared fluorescence imaging. Mice bearing subcutaneous PC3 tumors were injected intravenously with IRDye 800CW (Licor) labeled CPR01.1 (200 L). After 72 hours, fluorescence images were acquired on an IVIS Spectrum imaging system. The mouse was then sacrificed by cervical dislocation and images of isolated organs were acquired using the same settings.

(136) Results:

(137) The images show a preferential and selective accumulation of CPR01.1 in the tumor. The healthy organs are virtually negative for any uptake of CPR01.1 (FIG. 9). The signal recorded in the liver is consistent with the expected hepatic excretion route of an IgG labelled with a new-infrared dye.

Example 9

Immunofluorescence Staining of Xenograft Tumour Sections with the CPR01.1 Antibody

(138) Dual staining for the D domain of tenascin-C and the murine vascular marker CD31 Willebrand factor was performed on sections from the human xenograft melanoma A375, the human xenograft glioblastoma U87 and the murine xenograft teratocarcinoma F9. 10 m sections from freshly frozen tumour samples were defrosted at room temperate and fixed with ice-cold acetone, rehydrated in PBS and blocked with 3% BSA. Sections were first incubated with affinity-purified SIP formatted antibody fragments (final concentration 5 mg/ml), followed by an anti-Human IgE antibody (DAKO, 2.5 g/ml final concentration) and the rat anti-Mouse CD31 endothelial marker antibody (BD Pharmigen, 30 ng/mL final concentration). Bound SIPs were detected with a goat anti-rabbit Alexa 488 (Molecular Probes), whilst the CD31 endothelial marker was detected using donkey anti-rat IgG Alexa 594 (Molecular probes). DAPI was used for nuclei staining. The anti-hen egg Lysozyme antibody ScFv(KSF) was used as an isotype-negative control for the staining and the anti-fibronectin EDA domain antibody SIP(F8) (WO2008/120101) was used as a positive control since the presence of the EDA domain in these tissues has previously been confirmed.

(139) Results:

(140) The antibody anti-D domain of Tenascin-C (CPR01.1) in SIP format was able to stain xenograft tumours derived from both murine and human tumour cell lines (FIG. 10).

SEQUENCE LISTING

(141) Amino Acid Sequences of Antibody CT01 specific for Human Lysozyme

(142) TABLE-US-00009 SEQIDNO:1 (CT01-VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPA PRARFDYWGQGTLVTVSS SEQIDNO:2 (CT01-VL) SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSPTPAPGVVFG GGTKLTVLG SEQIDNO:3 (CT01-VHCDR1) GFTFSSYAMS SEQIDNO:4 (CT01-VHCDR2) AISGSGGSTYYADSVKG SEQIDNO:5 (CT01-VHCDR3) PAPRARFDY SEQIDNO:6 (CT01-VLCDR1) QGDSLRSYYAS SEQIDNO:7 (CT01-VLCDR2) GKNNRPS SEQIDNO:8 (CT01-VLCDR3) NSSPTPAPGVV
Amino Acid Sequences of Antibody FF01 specific for Human Neutrophil Elastase

(143) TABLE-US-00010 SEQIDNO:9 (FF01-VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA IKGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVT WNNYFDYWGQGTLVTVSS SEQIDNO:10 (FF01-VL) SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSPDGGRGVVFG GGTKLTVLG SEQIDNO:85 (FF01-alternativeaminoacidsequenceoftheVL domain) SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSPDGGRGVVFG GGTKLTVL SEQIDNO:11 (FF01-VHCDR1) GFTFSSYAMS SEQIDNO:12 (FF01-VHCDR2) AIKGSGGSTYYADSVKG SEQIDNO:86 (FF01-alternativeaminoacidsequenceoftheVH CDR2) AIKGSGGSTY SEQIDNO:13 (FF01-VHCDR3) VTWNNYFDY SEQIDNO:14 (FF01-VLCDR1) QGDSLRSYYAS SEQIDNO:15 (FF01-VLCDR2) GKNNRPS SEQIDNO:16 (FF01-VLCDR3) NSSPDGGRGVV
Amino Acid Sequences of Antibody 2PC10 specific for Human TIMP-1

(144) TABLE-US-00011 SEQIDNO:17 (2PC10-VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSAAMSWVRQAPGKGLEWVSA ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA PMFDYWGQGTLVTVSS SEQIDNO:18 (2PC10-VL) EIVLTQSPGTLSLSPGERATLSCRASQSVSTHLLAWYQQKPGQAPRLLIY GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWGLTPAMFG QGTKVEIK SEQIDNO:19 (2PC10-VHCDR1) GFTFSSAAMS SEQIDNO:20 (2PC10-VHCDR2) AISGSGGSTYYADSVKG SEQIDNO:21 (2PC10-VHCDR3) APMFDY SEQIDNO:22 (2PC10-VLCDR1) RASQSVSTHLLA SEQIDNO:23 (2PC10-VLCDR2) GASSRAT SEQIDNO:24 (2PC10-VLCDR3) QQWGLTPAM
Amino Acid Sequences of Antibody CPR01 specific for the D Domain of Human Tenascin-C

(145) TABLE-US-00012 SEQIDNO:25 (CPR01-VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA IKARGGLTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGG APFDYWGQGTLVTVSS SEQIDNO:26 (CPR01-VL) SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSPLNRLAVVF GGGTKLTVLG SEQIDNO:87 (CPR01-alternativeaminoacidsequenceoftheVL domain) SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSPLNRLAVVFG GGTKLTVL SEQIDNO:27 (CPR01-VHCDR1) GFTFSSYAMS SEQIDNO:28 (CPR01-VHCDR2) AIKARGGLTYYADSVKG SEQIDNO:29 (CPR01-VHCDR3) GGAPFDY SEQIDNO:30 (CPR01-VLCDR1) QGDSLRSYYAS SEQIDNO:31 (CPR01-VLCDR2) GKNNRPS SEQIDNO:32 (CPR01-VLCDR3) NSSPLNRLAVV
Amino Acid Sequences of Antibody SW01 specific for the IIICS Isoform of Human Fibronectin

(146) TABLE-US-00013 SEQIDNO:33 (SW01-VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKNR YIFDYWGQGTLVTVSS SEQIDNO:34 (SW01-VL) SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSPKAPRPVVFG GGTKLTVLG SEQIDNO:35 (SW01-VHCDR1) GFTFSSYAMS SEQIDNO:36 (SW01-VHCDR2) AISGSGGSTYYADSVKG SEQIDNO:37 (SW01-VHCDR3) NRYIFDY SEQIDNO:38 (SW01-VLCDR1) QGDSLRSYYAS SEQIDNO:39 (SW01-VLCDR2) GKNNRPS SEQIDNO:40 (SW01-VLCDR3) NSSPKAPRPVV
Amino Acid Sequences of Antibody CH01 specific for Human MMP3

(147) TABLE-US-00014 SEQIDNO:41 (CH01-VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSPYAMSWVRQAPGKGLEWVSA ITGQGGVTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKIS SFHFDYWGQGTLVTVSS SEQIDNO:42 (CH01-VL) EIVLTQSPGTLSLSPGERATLSCRASQSVSSHHLAWYQQKPGQAPRLLIY DASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQPRGAPTTFG QGTKVEIK SEQIDNO:43 (CH01-VHCDR1) GFTFSPYAMS SEQIDNO:44 (CH01-VHCDR2) AITGQGGVTYYADSVKG SEQIDNO:45 (CH01-VHCDR3) ISSFHFDY SEQIDNO:46 (CH01-VLCDR1) RASQSVSSHHLA SEQIDNO:47 (CH01-VLCDR2) DASSRAT SEQIDNO:48 (CH01-VLCDR3) QQPRGAPTT

(148) Amino Acid Sequences of Antibody L19 specific for the ED-B Domain of Human Fibronectin

(149) TABLE-US-00015 SEQIDNO:49 (L19-VH) GluValGlnLeuLeuGluSerGlyGlyGlyLeuVal GlnProGlyGlySerLeuArgLeuSerCysAlaAla SerGlyPheThrPheSerSerPheSerMetSerTrp ValArgGlnAlaProGlyLysGlyLeuGluTrpVal SerSerIleSerGlySerSerGlyThrThrTyrTyr AlaAspSerValLysGlyArgPheThrIleSerArg AspAsnSerLysAsnThrLeuTyrLeuGlnMetAsn SerLeuArgAlaGluAspThrAlaValTyrTyrCys AlaLysProPheProTyrPheAspTyrTrpGlyGln GlyThrLeuValThrValSerSer SEQIDNO:50 (L19-VL) GluIleValLeuThrGlnSerProGlyThrLeuSer LeuSerProGlyGluArgAlaThrLeuSerCysArg AlaSerGlnSerValSerSerSerPheLeuAlaTrp TyrGlnGlnLysProGlyGlnAlaProArgLeuLeu IleTyrTyrAlaSerSerArgAlaThrGlyIlePro AspArgPheSerGlySerGlySerGlyThrAspPhe ThrLeuThrIleSerArgLeuGluProGluAspPhe AlaValTyrTyrCysGlnGlnThrGlyArgIlePro ProThrPheGlyGlnGlyThrLysValGluIleLys (SEQIDNO:51) L19CDR1VH-SerPheSerMetSer (SEQIDNO:52) L19CDR2VH-SerIleSerGlySerSerGlyThrThr TyrTyrAlaAspSerValLys (SEQIDNO:53) L19CDR3VH-ProPheProTyrPheAspTyr (SEQIDNO:54) L19CDR1VL-ArgAlaSerGlnSerValSerSerSer PheLeuAla (SEQIDNO:55) L19CDR2VL-TyrAlaSerSerArgAlaThr (SEQIDNO:56) L19CDR3VL-GlnGlnThrGlyArgIleProProThr
Amino Acid Sequences of Antibody F16 specific for the A1 Domain of Human Tenascin-C

(150) TABLE-US-00016 SEQIDNO:57 (F16-VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYGMSWVRQAPGKGLEWVSA ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAH NAFDYWGQGTLVTVSR SEQIDNO:58 (F16-VL) SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSVYTMPPVVFG GGTKLTVLG SEQIDNO:88 (F16-alternativeaminoacidsequenceoftheVL domain) SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSVYTMPPVVFG GGTKLTVL (SEQIDNO:59) F16CDR1VH-RYGMS (SEQIDNO:60) F16CDR2VH-AISGSGGSTYYADSVKG (SEQIDNO:61) F16CDR3VH-AHNAFDY (SEQIDNO:62) F16CDR1VL-QGDSLRSYYAS (SEQIDNO:63) F16CDR2VL-GKNNRPS (SEQIDNO:64) F16CDR3VL-NSSVYTMPPVV
Amino Acid Sequences of Antibody G11 specific for the C Domain of Human Tenascin-C

(151) TABLE-US-00017 SEQIDNO:65 (G11-VH) EVQLVESGGGLVQPGGSLRLSCAASGFTFSGSRMGWVRQAPGKGLEWVSA INEEGGQTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKHP PHRPFDYWGQGTLVTVSR SEQIDNO:66 (G11-VL) SSELTQDPAVSVALGQTVRITCQGDSLRLYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSHGPRRPVVFG GGTKLTVLG scFvVH-VLdomainlinkersequence (SEQIDNO:67) GGGGSGGGGSGGGG DiabodyVH-VLdomainlinkersequence (SEQIDNO:68) GGSGG AlternativeVHdomainsequenceforantibodyCT01 (SEQIDNO:69) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPA PRARFDYWGQGTLVTVS AlternativeVHdomainsequenceforantibodyFF01 (SEQIDNO:70) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA IKGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKVT WNNYFDYWGQGTLVTVS AlternativeVHdomainsequenceforantibody2PC10 (SEQIDNO:71) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSAAMSWVRQAPGKGLEWVSA ISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKSA PMFDYWGQGTLVTVS AlternativeVHdomainsequenceforantibodyCPR01 (SEQIDNO:72) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA IKARGGLTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGG APFDYWGQGTLVTVS AlternativeVLdomainsequenceforantibodySW01 (SEQIDNO:73) SELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGKN NRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSPKAPRPVVFGG GTKLTVLG
Amino Acid Sequences of Antibody FF02 specific for Human Neutrophil Elastase

(152) TABLE-US-00018 SEQIDNO:74 (FF02-VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA IKGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWN WNLEFDYWGQGTLVTVSS SEQIDNO:75 (FF02-VL) SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSGLIWPRVVFG GGTKLTVLG SEQIDNO:76 (FF02-VHCDR1) GFTFSSYAMS SEQIDNO:77 (FF02-VHCDR2) AIKGSGGSTYYADSVKG SEQIDNO:78 (FF02-VHCDR3) WNWNLEFDY SEQIDNO:79 (FF02-VLCDR1) QGDSLRSYYAS SEQIDNO:80 (FF02-VLCDR2) GKNNRPS SEQIDNO:81 (FF02-VLCDR3) NSSGLIWPRVV SEQIDNO:82 (FF02inSIPformat) ThelinkerlinkingtheVHandVLdomainsofFF02 isunderlined.ThesequencelinkingtheVLdomain totheCH4domainofhumanIgEisshowninbold andunderlined. EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA IKGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKWN WNLEFDYWGQGTLVTVSSGGGGSGGGGSGGGGSSELTQDPAVSVALGQTV RITCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGN TASLTITGAQAEDEADYYCNSSGLIWPRVVFGGGTKLTVLGSGGSGGPRA APEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHS TTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAV SVNPESSRRGGC SEQIDNO:83 (LinkersequencelinkingVLdomaintoCH4domain ofhumanIgE) SGGSG SEQIDNO:84 (AminoacidsequenceoftheCH4domainofhuman IgE) GPRAAPEVYAFATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPD ARHSTTQPRKTKGSGFFVFSRLEVTRAEWEQKDEFICRAVHEAASPSQTV QRAVSVNPESSRRGGC
Amino Acid Sequences of Antibody CPR01.1 specific for the D Domain of Human Tenascin-C

(153) TABLE-US-00019 SEQIDNO:89 (CPR01.1-VH) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA IKGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGG APFDYWGQGTLVTVSS SEQIDNO:90 (CPR01.1-VL) SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYGK NNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCNSSLRGTLPVVFG GGTKLTVLG SEQIDNO:27 (CPR01.1-VHCDR1) GFTFSSYAMS SEQIDNO:91 (CPR01.1-VHCDR2) AIKGSGGSTYYADSVKG SEQIDNO:29 (CPR01.1-VHCDR3) GGAPFDY SEQIDNO:30 (CPR01.1-VLCDR1) QGDSLRSYYAS SEQIDNO:31 (CPR01.1-VLCDR2) GKNNRPS SEQIDNO:92 (CPR01.1-VLCDR3) NSSLRGTLPVV SEQIDNO:93 (CPR01.1,FullScFvsequence) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA IKGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGG APFDYWGQGTLVTVSSGGGGSGGGGSGGGGSSELTQDPAVSVALGQTVRI TCQGDSLRSYYASWYQQKPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTA SLTITGAQAEDEADYYCNSSLRGTLPVVFGGGTKLTVLG