LU100158B1

LU100158B1 - Immoglobulin or fragment thereof for detection of tyrosine-23 phosphorylated annexin a2

Info

Publication number: LU100158B1
Application number: LU100158A
Authority: LU
Inventors: Ursula Rescher; Volker Gerke
Original assignee: Univ Muenster Westfaelische Wilhelms
Priority date: 2017-04-03
Filing date: 2017-04-03
Publication date: 2018-10-15

Abstract

The present application relates to an immunoglobulin capable of binding to tyrosine-23 phosphorylated annexin A2, methods for detection said tyrosine-23 phosphorylated annexin A2 and the use of said immunoglobulin in a diagnostic or therapeutic method. The present application also relates to methods of production of an immunoglobulin capable of binding to tyrosine-23 phosphorylated annexin A2 and pharmaceutical and diagnostic compositions comprising said immunoglobulin

Description

IMMUNOGLOBULIN OR FRAGMENT THEREOF FOR DETECTION OF TYROSINE-23 PHOSPHORYLATED ANNEXIN A2

FIELD OF THE INVENTION

[0001] The present invention relates to an immunoglobulin or fragment thereof capable of binding to tyrosine-23 phosphorylated annexin A2, an in vitro method for detection of a tyrosine-23 phosphorylated annexin A2 and the use of said immunoglobulin or fragment thereof in a diagnostic or therapeutic method. The present invention further relates to a nucleic acid encoding for an immunoglobulin or fragment thereof capable of binding to tyrosine-23 phosphorylated annexin A2, a vector which comprises said nucleic acid and a host transformed or transfected with said vector. The present invention also relates to a method of production of an immunoglobulin or fragment thereof capable of binding to tyrosine-23 phosphorylated annexin A2 and a diagnostic composition comprising said immunoglobulin or fragment thereof. Also provided by the present invention is a kit comprising an immunoglobulin or fragment thereof capable of binding to tyrosine-23 phosphorylated annexin A2, a nucleic acid encoding for said immunoglobulin or fragment thereof, a vector comprising said nucleic acid, or a host transformed or transfected with said vector.

BACKGROUND OF THE INVENTION

[0002] Annexins are a family of Ca2+/lipid-binding proteins that differ from most other Ca2+-binding proteins in their Ca2+-binding sites. These have a unique architecture that allows them to dock onto membranes in a peripheral and reversible manner. The conserved Ca2+-and membrane-binding module is the annexin core domain, which consists of four so-called annexin repeats, each of which is 70 residues in length. It is highly ot-helical and forms a compact, slightly curved disc that has a convex surface harboring the Ca2+- and membranebinding sites and a concave side that points away from the membrane and is thereby available for other types of interaction/regulation. The N-terminal region precedes the core domain and is diverse in sequence and length. It mediates regulatory interactions with protein ligands and regulates the annexin-membrane association (reviewed by Gerke and Moss, 2002; Raynal and Pollard, 1994). Although the N-terminal domain has long been considered a separately folded entity, recent crystal structures reveal that, at least in annexin

Al, part of it can integrate into the folded core. Ca2+ (and probably membrane) binding can then trigger exposure of the N-terminal region, making it available for additional interactions/activities (Rosengarth and Luecke, 2003). The activity of the exposed N-terminal region could thus be tightly controlled through Ca27membrane binding.

[0003] The annexin family comprises >500 different gene products expressed in most phyla and species (reviewed by Morgan and Fernandez, 1997). In vertebrates, 12 annexin subfamilies (AI-AI 1 and A13), which have different splice variants, have been identified. These have different N-terminal domains and differently positioned Ca27membrane-binding sites within the core domain. Analyses of the biochemical properties and subcellular localizations of annexins, and later studies of the effects of anti-annexin antibodies and annexin mutants, mainly in permeabilized cell systems, have allowed several potential physiological functions to be assigned to different annexins. Most of these take into account their regulated binding to membranes and a scaffold role at certain membrane domains is a common theme. Proposed to act as membrane-membrane or membrane-cytoskeleton linkers, annexins have been implicated in Ca2+-regulated exocytotic events, certain aspects of endocytosis and stabilization of specific domains of organelle membranes and the plasma membrane. However, other potential functions have been put forward - for example, those taking into account the RNA-binding capacity of some annexins (Filipenko et al. 2004; Vedeler and Hollas, 2000), their regulated nuclear localization (Eberhard et al., 2001; Mizutani et al., 1992; Tomas and Moss, 2003) or specific nucleotide-binding activities (Banderowicz-Pikula et al., 2001; Caohuy et al., 1996). Because some annexins occur extracellularly, they might also function outside the cell. Detailed discussions of postulated annexin functions and their structure and biochemistry can be found elsewhere (Gerke and Moss, 2002; Bandorowicz-Pikula, 2003).

[0004] Annexin A2 (ANXA2), one member of the annexin family, was initially identified as a major substrate of pp60vsrc, a tyrosine-specific protein kinase encoded by the v-src oncogene of Rous Sarcoma Virus, (Erikson and Erikson, 1980) and thus has been discussed to play a role in cellular differentiation and/or transformation. It has been shown that Annexin A2 plays a role in the organization of membrane domains and membrane-cytoskeleton contacts (for a review, see Gerke et al., 2005). In addition to interacting with Ca2+ and membrane lipids, annexin A2 is able to bind G- and F-actin, it inhibits filament elongation at the barbed ends and it is recruited to sites of actin assembly at cellular membranes (Hayes et al., 2004, for a review, see Rescher and Gerke, 2004). Hence, the protein has been linked to actin remodelling processes at cellular membranes, such as those occurring during the clustering of certain membrane receptors or during the formation of actin tails on mobile macropinosomes (Merrifield et al,, 2001; Olifcrenko et al., 1999). These functions are probably linked to the dynamic organization of cholesterol-rich membrane microdomains to which annexin A2 can be recruited via a direct interaction with phosphatidylinositol (4,5)-bisphosphate (Hayes et al., 2006; Rescher et al., 2004).

[0005] As annexin A2 generally acts in cellular differentiation and/or transformation it has been shown to act in the context with many different diseases. For example, annexin A2 has also been shown to play a role in the production of infectious hepatitis C virus particles. Hepatitis C virus (HCV) is an important human pathogen affecting 170 million chronically infected individuals. In search for cellular proteins involved in HCV replication, a purification strategy for viral replication complexes has been developed and annexin A2 has been identified as an associated host factor. The silencing of annexin A2 expression resulted in a significant reduction of extra- and intracellular virus titers. Colocalization studies with individually expressed HCV nonstructural proteins indicated that NS5A specifically recruits annexin A2, probably by an indirect mechanism. By the deletion of individual NS5A subdomains, a domain III was identified as being responsible for ANXA2 recruitment. These data were considered to identify annexin A2 as a novel host factor contributing, with NS5A, to the formation of infectious HCV particles. J Virol. 2010 Jun;84(11):5775-89. Epub 2010 Mar 24. Annexin A2 has also been shown to Regulate Phagocytosis of Photoreceptor Outer Segments in the Mouse Retina (Ah-Lai Law et al., accepted June 25, 2009, Monitoring Editor: Jean E. Gruenberg) Moreover, phosphotyrosine proteomic screening revealed phosphorylation of the lipid-, calcium-, and actin-binding protein, annexin A2 (ANXA2) at Tyr23 as a major event preceding ts-v-Src kinase-induced cell scattering and the switch from an epithelial to mesenchymal phenotype (Marjo de Graauw, Bob van de Water).

[0006] Annexin A2 has also been identified as a major insulin receptor substrate that is functionally linked to the insulin-induced changes in the actin cytoskeleton. It has been shown that insulin-induced alterations in cell morphology and adhesion correlate with annexin A2 tyrosine-23 phosphorylation (Rescher et al., 2008, J. of Cell Science). One phosphorylation site of annexin A2 is located at Tyrosine-23 in the unique N-terminal domain, which is expected to face the cytosol in membrane-bound annexin (for a review, see Gerke et al., 2005). It has been shown, that the tyrosine-23 phosphorylation of annexin A2 (pY23ANXA2) is involved in the Rho/ROCK- mediated generation of contractile force leading ultimately to cell detachment, a morphological hallmark of cell transformation. Furthermore, it has been shown that a tyrosine-23 phosphorylation-dependent cell-surface localization of annexin A2 is required for invasion and métastasés of pancreatic cancer (Zheng et al. PLoS ONE, May 2, 2011).

[0007] As stated above, it is known that annexin A2, and particularly its tyrosine-23 phosphorylated variation, plays a key role in crucial biological regulating processes. However, the detection of tyrosine-23 phosphorylated annexin A2, throughout the entire prior art, is characterized by a laborious process wherein at least two antibody detection steps are necessary. Usually the first detection step comprises the detection of proteins being tyrosine phosphorylated with an anti-phosphotyrosine antibody, and the second detection step comprises a monoclonal annexin A2 antibody in combination with immunoprécipitation or mass spectroscopy. However, localization of tyrosine-23 phosphorylated annexin A2 in cell or tissues is no possible. Thus, it is highly desired to detect tyrosine-23 phosphorylated annexin A2 in a fast, easy to handle and specific manner by using an immunoglobulin capable of binding to pY23ANXA2 against in order to elucidate the distribution and quantity of tyrosine-23 phosphorylated annexin A2 in a tissue. In particular, said immunoglobulin would be useful for qualitative and quantitative detection of pY23ANXA2 in a sample obtained from a subject. Accordingly, the technical problem underlying the present application is to comply with the needs set out above. Specifically, the technical problem is to provide means and methods for the in vitro detection of tyrosine-23 phosphorylated annexin A2.

[0008] In sum, there is a need to provide new, alternative means and methods that help to detect the tyrosine-23 phosphorylated annexin A2 variant. Further technical problems solved by the present invention will become apparent from the descriptions, examples and figures that follow. The present invention complies with the needs described herein and provides as a solution to the technical problem an immunoglobulin or fragment thereof capable of binding to tyrosine-23 phosphorylated annexin A2. The embodiments which characterize the present invention are described herein, shown in the Figures, illustrated in the Examples, and reflected in the claims.

SUMMARY OF THE INVENTION

[0009] The present invention is based on the surprising finding that the immunoglobulin or fragment thereof as described by the present invention is capable of specifically binding to tyrosine-23 phosphorylated annexin A2. In this regard the inventors of the present invention used the tyrosine-23 phosphorylated N-terminal peptide sequence STPPSApYGSVKA (SEQ ID NO: 11) of human annexin A2 (UniProt. Number P07355, SEQ ID NO: 19) which corresponds to amino acid positions 18 to 29 of said human annexin A2. To said phosphor-peptide a cysteine residue was added for conjugation (STPPSApYGSVKA-C) and conjugated to KLH (Keyhole Limpet Heamocyanin) for immunization of rats and to OVA (Ovalbumin) for testing. After generation of hybridoma cell lines using rat lymphocytes, hybridoma supernatants were analyzed by ELISA on ANXA2 peptide, phosphorylâtes ANXA2 peptide and irrelevant peptide. Monoclonal antibody (mAB) of hybridoma subclone NZ-8H7-H5 showed best result in ELISA (Figure 8) and could be identified as a specific immunoglobulin capable of binding to tyrosine-23 phosphorylated annexin A2. Moreover, the sequence of the identified monoclonal immunoglobulin, in particular the sequences of the VL and VH variable region, could be decoded, providing the specific amino acids residues responsible for binding of said immunoglobulin to tyrosine-23 phosphorylâtes annexin A2.

[0010] To test the applicability of this monoclonal antibody in immunoblotting experiments, a previously established Baby Hamster Kidney (BHK) fibroblast cell line stably overexpressing the human insulin receptor (BHK-IR) was utilized (BHK-IR, Meller et al., 1995). Stimulation of this cell line with insulin results in a strong phosphorylation of ANXA2 on Y23 (Rescher et al., 2008) BHK-IR were stimulated for the indicated periods of time with 1 pg/ml insulin. Cytosolic lysates were resolved by SDS-PAGE and blotted for Y23-phosphorylated ANXA2. No signal was detected in unstimulated cells, whereas a strong signal was already detected after 5 min of insulin stimulation. This band became stronger after 30 min and peaked around 60 min. Total ANXA2 levels remained unchanged (Figure 1). Western Blot analysis of ANXA2 immunoprecipitated with HH7 from unstimulated and insulin-stimulated BHK-IR cells confirmed that ANXA2 was strongly tyrosine-phosphorylated upon stimulation with insulin (Figure 2).

[0011] Treatment of MDCK cells with H202 in the presence of a tyrosine-phosphatase inhibitor such as orthovanadate results in strong cellular phosphorylation (Wu et al., 2000). Probing of ANXA2 immunoprecipitated from MDCK cells with NZ-8H7-H5 mAB revealed no signal in untreated cells, whereas a strong signal was detected in cells treated with both agents. Addition of HGF, a growth factor activating tyrosine-based signaling cascades through the receptor-tyrosine kinase c-Met, did not further increase the amount of tyrosine-phosphorylated ANXA2 (Figure 3).

[0012] To test whether NZ-8H7-H5 mAB may be suitable for immunoprécipitation, ANXA2 from insulin-treated BHK-IR cells was immunoprecipitated using NZ-8H7-H5 and sheep a-rat coupled Dynabeads. Y23-phosphorylated ANXA2 was only precipitated under mild denaturing conditions, i.e. in the presence of 0.1% SDS (Figure 4). To test the specificity of NZ-8H7-H5 mAB for the tyrosine-phosphorylated target sequence in the physiological context, i.e. the presentation as part of the whole protein sequence, in the cell, phosphorylated by cellular kinases, GFP-tagged ANXA2 with mutated Y23 residue was expressed in BHK-IR cells and immunoprecipitated 1 h after stimulation with insulin. While ANXA2-GFP was successfully precipitated in all cases, only the wild type N-terminal sequence was detected with NZ-8H7-H5. The phospho-mimicking and the phosphorylation-deficient mutants Y23E and Y23F, respectively, showed no signal after stimulation (Figure 5).

[0013] Immunofluorescence staining using NZ-8H7-H5 mAB resulted in a weak background signal in untreated cells, whereas strongly stained small foci were detected in insulin-treated BHK-IR cells (Figure 6). In H202/vanadate/HGF-treated MDCK cells, cell-cell contact sites and the plasma membrane appeared positive (Figure 7). Further, variable regions of expressed NZ-8H7-H5 mAB could be sequenced and the corresponding VL and HL regions comprising Complementary Determining Regions (CDRs) are depicted in SEQ ID NO: 1-8.

[0014] In sum, the present invention relates to a new and very specific immunoglobulin (or fragment thereof) capable of binding to tyrosine-23 phosphorylated annexin A2, wherein said immunoglobulin or fragment thereof is suitable for qualitative and/or quantitative detection of tyrosine-23 phosphorylâtes Annexin A2 in vitro and jn vivo.

[0015] Accordingly, in a first aspect, the present invention relates to an immunoglobulin or fragment thereof capable of binding to tyrosine-23 phosphorylated annexin-2, wherein said immunoglobulin or fragment thereof comprises a VL region comprising Complementary Determining Regions (CDRs) CDR-L1, CDR-L2 and CDR-L3 as depicted in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and/or wherein said immunoglobulin or fragment thereof comprises a VH region comprising Complementary Determining Regions (CDRs) CDR-H1, CDR-H2 and CDR-H3 as depicted in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. It is envisaged that the provided immunoglobulin or fragment thereof has interspecies specificity. In particular, it is envisaged that said immunoglobulin or fragment thereof is capable of binding to human, dog, hamster, mouse, rat, cow, and/ or sheep tyrosine-23 phosphorylated annexin A2. Preferably, said tyrosine-23 phosphorylated annexin A2 comprises an amino acid sequence as depicted in any of SEQ ID NOs: 11 to 18. Accordingly, said immunoglobulin or fragment thereof is capable of binding to the peptide sequences as depicted in SEQ ID NOs: 11 to 18. It is further envisaged that the immunoglobulin or fragment thereof comprises a VL region as depicted in SEQ ID NO: 7 and/or a VH region as depicted in SEQ ID NO: 8. Preferably, the immunoglobulin of the present invention is a monoclonal antibody. It is also envisaged that said immunoglobulin or fragment thereof is humanized.

[0016] It is further envisaged that the immunoglobulin or fragment as provided herein is suitable for qualitative and/or quantitative detection of tyrosine-23 phosphorylated annexin A2 in vitro. In this regard it is envisaged that said detection is conducted by way of Western Blot, immunohistochemistry, immunophenotyping, fluorescence-activated cell scanning (FACS), Enzyme Linked Immunosorbent Assay (ELISA), Enzyme Linked Immuno Spot Assay (ELISPOT), radioimmunoassay, immunoprécipitation or co-immunoprecipitation. It is further envisaged that the immunoglobulin or fragment thereof as provided herein is for use in a diagnostic method and/or for use in a therapeutic method.

[0017] According to another aspect, the present invention relates to an in vitro method for detection of a tyrosine-23 phosphorylated annexin A2, wherein an immunoglobulin or fragment thereof according to the present invention is applied to a sample. Preferably, said sample is a tissue sample, a body fluid sample or a cell culture sample. It is particularly envisaged that said body fluid sample is a full-blood sample, a serum sample, bronchoalveaolar fluid or a synovial fluid.

[0018] In another aspect, the present invention further provides for a nucleic acid sequence encoding an immunoglobulin or fragment thereof according to the present inventon. Said nucleic acid sequence preferably comprises the sequence of SEQ ID NO: 9 and/or SEQ ID NO: 10.

[0019] According to another aspect, the present invention provides for a vector which comprises a nucleic acid sequence as defined herein. In this regard it is envisaged that said vector further comprises a regulatory sequence which is operably linked to said nucleic acid sequence. Preferably, said vector is an expression vector.

[0020] The present invention also relates to a host transformed or transfected with a vector according to the present invention.

[0021] In another aspect, the present invention provides for a method for the production of an immunoglobulin or a fragment thereof as defined herein, said method comprising culturing a host as defined herein under conditions allowing the expression of the immunoglobulin or fragment thereof as defined herein and recovering the produced immunoglobulin or fragment thereof from the culture.

[0022] According to another aspect, the present invention also refers to a method for the production of an immunoglobulin or fragment thereof as defined herein, said method comprising: (a) immunizing an animal with a molecule comprising the amino acid sequence as depicted in any of SEQ ID NOs: 11 to 18, and (b) obtaining an immunoglobulin or fragment thereof being capable of binding to tyrosine-23 phosphorylated annexin A2.

[0023] In another aspect, the present invention also refers to an immunoglobulin or fragment thereof according to the present invention obtainable by any of the methods as described herein.

[0024] According to another aspect, the present invention also provides for a diagnostic composition comprising an immunoglobulin or a fragment according to the present invention, preferably comprising a pharmaceutically or diagnostically acceptable excipient. Also provided is a kit comprising an immunoglobulin or fragment thereof as defined herein, a nucleic acid molecule as defined herein, a vector as defined herein, or a host as defined herein.

DETAILED DESCRIPTION OF THE INVENTION *** [0025] Unless otherwise stated, the following terms used in this document, including the description and claims, have the definitions given below. Those skilled in the art will recognize, or be able to ascertain, using not more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0026] It is to be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0027] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the methods and uses described herein. Such equivalents are intended to be encompassed by the present invention.

[0028] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integer or step. When used herein the term "comprising" can be substituted with the term "containing" or sometimes when used herein with the term "having".

[0029] When used herein "consisting of' excludes any element, step, or ingredient not specified in the claim element. When used herein, "consisting essentially of' does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. In each instance herein any of the terms "consisting", "consisting of and "consisting essentially of' may be replaced with either of the other two terms.

[0030] As used herein, the conjunctive term "and/or" between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by "and/or", a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or" as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term "and/or" as used herein.

[0031] As described herein, “preferred embodiment” means “preferred embodiment of the present invention”. Likewise, as described herein, “various embodiments” and “another embodiment" means “various embodiments of the present invention” and “another embodiment of the present invention”.

[0032] The word “about” as used herein refers to a value being within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about" can mean within 1 or more than 1 standard deviation, per the practice in the art. The term “about” is also used to indicate that the amount or value in question may be the value designated or some other value that is approximately the same. The phrase is intended to convey that similar values promote equivalent results or effects according to the invention. In this context “about" may refer to a range above and/or below of up to 10%. The word “about" refers in some embodiments to a range above and below a certain value that is up to 5%, such as up to up to 2%, up to 1%, or up to 0.5 % above or below that value. In one embodiment “about" refers to a range up to 0.1 % above and below a given value.

[0033] Several documents are cited throughout the text of this disclosure. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0034] The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art. *** [0035] The present invention aims at providing means and methods for directly detecting tyrosine-23 phosphorylated annexin A2 (pY23ANXA2). In particular, the inventors found out that an immunoglobulin or fragment comprising a VL region comprising Complementary Determining Regions (CDRs) CDR-L1, CDR-L2 and CDR-L3 as depicted in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and/or comprising a VH region comprising Complementary Determining Regions (CDRs) CDR-H1, CDR-H2 and CDR-H3 as depicted in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6 (clone No. NZ-8H7-H5 as described herein) is capable of specifically binding to tyrosine-23 phosphorylated annexin A2 in vitro. The term “in vitro” as used herein includes any detection methods that take place outside a living organism, also comprising any methods carried out in situ. Accordingly, the immunoglobulin or fragment thereof according to the present invention is also capable of binding the tyrosine-23 phosphorylated annexin A2 directly in a tissue.

[0036] The term “capable of binding” as used herein refers to the specificity of said immunoglobulin or fragment thereof to interact with tyrosine-23 phosphorylated annexin A2. As shown by the data of the present invention (Figure 8), said immunoglobulin or fragment thereof has particular binding specificity to tyrosine-23 phosphorylâtes annexin A2, i.e. specifically recognizes pY23ANXA2 while non-phosphorylated annexin A2 or other irrelevant peptides are bound to only low extent. Thus, the term “specifically” in this context means that the immunoglobulin or fragment thereof binds to a tyrosine-23 phosphorylated annexin A2 protein, but does not essentially bind to a non-phosphorylated annexin A2 or another protein. The term “another protein” includes any protein including proteins closely related to.

[0037] Although immunoglobulins against annexin A2 have been described in the art, the detection of tyrosine-23 phosphorylated annexin A2 throughout the prior art is characterized by a laborious process wherein at least two antibody detection steps are necessary. Usually the first detection step comprises the detection of proteins being tyrosine phosphorylated with an anti-phosphotyrosine antibody, and the second detection step comprises a monoclonal annexin A2 antibody in combination with immunoprécipitation or mass spectroscopy. Thus, the advantage of the present invention can be seen in the provision of an immunoglobulin or fragment thereof comprising defined binding domains and capable of specifically binding to tyrosine-23 phosphorylated annexin A2. In particular, the immunoglobulin or fragment thereof according to the present invention exhibits cross-species specificity, thereby specifically recognizing human, dog, rat, mouse, hamster, cow and sheep tyrosine-23 phosphorylated annexin A2.

[0038] The term "specifically recognizing" as used herein can be equally replaced by the term “reacting with” and means in accordance with the present invention that the immunoglobulin or fragment thereof is capable of specifically interacting with and/or binding to human annexin A2 (SEQ ID NO: 19). In particular, immunoglobulin or fragment thereof according to the present invention reacts with the amino acid sequence corresponding to positions 18 to 29 of human Annexin A2, i.e. the tyrosine-23 phosphorylated sequence as depicted in SEQ ID NO: 11. However, due to sequence homologies, the immunoglobulin or fragment thereof according to the present invention equally binds to the corresponding amino acid sequence of mouse (SEQ ID NO: 12), rat (SEQ ID NO: 13), hamster (SEQ ID NO: 14), dog (SEQ ID NO: 15), cow (SEQ ID NO: 16), and sheep (SEQ ID NO: 17). Equally, it is envisaged that the immunoglobulin or fragment thereof according to the present invention binds to the corresponding amino acid sequence of pig (SEQ ID NO: 18). Accordingly, the immunoglobulin or fragment thereof according to the present invention has interspecies specificity.

[0039] The term “interspecies specificity" as used herein means that the immunoglobulin or fragment thereof as disclosed by the present invention is capable to cross-react with an annexin A2 protein from a species different from that against which the immunoglobulin or fragment thereof was generated. In particular, "interspecies specificity" means binding of the same (annexin A2) target molecule in humans and non-human species. Thus, "interspecies specificity", also called "cross-species specificity" is to be understood as a reactivity to the same molecule (tyrosine-23 phosphorylated annexin A2) expressed in different species, but not to a molecule other than tyrosine-23 phosphorylated annexin A2. For example, it is envisaged that an immunoglobulin or fragment thereof directed against human tyrosine-23 phosphorylated annexin A2 would cross-react with mouse or rat tyrosine-23 phosphorylated annexin A2, but not with, e.g., human annexin A1 or human non-tyrosine-23 phosphorylated annexin A2. In sum, cross-species specific immunoglobulins or fragment thereof directed against tyrosine-23 phosphorylated annexin A2 are preferably contemplated by the present invention. For example, it is apparent that the mouse, rat or hamster tyrosine-23 phosphorylated annexin A2 amino acid sequence is quite similar, i.e., at many positions it is identical, to the human tyrosine-23 phosphorylated annexin A2 amino acid sequence, which itself in the positions corresponding to positions 18 to 29 of the human annexin 2 protein (SEQ ID NO: 19) is identical to the dog, cow, and sheeps tyrosine-23 phosphorylated annexin A2 amino acid sequence. Preferably, an immunoglobulin or fragment thereof according to the present invention binds to human (SEQ ID NO: 11), dog (SEQ ID NO: 15), cow (SEQ ID NO: 16) and sheep (SEQ ID NO: 17) tyrosine-23 phosphorylated annexin A2, in particular to the region corresponding to the amino acid residues 18 to 29 of the amino acid sequence shown in SEQ ID NO: 19, and equally binds to mouse (SEQ ID NO: 12), rat (SEQ ID NO: 13) and hamster (SEQ ID NO: 14) tyrosine-23 phosphorylated annexin A2, i.e. to the region corresponding to the amino acid residues 18 to 19 sequence shown in SEQ ID NO: 19. Thus, the immunoglobulin or fragment thereof according to the present invention recognizes human tyrosine-23 phosphorylated annexin A2 and is preferably cross-reactive with dog, hamster, rat, mouse, cow and sheep tyrosine-23 phosphorylated annexin A2.

[0040] It does not matter, how this specific detection is reached, which is to say the immunoglobulin or fragment thereof according to the present invention might be different kind as long as they have the ability to specifically detect tyrosine-23 phosphorylated annexin A2 as for example the immunoglobulin of clone No.NZ-8H7-H5 does.

[0041] The monoclonal antibody of clone No. NZ-8H7-H5 as disclosed herein is characterized by comprising a VL region comprising Complementary Determining Regions (CDRs) CDR-L1, CDR-L2 and CDR-L3 as depicted in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and/or comprising a VH region comprising Complementary Determining Regions (CDRs) CDR-H1, CDR-H2 and CDR-H3 as depicted in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. More precisely, the monoclonal antibody of clone No. NZ-8H7-H5 as described herein comprise a VL region comprising four framework regions (FR1, FR2, FR3, and FR4) as depicted in SEQ ID NOs: 20 to 23 and a VH region comprising four framework regions (FR1, FR2, FR3, and FR4) as depicted in SEQ ID NOs: 24 to 27. In particular, the monoclonal antibody of clone No. NZ-8H7-H5 as described herein is characterized by a VL region as depicted in SEQ ID NO: 7 and a VH region as depicted in SEQ ID NO: 8.

[0042] According to the present invention, the tyrosine-23 phopshorylated annexin A2 to which the immunoglobulin or fragment according to the present invention binds preferably comprises an amino acid sequence as depicted in any one of SEQ ID NO: 11 to 18. In particular, the immunoglobulin or fragment according to the present invention specifically binds/interacts with a given target epitope comprising any one of SEQ ID NOs: 11 to 18. An “epitope” is antigenic and thus the term epitope is sometimes also referred to herein as “antigenic structure” or “antigenic determinant”. Accordingly, the immunoglobulin or fragment thereof according to the present invention comprises an "antigen-interaction-site". The term "antigen-interaction-site" defines, in accordance with the present invention, a motif of the immunoglobulin or fragment thereof which is able to specifically interact with a specific antigen or a specific group of antigens, e.g. the identical antigen in different species. Said binding/interaction is also understood to define a "specific recognition". In context of the present invention, the term "epitope" refers to a site on the tyrosine-23 phosphorylated annexin A2 to which the immunoglobulin or fragment thereof as described elsewhere herein is produced and/or binds.

[0043] According to the present invention, the annexin A2 epitope comprising any one of SEQ ID NOs: 11 to 18 to which the immunoglobulin or fragment according to the present invention binds is a linear epitope or a conformation epitope. A "linear epitope" is an epitope where an amino acid primary sequence comprises the epitope recognized. A linear epitope typically includes at least 3, and more usually, at least 5, for example, about 8 to about 10 amino acids in a unique sequence. A "conformational epitope", in contrast to a linear epitope, is an epitope wherein the primary sequence of the amino acids comprising the epitope is not the sole defining component of the epitope recognized (e.g., an epitope wherein the primary sequence of amino acids is not necessarily recognized by the antibody defining the epitope).

Typically a conformational epitope comprises an increased number of amino acids relative to a linear epitope. With regard to recognition of conformational epitopes, the immunoglobulin or fragment thereof as described herein recognizes a 3-dimensional structure of the antigen, i.e. in view of the present invention a 3-dimensional structure of the tyrosine-23 phosphorylated annexin A2 comprising any one of SEQ ID NOs: 11 to 18. For example, when a protein molecule folds to form a three dimensional structure, certain amino acids and/or the polypeptide backbone forming the conformational epitope become juxtaposed enabling the antibody to recognize the epitope. Methods of determining conformation of epitopes include but are not limited to, for example, X-ray crystallography, 2-dimensional nuclear magnetic resonance spectroscopy and site-directed spin labeling and electron paramagnetic resonance spectroscopy.

[0044] The term “does not essentially bind” means that the immunoglobulin or fragment thereof capable of binding to tyrosine-23 phosphorylated annexin A2 of the present invention does not bind another protein, i.e., shows a cross-reactivity of less than 30%, preferably 20%, more preferably 10%, particularly preferably less than 9, 8, 7, 6 or 5% with another protein. Specific binding is believed to be effected by specific motifs in the amino acid sequence of the binding domain and the antigen bind to each other as a result of their primary, secondary or tertiary structure as well as the result of secondary modifications of said structure. The specific interaction of the antigen-interaction-site with its specific antigen may result as well in a simple binding of said site to the antigen. Moreover, the specific interaction of the antigen-interaction-site with its specific antigen may alternatively result in the initiation of a signal, e.g. due to the induction of a change of the conformation of the antigen, an oligomerization of the antigen, ect. According to the present invention, said binding domain is an immunoglobulin or fragment thereof capable of specifically binding to tyrosine-23 phosphorylated annexin A2, comprising a VL region comprising Complementary Determining Regions (CDRs) CDR-L1, CDR-L2 and CDR-L3 as depicted in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and/or wherein said immunoglobulin or fragment thereof comprises a VH region comprising Complementary Determining Regions (CDRs) CDR-H1, CDR-H2 and CDR-H3 as depicted in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

[0045] Typically, binding is considered specific when the binding affinity is higher than lO^M. Preferably, binding is considered specific when binding affinity is about 10'11 to 10‘8 M (KD), preferably of about 10'11 to 10‘9 M. If necessary, nonspecific binding can be reduced without substantially affecting specific binding by varying the binding conditions. Whether immunoglobulin or fragment thereof according to the present invention specifically reacts as defined herein above can easily be tested, inter alia, by comparing the reaction of said immunoglobulin or fragment thereof with a tyrosine-23 phosphorylated annexin A2 protein with the reaction of said immunoglobulin or fragment thereof with (an) other protein(s).

[0046] The immunoglobulin of the present invention is a polypeptide, including fragments thereof, preferably biologically active fragments, and peptides, usually having less than 30 amino acids, comprising one or more amino acids coupled to each other via a covalent peptide bond (resulting in a chain of amino acids). The term "polypeptide" or “protein” as used herein describes a group of molecules, which consist of more than 30 amino acids. Polypeptides may further form multimers such as dimers, trimers and higher oligomers, i.e. consisting of more than one polypeptide molecule. Polypeptide molecules forming such dimers, trimers etc. may be identical or non-identical. The corresponding higher order structures of such multimers are, consequently, termed homo- or heterodimers, homo- or heterotrimers etc. An example for a hereteromultimer is an antibody molecule, which, in its naturally occurring form, consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The term "polypeptide" as used herein also refers to naturally modified polypeptides, wherein the modification is effected e.g. by post-translational modifications like glycosylation, acetylation, phosphorylation and the like. Such modifications are well known to those skilled in the art.

[0047] A preferred binding molecule of the present invention is an immunoglobulin, also called antibody, or a fragment thereof. An “antibody" when used herein is a protein comprising one or more polypeptides (comprising one or more binding domains, preferably antigen binding domains) substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as myriad immunoglobulin variable region genes.

[0048] In particular, an “antibody" when used herein, is typically tetrameric glycosylated proteins composed of two light (L) chains of approximately 25 kDa each and two heavy (H) chains of approximately 50 kDa each. Two types of light chain, termed lambda and kappa, may be found in antibodies. Depending on the amino acid sequence of the constant domain of heavy chains, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, and several of these may be further divided into subclasses (isotypes), e.g., lgG1, lgG2, lgG3, lgG4, lgA1, and lgA2. An IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called a J chain, and contains 10 antigen binding sites, while IgA antibodies comprise from 2-5 of the basic 4-chain units which can polymerize to form polyvalent assemblages in combination with the J chain. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each light chain includes an N-terminal variable (V) domain (VL) and a constant (C) domain (CL). Each heavy chain includes an N-terminal V domain (VH), three or four C domains (CHs), and a hinge region. The constant domains are not involved directly in binding an antibody to an antigen, but can exhibit various effector functions, such as participation of the antibody dependent cellular cytotoxicity (ADCC). If an antibody should exert ADCC, it is preferably of the lgG1 subtype, while the lgG4 subtype would not have the capability to exert ADCC. The pairing of a VH and VL together forms a single antigen-binding site. The CH domain most proximal to VH is designated as CH1. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. The VH and VL domains consist of four regions of relatively conserved sequences called framework regions (FR1, FR2, FR3, and FR4), which form a scaffold for three regions of hypervariable sequences (complementarity determining regions, CDRs). The CDRs contain most of the residues responsible for specific interactions of the antibody with the antigen. CDRs are referred to as CDR 1, CDR2, and CDR3. Accordingly, CDR constituents on the heavy chain are referred to as H1, H2, and H3, while CDR constituents on the light chain are referred to as L1, L2, and L3.

[0049] The term "variable" refers to the portions of the immunoglobulin domains that exhibit variability in their sequence and that are involved in determining the specificity and binding affinity of a particular antibody (i.e., the "variable domain(s)"). Variability is not evenly distributed throughout the variable domains of antibodies; it is concentrated in sub-domains of each of the heavy and light chain variable regions. These sub-domains are called "hypervariable" regions or "complementarity determining regions" (CDRs). The more conserved (i.e., non-hypervariable) portions of the variable domains are called the "framework" regions (FRM). The variable domains of naturally occurring heavy and light chains each comprise four FRM regions, largely adopting a ß- sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the ß -sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRM and, with the hypervariable regions from the other chain, contribute to the formation of the antigen- binding site (see Kabat et al., loc. cit.). The constant domains are not directly involved in antigen binding, but exhibit various effector functions, such as, for example, antibody- dependent, cell-mediated cytotoxicity and complement activation.

[0050] The terms "CDR", and its plural "CDRs", refer to a complementarity determining region (CDR) of which three make up the binding character of a light chain variable region (CDR-L1, CDR-L2 and CDR-L3) and three make up the binding character of a heavy chain1 variable region (CDR-H1, CDR-H2 and CDR-H3). CDRs contribute to the functional activity of an antibody molecule and are separated by amino acid sequences that comprise scaffolding or framework regions. The exact definitional CDR boundaries and lengths are subject to different classification and numbering systems. CDRs may therefore be referred to by Kabat, Chothia, contact or any other boundary definitions, including the numbering system described herein. Despite differing boundaries, each of these systems has some degree of overlap in what constitutes the so called "hypervariable regions" within the variable sequences. CDR definitions according to these systems may therefore differ in length and boundary areas with respect to the adjacent framework region. See for example Kabat, Chothia, and/or MacCallum et al., (Kabat et al., loc. cit.; Chothia et al., J. Mol. Biol, 1987, 196: 901; and MacCallum et al, J. Mol. Biol, 1996, 262: 732). However, the numbering in accordance with the so-called Kabat system is preferred.

[0051] The term "amino acid" or "amino acid residue" typically refers to an amino acid having its art recognized definition such as an amino acid selected from the group consisting of: alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine (Cys or C); glutamine (Gin or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (He or I): leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); pro line (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic, or rare amino acids may be used as desired. Generally, amino acids can be grouped as having a nonpolar side chain (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, Val); a negatively charged side chain (e.g., Asp, Glu); a positively charged sidechain (e.g., Arg, His, Lys); or an uncharged polar side chain (e.g., Asn, Cys, Gin, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0052] The term "hypervariable region" (also known as "complementarity determining regions" or CDRs) when used herein refers to the amino acid residues of an antibody which are (usually three or four short regions of extreme sequence variability) within the V-region domain of an immunoglobulin which form the antigen-binding site and are the main determinants of antigen specificity. There are at least two methods for identifying the CDR residues: (1) An approach based on cross-species sequence variability (i. e., Kabat et al., loc. cit.); and (2) An approach based on crystallographic studies of antigen-antibody complexes (Chothia, C. et al., J. Mol. Biol. 196: 901-917 (1987)). However, to the extent that two residue identification techniques define regions of overlapping, but not identical regions, they can be combined to define a hybrid CDR. However, in general, the CDR residues are preferably identified in accordance with the so-called Kabat (numbering) system.

[0053] The term "framework region" refers to the art-recognized portions of an antibody variable region that exist between the more divergent (i.e., hypervariable) CDRs. Such framework regions are typically referred to as frameworks 1 through 4 (FR1, FR2, FR3, and FR4) and provide a scaffold for the presentation of the six CDRs (three from the heavy chain and three from the light chain) in three dimensional space, to form an antigen-binding surface.

[0054] The term "canonical structure" refers to the main chain conformation that is adopted by the antigen binding (CDR) loops. From comparative structural studies, it has been found that five of the six antigen binding loops have only a limited repertoire of available conformations. Each canonical structure can be characterized by the torsion angles of the polypeptide backbone. Correspondent loops between antibodies may, therefore, have very similar three dimensional structures, despite high amino acid sequence variability in most parts of the loops (Chothia and Lesk, J. Mol. Biol., 1987, 196: 901; Chothia et al, Nature, 1989, 342: 877; Martin and Thornton, J. Mol. Biol, 1996, 263: 800, each of which is incorporated by reference in its entirety). Furthermore, there is a relationship between the adopted loop structure and the amino acid sequences surrounding it. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues residing at key positions within the loop, as well as within the conserved framework {i.e., outside of the loop). Assignment to a particular canonical class can therefore be made based on the presence of these key amino acid residues. The term "canonical structure" may also include considerations as to the linear sequence of the antibody, for example, as catalogued by Kabat (Kabat et al, loc. cit.). The Kabat numbering scheme (system) is a widely adopted standard for numbering the amino acid residues of an antibody variable domain in a consistent manner and is the preferred scheme applied in the present invention as also mentioned elsewhere herein. Additional structural considerations can also be used to determine the canonical structure of an antibody. For example, those differences not fully reflected by Kabat numbering can be described by the numbering system of Chothia et al and/or revealed by other techniques, for example, crystallography and two or three-dimensional computational modeling. Accordingly, a given antibody sequence may be placed into a canonical class which allows for, among other things, identifying appropriate chassis sequences {e.g., based on a desire to include a variety of canonical structures in a library). Kabat numbering of antibody amino acid sequences and structural considerations as described by Chothia et al., loc. cit. and their implications for construing canonical aspects of antibody structure, are described in the literature.

[0055] CDR3 is typically the greatest source of molecular diversity within the antibodybinding site. H3, for example, can be as short as two amino acid residues or greater than 26 amino acids. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a review of the antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988. One of skill in the art will recognize that each subunit structure, e.g., a CH, VH, CL, VL, CDR, FR structure, comprises active fragments, e.g., the portion of the VH, VL, or CDR subunit the binds to the antigen, i.e., the antigen-binding fragment, or, e.g., the portion of the CH subunit that binds to and/or activates, e.g., an Fc receptor and/or complement. The CDRs typically refer to the Kabat CDRs, as described in Sequences of Proteins of immunological Interest, US Department of Health and Human Services (1991), eds. Kabat et al. Another standard for characterizing the antigen binding site is to refer to the hypervariable loops as described by Chothia. See, e.g., Chothia, et al. (1992; J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J. 14:4628-4638. Still another standard is the AbM definition used by Oxford Molecular's AbM antibody modelling software. See, generally, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). Embodiments described with respect to Kabat CDRs can alternatively be implemented using similar described relationships with respect to Chothia hypervariable loops or to the AbM-defined loops.

[0056] The sequence of antibody genes after assembly and somatic mutation is highly varied, and these varied genes are estimated to encode 1010 different antibody molecules (Immunoglobulin Genes, 2nd ed., eds. Jonio et al., Academic Press, San Diego, CA, 1995). Accordingly, the immune system provides a repertoire of immunoglobulins. The term "repertoire" refers to at least one nucleotide sequence derived wholly or partially from at least one sequence encoding at least one immunoglobulin. The sequence(s) may be generated by rearrangement in vivo of the V, D, and J segments of heavy chains, and the V and J segments of light chains. Alternatively, the sequence(s) can be generated from a cell in response to which rearrangement occurs, e.g., in vitro stimulation. Alternatively, part or all of the sequence(s) may be obtained by DNA splicing, nucleotide synthesis, mutagenesis, and other methods, see, e.g., U.S. Patent 5,565,332. A repertoire may include only one sequence or may include a plurality of sequences, including ones in a genetically diverse collection.

[0057] When used herein the term "antibody" does not only refer to an immunoglobulin (or intact antibody), but also to a fragment thereof, and encompasses any polypeptide comprising an antigen-binding fragment or an antigen-binding domain. Preferably, the fragment such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function. Typically, such fragments would comprise an antigen-binding domain and have the same properties as the antibodies described herein. Accordingly, said fragment is preferably also capable to inhibit dephosphorylation of tyrosine-23 phosphorylated annexin A2.

[0058] The term “antibody" as used herein includes tyrosine-23 phosphorylated annexin A2 antibodies that compete for binding to the same epitope as the epitope bound by the antibodies of the present invention, preferably obtainable by the methods for the generation of an antibody as described herein elsewhere. As mentioned herein, the epitope is contained in an amino acid sequence corresponding to the amino acid sequence between amino acids 18 and 29 of the amino acid sequence shown in SEQ ID NO: 19. In particular, the epitope comprises an amino acid sequence as depicted in SEQ ID NOs: 11-18. To determine if a test antibody can compete for binding to the same epitope as the epitope bound by the tyrosine-23 phosphorylated annexin A2 antibodies of the present invention, a cross-blocking assay eg., a competitive ELISA assay can be performed (see Figure 8). In an exemplary competitive ELISA assay, tyrosine-23 phosphorylated annexin A2 coated wells of a microtiter plate, or tyrosine-23 phosphorylated annexin A2 coated sepharose beads, are pre-incubated with or without candidate competing antibody and then a biotin-labeled anti- tyrosine-23 phosphorylated annexin A2 antibody of the invention is added. The amount of labeled antityrosine-23 phosphorylated annexin A2 antibody bound to the tyrosine-23 phosphorylated annexin A2 antigen in the wells or on the beads is measured using avidin-peroxidase conjugate and appropriate substrate.

[0059] Alternatively, the anti-tyrosine-23 phosphorylated annexin A2 antibody according to the present invention can be labeled, i.e. the amount of labeled anti-tyrosine-23 phosphorylated annexin A2 antibody that binds to the antigen will have an inverse correlation to the ability of the candidate competing antibody (test antibody) to compete for binding to the same epitope on the antigen, i.e., the greater the affinity of the test antibody for the same epitope, the less labeled anti- tyrosine-23 phosphorylated annexin A2 antibody will be bound to the antigen-coated wells. A candidate competing antibody is considered an antibody that binds substantially to the same epitope or that competes for binding to the same epitope as an anti-tyrosine-23 phosphorylated annexin A2 antibody of the invention if the candidate competing antibody can block binding of the anti-tyrosine-23 phosphorylated annexin A2 antibody by at least 20%, preferably by at least 20-50%, even more preferably, by at least 50% as compared to a control performed in parallel in the absence of the candidate competing antibody (but may be in the presence of a known noncompeting antibody). It will be understood that variations of this assay can be performed to arrive at the same quantitative value.

[0060] Thus, in a preferred embodiment, the immunoglobulin or fragment thereof according to the present invention is labeled, comprising e.g. radioactive or fluorescent label or some other detectable and measurable (detection) label. As used herein, the terms “label” or “detection label” refers to a detectable marker that may be detected by photonic, electronic, opto-electronic, magnetic, gravity, acoustic, enzymatic, or other physical or chemical means. The term “labeled” as used herein refers to incorporation of such a detectable label. Comprised are for example fluorescent compounds such as fluorescein, Texas Red or rhodamine, gold beads, biotin, particular epitope tags, enzymes or coenzymes such as FAD that produces a colored compound, bio- and chemiluminescent materials such as luciferin, luminol or DPD, and enzyme inhibitors such as phosphonates, just to name some. The labels used according to the present invention depend on the desired application of the specific immuno-labeled compound, i.e. the applied detection method, and are well known to those skilled in the art.

[0061] The term “antibody" also includes but is not limited to polyclonal, monoclonal, monospecific, polyspecific such as bispecific, non-specific, humanized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, grafted, and in vitro generated antibodies, with a monoclonal antibody being preferred. Accordingly, the term "antibody" also relates to a purified serum, i.e., a purified polyclonal serum. Accordingly, said term preferably relates to a serum, more preferably a polyclonal serum and most preferably to a purified (polyclonal) serum. The antibody/serum is obtainable, and preferably obtained, for example, by the method or use described herein and illustrated in the appended Examples. "Polyclonal antibodies" or "polyclonal antisera" refer to immune serum containing a mixture of antibodies specific for one (monovalent or specific antisera) or more (polyvalent antisera) antigens which may be prepared from the blood of animals immunized with the antigen or antigens.

[0062] Furthermore, the term "antibody" as employed in the invention also relates to derivatives or variants of the antibodies described herein which display the same specificity as the described antibodies. Examples of "antibody variants" include humanized variants of non-human antibodies, "affinity matured" antibodies (see, e.g. Hawkins et al. J. Mol. Biol. 254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832- 10837 (1991)) and antibody mutants with altered effector function (s) (see, e.g., US Patent 5, 648, 260). The terms "antigen-binding domain", "antigen-binding fragment" and “antibody binding region” when used herein refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between antibody and antigen. The part of the antigen1 that is specifically recognized and bound by the antibody is referred to as the "epitope" as described elsewhere herein. As mentioned above, an antigen-binding domain may typically comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH); however, it does not have to comprise both. Fd fragments, for example, have two VH regions and often retain some antigen-binding function of the intact antigen-binding domain. Examples of antigen-binding fragments of an antibody include (1 ) a Fab fragment, a monovalent fragment having the VL, VH, CL and CH1 domains; (2) a F(ab')2 fragment, a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) a Fd fragment having the two VH and CH1 domains; (4) a Fv fragment having the VL and VH domains of a single arm of an antibody, (5) a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which has a VH domain; (6) an isolated complementarity determining region (CDR), and (7) a single chain Fv (scFv). Although the two domains of the Fv fragment, VL and VH> are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are evaluated for function in the same manner as are intact antibodies.

[0063] The immunglobulin or fragment thereof according to the present invention is capable of binding to tyrosine-23 phosphorylated annexin A2, wherein said immunoglobulin or fragment thereof comprises a VL region comprising Complementary Determining Regions (CDRs) CDR-L1, CDR-L2 and CDR-L3 as depicted in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and/or wherein said immunoglobulin or fragment thereof comprises a VH region comprising Complementary Determining Regions (CDRs) CDR-H1, CDR-H2 and CDR-H3 as depicted in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6. Preferably, said immunoglobulin or fragment thereof comprises a VL region comprising four framework regions (FR1, FR2, FR3, and FR4) as depicted in SEQ ID NO: 20 to 23 and/or a VH region comprising four framework regions (FR1, FR2, FR3, and FR4) as depicted in SEQ ID NO: 24 to 27. More preferably, said immunoglobulin or fragment thereof comprises a VL region as depicted in SEQ ID NO: 7 and /or a VH region as depicted in SEQ ID NO: 8.

[0064] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post- translation modifications (e.g., isomerizations, amidations) that may be present in minor1 amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al., Nature, 256: 495 (1975), or may be made by recombinant DNA methods (see, e.g., U. S. Patent No. 4,816, 567). The"monoclonal antibodies"may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991), for example.

[0065] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain (s) is (are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U. S. Patent No. 4,816, 567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include "primitized" antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc.) and human constant region sequences.

[0066] The immunoglobulin or fragment thereof according to the present invention may further comprise humanized or human.

[0067] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F (ab') 2 or other antigen-binding subsequences of antibodies) of mostly human sequences, which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (also CDR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, "humanized antibodies" as used herein may also comprise residues which are found neither in the recipient antibody nor the donor antibody. These modifications are made to further refine and optimize antibody performance. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321: 522-525 (1986); Reichmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992).

[0068] Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab’, F (ab') 2 or other antigen- binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.

[0069] Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domain, in which all or substantially all of the CDR regions correspond to those of a nonhuman immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-329 (1988) and Presta, Curr. Opin. Struct. Biol. 2 : 593-596 (1992).

[0070] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers, Jones et al., Nature 321: 522-525 (1986); Riechmann et al., Nature 332: 323-327 (1988); Verhoeyen et al.,

Science 239: 1534-1536 (1988), or through substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U. S. Patent No. 4, 816, 567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0071] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody. Sims et al., J. Immunol.) 151: 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies. Carter et al., Proc. Natl. Acad. Sci. USA, 89 : 4285 (1992); Presta et al., J. Immunol., 151: 2623 (1993).

[0072] It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i. e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen (s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0073] Various forms of the humanized antibody are contemplated. For example, the humanized antibody may be an antibody fragment, such as an Fab, which is optionally conjugated with one or more cytotoxic agent (s) in order to generate an immunoconjugate.

Alternatively, the humanized antibody may be an intact antibody, such as an intact IgGI1 antibody.

[0074] As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ- line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-258 (1993); Bruggermann et al., Year in Immun., 7: 33 (1993); U. S. Patent Nos. 5,591, 669 and WO 97/17852.

[0075] Alternatively, phage display technology can be used to produce human antiobdies and antibody fragments in vitro, from immunoglublin variable (V) domain gene repertoires from unimmunized donors. McCafferty et al., Nature 348: 552-553 (1990); Hoogenboom and Winter, J. Mol. Biol. 227: 381 (1991). According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., Johnson, Kevin S. and Chiswell, David J., Curr. Opin Struct. Biol. 3: 564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature 352: 624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the techniques described by Marks et al. , J. Mol. Biol. 222: 581-597 (1991), or Griffith et al. , EMBO J. 12 : 725-734 (1993). See also, U. S. Patent. Nos. 5,565, 332 and 5,573, 905.

[0076] The techniques of Cole et al., and Boerner et al., are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer

Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. 147 (1) : 86-95 (1991). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resemble that seen in human in all respects, including gene rearrangement, assembly and antibody repertoire. This approach is described, for example, in U. S. Patent Nos. 5,545, 807; 5,545, 806,5, 569,825, 5,625, 126,5, 633,425, 5,661, 016 and in the following scientific publications: Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368 : 856-859 (1994); Morrison, Nature 368: 812-13 (1994), Fishwild et al., Nature Biotechnology 14 : 845-51 (1996), Neuberger, Nature Biotechnology 14: 826 (1996) and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995). Finally, human antibodies may also be generated in vitro by activated B cells (see U. S. Patent Nos 5,567, 610 and 5,229, 275).

[0077] The term "human antibody" includes antibodies having variable and constant regions corresponding substantially to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (See Kabat, et al. (1991 ) loc. cit.). The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and in particular, CDR3. The human antibody can have at least one, two, three, four, five, or more positions replaced with an amino acid residue that is not encoded by the human germline immunoglobulin sequence.

[0078] As used herein, "in vitro generated antibody" refers to an antibody where all or part of the variable region (e.g., at least one CDR) is generated in a non-immune cell selection (e.g., an in vitro phage display, protein chip or any other method in which candidate sequences can be tested for their ability to bind to an antigen). This term thus preferably excludes sequences generated by genomic rearrangement in an immune cell.

[0079] A "bispecific" or "bifunctional antibody" is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab’ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). In one embodiment, the bispecific antibody comprises a first binding domain polypeptide, such as a Fab' fragment, linked via an immunoglobulin constant region to a second binding domain polypeptide.

[0080] Numerous methods known to those skilled in the art are available for obtaining antibodies or antigen-binding fragments thereof. For example, antibodies can be produced using recombinant DNA methods (U.S. Patent 4,816,567). Monoclonal antibodies may also be produced by generation of hybridomas (see e.g., Kohler and Milstein (1975) Nature, 256: 495-499) in accordance with known methods, as described also in the experimental part of the present invention. Hybridomas formed in this manner are then screened using standard methods, such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (BIACORE™) analysis, to identify one or more hybridomas that produce an antibody that specifically binds with a specified antigen (see Figure 8). Any form of the specified antigen may be used as the immunogen, e.g., recombinant antigen, naturally occurring forms, any variants or fragments thereof, as well as antigenic peptide thereof. One exemplary method of making antibodies includes screening protein expression libraries, e.g., phage or ribosome display libraries. Phage display is described, for example, in Ladner etal., U.S. Patent No. 5,223,409; Smith (1985) Science 228:1315-1317; Clackson et al. (1991) Nature, 352: 624-628; Marks et al. (1991) J. Mol. Bio!., 222: 581-597WO 92/18619; WO 91/17271; WO 92/20791 ; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809.

[0081] In addition to the use of display libraries, the specified antigen can be used to immunize a non-human animal, e.g., a rodent, e.g., a mouse, hamster, or rat. In one embodiment, the non-human animal includes at least a part of a human immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using the hybridoma technology, antigen-specific monoclonal antibodies derived from the genes with the desired specificity may be produced and selected. See, e.g., XENOMOUSE™, Green etal. (1994) Nature Genetics 7:13-21, US 2003- 0070185, WO 96/34096, and W096/33735.

[0082] In another embodiment, a monoclonal antibody is obtained from the non-human animal, and then modified, e.g., humanized, deimmunized, chimeric, may be produced using recombinant DNA techniques known in the art. A variety of approaches for making chimeric antibodies have been described. See e.g., Morrison et al., Proc. Natl. Acad. ScL U.S.A. 81:6851 , 1985; Takeda et al., Nature 314:452, 1985, Cabilly et al., U.S. Patent No. 4,816,567; Boss et al., U.S. Patent No. 4,816,397; Tanaguchi et al., EP 171496; EP 173494, GB 2177096. Humanized antibodies may also be produced, for example, using transgenic mice that express human heavy and light chain genes, but are incapable of expressing the endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR-grafting method that may be used to prepare the humanized antibodies described herein (U.S. Patent No. 5,225,539). All of the CDRs of a particular human antibody may be replaced with at least a portion of a non-human CDR, or only some of the CDRs may be replaced with non-human CDRs. It is only necessary to replace the number of CDRs required for binding of the humanized antibody to a predetermined antigen.

[0083] Humanized antibodies or fragments thereof can be generated by replacing sequences of the Fv variable domain that are not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for generating humanized antibodies or fragments thereof are provided by Morrison (1985) Science 229:1202-1207; by Oi et al. (1986) BioTechniques 4:214; and by US 5,585,089; US 5,693,761; US 5,693,762; US 5,859,205; and US 6,407,213. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable domains from at least one of a heavy or light chain. Such nucleic acids may be obtained from a hybridoma producing an antibody against a predetermined target, as described above, as well as from other sources. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

[0084] In certain embodiments, a humanized antibody is optimized by the introduction of conservative substitutions, consensus sequence substitutions, germline substitutions and/or backmutations. Such altered immunoglobulin molecules can be made by any of several techniques known in the art, (e.g., Teng et al., Proc. Natl. Acad. Sci. U.S.A., 80: 7308-7312, 1983; Kozbor etal, Immunology Today, 4: 7279, 1983; Olsson et al., Meth. Enzymol., 92: 3-16, 1982), and may be made according to the teachings of WO 92/06193 or EP 239400).

[0085] An antibody or fragment thereof may also be modified by specific deletion of human T cell epitopes or "deimmunization" by the methods disclosed in WO 98/52976 and WO 00/34317. Briefly, the heavy and light chain variable domains of an antibody can be analyzed for peptides that bind to MHC Class II; these peptides represent potential T-cell epitopes (as defined in WO 98/52976 and WO 00/34317). For detection of potential T-cell epitopes, a computer modeling approach termed "peptide threading" can be applied, and in addition a database of human MHC class II binding peptides can be searched for motifs present in the VH and VL sequences, as described in WO 98/52976 and WO 00/34317. These motifs bind to any of the 18 major MHC class II DR allotypes, and thus constitute potential T cell epitopes. Potential T-cell epitopes detected can be eliminated by substituting small numbers of amino acid residues in the variable domains, or preferably, by single amino acid substitutions. Typically, conservative substitutions are made. Often, but not exclusively, an amino acid common to a position in human germline antibody sequences may be used. Human germline sequences, e.g., are disclosed in Tomlinson, et at. (1992) J. Mol. Biol. 227:776-798; Cook, G. P. etai. (1995) Immunol. Today Vol. 16 (5): 237-242; Chothia, et al. (1992) J. Mol. Biol. 227:799-817; and Tomlinson et al. (1995) EMBO J. 14:4628-4638. The V BASE directory provides a comprehensive directory of human immunoglobulin variable region sequences (compiled by Tomlinson, LA. étal. MRC Centre for Protein Engineering, Cambridge, UK). These sequences can be used as a source of human sequence, e.g., for framework regions and CDRs. Consensus human framework regions can also be used, e.g., as described in U.S. Patent No. 6,300,064.

[0086] It is known that an antibody may exert effector functions. Accordingly, it is envisaged that an antibody of the invention can exert one or more effector functions due to its immunoglobulin constant or Fc region. Alternatively, in certain embodiments it is envisaged that an antibody can contain an altered immunoglobulin constant or Fc region. For example, an antibody produced in accordance with the teachings herein may bind more strongly or with more specificity to effector molecules such as complement and/or Fc receptors, which can control several immune functions of the antibody such as effector cell activity, lysis, complement-mediated activity, antibody clearance, and antibody half-life. Typical Fc receptors that bind to an Fc region of an antibody (e.g., an IgG antibody) include, but are not limited to, receptors of the FcyRI, FcyRII, and FcyRIII and FcRn subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc receptors are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92, 1991; Capel et al., Immunomethods 4:25-34,1994; and de Haas et al., J. Lab. Clin. Med. 126:330-41, 1995).

[0087] Techniques for the production of antibodies, including polyclonal, monoclonal, humanized, bispecific and heteroconjugate antibodies are known to those skilled in the art.

[0088] The term "immunizing" refers to the step or steps of administering one or more antigens, in case of the present invention a tyrosine-23 phosphorylated annexin A2 protein or one or more immunogenic fragments thereof, to a non-human animal so that antibodies can be raised in the animal. The terms "antigen "and "immunogen" are used interchangeably herein to refer to a molecule or substance which induces an immune response (preferably an antibody response) in an animal, preferably a non-human animal immunized therewith (i.e. the antigen is "immunogenic" in the animal). In case of the present invention when an antibody is generated against tyrosine-23 phosphorylated annexin A2 the antigen is preferably a tyrosine-23 phosphorylated annexin A2 protein, immunogenic peptide or fragment thereof. More preferably, the antigen is a phophorylated peptide comprising any one of the amino acid sequences as depicted in SEQ ID Nos: 11 to 18, which correspond to postitions 18 to 29 of the human ANXA2 protein (SEQ ID NO: 19). The tyrosine-23 phosphorylated annexin A2 antigen may be naturally-occurring or recombinantly produced.

[0089] Preferably, the antigen used for immunizing a non-human animal is a purified antigen. A "purified" antigen is one which has been subjected to one or more purification procedures. The purified antigen may be "homogeneous", which is used herein to refer to a composition comprising at least about 70% to about 100% by weight of the antigen of interest, based on total weight of the composition, preferably at least about 80% to about 100% by weight of the antigen of interest.

[0090] Generally, immunizing comprises injecting the antigen or antigens into the nonhuman animal. Immunization may involve one or more administrations of the antigen or antigens. Specifically, the non-human animal is preferably immunized at least two, more preferably three times with said polypeptide (antigen), optionally in admixture with an adjuvant. An "adjuvant" is a nonspecific stimulant of the immune response. The adjuvant may be in the form of a composition comprising either or both of the following components: (a) a substance designed to form a deposit protecting the antigen (s) from rapid catabolism (e.g. mineral oil, alum, aluminium hydroxide, liposome or surfactant (e.g. pluronic polyol) and (b) a substance that nonspecifically stimulates the immune response of the immunized host animal (e.g. by increasing lymphokine levels therein).

[0091] Exemplary molecules for increasing lymphokine levels include lipopolysaccaride (LPS) or a Lipid A portion thereof; Bordetalla pertussis; pertussis toxin; Mycobacterium tuberculosis; and muramyl dipeptide (MDP). Examples of adjuvants include Freund's adjuvant (optionally comprising killed M. tuberculosis; complete Freund’s adjuvant); aluminium hydroxide adjuvant; and monophosphoryl Lipid A-synthetic trehalose dicorynomylcolate (MPL-TDM). The "non-human animal" to be immunized herein is preferably a rodent. A "rodent" is an animal belonging to the rodentia order of placental mammals. Exemplary rodents include mice, rats, guinea pigs, squirrels, hamsters, ferrets etc, with mice being the preferred rodent for immunizing according to the method herein. Other non-human animals which can be immunized herein include non-human primates such as Old World monkey (e.g. baboon or macaque, including Rhesus monkey and cynomolgus monkey ; see US Patent 5, 658, 570) ; birds (e.g. chickens); rabbits; goats; sheep; cows; horses; pigs; donkeys; dogs etc.

[0092] The antibody that can be obtained by the preferred method is a polyclonal antibody or polyclonal serum (e.g., obtainable from a rodent, more preferably from a rabbit, goat or sheep) or, if antibody-producing cells are isolated from the non-human animal, a monoclonal antibody (e.g., obtainable from a rodent, more preferably from a mouse, rat or sheep) can be produced as is commonly known in the art and described herein.

[0093] Accordingly, the present invention also provides for a method for making monoclonal antibodies comprising the following steps: (a) immunizing an animal with two or more different antigens (i.e., fragments from one and the same polypeptide, in case of the present invention tyrosine-23 phosphorylated annexin A2) so as to generate polyclonal antibodies against each antigen in the animal; (b) preparing monoclonal antibodies using immune cells of the immunized animal which produce said polyclonal antibodies; and (c) screening said monoclonal antibodies to identify one or more monoclonal antibodies that bind to each antigen. In the screening step, one finds at least one monoclonal antibody against at least two different antigens. Preferably, at least one monoclonal antibody is found for each antigen with which the animal was immunized.

[0094] Preferably, the animal is immunized with a composition comprising a mixture of the two or more different antigens; and step (b) comprises fusing immune cells from the immunized animal with myeloma cells in order to generate hybridoma cell lines producing the monoclonal antibodies.

[0095] Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Thus, the modifier "monoclonal"indicates the character of the antibody as not being a mixture of discrete antibodies. For example, the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature. 256: 495 (1975), or may be made by recombinant DNA methods (U. S. Patent No. 4,816, 567). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization.

[0096] Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986). The immunizing agent will typically include the antigenic protein or a fusion variant thereof. Generally either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphoctyes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Goding, Monoclonal Antibodies: Principles and Practice, Academic Press (1986), pp. 59-103.

[0097] Immortalized cell lines are usually transformed mammalian cell, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[0098] Preferred immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, preferred are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 cells (and derivatives thereof, e.g. , X63-Ag8-653) available from the American Type Culture Collection, Manassus, Virginia USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0099] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprécipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). The culture medium in which the hybridoma cells are cultured can be assayed for the presence of monoclonal antibodies directed again desired antigen. Preferably, the binding affinity and specificity of the monoclonal antibody can be determined by immunoprécipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked assay (ELISA). Such techniques and assays are known in the in art. For example, binding affinity may be determined by the Scatchard analysis of Munson et al., Anal. Biochem., 107: 220 (1980).

[0100] After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in a mammal.

[0101] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0102] Monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U. S. Patent No. 4,816, 567, and as described above. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, in order to synthesize monoclonal antibodies in such recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol., 5: 256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188(1992).

[0103] In a further embodiment, antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature, 348: 552-554 (1990). Clackson et al., Nature, 352: 624-628 (1991) and Marks et al., J. Mol. Biol., 222: 581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bioll'eclanology, 10: 779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21: 2265-2266 (1993) ). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

[0104] The DNA also may be modified, for example, by substituting the coding sequence for human heavy-and light-chain constant domains in place of the homologous murine sequences (U. S. Patent No. 4,816, 567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81: 6851 (1984) ), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Typically such nonimmunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.

[0105] The monoclonal antibodies described herein may by monovalent, the preparation of which is well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and a modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues may be substituted with another amino acid residue or are deleted so as to prevent crosslinking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art. Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide-exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0106] According to the present invention, in certain circumstances there are advantages to using antibody fragments, rather than whole antibodies. Smaller fragment size allows for rapid clearance, and may lead to improved access to solid tumors. Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., J Biochem Biophys. Method. 24: 107-117 (1992); and Brennan et al., Science 229: 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F (ab') 2 fragments (Carter et aL, BiolTechnology 10 : 163-167 (1992)). According to another approach, F (ab') 2 fragments can be isolated directly from recombinant host cell culture. Fab and F (ab') 2 with increase in vivo half-life is described in U. S. Patent No. 5,869, 046. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U. S. Patent No. 5,571, 894 and U. S. Patent No. 5,587, 458. The antibody fragment may also be a "linear antibody", e.g., as described in U. S. Patent 5,641, 870. Such linear antibody fragments may be monospecific or bispecific.

[0107] The term "immune cells" as used herein refers to cells which are capable of producing antibodies. The immune cells of particular interest herein are lymphoid cells derived, e.g. from spleen, peripheral blood lymphoctes (PBLs), lymph node, inguinal node, Peyers patch, tonsil, bone marrow, cord blood, pleural effusions and tumor-infiltrating lymphocytes (TIL). By "screening" is meant subjecting one or more monoclonal antibodies (e.g., purified antibody and/or hybridoma culture supernatant comprising the antibody) to one or more assays which determine qualitatively and/or quantitatively the ability of an antibody to bind to an antigen of interest. By "immuno-assay" is meant an assay that determines binding of an antibody to an antigen, wherein either the antibody or antigen, or both, are optionally adsorbed on a solid phase (i. e., an "immunoadsorbent" assay) at some stage of the assay. Exemplary such assays include ELISAs, radioimmunoassays (RIAs), and FACS assays.

[0108] “Bispecific antibodies” (BsAbs) according to the present invention are antibodies that have binding specificities for at least two different epitopes, including those on the same or another protein. Alternatively, one arm can be armed to bind to the target antigen, and another arm can be combined with an arm that binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG (FcyR) such as FcyRI (CD64), FcyRII (CD32) and FcyRin (CD16), so as to focus and localize cellular defense mechanisms to the target antigen-expressing cell. Such antibodies can be derived from full length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies). Bispecific antibodies may also be used to localize cytotoxic agents to cells which express the target antigen. Such antibodies possess one arm that binds the desired antigen and another arm that binds the cytotoxic agent (e.g., methotrexate). Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities. Millstein et al., Nature, 305: 537-539 (1983). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829 and in Traunecker et al., EMBO J. , 10: 3655-3659(1991).

[0109] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.

[0110] In a preferred embodiment of this approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecules provides for an easy way of separation. This approach is disclosed in WO 94/04690. For further details of generating bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121: 210 (1986).

[0111] According to another approach described in WO 96/27011 or US 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan).

Compensatory "cavities" of identical or similar size to the large side chains (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.

[0112] Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab’)2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab’fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes.

[0113] Fab’fragments may be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175: 217-225 (1992) describes the production of fully humanized bispecific antibody F (ab') 2 molecules. Each Fab’fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells overexpressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets. Various techniques for making and isolating bivalent antibody fragments directly from recombinant cell culture have also been described. For example, bivalent heterodimers have been produced using leucine zippers. Kostelny et al., J. Immunol., 148 (5): 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab'portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993) has provided an alternative mechanism for making bispecific/bivalent antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light- chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific/bivalent antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Imnzunol., 152: 5368 (1994). Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol.! 147: 60 (1991). Exemplary bispecific antibodies may bind to two different epitopes on a given molecule. Alternatively, an anti-protein arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g., CD2, CD3, CD28 or B7), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16) so as to focus cellular defense mechanisms to the cell expressing the particular protein. Another bispecific antibody of interest binds the protein of interest and further binds Human Serum Albumine.

[0114] The "diabody" technology described by Hollinger et al. , Proc. Natl. Acad. Sci. USA, 90. 6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a VH connected to a VL by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. , 152: 5368 (1994). Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).

[0115] A multivalent antibody may be internalized (and/or catabolized) faster than a bivalent antibody by a cell expressing an antigen to which the antibodies bind. The antibodies of the present invention can be multivalent antibodies (which are other than of the IgM class) with three or more antigen binding sites (e.g. tetravalent antibodies), which can be readily produced by recombinant expression of nucleic acid encoding the polypeptide chains of the antibody. The multivalent antibody can comprise a dimerization domain and three or more antigen binding sites. The preferred dimerization domain comprises (or consists of) an Fc region or a hinge region. In this scenario, the antibody will comprise an Fc region and three or more antigen binding sites amino-terminal to the Fc region. The preferred multivalent antibody herein comprises (or consists of) three to about eight, but preferably four, antigen binding sites. The multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain (s) comprise two or more variable domains. For instance, the polypeptide chain (s) may comprise VDI (X1n-VD2-(X2)n-Fc, wherein VDI is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent an amino acid or polypeptide, and n is 0 or 1. For instance, the polypeptide chain (s) may comprise: VH-CHI-flexible linker-VH-CHI-Fc region chain; or VH-CHI-VH-CHI-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for instance, comprise from about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptides contemplated here comprise a light chain variable domain and, optionally, further comprise a CL domain.

[0116] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U. S. Patent No. 4,676,980. Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in US Patent No. 4,676, 980, along with a number of cross-linking techniques.

[0117] For additional antibody production techniques, see Antibodies: A Laboratory Manual, eds. Harlow et al., Cold Spring Harbor Laboratory, 1988. The present invention is not necessarily limited to any particular source, method of production, or other special characteristics of an antibody.

[0118] The antibody of the present invention is preferably an “isolated” antibody. "Isolated" when used to describe antibodies disclosed herein, means an antibody that has been identified, separated and/or recovered from a component of its production environment. Preferably, the isolated antibody is free of association with all other components from its production environment. Contaminant components of its production environment, such as that resulting from recombinant transfected cells, are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the antibody will be purified (1) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0119] Amino acid sequence modifications of the Syndeca-4 antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the tyrosine-23 phosphorylated annexin A2 antibody are prepared by introducing appropriate nucleotide changes into the tyrosine-23 phosphorylated annexin A2 antibody nucleic acid, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of, residues within the amino acid sequences of the tyrosine-23 phosphorylated annexin A2 antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final construct possesses the desired characteristics. The amino acid changes also may alter post-translational processes of the tyrosine-23 phosphorylated annexin A2 antibody, such as changing the number or position of glycosylation sites. A useful method for identification of certain residues or regions of the tyrosine-23 phosphorylated annexin A2 antibody that are preferred locations for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells in Science, 244: 1081-1085 (1989). Here, a residue or group of target residues within the tyrosine-23 phosphorylated annexin A2 antibody are identified (e. g. , charged residues such as arg, asp, his, lys, and glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine) to affect the interaction of the amino acids with epitope. Those amino acid locations demonstrating functional sensitivity to the substitutions then are refined by introducing further or other variants at, or for, the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is conducted at a target codon or region and the expressed tyrosine-23 phosphorylated annexin A2 antibody variants are screened for the desired activity. Amino acid sequence insertions include amino-and/or carboxyl-terminal fusions ranging in length from one, two, three, four, five, six, seven, eight, nine or ten residues to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An insertional variant of the tyrosine-23 phosphorylated annexin A2 antibody molecule include the fusion to the N-or C-terminus of the antibody to an enzyme or a fusion to a polypeptide which increases the serum half-life of the antibody.

[0120] Another type of variant is an amino acid substitution variant. These variants have at least one, two, three, four, five, six, seven, eight, nine or ten amino acid residues in the tyrosine-23 phosphorylated annexin A2 antibody molecule replaced by a different residue. The sites of greatest interest for substitutional mutagenesis include the CDRs of the heavy and/or light chain, in particular the hypervariable regions, but FR alterations in the heavy and/or light chain are also contemplated. For example, if a CDR sequence encompasses 6 amino acids, it is envisaged that one, two or three of these amino acids are substituted. Similarly, if a CDR sequence encompasses 15 amino acids it is envisaged that one, two, three, four, five or six of these amino acids are substituted.

[0121] Generally, if amino acids are substituted in one or more or all of the CDRs of the heavy and/or light chain, it is preferred that the then-obtained “substituted” sequence is at least 60%, more preferably 65%, even more preferably 70%, particularly preferable 75%, more particularly preferable 80% identical to the “original” CDR sequence. This means that it is dependent of the length of the CDR to which degree it is identical to the “substituted” sequence. For example, a CDR having 5 amino acids is preferably 80% identical to its substituted sequence in order to have at least one amino acid substituted. Accordingly, the CDRs of the tyrosine-23 phosphorylated annexin A2 antibody may have different degrees of identity to their substituted sequences, e.g., CDRL1 may have 80%, while CDRL3 may have 90%.

[0122] Preferred substitutions (or replacements) are conservative substitutions. However, any substitution (including non-conservative substitution or one or more from the “exemplary substitutions listed in Table 1, below) is only envisaged as long as the tyrosine-23 phosphorylated annexin A2 antibody retains its capability to detect tyrosine-23-phosphorylated annexin A2. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". If such substitutions result in a change in biological activity, then more substantial changes, denominated "exemplary substitutions" in Table 1, or as further described below in reference to amino acid classes, may be introduced and the products screened for a desired characteristic.

[0123] Substantial modifications in the biological properties of the antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe. Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Any cysteine residue not involved in maintaining the proper conformation of the tyrosine-23 phosphorylated annexin A2 antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond (s) may be added to the antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment). A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e. g. a humanized or human antibody). Generally, the resulting variant (s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e. g. 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e. g. binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and, e.g., human tyrosine-23 phosphorylated annexin A2. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development. TABLE I Amino Acid Substitutions

[0124] Another type of amino acid variant of the antibody alters the original glycosylation pattern of the antibody. By altering is meant deleting one or more carbohydrate moieties found in the antibody, and/or adding one or more glycosylation sites that are not present in the antibody. Glycosylation of antibodies is typically either N-linked or 0-linked. N- linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X- threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. 0-linked glycosylation refers to the attachment of one of the sugars N- aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for 0-linked glycosylation sites). Other modifications of the antibody are contemplated herein. For example, the antibody may be linked to one of a variety of nonproteinaceous polymers, e. g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. The antibody also may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) microcapsules, respectively), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980). The tyrosine-23 phosphorylated annexin A2 antibodies disclosed herein may also be formulated as immunoliposomes. A "liposome" is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al. , Proc. Natl Acad. Sci. USA, 77: 4030 (1980); U. S. Pat. Nos. 4, 485, 045 and 4,544, 545; and W097/38731 published October 23, 1997. Liposomes with enhanced circulation time are disclosed in U. S. Patent No. 5,013, 556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al. J. Biol. Chem. 257: 286-288 (1982) via a disulfide interchange reaction. A chemotherapeutic agent is optionally contained within the liposome. See Gabizon et al. J. National Cancer Inst. 81 (19) 1484 (1989).

[0125] According to the present invention, the immunoglobulin or fragment thereof as described herein is suitable for qualitative and/or quantitative detection of tyrosine-23 phosphorylâtes annexin A2 in vitro. The term “qualitative” as used herein refers to the detection of the tyrosine-23 phosphorylâtes annexin A2 as such (positive or negative), wherein the “quantiative" detection refer to the amount tyrosine-23 phosphorylâtes annexin A2. Said detection is preferably conducted by way of Western Blot, immunohistochemistry, immunophenotyping, fluorescence-activated cell scanning (FACS), Enzyme Linked Immunosorbent Assay (ELISA), Enzyme Linked Immuno Spot Assay (ELISPOT), radioimmunoassay, immunonephelometry, Enzyme (EIA) immunoassay, Fluorescent immunoassay (FIA), chemiluminescent immunoassays, immunoprécipitation or co-immunoprecipitation, known to those skilled in the art. However, also any other detection method known from the prior art can be used.

[0126] Immunoprécipitation is the simplest immunoassay method and measures the quantity of precipitate, which forms after the reagent antibody (precipitin) has incubated with the sample and reacted with its respective antigen to form an insoluble aggregate. Immunoprécipitation reactions may be qualitative or quantitative. When using particle immunoassays, several antibodies are linked to the particle, and the particle is able to bind many antigen molecules simultaneously. This greatly accelerates the speed of the visible reaction. Immunonephelometry works by allowing for an immediate union of antibody and antigen which form immune complexes that are too small to precipitate. However, these complexes will scatter incident light and can be measured using an instrument called a nephelometer. The antigen concentration can be determined within minutes of the reaction. Radioimmunoassay (RIA) is a method employing radioactive isotopes to label either the antigen or antibody. This isotope emits gamma raysare, which are usually measured following removal of unbound (free) radiolabel. The major advantages of RIA, compared with other immunoassays, are higher sensitivity, easy signal detection, and well-established, rapid assays. The major disadvantages are the health and safety risks posed by the use of radiation and the time and expense associated with maintaining a licensed radiation safety and disposal program. For this reason, RIA has been largely replaced in routine clinical laboratory practice by enzyme immunoassay. Enzyme (EIA) immunoassay was developed as an alternative to radioimmunoassay (RIA). These methods use an enzyme to label either the antibody or antigen. The sensitivity of EIA approaches that for RIA, without the danger posed by radioactive isotopes. One of the most widely used EIA methods for detection of infectious diseases is the enzyme-linked immunosorbent assay (ELISA). Fluorescent immunoassay (FIA) refers to immunoassays which utilize a fluorescent label or an enzyme label which acts on the substrate to form a fluorescent product. Fluorescent measurements are inherently more sensitive than colorimetric (spectrophotometric) measurements. Therefore, FIA methods have greater analytical sensitivity than EIA methods, which employ absorbance (optical density) measurement. Chemiluminescent immunoassays utilize a chemiluminescent label. Chemiluminescent molecules produce light when they are excited by chemical energy. These emissions are measured by a light detector. Immunohistochemistry or IHC refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction. Alternatively, the antibody can also be tagged to a fluorophore, such as fluorescein or rhodamine. IHC is an excellent detection technique and has the tremendous advantage of being able to show exactly where a given protein is located within the tissue examined. It is also an effective way to examine the tissues .This has made it a widely-used technique in the neurosciences, enabling researchers to examine protein expression within specific brain structures. Its major disadvantage is that, unlike immunoblotting techniques where staining is checked against a molecular weight ladder, it is impossible to show in IHC that the staining corresponds with the protein of interest. For this reason, primary antibodies must be well-validated in a Western Blot or similar procedure. The technique is even more widely used in diagnostic surgical pathology for typing tumors (e.g. immunostaining for e-cadherin to differentiate between DCIS (ductal carcinoma in situ: stains positive) and LCIS (lobular carcinoma in situ: does not stain positive).

[0127] Accordingly, the present invention refers to the immunoglobulin or a fragment thereof as described herein for use in a diagnostic method. Also provided is an in vitro method for detection of a tyrosine-23 phosphorylated annexin A2, wherein an immunoglobulin or fragment thereof according to the present invention is applied to a sample. Also provided is the use of an immunoglobulin or a fragment thereof according to the present invention for detection of tyrosine-23 phosphorylâtes annexin A2 in vitro and in vivo.

[0128] Further, the present invention relates to a method of diagnosing a tyrosine-23 phosphorylated annexin A2 related disease in a subject, comprising: a) administering to a sample obtained from said subject an immunoglobulin or fragment thereof according to the present invention, b) determining the amount of tyrosine-23 phosphorylated annexin A2 in said sample by any of the quantification methods described here, and c) comparing the results received in step b) to reference data. A lower or equal amount of tyrosine-23 phosphorylâtes annexin A2 in comparison to the reference data indicates that said subject is not suffering from a tyrosine-23 phosphorylated annexin A2 related disease. A higher amount of tyrosine-23 phosphorylâtes annexin A2 in comparison to the reference data indicates that said subject is suffering from a tyrosine-23 phosphorylated annexin A2 related disease. Preferred tyrosine-23 phosphorylated annexin A2 related diseases in this regard are diseases associated with an increased generation of plasmin. As shown inter alia in Deora et al., J. Biol. Chem. 2004, 279(42):43411-8, annexin A2 is a profibrinolytic co-receptor for plasminogen and tissue plasminogen activator that stimulates activation of the major fibrinolysin, plasmin, at cell surfaces. Thus, it is expected that tyrosine-23 phosphorylation of annexin A2 may directly influence diseases associated with increased plasmin generation. Thus, said diseases are within the scope of the present invention. Appropriate “reference data” or an “appropriate reference value” in this regard are/is for example the amount of tyrosine-23 phosphorylated annexin A2 determined in a sample obtained from a subject known to not suffer from a tyrosine-23 phosphorylated annexin A2 related disease.

[0129] The term "disease" or “disorder” as used herein refers to any impairment of the normal state of a living animal subject or one of its parts that interrupts or modifies the performance of vital functions that is typically manifested by distinguishing signs and symptoms. Thus, a "disease" or “disorder” refers to any physical state of a subject connected with incorrectly functioning organ, part, structure, or system of the body resulting from the effect of genetic or developmental errors, infection, poisons, nutritional deficiency or imbalance, toxicity, or unfavorable environmental factors, illness, sickness, or ailment. For example, a disease may include, but is not limited to, cancer diseases, cardiovascular diseases, neurodegenerative diseases, immunologic diseases, autoimmune diseases, inherited diseases, infectious diseases, bone diseases, and environmental diseases.

[0130] The term “tyrosine-23 phosphorylated annexin A2 related disease” as used herein refers to any disease or disorder which originates from or is directly associated with an increase phosphorylation of annexin A2 in the tyrosine-23 position of annexin A2. In particular, tyrosine-23 phosphorylated annexin A2 related diseases according to the present invention are diseases associated with an increased generation of plasmin. In this regard it has been assumed that degradation of connective tissue by the enzyme plasmin plays an important role in the spread of various diseases. The associated diseases include cancer, cardiovascular diseases and inflammation, such as viral infections (Kwaan et al., Cancer Treat Res. 2009;148:43-66; Pamela et al., Arterioscler Thromb Vase Biol. 1999;19:499-504). Accordingly, tyrosine-23 phosphorylated annexin A2 related diseases according to the present invention also comprise said diseases.

[0131] The term “subject" as used herein, refers to a mammal, including a human, or a nonhuman animal. Thus, the methods, uses and compositions described herein are generally applicable to both human and non-human mammals. The non-human animal may be any one of mouse, rat, guineas pig, rabbit, cat, dog, monkey, horse, or human. A non-human animal may also represent a model of a particular disease or disorder. Alternatively, a nonhuman animal may represent a domesticated pet and/or livestock in need of treatment of a disease (for example a dog, cat, goat, bovine, ovine, etc.). As explained elsewhere herein, a sample may be analyzed that has been obtained from said subject, which is typically a living organism. Where the subject is a living human who may receive treatment for a disease or disorder as described herein, it is also addressed as a “patient”.

[0132] The term “detection” or “detecting” when used herein includes variations like determining, qualifying, or semi-qualifying. The term “detect” or “detecting”, as well as the term “determine” or “determining” when used in the context of tyrosine-23 phosphorylated annexin A2 refers to any method that can be used in combination with the immunoglobulin or fragment thereof according to the present invention to identify the presence of tyrosine-23 phosphorylated annexin A2 released or expressed by a cell. When used herein in combination with the words “level”, “amount” or “value”, the words “determine” or “determining" or “detect” or “detecting” are understood to refer to a quantitative as well as a qualitative level. In some embodiments the determination of tyrosine-23 phosphorylated annexin A2 in a sample may be a method of determining the level (quantitative or semi-quantitative) of tyrosine-23 phosphorylated annexin A2 by comparing the level of tyrosine-23 phosphorylated annexin A2 in the sample with the level of tyrosine-23 phosphorylated annexin A2 standard. “Determining” or “quantifying" the amount of tyrosine-23 phosphorylated annexin A2 in a biological sample can be carried out by way of any suitable technique available and known to those skilled in the art. Essentially, “determining” or “quantifying” the amount of tyrosine-23 phosphorylated annexin A2 in a sample comprises the use of an immunoglobulin or fragment thereof having binding specificity to tyrosine-23 phosphorylated annexin A2 as disclosed by the present invention. As described elsewhere herein, examples of suitable immunoassay techniques in this regard are radiolabel assays such as a Radioimmunoassay (RIA) or enzyme-immunoassay such as an Enzyme Linked Immunosorbent Assay (ELISA), Luminex®-assays, precipitation (particularly immunoprécipitation), a sandwich enzyme immune test, an electro-chemiluminescence sandwich immunoassay (ECLIA), a dissociation-enhanced lanthanide fluoro immuno assay (DELFIA), a scintillation proximity assay (SPA), turbidimetry, nephelometry, latex-enhanced turbidimetry or nephelometry, or a solid phase immune test. Further methods known in the art (such as gel electrophoresis, 2D gel electrophoresis, SDS polyacrylamid gel electrophoresis (SDS-PAGE), and Western Blotting, can be used alone or in combination with labelling or other detection methods as described herein.

[0133] The term “sample” when used as regards the methods of the present invention relates to a material or mixture of materials, typically but not necessarily in liquid form, containing one or more analytes of interest. Preferably, the sample of the present invention is a biological sample. The term "biological sample", as used herein, refers to a sample obtained from an organism or from components (e.g., cells) of an organism. The sample may be of any biological tissue or fluid. Frequently the sample will be a "clinical sample" which is a sample derived from a patient. The sample as used according to said method includes but is not limited to a tissue sample, preferably a tissue extract sample, a body fluid sample or a cell culture sample. Preferably said body fluid sample is a full-blood sample, a serum sample, bronchoalveaolar fluid or a synovial fluid. However, also plasma samples, urine samples, fecal samples, saliva samples, tracheal secretion samples, or tear fluid samples are applicable in this regard. Methods for collecting various biological samples are well known in the art.

[0134] According to another aspect, the present invention relates to a method for the production of an immunoglobulin or fragment thereof according to the present invention, said method process comprising, culturing a host as defined elsewhere herein under conditions allowing the expression of the immunoglobulin or fragment thereof as defined herein and recovering the produced immunoglobulin or fragment thereof from the culture. Likewise, the present invention relates to the use of a polypeptide comprising any of the amino acid sequences as depicted in SEQ ID Nos: 11 to 18 for the production of an immunoglobulin or fragment thereof according to the present invention.

[0135] Said host may be produced by introducing the vector of the invention or the nucleic acid molecule of the invention as described elsewhere herein into the host. The presence of at least one vector or at least one nucleic acid molecule in the host may mediate the expression of a gene encoding the above described immunoglobulin or fragment thereof. The described nucleic acid molecule or vector of the invention, which is introduced in the host may either integrate into the genome of the host or it may be maintained extrachromosomally. The host can be any prokaryote or eukaryotic cell. The term "prokaryote" is meant to include all bacteria, which can be transformed or transfected with DNA or RNA molecules for the expression of a protein of the invention. Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. The term "eukaryotic" is meant to include yeast, higher plant, insect and preferably mammalian cells. Depending upon the host employed in a recombinant production procedure, the protein encoded by the polynucleotide of the present invention may be glycosylated or may be non-glycosylated. Especially preferred is the use of a plasmid or a virus containing the coding sequence of the bispecific single chain antibody molecule of the invention and genetically fused thereto an N-terminal FLAG-tag and/or C-terminal His-tag. Preferably, the length of said FLAG-tag is about 4 to 8 amino acids, most preferably 8 amino acids. An above described polynucleotide can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art. Furthermore, methods for preparing fused, operably linked genes and expressing them in, e.g., mammalian cells and bacteria are well-known in the art (Sambrook, loc cit).

[0136] Preferably, said the host is a bacterium or an insect, fungal, plant or animal cell. It is particularly envisaged that the recited host may be a mammalian cell. Particularly preferred host cells comprise CHO cells, COS cells, myeloma cell lines like SP2/0 or NS/0. As illustrated in the examples of WO 2008/1 19567 for other molecules of the same class, particularly preferred are CHO-cells as hosts. More preferably said host cell is a human cell or human cell line, e.g. per.c6 (Kroos, Biotechnol. Prog., 2003, 19:163-168). In a further embodiment, the present invention thus relates to a process for the production of the immunoglobulin or fragment thereof of the invention, said process comprising culturing a host of the invention under conditions allowing the expression of said immunoglobulin or fragment thereof of the invention and recovering the produced polypeptide from the culture. The transformed hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth. The immunoglobulin or fragment thereof of the invention can then be isolated from the growth medium, cellular lysates, or cellular membrane fractions. The isolation and purification of the, e.g., microbially expressed immunoglobulin or fragment thereof may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies directed, e.g., against a tag of the immunoglobulin or fragment thereof of the invention or as described in the appended examples.

[0137] The conditions for the culturing of a host, which allow the expression are known in the art to depend on the host system and the expression system/vector used in such process. The parameters to be modified in order to achieve conditions allowing the expression of a recombinant polypeptide are known in the art. Thus, suitable conditions can be determined by the person skilled in the art in the absence of further inventive input. Once expressed, the immunoglobulin or fragment thereof of the invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like; see, Scopes, "Protein Purification", Springer-Verlag, N.Y. (1982).

[0138] The term “host” is intended to refer to an organism or a cell into which a vector such as a cloning vector or an expression vector as described elsewhere herein has been introduced. The organism or cell can be prokaryotic or eukaryotic. Preferably, the “host” is an isolated host cells, e.g., host cells in culture. The term “host cell” merely signifies that the cells are modified for the (over)-expression of the immunoglobulin or fragment thereof according to the present invention and include B-cells that originally express these binding molecule and which cells have been modified to over-express the binding molecule by immortalization, amplification, enhancement of expression etc. It should be understood that the term “host” is intended to refer not only to the particular subject organism or cell but to the progeny of such an organism or cell as well. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent organism or cell, but are still included within the scope of the term “host.” [0139] When using such recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E coli. The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human gamma3 (Guss et al., EMBO J. 5: 15671575(1986)).

[0140] Accordingly, also provided herein is a host transformed or transfected with a vector as defined according to the present invention. Many suitable vectors are known to those skilled in molecular biology, the choice of which would depend on the function desired and include plasmids, cosmids, viruses, bacteriophages and other vectors used conventionally in genetic engineering. Methods which are well known to those skilled in the art can be used to construct various plasmids and vectors; see, for example, the techniques described in Sambrook et al. (loc cit.) and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1989), (1994). Alternatively, the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells. As discussed in further details below, a cloning vector was used to isolate individual sequences of DNA. Relevant sequences can be transferred into expression vectors where expression of a particular polypeptide is required. Typical cloning vectors include pBluescript SK, pGEM, pUC9, pBR322 and pGBT9. Typical expression vectors include pTRE, pCAL-n-EK, pESP-1, pOP13CAT.

[0141] Preferably said vector comprises a nucleic acid sequence which is a regulatory sequence operably linked to said nucleic acid sequence defined herein. The term "regulatory sequence" refers to DNA sequences, which are necessary to effect the expression of coding sequences to which they are ligated. The nature of such control sequences differs depending upon the host organism. In prokaryotes, control sequences generally include promoter, ribosomal binding site, and terminators. In eukaryotes generally control sequences include promoters, terminators and, in some instances, enhancers, transactivators or transcription factors. The term "control sequence" is intended to include, at a minimum, all components the presence of which are necessary for expression, and may also include additional advantageous components. The term "operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. In case the control sequence is a promoter, it is obvious for a skilled person that double-stranded nucleic acid is preferably used.

[0142] Thus, the recited vector is preferably an expression vector. An "expression vector" is a construct that can be used to transform a selected host and provides for expression of a coding sequence in the selected host. Expression vectors can for instance be cloning vectors, binary vectors or integrating vectors. Expression comprises transcription of the nucleic acid molecule preferably into a translatable mRNA. Regulatory elements ensuring expression in prokaryotes and/or eukaryotic cells are well known to those skilled in the art. In the case of eukaryotic cells they comprise normally promoters ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the Pi_, lac, trp or tac promoter in E. coli, and examples of regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.

[0143] Preferable, the vector according to the present invention comprises a nucleic acid sequence comprising one or more of the sequences as depicted in SEQ ID NO: 9 and SEQ ID NO: 10 or parts thereof. Preferably, the vector comprises a regulatory sequence which is operably linked to said nuclei acid sequences.

[0144] Beside elements, which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. Furthermore, depending on the expression system used leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the recited nucleic acid sequence and are well known in the art; see e.g. WO 2008/1 19567. The leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product; see supra. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNAI , pcDNA3 (ln-vitrogene), pEF-DHFR, pEF-ADA or pEF-neo (Mack et al. PNAS (1995) 92, 7021 - 7025 and Raum et al. Cancer Immunol Immunother (2001) 50(3), 141 -150) or pSPORTI (GIBCO BRL).

[0145] Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming of transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and as desired, the collection and purification of the bispecific single chain antibody molecule of the invention may follow; see, e.g., the appended examples. An alternative expression system, which can be used to express a cell cycle interacting protein is an insect system. In one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The coding sequence of a recited nucleic acid molecule may be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of said coding sequence will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat. The recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which the protein of the invention is expressed (Smith, J. Virol. 46 (1983), 584; Engelhard, Proc. Nat. Acad. Sci. USA 91 (1994), 3224-3227).

[0146] Additional regulatory elements may include transcriptional as well as translational enhancers. Advantageously, the above-described vectors of the invention comprise a selectable and/or scorable marker. Selectable marker genes useful for the selection of transformed cells and, e.g., plant tissue and plants are well known to those skilled in the art and comprise, for example, antimetabolite resistance as the basis of selection for dhfr, which confers resistance to methotrexate (Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149); npt, which confers resistance to the aminoglycosides neomycin, kanamycin and paromycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995) and hygro, which confers resistance to hygromycin (Marsh, Gene 32 (1984), 481 -485). Additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman, Proc. Natl . Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate isomerase which allows cells to utilize mannose (WO 94/20627) and ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylase inhibitor, 2-(difluoromethyl)- DL-ornithine, DFMO (McConlogue, 1987, In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory ed.) or deaminase from Aspergillus terreus which confers resistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59 (1995), 2336-2338). Useful scorable markers are also known to those skilled in the art and are commercially available. Advantageously, said marker is a gene encoding luciferase (Giacomin, PI. Sci. 1 16 (1996), 59-72; Scikantha, J. Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett. 389 (1996), 44-47) or ß-glucuronidase (Jefferson, EMBO J. 6 (1987), 3901 -3907). This embodiment is particularly useful for simple and rapid screening of cells, tissues and organisms containing a recited vector.

[0147] The present invention also provides for a nucleic acid sequence encoding for an immunoglobulin or fragment thereof according to the present invention. Said nucleic acid sequence preferably comprises the sequence of SEQ ID NO: 9 and/or SEQ ID NO: 10 or parts of said sequences. The recited nucleic acid molecules can be used alone or as part of a vector to express the immunoglobulin or fragment thereof of the invention in cells, for, e.g., purification but also for gene therapy purposes, as described elsewhere herein. The nucleic acid molecules or vectors containing the DNA sequence(s) encoding any one of the above described immunoglobulin or fragment thereof of the invention is introduced into the cells which in turn produce the polypeptide of interest. Gene therapy, which is based on introducing therapeutic genes into cells by ex-vivo or in-vivo techniques is one of the most important applications of gene transfer. Suitable vectors, methods or gene-delivery systems for in-vitro or in-vivo gene therapy are described in the literature and are known to the person skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539; Schaper, Cire. Res. 79 (1996), 91 1 - 919; Anderson, Science 256 (1992), 808-813; Verma, Nature 389 (1994), 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995), 1077-1086; Onodera, Blood 91 (1998), 30-36; Verma, Gene Ther. 5 (1998), 692-699; Nabel, Ann. N.Y. Acad. Sci. 81 1 (1997), 289-292; Verzeletti, Hum. Gene Ther. 9 (1998), 2243-51 ; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, US 5,580,859; US 5,589,466; or Schaper, Current Opinion in Biotechnology 7 (1996), 635-640; dos Santos Coura and Nardi Virol J. (2007), 4:99.

[0148] The recited nucleic acid molecules and vectors may be designed for direct introduction or for introduction via liposomes, or viral vectors (e.g., adenoviral, retroviral) into the cell. Preferably, said cell is a germ line cell, embryonic cell, or egg cell or derived there from, most preferably said cell is a stem cell. An example for an embryonic stem cell can be, inter alia, a stem cell as described in Nagy, Proc. Natl . Acad. Sci. USA 90 (1993), 8424-8428.

[0149] Alternatively, the present invention relates to a method for the production of an immunoglobulin or fragment thereof as defined herein, said method comprising: (a) immunizing an animal with a molecule comprising the peptide as depicted in any of SEQ ID Nos: 11 to 18, and (b) obtaining an immunoglobulin or fragment thereof being capable of binding to tyrosine-23 phosphorylated annexin A2.

It is envisaged that the immunized animal is a non-human animal. Suitable animals and immunization techniques are described elsewhere herein.

[0150] Given the above, the present invention provides for a monoclonal or polyclonal antibody obtainable by the afore-described methods for the production of an antibody, i.e., by immunizing a non-human animal as described before. Hence, the term “antibody" when used herein also encompasses an antibody (monoclonal or polyclonal) obtainable by the methods for the generation of an antibody against tyrosine-23 phosphorylated annexin A2, preferably human tyrosine-23 phosphorylated annexin A2.

[0151] While it is possible to administer the immunoglobulin or fragment thereof according to the present invention as described herein above directly without any formulation to a subject or a sample, in one aspect of the present invention the compounds are preferably employed in the form of a pharmaceutical or diagnostic formulation composition, comprising a pharmaceutically or diagnostically acceptable carrier, diluent or excipient and any of the immunoglobulin or fragment thereof according to the present invention. Accordingly, the present invention relates to a diagnostic or pharmaceutical composition comprising any of the immunoglobulin or fragment thereof as described herein and a pharmaceutically or diagnostically acceptable excipient. Moreover, the present invention relates to the use of an immunoglobulin or fragment thereof as disclosed herein for the preparation of a diagnostic or pharmaceutical composition for diagnosing or treating a tyrosine-23 phosphorylated annexin A2 related disease. In particular, said tyrosine 23 phosphorylated annexin A2 related disease is a diseases associated with an increased generation of plasmin.

[0152] The term “diagnostic composition” when used herein refers to a composition comprising any one of the immunoglobulins or fragment thereof of the present invention and a pharmaceutically or diagnostically acceptable carrier, diluent or excipient, which can be applied for used in diagnosis. The term “pharmaceutical composition” when used herein refers to a composition comprising any one of the immunoglobulins or fragment thereof of the present invention and a pharmaceutically or diagnostically acceptable carrier, diluent or excipient, which can be applied in treatment. The carrier used in combination with the antibody of the present invention is water-based and forms an aqueous solution. An oil-based carrier solution containing the compound of the present invention is an alternative to the aqueous carrier solution. Either aqueous or oil-based solutions further contain thickening agents to provide the composition with the viscosity of a liniment, cream, ointment, gel, or the like. Suitable thickening agents are well known to those skilled in the art. Alternative embodiments of the present invention can also use a solid carrier containing the diagnostic compound for use in diagnosis as disclosed elsewhere herein. This enables the alternative embodiment to be applied via a stick applicator, patch, or suppository. The solid carrier further contains thickening agents to provide the composition with the consistency of wax or paraffin.

[0153] Pharmaceutically or diagnostically acceptable excipients according to the present invention include, by the way of illustration and not limitation, diluent, disintegrants, binding agents, adhesives, wetting agents, polymers, lubricants, gliands, substances added to mask or counteract a disagreeable texture, taste or odor, flavors, dyes, fragrances, and substances added to improve appearance of the composition. Acceptable excipients include lactose, sucrose, starch powder, maize starch or derivatives thereof, cellulose esters of alkanoic acids, cellulose alkyl esters, talc, stearic acid, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulfuric acids, gelatin, acacia gum, sodium alginate, polyvinyl-pyrrolidone, and/or polyvinyl alcohol, saline, dextrose, mannitol, lactose, lecithin, albumin, sodium glutamate, cysteine hydrochloride, and the like. Examples of suitable excipients for soft gelatin capsules include vegetable oils, waxes, fats, semisolid and liquid polyols. Suitable excipients for the preparation of solutions and syrups include, without limitation, water, polyols, sucrose, invert sugar and glucose. Suitable excipients for injectable solutions include, without limitation, water, alcohols, polyols, glycerol, and vegetable oils. The diagnostic compositions can additionally include preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, buffers, coating agents, or antioxidants. Suitable pharmaceutical and diagnostic carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference text in this field. The use of the diagnostic composition of the present invention as a diagnostic kit for diagnosing inflammatory disease associated with phagocyte and/or epithelial cell activation and overexpression and accumulation of S100A9 is also encompassed by the present invention.

[0154] Examples of suitable pharmaceutical carriers are well known in the art and include solutions, e.g. phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions, liposomes, etc. Compositions comprising such carriers can be formulated by well known conventional methods. Formulations can comprise carbohydrates, buffer solutions, amino acids and/or surfactants. Carbohydrates may be non-reducing sugars, preferably trehalose, sucrose, octasulfate, sorbitol or xylitol. Such formulations may be used for continuous administrations which may be intravenuous or subcutaneous with and/or without pump systems. Amino acids may be charged amino acids, preferably lysine, lysine acetate, arginine, glutamate and/or histidine. Surfactants may be detergents, preferably with a molecular weight of >1 .2 KD and/or a polyether, preferably with a molecular weight of >3 KD. Non-limiting examples for preferred detergents are Tween 20, Tween 40, Tween 60, Tween 80 or Tween 85. Non-limiting examples for preferred polyethers are PEG 3000, PEG 3350, PEG 4000 or PEG 5000. Buffer systems used in the present invention can have a preferred pH of 5-9 and may comprise citrate, succinate, phosphate, histidine and acetate. The compositions of the present invention can be administered to the subject at a suitable dose which can be determined e.g. by dose escalating studies by administration of increasing doses of the bispecific single chain antibody molecule of the invention exhibiting cross- species specificity described herein to non-chimpanzee primates, for instance macaques. As set forth above, the bispecific single chain antibody molecule of the invention exhibiting cross-species specificity described herein can be advantageously used in identical form in preclinical testing in non-chimpanzee primates and as drug in humans.

[0155] The carrier used in combination with the the immunoglobulins or fragment thereof of the present invention is preferably water-based and forms an aqueous solution. An oil-based carrier solution containing the conjugate of the present invention is an alternative to the aqueous carrier solution. Either aqueous or oil-based solutions further contain thickening agents to provide the composition with the viscosity of a liniment, cream, ointment, gel, or the like. Suitable thickening agents are well known to those skilled in the art. Alternative embodiments of the present invention can also use a solid carrier containing the the immunoglobulins or fragment thereof of the present invention. This enables the alternative to conjugate disclosed elsewhere herein via a stick applicator, patch, or suppository. The solid carrier further contains thickening agents to provide the conjugate with the consistency of wax or paraffin.

[0156] The biological activity of the pharmaceutical composition defined herein can be determined for instance by cytotoxicity assays, as described in the following examples, in WO 99/54440 or by Schlereth et al. (Cancer Immunol. Immunother. 20 (2005), 1 - 12). "Efficacy" or "in vivo efficacy" as used herein refers to the response to therapy by the pharmaceutical composition of the invention, using e.g. standardized NCI response criteria. The success or in vivo efficacy of the therapy using a pharmaceutical composition of the invention refers to the effectiveness of the composition for its intended purpose, i.e. the ability of the composition to cause its desired effect, i.e. depletion of pathologic cells, e.g. tumor cells. The in vivo efficacy may be monitored by established standard methods for the respective disease entities including, but not limited to white blood cell counts, differentials, Fluorescence Activated Cell Sorting, bone marrow aspiration. In addition, various disease specific clinical chemistry parameters and other established standard methods may be used. Furthermore, computer-aided tomography, X-ray, nuclear magnetic resonance tomography (e.g. for National Cancer Institute-criteria based response assessment [Cheson BD, Horning SJ, Coiffier B, Shipp MA, Fisher Rl, Connors JM, Lister TA, Vose J, Grillo-Lopez A, Hagenbeek A, Cabanillas F, Klippensten D, Hiddemann W, Castellino R, Harris NL, Armitage JO, Carter W, Hoppe R, Canellos GP. Report of an international workshop to standardize response criteria for non-Hodgkin’s lymphomas. NCI Sponsored International Working Group. J Clin Oncol. 1999 Apr;17(4):1244]), positron-emission tomography scanning, white blood cell counts, differentials, Fluorescence Activated Cell Sorting, bone marrow aspiration, lymph node biopsies/histologies, and various cancer specific clinical chemistry parameters (e.g. lactate dehydrogenase) and other established standard methods may be used. Another major challenge in the development of drugs such as the pharmaceutical composition of the invention is the predictable modulation of pharmacokinetic properties. To this end, a pharmacokinetic profile of the drug candidate, i.e. a profile of the pharmacokinetic parameters that effect the ability of a particular drug to treat a given condition, is established. Pharmacokinetic parameters of the drug influencing the ability of a drug for treating a certain disease entity include, but are not limited to: half-life, volume of distribution, hepatic first-pass metabolism and the degree of blood serum binding. The efficacy of a given drug agent can be influenced by each of the parameters mentioned above.

[0157] The pharmaceutical compositions according to the invention may be supplied as a kit comprising a container that comprises the immunoglobulins or fragment thereof of the present invention according to the invention. Accordingly, the present invention also relates to a kit comprising an immunoglobulin or fragment according to the present invention, a nucleic acid molecule as defined herein, a vector as defined herein, or a host as defined herein. Thus, it also envisaged to use the diagnostic composition of the present invention as a diagnostic kit for the diagnosis of a tyrosine-23 phosphorylated annexin A2 related disease.

[0158] In some embodiments the term “kit" when used herein refers to an assembly of useful compounds and other means like solid support plates or test stripes for detecting a tyrosine-23 phosphorylated annexin A2 related disease in a mammalian sample. A kit may include the diagnostic composition of the present invention or the nucleic acid molecules, the vector, or a host as defined elsewhere herein. Other components such as buffers, controls, and the like, known to those skilled in art, may be included in such test kits. The relative amounts of the various reagents can be varied, to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay. Particularly, the reagents can be provided as dry powders, usually lyophilized, which on dissolution will provide for a reagent solution having the appropriate concentrations for combining with a sample. The present kit may further include instructions for carrying out one or more methods of the present invention, including instructions for using standard and/or composition of the present invention that is included with the kit. In some embodiments the diagnostic kit comprises a monoclonal antibody exclusively binding to a tyrosine-23 phosphorylated annexin A2. The antibodies used in said kit can be present in bound or soluble from.

[0159] According to another aspect, the present invention refers to the immunoglobulin or fragment thereof according to the present invention for use in treatment. In particular, the present invention refers to the immunoglobulin or fragment thereof according to the present invention for use in the treatment of a tyrosine-23 phosphorylated annexin A2 related disease, such as a disease associated with an increased generation of plasmin. Disease which are associated with or originate from phosphorylation of annexin A2 are described elsewhere herein. Also provided is a method for treating a subject suffering from a tyrosine-23 phosphorylated annexin A2 related disease, said method comprising administering a therapeutically effective amount of the immunoglobulin or fragment thereof according to the present invention to a subject in need thereof. In some embodiments the immunoglobulin or fragment thereof according to the present invention is directed to inhibit or neutralize tyrosine-23 phosphorylated annexin A2.

[0160] The immunoglobulin or fragment thereof according to the present invention in this regard can be formulated in a variety of useful formats for administration by a variety of routes. Concentrations of the immunoglobulin or fragment thereof according to the present invention will be such that a therapeutically effective dose of the immunoglobulin or fragment thereof is included in the formulation, e.g., a pharmaceutical composition comprising a therapeutically effective dose of the immunoglobulin or fragment thereof according to the present invention and a carrier. Said pharmaceutical compositions are described elsewhere herein. The dosage regimen will be determined by the attending physician and clinical factors. As it is well known in the medical arts, dosages for any one patient depend upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs administered concurrently. Determination of the appropriate concentration of the immunoglobulin or fragment thereof according to the present invention would be readily apparent to those of ordinary skill in the art.

[0161] In some embodiments, the immunoglobulin or fragment thereof according to the present invention may be formulated, for example, for intravenous, oral, sublingual, intranasal, intraocular, rectal, transdermal, intradermal, subcutaneous, mucosal, topical, or parenteral administration. Thus, the immunoglobulin or fragment thereof according to the present invention may be administered in pharmaceutically acceptable formulations and in substantially non-toxic quantities.

[0162] The terms “treat”, “treatment" or “treating” as used herein refers to the medical therapy of any human or other animal subject in need thereof. The terms “treat”, "treatment” “treating” further means to reduce, stabilize, or inhibit the progression of a disease and/or symptoms associated therewith in a subject. Said subject is expected to have undergone physical examination by a medical or veterinary medical practitioner, who has given a tentative or definitive diagnosis which would indicate that the use of said specific treatment is beneficial to the health of said human or other animal subject. The timing and purpose of said treatment may vary from one individual to another, according to the status quo of the subject's health. Thus, said treatment may be prophylactic, palliative, symptomatic and/or curative. In terms of the present invention, prophylactic, palliative, symptomatic and/or curative treatments may represent separate aspects of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0163] Figure 1: Western blotting. BHK-IR cells were starved overnight and stimulated with 1 pg/ml insulin as indicated. 50 pg protein of PNS/sample was separated by SDS-PAGE blotted and stained with antibodies as indicated (rat mAb NZ-8H7-H5, 1:100, BD mouse mAb (X-ANXA2 1:4000).

[0164] Figure 2: Western blotting. ANXA2 immunoprecipitated with mouse mAb a-ANXA2 HH7 from BHK-IR cells stimulated as indicated was resolved by SDS-PAGE and Western blotting with antibodies as indicated.

[0165] Figure 3: Western blotting. MDCK cells starved overnight were stimulated for 1 h as indicated (0.15 mM H2O2, 100 μΜ Vanadate, 50 ng/ml HGF). ANXA2 was immunoprecipitated with mouse mAb α-ΑΝΧΑ2 HH7. -Ab +Lys: Lysate incubated with sheep a-mouse dynabeads w/o primary antibody, +Ab - Lys: HH7-coupled sheep a-mouse dynabeads, no lysate incubation.

[0166] Figure 4: Immunoprécipitation. BHK cells were starved overnight and stimulated with 1.25 pg/ml insulin. PNS was prepared using lysis buffer with or without 0.1% SDS added. 200 pg lysates protein was used to immunoprecipitate ANXA2 with NZ-H87-H5. Membrane was first incubated with NZ-H87-H5 to detect pTyR23-ANXA2, then reprobed with BD a-ANXA2 to detect total ANXA2.

[0167] Figure 5: Specificity for epitope. BHK-IR cells transiently expressing GFP-tagged ANXA2 mutants were starved overnight and stimulated for 1 h with 5 pg/ml insulin. 400 pg of PNS was used to immunoprecipitate GFP-tagged ANXA2 with rabbit pAb a-GFP antibody. Immunodetection of precipitated GFP-tagged ANXA2 was performed with the indicated antibodies.

[0168] Figure 6: Immunofluorescence microscopy. BHK-IR cells were starved overnight and stimulated for 1 h with 1 pg/ml insulin. Cells were fixed with MetOH. NZ-8H7-N5 was used as primary antibody and FITC-coupled goat a-rat antibody as secondary antibody.

[0169] Figure 7: Immunofluorescence microscopy. MDCK cells were stimulated for 1 h with 50 ng/ml HGF, 100 pM vanadate and 015 mM H202. Cells were fixed with MetOH. NZ-8H7-N5 was used as primary antibody and FITC-coupled goat a-rat antibody as secondary antibody.

[0170] Figure 8: ELISA assay. Hybridoma supernatants were analyzed by ELISA on annexin A2 peptide, phosphorylated annexin-2 peptide and, as a control, irrelevant peptide. The negative control was buffer on annexin A2 and irrelevant peptides, respectively. The positive control was anti-annexin A2 immune serum and anti-irrelevant peptide immune serum on annexin A2 and irrelevant peptides, respectively. The specificity of the monoclonal antibody clone NZ-8H7-H5 could be clearly proven.

EXAMPLES

[0171] The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration and the present invention is limited only by the claims. It will be clear to a skilled person in the art that the invention may be practiced in other ways than as particularly described in the present description and examples. Numerous modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the appended claims.

[0172] Generation of rat monoclonal NZ-8H7-N5 anti-pTvr23 ANXA2 antibody

Posphorylated and non-phosphorylated ANXA2 peptides comprising amino acids 18-29 of the human ANXA2 sequence (STPPSAYGSVKA) were synthesized, and a cystein residue was added for conjugation (STPPSAYGSVKA-C). Peptides were conjugated to KLH for immunization and to OVA for testing. To generate monoclonal antibodies, 5 rats were immunized and the sera were tested by ELISA on ANXA2 peptide, phosphorylated ANXA2 peptide and on an irrelevant peptide. Rat lymphocytes were fused for generation of hybridomas. Hybridoma supernatants were analyzed by ELISA on ANXA2 peptide, phosphorylated ANXA2 peptide, and on an irrelevant peptide. Cells of the hybridoma subclone NZ-8H7-N5 were cultured in DMEM, 10% heat inactivated FCS, 1% Penicillin/Streptomycin.

[0173] Purification of NZ-8H7-H5 from hybridoma supernatant

The antibody was purified using the Thiophilic Adsorption Kit (Pierce) according to the manufacturer's protocol and yielded 0.5 mg/ml supernatant, 0.5 mg/ml eluate.

[0174] Cell culture BHK-IR and MDCK cells were cultivated at 37°C and 7% C02 in high glucose DMEM (Invitrogen) supplemented with 10% heat-inactivated FCS (PAA), 2 mM L-glutamine, 10 U/ml penicillin and 10 pg/ml Streptomycin.

[0175] Transfection of plasmids

Cells were transfected with Effectene (Qiagen) as recommended by the manufacturer. BHK-IR were starved overnight (DMEM, glutamine, Pen/Strep, w/o FCS) and stimulated for the indicated periods of time with 1 pg/ml insulin (human recombinant, Sigma).

[0176] Induction of tvrosine phosphorylation MDCK cells were starved overnight and stimulated for 1 h with 0.15 mM H202, 100 μΜ Orthovanadate , 50 ng/ml HGF (Peprotech).

[0177] Antibodies and plasmids

The following antibodies and plasmids were used: rat mAb NZ-8H7-H5, mouse mAb a-ANXA2 from BD Biosciences, mouse mAb a-ANXA2 HH7 (Ref), rabbit pAb a-GFP from Invitrogen, sheep a-mouse and a-rabbit Dynabeads from Invitrogen, FITC-coupled goat a-rat mAb from Jackson ImmunoResearch.

[0178] Cell Ivsates 107 cells were resuspended in 250-500 μΙ cold lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCI, 1% Triton X-100, 10% glycerol, 1 mM EDTA) supplemented with inhibitors (2 mM Na-Orthovanadate, 10 mM NaF, 2 mM PMSF, 1 mg/ml aprotinin, 1 pg/ml leupeptin, or 1x Phos-Stop cocktail from Roche and 1x protease-inhibitor cocktail from Roche). Cells were lysed by 20 pulses in a pre-cooled (-20°C) block sonifier (amplitude 80/cycle 0.5) and incubated for 30 min on ice. Post nuclear supernatants (PNS) were obtained by centrifugation (800 g, 10 min, 4°C).

[0179] Immunoprécipitation 50 μΙ_ Dynabeads coated with secondary antibody were washed 3 x with 500 μΙ PBS, 0.1% BSA using a magnetic separator, and coated with ms mAb n-anxaA2 antibody HH7 diluted in PBS, 01% BSA at 4°C. Coupled beads were washed 2x with PBS, 01% BSA, 1X with 500 μΙ lysis buffer w/o inhibitors and incubated with PNS (BHK-IR: 200 μg protein/sample, MDCK: 150 μg protein/sample) for 1.5 h at 4°C. Beads were washed 3x with 500 μΙ lysis buffer w/o inhibitors, resuspended in 25 μΙ 4x sample buffer, and boiled at 95°c for 10 min. Beads were removed with magnetic separator and the supernatants were subjected to SDS-PAGE. Antibody-coupled beads w/o lysate (+Ab, -Lys) and lysates incubated with Dynabeads w/o primary antibody (-Ab, +Lys) served as negative controls.

[0180] Confocal immunofluorescence imaging

Cells were washed with cold PBS and fixed with 4% (w/v) paraformaldehyde in PBS for 10 min. Cells were rinsed 3x with PBS, and permeabilized with 0.2 % Triton X-100 in PBS. Cells were blocked for 15 min at RT with 2% BSA in PBS and incubated overnight with primary antibody diluted in 2% BSA in PBS. Cells were rinsed 3x with PBS and incubated with secondary antibody diluted in 2% BSA for 30 min at RT. After washing 3x with PBS, coverslips were mounted on glass slides with Mowiol 4-88 supplemented with 1 % N-propyl-gallate as antifading agent.

[0181] Discontinous SDS-PAGE and Western blotting

Protein samples were boiled for 10 min at 95°C with sample buffer and loaded onto SDS-containing gels (acrylamide concentration in stacking gel 5%, in resolving gel 15%). Protein bands were transferred for 1 h at 300 mA onto MetOH-activated PVDF membranes by Western blotting using a Mini trans-blot tank system (Bio-Rad). Transfer buffer: 25 mM Tris, 192 mM glycin, 20% MetOH. Membranes were washed 3x with TBS-T (20 mM Tris-HCI, pH 7.5, 150 mM NaCI, 0.2 % Tween 20) and blocked with 5% skimmed milk powder in TBS-T for 15 min. For immunodetection, membranes were incubated overnight at 4°C with primary antibody diluted in TBST, 5% skimmed milk powder. Membranes were washed 3x with TBS-T and incubated for 1 h with secondary antibody in TBS-T, 5% milk powder.

[0182] Results

To test the applicability of this antibody in immunoblotting experiments, a previously established Baby Hamster Kidney (BHK) fibroblast cell line stably overexpressing the human insulin receptor (BHK-IR) was utilized (BHK-IR, Mailer et al., 1995). Stimulation of this cell line with insulin results in a strong phosphorylation of ANXA2 on V23 (Rescher et al., 2008) BHK-IR were stimulated for the indicated periods of time with 1 pg/ml insulin. Cytosolic lysates were resolved by SDS-PAGE and blotted for Y23-phosphorylated ANXA2. No signal was detected in unstimulated cells, whereas a strong signal was already detected after 5 min of insulin stimulation. This band became stronger after 30 min and peaked around 60 min. Total ANXA2 levels remained unchanged (Figure 1). Western Blot analysis of ANXA2 immunoprecipitated with HH7 from unstimulated and insulin-stimulated BHK-IR cells confirmed that ANXA2 was strongly tyrosine-phosphorylated upon stimulation with insulin (Figure 2). Treatment of MDCK cells with H202 in the presence of a tyrosine-phosphatase inhibitor such as orthovanadate results in strong cellular phosphorylation (Wu et al., 2000). Probing of ANXA2 immunoprecipitated from MDCK cells with NZ-8H7-H5 revealed no signal in untreated cells, whereas a strong signal was detected in cells treated with both agents. Addition of HGF, a growth factor activating tyrosine-based signaling cascades through the receptor-tyrosine kinase c-Met, did not further increase the amount of tyrosine-phosphorylated ANXA2 (Figure 3). To test whether NZ-8H7-H5 may be suitable for immunoprécipitation, ANXA2 from insulin-treated BHK-IR cells was immunoprecipitated using NZ-8H7-H5 and sheep a-rat coupled Dynabeads. Y23-phosphorylated ANXA2 was only precipitated under mild denaturing conditions, i.e. in the presence of 0,1% SDS (Figure 4) . To test the specificity of NZ-8H7-H5 for the tyrosine-phosphorylated target sequence in the physiological context, i.e. the presentation as part of the whole protein sequence, in the cell, phosphorylated by cellular kinases, GFP-tagged ANXA2 with mutated Y23 residue was expressed in BHK-IR cells and immunoprecipitated 1 h after stimulation with insulin. While ANXA2-GFP was successfully precipitated in all cases, only the wild type N-terminal sequence was detected with NZ-8H7-H5. The phospho-mimicking and the phosphorylation-deficient mutants Y23E and Y23F, respectively, showed no signal after stimulation (Figure 5) . Immunofluorescence staining using NZ-8H7-H5 resulted in a weak background signal in untreated cells, whereas strongly stained small foci were detected in insulin-treated BHK-IR cells (Figure 6). In H202/vanadate/HGF-treated MDCK cells, cell-cell contact sites and the plasma membrane appeared positive (Figure 7).

[0183] Test of specificity of rat against annexin-2 monoclonal antibodies in ELISA» assay

To confirm specificity of the monoclonal antibodies, hybridoma supernatants were analyzed by ELISA. Annexin-2 peptide, phosphorylated Annexin-2 peptide and, as a control, irrelevant peptide were tested. The columns designated “negative control” was buffer on Annexin-2 and irrelevant peptides, respectively. The columns designated “positive control” was anti-Annexin-2 immune serum and anti-irrelevant peptide immune serum on Annexin-2 and irrelevant peptides, respectively. The specificity of the monoclonal antibody clone NZ-8H7-H5 could be clearly proven (Figure 8).

[0184] Variable region sequencing of rat against annexin-2 monoclonal antibodies

The procedure included standard dye-terminator capillary sequencing of cDNA (VL cDNA SEQ ID NO: 9, HL cDNA SEQ ID NO: 19) that was generated from extracted mRNA using standard RT-PCR protocol (aldevron). Cycle sequencing was performed using BogDye® Terminator v3.1 Cycle Sequencing kits under s standard protocol provided by Life Technologies ®. All data were collected using a 3730x1 DNA Analyzer system and the Unified Data Collection software procided by Life Technologies® for operation of the 3730x1 DNA Analyzer and to collect data produced by the 3730x1 DNA Analyzer. Sequence assembly was performed using CodonCode Aligner (CodonCode Cooperation). Mixed base calls were resolved by automatically assigning the most prevalent base call to the mixed base calls. Prevalence is determined by both frequency of a base call and the individual quality of the base calls.

Claims

An immunoglobulin or fragment thereof capable of binding to tyrosine-23 phosphorylated annexin A2, wherein the immunoglobulin or fragment thereof comprises a VL region comprising complementarity determining regions (CDRs) CDR-L1, CDR-L2 and CDR-L3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, and / or wherein the immunoglobulin or fragment thereof comprises a VH region comprising complementarity determining regions (CDRs) CDR-H1, CDR-H2 and CDR-H3 as shown in SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO: 6.

The immunoglobulin or fragment thereof of claim 1, wherein the immunoglobulin or fragment thereof has interspecies specificity.

3. An immunoglobulin or fragment thereof according to claim 1 or 2, wherein the immunoglobulin or fragment thereof is capable of binding human, dog, hamster, mouse, rat, cow, and / or Schaaf tyrosine-23 phosphorylated annexin A2.

4. An immunoglobulin or fragment thereof according to any one of claims 1 to 3, wherein the tyrosine-23 phosphorylated annexin A2 comprises an amino acid sequence as shown in any one of SEQ ID Nos 11 to 18.

5. The immunoglobulin or fragment thereof according to claims 1 to 4, wherein the immunoglobulin or fragment thereof has a VL region as shown in SEQ ID NO. 7.

6. An immunoglobulin or fragment thereof according to claims 1 to 5, wherein the immunoglobulin or fragment thereof has a VH region as shown in SEQ ID no. 8.

The immunoglobulin or fragment thereof according to claims 1 to 6, wherein the immunoglobulin is a monoclonal antibody.

The immunoglobulin or fragment thereof according to claims 1 to 7, wherein the immunoglobulin or fragment thereof is humanized.

9. immunoglobulin or fragment thereof according to claims 1 to 8, wherein the immunoglobulin or fragment thereof is suitable for qualitative and / or quantitative detection of tyrosine-23 phosphorylated annexin A2 in vitro.

An immunoglobulin or fragment thereof according to claim 9, wherein the detection is performed by Western blot, immunohistochemistry, immunophenotyping, fluorescence activated cell analysis (FACS), enzyme immunoassay (ELISA), enzyme immunoassay (ELISPOT), radioimmunoassay, immunoprecipitation or coimmunoprecipitation becomes.

11. An immunoglobulin or fragment thereof according to any one of claims 1 to 10 for use in a diagnostic method.

12. An immunoglobulin or fragment thereof according to any one of claims 1 to 11 for use in a therapeutic method.

13. An in vitro method for detecting a tyrosine-23 phosphorylated annexin A2, wherein an immunoglobulin or fragment thereof according to any one of claims 1-10 is applied to a sample.

The method of claim 13, wherein the sample is a tissue sample, a body fluid sample or a cell culture sample.

The method of claim 14, wherein the body fluid sample is a whole blood sample, a serum sample, bronchoalveolar fluid or a synovial fluid.

A nucleic acid sequence encoding an immunoglobulin or fragment thereof as defined in any one of claims 1 to 10.

17. Nucleic acid sequence according to claim 16 comprising the sequence of SEQ ID NO: 9 and / or SEQ ID NO: 10.

A vector comprising a nucleic acid sequence as defined in claim 16 or 17.

The vector of claim 18, wherein the vector further comprises a regulatory sequence operably linked to the nucleic acid sequence as defined in claim 16 or 17.

The vector of claim 19, wherein the vector is an expression vector.

21. A host transformed or transfected with a vector as defined in any one of claims 18 to 20.

22. A method for the production of an immunoglobulin or a fragment thereof according to any one of claims 1 to 10, wherein the method comprises cultivating a host as defined in claim 21 under the conditions comprising the expression of an immunoglobulin or fragment thereof as in any one of claims 1 to 10 defines and restores the produced immunoglobulin or the fragment thereof from the breeding.

23. A method for producing an immunoglobulin or fragment thereof according to any one of claims 1 to 10, the method comprising: (a) immunizing an animal with a molecule comprising the amino acid sequence as shown in any one of SEQ ID Nos: 11 to 18, and ( b) obtaining an immunoglobulin or fragment thereof which is capable of binding to tyrosine-23 phosphorylated annexin A2.

24. An immunoglobulin or fragment thereof obtainable by the method according to claim 22 or 23.

25. A diagnostic composition comprising an immunoglobulin or a fragment thereof according to any one of claims 1 to 10 and a pharmaceutically or diagnostically acceptable excipient.

A kit comprising an immunoglobulin or fragment thereof as defined in any of claims 1 to 101, a nucleic acid molecule as defined in claim 16 or 17, a vector as defined in any one of claims 18 to 20, or a host as defined in claim 21.