MXPA98009732A - Antibodies for the fibronectin ed-b domain, its construction and u - Google Patents

Abstract

The present invention relates to an isolated antibody or antibody fragment that is specific for and binds directly to the oncofetal domain ED-B of fibronectin (FN), characterized in that it has a dissociation constant (kd) of 6 x 10-8M, for ED-B FN when measured as a purified monomer

Description

ANTIBODIES FOR THE FIBRONECTIN ED-B DOMAIN, ITS CONSTRUCTION AND USES BACKGROUND This invention relates to specific binding members for a fetal fibronectin isoform, ED-B, which is also expressed in the neovasculature in tumor development, as demonstrated both by immunocytochemistry and by tumor labeling in vivo. It also relates to materials and methods that relate to such specific binding members. The main goal of most existing forms of tumor therapy is to destroy as many tumor cell constituents as possible. The limited success that has been experienced with chemotherapy and radiotherapy is related to the relative lack of specificity of the treatment and the tendency to toxic side effects on normal tissues. One way in which the selectivity of the anti-tumor therapy can be improved is to deliver an agent for the tumor through a binding protein, which usually comprises a binding domain of an antibody, with specificity for a marker antigen expressed in the surface of the tumor, but absent from normal cells. This form of targeted therapy, freely termed as "bullets" REF: 28797 magicas ", has been exemplified mainly by monoclonal antibodies (mAbs) of rodents which are specific for what are called tumor-associated antigens that are expressed on the cell surface, such mAbs can be chemically conjugated to the cytotoxic portion ( for example a toxin or a drug) or it can be produced as a recombinant fusion protein, where the genes coding for the mAb and the toxin bind together and are expressed in a battery The focus of the "magic bullet" has had a limited effect, although significant in the treatment of human cancer, most notably in the marking of tumors of lymphoid origin, where the malignant cells have a freer access to the therapeutic dose in circulation. solid tumors, remains a serious clinical problem, where only a tiny portion of the total cell mass, predominantly the cells in the The outermost periphery of the tumor is exposed to therapeutic immunoconjugates in the circulation; these peripheral targets form what is termed a "binding site barrier" into the tumor (Juweid et al, 1992, Cancer Res. 52 5144-5153). Within the tumor, the architecture of the tissue is usually too dense, with fibrous stroma and closely packed tumor cells to allow the penetration of molecules in the range of antibody size. In addition, it is known that tumors have a high interstitial pressure due to the lack of lymphatic drainage, which also prevents the inflow of exogenous molecules. For a recent review of the factors that affect the uptake of therapeutic agents within tumors see Jain, R (1994), Sci, Am. 271 58-65. Although there are obvious limitations to the treatment of solid tumors through the labeling of antigens associated with tumors, these tumors have a common feature which provides an alternative antigenic target for antibody therapy. Once they have grown beyond a certain size, the tumors are universally dependent on an independent blood supply to adapt the oxygen and nutrients to sustain growth. If this blood supply can be interfered with or if it can be oppressed, there is a realistic potential to kill thousands of tumor cells in the process due to lack of supply. As the tumor develops, it undergoes a change to an angiogenic phenotype, which produces a diverse array of angiogenic factors which act on neighboring capillary endothelial cells, inducing them to proliferate and migrate. The structure of these newly formed blood vessels is highly disorganized, with blind terminations and fenestrations that lead to increased leakage, in stark contrast to the ordered structure of the capillaries in normal tissue. Induction of angiogenesis is accompanied by ascending regulation of the expression of certain cell surface antigens, many of which are common to the vasculature of normal tissues. The identification of antigens which are unique to the neovasculature of tumors has been the main limiting factor in the development of a generic treatment for solid tumors through vascular labeling. The antigen which is the object of the present invention solves this problem directly. During tumor progression, the extracellular matrix of the surrounding tissue is remodeled through two main processes: (1) proteolytic degradation of extracellular matrix components of normal tissue, and (2) de novo synthesis of extracellular matrix components both by tumor cells as by stromal cells activated by tumor-induced cytosines. These two processes, in a stable state, generate a "tumor extracellular matrix", which provides a more adequate environment for the progress of the tumor and its qualitative and quantitative differentiation in comparison with normal tissues. Among the components of this matrix are the large isoforms of tenacin and fibronectin (FN); the description of these proteins as isoforms recognizes their extensive structural heterogeneity which is carried out at the transcriptional, post-transcriptional and post-translational level (see below). It is one of the isoforms of fibronectin, the so-called B + isoform (B-FN), which is the subject of the present invention. Fibronectins (FN) are high molecular weight, multifunctional glycoproteins, constituents of both the extracellular matrix and body fluids. They are involved in many different biological processes such as the establishment and maintenance of normal cell morphology, cell migration, hemostasis and thrombosis, wound healing and oncogenic transformation (for reviews see Alitalo et al., 1982; Yamada, 1983 Hynes, 1985; Ruoslahti et al., 1988; Hynes, 1990; Owens et al., 1986). The structural diversity in the FNs is carried out around an alternative splice of three regions (ED-A, ED-B and IIICS) of the primary FN transcript (Hynes, 1985; Zardi et al., 1987) to generate at minus 20 different isoforms, some of which are differentially expressed in tumors and tumor tissue. In the same way that it is regulated in a specific manner in tissue and in development, it is known that the splicing pattern of FN-pre-mRNA is deregulated in transformed cells and in malignant cancers (Castellani et al., 1986; Borsi et al. al, 1987, Vartio et al., 1987, Zardi et al, 1987, Barone et al, 1989, 'Carnemolla et al, 1989, Oyama et al, 1989, 1990, Borsi et al, 1992b). In fact, the FN isoforms containing the ED-A, ED-B and IIICS sequences are expressed to a greater degree in transformed cells and malignant tumors compared to normal cells. In particular, the isoform containing the sequence ED-B (isoform B +), is highly expressed in fetal and tumor tissue as well as during wound healing, but its expression is restricted in normal adult tissue (Norton et al, 1987; Schwazbauer et al, 1987, Gutman and Kornblihtt, 1987, Carnemolla et al, 1989, ffrench-Constant et al, 1989, ffrench-Constant and Hynes, 1989, Laitinen et al, 1991.) B + FN molecules are undetectable in mature vessels, but they are upregulated in angiogenic blood vessels in normal development (for example in the development of the endometrium), pathological (for example in diabetic retinopathy) and in the development of tumors (Castellani et al, 1994). The ED-B sequence is a repeated complete type III homology encoded by a single exon and comprising 91 amino acids. In contrast to the alternative spliced IIICS isoform, which contains a specific binding site for the cell type, the biological function of the A + and B + isoforms is still a research topic (Humphries et al, 1986). The presence of the B + isoform itself constitutes a neoantigen induced by tumor, but, furthermore, the expression of ED-B exposes a normally critical antigen within the repeat section 7 type III (which precedes ED-B); since this epitope is not exposed to FN molecules that lack ED-B, it follows that the expression of ED-B induces the expression of neoantigens both directly and indirectly. This critical antigenic site forms the target of monoclonal antibody (mAb) designated BC-1 (Carnemolla et al, 1992). The specificity and biological properties of this mAb have been described in EP 0 344 134 Bl and can be obtained from the hybridoma deposited in the European Collection of Animal Cell Cultures, Porton Down, Salisbury, United Kingdom, under number 88042101. The mAb has been used successfully to localize angiogenic blood vessels of tumors without cross-reactivity to mature vascular endothelium, illustrating the potential of FN isoforms for vascular labeling using antibodies. However, there are certain caveats to the specificity of mAb BC-1. The fact that BC-1 does not directly recognize the B + isoform has raised the question of whether in some tissues, the epitope recognized by BC-1 can be unmasked without the presence of ED-B and therefore indirectly lead to undesired cross-reactivity of BC-1. In addition, BC-1 is strictly specific for the human B + isoform, which means that animal studies on the biodistribution and localization of BC-1 tumors are not possible. Although polyclonal antibodies have been produced for recombinant fusion proteins containing the B + isoform (Peters et al, 1995), they are only reactive with FN which has been treated with N-glycanase to unmask the epitope. A further general problem with the use of mouse monoclonal antibodies is the human response against mouse antibodies (HAMA) (Schroff et al, 1985; Dejager et al, 1988). HAMA responses have a variety of effect, from neutralization of the administered antibody leading to a reduced therapeutic dose, to allergic responses, such as malaise and kidney damage. Although polyclonal antisera reactive with recombinant ED-B have been identified (see above), the isolation of mAbs with the same specificity as BC-1 subsequent to immunization of mice has generally proved difficult because of human and mouse ED-proteins. B are virtually 100% homologous in sequence. Therefore, the human protein has an appearance similar to an autoantigen for the mouse which then does not mount an immune resistance to it. In fact, in more than 10 years of intense research in this field, only one mAb with indirect reactivity to the FN B + isoform (BC-1) has been identified, and there is none that directly recognizes ED-B. It is almost as significant that the specificity BC-1 is for a steric epitope exposed as a sonsequence of ED-B, instead of being part of ED-B itself, which is likely to be absent from mouse FN and therefore both are not considered as "proper" by the mouse immune system. The embodiment of the present invention has been carried out using an alternative strategy to that previously used and where prior immunization with fibronectin or ED-B is not required: antibodies with specificity for the ED-B isoform have been obtained as Fvs of unique chain (scFvs) from libraries. of human antibody variable regions displayed on the surface of filamentous bacteriophages (Nissim et al., 1994; see also O92 / 01047, O92 / 20791, WO93 / 06213, 093/11236, W093 / 19172). We have found that by using an antibody phage library that is specific, scFvs can be isolated either by direct selection in recombinant FN fragments containing the ED-B domain or by recombinant ED-B itself when these antigens are coated on a surface solid ("panning technique"). These same antigen sources have also been used successfully to produce "second generation" scFvs with improved properties in relation to the original clones in a process of "affinity maturation". We have found that isolated scFvs react strongly and specifically with the B + isoform of human NF without pretreatment with N-glycanase. In antitumor applications, the antigen binding domains of the human antibody provided by the present invention have the advantage of not being subject to the HAMA response. Furthermore, as exemplified herein, they are useful in immunohistochemical analysis of tumor tissue, both in vi tro and in vivo. These and other uses are discussed further herein and are apparent to a person usually familiar with the art.

TERMINOLOGY Specific union member This describes a member of a pair of molecules which have binding specificity to each other. The members of a specific binding pair can be derived naturally or can be synthetically produced totally or partially. A member of the pair of molecules has an area on its surface, or a cavity, which specifically binds and therefore is complementary to a particular spatial and polar organization of the other member of the pair of molecules. Therefore, the members of the pair have the property of specifically joining together. Examples of specific binding pair types are antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor of a ligand, enzyme-substrate.

Antibody This describes an immunoglobulin, either natural or partially or completely synthetically produced. The term also encompasses any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. This can be derived from natural sources, or it can be synthetically produced partially or completely. Examples of antibodies are the immunoglobulin isotypes and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies (diabodies). It is possible to take monoclonal antibodies and other antibodies and use recombinant DNA technology techniques to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the variable region of the immunoglobulin or regions of complementarity determination (CDR), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for example, EP-A-184187, GB 2188638A 'or EP-A-239400. A hybridoma or other cell that produces an antibody can be subjected to genetic mutation or other changes, which may or may not alter the binding specificity of the antibodies produced. Since the antibodies can be modified in numerous ways, the term "antibody" should be considered to encompass any specific binding member or substrate having a binding domain with the required specificity. Therefore, this term encompasses antibody fragments, derivatives, functional equivalents and antibody homologs, which include any polypeptide consisting of an immunoglobulin binding domain., either natural or synthetic, completely or partially. Therefore, chimeric molecules comprised of an equivalent immunoglobulin binding domain, fused to another polypeptide, are included. The cloning and expression of chimeric antibodies is described in EP-A-0120694 and EP-A-0125023. It has been shown that fragments of a complete antibody can perform the function of binding antigens. Examples of binding fragments are: (i) the Fab fragment consisting of the domains of VL, VH, CL and CH1; (ii) the Fd fragment consisting of the VH and CH1 domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment (Ward et al., 1989) which consists of a VH domain; (v) isolated CDR regions; (vi) F (ab ') 2 fragments, a bivalent fragment comprising the bound Fab fragments; (vii) single chain Fv molecules (scFv), where a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site (Bird et al, 1988; Huston et al, 1988) (viii) bispecific single chain Fv dimers (PCT / US92 / 09965) and (ix) "diabodies", multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804; Holliger et al, 1993). The diasubodies are multimers or polypeptides, each polypeptide comprises a first domain that is constituted by a binding region of an immunoglobulin light chain and a second domain consisting of a binding region of an immunoglobulin heavy chain, the two domains are joined ( for example by a peptide linker) but are unable to associate with each other to form an antigen binding site: the antigen binding sites are formed by the association of the first domain of a polypeptide within the multimer, with the second domain of the other polypeptide within the multimer (WO94 / 13804). When bispecific antibodies are to be used, these can be conventional bispecific antibodies, which can be manufactured in many ways (Holliger and Winter, 1993), for example they can be prepared chemically or from hybrid hybridomas, or can be from any of the bispecific fragments mentioned above. It may be preferable to use scFv dimers or diabodies instead of whole antibodies. Diabodies and scFv can be constructed without an Fc region, using only variable domains, potentially reducing the anti-idiotypic reaction effects. Other forms of bispecific antibodies include the single-chain "Janusins" ("Janusins") described in Traunec er et al, (1991). Bispecific diabodies, as opposed to complete biospecific antibodies, may also be particularly useful because they can be easily constructed and expressed in E. coli. Diabodies (and many other polypeptides such as antibody fragments) of appropriate binding specificities can be easily selected using the phage display (WO94 / 13804) from libraries. If one arm of the diabody should be kept constant, for example, with a specificity directed against the X antigen, then the library can be manufactured where the other arm is varied and an antibody of appropriate specificity is selected.

Antigen binding domain This describes the part of an antibody which comprises the area which binds specifically to, and is complementary to, part or all of an antigen. When an antigen is large, an antibody can only bind to a defined part of the antigen, part of which is called an epitope. An antigen binding domain can be provided by one or more variable antibody domains. Preferably, an antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

Specific This refers to the situation in which a member of a specific binding pair shows no significant binding to molecules other than its specific binding partner. The term is also applicable when, for example, an antigen binding domain is specific for a particular epitope which is transported by numerous antigens, in which case the specific binding member presenting the antigen binding domain will be able to bind to the various antigens that present the epitope.

Functionally equivalent variant form This refers to a molecule (the variant) which, although having different structurals with another molecule (the original) retains some important homology and also at least part of the biological function of the original molecule, for example, the ability to bind to a particular antigen or epitope. The variants may be in the form of fragments, derivatives or mutants. A variant, derivative or mutant can be obtained by modification of the original molecule by the addition, deletion, substitution or insertion of one or more amino acids, or by binding to another molecule. These changes can be made at the nucleotide or at the protein level. For example, the encoded polypeptide can be a Fab fragment which then binds to an Fc tail from another source. Alternatively, a label such as an enzyme, fluorescein, etc. can be attached.

Brief description of the present invention In accordance with the present invention, a specific binding member is provided which is specific for the oncofetal ED-B domain of fibronectin (FN). The specific binding members according to the invention are linked to the ED-B domain directly. In one embodiment, a specific binding member is attached, after treatment of FN with the thermolysin protease to one or all of the FN containing ED-B. In a further embodiment, a specific binding member binds to any or all of FN containing repeated sections of type III which include domain ED-B. The known FNs are identified in two documents by Carnemolla et al., 1989; 1992). The reference to "all FNs containing ED-B" can be considered as a reference to all the FNs identified in these documents that contain ED-B. Preferably, the specific binding member binds to human ED-B and preferably B + FN of at least one additional species, such as mouse, rat and / or chicken. Preferably, the specific binding pair member is capable of binding to both ED-B of human fibronectin and ED-B of non-human fibronectin, such as that of mouse, which allows testing and analysis of the ED-B member in an animal model The specific binding pair members according to the present invention bind to ED-B fibronectin without competing with the deposited BC-1 antibody, publicly available, discussed elsewhere in this document. BC-1 is strictly specific for the human B + isoform. The specific binding pair members according to the present invention do not bind to the same epitope as BC-1. The binding of a specific binding member according to the present invention to B + FN can be inhibited by the ED-B domain. In one aspect of the present invention, the binding domain has, when measured as a purified monomer, the dissociation constant (Kd) of 6 x 10"8 M or less for ED-B FN.

In one aspect of the present invention, the binding domain is reactive with, ie, capable of binding to fibronectin ED-B without the ED-B treatment of fibronectin with N-glycanase. Specific binding pair members according to the present invention may be provided as isolated or in purified form, ie, in a preparation or free formulation of other specific binding pair members, eg, antibodies or antibody fragments, or free of other specific binding pair members amenable to binding to ED-B fibronectin. Preferably, the specific binding members according to the present invention are provided in substantially pure form. They can be "monoclonal" in the sense of coming from a single clone, instead of being restricted to antibodies obtained using traditional hybridoma technology. As discussed, specific binding pair members according to the present invention can be obtained using bacteriophage display technology and / or expression in recombinant, eg, bacterial host cells. There is no prior description of a specific monoclonal binding pair member which binds directly to ED-B fibronectin. Preferably, the specific binding member comprises an antibody. The specific binding member may comprise a polypeptide sequence in the form of an antibody fragment such as a single Fv chain (scFv). Other types of antibody fragments such as Fab, Fab ', F (ab') 2, Fabc, Facb or a diabody can also be used (Winter and Milstein, 1991; W094 / 13804). The specific binding member may be in the form of a complete antibody. The entire antibody can be in any of the forms of the antibody isotypes, for example, IgG, IgA, IgD, IgE and IgM and any of the forms of the isotype subclasses, for example, IgGl or IgG4. The antibody can be of any origin, for example, human, mouse, sheep or lamb. Other derivations will be clear to those familiar with the art. Preferably, the antibody is of human origin. By "human" is meant an antibody that has been partially or completely derived from a human cDNA, protein or peptide library. The term includes humanized peptides and proteins of non-human origin that have been modified in order to impart human characteristics to the antibody molecule and thereby allow the molecule to surpass the defenses of the human immune system. The specific binding member may also be in the form of a genetically engineered antibody, e.g., a bispecific antibody molecule (or a fragment such as F (ab ') 2) which has an antigen-binding arm (is say, a specific domain) against ED-B fibronectin as described, and another arm against a different specificity, or a bivalent or multivalent molecule. In addition to the antibody sequences, the specific binding member may comprise other amino acids, for example, which form a peptide or polypeptide, or to impart to the molecule another functional feature in addition to the ability to bind antigen. For example, the specific binding member may comprise a tag, an enzyme or a fragment thereof, and so on. The binding domain may comprise part or all of a VH domain encoded by a germline segment or a rearranged gene segment. The binding domain may comprise part or all of the kappa domain VL or a domain the bda VL. The binding domain may comprise a germline gene sequence VH1, VH3 or VH4, or a rearranged form thereof. A specific binding member according to the present invention may comprise a heavy chain variable region ("VH" domain) derived from the human germ line DP47, the sequence of which is shown in Figure 1 (a), residues 1 to 98. The nomenclature "DP" is described in Tomlinson et al, (1992). The amino acid sequence of CDR3 can be Ser Leu Pro Lys. The amino acid sequence of CDR3 can be Gly Val Gly Ala Phe Arg Pro Tyr Arg Lys His Glu. Therefore, the binding domain of a specific binding member according to the present invention can include a VH domain comprising the amino acid sequences shown in Figure Ka) for CGS-1 and CGS-2. The binding domain may comprise a light chain variable region ("VL" domain) derived from the human germline DPL16, the sequence of which is shown in Figure 1 (b) as codons 1-90. The VL domain may comprise a CDR3 Asn Ser Ser Pro Val Val Leu Asn Gly Val Val sequence. The VL domain may comprise a CDR 3 Asn Ser Ser Pro Phe Glu His Asn Leu Val Val sequence. The specific binding members of the invention may comprise functionally equivalent variants of the sequences shown in Figure 1, for example, one or more amino acids have been inserted, deleted, substituted or added, with the proviso that a property such as establishes in the present. For example, the CDR3 sequence can be altered, or one or more changes can be made to the framework regions, or the framework region can be replaced with another framework region or a modified form, with the proviso that the specific union binds to ED-B.

One or more CDRs can be used from the VL or VH domain of an antigen binding domain or an antibody described herein in what is referred to as a "CDR graft" in which one or more CDR sequences of a first antibody they are placed within a framework of non-antibody sequences, for example, as described in EP-B-0239400. The CDR sequences for CGS-1 and CGS-2 are shown in Figure 1 (a) and Kb). A specific binding member according to the invention may be one which competes with an antibody or scFv described herein for its binding to ED-B fibronectin. Competition among union members can easily be determined in vi tro, for example by marking a specific indicator molecule to a union member which can be detected in the presence of another unmarked member or union members, to allow the identification of members. of specific binding which bind to the same epitope or to an overlapping epitope. A specific binding member according to the present invention can be used in a method comprising causing or allowing the binding of the specific binding member to its epitope. The binding can be subsequent to the administration of the binding member of specific to a mammal, for example, a human or rodent such as a mouse.

The present invention provides the use of a specific binding member such as the above for use as a diagnostic reagent for tumors. The experimental evidence in animal models described below shows that the binding members according to the present invention are useful for in vivo tumor localization. Preferred specific binding members according to the present invention include those which bind to human tumors, for example, in a section cut by cryostat, which shows an invasive and angiogenic phenotype, and those which bind to embryonic tissues. , for example in a section cut with a cryostat. The binding can be demonstrated by immunocytochemical staining. In a preferred embodiment, the specific binding member does not bind, or does not bind significantly to tenacin, an extracellular matrix protein. In another preferred embodiment, the specific binding member does not bind, or does not bind significantly to normal human skin, for example, in a cryostat section and / or as demonstrated using immunocytochemical staining. Additional embodiments of the specific binding members according to the present invention do not bind, or do not bind significantly to one or more normal tissues (eg, in cryostat section and / or as demonstrated using immunocytochemical staining) which are selected from liver, vessel, kidney, stomach, small intestine, large intestine, ovaries, uterus, bladder, pancreas, suprarenal glands, skeletal muscle, heart, lung, thyroid and brain. A specific binding member for ED-B can be used as an in vivo marker agent which can be used to specifically demonstrate the presence and localization of tumors expressing or associated with fibronectin ED-B. It can be used as an image forming agent. The present invention provides a method for determining the presence of a cell or tumor that expresses or is associated with fibronectin ED-B expression, the method comprising contacting cells with a specific binding member as provided and determining the binding of the specific binding member to the cells. The method can be carried out in vivo or in vi tro in a test sample of cells removed from the body. The reactivities of antibodies in a sample of cells can be determined by any appropriate means. One possibility is the marking with individual indicator molecules. Indicator molecules can generate, directly or indirectly, detectable and, preferably, measurable signals. The binding of indicator molecules can be covalently, directly or indirectly, for example, via a peptide bond, or non-covalently. The binding via a peptide bond can result from the recombinant expression of a fusion gene encoding the antibody and an indicator molecule. A favored mode is by the covalent attachment of each antibody with an individual fluorochrome, a phosphor dye or laser with spectrally isolated absorption or emission characteristics. Suitable fluorochromes include fluoroscein, rhodamine, chondroitrin, and Texas red. Suitable chromogenic dyes include diaminobenzidine. Other indicators include macromolecular colloidal particles or particulate material such as latex sphere having color, magnetic or paramagnetic agents, and biologically or chemically active particles that may directly or indirectly cause detectable signals to be observed visually, electronically detected or otherwise recorded. . These molecules can be enzymes which catalyze reactions that develop or change colors or that cause changes in electrical properties, for example. They can be molecularly excitable such as the electronic transitions between energy states that result in characteristic spectral emissions or absorption. They can include chemical entities used together with biosensors. Biotin / avidin or biotin / streptavidin and alkaline phosphatase detection systems may be used.

The way to determine the binding is not a feature of the present invention and those familiar with the art are able to choose an appropriate mode according to their preference and general knowledge. The signals generated by individual antibody-reporter conjugates can be indicated to derive quantifiable absolute or relative data of the relevant antibody binding in cell samples (normal and test). In addition, a general nuclear stain such as propidium iodide can be used to enumerate the total cell population in a sample, which allows to provide quantitative ratios of individual cell populations in relation to total cells. When radionucleotides such as 12SI, 1: L1In or 99mTc bind to an antibody, if this antibody is located preferentially in a tumor rather than in normal tissues, the presence of a radiolabel in the tumor tissue can be detected and quantified using a gamma camera. . The quality of the obtained tumor image correlates directly with the signal: noise ratio. Antibodies can be used as diagnostic agents to map newly vascularized tumors and can also be used, for example, in modified form, to deliver cytotoxic agents or to activate coagulation within new vessels, and thus kill for lack of supply the developing tumor suppressing oxygen and nutrients and constituting an indirect form of therapy against tumors. The present invention also provides the use of a specific binding member as in the foregoing for use as a therapeutic reagent, for example, when it is coupled, joined or genetically engineered as a fusion protein to possess an effector function. A specific binding member according to the present invention can be used to label a toxin, radioactivity, T cells, killer cells or other molecules to a tumor that expresses or is associated with the antigen of interest. Accordingly, further aspects of the invention provide methods of treatment comprising the administration of a specific binding member as provided, pharmaceutical compositions comprising such a specific binding member, and the use of such a specific binding member in the manufacture of a medicament for administration, for example in a method for making a medicament or pharmaceutical composition comprising formulating the specific binding member with a pharmaceutically acceptable excipient. In accordance with the present invention, the provided compositions can be administered to individuals. Preferably, the administration is in a "therapeutically effective amount", this is sufficient to show benefit to the patient. Such a benefit can be at least a decrease in at least one symptom. The actual amount administered and the regimen and course at the time of administration will depend on the nature and severity of the disease being treated. The prescription of treatment, for example decisions regarding dosage, etc., is within the responsibility of general practitioners and other doctors of medicine. Appropriate doses of antibody are well known in the art; see Ledermann et al., (1991); Bagshawe K. D. et al. (1991). The composition can be administered alone or in combination with other treatments, either simultaneously or sequentially based on the condition being treated. The pharmaceutical compositions according to the present invention, and for use according to the present invention may comprise in addition to the active ingredient, a carrier, carrier, buffer, stabilizer or other pharmaceutically acceptable materials well known to those familiar with the art. Such materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The precise nature of the carrier or other carrier will depend on the route of administration, which may be oral, or by injection, for example intravenously.

The pharmaceutical compositions for oral administration may be in the form of a tablet, capsule, powder or liquid. A tablet can be constituted of a solid carrier such as gelatin or an adjuvant. The liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline, dextrose or other solution of saccharides or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. For intravenous injection, or injection at the absorption site, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is free of pyrogens and has adequate pH, isotonicity and stability. Those familiar with the pertinent technique are able to prepare suitable solutions using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. They may be included as required preservatives, stabilizers, buffers, antioxidants and / or other additives. A specific binding member according to the present invention can be made by the expression of coding nucleic acid. The nucleic acid encoding any specific binding member as provided by itself forms an aspect of the present invention, as well as a method of producing the specific binding member, which method comprises the expression of the coding nucleic acid therefor. . Expression can be conveniently carried out by culturing under appropriate conditions appropriate recombinant host cells containing the nucleic acid. The nucleic acid can encode any of the amino acid sequences of the antibody antigen binding domains described herein or any functionally equivalent form. Changes can be made at the nucleotide level by addition, substitution, deletion or insertion of one or more nucleotides, changes which may or may not be reflected at the amino acid level, based on the degeneracy of the genetic code. Systems for cloning and expressing a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells and many others. A common, preferred bacterial host is E. coli. The expression of antibodies and antibody fragments in prokaryotic cells such as E. coli is well established in the art. For a review, see, for example, Plückthun, (1991). Expression in eukaryotic cells in culture is also available to those familiar in the art as an option for the production of a specific binding member, see for recent reviews, for example, Reff, (1993); Trill et al. (nineteen ninety five). Suitable vectors can be chosen or constructed to contain the appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, extender sequences, marker genes and other sequences as appropriate. The vectors can be plasmids, viral for example, phage or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for nucleic acid manipulation, for example in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis, are described in detail in Short Protocols in Molecular Biology, second edition, Ausubel et al. eds. , John Wiley & Sons, 1992. The descriptions of Sambrook et al. and Ausubel et al. they are incorporated herein by reference.

Therefore, a further aspect of the present invention provides a host cell containing nucleic acid as described herein. A further aspect provides a method comprising introducing such nucleic acid into a host cell. The introduction can use any available technique. For eukaryotic cells, the adesuted techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection and transduction using retroviruses or other viruses, for example, vaccinia or for insect cells, baculovirus. For bacterial cells, suitable techniques can include transformation into calcium chloride, electroporation and transfection using bacteriophage. Introduction can be followed by causing or allowing expression of the nucleic acid, for example, by culturing host cells under conditions for gene expression. In one embodiment, the nucleic acid of the invention is integrated into the genome (eg, chromosome) of the host cell. The integration can be promoted by inclusion of sequences which promote recombination with the genome, according to standard techniques. The continuous production of a specific binding member can be used, for example, in any of the manners described herein, for example in the formulation of a pharmaceutical or diagnostic product, such as a computer comprising, in addition to the member of specific binding, one or more reagents to determine the binding of the member to the cells, as described. Additional aspects of the invention and modalities will be apparent to those familiar with the art. In order that the present invention be fully understood, the following examples are provided by way of exemplification only and not in a limiting manner. Reference is made to the following figures: Figure 1 shows aligned amino acid sequences of VH and VL of scFvs CGS-1 and CGS-2. Figure Ka) shows the VH sequences; Figure 1 (b) shows the VL sequence. The CDRs (1, 2 and 3) are indicated. Most of the VH homologous human germline for both scFvs is the DP47 segment of the VH3 family; The VL segment of both clones is DPL16, the light chain used to construct the original scFv library (Nissim et al, 1994). The residues that differentiate the two clones from each other are underlined. Figure 2: Figure 2A shows a model of the domain structure of the human FN subunit. The IIICS, ED-A and ED-B regions of variability are indicated, due to the alternative alignment of pre-FN mRNA. The figure also indicates internal homologies as well as the main thermolysin digestion products contained in ED-B (Zardi et al, 1987). Figure 2B shows the SDS-PAGE test of 4-18% plasma and WI38VA FN and its thermolysin digests stained with Coomassie blue and immunoblots probed with BC-1, IST-6, CGS-1 and CGS-2. Undigested plasma FN (lane 1) and digested in plasma FN using thermolysin at 1 μg / mg FN (lane 3) and 10 μg / mg FN (lane 4). The undigested WI38VA FN (lane 2) and digested using thermolysin at 1 μg / mg (lane 5), 5 μg / mg (lane 6) and 10 μg / mg (lane 7) of FN. The numbers on the right side indicate the main thermolysin digestion products shown in Figure 2A. The values to the left indicate the molecular weight markers in kilodalton units (kD). Figure 3: Figure 3A shows the repeated sequences of FN type III contained in the fusion and recombinant proteins expressed in E. coli, and the reactivity of these proteins with CGS-1 and CGS-2 and with the mAb BC-1 and IST-6. Figure 3B shows the gel stained with Coomassie blue and throughout the immunoblots probed with CGS-1, CGS-2, BC-1, IST-6. The numbering of the lanes corresponds to that of the peptide constructs in the upper part of the figure. The values to the left indicate the molecular weight markers in kD. Figure 4: Infrared mouse imager; The mouse imager used for marking experiments consists of a black non-fluorescent box equipped with a halogen and tungsten lamp with specific excitation and emission filters for the CY7 infrared fluorophore and a computer-controlled 8-bit monochromatic CCD camera. Figure 5: labeling of fluorescently labeled antibody fragments for mouse F5 teratocarcin using monomeric scFv (CGS-1) and scFv (CGS-1) 2 dimer targeting for B-FN. The dimeric scFv (DI.3) 2 with an isozyme-binding specificity was used as a negative control. Figure 6: Objective establishment of fluorescently labeled antibody fragments for mouse F9 teratocarcinoma using the affinity of mature scFv (CGS-2) and the lower affinity of scFv (28SI) directed to the same B-FN epitope. Target establishment is detected in both large tumors (approximately 0.6 grams), covered at 48 hours with a black scab that partially obscures the image, as well as small tumors (approximately 0.2 grams). All documents mentioned herein are incorporated by reference.

List of examples Example 1 - Isolation of human scFvs specific for the ED-B domain of human FN.

Example 2 - Affinity maturation of human scFvs specific for the ED-B domain of human FN. Example 3 - Affinity specificity of matured scFvs for fibronectins containing ED-B. Example 4 - Use of scFvs against ED-B matured for affinity by immunocytochemical staining of human and mouse tumor sections. Example 5 - Use of scFvs against ED-B, matured by affinity in the establishment as an in vivo target of human tumors. Example 6 - Target establishment of mouse F9 teratocarsinoma xenografted in athymic mice.

Example 1 - Isolation of human scFvs specific for the ED-B domain of human FN.

The human scFv phage library (Nissim et al, 1994) was used for the selection of recombinant antibodies. Two different forms of ED-B isoform were used as a source of antigen for selection and, in both cases, the isoform was recombinant human protein. Recombinant FN peptides containing repeat sections type III 2-11 (B-) and 2-11 (B +) are expressed in Es cheri chi a coli.

A construsus is produced using F? S DNA? of clones pFH154 (Kornblihtt et al 1985),? F10 and? F2 (Carnemolla et al, 1989). The constructs of AD? C, which covers bases 2229-4787, (Kornblihtt et al, 1985) are inserted into vector pQE-3/5 using the QIAexpress equipment from Qiagen (Chatsworth, CA). Recombinant F? -III 2-11 (B-) and (B +) are purified by immunoaffinity chromatography using mAb 3E3 conjugated with Sepharose 4B (Pharmacia). Fragments of AD? for the preparation of F fragments? recombinants containing the repeated sections of type III homology 7B89, 789, ED-B and F? -6 produced by amplification of polymerase chain reaction (PCR) using AD? UltMA polymerase (Perkin Elmer), using AD? c of F clones? 2-11 (B +) and F? 2-11 (B-) as a template. The primers are designed to allow the cloning of PCR products into pQE-12 using the QIAexpress (Quiagen) equipment. Subsequently they are transformed into E. coli and are expressed. All AD? C clones were sequenced using the sequencing kit Sequenase 2.0 AD? (USB). The recombinant proteins were purified by Ni-? TA chromatography (IMAC) according to the manufacturer's instructions (Qiagen), using the hexahistidine tag in the carboxy terminal part of the F? Fragments. The ED-B-3Gal fusion protein is prepared by cloning cDNA for ED-B into the bacteriophage vector gtll to provide the clone? ED-B. The clone? ChF? 60 (which contains part of the ED-B sequence) was derived as a pchFN.sub.SO protein from cloned chicken FN (Norton et al., 1987). For the selection of the human scFv phage library, three rounds of panning technique were performed for each of the two different recombinant antigens (7B89 and ED-B). The antigens were coated on immunotubes (Nunc; Maxisorp, Roskilde, Denmark) overnight at 50 μg / ml in PBS (20 mM phosphate buffer, NaCl 0. 15 M, pH 7.2). The first antigen was the 7B89 fragment of recombinant FN, in which the ED-B domain is flanked by the repeat sections with homology to adjacent N type III; this was coated at 4 ° C during the night. The second antigen used was recombinant ED-B (Zardi et al, 1987) with a hexahistidine label in the carboxy terminal part; this protein does not contain lysine residues, so that the amino terminal group of the first amino acid is available for covalent immobilization specific to the ED-B site for the ELISA reaction plates (Nunc; Covalink). The coating was carried out overnight at room temperature. After three rounds of panning technique, the eluted phage is infected in E. coli HB2151 cells and plated as described (Nissim et al., 1994). After each round of screening, 95 single ampicillin-resistant colonies are analyzed to identify antigen-specific scFvs by ELISA. The clones which provide the highest ELISA signals on the antigens used for the panning technique are selected for further analysis and for affinity maturation. These clones also demonstrate that specific staining of sections of glioblastoma multiforme and breast tumors is provided by immunocytochemical staining, described in more detail in Example 4.

Example 2 - Affinity maturation of human scFvs specific for the ED-B domain of human FN. 35GE clones (from the selection with 7B89) and 28SI (from the selection with the ED-B domain alone) were selected as candidate antibodies for affinity maturation. In order to diversify the light chains as a means to improve affinity, we then explored the affinity maturation strategy if already based on the randomization of the six sentral residues (DSSGNH) of the CDR3 light chain using degenerate oligonucleotides and PCR (figure 1), which provides a potential diversity of the sequence of 206 = ß 4 x 107. This region (together with the heavy chain CDR3) is located at the antigen binding site (Padlan, 1994). We also mutated the arginine residue that directly precedes the chain of six residues to serine, in order to avoid the possibility of electrostatic effects that dominate the selection. The plasmid from a single bacterial colony expressing the "original" scFv fragment was amplified by PCR with LMB3 primers (5 'CAG GAA ACA GCT ATG AC 3') and CDR3-6-VL-FOR (5 'CTT GGT CCC TCC GCC GAA TAC CAC MNN MNN MNN MNN Mn N AGA GGA GTT ACA GTA ATA GTC AGC CTC 3 ') (94C [1'] - 55C [1 '] - 72C [1'30"3/25 cislos; see Marks et al., 1991, for buffers and conditions.) The resulting product was purified by gel (in order to remove traces of the plasmid containing the original scFv gene) and used as a template for a second amplification step with LMB3 primers. and Jl-? ot-FOR (5 'ATT GCT TTT CCT TTT TGC GGC CGC GCC TAG GAC GGT CAG CTT GGT CCC TCC GCC 3') (94C [1 '] - 55C [1'] - 72C [1'30" ], 25 cycles). The untreated PCR product, which runs as a single band of correct molecular weight on agarose gel, is purified directly from the PCR mixture using Spin-Bind (FMC, Rockland, ME, USA), subjected to double digestion with? col /? otl and gel-bound with phagemid pHE? l digested with Ncol /? otl (Hoogenboom et al., 1991) containing a false insert? col /? otl to facilitate vector separation with double digestion of the vector with a single digestion. The vector is prepared with a maximum plasmid preparation kit Qiagen (Chatsworth, CA, USA) approximately 5 μg of the digested plasmid and the insert were used in the ligation mixture, which is extracted once with phenol, once with phenol / chloroform / isoamyl alcohol (25: 25: 1), and then precipitated with ethanol using glycogen (Boehringer, Mannheim, Germany) as a carrier, and dried under vacuum at high speed. The pellet or pellet was resuspended in 20 μl of water and subjected to electroporation in electrocomposite E. coli TGl cells (Gibson, 1984). Typically we use electrocompetent cells with a titer of 109 transformants / μg if glycerol concentrates are used, or 1010 transformants / μg with freshly prepared electrocompetent cells. This typically provides > 107 clones with the procedure indicated here. The maturation library is then processed as the Nissim library (Nissim et al., 1994) to produce phage particles, which were used for a round of selection in immunotubes using 7B89 (10 μg / ml) as antigen, followed by a round of kinetic selection (Hawkins et al., 1992). This selection step was performed by incubating biotinylated 7B89 (10 nM) with the phage suspension (approximately 1012 tu) in PBS with 2% milk (2% MPBS) from the first round of selection for 5 minutes, and then adding 7B89 not biotinylated (1 μM) and letting the competition proceed for 30 minutes. Subsequently, 100 μl of streptavidin-coated dinaspheres (Dynal: M480) pre-blocked in 2% MPBS were added to the reaction mixture, mixed for 2 minutes and then captured in a magnet and washed 10 times with alternating washings of (PBS). + 0.1% Tween-20) and PBS. The phage was eluted from the spheres with 0.5 ml of 100 mM triethylamine. This solution was subsequently neutralized, with 0.25 ml of 1 M Tris, pH 7.4 and was used to infect HB2151 cells that grow exponentially (Nissim et al., 1994). 95 single colonies resistant to ampicillin were used to produce supernatants containing scFv, which were analyzed by immunohistochemical ELISA and via the BIAcore nucleus to identify the best binders. They were then subcloned between the Sfil / Notl sites of the expression vector pDN268 (Neri et al., 1996), which adds a phosphorylatable tag, the FLAG epitope and a hexahistidine tag at the C-terminus of the scFv. Colonies alone in the relevant antibodies subcloned in pDN268 are grown at 37 ° C in 2 x TY containing 10 mg / l of ampicillin and 0.1% glucose. When the cell culture reached an OD600 = 0.8, IPTG was added to a final consentration of 1 mM and the growth continued for 16-20 h at 30 ° C. After centrifugation (Sorvall GS-3 rotor, 7000 rpm, 30 minutes), the supernatant was filtered, consented and subjected to charge buffer exchange (50 mM phosphate, pH 7.4, 500 mM NaCl, 20 mM imidazole) using a Minisette tangential flow device (Filtron).

The resulting solution is loaded in 1 ml of Ni-NTA resin (Qiagen), washed with 50 ml of loading buffer and eluted with elution buffer (50 mM phosphate, pH 7.4, 500 mM NaCl, 100 mM imidazole). The purified antibody was analyzed by SDS-PAGE (Laemmli, 1970) and dialyzed against PBS at 4 ° C. Purified scFv preparations were further processed by gel filtration using an FPLC apparatus equipped with an S-75 column, since it is known that multivalent scFv fragments can show good artificial binding in BIAnucleus (Jonsson et al., 1991) by virtue of its avidity effects (Nissim et al., 1994; Crothers and Me zger, 1972). The antibody concentration of purified monomer fractions was determined spectrophotometrically with FPLC assuming an absorbance at 280 nm of 1.4 units for a scFv solution of 1 mg / ml. The binding of monovalent scFv at various concentrations in the range of 0.1 - 1 μM in PBS was measured on a BIAnucleus machine (Pharmacia Biosensor), using the following antigens: (i) 1000 resonance units (UR) of fragment F? biotinylated recombinant of 7B89 immobilized on a wafers coated with streptavidin which binds specifically to 250 RU of scFv; (ii) 200 RU of recombinant ED-B, chemically immobilized at the N-terminal amino group, which binds specifically to 600 RU of scFv; (iii) 3500 RU of WI38VA of fibronectin rich in ED-B (see Example 3), which binds speci fi cally to 150 RU of scFv. The kinetic analysis of the data is done in accordance with. the instructions of the manufacturers. Based on the qualitative analysis BIAnucleus of the supernatants containing antibody, we selected a matured version in terms of affinity of each clone scFv: the clone CGS-1 of the selection with fragment 78B9 and CGS-2 of the selection with the fragment Recombinant FN of ED-B. The association rate constants (kon) and dissociation rate constants (koff) are shown in Table 1, together with the equilibrium dissociation constants (Kd) calculated from both scFv and the original clone 28SI. Although both clones CGS-1 and CGS-2 have Kd in the nanomolar range, the clone CGS-2 shows greater improvement over its original clone, which provides a Kd of 1 nM (improved from 110 nM) with respect to the all of the three proteins tested in the detector wafer (Table 1). The improvement is mainly due to a slower kinetic dissociation constant (~ 10 ~ 4 s "1), measured with monomer antibody preparations (not shown) .The maturation strategy seems to be general, and has provided antibodies of improved affinity against protein that binds to maltose, cytochrome C, the extracellular domain of mouse endoglin (DN, L. Wyder, R. Klemenz), cytomegalovirus (AP, G. Neri, R. Botti, PN), the HMGI-C marker protein of nuclear tumor (AP, P. Soldani, V.

Giansotti, P.N.) and the alsaline phosphatase plasentaria marker of ovarian tumor (M. Deonarain and A.A. Epenetos). Therefore, the strategy seems to be at least as effective as other maturation strategies (Marks et al., 1992, Low et al., 1996) and provides antibodies with affinities similar to those derived from very large phage antibody libraries. (Griffiths et al., 1994; Vaughan et al., 1996). CGS-1 and CGS-2 clones matured for affinity were sequenced and aligned to a database of human germline antibody V (V-BASE) genes and then translated using MacVector programming elements. The VH gene of both clones is the most homologous to the human DP47 germline (VH3), and each clone also has a different VH CDR3 sequence (Figure 1). The VL gene of both clones has the germline DPL16 used in the construction of the human synthetic scFv repertoire described in Nissim et al, 1994. The CDR3 sequences of VL differ from each other in four out of six of the randomized residues (figure lb).

TABLE 1 Kinetic and dissociation constants of monomeric scFv fragments CGS-1 and CGS-2 towards proteins containing the ED-B domain Antigen: ED-B 7B89 FN 138VA ScFv: CGS-1 S128 CGS-2 CGS-1 S128 CGS-2 CGS-1 S128 CGS-2 koffics "1) * 7.0xl0" 3 2.7X10"2 1.5X10-4 3.9xl0 -3 3.0xl0'2 2.3xl0"4 5.0X10-3 7.1X10-2 ß.5_LTi ik ^ íM ^ s'1) * 1.3xl05 2.5X105 1.3xl05 '1. lxlO5 2.9xl0s l.lxlO5 4.1X105 1.2xl06 2.9 x3 w Kd (M) * 5.4X10"8 l.lXlO" 7 l.lxlO "9 3.5xl0" 8 l.OxlO "7 2.1X10" 9 1.2X10"8 5.9xl0" 8 2 &_L? Legend for Table 1 The experiments were performed as described in the Materials and methods section. * The values koff and kon are accurate up to +/- 30%, based on the precision of determinations of concentration and in relation to the slight difference of results obtained when different regions of the sensorgrams are used for the adjustment procedure. Kd = koff / kon.

Example 3 - Affinity specificity of matured scFvs for fibronectins containing ED-B.

The immunoreactivity of the two scFv, CGS-1 and CGS-2 matured in affinity was determined by ELISA and compared directly to mAb BC-1 (which recognizes the B-FN isoform) and mAb and IST-6, which only recognizes the isoforms of FN that lack ED-B (Carnemolla et al., 1989, 1992). The characterization of these mAbs has been previously reported (Carnemolla et al, 1989, 1992). The fine specific analysis was subsequently carried out using an extensive panel of fragments of F? derivatives by treatment with thermolysin and recombinant fusion proteins. The antigens used for ELISA and for immunoblotting were prepared as follows. FN was purified from human plasma and from the conditioned medium of the WI38VA13 cell line, as previously reported (Zardi et al, 1987). The purified FN were digested with thermolysin (type X protease, Sigma Chemical Co.) as reported by Carnemolla et al (1989). FN fragments native to HOkD (B-) and native 120 kD FN were purified.

(B +) (see Figure 2) from a Fn digest as previously reported (Borsi et al, 1991). The large isoform of tenacin C is purified as reported by Saginati et al (1992). The recombinant proteins are expressed and purified as described in Example 1. Analysis was carried out by SDS-PAGE and Western blotting as described by Carnemolla et al (1989). All antigens used in ELISA were diluted in PBS between 50-100 μg / ml and coated at 4 ° C overnight on Immuno-Plate wells (Nunc, Roskilde, Denmark). The unbound antigen was removed with PBS and the plates were subsequently blocked with PBS containing bovine serum albumin (BSA) at 3% (w / v) for 2 h at 37 ° C. This is followed by four washes with PBS containing 0.05% Tween 20 (PBST). Subsequently, the antibodies were allowed to bind at 37 ° C for 1.5 h; scFv were incubated with an antiserum directed against the tag sequence: mAb M2 [Kodak, New Haven CT] for the FLAG tag or 9E10 [ATCC, Rockville, MD] for the myc tag. The control antibodies tested were mAb BC-1 and IST-6. After four washes with PBST, the plates were incubated for 1 h at 37 ° C with goat IgG against biotinylated mouse diluted 1: 2000 (in PBST + 3% BSA) (Bio-SPA Division, Milan, Italy). The washings were repeated and biotinylated streptavidin-alkaline phosphatase complex (Bio-SPA Division, Milan, Italy) (diluted 1: 800 in PBST containing 2 mM MgCl 2) was added for 1 h at 37 ° C. The reaction was developed using phosphatase substrate tablets (Sigma) in 10% diethanolamine, pH 9.8 and a reading of the optical density at 405 nm was taken. The results are presented below in Table 2.

Table 2 CGS-1 CGS-2 BC-1 IST-6 Plasma FN 0.07 0.04 0.09 1.73 WI38VA FN 1.16 0.72 1.20 1.12 nllO kD (B-) 0.03 0.01 0.05 1.20 nl20 kD (B +) 0.82 0.81 1.20 0.02 rec FN7B89 1.11 1.02 1.02 0.01 rec FN789 0.01 0.01 0.05 rec 1. ED 1.21 1.32 0.15 0.04 rec FN-6 0.01 0.01 0.08 0.03 Tenasciña 0.01 0.02 0.06 0.02 The immunoreactivity of scFv and monoclonal antibodies with antigens derived from fibronectin was measured by ELISA. The values represent the OD measured at 405 nm after subtracting the background signal. The data are the average of four experiments that show a maximum standard deviation of 10%. The identity of the different forms of fibronectin used in the experiments is as follows: Plasma FN = human plasma fibronectin; WI38-VA FN = fibronectin supernatant of fibroblasts transformed with SV 40 (Zardi et al, 1987); nllOkD = domain 4 of FN treated with thermolysin, without ED-B; nl20kD = domain 4 of F? treated with thermolysin, which contains ED-B; rec FN7B89 = ED-B domain flanked by repeated sections of monogy with F? adjacent type III; .rec FN789 = repeated sections of homology with FN type III, with an ED-B domain; rec ED-B = recombinant ED-B alone; rec FN6 = domain 6 of recombinant FN. Both CGS-1 and CGS-2 recognize the recombinant ED-B peptide, as well as all the native or recombinant fragments of FN containing the ED-B sequence, and at the same time do not bind to any of the FN fragments. who lack ED-B. In addition, CGS-1 and CGS-2 do not react with tenacin, (which comprises fifteen repeated sections of type III homology: Siri et al, 1991) and plasma FN, which does not contain detectable constellations of the ED-B sequence in the thermolysin digestion products (Zardi et al, 1987). In contrast, CGS-1 and CGS-2 react strongly with F? purified from the WI38VA cell line transformed with SV40. Approximately 70-90% of F molecules? of this cell line contain ED-B, as shown by digestion with thermolysin and experiments with nuclease SI using F? purified and total RNA prepared from the cell line (Zardi et al, 1987; Borsi et al, 1992). The specificity of the scFvs for the ED-B component of FN is further demonstrated by the use of soluble recombinant ED-B to inhibit the binding of CGS-1 and / or CGS-2 to FN in WI38VA cells (data not shown). The data confirm that CGS-1 and CGS-2 only react specifically with FN derivatives containing the ED-B domain. They still show the same reactivity as mAb BC-1, except in the case of recombinant ED-B, which is not recognized by BC-1. The intensity of the ELISA signals obtained in relation to the mAb controls reflect the high specificity of the two scFvs for the antigens containing ED-B. The specificity of CGS-1 and CGS-2 was further investigated in immunoblots using plasma FN and WI38VA cells and digested by thermolysin thereof. Upon thermolin digestion, the FN of WI38VA cells (most of which contains ED-B) generates a 120 kd fragment (containing ED-B) and a minor fragment of 110 kD, which lacks ED -B (figure 2A, Zardi et al, 1987). The additional digestion of the 120 kD domain generates two fragments: an 85 kD fragment which contains almost the entire sequence of ED-B in its carboxy terminal part, and a sequence of 35 kD (Figure 2A; Zardi et al. 1987). On the left side of Figure 2B is a Coomassie stained gel of the protein fractions analyzed by immunoblotting. The plasma FN (lane 1) and thermolysin digests of the protein (lane 3, which contains the 110 kD protein, and lane 4, which contains the digested HOkD protein) are not recognized by CGS-1 and CGS- 2 .. In contrast, the ED-B rich FN of WI38VA cells, both intact (lane 2) and after increased digestion with thermolysin (lanes 5, 6 and 7) is recognized by both scFv fragments. The smallest fragment derived from FN that can be specifically recognized by CGS-1 is the 120 kD protein (which spans repeated sections III 2-11 inclusive), while CGS-2 is able to recognize the 85 kD fragment that covers the repeated sections 2-7 in addition to the N-terminal part of ED-B (Figure 2B; Zardi et al, 1987). These results indicate that the two scFvs are reactive to different epitopes within the ED-B sequence. The binding of CGS-2 to the 85 kD domain indicates that the epitope for this clone is in the amino terminal part of ED-B. In contrast, the loss of binding of CGS-1 when the 120 kD domain is digested to 85 kD, shows that it recognizes an epitope located more towards the carboxy terminal part of the ED-B molecule. The fine specificity of CGS-1 and CGS-2 was further investigated by immunoblotting using recombinant FN fragments and fusion proteins with or without the ED-B sequence. F? Fusion proteins were prepared as described by Carnemolla et al (1989). The results of these experiments are shown in Figure 3; for the association of the schematic diagram to the structure of the human FN domains, see Carnemolla et al, 1992. The obtained binding profiles are confirmed essentially with those previously found by ELISA and immunoblots on purified FN and proteolytic cleavage products: CGS- 1 and CGS-2 are strongly reactive with fragments of F? containing ED-B (lanes 2 and 4), but show no reactivity to FN sequences lacking ED-B (lanes 1 and 3). CGS-1 does not react with the ED-B fusion protein both human (lane 5) and chicken (lane 6), whereas CGS-2 reacts strongly with both fragments (figure 3). This result may reflect certain conformational constraints of the epitope in FN containing ED-B recognized by CGS-1; it is possible, for example, that the epitope is sensitive to denaturation or does not present correctly when fractionated by SDS-PAGE and transferred to a solid support such as nitrocellulose. Taken together, these results demonstrate that CGS-1 and CGS-2 bind strongly and specifically to FNs containing ED-B, in regions different from each other and distinct from the structure of ED-B, which is recognized by mAb BC -1.Example 4 - Use of scFvs against ED-B matured for affinity by immunocytochemical staining of human and mouse tumor sections.

Both CGS-1 and CGS-2 have been used to immunolocalize FN molecules containing ED-B in various normal and neoplastic human tissues. For normal tissue, the skin was chosen, since it is known that the B-FN isoform is expressed in macrophages and fibroblasts during the healing of cutaneous wounds (Carnemolla et al, 1989, Brown et al, 1993). The two human tumors previously selected have been analyzed for their specificity of mAb staining against fibronectin: glioblastoma multiforme has been studied in detail because the endothelial cells in the vessels of this tumor are in a highly proliferative state with increased angiogenetic processes that they include the expression of B-FN isoforms (Castellani et al, 1994). In addition, studies using a panel different from the normal hyperplastic and neoplastic human breast tissues have provided additional evidence of a correlation between angiogenesis and B-F expression. (Kaczmarek et al, 1994). For the experiments described here, the immunohistochemical staining of CGS-1 and CGS-2 has been compared with that of mAb BC-1 (which recognizes the BF isoform?) And other mAbs that are known to react with any or all of the isoform variants of FN (IST-4) or only with the FN isoforms lacking ED-B (IST-6). The characterization of the totality of these control antibodies has been previously reported (Carnemolla et al, 1989, 1992).

Normal and neoplastic tissues were obtained from samples taken during surgery. It has already been established that tissue preparation and fixation is critical for accurate and sensitive detection of molecules containing FN (Castellani et al, 1994). For immunohistochemistry, cryostat sections 5 μm thick were air dried and fixed in cold acetone for 10 minutes. Immunostaining was performed using a staining kit with streptavidin-biotin complex and alkaline phosphatase (Bio-SPA Division, Milan, Italy) and naphthol-AS-MX-phosphate and Fast Red TR (Sigma). Gill's hematoxylin was used as contratinction, followed by assembly in glycergel (Dako, Carpenteria, CA) as previously reported by Castellani et al, 1994. In order to analyze the additional specificity in experiments where positive tissue staining is obtained , the specificity by ED-B was demonstrated by preincubation of antibodies with the recombinant ED-B domain, followed by detection as previously described. The results of these experiments show in general that both CGS-1 and CGS-2 react with the same histological structures as mAb BC-1. The staining pattern obtained with gel using CGS-1, CGS-2 and BC-1 reflect the absence of ED-B from the FN that expresses in the dermis. In sections of invasive duct calcinoma sections, CGS-1, CGS-2 and BC-1 show a restricted distribution of staining, confined to the border between the neoplastic cells and the stroma. This is consistent with the fact that although the total FN is homogeneously distributed through the tumor stroma, the expression of B-FN is confined to certain regions, and these areas are those that have previously been located successfully (in 95% of the cases) in invasive ductal carcinoma using mAb BC-1 (Kaczmarek et al, 1994). The previous findings in the BC-1 staining of glioblastoma multiforme tumor have been confirmed. Castellani et al (1994) have observed a typical staining pattern of vascular structures similar to glomerular ones and in our experiments, it has been demonstrated that CGS-1 and CGS-2 provide qualitatively identical results. However, there is an important difference between CGS-1 and CGS-2 and mAb BC-1: two human scFv have been shown to bind both chicken and mouse B-FN, while BC-1 is strictly specific for humans. CGS-2 reacts with chicken embryos (data not shown) and both CGS-1 and CGS-2 react with mouse tumors. Staining by CGS-1 of vascular structures and cuts of mouse F9 keratocarcinoma has also been demonstrated. By contrast, all normal mouse tissues tested (liver, vessel, kidney, stomach, small intestine, large intestine, ovary, uterus, bladder, pancreas, suprarenal glands, skeletal muscle, heart, lung, thyroid and brain) show a staining reaction negative with CGS-1 and CGS-2 (data not shown). Structures stained in F9 keratocarcinoma slices have been shown to be specific for ED-B by using the recombinant domain of ED-B to completely inhibit the staining obtained (data not shown).

Example 5 - Use of scFvs against ED-B, matured by affinity in the establishment as an in vivo target of human tumors.

The SKMEL-28 human melanoma cell line was used to develop xenografted tumors in 6-10 week old nude mice (Balb-c or MF-1; Harían UK), by injecting 1 x 107 cells / mouse subcutaneously into a flank. Mice exhibiting tumors were injected into the tail vein with 100 μl of scFv1-Cy711 mg / ml solution in PBS when the tumors reached a diameter of approximately 1 cm. The labeling of recombinant antibodies with CY7 is obtained by adding 100 μl of 1 M sodium bicarbonate, pH = 9.3, and 200 μl of CY7-bis-OSu (Amersham, Cat. Nr. PA17000, 2 mg / ml in DMSO) to 1 ml of antibody solution in PBS (1 mg / ml). After 30 minutes at room temperature, 100 μl of Tris MM, pH = 7.4, is added to the mixture and the labeled antibody is separated from the unreacted dye using disposable PD10 columns (Pharmacia Biotech, Piscataway, NJ, USA) balanced with PBS. Eluted green antibody pressures are concentrated to approximately 1 mg / ml using Centricon-10 tubes (Amicon, Beverly, MA, USA). The proportion of marking achieved is generally close to one molecule of CY7: an antibody molecule. This is estimated spectroscopically with 1 cm cuvettes, assuming that an antibody solution of 1 mg / ml provides an absorption of 1.4 units at 280 nm, and that the molar expression coefficient of CY7 at 747 nm is 200,000 (M ^ cm "1) and neglecting the absorption of CY7 at 280 nm Immunoreactivity in the antibody samples after labeling is confirmed by either band shift (? eri et al., 1996b), affinity chromatography on a column of antigen or by core analysis, mice were imaged with a mouse image producer constructed at regular time intervals under anesthesia by inhalation of an oxygen / fluorothane mixture. each sample, in order to determine the repeatability of the results.The procedures were carried out in accordance with the State project license.

United "Tumor Targeting" granted to D.? Eri (United Kingdom PPL 80/1056). The infrared mouse imager is constructed as a modification of the photodetection system of Folli et al. (1994), which allows the use of the infrared CY7 fluorophore. The infrared illumination was chosen in order to obtain a better penetration of the tissue. The fluorescence of CY7 (> 760 nm) is invisible to humans and requires the use of a computer-controlled CCD camera. The image generator consists of a light-tight box, painted black, and for a 100 W tungsten and halogen lamp, placed with a 50 mm diameter excitation filter designed specifically for CY7 (Chroma Corporation, Brattleboro, VT; USA; 673-748 nm). The resulting illumination beam is, with a good approximation, homogeneous over an area of a size of 5 x 10 cm, in which the mouse is placed for image generation. Fluorescence is detected in an 8-bit monochromic Pulchix CCD camera, equipped with a C-mount lens and a 50 mm emission filter (Chroma Corporation, Brattleboro, VT, USA; 765-855 nm), interconnected with an ImageDOK system (Kintec Imaging Ltd., Liverpool, United Kingdom). This system consists of a computer, equipped with an image sensor and programming elements for the capture and integration of sequential images. Typically, three sequential images acquired 50 ms each are used in the averaging process; This number is kept constant for the series of images of an animal, to allow the direct preparation of establishment as target of tumors at different points in time. The images in the TIFF format are then converted to PICT files using a graphics converter program, and they are made using the MacDraw Pro program with a Power Macintosh 7100/66 computer. Figure 4 shows a diagram of the design of this device. These experiments show that both scFv localize the tumor when visualized at the macroscopic level. The microscopic demonstration of establishment as target of neovasculature of developing tumors with the two scFv against EDB is detailed below. Athymic and / or SCID mice exhibiting SKMEL-28 xenografted human melanoma or a flank mouse F9 keratocarcinoma are injected with either unlabeled scFv fragments labeled FLAG, or biotinylated scFv fragments. Mice are sacrificed at different times in time after injection, obtaining tumor and non-tumor sections which are then stained with conventional immunohistochemistry protocols, using M2 antibody against FLAG (Kodak, 181) or streptavidin-based detection reagents . The establishment as an objective is usually obtained at 12 hours after the injection. Both CGS1 and CGS2 are shown to bind to the neovasculature in both the xenografted human tumor and mouse keratocarcinoma.

^ ^ Example 6 - Objective establishment of mouse F9 teratocarcinoma xenografted in athymic mice.

We developed solid tumors in the flank of athymic tumors by cutaneous injection of 4 x 106 cells of mouse F9 teratocarcinoma. This tumor grows very rapidly in mice, reaching 1 cm in diameter in about a week after injection, and is highly vascularized. To generate images of the establishment as objective of the antibodies, we used a modification of the photodetection methodology of Folli et al (1994), which allows a kinetic evaluation of the establishment as objective of the tumor and of the antibody clearance in the same animal from which an image is taken. of various points in time, this is discussed in detail before (see Figure 4). To target the tumor and to facilitate the detection of antibodies, scFv (CGS-1), scFv (CGS-2) and scFv (DI.3) are added to lysozyme (McCafferty et al., 1990) with a homodimerization tag (Pack et al., 1993) by subcloning antibodies to the Sfil / Notl sites of the expression vector pGIN50. This vector is a derivative of pDN268 (Neri et al., 1996b), in which the His6 sequence of the tag is replaced by the sequence: GGC LTD TLQ AFT DQL EDE KSA LQT EIA HLL KEK EKL EFI LAA H, which contains a cysteine residue in the amphipathic helix of the Fos protein for the covalent homodimerization of the antibody fragments (Abate et al., 1990). Complete covalent dimerization is not obtained: approximately 30-50% of the antibody fragments consist of covalently bonded dimers. Antibody fragments were purified by affinity chromatography on columns obtained by coupling chicken egg lysozyme (Di.3) or 7B89 (antibodies against ED-B, Carnemolla et al., 1996) to Sepharose activated with CNBr (Pharmacia Biotech, Piscataway, NJ, USA). The supernatants are loaded onto affinity supports, which are subsequently washed with PBS, with PBS + 0.5 M NaCl and eluted with 100 mM Et3N. The antibodies are subsequently dialyzed against PBS. The antibodies were labeled as described above after they were injected into the tail vein of tumor-bearing mice with 100 μl of scFv1-Cy71 1 mg / ml solution in PBS, when the tumors reached a diameter of approximately 1 cm. As shown in Figure 5, scFv (CGS-1) is located in the tumor for up to three days, although there is also a rapid depuration of the tumor during this period. However, there is also part of the femur stain. The operation of the target establishment of CGS-1 to the tumor is markedly improved by introducing an amphipathic helix containing a cysteine residue in the C-terminal portion to promote dimerization of the antibody (Pack et al., 1993). In fact, the location of dimeric scFv (CGS-2) 2 does not seem to decrease significantly from 24 to 72 hours. In contrast, a negative control (the dimeric antibody scFv (DI.3) 2, antibody against lysozyme), shows a rapid clearance and there is no detectable location on the tumor or femur. ScFv (28SI) shows a weak establishment as a tumor target at 6 hours (not shown), but none is detectable at 24 hours or later (Figure 6). Affinity maturation leads to a remarkably improved establishment as an objective; therefore scFv (CGS-2) targets small and large F9 tumors efficiently, either as a monomer (Figure 6) or as a dimer (not shown). After 2 days, the percent injected dose of antibody per gram of tumor is found to be approximately 2 for the scFv monomer (CGS-2) and 3-4 for the scFv dimer (CGS-2). The dose delivered to the tumor by scFv (CGS-2) is also higher than for scFv (CGS-1) (Figures 5 and 6), correlating with their respective affinities (Table 1). However, both scFv (28SI) and scFv (CGS-2) appear to be susceptible to proteolytic cleavage and show high uptake in the liver (Figure 6), whereas scFv (CGS-1) antibodies are significantly more stable and show a uptake in the liver much slower (figure 5).

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(B) STREET: Via Roma 22 (C) CITY: SIENA (E) COUNTRY: ITALY (F) POSTAL CODE (ZIP): 53100 (ii) TITLE OF THE INVENTION: ANTIBODIES FOR THE ED-B DOMAIN OF FIBRONECTIN, ITS CONSTRUCTION AND USES (iii) NUMBER OF SEQUENCES: 12 (iv) COMPUTER LEADABLE FORM: (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (EPO) (v) CURRENT REQUEST DATA: REQUEST NUMBER: PCT / GB97 / 01412 (2) INFORMATION FOR SEC. FROM IDENT. DO NOT: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 1: Ser Leu Pro Lys 1 (2) INFORMATION FOR SEC. FROM IDE? T. ? O: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 2 Gly Val Gly Ala Phe Arg Pro Tyr Arg Lys His Glu 1 5 10 (2) INFORMATION FOR SEC. FROM IDENT. NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: ADNC (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO-: 3 CAGGAAACAG CTATGAC 17 (2) INFORMATION FOR SEC. FROM IDENT. NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 51 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDE? T. ? O: 4 CTTGGTCCCT CCGCCGAATA CCACMNNMNN MNNM? NMN? M NNAGAGGAGT TACAGTAATA GTCAGCCTC 69 (2) INFORMATION FOR SEC. FROM IDENT. NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: ADNC (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 5 ATTGCTTTTC CTTTTTGCGG CCGCGCCTAG GACGGTCAGC TTGGTCCCTC CGCC 54 (2) INFORMATION FOR SEC. FROM IDENT. NO 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 43 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO 6: Gly Gly Cys Leu Thr Asp Thr Leu Gln Wing Phe Thr Asp Gln Leu Glu 1 5 10 15 Asp Glu Lys Ser Ala Leu Gln Thr Glu lie Ala His Leu Leu Lys Glu 25 30 Lys Glu Lys Leu Glu Phe lie Leu Ala Wing His 35 40 (2) INFORMATION FOR SEC. FROM IDENT. NO: 7 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. DO NOT: Pro Val Val Leu Asn Gly Val Val (2) INFORMATION FOR SEC. FROM IDENT. NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 8 Pro Phe Glu His Asn Leu Val Val 1 5 (2) INFORMATION FOR SEC. FROM IDENT. NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 113 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (vi) ORIGINAL SOURCE: (B) CEPA: CGS1 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 9: Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Wing Val Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala lie Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Wing Arg Ser Leu Pro Lys Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Arg (2) INFORMATION FOR SEC. FROM IDENT. NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 121 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear(ii) TYPE OF MOLECULE: protein (vi) ORIGINAL SOURCE: (B) CEPA: CGS2 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 10: Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Wing Wing Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala lie Ser Gly Ser Gly Ser Thr Tyr Tyr Wing Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Wing Arg Gly Val Gly Wing Phe Agr Pro Tyr Arg Lys His Glu Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Arg 115 120 (2) INFORMATION FOR SEC. FROM IDENT. NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 109 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (vi) ORIGINAL SOURCE: (B) CEPA: CGS1 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 11; Be Ser Glu Leu Thr Gln Asp Pro Wing Val Ser Val Wing Leu Gly Gln 1 5 10 15 Thr Val Arg lie Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Wing 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Wing Pro Val Leu Val lie Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Lie Pro Asp Arg Phe Ser Gly Ser 50 55 60 Being Ser Gly Asn Thr Wing Being Leu Thr lie Thr Gly Wing Gln Wing Glu 65 70 75 80 Asp Glu Wing Asp Tyr Tyr Cys Asn Ser Ser Pro Val Val Leu Asn Gly 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 100 (2) INFORMATION FOR SEC. FROM IDENT. NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 109 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (vi) ORIGINAL SOURCE: (B) CEPA: CGS2 (xi) DESCRIPTION OF THE SEQUENCE: SEC. FROM IDENT. NO: 12: Be Ser Glu Leu Thr Gln Asp Pro Wing Val Ser Val Wing Leu Gly Gln 1 5 10 15 Thr Val Arg lie Thr Cys Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Wing 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Wing Pro Val Leu Val lie Tyr 35 40 45 Gly Lys Asn Asn Arg Pro Ser Gly Lie Pro Asp Arg Phe Ser Gly Ser 50 55 60 Being Ser Gly Asn Thr Wing Being Leu Thr lie Thr Gly Wing Gln Wing Glu 65 70 75 80 Asp Glu Wing Asp Tyr Tyr Cys Asn Ser Ser Pro Phe Glu His Asn Leu 85 90 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates. Having described the invention as above, property is claimed as contained in the following:

Claims (29)

1. A process for the production of a specific binding member isolated from synthetic molecular repertoires which is specific for and binds directly to the oncofetal domain of ED-B fibronectin (FN), the process is characterized in that it comprises the expression of a nucleic acid which codes for such a specific binding member.

2. The process according to claim 1, characterized in that the process comprises: a) analyzing a library of peptides or proteins expressed in the phage with a recombinant fibronectin fragment containing the ED-B domain derived from the fibronectin protein; b) infecting bacterial host cells with positive clones; c) submit the positive phage clones to an affinity maturation process; d) repeating steps a) and b) to select positive phage clones with improved affinity for antigen; e) infecting host cells with positive clones and purifying antibody molecules from such host cells.

3. The process according to claim 2, characterized in that step a) comprises analyzing a phage scFv library with recombinant antigen derived from the fibronectin protein.

4. The process according to claim 3, characterized in that the phage library expresses the scFv of human origin.

5. The process according to claim 2, characterized by, in step a), the phage clones are analyzed with recombinant antigens 7B8 or E ^ -B.

6. A specific binding member isolated from synthetic molecular repertoires, characterized in that it is specific for and binds directly to the oncofetal domain ED-B of fibronectin (FN), produced by a process according to claim 1 or 2.

7. A specific binding member, according to claim 6, characterized in that it comprises an antibody with antigen-binding domain.

8. A specific binding member, according to claim 7, characterized in that the antibody with antigen-binding domain is of human origin.

9. A specific binding member according to any of claims 6 to 8, characterized by binding to all FNs containing ED-B after treatment of FN with the proteolysin thermolysin.

10. A specific binding member according to any of claims 6 to 9, characterized in that it binds to all recombinant FNs containing repeat sections of type III homology which include the domain ED-B.

11. A specific binding member, according to any of claims 6 to 10, whose binding to B-FN is inhibited by the ED-B domain.

12. A specific binding member, according to any one of the preceding claims, characterized in that it binds to B-FN of human, mouse, rat, chicken and any other species in which the domain ED-B is conserved.

13. A specific binding member, according to any of the preceding claims, characterized in that it binds to B-FN without treatment of the NF with N-glycanase.

14. A specific binding member, according to any one of the preceding claims, characterized in that it has a variable heavy chain (VH) region of the sequence derived from the human germ line DP47 (codon 1 Glu-codon 98 Arg, inclusive, in Figure 1) and the sequence CDR3 Ser Leu Pro Lys.

15. A specific binding member, according to any of claims 6 to 13, characterized by having a variable heavy chain (VH) region of the sequence derived from the human germ line DP47 (codon 1 Glu-codon 98 Arg, inclusive, in Figure 1) and the sequence CDR3 Gly Val Gly Ala Phe Arg Pro Tyr Arg Lys His Glu.

16. A specific binding member, according to any of claims 6 to 13, characterized by having a variable ligand chain region (VL) of the sequence derived from the human germline DPL16 (codon 1 Ser-codon 90 Ser, including, in figure 1) and the rest of the CDR3 sequence as Pro Val Val Leu Asn Gly Val Val.

17. A specific binding member, according to any of claims 6 to 13, characterized by having a variable light chain (VL) region of the sequence derived from the human germline DPL16 (codon 1 Ser-codon 90 Ser, inclusive, in Figure 1) and the remainder of the CDR3 sequence as Pro-Ghe His Asn Leu Val Val.

18. A specific binding member, according to any of claims 6 to 13, characterized in that it has a variable heavy chain (VH) region of the sequence derived from the human germ line DP47 (codon 1 Glu-codon 98 Arg, inclusive, in Figure 1) and the CDR3 sequence.

19. A specific binding member, according to any of the preceding claims, characterized by, when measured as a purified monomer, has a dissociation constant (Kd) of 6 x 10 ~ 9M or less, for ED-B of FN.

20. A specific binding member, according to any of the preceding claims, characterized in that the binding member comprises a scFv molecule.

21. A specific binding member, according to any of the preceding claims, characterized in that the binding member comprises a dimeric scFv molecule.

22. A specific binding member, according to any of the preceding claims, characterized in that the binding member is an antibody fragment according to claims 14-18.

23. A pharmaceutical composition characterized in that it comprises a specific binding member according to any of the preceding claims in an effective amount, together with a pharmaceutically acceptable excipient.

24. A nucleic acid characterized in that it encodes a specific binding member according to any of claims 6 to 22.

25. A phage, characterized by coding for a specific binding member according to any of claims 6 to 22.

26. A host cell, characterized by porgue is transformed or transfected with a nucleic acid according to claim 24.

27. A specific binding member, according to any of claims 6 to 22, characterized in that it is used in therapy.

28. The use of a specific binding member, according to any of claims 6 to 22, characterized by being used in the manufacture of a medicament for imaging or targeting tumors.

29. A diagnostic equipment characterized in that it comprises a specific binding member according to any of claims 6 to 22 and one or more reagents that allow the determination of the binding of the member to the cells.