HK1092375B - Use of radiolabelled antibody l19 against fibronectin ed-b in the preparation of a medicament for targeting of tumor vasculature - Google Patents

Description

Pharmaceutical use of radiolabeled anti-fibronectin ED-B antibody L19 to target tumor vasculature

The present invention relates to targeting tumor vessels using radiolabeled antibody molecules. In particular, the invention relates to the use of antibody molecules that bind the ED-B of fibronectin, which have demonstrated effectiveness in tumor targeting. In various embodiments of the invention, the antibody molecules are used in different molecular forms. In certain embodiments, the antibody molecule comprises human IgG 1. In other embodiments, the antibody molecule is a small immunoglobulin (mini-immunoglobulin), such as produced by fusing a scFv antibody molecule to the CH4 constant region of a secretory IgE isotype that naturally contains a cysteine at its COOH terminus for forming a covalently linked dimer. Blood clearance, in vivo stability and other advantageous properties are used in different aspects and embodiments of the invention, e.g. in tumor targeting. The different in vivo behavior of different antibody molecular forms can be used for different diagnostic and/or therapeutic purposes, depending on clinical needs and disease conditions.

Although monoclonal antibodies (mAbs) of non-human origin have great potential as therapeutic agents, few clinical trials have been conducted due to their immunogenicity (1 Shawlert et al, 1985; 2 Miller et al, 1983), poor pharmacokinetic properties (3 Hakimi et al, 1991; 4 Stephens et al, 1995), and ineffectiveness in restoring effector function (5 Riechmann et al, 1988; 6 Junghens et al, 1990). Recent observations for the isolation of human antibody fragments from phage display libraries (7McCafferty et al, 1990; 8 Lowman et al, 1991; reviewed in 9 Nilsonn et al, 2000 and 10 Winter et al, 1994) solved these problems, reviving the study and reignition of the use of these agents in the treatment of major diseases. Indeed, these molecules should serve as ideal building blocks for new diagnostic and therapeutic tools (11Reichert, 2001; 12 Huls et al, 1999). In addition, these antibodies can be "matured" to achieve an affinity in the picomolar range (13Pini et al, 1998), which is at least desirable, if not necessary, for their clinical use.

However, clinical use of human antibody fragments requires highly specific targets for selective delivery of diagnostic or therapeutic agents. In the case of tumors, the most commonly used target is a cell surface antigen, which is usually neither abundant nor stable. However, during tumor progression, the microenvironment surrounding the tumor cells is extensively modified to create a "tumor environment" that is a target for antibody-based tumor therapy (14 Neri and Zardi, 1998). Indeed, the notion that altered tumor microenvironments are themselves carcinogens that can be targeted is increasingly gaining consensus. Molecules capable of delivering therapeutic agents efficiently to the tumor microenvironment thus represent a promising and important new tool for cancer therapy (15 Bissel, 2001; 14 Neri and Zardi, 1998).

Fibronectin (FN) is an extracellular matrix (ECM) component that is widely expressed in a variety of normal tissues and body fluids. Different FN isoforms may be produced by alternative splicing of FN pre-mRNA, a process regulated by cytokines and extracellular pH (16 Balza et al, 1988; 17Carnemolla et al, 1989; 18 Borsi et al, 1990; 19 Borsi et al, 1995). The complete type III repeat ED-B, also known as exo (extra type) type III repeat B (eiiib), may or may not be completely included in the FN molecule (20 Zardi et al, 1987). ED-B is highly conserved across species, with 100% homology in all mammals studied to date (human, rat, mouse, dog), and 96% homology to the analogous domain of chicken. FN isoforms containing ED-B (B-FN) are not detectable immunohistochemically in normal adult tissues, except in tissues undergoing physiological remodeling (e.g., endometrium and ovary) and during wound healing (17Carnemolla et al, 1989; 21ffrench-Constant, et al, 1989). In contrast, it is highly expressed in tumor and embryonic tissues (17Carnemolla et al, 1989). In addition, B-FN has been shown to be a marker of angiogenesis (22Castellani et al, 1994), and endothelial cells that invade tumor tissue migrate along ECM fibers containing B-FN (23Tarli et al 1999).

Selective targeting of tumor vessels using human recombinant antibody scFv (L19) (13Pini et al, 98) specific for the B-FN isoform (24 Carnemolla et al, 1996; 23Tarli et al, 99; 25 Viti et al, 99; 26 Neri et al, 97; 27 Demartis et al, 2001) has been described. The antibodies are useful in vivo diagnostics (immunoscintigraphy) and therapeutic methods for the selective delivery of therapeutic radionuclides or toxic substances to tumor vessels. In addition, Birchler et al (281999) showed that scFv (L19) chemically coupled to a photosensitizer selectively accumulated in newly formed blood vessels of the angiogenic rabbit corneal model and mediated complete and selective occlusion of ocular neovessels upon irradiation with near infrared light.

More recently, Nilsson et al (292001) reported that immunoconjugates of scFV (L19) with the extracellular domain of tissue factor mediated selective infarction in different types of murine tumor models. Furthermore, fusion proteins of scFv (L19) with IL-2 or IL-12 have been shown to enhance the therapeutic efficacy of these two cytokines (30 Hall et al, submitted; 31 Carnemolla et al, 2002). A description of the use of the fusions in the treatment of pathological angiogenic lesions, including tumors, is also found in WO 01/62298. Finally, since L19 responded equally well to mouse and human ED-B, it was used in preclinical and clinical studies.

See also PCT/GB97/01412, PCT/EP99/03210, PCT/EP01/02062 and PCT/IB 01/00382.

Different antibody formats show different manifestations in vivo stability, clearance and tumor targeting properties (32 Wu et al, 2000). Small immunoglobulins or Small Immunoproteins (SIP) are described in (33 Li et al, 1997).

The present invention is based on the preparation, identification and in vivo biodistribution studies of different forms of L19 human antibody molecules (i.e., scFv, small immunoglobulins and intact IgG1), and labeling with radioisotopes.

Brief Description of Drawings

Figure 1 shows a model illustrating the structure of different proteins. A: domain structural models of FN subunits. Protein sequences undergoing alternative splicing are shown in grey. As shown, the epitope of recombinant antibody L19 is located in repeat ED-B. B-D: schematic of constructs used to express L19(scFv) (B), L19-SIP (C) and L19-IgG1/K, respectively.

FIG. 2 shows the growth curve of SK-MEL-28 tumor in nude mice (triangles)) And growth curves of F9 tumor in 129 mouse strain (shown by circles). Volume (mm)³) Plotted as a function of time (days). Each data point is the mean of six mice ± SD.

Figure 3 shows the results of size exclusion chromatography on different forms of L19. Size exclusion chromatography (Superdex 200) profiles of L19 in scFv, small immunoglobulin and IgG1 format after radioiodination are shown in panel A, B and panel C, respectively. D. Panels E and F show a graphical representation of size exclusion chromatography (Superdex 200) of plasma at the times indicated following intravenous injection of L19 in the form of radioiodinated scFv, small immunoglobulin and IgG1, respectively. When the plasma was loaded at different times after injection, no changes in the L19-SIP or L19-IgG1 profiles were detected, whereas a second peak with a higher molecular weight was observed 3 hours after injection of L19(scFv) 2.

FIG. 4 shows the results of a biodistribution experiment in SK-MEL 28 tumor bearing mice using different radioiodinated forms of the L19 antibody molecule. Changes in% ID/g in tumor (FIG. 4A) and blood (FIG. 4B) at the times indicated after intravenous injection were reported. In FIG. 4C, the tumor-blood ratio is plotted against% ID/g. The curves for L19(scFv) are indicated by diamonds, L19 small immunoglobulins by squares and L19 IgG1 by triangles.

Figure 5 shows the results of biodistribution experiments in mice bearing F9 tumors using radioiodinated L19(scFv) (squares) and L19 small immunoglobulin (diamonds). Changes in% ID/g in tumor (A) and blood (B) at the different times indicated after intravenous injection were reported.

FIG. 6 shows the change in U251 tumor area (mm) over time (days) following injection of saline and I-131-L19-SIP, respectively.

The present invention relates to a specific binding member (specific binding member) that binds the ED-B of human fibronectin, wherein said specific binding member is selected from the group consisting of⁷⁶Br、⁷⁷Br、¹²³I、¹²⁴I、¹³¹I and²¹¹at is a group of one or more isotopic radiolabels. The invention also provides methods of producing such specific binding members, and their use in diagnosis and therapy.

The specific binding members of the invention show beneficial properties in animal experiments, such as higher dose delivery to tumors relative to red bone marrow, and high tumor accumulation properties.

In one aspect, the invention provides a specific binding member which binds the ED-B of human fibronectin and comprises an L19VH domain and a VL domain, optionally an L19VL domain, wherein the specific binding member comprises a binding domain which binds to epsilon_S2-CH4, or said building block comprises an intact IgG1 antibody molecule, and wherein said specific binding building block is selected from the group consisting of⁷⁶Br、⁷⁷Br、¹²³I、¹²⁴I、¹³¹I and²¹¹at is in the group of isotopic radiolabels. Preferably the radioisotope is¹²³I or¹³¹I, most preferably¹³¹I。

A radiolabel or radiolabelled molecule may be attached to the specific binding member and may be labelled at, for example, a tyrosine, lysine or cysteine residue.

The L19VH and L19VL domain sequences are described in Pini et al (1998) j.biol.chem.273: 21769-21776, wherein the VH domain sequence is: EVQLLESGGGLVQPGGSLRL SCAASGFTFS SFSMSWVRQA PGKGLEWVSSISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAEDTAVYYCAKPF PYFDYWGQGT LVTVSS, VL domain sequence is: EIVLTQSPGT LSLSPGERAT LSCRASQSVS SSFLAWYQQKPGQAPRLLIY YASSRATGIP DRFSGSGSGT DFTLTISRLEPEDFAVYYCQ QTGRIPPTFG QGTKVEIK, those sequences fully incorporated herein by reference to the article by Pini et al are considered to be listed herein.

Typically, the VH domain is paired with a VL domain to provide an antibody antigen binding site. In a preferred embodiment, the L19VH domain is paired with the L19VL domain, thereby forming an antibody antigen binding site comprising the L19VH and VL domains. In other embodiments, the L19VH is paired with a VL domain other than L19 VL. The promiscuity of light chains is well established in the art.

One or more CDRs may be taken from the L19VH or VL domain and incorporated into a suitable framework. Such operation is discussed further below. L19VH CDRs 1, 2 and 3 are shown in seq id NOs: 1.2 and 3. L19VL CDRs 1, 2 and 3 are shown in SEQ ID NOs: 1.2 and 3.

In a preferred embodiment, the specific binding member is L19-SIP, most preferably¹²³I-tagged L19-SIP (referred to herein as I-123-L19-SIP) or¹³¹I-labeled L19-SIP (referred to herein as I-131-L19-SIP).

The sequences listed herein and useful for the VH and VL domains of the specific binding member for ED-B and variants of the CDRs thereof may be obtained by sequence alteration or mutation and screening methods.

Variable domain amino acid sequence variants of any of the VH and VL domains specifically disclosed herein may be employed in accordance with the teachings of the present invention. A particular variant may comprise one or more amino acid sequence alterations (additions, deletions, substitutions, and/or insertions of amino acid residues) and may be less than about 20 alterations, less than about 15 alterations, less than about 10 alterations, or less than about 5 alterations, 4, 3, 2, or 1 alteration. Changes may be made in one or more framework regions and/or one or more CDRs.

The specific binding member of the present invention may be one which competes for binding to antigen with a specific binding member which binds both ED-B and comprises an antigen binding site formed by the L19VH domain and the L19VL domain. Competition between binding members can be readily assayed in vitro, for example using ELISA and/or by labelling one binding member with a specific reporter molecule, which can be detected in the presence of the other unlabelled binding member, thereby allowing the identification of specific binding members that bind the same epitope or overlapping epitopes.

Thus, a further aspect of the invention is the use of a specific binding member comprising the antigen binding site of a human antibody, which competes with L19 for binding to ED-B.

Specific binding members of the invention may bind ED-B with a binding affinity of at least L19, the binding affinities of the different specific binding members being compared under appropriate conditions.

In addition to antibody sequences, specific binding members of the invention may comprise other amino acids, for example to form peptides or polypeptides (such as folded domains), or to confer other functional properties to the molecule in addition to its ability to bind antigen. The specific binding member of the invention may carry a detectable label or may be conjugated to a toxin or enzyme (e.g. via a peptide bond or linker).

In the treatment of disease or damage resulting from pathological angiogenesis, a specific binding member of the invention may be conjugated to a toxic molecule, for example a biocidal or cytotoxic molecule selected from interleukin-2 (IL-2), doxorubicin, interleukin-12 (IL-12), interferon-gamma (IFN-gamma), tumour necrosis factor alpha (TNF α) and tissue factor (preferably truncated tissue factor, for example truncated to residues 1-219). See, for example, WO 01/62298.

The specific binding members of the invention may be used in a method of treatment or diagnosis of the human or animal body, such as a method of treatment (which may include prophylactic treatment) of a disease or condition in a human patient, which method comprises administering to said patient an effective amount of a specific binding member of the invention. Preferably, the specific binding members of the invention are administered to the patient by parenteral administration. Conditions treatable in accordance with the invention include tumours (particularly solid tumours) and other lesions of pathological angiogenesis, including rheumatoid arthritis, diabetic retinopathy, age-related macular degeneration and haemangiomas.

The specific binding member is well suited for use with a member selected from the group consisting of⁷⁶Br、⁷⁷Br、¹²³I、¹²⁴I、¹³¹I and²¹¹at and then used in radiodiagnosis and radiotherapy.

In a further aspect the invention provides a method of producing a specific binding member of the invention comprising administering to a subject in need thereof a composition selected from the group consisting of⁷⁶Br、⁷⁷Br、¹²³I、¹²⁴I、¹³¹I and²¹¹at is labeled with a specific binding member.

For direct radiolabeling of specific binding members, the tyrosine component of the molecule may be targeted. In this particular procedure, a halide such as Br^-、I^-、At^-By oxidation with suitable oxidizing agents in the presence of Active Pharmaceutical Ingredients (API), e.g.(coated tubes), iodo-Beads, Chloramine-T (Chloramine-T) (sodium salt of N-chloro-p-methylbenzenesulfonamide), and the like.

Indirect labeling with, for example, bromine, iodine, or astatine may be accomplished by pre-labeling a bifunctional halogen carrier, preferably derived from, for example, benzoic acid derivatives, Bolton-Hunter derivatives, benzene derivatives, and the like. The vector may be transformed into an activated species conjugated to the epsilon amino group of a lysine residue or to the N-terminus of an API. This indirect method also provides a synthetic route to chemoselectively radiolabel peptide compounds at the sulfhydryl group of the cysteine component. The cysteine-bridged molecule can first be reduced with a suitable reducing agent such as tin chloride (divalent), Tris (2-carboxyethyl) phosphine (TCEP) to produce a free cysteine SH group that can react with the halogen carrier. As the bound functional group, maleimide and α -bromoacetamide derivatives can be applied.

The method of producing a specific binding member of the invention may comprise expressing a nucleic acid encoding the specific binding member prior to labelling the specific binding member. Thus, as an early step, the method of producing the specific binding member may optionally comprise causing or allowing expression of the encoding nucleic acid (i.e. the nucleic acid comprising the sequence encoding the specific binding member). Such a method may comprise culturing the host cell under conditions to produce the specific binding member.

The production process may comprise isolation and/or purification steps of the product. The specific binding member may be isolated and/or purified after expression from the nucleic acid and/or recovered from the host cell. The isolation and purification may be performed prior to labeling. Alternatively or additionally, the specific binding member may be isolated and/or purified after labelling.

The method of production may comprise formulating the product into a composition comprising at least one additional ingredient, such as a pharmaceutically acceptable excipient. Thus, the (labelled) specific binding member may be formulated as a composition comprising at least one additional ingredient, such as a pharmaceutically acceptable excipient.

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

Term(s) for

Specific binding member (specific binding member)

This term describes the building blocks (members) of a pair of molecules that have binding specificity for each other. The member of a specific binding pair may be naturally occurring or wholly or partially synthetically produced. One member of a pair of molecules has a region or cavity on its surface that specifically binds to and is thus complementary to a particular spatial and polar configuration of the other member of the pair. The paired building blocks thus have the property of binding specifically to each other. Types of specific binding pairs are, for example, antigen-antibody, biotin-avidin, hormone-hormone receptor, receptor-ligand, enzyme-substrate. The present application relates to antigen-antibody type reactions.

Antibody molecules

This term describes naturally occurring or partially or wholly synthetically produced immunoglobulins. The term also encompasses any polypeptide or protein comprising an antibody binding domain. Antibody fragments comprising an antigen binding domain are for example Fab, scFv, Fv, dAb, Fd; and diabodies (diabodies). The invention relates to intact IgG1 antibody molecules and antibodies comprising epsilon as described_S2-CH 4.

Recombinant DNA technology can be used to produce other antibody molecules from an original antibody molecule that retain the specificity of the original antibody molecule. These techniques may involve introducing DNA encoding the immunoglobulin variable region or Complementarity Determining Region (CDR) of an antibody into the constant region, or constant region plus framework regions, of a different immunoglobulin. See, for example, EP-A-184187, GB 2188638A or EP-A-239400.

Since antibodies can be modified in many ways, the term "antibody molecule" should be understood to encompass any specific binding member or substance of the antigen-binding domain of an antibody having the desired specificity. Thus, this term encompasses antibody fragments and derivatives, including any polypeptide comprising an immunoglobulin antigen-binding domain, whether natural or wholly or partially synthetic. The term therefore includes chimeric molecules comprising an immunoglobulin binding domain or equivalent fused to another polypeptide. Cloning and expression of chimeric antibodies is described in EP-A-0120694 and EP-A-0125023.

Antigen binding domains

This term describes a particular portion of an antibody molecule that comprises a region that specifically binds to and is complementary to part or all of an antigen. In the case of larger antigens, the antibody may bind only a specific portion of the antigen, which portion is referred to as an epitope. The antigen binding domain may be provided by one or more antibody variable domains (e.g. so-called Fd antibody fragments consisting of a VH domain). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH).

Specificity of

This term may be used to describe a state in which one member of a specific binding pair does not have any significant binding to a molecule other than its specific binding partner. The term is also applicable to, for example, situations where the antigen binding domain is specific for a particular epitope carried by multiple antigens, in which case the specific binding member carrying the antigen binding domain is capable of binding to the various antigens carrying that epitope.

Included

The term is generally meant to include, i.e., to allow the presence of, one or more features or components.

Separated from each other

This term means that the specific binding members of the invention or the nucleic acids encoding these binding members are generally in the state as specified in the invention. The building blocks and nucleic acids are free or substantially free of their natural attendant materials, such as materials found in their natural environment or in the environment in which they are prepared (e.g., cell culture) when such preparations are prepared in vitro or in vivo by recombinant DNA techniques. The building blocks and nucleic acids may be formulated with diluents or adjuvants and still be considered separate for practical purposes, for example the building blocks will usually be mixed with gelatin or other carriers if used to coat microtiter plates for immunoassays, or pharmaceutically acceptable carriers or diluents when used for diagnostic or therapeutic purposes. Specific binding members may be native or glycosylated by heterologous eukaryotic cell systems, such as CHO or NSO (ECACC 85110503) cells, or they may be non-glycosylated (e.g. if produced by expression in prokaryotic cells).

By "substantially as listed" is meant that the relevant CDR or VH or VL domain of the invention is identical or highly similar to the sequence in the particular regions listed herein. "highly similar" covers that 1 to 5, preferably 1 to 4 such as 1 to 3 or 1 or 2 or 3 or 4 substitutions may be made in the CDR and/or VH or VL domain.

The structures carrying the CDRs of the invention are typically antibody heavy or light chain sequences, or substantial portions thereof, in which the CDRs are located at positions corresponding to the CDRs of naturally occurring VH and VL antibody variable domains encoded by rearranged immunoglobulin genes. The structure and position of immunoglobulin variable domains can be determined as described by reference to (Kabat, E.A. et al, Sequences of Proteins of immunological interest.5th edition. US Department of Health and Human services.1991, and Internet (http:// immunological. by. nwu. edu. or using any search tool to find "Kabat") available updates.

Preferably, the CDR amino acid sequences substantially as set out herein are carried as CDRs in a human variable domain or a substantial part thereof. L19VH CDR3 and/or L19VL CDR3 sequences substantially as set out herein may be used in preferred embodiments of the invention, preferably each sequence is carried as a CDR3 in a human heavy or light chain variable domain (as the case may be) or a substantial portion thereof.

The essential part of an immunoglobulin variable domain comprises at least 3 CDR regions, and between them a framework region. Preferably, the portion further comprises at least about 50% of either or both of the first and fourth framework regions, the 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. Other residues at the N-terminus or C-terminus of the essential portion of the variable domain may be those not normally associated with naturally occurring variable domain regions. For example, construction of a specific binding member of the invention by recombinant DNA techniques may result in the introduction of N-or C-terminal residues encoded by linkers introduced for the convenience of cloning or other manipulation steps. Other manipulation steps include the introduction of linkers to link the variable domains of the invention with additional protein sequences, including immunoglobulin heavy chains, other variable domains, or protein markers, as discussed in more detail below.

In the IgG1 antibody molecules of the invention, the VL domain may be attached C-terminally to the antibody light chain constant region comprising a human ck or C λ chain, preferably a ck chain.

In addition to using⁷⁶Br、⁷⁷Br、¹²³I、¹²⁴I、¹³¹I and/or²¹¹In addition to the At label, the specific binding member of the invention may be labeled with another detectable or functional label. The detectable label is described below and includes radioactive labels such as the radioactive isotopes technetium, indium, yttrium, copper, lutetium or rhenium, in particular^94mTc、^99mTc、¹⁸⁶Re、¹⁸⁸Re、¹¹¹In、⁸⁶Y、⁸⁸Y、¹⁷⁷Lu、⁶⁴Cu and⁶⁷cu, which can be attached to the antibodies of the invention using conventional chemistry in the field of antibody imaging as described herein. Other radioisotopes that may be used include²⁰³Pb、⁶⁷Ga、⁶⁸Ga、⁴³Sc、⁴⁷Sc、¹¹⁰mIn、⁹⁷Ru、⁶²Cu、⁶⁸Cu、⁸⁶Y、⁸⁸Y、⁹⁰Y、¹²¹Sn、¹⁶¹Tb、¹⁵³Sm、¹⁶⁶Ho、¹⁰⁵Rh、¹⁷¹Lu、⁷²Lu and¹⁸F。

labels also include enzyme labels such as horseradish peroxidase. Labels also include chemical moieties such as biotin which can be detected by binding a specifically associated detectable moiety such as labeled avidin.

An example of a labeling scheme is as follows:

for direct radiolabelling of specific binding members, cysteine-bridged molecules are first reduced by a suitable reducing agent such as tin chloride (bivalent), Tris (2-carboxyethyl) phosphine (TCEP) to give a free cysteine SH group which can be reacted with an isotope such as Tc or Re. In this particular procedure, permetalate from the instant generator system is reduced by a reducing agent such as tin chloride (divalent) in the presence of an ancillary ligand such as sodium tartrate and API (see experimental section below for details).

Indirect labeling with, for example, indium, yttrium, lanthanide or technetium and rhenium may be carried out by pre-binding a chelating ligand, preferably derived from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), cyclohexyl 1, 2-diaminetetraacetic acid (CDTA), ethylene glycol-O, O ' -bis (2-aminoethyl) -N, N ' -diacetic acid (HBED), triethylenetetraminehexaacetic acid (TTHA), 1, 4, 7, 10-tetraazacyclododecane-N, N ', N "-tetraacetic acid (1, 4, 7, 10-tetraazacyclododecane-N, N ', N" -tetraacetic acid, DOTA), 1, 4, 7-triazacyclononane-N, N ', N "-triacetic acid (1, 4, 7-triazacyclononane-N, N ', N' -triacylglycoic acid, NOTA), 1, 4, 8, 11-tetraazacyclotetradecane-N, N ', N ", N' '' -tetraacetic acid (1, 4, 8, 11-tetraazacyclotetradecane-N, N ', N' '' -tetracetic acid, TETA), mercaptoacetyldiglycine (MAG 2), mercaptoacetyltriglycine (MAG 3), Mercaptoacetylglycylcysteine (MAGC), cysteinylglycylcysteine (CGC). The chelating ligand has a suitable coupling group such as an active ester, maleimide, thiocarbamate or alpha-halogenated acetamide moiety. For the purpose of complexing chelating ligands with amine groups, e.g. epsilon-NH of lysine residues₂-group binding without prior reduction of the L-19-SIP compound.

The method of labelling a specific binding member may comprise labelling a polypeptide comprising a polypeptide selected from the group consisting of⁷⁶Br、⁷⁷Br、¹²³I、¹²⁴I、¹³¹I and²¹¹at is conjugated to the lysine residue or the N-terminus of the specific binding member and to the cysteine residue. The method may comprise conjugating the halogen carrier to a lysine or cysteine residue of the specific binding member, or to the N-terminus of the specific binding member. (i) Cysteine residues and (ii) lysine residues or the N-terminal either or bothTo be labelled with the same or different radioisotopes in accordance with the invention.

The specific binding members of the invention are designed for use in a method of diagnosis or treatment of a human or animal subject, preferably a human. The specific binding members are particularly useful in radiotherapy and radiodiagnostic methods.

Thus, the invention further provides a method of treatment comprising administration of a specific binding member as provided herein, a pharmaceutical composition 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 of manufacture of a medicament or pharmaceutical composition, comprising formulation with said specific binding member together with a pharmaceutically acceptable excipient.

Clinical indications for which the specific binding members of the invention may be used to provide therapeutic benefit include tumours such as any solid tumour, and other lesions resulting from pathological angiogenesis, including rheumatoid arthritis, diabetic retinopathy, age-related macular degeneration and haemangiomas.

Specific binding members of the invention may be used in methods of treatment of the human or animal body, such as methods of treatment (including prophylactic treatment) of a disease or condition in a human patient, which method comprises administering to the patient an effective amount of a specific binding member of the invention. Preferably, the treatment is radiation treatment. Conditions treatable in accordance with the invention are discussed elsewhere herein.

The specific binding members of the invention are useful in SPECT imaging, PET imaging, and therapy. Preferred isotopes for SPECT imaging include¹²³I and¹³¹I. preferred isotopes for PET are¹²⁴I。¹³¹I is a preferred isotope for use in therapy.

Since different isotopes for one element of imaging and therapy are used, the biodistribution of the respective immunoconjugates is the same. This and the use¹¹¹In-labeled derivatives were imaged to predict the respective⁹⁰Y markOther protocols for the biodistribution of therapeutic derivatives are advantageous, since the corresponding¹¹¹In and⁹⁰the biodistribution of the Y-labelled derivatives may vary. See carrasquallo j.a.et al (1999) J nuclear Med 40: 268-276.

Thus, the invention further provides a method of treatment comprising administration of a specific binding member as provided herein, a pharmaceutical composition 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 of manufacture of a medicament or pharmaceutical composition, comprising formulation with said specific binding member together with a pharmaceutically acceptable excipient.

According to the present invention, the provided compositions can be administered to an individual. Administration is preferably in a "therapeutically effective amount," which is an amount sufficient to show benefit to the patient. The benefit may be at least an improvement in at least one symptom. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the condition being treated. The determination of treatment prescriptions, e.g., dosages, etc., is within the responsibility of the general practitioner and other physicians. Suitable dosages of antibodies are well known in the art; see Ledermann j.a.et al (1991) Int j.cancer 47: 659 and 664; bagshawe k.d.et al (1991) antibodies, Immunoconjugates and Radiopharmaceuticals 4: 915 and 922.

The compositions may be administered alone or in combination with other therapies, simultaneously or sequentially, depending on the condition to be treated.

Specific binding members of the invention, including those comprising an antibody antigen-binding domain, may be administered to a patient in need of treatment by any suitable route, typically by injection into the bloodstream and/or directly into the treatment site, e.g. a tumour. Preferably, the specific binding member is administered parenterally. The precise dosage will depend on many factors, such as the route of treatment, the size and location of the area to be treated (e.g., tumor), the precise nature of the antibody (e.g., intact IgG1 antibody molecule, small immunoglobulin molecule), and the nature of any detectable label or other molecule attached to the antibody molecule. Typical antibody doses are 10-50 mg.

This is the dose for a single treatment of an adult patient, and can be adjusted appropriately for children and infants, or the doses of other antibody forms can be adjusted proportionally according to molecular weight. Treatment may be repeated once a day, twice a week, at weekly intervals, or one month, at the discretion of the physician.

The specific binding member of the invention is typically administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the specific binding member.

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

For intravenous injection or injection at the site of pathology, the active ingredient is in the form of an aqueous solution acceptable for parenteral administration, pyrogen-free and with suitable pH, isotonicity and stability. Suitable solutions may be prepared by those skilled in the art using, for example, isotonic media such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included as desired.

The compositions may be administered alone or in combination with other therapies, simultaneously or sequentially, depending on the condition to be treated. Other treatments may include the administration of suitable doses of analgesic drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or antiemetics.

The present invention provides a method comprising causing or causing a specific binding member provided by the present invention to bind to ED-B. As mentioned, such binding may take place in vivo, e.g. after administration of the specific binding member or nucleic acid encoding the specific binding member, or in vitro, e.g. in ELISA, Western blot, immunocytochemistry, immunoprecipitation or affinity chromatography.

The amount of binding of the specific binding member to ED-B can be determined. Quantification may be with respect to the amount of antigen in the test sample, which is of diagnostic interest.

The reactivity of the antibody to the sample can be determined by any means. Can be determined by Radioimmunoassay (RIA). The radiolabeled antigen is mixed with unlabeled antigen (test sample) and allowed to bind to the antibody. The bound antigen is physically separated from the unbound antigen and the amount of radioactive antigen bound to the antibody is determined. The more antigen in the test sample the less radioactive antigen binds to the antibody. Competitive binding assays for nonradioactive antigens may also be performed using antigens or analogs linked to reporter molecules. The reporter may be a fluorescent dye, a phosphorous or laser dye with spectrally separated light absorbing and light emitting properties. Suitable fluorescent dyes include fluorescein, rhodamine, phycoerythrin, and texas red. Suitable chromogenic dyes include diaminobenzidine.

Other reporter molecules include macromolecular colloidal particles or particulates such as colored, magnetic or paramagnetic latex beads, and biologically or chemically active agents that can directly or indirectly elicit a detectable signal that can be visualized, electronically detected, or otherwise recorded. These molecules may be, for example, enzymes which catalyze reactions which produce or change color or which cause changes in electronic properties. They may be molecularly excitable, whereby electronic transitions between energy states result in characteristic spectral absorption or emission. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be used.

The signal generated by a single antibody-reporter conjugate can be used to obtain quantifiable absolute or relative data of the binding of the relevant antibody in the sample (normal and test samples).

The invention further relates to a specific binding member which competes for binding to ED-B with any specific binding member which binds both antigen and a VH domain comprising a V domain comprising CDRs having amino acids substantially as set out herein, preferably a VH domain comprising VH CDR3 shown in SEQ id no: 3. Competition between binding members can be conveniently assayed in vitro, for example by labelling one binding member with a specific reporter molecule, which is detectable in the presence of other unlabelled binding members, thereby allowing the identification of specific binding members that bind the same epitope or overlapping epitopes. Competition can be determined, for example, using an ELISA as described in cartemolla et al (241996).

As described above, the method of producing a specific binding member of the invention may comprise expressing the encoding nucleic acid and may optionally comprise culturing the host cell under conditions in which the specific binding member is produced. The specific binding members and encoding nucleic acid molecules and vectors according to or used in accordance with the invention may be isolated and/or purified in substantially pure or homogeneous form from, for example, their natural environment, in the case of nucleic acids, none or substantially none of which is derived from a nucleic acid or gene other than the sequence encoding a polypeptide having the desired function.

The nucleic acids used in the present invention may comprise DNA or RNA and may be wholly or partially synthetic. The nucleotide sequences listed herein encompass DNA molecules having a particular sequence, as well as RNA molecules having a particular sequence, wherein U replaces T, unless otherwise specified.

Systems for cloning and expressing polypeptides in a variety of different host cells are well known in the art. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus systems. Mammalian cell lines expressing heterologous polypeptides available in the art include chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells, and many others. A generally 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, e.g., Pluckthun, a.bio/Technology 9: 545-551(1991). Expression in cultured eukaryotic cells can also be used by those skilled in the art to generate specific binding members, see recent reviews such as Ref, M.E (1993) curr. opinion biotech.4: 573-; trill j.j.et al (1995) curr.opinion Biotech 6: 553-560.

Suitable vectors may be selected or constructed containing appropriate regulatory sequences including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and other suitable sequences. The vector may be a plasmid, a virus such as a phage or a phagemid. See, for example, Molecular Cloning: a Laboratory Manual: 3nd edition, Sambrook et al, 2001, Cold Spring Harbor Laboratory Press. Many known techniques and Protocols for nucleic acid manipulation, such as preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and protein analysis are described in detail in Current Protocols in molecular biology, Second Edition, Ausubel et al, eds., John Wiley & Sons, 1992. The teachings of Sambrook et al and Ausubel et al are incorporated herein by reference.

The method of producing a specific binding member of the invention may further comprise introducing the encoding nucleic acid into a host cell. The introduction can be performed using any available technique. For eukaryotic cells, suitable techniques may include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses such as vaccinia, or for insect cells using baculovirus. For bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using phage.

Expression of the nucleic acid may be caused or allowed to occur after introduction, for example by culturing the host cell under conditions which allow expression of the gene.

In one embodiment, the nucleic acid of the invention is integrated into the genome (e.g., chromosome) of the host cell. Integration can be facilitated by including sequences in the genome that facilitate recombination according to standard techniques.

Other aspects and embodiments of the invention will be apparent to those skilled in the art from consideration of the present disclosure, including the examples set forth below. The methods of synthesis and labelling of specific binding members of the invention are exemplified fully in the examples below. These examples are intended to be illustrative of the invention only and not to be limiting. All documents mentioned in this specification are incorporated by reference.

Examples of aspects and embodiments of the invention

1. Preparation and characterization of the specific binding members of the invention

The following example uses the radiolabeled peptide compound L19-SIP.

1.1: synthesis of I-131-L19-SIP (Chloramine-T method)

230. mu.l of PBS (0.2M PBS, pH7.4) containing 200. mu. g L19-SIP was placed in a reaction flask, and reacted with 185MBq 2¹³¹I]NaI was mixed and reacted with 30. mu.L of freshly prepared chloramine-T solution (2mg/mL) in 0.2M PBS (pH 7.4). After 1 min, 50. mu.L of Na was added₂S₂O₅Solution (10mg/mL in PBS 0.2M, pH 7.4).¹³¹The I-labeled L19-SIP was purified by gel chromatography using a NAP-5 column (Amersham, eluent: PBS) previously blocked with 5ml of 0.5% bovine serum albumin in PBS.

Yield of radioactive chemical: 45.7 percent.

Purity of radioactive chemical product: 88.3% (SDS-PAGE).

Specific activity: 31.7 MBq/nmol.

Immunoreactivity: 76 percent.

1.2: synthesis of I-131-L19-SIP (iodogen method)

800 μ g L19-SIP dissolved in 800 μ l PBS (0.2M PBS, pH7.4) and 500MBq [ deg. ]¹³¹I]NaI was mixed and placed in a reaction flask (iodogen tube, PierceInc.). The mixture was gently shaken at room temperature for 30 minutes.¹³¹The I-labeled L19-SIP was purified by gel chromatography using a NAP-5 column (Amersham, eluent: PBS) previously blocked with 5ml of 0.5% bovine serum albumin in PBS.

Yield of radioactive chemical: 93.2 percent.

Purity of radioactive chemical product: 91.1% (SDS-PAGE).

Specific activity: 46.6 MBq/nmol.

Immunoreactivity: 78 percent.

1.3: synthesis of I-123-L19-SIP (iodogen method)

230. mu.l of PBS (0.2M PBS, pH7.4) containing 200. mu. g L19-SIP was mixed with 200MBq [2 ], [ solution ] of¹²³I]NaI was mixed and placed in a reaction flask (iodogen tube, Pierce Inc.). The mixture was gently shaken at room temperature for 30 minutes.¹²³The I-labeled L19-SIP was purified by gel chromatography using a 5ml 0.5% bovine serum albumin-blocked NAP-5 chromatography column (Amersham, eluent: PBS) previously used in PBS.

Yield of radioactive chemical: 81.6 percent.

Purity of radioactive chemical product: 89.6% (SDS-PAGE).

Specific activity: 61.2 MBq/nmol.

Immunoreactivity: 84 percent.

1.4: synthesis of I-124-L19-SIP (iodogen method)

230. mu.l of PBS (0.2M PBS, pH7.4) containing 200. mu. g L19-SIP was mixed with 50MBq [2 ], [ solution ] of¹²³I]NaI was mixed and placed in a reaction flask (iodogen tube, Pierce Inc.). The mixture was gently shaken at room temperature for 30 minutes.¹²⁴The I-labeled L19-SIP was purified by gel chromatography using a 5ml 0.5% bovine serum albumin-blocked NAP-5 chromatography column (Amersham, eluent: PBS) previously used in PBS.

Yield of radioactive chemical: 84.5 percent.

Purity of radioactive chemical product: 89.6% (SDS-PAGE).

Specific activity: 22.8 MBq/nmol.

Immunoreactivity: 86 percent.

1.5: (3- (4-hydroxy-3-, ") ¹³¹ I]Synthesis of iodo-phenyl) -propionate) -L19-SIP

Mu.g of (3- (4-hydroxy-phenyl) -N- (sulfonato-succinimidyl) propionate) was dissolved in 1mL of DMSO. Mu.l of chloramine-T (5mg/ml in PBS) was mixed with 74MBq [ solution ]¹³1I]NaI was mixed and neutralized with 15. mu.L PBS (0.2M, pH 7.4). Mu.l of a solution of (3- (4-hydroxy-phenyl) -N- (sulfonato-succinimidyl) propionate was added to chloramine-T/, [ solution ]¹³¹I]NaI solution and the mixture was allowed to react for 1 minute. Adding 40 μ L of Na₂S₂O₅(10mg/mL in PBS 0.2M, pH7.4), followed immediately by the addition of 230. mu.l of borate buffer (0.2M PBS, pH8.5) containing 200. mu. g L19-SIP.

(3- (4-hydroxy-3-)¹³¹I) Iodo-phenyl) -propionate) -L19-SIP was purified by gel chromatography using NAP-5 chromatography column previously blocked with 5ml PBS containing 0.5% bovine serum albumin (Amersham, eluent: PBS).

Yield of radioactive chemical: 37.2 percent.

Purity of radioactive chemical product: 94.6% (SDS-PAGE).

Specific activity: 10.3 MBq/nmol.

Immunoreactivity: and 69 percent.

1.6: MIRD calculation for I-131-L19-SIP

Based on the biodistribution data in tumor-bearing mice, the absorbed dose of L19-SIP labeled I-131 can be calculated by the MIRD formula (MIRD formalisms). Biokinetic modeling was performed using the% ID data of I-131-L19-SIP in mice bearing human glioblastoma (U251). Residence time is calculated by integrating the area under the bi-exponential and mono-exponential function curves from zero to infinity, including the biological and physical half-lives of the compounds.

Considering mouse organs as both radiation source and radiation target, the S value of the MIRDOSE 3.1 software can be used to estimate the I-131-L19-SIP dose as self-absorbed dose to self-organs (no radioactive cross-emission).

Mouse organ dose (mGy/MBq):

liver 50

Kidney 160

Spleen 50

Lung 220

Ovary 180-410 (dependent on ovulation cycle status and ED-B expression)

Uterus 600 (dependent on ovulation cycle status and ED-B expression)

Testis 55

Blood 130

Red bone marrow 50 (calculated based on blood dose)

Tumor 940 (calculated for 100mg tumor)

Using the residence time calculated in the MIRDOSE 3.1 program, the body absorbed dose of I-131-L19-SIP can be estimated.

Human organ dose (mGy/MBq):

adrenal gland 9.46E-02

Brain 1.64E-02

Mammary gland 7.33E-02

Gallbladder 1.00E-01

LLI wall 4.47E-01

Small intestine 1.10E-01

Stomach 9.45E-02

ULI wall 2.11E-01

Heart wall 6.22E-02

Kidney 1.86E-01

Liver 7.46E-02

Lung 7.04E-02

Muscle 8.71E-02

Ovary 8.07E-01

Pancreas 1.15E-01

Red bone marrow 9.11E-02

Bone surface 9.74E-02

Skin 7.20E-02

Spleen 7.11E-02

Testis 2.38E-01

Thymus 8.55E-02

Thyroid gland 8.54E-02

Bladder wall 7.19E-01

Uterus 4.72E-01

Whole body 8.78E-02

EFF DOSE EQUIV 3.60E-01

EFF DOSE 3.21E-01

It was concluded that the red bone marrow and reproductive organs (ovary/uterus and testis) are dose limiting organs. However, a therapeutic window based on dosimetry calculations appears to be advantageous and promising. The ratio of tumor dose to red bone marrow dose was found to be 18. Therefore, I-131-L19-SIP showed that the dose delivered to the tumor was 18 times the dose delivered to the red bone marrow, which was significant.

1.7: tumors after a single intravenous injection of I-131-L19-SIP into tumor-bearing nude mice Study of treatment

A single intravenous injection of I-131-L19-SI was made into U251 (glioblastoma) bearing nude mice (approximately 27g in weight). Study doses were 37MBq and 74MBq, respectively. In addition, a group of control animals (single injection of saline) was studied. Tumor size (mm) was measured using a caliper during the time after injection²)。

The growth of the tumors in nude mice monitored after a single intravenous injection of saline and I-131-L19-SIP, respectively, is shown in FIG. 6.

Animals injected with a single 74MBq I-131-L19-SIP per animal showed a significant effect on U251 tumor growth, arresting it for 18 days. The same was true for the low dose group (37MBq), except that the low dose group started a slight tumor growth on the last 5 days. In contrast, tumors in the control group continued to grow throughout the observation period.

The results of this study show the excellent potential of I-131-L19-SIP for the treatment of solid tumors.

1.8: I-123-L19-SIP imaging following a single intravenous injection of tumor-bearing nude mice

The substance of the present invention was injected intravenously at a dose of about 9.25MBq into nude mice (approximately 25g in body weight) carrying F9 (teratocarcinoma). Gamma camera imaging was performed at various times after administration of the substance.

Tumors can be unambiguously described by planar scintigraphy (planar scintigraphy) of I-123-L19-SIP in F9 (teratocarcinoma) -bearing nude mice at 4 and 24 hours post-injection. At 4 hours post-injection, only a weak background (not bound to specific organs, but obtained from blood) was detectable elsewhere in the body, except for strong uptake by the tumor. Although the signal remains in the tumor, over time, the background signal in other parts of the body disappears. Thus, only tumors were detectable 24 hours after injection.

The results of this study show the excellent potential of I-123-L19-SIP for imaging solid tumors.

2. Further examples and experiments

Materials and methods

scFv preparation and expression of scFv, Small Immunoglobulin (SIP) and IgG1 constructs

scFv (L19) (FIG. 1A) was affinity matured (Kd 5.4X 10) specifically for the ED-B domain of fibronectin^-11M) antibody fragment (13Pini et al, 1998). scFv (D1.3) (7 McCaffertyet al.; 26 Neri et al, 1997) was used as a control, a mouse scFv against chicken egg white lysozyme. These scFvs were expressed in E.coli strain HB2151(Maxim Biotech, San Francisco CA) as described by Pini et al (341997).

Small immunoglobulins

To construct the L19 mini-immunoglobulin (L19-SIP) gene (FIG. 1C), the DNA sequence encoding scFv (L19) was amplified by Polymerase Chain Reaction (PCR) using Pwo DNA polymerase (Roche) according to the manufacturer's instructions, primers BC-618(gtgtgcactcggaggtgcagctgttggagtctggg-SEQ ID NO: 8) and BC-619(gcctccggatttgatttccaccttggtcccttggcc-SEQ ID NO: 9) containing ApaLI and BspE I restriction sites, respectively. The amplified product was inserted into the pUT-epsilon SIP vector ApaLI/BspEI, thus providing the scFv gene having a secretion signal required for secreting the protein in the extracellular medium. The pUT-epsilon SIP vector was derived from the previously described pUT-SIP-long (33 Li et al, 1997) and is the CH4 domain (epsilon) of the human IgE secretory isoform IgE-S2_S2-CH 4; 35 Batista et al, 1996) in place of the human constant γ 1-CH3 domain. CH4 is a domain, ε, that dimerizes the IgE molecule_S2Isoforms contain a cysteine at the carboxy terminus, which passes through the interchainDisulfide bonds stabilize the IgE dimer. In the final SIP molecule, ScFv (L19) is related to ε_S2the-CH 4 domains are linked by a short GGSG linker. The SIP gene was then excised from plasmid pUT-epsilon SIP-L19 using HindIII and EcoR restriction enzymes and cloned into The mammalian expression vector pcDNA3(Invitrogen, Groningen, The Netherlands) containing The Cytomegalovirus (CMV) promoter to obtain construct pcDNA 3-L19-SIP.

The DNA sequence encoding scFv (D1.3) was amplified using primers BC-721(ctcgtgcactcgcaggtgcagctgcaggagtca-SEQ ID NO: 10) and BC-732(ctctccggaccgtttgatctcgcgcttggt-SEQ ID NO: 11) and inserted into the pUT-epsilon SIP vector ApaLI/BspEI. The D1.3-SIP gene was then excised from pUT-epsilon SIP-D1.3 with HindIII and EcoRI restriction enzymes and cloned into pcDNA3 to obtain the construct pcDNA 3-D1.3-SIP.

These constructs were used to transfect SP2/0 murine myeloma cells (ATCC, American Type Culture Collection, Rockville, Md., USA) using the FuGENE 6 transfection reagent (Roche) according to the manufacturer's optimized protocol for adherent cells. Transfectomas were grown in DMEM supplemented with 10% FCS and selected using 750. mu.g/ml Geneticin (G418, Calbiochem, san Diego, Calif.).

IgG1

To prepare intact IgG1, the variable region of the L19 heavy chain (L19-VH) was excised from the previously described L19-pUT epsilon SIP with HindIII and XhoI along with its secretory peptide sequence and inserted into a pUC-IgG1 vector containing the entire human gamma 1 constant heavy chain gene. The recombinant IgG1 gene was then excised from pUC-IgG1-L19-VH with HindIII and EcoRI and cloned into pcDNA3, resulting in construct pcDNA3-L19-IgG 1.

To prepare the complete L19 light chain, L19-VL was amplified by PCR from L19-pUT-epsilon SIP (described above) using primers BC-696(tggtgtgcactcggaaattgtgttgacgcagtc-SEQ ID NO: 12) and BC-697(ctctcgtacgtttgatttccaccttggtcc-SEQ ID NO: 13) containing ApaLI and BsiWI restriction sites, respectively. After digestion with ApaLI and BsiWI, the amplification product was inserted into the vector pUT-SEC-hC kappa containing the secretory signal sequence and the human constant region kappa light chain sequence. The recombinant light chain gene was then excised from pUT-SEC-hC kappa-L19-VL using HindIII and XhoI and inserted into the pCMV2 delta mammalian expression vector (obtained from the pcDNA3 vector by removal of the G418 resistant gene) to obtain the construct pCMV2 delta-L19-kappa.

Equimolar amounts of these constructs were used to co-transfect SP2/0 murine myeloma cells as described above. The Geneticin-selected clones were screened in ELISA for the ability to secrete intact heavy and light chain chimeric immunoglobulins.

All DNA constructs were purified using the Maxiprep system from Qiagen (Hilden, Germany) and the DNA sequences of both strands of the constructs were confirmed using the ABI PRISM dRhodamine terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, Foster City, Calif.). All Restriction Enzymes (RE) were obtained from Roche Diagnostics (Milan, Italy) except BsiWI (New England Biolabs, Beverly, Mass.). After RE digestion, the inserts and vectors were recovered from the agarose gel using the Qiaquick method (Qiagen).

Purification and quality control of antibodies

Immunoaffinity chromatography was performed to purify the different antibodies as described by Carnemolla et al (241996).

ED-B conjugated to Sepharose 4B (amersham pharmacia biotech, Uppsala, Sweden) according to the manufacturer's instructions (24 cartemolla et al, 96) was used for immunopurification of all the different L19 antibody formats, and chicken egg white lysozyme (Sigma, st.louis, USA) conjugated to Sepharose 4B (amersham pharmacia) was used for purification of D1.3 antibody.

The immunopurified antibody forms L19-SIP and L19-IgG1 were dialyzed against PBS, pH7.4 at +4 ℃ without further purification. Since scFv from immunoaffinity chromatography consists of both monomeric and dimeric forms, another purification step is required to isolate the latter as described by Demartis et al. (272001). Batches of different antibody formats were prepared and analyzed using SDS-PAGE, immunohistochemistry, size exclusion chromatography (Superdex 200, amersham pharmacia Biotech) and ELISA experiments under reducing and non-reducing conditions.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PA GE), enzyme-linked immunosorbent assay (ELISA), size exclusion chromatography and immunohistochemistry

Screening ELISA experiments were performed on conditioned media as described by Carnemolla et al (241996). To disclose the expression of different L19 antibody formats, a recombinant fragment 7B89 containing the ED-B domain of FN (24 cartemolla et al, 1996), including the epitope recognized by L19, was immobilized on Maxisorp immunoplates (Nunc, Roskilde, Denmark). To detect the D1.3 antibody in ELISA experiments, chicken egg white chicken lysozyme (Sigma) was immobilized on the surface of EIA plate NH2 (Costar, Cambridge, MA). Peroxidase-conjugated rabbit anti-human IgE (Pierce, Rockford, IL) diluted according to the manufacturer's instructions was used as a secondary antibody to detect SIP. Peroxidase-conjugated rabbit anti-human IgG (pierce) was used in the case of IgG 1. For the scFv containing the tag sequence FLAG, a mouse anti-human FLAG monoclonal antibody (M2, Kodak) and a peroxidase-conjugated goat anti-mouse antibody (Pierce) were used as secondary and tertiary antibodies, respectively. In all cases, immunoreactivity of the immobilized antigen was detected using the peroxidase substrate abts (roche) and absorbance at 405nm was measured.

Superdex200 (Amersham Pharmacia) column was used to analyze the gel filtration mode of the purified antibody under native conditions, using fast protein liquid chromatography (FPLC; Amersham Pharmacia).

Immunohistochemistry was performed on cryostat sections of different tissues as described in Castellani et al (221994) and 4-18% gradient SDS-PAGE was performed under reducing and non-reducing conditions as described in Carnemolla et al (171989).

Animals and cell lines

Athymic nude mice (8 week old nude/nude CD1 female) were obtained from Harlan Italy (Correzzana, Milano, Italy), 129(SvHsd clone) strain mice (8-10 weeks old, female) were obtained from Harlan UK (Oxon, England). SmallMurine embryonic teratocarcinoma cells (F9), human melanoma-derived cells (SK-MEL-28) and mouse myeloma cells (SP2/0) were purchased from American type culture Collection (Rockville, Md.). To induce tumors, nude mice were injected subcutaneously with 16X 10⁶SK-MEL-28 cells, 129 strain mice injected with 3X 10⁶F9 cells. Tumor volume was calculated using the following formula: (d)² x Dx 0.52, where D and D are the short and long diameters (cm) of the tumor, respectively, as determined by caliper. Animals were housed, handled and sacrificed according to the national laws on animal protection for scientific research purposes (italian law No.116, 27 January, 1992). Radioiodination of recombinant antibodies

Radioiodination of proteins according to the Chizzonite Indirect method (36 Riske et al, 1991), iodine-labeled tubes (Pierce) pre-coated with IODO-GEN were used in combination with Na according to the manufacturer's instructions¹²⁵I (NEN Life Science Products, Boston, MA). In the reported experiments, 1.0mCi of Na was used for 0.5mg of protein¹²⁵I. Radiolabeled molecules were pretreated with 0.25% BSA and equilibrated in PBS on a PD10(Amersham Pharmacia) column from free¹²⁵And (I) separating. The radioactivity of the samples was determined using a Crystal gamma counter (Packard Instruments, Milano, Italy). Immunoreactivity of the radiolabeled protein was performed on a 200. mu.l ED-B Sepharose column saturated with PBS containing 0.25% BSA. A known amount of radioiodinated antibody (200 μ l PBS containing 0.25% BSA) was applied to the top and allowed to pass into the column. The column was then rinsed with 1.5ml PBS containing 0.25% BSA to remove non-specifically bound antibody. Finally, the immunoreactive conjugate was eluted using 1.5ml of 0.1M TEA, pH 11. The radioactivity of the unbound and bound material was counted and the percentage of immunoreactive antibodies was calculated. The immunoreactivity was always higher than 90%.

To further analyze the radioiodinated antibodies, a known amount of 200. mu.l of radiolabeled protein was loaded onto a Superdex200 column. The retention volumes of the different proteins did not change after radioiodination. For the three radioiodinated L19 antibody formats and their negative controls, the radioactivity recovered from the Superdex200 column was 100% (fig. 3A, 3B and 3C).

Biodistribution test

To prevent¹²⁵Non-specific accumulation of I in the stomach and concentration in the thyroid, 20mg sodium perchlorate (CarloErba, Italy) in water was orally administered to mice 30 minutes prior to injection of radiolabeled antibody. This procedure was repeated at 24 hour intervals during the biodistribution experiments. Tumor bearing mice were injected in tail vein with 100 μ l saline containing 0.1nmole of a different radiolabeled antibody (6 μ g for scFv, 8 μ g for SIP, 18 μ g for IgG). Three animals were sacrificed at each time point, different organs including tumors were excised, weighed, counted in a gamma counter, then fixed with PBS, 5% formaldehyde in ph7.4, and subjected to microautoradiography as described by Tarli et al (231999).

Blood samples were taken to prepare plasma to determine the stability of the radiolabeled molecules in the bloodstream, using the immunoreactivity test and gel filtration assay already described. In both cases 200. mu.l of plasma were used. The radioactivity content of the different organs is expressed as a percentage of injected dose/gram (% ID/g). The parameters of blood clearance of radioiodinated antibodies were fitted using the least squares method using MacIntosh Program Kaleidagraph (Synergy Software, reading PA, USA) and the equation:

X(t)＝A exp(-(αt))+B exp(-(βt))，

wherein X (t) is the% ID/g of radiolabeled antibody at time t. This equation describes a bi-exponential blood clearance pattern, where the amplitude of the alpha phase is defined as Ax 100/(A + B) and the amplitude of the beta elimination phase is defined as Bx 100/(A + B). α and β are rate parameters relating to the half-life of the respective blood clearance period. T1/2(α phase) ═ ln2/α ═ 0.692./α, and T1/2(β phase) ═ ln2/α ═ 0.692./α. X (0) is assumed to equal 40%, corresponding to a blood volume of 2.5ml per mouse.

Results

Antibody preparation

The variable regions of the different antibody formats of L19(scFv, small immunoglobulin and intact human IgG1) were used (13Pini et al, 1998) and their properties to target tumor vessels in vivo.

Figure 1 shows constructs for expressing different forms of the L19 antibody. Similar constructs were made using variable regions of scFv specific for non-relevant antigens (D1.3; 7 McCafferty; 26 Neri et al, 1997).

To obtain SIP and IgG1, SP2/0 murine myeloma cells were transfected with the construct shown in FIG. 1 and stable transfectomas were selected using G418. The best producers were determined by ELISA and these clones were expanded for antibody purification. Purification of all three of these L19 antibody forms was based on immunoaffinity chromatography, using recombinant ED-B conjugated to Sepharose. The yield was approximately 8mg/L for scFv (L19), 10mg/L for L19-SIP and 3mg/L for L19-IgG 1. For the control protein, scFv specific for hen egg lysozyme (D1.3) was used, and the variable region of scFv D1.3 was used to construct D1.3-SIP. Both antibodies were purified on hen egg lysozyme conjugated to Sepharose. The yields were 8 and 5mg/l, respectively. As a control for L19-IgG1, commercially available human IgG 1/kappa (Sigma) was used.

The three purified forms of L19 were analyzed by SDS-PAGE under reducing and non-reducing conditions. For scFv (L19), the apparent molecular weight was approximately 28kDa under both reducing and non-reducing conditions as expected (not shown). L19-SIP showed a molecular weight of approximately 80kDa under non-reducing conditions and a molecular weight of approximately 40kDa under reducing conditions. The results indicate that more than 95% of the native molecules are present as covalently linked dimers. L19-IgG1 showed a major band of approximately 180kDa under non-reducing conditions, as expected, while two bands corresponding to a heavy chain of approximately 55kDa and a light chain of approximately 28kDa under reducing conditions were shown. The elution pattern of the three L19 antibody forms was obtained by analysis by size exclusion chromatography (Superdex 200). In all three cases, a single peak representing 98% or more with a normal distribution was detected. Apparent molecular weight vs scFv (L19) using a standard calibration curve₂60kDa, 80kDa for L19-SIP and 180kDa for L19-IgG 1. In addition, molecular aggregates, which are usually present in recombinant protein preparations and can invalidate the results obtained in vivo studies, are proven to be absent. SDS-PAGE and size exclusion chromatography (Superdex 200) of the purified control proteins gave similar results.

Immunohistochemical analysis was performed on cryostat sections of SK-MEL-28 human melanoma induced in nude mice and F9 murine teratocarcinoma induced in 129 strain mice using these three different L19 antibody formats. The best results are obtained at concentrations as low as 0.25-0.5 nM. All three purified L19 antibodies recognized the same structure.

In vivo stability of radiolabeled L19 antibody form

For in vivo biodistribution studies, SK-MEL-28 human melanoma and F9 murine teratocarcinoma were used. SK-MEL-28 tumors had a relatively slow growth rate, whereas F9 tumors grew rapidly (FIG. 2). Thus, the use of SK-MEL-28 tumors enabled long-term experiments (up to 144 hours), while F9 tumors were induced for short-term biodistribution studies (up to 48 hours). When the tumor is about 0.1-0.3 cm³All biological profiling experiments were performed. For comparison of the various antibody formats, an equimolar amount (0.1nmol) of antibody was injected in 100. mu.l sterile saline. Prior to injection, radioiodinated compounds were filtered through 0.22 μm, and immunoreactivity and gel filtration patterns were examined (see "materials and methods"). The immunoreactivity of radiolabeled proteins was always over 90%.

FIGS. 3A-C report a schematic representation of a gel filtration assay (Superdex 200) of the radioiodinated L19 antibody format.

Blood samples were taken from the treated animals at different time points after injection, and the radioactivity present in the plasma was analyzed for immunoreactivity and by gel filtration chromatography. The gel filtration plot shows that all three forms of the L19 antibody have a single major peak in the molecular weight of the injected protein. Only the graphical representation of scFv showed another peak with higher molecular weight, suggesting aggregate formation (FIGS. 3D-F). Furthermore, the formation of large molecular weight aggregates not eluted from the Superdex200 column was observed for scFv (L19) 2. In fact, the loaded radioactive yield of scFv (L19)2 was about 55% when both L19-SIP and L19-IgG were recovered from the Superdex200 column at 90-100% of the radioactivity used. The retained radioactivity was recovered only after washing the column with 0.5M NaOH, indicating that large aggregates were blocked on the column (table 1).

Table 1 also reports the results of immunoreactivity tests performed on plasma (see "materials and methods"). During the experiment, L19-SIP and L19-IgG1 maintained the same immunoreactivity in plasma as the starting reagent. In contrast, the immunoreactivity of scFv (L19)2 in plasma was reduced to less than 40% at 3 hours post injection.

Comparative biodistribution experiments

Tables 2a, b, c and FIG. 4 report the results obtained in a biodistribution experiment using a radiolabeled L19 antibody in SK-MEL-28 tumor-bearing mice.

Tables 2a, b, c show the mean% ID/g (± SD) of tissues and organs including tumors at various times after intravenous injection of radiolabeled antibody.

FIG. 4 shows the% ID/g change of different antibody formats in tumor (A) and blood (B) at different times of the experiment, and the ratio (C) between the% ID/g in tumor and blood. All three forms of the L19 antibody selectively accumulated in the tumor, and the ratios of% ID/g of tumor to other organs are reported in table 3.

As demonstrated by microscopic autoradiography, the antibody accumulated only in tumor vessels, but not specifically on vessels of normal organs. In contrast, no specific accumulation of radioiodinated control molecules was found in tumor or normal tissues (table 2a, b, c).

All three of these L19 antibody forms showed primarily kidney-mediated clearance as determined by counting urine samples. As expected, scFv (L19)2 cleared more rapidly and intact L19-IgG1 was slower. A double exponential function curve fit yields half-life values as reported in table 4.

FIG. 5 shows the change in% ID/g (+ -SD) of tumor and blood obtained with radioiodinated scFv (L19)2 and L19-SIP using the F9 teratocarcinoma tumor model. Due to the high angiogenic activity of F9 teratocarcinoma, the accumulation of radioactive molecules in this tumor was 3-4 fold higher than in SK-MEL-28 tumors at 3 and 6 hours after intravenous injection and continued to be higher for the 48 hours of the experiment. For SK-MEL-28 tumors, specific accumulation in tumor vessels was confirmed by microscopic autoradiography, and no specific tumor accumulation was observed after injection of control molecules. Table 5 reports the% ID/g of L19(scFv) and L19SIP at different times post injection in F9 tumor and other organs.

Synthesis of reduced L19-SIP

To 422. mu.l of PBS solution containing 375. mu.g (5nmol) L19-SIP was added 50. mu.l of TCEP-solution (14.34mg TCEPxHCl/5ml Na₂HPO₄Aqueous solution, 0.1M, pH 7.4). The reaction mixture was gently shaken at 37 ℃ for 1 hour. Reduced L19-SIP was purified by gel chromatography using a NAP-5 column (Amersham, Elurant: PBS). SDS-PAGE analysis of the isolated product confirmed the quantitative conversion of L19-SIP to reduced L19-SIP.

Yield: 100.3. mu.g/200. mu.l PBS (26.7%).

Synthesis of Tc-99m-L19-SIP

3.0mg of L-disodium tartrate was placed in a bottle, followed by addition of 200. mu.l PBS containing 100.3. mu.g reduced L19-SIP and application of the solution to 100. mu. lNa₂HPO₄-dilution with aqueous buffer (1M, pH 10.5). Mu.l Tc-99m generator (generator) eluent (24 hours) and 10. mu.l SnCl were added₂Solution (5mg SnCl)₂1ml0.1M HCl). The reaction mixture was shaken at 37 ℃ for 0.5 hour. Tc-99 m-labeled L19-SIP was purified by gel chromatography using a NAP-5 column (Amersham, eluent: PBS).

Yield of radioactive chemical: 35.6 percent.

Purity of radioactive chemical product: 90.2% (SDS-PAGE).

Specific activity: 26.4 MBq/nmol.

Immunoreactivity: 91.4 percent.

Tc-99m-MAG ₂ Synthesis of-L19-SIP carboxymethyl-t-butyl disulfide

A solution of 21.75ml (0.312mol) of 1-mercaptoacetic acid, 43.5ml (0.312mol) of triethylamine and 100g (0.312mol) of N- (tert-butylmercapto) -N, N' -di-BOC-hydrazine in 1L EtOH (abs.) is refluxed (N₂Gas) for 60 hours. EtOH was evaporated under reduced pressure to a final volume of about 200 ml. The residue was poured into 1.8L H₂O and the pH of the resulting suspension was adjusted to 7.14 using 5 moles NaOH. The di-BOC-hydrazine was filtered off and the pH of the resulting solution was adjusted to 2.2 using semi-concentrated HCl. With 600ml CH₂Cl₂The crude extract was extracted three times from water. The combined organic layers were passed over MgSO₄Drying and evaporation of the solvent under reduced pressure gave 41.1g (80%) of a yellow oil. The starting material is sufficiently pure for further synthesis.

N- (benzyloxycarbonyl-Gly) Gly t-butyl ester (Z- (N-Gly) Gly t-butyl ester

The reaction mixture containing 35.02g (114mmol) of Z-Gly-O succinimide and 15g (114mmol) of Gly-O-^t1.4L CH of Bu₂Cl₂Solution in N₂Stirring at room temperature for 20 hours under air. The organic layer was washed 3 times with 250ml of 1% aqueous citric acid and 200ml of half-saturated NaHCO₃The aqueous solution was washed 2 times and 1 time with 200ml water. The organic layer was passed over anhydrous MgSO₄And (5) drying. Evaporation of CH under reduced pressure₂Cl₂Yield 36.5g (99%) of Z-Gly-Gly-O-^tBu yellow oil. This crude extract is sufficiently pure for further synthesis.

Gly-Gly t-butyl ester

36.5g (113mmol) of Z-Gly-Gly-O^tBu was dissolved in 1L THF, followed by the addition of 3.65g palladium on charcoal (10%). The mixture is reacted with hydrogen₂The mixture was stirred at room temperature for 3 hours under a gas (1 atm). Applying the suspension with N₂Purification, filtration (PTFE filter: 0.45 μm) and concentration of the filtrate under reduced pressure gave 20.3g (95%) Gly-Gly-O-^tBu yellow oil. This crude extract is sufficiently pure for further synthesis.

Carboxymethyl-t-butyldithioglycylglycine t-butyl ester

430ml of CH containing 23.85g (115.6mmol) DCC₂Cl₂The solution was added dropwise to a solution containing 21.76g (115.6mmol) of Gly-Gly-O-^tBu, 20.84g (115.6mmol) carboxymethyl-t-butyl disulfide and 13.3g (115.6mmol) NHS in 1L CH₂Cl₂In solution. The resulting suspension is in N₂Stir overnight at room temperature under air. After filtration, the resulting solution was taken up in 400ml of half-saturated NaHCO₃The aqueous solution was washed 3 times with 400ml of water and 1 time. The dried organic layer (MgSO)₄) Evaporated under reduced pressure. The crude extract was purified by chromatography on silica gel using a solvent gradient ranging from CH₂Cl₂MeOH 99:1 to CH₂Cl₂MeOH 98.5: 1.5. 26.1g (64%) of a yellow oil are isolated.

Mercaptoacetyl glycyl glycine

26.32g (75.09mmol) carboxymethyl-t-butyldithioglycylglycine t-butyl ester in N₂Dissolved in 233ml TFA under air. The resulting solution was stirred at room temperature for 20 minutes. Under reduced pressure (5-10X 10)^-2mbar) and the resulting oil was dried for an additional 2 h under stirring (5-10X 10)^-2mbar). Add 250ml Et₂After O a white powder precipitated and the suspension was stirred for 3 hours. The product is filtered off and resuspended in 100ml Et₂And (4) in O. The resulting suspension was stirred overnight, the product was filtered off and the material was dried at room temperature under reduced pressure, yielding 20.46g (92.5%) of a white powder.

Mercaptoacetyl glycyl glycine NHS ester

Mercaptoacetylglycylglycine (1g, 3.4mmol) and N-hydroxysuccinimide (391mg, 3.4mmol) were mixed in a dry round-bottomed flask and dissolved in anhydrous DMF (4 ml). To anhydrous dioxane (2ml) containing DCC (700mg, 3.4mmol) was added while stirring the mixture. Precipitate formation (DCU) started within 15 minutes. After 1 hour, the precipitate was removed by vacuum filtration. The precipitate was washed with cooled dioxane. The dioxane was removed from the filtrate. The product was precipitated from the remaining DMF solution by addition of diethyl ether. The product was isolated by filtration, washed with cooled diethyl ether and dried in a vacuum desiccator. Yield: 1.33 (99%).

Tc-99m-MAG₂Synthesis of-epsilon-HN (Lys) -L19-SIP

Mu.l PBS containing 200. mu.g (2.66nmol) of non-reduced L19-SIP was diluted with 300. mu.l sodium borate buffer (0.1M, pH8.5) and dialyzed twice with 200ml phosphate buffer (0.1M, pH8.5), 1 hour each time, using Slide-A-Lyzer10,000MWCO (Pierce Inc., Rockford, IL, U.S. A.). Mu.l of mercaptoacetylglycylglycine NHS ester solution (0.50mg dissolved in 500. mu.l of phosphate buffer, 0.1M, pH8.5) was added and the reaction mixture was heated at 37 ℃ for 3 hours. The reaction mixture was dialyzed 2 times for 1 hour with 200ml of phosphate buffer (0.1M, pH8.5) and 1 time for 17 hours (overnight) using Slide-A-Lyzer10,000MWCO (Pierce Inc., Rockford, IL, U.S. A.). 3.0mg L-disodium tartrate was added to the flask followed by 90. mu.l Tc-99m generator eluent (daily elution) and 25. mu.l SnCl₂Solution (5mg SnCl)₂1ml0.1M HCl). The reaction mixture was shaken at 37 ℃ for 0.5 hour. Tc-99 m-labeled L19-SIP was purified by gel chromatography using a NAP-5 column (Amersham, eluent: PBS).

Yield of radioactive chemical: 55.1 percent.

Purity of radioactive chemical product: 94.5% (SDS-PAGE).

Specific activity: 15.2 MBq/nmol.

Immunoreactivity: 81.1 percent.

Synthesis of Re-188-L19-SIP

3.0mg of L-disodium tartrate was placed in a vial, followed by addition of 310. mu.l PBS containing 150. mu.g reduced L19-SIP-SH and the solution was taken up with 100. mu.l Na₂HPO₄Aqueous buffer (1M, pH 10.5) diluted. 100 μ l Re-188 generator eluent and 50 μ l SnCl were added₂Solution (5 mgSnCl)₂1ml0.1M HCl). The reaction mixture was shaken at 37 ℃ for 1.5 hours. Re-188 labeled L19-SIP was purified by gel chromatography using a NAP-5 column (Amersham, eluent: PBS).

Yield of radioactive chemical: 34.8 percent

Purity of radioactive chemical product: 97.2% (SDS-PAGE)

Specific activity: 13.5MBq/nmol

Immunological activity: 91.7 percent

Synthesis of reduced L19-SIP for specific binding of EDTA, CDTA, TETA, DTPA, TTHA, HBED, DOTA, NOTA, DO3A, and similar types of chelators to cysteine SH group

Mu.l of TCEP solution (14.34mg TCEPxHCl/5ml Na)₂HPO₄Aqueous solution, 0.1M, pH7.4) was added to 422 μ L PBS solution containing 375 μ g (5nmol) L19-SIP. The reaction mixture was gently shaken at 37 ℃ for 1 hour. The reduced L19-SIP was purified by gel chromatography using a NAP-5 column (Amersham, eluent: sodium acetate buffer, 0.1M, pH 5.0). SDS-PAGE analysis of the isolated product confirmed the quantitative conversion of L19-SIP to reduced L19-SIP.

Yield: 105.7. mu.g/200. mu.l (28.2%).

Synthesis of In-111-MX-DTPA-maleimide-S (Cys) -L19-SIP-R (R ═ reduced)

Mu.l of sodium acetate buffer (0.1M, pH5) containing 105. mu.g (2.8nmol) of reduced L19-SIP was reacted with 50. mu.l of dissolved 1, 4, 7-triaza-2- (N-maleimidovinylpp-amino) benzyl-1, 7-bis (carboxymethyl) -4-carboxymethyl 6-methylheptane (0.25mg of DTPA-maleimide in 500. mu.l of 0.1M pH5 sodium acetate buffer) at 37 ℃ for 3 hours. The reaction mixture was dialyzed 2 times against 200ml of sodium acetate buffer (0.1M, pH6), 1 hour each, using SlideA-Lyzer10,000MWCO (Pierce Inc., Rockford, IL, U.S. A.).

Adding 80 μ l of [ In-111 ]]InCl₃Solution (HCl, 1N, 40MBq, Amersham Inc.) and the reaction mixture was heated at 37 ℃ for 30 minutes.

In-111 labeled DTPA-maleimide-S (Cys) -L19-SIP was purified by gel chromatography using NAP-5 column (Amersham, eluent: PBS).

Yield of radioactive chemical: 51.6 percent

Purity of radioactive chemical product: 97.2% (SDS-PAGE)

Specific activity: 7.9MBq/nmol

Immunoreactivity: 88.5 percent

Synthesis of MX-DTPA-maleimide (1, 4, 7-triaza-2- (N-maleimidovinylp-amino) benzyl-1, 7-bis (carboxymethyl) -4-carboxymethyl 6-methylheptane)

512mg (1mmol) of { [3- (4-amino-phenyl) -2- (bis-carboxymethyl-amino) -propyl]- [2- (bis-carboxymethyl-amino) -propyl]-amino } -acetic acid (Macrocyclics inc. dallas, TX, u.s.a.) and 707mg (7mmol) triethylamine were dissolved in 3ml anhydrous DMF. 1ml of anhydrous DMF containing 400mg (1.5mmol) of 2, 5-dioxo-pyrrolidin-1-yl 3- (2, 5-dioxo-2, 5-dihydro-pyrrol-1-yl) -propionic acid (Aldrich) was added dropwise. The solution was stirred at 50 ℃ for 5 hours. 30ml of diethyl ether were slowly added. The reaction mixture was further stirred for 30 minutes. The precipitate was collected by filtration. The crude extract was purified by RP-HPLC (acetonitrile-: water-: trifluoroacetic acid/3: 96.9:0.1 to 99.9:0: 0.1). Yield: 61% (405mg, 0.61 mmol). MS-ESI: 664M⁺+1。

Synthesis of In-111-MX-DTPA- ε -HN (Lys) -L19-SIP

Mu.l PBS containing 200. mu.g (2.66nmol) of non-reduced L19-SIP was diluted with 300. mu.l sodium borate buffer (0.1M, pH8.5) and dialyzed 2 times, 1 hour each, against Slide-A-Lyzer10,000MWCO (Pierce Inc., Rockford, IL, U.S. A.) with 200ml sodium borate buffer (0.1M, pH 8.5). Mu.l of a 1, 4, 7-triaza-2- (p-isothiocyanato) benzyl-1, 7-bis (carboxymethyl) -4-carboxymethyl-6-methylheptane (MX-DTPA) solution (0.33mg of MX-DTPA dissolved in 500. mu.l of sodium borate buffer 0.1M, pH8.5) was added and the reaction mixture was heated at 37 ℃ for 3 hours. The reaction mixture was dialyzed against 200ml of sodium acetate buffer (0.1M, pH6.0) for 2 times 1 hour and 1 time 17 hours (overnight) using Slide-A-Lyzer10,000MWCO (Pierce Inc., Rockford, IL, U.S. A.).

Adding 80 μ l of [ In-111 ]]InCl₃Solution (HCl, 1N, 40MBq, Amersham Inc.) and the reaction mixture was heated at 37 ℃ for 30 minutes. In-111-labeled MX-DTPA-. epsilon. -HN (Lys) -L19-SIP was purified by gel chromatography using NAP-5 column (Amersham, eluent: PBS).

Yield of radioactive chemical: 72.4 percent

Purity of radioactive chemical product: 80.3% (SDS-PAGE)

Specific activity: 8.8MBq/nmol

Immunoreactivity: 77.5 percent

Synthesis of In-111-DOTA-C-benzyl-p-NCS-epsilon-HN (Lys) -L19-SIP

Mu.l PBS containing 200. mu.g (2.66nmol) of non-reduced L19-SIP was diluted with 300. mu.l sodium borate buffer (0.1M, pH8.5) and dialyzed 2 times, 1 hour each, against Slide-A-Lyzer10,000MWCO (Pierce Inc., Rockford, IL, U.S. A.) with 200ml sodium borate buffer (0.1M, pH 8.5). To the solution was added 50 μ l1, 4, 7, 10-tetraaza-2- (p-isothiocyanato) benzylcyclododecane-1, 4, 7, 10-tetraacetic acid (benzyl-p-SCN-DOTA, Macrocyclics inc., Dallas TX, u.s.a.) solution (1.5mg benzyl-p-SCN-DOTA dissolved in 5ml sodium borate buffer, 0.1M, ph8.5) and the reaction mixture was heated at 37 ℃ for 3 hours. The reaction mixture was dialyzed 2 times for 1 hour with 200ml of sodium acetate buffer (0.1M, pH6.0) and 1 time for 17 hours (overnight) using Slide-A-Lyzer10,000MWCO (Pierce Inc., Rockford, IL, U.S. A.).

Adding 80 μ l of [ In-111 ]]InCl₃Solution (HCl, IN, 40MBq, Amersham Inc.) the reaction mixture was heated at 37 ℃ for 30 minutes. In-111-labeled DOTA-C-benzyl-p-NCS-. epsilon. -HN (Lys) -L19-SIP was purified by gel chromatography using NAP-5 column (Amersham, eluent: PBS).

Yield of radioactive chemical: 70.8 percent

Purity of radioactive chemical product: 92.1% (SDS-PAGE)

Specific activity: 10.1MBq/nmol

Immunoreactivity: 75.1 percent of

Synthesis of Y-88-MX-DTPA- ε -HN (Lys) -L19-SIP

Mu.l PBS containing 200. mu.g (2.66nmol) of non-reduced L19-SIP was diluted with 300. mu.l sodium borate buffer (0.1M, pH8.5) and dialyzed 2 times, 1 hour each, against Slide-A-Lyzer10,000MWCO (Pierce Inc., Rockford, IL, U.S. A.) with 200ml sodium borate buffer (0.1M, pH 8.5). Mu.l of MX-DTPA solution (0.33mg of MX-DTPA dissolved in 500. mu.l of sodium borate buffer, 0.1M, pH8.5) was added, and the reaction mixture was heated at 37 ℃ for 3 hours. The reaction mixture was dialyzed 2 times for 1 hour against 200ml of sodium acetate (0.1M, pH6.0) buffer and 1 time for 17 hours (overnight) using Slide-A-Lyzer10,000MWCO (Pierce Inc., Rockford, IL, U.S. A.).

Adding 100 mul of [ Y-88 ]]YCl₃Solution (HCl, 1N, 75MBq, Oak Ridge national lab.) and the reaction mixture was heated at 37 ℃ for 30 minutes. Y-88-labeled MX-DTPA-. epsilon. -HN (Lys) -L19-SIP was purified by gel chromatography using NAP-5 column (Amersham, eluent: PBS).

Yield of radioactive chemical: 68.1 percent of

Purity of radioactive chemical product: 91.5% (SDS-PAGE)

Specific activity: 11.4MBq/nmol

Immunoreactivity: 70.5 percent

Synthesis of Lu-177-DOTA-C-benzyl-p-NCS-epsilon-HN (Lys) -L19-SIP

Mu.l PBS containing 200. mu.g (2.66nmol) of non-reduced L19-SIP was dissolved with 300. mu.l sodium borate buffer (0.1M, pH8.5) and dialyzed 2 times, 1 hour each, with 200ml sodium borate buffer (0.1M, pH8.5) using Slide-A-Lyzer10,000MWCO (Pierce Inc., Rockford, IL, U.S. A.). Mu.l of benzyl-p-SCN-DOTA solution (1.5mg dissolved in 5ml sodium borate buffer 0.1M, pH8.5) was added and the reaction mixture was heated at 37 ℃ for 3 hours. The reaction mixture was dialyzed 2 times for 1 hour with 200ml of sodium acetate buffer (0.1M, pH6.0) and 1 time for 17 hours (overnight) using Slide-A-Lyzer10,000MWCO (Pierce Inc., Rockford, IL, U.S. A.).

Adding 200 μ l of [ Lu-177 ]]LuCl₃Solution (HCl, 1N, 80MBq, NRH-Petten, Netherlands) and the reaction mixture was heated at 37 ℃ for 30 minutes. Lu-177 labeled DOTA-C-benzyl-p-NCS-E-HN (Lys) L19-SIP was purified by gel chromatography using NAP-5 column (Amersham, eluent: PBS).

Yield of radioactive chemical: 72.2 percent

Purity of radioactive chemical product: 94.9% (SDS-PAGE)

Specific activity: 18.3MBq/nmol

Immunoreactivity: 73.4 percent

Organ distribution and excretion of In-111-MX-DTPA-L19-SIP following a single intravenous injection into tumor-bearing nude mice

The labeled peptides of the invention were injected intravenously at a dose of about 37kBq into animals bearing F9 (teratocarcinoma) (body weight about 25 g). The radioactivity in the various organs and in the excreta at various times after administration of the substances was determined using a gamma counter.

The biodistribution (mean. + -. SD, 3 animals) of In-111-MX-DTPA-L19-SIP In F9 (teratocarcinoma) -bearing nude mice is shown In Table 6.

Organ distribution and excretion of Tc-99m-L19-SIP following a single injection into tumor-bearing nude mice

The labeled peptide was injected intravenously at a dose of about 56kBq into animals carrying F9 (teratocarcinoma) (body weight of about 25 g). The concentration of radioactivity in each organ and the radioactivity in the excreta at different times after administration of the substance were determined using a gamma counter. In addition, tumor blood ratios were established at different times based on the concentration of the peptide in the tumor and blood.

The biodistribution (mean. + -. SD, 3 animals) of Tc-99m-L19-SIP in F9 (teratocarcinoma) -bearing nude mice is shown in Table 7.

The tumor blood ratios (mean. + -. SD, 3 animals) of Tc-99m-L19-SIP in F9 (teratocarcinoma) -bearing nude mice are shown in Table 8.

Radiolabeled peptides have been shown to have advantageous properties in animal experiments. For example, Tc-99m-L19-SIP and In-111-MX-DTPA- ε -HN (Lys) -L19-SIP showed high tumor accumulation at 1 hour after injection (p.i.) of 17.2(Tc-99m) or 12.9 (In-111)% injected dose/g (ID/g). Significant tumor retention of 9.4(Tc-99m) or 13.0 (In-111)% ID/g was observed at 24 hours post p.i. injection. Thus, tumor uptake is significantly higher than other known In-111 or Tc-99m labeled antibody fragments (e.g., Kobayashi et al, J.Nuc.Med., Vol.41(4), pp.755-762, 2000; Verhaar et al, J.Nuc.Med., Vol.37(5), pp.868-872, 1996). Blood clearance of the compound yielded tumor/blood ratios of 13:1 and 6:1, respectively, 24 hours after injection.

Most notably, at 24 hours post p.i. injection, In-111-MX-DTPA-epsilon-hn (lys) -L19-SIP showed significantly lower renal uptake and retention (22.5% ID/g) compared to other high retention (120% ID/g) In-111 labeled recombinant antibody fragments as described by, for example, Kobayashi et al. Renal retention is a very common problem, often preventing the use of lanthanide-labeled compounds in radiotherapy.

The experimental results show the excellent potential of the radioimmunoconjugates described in the present invention for diagnostic and therapeutic applications, preferably to patients by parenteral administration.

Discussion of the related Art

The observation that cytotoxic anticancer drugs are more efficiently localized to normal tissues than to tumors (37Bosslet et al, 1998) prompted the start of studies on the possibility of selective drug delivery to tumors. However, effective targeting of tumors has two main requirements: 1) a specific, abundant, stable and readily available target site within the tumor for ligand molecules from the bloodstream, and 2) ligand molecules with suitable pharmacokinetic properties that can readily diffuse from the bloodstream to the tumor, and that have a high affinity for the target site to ensure its efficient and selective accumulation in the tumor.

Due to its distinctive characteristics, the tumor microenvironment is a possible pan-tumor (pan-tumor) target. In fact, tumor progression induces (and subsequently requires) significant alterations in tumor microenvironment components, particularly those of the extracellular matrix (ECM). The molecules that make up the ECM of solid tumors differ in number and mass from those of normal ECM. In addition, many of these tumor ECM components are common to all solid tumors and are responsible for general properties and functions such as cell infiltration (infiltration of normal cells into tumor tissue and cancer cells into normal tissue) and angiogenesis. Among the numerous molecules that make up the modified tumor ECM, the inventors focused on FN isoforms that contain an ED-B domain (B-FN.

B-FN is widely expressed in the ECM of all solid tumors tested to date and is persistently associated with the angiogenic process (22Castellani et al, 1994), but is undetectable in normal adult tissues (17 cartemolla et al, 1989). Targeted delivery of therapeutic agents to the endo-subcutaneous ECM overcomes the problems associated with interstitial hypertension of solid tumors (38 Jain et al 1988; 39 Jain, 1997; 40Jain RK, 1999).

L19(13 Pini et al 1998; 25 Viti, Canc. Res., 23Tarli, et al, 1999), a peptide having high affinity for the ED-B domain of FN (Kd 5.4X 10)^-11M) that selectively and efficiently accumulates around tumor neovessels in vivo and is capable of selectively transporting and accumulating any therapeutic molecule conjugated thereto to and in tumors (28 Birchler et al, 1999; 29Nilsson, et al, 2001; 30 Hall et al.2002; 31 cartemolla et al, 2002). The ability of L19 to selectively target tumors has been demonstrated in patients using scintigraphic techniques.

The present specification reports the labeling of Small Immunoprotein (SIP) with radioisotopes, the use of radiolabeled SIP, and the tumor vascular targeting potency and pharmacokinetic properties of three different L19 human antibody formats, scFv, small immunoglobulin/small immunoprotein and intact human IgG 1.

SIP molecules are fused to the secreted isoform S of human IgE by scFv (L19)₂And epsilon CH4 domain. The epsilon CH4 is a domain that dimerizes the IgE molecule, the S₂Isoforms contain cysteines at the COOH terminus that covalently stabilize the dimer through interchain disulfide bonds (35 Batista et al, 1996). The IgE binding site of Fc ε RI is located in the CH3 domain (41Turner and Kinet, 1999; 42 Vangelisa et al, 1999; 43 Garman et al, 2000), so that scFv fused to ε CH4 domain does not activate any signal production that leads to hypersensitivity reactions according to embodiments of the present invention.

The expression of these three forms in two different tumor models in mice (murine F9 teratocarcinoma and human SK-MEL-28 melanoma) has been studied. F9 teratocarcinoma is a rapidly growing tumor that kills animals within about 2 weeks once implanted. SK-MEL-28 tumors, on the other hand, exhibit a biphasic growth curve, an early fast-growing phase followed by a second slower-growing phase. The amount of ED-B in F9 teratocarcinoma has previously been shown to remain stable during tumor growth (23Tarli, et al, 1999); in contrast, ED-B accumulates in SK-MEL-28 melanoma, in proportion to tumor growth capacity (23Tarli et al, 1999), with abundant ED-B found in the first phase and lesser amounts in the second phase. The use of SK-MEL-28 melanoma makes it possible to study biodistribution over a long period of time without significant changes in the tumour mass that could lead to misjudgement (FIG. 2).

Comparative studies of the stability of the three L19 antibody forms in vivo showed that L19-SIP and L19-IgG1 maintained the same immunoreactivity and molecular weight in plasma during the experiment (144 hours) as before injection. In contrast, scFv (L19) rapidly lost its immunoreactivity in plasma and produced aggregates that were too large to enter the gel filtration column. This aggregation of scFv is likely related to the tumor to lung% ID/g ratio, as aggregates can accumulate in the microvasculature of the lung (table 3). For all three forms, blood clearance is primarily mediated through the kidney, showing a biphasic curve with alpha and beta phases as reported in table 4, which is inversely proportional to molecular size.

The accumulation of the different antibody forms in the studied tumors is a consequence of the clearance rate and in vivo stability of the molecules. Using scFv, a maximum in the percentage injected dose/g (% ID/g) was observed 3 hours after injection of radiolabeled antibody, and then rapidly decreased. Using SIP, the% ID/g in tumors is 2-5 fold higher than that of scFv, reaching a maximum 4-6 hours after injection. This pattern was observed in both F9 and SK-MEL-28 tumors. In contrast, the accumulation of IgG1 in the tumor continued to increase during the experiment. However, due to its slow clearance, the tumor blood ratio of% ID/g after 144 hours was only about 3, in contrast to 10 for scFv and 70 for SIP after the same time (FIG. 4).

The same unique properties of in vivo stability, clearance and tumor targeting properties demonstrated by the three antibody formats studied in this invention can be used for different diagnostic and/or therapeutic purposes, depending on clinical needs and disease conditions. For example, radiolabeled antibodies that show good tumor-organ and tumor-blood ratios immediately after injection are necessary for in vivo diagnostic immunoscintigraphy, primarily because of the short half-life of the isotopes used in this assay.

There are different approaches to using antibodies as carriers for therapeutic agents: delivering a substance that exhibits its therapeutic effect after reaching its target site (e.g., a photosensitizer that is activated only at the target site), and for that reason is correlated with the absolute amount delivered to the tumor; the delivery of substances that exert their therapeutic and toxic effects before reaching their target site (e.g., the β emitter Yttrium-90) requires special attention to the ratio of the area under the tumor to blood accumulation curve as a function of time to minimize systemic toxicity and maximize the anti-tumor therapeutic effect.

For example, L19-SIP appears to provide the best coordination of molecular stability, clearance and tumor accumulation. Similar fusion proteins consisting of scFv antibody fragments bound to a dimerization domain have been described (44 Hu et al, 1996; 33 Li et al, 1997), but in both cases human γ 1CH3 was used as dimerization domain. Human epsilon_S2The use of the CH4 domain provides an easy way to obtain covalent stabilization of the dimer. In addition, the disulfide bond formed by the C-terminal cysteine residue can be readily reduced under sufficiently mild conditions to protect the overall structure of the molecule, thus providing an easily accessible reactive group for radiolabeling or chemical conjugation. This feature appears to have particular potential for clinical use.

The three step procedure of removal of circulating antibodies was not only useful for therapeutic purposes, but also for diagnostic immunoscintigraphy (45 Magnani et al 2000), although the accumulation of L19-IgG1 was largely in the tumor, which was offset by the slow blood clearance rate.

Reference to the literature

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35.Batista et al.J.Exp.Med.，184：2197-205，1996。

36.Riske et al.J.Biol.Chem.，266：11245-11251，1991。

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39.Jain.Vascular and interstitial physiology of tumors.Role in cancerdetection and treatment.In：R.Bicknell，C.E.Lewis and N.Ferrara(eds).Tumour Angiogenesis，pp.45-59.New York：Oxford University Press，1997。

40.Jain.Annu.Rev.Biomed.Eng.，1：241-263，1999。

41.Turner and Kinet.Nature，402Suppl.，B24-B30，1999。

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TABLE 1 immunoreactivity of radiolabeled antibodies at various time points after intravenous injection (I)^*) And radioactivity recovery (R) from superdex200

Immunoreactivity (%) in plasma and radioactive recovery (%) from Superdex200 were determined as described in materials and methods.

^*: for standardization, immunoreactivity assay results refer to the percentage value of immunoreactivity prior to intravenous injection.

Nd: not determined

TABLE 2a biodistribution experiment of radiolabeled L19 and D1.3 antibody fragments in SK-MEL-28 tumor-bearing mice

L19(scFv)

	3h	6h	24h	48h	72h	144h
							Tumor(s)	2.47±0.65	2.01±0.72	1.62±0.43	0.95±0.14	0.68±0.04	0.32±0.14
Blood, blood-enriching agent and method for producing the same	1.45±0.58	0.54±0.12	0.10±0.03	0.04±0.01	0.03±0.02	0.03±0.01
							Liver disease	0.48±0.20	0.18±0.05	0.04±0.01	0.02±0.00	0.02±0.01	0.02±0.00
Spleen	0.67±0.28	0.27±0.04	0.07±0.02	0.03±0.00	0.02±0.01	0.02±0.00
							Kidney (A)	4.36±0.32	1.67±0.08	0.16±0.01	0.06±0.01	0.04±0.02	0.03±0.00
Small intestine	0.77±0.21	0.57±0.05	0.24±0.06	0.17±0.04	0.12±0.05	0.09±0.01
							Heart and heart	0.77±0.20	0.31±0.07	0.07±0.02	0.02±0.00	0.02±0.01	0.02±0.00
Lung (lung)	2.86±0.34	1.50±0.67	1.07±0.42	0.73±0.39	0.55±0.11	0.51±0.22

D1.3(scFv)

	3h	6h	24h	48h	72h	144h
							Tumor(s)	1.03±0.74	0.87±0.42	0.15±0.10	0.07±0.02	Nd	Nd
Blood, blood-enriching agent and method for producing the same	1.52±0.86	0.81±0.13	0.02±0.00	0.01±0.00	Nd	Nd
							Liver disease	1.19±0.65	0.66±0.26	0.14±0.04	0.03±0.08	Nd	Nd
Spleen	1.05±0.88	0.42±0.33	0.07±0.02	0.05±0.01	Nd	Nd
							Kidney (A)	3.01±2.48	1.83±0.76	0.48±0.01	0.18±0.05	Nd	Nd
Small intestine	0.56±0.54	0.56±0.13	0.17±0.03	0.02±0.01	Nd	Nd
							Heart and heart	0.86±0.54	0.55±0.84	0.02±0.01	0.01±0.00	Nd	Nd
Lung (lung)	1.28±0.65	1.06±0.88	0.04±0.01	0.03±0.01	Nd	Nd

Results are expressed as percent injected antibody dose per gram of tissue (% ID/g). + -. SD

nd: not determined

TABLE 2b biodistribution assay of radiolabeled L19-SIP and D1.3-SIP in SK-MEL-28 tumor-bearing mice

L19 SIP

	3h	6h	24h	48h	72h	144h
							Tumor(s)	5.23±0.65	6.14±2.23	4.20±2.47	2.57±0.31	2.33±0.90	1.49±0.65
Blood, blood-enriching agent and method for producing the same	9.82±0.68	5.03±0.52	1.39±0.06	0.29±0.04	0.08±0.02	0.02±0.01
							Liver disease	2.65±0.14	1.74±0.31	0.50±0.06	0.19±0.01	0.10±0.02	0.05±0.01
Spleen	3.76±0.36	2.43±0.24	0.71±0.05	0.26±0.04	0.13±0.01	0.17±0.18
							Kidney (A)	7.33±0.91	3.87±0.21	1.09±0.05	0.30±0.04	0.14±0.02	0.06±0.01
Small intestine	1.45±0.24	1.44±0.29	1.06±0.43	0.56±0.08	0.40±0.08	0.18±0.00
							Heart and heart	4.16±0.24	2.15±0.08	0.52±0.05	0.13±0.03	0.06±0.01	0.02±0.01
Lung (lung)	7.72±0.60	5.41±0.55	1.81±0.40	0.59±0.29	0.19±0.03	0.05±0.01

D1.3SIP

	3h	6h	24h	48h	72h	144h
							Tumor(s)	3.80±0.30	1.65±0.12	0.70±0.00	0.26±0.01	0.07±0.01	0.04±0.03
Blood, blood-enriching agent and method for producing the same	10.40±0.81	4.45±0.14	1.21±0.01	0.32±0.00	0.08±0.01	0.06±0.02
							Liver disease	4.05±0.98	2.73±0.33	1.43±0.07	0.51±0.21	0.15±0.08	0.02±0.01
Spleen	3.31±0.66	1.76±0.50	0.82±0.12	0.46±0.20	0.15±0.05	0.04±0.02
							Kidney (A)	8.41±0.49	4.64±0.06	1.47±0.05	0.36±0.20	0.16±0.03	0.06±0.01
Small intestine	2.03±0.55	1.06±0.20	1.02±0.06	0.14±0.03	0.08±0.02	0.12±0.04
							Heart and heart	3.28±0.20	1.81±0.02	0.29±0.01	0.06±0.00	0.05±0.01	0.04±0.01
Lung (lung)	6.16±0.28	4.52±0.07	1.16±0.05	0.09±0.00	0.06±0.01	0.05±0.01

Results are expressed as percent injected antibody dose per gram of tissue (% ID/g). + -. SD

nd: not determined

TABLE 2c biodistribution assay of radiolabeled L19-IgG1 and hIgG1 κ in SK-MEL-28 tumor bearing mice

L19 IgG1

	3h	6h	24h	48h	72h	144h
							Tumor(s)	4.46±0.08	5.39±1.01	6.70±2.10	7.80±2.51	8.90±2.52	11.22±3.19
Blood, blood-enriching agent and method for producing the same	16.04±0.81	12.02±1.65	8.31±1.77	5.12±1.42	5.02±3.81	4.87±0.26
							Liver disease	6.03±0.37	6.77±0.53	2.41±0.35	1.45±0.41	1.26±0.71	1.09±0.16
Spleen	6.63±1.34	6.37±1.37	2.51±0.47	2.01±0.32	1.80±1.02	1.51±0.29
							Kidney (A)	6.47±0.39	5.12±0.47	3.07±0.35	1.73±0.63	1.54±1.14	1.12±0.44
Small intestine	1.60±0.39	1.35±0.65	1.43±0.19	1.13±0.32	1.13±0.98	0.97±0.47
							Heart and heart	5.63±0.67	4.77±0.52	2.87±0.45	1.48±0.51	1.32±1.09	0.92±0.37
Lung (lung)	6.55±0.65	5.15±0.62	4.16±0.66	2.28±0.80	1.98±1.60	1.42±0.45

hIgG1κ

	3h	6h	24h	48h	72h	144h
							Tumor(s)	Nd	3.28±0.38	4.00±0.22	2.78±0.20	Nd	2.32±0.26
Blood, blood-enriching agent and method for producing the same	Nd	10.12±0.35	7.87±0.25	6.24±0.34	Nd	5.41±0.51
							Liver disease	Nd	4.02±0.09	2.06±0.10	1.90±0.24	Nd	1.28±0.03
Spleen	Nd	4.47±0.28	1.82±0.01	1.42±0.19	Nd	1.24±0.03
							Kidney (A)	Nd	5.40±0.19	2.56±0.06	2.08±0.22	Nd	1.30±0.15
Small intestine	Nd	0.72±0.07	0.46±0.05	0.36±0.03	Nd	0.31±0.01
							Heart and heart	Nd	3.80±0.15	2.52±0.21	0.99±0.18	Nd	1.48±0.13
Lung (lung)	Nd	4.82±0.92	3.64±0.08	1.75±0.32	Nd	1.09±0.13

Results are expressed as percent injected antibody dose per gram of tissue (% ID/g). + -. SD

nd: not determined

TABLE 4 kinetic parameters of blood clearance of the three L19 antibody forms

^a): relative number of two half-life moieties

TABLE 5 biodistribution assay of radiolabeled L19(scFv) and L19SIP in mice bearing F9 tumor

L19(scFv)

	3h	6h	24h	48h
					Tumor(s)	10.46±1.75	8.15±2.63	3.18±0.83	2.83±0.71
Blood, blood-enriching agent and method for producing the same	2.05±0.38	1.88±1.14	0.17±0.01	0.06±0.02
					Liver disease	1.62±1.67	0.73±0.51	0.07±0.01	0.04±0.02
Spleen	1.53±0.36	0.90±0.54	0.11±.0.01	0.05±0.01
					Kidney (A)	12.70±0.73	4.37±0.98	0.24±0.03	0.18±0.08
Small intestine	0.68±0.15	0.95±0.23	0.24±0.01	0.17±0.06
					Heart and heart	1.35±0.21	0.81±0.38	0.08±0.02	0.04±0.01
Lung (lung)	2.88±0.29	2.06±0.69	0.38±0.60	0.33±0.05

L19 SIP

	3h	6h	24h	48h
					Tumor(s)	17.46±1.93	16.65±2.59	15.32±2.17	12.00±1.91
Blood, blood-enriching agent and method for producing the same	13.51±0.57	9.62±1.18	1.73±0.02	1.14±0.20
					Liver disease	2.81±0.37	2.39±0.13	0.54±0.14	0.32±0.00
Spleen	3.42±0.26	2.66±0.27	0.61±0.09	0.37±0.01
					Kidney (A)	9.18±0.76	5.85±0.50	1.16±0.05	0.76±0.06
Small intestine	0.95±0.03	1.36±0.21	0.83±0.11	1.04±0.14
					Heart and heart	4.64±0.24	3.67±0.46	0.67±0.06	0.46±0.14
Lung (lung)	5.61±0.01	5.93±0.57	1.66±0.19	0.91±0.08

Results are expressed as percent injected antibody dose per gram of tissue (% ID/g). + -. SD

nd: not determined

TABLE 6

TABLE 7

TABLE 8

	1h p.i.	3h p.i.	24h p.i.
				Blood ratio of tumor	1.01±0.33	2.54±0.74	12.81±4.03

Sequence listing

<110> Feilogen shares Co

SCHERING AG

<120> targeting of tumor vasculature using radiolabeled antibody L19 against fibronectin ED-B

<130>SMWFP6177075

<140>EP 03255633.4

<141>2003-09-10

<140>US 60/501,881

<141>2003-09-10

<150>

<151>

<160>13

<170>PatentIn version 3.1

<210>1

<211>5

<212>PRT

<213>Homo sapiens

<400>1

<210>2

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<213>Homo sapiens

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<213>Homo sapiens

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<213>Homo sapiens

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<210>5

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<213>Homo sapiens

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<211>10

<212>PRT

<213>Homo sapiens

<400>6

<210>7

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<213>Artificial sequence

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<223>Linker peptide

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Claims (20)

1. A specific binding member that binds human ED-B, wherein said specific binding member is for use¹³¹I isotopically labelled, and comprising an antigen binding site comprising an antibody VH domain and an antibody VL domain, wherein said antibody VH domain is selected from the group consisting of the L19VH domain and a VH domain comprising VH CDR1, VH CDR2 and VH CDR3, wherein said VH CDR3 is SEQ ID NO:3, and VH CDR1 is SEQ ID NO: 1, and VH CDR2 is L19 VHCDR1 of SEQ ID NO: 2L 19VH CDR 2; whereinThe antibody VL domain is selected from the group consisting of the L19VL domain and a VL domain comprising VL CDR1, VL CDR2 and VL CDR3, wherein the VL CDR3 is SEQ ID NO: 6, and a VL CDR1 of SEQ ID NO: 4, and a VL CDR2 of SEQ ID NO: 5, L19VLCDR 2; wherein the specific binding member comprises a small immunoglobulin comprising an epsilon_S2-CH4 fusing and dimerizing the antibody VH domain and antibody VL domain.

2. The specific binding member of claim 1 comprising an antibody VH domain comprising a VH domain having the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2 and SEQ ID NO:3, which specific binding member competes with the ED-B binding domain of an antibody comprising the L19VH domain and the L19VL domain for binding to ED-B.

3. A specific binding member according to claim 1 or 2 comprising the L19VH domain.

4. The specific binding member according to claim 3 comprising the L19VL domain.

5. A specific binding member according to claim 1 or 2 wherein the antibody VH domain and antibody VL domain are located at an angle to epsilon_S2-CH4 fused scFv antibody molecule.

6. The specific binding member of claim 5, wherein the scFv antibody molecule is conjugated to epsilon via a linker peptide_S2-CH4 fusion, wherein the linker peptide has the amino acid sequence GGSG.

7. A method of producing a specific binding member according to any one of claims 1 to 6, said method comprising administering to a subject in need thereof a therapeutically effective amount of a compound of formula I¹³¹I isotope labeling of the specificityA sexual coupling member.

8. The method of claim 7, wherein said labeling comprises oxidation in the presence of said specific binding member¹³¹I^-A halide of (a).

9. The method of claim 7, wherein said labeling comprises labeling a cell containing¹³¹I an activated bifunctional halogen carrier of a radioisotope is conjugated to a lysine or cysteine residue of the specific binding member or to its N-terminus.

10. A method according to any one of claims 7 to 9, wherein the method comprises expressing the nucleic acid encoding the specific binding member prior to labelling.

11. The method of claim 10, comprising culturing the host cell under conditions to produce the specific binding member.

12. The method of any one of claims 7-9, further comprising isolating and/or purifying the specific binding member.

13. A method according to any one of claims 7 to 9, further comprising binding said specific binding member to ED-B or a fragment of ED-B in vitro.

14. A composition comprising a specific binding member according to any one of claims 1 to 6 for use in a method of treatment of the human or animal body by therapy.

15. The composition of claim 14 for use in a method of treating a lesion of pathological angiogenesis.

16. The composition of claim 14 for use in a method of treating a tumor.

17. A composition comprising a specific binding member according to any one of claims 1 to 6 for use in a diagnostic method.

18. Use of a specific binding member according to any one of claims 1 to 6 in the manufacture of a medicament for the treatment of lesions of pathological angiogenesis.

19. Use of a specific binding member according to any one of claims 1 to 6 in the manufacture of a medicament for the treatment of tumours.

20. Use of a specific binding member according to any one of claims 1 to 6 in the manufacture of a diagnostic agent.