WO1990006323A2

WO1990006323A2 - Chimeric proteins incorporating a metal binding protein

Info

Publication number: WO1990006323A2
Application number: PCT/US1989/005424
Authority: WO
Inventors: Hubert J. P. Shoemaker; John Ghrayeb; Lee K. Sun
Original assignee: Centocor, Inc.
Priority date: 1988-11-29
Filing date: 1989-11-29
Publication date: 1990-06-14
Also published as: WO1990006323A3

Abstract

A chimeric protein comprising a protein having an affinity for a biological target linked through a peptide linkage to a metal binding protein or functional domain thereof not normally associated with the protein are described. The protein can be an immunoglobulin specific towards tumor or profileration associated antigens or specific towards markers of cardiovascular disease. The chimeric proteins of the present invention are useful in in vivo diagnostic techniques such as immunoscintigraphy and also as an immunotherapeutic agent.

Description

CHIMERIC PROTEINS INCORPORATING A

METAL BINDING PROTEIN

Background

Generally, two approaches have been taken to label proteins with radiometals for radiodiagnostic and radiotherapeutic procedures. The first is the direct labelling method by which a radiometal is bound to the protein molecule itself. The second is the indirect labelling method by which a bifunctional agent is coupled to the protein and the radiometal is attached to the protein through the bifunctional agent.

Various attempts have been made to label antibodies with radiometals by the indirect

approach. For example, Khaw et al. , Scie nce 209:

295-297 (1980) disclose the labeling of antibodies to cardiac myosin with indium-111 via the bifunctional agent diethylenetriaminepentaacetic acid and use of the labeled antibodies to image for myocardial infarction. In addition, Krejarek et al.,

Bicohem, Biopys . Res. Commun. 77: 581-585 (1977),

Fritzberg et al. , J . Nucl. Med., 27: 957 (1986), and Childs et al., J.Nucl . Med, 26:293 (1985) teach a chelating agent conjugated onto a protein and subsequent attachment of a radiometal through the chelating agent.

Tolman et al. in U.S. Patent No. 4,732,864, describe conjugates of target seeking biologically active molecules which are trace-labelled by

covalent conjugation to bifunctional metal chelates which are suitable for diagnostic or therapeutic applications. Tolman et al. teach the use of antibodies or other proteins as the target-seeking biologically active molecules and metallothionein as a possible metal chelate.

Various degrees of success have been achieved with indirect labelling methods. However, the labelled product is often difficult to prepare, has reduced immunoreactivity and is unstable in vivo. Further, techniques for purifying the labelled product before use are often required. A need exists for improved methods of stably labelling proteins with radiometals for radiodiagnostic and radiotherapeutic procedures.

Summary of the Invention

This invention pertains to chimeric proteins which can be stably labelled with a metal,

preferably a radiometal, for diagnostic and

therapeutic procedures and to the chimeric proteins so-labelled. The chimeric proteins comprise a protein having an affinity for a biological target linked to a metal binding protein (or the metal binding domain of a protein) which is not normally associated with the protein (i.e. derived from another protein or peptide). The linkage is a peptide linkage such that the two components form a contiguous polypeptide. Incorporation of the metal binding region into a protein allows a protein to be labelled with a metal. The labelled chimeric proteins are useful as diagnostic and therapeutic agents, preferably radiodiagnostic and radio- therapeutic agents. The chimeric proteins of this invention are produced by incorporating a metal binding protein (or domain thereof) into the amino acid sequence of the protein specific for the biological target by genetic engineering techniques. Proteins specific for a biological target can include biological receptors such as immunoglobulins and immunoglobulin fragments and ligands for

biological receptors such as hormones and growth factors.

In particular, this invention pertains to recombinant immunoglobulins having a metal binding protein incorporated into one or more of their constituent chains and to immunoglobulins labelled with radiometals through the metal binding protein. The recombinant immunoglobulins can be stably labelled with a metal or radiometal for immunodiagnostics and immunotherapeutic procedures.

Several advantages are derived from incorporating a metal binding protein or functional domain thereof into the protein for labelling with a metal. The labelling of the protein is less labor intensive since the metal has an affinity for the metal binding region. The metal-protein complex is general more stable because of the affinity of the incorporated binding domain for the metal. Third, the need for additional coupling reagents which could evoke immune responses is eliminated.

Brief Description of Drawings

Figure 1 shows the construction of a plasmid

(hCγ4/MT) containing DNA encoding the human constant region of an immunoglobulin and human

metallothionein.

Figure 2 shows the construction of an expression vector containing the DNA encoding the variable region of the 17-1A tumor-specific antibody (17-1A V_H ) and DNA encoding the human constant region of an immunoglobulin linked to the DNA encoding human metallothionein (hCγ₄/MT).

Figure 3 is a graph showing the binding of radioiodinated murine 17-1A to HT29 cells in the presence of a purified IgG of 17-1A(o) or G4K/MT(o).

Figure 4 is a gel filtration HPLC chromatogram of Tc-99m-IgG2a.

Figure 5 is a gel filtration HPLC chromatogram of Tc-99m-IgG4K/MT. Detailed Description

The chimeric proteins of this invention are proteins which have an affinity for a biological target, into which a metal binding domain has been incorporated. The metal binding domain is one which is not normally associated with the protein and allows the chimeric protein to be stably labeled with a trace-metal, preferably a radiometal.

A metal is any metal capable of binding to a metal binding region which can either be detected in vivo or serve as a toxin to target cells. Metal binding proteins are proteins having an affinity for metals such as metallothionein. Examples of

radiometals which can be used in the present

invention are technetium-99m, In-111, Cu-67, Pd-109, Pd-103, Re-188, Au-198, Au-199, Ru-97, Hg-197,

Ag-111, Bi-212, Os-191 and Pb-203. The radiometals Tc-99m, In-111, Cu-67, and Pb-203 can be used in diagnostic applications and Cu-67, Pd-109, Re-188, and Au-199 can be used in therapeutic applications. An example of a non-radioactive metal which can serve as a toxin is zinc.

The chimeric protein can be produced by genetic engineering techniques. In general, DNA encoding the protein is ligated to the DNA encoding the metal binding protein or a sufficient protion of the metal binding domain. A sufficient portion of the metal binding domain is that portion necessary for

labelling with a radiometal. The DNA construct is designed so that the the resulting DNA construct is inserted into an expression vector which is

incorporated into an appropriate host cell to produce the chimeric protein. In preferred embodiments, the chimeric protein is an immunoglobulin protein into which a metal binding domain has been incorporated. Depending on the intended diagnostic or therapeutic use, the immunoglobulins can be specific for various

biological targets. For tumor diagnosis and

therapy, the immunoglobulins can be specific for tumor- or proliferation-associated antigens. These include antigens associated with gastrointestinal, breast, ovarian, lung and renal cancer cells. Some specific examples are the antigens defined by antibodies 17-1A (gastrointestinal tumors), 0C125 (ovarian carcinoma), 0V-TL3 (ovarian carcinoma), 103D2 (breast) and 123. C3 (renal carcinoma). Others include 72.3 (colon), DF3, 115D8, RC38 (renal), G250 (renal) and 55-2A.

In the cardiovascular field, the immunoglobulins can be specific for a variety of markers of cardiovascular disease. Some examples include RllDlO (myosin-specific) which can be used to detect and localize and evaluate myocardial infarction, 7E3, 10E5, S12 (platelet- specific) and T2G1, 59D8 and GC4 (fibrin- specific) which can be used to detect thromboses and atherosclerotic plaque.

In general, the metal binding protein is incorporated into at least one of the constituent chains of the immunoglobulin, preferably a heavy chain. This can be accomplished by preparing a DNA construct comprising, at minimum, a DNA segment which encodes (1) at least the functional portion of the variable region of an immunoglobulin chain l inke d to (2) a DNA segment encoding the metal binding protein or its functional domain.

Preferably, the DNA encoding the metal binding protein is linked to DNA encoding at least a portion of the constant region.

The DNA construct is assembled in or inserted into a DNA expression vector by standard techniques. Recipient cells capable of expressing the encoded product are then transfected with the DNA construct. Typically, the recipient cells are also transfected with the DNA encoding the counterpart chain. The transfected recipient cells are cultured and the expressed immunoglobulins or immunoglobulin chains are recovered.

Genes encoding the variable region of Ig light and heavy chains can be obtained from lymphoid cells which produce the antibodies specific for the desired target antigen. For example, the hybridoma cell lines which produce antibodies against the specific tumor antigens provide a source of

immunoglobulin variable region genes against those antigens. Cell lines can be produced by challenging a rodent with a tumor cell or a tumor antigen containing cell component or fraction, forming fused hybrid cells between antibody producing cells and a myeloma cloning the hybrid and selecting clones which produce antibody against tumor-associated antigen. See U.S. Patent No. 4,172,124 Koprowski et al. Preferred constant regions of the immunoglobulins are of human origin. Human constant regions can be obtained from antibody producing cells by standard cloning techniques. Alternatively, because genes representing the two classes of light chains and the five classes of heavy chains have been cloned, constant regions of human origin are readily available from these clones.

Genes encoding the metal binding region can be obtained from a DNA clone of the metal binding region or can be synthesized using standard

synthesis techniques. The preferred metal binding protein is metallothionein; however, other metal binding proteins or oligo- or polypeptides such as lys-cys-thr-cys-cys-ala can also be used.

Preferably, the metal binding protein is a human protein because this reduces immunogenicity.

The DNA construct encoding Immunoglobulin chain/metal binding protein and the counterpart chain can be assembled in two different expression vectors which can be used to cotransform a recipient cell. In this protocol, each vector contains two selectable genes-one for selection in a bacterial system and one for selection in a eukaryotic system- each vector having a different pair of such genes. These vectors allow production and amplification of the DNA constructs in bacterial systems and

subsequent cotransfection of eukaryotic cells and selection of the cotransfected cells. Examples of selectable genes for the bacterial system are the genes which confer ampicillin and the gene which couples chloramphenicol resistance. Two selectable genes for selection of eukaryotic transfectants are preferred: (i) the xanthine-guanine phosphoribosyl- transferase gene (gpt), and (ii) the phosphotrans- ferase gene from Tn5 (designated neo). Selection with gpt is based on the ability of the enzyme encoded by this gene to use xanthine as a substrate for purine nucleotide synthesis; the analogous endogenous enzyme cannot. In a medium c ontaining xanthine and mycophenolic acid which blocks the conversion of inosine monophosphate to xanthine monophosphate, only cells expressing the gpt gene can survive. The product of the neo blocks the inhibition of protein synthesis in eukaryotic cells caused by the antibiotic G418 and other antibiotics of its class. The two selection procedures can be used simultaneously or sequentially to select for the expression of immunoglobulin chain genes

introduced on two different DNA vectors into a eukaryotic cell.

The preferred recipient cell line for

production of the immunoglobulins of this invention is a myeloma cell. A myeloma cell can synthesize, assemble and secrete immunoglobulins encoded by transfected Ig genes. Further, it possesses the mechanism for glycosylation of the immunoglobulin. A particularly preferred recipient cell is the myeloma cell Sp2/0. This cell produces only immunoglobulin encoded by the transfected immunoglobulin genes. Myeloma cells can be grown in culture or in the peritoneum of mice where secreted immunoglobulin can be obtained from ascites fluid. Other lymphoid cells such as B lymphocytes or hybridoma cells can serve as suitable recipient cells.

Several methods exist for transfecting lymphoid cell with vectors containing DNA coding for the Immunoglobulin chain/metal binding protein hybrid. A preferred way of Introducing DNA into lymphoid cells is by electroporation. In this method, the cells and appropriate expression vectors are placed in an electroporation apparatus in media and

subjected to an electric field. After electroporation, the transfectants are selected in a growth medium. Other techniques which can be used to introduce DNA into many cell types are calcium phosphate precipitation, diethylaminoethyl

(DEAE) -dextran, or protoplast fusing. See Ausebel, F. M . et al., Current Protocals in Molecular Biology, Greene Publishing Associates, N.Y., (1988).

DNA constructs in appropriate vectors can be expressed in non-animal cells such as bacteria.

When expressed in bacteria, the immunoglobulin chains become part of inclusion bodies. Thus, the chains must be isolated and purified and then assembled into immunoreactive, radiometal-binding immunoglobulin molecules.

Radiolabeled proteins can be used in immunoscintigraphy. One important use is the imaging of tumors. Antibody fragments are preferred for most immunoscintigraphic techniques. Labeled Fab' fragments of tumor specific antibodies can be prepared and used to image primary or secondary tumors or myocardial infarctions. The preferred radioisotope of immunoscintigraphy is technetium-99m which has a single photon energy of 140 keV, a half-life of about 6 hours, and is readily available from a ⁹⁹Mo - ^99mTc generator.

The radiolabeled Fab' fragment is introduced into a subject. After it is introduced into the subject, sufficient time is allowed for the labeled

Fab' fragment to accumulate at the site of the tumor or myocardial infarcts. The subject is then scanned with a gamma camera to detect the gamma emission of the technetium-99m and to thereby obtain an image of the myocardial infarction or tumor. In this way, a tumor or infarction can be localized and its size can be determined.

Alternatively, radiolabeled proteins can be used in radioimmuno therapy by selectively delivering radioisotopes to cells in vivo. The preferred radioisotope for radioimmunotherapy is rhenium since it is a beta emitter which can kill target cells.

Non-radioactive metals which are toxic to the target cells can also be used for immunotherapy. Tumor specific labeled antibodies are introduced into a subject wherein the labeled antibodies selectively seek out and destroy cancer cells.

The invention is further illustrated by the following examples. EXAMPLE 1

Methods

Oligodeoxynucleotide Synthesis

The oligonucleotides were prepared using the phosphoramidite method on Applied Biosystems DNA Synthesizer model 380A. The crude product was purified on a 20% polyacrylamide gel. The purified DNA was used as a primer in DNA sequencing for use in cloning. The purified material was phosphorylated and annealed to form double-stranded fragments.

DNA Sequencing

DNA fragments were subcloned into M13mp18 RF vector. DNA sequences were determined by the dideoxy sequencing method using appropriate primers.

Transfer of DNA into myeloma cells by electroporation

Transfer of DNA into cells was carried out by electroporation using Bio-Rad Gene Pulser Transfection Apparatus. Approximately 5x10⁶ cells in 0.8ml of Dulbecco's phosphate-buffered saline (PBS) containing 30 ug of pSV₂184ΔHneo17-1AV_k.hC_k or pSV₂ ΔHgpt17-1AV_h -hCγ₄/MT were subjected to an electric field of 0.2 kv and a capacitance of 960 uFD at 4°C The cells were diluted with DMEM supplemented with 15% FBS and plated out in one 96-well microliter plate. After 48 hours, the transfected cells were selected in growth medium containing 1 mg/ml of G418 for the expression of the light chain. For heavy chain expression, transfected cells were selected in growth medium containing 1 ug/ml of mycophenolic acid, 50 ug/ml of xanthine and 2.5 ug/ml of

hypoxanthine.

Quantitation of antibody productio n

A stably transfected cell line G4K/MT was established and analyzed. Tissue culture

supernatant was analyzed for IgG content by particle concentration fluorescence immunoassay using

standard curves generated with purified human IgG. The assays were carried out with an automated instrument (Pandex Laboratories, Mundelein, IL).

Concentration of IgG production was estimated to be 5 ug/ml by using polystyrene beads coated with goat anti-human IgG (Fc) antibody and fluorescein- conjugated goat anti-human IgG (Fc) antibody.

Antibody purificati on

Stable transfected cell lines were carried in DMEM containing G418 at 1 mg/ml , myc opheno l ic at

1 ug/ml , xanthine at 50 ug/ml, hypoxanthine at 2.5 ug/ml, and ZnCl₂ at 0.14 ug/ml, supplemented with 15% fetal bovine serum.

Tissue culture supernatant was passed through an affinity column of Sepharose-bound goat

anti-human IgG (Fc) antibody and adsorbed antibody was eluted in 0.1M glycine at pH 2.2 containing 100 uM ZnCl₂. Purified IgG was dialyzed against 50mM phosphate buffer at pH 6.5 containing ImM

ethylenadiamine tetraacetic acid (EDTA) and 100uM ZnCl₂.

Radioiodination of purified murine IgG 17-1A (for immunoassay

Murine IgG 17-1A was purified from ascites fluid by chromatography on staphylococcal protein

A-Sepharose. Bound IgG was eluted with 50mM sodium citrate buffer at pH 3.5. Fifty-five ug of the purified IgG was labelled with 800 uCi of Na ¹²⁵I using lodo-Beads (Pierce Chemical Company, Rockford,

IL) . Aften 10 minutes, the reaction was quenched with bovine serum albumin (PBS) containing phosphate buffered saline 5% (BSA). Free iodine was removed with a prepacked PD-10 column (Pharmacia). Specific activity was 18,000 cpm/ng of protein.

Binding inhibition immunoassay

A monolayer of HT-29 human colorectal carcinoma cells were harvested with trypsin, washed and resuspended in growth medium. Cells were then seeded into 96-well microliter plates at

2x10⁵ cells per well in 100 ul aliquots. After incubation at 37° for 17 hours, the cells were refed with 100 ul of cold medium and kept at 4°C for 30 minutes. Each well was then incubated at 4°C for 2 hours with medium containing 450,000 cpm of

iodinated murine 17-1A and different concentrations of cold competing IgG in a final volumn of 100ul. The cells were then washed three times with warm medium and cell-bound radioactivity was measured in a gamma counter.

Procedures Condtruction of the heavy chain expression vector pSV₂ΔHgpt 17-1A V_hC-hγ4/MT

Plasmid phCγ₄ containing, the human Cγ₄ genomic DNA was used to derive the IgG/metallothionein (MT) fusion gene. Plasmid phCγ₄ was partially digested with restriction endonuclease SacII and ligated with synthetic 24-mer oligonucleotide,

5'-GGGAATTCAAGCTTCTCGAGCCGC-3' . hCγ₄/CAS with EcoRI, Hindlll, and Xhol sites in the middle of the CH2 region was thus created to allow for the

insertion of DNA fragments with appropriate ends. The SacII recognition sequence, CCGCGG , is located at nucleotide 1238-1243 downstream from the Sail site of phCγ₄.

phMTII-3, ( Karin et al. Nuc. Acid Res. 10,

3165-73, 1982) a cDNA clone of human

metallothionein, was digested with BamHI and PvuII to release a 178 bp fragment containing the coding sequences for amino acid residues 2 to 58 of the 61 amino acid-peptide. The 5' and 3' ends were

modified to EcoRI and Xhol, respectively, using commercial linkers. The resulting 197 bp DNA fragment was then inserted at the EcoRI and Xhol sites of hCγ₄/CAS to give hCγ₄/MT. The junction of immunoglobulin and MT DNA was verified by DNA sequencing using a synthetic primer

(5'-CGTGGAGGTGCATAATGC-3') which is homologous to the region 10 bp upstream from the SacII site. As shown in Figure 1, hC7,MT contains 8 extra amino acids in addition to the 57 amino acids derived from hMT.

The functionally rearranged heavy chain

variable gene of 17-lA was isolated and cloned into an expression vector containing genomic DNA encoding human 3 constant region to give

pSV₂ΔHgpt17-1AV_H -hCγ₃ (Sun et al. Proc. Natl. Acad. Sci. USA, 84:214-218 (1987). The 7.5 kb human Cγ₃ was replaced with the 2.7 kb Cγ₄/MT DNA fragment to give pSV₂ΔHgpt 17-1A V_H-hCγ₄MT.

Transfection of DNA into mouse myeloma Sp2/0 cells The heavy and light chain vectors were used to sequentially transfect the nonproducing mouse myeloma Sp2/0 cells. Stable light chain-producing cell lines were established first. The heavy chain construct was then transfected into the light chain producer. The stable transfected lines obtained were carried in growth medium containing selections for both gpt and neo genes. The efficiency of electroporation was approximately 10 at each step.

The cell line G4K/MT was chosen for further analysis. Antibody concentration in the culture supernatant of G4K/MT was estimated to be 5 ug/ml as measured by particle concentration fluorescence immunoas say.

EXAMPLE 2 Evaluation of Technetium (Tc-99m) labelled IgG4K/MT

Tc -99m Radiolabelling of Murine IgG2a (17-1A) and Tg G4K/MT

All samples were equilibrated at room

temperature for 60 minutes. The labelling was accomplished by the transfer method described in

PCT Application No. 88/07382, published October 6, 1988, the teachings of which are hereby incorporated by reference. A stannous chloride/D - glucarate composition vial was prepared as described in PCT Application WO88/07382, the teachings of which are hereby incorporated by reference. This stannous chloride/D-glucarate composition was reconstituted with 1ml (10mCi) Tc-99m pertechnetate. After 10 minutes, 0.10 ml of the contents of the stannous chloride/D-glucarate vial was transferred to 0.10 ml IgG2a (1 mg/ml) in a second vial. The reac ti on mixture was assayed by thin layer chromatography (ITLC; 0.1M citrate buffer, pH 5).

A labelling study at 37°C (1-5 hours) showed that Tc-99m incorporation for IgG2a ranged from 1.7% to 0% while IgG4K/MT ranged from 42.5% to 65.0% (see Table 1). These Tc-99m labelled products (i.e., IgG2a at 37°C for 5 hours and IgG4K/MT at 37° for 5 hours) were also analyzed by gel filtration high performance liquid chromatography (HPLC). The results showed that reaction of IgG2a with reduced Tc-99m at 37° produced pertechnetate in radioscan at 13.97 minutes (Figure 4). Under similar reaction conditions, reaction of IgG4K/MT with reduced Tc-99m produced mostly Tc-99m labelled IgG4K/MT in

radioscan at 8.75 minutes (Figure 5).

Thus, the results Indicate that Tc-99m

corporation into murine IgG4K/MT can be accomplished by warming the antibody to 37° in the presence of Tc-99m glucarate. Under the same conditions, the IgG2a (17-1A) is not labelled with Tc-99m.

Equivalents

Those skilled in the art will know, or be able to ascertain using no more than routine

experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. A chimeric protein comprising a protein having an affinity for a biological target linked to at least one metal binding protein or the functional domain thereof, which is not normally associated with the protein wherein the linkage is a peptide linkage which forms a contiguous polypeptide.

2. A chimeric protein according to Claim 1,

wherein the protein having an affinity for a biological target is selected from the group consisting of hormones, ligands, growth

factors, biological receptors, and

immunoglobulins.

3. A chimeric protein according to Claim 1,

wherein the metal binding protein is

metallothionein.

4 . A metal lab e l led chimeric protein comprising a protein having an affinity for a biological target linked to at least one metal binding protein or the functional domain thereof, which is not normally associated with the protein, wherein the linkage is a peptide linkage which forms a contiguous polypeptide and the metal binding protein or the functional domain thereof having a metal bound thereto.

5. A metal-labelled chimeric protein according to Claim 4, wherein the metal is a radiometal.

6. A metal-labelled chimeric protein according to Claim 5, wherein the radiometal is selected from the group consisting of Tc-99m, Re-188, Cu-67, Pd-103, In-111, Pb-203, Ru-97, Hg-197, Ag-111, Au-198, Bi-212, Os-191, Pd-109, and Au-199.

7. A metal-labelled chimeric protein according to Claim 5, wherein the radiometal is selected from the group consisting of Tc-99m and Re-188.

8. A radiolabelled chimeric protein according to Claim 5, wherein the radiometal is Tc-99m.

9. A metal-labelled chimeric protein according to Claim 4, wherein the metal is a non-radioactive metal.

10. A metal-labelled chimeric protein according to Claim 9, where the non-radioactive metal is zinc.

11. A recombinant immunoglobulin comprising

an antigen binding region specific for a tumor associated or proliferation- associated antigen linked to at least one metal binding protein, or functional domain thereof, which is not normally associated with the immunoglobulin wherein the linkage is a peptide linkage which forms a contiguous polypeptide.

12. A recombinant immunoglobulin according to Claim

11, wherein the tumor-associated antigen is selected from the group consisting of breast, ovarian, lung, and renal cancer antigens.

13. A recombinant immunoglobulin according to Claim

12, wherein the tumor-associated antigen is defined by an antibody selected from the group consisting of 17-1A, OC125, OV-TL3, 103D2, 123.C3, 72.3, DF3, 115D8, RC38, G250, and

55-2A.

14. A recombinant immunoglobulin according to Claim

13, wherein the tumor-associated antigen is defined by the antibody 17-1A.

15. A recombinant immunoglobulin according to Claim 11, wherein the metal binding protein is metallothionein.

16. A metal-labelled recombinant immunoglobulin

having an affinity for a biological target linked to at least one metal binding protein or the functional domain thereof, which is not normally associated with the immunoglobulin, wherein the linkage is a peptide linkage which forms a contiguous polypeptide and the metal binding protein or the functional domain thereof having a metal bound thereto.

17 . A me tal-labelled recominant immunoglobulin

according to Claim 16, wherein the metal is a radiometal.

18. A metal-labelled recombinant immunoglobulin according to Claim 17, wherein the radiometal is selected from the group consisting of

Tc-99m, Re-1,88, Cu-67, Pd-103, In-111, Pb-203, Ru-97, Hg-197, Ag-111, Au-198, Bi-212, Os-191, Pd-109, and Au-199.

19. A metal-labelled recombinant immunoglobulin

according to Claim 17, wherein the radiometal is selected from the group consisting of Tc-99m and Re-188.

20. A metal-labelled recombinant immunoglobulin

according Claim 16, wherein the radiometal is Tc-99m.

21. A metal-labelled recombinant immunoglobulin

according to Claim 16, wherein the metal Is a non-radioactive metal.

22. A metal-labelled recombinant immunoglobulin

according to Claim 21, where the non- radioactive metal is zinc.

23. A recombinant immunoglobulin comprising an

antigen binding region specific for a marker of cardiovascular disease linked to at least one metal binding protein or functional domain thereof which is not normally associated with the immunoglobulin wherein the linkage is a peptide linkage which forms a contiguous polypeptide.

24. A recombinant immunoglobulin according to Claim

23, wherein the marker of cardiovascular disease is selected from the group consisting of platelets, myosin, and fibrin.

25. A recombinant immunoglobulin according to C l aim

24, wherein the marker of cardiovascular disease is defined by an antibody selected from the group consisting of 7E3, 10E5, S12, and R11D1O.

26. A recombinant immunoglobulin according to Claim 23, wherein the metal binding protein is metallothionein.

27. A method for radiolabelling a recombinant

immunoglobulin, comprising:

a. providing an immunoglobulin specific for a tumor-associated, proliferation-associated, or cardiovascular disease-associated antigen, linked to at least one metal binding protein or functional domain thereof, not normally as- sociated with the immunoglobulin, wherein the linkage is a peptide linkage forming a contiguous polypeptide;

b. contacting the contiguous polypeptide with a radiometal under conditions which allow binding to occur.

28. A method for radiolabelling a recombinant immunoglobulin according to Claim 27, wherein the radioimetal is selected from the group consisting of Tc-99m, Re-188, Cu-67, Pd-103, In-111, Pb-203, Ru-97, Hg-197, Ag-111, Au-198,

Bi-212, Os-191, Pd-109, and Au-199.

29. A method for radiolabelling a recombinant

immunoglobulin according to Claim 24, wherein the radioimetal is selected from the group consisting of Tc-99m and Re-188.

30. A method for radiolabelling a recombinant

immunoglobulin according to Claim 27, wherein the radiometal is Tc-99m.

31. A recombinant immunoglobulin comprising a

antigen binding region specific for a 17-1A antigen linked to at least one metallothionein region, or functional domain thereof which is not normally associated with the immunoglobulin wherein the linkage is a peptide linkage which forms a contiguous polypeptide.

32. A kit for radiolabelling a recombinant immunoglobulin, comprising:

a sealed, sterile vial containing a recombinant immunoglobulin comprising an antigen binding region specific for a tumor- associated, proliferation-associated, or cardiovascular disease-associated antigen linked to at least one metal binding protein, or functional domain thereof, which is not normally associated with the immunoglobulin wherein the linkage is a peptide linkage which forms a contiguous polypeptide.

33. A kit for radiolabeling a recombinant immunoglobulin according to Claim 32, wherein the tumor- associated antigen is defined by the antibody 17-1A.

34. A method of obtaining a scintigraphic image of a tumor or a myocardial infarction in a subject, comprising:

preparing a radiolabelled immunoglobulin specific for the tumor or myocardial infarction;

introducing the radiolabelled immunoglobulin into subject;

allowing the radiolabelled immunoglobulin to localize at the tumor or myocardial infarction; and

scanning the subject with a gamma camera to obtain an image of the tumor.

35. A method of obtaining a scintigraphic image according to Claim 34, wherein the radio- labelled immunoglobulin is specific for a tumor-associated antigen defined by the antibody 17-1A.

36. A method of tumor immunotherapy, comprising:

preparing a radiolabelled Immunoglobulin specific for a tumor-associated antigen;

introducing the radiolabelled immunoglobulin into a subject afflicted with a tumor.

37. A method of tumor immunotherapy according to Claim 36, wherein the radiolabelled immunoglobulin is specific for a tumor-associated antigen defined by the antibody 17-1A.