WO1995004753A1

WO1995004753A1 - Compositions of fusion proteins containing metallothionein and targeting-protein structural components

Info

Publication number: WO1995004753A1
Application number: PCT/US1994/008689
Authority: WO
Inventors: Paul Zamora; Jeffery K. Griffith
Original assignee: University Of New Mexico
Priority date: 1993-08-11
Filing date: 1994-08-04
Publication date: 1995-02-16

Abstract

Components of the primary amino acid sequence of a metallothionein are genetically incorporated into proteins by recombinant DNA techniques to produce a hybrid molecule comprising an effector protein continuous with the metallothionein. The sulfhydryl and lysine residues of the metallothionein portion of the hybrid molecule provide binding sites for ligands such as radionuclides, contrast agents, magnetic resonance agents, fluorochromes, and enzymes. The labeled hybrid molecule is useful for the diagnosis and localization of disease lesions and is a cost-effective method of producing key ingredients for immunoassays including immunosorbant assay, immunoblot, immunodot, immunohistochemistry, and/or flow cytometry.

Description

COMPOSITIONS OF FUSION PROTEINS CONTAINING METALLOTHIONEIN AND TARGETING-PROTEIN STRUCTURAL COMPONENTS

Field of the Invention

The present invention relates to fusion proteins for diagnostic and therapeutic use which contain primary sequences of an effector molecule and a cysteine-containing a ino acid sequence, particularly a primary sequence of a metaUothionein. The sulfhydryl groups of the cysteine residues serve to bind therapeutic or detectable probes such as radionuclides, while the effector sequence localizes the protein on a predetermined target. The invention also relates to methods for the genetic construction of such proteins.

Background of the Invention

A number of methods have been developed to label proteins with radioactive and metal probes such as 99m-Tc for subsequent use in diagnostic imaging and disease therapy. In general, the methods for protein labeling with diagnostically useful probes such as 99m-Tc can be broken down into two methods: direct labeling of the protein and labeling of the protein through chelation.

Hnatowich has reviewed both methods with particular application to antibodies (Hnatowich, Nucl. Med. Biol.. 17, 49- 55, 1990; Hnatowich, Semin. Nucl. Med.. 20, 80-91, 1990a) . For purposes of illustration, 99m-Tc will be used as an example of a diagnostically useful probe, although similar principles apply to other types of probes. The direct labeling procedures involve either 1) reduction of the protein and the subsequent or simultaneous addition of 99m-Tc or 2) reduction of pertechnetate and subsequent addition of the protein. Direct labeling methods have been applied primarily to antibodies, although other types of proteins have also been so labeled. While this method is widely used it suffers from a number of problems including the requirement that the protein be able to withstand chemical reduction with reducing agents such as stannous ions or dithionite, and a lack of stability of the chemical bond between the protein and the label. Furthermore, this method requires that the protein to be labeled meet at least two critical criteria: 1) the protein must contain reducible disulfide bonds, and 2) immunological and/or biological activity of the protein must not be compromised by the chemical reduction process or the subsequent labeling. While antibodies meet such criteria, there are a large number of other potentially useful proteins which either do not contain reducible disulfide bonds or which have disulfide bonds which are critical to biological activity. Thus, a need exists for improved methods for the labeling of proteins with useful probes such as technetium. The labeling through chelation methods involves covalent binding of a chelator to the protein and subsequent attachment of the probe through the chelate. The chelates can be attached to the protein through free amine groups or sulfhydr- yl groups on the amino acid side chains of the proteins. Using the labeling through chelation technique, 111-In and to a lesser extent 99m-Tc have been used to label a large number of proteins. Examples of chelating groups that have been used or can be so used include polydentate carboxylic acids (EDTA, DPTA, and the like) , polydentate polya ines (amino oxime) , chelates containing both amide and sulfur groups [N,N'-bis(mercaptoacetamino) ethylenediamine; diaminodithiol] , and chelates containing carboxyl and sulfur groups (dimercaptosuccinic acid) . The chelator may be labeled prior to conjugation or subsequent to conjugation. As with the direct labeling methods, the labeling through chelation methods have been widely applied to antibodies and to a lesser degree to other proteins. The labeling through chelation method suffers from the fact that it introduces chemical modifications into the protein via the covalent binding of the chelate. This chemical modification of the protein can severely alter the immunological and biological behavior of the protein.

In an approach similar to that of introducing chelate groups into proteins, metaUothionein has been chemically conjugated to monoclonal antibodies by using sulfo-succini- midinyl-4-[4-maldeimidonmethyl]-cyclohexane-l-carboxylate (see Brown et al., Anal. Biochem.. 172, 22-28, 1988) to chemically bond the metaUothionein to the antibody. The chemically- conjugated metaUothionein/antibody conjugate was then labeled with 99m-Tc. Chemical conjugation methods, however, have several drawbacks, including the potential for the formation of ag- gregates and colloids, potential difficulties associated with manufacturing preparations which involve a chemical conjugate, and in the case of metaUothionein the introduction of bulky side groups which can sterically hinder or otherwise compromise the immunological and/or biological activity of the antibody. In spite of the variety of methods of introducing useful probes into proteins, each suffers from drawbacks and limitations, and there continues to be a need for methods which can introduce probes into proteins with a minimum of detrimental modification of the effector or targeting protein. This invention uses fusion proteins, e.g. chimeric molecules, produced by recombinant DNA procedures to address this need.

Discussion of Related Art

There are now several examples of fused gene products produced by recombinant DNA technology. One such example is the production of cloned toxin/ligands (reviewed in Soria, Pharmacol. Res. _f 21, suppl. 2, 35-46, 1989). These fusion proteins have been envisioned as useful in disease therapy. For example, Soria (Pharmacol. Res.. 21, suppl. 2, 35-46, 1989) has cited the following examples of fusion gene products: a) Truncated diphtheria toxin fragment fused to melanocyte-stimulating hormone (MSH) . The toxin-hormone chimeric gene directed the expression of a fusion protein that retained the ADP-ribosyltransferase activity and the lipid-associating do¬ mains of diphtheria toxin; however, the diphtheria toxin recep- tor-binding domain was replaced with MSH sequences. The chimeric toxin was found to be toxic for MSH receptor-positive human mela¬ noma cells. b) Lymphyokine fused to bacterial toxin. Targeting of cloned toxin/ligand fused products based on lymphyokine-coding genes (IL-2 and IL-6) and either diphtheria toxin or 2-Pseudo- monas exotoxin has been reported. Additionally, a fused gene product of transforming growth factor alpha and Pseudomonas exotoxin has also been produced. c) Cell surface receptor fused to bacterial toxin. CD4 is a cell surface receptor on T-lymphocytes which has been shown to serve as the receptor for the AIDS virus. The cloned HIV- binding portion of the human CD-4 molecule fused to cloned Pseudomonas exotoxin was shown to bind to the viral protein gp 120 both in solution and of the cell surface. Cells expressing the HIV envelope protein on their cell surfaces were selectively killed by this chimeric protein.

Butt et al (J . Biol. Che . , 263, 16364-16371, 1988, incorporated herein by reference) have produced a ubiquitin- metallothionein fusion protein which was expressed in yeast. This fusion protein was genetically produced for the analysis of ubiquitin functions. Ubiquitin is a 76-amino acid protein that is present in the cytoplasm of all eukaryotic cells. Its ubiquitous distribution in cells makes it totally unsuitable as a targeting agent, as does its reactivity with a wide range of cellular proteins. In the study of Butt et al., metaUothionein was used as a "typical cellular protein" to study ubiquitin function; similar studies have used other amino-terminally ubiquitinated proteins expressed in yeast such as ubiquitin-beta- galactosidases, ubiquitin carboxyl extension genes, and ubi- quitin-alpha subunit gene of GTP binding regulatory protein (see Butt et al, J. Biol. Chem.. 263, 16364-16371, 1988).

Sano et al, Proc. Natl. Acad. Sci. USA .89.: 1534-1538 (1992) describe a streptavidin-metallothionein fusion protein only useful for labelling biological materials containing unhindered biotin. The biotinylation process requires biotinyla- tion of the target material prior to labeling or conjugating the target, and precludes in vivo use of the fusion protein. Summa y of the Disclosure

In contrast to the technology described in related art, such as the technology described by Butt et al. , the present invention does not relate to fusion of metaUothionein to ubiquitin or other protein normally expressed in the cytoplasm, nor does it relate to chemical bonding of a probe to an effector protein, either directly or through a chelate. Rather, the invention relates to the genetic construction of fusion proteins comprising effector sequences, particularly cell secreted products which have localizing capacity, fused to amino acid sequences containing free sulfhydryl side chains for binding the desired probe, and the use of such proteins after labelling with a suitable probe in diagnostic and therapeutic procedures.

In particular, the invention comprises processes for the preparation of recombinant DNA derived proteins which are fused gene protein products comprising a probe-binding amino-acid sequence such as a metaUothionein fused to an effector amino acid sequence having specific affinity for a predetermined target, such as tissue plasminogen activator (TPA) , and products of the processes. The sulfhydryl side chains of the cysteine residues which are a part, for example, of the primary structure of the metaUothionein, are contemplated as especially useful for binding detectable metal probes, particularly those metals commonly used in diagnostic imaging. The metallothionein/protein complexes are useful, via the probe, in a variety of qualitative, semi-quantitative, and quantitative assays and also can be used in therapeutic treatments, as in the formulation of pharmaceuti¬ cals.

The invention accordingly provides a method for producing hybrid proteins which contain a metaUothionein, portions of metaUothionein, and/or other amino acid sequences which contain metal binding sites similar to those found in a metaUothionein. The invention further provides a hybrid protein containing components of a metaUothionein which can be labeled with a detectable probe through the sulfhydryl groups for subsequent use as a radiopharmaceutical or pharmaceutical. The invention additionally provides a hybrid protein containing components of a metaUothionein which can be labeled with a detectable probe through the sulfhydryl groups for subsequent use as an in vitro diagnostic reagent. In particular, the invention provides a convenient method for constructing a therapeutic or diagnostic complex comprising an effector component and a probe- binding component wherein substantially the sole biological activity of the effector component is the binding affinity for the predetermined target; by the process of the invention, the foreign DNA introduced into the host consists essentially of a DNA sequence encoding for a fusion protein wherein the effector polypeptide portion is intentionally compromised to eliminate domains which are detrimental or superfluous to the intended use of the complex, and to retain only those domains which have binding affinity for the target. Genetic construction of such an intentionally-compromised complex permits the ready production of therapeutic and diagnostic agents having, for example, low allergenicity, reduced cross-reactivity, high specificity, and high avidity, in contrast to chemically-constructed complexes, which cannot easily or accurately be modified.

Definitions

The terms "hybrid protein", "chimeric protein", and "(gene) fusion protein" are herewithin equivalent terms.

The term "protein" as used herein includes any oligopeptide, polypeptide, or protein which is naturally produced by viruses, bacteria, animal cells or plant cells; or is produced by chemical synthesis, genetic engineering by recombinant DNA technology, hybridoma technology, or technology involving the use of transgenic animals.

The term "recombinant DNA derived proteins" herein includes proteins which are obtained by a) selection of cellular DNA or DNA containing elements (including whole chromosomes or parts of chromosomes) from one type of cell, b) introduction of the selected DNA into the genome of a second host cell, and c) expression of the desired protein in host viruses, cells, or animals. Suitable processes for the selection of DNA, the selection of the host, and expression of the desired protein are discussed in Butt et al. , supra.

"MetaUothionein" as used herein broadly refers to any polypeptide which contains large amounts of cysteine residues with associated free sulfhydryl groups capable of binding metal probes or other probes which are reactive with or can be modified to be reactive with the sulfhydryl groups. The metallothioneins, and the genes that produce them, may be obtained from lower organisms such as bacteria and yeasts, or from plant or animal sources; the term includes truncated forms, elongated forms, and forms which are chemically synthesized to contain metal binding sites similar to those found in metaUothionein.

The term "fused gene products containing metaUothi¬ onein and an effector molecule" as used herein includes hybrid or chimeric molecules which have within the primary amino acid sequence amino acids corresponding, or corresponding in part, to metaUothionein and the effector protein.

The term "effector protein" as used herein includes any type of enzyme, lymphokine, cytokine, hormone, factor, antibody, or other protein or fragment thereof which can bind specifically to a naturally-occurring, unmodified target cell or target substrate, in vivo or in vitro. Examples of such proteins are tissue plasminogen activator, urokinase, pro-urokinase, inter- leukin-1, interleukin-2, interleukin-3, interleukin-6, granulo- cyte stimulating factor, macrophage stimulating factor, tumor necrosis factor, pituitary hormones (TSH, LH, FSH) , chorionic gonadotropin, platelet-derived growth factor, transforming growth factor alpha, transforming growth factor beta, gamma interferon, alpha interferon, beta interferon, soluble CD4, fibroblast growth factor, insulin-like growth factor I, insulin-like growth factor H, growth hormone, antibodies which recognize tumor-associated antigens, antibodies which recognize clot (thrombus) related components, antibodies which recognize components of inflammatory lesions, antibodies which recognize components of bacterial and/or viral infections, and the like..

The term "detectable probe" as used herein refers to any type of material which binds to metaUothionein and which can be detected, such as by known qualitative, semi-quantitative, or quantitative methods. Examples of such probes include heavy metals, radionuclides, enzymes, fluorochromes, and paramagnetic compounds. The radionuclides 99m-Tc, 67-Cu, and 188-Re are contemplated to be of particular utility.

The terms "qualitative, semi-quantitative, and quantitative assays" refers to assays which are based on the binding of an effector protein portion of the fusion product to a cellular or molecular target, and the binding monitored via the detectable probe.

The term "pharmaceutical" refers to a product which is suitable for use in humans or in animals and which may be used in various clinical applications such as disease localization, diagnosis, or management. The term "host cell" as used herein collectively refers to prokaryotic and eukaryotic cells, and also viruses containing the foreign DNA encoding for the fusion protein of the invention.

Detailed Description of the Invention

Incorporation of metaUothionein into the primary sequence of the effector molecule.

Exemplary of the invention, the primary amino acid sequence of a metaUothionein is genetically introduced into an effector molecule. MetaUothionein, however, is but one example of a primary sequence with probe binding cysteine residues which can be genetically fused into a protein which has targeting capacity according to the invention. The invention comprises the genetic construction of a gene encoding fusion protein which has incorporated into its primary sequence probe-binding sites so that the components of a metaUothionein or other probe-binding sequence are continuous with the chosen effector. The probe binding portion of the fusion protein may contain the entire amino acid sequence of the metaUothionein; however, the probe binding portion may also instead contain various molecular variants of a metaUothionein such as truncated sequences, multiple copies of the entire amino acid sequence, multiple copies of truncated portions, or amino acid sequences produced by synthetic means including those generated from synthetically produced DNA based on or similar to metaUothionein.

The invention thus comprises a genetically constructed fusion (hybrid) molecule which has in its primary sequence a) metaUothionein and b) an effector, wherein the metaUothionein portion of the molecule can be used to bind probes and the effector is an oligopeptide, peptide, protein, or glycoprotein which is conveniently a cell-secreted product with localizing capability; i.e., the fusion product is specific for the target. Preferably, the genetic construction process compromises the effector component to eliminate domains having unwanted biolog¬ ical activity, as noted above. Examples of metallothioneins that can be used in the practice of the invention.

Yeasts and animal metallothioneins are known to have a high concentration of cysteine (approximately 30% [by weight] by amino acid content) and lysine residues, and to have high affinity for certain metals, notably Cd, Zn, Hg, Ag, and Sn. The strong metal affinity is due in large part to the high sulfhydryl content attributable to cysteine. Metallothioneins are widely distributed in the body of both man and animals, having been isolated from kidney, liver, spleen, intestine, heart, brain, lung, and skin. This invention contemplates the use of metal¬ lothioneins from any animal or microbial source. Metal¬ lothioneins or other amino acid probe-binding sequences contain¬ ing at least about 20% by amino acid content cysteine residues are preferred. Exemplary metaUothionein sequences suitable for fusion with the effector according to the invention are set forth in U.S. Patent 4,732,864 and references cited therein, incorpo¬ rated herein by reference.

Several plant proteins have been reported to have a high content of cysteine and to have metal binding properties similar to animal metaUothionein. Some of these proteins are lectins, like those present in cereals and members of the solanaceae family (potato, tomato, thorn apple) . Others are highly abundant polypeptides with antifungal properties, the thionins, which are present in monocots as well as in dicots. Cereal seeds contain purothionins which are toxic to animals and have thioredoxin activity. This invention contemplates the use of such plant derived proteins with properties analogous to metaUothionein. While the primary sequence used to confer probe binding capacity to the fusion protein as described in this invention may be of animal, non-animal, or synthetic origin, the preferred embodiment involves the use of human or rodent metaUothionein.

A number of fused proteins have been described resulting in the production of a toxin/effector molecule. The fusion proteins of this invention are unique and distinct from previously described toxin/effector molecules in that a) the preferred metaUothionein sequence is not a toxin nor is it toxic, b) the metaUothionein or other probe-binding amino acid sequence is used to carry a probe, and c) the resultant fusion protein containing metaUothionein is useful in diagnostics to detect a target, such as in diagnostic imaging, or in therapeu¬ tics, such as in the treatment of cancerous cells with radio¬ active or toxic chemicals.

Advantages of the effector molecules genetically fused to metaUothionei . There are a number of advantages in the preparation of a diagnostic or therapeutic agent which contains probe binding sequences in its primary sequence as is described in this invention. The fusion proteins described in this invention can be produced in organisms including viruses, prokaryotic cells, eukaryotic cells, and animals. The production organisms are known to those skilled in the art and in preferred embodiments are E. coli cells and Chinese hamster ovary cells. Expression of the hybrid molecules described in this invention in E. coli cells is particularly attractive as the rapid growth rate and simplicity in large scale production would considerably simplify manufacturing and purification of the hybrid molecule.

In regard to mammalian cells such as Chinese hamster ovary cells, a direct incorporation of the metaUothionein gene should allow for a controlled or accelerated rate of production via heavy metal or glucocorticoid induction. Mammalian cell metaUothionein genes are inducible by heavy metals such as cadmium, and by glucocorticoids such as dexamethasone. Promoters from mouse and human metaUothionein genes have been linked to heterologous protein-coding sequences, introduced to host cells, and then tested for inducibility in response to cadmium or dexamethasone. When the human metaUothionein promoter was linked to the Herpes simplex thymidine kinase (HSV-tk) structural gene sequences and introduced to fibroblasts, the hybrid genes were inducible by cadmium and/or dexamethasone (Karin et al., Cell. 36, 371-379, 1984 incorporated herein by reference) . Similarly, the mouse metaUothionein promotor was linked to human growth hormone (hGH) protein-coding sequences. The hybrid gene was placed in a bovine papilloma virus (BPV) vector and intro¬ duced to mouse C 127 cells (Pavalakis and Hamer, Proc. Nat. Acad. Sci. USA. 80, 397-401, 1983 incorporated herein by reference). In this system hGH was induced by cadmium but not dexamethasone. It should be pointed out that in these systems that the resultant protein that was produced did not contain metaUothionein.

The direct incorporation of metaUothionein into the primary sequence for the specific of probe binding should considerably simplify the manufacturing and thereby significantly reduce processing costs. In the method described in this invention only the metaUothionein/effector molecule need be purified. In other systems involving covalent conjugations the effector molecule must be purified, chemically conjugated to a probe binding compound, and the conjugate separated from the unbound non-conjugated materials.

Types of probes that can be used in this invention.

In one of the preferred embodiments of this invention, 99m-Tc is used as the probe. In general, detectable probes, which can bind to the metaUothionein are useful. There are a number of probes which are contemplated in this invention and include the radioactive isotope of technetium, copper, gallium, ruthenium, rhenium, yttrium, gadolinium, or cadmium. The probe may also be fluorescent material and contain fluorescein, rhoda ine, or fluorescent chelates of rare earth metals especial¬ ly europium. The probe may also be a paramagnetic ion such as gadolinium, iron, manganese, copper, nickel, or a lanthanide element of the atomic numbers 57-70 or a transition metal of atomic numbers 21-29, 42, or 44. The specific type of fluorochromes encompassed by this invention are organic molecules which would react with reactive groups of a metaUothionein through thiol residues. Examples of organic fluorochromes which specifically react with sulfhydryl residues include but are not limited to fluorobenzoxadiazoles and bromobimanes. Alternatively, fluorochromes such as fluorescein and rhodamine may also be used. A number of enzymes have been used as detection probes and include horseradish peroxidase, alkaline phosphate, acid phosphatase, glucose oxidase, urease, and others known to those skilled in the art. These enzymes are useful for direct chemical conjugation to unmodified target cell products.

In regard to labeling with 99m-Tc, direct labeling procedures are expected to be particularly useful in labeling the metaUothionein portion of the hybrid molecules with minimal effects on the effector portion of the hybrid. The direct labeling procedures which may be so used include either 1) reduction of the protein and the subsequent or simultaneous addition of 99m-Tc or 2) reduction of the protein and the subsequent or simultaneous addition of the protein. Rhodes et al. (J. Nucl. Med. 27, 685-693, 1986 incorporated herein by reference) describe a method labeling of the protein through sulfhydryl groups which may be of particular value in this regard.

In the practice of another application of the inven¬ tion, the probe-labeled metallothionein/effector molecule is used as the critical component of clinical laboratory test kits. In this regard, labeling with fluorochromes or enzymes is particu¬ larly attractive. Examples of fluorochromes which can be used in this regard are fluorobenzoxadiazoles, bromobimanes, fluores¬ cein and rhodamine. A number of enzymes have been used as detection probes and include horseradish peroxidase, alkaline phosphate, acid phosphatase, glucose oxidase, urease, and others known to those skilled in the art. These enzymes have previously been used directly for chemical conjugation, or complexed via chemically conjugated bridging compounds such as biotin and/or avidin or strepavidin.

Use of 99m-Tc as a diagnostically useful probe.

A number of methods may be used to radiolabel the fusion proteins which are described in this invention. A method that maybe of particular utility to label the chimeric metallo- thionein tissue plasminogen activator protein (MT-TPA) , for example, is that described for the labeling of metaUothionein chemically conjugated to mouse monoclonal antibody B72.3 (Brown et al., Anal. Biochem. , 172, 22-28, 1988 incorporated herein by reference) . 99m-Tc glucoheptate (Glucoscan, Dupont Company) is prepared by adding 1 ml of 50-100 mCi of 99m-Tc (as pertechne- tate) to a single vial of gluceptate and allowing the reaction mixture to sit for 10 minutes. The 99m-Tc gluceptate is then added to a buffered solution of MT-TPA and the chelation reaction is allowed to proceed. At the end of the reaction period, the 99m-Tc-labeled MT-TPA is separated from the 99m-Tc gluceptate by passing it through a PD-10 column. The void volume contains the 99m-Tc-labeled MT-TPA. The amount of bound activity is deter¬ mined using a dose calibrator.

A number of other methods for the direct labeling of proteins with 99m-Tc have been described and would be known to those skilled in the art, and may be applied to this invention. One that is expected to be of particular utility in this regard is described in Rhodes et al (J. Nucl. Med. , 27, 685-693, 1986 incorporated herein by reference) .

After radiolabeling, the proportion of firmly bound 99m-Tc associated with the fusion protein is readily determined by methods known to those skilled in the art, such as thin layer chromatography in either acetone or saline wherein the firmly bound probe remains at the origin and the loosely bound material migrates with the solvent front. Additionally, after labeling, the percent bioreactive fraction may be determined using methods according to the nature of the effector molecule. For example, in the case of MT-TPA, fibrin clots are useful as substrates in determination of the amount of radioactive MT-TPA bound.

Types of effector molecules usςful in the practice of this invention.

In one of the preferred embodiments of this invention the effector molecule is tissue plasminogen activator. This invention also contemplates use of other effector molecules including enzymes other than tissue plasminogen activator, or a lymphokine, cytokine, hormone, factor, or antibody which can bind specifically to a target cell or target substrate. These effector molecules include, but are not limited to urokinase, pro-urokinase, interleukin-1, interleukin-2, interleukin-3, interleukin-6, granulocyte stimulating factor, macrophage stimulating factor, tumor necrosis factor, pituitary hormones (TSH, LH, FSH) , chorionic gonadotropin, platelet-derived growth factor, transforming growth factor alpha, transforming growth factor beta, gamma interferon, alpha interferon, beta interferon, soluble CD4, fibroblast growth factor, insulin-like growth factor I, insulin-like growth factor II, growth hormone, and the like. The metaUothionein sequence may be fused to the effector molecule at either the carboxy-terminal or amino-terminal end of the molecule.

In one of the preferred embodiments, the selected metaUothionein sequence is fused to the carboxyl-terminal end of the portion of tissue plasminogen activator which is as¬ sociated with enzymatic activity. The fusion at this end of the tissue plasminogen activator molecule is designed to minimize enzymatic activity of the molecule and provide maximal fibrin binding which is dominant on the N-terminal portion of the protein.

In another preferred embodiment, the selected metaUo¬ thionein sequence is fused not at the carboxyl-terminal end of tissue plasminogen activator, but 10-20 upstream amino acid sequences. The results of this preferred embodiment is a fusion molecule which has effector binding plus probe binding segments, but which has spliced out of it the enzymatic portion of the native enzyme. The intentional "compromising" of the enzymatic site does not limit the localizing utility of the fusion protein and the lack of a functional enzyme site minimizes the probabili¬ ty of autolytic degradation. The construction of fusion proteins based on compromised effector molecules as well as from molecules which are simply tagged on to the end of an effector molecule is within the scope of the invention. The aforementioned embodiment constructing fusion proteins based on compromised effector proteins, i.e., those which retain binding activity but lack non-localizing amino acid sequences, takes advantage of the fact that many effector molecules have portions which are irrelevant to localizing capacity, and as in the aforementioned embodiment can be eliminated from the fusion protein. Enzymes like tissue plasminogen activator and urokinase have fibrin binding domains and enzymatic domains. The enzymatic domain is largely irrele¬ vant to the localizing capacity and can be eliminated in the types of fusion proteins contemplated in this invention without loss of fibrin binding. Polypeptide hormones like TSH, LH, and FSH are composed of two subunits. One of the subunits is responsible for specific binding while the other is responsible for activation of adenylate cyclase. Construction of a fusion protein composed of the specific binding subunit of TSH, for example, and metaUothionein is an example of a fusion protein of the type contemplated in this invention. Antibodies are further examples of relatively large molecules composed of several subunits which have highly specific effector regions. Antibodies have associated with them many complex biological properties, nearly all of which do not directly relate to binding to antigens. The antigen binding regions of antibodies are relatively small, and can be genetically engineered into small, single chain antibody sequence such that the final construct retains both antigenic specificity and the probe binding capacity of metaUothionein. In another of the preferred embodiments, the effector molecule is altered prior to the fusion to eliminate or compro¬ mise undesirable characteristics. Many effector molecules have within them two separable biological function domains. One domain is responsible for receptor binding and the other for transmission of a biological signal of enzymatic function. Elimination, truncation, point mutation, or replacement of the domain associated with signal (enzyme function) transduction would still produce an effector molecule with selective binding. Use of such a compromised effector molecule fused to a metallo- thionein is an unique aspect of this invention. Isolation of the chimeric proteins of this invention.

The chimeric proteins produced using this invention can be isolated and purified by a number of methods known to those skilled in the art. An illustration of such methods is provided as follows for hybrid metallothionein/TPA(MT-TPA) molecules. MT-TPA molecules are extracted from cellular pastes in an extraction buffer containing Tris, EDTA, lysozy e, and deoxycholate and the inclusion bodies collected by centrifugation. The inclusion bodies are then lysed in Tris buffer containing Tween 80 and 8 M urea. The extract is then diluted into Tris buffer containing Tween 80 and both reduced and oxidized glutathione. Residual denaturants are removed by dialysis.

Isolation of the MT-TPA can be accomplished by affinity adsorption methods known to those skilled in the art for isolation of TPA or metaUothionein. Examples of solid phase affinity supports which are useful in this regard are: a) zinc chelate-agarose or lysine-Sepharose as applied to the TPA portion of the hybrid molecule, b) thiopropyl Sepharose 6B as applied to isolating cysteine containing proteins, c) monoclonal antibodies which bind either TPA or metaUothionein covalently bound to Sepharose or other solid phase. Other types of chromatography may also be used to isolate the MT-TPA including, for example, ion exchange, hydrophobic interaction, molecular sieving, and combinations of chromatographic techniques, such as will be apparent to those skilled in the art. In an exemplary procedure, the final preparation containing the isolated MT-TPA is concentrated to at least 1 mg/ml under positive pressure (nitrogen) by membrane concentration (nominal molecular weight cut-off 30,000 daltons) and filtered through a 0.22 micron filter to remove colloidal material and to result in a sterile preparation. The final preparation of MT-TPA is aliquoted and stored refrigerated, frozen, or lyophilized. The final preparation may include buffers, salts, stabilizers, or other additives which would be known to those skilled in the art.

Similar isolation methods can be applied to other types of fusion proteins produced according to this invention, and in part depend on the type and nature of the effector molecule used in the construction of the fusion protein.

Binding of probes to the fusion proteins of this invention.

The methods used to bind the detectable probes to the fusion proteins of this invention would depend on the type of probe used, but in any case would be known to those skilled in the art.

In the case of the binding of radionuclides with high affinities for metaUothionein such as Cu-67, binding can be accomplished by adding aqueous Cu(I)-67 or CU(II)-67 as a chloride salt under anaerobic conditions, allowing time for the Cu-67 to bind, and then separating the unbound Cu-67 from the bound Cu-67 by use of chelation matrices or chromatography (molecular sieve, ion exchange, or the like) .

In the case of the binding of radionuclides such as Tc- 99m, the radiometal may require reduction to a lower oxidation state to obtain binding to the fusion proteins of this invention. In the case of Tc-99m, the oxidation state can be reduced from Tc(VII) to Tc(V) or TC(IV) by use of a chemical reducing agent such as stannous chloride, stannous tartrate, stannous fluoride, or other chemical reducing agent known to those skilled in the art. The chemical reducing agent may be used with or in combination with a variety of radiometal complexing agents such as glucoheptate, citric acid, tartrate, tartrate/phthalate, ethylene diamine tetraacetic acid, or other stabilizing and/or complexing agents known to those skilled in the art. The chemical reducing agent and complexing agent is added the order of addition would generally be to add the Tc-99m (in.the form of sodium pertechnetate) to the reducing agent/complexing agent, and then to add that mixture to the fusion protein. Methods used to bind fluorochromes and enzymes to the fusion proteins of this invention are widely used and well known to those skilled in the art. The fluorescent bimane compounds can be conjugated directly to the fusion proteins of this invention by dissolving the bimane in acetonitrile, adding the dissolved bimane to the protein, and subsequently separating the bound fluorochrome/protein complex from the unbound fluorochrome by molecular sieve chromatography. Enzymes can be complexed to the fusion proteins of this invention by use of bifunctional crosslinking agents wherein the crosslinking agent is complexed first to the enzyme and then to the fusion protein through the thiol groups present in the metaUothionein portion of the fusion protein.

Preparation of diagnostic test kits of this invention.

Diagnostic test kits containing fusion protein such as metallothionein/TPA as described in this invention can be prepared in a form ready for labeling with a detectable probe such as technetium-99m for subsequent administration to a patient, preferably parenterally. These diagnostic test kits form a part of this invention. The kits are preferably sterile, apyrogenic, and contain either lyophilized, frozen, or refrigera- ted fusion protein such as metallothionein/TPA preparations. A typical kit contains between about 0.1 mg and 10 mg of fusion protein such as metallothionein/TPA protein. Methods for freezing or lyophilizing are known to those familiar with the art. The fusion protein is preferably provided as a penultimate product, e.g., a pharmaceutical intermediate, and is labelled to provide the final radiopharmaceutical or other diagnostic probe immediately prior to use.

Other components of the diagnostic kit may include alcohol swabs, needles, syringes, a 10-ml vial containing an aqueous solvent, and/or a detailed package insert including directions for the kit's use. The technetium-99m or other radioactive probe is typically not supplied with the kit but is obtained from a radiopharmacy or other source familiar with the preparation and dispensing of technetium-99m. The preparation and dispensing of technetium-99m is a routine practice in diagnostic imaging and is known to those familiar with the art. Each component of the kit is supplied sterile, apyrogenic, and in accord with all relevant Federal regulations and guidelines governing the manufacturing of radiophar aceu- ticals and biologicals. The metallothionein/TPA proteins are conveniently prepared as described in example III or by exposure to stannous ions as detailed in Rhodes et al. (J. Nucl.' Med.. 27, 685-693, 1986 incorporated herein by reference) and placed in vials. The contents of the vials are preferably frozen and/or lyophilized, and the vials purged with nitrogen or argon prior to capping to remove atmospheric oxygen which could inhibit the subsequent labeling with technetium. The essential component in the vials is a predetermined amount of fusion protein such as metallothionein/TPA, but the vials generally also contain suitable buffers and salts. The exact amount of metal- lothionein/TPA to be dispensed is determined empirically using methods known to those skilled in the art, depending upon the intended use. Similarly, the amount and volume of buffers and salts is known to those skilled in the art and depends in large part on the amount of metallothionein/TPA used. For actual exemplary use in a clinical situation and upon request by a nuclear medicine physician, a radiopharmacist compounds the radiopharmaceutical according to instructions supplied with the kit in the form of a package insert included with the kit.

Contemplated use in diagnostic imaging.

In the practice of the present invention, the diagnos¬ tic imaging of disease or disorder lesions is contemplated. The type of diagnostic imaging contemplated determines the specific effector molecule used. By way of example, diagnostic imaging with MT-TPA is useful, for example, in the localization of thrombi, thromboemboli, blood clots, and bleeding lesions.

By further example, to employ MT-TPA in a diagnostic imaging procedure, a diagnostic test kit according to the invention is used by a physician or pharmacist to prepare the probe labeled metallothionein/TPA, as described above. The probe-labeled metallothionein/TPA is then administered in- travenously to a patient. After administration of the labeled metallothionein/TPA a period of time is allowed to elapse prior to performing the intended diagnostic procedure, e.g. , radioscin- tigraphy or magnetic resonance imaging. This elapsed time between administration of the drug and the imaging procedure provides the labeled metallothionein/TPA time to accumulate to a sufficient extent at the locality or localities of thrombi. Suitable radioactive doses of technetium-99m are typically between about 2 and 20 millicuries and the dosage of the labeled metaUothionein/ TPA between about 0.1 and 10 milligrams per patient.

Similar approaches as detailed above are applicable to other hybrid fusion proteins for imaging of other types of diseases. One particular disease state that is specifically contemplated for diagnostic imaging using reagents and methods described in this invention is cancer. Other disease states which are contemplated for diagnostic imaging according to this invention are inflammatory lesions and infections. In regard to these lesions, effector molecules composed of lymphokines, cytokines, growth factors, and antibodies are contemplated as being particularly useful.

Also contemplated in the application of this invention is the use of therapeutic probes. Radioactive probes which are now of particular use in therapy include 188-Re, 187-Re, and 67- Cu. The therapeutic probe is not necessarily radioactive and may consist of, for example: toxic metals such as cadmium or mercury for passive therapy; a fluorescent or photosensitive metal or organic compound for use in laser therapy; or an enzyme such as peroxidase for localized oxidative cell killing in cancer therapy.

Regardless of the type of therapeutic probe used, the probe is bound to the metaUothionein portion of the hybrid fusion protein via free sulfhydryl groups and is delivered (targeted) to the site of the disease lesion by the effector portion of the hybrid molecule. EXAMPLES

These examples outline the procedures and methods used in making a preferred embodiment consisting of a hybrid protein molecule containing the sequences of both metaUothionein and tissue plasminogen activator. The principles outlined in this example are applicable to other hybrid constructions using comparable methods as will be apparent to those skilled in the art.

EXAMPLE I Construction of metaUothionein/tissue plasminogen activator.

To construct a chimeric gene from the elements of the E. Coli plasmid pNS3 and the cloned genes encoding human tissue plasminogen activator (TPA) and hamster metallothionein-2 (MT) the following protocol is used. The resulting chimeric gene directs regulated synthesis in E. coli of a hybrid protein including the entire sequence or MT and all but the C-terminal 20 amino acid residues of TPA.

Step One: Bacterial Expression of TPA The complete nucleotide sequence of the mRNA that encodes human TPA has been determined and is known to those skilled in the art (for example, the sequence is available on GenBank database) . The TPA protein-encoding sequence begins at nucleotide 76 and ends at nucleotide 1765. There are unique cleavage sites for restriction enzymes EcoNI and Smal at nucleotides 23 and 1704, respectively, and cleavage sites for the restriction enzyme Bglll at nucleotides 179 and 2153.

Key aspects of the selection of the TPA gene coding sequence are shown in Figure 1. The TPA gene is linearized 53 nucleotides 5' to the translation initiation codon with restric- tion enzyme EcoNI. Controlled hydrolysis with enzyme Bal31 is used to resect about 53 nucleotides between the EcoNI site and the translation start codon. The resected DNA is then treated with the enzyme Bglll under conditions where only about 50% of all sites are cleaved. This generates a population of fragments with a cleaved Bglll site at the 3' end of the TPA coding strand, some of which will contain the complete TPA coding sequence and have the sequence ATGG at the 5' end.

This population is ligated into the cloning vector pNS3 (Figure 2) which has been previously cleaved with the enzymes Smal and BamHI. Plasmid pNS3 is a derivative of the common cloning vector pBR322 which contains the PL prompter of bacterio- phage lambda, two synthetic ribosome binding sites (RBS) and adjacent cleavage sites for enzymes Smal and BamHI. Plasmid pNS3 confers resistance to the antibiotic ampicillin. Enzyme Smal cleaves between the C and G residues in the sequence CCCGGG. TPA genes resected up to the ATGG sequence, when ligated to the CCC sequence resulting from the digestion of pNS3 with Smal, create a unique new Ncol site (CCATGG) which are diagnostic for the construction. Enzyme BamHI generates a "sticky end" which is complementary to that produced by Bglll. The pNS3-TPA chimeras are introduced into calcium- chloride treated E. coli cells containing the lambda phage CI857 gene (selected for resistance to ampicillin at 30°C) . The CI857 gene encodes a temperature sensitive repressor of the PL promoter. Thus, the TPA gene is not expressed unless the temperature is raised to 42°C. Mini-lysates of plasmid DNA are resolved by electrophoresis in agarose gels. Clones with plasmids having the unique Ncol cleavage site are grown at 42°C to induce expression of TPA. TPA expression is then quantitated by immunological techniques.

Step Two: Construction of Hybrid TPA-MT Genes. The key steps in the construction of the hybrid TPA-MT genes are outlined in Figure 3. The pNS3-TPA chimeric gene is digested with restriction enzymes Smal, which will cleave the plasmid between the C and G of the CCCGGG recognition site at nucleotide 1704 of the TPA sequence to leave 2 nucleotides of codon. The plasmid is then digested with enzyme Pstl, which cleaves within the ampicillin resistance gene of the pNS3 sequence. Recombinant plasmid pCHMT-2 encodes the complete hamster MT coding sequence and the same ampicillin resistance gene as plasmid pNS3. Plasmid pCHMT-2 will be linearized at the MT translation start site with enzyme Ncol producing a "sticky end" with the sequence CATG. This will be filled-in using the Klenow fragment of DNA poly erase I and all four deoxyribonucleo- tide triphosphates to make a blunt-ended fragment.

The pCHMT-2 is cut within the ampicillin resistance gene with enzyme Pstl. The appropriate pNS3-TPA and pCHMT-2 fragments are purified, ligated and introduced into calcium- chloride treated E. coli containing the CI857 gene as described above selecting for resistance to ampicillin. Since pNS3 and pCHMT-2 contain the same ampicillin resistance gene, ligation of the two fragment's complementary Pstl sites reconstitutes the ampicillin resistance gene. The junction of the Smal cut TPA sequence and the filled-in Ncol site of the MT sequence produces an in-frame fusion and regenerates the Ncol cleavage site.

Since bacterial expression of MT confers heavy metal resistance, the chimeric genes should also confer heavy metal resistance at 42°C when the CI857 transcriptional repressor is inactive, but not at 30°C when the CI857 transcription repressor is functional.

Claims

WHAT IS CLAIMED IS:

1. Recombinant DNA encoding for expression of a fusion protein comprising a probe-binding amino acid sequence and an effector amino acid sequence fused to the probe-binding sequence for selectively binding the fusion protein to a predetermined target.

2. The recombinant DNA of claim l, wherein the probe- binding amino acid sequence includes cysteine residues having sulfhydryl groups for binding the probe.

3. The recombinant DNA of claim 2, wherein the probe- binding sequence comprises the entire amino acid sequence of any metaUothionein, a truncated sequence of a metaUothionein, a multiple copy of the entire amino acid sequence of a metaUothio¬ nein, a multiple copy of a truncated sequence of a metallothio- nein, a metaUothionein sequence generated from synthetically produced DNA, or a functional equivalent thereof.

4. The recombinant DNA of claim 1, wherein the effector comprises an oligopeptide, a peptide, a protein, or a glycoprot- ein.

5. The recombinant DNA of claim 1, wherein the effector comprises tissue plasminogen activator.

6. The recombinant DNA of claim 1, wherein the effector comprises an enzyme, a lymphokine, a cytokine, a hormone, a factor, or an antibody.

7. The recombinant DNA of claim 1, wherein the effector is urokinase or prourokinase.

8. The recombinant DNA of claim 1, wherein the effector is interleukin-1, interleukin-2, interleukin-3, interleukin-6, granulocyte stimulating factor, macrophage stimulating factor, gamma interferon, alpha interferon, beta interferon, or tumor necrosis factor.

9. The recombinant DNA of claim 1, wherein the effector is a pituitary hormone, chorionic gonadotropin, or a subunit of a pituitary hormone or chorionic gonadotropin.

10. The recombinant DNA of claim 1, wherein the effector is a monoclonal antibody, a fragment of a monoclonal antibody, or a fragment of a monoclonal antibody modified by a recombinant DNA technique.

11. The recombinant DNA of claim 1, wherein the effector is an antibody to a human disease lesion.

12. The recombinant DNA of claim 1, wherein the effector is an antibody to a human blood component.

13. The recombinant DNA of claim 12, wherein the blood component is fibrin.

14. The recombinant DNA of claim 1, wherein substan¬ tially all the biologically active effector domains are those which have affinity for the predetermined target.

15. A transformed or transfected host cell containing the recombinant DNA of claim 1 capable of expressing the DNA to produce the fusion protein.

16. The host cell of claim 15, wherein the probe- binding amino acid sequence of the fusion protein includes cysteine residues having sulfhydryl groups for binding the probe.

17. The host cell of claim 15, wherein the probe- binding sequence of the fusion protein comprises the entire amino acid sequence of any metaUothionein, a truncated sequence of a metaUothionein, a multiple copy of the entire amino acid sequence of a metaUothionein, a multiple copy of a truncated sequence of a metaUothionein, a metaUothionein sequence generated from synthetically produced DNA, or a functional equivalent thereof.

18. The host cell of claim 15, wherein the effector sequence of the fusion protein comprises an oligopeptide, a peptide, a protein, or a glycoprotein.

19. The host cell of claim 15, wherein the effector sequence of the fusion protein comprises tissue plasminogen activator.

20. The host cell of claim 15, wherein the effector sequence of the fusion protein comprises an enzyme, a lymphokine, a cytokine, a hormone, a factor, or an antibody.

21. The host cell of claim 15, wherein the effector sequence of the fusion protein is urokinase or prourokinase.

22. The host cell of claim 15, wherein the effector sequence of the fusion protein is interleukin-1, interleukin-2, interleukin-3, interleukin-6, granulocyte stimulating factor, macrophage stimulating factor, gamma interferon, alpha inter- feron, beta interferon, or tumor necrosis factor.

23. The host cell of claim 15, wherein the effector sequence of the fusion protein is a pituitary hormone, chorionic gonadotropin, or a subunit of a pituitary hormone or chorionic gonadotropin.

24. The host cell of claim 15, wherein the effector sequence of the fusion protein is a monoclonal antibody, a fragment of a monoclonal antibody, or a fragment of a monoclonal antibody modified by a recombinant DNA technique.

25. The host cell of claim 15, wherein the effector sequence of the fusion protein is an antibody to a human disease lesion.

26. The host cell of claim 15, wherein the effector sequence of the fusion protein is an antibody to a human blood component.

27. The host cell of claim 26, wherein the blood com¬ ponent is fibrin.

28. The host cell of claim 15, wherein substantially all the biologically active domains on the effector sequence are those which have affinity for the predetermined target.

29. Agenetically-constructed fusionprotein comprising an amino-acid sequence having probe-binding sites fused to an effector amino acid sequence wherein substantially all the biologically active domains have affinity for the predetermined target.

30. The fusion protein of claim 29, wherein the probe- binding sequence of the fusion protein comprises the entire amino acid sequence of any metaUothionein, a truncated sequence of a metaUothionein, a multiple copy of the entire amino acid sequence of a metaUothionein, a multiple copy of a truncated sequence of a metaUothionein, a metaUothionein sequence generated from synthetically produced DNA, or a functional equivalent thereof.

31. The fusion protein of claim 29, wherein the effector sequence of the fusion protein comprises an oligopep- tide, a peptide, a protein, or a glycoprotein.

32. The fusion protein of claim 29, wherein the effector sequence of the fusion protein comprises tissue plas- minogen activator, an enzyme, a lymphokine, a cytokine, a hormone, a factor, an antibody, urokinase, prourokinase, inter- leukin-1, interleukin-2, interleukin-3, interleukin-6, granuloc- yte stimu-lating factor, macrophage stimulating factor, gamma interferon, alpha interferon, beta interferon, tumor necrosis factor, a pituitary hormone, chorionic gonadotropin, a subunit of a pituitary hormone or chorionic gonadotropin, a monoclonal antibody, a fragment of a monoclonal antibody, a fragment of a monoclonal antibody modified by a recombinant DNA technique, an antibody to a human disease lesion, or an antibody to a human blood component, and wherein the effector has been intentionally compromised during genetic construction of the protein to remove substantially all biologically active domains other than those having affinity for the predetermined target,

33. The fusion protein of claim 32, wherein the effector sequence of the fusion protein is tissue plasminogen activator.

34. The fusion protein of claim 29, further including a therapeutic or diagnostic probe bound to the fusion protein.

35. The fusion protein of claim 34, wherein the bound probe is a detectable probe comprising a radioactive isotope of technetium, copper, gallium, ruthenium, rhenium, yttrium, gadolinium, cadmium, or mercury.

36. The fusion protein of claim 35, wherein the detec¬ table probe comprises a fluorescent ion of an element or fluoroc- hrome.

37. The fusion protein of claim 36, wherein the f- luorescent or fluorochrome is fluorescein, rhodamine, or a fluorescent chelate of a rare earth metal.

38. The fusion protein of claim 36, wherein the fluorescent or fluorochrome is a fluorescent chelate of europium, a fluorobenzoxadiazole, or a bromobimane.

39. The fusion protein of claim 29, wherein the bound probe is a detectable probe comprising a paramagnetic ion or ions or a complex thereof.

40. The fusion protein of claim 39, wherein the paramagnetic ion or ions or complex thereof is one or more of gadolinium, iron, manganese, copper, nickel, or a lanthanide element of the atomic number 57-70, or a transition metal of the atomic number 21-29, 42, or 44.

41. The fusion protein of claim 30, wherein the effector sequence of the fusion protein comprises an oligopep- tide, a peptide, a protein, or a glycoprotein.

42. The fusion protein of claim 30, wherein the effector sequence of the fusion protein comprises tissue plas¬ minogen activator, an enzyme, a lymphokine, a cytokine, a hormone, a factor, an antibody, urokinase, prourokinase, inter- leukin-1, interleukin-2, interleukin-3, interleukin-6, granuloc- yte stimulating factor, macrophage stimulating factor, gamma interferon, alpha interferon, beta interferon, tumor necrosis factor, a pituitary hormone, chorionic gonadotropin, a subunit of a pituitary hormone or chorionic gonadotropin, a monoclonal antibody, a fragment of a monoclonal antibody, a fragment of a monoclonal antibody modified by a recombinant DNA technique, an antibody to a human disease lesion, or an antibody to a human blood component.

43. The fusion protein of claim 42, wherein the effector is tissue plasminogen activator.

44. The fusion protein of claim 30, further including a therapeutic or diagnostic probe bound to the fusion protein.

45. The fusion protein of claim 44, including a detectable probe bound to the fusion protein comprising a radioactive isotope of technetiu , copper, gallium, ruthenium, rhenium, yttrium, gadolinium, cadmium, or mercury; a fluorescent or fluorochrome; or a paramagnetic ion or ions or a complex thereof.

46. The fusion protein of claim 45, wherein the probe is a paramagnetic ion or ions or complex thereof comprising one or more of gadolinium, iron, manganese, copper, nickel, or a lanthanide element of the atomic number 57-70, or a transition metal of the atomic number 21-29, 42, or 44.

47. The fusion protein of claim 45, wherein the probe is a fluorescent or fluorochrome comprising fluorescein, rhodamine, a fluorescent chelate of a rare earth metal, a fluorescent chelate of europium, a fluorobenzoxadiazole, or a bromobimane.

48. A lyophilized preparation of the fusion protein of claim 29.

49. A method for producing a fusion protein having a probe-binding sequence and an effector sequence fused to the probe-binding sequence for selectively binding the fusion protein to a predetermined target, comprising transforming or transfect- ing a host cell with the recombinant DNA of claim 1 so that the cell produces the fusion protein.

50. The method of claim 49, wherein the probe-binding sequence of the fusion protein comprises the entire amino acid sequence of any metaUothionein, a truncated sequence of a metaUothionein, a multiple copy of the entire amino acid sequence of a metaUothionein, a multiple copy of a truncated sequence of a metaUothionein, a metaUothionein sequence generated from synthetically produced DNA, or a functional equivalent thereof.

51. The method of claim 50, wherein the effector sequence of the fusion protein comprises tissue plasminogen activator, an enzyme, a lymphokine, a cytokine, a hormone, a factor, an antibody, urokinase, prourokinase, interleukin-1, interleukin-2, interleukin-3, interleukin-6 , granulocytestimu-1- ating factor, macrophage stimulating factor, gamma interferon, alpha interferon, beta interferon, tumor necrosis factor, a pituitary hormone, chorionic gonadotropin, a subunit of a pituitary hormone or chorionic gonadotropin, a monoclonal an¬ tibody, a fragment of a monoclonal antibody, a fragment of a monoclonal antibody modified by a recombinant DNA technique, an antibody to a human disease lesion, or an antibody to a human blood component.

52. A diagnostic method for localizing or imaging a disease or disorder lesion in a mammal, comprising administering to the mammal the fusion protein of claim 29, wherein the effec¬ tor is specific to the lesion and the probe is detectable, in an amount sufficient for detection of the probe.

53. A method for the treatment of a disease or disorder in a mammal, comprising administering to the mammal the fusion protein of claim 29 wherein the effector is specific to the disease and the probe is therapeutic, in a therapeutic amount.

54. The method of claim 53, wherein the disease or disorder is thrombosis, thromboembolism, or blood clots, and the effector is tissue plasminogen factor.

55. The method of claim 52, wherein the disease is cancer.

56. The method of claim 53, wherein the disease is cancer.

57. The method of claim 53, wherein the disease or disorder is inflammatory.

58. The method of claim 53, wherein the disease or disorder is infectious.

59. The method of claim 53, wherein the therapeutic probe comprises a radioactive isotope, a toxic metal, a fluores- cent or photosensitive metal or organic compound suitable for use in laser therapy, or an enzyme having localized oxidative cell killing properties.

60. A diagnostic kit for in vivo or in vitro diagnosis of mammalian diseases or disorders comprising a diagnostic amount of the fusion protein of claim 29 as a diagnostic reagent inter¬ mediate.

61. The diagnostic kit of claim 60, packaged for clinical, laboratory, home, or medical office use.

62. A diagnostic or therapeutic clinical kit including the probe-labeled fusion protein of claim 30.

63. The kit of claim 62, wherein the probe is a fluorochrome or enzyme.

64. A diagnostic kit for in vivo or in vitro diagnosis of mammalian diseases or disorders comprising a diagnostic amount of the fusion protein of claim 30 as a diagnostic reagent inter¬ mediate.