This invention relates to novel reagent conjugates and a novel multi-step process for delivering active compounds to target analytes of interest in a patient for diagnostic and therapeutic purposes. According to the process, two novel reagents are bound to each other by linkage of the sequence-specific components they contain. The first reagent, which is comprised of a target recognition component and a first sequence-specific component, is introduced into the patient and allowed to achieve maximal localization on the target cells. The second reagent, which is comprised of an active compound component and a second sequence-specific component is then introduced into the patient, thereby forming a complex with the first reagent via the recognition and binding of the sequence-specific components of the two reagents to form the reagent conjugate of the invention. The active compound component is thereby efficiently and specifically delivered to the target analyte.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention relates to novel reagent conjugates and novel multi-step processes to deliver active compounds to target analytes. The novel reagent conjugates and processes of the invention have broad application in the medical and research field for imaging and for delivery of an unlimited range of compounds to the patient for therapeutic purposes.

The two reagents which make up the reagent conjugate of the invention each consist of two components which may be directly or indirectly linked to each other. The first reagent is comprised of a target recognition component and a first sequence-specific component. The second reagent is comprised of an active compound component and a second sequence-specific component which recognizes and specifically binds to the first sequence-specific component. This second sequence-specific component rapidly finds and attains optimal binding with the first sequence-specific component, thereby linking the two reagents to form the reagent conjugate of the invention.

Each sequence-specific component of the invention consists of an array of covalently linked units which specifically recognizes and binds to a complementary array of similar units comprising the sequence-specific component of the corresponding reagent. These sequence-specific components include, but are not limited to, DNA, DNA-like polymers, RNA, RNA-like polymers, and synthetic polymers with natural or modified ribo- or deoxyribonucleotide bases. The sequence-specific components of the invention may also consist of proteins (especially those which form alpha-helices in solution), leucine zippers, and any other naturally occurring or synthetic polymer that can form specific and stable linkages with other like polymers.

The novel reagent conjugates can be used, for example, to radio-image sites of tumor concentration. Tumor radio-imaging methods are disclosed which employ the novel reagent conjugates of the invention. Application of the instant invention is not limited to the detection of tumors, however, but has broad and unlimited application in diverse areas of medical research and diagnostics to detect any target analyte of interest.

Another aspect of the invention relates to a therapeutic treatment method which comprises the delivery of therapeutic drugs to specific target sites in vivo, e.g. viruses, virus-infected cells, tumor cells, bacterial cells, pathogens, fungi, parasites, and abnormal eukaryotic cells.

Kits for detecting or delivering therapeutic agents to such sites are another aspect of the invention.

In the preferred embodiment, the method of the invention comprises the introduction into the patient of an effective amount of the first reagent, which is comprised of a target recognition component and a first sequence-specific component. The first reagent then equilibrates for an effective amount of time to achieve maximal localization on the target analyte, for example, a cell surface epitope of interest. An effective amount of a second reagent, which is comprised of an active compound component and a second sequence-specific component is then introduced into the patient by an appropriate method. The second sequence-specific component rapidly finds and binds to the first sequence-specific component, thereby delivering the active compound to the target.

In other embodiments of the invention the components of the first and/or second reagents may be administered separately, in a step-wise fashion using a multi-step procedure.

There are several important advantages to be obtained by utilizing the multi-step method of the invention in which two sequence-specific components recognize and bind to each other to link the reagents of the invention. The use of the sequence-specific components to form the reagent conjugate of the invention results in highly specific delivery of the active compound to the target. For applications in which this delivery system forms part of an in vivo detection or imaging system, use of the invention results in superior images and improved detection sensitivity. For therapeutic applications, use of the invention should result in improved efficiency or effectiveness in delivering therapeutic compounds to targets.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a multi-step procedure (See FIG. 1, see Original Patent) for delivering active compounds to target analytes in which a high level of specificity is achieved by the recognition and binding of the sequence-specific components of the two reagents of the invention to each other. This recognition and binding event links the two reagents to form the reagent conjugate of the invention, thereby delivering the active compound to the target analyte.

According to the preferred embodiment of the invention, an effective amount of a first reagent, which is comprised of a target recognition component and a first sequence-specific component, is introduced into a patient by intravenous, subcutaneous, lymphatic, intramuscular, intraperitoneal, or cerebrospinal routes, or by other available methods for introducing macromolecules into an organism. The first reagent then equilibrates for an effective amount of time to achieve maximal localization on the target analyte. An effective amount of a second reagent, which is comprised of an active compound component and a second sequence-specific component, is then introduced into the patient by an appropriate method. The second sequence-specific component rapidly finds and specifically binds to the corresponding first sequence-specific component, which is bound to the first reagent, thereby linking the two reagents to form the reagent conjugate of the invention. Thus, the active compound is delivered with great specificity and efficiency to the target.

In other embodiments of the invention, the components of the first and/or second reagents may be administered separately, in a step-wise fashion using a multi-step procedure. For example, in the case where the components of the first reagent are administered step-wise, the target-recognition component of the first reagent is introduced into the patient and allowed to equilibrate, as in the preferred embodiment. An appropriate quantity of the first sequence-specific component is then added, such that the sequence-specific component is able to find and non-covalently bind to all or most of the target analyte-bound target recognition component, thereby resulting in the formation of the target analyte-bound first reagent in vivo. An effective amount of the second reagent, comprising the second sequence-specific component and the active compound component, may then be administered in a single step and allowed to bind to the target analyte-bound first reagent. If desired. the second reagent may also be added in a step-wise fashion, in which case the second sequence-specific component is administered after the first reagent has been formed, or has reached equilibration with the target, and the second sequence-specific component is allowed to bind to the first sequence-specific component. An effective amount of the active compound component of the second reagent is then added and allowed to bind to the second sequence-specific component which has already been localized to the target analyte-bound first reagent, thereby forming the reagent conjugate of the invention and specifically delivering the active compound to the target site.

In some embodiments of the invention, the individual components of the reagents may be delivered in such a form that they are able to directly bind to each other, i.e., the target recognition component can directly bind to the first sequence-specific component and the active compound component can directly bind to the second sequence-specific component in vivo. For example, the target recognition component may contain a covalently or non-covalently bound avidin moiety and the first sequence-specific component may contain a covalently or non-covalently bound biotin moiety. The binding of the avidin group on the target recognition component to the biotin moiety of the first sequence-specific component will effectively deliver the first sequence-specific component to the target analyte-bound target recognition component, thereby forming the first reagent in vivo. This use of biotin-avidin binding is in direct contrast to previously described delivery systems (see, for example, Goodwin, et al., Journal of Nuclear Medicine (1987) 28:722), where biotin-avidin interactions were used to directly link the target recognition and active compound components of the delivery system.

In other embodiments of the invention, an additional element is required to mediate the binding of the individual components. An example of such an embodiment would be the case where the target recognition component contains a biotin moiety and the first sequence-specific component also contains a biotin moiety. In this case, the target recognition component would be administered and allowed to equilibrate. In a second step, avidin, a biotin-binding protein, would be introduced and allowed to bind to the biotin moiety on the target recognition component. A third step would then consist of introducing the biotinylated first sequence-specific component, which binds to the avidin on the target analyte-bound target-recognition component to form the first reagent. The avidin thus mediates the attachment of the two components of the first reagent in vivo such that they are indirectly bound to each other. As with the first reagent, the components of the second reagent may also be added in a step-wise fashion and may be directly or indirectly linked to each other.

The indirect linkage of the reagent components may also be mediated by interactions between proteins, modified or unmodified, naturally occurring or synthetic nucleic acids, proteins and modified or unmodified, naturally occurring or synthetic nucleic acids, hormones and receptors, small molecular weight organic compounds, metabolites, or any other components that are capable of forming specific and stable linkages.

The term "target recognition component" refers to any substance which is capable of recognizing and binding to the target analytes of the invention. Among the common target recognition components useful according to the invention are antibody fragments, antibody fragment multimers, metabolites, hormones, modified or unmodified, naturally occurring or synthetic nucleic acids or nucleic acid-like polymers. Other target recognition components which can be used in accordance with the invention are naturally occurring or synthetic proteins or other organic polymers with binding specificity and affinity comparable to, or exceeding, that found using antibodies and antibody fragments.

The target recognition component can be used to specifically detect and bind to unique aspects of the analyte. The target analyte of the invention may be comprised of a molecule of small or high molecular weight, a molecular complex. a pathogen, or a biological system, such as a virus, a cell. or a group of cells. For example, target analytes may be comprised of proteins, lipids, carbohydrates, polysaccharides, lipopolysaccharides, protein complexes, and nucleic acids or segments thereof, either single-stranded or double-stranded, which may be found on, or within, a cell, a virus, viral components such as cores or capsids, bacteria of different types, tissue cells, pathogenic or non-pathogenic components of a cell, within the cell or on the cell surface, and the like. Some examples of the types of cells that may be detected in accordance with the invention are bacterial cells, fungal cells, virus-infected cells, abnormal eukaryotic cells, and cancer cells, including those found in tumors, such as melanoma or lymphoma cells, as well as those found in body fluids. Bacteria, either whole cells or fragments thereof, such as cell walls or other recognizable portions, including both gram positive and gram negative bacteria, may also be detected. Fungi and other microorganisms are also detectable, as well as animal cells, e.g. mammalian cells. Among the most common target analyte proteins are the structural proteins, enzymes, immunoglobulins, receptors, or fragments thereof. Among the most common nucleic acids which can be detected are DNA and RNA, including, but not limited to, tRNA, mRNA, rRNA, and the like. Cellular components characteristic of a particular tissue or organ can also be targeted, thereby allowing tissue- or organ-specific delivery of the active compounds of the invention. However, the method of the invention can be used to detect an unlimited range of target analytes. Any protein, lipid, carbohydrate, polysaccharide, polymer, metabolite, or nucleic acid characteristic of a particular tissue, organ, pathogen, or disease state may be detected. In the preferred embodiments of the invention, the target recognition component recognizes cell surface moieties such as proteins, lipids. polysaccharides, lectins or nucleic acids.

The term "active compound component" refers to any substance which is capable of performing a diagnostic or therapeutic function in accordance with the invention. The active compound component is delivered to the patient through the method of the invention either for diagnostic purposes, to detect the presence of the target analytes, or for therapeutic purposes, to treat the patient by delivering therapeutic compounds to specific sites. Examples of active compound components useful in accordance with the invention include, but are not limited to, radioisotopes, complexes of radioisotopes bound to chelator moieties, moieties containing directly or indirectly linked radioactive ions, toxins, cytokines, chemotherapeutic agents, synthetic drugs, differentiation inducers. radio-therapeutic compounds, radio-diagnostic compounds, proteins, hormones, polysaccharides, oligosaccharides, receptors, enzymes, enzyme inhibitors, enzyme substrates, enzyme cofactors, metabolites, vitamins, anti-metabolites, electron dense materials, metal-containing compounds, and naturally occurring or synthetic, modified or unmodified nucleic acids, including those composed of chemically or enzymatically modified bases, sugars or linking groups. Anti-tumor agents, such as doxorubicin and cisplatin, anti-microbial agents, such as aminoglycosides and antibiotics, anti-fungal agents, anti-viral agents, and anti-parasitic agents are also useful as active compound components of the invention. Liposomes with surface moieties that enable them to recognize and bind to the sequence-specific component of the second reagent may be used to encapsulate any or all of the above-identified active compound components, thereby delivering the active compound components to the target.

The term "sequence-specific component" in accordance with the invention, refers to those polymers which recognize a corresponding unit on another polymer and bind to this unit through the formation of hydrogen bonds, ionic pairs, or through hydrophobic interactions. The first reagent comprises the first sequence-specific component. The second reagent comprises the second sequence-specific component.

Among the most likely sequence-specific components useful according to the method of the invention are DNA, DNA-like polymers, RNA, RNA-like polymers, including but not limited to, modified and unmodified purines and pyrimidines, polypeptides (especially those which form alpha-helices in solution, such as leucine zippers), proteins, as well as synthetic polymers with modified or unmodified nucleotide bases. However, the method of the invention can be applied to any polymer with a given sequence of units which can recognize and specifically bind to a particular sequence of units on another polymer.

A nucleic acid sequence-specific component in accordance with the invention, may comprise any nucleotide sequence, provided that it is long enough to provide stable and specific annealing with a complementary nucleotide sequence under the desired conditions, that it be complementary to the sequence-specific component in the other reagent of the reagent conjugate, and that it be sufficiently different from other nucleotide sequences that may be present so as to avoid the likelihood of binding to non-target sequences. The sequence-specific components useful in accordance with the invention are preferably single-stranded oligonucleotides from about 8 to about 50 bases long, most preferably, from about 8 to about 20 bases in length. This characteristic ensures effective and rapid association between corresponding polymers. A shorter length than that set forth will result in a less effective association between these polymers, while a polymer length greater than that set forth will not result in any additional selectivity of binding. Further, a longer sequence-specific component is more expensive to produce. Other polymer types and lengths are also useful according to the invention. The optimal lengths of these other polymers will depend upon the particular polymer chosen and the specific application for which it will be employed.

In the preferred embodiment, the sequence-specific components are nuclease-resistant oligonucleotides, preferably homopolymers, such as the 2'-modified nucleic acids described in U.S. patent application Ser. No. 08/032,769, filed on Mar. 16, 1993, which is a continuation of U.S. patent application Ser. No. 446,235, filed on Dec. 4, 1989, now abandoned to Brakel, et al, entitled "Modified Nucleotide Compounds." assigned to the instant assignee, the disclosure of which is incorporated by reference herein and made a part hereof. These modified oligonucleotides are more resistant to degradation by nucleases present in serum.

The sequence-specific components may be directly or indirectly linked to the target recognition or active compound components of the two reagents of the invention in vivo or in vitro. Attachment of the two components of each reagent may be direct, by covalent attachment of the two entities. Alternatively, the two components of each reagent may be indirectly joined to each other, such as by non-covalent binding of specific moieties on each component to one or more mediating compounds. They may be introduced into the patient simultaneously with the other components of the reagents either in complexed form, as would be the case when they are joined in vitro, or as individual entities, as when they are joined to the other reagent components in vivo.

The active compound components and target recognition components as defined in this invention are linked by the binding of the first and second sequence-specific components to each other. Alternatively, multiple linkages may be formed in accordance with the invention, such as by the attachment of multiple sequence-specific components to the same target recognition or active compound component, or by the sequential binding or layering of multiple pairs of sequence-specific components to each other to form a linkage between the target recognition component and the active compound component. The formation of multiple linkages may be advantageous with regard to achieving increased sensitivity or specificity for a particular diagnostic or therapeutic effect.

Various methodologies may be used to link the target recognition components and the active compound components of the invention to the first and second sequence-specific components, respectively. In the case where the sequence-specific component is an oligonucleotide, a linker as described in U.S. Pat. Nos. 4,711,955 (issued on Dec. 8, 1987) and 5,328,824 (issued on Jul. 12, 1994), both of which are assigned to Yale University, the disclosures of which are incorporated by reference herein and made a part hereof, maybe used to attach the oligonucleotide to the target recognition or active compound components of the reagent. For example, the linker may be covalently attached to oligonucleotides which have been derivatized such that they contain terminal allylaminouracil groups with accessible amino groups, or terminal thiol groups, as described in Engelhardt et al., U.S. Pat. No. 5,241,060, "Base Moiety-Labeled Nucleotide," issued on Aug. 31, 1994, based on Ser. No. 07/532,704, filed on Jun. 4, 1990, which is a divisional of Ser. No. 07/140,980, filed Jan. 5, 1988, abandoned, which is a continuation of Ser. No. 06/674,352, filed Nov. 21, 1984, abandoned, which is a continuation application of Ser. No. 06/391,440, filed Jun. 23, 1982, titled "Modified Nucleotides, Methods of Preparing and Utilizing and Compositions Containing the Same," also abandoned. Also issued in the same aforementioned patent family is Engelhardt et al., U.S. Pat. No. 5,260,433, "Saccharide Specific Binding System Labeled Nucleotides," that issued on Nov. 9, 1993 as a divisional of the aforementioned Ser. No. 07/140,980. Ser. No. 391,440, filed on Jun. 23, 1982, entitled "Modified Nucleotides, Methods of Preparing and Utilizing and Compositions Containing the Same," to D. Engelhardt, et al., now abandoned, which was re-filed as U.S. patent application Ser. No. 674,352 on Nov. 21, 1984, now abandoned, which was re-filed as pending U.S. patent application Ser. No. 140,980 on Jan. 5, 1988, abandoned in favor of continuation application Ser. No. 07/685,982, filed on Apr. 15, 1991. All of the foregoing patent disclosures are incorporated by reference herein and made a part hereof. Thus, in the case where the sequence-specific components of the invention are comprised of oligonucleotides, the methods taught in the above-cited references may be used to attach the target recognition component and the active compound component to their respective sequence-specific components.

Alternatively, oligonucleotides that are attached to linkers which terminate in an N-hydroxysuccinamidyl ester, imidoester, or an imidazolyl carbamate group may be reacted with the amino groups on the target recognition component to yield the first reagent of the invention.

Still another method for attaching an oligonucleotide sequence-specific component to a target recognition or active compound component would be to use an oligonucleotide which is attached to a linker arm that terminates in an amine or a hydrazine group which could then react with the amine groups of the other reagent components.

When the sequence-specific components of the invention are comprised of oligonucleotides and when the above-cited means of attachment are used, the linker arms of the invention are preferably from about 8 to about 30 atoms in length. This length is optimal for separation of the sequence-specific components from either the active compound component or the target recognition component. A linker arm containing fewer atoms could reduce the association between corresponding polymers, interfere with target recognition, or diminish the effectiveness of the active compound component.

Other methods useful in accordance with the invention for covalently attaching polynucleotides to proteins are described in the literature. These methods include carbodiimide cross-linking (Halloran, M. K., J. Immunol. 373 (1966), cross-linking in the presence of formaldehyde (Manning, J. E., et al., Chromosoma 53: 107-117 (1975)), treatment with 4-azidophenyl glyoxal (Politz, S. M., Biochemistry 20: 372-378 (1981), and oxidation of the 2' and 3' hydroxy groups of a polyribonucleotide or a polydeoxyribdnucleotide with a 3'-terminal ribonucleotide, followed by a Schiff's base reaction with the amine groups of a protein and borohydride reduction (Sodja, A., et al., Nucleic Acids Research 5: 383-401 (1978)). Other methods include direct bromination of DNA (Jones, A. S., Nature 183: 1603 (1959) followed by reaction with diaminohexane and coupling via protein carboxyl functions (Lowe, C. R., Eur. J. Biochem 73: 265-274 (1977)), or by mercuration of cytosine moieties (Dale, R. M. K., et al, PNAS 70: 2236-2242(1973)) followed by halogenation (Dale, R. M. K., et al, Nucleic Acids Res. 2: 915-930 (1975)).

According to the preferred embodiment of the invention, the first reagent is comprised of a target recognition component which is an Fab' fragment of an IgG and a first sequence-specific component which is an oligonucleotide with about 8 to about 20 bases. The target recognition component is covalently joined to the first sequence-specific component through an appropriate length linker arm, in accordance with the preferred method of the invention. In this embodiment, the linker, which contains either an iodoacetamide or a bromoacetamide group, is attached to the Fab' fragment on one end and to the 3' terminus of the oligonucleotide on the other end, thereby forming the first reagent of the invention.

Other oligonucleotides, ranging from about 8 to about 50 bases in length may be used as sequence-specific components, in accordance with the invention. These oligonucleotides may be prepared by phosphoramidite chemistry using an automated oligonucleotide synthesizer.

In other embodiments, a 5' terminal amino group may be introduced onto the oligonucleotide sequence-specific component, as described by Agrawal et al. (Agrawal. S., Christodolou, C. and Gait, M. J. (1986) Nucl. Acids. Res. 14: 6227). An activated ester reaction in which the 5' terminal amino group of the oligonucleotide is reacted with a terminal carboxylic acid on a linker may be used to form the first reagent of the invention.

In the preferred embodiment, the first reagent, comprising a complex of the target recognition component and the first sequence-specific component, as described hereinabove, is administered to the patient in a single step, such as by intravenous, subcutaneous, intramuscular, intrabronchial, intrapleural, intraarterial, intraperitoneal, lymphatic, or cerebrospinal routes, or using any other available system for introducing macromolecules into an organism. The dosage at which the first reagent is administered to the patient depends upon the target analyte, as well as upon the particular target recognition and sequence-specific components which are employed.

The equilibration period for the first reagent is that length of time required to achieve maximal localization at the target site. One advantage of the instant invention is that it provides for a delivery system which includes a lengthy equilibration period for the first reagent without unnecessarily exposing the patient to harmful agents for long periods of time. The second reagent can bind quickly and efficiently to the fully equilibrated first reagent, further minimizing the time and amount of patient exposure to potentially harmful active compounds. At the end of the equilibration period, according to a preferred embodiment, the first reagent circulating in the blood may be removed or cleared to prevent it from binding to the second reagent. The method of the invention is not limited to the use of a clearing step. However, background may be reduced, resulting in a clearer image upon radioimaging, when radioimaging is desired in accordance with the invention, if a clearing step is included in the procedure.

Clearance of excess first reagent from the patient may be effected by a reagent similar to the second reagent. except that the active compound component contained in the second reagent is replaced by a different compound, e.g., human transferrin, according to the preferred embodiment, such that clearance is effected by the binding of the second sequence-specific component in the clearing agent to the first reagent via its complementary sequence-specific component (See FIG. 2, see Original Patent). Alternatively, the clearing agent may consist of target analyte or target analyte-like moieties which are attached to transferrin such that the clearing agent binds to the excess. circulating target recognition component of the first reagent. This type of clearing agent is similar to that described by Goodwin, et al., Journal of Nuclear Medicine (1988) 29: 226-234.

In other embodiments of the invention, the target recognition component and the first sequence-specific component may be administered to the patient sequentially, in separate steps such that these components join non-covalently in the patient to form the first reagent. In these cases, adequate time periods are allowed for optimal binding and equilibration of the target recognition component and the target analyte and, subsequently, for binding and equilibration of the first sequence-specific component to the bound target recognition component. Clearance of excess target recognition or first sequence-specific components can be effected using one or more types of clearing agents described in the preferred embodiment of the invention, or any other type or kind of clearing agent useful for the purpose desired. Clearance may be accomplished in a single step, either following the equilibration of the target recognition component, or following the formation of the first reagent, or in multiple steps, following the equilibration of each component type.

After an appropriate period of time has elapsed for equilibration, formation, and clearance of the first reagent, the second reagent is administered to the patient. In the preferred embodiment, the second reagent is comprised of an active compound component and a second sequence-specific binding component, as defined hereinabove, connected by a covalently bound linker arm. In other embodiments, the components of the second reagent are non-covalently attached to one another and may be added as a preformed second reagent. Alternatively, the components of the second reagent may be administered sequentially in a multi-step procedure, wherein the active compound component and the second sequence-specific component are joined in the patient to form the second reagent of the invention. In this embodiment, the second sequence-specific component rapidly finds and binds to the first sequence-specific component which is directly or indirectly attached to the target recognition component, now bound to the target analyte. The active compound component is then added and allowed to bind to the second sequence-specific component, thereby forming the second reagent of the invention and completing the formation of the reagent conjugate.

The formation of the reagent conjugate of the invention is thus dependent upon the presence of the target analyte. The reagent conjugate thereby formed may be used for diagnostic applications, i.e., for imaging, with high sensitivity, specificity, and accuracy, or for therapeutic applications, by virtue of the therapeutic action of the active compounds thereby delivered.

The sequence-specific component of the second reagent in the preferred embodiment is comprised of an oligonucleotide which is complementary to the oligonucleotide which forms the sequence-specific component of the first reagent (See FIG. 2, see Original Patent). In the preferred embodiment, this first sequence-specific component is a 2'-modified oligoguanidylate which is about 20 bases in length. The second sequence-specific component is a 2'-modified oligocytidylate which has modified termini that render it resistant to diesterases in the serum. This second sequence-specific component binds rapidly and specifically to the corresponding first sequence-specific component in the first reagent. Modified homopolymers are the preferred sequence-specific components of this invention since they are resistant to nucleolytic degradation and they bind rapidly to complementary oligonucleotides. A reactive amino group is introduced onto the 3' terminus of the oligonucleotide to facilitate covalent attachment of this entity to a suitable linker arm. Derivatization of the oligonucleotide is accomplished using an appropriate type of controlled pore glass bead reaction column. In the preferred embodiment, a linker arm similar to that used to assemble the first reagent, except that it terminates in an N-hydroxy-succinimide group, forms a covalent bond with the derivatized oligonucleotide sequence-specific component of the second reagent. The oligonucleotide is then reacted with a derivatized 1,2-diaminocyclohexanetetraacetate. DCTA, chelator as more fully set forth in U.S. Pat. No. 4,707,352, entitled "Method of Radioactively Labeling Diagnostic and Therapeutic Agents Containing a Chelating Group," issued Nov. 17, 1987; U.S. Pat. No. 4,767,609, entitled "Therapeutic and Diagnostic Processes Using Isotope Transfer to Chelator-Target Recognition Molecule Conjugate," issued on Aug. 30, 1988, and U.S. Pat. No. 4,772,548, entitled "Radioisotopic Assay Using Isotope Transfer to Chelator-Target Recognition Molecule Conjugate," issued on Sep. 20, 1988, all to J. Stavrianopoulos, assigned to the instant assignee, and the disclosures of which are incorporated by reference herein and made a part hereof.

In the preferred embodiment, a radioactive metal ion having a short half-life, such as the beta emitter .sup.90Y is used as the active compound component for diagnostic imaging (See FIG. 2). However, other radioactive metal ions may be used, depending upon whether it is desired to image a tissue or organ, or to irradiate and kill undesirable cells or pathogens, for example, viruses, bacteria, or cancerous cells. Additional metal ions, which are also useful in accordance with the invention for diagnostic imaging include, but are not limited to the gamma emitters, .sup.111In and .sup.99mTc, and the positron emitters, .sup.49Cr, .sup.52Fe, .sup.55Co, .sup.56Mn, .sup.61Cu, .sup.68Ga, and .sup.90Mo. According to the instant invention, these metal ions may be used simultaneously, or individually, in any desired combination, to perform single or multiple radio-imaging assays. Metal ions which are useful for tumor therapy in accordance with the invention may be selected from a broad range of radio-therapeutic agents, which include, but are not limited to, .sup.61Co, .sup.90y, .sup.106Rh, .sup.149Nd, .sup.150Pm, .sup.188Re, and .sup.212Bi.

These metal ions, in accordance with the preferred embodiment of the invention, are strongly bound to a chelator moiety of the second reagent immediately before administration into the patient. Thus, the active compound component remains stable in the system, thereby maximizing its effectiveness after delivery. However, the radioisotopes may also be bound to a chelator moiety of the second sequence-specific component in vivo.

The use of the chelator in accordance with the invention, reduces the non-specific background, which becomes important at the time when the cells are radioimaged, and minimizes the exposure of non-target tissues and organs to radioisotopes.

In order to guarantee specific targeting of chelated radioactive metals delivered using the method of the invention, the chelator molecule must exhibit the following properties: (1) The chelator must exhibit a high binding affinity for the metal under consideration. This will ensure that the radioisotope is specifically bound to the conjugated protein at the position of the chelator molecules, rather than randomly associated with charged residues along the polynucleotide or polypeptide chain; (2) The disassociation rate of the radioactive metals from the chelator molecule must be low in order to minimize the rate at which the radioactive metals become available for random binding to other non-specific proteins in vivo; (3) The chelator must be capable of conjugation to the sequence-specific component of the second reagent under mild conditions, so as to avoid, or minimize inactivation of the second sequence-specific component; and (4) The presence of the chelator molecule in the reagent conjugate must not interfere with the function of the target recognition or sequence-specific components.

The preferred chelator for purposes of the instant invention is 1,2-diaminocyclohexanetetraacetate (DCTA), since this compound has a higher binding affinity than that of comparable chelators. Other chelators which are useful according to the invention are p-benzylamino-TETA (p-benzylamino 1,4,8,11 tetra aza cyclohexyltetradecane, N, N', N'', N'''-tetra acetic acid) and its derivatives, as described by Moi et al. (1985) Analyt. Biochem. 148: 249; EDTA (ethylenediamine tetraacetic acid) and its derivatives, and DTPA (diethylenetriamine pentaacetic acid) as more fully set forth in U.S. Pat. No. 4,622,420 to Meares et. al. Other chelating groups can be used for purposes of the invention and are bonded to the metal ions through appropriate means.

In accordance with the preferred method of the invention, the chelator is attached to a linker arm before the linker is joined to the 3'-amino terminus of the oligonucleotide used as the sequence-specific component of the second reagent, as more fully described in U.S. Pat. Nos. 4,707,352, 4,767,609, and 4,772,548, as cited hereinabove and incorporated by reference herein.

Metal ions may be non-covalently bound to the chelator using procedures described in U.S. Pat. No. 4,707,352 as cited hereinabove. The chelated metal ions are separated from excess unchelated ions by column chromatography and administered after an appropriate time period and at an appropriate dosage, based upon the desired diagnostic or therapeutic use.

The method of the invention can be used in conjunction with any of the conventional imaging systems involving radioactivity, i.e. to detect gamma rays or X-rays. The preferred radionuclides useful for the invention emit positron or gamma rays. Thus, the most preferred metal ions useful in accordance with the invention include .sup.111In, .sup.68Ga, .sup.55Co, and .sup.61Cu. Emitted gamma rays may be detected using a variety of techniques. Positron emission tomography is the preferred method of radioimaging positrons in accordance with the invention. Any other available means of radioimaging radioactive compounds such as scintillography, may also be used in accordance with the invention.

The method of the invention has the additional advantage that only small amounts of the unbound active compound component remain in the system at the time of imaging, due, in part, to the complete specificity of the recognition and binding between the two sequence-specific components, as taught in the instant invention. Additionally, if there is any unbound active compound component, it may be cleared, in conjunction with the active compound component associated with the sequence-specific component, through the renal system prior to imaging. The incorporation of a clearance step into the procedure results in lower backgrounds, thereby yielding a clearer image when radioimaging is performed. In addition, any system can be tailored, based upon the instant invention, to detect or treat an unlimited range of targets, since one skilled in designing such systems can utilize an unlimited range of target recognition components, active compound components, and sequence-specific components for the purposes of the invention.

The present invention lends itself readily to the preparation of kits comprising one or more of the elements necessary to perform the intended diagnostic or therapeutic functions.
 

Claim 1 of 14 Claims

1. A method for visualizing specific target analytes in a subject which comprises: (1) providing a reagent conjugate useful for delivering active compounds to specific target analytes into a subject, said conjugate comprising (a) a first reagent comprising a specific target recognition component which comprises a Fab' antibody fragment which is covalently attached to a first sequence-specific component comprising a first oligonucleotide comprised of 2'-chlorodeoxy-guanosine which is about 20 bases long; and (b) a second reagent comprising an active compound component linked to a second sequence-specific component which specifically recognizes and binds to said first sequence-specific component, said second reagent-comprised of .sup.90y bound to a 1,2-diaminocyclohexane-tetraacetate (DCTA) chelating moiety, said second sequence-specific component comprising a second oligonucleotide, said DCTA chelating moiety being covalently attached through the allylamino group at the 5-position of a cytidyl residue in said second oligonucleotide which comprises the sequence [(2'-chloro-deoxycytosine).sub.3(2'-chloro-deoxycytosine-5-allylamine)].s- ub.5, and wherein said first and second sequence-specific components (a) and (b) have been rendered nuclease resistant by the presence of one or more nucleotide analogs to said oligonucleotides; (2) introducing into said subject an effective amount of said reagent conjugate; and (3) visualizing said specific target analytes.

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