EP0680340A1

EP0680340A1 - Amplified direction of effector groups to specific target cells in an animal

Info

Publication number: EP0680340A1
Application number: EP94907865A
Authority: EP
Inventors: Amin I. Kassis
Original assignee: Harvard College
Current assignee: Harvard College
Priority date: 1993-01-21
Filing date: 1994-01-21
Publication date: 1995-11-08
Also published as: WO1994016734A1; CA2154471A1; AU6126794A; JPH08508713A; IL108388A0

Abstract

Targeting certain effector molecules to specific living cells, such as a cancer cells, or their constituents in an animal including a human. In the method of the invention sequential formation of specific binding pairs targets effector molecules in an amplified fashion to a particular target cell. Initially, a specific binding pair if formed between a target molecule on the target cell and a first reagent. This specific binding pair formation results from specific binding between the target molecule and a first functional group on the first reagent. Sequential specific binding pairs are then formed between the first reagent and a second reagent, the second reagent and a third reagent etc.. These sequential specific binding pairs form because each reagent has a second functional group that specifically binds to a first functional group present in the next reagent in the sequence. Amplification occurs because the first and second functional groups on each reagent are multivalent. Generally, the last reagent in the sequence has a first functional group, but has an effector group in place of the second functional group. The effector group may be either a diagnostic marker or a therapeutic molecule.

Description

AMPLIFIED DIRECTION OF EFFECTOR GROUPS TO SPECIFIC TARGET CELLS IN AN ANIMAL

BACKGROUND OF THE INVENTION

Field Of The Invention

The invention relates to the optimization of diagnostic and therapeutic uses of specific binding molecules, such as monoclonal antibodies. More particularly, the invention relates to use of such molecules to direct effector groups in an amplified fashion to specific target cells in an animal, including a human.

Summary Of The Related Art

Monoclonal antibodies (MAbs) have high specificity and high affinity and/or avidity for their antigens. Because of this, Goldenberg et al., New-

England J. Med. 298.: 1384- 1386 (1978); Mach et al., Immunol. Today 2:239-249 ( 1981 ); and Eppanetos et al., Lancet 2:999-1006 ( 1982), among others, have considered MAbs particularly attractive as selective carriers of diagnostic and/or toxic agents. Ghose and Blair, J. Natl. Cancer Inst. 6J.:657-676 ( 1978); Eckelman et al.,

Cancer Res. 40:3036-3042 (1980); Pressman, Cancer Res. 40:2960-2964 ( 1980) and many others teach the use of MAbs for diagnosis in humans, using, e.g.. ^99mTc or ^ιnIn, or for therapy in humans, using beta emitters such as ¹³¹I, ¹⁸⁶Re, ⁹⁰Y,

165_Dy5 67_{Cu5 Qr} 109p_{d) Qr a}j_{pha em}j_{tters suc}h _as 2H_{At or} 212_βi More recently, however, problems such as low percent maximum injected dose per gram of target tissue, slow clearance and nonuniform distribution within tumors have led Fischman et al., J. Nucl. Med. 30^*191 1-1915 ( 1989) and others to question the future of MAbs in radioimmunodiagnosis and radioimmunotherapy. In addition, direct radiolabelling of MAbs can inadvertently and adversely affect their structure, charge immunological function, kinetics of biodistribution and targeting potential. Badger et al., Cancer Res. 45:1536-1544 ( 1985) discloses that having two or more iodine atoms per MAb molecule can seriously alter the biological residence time in the bloodstream and the immunological function of the MAb. Endo et al., J. Immunol. Methods 104:243- 248 ( 1987) teaches that coupling of even a few drug molecules to amino groups of a MAb can decrease its antigen binding capacity.

These limitations demonstrate the need for better labelling procedures that maintain MAb structural integrity, as well as better targeting approaches that do not depend so heavily on the preservation of structural integrity of each and every MAb molecule. Various approaches have been addressed to these needs. Goodwin et al., J. Nucl. Med. 29:226-234 (1988) discloses the use of chelated ^nlIn as label, in conjunction with bifunctional antibodies having one valency directed to a target tumor-associated antigen and the other valency directed to a ¹¹¹In-chelate. Paganelli et al., Int. J. Cancer 2:121-125 ( 1988) teaches the use of biotinylated antibodies with radiolabelled avidin as a detectable marker. Khawli et al., J. Nucl. Med. 29:923 ( 1988) discloses the use of avidin- conjugated antibodies with a radiobiotin derivative marker.

These approaches, however, have also demonstrated shortcomings. For example, Garlick and Giese. J. Biol. Chem.263:210-215 ( 1988) teaches that certain radiolabeled biotin derivatives exhibit a reduced affinity for avidin.

There is, therefore, a need for new methods for directing effector molecules to specific target cells that do not rely upon strict structural integrity of all MAb molecules used. Preferably, such methods should allow more than one effector molecule to be directed to each target molecule.

BRIEF SUMMARY OF THE INVENTION

The invention relates to targeting certain effector molecules to specific living cells, such as cancer cells, or their constituents in an animal, including a human. In the method of the invention sequential formation of specific binding pairs targets effector molecules in an amplified fashion to a particular target cell. Initially, a specific binding pair if formed between a target molecule on the target cell and a first reagent. This specific binding pair formation results from specific binding between the target molecule and a first functional group on the first regent. Sequential specific binding pairs are then formed between the first reagent and a second reagent, the second reagent and a third reagent, and so on. These sequential specific binding pairs form because each reagent has a second functional group that specifically binds to a first functional group present in the next reagent in the sequence. Amplification occurs because the first and second functional groups on each reagent are multivalent. Generally, the last reagent in the sequence has a first functional group, but has an effector group in place of the second functional group. The effector group may be either a diagnostic marker or a therapeutic molecule.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a bound complex formed by the method according to the invention carried out through the third reagent step, using a diagnostic effector group. In this figure, T is a target cell, C is a chemical constituent thereon, FI and F2 are first and second functional groups respectively, N is a nuclide, and the encircled numbers 1, 2 and 3 are first, second and third reagents, respectively. Dots indicate specific binding while straight lines extending from reagents indicate covalent conjugation.

Figure 2 shows a bound complex formed by the method according to the invention carried out through the third reagent step, using a therapeutic effector group. Symbols are as in Figure 1, except that R is a radionuclide.

Figure 3 shows a bound complex formed by an embodiment of the method according to the invention in which a therapeutic drug is an effector group on a third reagent, and an enzyme capable of releasing that drug from the third reagent is administered after formation of the bound complex. The left panel shows the bound complex prior to enzyme addition, the right panel shows the bound complex and vicinity thereafter. Symbols are as in Figure 1 , except that D is a therapeutic drug and encircled E is an enzyme capable of releasing the drug from the third reagent.

Figure 4 shows a bound complex formed by an embodiment of the method according to the invention in which an enzyme that catalyzes conversion of a specific inactive prodrug to an active drug is conjugated to a third reagent, and the specific prodrug is added after formation of the bound complex. Symbols are as in Figure 1 , except that encircled P is an inactive prodrug and D is an active drug.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to a method for optimizing diagnostic and therapeutic uses of monoclonal antibodies (MAbs) and other specific binding ligands in animals afflicted with various diseases. More particularly, the invention relates to a method of targeting certain effector molecules to living cells, such as cancer cells, or their constituents in an animal, including a human. The method according to the invention provides certain advantages over currently available methods for targeting effector molecules to specific cells. First, the method does not depend so heavily on preserving the immunointegrity of each and every antibody molecule, since it allows wasting of a significant proportion of the first reagents administered. These first reagents that have been damaged during chemical manipulation will simply fair to bind the target and will be swept from the animal's circulation. Since the first reagent does not carry expensive or toxic effector material, it can be administered at saturating levels. This provides an additional advantage of increasing the uniformity of its distribution within the target. Finally, since the first reagent does not carry the effector molecule in cases where that reagent is a murine MAb, the formation of effector group-containing immune complexes via the HAMA response is avoided. In various embodiments, the method of the invention uses the sequential formation of specific binding pairs to target effector molecules in amplified fashion to particular target cells or cell constituents in an animal, including a human. In these various embodiments, a first specific binding pair is formed between a target molecule on a cell and a first reagent having a first functional group that specifically binds to that target molecule. In a preferred embodiment, this first reagent will be an antibody, most preferably a monoclonal antibody, that specifically binds the target molecule on the cell and the first functional group will be an antigen binding site. Those skilled in the art, however, will recognize that certain target molecules, such as ligands or receptors, can be specifically bound by their respective receptors or ligands, thus making such respective receptors or ligands acceptable as first reagent with the ligand or receptor binding site being the first functional group. Whether the first reagent is an antibody, receptor or ligand, it will be conjugated, preferably covalently, to a second functional group that allows the first reagent to form a specific binding pair with a second reagent. For various first reagents according toe the invention, preferred second functional groups include double stranded oligonucleotide (DSO), biotinylated DSO (BN-DSO), avidin-conjugated DSO (AV-DSO), single strand oligonucleotide-conjugated DSO (SSO-DSO), biotinylated SSO (BN-SSO), DNA intercalating agents (INT), biotinylated INT (SSO-INT), biotin that is conjugated to a peptide, protein or carbohydrate or otherwise rendered multimeric (BN), avidin (AV), single stranded oligonucleotide (SSO), and avidin-conjugated SSO (AV-SSO). These functional groups and their specific binding partners are shown in Table I below. In various embodiments, the reagents according to the invention may have first and second functional or effector groups directly linked together, or they may be connected via another molecule, such as human serum albumin, or any other non-antigenic molecule.

TABLE I Functional Groups And Their Specific Binding Partners

Functional Group Specifically Binds To:

DSO INT BN-DSO AV and/or INT AV-DSO BN and/or INT SSO-DSO c o m p l i m e n t a r y S S O (CSSO) and/or INT

INT DSO

BN-INT AV and/or DSO

AV-INT BN and/or DSO

SSO-INT CSSO and/or DSO

BN AV

AV BN

SSO CSSO

AV-SSO BN and/or CSSO

BN-SSO AV and/or CSSO

In the various embodiments of the method according to the invention, the first reagent is administered to an animal, preferably parenterally, and allowed to form a specific binding pair with a target molecule on a cell, to which the first functional group of the first reagent specifically binds. Any first reagent that has neither formed a specific binding pair with the target molecule nor yet been excreted from the body will be in the circulation of the animal. In these various embodiments of the method of the invention, a second reagent is then administered to the animal. Administration of the second reagent preferably involves the use of an amount of second reagent that exceeds (in molar terms) the amount of the first reagent remaining in the circulation at the time that the second reagent is administered.

The second reagent forms a specific binding pair with the first reagent, which is bound to the target molecule on a cell. The ability of the first and second reagents to form a specific binding pair arises in the following manner.

The second functional group of the first reagent is selected, for example, from the functional groups shown in the left hand column of Table I. The first functional group of the second reagent is selected, for example, from the functional groups shown in the right hand column of Table I, and is a functional group to which the second functional group of the first reagent specifically binds, as shown in Table I. Thus, the second functional group of the first reagent specifically binds to the first functional group of the second reagent, resulting in the formation of a specific binding pair between the first and second reagents. In one embodiment of the method according to the invention, the second reagent does not have a second functional group, but rather has an effector group. The effector group can have either a diagnostic function or a therapeutic function. For example, diagnostic effector groups useful in the method according to the invention include nuclides and radionuclides. Therapeutic effector groups useful in the method according to the invention include therapeutic enzymes, drugs, toxins, radionuclides, nuclides, enzymes that catalyze the conversion of a prodrug into an active drug, and antisense oligonucleotides. In this embodiment, a third reagent may optimally be administered to the animal after allowing the second reagent to form a specific binding pair with the first reagent, such that the amount of third reagent exceeds the amount of second reagent remaining in the circulation of the animal at the time that the third reagent is administered. When the effector group of the second reagent is a drug, therapeutic enzyme, toxin, radionuclide with short range emission or antisense oligonucleotide, this third reagent will comprise an enzyme that is capable of cleaving the chemical bond between the effector group and the second reagent. When the effector group of the second reagent is an enzyme capable of catalyzing conversion of a prodrug to an active drug, then the third reagent is the prodrug that is a substrate for the enzyme.

In an alternative embodiment, the second reagent has a second functional group that allows it to form a specific binding pair with a third reagent. The second functional group of the second reagent is selected, for example, from the functional groups shown in the left hand column of Table I.

In yet another embodiment of the method according to the invention, the method is carried out as described above, using the alternative embodiment in which the second reagent has a second functional group that allows it to form a specific binding pair with a third reagent. After the second reagent has formed a specific binding pair with the first reagent, a third reagent is administered, preferably parenterally, to the animal, such that the total amount of the third reagent exceeds the total amount of the second reagent that is in the circulation of the animal at the time that the third reagent is administered. This third reagent is capable of forming a specific binding pair with the second reagent, because the third reagent has a first functional group to which the second functional group of the second reagent specifically binds. This first functional group of the third reagent may be selected, for example, from the functional groups shown in the right hand column of Table I, and will be chosen to specifically bind to the second functional group of the second reagent, as shown in Table I.

In one embodiment of the method according to the invention, the third reagent does not have a second functional group, but rather has an effector group. The effector group can have either a diagnostic function or a therapeutic function. For example, diagnostic effector groups useful in the method according to the invention include nuclides and radionuclides. Therapeutic effector groups useful in the method according to the invention include therapeutic enzymes, drugs, toxins, radionuclides, nuclides, enzymes that catalyze the conversion of a prodrug into an active drug, and antisense oligonucleotides. In this embodiment, a fourth reagent may optionally be administered to the animal after allowing the second reagent to form a specific binding pair with the first reagent, such that the amount of fourth reagent exceeds the amount of third reagent remaining in the circulation of the animal at the time that the fourth reagent is administered. When the effector group of the third reagent is a drug, toxin, radionuclide with short range emissions, therapeutic enzyme or antisense oligonucleotide, this fourth reagent will comprise an enzyme that is capable of cleaving the chemical bond between the effector group and the third reagent, thereby releasing the effector group. When the effector group of the third reagent is an enzyme capable of catalyzing the conversion of a prodrug to an active drug, then the fourth reagent is the prodrug that is a substrate for the enzyme.

In an alternative embodiment, the third reagent has a second functional group that allows it to form a specific binding pair with a fourth reagent. The second functional group of the third reagent is selected, for example, from the functional groups shown in the left hand column of Table I. In this embodiment, after the third reagent has formed a specific binding pair with the second reagent, the fourth reagent is administered to the animal, preferably parenterally, such that the total amount of the fourth reagent exceeds the total amount of the third reagent that is in the circulation of the animal at the time that the fourth reagent is administered. This fourth reagent is capable of forming a specific binding pair with the third reagent, because the fourth reagent has a first functional group to which the second functional group of the third reagent specifically binds. This first functional group of the fourth reagent may be selected, for example, from the functional groups shown in the right hand column of Table I, and will be chosen to specifically bind to the second functional group of the third reagent, as shown in Table I.

In one embodiment of the method according to the invention, the fourth reagent does not have a second functional group, but rather has an effector group. In this embodiment, a fifth reagent may optionally be administered to the animal after allowing the fourth reagent to form a specific binding pair with the third reagent, such that the amount of fifth reagent exceeds the amount of fourth reagent present in the circulation of the animal at the time that the fifth reagent is administered. When the effector group of the fourth reagent is a drug, toxin, radionuclide, nuclide, therapeutic enzyme, or antisense oligonucleotide, this fifth reagent will comprise an enzyme that is capable of cleaving the chemical bond between the effector group and the fourth reagent, thereby releasing the effector group. When the effector group of the fourth reagent is an enzyme capable of catalyzing the conversion of a prodrug to an active drug, then the fifth reagent is the prodrug that is a substrate for the enzyme.

In an alternative embodiment, the fourth reagent has a second functional group that allows it to form a specific binding pair with a fifth reagent. The second functional group of the fourth reagent is selected, for example, from the functional groups shown in the left hand column of Table I. In this embodiment, after the fourth reagent has formed a specific binding pair with the third reagent, the fifth reagent is administered to the animal, preferably parenterally, such that the total amount of the fifth reagent exceeds the total amount of the fourth reagent that is in the circulation of the animal. This fifth reagent is capable of forming a specific binding pair with the fourth reagent, because the fifth reagent has a first functional group to which the second functional group of the fourth reagent specifically binds. This first functional group of the fifth reagent may be selected, for example, from the functional groups shown in the right hand column of Table I, and will be chosen to specifically bind to the second functional group of the fourth reagent, as shown in Table I.

In one embodiment of the method according to the invention, the fifth reagent does not have a second functional group, but rather has an effector group. In an alternative embodiment, the fifth reagent has a second functional group that allows it to form a specific binding pair with an additional reagent. Those skilled in the art will recognize that his amplification system can be extended beyond a fifth reagent to include a larger number of reagents, using the types of functional and effector groups described herein. In any of the above-mentioned embodiments of the method according to the invention, the point of the method is to target diagnostic or therapeutic effector molecules, to the vicinity of the target cell. The method according to the invention both creates and amplifies a signal in the vicinity of the target cell. The signal is created by specifically binding to the target cell a chemical constituent that can be detected or that can be specifically bound by yet another chemical constituent, including an effector molecule. The signal is amplified by having multiple copies of this chemical constituent on the reagents used in the method of the invention.

An example of such signal creation and amplification using a diagnostic effector group is shown in Figure 1. In that figure, a target cell, T, has a chemical constituent, C, that is specifically bound by a first functional group, FI, of a first reagent, 1. Then, multiple copies of a second functional group, F2, on the first reagent are specifically bound by a first functional group, FI , on a second reagent, 2. Similarly, multiple copies of the second functional group, F2, on the second reagent are bound by a first functional group, FI , on a third reagent, 3, which in turn has multiple detectable nuclides attached. A signal is created, since the signal C has been converted to a detectable signal N. The signal has been amplified, since a single copy, in this case, of signal C has been replaced by many copies of signal N. As shown in Figure 2, this system works equally well for use with a therapeutic effector molecule, in this case a therapeutic radionuclide, R. In this use, many therapeutic effector groups are brought into the vicinity of the target cells. If, for example, the target cell is a cancer cell, such a high concentration of effector groups, in this case radionuclides, would greatly improve the chances of killing the target cancer cell.

In other cases, it is desirable for the final effector group to be acted upon by an outside molecule that is not part of the bound complex attached to the target cell. For example, Figure 3 illustrates a situation in which the effector group is a chemotherapeutic drug, D, that must be released from reagent 3 to be taken up by the target cell, T, in this case a cancer cell. By first delivering many copies of the drug to the vicinity of the cancer cell, then providing an enzyme that can release the drug from the reagent, a high concentration of the drug is localized in the vicinity of the cancer cell.

Alternatively, localization of high concentrations of a drug in the vicinity of a target cell can be achieved by conjugating to a final reagent an enzyme that is capable of catalyzing conversion of a prodrug to an active drug, as shown in Figure 4. In that figure, the enzyme, E. is conjugated to a third reagent, 3, and the inactive prodrug, P, is converted to an active drug, D, only in the vicinity of the target cell, T. As used herein, the terms set forth below are intended to have the following meanings, for purposes of defining the invention.

A specific binding pair is a pair of chemical constituents having a binding affinity for each other of at least 10⁶/_mole, _or greater, including affinities beyond about 10¹⁵//mole. Examples illustrating this range are the specific binding pairs DSO-INT, having a binding affinity of about 10^*7_mole, and AV-BIO, having a binding affinity of at least 10^lδ/mole. A number of specific binding pairs within this range are set forth in Table I, above.

Specific binding, for purposes of the invention, is binding between two chemical constituents with an affinity of at least about 10⁶/mole, including binding with affinities beyond about 10¹⁰/mole.

A first reagent, for purposes of the invention, is a molecule that specifically binds to a chemical constituent on a target cell. Examples of such first reagents are antibodies that specifically bind antigens on the cell surface, ligands that specifically bind receptors on the cell surface, and receptors that specifically bind ligands on the cell surface. The term "antibody" is intended to include monoclonal, chimeric, humanized and human antibodies, or fragments thereof, including Fab, F(ab)₂' and F(v) fragments. Such first reagents have a first functional group that specifically binds to the chemical constituent on the target cells, and a second functional group that specifically binds to a second reagent.

A functional group is intended to mean a chemical constituent that mediates specific binding between a chemical constituent on a target cell and a first reagent, or between a first reagent and a second reagent, or between a second reagent and either a third reagent or an effector cell, and so on. As described herein, the second functional group of the first reagent specifically binds to the first functional group of the second reagent, the second functional group of the second reagent, if present, specifically binds to the first functional group of a third reagent or to an effector cell, the second functional group of the third reagent, if present, specifically binds to the first functional group of a fourth reagent or to an effector cell, and so on. Preferably, all reagents will be multivalent, i.e.. will have multiple copies of first and second functional groups, except that certain preferred first reagent may have as few as one or two first functional groups. Preferred functional groups are set forth in the left hand column of Table I. Some second, third, fourth, fifth, etc., reagents may have only a first functional group, with the second functional group being replaced by an effector group. The effector group is a chemical constituent that confers upon a reagent a therapeutic or diagnostic function at or near the site of the target cell. Effector groups conferring diagnostic function include nuclides such as gadolinium and fluorine, which are detectable by MRI, and radionuclides, such as position emitters detectable by PET (e.g.. carbon-1 1 , oxygen-15, fluorine-18) and gamma emitters detectable by tomography (e.g.. technetium-99. iodine-123, iodine-131 , gallium-67, indium- I l l ). Effector groups conferring therapeutic function include drugs, toxins, radionuclides, therapeutic enzymes and proteins, antisense oligonucleotides and enzymes capable of catalyzing conversion of prodrugs to active drugs. Drugs include, but are not limited to common chemotherapeutic agents and antibiotics. Therapeutic enzymes and proteins include, but are not limited to, tissue plasminogen activator, streptokinase and human DNase. Antisense oligonucleotides include oligonucleotides having nuclease-resistant internucleotide linkages, such as phosphorothioate, phosphorodithioate, alkylphosphonate, phosphoramidate, and phosphotriester linkages, among other linkages and modifications well known in the art. Enzymes capable of catalyzing conversion of a prodrug to an active drug include carboxypeptidase-19, which cleaves phenylalanine-linked methotrexate to yield active methotrexate, carboxypeptidase G2, which can cleave glutamate-linked bis-chlorobenzoic acid mustard to yield the active mustard, an alkylating agent, B-lactamase, which cleaves desacetyl vinblastine hydrazide-linked cephalosporine 20, and alkaline phosphatase, which dephosphorylates (and thus activates) phosphorylated anti-tumor drugs, such as mitomycin and etoposide. The following Examples are intended to further illustrate certain preferred embodiments of the invention and are not limiting in nature.

Example I

Preparation Of A First Reagent Having

Biotin As a Second Functional Group

Anti-human mammary cancer MAb B72.3, an IgG_χ antibody that reacts avidly with glycoprotein TAG72 is obtained by culturing in vitro the hybridoma cell line producing it (ATCC MB8108) in a Mini Flo-Path™ bioreactor (Amicon, Danvers, MA). The MAb is dissolved in 0.1 M NaHCO_s 0.2 M NaCl (pH 8.5) at a concentration of 2 mg/ml. Biotinyl-N-hydroxysuccinimide ester (Sigma, St. Louis, MO) is dissolved in dimethylformamide at 2-10 mg/ml, and added in 5-25 μl aliquots to the MAb solution, which is then allowed to stand at room temperature for 1 hour without stirring. The mixture is then dialyzed overnight against 0.15 M NaCl with several changes and finally is dialyzed against phosphate buffered saline (PBS), aliquotted, and stored frozen at -20° C. The MAb binds about 1 to 20 biotin groups per MAb molecule.

Example 2 Preparation Of A First Reagent Having A DNA Intercalator As A Second Functional Group

The carbohydrate moieties on the IgG sample ( 1-10 mg/ml, 30 mM acetate buffer pH 5.0) are oxidized by the addition of sodium periodate (100-200 μg in 100-200 μg water) in a 1:15 Ab:periodate molar ratio. The mixture is kept on ice for 90 minutes and the oxidized IgG run on a Sephadex G-25 column. To the contents within the void volume, ethidium bromide (400 μl of a 1 mg/ml water) is added and the mixture kept in the dark at room temperature for 4 days. During this period, sodium cyanoborohydride ( 1 mg/ml water) was added in 2 portions, 4 hours apart (cyanoborohydride to ethidium molar ratio of 1:1 ). The sample is then filtered on G-25 (elution buffer 25 mM Hepes, pH 7.3). Protein is determined using BCA reagent (Pierce) while conjugated ethidium is assayed by measuring the absorbance at 480 nm. Finally, the ability of the conjugated ethidium to intercalate with DNA was determined by measuring the fluorescence intensity of the sample in the presence of 15 μg calf thymus DNA (520 nm emission, 590 excitation). The data indicate that when ethidium is added to IgG (100: 1 molar ratio), 30 moles of ethidium are conjugated per mole of IgG. In addition, the fluorescent signal of the ethidium-IgG conjugate is enhanced 3-4 times in the presence of calf thymus DNA indicating the ability of the conjugated ethidium to intercalate with DNA.

Example 3 Preparation Of A Reagent Having DSO

As First And Second Functional Groups

Human serum albumin (HSA) (Sigma, St. Louis, MO) is digested to yield 7 CNBr fragments (HSA-f) as follows. First 100 mg HSA is denatured in 10 ml 0.5 M triethanolamine acetate buffer (pH 8.1 ) containing 6 M guanidine-HCl and 0.1% EDTA for 30 minutes at 50° C, then reduced by adding 200 mg dithiothreitol (DTT) and incubating at 50° C for 4 hours. Iodoacetamide is then added to 260 mM final concentration to alkylate the protein and prevent mixed disulf ide formation. The reaction is then allowed to proceed in the dark for 20 minutes at room temperature, and is stopped by the addition of excess DTT. The reaction mixture is then dialyzed against distilled water and incubated at 4° C overnight under nitrogen-saturated 70% formic acid with 100 mg cyanogen bromide (CNBr) to produce HSA-f (seven fragments) which is then separated by

HPLC using a TSK G 3000 SW column (0.75 x 60 cm) pre-equilibrated and eluted with 35% CH₃CN in 0.1 ;% trifluoroacetic acid at a flow rate of 1 ml/minute.

Two complementary 40-mer oligonucleotide phosphorothioate strands are prepared on a Model 8700 automated synthesizer (Milligen-Biosearch, Burlington, MA) using H-phosphonate chemistry on controlled pore glass (CPG), followed by oxidation with 0.2 M sulfur in carbon disulfide/pyridine/triethylamine (9:9:1 , v/v). Synthesis is carried out on a 5x 10 micromolar scale. Oligonucleoside phosphorothioates are purified by low pressure ion exchange chromatography (DEAE-cellulose, DE-50 Whatman), followed by reverse phase chromatography (C₁₈) and dialysis. A detailed description of the H-phosphonate approach to synthesizing oligonucleoside phosphorothioates is given in Agrawal and Tang, Tetrahedron Letters 31:7541 -7544 ( 1990). One strand is prepared with the Amino-Link™ (Clontech, Palo Alto, CA) at the 5' or 3' end. The oligos are then mixed at a 1 :1 molar ratio, heated in a water bath to 90° C and cooled slowly to room temperature to yield duplexes. The duplexes are then reacted with succinimidyl 4-(p-maleimidophenyl) butyrate in 0.1 M phosphate buffer, ImM EDTA (pH 8.0). The maleimide-conjugated butyramide oligos are then added to HSA-f, following mild reduction in 2-mercaptoethanol. The mixture is incubated overnight at 0 to 4° C, then separated by HPLC, as described for HSA- f, above.

Example 4

Preparation Of A Reagent Having Biotin

First Functional Groups And A Radioisotope

Chelator As Second Effector Groups

DTPA (diethylenetriaminepentaacetic acid, Aldrich; 7.1 mgs/ml) and

NHS-biotin (N-hydroxysuccinimidobiotin ester, Sigma; 6.8 mgs/ml) were co- dissolved in DMF (N,N-dimethylformamide, anhydrous, Aldrich). Aliquots ( 10- 60 μl) were added to HSA (human serum albumin, Sigma; 1.32 mgs/ml in 0.1 M NaHC0₃, 0.2 M NaCl, pH 8.5) at molar ratios of between 5 and 40. The reactions were carried out in glass tubes which had been washed in 6N HNO_s and aqueous buffers were stored over chelating resin (Iminodiacetic acid, Sigma). To determine the extent of biotinylation, tritiated biotin (d-[8,9-³H] biotin, succinimidyl ester, Amersham; 100,000 cpm) was added to each reaction tube. After one hour at room temperature, an aliquot of each solution was counted, and the solutions were dialyzed into 0.15 M NaCl to remove unbound biotin. Aliquots of the dialysate were counted and the final biotin/HSA ratios determined by multiplying the fraction of counts recovered in the dialysate by the initial biotin:HSA ratio. To determine the number of moles DTPA conjugated to each mole HSA 10 μl of each of the reaction mixtures was added to 120 μl 0.1 M sodium citrate, pH 6.0, containing luCi ^luInCl₃ (Dupont-NEN). After one hour at room temperature, 5 μl aliquots were spotted on silica strips and run in sodium citrate, pH 8.0. The strips were cut into origin and front sections and counted. The number of moles DTPA per mole HSA was calculated by multiplying the fraction of counts at the origin by the initial DTPA:HSA ratio. The protein concentration of each reaction mixture was determined using the Pierce BCA Method.

There was an apparent linear relationship between the number of moles added and the number of moles bound for both DTPA and biotin within the molar ratio range of 5 and 40.

Example 5

Preparation Of A Third Reagent Having

A ^mIn Effector Group

Biotin- or DSO-conjugated HSA-f is prepared as described in Examples 3 and 4. Diethylenetriamine pentaacidic dianhydride (DTPA) (Aldrich,

Milwaukee, WI) is dissolved in DMSO and conjugated to biotin- or DSO- conjugated HSA-f (2 mg/ml in 0.1 M NaHCO_s, pH 8.2) at a molar ratio of 5:1. After 1 hour at room temperature, unconjugated DTPA is separated from DTPA- and biotin- or DSO-conjugated HSA-f by HPLC under conditions described in Example 3. Next, ^mIn acetate is prepared by addition of 1M sodium acetate to an equal volume of ^mIn radionuclide solution for a final acetate concentration 0.5 M, and pH is adjusted to 5.5-6.0 using 2 M NaOH. The ^u:*In acetate is mixed 1:1 with the conjugated HSA-f in 0.01 M acetate buffer (pH 6.0) and left at room temperature for 30 minutes. Formed third reagent is separated from free ^luIn by HPLC under conditions described in Example 3.

Example 6 Preparation Of A Reagent Having A ""Tc Effector Group

Biotin- or DSO-conjugated HSA-f is prepared according to Examples 3 and 4. One ml of a 1 mg/ml conjugated HSA-f solution is added to 0.67 ml of a solution containing 5 mM SnCl₂, 40 mM potassium phhalate and 13.4 m sodium potassium tartrate (pH 5.6). The mixture is incubated at room temperature for 24 hours, then I mCi pertechnetate is added and incubation at room temperature is continued for 1 hour.

Example 7

Preparation Of A Reagent Having A

Radioiodine Effector Group

Radioactive iodine (0.1 -1.0 mCi Na¹²³I or Na¹²⁵I) is mixed in a 5:1 ratio with DSO-conjugated HSA-f ( 1 to 10 mg) in PBS and transferred to a glass tube coated with 1 mg lodogen. After 5 minutes at room temperature, the formed reagent is separated from free iodine by HPLC, under conditions described in Example 3.

Example 8

Preparation Of A Reagent Having A ²¹¹At Effector Group

Biotin- or DSO-conjugated HSA-f is prepared according to Examples 3 and 4. The radioastato group is then added using either N-succinimidyl-p- p[²¹¹At]-Astatobenzoate, as described by Khawli and Kassis, Nucl. Med. Biol.

16:727-733 ( 1989) or N-(m-[²¹¹At]-astatomaleimide) as described by Khawli et al., Proceedings of the Third International Conference on Monoclonal Antibody Immunoconjugates for Cancer, p. 126 ( 1988). The [²¹¹At]-labeled HSA-f is separated from free ²¹¹At on a G25 Sephadex column. Example 9

Direction Of A ^Tc Effector Group

To Tumor Cells In A Mouse

The LS 174T human adenocarcinoma (colon) cell line, which expresses TAG72, is grown as a solid subcutaneous tumor in nude mice by injecting 10⁷ cells subcutaneously into the flanks of 5 to 8 week old male Swiss-nu/nu nude mice. The tumors are allowed to grow to 3 to 5 mm in size. The mice are then injected intravenously with a first reagent having a first functional group that is a TAG72-binding site and a second functional group that is biotin. This reagent is prepared as described in Example 1 and is administered at a concentration of 10-100 mg/kg body weight. After 1 -2 days, the mice are injected with 10-100 μg avidin (Sigma,

St. Louis, MO), which is a second reagent having the biotin binding sites of avidin as first and second functional groups. After 24-28 additional hours, the mice are injected with 10-100 mg/kg of a third reagent having a first functional group that is biotin and an effector group that is ^99mTc. This third reagent is prepared as described in Example 6. The localization of the ^99mTc tumor is confirmed by tomography, within 24 hours.

Example 10 Direction Of A ^ιnIn Effector Group

To Tumor Cells In A Mouse

Nude mice bearing a 3 to 5 mm LS 174T tumor are prepared as described in Example 9. The mice are then injected with 10-100 mg/kg of a first reagent having a first functional group that is a TAG72 binding site and a second functional group that is avidin. After 24-48 hours, the mice are injected with

1 -5 mg/kg of a second reagent having a first functional group that is biotin and a second effector group that is DTPA. This reagent is prepared according to Example 3. After 1 -6 additional hours, the mice are injected with ^lnInCl₃. After 1 -48 additional hours, localization of the ^luIn effector group to the tumor site is confirmed using tomography.

Claims

WHAT IS CLAIMED IS:

1. A method of targeting an effector group in an amplified manner to a pre¬ selected chemical constituent in the body of an animal, the method comprising the steps of:

(a) administering to the animal a first reagent comprising a first functional group and a second functional group, wherein the first functional group of the first reagent specifically binds to the pre-selected chemical constituent;

(b) allowing the first reagent to form a specific binding pair with the pre-selected chemical constituent; (c) successively administering to the animal at least one cycling reagent comprising a first functional group and a second functional group, wherein the first functional group of the first cycling reagent administered specifically binds in an amplified manner to the second functional group of the first reagent; (d) allowing the first cycling reagent to form a specific binding pair with the first reagent and then allowing successively administered cycling reagents to form specific successive binding pairs in an amplified manner;

(e) then administering to the animal an effector reagent comprising a first functional group and an effector group wherein the first functional group of the effector reagent specifically binds to the second functional group of the at least one cycling reagent;

(f ) allowing the effector reagent to form a specific binding pair with the at least one cycling reagent; and wherein each specific binding pair is defined as having a binding affinity greater than 10⁶/M.

2. The method according to claim 1 , wherein each functional group is selected from the group consisting of antibody, antigen, antibody fragment, cell receptor, ligand to a cell receptor, DSO, BN-DSO, AV-DSO, SSO-DSO, BN-SSO, INT, BN-INT, AV-INT, SSO-INT, BN, AV, SSO, AV-SSO and CSSO.

3. The method according to claim 1 , wherein the cycling reagent further comprises a carrier molecule chemically bound to at least one functional group.

4. The method according to claim 3, wherein the effector reagent further comprises a carrier molecule chemically bound to at least one functional group and at least one effector group.

5. The method according to claim 4, wherein the carrier molecule is a protein, peptide, glycoprotein, sugar, or lipid.

6. The method according to claim 1, wherein the effector group is selected from the group consisting of nuclides, radionuclides, drugs, enzymes that catalyze conversion of prodrugs to active drugs, and antisense oligonucleotides.

7. The method according to claim 1 , further comprising the step of administering to the animal an enzymatic reagent which catalyzes release of the effector group from the effector reagent, wherein the effector group is a drug or antisense oligonucleotide.

8. The method according to claim 1 , further comprising the step of administering to the animal a prodrug reagent, wherein the effector group is an enzyme that catalyzes conversion of the prodrug reagent to an active drug.

9. A reagent comprising a first functional group and a second functional group, said first functional group comprising a member selected from the group consisting of antibody antigen binding site, avidin, and biotin and said second functional group comprising a member selected from the group consisting of double stranded oligonucleotide, single stranded oligonucleotide, and intercalating agent.

10. The reagent of claim 9 wherein said first functional group comprising an antibody antigen binding site and said second functional group comprising double stranded oligonucleotide.

1 1. The reagent of claim 9 wherein said first functional group comprising an antibody antigen binding site and said second functional group comprising single stranded oligonucleotide.

12. The reagent of claim 9 wherein said first functional group comprising

* 5 avidin and said second functional group comprising double stranded oligonucleotide.

13. The reagent of claim 9 wherein said first functional group comprising avidin and said second functional group comprising single stranded oligonucleotide.

10 14. The reagent of claim 9 wherein said first functional group comprising avidin and said second functional group comprising intercalating agent.

15. The reagent of claim 9 wherein said first functional group comprising biotin and said second functional group comprising double stranded oligonucleotide.

15 16. The reagent of claim 9 wherein said first functional group comprising biotin and said second functional group comprising single stranded oligonucleotide.

17. The reagent of claim 9 wherein said first functional group comprising biotin and said second functional group comprising intercalating agent.