WO1992005804A1

WO1992005804A1 - Chelating agents

Info

Publication number: WO1992005804A1
Application number: PCT/US1991/007016
Authority: WO
Inventors: Wolfgang J. Wrasidlo; Michael H. Silveira
Original assignee: Brunswick Corporation
Priority date: 1990-09-27
Filing date: 1991-09-26
Publication date: 1992-04-16
Also published as: JPH06505795A; CA2092434A1; EP0554358A4; EP0554358A1

Abstract

Metal chelates and therapeutic or diagnostic conjugates of site-specific compounds, metal chelates, and metal ions are described. The metal chelates consist of at least two molecular components linked together. One molecular component is a chelate having a high affinity for metal ions and a high Ks value. The other molecular component is a chelate having a low Ks value and the ability to undergo rapid metal exchange.

Description

CHELATING AGENTS TECHNICAL FIELD

The technical field of the present invention relates to chelatin agents, particularly bifunctional chelating agents, and to conjugates of site-specific compounds, chelating agents, and metal ions, including radioisotopes, for use as therapeutic agents in radiation therapy.

In one aspect, this invention relates to a method for treating cellular disorders, particularly cancer, which employs a chelating agent for a metal ion, such as a radioisotope, conjugated to a site-specific compound or target cell binding protein, such as a monoclonal antibody.

In another aspect, it relates to the use of metal chelate conjugated site-specific compounds or target cell binding proteins, such as monoclonal antibodies, for diagnostic purposes.

BACKGROUND ART Radiation therapy has long been used in the treatment of cancer and other localized lesions. The attractiveness of radiation lies in the ability to focus the energy of the radioactive particles or energy on a specific, small target. Radiation is, therefore, a treatment which minimizes overall bodily exposure to the therapeutic agent, in contrast to the general systemic exposure of chemotherapeutic agents. One drawback to radiation treatments is that the radiation passes through other tissue in the process of destroying targeted cancer cells. As a result, the effects are more general than the limited bodily exposure would indicate, so that radiation therapy can be very devastating to the patient and contraindicated for weakened individuals.

Much of the problem associated with radiation therapy results from the inability of current delivery procedures to utilize the optimum types of radiation for treatments. The physical properties of the radioactive substances available for use place certain restrictions on the administration techniques. External administration, for example, is necessarily limited to the use of beta or gamma radiation because of the long distances the radiation travels to its target. Alpha radiation of sufficient energy to travel these distances is generally not appropriate due to the heavy damage which it would inflict on the tissues through which it passes on the way to the target, and the exceptional difficulties in handling the appropriate isotopes. Nevertheless, alpha particle radiation is the ideal type of killing radiation for most treatments intended to be cytotoxic because of its short penetration distance and its exceptional killing effectiveness. As a result, rather low doses of the very large alpha particles are sufficient for imposing lethal damage on the cells through which they pass. Alpha particle treatments have not proven optimum under any administrative technique, however, because of the difficulty in developing the requisite combination of alpha particle energy, suitable half life length, as well as targeting and handling problems. The alternative to external application of radiation in the treatment of cancer tumors is internal application, i.e. internal specific targeting of the radiation, such as by injection. In such treatments the effective penetration of the radiation is preferably short to avoid undue exposure to surrounding tissues and the half life of the radioactive molecule is preferably short to minimize the total bodily exposure to freely circulating or nonspecifically bound radioactive treatment compounds and degradation products thereof. The first application of this technique was the administration of radioactive iodine for thyroid tumors. This was effective due to the specific uptake of iodine into the thyroid and the reasonably short half lives of iodine isotopes. However, this is a very limited therapy and not applicable to other types of cancer.

The development of site-specific compounds or target cell binding proteins, such as monoclonal antibodies, polyclonal antibodies, monoclonal antibody fragments, and binding proteins, which can be targeted to specific cells with a high degree of specificity, has made it possible to selectively deliver therapeutic agents to specific targeted cells. Current internal targeted therapies, therefore, center primarily around the use of such monoclonal antibodies as the targeting mechanism, to deliver the radio isotope combined therewith to the specific target. For example, U.S. Patent 4,454,106, issued to Gansow et al . , teaches a procedure whereby a radioactive metal, such as bismuth-212 (Bi²¹²) chelated with a derivative of diethylene triamine pentaacetic acid (DTPA) , is conjugated with a monoclonal antibody for internal targeted therapy. Since the radioactive molecules with the most desirable properties are generally metals, an appropriate caging agent such as a chelate is attached to the antibody for binding the radioactive metal. The problems associated with these types of compounds stem primarily from the weak strength of the chelating bond, which results in the disassociation of the chelate from the antibody and the loss by the chelate of binding affinity for its isotope, all of which can result in freely circulating radioisotopes inside the patient's body.

The major problem in the development of antibody targeted therapies has been the difficulty in effecting a stable attachment of the radioisotope to the targeted molecule. Most highly advanced in this general area are the complexes synthesized for radioactive imaging. Such compounds involve covalently bound or chelated beta or gamma emitting isotopes. The best of these compounds utilize very stably bound radioisotopes with short half lives and which are rapidly removed from the body.

Targeting radioisotope complexes for cell killing has unique problems which have not been effectively solved at this time. High energy alpha, beta and gamma emitting isotopes are undesirable due to the long range of their effects. Radioisotopes with long half lives are not acceptable because of their long term of action, unless they can be rapidly diluted and eliminated, e.g. for tritium. Such molecules, however, which undergo rapid exchange with the surrounding molecular environment, are not likely to remain targeted long enough to be effective. Alpha particle emitting isotopes, with the proper energy and half life, are few in number and extremely difficult to entrap and bind to targeted molecules.

The only alpha emitting targeted complex yet prepared with even minimal efficacy is that of Bi²¹², chelated with DTPA attached to an antibody. DTPA-bismuth has a stability constant (K_s) of 20.5, which is the best currently available for this purpose. However, treatments with this therapeutic complex are not very effective due to the rapid loss of bismuth from the chelator. Partial compensation for this shortcoming has been achieved by direct injection of the complex into the target organ. Targeting is still minimal as this is not a very desirable method of administration. Also, the possibility of simultaneous treatment of metastasized cancer cells is virtually nonexistent.

Another aspect of the design of such complexes is the specificity of the entrapping compound. Many chelating and caging compounds may hold various ions. This can be a problem in the physiological milieu of the body which contains considerable concentrations of mono- and divalent ions of various types. If these physiological ions are bound by the entrapping molecule and the kinetics are at all favorable for exchange, the metal ion, such as a radioisotope, is likely to be rapidly freed. It is thus desirable to use entrapping compounds which have a high degree of specificity for the metal ions being delivered.

The choice of an appropriate radioactive isotope is another critical aspect of targeted radiation therapy. External treatments generally utilize long lived isotopes for economy, regulating bodily dosage by exposure time. However, when the radioactive molecule is administered internally, degradation of the therapeutic compound is likely to occur, thus necessitating the use of one having a short half life. Ideally, the half life preferably provides just ample time for targeted cell killing. The primary problem with such short and precise half lives is that it is difficult to produce the therapeutic compound, administer it and allow time for cell killing before the radioactivity has decayed below effective levels. Since most potential candidates require a cyclotron for production, they cannot be seriously considered for regular use. Of those possible candidates that do not require the production in a cyclotron, Bi²^ and Pb²^² have received considerable attention because they have acceptable emission energy and half life characteristics. Bi²¹², however, is the only one that has been utilized because it is the only alpha emitter among the candidates which has been chelated with any reasonable effectiveness. A much better candidate in terms of on-site generation and isolation capabilities is Pb²¹² as generated from Th²²⁸. Lead, however, has proven to be extremely difficult to effectively bind to a targeting molecule. DISCLOSURE OF INVENTION

The present invention discloses novel metal chelating agents and novel^' therapeutic or diagnostic conjugates of site-specific compounds, chelating agents, and metal ions, such as radioisotopes. The invention also discloses a method for producing a radiolabeled material attached to a site-specific compound, such as a monoclonal antibody, for immunotherapy of cancers and other uses. The radiolabeled material is held in an entrapping compound such as a chelating or caging agent which is bound to the site-specific compound. Macrocycles A, acrobicycles B, and macrotricycles C and D are good candidates as chelating agents for metal ions.

Macrocycles contain two dimensional cavities lined with atoms that bind cations and have been studied extensively. Macrobicycles and macrotricycles, collectively known as macropolycycles, have been studied more recently. They contain three-dimensional intramolecular cavities lined with various binding sites, have the ability to form inclusion complexes and bind selectively spherical metallic cations. They are molecules of intermediate size, mesomolecules which may display a multitude of new properties and hence have given rise to a new field of supramolecular chemistry.

Cryptands lined with oxygen, sulfur, and nitrogen binding sites may be synthesized from commercially available macrocycles. Their size is governed by the length of the bridges and increases stepwise along the series 1 - 7.

[1.1.1.], m=n=0 9, X=NCH₃ Λ Λ_j [2.2.C8], X=0; Y=CH₂ 2, [2.1.1.], m=0, n=1 19, X=NCH₂COOH 22, X=0; Y=0, NCH₃ 3, [2.2.1. ], m=1, n=0 or NCH₂CH₂COOH j , χ=θ; Y=NCH₃

4, [2.2.2.], m=n=1 14, X=NCH₃;Y=0

5, [3.2.1.], m=1, n=2 16, X=0; Y=S

6, [3.3.2.], m=2, n=1 2 , X=S; γ=o

7, [3.3.3.], m=n=2 18, X=Y=S 8, HC (CH₂OCH2CH2-OCH₂CH2θCH2)3 CH 20, X=0; Y=0, NCH₂COOH X=Q; Y=NCH₂CH₂COOH

15 22 =n-G_| oH₂-_], n- ι ι H₂₃, or n-C-₁4H₂g

Complexing studies of these ligands show that they are capable of dissolution of salts in organic solvents in which they are otherwise insoluble, as well as solubilization of insoluble salts in water. Crystal structure determination of a number of cryptates confirm that the cation is located inside the molecular cavity. They are considerably more stable and their dissociation rates are several orders of magnitude slower than those of chelates formed by macrocycles or polyanionic ligands like ethylene diamine tetraacetic acid (EDTA) , diethylene triamine pentaacetic acid (DTPA) , meso-2,3-dimercapto succinic acid, and the like. The selectivity in binding of cryptand is based on the principle of cavity size selectivity, the preferred cation being that whose size most closely fits the cavity. Replacement of oxygen binding sites with nitrogen and sulfur atoms decreases both the stability as well as selectivity. Cryptands have been used to decorporate radioactive stroncium-85 and radium-224 from rats and hence the macropolycycles should withstand the recoil energy of alpha emitters.

Macrotricycles g2_ - 2 L of type C have been used for complexing alkali cations and alkaline earth cations and found to form non symmetric mononuclear as well as symmetric binuclear complexes.

23 24, X=CH₂ g§, X=0 26 27, X=NH

These complexes have either 1:1 or 2:1 cation:ligand stoichiometry respectively. The ligands 24 - 26 from mononuclear, binuclear as well as heteronuclear bimetallic cryptates with silver nitrate and lead nitrate. Ligand 25, when treated with equimolar quantities of AgN0₃ and

Pb(N0₃)₂ forms an equilibrium mixture of two homonuclear complexes

[2 Ag⁺ 25] and [2 Pb⁺ 25], and of the heteronuclear complex [Ag⁺ Pb⁺ 25]. Thus one can expect similar macrotricycles to form heteronuclear complexes with two different alpha emitters, such as Pb²¹² and Bi²¹².

Conjugated to these cryptands and macrotricycles are chelating agents having a low K_s value and the ability to undergo rapid metal exchange. These novel chelating agents which result, have the properties of rapid uptake of metal ion and high stability.

It is an object of the present invention to provide novel chelating agents.

It is another object of the present invention to provide novel therapeutic or diagnostic conjugates of site-specific compounds, chelating agents, and metal ions, such as radioisotopes.

It is a further object of the present invention to provide an effective method of treating cellular disorders employing novel therapeutic or diagnostic conjugates of site-specific compounds, chelating agents, and metal ions, such as radioisotopes. It is still another object of the present invention to provide a method of selectively targeting lethal doses of radiation to diseased cells which causes little or no destruction of healthy cells.

MODES FOR CARRYING OUT THE INVENTION

The present invention discloses novel metal chelate conjugates and novel therapeutic or diagnostic conjugates of site-specific compounds, chelating agents, and metal ions, such as radioisotopes.

Chelating compounds and other caging agents are known in the prior art for entrapping lead. There are a few classes of these compounds and any number of specific embodiments within each class. These specific embodiments are normally derivatives or modifications of known core molecules. Such chemical changes are preferred so that any entrapping molecule can meet the strict requirements for the Pb²¹² delivery complex. Examples of these classes of compounds include: metal organics

metal carbonyls

crown ethers and crown azos, such as 1,4,10,13-tetraoxa- 7,16-diazacyclooctadecane (TDCOD)

-SH . polythiols, such as mercaptanes HOOC-R-SH-M SH ^'' complexes, such as diethylene-triamine- pentaacetic acid (DTPA)

polyporphyrins

cages, such as amino capten

and cryptates, including binuclear inclusion complexes

(Y = Antibody reactive group.) The novelty of the present invention lies in the combination of two or more of the above chelating agents to form a novel chelating agent. One of the chelating agents has a high affinity for metal ions, including radioisotopes, and a high K_s (stability constant) value. This component is characteristically a strongly metal binding ligand with spacing for various metal ions and forms stable interaction with the chelated metal. It may even bind more than one metal ion. The thiol analogs of many of the above listed classes of chelating agents exhibit these characteristics.

To this high affinity chelating agent is attached one or more chelating agents having a low K_s but a fast uptake of metal ion, such as ethylene diamine tetraacetic acid (EDTA) , diethylene triamine pentaacetic acid (DTPA), 2,3- dimercapto-1-propane sulfonic acid (2,3 DMPS) , and meso- 2,3-dimercaptosuccinic acid (2,3 DMSA) . These components are characterized by the ability to undergo a rapid metal exchange and diminished stability when the metal is chelated.

Examples of the novel, bifunctional, chelating agents of the present invention are 1,4,10,13-tetraoxa-7,16- diazacyclooctadecane (TDCOD) combined with two molecules of di ercaptosuccinic acid, as seen below in Example 1 and

TDCOD combined with one or two molecules of DTPA.

In three dimensional terms, the novel chelator of the present invention comprises a macrocycle or macropolycycle containing a two- or three-dimensional intramolecular cavity (the high affinity chelating agent or binding group) having one or more projecting arms (the chelating agent having the low K_s value) . The metal ion is chelated by the agent having the fast uptake characteristic, the arm, and is then attracted into the cavity of the agent having the high affinity cahracteristics. The two or three chelating agents become folded, resulting in intramolecular interaction in the form of coordinate bonding, to form a stable chelating agent having a very high K_s value.

In some cases, when in the folded state and the metal ion chelated is Pb, none of the standard assays for Pb show the release of Pb from the novel chelating agent of the present invention. These highly stable, novel chelators are useful in vivo because it is not likely that the metal ion will leave the chelate prior to delivery by the site specific compound to the desired site. Once delivered to the appropriate site, the normal radioactive decay occurs, destroying the target cells while untargeted cells out of range of the radiation are not harmed. Once the decay process is completed, the normal body processes can be used to remove the metal ion and its chelator.

The chelate containing the cavity and its one or more arms may be linked directly or via linking groups inserted to connect the two agents. Once these agents are linked to form the novel chelator of the present invention, site specific compounds, such as monoclonal antibodies, can be attached to one or the other of the component chelating agents, again, via either direct linkage or linking groups. The monoclonal antibodies may be attached either before or after the metal ion is chelated. Preferred for the practice of this invention are two groups of caging molecules, captens and cryptates to which may be attached chelating agents with lower K_s values, such as EDTA, DTPA, DMPS, and DMSA. The captens and cryptates hold metal ions, including radioisotopes, within a physically defined space interior to the molecule. The strength of the entrapment of the metals in these compounds derives from the ionic bonds to the metal ions and also from the molecular structure of the surrounding molecule which is actually built around the metal ion during synthesis, rather than merely chelated afterwards. The cryptates are the more complex of the two groups and also the more three-dimensional in terms of the molecular caging. The major advantages of these compounds is that they have the ability to provide high _s values, slow exchange kinetics, and the potential for the engineering of a high degree of specificity for specific metal ions, such as Pb²¹² and Bi²¹².

While in the past, Pb²¹² has proven extremely difficult to effectively bind to a targeting molecule, several additional considerations make Pb²¹² the isotope of choice for use in radiation therapy of cancers. For example, Pb²¹² provides a number of decay pathways resulting in alpha particle emissions, and can be readily produced from elements of natural origin, i.e., not needing a cyclotron for production. With respect to the two primary considerations for isotopes for internal targeting treatment of cancer cells, i.e. half life and ease of isolation, the decay pathway from τh²²⁸ to pb²⁰⁸ is the preferred source for the isotope. pb²¹² has a half life of 10.6 hours before decaying into stable Pb²⁰⁸. While perhaps on the short side of ideal, this is a very acceptable length of time to allow for production and purification of the therapeutic complex, administration to the patient and exposure time to the cancer cells. As an additional advantage, Pb²¹² emits an alpha particle with an ideal energy for localized cell killing. This alpha particle has about 8.8 Mev of energy which translates into a tissue penetration depth of about 80 μm, equivalent to about 4-5 tumor cell diameters. Thus, the alpha particle from Pb²¹² has the ability to kill cells only very locally to the site of the attachment. Pb²¹² lends itself to simple, efficient, and economical production and isolation for use in any desired application. The novel, bifunctional, chelators of the present invention have overcome the difficulties previously encountered in binding lead to a targeting molecule and hence have made it possible to use Pb²¹² as the metal ion of choice.

Such compounds designed to contain only lead can be attached to monoclonal antibodies in the empty state (no entrapped metal ion) and then used to entrap Pb²^². Alternatively, the lead may be first chelated to the entrapping compound which is then in turn bound to the monoclonal antibodies.

If the Pb²¹² is to be used as a therapeutic agent in radiation therapy, one such method pursuant to one embodiment of this invention, involves dissolving the Pb²¹² in a suitable antibody-chelating complex solution to entrap the Pb²¹² in the complex. For therapeutic use by internal administration, the chelating complex is thereafter filtered and purified for subsequent administration.

Specifically, the Pb²¹² antibody-chelating complex, to which antibody has already been conjugated by means such as interfacial condensation or any other convenient means known to the art, is preferably first separated from free uncombined isotopes. While the solution may be centrifuged, a preferred method for effecting this operation is to filter the solution through an ion exchange column such as a column of Amberlite IRA-400, Dowex 1 or equivalent ion exchange resin, and then into a second ion exchange column such as a Sephadex G-25 or G-50 cartridge for good effective separation of the complex from free isotopes. Thereafter, the purified complex is filtered to remove any solids therefrom, and sterilized. After analyzing the material to verify the radioactivity, the material is then ready for injection into a patient. Because of its short half life, the Pb²¹², once generated, is preferably processed and administered without delay. While Pb²¹² is preferred for use in the present invention, this invention also contemplates the use of metal alpha, beta, gamma or positron emitters. Auger electron emitters, and fluorescing lanthanides for therapeutic and diagnostic use. The chelating agents of the present invention have the advantage of rapid metal ion uptake. Once picked up, the entrapping chelator folds itself so that the metal ion is then tightly bound by the highly stable chelator. The metal ion is thus bound in such a way that little exchange with extraneous metal ions will take place. The best of these compounds result in very stably bound radioisotopes with short half lives and which are rapidly removed from the body.

The use of site-specific compounds or target cell binding proteins, such as monoclonal antibodies, polyclonal antibodies, monoclonal antibody fragments, and binding proteins, which can be targeted to specific cells with a high degree of specificity, in combination with the novel, bifunctional, chelating agents of the present invention makes it possible to selectively deliver therapeutic agents to specific targeted cells. The chelating agents combined with monoclonal antibody can be used either in vivo or in vitro for such things as diagnostic evaluations, treatment of cancers and other diseases, and other radio-immuno therapy.

One embodiment of the present invention contemplates the use of the bifunctional chelating agents of the present invention as detoxifying agents. In this embodiment, no monoclonal antibody or other targeting molecules are necessary. Instead, the chelating agent without metal ion is administered either in vivo or in vitro as the case may be. The chelating agent then picks up unwanted metal ions. Thereafter the chelated metal and chelator are removed.

In another embodiment, the present invention contemplates an in vivo diagnostic procedure which comprises introducing a metal chelate conjugated monoclonal antibody into the body, allowing sufficient time for the conjugate to localize and identifying the degree and location of localization, if any. The present invention also contemplates in vitro analytical procedures employing a chelate conjugated monoclonal antibody. The conjugated antibody of the present invention is substantially free of adventitiously or weakly chelated metal.

The metal chelate conjugated antibodies of the present invention may be administered in vivo in any suitable pharmaceutical carrier, including a physiologic normal saline solution. The concentration of metal chelate conjugated antibodies within the solution is preferably a matter of choice. Levels ranging from 10 to 100 mg per ml are readily attainable but the concentrations may vary depending upon the specifics of the application. Appropriate concentrations of biologically active materials in a carrier are routinely determined in the art.

The effective dose of radiation or metal content to be utilized for any application is likely to depend upon the particulars of that application. In treating tumors, for example, the dose is likely to depend, inter alia , upon tumor burden, accessibility, and the like. Somewhat similarly, the use of metal chelate conjugated antibodies for diagnostic purposes is likely to depend, inter alia , upon the sensing apparatus employed, the location of the site to be examined, and the like. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

SVJUTIT ^•___! 1

Reaction of crown ether with dimercapto succinic acid

The following reaction results in the formation of a chelating agent of the present invention which contains a two dimensional cavity having two arms attached on diagonally opposite atoms. These arms are made up of polyanionic complexing agents. 23 mg (0.01 moles) of 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (TDCOD) was dispersed in 1 ml acetonitrile and 37 mg (0.02 moles) of dimercaptosuccinic acid (DMSA) was added. Then 1 ml of dioxane and 38 mg (0.02 moles) ECDI (l-[3- (dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride) was added and the yellow suspension stirred with nitrogen blanket and alternatively evacuated with line vacuum. The reaction was allowed to run for 3 days and a deep yellow solution was separated from a brown paste and was evaporated.

The brown sticky semisolid was dried and 12 mg was dissolved in 1 ml of water followed by addition of 5 mg ECDI at pH 5. Then 200 μl was added to 1 ml 9.2.27 at pH 6.5. The mixture was stirred over night and separated by NAP-5. Then 2 mg Pb0₂ was added and the mixture analyzed for Pb over various time intervals. The results are shown in Table 1 below.

TABLE 1 Time Pb (ppm) 15 in 11

4 hours 23

22 hours 10

The 22 hour sample was first NAP-5 filtered and then analyzed for Pb. It showed a 2.5:1 Pb to antibody ratio. Approximately 1 ml of this sample was treated with H₂S from an acetamide-HCl generator. After spinning the sample for 2 minutes at 14,000 rpm, the supernatant had 8 ppm Pb. The loss of 2 ppm of Pb is presumably due to DMSA binding. EXAMPLE 2

Reaction of Dimercapto succinic acid with crown TDCOD and

9. 2. 27 The following reaction results in the formation of a chelating agent of the present invention which contains a two dimensional cavity having two arms attached on diagonally opposite atoms. These arms are made up of polyanionic complexing agents. 57 mg TDCOD was mixed with 80 mg of DMSA in 2 ml dimethylacetate (DMAc) under nitrogen with heating and 38 mg ECDI was added. The bright orange solution was stirred over night. Then 16 mg ECDI was added and the mixture warmed, stirred for 15 minutes and 200 μl was added to 11.5 mg/ml of 9.2.27 antibody. After adjusting the pH to 6.5, when adding the DMSA-crown 18 the pH of the monoclonal antibody dropped to 5.4, the mixture was allowed to react. After reaction, the mixture was purified with a Nick column. All yellow color was retained on the column and the filtrate was colorless. EXAMPLE 3

Reaction of DTPA with TDCOD and 9. 2. 27

The following reaction results in the formation of a chelating agent of the present invention which contains a two dimensional cavity having two arms attached on diagonally opposite atoms. These arms are made up of polyanionic complexing agents. 71 mg (0.02 moles) of DTPA and 26 mg (0.01 moles) of TDCOD were mixed and 1 ml of DMAc was added. Then 30 μl of triethylamine (TEA) was added and the mixture was stirred. Then another 1 ml DMAc was added. After 1 hour, another 30 μl (30 mg) of TEA was added. The mixture was heated until clear. A 100 μl aliquot was removed from the clear solution and added to 1 ml (11.9 mg) 9.2.27. Then 5 μl TEA was added (pH 8.5) and the clear solution was stirred for 2 hours. The solution was then NAP-5 purified. The resulting solution contained 5.8 mg/ml of protein. 1 mg of Pb0₂ was added to a portion of the antibody containing solution and after spinning down, the supernatant was analyzed. After 0.5 hours, 9 μg Pb was found. After 2.5 hours, 24 μg Pb was found.

60 ppm of PbN0₃ was added to a second portion of the antibody containing solution. After 30 minutes the solution was NAP-5 purified and analyzed via atomic absorption. 15 ppm Pb were found indicating a 3:1 Pb to antibody ratio. The sample was repurified by NAP-5 and 7 ppm Pb were found.

EXAMPLE 4 Reaction of 9. 2. 27 with TDCOD-DTPA

The following reaction results in the formation of a macropolycyclic chelating agent of the present invention in which a macrocycle is connected at diagonally opposite atoms by a bridge made up of a polyanionic complexing agent. 11.5 mg 9.2.27 was dissolved and the pH adjusted to 8.5 and 11 mg of TDCOD-DTPA, made as in Example 3 except that the molar ratio of TDCOD to DTPA is 1:1, was added in 50 μl of DMAc (paste) . The clear, colorless mixture was stirred for 1 hour in the presence of 3 mg ECDI and then gel purified using NAP-5. HPLC showed 20% conjugated antibody in multimers.

The part of the purified sample was mixed with 3 mg Pb0₂ for 15 minutes then centrifuged for 2 minutes at 30,000 rp . The supernatant was analyzed via atomic absorption which showed that 15 ppm of Pb was taken up by the mAb-TDCOD-DTPA complex. A control sample of 9.2.27 and 3 mg of Pb0₂ dissolved together showed that 0 ppm of Pb was taken up by the antibody. Another part of the purified mAb-TDCOD-DTPA complex was reacted with 3 mg of Pb0₂ for 2 hours. The mixture was centrifuged and stored cold. This sample had 29 ppm Pb. After running over NAP-5 (250 μl) the sample had 6 ppm Pb.

A third sample was stored 3 days after reacting with ^pk°2 ^anc then the excess Pb was centrifuged. The supernatant had 97 ppm Pb.

EXAMPLE 5 Reaction of DTPA-crown 18 with 9. 2. 27 8 mg of DTPA-crown 18 was reacted with 11.5 mg of 9.2.27 at pH 7.4 for 10 minutes. After NAP-5 purification of 0.5 ml, eluted with 1 ml, the purified product was reacted with 5 mg PbN0₃ and again NAP-5 purified. The excess PbN0₃ precipitated as PbCl₂ and was centrifuged. The NAP-5 purified sample had 104 ppm of Pb resulting in a 33:1 Pb to antibody ratio.

EXAMPLE 6 Reaction of DTPA-crown 18 with 9. 2. 27 conjugate and Pb 11.5 mg (7.4 x 10"⁸ moles) of 9.2.27 antibody and 5.0 mg (7.4 x 10~⁸ moles) of DTPA-crown 18 were dissolved in 120 μl dry DMAc and 5 μl TEA and mixed for 1 hour. The clear colorless liquid was gel filtered through G-25 sephadex.

An 0.5 ml portion of this conjugate was mixed with 1 mg Pb0₂ and 0.5 ml was mixed with PbN0₃. Both samples were NAP-5 gel filtered and measured for Pb after 3 hours of mixing. The Pb0₂ sample contained 26 ppm Pb and the

PbN0₃ sample contained 67 ppm Pb.

After G-50 chromatography during which 100 μl of the

PbN0₃ sample was diluted with 400 μl of PBS and eluted with 400 μl of PBS, atomic absorption indicated that 2 ppm of Pb remained. This indicated that there was a 3:1 Pb to antibody ratio.

The next day the PbN0₃ sample showed a reading of 31 ppm Pb and after a week the readings indicated 45 ppm of Pb. -21-

EXAMPLE 7

Antibody- Complex Reaction

₃

V

NH -HCl NH -HCl [cage] -NH-C- (CH₂ ) -C-0-CH₃ isolate and purify

mAb-NH.

V

NH-HCl NH-HCl II II [cage]-NH-C-(CH₂)₄-C-NH-mAb purify

PbO

NH-HCl NH-HCl II II

[Pb-cage] NH-C-(CH₂)₄-C-NH-mAb

These linkages are stable to pH 10.5 and this complex is likely to be quite suitable for in vivo stability.

EXAMPLE 8

Compound 3^ was synthesized as shown below.

b , Y = O

34a 34c c_. . Y = NTos 35 , Y = NH

Monoprotection of the diamine 2^ using benzyl chloroformate in benzene (diamine:cholorformate 1:1) gave compound 2 . Condensation of ^ with the diacyl dichloride 30a. 30b. and 30c afforded respectively the diamides 31a, 31b. and 31c) , which were then converted into 32a. 32b, and 32c with hydrogen bromide in acetic acid (48%) . The yields were about 90% for the two steps.

The next step was performed under high dilution conditions following a procedure very similar to the one used for the synthesis of the previously described macrobicyclic systems. Condensation of 32a, 32b, and 32c with the dichlorides 30a. 30b, and 30c respectively (in benzene and in the presence of three equivalents of triethylamine) afforded the macrotricyclic tetracarboxamides 33a, 33b, and 33c. These tetramides 3^ were reduced by diborane in tetrahydrofuran by refluxing for 10 hours. Hydrolysis of the resulting product with 6 N HC1, refluxing for about 10 hours, followed by passage of the aqueous solutions over a quarternary ammonium resin column in its hydroxide form afforded the macrotricyclic tetramides 34a (yield 90%) , 34b (yield 90%) , and 34c (yield 90%) . Removal of the tosyl groups of 34c by treatment with sodium in liquid ammonia:ethylamine (1:1) gave 3 L (yield 70%) . Chelating agents having low K_s values, such as polyanionic complexing agents may be attached to compound ^5, resulting in the formation of a chelating agent of the present invention.

EXAMPLE 9 Various compounds synthesized following the process in Example 8 can be converted into other compounds suitable for attachment of monoclonal antibodies and compounds capable of fast uptake of Pb²¹². One such application is shown below. The following reaction results in the formation of a chelating agent of the present invention which contains a macropolycycle containing a three dimensional cavity having an arm attached on the bridge connecting the macrocyclic segments. An analogue of the same, which is not shown here, could be synthesized wherein the arm is made up of a polyanionic complexing agent .

DCC, N— hydroxy succm —imi —αe - ,> solvent

39 40

MAb

<ιl

42

For compound 42 : n=1, 2, 3, and the like Y=CH₂, O, N, S, and the like D=0, NH, S, and the like

E=0, NH, S, and the like X=CH₂, O, S, -Z-EDTA, -Z-DTPA, -Z-DMSA, and the like

Z=the spacer between X and the polyanionic complexing agents like EDTA or DTPA

Compound 32b is reacted under high dilutions conditions with the diacid chloride 30c in the presence of triethyl a ine as a base to give compound 3 ^. Reduction of the tetracarboxamide functionality with lithium aluminum hydride under refluxing conditions in the presence of tetrahydrofuran as a solvent gives the compound 32_,. Deprotection with liquid ammonia and ethyl amine in a ratio of 1:1 yields compound 3J Conversion of the acid functionality into a good leaving group followed by treatment with dicyclohexyl carbodiimide and N-hydroxy succinimide yields compound 40. which is now compatible for reaction with a monoclonal antibody 4_^1. The resulting product bears a site specific delivery and a macropolycycle capable for functioning as a receptor for an alpha emitter like Pb²¹², examples of which can be seen at 42.

EXAMPLE 10 The synthesis of a polyanionic complexing agent (EDTA) - macrocyclic (18-crown-6)-monoclonal antibody conjugate 4_6 is outlined below. The following reaction results in the formation of a chelating agent of the present invention which contains a two dimensional cavity having one arm. The arm is made up of polyanionic complexing agents.

EDTA - Macrocycle - MAb Conjugate 46

The ethyl triester of EDTA on conversion to its N- hydroxy succinimide derivative under aqueous conditions using EDCI as a coupling agent reacts with the monoprotected derivative of 18-crown-6 29_, to give compound 44. Hydrolysis of the ethyl esters and the benzyl chloroformate functionality in one step with HBr (48%) followed by reaction with bromo acetyl bromide in the presence of an appropriate solvent yields compound 45_. which reacts with a monoclonal antibody to give conjugate 46 capable of complexing lead to yield a radio immunoconjugate.

Variation of this synthesis permits the use of other complexing agents like DTPA, DMSA, and the like. The length of the arm between the macrocycle and the functionality that reacts with the monoclonal antibody can be varied and so can the functionality that reacts with the a ino groups on the lysines of the monoclonal antibody. It would then be possible to synthesize an azido, diazo, isothiocyante, isocyanate, thiol, hydrazide, and the like instead of alpha bromo acetate. EXAMPLE 11 The preparation of a radio-immunoconjugate which is composed of an 18-crown-6 as well as DMSA as complexing agents is illustrated below. The following reaction results in the formation of a chelating agent of the present invention which contains a two dimensional cavity having two arms attached on diagonally opposite atoms. These arms are made up of polyanionic complexing agents.

Radio— Immuno

Conjugate

Compound 2^ on reaction with a two molar ratio of acrylonitrile followed by reduction with diborane gives the diamine £7. The diamine on reaction with compound 48 derived from meso-2,3-dimercapto succinic anhydride gives compound 4 . Further reaction with the reagent 5O and treatment of the resulting compound jy, with hydrazine hydrate gives the hydrazide §2_. The monoclonal antibody used on oxidation of the carbohydrate portion with sodium metaperiodate to generate aldehydic functionalities reacts with compound ^2 to give a conjugate capable of complexing pj-,212 to give the desired radio immunoconjugate.

EXAMPLE 12 The synthesis of a complexing agent in which a macrocycle unit is cyclized with a DTPA unit is shown below. The following reaction results in the formation of a macropolycyclic chelating agent of the present invention in which a macrocycle is connected at diagonally opposite atoms by a bridge made up of a polyanionic complexing agent.

Radio— Immuno Conjugate

The 18-crown-6 unit has an arm with an azido functionality which on photolysis reacts with a monoclonal antibody and the resulting immunoconjugate is capable of complexing with an alpha emitter.

Thus compound 5£ on reaction with DTPA under high dilution conditions undergoes cyclization to give compound 5j . The tosylate, on displacement with sodium azide to incorporate an azido functionality 5> which on photolysis in the presence of a monoclonal antibody, yields compound 57. This conjugate is capable of complexing with an alpha emitter. EXAMPLE 13

The synthesis of another conjugate is shown below. Chelating agents having low K_ε values, such as polyanionic complexing agents may be attached to compound 3, resulting in the formation of a chelating agent of the present invention.

An aromatic nitro functionality on a macrobicycle 58 functions as a starting material. Hydrogenation using Pd/C (10%) as a catalyst gives the aromatic amine 59. Diazotisation using nitrous acid generated by the action of hydrochloric acid on sodium nitrite gives the diazonium salt 6_g. This is coupled with tyrosine of a monoclonal antibody to give compound § or converted into an azide 2_ by reaction with sodium azide an photolysed in the presence of monoclonal antibody to give the desired conjugate 6_,3. Compounds 61. and 6^ are both usable in radio im uno therapy.

EXAMPLE 14 The synthesis of a chelating agent that reacts with a modified monoclonal antibody is shown below. The following reaction results in the formation of a chelating agent of the present invention which contains a macropolycycle containing a three dimensional intermolecular cavity having an arm attached on the bridge -30-

connecting the macrocyclic segments.

66

64

NH₂0H-HC1

Y= 0, S, CH₂, and the like

Compound 6 on reaction with commercially available anhydride 6J> gives the corresponding amide ee_. Removal of the protecting groups with hydroxyl amine hydrochloride gives a thiol which is capable of undergoing a Michael addition reaction to a modified monoclonal antibody 67. The modified monoclonal antibody is then treated to form compound 68.

EXAMPLE 15

Pb Cage Experiment Tests were conducted to determine the stability of the novel chelating agents of the present invention, particularly in terms of their ability to keep lead bound under adverse conditions.

This example involves the use of a slurry of amino hexyl sepharose bound to a cage with a structure as in

Example 3. Lead nitrate was added to the gel, approximately 200 μg total, with almost complete absorption onto the cage. The gel, after extensive washes with deionized water was suspended to 1 ml volume and Pb content was based on absorbed material versus washes. Therefore since 0 ppm was recorded in the wash water, approximately 200 μg remained in the gel. A first series of tests was conducted to measure how much lead remained bound after treating the cages with water, the control, MnCl, which attempts to displace lead in the cage, and DTPA, a chelating agent which attempts to pull lead out of the cage. Deionized water was used to dilute 40 μg of Pb in complex form to 1 ml final volume at 80°C. Deionized water was also used to dilute 40 μg of Pb in complex form and 80 μg of MnCl to 1 ml final volume at 80°C. Finally, deionized water was used to dilute 40 μg of Pb in complex form and 80 μg of DTPA to 1 ml final volume at 80°C. Aliquots were taken at 0, 1, 3, and 24 hours. Samples were spun down to remove the cages, including the cage bound Pb, and the Pb content of the supernatant was measured in μg via atomic absorption. The results are shown below in Table 2. TABLE 2

Time (Hrs) Cage + H₂0 Cage + MnCl Caσe + DTPA

0 0 ^~ 2 2

1 0 2 2

3 0 2 2 24 1 2 3

The results indicated that very little lead was displaced from the cage under the experimental conditions.

To study the pH stability of the cage, a quantity of

14 μg of the Pb complex was added to water solutions (total volume 1 ml) having pH 7, 8, 9, 10, 11, 12, and 13 respectively. Aliquots were taken at 0, 1, and 24 hours. Samples were spun down to remove the cages, including the cage bound Pb, and the Pb content of the supernatant was measured in μg via atomic absorption. Lead release was immediate at pH 12 and 13 so no further tests were done at those pHs after the initial test. The results are shown below in Table 3.

TABLE 3

12 13_

13 13

The results indicated that the cages were very stable from pH 7 to 10 and that stability decreased by about 50% at pH 11 and by almost 100% at a pH above 12.

A final study was conducted using a competitive chelating agent, DMSA, to look at cage stability over time. Deionized water was used to dilute 12 μg of Pb in complex form and 60 μg of dimercapto succinic acid to 1 ml final volume. Aliquots were taken at 0 and 1 hours and 5 days. Samples were spun down to remove the cages, including cage bound Pb, and the Pb content of the supernatant was measured in ppm via atomic absorption. The results are shown below in Table 4.

TABLE 4 Time Pb fppnO

0 hour 3

1 hour 3 5 days 5

The results indicated that the cages remained fairly stable over time and that little lead was removed.

EXAMPLE 16

Pb-DTPA-9. 2. 27 Complex 11.5 mg (7.4 x 10^~8 moles) of 9.2.27 monoclonal antibody (mAb) was added to a 2 ml Eppendorff vial and 1.4 mg (3.7 x 10^~6 moles) of DTPA (50 molar excess) was added.

The pH ranged between 7.8 and 9.2 after adjustment. After

10 minutes 0.5 ml was purified by NAP-5. OD 280 = 5.3 mg/ml.

A second 0.5 ml sample was NAP-5 purified after 30 minutes (OD 280 = 4.2 mg/ml).

The second sample was reacted with excess Pb(IV) oxide for 20 minutes and centrifuged for 8 minutes at 30,000 rp . The supernatant had 18 ppm Pb (8.8 x 10~⁶ moles/ml) which is equivalent to a 3:1 Pb to antibody ratio.

An 0.5 ml sample was recentrifuged and stored for further testing. After three hours of storage, the supernatant had 17 to 18 ppm of Pb. After 5 hours of storage, the sample was NAP purified using 2.7 mg/ml and had 5 ppm Pb (2.41 x 10~⁸ moles/ml) which is equivalent to a 2:1 Pb to antibody ratio. After allowing the sample to sit over a weekend in the refrigerator, the sample was run again through an NAP-5 column. Atomic absorption analysis revealed that 2 ppm of Pb remained.

The process was repeated when 2 ml of 9.2.27 monoclonal antibody (mAb) was added to an

Eppendorff vial and 3.5 mg (9.25 x 10~⁶ moles) of DTPA was added as well. The pH ranged between 7.8 and 9.2 after adjustment.

The sample was then reacted with 10 μl saturated

PbN0₃ for 20 minutes and centrifuged for 8 minutes at

30,000 rpm. The supernatant had 47 ppm Pb (2.35 x 10~⁷ moles/ml) which is equivalent to a 7:1 Pb to antibody ratio. This sample was stable for several days with the

Pb measurement remaining at 47 ppm.

EXAMPLE 17

Complexing rate of PbU2 with 4, 7 ', 13, 1 6, 21, 24-Hexaoxa-l, 10- diazabicyclo [ 8. 8. 8] hexacosane and benzo derivative

7 mg of cryptate was dissolved in 5 ml of water and 5 mg of Pb0₂ was added with stirring. After spinning (3,000 rpm for 10 min.), 200 μl of supernatant was periodically removed and tested for Pb using atomic absorption, The results are shown in the table below.

TABLE 6

Time (hours^ ppm Pb

0 1 1 2 3 4 4 6 6 8 7 13 23 20

EXAMPLE 18

Lead Chelating Complex The following table represents the results when various chelating agents are used to chelate lead. The chelators were ethylene diamine tetraacetate (EDTA) , diethylene triamine pentaacetic acid (DTPA), 2,3- dimercapto-1-propane sulfonic acid (2,3 DMPS) , and meso- 2,3-dimercaptosuccinic acid (2,3 DMSA). All lead levels are listed in ppm (parts per million) . Levels greater than 70 ppm are considered offscale and are not as accurate. Even at levels of 1 mg of lead oxide there is very little difference in lead concentrations, indicating that uptake of lead is determined by the chelator's reaction capability.

TABLE 7

Assay for in vitro activity

The conjugate prepared in Example 3 is evaluated for in vitro cytotoxic activity using the following procedure.

10⁴ M21-UCLA melanoma cells per well are allowed to grow in 96-well plates in a 10% C0₂ atmosphere at 37°C in 100 μl of RPMI 1640, 10% fetal bovine serum (FBS) medium. After 24 hours, the nutrient is taken off and replaced by 100 μl of RPMI 1640, containing varying concentrations of the metal chelate immunoconjugate. The metal chelate immunoconjugate is formed using TDCOD-DTPA, Pb0₂, and 9.2.27 monoclonal antibody, as indicated in Example 3. The metal chelate immunoconjugate containing nutrient is removed 30 minutes later and the wells are rinsed three times with the original medium and the cells are allowed to continue to grow in the original medium. After 24 hours, 10 μl of 1 μCi ³H-thymidine containing medium is added in order to measure thymidine uptake. Thymidine is incorporated into DNA and thymidine uptake is used to measure DNA synthesis which relates to cell viability. After another day of growth the plates are shock frozen, then thawed and the individual well contents passed through glass fiber filters. The radioactivity is determined and taken as a measure of cell viability.

The results of the in vitro assay show a high level of cytotoxicity activity for the conjugate.

EXAMPLE 20 Assay for in vivo binding specificity and affinity The conjugate prepared in Example 3 is evaluated for in vivo binding specificity and affinity by the following procedure.

Thy us deficient BALBc (nude/nude) mice are subcutaneously injected with 2xl0⁶ M21-UCLA melanoma cells. After two weeks, 35 μg of metal chelate immunoconjugate is injected into the tail vein. After 48 hours the animals are sacrificed and the radioactivity in individual organs is determined. The metal chelate immunoconjugate is formed using TDCOD-DTPA, Pb0₂, and 9.2.27 monoclonal antibody, following the procedures discussed in Example 3. The in vivo biodistribution data obtained with tumor bearing nude mice also shows that the conjugate has a high degree of binding specificity and affinity. Further, the data indicates that any unbound conjugate is cleared from the body as indicated by the low levels of conjugate found in the blood, liver, kidney, spleen and intestine.

INDUSTRIAL APPLICABILITY The bifunctional chelating agents of the present invention are useful as therapeutic agents in radiation therapy.

They can be used for treating cellular disorders, particularly cancer, by employing a chelating agent for a metal ion, such as a radioisotope, conjugated to a site- specific compound or target cell binding protein, such as a monoclonal antibody. They can also be used either for in vitro or in vivo diagnostic purposes.

Claims

1. A metal ion chelating agent comprising a first molecular component, wherein said first molecular component is a chelate having a high affinity for metal ions and a high K_s value, linked to at least one second molecular component, wherein said second molecular component is a chelate having a low K_s value and the ability to undergo rapid metal exchange.

2. The metal ion chelating agent of claim 1, wherein said chelating agent exhibits high stability after binding one or more metal ions.

3. The metal ion chelating agent of claim 1, wherein said first molecular component is linked to two of said second molecular components.

4. The metal ion chelating agent of claim 1, wherein said second molecular component chelates a metal ion, and said metal ion is then chelated to said first molecular component in such a way that said second molecular component becomes folded and forms intermolecular interactions in the form of coordinate bonds with said first molecular component.

5. The metal ion chelating agent of claim 1, wherein said second molecular component is a member of the group consisting of metal organics, metal carbonyls, polythiols, complexes.

6. The metal ion chelating agent of claim 5, wherein said second molecular component is a member of the group consisting of ethylene diamine tetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), 2,3- dimercapto-1-propane sulfonic acid (2,3 DMPS), and meso- 2,3-dimercaptosuccinic acid (2,3 DMSA).

7. The metal ion chelating agent of claim 1, wherein said first molecular component is a member of the group consisting of crown ethers, crown azos, polyporphyrins, cages, and cryptates.

8. The metal ion chelating agent of claim 7, wherein said first molecular component is a member of the group consisting of 1,4,10,13-tetraoxa-7,16- diazacyclooctadecane (TDCOD) , cages, and cryptates.

9. The metal ion chelating agent of claim 1, wherein said metal ion chelating agent chelates metal ions; said metal ions being members of the group consisting of transition metals, alkali earth metals, and lanthanides.

10. The metal ion chelating agent of claim 1, wherein said metal ion chelating agent chelates radioisotopes; said radioisotopes being members of the group consisting of alpha emitters, beta emitters, gamma or positron emitters, Auger electron emitters, and fluorescing lanthanides.

11. The metal ion chelating agent of claim 10, wherein said radioisotopes are members of the group consisting of Pb²¹² and Bi²¹².

12. A composition for producing localized cytotoxic effects on targeted cells comprising a conjugate of a metal ion chelating agent comprising a first molecular component, wherein said first molecular component is a chelate having a high affinity for metal ions and a high K_s value, linked to at least one second molecular component, wherein said second molecular component is a chelate having a low K_s value and the ability to undergo rapid metal exchange; chelated to one or more metal ions; and linked to a target cell binding protein; said conjugate having binding specificity and binding affinity for said target cells and having chelating agent activity.

13. The composition of claim 12 wherein said chelating agent exhibits high stability after binding one or more metal ions.

14. The composition of claim 12, wherein said first molecular component is linked to two of said second molecular components.

15. The composition of claim 12, wherein said second molecular component chelates a metal ion, and said metal ion is then chelated to said first molecular component in such a way that said second molecular component becomes folded and forms intermolecular interactions in the form of coordinate bonds with said first molecular component.

16. The composition of claim 12, wherein said second molecular component is a member of the group consisting of metal organics, metal carbonyls, polythiols, complexes.

17. The composition of claim 16, wherein said second molecular component is a member of the group consisting of ethylene diamine tetraacetic acid (EDTA) , diethylene triamine pentaacetic acid (DTPA), 2,3- dimercapto-1-propane sulfonic acid (2,3 DMPS), and meso- 2,3-dimercaptosuccinic acid (2,3 DMSA).

18. The composition of claim 12, wherein said first molecular component is a member of the group consisting of crown ethers, crown azos, polyporphyrins, cages, and cryptates.

19. The composition of claim 18, wherein said first molecular component is a member of the group consisting of 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (TDCOD) , cages, and cryptates.

20. The composition of claim 12, wherein said metal ion chelating agent chelates metal ions; said metal ions being members of the group consisting of transition metals, alkali earth metals, and lanthanides.

21. The composition of claim 12, wherein said metal ion chelating agent chelates radioisotopes; said radioisotopes being members of the group consisting of alpha emitters, beta emitters, gamma or positron emitters, Auger electron emitters, and fluorescing lanthanides.

22. The composition of claim 21, wherein said radioisotopes are members of the group consisting of Pb²¹² and Bi²¹².

23. The composition of claim 12, wherein said target cell binding protein is a member of the group consisting of monoclonal antibodies, polyclonal antibodies, antibody fragments, and binding proteins.

24. The composition of claim 12, wherein said target cell binding protein is a monoclonal antibody.

25. The composition of claim 12, wherein said target cell binding protein is a fragment of a monoclonal antibody.

26. A method of producing localized cytotoxic effects on target cells which comprises combining an effective amount of a composition for producing localized cytotoxic effects on target cells, and a pharmaceutically acceptable carrier therefor; said composition comprising a conjugate of a metal ion chelating agent comprising a first molecular component, wherein said first molecular component is a chelate having a high affinity for metal ions and a high K_s value, linked to at least one second molecular component, wherein said second molecular component is a chelate having a low K_s value and the ability to undergo rapid metal exchange; chelated to one or more metal ions; and linked to a target cell binding protein; said conjugate having binding specificity and binding affinity for said target cells and having chelating agent activity; and administering said combined composition and pharmaceutically acceptable carrier to said target cells.

27. The method of claim 26, wherein said target cells are hosted in a mammalian host.

28. The method of claim 26, wherein said combined composition and vehicle is administered by a technique selected from the group consisting of topical administration, intravenous administration subcutaneous administration, and intraperitoneal administration.

29. The method of claim 26, wherein said chelating agent exhibits high stability after binding one or more metal ions.

30. The method of claim 26, wherein said first molecular component is linked to two of said second molecular components.

31. The method of claim 26, wherein said second molecular component chelates a metal ion, and said metal ion is then chelated to said first molecular component in such a way that said second molecular component becomes folded and forms intermolecular interactions in the form of coordinate bonds with said first molecular component.

32. The method of claim 26, wherein said second molecular component is a member of the group consisting of metal organics, metal carbonyls, polythiols, complexes.

33. The method of claim 32, wherein said second molecular component is a member of the group consisting of ethylene diamine tetraacetic acid (EDTA) , diethylene triamine pentaacetic acid (DTPA), 2,3-dimercapto-l-propane sulfonic acid (2,3 DMPS), and meso-2,3-dimercaptosuccinic acid (2,3 DMSA) .

34. The method of claim 26, ^' wherein said first molecular component is a member of the group consisting of crown ethers, crown azos, polyporphyrins, cages, and cryptates.

35. The method of claim 34, wherein said first molecular component is a member of the group consisting of

1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (TDCOD) , cages, and cryptates.

36. The method of claim 26, wherein said metal ion chelating agent chelates metal ions; said metal ions being members of the group consisting of transition metals, alkali earth metals, and lanthanides.

37. The method of claim 26, wherein said metal ion chelating agent chelates radioisotopes; said radioisotopes being members of the group consisting of alpha emitters, beta emitters, gamma or positron emitters. Auger electron emitters, and fluorescing lanthanides.

38. The method of claim 37, wherein said radioisotopes are members of the group consisting of Pb²¹² and Bi²¹².

39. The method of claim 26, wherein said target cell binding protein is a member of the group consisting of monoclonal antibodies, polyclonal antibodies, antibody fragments, and binding proteins.

40. The method of claim 26, wherein said target cell binding protein is a monoclonal antibody.

41. The method of claim 26, wherein said target cell binding protein is a fragment of a monoclonal antibody.

42. An in vivo diagnostic method which comprises combining an effective amount of a composition capable of combining with target cells in a body, and an acceptable carrier therefor; said composition comprising a conjugate of a metal ion chelating agent comprising a first molecular component, wherein said first molecular component is a chelate having a high affinity for metal ions and a high K_s value, linked to at least one second molecular component, wherein said second molecular component is a chelate having a low K_s value and the ability to undergo rapid metal exchange; chelated to one or more metal ions; and said conjugate having binding specificity and binding affinity for said target cells and having chelating agent activity; administering said combined composition and acceptable carrier to said target cells; and detecting the presence of said metal ion chelating agent.

43. An in vitro diagnostic method which comprises combining an effective amount of a composition capable of combining with target cells in a test medium, and an acceptable carrier therefor; said composition comprising a conjugate of a metal ion chelating agent comprising a first molecular component, wherein said first molecular component is a chelate having a high affinity for metal ions and a high K_s value, linked to at least one second molecular component, wherein said second molecular component is a chelate having a low K_g value and the ability to undergo rapid metal exchange; chelated to one or more metal ions; and said conjugate having binding specificity and binding affinity for said target cells and having chelating agent activity; administering said combined composition and acceptable carrier to said target cells; and detecting the presence of said metal ion chelating agent.

44. A method for the preparation of a metal ion chelating agent comprising the steps of reacting a first molecular component, wherein said first molecular component is a chelate having a high affinity for metal ions and a high K_s value, with at least one second molecular component, wherein said second molecular component is a chelate having a low K_s value and the ability to undergo rapid metal exchange; and recovering the product of said reaction.

45. A method for the preparation of a composition for producing localized cytotoxic effects on targeted cells comprising a conjugate of a metal ion chelating agent; linked to a target cell binding protein; said target cell binding protein containing at least one active binding site, comprising: (a) reacting a first molecular component, wherein said first molecular component is a chelate having a high affinity for metal ions and a high K_s value, with at least one second molecular component, wherein said second molecular component is a chelate having a low K_s value and the ability to undergo rapid metal exchange;

(b) recovering the product of said reaction, namely said metal ion chelating agent;

(c) providing an aqueous solution of said target cell binding protein to form an aqueous phase;

(d) providing said metal ion chelating agent in a phase immiscible with said aqueous phase and forming an interface therewith;

(e) conducting an interfacial condensation of one or more of said target cell binding proteins and one or more of said metal ion chelating agents to form one or more covalent bonds between said target cell binding proteins and said metal ion chelating agents while protecting said active binding sites of said target cell binding proteins from the condensation reaction, said interfacial condensation resulting in the formation of a conjugate of said compounds;

(c) reacting said metal ion chelating agent of said conjugate with a metal ion to form a metal ion chelate - target cell conjugate; and

(g) recovering said metal ion chelate - target cell conjugate.

46. A method for the preparation of a composition for producing localized cytotoxic effects on targeted cells comprising a conjugate of a metal ion chelating agent; linked to a target cell binding protein; said target cell binding protein containing at least one active binding site, comprising:

(a) reacting a first molecular component, wherein said first molecular component is a chelate having a high affinity for metal ions and a high K_s value, with at least one second molecular component, wherein said second molecular component is a chelate having a low K_s value and the ability to undergo rapid metal exchange;

(b) recovering the product of said reaction, namely said metal ion chelating agent; (c) reacting said metal ion chelating agent with a metal ion to form a metal ion chelate;

(d) providing an aqueous solution of said target cell binding protein to form an aqueous phase;

(e) providing said metal ion chelate in a phase immiscible with said aqueous phase and forming an interface therewith;

(f) conducting an interfacial condensation of one or more of said target cell binding proteins and one or more of said metal ion chelates to form one or more covalent bonds between said target cell binding proteins and said metal ion chelates while protecting said active binding sites of said target cell binding proteins from the condensation reaction, said interfacial condensation resulting in the formation of a conjugate of said compounds; and

(g) recovering said conjugate of said compounds.

47. A method for removing metal ions which comprises combining an effective amount of a conjugate of a metal ion chelating agent, and an acceptable carrier therefor; said conjugate of a metal ion chelating agent comprising a first molecular component, wherein said first molecular component is a chelate having a high affinity for metal ions and a high K_s value, linked to at least one second molecular component, wherein said second molecular component is a chelate having a low K_g value and the ability to undergo rapid metal exchange; said conjugate having chelating agent activity; and administering said combined composition and acceptable carrier.

48. The method of claim 47, wherein said chelating agent exhibits high stability after binding one or more metal ions.

49. The method of claim 47, wherein said first molecular component is linked to two of said second molecular components.

50. The method of claim 47, wherein said second molecular component chelates a metal ion, and said metal ion is then chelated to said first molecular component in such a way that said second molecular component becomes folded and forms intermolecular interactions in the form of coordinate bonds with said first molecular component.

51. The method of claim 47, wherein said second molecular component is a member of the group consisting of metal organics, metal carbonyls, polythiols, complexes.

52. The method of claim 51, wherein said second molecular component is a member of the group consisting of ethylene diamine tetraacetic acid (EDTA) , diethylene triamine pentaacetic acid (DTPA), 2,3-dimercapto-l-propane sulfonic acid (2,3 DMPS), and meso-2,3-dimercaptosuccinic acid (2,3 DMSA) .

53. The method of claim 47, wherein said first molecular component is a member of the group consisting of crown ethers, crown azos, polyporphyrins , cages, and cryptates.

54. The method of claim 53, wherein said first molecular component is a member of the group consisting of 1,4,10,13-tetraoxa-7,16-diazacyclooctadecane (TDCOD) , cages, and cryptates.

55. The method of claim 47, wherein said metal ion chelating agent chelates metal ions; said metal ions being members of the group consisting of transition metals, alkali earth metals, and lanthanides.

56. The method of claim 47, wherein said metal ion chelating agent chelates radioisotopes; said radioisotopes being members of the group consisting of alpha emitters, beta emitters, gamma or positron emitters, Auger electron emitters, and fluorescing lanthanides.

57. The method of claim 56, wherein said radioisotopes are members of the group consisting of Pb²¹² and Bi²¹².

58. A metal ion chelating agent comprising a first molecular component, wherein said first molecular component is a chelate having a high affinity for metal ions and a high K_s value, linked to a second molecular component, wherein said second molecular component is a chelate having a low K_s value and the ability to undergo rapid metal exchange; said second molecular component is linked to said first molecular component in two places forming a bridge between two sections of said first molecular component.

59. The metal ion chelating agent of claim 58 wherein said chelating agent exhibits high stability after binding one or more metal ions.

60. The metal ion chelating agent of claim 58, wherein said second molecular component chelates a metal ion, and said metal ion is then chelated to said first molecular component in such a way that said second molecular component becomes folded and forms intermolecular interactions in the form of coordinate bonds with said first molecular component.

61. The metal ion chelating agent of claim 58, wherein said second molecular component is a member of the group consisting of metal organics, metal carbonyls, polythiols, complexes.

62. The metal ion chelating agent of claim 61, wherein said second molecular component is a member of the group consisting of ethylene diamine tetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), 2,3- dimercapto-1-propane sulfonic acid (2,3 DMPS), and eso- 2,3-dimercaptosuccinic acid (2,3 DMSA).

63. The metal ion chelating agent of claim 58, wherein said first molecular component is a member of the group consisting of crown ethers, crown azos, polyporphyrins, cages, and cryptates.

64. The metal ion chelating agent of claim 63, wherein said first molecular component is a member of the group consisting of I,4,l0,l3-tetraoxa-7,l6- diazacyclooctadecane (TDCOD) , cages, and cryptates.

65. The metal ion chelating agent of claim 58, wherein said metal ion chelating agent chelates metal ions; said metal ions being members of the group consisting of transition metals, alkali earth metals, and lanthanides.

66. The metal ion chelating agent of claim 58, wherein said metal ion chelating agent chelates radioisotopes; said radioisotopes being members of the group consisting of alpha emitters, beta emitters, gamma or positron emitters. Auger electron emitters, and fluorescing lanthanides.

67. The metal ion chelating agent of claim 66, wherein said radioisotopes are members of the group consisting of Pb²¹² and Bi²¹².

68. The metal ion chelating agent of claim 58, wherein one or more third molecular components is linked to said bridge formed by said second molecular component.

69. The metal ion chelating agent of claim 68, wherein said third molecular component is a chelate having a low K_s value and the ability to undergo rapid metal exchange.

70. The metal ion chelating agent of claim 68, wherein said third molecular component chelates a metal ion, and said metal ion is then chelated to said first molecular component in such a way that said third molecular component becomes folded and forms intermolecular interactions in the form of coordinate bonds with said first molecular component.