WO1998004293A1

WO1998004293A1 - Improved detection and therapy of lesions with biotin-chelate conjugates

Info

Publication number: WO1998004293A1
Application number: PCT/US1997/013285
Authority: WO
Inventors: Gary L. Griffiths; Hans J. Hansen; Habibe Karacay
Original assignee: Immunomedics, Inc.
Priority date: 1996-07-31
Filing date: 1997-07-31
Publication date: 1998-02-05
Also published as: AU4047497A

Abstract

An improved method of detecting and/or treating lesions in a patient in which a pre-targeting approach is used wherein the total amount of radionuclide delivered to a target cell, tissue, or pathogen is dramatically increased. In this method, the chelate conjugate may be purified by chromatography after chelate formation, may contain multiple chelates or a blood transit-modifying linker or added within the chelate conjugate, or both; or a combination of these. The improved chelate conjugates can be used as detection of therapeutic agents to detect or treat the targeted cell, tissue, or pathogen.

Description

IMPROVED DETECTION AND THERAPY OF LESIONS WITH BIOTIN-CHELATE CONJUGATES

BACKGROUND OF THE INVENTION

Cross Reference to Related Applications

This application is a continuation-in-part of co- pending application Ser. No. 08/409,960, filed March 23, 1993, which is a continuation of application Ser. No. 08/062,662, filed May 11, 1993 (now abandoned).

Field of the Invention

The present invention relates to methods for detecting and treating pathological conditions with a multi-step process using improved detection or therapeutic chelate conjugates comprised of biotin, chelator, and optional linker.

Description of the Prior Art

Antibodies against different determinants associated with pathological and normal cells, as well as those associated with pathogenic microorganisms, have been used for the detection and treatment of a wide variety of pathological conditions or lesions. The targeting antibody is conjugated to an appropriate detection or therapeutic agent as described, for example, in Hansen et al . , U.S. patent No. 3,927,193 and Goldenberg, U.S. patent Nos. 4,331,647, 4,348,376, 4,361,544, 4,468,457,

4,444,744, 4,460,459, 4,460,561, 4,624,846, 5,482,698, and 4,818,709, and U.S. patent application Serial Nos. 07/933,982, 08/062,662, and 08/486 , 166 , the disclosure of all of which are incorporated herein by reference. Detection and therapeutic methods have been developed that utilize such targeting antibodies and other binding proteins. The effectiveness of these methods, however, is limited by the absolute amount of detection or therapeutic agent, such as a nuclide, that is delivered to a target. Effectiveness also is limited by the target : background ratios achieved for delivery of detection or therapeutic agent to a target .

An important therapeutic method that uses antibody targeting is radioimmunotherapy. It cannot be overstressed that the fundamental limitation to radioimmunotherapy is the low absolute amount of therapy nuclide that can be delivered to a target . Poor targeting of a nuclide, determined as a poor target :back- ground ratio, contributes to the problem by restricting the amount of delivered nuclide still further, according to the ability of non-targeted tissue to withstand deleterious effects of the nuclide present there due to background binding . An important technical development which increases the target : background ratios when using detection or therapeutic agents is the "pre-targeting" approach, as described, for example, in Goodwin et al . , U.S. Patent No. 4,863,713; Goodwin et al . , J. Nucl . Med. 29:226, (1988); Hnatowich eta]., J. Nucl . Med. 28:1294, (1987); Oehr et al . , J. Nucl . Med . 29:728, (1988); Klibanov et al . , J. Nucl . Med. 29:1951, (1988); Sinitsyn et al . , J. Nucl . Med . 30:66, (1989); Kalofonos e t al . , J. Nucl . Med . 31:1791, (1990); Schechter et al . , In t . J. Cancer 48 -. 167 , (1991); Paganelli et al . , Cancer Res . 51:5960, (1991); Paganelli et al . , Nucl . Med. Commun . 12:211, (1991); Stickney et al . , Cancer Res . 51:6650, (1991); Yuan et al . , Cancer Res . 51:3119, (1991); Gustavson et al., U.S. Patent Νos . 5,283,342, 5,630,996, 5,541,287 and 5,578,287, and Axworthy et al . , U.S. Patent Νos. 5,624,896, 5,608,060 and 5,616,690 all incorporated herein in their entireties by reference. The pre-targeting approach decouples the therapy agent delivery step from the antibody targeting step. An example of pre-targeting in the context of tumor radiotherapy is the administration of a conjugate comprising a tumor specific antibody and a second binder to a cancer patient. The conjugate circulates within the patient's body and some of the conjugate binds to the tumor by affinity between the antibody moiety and a tumor-specific antigen on tumor cells. Conjugate that does not adhere to the tumor clears gradually over time, preferably by administration of a specific clearing agent .

After clearance of excess tumor specific antibody- conjugate, a therapeutic agent -chelate conjugate that binds to the second binder moiety of the conjugate is administered. The therapeutic agent -chelate conjugate localizes to tumor by virtue of its affinity to the second binder in the antibody- conjugate that has been pre-targeted to tumor. The pre-targeting approach uses two binding partners, such as avidin and biotin, to indirectly localize a detection or therapeutic agent to target tissue. For example, in a "2-step" procedure, a targeting antibody- avidin conjugate, having an avidin as "second binder", first is injected into a patient and the antibody-avidin localizes to a target tissue. Then, in a second step, biotin conjugated to a detection or therapeutic agent is injected and binds to the avidin pre-localized to the target tissue. Timing of the second injection after the first one is critical. Injecting a biotin chelate conjugate too early will increase the proportion of biotin chelate conjugate that remains in the bloodstream. On the other hand, injecting the biotin chelate conjugate very late may decrease the amount of biotin that localizes to a tumor because of reduced retention of the targeting protein at the tumor with time. This results in decreased accretion of the therapeutic agent, which is part of the chelate conjugate, at the tumor

A "3 -step" procedure also has been developed to improve target to background ratios of antibody-avidm (or biotm) conjugate that becomes localized to target tissue. In this method, administration of a targeting antibody-avidin (or biotm) conjugate is followed by administration of a clearing agent to remove unbound conjugate. For example, when antibody-avidm is used as a tumor specific antibody-conjugate in radiotherapy, non- targeted circulating antibody-avidm can be cleared by injecting a biotmylated polymer such as biotmylated human serum albumin. This type of agent forms a high molecular weight species with circulating antibody-avidm conjugate. The high molecular weight species is quickly recognized by the hepatobiliary system and deposited primarily in the liver. With circulating antibody- avidin conjugate removed, non-targeted biotm-chelator-radio- nuclide, which is administered m the last step, is rapidly eliminated. The radionuclide is m circulation for only a short time prior to renal clearance Thus, this procedure causes considerably less bone marrow toxicity to the patient compared to complexmg the radionuclide directly to an antibody However, this method does not address the fundamental problem of insufficient radionuclide delivery to the tumor.

The effectiveness of nuclide therapy as outlined above is limited by the accretion of therapeutic nuclide to the tumor site. Unfortunately, carrier molecules that lack a functioning detection or therapeutic agent typically are in great excess over functional carrier molecules. In fact, much less than one percent of targeted antibodies usually receive an atom of isotope If all, or virtually all targeted antibodies could receive one or more atoms of a therapeutic isotope, the effectiveness of these methods could be improved greatly.

One cause of the insufficient nuclide problem is that the nuclide chelator conjugates used have very low specific activities. The low specific activity of these conjugates often is a consequence of difficulties in purifying a protein-nuclide chelate conjugate away from protein that does not have a radionuclide complexed to it.

Another cause of the low accretion problem is that the body residence time of a diagnostic or therapeutic agent, expressed as its half-life, i.e. how long half of a parenterally injected dose stays inside the body, is uncontrolled. An excessively long body residence time will unnecessarily expose untargeted body tissues to the detection or therapeutic agent and cause side effects. These side effects limit how much radionuclide can be used. On the other hand, if the body residence time is too short, then the injected agent will not have enough time to saturate and bind all pre-targeted antibody- conjugate. Consequently, less detection or therapeutic agent is delivered to the target .

Prior art methods that utilize protein-based detection or therapeutic agents often have excessive body residence times. This is because a proteinaceous detection or therapeutic agent is too large for filtration clearance by the kidney. Most proteins of greater than 40,000 molecular weight are slowly cleared by the liver and other tissues of the reticuloendothelial system, or reabsorbed after catabolism in the kidney.

On the other hand, a small detection or therapeutic agent having a molecular weight less than about 40,000 daltons can be quickly removed by kidney filtration. In fact, pretargeting methods that utilize small compounds, in particular compounds less than about 10,000 daltons, often are limited by too short a body residence time.

The ideal detection or therapeutic chelate conjugate for use in these methods has a small size and is quickly cleared by the kidney, yet has a body residence time sufficient for it to saturate target sites. The ideal body residence time is determined by a number of factors, including the nature of the target, targeting conjugate, kidney health, half-life of detection or therapeutic agent, and binding moieties used to bind the detection or therapeutic agent to the targeted site. Unfortunately, known methods do not provide convenient control of body residence time for delivery of detection or therapeutic agent . A means to control body residence time would give significant clinical benefits, particularly with pre- targeting strategies that are sensitive to the timing of injections . Kidney resorption capacity can be altered by chemical agents for controlling body residence time of therapeutic proteinaceous agents. For example, US patent No. 5,380,513 discloses the use of cationic compounds such as lysine, that promote urinary excretion of protein. Although basic amino acids have been used to block kidney resorption of low molecular weight peptides as well as protein, the effects of these chemical agents are generalized and not limited to specific medicinal reagents. Fewer side effects would result if kidney resorption capacity only could be modified for a particular medicinal reagent.

As summarized above, the new pretargeting methods solve the target : background ratio problem but do not alleviate the fundamental problem of low accretion. Although multi-step procedures can specifically locate antibody or other binding protein onto target surfaces, in reality, few of the located proteins receive a detection or therapeutic agent. If all, or substantially all pre-targeted protein could receive at least one detection or therapeutic agent, then clinical effectiveness could be greatly improved.

SUMMARY OF THE INVENTION

An object of the present invention is to provide methods that deliver higher amounts, higher specific activities, and higher target :nontarget ratios of detection or therapeutic agents to a targeted site. Another object of the invention is to improve multi- step processes for detecting and treating pathological conditions by providing greater control of the residence time of administered detection and therapeutic agents in the body.

A further objective of the present invention is to provide a detection or therapeutic agent with an increased specific activity, and to allow delivery of higher amounts of detection or therapeutic agents to a targeted cell, tissue, or pathogen.

These and other objectives of the invention are achieved by providing a method for detecting or treating a target cell, tissue or pathogen in a patient, the method comprising pre-targeting the target cell, tissue, or pathogen with avidin, using a targeting protein that specifically binds a marker substance on the target cell, tissue, or pathogen and to which avidin is bound either directly as an avidin-targeting protein conjugate, or indirectly via non-covalent binding of avidin to a biotin-targeting protein conjugate; parenterally injecting a detection or therapeutic composition comprising a chelate conjugate of biotin, a chelator, and a chelatable detection or therapeutic agent, and allowing the composition to accrete at the targeted cell, tissue, or pathogen; wherein the chelate conjugate is purified by chromatography after chelate formation, or further comprises a blood transit -modifying linker or addend that is covalently bound within the chelate conjugate, or both; and using the detection or therapeutic agent to detect or treat the targeted cell, tissue, or pathogen.

Objectives of the invention also are achieved by providing a method for detecting or treating a target cell, tissue or pathogen in a patient, the method comprising pre-targeting the target cell, tissue, or pathogen with avidin, using a targeting protein that specifically binds a marker substance on the target cell, tissue, or pathogen and to which avidin is bound either directly as an avidin-targeting protein conjugate, or indirectly via non-covalent binding of avidin to a biotin-targeting protein conjugate; parenterally injecting a detection or therapeutic composition comprising a biotin- ultiple chelate conjugate and a chelatable metal ion detection or therapeutic agent, and allowing the composition to accrete at the targeted cell, tissue, or pathogen; wherein the biotin-multiple chelate conjugate comprises at least five chelates, and using the detection or therapeutic agent to detect or treat the targeted cell, tissue, or pathogen.

Also provided is a sterile injectable composition for human use comprising a detection or therapeutic composition comprised of a chelate conjugate of biotin, chelating agent, and chelatable therapeutic or detection agent; or biotin, chelating agent, blood transit modifying linker or addend, and chelatable therapeutic or detection agent.

Upon further study of the specification and appended claims, further objects and advantages of this invention will become apparent to those skilled in the art.

DETAILED DESCRIPTION

Insights leading to the present invention stemmed from kinetic analyses of therapeutic agent delivery in a pre-targeting system. The major limitation of radioimmunotherapy ("RAIT") has been that low amounts of therapeutic agent, such as a nuclide accrete to target sites in a patient. This limitation is compounded by the target-to-tissue ratio in standard RAIT protocols. In a standard RAIT protocol, radionuclide toxicity limits how much circulating radionuclide attached to antibody can be delivered to a target. In an ideal kinetic situation, a large amount of radionuclide is introduced into a patient, a large proportion of the introduced nuclide binds to target and excess nuclide quickly is removed from the body.

Decoupling the antibody and nuclide delivery steps by using a pretargeting method improves the proportion of nuclide that binds specifically to target tissue but does not address the related problem of low absolute target accretion. Furthermore, even in the newer methods, fewer than ten percent of target antibodies typically receive a desired nuclide in the context of radiotherapy. Consequently, these methods could be improved greatly if a means were found to bind at least one functional detection or therapeutic agent to each pre-targeted protein.

As an example, 3.25 X 10¹⁴ molecules of antibody- avidin conjugate were found to bind per gram of tumor in a nude mouse/human xenograft animal model of colorectal cancer. About one in ten of the theoretical biotin binding sites placed at the tumor via the antibody-avidin conjugate were subsequently targeted with biotin moieties. In addition, due to low specific activity of the biotin-chelate conjugate, only about one in 350 of those biotin residues was actually associated with a radionuclide atom. The present inventors realized that the amount of radionuclide delivered to a target could be greatly increased by employing a biotin (or avidin) conjugate which is associated with at least one atom of radionuclide. The inventors discovered several means to achieve this, including purification of radionuclide after chelate formation and placing multiple chelates in one conjugate. In addition, the inventors discovered that the amount of radionuclide accreting at a target can be increased by optimizing the body residence time of the conjugate. The invention provides typically a 100 to 1000 fold increase in the delivery of isotope to a tumor by combining one or more of these techniques with selection of a suitable isotope and of a good first step agent such as strepavidin-antibody to localize the isotope. The increase in total amount of delivered radionuclide made possible by the invention, together with a dramatic increase in "therapeutic ratio" provided by the pretargeting approach allows successful transfer of radioimmunotherapy from animal subjects to human subjects .

Accordingly, the invention contemplates increasing the absolute amount of radiolabelled biotin (or avidin) that accretes to targets by alternate means of (1) purifying isotope after chelate formation, (2) linking multiple radiolabels onto one binding site via a polymeric chelate substituted with a single biotin (or avidin) , termed "multiple chelate conjugate" and (3) saturating pre-targeted antibody-avidin binding sites by controlling the blood clearance of biotin-chelate- nuclide .

Purifying the radionuclide-chelator-biotin conjugate is a preferred means to solve the low specific activity problem. Due to a nuclide 's cationic nature, the small size of biotin, and the small size of most chelators, a chelate-biotin conjugate that contains the nuclide can be conveniently purified away from a conjugate that lacks the desired nuclide. Consequently, a therapeutic agent composition of much higher specific activity can be delivered to a target.

During use within the targeting procedures, the detection or therapeutic chelate conjugate preferably is purified immediately prior to use. When a relatively stable detection or therapeutic agent is used, however, purification to increase its specific activity can be performed prior to storage .

Linking multiple radiolabels onto one binding site via a multiple chelate binding partner to increase radionuclide accretion can be carried out with many different synthetic polymers and naturally occurring molecules using reactions that are known to the skilled artisan. When using a synthetic polymer, the polymer backbone size can be selected from a wide variety of sizes such as from 200 daltons in molecular weight up to 2,000,000 daltons in molecular weight and can be a polyamino acid, polysugar, polyacid, polyamine, or polyalcohol . The polymer comprises at least two chelates. Preferably the polymer comprises at least 5 chelates and more preferably at least 25 chelates. The polymer may be selected according to the pharmacokinetic properties that it brings to the final radiolabeled biotin conjugate. When using a naturally occurring molecule to form the multiple chelate conjugate, the molecule is large enough to contain at least 2 natural chelating sites, preferably at least 3 natural sites, and more preferably at least 5 sites. Most preferred in this context is metallothionein. Biotin can be attached to an amino group of metallothionein and radiometals that bind strongly to sulfur atoms are attached to the apometallothionein-biotin conjugate. Such a metallothionein agent can bind up to seven atoms of elements such as copper, zinc, rhenium and similar thiophilic metals. Metallothionein is preferred because it is non-immunogenic and, as a thioprotein, will not bind well to all metals. A desired consequence of metallothionein¹ s metal specificity is that iron contamination of metallothionein radionuclide conjugates such as those made from radiorhenium is much less than that of prior art chelators such as EDTA-type agents. Accretion of radionuclide to a target site, according to one embodiment of the invention, can be increased by modifying a chelate conjugate to alter its renal clearance rate and thus its body residence time. Starting with a small binder (biotin) to bind the pre- targeted antibody-conjugate and a small chelator, a "linker" moiety to link biotin with chelator can be designed or chosen having a predictable kidney clearance ratio. The present inventors discovered that renal clearance of a chelate conjugate that comprises a detection agent could be adjusted by designing a linker having a particular composition because the linker can dominate the overall blood clearance characteristics of the chelate conjugate. The selection of a linker is determined by analyzing the ionic, hydrophobic, and steric characteristics needed to match kidney physiology and to delay or facilitate the excretion of the detection or therapeutic agent by the kidney. A particular linker also may be chosen on the basis of resistance to proteases and to biotinidase. The excretion of a substance that is filtered, but not reabsorbed or secreted by the kidney is controlled by the sieving properties of the kidney glomerular wall. How well the kidney excretes such a substance can be determined by measurements of the clearance of a test molecule, relative to some "freely permeable" reference polymer such as inulin, as summarized by Brenner et al . in Am . J. Physiol . 234: F455-60 (1978), which is herein incorporated in its entirety by reference. The ratio of urinary clearance of a test solute to a reference solute is equal to the ratio of the concentration of the test solute in Bowman's space to its concentration in plasma water. This ratio is called the "fractional clearance" of the test solute. The fractional clearance varies from 0, when test molecules cannot enter the kidney filtrate, to 1 when they encounter no measurable restriction to filtration. A chelate conjugate that has a fractional clearance near 1 is quickly cleared by the kidney (if not reabsorbed by the kidney) and has a short body residence time, l e. fast renal clearance. A chelate conjugate that has a fractional clearance near zero typically is cleared slowly by the liver or other tissue, and has a longer body residence time, i.e slow renal clearance In practice, it is preferred to modify the chelate conjugate by selection of a linker that gives the chelate conjugate a fractional clearance of an intermediate value between 1 and 0. More preferred is a fractional clearance between 0.2 and 0.8.

A "chelator", as used in the present invention, is a polyfunctional compound having a chelating group and at least one pendant functional group The chelating group can chelate a radionuclide. The pendant functional group may be the same as or different from the chelating group, and can covalently bind to a linker or to biotin Chelating agents may be represented by the formula A(F)_n wherein A represents the chelating moiety, F represents a pendant functional group, and n is an integer from 1 to 3 , preferably 1.

Groups that are capable of chelating radionuclides include macrocycles, linear ligands, or branched ligands. Examples of macrocyclic chelating moieties include polyaza- and polyoxamacrocycles , and poly (thioalkyl) azamacrocycles . Examples of polyazamacrocyclic moieties include those derived from compounds such as the preferred 1,4,7,10- tetraazacyclododecane-N,N' ,N' ' ,N" ' ' -tetraacetic acid (herein abbreviated as DOTA) ; 1,4,7,10- tetraazacyclotπdecane-N, N' ,N' ' ,N' ' ' -tetraacetic acid, 1,4,8, ll-tetraazacyclotetradecane-N,N' , N' ' ,N' ' ' - tetraacetic acid; and 1, 5 , 9 , 13-tetraazacyclohexadecane- N,N' ,N' ' ,N' '^• -tetraacetic acid. Examples of linear or branched chelating moieties include those derived from compounds such as ethylenediaminetetraacetic acid (herein abbreviated as EDTA) and diethylenetriamine- pentaacetic acid (herein abbreviated as DTPA) .

Chelating moieties having carboxylic acid groups, such as the above examples may be derivatized to convert one or more carboxylic acid groups to amide groups. Synthetic methods for the preparation of chelating agents useful in the practice of the invention are known. See for example, Cox et al . , J. Chem . Soc . , Perkin Trans . 1: 2567-76 (1990) and Moi et al . , J. Am. Chem . Soc . 110: 6266-7 (1988).

The present invention also contemplates chelators that bind soft acid metal cations. Such chelators include polypeptides such as metallothionein and metallothionein fragments, porphyrins, amine- terminating bifunctional chelating reagents and other molecules that use nitrogen and/or thiol ligands (e.g. N₂S₂; N₃S ligands) to bind soft acid metal cations such as rhenium-188, cadmium, silver, mercury, copper, and zinc. A "soft acid metal cation" is one which preferentially binds with a nitrogen or sulfur containing ligand, especially a thiol, rather than an oxygen containing ligand.

Many short peptides can be made and are contemplated for use in this invention. In fact, virtually all combinations of amino acids that are shorter than 400 amino acid residues long could be employed for some purposes as a linker. Preferred are short polypeptides of less than about 50 amino acid residues, and most preferably less than ten residues. Especially preferred are peptides that contain a basic amino acid such as lysine, which, for some nuclides, can balance out a negatively charged chelate.

D-amino acids are preferred for chelate conjugates having good serum stability. Peptide bonds formed from the D-amino acids are much less susceptible to proteolysis, and chelate conjugates made from D-amino acids are longer lived in the circulation (i.e. have longer "body residence" times) . Also, chelate conjugates formed from D-amino acids are more resistant to biotinidase activity.

Examples of biotin-polypeptide-chelator chelate conjugates include:

biotin-D-Phe-D-Lys-NH₂

(I) NH-CO-DOTA; biotin-D-Lys-NH₂ (II) NH-CO-DOTA; biotin-D-Ser-D-Lys-NH₂

(III) NH-CO-DOTA; biotin-D-Lys-NH_:

(IV) NH-CO-DOTA; and biotin-D-Phe-D-Phe-D-Lys-NH₂ (V) NH-CO-DOTA.

In some cases a peptide can be used as a combined chelator and linker. Among the useful polypeptides in this context are metallothioneins and peptide fragments of metallothioneins such as the metallothionein C- terminal fragment KCTCCA, and glutathione derivatives. Metallothioneins are described in Metallothioneins : Proceedings of the First International Meeting on Metallothionein and Other Low Molecular Weight Metal - Binding Proteins, Zurich, July 17-22, 1978, p. 46-92, ed. by Kagi and Norberg, Birkhause Verlag Basel, (1979) which is incorporated herein by reference. Metallothionein fragments are preferred in the practice of this invention as are functionally similar polypeptides having at least about six amino acid residues .

Metallothioneins, their peptide fragments, and glutathione derivatives can chelate a wide variety of metal ions with high affinity. Because the sulfhydryl moieties in these molecules are bound to metal ions, they are generally not available to serve as functional groups for direct conjugation to biotin or indirect conjugation through a linker, but other groups, such as -NH₂, -OH and -C00H groups are available for this purpose .

Metallothioneins, metallothionein fragments, and glutathione derivatives can be covalently conjugated to targeting proteins using reagents and methods which utilize these groups, while essentially not interfering in the polypeptide ' s metal -binding capability.

A ine-terminating bifunctional chelating reagents (BFC) contain pendant thiol and amine groups which are suitably disposed to tightly bind radioactive metals such as ¹⁸⁶Re, ¹⁸Re, ^mAg, and ⁶⁷Cu . Conjugation of a BFC to a carbohydrate linker is achieved through an amine or hydrazine function on the BFC, which can respectively form an imine Schiff base) or hydrazone linkage to an aldehyde or ketone function on the carbohydrate after its oxidation. Imine linkages can be stabilized by reduction with a reducing agent such as sodium cyanoborohydride . During the conjugation step the thiol group of the chelator moiety is masked as a thiol ester or disulfide, and is deprotected after the preparation of the chelate conjugate.

The BFCs can be described by the general structures la, lb, and Ic:

In general structure la, X is CH, or X and Z taken together can be CO; Y is CR⁴R⁵, CH₂CR⁴R⁵ or (CH₂)₂CR⁴R⁵ where R⁴ and R⁵ are the same or different and are selected from the group consisting of hydrogen, C, to C_2ϋ straight chain, branched or cyclic alkyl, optionally substituted with hydroxy, alkyloxy, nitro, halogen and the like, C₄ to C₂₀ homoaryl or heteroarylaryl , heteroaryl being one or more aromatic rings containing one or two oxygen, nitrogen or sulfur atoms, the aryl group being optionally substituted with hydroxy, alkyloxy, nitro, halogen and the like; Z can be any group capable of reacting with an oxidized carbohydrate, or Z can be H; R¹ is a thiol protecting group; R² and R³ can be the same or different, and each is selected from the group consisting of hydrogen, C, to C₂₍₎ straight chain, branched or cyclic alkyl, optionally substituted with hydroxy, alkyloxy, nitro, halogen and the like, C₄ to C_2ϋ homoaryl or heteroarylaryl, heteroaryl being one or more aromatic rings containing one or two oxygen, nitrogen or sulfur atoms, the aryl group being optionally substituted with hydroxy, alkyloxy, nitro, halogen and the like, where the alkyl and aryl groups comprise metal -ligating moieties selected from the group consisting of sulfhydryl, amine and carboxylic acid or their protected derivatives; R² and R³ also can be any group capable of reacting with oxidized carbohydrate groups such as amines, hydrazides and the like. When the linker is not a carbohydrate, other reactions can be used that lead to a covalent bond with the BFC, and which are known to the skilled artisan.

In formula (lb) D is H or CH₂SR' ; E can be any group capable of reacting and/or complexing with the linker; R¹ is a thiol protecting group, and m is 0, l, 2, or 3. In formula (Ic) Q can be any group capable of reacting and/or complexing with the linker; R¹ is a thiol protecting group; and each n independently is 2 or 3. Representative examples of la, lb, and Ic are provided in the Examples below. In some of these examples, the linker-binding group is shown as, but not limited to, a hydrazide. Only the thiol-protected versions of the structures are shown (with ' R' being acyl , benzoyl or 2-thiopyridyl) , although metal - complexation will involve thiol-deprotected complexes. The synthesis of the BFCs can be achieved by methods that are well known in the art.

The thiol protecting group used in the BFC can be any organic or inorganic group which is readily removed to regenerate the free sulfhydryl in the presence of linker. Examples of suitable protecting groups include thiol esters, thiocarbamates and disulfides. In a preferred embodiment the thiol protecting group is a benzoate thioester. Those skilled in the art are familiar with procedures for protecting and deprotecting thiol groups. For example, benzoate thioesters may be deprotected under mild and selective conditions using hydroxylamine . The detection or therapeutic agent chelate conjugate binds pre-localized antibody- conjugate by virtue of the avidin-biotin binding reaction between an avidin and the biotin of the detection or therapeutic agent chelate conjugate. "Biotin" as a binding partner, can be, inter alia , modified by the addition of one or more addends, usually through its free carboxyl residue. Useful biotin derivatives include active esters, amines, hydrazides and thiol groups that are coupled with a complimentary reactive group such as an amine, an acyl or alkyl group, a carbonyl group, an alkyl halide or a Michael-type acceptor on the appended compound or polymer. Some biotin derivatives, e.g. NHS-LC-biotin, are commercially available and can be attached to a bifunctional chelator such as ITC-Bz-DTPA. Shih et al . , in U.S. patent No. 5,057,313, hereby incorporated in its entirety by reference, describes reactions for forming conjugates between amino and carboxy functionalized molecules, and between amino or hydrazide groups and aldehyde or ketone carbonyls resulting from oxidative cleavage of carbohydrates. These reactions are suitable for conjugating biotin with a linker and for conjugating biotin directly with a chelator.

Unfortunately, a biotin-amide linkage has been found to be unstable, even in vitro in the presence of human serum. D-amino acids and D-amino acid peptides can form more stable linkages between biotin and chelators and are preferred alternatives.

The advantages of incorporating one or more D- amino acid containing bonds between chelator and biotin can be summarized as follows : 1) The peptide bond between biotin and a D-amino acid is stable towards biotinidase. This prevents undesired separation of biotin from the detection or therapeutic agent in human serum.

2) One or more D-amino acids can be used to link biotin and a chelator. The peptide ' s hydrophobicity can be adjusted for example, by increasing the number of phenylalanines or other hydrophobic residues within the linker to achieve a desired body residence time.

3) The number of chelators per biotin can be increased by coupling more chelators, through multiple amino acid residues, in the linker. The primary amino acid residues of lysine are a preferred embodiment in this context. By attaching one chelator per lysine residue, and incorporating multiple lysine residues in the biotin-peptide the specific activity of the chelate conjugate can be increased. 4) A biotinylated peptide can be synthesized easily by a manual solid phase technique or by an automated peptide synthesizer.

As used herein "avidin" includes all of its biological forms either in their natural states or in their modified forms. Avidin, found in egg whites, has a very high binding affinity for biotin, which is a B- complex vitamin (Wilcheck et al., Anal. Bioche , 171:1, 1988) . Streptavidin, derived from Streptomyces avidinii , is similar to avidin, but has lower nonspecific tissue binding, and therefore often is used in place of avidin. Modified forms of avidin which have been treated to remove the protein's carbohydrate residues ( "deglycosylated avidin"), and/or its highly basic charge ("neutral avidin"), for example, also are useful in the invention. Both avidin and streptavidin have a tetravalency for biotin, thus permitting amplification when the former bind to biotin. Four detection or therapeutic agents, such as nuclides, can be attached to each targeting protein by the methods of the present invention.

Among the radionuclides and labels useful in the methods of the present invention, gamma-emitters, positron-emitters, x-ray emitters and fluorescence- emitters are suitable for localization and/or therapy, while beta and alpha-emitters and electron and neutron- capturing agents, such as boron and uranium, also can be used for therapy. In addition to nuclides, cytotoxic drugs that can become chelated are known to those skilled in the art and are useful for the present invention. Suitable compounds can be found in compendia of drugs and toxins, such as the Merck Index, Goodman and Gilman, and the like, and in the references cited above. Useful diagnostic radionuclides include Ruthenium- 95, Ruthenium-97, Ruthenium- 103 , Ruthenium-105 , Technetium-99m, Mercury-197, Gallium-67, Gallium-68, Osmium-191, Indium-Ill, Indium-113 and Lead-203. Useful therapeutic radionuclides include Antimony- 119, Actιmum-225, Rhemum-186, Rhenιum-188, Rhenιum-189, Sιlver-111, Platmum-197, Palladιum-103 , Palladium- 109, Copper-67, Yttrιum-90, Scandιum-47, Samarιum-153 , Lutetιum-177, Rhodιum-105, Praseodymιum-142 ,

Praseodymium- 14 , Terbιum-161, Holmιum-166, Lead-212, Bιsmuth-212, Gold-198 and Gold-199.

A chelate is formed between the chelator and a chelatable detection or therapeutic agent such as a nuclide, using methods known in the art. Typically, the chelator is dissolved in a buffered aqueous medium and a purified radionuclide is added The pH may be selected to optimize chelate formation. For example, when chelation s achieved by acetate groups binding to the metal ion (as is the case for various acetic acid compounds), the pH may be adjusted (using, for example, aqueous tetramethylammonium acetate) , to obtain a pH of about 3 to about 6, and more preferably about 5 to provide a preponderance of ionized carboxylate (-C00-) groups, and thereby yield a chelating species which is anionic. Furthermore, the reaction mixture temperature may be adjusted, for example to 37°C for 30 minutes, to accelerate the chelation reaction After a period of time or upon completion of the reaction, an excess of an appropriate quenching agent, such as DTPA

(diethylenetπaminepentaacetic acid) may be added. The quenching agent acts to form anionic chelates with any radionuclide not yet chelated by the chelating agent. The resulting reaction mixture may then be purified to a high specific activity.

In order to exploit fully the pretargeting method, the chelate should have the highest specific activity possible. Purification is preferably carried out by chromatography and more preferably by ion exchange chromatography. A number of chromatographic techniques are known n the art and can be used. Methods for purifying chelate prior to conjugation with protein are known, as typified by Li et al . , Bioconj uga te Chem . 5: 101-4 (1994), which is herein incorporated in its entirety by reference. The preferred type of chromatographic material is determined by the properties of the chelator. When the chelator has a net charge near zero it becomes a cation upon chelate formation with a metallic nuclide and preferably is purified by cation exchange chromatography, and most preferably with DEAE cellulose prepared in an acetate form. In a preferred embodiment a DOTA-peptide -biotin chelator conjugate having a net charge of (-3) at physiological pH is used to chelate a metal (+3) cation and form a neutral chelate conjugate. The metal - containing chelate conjugate is purified from non- metal -containing chelator by anion exchange chromatography .

For optimizing the body residence time of a radionuclide conjugate, a skilled artisan can estimate what size of polymer and what kinds of charges are most suitable for the linker, based on the predicted size, i.e. the effective molecular radius, of the completed chelate conjugate. Alternately, fractional clearances can be determined by experimentation.

The term "effective molecular radius" refers to what size a molecule displays in solution. This value easily is determined by a skilled artisan. A preferred method to determine the effective molecular radius of a molecule is quantitative gel chromatography as taught by Chang et al . in Kidney Intern . 8: 212-18 (1975) and in Biophys . J. 15: 887-906 (1975).

Uncharged polymers such as dextran have a fractional clearance of 1 when they have an effective molecular radius of about 18 angstroms or less. Such polymers have a fractional clearance of about 0 when they have an effective molecular radius of about 44 angstroms or more. For purposes of the present invention, a non-charged polymer such as polyethylene glycol or dextran which is not reabsorbed by the kidney can be a linker. In this case the biotin-linker-chelator-nuclide conjugate should have an effective molecular radius of more than 18 angstroms in order to prolong its body residence time, compared to a small molecule that is filtered without restriction by the kidney. The chelate conjugate should, at the same time, have an effective molecular radius of less than about 44 angstroms in order that the kidney gradually clear it from blood. If the effective molecular radius is significantly more than 44 angstroms then the body residence time will be too long and a mechanism different from kidney clearance may dispose of the chelate conjugate.

The selected linker can have one or more negative charges. If a polyanionic linker, such as, for example, dextran sulfate is used, then the chelate conjugate size should be smaller to obtain the same degree of fractional clearance by the kidney. For example, a dextran polymer- based chelate conjugate having an effective molecular radius of 18 angstroms will have a fractional clearance of about 1 (quickly removed by passing through the kidney) , but a dextran sulfate-based chelate conjugate having an effective molecular radius of 18 angstroms will have a fractional clearance closer to 0.5, and will be removed from the blood more slowly. The selected linker can have one or more positive charges. A positively charged linker such as a short polypeptide having lysine and/or arginine residues will effect more rapid kidney clearance of the chelate conjugate . In practice, the size and charge of the complete conjugate made from biotin, linker, chelator, and chelated detection or therapeutic agent is considered when estimating fractional clearance. However, when biotin is used with the preferred chelator DOTA or other small chelator, the linker may dominate the chelate conjugate's blood residence time.

For a neutrally charged chelate conjugate, the linker should be chosen such that the chelate conjugate's effective molecular radius is between about 18 and about 42 angstroms. For anionic chelate conjugates, the effective molecular radius should be smaller, depending on how many excess negative charges are in the chelate conjugate. For very anionic chelate conjugates, the effective molecular radius should be between about 15 and about 32 angstroms. When the chelate conjugate is to have a net positive charge, the linker should be chosen to give a slightly greater effective molecular radius of the chelate conjugate in comparison to that for neutrally charged chelate conjugates.

Blood residence time also can be controlled by altering the linker's hydrophobicity . Polypeptides are preferred for this use. One or more hydrophobic amino acid residues such as phenylalanine, tryptophan, tyrosine, leucine, isoleucine, or valine in the peptide linker will increase the hydrophobicity of the chelate conjugate. Adding one or more hydrophobic residues to the chelate conjugate can both alter the effective molecular radius of the chelate conjugate and increase the chances that the chelate conjugate will interact with other proteins in the blood.

While not wishing to be bound by a particular theory of the invention, it is believed that one mechanism for prolonging the blood residence time of small chelate conjugates that contain hydrophobic residue (s) is the ability of the chelate conjugate to associate with serum albumin and escape filtration by the kidney. In this context, a chelate conjugate having one or more hydrophobic residues, such as for example, a long aliphatic chain, a phenyl ring, an imidazole, and the like, may have affinity for the hydrophobic binding site of serum albumin. Alternative chelate conjugates can be tested for their relative blood residence times by comparing how well they associate with serum albumin. A carrier that has hydrophobic residue (s) is particularly suited for the formation of a detection or therapeutic chelate conjugate that provides a detection or delivery agent over a longer time period.

The optimum body residence time for a given set of conditions can be determined experimentally. A factor in the optimum body residence time however, when using a nuclide detection or therapeutic agent, is the half-life of the isotope used. A very short half-life nuclide works best in combination with chelate conjugates that are cleared more rapidly by the kidney. A chelate conjugate that exhibits a small effective molecular radius and which is not very hydrophobic will exhibit a short body residence time.

Linkers that cause a fractional clearance of near 0 or that cause the chelate conjugate to bind to serum protein are more preferred for long half -life isotopes. This kind of reagent would behave like a time-release capsule and slowly localize at the target site.

The use of a hydrophobic linker can prolong the serum half life of the detection or therapeutic chelate conjugate. Consequently, an isotope of shorter tissue range (lower decay energy) and longer half -life can be employed to spare blood and marrow cells while delivering a sustained dose- level to a target. The methodology used in this approach, in essence, allows the choice of possible radiotherapeutic isotopes to be expanded considerably .

A physiological solution of the biotin-chelator conjugate is advantageously metered into sterile vials, e.g., at a unit dosage of about 0.1 - 500 mg of the chelator conjugate, and the vials are either stoppered, sealed and stored at low temperature, or lyophilized, stoppered, sealed and stored. The vial contents can be reconstituted with a solution containing the metal ion to be chelated. During use, the detection or therapeutic biotin- chelate conjugate is injected parenterally after a pre- targeted antibody conjugate has had time to localize to a targeted cell, tissue or pathogen. Alternatively, the biotin-chelate detection or therapeutic conjugate is injected after the clearing and localizing agent in a 3 - step method. Parenteral administration comprises intravenous, in t r aar t e r i al , int r ap 1 eural , intraperitoneal, intrathecal, subcutaneous and perfusion administration.

The improved method of the present invention can enhance detection (either by internal procedures or by external imaging) and/or enhance treatment of lesions, including cancers, infectious diseases, cardiovascular diseases and other pathological conditions as part of a pre-targeting procedure. Such pre-targeting procedures utilize targeting protein to locate a biotin detection or therapeutic agent to a target site. The targeting protein comprises a protein, peptide, polypeptide, glycoprotein, lipoprotein, or the like, e.g. hormone, ly phokine, growth factor, albumin, cytokine , enzyme, immune modulator, receptor protein, antibody and antibody fragment . Internal detection procedures in the context of the invention comprise intraoperative, intravascular or endoscopic, including laparoscopic, techniques, both surgically invasive and non-invasive. Examples of appropriate applications are provided in the above- referenced and incorporated Goldenberg patents and applications .

The cancer states include carcinomas, melanomas, sarcomas, neuroblastomas, leuke ias, lymphomas, gliomas and myelomas. The infectious diseases include those caused by invading microbes or parasites. As used herein, "microbe" denotes virus, bacteria, rickettsia, mycoplasma, protozoa, fungi and like microorganisms, "parasite" denotes infectious, generally microscopic or very small multicellular invertebrates, or ova or juvenile forms thereof, which are susceptible to antibody- induced clearance or lytic or phagocytic destruction, e.g., malarial parasites, spirochetes and the like, including helminths, while "infectious agent" or "pathogen" denotes both microbes and parasites.

The improved pre-targeting methods of the invention increase the sensitivity of the above recited detection procedures and increases the efficacy of the above recited therapeutic procedures.

Without further elaboration, it is believed that one skilled in the art can, from the preceding description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. In the following examples, all temperatures are set forth uncorrected in degrees Celsius; unless otherwise indicated, all parts and percentages are by weight.

Accordingly, these embodiments constitute improved methods and reagents for detection and therapy of cancer and other pathological conditions.

EXAMPLES

Example 1 - Coupling polypeptide and carbohydrate linkers to Biotin

A- To polypeptide via Lysine.

A polypeptide having at least one free amino group at a concentration of 10 mg/ml in a borate buffer, 0.1M, pH 8.5 is mixed with a 10 fold molar excess of an activated sulfosuccini ide ester of D-biotin. The reaction solution is stirred for 16 hours and kept at a temperature of 25°C. At the end of the reaction period, the modified polypeptide is separated from unbound biotin and other low molecular weight contaminants by size- exclusion or ion exchange chromatography. B- To polypeptide via Cvsteine.

A polypeptide at a concentration of 10 mg/ml, containing at least one free cysteine residue in phosphate buffer, pH 7.5 , is mixed with a 10 fold molar excess of biotin-maleimide (N-biotinyl-N- [6-maleimido hexanoyl] hydrazide) (Sigma Chem. Co.) . A DMSO co-solvent is added up to a 20% concentration to facilitate reactant solubility. The reaction solution is stirred for 1 hour at a temperature of about 25°C. At the end of the reaction period, the biotinylated polypeptide is separated from unbound biotin and other low molecular weight contaminants chro atographically.

C- To oxidized dextran

Dextran at a concentration of 1 mg/ml is treated with sodium metaperiodate to a final concentration of 0.03 mg/ml in phosphate buffered saline at room temperature for 4 hours. The oxidized dextran is purified from sodium metaperiodate by size -exclusion chromatography in phosphate buffer, 0.1 M, pH 7.5. The oxidized dextran (1 mg/ml) is reacted with biotin-hydrazide (Pierce Chemical Co.) in 0.1 M phosphate buffer, pH 7.5 for 6 hours at 37°C. After coupling, the formed hydrazone is reduced by the addition of sodium cyanoborohydride with stirring overnight. The biotinylated dextran is purified by size- exclusion chromatography on a G-25 Sephadex column.

Example 2- Preparation of Biotin-D-Phe-D-Lys-DOTA

N-hydroxysuccinimide ester of DOTA (1,4,7,10- tetraazacyclododecane N, N , N , N ^"-tetraacetic acid) is prepared by modification of a procedure described by Lewis et al . , Bioconjugate Chem. 5: 565-76 (1994) . Freshly prepared EDC (3 -ethyl-3 - [3 - (dimethyl - amino) propyl] carbodiimide) in H₂0 (12.25 g in 40μl, 64 μmol) is added to a solution of 60 mg (128 μmol) of trisodium DOTA (Parish Chemicals) and 27.7 mg (128 μmol) of sulfo-NHS (N-hydroxysuccinimide) in 960 μL of H₂0 at 4°C. The reaction mixture is stirred at 4°C for 30 minutes. The theoretical concentration of active ester in the reaction mixture is 64 mM.

The peptide biotin-D-Phe-D-Lys-NH₂ (16.6 mg, 32 μmol) is added to the above solution and the pH is adjusted to 8.5 with 6 M NaOH. The solution is stirred for 18 hours at 4°C. The product is isolated by purification on a preparative reverse-phase C18 column. Example 3 - Chelating Gadolinium into biotin-peptide-DOTA for MRI

A solution of biotinyl-peptide-DOTA is treated with a 0.01-1 mol solution of gadolinium cation in acetate buffer at a pH of 5 for 3 hours at 37°C. The metallated biotin-peptide-DOTA is separated from unincorporated metal chromatographically .

Example 4 - Construction of Chelate conjugates that contain a D-amino acid The peptides, 1) biotin- (D) -Lys-NH₂, 2) biotin- (D) - Phe- (D) -Lys-NH₂ and 3) biotin- (D) -Phe- (D) -Phe- (D) -Lys-NH₂ were synthesized manually by standard Fmoc procedures on a Rink resin (Advanced ChemTech; 0.56 mmol/g) according to published procedures. (1) Fmoc amino acids (advanced ChemTech) were assembled on Rink resin using HOBT and a DIC coupling procedure. After the last amino acid was attached, (+) -biotin was coupled using HBTU, HOBT and DIPEA 0.15 M in DNC except for DIPEA (0.75 M) . A minimum amount of N-methyl pyrrolidone was added to aid in solubilization . The peptides were cleaved from the resin with a solution of 91 % TFA, 4.5 % H₂0 and 4.5 % ethylmethyl sulfide. Subsequent to removal of the solvents, the crude peptides were treated with 0.3- 0.5x molar excess of ITC-Bz-DTPA in H₂0 at pH 8.6 at 37" C until all of the ITC-Bz-DTPA was consumed as monitored on an analytical HPLC column. The peptide bio- (D) -Phe- (D) - Phe- (D) -Lys-NH₂ required acetonitrile for solubilization in H₂0. Biotinylated peptide complexes and the unreacted peptides were isolated following purification on a preparative C-18 HPLC column using the gradient 0-70 % B in 40 min at 75 mL/min where solvent A was 0.1 % TFA in H₂0 and solvent B was 0.1 % TFA in 90 % acetonitrile. Yield: peptides 59-64 %, peptide-chelators 68-76 %. Mass spectrum analysis: MH+ : peptide (2) : 519, peptide (3) : 666, bio-pep-DTPA (1) : 912, bio-pep-DTPA (2) : 1059, bio- pep-DTPA 13): 1206. The structure of biotin-D-Lys-DTPA is shown below.

Chelate Formation From Radionuclide and Chelator:

InCl₃ or YC1₃, 200 μCi , in 0.05 M HC1 was diluted with 3 volumes of 0.5 M sodium acetate, pH 6. The chelator (8 X 10^"5 M) in 15 μL of 0.5 M sodium acetate, pH 6 was added and incubated at room temperature for 1 hour. Approximately 0.2 μCi of the sample was applied to ITLC (Gelman sciences) pre-spotted with 5 μL 1 % HSA and developed in 5:2:1 H₂0 : ethanol : ammonium hydroxide solvent system to determine the extent of labeling. The formed chelate moved with the solvent front. Ability to bind to streptavidin was demonstrated by treating the formed chelate with streptavidin. A shift to higher molecular weight region where streptavidin eluted on a size exclusion HPLC column indicated complete binding to streptavidin .

Example 8 - Purification of Yttrium- 90-Biotin-peptide- DOTA by Ion Exchange Chromatography

A sample of Yttrium-90-biotin-peptide-chelate is applied to an anion exchange resin, equilibrated in acetate buffer, pH 5.5. The neutral radiolabelled conjugate is eluted with water, in carrier-free form from the triply negatively charged free form (uncomplexed chelator) conjugate. It will be apparent to those skilled in the art that various modifications and variations can be made to the processes and compositions of this invention. Thus, it is intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.

Claims

WHAT IS CLAIMED IS:

1. A method of detecting or treating a target cell, tissue or pathogen in a patient, the method comprising:

(a) pre-targeting said target cell, tissue, or pathogen with avidin, using a targeting protein that specifically binds a marker substance on said target cell, tissue, or pathogen and to which avidin is bound either directly as an avidin-targeting protein conjugate, or indirectly via non-covalent binding of avidin to a biotin- targeting protein conjugate;

(b) parenterally injecting a detection or therapeutic composition comprising a biotin-chelator conjugate and a chelatable metal ion detection or therapeutic agent, and allowing the composition to accrete at the targeted cell, tissue, or pathogen; wherein said chelate conjugate is

1) purified by chromatography after chelation of said metal ion, or

2) further comprises a blood transit -modifying linker or addend that is covalently bound in said chelate conjugate, or both; and

(c) using the detection or therapeutic agent to detect or treat the targeted cell, tissue, or pathogen .

2. The method of claim 1, wherein said chromatography is anion exchange chromatography.

3. The method of claim 1, wherein said blood transit- modifying linker or addend is selected from the group consisting of polyethylene glycol, polypeptide, and dextran.

4. The method of claim 1, wherein said blood transit- modifying linker or addend has a fractional clearance by the kidney of less than 1.

5. The method of claim 1 wherein said blood transit- modifying linker or addend has an effective molecular radius of between 15 and 40 angstroms.

6. The method of claim 1, wherein said metal ion detection or therapeutic agent is an electron- or neutron- capturing agent.

7. The method of claim 1, wherein the metal of said metal ion therapeutic agent is selected from the group consisting of Antimony-119 , Actinium-225 , Rhenium-186, Rhenium-188, Rhenium-189, Silver-Ill, Platinum-197, Palladium-103 , Palladium-109 , Copper-67, Yttrium-90, Scandium-47, Samarium-153 , Lutetium-177, Rhodium-105, Praseodymium- 142 , Praseodymium-143 , Terbium-161, Holmium- 166, Lead-212, Bismuth-212, Gold-198 and Gold-199.

8. The method of claim 1, wherein the biotin and chelator in said chelate conjugate are joined through a linker that includes one or more D-amino acids.

9. A sterile injectable composition for human use comprising a detection or therapeutic composition comprised of

(i) a biotin-chelator conjugate and a chelatable metal ion therapeutic or detection agent, or (ii) a biotin-chelator conjugate having covalently bound thereto a blood transit-modifying linker or addend, and a chelatable metal ion therapeutic or detection agent .

10. A sterile injectable composition as claimed in claim 9, wherein the linkage between biotin and chelator includes one or more D-amino acids.

11. The method of claim 1, wherein the chelating agent is DOTA.

12. The method of claim 1, wherein said target tissue is cancerous, cardiovascular, infectious or inflammatory

13. The method of claim 1, wherein the method is external imaging or internal detection.

14. The method of claim 13, wherein said internal detection is during an operative, mtravascular or endoscopic procedure.

15. The method of claim 1, wherein said detection agent is a radionuclide or MRI enhancing agent

16. The method of claim 15, wherein the radionuclide or label is a gamma-, positron-, x-ray or fluorescence- emitter .

17. A method of detecting or treating a target cell, tissue or pathogen in a patient, the method comprising-

(a) pre- targeting said target cell, tissue, or pathogen with avidin, using a targeting protein that specifically binds a marker substance on said target cell, tissue, or pathogen and to which avidin is bound either directly as an avidin- argeting protein conjugate, or indirectly v a non-covalent binding of avidin to a biotin-target g protein conjugate;

(b) parenterally injecting a detection or therapeutic composition comprising a biot -multiple chelate conjugate and a chelatable metal ion detection or therapeutic agent, and allowing the composition to accrete at the targeted cell, tissue, or pathogen; wherein said biotm-multiple chelate conjugate comprises at least two chelates, and

(c) using the detection or therapeutic agent to detect or treat the targeted cell, tissue, or pathogen.

18. The method of claim 17, wherein said multiple chelate conjugate is a metallothionein.

19. The method of claim 17, wherein said multiple chelate conjugate comprises a synthetic polymer.