CA2471817A1 - Labeling targeting agents with gallium-68 and gallium-67 - Google Patents

Abstract

A method and compositions are described for labeling a targeting agent with Ga- 68, in which eluate from an acid-eluted Ge-68/Ga-68 generator is combined wi th a macrocycle-containing targetable agent. The labeling method and compositio ns disclosed ensure that a simple elution of gallium-68, taken directly from a generator, can be used without further manipulation to quantitatively label a macrocycle-containing targetable agent. The Ga-68 labeled targeting agent so produced is useful with specific targeting agents, and is most especially useful in a pretargeting method for positron emission tomographic detection.

Description

Label~~g Targeting Agents With ~~~liunn-b8 and Gallium-67 CrOSs-reference to Related Appiications This application claims priority from IJ.S. Provisional Serial No. 60/342,104, filed December 26, 2001, which iS incorporated herein in its entirety.
STATEMENT R~GARDIi\1G F~~?E~~A~.L~ SPONSO~D RESEARCH 41~
L)EVELOP1VIENT
Part of the w~rl~ performed during development of this inventioy utilized U.S.
Government funds. The U,S. Governirierit has certain rights in this invention.
Part of the word described in this invention was supported by a FIBS grant 1243 CA-X3424-Ol from the National institutes of I~ealth.
FIELD OF THE INVFaNT~ON
The present invention relates to improved methods for labeling targetiri~
agents, particularly with Ga-68, and the labeied targeting agents produced thereby, as well as their uses.
Bac~GRO~1N~ Q~ TAE INVENTIQN
Specific targeting agents bearing a detectable moiety offer the potential for earlier diagi~iosis of disease, if an increased amount of the tar~etirig-detectable conjugate is localized to a greater extent in tissue to be imaged compared to bacl~groui~d tissues. In practice, detectable agent in background tissue needs to be minimized while detectable agent in target tissue needy to be maximized. radioactive nuclides are preferred detectable agents and nuclides such as Tc-99m and ln-111 are widely used for scintigraphy and single photon emission computed tomography. However, there are limitations on the sensitivity of these detection modalities. For instance, even for specific targeting of tumor tissue in a patient, tumor nodules of less than 1 centimeter diameter may be very difficult to detect. A superior imaging modality is offered by positron emission tomagraphy (PET), which offers the promise of ~ dramatic increase in sensitivity, and therefore the ability to detect disease at an earlier stage.
What has mainly hampered development of PET into a routinely applied clinical diagnostic modality are the inadequate technidues related to radiolabeling of specific targeting agents with useful PET nuclides.
There are about 2Q nuclides of theoretical utility for PET in that they have positron decay and ~ suitable half life (minutes or longer). In practice, most of these nuclides are really unsuitable for a variety of reasons, including, several separate .reasons in the case of many of them. these reasons include but are not limited to, availability and cost of parent yuclides, nuclide preparation issues related to target preparation and bombardment, dandling and shipment of the nuclide, cyclotron size and energy etc., chemical separation issues, radiolabeling issues including on-site radiolabeling issues, and decay energy and properties of the PET nuclides themselves which often include other beta and/or gamma decay. These extensive and severe practical matters can essentially preclude most nuclides from consideration as viable PET agents, and the two most commonly considered nuclides for PET, fluorine-18 and gallium-68, retain tl~e best combination of features, and fewest problems among PET nuclides.
Galliuy-68 (Ga-68) has two great advantages over F-18 when considering both nuclides for FET usage. First, it is available frog a generator, which makes it available on site by a simple 'milking' process that can be carried out daily, or even hourly. This makes Ga-68 independent of the need fox a nearby cyclotron, as is needed for F-1 S.
Second, it is a radiometal and can be complexed by suitable chelating agents. In contradistinction, fluorine is mainly available as F-18 fluoride ion in aqueous solution and this must be taken into a dry organic environment prior to chemical manipulations to covalently attach it to targeting agents of choice, The half lives of both nuclides axe short (F-18 ~ 2 h; Ga-6$ ~ 68 minutes) and the intense positron energy emitted makes chemical manipulations fax from trivial and possibly e~trernely hazardous to technical personnel.
Given the chemistry needed for F-1 ~ attachment to targeting agents, the radiochemical processes can anl~ be carried out in custom-designed dedicated facilities, al~td these facilities must also be located near a e~clotron that produces the F-18 raw ~natexial.
Gallium-68 does not suffer from these drawbacks. It is available from a long-lived parent nuclide (germanium-68; half life 288 days) that can be adsorbed to various solid phases, from which the Ga-68 can then be selectively eluted. Thus, a Ga-68 generator can be fabricated, and several have been described (Ambe, 1988; Greene and '~'ucker, 1961; f,oc'h, 1980; Lewis 1981; I~anrahan, 1982; McElvaney, 1983; Neirinckx, 1980).
The most developed generator is one based on adsorption of the parent Ge-68 to a stannic oxide bed {Loc'h, 1980), from which the Ga-68 is eluted with dilute hydrochloric acid.
Alternate generators have utilized alumina as adsorbent and EFTA to elute the galliuru-68, which presents significant problems in conversion o~ Ga-6$-EFTA complex to other Species, given the 6$-minute half life of the nuclide. Tl~e invention described herein is preferably directed toward ger~er~tors of the i'trst type that can be eluted with acid or salt solutions, rather than with chelates such as EDTA.
Given the availability of Ga-6$ generators over many years, it is very surprising that no Ga-68-labeled targeting agents have been developed past the point o~rese~rch article material, and toward routine clinical use. It is one object o~the present invention to overcome the radiolabeling p~'oblerr~s that have prevented routine clinical pre~ar~tian of Ga=68- labeled t~t',~eting agents, Gallium is an ~r.~pl~oteric element, which is tQ say that it displays both basic and acidic reactive properties, and this considerably complicates manipulation o~radiogallium. In addiXio~, in dilute solution gallium tents to ~prrr~ non- ox poorly-chelated ~he~ical species. TI~e short-lived Ga-6~ eluted carries free from a generator is present in extremely diiute solution, typically under one picomole (1Q-~Z moles) per milliCurie. ~t cani _th.~refoie be p~r~icularly prone to the ~orrnation of gailates and other species (I~natowich, I975; Kdlprathipanja and Hnatowich, 1977). This is particularly so as the pH is raised and hydroxy or aqua- ions tend to replace chloride ions in the immediate vicinity of the gallium ions.

Ge-6SlGa-68 genexators of the stannous oxide type are usually eluted with a 10-12 mL
portion of ultra-pure 1 N hydrochloric acid, providing the Ga-68 daughter in highly dilute farm and in the presence of a large amount of hydrochloric acid. Without ~
purification step, there is also the possibility of eluting other extraneous metal ions along with the Ga-68, and each of these, even in nsnomoiax aruounts, would be typically in 100-10,000 rxrolax excess to the Ga-6$. Arlianic stannates, can also be eluted which cari also carnplic~te carrier-free xadialabeling methods. Once the Ga-68 is obtained, there is then challenge to bind it to a targeting species, in light of ah the above potential prablerus, and this has been approached in several distinct ways.
First, sortie workers decided that the c~~'riex-free Ga-68 needed to be ruixed with cold galliuru to prevent problerus seen at high dilution, and sa cold gallium was added to Ga-68 eluate prior to admixing with the rrratexial to be Ga-68 radiolabeled (Schuhrrrachex, 1995; I~livenyi, 1998). This is cumbersome, and also precludes the preparation of high specific activity Ga-68-labeled species, since the cold added gallium trust also be bound by any chelate added during iabeling.
second approach relied an the use of the 'txar~scl~elator' acetylacetone (2,4-pentanedione) in large excess to bind to xadiogallium, essentially using it to out-compete the hydxoxy/actua ions present in the adueaus solution (Lee, 1997; Wu 1997).
S-Unfortunately, this approach is not useful clinically since acetylacetone is a neurological toxin, teratogen and possible rr~utagen.
A third approach described the evaporation of the Ga-68 eluate from the generator to dryness under a flow of inert gas (Sun, 1996). This was done to reyove the excess HCl and to allow the reconstitution of the Ga-68 in another medium. Qne variation of the method also called for the addition of acetylacetone to protect the Ga-68 while the drying process was continuing (Green, 1993; Tsang, 1993).
Finally, a fourth approach recommended addition of extra concentrated I-tCl to tl~e G~-6~
generator eluate, until the HCl was 61~ in concentration (Kung, 1990). Then, the Ga-68 in concentrated HCI, was extracted with diethyl ether and reduced to dryness under a stream of nitrogen.
The most advanced technology for clinical application and use Qf Ga-6$ was developed over a three year period, and was based Qn the evaporation of a ,reduced elution volume of Ga-68 eluate ill 1 ~T HCl (Goodwin, 1994). Prior to evaporation the Ga-d8 was eluted from the Ge-68/Ga-68 generator through an AG1~8 ion exchange filter, and then evaporated on a rotary evaporator, prior to being reconstituted in 10 mM ICI.
Use of Ga-68 carries with it the following concerns: 1 ) The Ga-68 has a half life of only 68 minutes, and therefore any methodology used should be fast. 2) danger to technical personnel is high since tl~e Ga-68 nuclide decays with positron emission at 511 keV
making the emergent gamma-rays very difficult to block even with thick (> one inch) lead shielding. 3) In ~ clinical scenario, the Ga-68 rr~ust be obtained sterile and pyrogen-free, and this along with 2) above creates a preference far a method in which manipulations are kept to a ~nini~nurr~. 4) Clinical technical staff have limited chemistry expertise, and are under constant time pressure to produce other unrelated agents during a normal day. They cannot be expected to perform intricate manipulations of the above types in order to effect a Ga-~8 labeling.
The above sut~mary of work over ~. ~0-year or so period clearly indicates a need in the art for a viable, rapid and simple method for Ga-Gg labeling of specific targeting agents.
~?isadvanta~es in the previous methods of Ga-68 labeling lave prevented routine adoption of the acid-eluted Ge-68/Ge-68 generators in clinical PET, and lave subsisted far over 20 years.
SUMIYI~RY OF THE ~NV)~NT~ON
It is an abject of the invention to provide a Ga-68 labeling method (also useful with gahium-67) that is fast, safe and does net require complicated cDemical manipulations, along with compositions useful in the practice of the method. L~evelopyent of such a new methodology enables the wider use of Ge-68/Ga-68 in-house generators, which have not been widely used before due to bath the inherent difficulties in Dandling gallium-C~, and the difficulties in producing useful imaging agents containing gallium-68 in ~
reasonable time-frame while using only simple rrzethodologies. In turn, the present invention while allowing the ready use of Ga-68 eluate in a clinical setting, will lead to the greater adoption of PST-based procedures for identification of diseases, and thereby, the earlier and more accurate ~Ietection of said diseases. Again, in turn, this should lead to better to better treatment options and greater cure rates.
In one aspect, the invention combines ~. method of eluting, with an ~Gidic solution, stannic oxide or titanium dioxide based, or similar, Ge-GBIGa-68 generator.
The galliuzn-C8 is eluted such that the entire av~il~ble yield of Ga-68 is ~~ssed directly into a vial containing ~ solution or a lyophilised preparation of the chel~te-targeting agent conjugate tl~~t is to be labeled. Using the methods and compositions of this invention it is now possible to effect an approximately 80-I00~/o incorporation of G~-68 into a chelate-targeting agent conjugate, such that the labeled material can be used, optionally, without further purificstio~ proceduxes.~ The chelate-targeting agent conjugate can be compounded into bits, forrt~nlated, stabilized end ready-to-use, to accept the Ga-68 froze the gei~ez~stor, ~s eluted in acidic solution.
Another embodiment of the invention is a composition comprising a targeting agent labeled with Ga-6~; wherein skid Ga-6$ associates with a cl~zelste-containing conjugate comprising a macrocyclic ring. Such macrocyclic rings are exemplified by 1,4;7-trig..zacyclonon.ne-N,N',N"-triacetic acid ~~T~~ and 1,f,7,1~-_$_ tetraa~acyclododecaue-N,N',N",N"'-tetraacetic acid. Such macrocyclic ring moieties are linked to a useful targeting agent, for example a peptide that can target a site of disease, either directly or indirectly. ~'he gahium-6~ compositions thus formed may be used in pOSlt~'Q~ enlls510n tomographic detection rr~ethQds (with or without preceding, concomitant, or subsequent therapy). An example of a useful disease specific targeting method, wherein the current compositions and methods invention find application is as agents useful in pre-targeting methods for positron emission tomographic detection (with or without preceding, concomitant, or subsequent therapy). In this embodiment, a multi-speci~le targeting agent having at least one binding site for a hapten and at least one binding site .for a diseased tissue is administe~e~ to a mammalian patient in need thereof, once this mufti-specific targeting agent has rnaximi~ed its accumulation at the site of disease and its levels in non-target tissues and ei~culatiQn have dropped to an acceptably low level, the composition containing the gallium-6~ imaging nuclide is given.
The patient and hi~~her disease may then be imaged using positron emission tornograpby at times fr. om 1 S minutes to eight hours post-administration of the gahium-68-containing hapten. The general ri~etl~acl of this 'p~'etargetiug' embodiment is described in US patent #
5,256,39 (Barbet et al., 193).
A~otl~er embodiment l~ a co~x~pQSition comprising a ~nacrocyc~ie-targeting agent conjugate dissolved in sufficient p~-neutral buffer to enable spbstantially complete incorporation of Ga-f 8 when admixed with a solution of Ga-68 in dilute acid.
Such buffers are exemplified by relatively concentrated solutions (0.1 to 5 M) of acetate salts at pH 5-~. Another embodiment comprises a kit containing sufficient macrocyclic chelate-conjugated targeting agent together with sufficient ammonium acetate buffer to bind an added acidic Ga-68 solution. The kit of the invention is formulated such that Buff dent neutralizing buffer, e.g. ammonium acetate, is present to substantially neutralize the excess acid in which the Ga-68 is added, allowing facile complexatic~n by of the added Ga-6~ by the macxocyelic chelate-conjugated targeting agent.
$RI~F ~ESCR~~~'~ON OF THE DiRAW)fNGS
Fig. 1 is a typical size-exclusion ~IPLC trace of the complex 679-IgG (anti-HSG) admixed with the xadiolabeled peptide Ga-6~-IlVIP 2A.1. The y-axis pints radioactivity against time (~-axis). ~i~ recovered Ga-6S elutes as the complex between the antibody and the Ga-68-labeled peptide, near 8 minutes retention time, at an apparent molecular weight of appr~~ilnately 160,000 laaltons. Free Ga-68 peptide would elute at around 14 minutes xetentien time.
>pETA.ILED D~SCRIPTIUN C?F TAE PR.>GFI~~>p E1Y~80~.?IIV~ENTS
Unless otherwise specified, the terms "a" or "an" mean "one or raoxe".
Any Ghelate to be used within this invention must be capable o~bindix~~ Ga-5$, preferably in very dilute solutipn, quickly, near quantitatively, and preferably irreversibly Aver a 1-4 l~ pe~'iod. Preferred chelate~ of the invention axe exemplified by xn~cracyclic derivatives such as "NOTA", which is [1,4,7-triazacyclononane-N,N',N"-triacetic acid]
or"~QTA' ~1,4,7,1p-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid].
Apreferred peptide bearing a NaTA residue, end that is useful in spedfic targeting is shown in Formula I. It has the Immunornedics code number llYtP 244. Specific targeting is affected by use of a mufti-specific pretargeting agent, typically a bispeci~c monoclonal antibody that recognizes with at least one arm a tynor-associated antigen, and with at least one other arm a hapten. ~n this case, the hapten is represented by the substituent termed HSG, for ~ histaminyl-succinyl-glycyi- moiety (Formula ~I). Air example of such bispecific antibody would be hMN-~ 4 x G79-F(ab')2, wherein hMN-14 represents a humanized flab' arnl of an anti-carcinoembryonic antigen (anti-CEO) antibody, and 6'~9 represents ~ Fab' arm of an antibody raised against the MSG hapten.
t~ preferred peptide bearing a ~OT~ residue, and that is useful in bispecific ~r~tibody prefargeting methodology is IIV~I" 241, and is shown in Formulae IIh Similarly to IIVIP
244, it comprises a tetrapeptide l~~ckbone substituted with two I~SG sub-units, but in this case G~~'ies the DQT~1 macrocyclic chelate. In this e~c~mple, the DOTS chelate is linked to the a~~ino-terminal alpha amino acid residue (in this example phenylaiauine) via one of its carboxyl groups, whereas tl~e NOTA xnacrocyGle of IN.1F 244 is linked to its peptide baekb~ne via a discrete isothiocyanato-benzyl group attached to a single carbon an the macrocyclic ring. For the purposes of the current invention the manner of the coupling of the macrocyclic chelate to the peptide backbone carrier, is not important, as long as said rnacrocyclic chelate, once conjugated, can stih bind gallium.

Particular solutions o~ the above, and similar, Peptides can be prepared such that a tin Axide- ox titanium dioxide-based Ge-68/Ga-68 generator call be diluted directly into such solutions to ~f~'ect a facile Ga-68 labeling and produce an ~ger~t that xe~uixes no further puxi~catior~ prior tp use. Useful peptidyl solutions are ~r~pared in an appropriate buffer, useful iz'~ the pPI ~-6 range, such as ~u ammonium acetate buffer o~
appropriate corlcex~tratlc~~ into which Ga-68 iu Q.5 to 1 normal hydrochloric acrd generator eluer~t is eluted directly.
Formula I. Structure of the peptide teraned ~P 244.
H()(~C' ~-IOOC
COOH
NH N~''~~~~D-Ala-~ys(HSG)-Tyx-Lys(HSG)-NHS
Formula ~I. Structure of the hist~mix~yl-succir~Yi-glYcYl (HSG) sub-unit moiety.

~~~~r N ~N~
Fo~mul~e III. Structures of the hist~m~r~yl-suceinyl-glycyl (HSG) sub-unit moiety, the DO'I'A sub-ul~it .moiety ~xld the peptide ~ucp~porating tie di-HSG and ~no~y-DOTA sub-units, termed IMP 241.
IMP 241: DUTA-Phe-Lys(I~SG)-D-Ty~-Lys(IISG)-NlI2 t~ooc~ ~ ~COOt-z DOTA
N N
N N o HOOC
Iu a detailed example, NQTA-containing peptide IMP 244 is dissolved in 2 mL of arr~moniu~n acetate buffer, pH 7.2. The Ge-68/Ga-68 generator is eluted with a reduced l~ist~mi ~HSG~ inyl-glycyl-amount of hydrochloric acid (HCl) from that recoxximended by the manufacturer, in this case a Sn02-based generator (NEIL, Billerica, MA). Typically, this elution volume can be 2-5 mL instead of 20 niL recommended by this particular manufacturer. The lesser amount taxi be used without significant loss of recovered activity, by judicious ~electior~
of the central portion of the elution pea]c. The Ge-6$lGa-6$ generator may be optionally fitted with an anion-exchange membrane such as a Q51; cartridge (Sartorius AG, Goettingen, -Germany), to remove any eluted stannate packing material that may be eluted from the generatrar with the ~Ci. The H~1 used is of the purest form available (e.g.
Qptima grade HCl from llisher Scientific, Pittsburgh, p'A, diluted down to a Goneenlration of 0.5-1.0 N using 18 Me~Qkan purity deionized water.) tQ assist in maintaining Ga-6~
yield without ixttroducing the cor~taxnix~ating effect of other metals. The gerterator can be eluted with 1.N HCI, or with s~r~il~rly good recovery of expected Ga-6~, with 0.5 N f iCl.
The pH of tie final labeling xnixttire is typically between 4.5-4.8, depending upon whether 1 N ~r 0.~ N fiCl hay been used for generator ~lution<
The arxtount of NQT.A-peptide ZN1P 244; xieaded to effect satisfactory labeling within a 30 minute to 1 h time .frarr~e is generally above 1 x 10-~ moles, more preferably above 1 ~
10-8 ix~oles, and most preferably at or above 5 x 10-$ moles. Since, in this instance, the final.vQluxne is appr~xinlately 7 mL pf cpxnbiiied generator eluate axxd aruxrionip~rx acetate buffer, the lowest xuolarity of NQT~.-peptide needed for effective labeling is in the range of 1-10 micromolar. A preferred labeling temperature is somewhat elevated, from ambient into the 3S-45°C range. Labeling can also be accomplished at more elevated temperatures, for example at 50-100 degrees Celsius.
In an embodiment using the ~iVI~' 241 peptide, the DOTA-peptide is placed in a [p~eferably~ acid-Washed glass, plastic ox resin vial. Around 1.0 x 10-7 to 1.0 x 10-~ moles of the hOTA-peptide is a rr~ost preferred an count, althpugh Sorr~ewhat lesser or greater amounts may also be successfully used. This amount of peptide is generahy then mixed With approximately 1-2 mL of 2-5 M ammonium acetate buffer, pH S.0 to pH 8Ø
The composition may then be stored at dour degrees, frozen or lyophilized for future use, ox it may be used immediately. Such formulations can be ~reps~e~ in multiple sealed vials, and made under sterile, py~o~en-free conditions for manufacturing and general use purposes.
For gallium-68 r~dialabeling, an IIVII?' 241 composition prepared according to the above desct'~ptior~ is placed onto the outset valve of a germanium-68/gailium-68 generator. The vial cont~i~ing the peptide is shielded in lead, preferably 1-2 inches thicl~.
By use of plastic tubing the operator then elutes the generator with dilute hydrochioric acid from a remote position into the shielded vial of the DOTA-peptide composition.
Generally, the generator can be eluted using ~ hand pushed syringe containing the acid, or it can be conveniently eluted using ~ peristaltic pump. The elution tire is advantageously kept under 15 minutes, more preferably under eve minutes, given the 68-minute half life of the gallium-68 radionuclide. A needle or catheter, which can be metallic, but is preferably non-metallic, is used to maintain equal pressure inside and outside of the vial dining the elution.
The Gallium-6$ generator is eluted with between 1 and 10 mL of hydrochloric acid, preferably 2-5 mL, to ensure adequate yield of the gallium-6~ into the Follection vial, without unwanted over-dilution. the acid used is preferably of the highest purity possible in order t4 maintain goad recovered yield of gallium-68 over an extended time period, to maxixrai2e utility of tl~e generator. During elution, the first several m1J o~
generator eluent may be diiverted to waste, before the main fraction containing the gallium-6$
is harvested, The vial of Vii' 241 is then heated at 95°C, witb lead shielding, in a water-bath for 15 minutes. Gallium-68 xadiolabelings can also be lone in as little as five minutes, or can take up to an hour and still be useful, with conditions also being particularly dependent an the amount a~pepxide used in the fa~r~x~ulation end the anal pI~ of the labeling mixture, ~l~e ~refe~ed f nal pI~ is b~tweed 2 and ~; with the most preferred pH being between ~
and 5, y ~nacrocyclic cbelate-peptide conjugate can be labeled using the above siypli~ed techniques, and specific targeting agents such as octreotide, LHRI~, somatostatin and gastriy, ifbe~ring a r.~~c~ocyclic chel~te such as NOTA or DC~~A,- can be used in the invention. In a prefeired embodiment, peptides such as l~F 244 and IlVII?' 241, herein disclosed, are usefully labeled with Ga-68 and are tbcn used as Ga-68 complexes within pretargeti~g methods for Ga-~8 PET imaging, The pretargeting method combines the use of a primary disease targeting agent that is multispecific for both disease and a user-defined hapten, with the use of a later-administered hapten labeled with an imaging or therapy agent; in this instance with gallium-68. Preferably, the user-defined hapten is bivalent fox the mnltispecific targeting agent, and is able to cross-link tile former specifically at disease sites, producing an 'affinity enhancement effect'.
The hapten is designed by the user ~~x its utility is several aspects. Most importantly, the hapten is designed to clear living systems very rapidly. It is preferably very hydrophilic, even when bearing gallium. Exemplified in this disclasure axe the examples of the peptides IMP 244 and 241, both bearing two HSG sub-units, used for recognition by an anti-MSG antibody arni (termed 679), The HSG sub-units also impart hydrophilicity on the final hapten structure besides berg integral tQ the binding process.
I~owevex, it must be u~nclexstQQd tbat other sub-unit recognition units and other backbone sub-wits c~aulct be just ~~ e~siiy used within the scope of the invention. hox the latter, peptides axe useful, and any seqtience could be designed using st~udaxd methods of peptide syntheses. .Other bacl~iriQne sub=units, such as sugars and dextrans axe also contemplated as within the scope of the current invention.
I-~eretQ~ore it had been disclosed that use of a gern~aniuru-68/galiium-68 generator invtalved post-elution manipulation of the eluted gallium-6~, to concentrate the nuciide and keep it in a chelatable form. These procedures involved organic solvent-fram-acid e~tractions, low-pressure evaporations and other detailed hands-on techniques as outlined _ 17-in the b~c~grot~ud above. It must be understood that ~ commercially viable axed clinically acceptable method fox adoption of gallium-~8-labeled imaging agents fox PET is incompatible witb all these previously described, complex radiolabeling rr~ethods. This is true fox multiple ,reasons, most importantly because of the extreme hazards in handling the high-energy, high intensity Ga-6~ (positron energy 1900 MeV), and the need tQ
maintain sterility and apyrogenicity while working with the 68-minute l~al~
life gailium-6~. The invention, in its entirety and unlike previQUS art, represents a very simple integrated process fox pex~ornaing faciie labeling of macrocycle-(NQTAr- and DC~T1~)_ crantaining, disease-targeting conjugates with gallium-bg. The present invention is further illustrated by, though in no way limited tp, the following examples.
Examples:
Exam~le.1. Acid Elution of a Ge-68/Ga-68 Generator:
A Ge-6~/G~-68 generator is placed inside a half inch lead 'molycoddle' fox extra shielding, and this is further surrounded by a 2-inch thick lead wall. The inlet of the generator is fitted with sterile tubing and ~ 3-way stopcock. The two other ports of the stopcock are attached to a 10-mL sterile syringe and ~ source of ultra-lure 0.5 N
hydrochloric acid, respectively. The outlet port of the generator is fitted with sterile tubing and a (~h'S anion exchange membrane that had been previously washed with 0.~ N
hydrochloric acid. ~y means of the inlet syringe, a S-mL portion of the 0.5 N
hydrochloric acid is withdrawn from the stock solution, the stopcock is switched to allow access to the generator column, and the acid is hand-pushed through the generator. The elute containing the Ga-68 is collected in a lead-shielded acid-washed vial optionally already containing the NOTA-containing targeting agent to be Ga-~~
radiolabeled.
Example 2 Ga-68 Labeling of IMP 244 Peptide Using the.Above Elution Technique:
.~A. 5 x 10-s portion of the NC)T~-containing peptide F 244 is mired with 2 mL
o~4M
met~h~ree amzrloniuxn ~ce~~te tiu~fer, p~ '7.2~ in an void-washed vial. 'The Ga-68 ingrawth from the generator, S mCi, is eiu~ed directly intb the IIV.IF 244 solution usi~~ the techr~idues described in the previous ex~rnple; After brief mixings tl~e vial contents s~'e heeled 30 rz~int~tes at 45°C. The ir~,corpor;ttion o~G~-6$ into the Ice" 244 is measured at ~4~/a, after the ~0-ri~inute libeling time, by Size-excius~o~i'high-perfozuxiance li~~tid cl~Qi~atography (SE-I~PLC) on a via-Si1250 columin run in 0.21V~ ~hos~hate buffer, pH
6.8, with calurnn recovery determined, and detection by in-line Radiomatic detection using energy vc!indaws set :~o~ G~-~~. Corroborative data is obtained using instant thin-laYe~' chrorx~~logrsphy (ITl=.C) using silica gei-impre~nsted glsss fiber stz~ps (Gein~an Sciences, ~~n arbor, MI), deveioped in a 5:3:1 mixture of pyridine, acetic acid and water.

Example 3. Ga-68-IMP 244 and anti-HSG MAb Complex Formation:
An aliquot of the Ga-68-IMP 244 complex is mixed with a 20-fold molar excess of bispecific antibody (bsAb) hMN-14 x 679 F(ab')2 ~auti-CAA x anti-HSG~ in 0.2 M
phosphate buffered _saline, pH 7.2, and reapplied to tye above SE-HPIJC
analytical system. Tl~e radioactivity that eluted at a retention time of around 1.4.2 minutes in the last example was near-quantitatively shifted to a retention tine near 8.~ minutes after mixing with the bispecific antibody. Comparison to this retention time to those from application of molecular weight standards to the Sl~-IiPI~C under tl~e same Conditions indicate that the radioactivity leas shifted to a molecular weight near 200,000 Daltons.
Example 4. Stabilit~Studies on the Ga-68-IMP 244 Peptide:
a) To EDTATl~e G~.-68-TT1VIP 244 peptide is diluted into 0.2M sodiurrt pl~osph~te buffer containing 1 xnlV~ ~DTA and allowed to stand at x~QO~n te~~e~'ature. At periods up to 2-3 h npc~sl-incubation alis~uots were tested by size-exclusion high-performance li~ui~l chroxn~tograpby iu the above system. In this system, Ga-68-fNIP 244 elutes near 14.2 ~x~i~utes, while Ga=68-EFTA elutes near 13.~ minutes. Upon fu~her mixing of an aliquot of the Ga-68-1M~ 244 + EDTA mixture with a 20: ~ molar excess of hMN-14 x 679 F{ab')2 bis~b and re-analysis by SE-.PLC, the radioactivity peak that eluted at a retention time of around 14.2 minutes was near-quantitatively shifted to ~
retention time near 8,8 minutes, showing that the Ga-68-IMP 244 is stable to EDTA challenge over a mufti-hour period.
b) In Human Serum: A 100-uL sample of the Ga-b8-~P 244 is mixed with 2 rnL of whole human serum and incubated over a 3 h period at 37°C. Aliquots are taken at ir~tertr~edi~te times and analyzed by SE-HPLC. ~To change in retention tine from the arig~hal 14.2 minutes corresponding to Ga-6$-IMP 244 is seer, prpV~~~g no non-specific binding to any of the components that corrtprise hurr~al~ serum, and np loss c~
radioactivity ft'om Ga-68-IMP 244 to any of the components that comprise human serum.
Adcliti~anally, after 3h incubation, upan further mixing of an aliquot of the Ga-68-hVIP
244 in human serum m~Xture with a 20;1 ~Qlar excess ~fhIVIN-14 x 679 ~(ab')z bsAb and xe-analysis by ~~-PIPLC, the rac~iQ~.ctiyity peak t~~t eluted at ~
reterltic~n tide of around 14.2 minutes was near-quantitatively shifted to a retention tune near ~.8 rr~ix~ut~s.
This S~ovcWs that tl~~ Ga-6~ remains bound to the IMP 244 peptide, and the latter is still fur~ction~lly able tQ bird tQ the ~-14 ~ ~7~ F(ab')z bsA.~.
Example 5. Preparation of Ga-68-IMP 2~.1 In ~n acid washed tOmL CZ-Resin vial (West Ph~m~aceutic~l Services #19550022) are mixed l.OmL of 2.SM NH40Ac, pH 5.5 buffer, and 11.4uL of a stock solution of 2,2 x10-3 ~ IIVI~" 24I in O.S1VI NH40Ac, pfI 4, (2.5 x 10-8 mol of total peptide). The vial is stappered with an acid washed Flurotec stopper (West Pharmaceutical Services #1243), crimped closed and swirled to mix thoroughly. The Gallium-68 generator is then eluted with 6 mL 1N HCl (QPTIMA Fisher #A466-250, dilated to 1N with i 8 lVleglJh~n water), with the first two m~. of generator eiuent diverted to waste. With a vent needle (i 8G 1-1/4" Je~co i.v. Catheter Placement Unit, (VW~ #329t ~-8~0) in plane, the generator eluent is directed into the labeling vial through a non-metallic catheter. The vial is then heated at 95°C in ~ water-bath for 15 ,minutes. The anal pH of the labeling mixture is a~praximately 3.9. For r~.diaan~~ysis, an aliquot is withdrawn and diluted to 0.05 uCi/uL
with sterile saline, Ten-twelve uL a~ the dilution (containing approximately 0.5-0.6 uCi of G~.-68) is mixed with ~. 20-fold molar excess m679-~gCr anti-HSG antibody and a~~lied to ~ ~i~e-exclusion Rio-SiI- Sec-ZS4 co~ur~n equilibrated and rttii ~n 0.2M sodium ~bos~l~ate bu~fe~, ~~ 68, containing 0.02~/p ssad~um azide, ~t itnLlrilinute:
Colut~a~
recovery of applzed radia~ctivity is dome by ca~iecting the entire elu~nt froxrt the 20-rriii~ute r~ a~d.dQUrttin~ 3 x 1mL aliquots therefrd~n against 3 x 1., aliquots ef a.
standard; prepared by diluting the same volume of iabele~i peptide ~n 20yL
HPLC column elution buffer. S~-~P'LC Column tecaver~! upon success~~ x~dia~abeiing is >
$0°l0. Axe e~ar~p~e of the .radio-HPLC analysis ofthe Ga-68-I~F' 24t - 679 IgG MAb complex is shown in Figure 1.
~xaxnlile 6: Specific Localization of ~a-67-T1VIP 241 Using Bispecific Antibody Pretargetin~:

Gallium-67 was used iii l~.eu of gallium-68 due to the 6$-minute half life of the latter, and the inherent difficulties of quantitating the activity of gallium-68 in tissue due to significant reduction in radioactivity during the counting period. The bispecific antibody I-125-hMN-14 x 679-F(ab')2 (anti-CEA x anti-HSG, radiolabeled with iodine-125 as a tracer radionuclide) was injected into nude mice bearing GW39 human colon tumor xeno~rs~ks. 'f Wenty-four hours later, the Ga-67-IMf' 241 peptide, prepared according to the method described in ~xa~nple # 4, was injected. ~t specified times post-injection, five ~nim~~s ~e~ time-point were S~c~~~ced and tissues collected and counted for both I-1.25 and Ga-67 radioactivity, using dual energy windows for each respective r~dio~uclide.
I~~ta for I-125 (bs.~b) is shown in Table l, and data for Ga-67 (IMF 241.) is shown in T~b~le 2. Additloz~~lly, biodistribution of Ga-67-1M~ 241 given alone, without bsAb pz~et~rgeting is shpwn zz~ T~bie 3 (four animals per group irk this series).
In each Table, the data ,i~ presented in terms afpercexit injected dose per gram of tissue (~
st~r~d~rd deviation). Additionally, the bioclistributiozt dots. for I-1.25-N-14 x 679-F(ab')a axzd C,a-67-IMP 241, frorz~ the same e~~erimeut, is re~o~ted in tei~xls of t~tz~or-to non-tumor ~atia~ (~'/NT) (T~bles'4 ~ 5, fax I=125 and Ga=67, fespectively).
The uptake of the peptide mirrors uptake of bsAb in almost all tissues (Table 1 & 2).
Tumor=to-tissue ratios fog the Ga-G7-IM.~ 241 are better at later time-points, apparently without airy significant loss of Ga-67 counts in the target tumor. When the Ga-241 is given alone (Table 3), it essentially clears completely within the frst hour ~ost-injeGtiou, with no non-specie uptake in any tissue save residual kidney activity, proving that tl~~ Ga-67-DOTA complex on the IMP 241 peptide is stable enough for use with Ga-68. T/NT ratios are positive for the I-125-hMN-14 x 679-F(ab')Z from the earliest (one lour time-point measured) (Table 4), with stomach the lowest positive ratio, due to normal clearance of antibody-cleaved radioiodine in that organ. Most imporkantly, the data in Fable 5 slows T/hTT ratios far the prospective imaging agent analog at major time-points of interest, >3y two lours, the mean ratio is above 6;1 for tumor-to-blood, and is very ligh for all other tissues, even ~ 3.5;1 for tumor-to-kidney, meaning that excellent ixn~.ges wih be obtainable by this time, In fact, strongly positive tu~x~or-ta-nor-tenor ~'atios are seen for all organs from one hour post-injection onwards, pr~vin~
that this irx~aging system will be used for 1'ET with the 6~-minute half life gallium-68, Table 1. Biadistributioia of 1-125-~i~.N-14 x 679-F(ab')2 bispeci~e antibody.
Data in terms pf perGelt itijec~ed dose her ~,raxr~ oitissue (~ standa.~d deviation);
Tissue Tunes are post-iujectiQx~
of lieptide (~2~ h post-injection of bs.Ab) 1,~ 21 ~~ 41 Tumor 3.11,0 2.4~0.~ 1.5~O.G 2.50.9 2.511.2 Liver 0,T ~ 0,1 0.4 ~ 0.1 0.5 ~ Q:2 0,4 ~ 0. 0.5 ~ 0,1 ~.

Spleen 1,0 ~ 0,4 Q,G ~ 0.4 0.6 ~ 0,1 0,6 t 0,3 0,7 ~ 0;3 Kidney 0;70.1 ' 4.50.1. 0.50.1 0,40.1 0:40,1 ~.,~ngs 0,gt0.3. 0:40.1 0.40.1 0.40.1 0.50,1 Blood I.5~0.2 1.00.3 0.80.2 0.90.2 1.00.3 Stozn~cl 29 ~ Q.7 1.3 ~ 1.0 1.6 ~ 1.2 1.5 ~ 1.1 2.4 t 1.8 Srn. Intestine0.3 0.1 0.2 ~ 0.1 0,2 ~ 0.~ 0.2 ~ 0.~ 0.2 ~ 0.1 ~

Lg. Intestine0.3 0.0 0.3 ~ 0.1 0.3 ~ 0.1 0.3 ~ 0.1 0.3 ~ 4.2 ~

Bone O.S~ 0.1 0.20.1 0.30.1 0.20.1 0.20.1 ~'~ble 2. $iodistr~bution of Ga-67-IMP 241 given 24 hours after I-125-hMN-14 x G7~-F(~b')a biSpecifie antibody. Data in terrr~s of percent iujcatcd dose per grad of tissue (~
standard deviation).
tissue Tirn~ pest-injection of Ga-6~-IIY~P
241 pcpli'de ' 1li 2h 3h 4h ~b ~.urr~car 13,2~~.8 10.76.7 8.36.1 11.7~4.~ 16.08.7 Liver 1.7~Q,4 0:'70.3 0,~~0.3 0.90.6 1a0~0,5 ~pieen ~.4 ~ 0:5 0.6 ~ 0.5 0.6 ~ 0,2 0,~ ~ O.G 1,0 ~ 0.

I~ldla~y 6.3'~ ~,5 3.1 ~ 1.3 3:4 ~ 0:~ 3,~'~ Q.~ 37 ~ 1.5 ~

L;ungs 2.90.9 0.90.5 0,~~0.2 1.00.6 0.80.3 .

Blood ~.2 ~ 1,8 2.1 ~ 1.6 1.3 ~ 0.5 1.7 ~ 1.2 1,4 ~ 0.6 StQ~na.cla 0.4 ~ 0,2 0.2 ~ 0.1 0.1 ~ 0.0 0,4 ~ 0.6 0.1 ~ 0.1 _S~" ~testine0.7 ~ Q.1 0:4 ~ 0.3 0.2 ~ 0.0 0.2 ~ 0.6 0.5 ~ 0.4.

I,g, ~t~sti~e0.~ ~ 0.1 0:2 ~ O.i.0,2 ~ 0.1 0.2 ~ 0.1 0.3 ~ Q,2 Bone 2.1 ~ 1,S 0.5 ~ 0.4 0.5 ~ 0.2 0.6 ~ 0.2 0.4 ~ 0.

~~ble 3. Bioctistribntion of Ga-f7-In~F 24t given alone, With no bs.Ab pretargeting.
Tissue Time host-injection of Ga-67-AMP
241 peptide lt~ 2b 3b 4h 6h T~~n~ar 0.'10.4 0,30.1 0;20:1 0.20.0 0.10.0 Liver 0; ~ ~ 0.1 ~ 0.0 0, ~ ~ 0,1 ~ 0.0 0,1 t 0, 0.0 0.0 0 ~~leer~ 0.0 t 0.0 0.1 ~ 0, 01 ~ Q:0 0, I ~ 0.1 ~ 0.0 I. 0.0 d~~ey 2,2 ~ 0.$ l.'7 t 0.5 2.6 ~ 0.7 2.0 ~ 0,4 ~,~ ~ 0~5 tuns O,l t 0=1 0.1 ~ O: O.t ~ 0,0 0,0 ~ 0.0 0.I X0,0 I.

$laod O,t ~ Q;0 Q.l ~ 0,0 0.0 ~ 0.0 0.0 ~ 0.0 0,0 t 0,0 Ste~~ch 0,1 ~ 0,1 0.0 ~ 0,0 0.0 ~ 0.0 0,1 ~ 0,1 0.0 ~ 0,0 Sm, Intestine0.3 t 0.3 0.1 ~ 0. Q. l ~ 0.3 ~ 0,2 Q,1~ ~ 0.0 t 0~,1 I,g. Iiitestir~eO. t t 0.0 ~ 0.0 0.0 t 0.0 0,5 ~ 0.8 0.1 ~ 0.0 0.0 Bone 0.1t0~1 0.10.0 0.10.0 0.10.0 0.00.0 Table 4. Biad~str~butio~ ~f I-125-I~MN-14 x 679-F(ab')a bispecific antibody With data ~e~orte~ i~ teraxas of T/~T ratios.
Tu~nz~r-ts~-~'i~ne posh-i~jectiox~
Qf peptide (+24 h host-i~j~ctiQr~
o~bsl~b) tiss~~ xatio1 h 2 h 3 .h 4 h 6 h I-.fiver 4,8 ~ 5,5 ~ ~,4 3.G ~ l.~ G.4 ~ 2.1 5.~ ~ 3.3 2.3 S~,~eeri 3,52"1 4,63.3 2.70.9 4.f~2.1. 3.52.2 I~ic~ia~y 4,3 ~ 5.Q X1:4 3,4 ~ 1,~ ~.8 ~ 1.7 ~:0 ~ 3,7 ~,6 .

Lungs 4.3 ~ ~,~ ~ 2.0 3,g ~ ~.~ 5,7 X1.3 5,7 ~ 3.3 1,7 ' ~~ood 2,~ ~ 2.4 ~ 0.7 2.2 ~ '1;72.~ ~ Q.6 2.~ t 1.4 0.8 StQhi~ch; ~:~ ~ 2.5 ~ 1..~ I.~ ~ 1.0 1,$ ~ 0.5 1,9 ~ 1:8 0.8 s~. ~testi~e9.Q ~ ~ ~.9 x'2.37:7 ~ 1.9 12;~ ~ 1?~4 ~ ~~3 3:1 2.3 ~~. ~t~stiz~e9.~ ~ 7,~ ~ lf~ 5,2 ~ 1.6 8.7 ~ 0.9 5,~ ~' S.
3,9 ~or~e 7,6 ~ lfJ~y ~ 5,6 ~ ~.9 IO,'2 ~ 12:1 ~ 9.3 4.7 2.~ 2,$

Table 5. Biod~stributi0n Q~ G~-67-IMP 241 given 24 l7QllxS ~ftex ~-125-~MN-14 x 679-F(~b')a bispeci~'~c antibody.
Tumar-to- Time post-~njecti~n of peptide (+24 h post-injec~~On of bsArb) t~ss~e ratio1 lu 2 h 3 h 4 h 6 h Livex 8.0 ~ 15.9 ~ 8.9 13,1 ~ 16.2 ~ 16.0 t 10.5 3,$ 12.0 $.6 Spleen 10.7 ~ 26,6 ~ 2~,716,3 ~ 26.1 + 1T.4+ 12.7 7.2 13.1 23:4 I~idx~ey 2.3 + 3.5 ~ 2.2 2.4 ~ 1,8 3.1 ~ 1.1 4.3 ~ 2.2 1.6 ~~tl~g~ 4.5 ~ X3.1. t 11;4 ~ I3.5 ~ 20.2 ~ y2.5 1.9 '7.5 11.1 5.9 $lo~d , 1:7 ~~ 6.1. ~ 5.0 $~S + 9,5 ~.'~ ~ 12:1 18:0 (?.8 4,6 , ~tc~til~cli36.7 ~ 52~ ~ 17.6 73:6 ~ 56.9 ~ 112.5 ~
22.8 4~.4 . 36.0 60.7 SrnI~tes~l~e18,6 ~. 27.7 ~ 9.1 35:5 ~ 22.0 ~ 47.2 + 35:9 9.I 26.0 12.'7 L~. Intestine~,~,3 X4;2 ~ 55:072,2 t. 56.5 ~ 162,3 +
~ 44.1 . X9.0 21.7 270 ~o~~ 11.5 ~ 24,6 + I5.1~ 7,~ ~ 18.3 ~ 53.0 ~ 4I;7 ~ 1.0 13:7 3. ~.

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5,~39,439.Jurt~ 17, 1987.
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It will be apparent. to those skilled in the art that various modifications and variations can 'bs made to the products, compositions, n Methods, and processes of this invention. Thus, it is intended that the present invention cover such modifications and variations, provided they come within the scope of the appended claims and their equivalents.
-3?-The disclosure of ail publications cited above are expressly incorporated herein by reference in their entireties to the same extent ~~ if each were incorporated by reference i~~dividually.

Claims (37)

1. A targeting agent for positron en Fission tomography comprising a complex of gallium-68 with a macrocyclic chelating agent linked via a peptidyl spacer to one or more recognition units recognized by a multispecific targeting antibody.

2. The targeting agent of claim 1, wherein said Ga-68 associates with a 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA) or 1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA) moiety, linked via said peptidyl spacer to one or more said recognition units recognized by said multispecific targeting antibody.

3. The targeting agent of claim 1, wherein the macrocyclic chelating agent complexed with gallium-68 and linked via a peptidyl spacer to one or more recognition units recognized by said multispecific targeting antibody has the formula:

4. The targeting agent of claim 1, wherein the macrocyclic chelating agent complexed with gallium-68 and linked via a peptidyl spacer to one or more recognition units recognized by said multispecific targeting antibody has the formula:
DOTA-Phe-Lys(HSG)-D-Tyx-Lys(HSG)-NH2

5. A method of detecting a condition comprising: a) administering to a subject in need thereof a multispecific targeting agent having at least one binding site for a histaminyl-succinyl-glycyl moiety together with at least one binding site with affinity for an antigenic determinant on target tissue related to the condition to be detected, and b) sometime later, administering a Ga-68 radiolabeled targeting agent of claim 1, and c) imaging the subject using positron emission tomography.

6. The method of claim 5, further comprising treating the condition being detected.

7. The method of claim 5, wherein the condition being detected is cancer, thrombosis, stroke, atherosclerotic plaque, infection or inflammation.

8. The method as claimed in claim 5, wherein the multi-specific targeting agent is hMN-14 x 679 F(ab')2 [anti-carcinoembryonic antigen x anti-HSG].

9. A method of preparing a Ga-68 labeled targeting agent by combining eluate from an acid-eluted Ge-68/Ga-68 generator and a NOTA- or DOTA-containing targetable agent.

10. The method of claim 9, further comprising preparing a Ga-68 labeling targeting agent by directly eluting an acid-eluted Ge-68/Ga-68 generator into a vial containing a NOTA- or DOTA-targetable agent.

11. The method of claim 9, further comprising use of an anion-exchange step to remove unwanted tin, titanium or other metals from a generator eluate.

12. The method of claim 9, further comprising the use of a dilute acid for generator elution.

13. The method of claim 9, wherein a 0.5-1N hydrochloric acid solution is used for generator elution.

14. The method of claim 9, further comprising elution of the generator eluate into a pH
neutral ammonium acetate solution containing enough ammonium acetate to substantially establish the pH of the final labeling solution at between three and six.

15. The method of claim 9, wherein the NOTA- or DATA-containing targetable conjugate is selected frog NOTA- and DATA-conjugated peptides, polypeptides, proteins, carbohydrates, cytokines, hormones and cell receptor-biding agents.

16. The method of claim 9, wherein the NOTA-containing targetable conjugate is selected from peptides, polypeptides and proteins targeting somatostatin, LHRH, VIP, BLys and gastrin receptors.

17. The method of claim 9, wherein the NOTA-containing targetable conjugate is selected from peptides, polypeptides and proteins targeting disease-associated antigens.

18. The method of claim 9, wherein the NOTA-containing targetable conjugate targets a tumor-associated antigen.

19. A composition comprising a NOTA- or DOTA-targeting agent conjugate dissolved in sufficient pH neutral ammonium acetate to enable substantially complete incorporation of Ga-68 when admired with a solution of Ga-68 in dilute acid.

20. The composition of claim 19, wherein the NOTA- or DOTA-containing targetable conjugate is recognized by a primary targeting agent bearing at least one secondary recognition unit that recognizes an epitope of the NOTA- or DOTA-containing targetable conjugate.

21. The composition of claim 20, wherein the NOTA- or DOTA-containing targetable conjugate is recognized by a multispecific antibody bearing at least one secondary recognition ann that recognizes an epitope of the NOTA- or DOTA-containing targetable conjugate.

22. The composition of claim 21, wherein the multispecific antibody is comprised of a bispecific antibody or bispecific antibody fragment.

23. The composition of claim 22, wherein the NOTA or DOTA-containing targetable conjugate additionally contains at least one histaminyl-succinyl-glycyl- (HSG) recognition units attached to an epsilon lysine moiety and the bispecific antibody has at least ope arm that recognizes the histaminyl-succinyl-glycyl- subunit when attached to an epsilon lysine moiety,

24. The composition of claim 23, wherein the NOTA-containing targetable conjugate is NOTE-2-benzylthioureayl-D-Ala-Lys(HSG)-Tyr-Lys(HSG)-amide.

25. The composition of claim 24, wherein any of the amino acids of the peptidyl backbone are in either the L- or D- configuration.

26. The composition of claim 23, wherein the DOTA-containing targetable conjugate is DOTA-Phe-Lys(HSG)-D-Tyr-Lys(HSG)-amide.

27. The composition of claim 26, wherein any of the amino acids of the peptidyl backbone are in either the L- or D- configuration.

28. The composition of claim 23, wherein the NOTA or DOTA-containing targetable conjugate has a concentration of about 1 micromolar or above in the composition.

29. The composition of claim 21, wherein the multi-specific antibody is prepared by means of chemical cross-linking, quadroma technology, or as a construct of molecular biology.

30. The composition of claim 29, wherein the multi-specific antibody is murine, chimeric, humanized or human in origin.

31. A kit comprising sufficient macrocycle-conjugated targeting agent to bind added acidic Ga-68 together with sufficient ammonium acetate buffer to substantially neutralize excess acid, and allow Ga-68 complexation by the macrocycle.

32. The kit of claim 31, further comprising a Ge-68/Ga-68 generator equipped for acid elution.

33. The kit of claim 31, wherein the gallium-68 generator eluate, once added, essentially forms a quantitative radiolabeling of the kit contents within about one hour.

34. The kit of claim 31, wherein the components are provided in liquid form, frozen, or lyophilized.

35. The kit of claim 31, wherein the components are heated to between 45 and degrees Celsius for up to one hour to effect radiolabeling.

36. A method of detecting a condition comprising: a) administering to a subject in need thereof a multispecific targeting agent having at least one binding site for a histaminyl-succinyl-glycyl moiety together with at least one binding site with affinity for an antigenic determinant on target tissue related to the condition to be detected, and b) sometime later, administering a Ga-67 radiolabeled targeting agent of claim 1, and c) imaging the subject using single photon emission computed tomography.

37. A kit comprising sufficient macrocycle-conjugated targeting agent to bind added acidic Ga-67 together with sufficient ammonium acetate buffer to substantially neutralize excess acid, and allow Ga-67 complexation by the macrocycle.