Proc. Nati. Acad. Sci. USAVol. 85, pp. 4025-4029, June 1988Medical Sciences

Specific and stable labeling of antibodies with technetium-99m witha diamide dithiolate chelating agent

(radioimmunoimaging/active ester/preformed chelate conjugation to antibody fragments)

ALAN R. FRITZBERG*, PAUL G. ABRAMS, PAUL L. BEAUMIER, SUDHAKAR KASINA, A. CHARLES MORGAN,T. N. RAO, JOHN M. RENO, JAMES A. SANDERSON, A. SRINIVASAN, D. SCOTT WILBUR,AND JEAN-LUC VANDERHEYDENNeoRx Corporation, 410 West Harrison Street, Seattle, WA 98119

Communicated by Lewis H. Sarett, January 25, 1988

ABSTRACT Technetium-99m labeling of antibodies hasbeen suboptimal because of low affinity adventitious binding,nonspecific labeling, and loss of immunoreactivity. The dia-mide dithiolate ligand system (N2S2) forms highly stable,well-defined tetradentate complexes with Tc(V). Antibodiesand their fragments have been labeled by conjugation ofpreformed "mTc4,5-bis(thioacetamido)pentanoate active es-ter to protein amine groups to give a chemically known99"Tc-N2S2 complex covalently linked to antibody. Evalua-tions of the "'Tc-N2S2-bound antibodies and their fragmentshave shown high stability and retained immunoreactivity.

Successful targeting ofdiagnostic radionuclides to tumors notonly provides a tool to diagnose and stage cancer but alsodemonstrates feasibility for therapy where ligand systemscan be applied to therapy radionuclides. Early studies withradiolabeled antibodies utilized radioiodine (1231/1311) be-cause of extensive experience in protein radioiodination,covalent attachment, and ready availability of the radio-nuclide (1, 2). Improved tumor-to-nontumor ratios wereachieved with 1"'In compared to 131I by using diethylenetria-minepentaacetate (DTPA) bifunctional chelating agent tech-nology (3-5). 1311 and 1"'In have long half-lives of 8 days and67 hr, respectively. Recent studies utilizing antibody frag-ments have shown that tumor uptake and background tissueclearance can take place more rapidly with fragments,allowing the use of shorter half-life radionuclides such as99mTc (6).99mTc is the ideal diagnostic single-photon radionuclide

with a 6-hr half-life, 140 keV 'y radiation, no particulateradiation, and inexpensive, convenient availability. Theseattributes allow the routine administration of doses of 30 mCi(1 Ci = 37 GBq) that result in high photon-flux levelsfacilitating lesion detection by single-photon-emission com-puterized tomography. Despite these attractive aspects, thenumerous oxidation levels available and the proclivity of99mTc for nonspecific binding to proteins preclude easyadaptation to antibody labeling processes developed for otherradionuclides such as 1"'in.

Antibodies have been labeled with "mTc both directly andthrough, bifunctional chelating agents, but with suboptimalresults. Direct labeling involves metal binding to donor atomson the protein, resulting in multiple binding sites of low metalaffinity (7). Bifunctional chelating agents, including DTPA (8,9) and bis(semithiocarbazones) (10), have been studied for99mTc labeling but have been found to suffer from problemsof (i) nonspecific uptake because of the requirement forseveral ligands per antibody, (ii) adventitious binding of themetal to the protein, (iii) formation and binding of colloids to

the antibody (11), (iv) control of the oxidation level ofTc, and(v) low yields of specifically bound "mTc (9).We have applied a diamide dithiolate ligand system (N2S2)

to "mTc labeling of antibody fragment. This ligand systemforms highly stable tetradentate complexes with Tc (CmTc-N2S2) at the +5 oxidation level (12). Studies of a largenumber of compounds containing the N2S2 donor groupshowed that high yields of "mTc complexes of the predictedstructure were obtained (13). We have shown that otherpotential metal-binding groups, such as the carboxylategroup present in the N2S2 ligands, do not participate in metalbinding (14). This property enables labeling of antibody byformation of the technetium complex, then chemical conver-sion of the carboxylate group to an active ester, and finallyconjugation of the preformed complex to antibody. Prelimi-nary results of studies of this approach were included in areview of 99mTc labeling of antibodies (15).

MATERIALS AND METHODSPreparation of 99mTc-4,5-bis(thioacetamido)pentanoyl

(N2S2)-Conjugated Anti-Melanoma 9.2.27 F(ab')2 Fragment.To a mixture of 25 1.l of 4,5-bis(benzoylthioacetamido)pen-tanoic acid (1.0 mg/ml solution in 90% CH3CN) and 100 ,ul of1 M NaOH was added 100 mCi of sodium [99mTc]pertech-netate in 1.0 ml of saline (0.9% NaCl). Then 1.0 mg of sodiumdithionite (0.10 ml of a freshly prepared 10 mg/ml solution)was added, and the mixture was heated at 750C for 15 min.The pH was brought to about 6 with 0.10 ml of 1 M HCI and0.30 ml of 0.2 M sodium phosphate buffer (pH 6.0). Then 10.0mg of 2,3,5,6-tetrafluorophenol (0.10 ml of a 100 mg/mlsolution in 90% CH3CN) and 12.5 mg of 1-(3-dimethylami-nopropyl)-3-ethylcarbodiimide (0.10 ml of a 125.0 mg/mlsolution in 90% CH3CN) were added, and the solution washeated at 75°C for 30 min. The resulting tetrafluorophenylactive ester derivative of "mTc-4,5-bis(thioacetamido)pen-tanoate was purified by loading the reaction mixture on aconditioned C18 cartridge (J. T. Baker), washing with 2.0 mlof20% (vol/vol) ethyl alcohol/0.01 M sodium phosphate, pH7.0, eight times, and eluting with 100% CH3CN. The solventwas evaporated under a stream of N2. Then 0.5 ml of the9.2.27 F(ab')2 fragment (16) at 2.5 mg/ml and 0.50 ml of 0.2M sodium phosphate (pH 9.0) were added for conjugation.After 15 min at room temperature, 25 mg of lysine (0.25 ml ofa 250-mg/ml solution at pH 9.0) was added to quenchunreacted ester. The 99mTc-N2S2-9.2.27 F(ab')2 was purifiedby passage through a G-25 Sephadex column (Pharmacia)equilibrated with phosphate-buffered saline.

Abbreviations: N2S2, diamide dithiolate chelating system;99M Tc-N2S2-9.2.27 F(ab')2, 99mTc-4,5-bis(thioacetamido)pentanoyl-9.2.27 F(ab')2 fragments; DPTA, diethylenetriaminepentaacetate.*To whom reprint requests should be addressed.

4025

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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4026 Medical Sciences: Fritzberg et al.

C

HN NH

s sI IR R

R = H. CH3CO

Tc-

S S

Lo-

sodium I I1

+ 99sTCC_ dithionite 0 NXIIN N 0+O~~m~cO~ I o 4-)

S S

2,3,5,6-tetrafluorophenol,carbodiimide

0 F F0~~~~~~~~~0~~~~~~~~~

)L NH r'vAb

Ab-NH 0 I/ F F-0

FIG. 1. Synthesis of "mTc-N2S2-antibody by formation of 9'Tc-4,5-bis(thioacetamido)pentanoate, which is converted to an active esterby water-soluble carbodiimide and finally conjugated to antibody via acylation of amino groups.

An idiotypic antibody (NR-2-AD) specific to an individu-al's human B-cell lymphoma (17) was used in F(ab')2 form asa control antibody for the in vivo biodistribution studies. Itwas labeled as described for 9.2.27 F(ab')2.

Evaluation of ""'Tc-N2S2-9.2.27 F(ab')2 Stability. Thestability of the 9'9Tc binding to the antibody fragment wastested by incubation at 37TC in the presence of variouschallenge agents, including human serum, 10 mM DTPA, 10mM 2,3-bis(thioacetamido)propanoate, and 6 M urea. Sam-ples were removed at various times and analyzed by HPLC,isoelectric focusing electrophoresis, and NaDodSO4/PAGE.

Radiolabeled Cell Binding Assay. The immunoreactivity of"mTc-N2S2-9.2.27 F(ab')2 was assessed in an antigen-excesscell binding assay (18). Radioactivity bound to cells wasdetermined at increasing FMX-met (metastatic; ref. 19)melanoma cell levels until maximum binding was shown(antigen excess); then correction was made for nonspecificbinding by blocking of radiolabeled binding with excessunlabeled antibody.In Vivo Evaluation of ""'Tc-Labeled Antibody F(ab')2 Frag-

ments in Tumored Nude Mice. Nude mice (mean weight, 28.2g + 1.7 SD) were subcutaneously implanted with humanmelanoma xenograft A-375 mi/m in the left flank or side.Imaging and biodistribution studies were conducted in groupsof three and six mice, respectively, with 9'9Tc-labeledF(ab')2 fragments of melanoma-specific 9.2.27 antibody andnonspecific NR-2-AD antibody. Tumor weights were 335 mg+ 185 SD for the 9.2.27 F(ab')2 biodistribution and 222 + 67mg for the NR-2-AD F(ab')2. Tumor weights were 619 mg forthe 9.2.27 F(ab')2 and 543 mg (range, 543-720 mg) for theNR-2-AD F(ab')2 imaging studies. Imaged mice each re-ceived 50 ,ug of protein labeled with 1-1.5 mCi in anintravenous administration of 100 ,u via the tail vein. Micewere imaged serially for 16 hr after injection with a small-field-of-view (200 mm) scintillation camera (General Elec-tric). In the biodistribution studies, mice each received 10 ,ugof protein labeled with 100 uCi in an intravenously admin-istered dose of 100 ,ul. The animals were sacrificed 20 hr afterinjection; blood, tumor, tail (injection site), skin, heart, bone,lung, liver, spleen, stomach, neck (thyroid), kidneys, andintestine were isolated, blotted when appropriate, weighed,and assayed in a crystal scintillation detector well counter.Mean percent injected dose per g of body weight was

calculated by comparison with radioactivity of standardsamples of injectate.

1%

A

0

0 N\ II/N 0 F F

s s-~

FIG. 2. HPLC chromatograms ofcarboxylate (A) and active ester(B) forms of 9mTc-4,5-bis(thioacetamido)pentanoate (A) and its2,3,5,6-tetrafluorophenyl derivative (B). The column used was re-versed-phase Ultrasphere ODS (5 ,Am; Beckman). Conditions: 10%6(vol/vol) CH3CN/0.01 M phosphate, pH 7.0 (A); and 34%CH3CN/0.01 M phosphate, pH 7.0 (B), at 1.0 ml/min. Pairs of peaksresult from chelate ring epimers with respect to syn or anti alkylcarboxylate functionality relationship to the Tc oxo group (20). Thedecreased polarity because ofesterification is evident by the increasein organic solvent necessary to elute the complexes.

Proc. Natl. Acad. Sci. USA 85 (1988)

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Proc. Natl. Acad. Sci. USA 85 (1988) 4027

RESULTSStarting material, [9mTc]pertechnetate, was reduced bydithionite in basic solution in the presence of the N2S2 ligandto give 99mTc4,5-bis(thioacetamido)pentanoate, which wasconverted to the 2,3,5,6-tetrafluorophenyl active ester byreaction with the phenol and water-soluble carbodiimide(Fig. 1). HPLC chromatograms of carboxylate and activeester forms of the Tc complex are shown in Fig. 2. Thereduced polarity of the ester allowed purification by alow-pressure reversed-phase cartridge system. Conjugationto antibody lysine amino groups was carried out by additionof anti-melanoma 9.2.27 F(ab')2 and buffer to adjust the pHto 9.0. HPLC chromatograms ofcrude product and 'mTc-N2S2-9.2.27 F(ab')2 after gel filtration purification are shown inFig. 3. The yields of the carboxylate form of the complexwere essentially quantitative. The ester yields were about70%. Conjugation yields ranged from 50% to 70%o. Thus,overall yields were about 35% to 50%. Total time for theprocedure was about 3 hr.

Support for the aminoacylation reaction to give an amideconjugate was provided by pH dependency of the reaction(Fig. 4). The yield of 'Tc-labeled antibody was low atneutral pH and increased as the pH increased in a mannerexpected for reaction-rate acceleration because of increasedavailability of deprotonated amino groups. Routinely, con-jugations were run at pH 9 as a trade-off between yield andconcern for maintaining mild conditions with respect to therelatively unstable F(ab')2 fragments.The stability of the 99mTc label was evaluated by challenge

under several conditions. These included human serum,chelating agents 10 mM DTPA and 10 mM 2,3-bis(thioace-

A

B

C

FIG. 3. HPLC chromatograms of starting 9.2.27 F(ab')2 (spec-trophotometric trace in A), crude 9'Tc conjugation product (radio-metric trace in B), and purified 9I1Tc-N2S2-9.2.27 F(ab')2 (radio-metric trace in C). The column used was Zorbax GF-250 (DuPont).Conditions: 0.2 M sodium phosphate (pH 7.0) at 1.0 ml/min.Purification was by G-25 gel filtration.

CD

86an

55F

45~

35F

25[

15

7.0 7.5 8.0 8.5pH

9.0 9.5 10.0

FIG. 4. The dependency of the reaction of the 2,3,5,6-tetrafluorophenyl active ester derivative of FmTc-4,5-bis(thioaceta-mido)pentanoate with 9.2.27 Fab antibody fragment on pH. Theconjugation reaction was carried out at 1.0 mg/ml antibody fragmentconcentration with pH buffered as shown by phosphate. Nonspecificassociation of the active ester complex was minimized by addition of25 mg of lysine after 30 min at room temperature.

tamido)propanoate as the free thiol form of the propanoateform of the N2S2 ligand, and 6 M urea for denaturation of theantibody fragment. The challenge with the propanoate formof the ligand allowed identification of released "mTc radio-activity as (i) non-N2S2-associated, (ii) propanoate N2S2-chelated if exchangeable to an N2S2 complex, or (iii) simplynonspecifically associated pentanoate N2S2, the ligand formused for labeling. The results in Table 1 show insignificantlosses of 'mTc radioactivity under all challenge conditionsand, thus, high stability of the 99mTc label consistent with themetal bound in a stable complex and the complex covalentlyattached to the antibody.The effects ofantibody derivatization by preformed 'mTc-

N2S2 (pentanoate form) complex conjugation on 9.2.27F(ab')2 were assessed by isoelectric focusing electrophoresis(Fig. 5). The protein mass was detected by Coomassie bluestain, and radioactivity was detected by autoradiography. Nochange in migration after labeling was seen in bands bystaining. However, autoradiography indicated a shift to lowerpI of the radiolabeled species. The covalent attachment of99mTc would be expected to give a charge shift because of thenet -1 charge on the 99mTc-N2S2 complex.

Immunoreactivity was assessed by measuring the fractionof 'mTc radioactivity bound to FMX-met melanoma cells.With 'mTc-N2S2 (pentanoate form) attached to whole9.2.27, a mean immunoreactivity of 77 + 4.0% SD (6preparations) was found. Immunoreactivity appeared to be

Table 1. Radiolabel stability studies of 99mTc-N2S2-9.2.27 F(ab')2by challenge with various agents

99MTc-N2S2 bound to antibody afterinjection, %

Challenge 1 hr 3 hr 9 hr 24 hrControl 98.0 97.8 97.4 9.7C3N2S2* 97.0 97.0 %.2 94.7DTPA 98.1 98.1 97.2 96.5Serumt 100.0 100.0 100.0Urea 98.1 98.2 97.5

*N2S2 propanoate form.tSerum stability was studied with a separate preparation. No changewas observed in 24 hr by HPLC, NaDodSO4/PAGE, or isoelectricfocusing techniques.

0,

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4028 Medical Sciences: Fritzberg et al.

Ln

markers

F(ab')Tc-N2S2 F (ab' ) 2

9 9 serum andF2S2F(ab')2

Proc. Nail. Acad. Sci. USA 85 (1988)

Ln0Dco

FIG. 5. Isoelectric focusing electrophoresis analysis of 99mTc-N2S2-9.2.27 F(ab')2. Lanes show markers and Coomassie blue staining ofantibody F(ab')2, corresponding radioactivity of 991Tc-N2S2-conjugated F(ab')2, and a sample [lanes serum and 9'Tc-N2S2-F(ab')2] in whichthe preparation was mixed 1:1 with serum. The shift in the autoradiography bands compared to protein staining can be attributed to the minuscharge associated with the Tc complex. The isoelectric focusing was wide-gradient pH 3.5-9.5 in a 0.5-mm thick agarose gel (0.9% Isogel, LKB)containing 2.5% (wt/vol) Ampholines (LKB) and 10% sorbitol. Samples applied were 10 ,g of 9.2.27 F(ab')2 and 1:1 serum and labeled F(ab')2diluted 1:9 with phosphate-buffered saline before application. Volumes were 15 A.l. Specific activity of labeled F(ab')2 was 6.9 mCi/mg, andconcentration was 1.8 mg/ml.

comparable or better than that found with previous labelingmethodologies using 9.2.27-e.g., 68% for "'In-DTPA-conjugated (21) and 59% for 1251I and 1311-labeled by thechloramine-T method (22). Cell binding immunoreactivity of99mTc-N2S2-9.2.27 F(ab')2 was 74 ± 4.9% (11 preparations).

In vivo tumor targeting of 99mTc-N2S2-9.2.27 F(ab')2 wasassessed in nude mice bearing melanoma A-375 m/m xeno-grafts. The uptake in tumor and other organs is shown in Fig.6 at 20 hr after injection. The concentration, as shown by thepercent dose per g ofbody weight, was highest in tumor, withgood clearance from all other organs indicated. Also shownfor comparison are concentration values for99mTC-N2S2-NR-2-AD F(ab')2, an irrelevant idiotypic antibody to an individ-ual B-cell lymphoma. Values are somewhat higher in bloodand much lower in tumor for the control antibody fragment.Other organ concentrations are comparable for each. Thelocalization index as indicated by the tumor-to-blood ratio forspecific antibody 9.2.27 divided by the ratio oftumor to bloodfor nonspecific antibody NR-2-AD was 25.2. Images of99mTc-N2S2-9.2.27 F(ab')2 recorded 1, 4, and 16 hr afterinjection are shown in Fig. 7. Also included is an image of99mTc-N2S2-NR-2-AD F(ab')2 recorded 16 hr after injectionfor comparison. Uptake in tumor is seen on early images withmost of body background radioactivity except for kidneysclearing by 16 hr after injection. Kidneys are the primary

1 2

10.]

8 1

6 -

4 -

2 -

BL TA TU SK HE BO LU LI SP ST TH KI IN

TISSUE9.2.27 EM NR-2-AD

FIG. 6. Biodistribution values in percent injected dose per g oftissue with SD error bars of 99mTc-N2S2-9.2.27 F(ab')2 and -NR-2-AD F(ab')2 in mice bearing A-375 m/m xenografts 20 hr after injection.Specific tumor localization is seen by increased tumor uptake ofspecific (9.2.27) compared to irrelevant (NR-2-AD) antibody F(ab')2fragments. The specific tumor localization index was 25.2.

route of excretion. Images of the kidneys are seen intenselyat early times with significant clearing by 16 hr. The irrelevantF(ab')2 showed relatively increased blood pool activity at the16-hr time point.

DISCUSSIONThe results of these studies indicate that it is practical to labelantibodies or their fragments with 'mTc by using well-

hriM) 4 hr (M)

FIG. 7. -y camera images of mice bearing A-375 m/m melanomaxenografts recorded 1, 4, and 16 hr after injection with 9"'Tc-N2S2-9.2.27 F(ab')2 (labeled M) and 16 hr after injection with the irrelevant99mTc-N2S2-NR-2-AD F(ab')2 (labeled I). Uptake in the tumor andclearance from the vascular system is seen over the time periodsshown, with tumor and kidneys visible at 16 hr (M). The irrelevantimage (I) shows a tumor area similar in density to the blood pool,which was developed showing much higher blood background thanspecific antibody images.

LUJLO)

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Proc. Natl. Acad. Sci. USA 85 (1988) 4029

defined chemistry. The chemistry involves forming a stablecomplex with "mTc and subsequently conjugating the com-plex by using the standard active-ester approach under mildconditions. This has been done with a diamide dithiolateligand system. High stability and retention of immunoreac-tivity has been demonstrated for 9'9Tc-labeled antibodiesand their fragments by this approach. Cleavage of F(ab')2 toFab' was not seen, which has been problematic for otherapproaches that involved the reducing agent stannous ion(23). Other work in our laboratory has shown similar labelingresults for Fab or Fab'. The challenge studies of antibodyfragments labeled with the N2S2 complexes indicate that thelabel was stably bound. Together with rapid clearance ofantibody fragments and lack of normal tissue accumulation,optimal imaging can be achieved within periods appropriatefor the short half-life of the isotope. Encouraging results ofimaging sites of metastatic melanoma in patients by using the99'TcN2S2 preformed chelate approach have been reported(24).Rhenium has virtually identical structural properties in its

complexes compared to Tc (25). We have demonstrated thiswith penicillamine, an N,S ligand (26). Application of theN2S2-metal complex conjugation technology for radiother-apy is thus apparent from these studies, since isotopes ofrhenium, "8Re and "mRe, have favorable therapy propertiesbecause oftheir radiations. Preliminary studies of '"Re and18Re indicate that the same N2S2 diamide dithiolate tech-nology is applicable to the rhenium labeling of proteins andgives biodistribution properties that are virtually identical tothose of 99mTc (27).

The efforts of the NeoRx technological staff of both the chemistryand immunology divisions in carrying out the research are greatlyappreciated. The support of National Institutes of Health Grant CA40528 to A.R.F. and National Cancer Institute Contract N01-CM-67719 are gratefully acknowledged.

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