Bioconlugete Chem. 1991, 2, 333-330
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Preparation of Hydrazino-Modified Proteins and Their Use for the Synthesis of 99mTc-ProteinConjugates David A. Schwartz,l*t Michael J. Abrams,+Marguerite M. Hauser: Forrest E. Gaul,? Scott K. Larsen,t Donald Rauh,* and J o n A. Zubietas Johnson Matthey Pharmaceutical Research, 1401 King Road, West Chester, Pennsylvania 19380-1497, McNeil Pharmaceutical Research, Spring House, Pennsylvania 19477-0776, and Department of Chemistry, Syracuse University, Syracuse, New York 132444-4100. Received April 15, 1991
The syntheses and protein linking properties of succinimidyl4-hydrazinobenzoatehydrochloride (SHBH) and succinimidyl 6-hydrazinonicotinate hydrochloride (SHNH), two new heterobifunctional linkers which lead to hydrazino-modified proteins, are described. SHBH-modified proteins are unstable due to the presence of the phenylhydrazine moiety. This problem was overcome by synthesizing the hydrazinopyridine analogue SHNH, and the conjugates derived from this linker are stable. Tc(V) oxo precursors readily add to hydrazinopyridine-modified proteins to yield the desired 99mTc-radiolabeled protein. ggmTc-hydrazinopyridine-polyclonalIgG conjugates are useful agents for the imaging of focal sites of infection.
The use of radioisotopes conjugated to proteins for the diagnosis and therapy of diseases is of great current interest (1,2).A majority of the work has been directed toward conjugation to tumor-associated monoclonal antibodies of a- and 8-emitting isotopes (e.g. 1311, 67Cu,211At, 212Bi,and lsS/lseRe)for therapy of cancer and y-emitting "'In, and -Tc) for its diagnosis. Other isotopes (1311,1231, proteins hae been radiolabeled for the imaging of myocardial infarction (3)and also focal sites of infection ( 4 ) . Various linkers exist for conjugation of these radioisotopes to proteins. All of these are heterobifunctional molecules containing a moiety for chelation of the metal and a moiety for covalent attachment of the ligand backbone to the protein (e.g. an active ester or an isothiocyanate) (5, 6). -Tc is the preferred isotope for scintigraphic imaging applications (7) due to its favorable physical properties, cost, and availability. Rubin et al. (8) have reported the use of llIn-DTPA-polyclonal IgG conjugates for the imaging of focal sites of infection. We were especially interested in the synthesis of wmTc-polyclonal IgG conjugates for the imaging of focal sites of infection. The development of a readily preparable mTc conjugate would lead to a more widely available agent with better radiochemical properties. Previous efforts directed toward the conjugation of h T c to proteins include the use of diethylenetriaminepentaacetic acid (DTPA) (9), N2Sz (IO),and N3S (11) functionalized ligands and direct reduction (12)of the protein disulfide bonds to form free sulfhydryl groups. We initiated a program to develop a novel technology for the conjugation of "Tc to proteins and herein report our results. Nicholson and Zubieta have reported on the reactivity of rhenium(V1 oxo complexes and aromatic hydrazides and hydrazines to form rhenium diazenido species which have been characterized by X-ray crystallography (13). The known rapid reactivity of aromatic hydrazines and the proper rhenium precursor, the Re=N double bond character of the products identified in these crystal structures, which may confer in vivo stability, and the
* To whom correspondence should be addressed. + Johnson
Matthey Pharmaceutical Research.
I Syracuse
University.
* McNeil Pharmaceutical Research.
known reactivity similarities of rhenium and technetium (14) were characteristics which led us to examine the possibility of producing a WTc-aromatic hydrazine protein conjugate linked via a Tc-diazenido bond. The successful exploitation of the above outlined chemistry to produce a WTc-protein conjugate requires several components: (1)synthesis of an aromatic hydrazine linker, (2) efficient conjugation of this linker to a protein and a method for quantification of the modification, (3) stability of the hydrazine-modified protein, (4) choice of the proper technetium-99m precursor, ( 5 ) rapid and efficient radiolabeling, and (6) in vivo stability of the conjugate. EXPERIMENTAL PROCEDURES 'H NMR were recorded in DMSO-d6 on a 300-MHz Bruker AF-300 spectrometer. Elemental analyses were performed a t Atlantic Microlabs, Norcross, GA. The synthesis of SHNH (8) is described in ref 20. 4-[2-( tert-Butoxycarbonyl)hydrazino]benzoic Acid (3). To a stirred solution of 4-hydrazinobenzoic acid (5 g, 32.8 mmol) and triethylamine (9.5 mL, 68.2 mmol) in dimethylformamide (100 mL) was added dropwise a solution of BOC-ON (8.09 g, 32.8 mmol) in dimethylformamide. The reaction mixture was stirred a t room temperature for 3 h. Aqueous hydrochloric acid (10%) was added and subsequently the solution became cloudy. The solution was extracted with ethyl acetate, and the combined organic extracts were washed with water, dried over magnesium sulfate, filtered, and concentrated under reduced pressure to give a brown solid. The solid waa recrystallized from chloroform to give the desired product as a pale brown solid: yield 69% ;lH NMR 6 1.47 (a, 9 H), 6.77 (d, 2 H,Jab = 8.6 Hz), 7.85 (d, 2 H, Jab = 8.6 Hz).Anal. Calcd for C12H16N204: C, 57.13; H, 6.39; N, 11.10. Found: C, 57.02; H, 6.13; N, 11.61. Succinimidyl 4-[2-( tert-Butoxycarbony1)hydrazinolbenzoate (5). To a solution of 4-[2-(tert-butoxycarbonyl)hydrazino]benzoic acid (7.0 g, 27.8 mmol) and N-hydroxysuccinimide (3.19 g, 27.8 mmol) in dioxane (100 mL) was added dropwise a solution of dicyclohexylcarbodiimide (5.72 g, 27.8 mmol) in dioxane (35 mL). The reaction mixture was stirred a t room temperature for 16 h. After ca. 30 min a white precipitate formed (dicyclohexylurea). Acetic acid (0.5 mL) was added and stirring
1043-1802/B1/2B02-0333~02.50/00 lB9l Amerlcan Chemlcal Soclety
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Bloconjwte Chem., Vol. 2, No. 5, 1991
was continued for 1 h. The reaction mixture was filtered to remove the urea byproduct. The filtrate was concentrated under reduced pressure to give a brown solid which was treated with ether, and the solids were isolated by filtration to give a pale brown solid: yield 86% ; mp 185186.5 "C; 1H NMR d 1.47 (s, 9 H), 2.88 (s, 4 H), 6.85 (d, 2 H,J a b = 8.9 Hz), 8.04 (d, 2 H,J a b = 8.9 Hz). Anal. Calcd for C16HlsN306: C, 55.01;H,5.48;N,12.03. Found: C, 55.17;H, 5.84;N, 11.86. Succinimidyl 4-Hydrazinobenzoate Hydrochloride (7, SHBH). To a solution of hydrogen chloride in dioxane (prepared by bubbling hydrogen chloride into dioxane for ca. 5 min) was added succinimidyl 4-[2-(tertbutoxycarbonyl)hydrazino] benzoate (1.5g, 4.3mmol). The reaction mixture was stirred a t room temperature for 5 h. The reaction mixture was never homogeneous; however the color was initially pale brown and over 2 h became orange. The reaction mixture was filtered to give a pale yellow solid: yield 72% ;mp 203.5-205 "C; 'H NMR 6 2.87 ( ~ ,H), 4 7.05 (d, 2 H, J a b = 8.9 Hz), 7.97 (d, 2 H, 8.9 Hz). Anal. Calcd for CllH12ClN304: C, 46.25;H, 4.23;C1,12.40; N, 14.71. Found: C, 46.74;H, 4.38;C1, 12.24; N, 14.26. Protein Modification. A 4 molar excess of freshly dissolved SHNH (30 mM in DMF) was added dropwise to a stirred solution of IgG (1.0g in 20 mL of 0.1 M phosphate buffer, pH 7.8). The solution was stirred gently for 5 h a t room temperature protected from light. This was followed by dialysis against 10 mM citrate, pH 5.2,a t 4 "C (five buffer changes over 24 h). The mixture was then filtered through a 0.2-pm filter and the protein concentration was determined by the Bradford method. The solution was diluted to 5 mg/mL with buffer (100 mM NaCl, 20 mM citrate, 1% mannitol, pH 5.21,divided into 2OO-hL aliquots and stored a t -20 "C. The number of hydrazino groups was determined by converting the hydrazino groups to the corresponding hydrazone by reaction withp-nitrobenzaldehyde(15)and measuring the optical density a t 385 nm (extinction coefficient: 2.53 X lo4 L mol-' cm-9. Typically, approximately three hydrazino groups were present per IgG. Radiolabeling. Twenty millicuries of freshly prepared gsmTc-glucoheptonate (Du Pont Glucoscan kit) was added to 0.5 mL of a solution hydrazino-modified IgG (5.0mg/ mL in citrate buffer, pH 5.2)and the mixture was incubated for 60 min a t room temperature. Radiochemical purity was determined by using ITLC-sg chromatographic strips (Gelman Laboratories, Ann Arbor, MI) with 0.1 M sodium citrate (pH 5.5)as solvent. All preparations had a t least 90% radioactivity bound to the antibody. RESULTS AND DISCUSSION
The synthesis of alkyl hydrazide-protein conjugates has been reported by King et al. (15). They used a novel twostep method for the addition of hydrazide groups to proteins in which the protein is initially modified with an a-bromoacetamide followed by addition of a thiol hydrazide to produce the desired conjugate. Modification of this method for the addition of an aromatic hydrazine is not suitable for radiolabeling of disulfide-containing proteins with technetium-99m, as it has been demonstrated (12) that initial incubation of IgG with thiols (e.g. mercaptoethanol or dithiothreitol) causes nonspecific reduction of some disulfide bonds in the protein. In reactions of these proteins with labile technetium-99m precursors (e.g. 99mTc-glucoheptonate), nonspecific 90mTc binding occurs due to the thiophilic nature of technetium. To test our hypothesis that an aromatic hydrazinemodified protein would react with a wmTc(V) oxo species
Scheme I. Synthesis of 4-Hydrazinobenzoic Acid (SHBH)and 6-Hydrazinonicotinic Acid (SHNH)
2 X=N
3 x=c
s x=c 6 X-N
4 X-N
1
120
,,$
..._.. ........_... .. ...................._.. . ....... ................_.............. ...............
1
': I
0 SHBH p H 5.2 O SHNH p H 5.2 A SHNH p H 6 . 0 ,
,
0 0
..-0
g _ _ _ _ _ _ _ _ _ _ _ A_ - - - - -
-----.-------..-
1
2
Time (weeks)
Figure 1. Stability (4 "C) of 4-hydrazinobenzoic acid modified (SHBH)and 6-hydrazinonicotinicacid modified (SHNH)proteins.
we required a heterobifunctional linker containing crossreactive moieties, i.e. a hydrazine and an active ester. This was accomplished following the example of Keller and Rudinger (16). They synthesized a maleimido-hydrazide linker by the anhydrous acid (HCl(g)/dioxane) deprotection of a [2-(tert-butoxycarbonyl)hydrazido]maleimide leading to the desired stable hydrazinium salt. In our case we examined whether a [2-(tert-butoxycarbonyl)hydrazino] succinimido ester would react similarly under these conditions. Thus 4-[2-(tert-butoxycarbonyl)hydrazinolbenzoic acid (3) was converted to its succinimidyl ester 5 by using standard DCC coupling conditions (Scheme I). The ester was subsequently treated with anhydrous HCl/dioxane, which led to the precipitation and subsequent isolation of the desired hydrazinium salt, succinimidyl 4-hydrazinobenzoate hydrochloride (7,
SHBH). The conjugation of SHBH to polyclonal IgG proceeded smoothly. Addition of a 0.03 M solution of SHBH in DMF (4-24 equiv/mol IgG) to a buffered solution (phosphate, pH 7.8) of polyclongal IgG and subsequent incubation yielded the desired conjugate. Hydrazine substitution was quantified by using the colorimetric assay method of King et al. (15). Stability of the conjugate a t both 4 and -20 "C was poor. This was determined by repeated hydrazine and protein (Bradford assay) analyses. Figure 1 shows the decrease in hydrazine residue substitution over time. This instability is believed to be due to the presence of the phenylhydrazine moieties. Phenylhydrazine itself readily oxidizes in solution and its stability has been studied (17).
Synthesis of sqc-Protein Conjugates
Bioconjugafe Chem., Vol. 2, No. 5, 1991
335
7
6 -
a
m C 5
18.7 mg/mL IgG A 37 mg/mL IgG
I -
3
L
_
2
_
I
4
-
I
6
L
J
8
;O
ShUH (qo!es/moles
1
I
I
12
16
16
18
IqG)
Figure 2. Protein modification efficiency of succinimidyl6-h~-
drazinonicotinate hydrochloride (SHNH)at various linker and protein concentrations.
Jeon and Sawyer have recently reported on the hydroxideinduced synthesis of superoxide from dioxygen and phenylhydrazine, which may explain the loss of hydrazino moieties from the modified protein (18). To circumvent this problem the pyridine analogue was synthesized, as hydrazinopyridine moieties are stable entities, and also the reaction of ReVOC13(PPh3)2and 2-hydrazinophthalazine (a similar heterocycle) yielded stable Re-diazenido structures (13). The synthesis of succinimidyl 6-hydrazinonicotinate hydrochloride (8; SHNH) is outlined in Scheme I. 6-Hydrazinonicotinic acid (2) was synthesized by treating 6-chloronicotinic acid with 85?6 hydrazine hydrate for 4 h a t 80 "C, removal of excess hydrazine, acidification to pH 5.5, and isolation of the precipitated product. The hydrazine moiety was protected as its BOC derivative 4 (di-tert-butyl dicarbonate/Et3N/ DMF), and the succinimidyl ester 6 was synthesized under standard DCC coupling conditions (N-hydroxysuccinimide/DCC/DMF). Conversion of the BOC active ester to its hydrazinium salt under anhydrous acid conditions smoothly led to the desired product, 8 (SHNH). Conjugation of SHNH to polyclonal human IgG as described above gave the desired hydrazinopyridinemodified protein. Figure 2 describes the conjugation efficiency a t different linker and protein concentrations. As expected conjugation efficiency increased with increasing protein concentration. Over modification led to precipitation of the protein (molar substitution ratio (MSR) > 10). A t 35 mg/mL protein concentration and a 4-fold excess of linker the conjugation yield was >70% (MSR 2.8-3.0 hydrazine groups/IgG) with excellent protein recovery (>90% ). Treatment of the protein with 6-hydrazinonicotinic acid, the nonlinkable equivalent, gave negligible modification as determined by the hydrazone assay. Hydrazinopyridine-modified protein stored a t 4 "C showed no loss of hydrazine residues over several months and storage a t -20 "C gave no loss of hydrazine residues or protein precipitation over 13 months. The modified protein could be lyophilized and reconstituted without event. The choice of the proper technetium species for conjugation was critical. As predicted, a technetium(V) oxo species was required. 99mTc-glucoheptanoate which is produced by the stannous chloride reduction of pertechnetate in the presence of sodium glucoheptonate was examined initially. Characterization of this species has not been established unambiguously, but it is known to possess a Tc=O bond (IR v 950 cm-I) (19). Incubation of the modified protein (10 mM; 2-3 hydrazines/IgG) with 99mTc-glucoheptonate for 45-60 min a t room tempera-
1 r
4"
*
Y I
_
Figure 3. y-camera images of a group of rats with Lscherichia coli deep thigh infections: upper left quadrant, %"Tc-labeled
human polyclonal human IgG at 4 h; upper right and lower left quadrants, at 24 h; lower right quadrant, l*lIn-labelled human polyclonal IgG. ture gave >95 % technetium incorporation a t pH 5.2-6.2. In a control experiment polyclonal IgG was treated with 6-hydrazinonicotinic acid for 6 h and subsequently dialyzed (acetate buffer, pH 5.2). On addition of 99mTc-glucoheptonate there was 90 % radiochemical yield in 1 h a t room temperature. 99mTc-MAG-3(22) which is a Tc(V) oxo complex with an N2S2 donor set did not react with the hydrazino-modified protein. Incubation of hydrazino-modified polyclonal IgG with other 9 9 m T precursors ~ yielded little or no radioincorporation, e.g. Tc-pyrophosphate (28 9; radioincorporation), Tc-methylenediphosphonate (23) (14 5% ), pertechnetate (O%), and Tc(1II)DMSA (0%). The stability of the Tc-hydrazino-protein conjugate was studied by incubation of the conjugate with various chelators and human serum a t 37 "C for 24 h. The radiolabeled protein conjugate retained its radiolabel in the presence of DTPA (987; retention of label as determined by HPLC and ITLC analyses), sodium diethyldithiocarbamate (98 % ) or L-cysteine (92 5% ) (all 10 mM in PBS, pH 7.4), and human serum (95%). Imaging of focal sites of infection with the wmTc-hydrazinopyridine-polyclonal IgG conjugate gave images of equal quality to ll'In-DTPApolyclonal IgG conjugates, demonstrating the in vivo stability of the radiolabeled conjugate (see Figure 3). Biodistributions of the two conjugates were also similar (24). The exact nature of the bonding between the T c moiety and the hydrazinopyridine modified protein has not been unequivocally established. Abrams et al. (25)have recently isolated, characterized, and determined the crystal structure of [TcC12(C8N&) (PPh3)2]*0.75C7H8and demonstrated that it is a technetium-hydralazine complex. This structure shows the presence of a Tc=N bond (1.767 A), demonstrating the multiple bond character of this bond. A 99"Tc-hydrazinopyridine-modified conjugate of fragment E l , a protein isolated from digests of blood clots
336 Bbconjugate Chem., Vol. 2, No. 5, 1991
which has been shown to localize in blood clots in vivo, was prepared. This conjugate successfully imaged thrombi in rabbits and dogs (26). In summary, we have developed a novel linker for the ready modification of proteins with hydrazino groups and demonstrated the ability of these hydrazino-modified proteins to react with gsmTc-glucopheptonate. These radiolabeled proteins have demonstrated in vivo stability and localization in two in vivo models. The characterization of the protein-Tc bonding and the further utility of this linker and other hydrazinopyridine analogues are areas of active interest in our laboratories. ACKNOWLEDGMENT We thank Drs. G. Henson and R. Brooks for helpful discussions. We also thank Drs. H. Solomon, P. Kramer, A. J. Fischman, R. Callahan, A. Fuccello, L. Knight, and Mr. D.Reixinger for their collaborations. LITERATURE CITED (1) Schlom, J. (1986) Basic Principles and Applications of Monoclonal Antibodies in the Management of Carcinomas. Cancer Res. 46, 3225. Cobb, L. M., and Humm, J. L. (1986) Radio-
immunotherapy of malignancy using antibody targeted radionuclides. Br. J. Cancer 54, 863. (2) Rosenblum, M. G., Murray, J. L., Lamki, L., David, G., and Carlo, D. (1987) Comparative clinical pharmacology of ll1Inlabeled murine monoclonal antibodies. Cancer Chemother. Pharmacol. 20, 41. (3) Goethals, P., Coene, M., Slegers, G., Vogelaers, D., Everaert, J., Lemahieu, I., Colardyn, F., and Heyndrickx, G. R. (1990) Production of carrier-free gallium-66 and labeling of antimyosin antibody for positron imaging of acute myocardial. Eur. J. Nucl. Med. 16, 237. (4) Rubin, R. H., Young, L. S., Hansen, W. P., Nedelman, M., Wilkinson, R., Nelles, M. J., Callahan, R., Khaw, B.-A., and Strauss, H. W. (1988) Specific and Nonspecific Imaging of lacaliszed Fisher Immunotype 1 Pseudomonas aeurginosa Infection with Radiolabeled Monoclonal Antibody. J.Nucl. Med. 29, 651. (5) Meares, C. F. (1986) Chelating Agents for the Binding of Metal Ions to Antibodies. Nucl. Med. Biol. 13, 311. (6) Hnatowich, D. J., Layne, W. W., Childs, R. L., Lanteinge, D., Davis, M. A., Griffin, T. W., and Doherty, P. W. (1983) Radioactive Labeling of Antibody: A Simple and Efficient Method. Science 220, 613. (7) Pinkerton, T. C., Desilets, C. P., Hoch, D. J., Mikelsons, M. V., and Wilson, G. M. (1985) Bioinorganic Activity of Technetium Radiopharmaceuticals. J. Chem. Educ. 62, 965. (8) Rubin, R. H., Fischman, A. J., Needleman, M., Wilkinson, R., Callahan, R. J., Khaw, B.-A., Hansen, W. P., Kramer, P. B., and Stauss, H. W. (1989)Radiolabeled, Nonspecific, Polyclonal Human Immunoglobulin in the Detection of Focal Inflammation by Scintigraphy: Comparison with Gallium-67 Citrate and Technetium-99m-LabeledAlbumin. J.Nucl. Med. 30, 385. (9) Lanteingne, D., and Hnatowich, D. (1984) The Labeling of DTPA-coupled Proteins with ~ T cZnt. . J. Radiat. Zsot. 35, 617.
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(10) Rao,T. N., Adhikesavalu,D., Camerman, A., andFritzberg, A. R. (1990) Technetium(V) and Rhenium(V) Complexes of 2,3-Bis(mercaptoacetamide)propionate.Chelate Ring Stereochemistry and Influence on Chemical and Biological Properties. J. Am. Chem. SOC.112, 5798. (11) Fritzberg, A. R., Kasina, S., Vanderheyden, J. L., and Srinivasan,A. (1988)Metal-radionuclide-labeled proteins and glycoproteins and their preparation for diagnosis and therapy. Eur. Pat. Appl. EP 284071. (12) Mather, S. J., and Ellison, D. (1990) Reduction-Mediated Technetium-99mLabeling of MonoclonalAntibodies. J.Nucl. Med. 31, 692. (13) Nicholson, T., and Zubieta, J. (1988) Complexes of Rhenium with Benzoylazo and Related Ligands. Polyhedron 7 , 171. (14) Cotton, F. A., and Wilkinson, G. (1988)Advanced Inorganic Chemistry, 5th ed., p 847, John Wiley and Sons, Inc., New York. (15) King, T. P., Zhao, S. W., and Lam, T. (1986) Preparation of Protein Conjugates via Intermolecular Hydrazone Linkage. Biochemistry 25, 5774. (16) Keller, O., and Rudinger, J. (1975) Preparation and Some Properties of Maleimido Acids and Maleoyl Derivatives of Peptides. Helv. Chim. Acta 58, 531. (17) Vulterin, J., and Zyka, J. (1963) Investigation of Some Hydrazine Derivatives as Reductiometric Titrants. Talanta 10, 891. (18) Jeon, S., and Sawyer, D. T. (1990) Hydroxide-Induced Synthesis of the Superoxide Ion from Dioxygen and Aniline, Hydroxylamine or Hydrazine. Znorg. Chem. 29,4612. (19) Kieviet, W. (1981) Technetium radiopharmaceuticals: chemical characterization and tissue distribution of Tc-glucoheptonate using Tc-99m and carrier Tc-99. J.Nucl. Med. 22, 703. (20) Pak, K. Y., Dean, R. T., Mattis, J. A., Buttram, S., and Lister-James,J. (1988)Method for Labelling Antibodies with a Metal Ion. International Patent Appl. PCT/US88/01048. (21) Colombo,F., Matarrese, M., Bugaj, J.,Gerundini, P., Fazio, F., and Deutsch, E. (1988) Tc-99m Complexes of a,a-Disubstituted Hydroxy a-Carboxylic Acids. J. Nucl. Med. 29, (Supplement) 934. (22) Nosco, D., and Verbruggen, A. (1990) Technetium-99m complex for examining renal function. US4925650. (23) Martin, J. L., Yuan, J., Lunte, C. E., Elder, R. C., Heineman, W. R., and Deutch, E. (1989) Technetium-Diphwphonate Skeletal Imaging Agents: EXAFS Structural Studies in Aqueous Solution. Znorg. Chem. 28, 2901. (24) Abrams, M. J., Juweid, M., tenKate, C. I., Schwartz, D. A., Hauser, M. M., Gaul, F. E., Fuccello,A. J.,Rubin,R. H., Straw, H. W., and Fischman, A. J. (1990) Technetium-99m-Human Polyclonal IgG Radiolabeled via the Hydrazino Nicotinamide Derivative for Imaging Focal Sites of Infection in Rate. J. Nucl. Med. 31, 2022. (25) Abrams, M. J.,Larsen, S. K.,and Zubieta,J. (1990)Synthesis and Crystal and Molecular Structure of a Technetium-Hydralazino Complex, [TcCl*(CeHsN1)(PPhs)n].0,75C,Hs. Znorg. Chim. Acta 173, 133. (26) Knight, L. C., Abrams, M. J., Schwartz, D. A,, Hauser, M. M., Kollman, M., Zaydenberg, I., Gaul, F. E., and Maurer, A. H. (1990) Tc-99m Labeling of Fragment E l for Thrombus Imaging. J. Nucl. Med. 31 (Suppl.), 776.
