[CANCER RESEARCH 40, 3036-3042. August 1980]0008-5472/80/0040-OOOOS02.00

Radiolabeling of Antibodies1

William C. Eckelman, Chang H. Paik, and Richard C. Reba

Section of Radiopharmaceutical Chemistry, George Washington University Medical Center. Washington. D. C 20037

Abstract

The radiolabeling of antibodies is considered in terms of thechoice of radionuclide, the method of conjugation, and theeffect of conjugation on plasma clearance, lodination techniques are reviewed, but the major emphasis is placed on themethods of conjugating metallic radionuclides using bifunc-

tional chelating agents. The technique of producing clinicallyuseful radiolabeled antibodies by balancing altered substratespecificity caused by radiolabeling against accelerated plasmaclearance is discussed.

lodinated proteins have been used since the production ofradioisotopes of iodine in the early 1940's. The first use of 131I-

labeled proteins of high specific activity was in the insulinradioassay by Berson ef al. (6). Procedures for labeling proteins with other radionuclides were introduced at a much laterdate. Radiolabeled proteins can be divided into 2 groupsaccording to use. Those that have been used as in vivo imagingagents are relatively unsophisticated radiochemicals. For instance, many 99mTc-HSA2 preparations are clinically useful

vascular pool agents, yet in fact they contain large percentagesof radiolabeled small colloid (44). Since the radiolabeled colloidis cleared from the blood relatively slowly in relation to theduration of the clinical observation, this impure radiopharma-

ceutical serves the practical purpose of the blood pool agent.In addition, some radiopharmaceuticals have as their mechanism of localization the clearance of foreign substances fromthe body and therefore do not have strict structural requirements (16). These 2 factors have minimized the requirementfor biological, biochemical, physiological, and immunologicalintegrity. Much of the current use of in vivo radiotracers hasbeen through the pragmatic exploitation of easily producedand readily available combinations of radionuclide and substrate.

On the other hand, the in vitro use of radiotracers for radioim-

munoassay and radioreceptor assay demands a critical controlof the biological and immunological properties of the radiotra-

cer.The recent interest to study pharmacology, biochemistry,

and immunology in vivo has created a difficult challenge in newdrug design. The biological and pharmacological propertiesmust be as rigidly maintained as those required for the in vitrouses. Additionally, the target-to-nontarget distribution must be

sufficient to allow identification of the target structure by theuse of external detection. In conventional drug design, "themagic bullet" has been the goal, but in fact high blood levels

1Presented at the UICC Workshop on Radioimmunodetection of Cancer. July

19 to 21, 1979, Lexington. Ky. Supported in part by Grant CA 18675 awardedby the National Cancer Institute and Grant HL 19127 awarded by the Heart. Lungand Blood Institute.

' The abbreviations used are: HSA. human serum albumin; DTPA, diethyl-enetriaminepentaacetic acid; IDA, iminodiacetic acid; DTTA. diethylenetriamine-tetraacetic acid.

of drug have been the most expedient method for producinghigh concentration in the target structure. In the design ofdiagnostic radiopharmaceuticals, this latter approach is notpossible.

Choice of Radionuclide

The choice of radionuclide is dependent on its nuclear properties, including its physical half-life, its production factors, theavailable imaging device, and the effective half-life of the la

beled radiopharmaceutical (54). Most commercially availablescintillation cameras are designed to maximize the detectionefficiency and resolution of y-ray energy with a range of 100 to250 keV. y-Rays in this energy range have satisfactory tissuepenetration [the whole-body absorbed fraction for radiationdistributed in the whole body is 37% for 100-keV and 34% for250-keV y-rays (50)], but the energy is low enough to be easilycollimated. A 0.5-inch-thick sodium iodide crystal can absorbmore than 90% of 140-keV y-rays incident on the crystalsurface but less than 20% of 500-keV y-rays (3). The most

widely used radionuclides and those with the highest usefulphoton yield per absorbed radiation dose are 99mTc, 123I,131I,111In, and 67Ga. Depending on the effective half-life in blood

and the time of the function to be measured, in some instances,the physical half-life of the radionuclides 99mTc(6 hr) and 123I

(13 hr) may be too short to be used.A major consideration for the use of any radiolabeled com

pound in humans is the radiation dose to the patient. Most ofthe radionuclides are chosen because the equilibrium absorbeddose to the subject is small compared to the number of usefuly-rays. Because of this, many 13tl radiopharmaceuticals have

been replaced by a radiotechnetium compound. The absorbeddose constant (A,) is given for 5 radionuclides: 67Ga, 99mTc,11'In, 123I,and 131I(Table 1) (12). The absorbed dose constants

differ by approximately a factor of 6. However, depending onthe effective half-life, this difference could be enlarged consid

erably. The upper limit of radioactivity that can be injected willdepend on the equilibrium absorbed dose constant to someextent but probably to a greater degree on the effective half-life. If the biological half-life is longer than the physical half-life,then the short-lived radionuclides 67Ga, 99mTc, 123I, and '"In

would have a definite advantage. Radioiodide, the ultimatemetabolite of iodinated proteins, does have a long biologicalhalf-life (Table 2) (5).

Ideal Properties of the Radiolabeled Substrate

The ideal properties of a radiolabeled substrate are notuniversally agreed upon. The expression "tracer" is used

often, but this term should be reserved for those instanceswhen the biochemical and immunological properties of theradiolabeled substrate are identical to the parent compound.In this circumstance, the biochemical and immunological properties of the radiopharmaceutical will be identical to the parentcompound.

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Radiolabeling of Antibodies

Table 1

Relative dosimetry of radionuclides, and major sources of equilibrium absorbeddose for <"Ga."Radiation123l(f,/2

-13.0hr)°YK

electronK-X-rayK-X-rayK-X-rayAuger131l(f,/2

=8.06days)cftAÕ4Y»K-iceYi2TÕ4"1ln(t,

2 = 2.81days)dYIK-toeY2K-iceK-aX-rayK-aX-rayTcif,

2 =6hr)eYK

electron67Ga(f,/2

= 78.1hr)'YK

electronYYYY"Te.

'"In.'"/,A/,"0.830.130.470.240.132.190.070.900.060.820.010.060.020.890.090.940.050.470.240.880.090.380.290.240.020.160.043

N„mean number per disintegration; £,,meanA,,

equilibrium absorbed dose content ing-rad/fiCi6A, =0.365g.rad/(iCi-hr.cA, = 1.22g.rad//iCi-hr."A,

=0.90g.rad//iCi-hr.8A, =0.286g-rad/fiCi-hr.'A, =0.369g-rad//iCi-hr.9y-Ray radiation used in imaging.and

'31;£,0.1

5990.1270.0270.0270.0310.0010.0960.1920.2840.36490.3300.6370.7230.1

7290.1450.24790.2200.0230.0230.1

4090.1190.09390.0840.1

8590.2090.30090.394energy

in•hr.A,0.2830.0360.0270.0140.0080.0040.010.370.030.640.010.090.030.330.030.490.020.020.010.2630.0230.0750.0500.0940.0110.1030.036MeV

per particle;

Table 2Iodide pharmacokinetics"

% of administered dose

SourceorganWhole-body

excretionLiverIntestineBloodStomach1

hr7.91.415.414.713.66hr37.60.99.49.08.324hr76.10.21.61.61.420days87.10.2

Taken from Ref. 5.

Proteins in general are cleared from the blood slowly so that,when a radioactive protein is the substrate, the detection ofthe target radioactivity is difficult. However, for a substrate tobe useful, strict adherence to the requirement that all propertiesbe identical to the parent structure may not be necessary sincean accelerated blood clearance would be a desirable feature.In many individual instances, we prefer selected criteria, suchas the preceding example, to be more appropriate, and inparticular we believe this deviation from a perfect biologicaltracer to be an important strategy in the design of radiolabeledantibodies for tumor detection.

One example of the improved clinical efficacy which mayoccur between a useful radiopharmaceutical and a true tracer

is iodinated fibrinogen (23). If fibrinogen is iodinated under mildconditions at 0.5 iodine atom/molecule, the isotopie clottingability is 80%, but the blood clearance is slow (f1/2 of the latecomponent, 52 hr) and the average thrombus:blood ratio is 24at 24 hr. This preparation appears to be a satisfactory tracerof fibrinogen but a poor radiopharmaceutical when comparedto heavily iodinated fibrinogen. When fibrinogen is iodinated at25 iodine atoms/molecule, the isotopie clotting ability is reduced to 71 to 73%, but the blood clearance is faster (f1/2, 28hr for the ICI method) and the thrombus to blood ratio is 33 to50. The heavily iodinated fibrinogen is less of an ideal tracerbut a better radiopharmaceutical.

Conjugation of the Radionuclide to the Protein

Iodine

The chemical properties of iodine are a result of the decreasing potential, the larger atomic radii, the larger van der Waalsforces, and the increased polarizability found as the atomicnumber increases in the Group VII congeners (11 ).

Of the halogens, iodine is most likely to support a positivecharge and thus is the least reactive toward electrophilic addition or substitution. Iodine also forms the weakest bonds tocarbon (=60 kcal/mol for aromatic carbon-iodine bonds). TheI* ion does not exist alone but usually forms a complex with a

nucleophilic species such as water or pyridine. In electrophilicsubstitution, the most reactive aromatic compound is phenol,followed by aniline, methoxybenzene, and imidazole. The aniónof phenol seems to be the reactive species so that closeattention must be paid to the pKa value of the target structureand the pH of the reaction.

The iodinating agents most widely used to produce electrophilic addition or substitution are: iodine (10); iodine monoch-loride (37); chloramine-T (28); lactoperoxidase (38); electroly

sis (32); and prelabeled activated ligands (7).Iodination Using Iodine. The reaction mechanism for the

electrophilic aromatic substitution has been studied extensively. Reactions with phenols and imidazole generally showan isotope effect, indicating that the rate-determining step

involves the removal of hydrogen from the intermediate. In thiscase, the rate of catalysis seems to follow the nucleophilicitiesof the base used. Where no isotope effect is observed, as inthe iodination of dimethylaminobenzenesulfonic acid, the rateof reaction appears to be related to the basicity of the catalyst.This suggests that the formation of a complex iodinating agentmay be involved in a rate-determining step.

Later papers by Grovenstein ef al. (22) showed a definiteeffect of iodide on the rate of iodination. This effect should notbe observed unless l? is the reactive species.

O O OH

K,

These same authors observed that, as the iodide concentration decreased, the kinetic hydrogen isotope effect has alsodecreased, and the first step involving the attack of the iodinebecame rate determining. As many workers have suggested,at high concentrations of iodide, the removal of hydrogenbecame the rate-determining step.

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W. C. Eckelman et al.

In radioiodination, one-half of the radioisótopo is convertedto the iodide chemical form after the reaction of the positiveiodinating species. This limits the yield considerably.

lodination Using Iodine Monochloride. MacFarland (37)showed that ICI increased the iodination efficiency as compared to \2 solutions because all iodine atoms can be incorporated. Radioiodine is mixed with ICI, and then this solution isadded to the target molecule. Since carrier ICI is added, thespecific activity of the product is lower than that obtained withchloramine-T. Lambrecht ef al. (34) have proposed a recoilmethod for preparing carrier-free radioiodinated ICI using chlo

rine, but this is not in common use because of the complicatedtechniques involved.

The mechanism of this reaction has been studied by Berliner(4). He suggested that either H2OI+ or ICI might be the electro-

phile in the rate-determining step. By studying the reverse

reaction, i.e., deiodination, Batts and Gold (2) showed that thechloride concentration affected the rate of reaction and therefore that ICI must be the electrophile. The deiodination wasalso acid catalyzed, and the iodine released could be trappedby either chloride or dimedone. A substantial catalysis bychloride was observed, but the deiodination rate approached0 as the pH approached 7.

lodination Using Chloramine-T. Chloramine-T, the sodiumsalt of A/-chloro-p-toluenesulfonamide, has been used as an

oxidant in analytical chemistry since its discovery by F. D.Chattaway in 1905. At present, it is not widely used becausethe advantages over a standard iodine solution are few. It hasbeen used to oxidize thioglycolic acid and thioacetamide andtherefore could affect the target molecule if used in excess.Chloramine-T is unstable in light, but a 0.05 M solution in water

decreased in oxidizing power by only 0.02%/month whenstored in the dark. The major equilibria of chloramine-T asgiven by Jennings (29) are:

RNCINa = RNCr + Na*RNCr + H+ = RNCIH

2 RNCIH = RNCI2 + RNH2RNCI2 + H2O = RNHCI + HOCIRNHCI + H2O = RNH2 + HOCIHOCI= H++ ocr

Ka = 2.38 X 10~3K, = 6.1 X 1CT2Kft = 8 X 10~7K„= 4.9 X 10"8

Ka = 3.3 x 1CT8

The acid and RNCI2 exist at pH 1; at pH 2.6, the concentration of RNCr and RNCIH is equal; and at pH 4 RNCI~ predom

inates. The rate of reaction for the hydrolysis at pH 3.7 and25°is 10.10 liters/mol/sec, and the reverse reaction is 215

liters/mol/sec.In addition to being a powerful oxidizing agent, chloramine-

T can also act as a chlorinating agent in electrophilic substitution of aromatic molecules. Huguchi and Hussain (26) describethe active species as the N,A/-dichloro-p-toluenesulfonamide(dichloramine-T). The rate of disproportionate to producedichloramine-T at pH 6.4 is 1.3 liters/mol/sec and at pH 6.9

is 0.40 liter/mol/sec. When the amide was added to thereaction, the rate of disproportionation dropped to one-half of

the expected value. Furthermore, hypochlorous acid reactsmore slowly than does dichloramine-T under comparable conditions using p-cresol. This suggests that dichloramine-T is

probably the active chlorinating agent and not HOCI as previously proposed.

When phenol is used as the target molecule and the chlora-mine-T:phenol ratio is 10, chlorophenol was found in the re

action mixture after 3 min in a yield of 10 to 19% in the pHrange of 9.6 to 6.5. In the presence of radioiodide, the averageyield of iodophenol based on radioiodine was 50%, whereasthe average yield of chlorophenol based on phenol was about10%. In attempts to produce iodinated molecules with maximum specific activity, equimolar amounts of chloramine-T,

iodide, and target molecule may produce mixed chloroiodocompounds which would be difficult to separate and identifyfrom the desired radioiodine compounds. Chloramine-T acts

not only as an oxidizing agent for sodium iodide but also as anoxidizing agent and a chlorinating agent for the target molecule.3

The use of chloramine-T has been discouraged because of

these possible side reactions. Nevertheless, use of appropriatemolar ratios can produce satisfactory products even with themost sensitive target molecules. Freychet ef al. (18) havereported that either equimolar amounts of chloramine-T orchloramine-T added in small aliquots produces radiolabeled

insulin which retains its biological activity. Eckelman ef al. (15)report that bleomycin can be radiolabeled with the chloramine-

T method and that this sensitive molecule retains its antibacterial activity after iodination.

To use chloramine-T, the target molecule must contain a

tyrosine or imidazole moiety. The major advantage of iodinationusing chloramine-T is the simplicity and the reactivity whichallows the rapid preparation of high-specific-activity products.

lodination Using Lactoperoxidase. Iodide can be enzymat-ically oxidized to active iodine (38). Immunoglobulin and bovineserum albumin were iodinated to low specific activity; later,Thorell and Johansson (53) improved the method to preparehigh-specific-activity iodinated polypeptides and proteins. This

method is probably less destructive to the target molecule;however, the usual problems when dealing with enzymes arepresent, such as purification, determination of enzymatic activity, and separation from the reaction mixture. Carrier-free io

dide can be used, and therefore high specific activities areobtained. The lactoperoxidase can be immobilized on a Seph-arose column to prevent contamination of the radiopharmaceu-

tical by the enzyme.lodination Using Electrolysis. Electrolytic oxidation of iodide

has been suggested for the preparation of iodinated molecules(32). Although the reaction conditions are generally mild, carrier iodine is needed, and the reaction volumes are usuallylarger than that needed for other methods. Recently, Miller andWatkins (41) proposed a method of electrolytic iodination inacetonitrile that allows the iodination of molecules as unreactiveto electrophilic substitution as ethyl benzoate. This reactionapparently proceeds through a nitrogen iodine intermediate.

lodination Using Prelabeled Ligands. Bolton and Hunter (7)have proposed iodinated 3-(4-hydroxyphenyl)propionic acidN-hydroxysuccinimide ester as an indirect iodinating reagent

which avoids many of the problems of direct iodination. In thisindirect method of iodination, the active ester is iodinated andpurified from oxidizing and reducing agents before being mixedwith the target molecule. In this way, the impurities in the iodidesolution, the oxidizing agents such as chloramine-T, and thereducing agents such as metabisulfate do not come into contact with the target molecule. The reaction results in the formation of an amide bond with the lysine groups of the target

3 V. Jiang and W. C. Eckelman. unpublished observations.

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molecule. This allows the iodination of molecules that do notcontain tyrosine or with biological activity that might be loweredby alteration at the tyrosine moiety rather than at the lysinemoiety. The yield is usually lower than that obtained withchloramine-T because of competitive hydrolysis of the activeester. Although the Bolton-Hunter reagent is the most popular,

2 other prelabeled ligands, iodoaniline (24) which is convertedto a reactive diazonium ion and methyl-3,5-diiodo-p-hydroxy-

benzimidate (56), have been described as being equally effective reagents. This indirect iodination has been used for sometime in the preparation of iodinated tracers in radioimmunoas-

say. Iodinated histamine, tyramine, and tyrosine methyl estercan be reacted with carboxymethyl groups to produce aniodinated derivative (27).

Purification. In the past, high specific activities were attainedby using one or more iodine molecules per target molecule.Discouraged by the evidence that heavily iodinated species arepresent even at low molar ratios (49) and that chloro compounds might be present with chloramine-T and encouragedby the refinements of high-pressure liquid chromatography,most investigators iodinate low-molecular-weight compounds

at high target: iodide ratios and then separate the iodinatedtarget molecule to attain maximum specific activity. This reduces the possibility of producing heavily iodinated species.This specification is not always possible with radiolabeledproteins. Assuming that there is 1 iodine atom/molecule andthat the radioisotopes are carrier free, for 126I,the maximumtheoretical specific activity is 2200 Ci/mmol; for 131I, it is16,000 Ci/mmol; and for 123I it is 236,000 Ci/mmol. Thisspecific activity for 131I is not reached because as much as80% of the iodide is 127I.

Indium, Gallium, and Technetium

Direct labeling of a protein with a metallic radionuclidesuffers from 2 weaknesses (16): (a) the native functional groupsof the protein may be needed to interact with the active biological site; e.g., the functional groups may be needed for theantigen-antibody reaction. Should the radiolabel interfere with

this, the normal behavior of the molecule will be altered, andtracer studies will not be accurate; (b) the radionuclide maybind to the protein with insufficient affinity to produce a stablebond. Again, it will not be possible to follow the desired truebehavior of the labeled compound.

The importance of both of these factors is exemplified byradiolabeled bleomycin. Bleomycin is a mixture of closely related antibiotics that have been used successfully to treat avariety of cancers. Chemically, this antibiotic acts as a chelatingagent and has been shown to bind a number of divalent andtrivalent cations but with varying affinities (47). Although bleomycin chelates of indium, gallium, and copper have not demonstrated the necessary in vivo and in vitro stability, the cobaltbleomycin bond is more stable (46). Ideally, a stable techne-tium-labeled bleomycin would be the most efficacious complex.

However, inspection of the bleomycin structure indicates thatthe disaccharide moiety is the most likely chelating group but,unfortunately, at neutral pH this is a low affinity site for "Tc.

That this is so is based on work by Richards and Steigman(48), who demonstrated that the sugar moiety has a high affinityfor 99mTc at pH 10 to 12 but a weak affinity at neutral pH.

Therefore, the use of the native functional groups of bleomycin

to bind a tracer such as technetium results in a weak chelatewith poor stability.

Another factor for consideration is the change in biologicalactivity of a drug or biological derivative secondary to theaddition of a radiolabel. It has been shown that the chelation ofcopper to bleomycin destroys its ability to cleave strands ofDMA (1 ). When labeled with cobalt, the antibacterial activity ofbleomycin is diminished and becomes negligible when testedagainst the usually responsive Bacillus subtilis ATCC 6633(15). In this instance, the cobalt appears to alter the biologicaleffectiveness of the bleomycin because of its bond to thefunctional groups responsible for maintaining the antibioticintegrity of the parent drug.

Derivatization of the protein by linking a chelating agent to aspecific site on the protein may eliminate these problems.Goodwin ef al. (21) and Sundberg ef al. (51) synthesized anEDTA derivative containing a diazonium group [1-(p-benzene-diazonium)ethylenediamine-A/,/V,/V',A/'-tetraacetic acid]. This

chelating agent could be reacted with a molecule containingan activated benzene ring, i.e., phenol or aniline, and chelatederivatives of fibrinogen, albumin, and bleomycin have beenconstructed. These compounds were subsequently labeledwith111ln.

In other work, Heindel ef al. (25) prepared a series ofstructural analogs of tolbutamide, a drug known to stimulatethe synthesis and release of insulin by the pancreas. Thederivatives contained an /V,A/-dimethylaminoethylaminoethyl,an aminoethylaminoethyl, or a 3-carbethoxymethyl-1-toluene-

sulfonylurea derivative. To date, no practical applications forimaging have resulted from this research.

To determine whether biological fatty acids could be used totransport technetium for myocardial scintigraphy, fatty acidand long-chain hydrocarbon analogs containing a strong che

lating group were evaluated (14, 31). The chelating agentsinvolved in this investigation included DTPA, EDTA, and dieth-

ylenetriamine. DTPA was chosen as one of these agents sinceit is known to form stable technetium chelates both in vivo andin vitro. Furthermore, DTPA offers the advantages of allowingone to incorporate a well-defined structural molecule to the

chosen biological molecule. This chelate also occupies the 6coordinate sites of technetium, lessening the possibility ofbinuclear complexes found with chelating agents possessingfewer sites. Finally, the use of DTPA eliminates the possibleformation of bis compounds which may exist when a largeexcess of tridentate ligand is present.

An attempt to trace amino acid metabolism with a derivativewhich could firmly bind technetium has been reported byCastronovo et al. (9); this synthetic amino acid contained aphosphonic acid group. However, no successful clinical application has been obtained.

More recently, Gallery ef al. (8) and Loberg ef al. (36) haveproduced an analog of the myocardial antiarrythmic drug,lidocaine. Their chelating agent was IDA which characteristically binds metals strongly and easily reacts with functionalgroups on the biologically active molecule. To produce thislidocaine analog, IDA was reacted with io-chloro-u)-2,6-dime-

thylacetanilide. However, IDA is not a generally applicablechelating agent. Fields ef al. (17) have observed that the pKaof the nitrogen of an IDA derivative is crucial to the productionof a single radiochemical. If the pKa is approximately 6, radi-

ochemically pure technetium chelates can be prepared in

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aqueous solution, e.g. 99mTc-A/-(2,6-dimethylphenylcarbam-

oylmethyOiminodiacetic acid (Tc-HIDA). With higher pKa values,

other radiochemicals in addition to the bis chelate structure areobserved.

Many chemical reactions have been prepared to link chelat-

ing groups to proteins. Leung et al. (35) and Meares ef al. (40)used the diazonium reaction at pH 7 to 10. This resulted inbonds primarily to lysine but with some reaction with thehistidine moiety. Paik et al. (42) investigated the pH dependence of the diazonium reaction and observed increasing yieldsof diazohistidine compounds (=60%) as the pH rose to 11.5.They therefore reacted a diazo chelating agent with HSA toproduce a protein derivatized at histidine and lysine. Becauseof the high pH required for linking using the diazonium reaction,Goodwin ef al. (20) have suggested alkylation reactions thattake place at neutral pH. Presumably, these agents react onlywith sulfhydryl bonds in the protein. Recently, Paik ef al. (43)have suggested amidination as another method of conjugationthat produces a specific reagent for a single amino acid, lysine.Both of these methods were based on the hypothesis that theabsence of multiple sites of conjugation would simplify theblood clearance curve. Glutaraldehyde linkage has been usedby Pritchard ef al. (45) to prepare derivatives of transferrin.Glutaraldehyde is relatively nonspecific, causing cross-linking

and transferrin polymers. As a result, Ternynck and Avrameas(52) proposed that benzophenone could be reacted with onesubstrate at pH 6, that the conjugate could be purified, andthat the second substrate could be coupled at higher pH. Thisprevents the problems associated with glutaraldehyde coupling. Finally, the anhydride of DTPA has been used to preparebifunctional chelates (33, 55). The chemical structure of thisspecies is not well defined and may be a polymeric chelatingagent (30). None of the conjugation methods have been fullyvalidated.

The correct method of conjugation has not been defined.The ideal would be to use a technique which would properlybalance the degree of altered protein specificity (e.g., antibodybinding to antigen caused by modification) against acceleratedplasma clearance. A major reason for using chelating agentsis the possibility that the metabolism occurring in vivo willproduce a rapidly cleared chelate. Thus, if the protein degradation is such that the radioactivity is separated from theprotein and a water-soluble chelate is produced, the nontarget

background would be reduced by the rapid excretion of theradiolabeled chelate by the kidneys. On the other hand, metabolism of '31l-labeled proteins produces iodide which has a

relatively long effective half-life.The radiolabeling of the protein-conjugated chelating agent

is not a trivial matter. Because of the relatively small amountsof antibody available and the necessity of keeping the numberof chelating agents per protein small, chelate formation is notalways maximal. Care must be taken not to introduce othermetal contaminants or chelating agents.

Effects of the Labeling Technique on the Clearance Properties and in Vitro and in Vivo Exchange Properties

There has been little published concerning the in vitro and invivo stability of chelates and chelate-containing proteins. Morehas been published on the clearance of radioiodinated proteinsin relation to the retention of biological and immunologicalproperties. A widely referenced work in the determination of

the metabolism of 131Iinsulin in humans is by Berson ef al. (6).

In this paper, the authors showed a difference in blood clearance depending on whether the patient had been treatedpreviously with insulin. The investigators evaluated the biological and immunological properties of the radiolabeled proteinand thoroughly discussed the effects of subtle alterations ofproteins produced by isotopie labeling. Since that time, numerous studies have been reported so that an exhaustivereview is not possible. Rather, we limit the review to thosepapers in the radiopharmaceutical chemistry literature dealingwith the effects of the labeling technique on the in vivo behaviorof protein conjugated to chelates.

Leung ef al. (35) and Meares ef al. (40) have published aseries of papers describing the effect of different variables onthe blood clearance of the radiotracer. In vitro exchange studies showed that indium did not bind to transferrin when theradiolabeled EDTA conjugate of HSA was mixed with serum. Invitro studies using human serum showed only 4% I11ln trans

ferrin after 333 hr of incubation. About 7% free chelate wasalso found after 333 hr. In rabbits, the major portion of the "11n

radioactivity remains in the albumin fraction.Goodwin ef al. (20) observed that the level of conjugation

significantly affected the blood clearance. At 0.3 EDTA derivative/albumin molecule, the blood clearance approached thatof 125I-HSA, but at 1.5 EDTA derivatives/albumin molecule,

using the alkylation method of conjugation, the blood clearancewas accelerated.

The exchange rates observed by Goodwin ef al. are slowerthan those observed by Wagner and Welch (55) for a DTPAderivative of HSA. The rate of exchange for the DTPA derivativeof HSA with transferrin was 9% in 24 hr. Paik ef al. (43) studiedthe exchange of 111ln-DTPA, an 111ln-DTTA azoimidate derivative, and 111ln-DTTA azoimidate conjugated to albumin. Theformation of 111lntransferrin was 10% from '"In conjugated toalbumin, 12% from 111ln-DTPA, and 33% from '"In-DTTA

azoimidate derivative at 24 hr, similar to those observed byWagner and Welch. Paik ef al. also observed the same bloodclearance dependence on extent of conjugation as observedby Goodwin ef al. At 0.9 chelate molecule/albumin, lnln wascleared faster than was 125I-HSA between 24 and 48 hr. The

clearance between 0 and 2 hr was similar for the 2 radiolabeledcompounds. The use of monomer albumin, the use of theamidination conjugation method which assures a single type oflinkage, and purification of the "'In-labeled substrate did not

prevent accelerated plasma clearance. Apparently, the majorfactor controlling plasma clearance is the level of conjugationper protein molecule. Thus, these preliminary data support theapproach of balancing altered substrate specificity caused bymodification against accelerated plasma clearance.

Conclusion

The clinical utility of radiolabeled proteins, specifically radio-labeled antibodies, is limited by 2 major problems: (a) theidentification of an antibody that binds only to the target tumortissue; and (to) the development of a procedure to radiolabelthe protein such that the substrate specificity is retained butthe blood clearance is at a sufficiently rapid rate to allow earlyimaging.

The former is being addressed by the immunologists, and inthe case of carcinoembryonic antigen antibody rapid progress

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Radiolabeling of Antibodies

is being made (19). However, in the case of the latter, very fewsystematic studies are being performed. The first part of thetask is to develop methods for measuring substrate specificityin vivo. In a related problem in our laboratory, we developed aradioiodinated /8-adrenoceptor-blocking agent that gave a

heart to blood ratio of 20 in rats (13). However, when wecoinjected a known potent /8-adrenoceptor blocker, proprano-lol, we were unable to observe any displacement of the radio-iodine. We were not, in fact, observing /S-adrenoceptor bindingbut rather an interaction with a high-affinity or high-capacity

nonreceptor protein. Until proven otherwise, nonspecific binding must be considered as a real possibility since radioiodinated albumin was one of the first tumor-imaging agents used

in nuclear medicine (39). In vivo specificity studies must becarried out for radiolabeled antibodies. With these data in hand,the complicated task of choosing the appropriate method ofconjugation can be undertaken. Only then can the design ofradiolabeled antibodies with optimal targetinontarget ratios bea reality.

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