Nucl. Med. Biol. Vol. 13, No. 2, pp.195-201, 1986 Int. J. Radiat. Appl. Instrum. Part B Printed in Great Britain

0883-2897/86 $3.00+0.00 Pergamon Journals Ltd

Samuel E. SALPSRN, M.D.

Nuclear Medicine Service, VA Medical Center, San Diego, California, and University of California, San Diego, California, USA

Radiolabeling of antibodies with In-111 has now been acccnnplished to the point that it is highly reproducible, achieves excellent labeling efficiency, and does not damage the antibody. Use of In-111 for radioimmunodetection is advantageous because of the excellent imaging characteristics of the In-111, moderate radiation dose, ease of labeling, and appropriate half-life. The liabilities of the In-111 method include slightly greater cost of the radio- nuclide, slw clearance of backgramd sites, and a shelf life requiring it to be ordered on a weekly basis.

When all characteristics of the radionuclide are taken into account, it appears to be superior to I-131 for radioimmunoimaging. A controlled study using iodinated and Indium labeled antibodies in the same group of patients needs to be done to accurately access how well the two function for tumor detection.

Sadioimmun&tection got its beginning in the early 1950’s. At that time Dav Pressman and his coworkers built antibodies against a tumor and radiolabeled them. 8 The radiolabel used was an isotope cf iodine and thus began the long romance of iodine and antibodies which persists tw. Indeed the iodirbation of proteins predates Pressman and their co-workers by many years. It is simple, fundamentally correct in concept, and can bs done by almost anyone with a small amount cf experience In vitro the bond between the iodine and the protein is quite strong. As a consequence mm in vitro studies have taken place using radioiodinated antibodies, thus giving credibility to the use of the iodine as a tracer.

It was assumed that the radiolabel was as stable in vivo as it was in vitro. While it was knwn that free iodine came off poteins, this was thought to be associated with their catabolism. Few suspected that the iodine atom could be cleaved from its covalent position on the protein prior to the destruction of its carrier molecule. Eventually radioiodination of antibodies became dogma and people stoFped thinking about the possibilities of dehalcgenation of the protein prior to its catabolism At the time of this writing (January of 1986), iodine is still the most commonly used radionuclide for the labeling of antibodies for in vivo work.

The most commonly used isotops of iodine for radioimmunodetection is I-131.(2r3) While I-123 can be used as the radiomrmaceutical for radioimmunodetection, its half-life of 13.3 hours is disadvantageous fran a logistical sta@oint Further, the I-123 half- life is not sufficient to allow formation of maximal lesiorrto-background ratios if one is utilix~ intact antibody. Thus, I-123 is best suited for use with Fab or Fab’ antibody f ragmen&. Even in that case the facility manufacturing the radionuclide must be relatively close to the site of labeling and use of the radiopharmaceutical. 131-Iodine with its eight w half-life does not fall under the logistical constraints described for I-123 and thus has become the dominant isotope of iodine for radioimmunodetection.

195

196

A review of the literature, both in the animal model and in clinical studies which have used isotopes of iodine for radioimmunoimaging, will reveal on close inspection a degree of tracer lost fran the organism out of proportion to what one would expect C&B IgG molecule When our group noted this, we undertook labeling with metal chelates.

Abundant work has been done in recent years on the radiolabeling of proteins with metal isotopes employing the so-called bifunc

M onal chelation tee

m ique. Key in

this effort was the work of Krejcarek and Tucker and Claude Meares , et al. The technique is not difficult, however, it requires attention to detail. Basically, one begins with a metal chelating molecule (usually DIPN and Iju a series of chemical steps forms an anhydride of one of the chelating carboxyl groups This, when placed in an antibody environment, reacts with amine groups on the antibody. These are usually on lysine molecules At this poinb the remaining carboxyls attached to the antibody are capeble of chelating a metal ioh Following column purification to rid the material of any unreacted chelating group the antibody can be labeled by putting the radionuclide and the sidechain labeled antibody together in a reaction vial. Using this technique and applying one sidechain per antibody, one can achieve labeling efficiencies as high as 5 mCi per mg and maintain the radIoimmunoreactivity and in vivo integrity of the antibody. The resultant radio&armaceutical has a variety of advantages over radioiodine and at the same time has problems of its own We can now discuss the advantages and disadvantages of both radioiodine labeling and In-111 bifunctional labeling.

Table I is ox&rUc&d, in such fashion that one can ccmpare the suitability of In-111 and I-131 for radioimmunodetection work. It further indicates the advantages and disadvantages of each, especially from the physical standpoint. The advantages and disadvantages in vivo will be covered later.

The #@Cal half-life of In-111 is 2.8 days. This is in contrast to the 8 day physical half-life of I-131. Ihe In-111 half-life is such that at the time d peak tumor concentration, about 68-78% of the radio@armaceutical is yet to decay which is more than adequate for imaging, Indeed, if a 5 mCi dose is given, one can still image quite well at one week postadministration because of other &y&al characteristics of the radio- nuclide (which will be discussed below). In the case of I-131 the 8 e physical half- life would allow imaging at far later time points than In-111 prwided it formed a stable Ccmpolltld In actual fact however, imaging byond one week is probably not worthwhile given the need for a relativity quick anger. The @ton energies of In-111 are 173 and 247 KEY. Thus 2 @otons are pr&ced that are canpatible with the imaging charac- teristics of the half-inch thick crystal of a gamma camera. I-131 on the other hand produces several photons, the primary one being 364 KEK Sane cb the other @xWns from I-131 are of high energy, penetrate the collimator septa and degrade the image. Math- matically, approximately 1.8 useful photons are produced per disintegration of In-111 versus about 0.9 photons per disintegration of I-131. An examination of the energy of these &otons indicates that slightly more than half of the In-111 &otons ~oduced will be detected by the l/2 inch gamma camera crystal compared to only 1 in 5 of those produced by the 364 KEV photon of I-131. Should one be imaging with a 3/8 inch thick crystal, the efficiency of the 364 KEV I-131 @~&on drops even more dramatically. When the effects of both @ton production and crystal efficiency of their energies are taken into oonsideration for the two isotopes, there will be greater than 10 times the produc- tion d “effective” &otons from In-111 than will be achieved with I-13l. Stated differ- ently, based on physical characteristics alone, one should at time zero get the same count rate from 0.1 mCi of In-111 as one can get from 1.0 mCi of I-131 if one uses a gamma camera with a l/2 inch crystal. Since image quality is to sane degree deI~r&nt on the total nurmber cf events detected; given the same quantity of the two radionuclides, one is reduced to prolonged imaging time to achieve the same quality image with I-131 as one can achieve with 11~111.

This, of course, is based on statistics alone. The resolution of the image however, is not dependent only on statistic& Collimation is probably the dominent factor in spatial resolution of an image in today’s modern cameras. Lower energy isotolxs such as Itrlll are easier to collimate While very high slmtial resolution is probably not as important for radioimmunoimaging as it is for more delicate nuclear medicine procedures, it remains important that it at least be adequate. With medium energy collimation, it is certainly no more than adequate and with a high erwxqy oolli- mator it is less than that A high energy collimator must be used with I-131, and even then, septal penetration can occur fran the 637 and 723 KEV &oton~

!nB

LEI-

klvantages and Limits of In-111 and I-131 as Radiontiides for

2.

3.

4.

5.

6.

7.

8.

9. Rysical l./2Life

Photon energy

Photon ProcZlction

EEficiency

for l/2" crystal

CcUimation

Radiation

ckme/uCi

Sh

elf

life

CosMiCi

Ezlseoflabeling

10. mssihle specific activity

xL?A

2.8 days

8.05 days

173 KIN (89%)

80 KEV(3%), 284KW(5%),

247 KEV (94%)

364KFW(82%),

637KEv(7%),

723KW(2%)

1.8 useful @otons

0.9 useful @otons/

per disintegration

disintegration

65% (173KEn

20% (364KES0

35% (247KPS')

Mediunenergy

Bigh energy

Lcw production

of146, High prohction 6OOKW

220, 243KIW Beta

Beta

Average

Excellent

$30.

$10.

4Omib nopurifi-

60min,~ustccAunn

cation necessary

purify

10 uCi/ug achievable At least as high as In-111

withoutlossof huIImcP

reactivity

or change in

distribution

of molecule

Radioinmmoimaging

In-ill mst captiblewith M&b

kinetics

1~111 "cleaner"

making imge

productioneasier

I*lll&oton productiontwice

that of I-131

I*lll~ovi&s 5Xthenunber of

of effective

i.e. amted @mtons

In-lllallavs

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In-111 delivers larer rad &me/

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198

!Ihe radiation dose fran the 2 radioffiarmaceuticals is very difficult to equate because, while the distribution and excretion of 11~111 are highly l~edictable, the same factors are highly unpedictable for I-132 If the radionuclides both remained in the various tissues until all of the radioactive atoms decayed, and if equivalant (1 mCi) dosages were given, the radiation dose frcrn I-131 would be higher than frcan 11~111. This is due to the fact that the I-131 has an 8 a4y pwsical half-life and high production of 600 KEV Beta particles. Indeed it is this Beta factor which enables I-131 to be used for the theran of &pert&roidism and t&roid cancer. Iiavever, when one considers the fact that as much as half of I-131 associated with an intact antibody can be eliminated through the urine by 72 hours postadministration, one finds that it is possible for the the rad dose from I-131 to be even lower than for In-111. What cannot be predicted in these calculations, unfortunately, is the degree of the stabiliw of the I-Ul-antibody preparation It can vary fran labeling method to labeling methob and even within a given method There is also evidence suggesting that it rn* vary fran antibo& to antibody. This is not true for bifunctional chelation

When one considers shelf-life of the radionuclide in radioimmunoimaging, the advantage quickly falls to radioiodine 131. It is useful and convienent to be able to buy radioiodine in high quantity and high specific activity. The cost is cheaper if bought in bulk and &&ping problens are reduced since there are fewer chances for the postal service to cause grief. It &ould be remembered however, that this process can be a doubleedged sword. As the I-131 decays, the activity, per volume decreases This can cause a problem in iodinatioxb Nearly everyone who has had experience with radioiodine has experienced poor results in labeling with I-131 that has been on the shelf for awhile. The in vivo distribution has occasionally been affected under these circum- stances in some of our studies. On the other hand, a 5 mCi dose of 1llIn is useful for labeling through one half life for clinical studies and we have used it for animal work as late as 3 half-lives after its production The labeling efficiency remains high and the in vivo kinetics and distribution are what we have learned to expect from In-111 labeled antibodies.

The cost per mCi of In-111 and I-131 favors the I-131 at this time. This is especially true if one wishes to order a great deal of I-131 at any single shipment. Should radioimmunoimaging with In-111 be reduced to common clinical practice however, one could expect the cost of the radionuclide to drop accordingly based on more efficient production and industrial competition

Sase of labeling is important in any nuclear medicine procedure It now takes approximately 40 minutes to label with 11111~ If one uses the “kit” technique, one can expect a high labeling efficiency virtually every time prwiding the 1llIn has been prepared correctly. Ccrnpeting ions acquired in producing the radionuclide will occasion- ally make labeling impossible, Mever prcblems of this nature are becoming less frequent as production of “ultraclean” 1llIn is becaning routine The problems of I-l31 labeling are well known hwever. Using even the short methcds, one can expect to spend 60 minutes from start to finish. Further, with column purification, which is a necessary step in iodination, the introduction of mrogens or bacteria into the radiopharmaceutical are real.

Finally, there is the question of the specific activity achievable by the two techniques. Currently, we have no difficulty of achieving specific activities of 5 mCi per mg with -111. We have found, interestingly enough, that high specific activity is not necessarily desirable with radioimmunoimaging, and in fact carrier protein must be added to the radio~armaceutical for optimal clinical results. This will be discussed later when we address invivo advantages and disadvantages. The theoretical specific activity of I-131 labeled preparations may go higher than that of In-111 since it is necessary to limit the number of chelating groups to one per molecule High specific activity with 131-I is not necessarily desirable however since an wer&Mance cf 131-I on the antibody (on any protein, for that matter) seems to cause problems with its distribution. This is especially true if the labeling occurs 2 or 3 half-lives after production of the radionuclide

Naw let us look at the advantage and limits of the two radionuclides in the in vivo situation If one is to image with I-131 it is absolutely essential that one block the t&roid gland of the ptient with nonradioactive iodine prior to the administration

1.

Nee

d for thyroid blockade

No

2.InVivo Stability

Yes

3. Toxicity

4. Distribution

first 8 hrs

5. Distribution

after first 8 hrs

6. Stability in fa

ce of

Modulation

7. A

bili

ty

to doSPECIbased

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methods

9. Accuracy,

Specificity,

Sensitivity

Unrelated

to tracer

atimagingdoses

SmIE

P&e&ion in all

tissues to greater

extent than I-131

Stable

Better than I-131

basedcnphantan

testing

Depends onantibody

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.

Ye

s

No

Unrelated to tracer at

imaging doses if thyroid

blocked

San

e

Markeddehalogenation

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INdance of rapid dehalo-

genation

Depends on rapidity of

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thickness

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tion photon) too great.

Depends on antibody

EM

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Advantage Irrlll

Dehalogenation marked after

24 hours. Advantage In-111

Sane

No difference

at 8 hrs

Difficult to acoess. Liver

metastasesmq beeasierto detect

with I-131 but more work needs to

bedonetoprovesane

Advantage In-ill

Usually advantage In-111

Prcbableadvantage

In-111

No canparisons

ever preformed

with sameantibcdy in same

patient without circulating

antigen.

200

of the radiopharmaceutical. If this is not done the thyroid will normally trap between 10 and 30% cf the free iodine Fesent in the serum, If one administers a 3 mci dose and if there is 50% dehalogenation over a period of 72 hours, it is possible that the t&roid could trap as much as 500 uCi of I-131. This will deliver between 500 and 1000 rads to the normal wroid glarxl with possible untoward results. There is no need for t&roid blockade with In-111 sin- the radionuclide is not concentrated & the thyroid gland,

The stability of the two radionuclides in vivo has been alluded to previously. As indicateb it was dramatically different between the two isotopes with iodine caning off of the antibody at a variable rate. Gn the contrary, very little of the In-111 was removed fran the malecula Ihe radioiodine which does cane off the molecule is quickly lost in the urine prcrvided the t&roid is blocked.

The toxicity of radiolabeled antibodies varies with the conditions under which they are administered and has little to do with the nucleide attached, pro&led radiation effect is kept to a reasonable level. In our hands there has been very little in the way of allergic or other wpes of reactions We have noticed a slight rise in the transami- nase enzymes in about 20% of the people that we have infused. These are always back to baseline levels by 72 hours The etiology cb these ermyme rises are obscure and hwe not been associated with clinical disease stata In a few ptients, allergic-type reqnses characterized by hives and shattness of breath hwe occurred, but such reactions are rare. This become8 more of a problem in the murine system if antimouse antibodies are present CFA complexes formed with the administered antibodies seem to circulate freely in the vascular ann~rtments for a considertie time When circulating antigen is present in the form of an antimouse ant&o& however, the immune canplexes formed are quickly removed fran the circulation, @ma&y by the liver. These @ients do have reactions, yet the ones we have observed to date have been mild. Another place where reactions are very likely to occur is when antigen is on the surface of circulating formed elements of the blood, In our hands this has been specifically true of antigen on white cells (in the ON system) and on circulating T 1ymFhocytes in the lym&ana system, In these cases as the concentration d antiboqr rises, more and more d the cells are removed fran the circula- tion by the reticuloendothelial organs, especially the spleen, where it is wesumed thq are destroyed with release of their endocellular contents. The patients experience a dramatic reaction It is characterized by fever, chills, m vaniting, weakness, and a fall in blood pressure. The reaction occurs between between 1 and 2 hrs following administration of the antibody and it is generally over within an hour.

Thus it is especially important that all antibodie& in every system that will be used for radioimmuncdetectior4 be screened against formed elements of the blood Such reactions will occur regardless of the tracer used for labeling, Further work in our lab indicates that if the mass of antibody is kept low, reactions fran antigen fixation on circulating cells does not occur.

In the first 8 hrs f&lcwing administration of the radio&armaceutical, distri- bution of an iodinated and indium labeled antibody look the same. Indeed, when an antiboay is radiolabeled so that both tracers are on the same molecule, the early distri- butions are virtually identicaL With time, this breaks duvn with divergence of iodine and indium in every organ system with the possible exception of bloc& Using 114W-In, we have traced the fate of the Indium in the animal model and found that approximately 75% of the label has been lost from the tissues by 1 month, 2/3 via the GI tract, and about l/3 excreted in the urine

When modulation occurs in radioimmunoimaging (as it does in patients with !F cell lym&ana fallawing administration d the anti-F&l1 antibody, T-101) the immune complex is internalized by the malignant cell. When the radiolabel is In-111, the distribution of the radio@armaceutical is mimarily to the malignant lym@ nodes and halved skin sites. If I-131 is the radio@mumaceutical, there is rapid dehalogenation and the malignant nodes are not visualized (direct communication, Larson and Carresguillo). Thus it appears that internalization of the radiolabeled antibody bv cells is an event which leads to rapid dehal~nation Since animal data from our- lab stronalv indicates that intrwascular antibodv labeled with either indium or iodine have the s& kinetics (prcwided there is nothing -in the way of cros=reacting suhetance to alter the kinetics) it is the author’s opinion that internalization of the antibody is important in the dehalogenaticn process

201

The differences in the two radionuclidesthatwehavebeen discussing becomes quickly obvious when one attempts to do single photon emmision computed tomograpl’y (smcr) andotheK&ectronic manipilationoftheradionucli&a SPECT, tobeeffective, is dependent upon a high photon flux. As previously stated, this is generally not availablewhenusing of I-131as a radionuclide. Thisthecase with In-111. The high photonyield from In-lll,& shigh crystal interaction, and type of collimationmakes SPECT very easy. Indeed, mTc subtractiontechniques Based onphantcm testing) are said to be more optimal with In-111 than with I-131.

Finally, the effects of the above observations and speculations can only be interpretedinlight of the ability to detect tumor in patients At the moment it is virtually impossible to state which Kadionudlide has the advantage in this KS$lKd OUK

own clinical results with 11~111, in the detection of 5 tumortypes, using multiple antibodies, has indictedthatwe canpick upbetween and 75% of thelesionsthatarel l/2 cm in diameter or greater. This is highly dependent upon the antigan:antibody system. Resultswith iodinelabeledmomclonal andpolyclonal antibodieshavebeenjust as VaKiahle when the work of other investigators is observed. Ot~iously much remains to be done in the arena of radioimmunaletection if we aretoachieve an imaging technique with high specificity and sensitivity. However it appears oM.ousto this author that the radionuclide used in pursuit of thisexcellence shouldnotbe radioiodineandmay well be Irr-ill.

1. Presanan K. Cancer 153; 6:619-623.

2. Mach J-P, et al. N. Eng. J. Med. 303; 510, (1980)

3. Moshakis V, et al. BK. J. Can. 44:91-99, U.980)

4.Stecn P, et al. In &bUrUUiDCancerniacrmsis&U3Treatment. pp 245-253. hK:Raven Press, (1982)

5. Ralpern S, et al. Can. Res. 43:5347-5355, (1983)

6. Halpern S, et al. Radial. 155:493-499, (1985)

7. Krejcatek, G andTucker, K. Bio. Chem.Bio. PhysRes Commun. 71:581-585, (1977)

8. Meares, C, et al. Proc. N&l. Acad. Sci. USA. 73:3803-3806, (1976)