Radioiodinated iodobenzoyl conjugates of a monoclonal antibody Fab

5 Oct 1990 - Similarly, coinjection of either the p-iodo- benzoyl or m-iodobenzoyl conjugate of NR-LU-10 Fab with the Fab radioiodinated with ChT/radi...
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Radioiodinated Iodobenzoyl Conjugates of a Monoclonal Antibody Fab Fragment. In Vivo Comparisons with Chloramine-T-Labeled Fab D. Scott Wilbur,*J Stephen W. Hadley, Leah M. Grant, and Mark D. Hylarides NeoRx Corporation, 410 West Harrison Street, Seattle, Washington 98119. Received October 5, 1990

A comparative investigation of the biodistributions of radioiodinated p- and m-iodobenzoyl conjugates of a monoclonal antibody Fab fragment, NR-LU-10 Fab, and the same antibody Fab fragment radioiodinated by the chloramine-?' (ChT) method has been carried out in mice. Coinjected, dual-isotope studies in athymic mice with tumor xenografts have demonstrated that there are only minor differences in the in vivo distributions of the iodobenzoyl-labeled Fabs, except in the excretory organs, kidneys, and intestines, where major differences were observed. Similarly, coinjection of either the p-iodobenzoyl or m-iodobenzoyl conjugate of NR-LU-10 Fab with the Fab radioiodinated with ChT/radioiodide into BALB/c mice provided additional data that indicated that the two iodobenzoyl conjugates distributed similar in a number of selected tissues. The tissue-distribution differences of the regioisomeric iodobenzoyl conjugates in relation to the ChT-radioiodinated Fab were large for the stomach and neck, consistent with previous studies. The most notable difference between the two iodobenzoyl conjugates was the kidney activity, where the m-iodobenzoyl conjugate was similar to the directly labeled Fab, but the p-iodobenzoyl-conjugated Fab was higher by nearly a factor of 2.

INTRODUCTION Monoclonal antibody Fab fragments have a rapid blood clearance and have good tumor-to-surrounding-tissue ratios at 4-12 h postinjection, which makes their use favorable for radioimmunoscintigraphy of cancers ( 1 ) . Indeed, clinical studies have shown that imaging of tumors can be achieved when monoclonal antibody Fab or Fab' fragments are radiolabeled with technetium-99m (tip = 6.02 h, y = 140 keV) as early as 2-6 hours postinjection ( 2 , 3 ) . However, due to the Fab's propensity to localize in the kidneys and the hepatobiliary excretion observed for some technetium-99m-labeled antibodies ( 4 ) ,there can be questions concerning the masking of cancer sites by radioactivity localized in the region of the excretory tissues. By 30 h postinjection much of the radioactivity in the kidneys and colon has cleared, unfortunately the short half-life of technetium-99m makes imaging at that time difficult. Application of iodine-123 (tip = 13.1 h, y = 159 keV) labeled monoclonal antibody Fab fragments, which can be imaged at 30 h postinjection, could provide additional information about cancer sites in the abdominal region. While radioiodinated antibodies can be readily obtained by standard radioiodination procedures (5),the application of radioiodinated antibodies to the diagnosis and therapy of cancer has been limited by an apparent in vivo deiodination (6,7).Radioiodinated antibodies studied in vitro and in serum are generally quite stable, suggesting that deiodination occurs via an enzymatic process, presumably deiodinases involved in thyroid hormone biochemistry (81 1 ) . The problem of in vivo instability of radioiodinated antibodies led us, and other investigators, to study alternative methods of antibody radioiodination (12-18). Thus, we have studied a number of radioiodinated conjugates of monoclonal antibodies which were designed to stabilize the radioiodine in vivo ( 19-21). In the reagents tested, stabilization of the radioiodine was accomplished by attachment of the radioiodine to a nonactivated aromatic ring in the position para to the functionality used 1 Address correspondence to D. Scott Wilbur, Dept. of Radiation Oncology, RC-08, University of Washington Medical Center, 1959 N.E. Pacific St., Seattle, WA 98195.

1043-1802/91/2902-0111$02.50/0

in the antibody conjugation. Indeed, these p-iodophenyl (PIP) antibody conjugates have been found to be quite stable toward in vivo deiodination. Similar studies by Zalutsky et al. (12-14,22) have demonstrated that m-iodobenzoyl antibody conjugates are also stable toward in vivo dehalogenation. Further, Garg et al. (24) have reported that significant differences were observed between the in vivo distributions of dual-labeled m- and p-iodobenzoyl conjugates of an intact antibody, 81C6, and aF(ab')2 fragment of another antibody, OC 125. However, there were no reports in the literature delineating the differences between m- and p-iodobenzoyl conjugates on antibody Fab fragments. Due to these factors, it was not evident a priori which radioiodinated conjugate should be used in imaging studies employingradioiodinated antibody Fab fragments. Reported herein are the results of an investigation which was designed to help determine whether radiolabeling with the m- or p-iodobenzoyl conjugates of a pancarcinoma antibody fragment, NR-LU-10 Fab? affected the distribution of radioactivity. To alleviate the animal-to-animal variation in the in vivo distribution studies, dual-labeled (lZ5Iand 1311),coinjected preparations of m-iodobenzoyland p-iodobenzoyl-conjugated NR-LU-10 Fab were employed. Additionally, comparative studies, where NRLU-10 Fab was radioiodinated with either of the iodobenzoyl conjugates and (separately) radioiodinated directly by use of a modified radioiodide and chloramine-?' (ChT) labeling method (25),were conducted to assess the relative amounts of deiodination. EXPERIMENTAL PROCEDURES Materials. All reagents used were analytical reagent grade or better and were used as obtained. HPLC solvents were obtained as HPLC grade and were filtered (0.2 pm) prior to use. Radioiodine was obtained from Du Pont/ The NR-LU-10 monoclonal antibody is a pancarcinoma antibody of the IgGzb subclass which reacts with a glycoprotein antigen of ca. 40 kDa. The antigen is found on several carcinomas such as colon, breast, ovarian, prostate, and lung. Radiolabeled NR-LU-10 Fab is currently undergoing clinical investigation for application to imaging of several different carcinomas in patients. 0 1991 American Chemical Society

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NEN (Billerica, MA) as high-concentration, high specific activity solutions in 0.1 N NaOH. Phosphate-buffered saline was purchased from Gibco Laboratories (Baltimore, MD) as Dulbecco's phosphate-buffered saline (#310-4190) or from Whittaker Bioproducts, Inc. (Walkersville, MD). General Procedures. High-performance liquid chromatography (HPLC) was conducted on either a gradient system consisting of two Beckman Model llOB pumps, a Beckman Model 153UV detector, a Rheodyne Model 7125 injector, and a Beckman Model 170 radioisotope detector or an isocratic system identical with the gradient except having only one solvent pump. All separations were run at a flow rate of 1mL/min. Spectrophotometric analysis of the HPLC effluent was performed with UV detection at 254 nm and in-line y-radiometric detection. Compounds 1-4 were analyzed by reverse-phase chromatography, and the Fab conjugates 5 and 6 were analyzed by size-exclusion chromatography. Reverse-phase chromatography was conducted on a 4.6 mm X 12.5 cm, C-18 column (Whatman) using a binary gradient solvent system. Eluant A in the gradient was a mixture of 98% MeOH and 2 % of a 1% HOAc/H20 solution. Eluant B used in the gradient was a mixture of 20% MeOH and 80% of a 1% HOAc/H20 solution. The gradient was begun by eluting for 2 min at 50% of each eluant. After 2 min the solvent mixture was increased in eluant A over the next 8 min to a final composition of 98 ?6 eluant A and 2% eluant B. The eluant mixture remained at that composition for the next 10 min to complete the gradient. Retention times under these conditions were as follows: free radioiodide eluted at 1.4 min (solvent front), compound 1 eluted at 14.6 min, compound 2 eluted at 15.1 min, compound 3 eluted at 3.4 min, and compound 4 eluted at 4.2 min. Size-exclusionchromatography was conducted on a Zorbax Bio Series GF-250 9.4 mm X 24 cm column (Du Pont) with 0.2 M phosphate, pH 6.8, as the eluant. All radioiodinated Fab fragments eluted at 9.8 min. Less than 5 % of the protein aggregates were detectable by HPLC analysis of the radioiodinated Fabs. Thin-layer chromatography of protein conjugates was conducted on silica gel impregnated glass-fiber strips (ITLC, Gelman) eluting with 80% MeOH/H20. In this system, the protein is denatured at the origin and the free iodide or radioiodinated small molecules that are present move with the solvent front. Radiochemical purity of the radiolabeled Fab preparations was defined as the percent protein-bound activity, which was assessed by dividing the counts at the TLC origin by the total TLC counts. Radioactive samples were counted in a Packard Autogamma 5650 instrument. Protein concentrations in solution were measured by UV spectrophotometry at 280 nm. The concentrations of the radioiodinated Fab fragments were obtained by using the Beer-Lambert equation with the molar absorptivity being equal to 1.53. Athymic (nude) mice were obtained from Simonsen Laboratories, Inc. (Gilroy, CA) and were used at 10-12 weeks of age. Female nude mice (nu/nu),weighing 20-27 g (23.8g f 1.4 g average body weight, N = 16),were housed in microisolator caging with bonnet filter tops and maintained on sterilized chow and water in a controlled environment. Tumor xenografts were derived from subcutaneous flank implantation of 2.5 X lo6LS-180 cultured human colon tumor cells.3 The tumors were allowed to grow for 7-10 days to produce tumors which had an average 3 The LS-180 tumor model is a human colon carcinoma xenograft derived from the LS-174T tumor line.

Wilbur et al.

Table I. Tissue Distribution of Radioiodine (% ID/g) at 4 and 20 h Postcoinjection of the NR-LU-IOFab Conjugates of pIodobenzoy1 ([ *s11]-5)and m-Iodobenzoyl (['%1]-6)in Athymic Mice 4h 20 h [1311]-5 [lZsI]-6 tissues [1311]-5 [1zsI]-6 blood 1.58 f 0.18 1.46 f 0.27 0.07 f 0.02 0.10 f 0.03 tumor 7.45 1.39 7.24 1.40 4.51 f 1.65 4.46 f 1.67 skin 1.14 f 0.10 1.06 f 0.10 0.05 f 0.02 0.06 f 0.02 muscle 0.44 f 0.06 0.43 f 0.07 0.03 f 0.01 0.03 f 0.01 lung 5.83 f 2.87 5.59 2.73 0.77 f 0.77 0.66 f 0.53 liver 0.73 f 0.12 0.74 f 0.12 0.07 f 0.01 0.12 f 0.04 0.53 f 0.12 0.57 f 0.12 0.04 f 0.02 0.10 f 0.03 spleen stomach 0.80 i 0.41 0.77 f 0.41 0.04 f 0.03 0.05 f 0.04 neck (thyroid) 1.00 f 0.13 0.99 f 0.15 0.13 f 0.05 0.17 f 0.08 14.11 1.18 8.25 0.84 0.27 f 0.07 0.15 f 0.05 kidneys intestines 1.18 f 0.19 0.61 f 0.08 0.03 f 0.02 0.03 f 0.01 ~

~

~

*

~~~~

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*

*

tumor weight of 201 f 100 mg (a range of weights from 61 to 400 mg). BALB/c mice were obtained from Charles River Laboratories, Inc. (Wilmington, MA) and were used at 10-12 weeks of age. Female BALB/c mice weighed 19-24 g (22.6 g f 3.1 g average body weight, N = 16, and 21.3 g f 1.0 g average body weight, N = 16, for the two experiments, respectively). Preparationof N-SuccinimidylpTri-(n-butylstanny1)benzoate(1) and Preparationof N-Succinimidyl m-(Tri-n-butylstanny1)benzoate(2). The synthesis of the para-substituted aryltin reagent 1 was accomplished in three synthetic steps involving the palladium-catalyzed metal-halogen exchange of methyl p-bromobenzoate, followed by methyl ester hydrolysis and subsequent formation of the succinimidoester as previously described (16). The synthesis of the meta-substituted aryltin reagent 2 was accomplished in a manner similar to that previously reported by Zalutsky and Narula (13). Preparation of Radioiodinated N-Succinimidyl pIodobenzoate (3) and N-Succinimidyl m-Iodobenzoate (4). Radioiodination of the regioisomeric N-succinimidyl (tri-n-butylstanny1)benzoates was conducted in a manner similar to that previously described (16). To exemplify the labeling conditions, reaction of meta positional isomer is described below. Into a reaction vial fitted with a septum was placed a 50 pL solution containing 12.5 pg (0.025 mmol) of N-succinimidyl m-(tri-n-butylstanny1)benzoatein 5 % HOAc/ MeOH, a 10 pL solution containing 10 pg (0.075mmol) of N-chlorosuccinimide (NCS) in MeOH, and 10 pL of phosphate-buffered saline. To this mixture was added 2 pL (1.2 mCi) of Na125I in 0.1 N NaOH. After 5 min at room temperature the reaction was quenched by the addition of a 10pL solution containing 0.72 mg/mL (0.076 mmol) NazS205 in H2O. The crude product was a mixture of three radioactive components: 48% of [1251]-4,34% of m-[125]iodobenzoicacid, and 4% of [1251]iodide,by radioHPLC analysis. In a like manner, radioiodination of 1 with NCS/l311 affordeda77 % radiochemical yieldof [1311]-3,5% ofp-[131]iodobenzoic acid, and 1176 [1311]iodide. Preparation of [ lZ5I]-3,employed in the second biodistribution (Table 11), afforded a radiochemical yield of 85%, with 5% of p-[1251]iodobenzoicacid and 2 % of [1251]iodidebeing present. The methanol was evaporated from the crude mixture by passing a stream of N2 gas through the reaction vial. To contain any volatile radioiodine, the reaction vial was stoppered with a septum seal, NPwas introduced by means of a needle inlet, and a needle outlet was attached to a syringe barrel containing granulated charcoal. The crude

Iodobenroyl Antibody Fab Conjugate Evaluations

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Radioiodination of NR-LU-10 Fab with Chloramine-T. To a reaction vial was added a 60-pL aliquot of NalslI, 100 pL of PBS, and 25 pL (500 pg, 0.01 mmol) of NR-LU-10 Fab in PBS. Followingmixing, a 10pL solution containing 1 mg/mL chloramine-T (10 pg, 0.046 mmol) was added. After 10min at room temperature, the reaction was quenched with 6 pL of a 0.72 mg/mL solution of NaHS03 (0.046 mmol) in H20. The crude product was purified by chromatography on a Sephadex G-25 (PD-10) column eluting with PBS. The preparation used in the second biodistribution was obtained in a radiochemical yield of 52%, a specific activity of 0.75 mCi/mg, and a final purity of 98.8%. The preparation used in the third Table 111. Tissue Distribution of Radioiodine (%ID/g)at 4 biodistribution (Table 111) had a radiochemical yield of and 16 h Postinjection of the NR-LU-10 Fab Conjugate of m-Iodobenzoyl ([lX61]-6)and [1311]ChT-Labeled NR-LU-10 95%,a specific activity of 0.73 mCi/mg, and a radiochemFab in BALB/c Mice ical purity of 99.3 % . 4h 16 h Injection Mixtures for Animal Biodistributions. Radioiodinated NR-LU-10 Fab mixtures were diluted to tissues [ 12611-6 [ "'I]C hT [Y 1 - 6 ['"I] ChT obtain ca. 100 pL of injectate per mouse. The injectate blood 1.60 i 0.34 2.24 f 0.62 0.20 f 0.07 0.25 f 0.06 'solution for the initial biodistribution study (Table I) lung 1.28 f 0.40 1.96f 0.39 0.19 f 0.06 0.24 f 0.05 contained 5 pg of NR-LU-10 Fab labeled with 1.0 pCi lZ5I stomach 0.64f 0.11 6.52 f 1.46 0.04f 0.01 0.36 f 0.16 and 2.1 pCi lslI in each 100-pL aliquot. neck (thyroid) 0.83 f 0.13 5.30 f 2.69 0.19f 0.12 5.11 f 3.58 kidney 8.96f 1.05 7.72f 0.88 0.52 f 0.16 0.39 f 0.05 The injectate solution for the second biodistribution study (Table 11)contained lOpg of NR-LU-10Fab labeled evaporated product was used in the subsequent conjuwith 2.7 pCi IZ5Iand 3.7 pCi 1311in each 100-pL aliquot. gation reactions without further purification. (Previous The injectate in the third biodistribution study (Table studies4 had indicated that purification of the crude ra111) contained 9.1 pg of NR-LU-10 Fab labeled with 2.9 dioiodinated benzoate 3 was not necessary prior to pCi lZ5Iand 3.7 pCI 1311in each 100-pL aliquot. conjugation to the antibody, as immunoreactivities and tissue distributions were not altered from the purified Animal Biodistribution Studies. Biodistribution examples.) studies were conducted by administering the above solutions of radiolabeled protein via the lateral tail vein Conjugation of Radioiodinated 3 and 4 to Antiin the mice. The tumored athymic mice and BALB/c bodies. To the vial containing evaporated crude radiomice were each marked for identification and were handled iodinated 3 or 4 was added a solution containing 200 pL in the same manner. Replicate (4X) 5-mL aliquots of the (1mg) of NR-LU-10 Fab (50pL of a 20 pg/pL PBS solution injectate were prepared and counted to serve as standards of Fab and 150 pL of a 1.0 M, pH 9.25, solution of Na2for the calculation of the total injected dose. Mice were cos). After 5 min at room temperature, the crude labeled restrained, weighed, and injected; weighing the syringe protein was assessed by TLC to obtain the conjugation yield (percent bound to protein) and was purified via a before and after to determine the volume injected. At Sephadex G-25 column (PD-10, Pharmacia) eluting with designated times postinjection, groups of mice (eight PBS. The early-eluting radioactive fractions were colanimals) were sacrificed by cervical dislocation and lected and pooled to yield purified conjugate 5 or 6. dissected. Blood samples were collected just prior to sacrificing the mouse by collecting a sample from retroProtein-bound activity (final purity) was assessed by TLC orbital bleeding into a Pasteur pipet, transferring the blood analysis. HPLC analyses were conducted to assess aggregate formation (i.e. higher molecular weight species) into a preweighed tube, and weighing the tube. Tails were removed and counted as an indicator of the completion and to evaluate relative retention times. of injection (datanot shown). Ten additional tissues were The preparations of radioiodinated 5 and 6 employed excised from the mice. These tissues were tumor, skin in the initial biodistribution study (Table I) gave the (from back), muscle, lung, liver, spleen, neck (soft tissue following results. Conjugation of NR-LU-10 Fab with from the ventral portion of the neck), kidney and intestine. [1311]-3afforded 1027 pCi (56% overall radiochemical Bone (femur) was also excised, but the counts were too yield), with a specific activity of 0.82 mCi/mg and a ralow, and the variability too high, to lead to any conclusive diochemicalpurity of 98.3 % by TLC analysis. Conjugation analyses. In the studies employing BALB/c mice fewer of NR-LU-10 Fab with [lZ5I]-4afforded 415 pCi (35% tissues were excised, evaluating those that were different overall radiochemical yield) with a specific activity of 0.41 in the first biodistribution. The tissues excised in BALB/c mCi/mg and a radiochemical purity of 97.3% by TLC mice were lungs, liver, kidneys, stomach, and neck. The analysis. organs were removed and counted intact. Tissue samples The preparation of [1251]-3-conjugatedNR-LU-10 Fab used for in vivo comparison with [1311]iodide/ChT-labeled were blotted, weighed using an analytical balance, placed in plastic test tubes, and counted in a y-scintillation NR-LU-10 Fab (Table 11)afforded an overall radiochemcounter. ical yield of 4695, a specific activity of 1.76 mCi/mg, and a final purity of 99.4 76. Dual-channel counting was carried out using a lowThe second preparation of [1251]-4-conjugatedNR-LUenergy window (20-80 keV) for iodine-125 and a high10 Fab used for in vivo comparison with [13lI]iodide/ChTenergy window (240-400 keV) for iodine-131. A 19% labeled NR-LU-10 Fab (Table 111) was carried out in the spillover of counts from lS1I was measured in the l25I same manner as the first preparation and afforded a 31 % window. All 1251 counts were corrected (after background overall radiochemical yield, a specific activity of 0.57 mCi/ subtraction) for spillover. Data analysis included calcumg, and a 96.4% purity by TLC analysis. lation of the percent injected dose per gram (5% ID/g) for each isotope in each tissue, an average and standard 4 Unpublished results. deviation for the % ID/g in each set of eight animals, the

Table 11. Tissue Distribution of Radioiodine (%ID/g)at 4 and 16 h Postcoinjection of the NR-LU-10 Fab Conjugate of pIodobenzoy1 ( [ 1 q - 5 ) and [~SII]Iodide/ChT-Labeled NR-LU-10 Fab in BALB/c Mice 4h 16 h tissues [1%1]-5 [lSII]ChT ['%I]-5 [13'I]ChT blood 2.73 i 0.49 3.13f 1.10 0.34 f 0.04 0.27 f 0.07 lung 2.93 f 0.77 3.34 f 1.07 0.40f 0.12 0.36 f 0.10 stomach 1.67 i 2.70 10.26 f 6.36 0.07 f 0.02 0.41 f 0.18 neck 0.95 f 0.22 9.01f 4.54 0.46 f 0.25 12.36 f 10.59 (thyroid) kidney 24.52f 4.38 11.03f 2.23 1.56 f 0.45 0.50 i 0.17

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Scheme I. Scheme for Radioiodination of Monoclonal Antibodies Using Iodobenzoyl Conjugates n

0

0

3 (para) 4 (meta) Protein-NH2

C-NH-Protein

5 (para) 6 (meta)

ratios of the 9; ID/g for the two isotopes (1251/1311) in the tissues of each animal, and the average ratio and standard deviation for each tissue. Statistical analysis of the data were conducted [with the computer program Statworks (Cricket Software, Philadelphia, PA)] on % ID/g data for 1251 and 1311from the eight animals in each group by using paired analyses. Criterion of significance was set at p < 0.05. RESULTS

The synthetic scheme for radioiodination of antibodies (or other proteins) with iodobenzoyl conjugates is given in Scheme I. The synthesis of the aryltin reagent 1 by palladium-catalyzed metal-halogen interchange has been carried out many times in these laboratories and usually gives 25-35% overall yields. Contrary to this, several attempts to employ the palladium-catalyzed reaction of hexabutylditin and methyl m-bromobenzoateto synthesize meta isomer 2 failed. An alternative synthetic method used for preparation of 1 was aryl lithiation at -100 "C (261, followed by stannylation with tri-n-butylstannyl chloride to provide 2 in 36% overall yield. Radioiodination of the two aryltin intermediates was comparable. The radioiodination yields were higher for para isomer 3 than for meta isomer 4, but this was easily attributed to the fact that the starting aryltin compound 2 was not as pure (free of stannylbenzoic acid) as was aryltin intermediate 1. Even trace quantities of free trin-butylstannylbenzoic acid will cause an appreciable loss of yield, because the stannylbenzoic acid reacts faster than the corresponding N-succinimidyl ester. The in vivo comparison of the para and meta positional isomers of radioiodinated iodobenzoyl-antibody conjugates was conducted in three separate animal biodistribution studies. To distinguish the in vivo distribution of Fab conjugated with 3 from that conjugated with 4, iodine125 ( t l p = 60.14 d, X-ray = 27 keV) and iodine-131 (tip = 8.04 d, y = 364 keV) were used in dual-labeled experiments. In the initial study, the Fab fragment of NR-LU-10, an anticarcinoma antibody,z was radioiodinated with N-succinimidyl p-iodobenzoate ([ 1311]-3)and N-succinimidyl m-iodobenzoate ( [ lZ5I]-4)in two separate vessels. Following radioiodination, the radioiodinated compounds were conjugated to NR-LU-10 Fab. Once conjugated, the two preparations were purified by gelpermeation chromatography and combined prior to injection into the mice. The biodistributions of the coinjected Fab conjugates [lS1I]-5and [125]-6were assessed in athymic mice bearing LS-180colon carcinoma xenografts.3 Animal biodistributions were obtained at 4 and 20 h postin-

jection with eight animals being sacrificed at each time point. A compilation of the data obtained is shown in Table I. A second biodistribution study compared the in vivo stability of p-iodobenzoyl Fab conjugate ([1251]-5) with coinjected radioiodinated NR-LU-10 Fab labeled by using [1311]iodideand chloramine-T. A limited biodistribution study was conducted in BALB/c mice, since information on the localization of radioiodine in a few specific tissues was sought. Thus, two groups of eight BALB/c mice were injected with a mixture of [1251]-5and [l3l1]iodide/ChTlabeled Fab and were sacrificed at 4 and 16 h postinjection. The stability of the radiolabels with regard to in vivo deiodination was of interest, so tissues where free radioiodide accumulates, neck tissue (containing thyroid) and stomach, were examined. The large differences in the kidney values obtained in the initial study prompted evaluation of that tissue as well. The lung was also included because the values obtained in the initial biodistribution study were higher than those normally obtained in similar studies. The biodistribution data obtained are shown in Table 11. A third biodistribution study compared the in vivo distribution of m-iodobenzoyl NR-LU-10 Fab conjugate ([1251]-6)coinjected with NR-LU-10 Fab radioiodinated by using [1311]iodide/ChT. The biodistributions were conducted in BALB/c mice, as described previously. The biodistribution data obtained are presented in Table 111. DISCUSSION

Our interest in development of radiolabeled antibodies for diagnostic imaging prompted an investigation of the p- and m-iodobenzoyl radiolabels on a pancarcinoma Fab fragment, NR-LU-10 Fab, currently in clinical trials for detection of a number of different carcinomas. In the investigation we sought to obtain data on the in vivo distributions of NR-LU-10 Fab when radiolabeled as p-iodobenzoyl and m-iodobenzoyl conjugates. The biodistribution data was sought to help determine if there were differences which would point to the choice of one of these reagents for use in clinical trials. The data obtained from the initial biodistributions of coinjected m-[1251]iodobenzoyl-and p-[1311]iodobenzoyllabeled Fab (Table I) show that the two radioiodine nuclides, and presumably the radiolabeled Fabs, have very minor differences in tissue distribution, except in the kidneys and intestines. In these two tissues isotopic ratios of 1.7 and 1.9 for [1311]-5/[1251]-6 were obtained, respectively, at 4 h postinjection. At 20 h postinjection the ratio of [1311]-5/[1251]-6in the kidneys remained at 1.8,whereas no difference in isotopes was noted in the intestines at that time. It should be noted that at the later time point isotopic ratios in the tissues varied more than those at the 4-h time, presumably due to the fact that there were low number of counts in many tissues. The only tissue that retained much of its activity by 20 h was the tumor. The isotopic ratios in the tumor were found to be nearly identical at both time points studied, suggesting that NRLU-10 Fab radioiodinated with either reagent would be equally effective at tumor localization. These results appear to be in contrast to those reported by Zalutsky et al. (23, 24), but we believe that this may be due to the nature of the antibody and tumor type under study. Interestingly, even though the differences in tissue distribution were small in this study, analyses of the data using a paired Student's t test (on paired data from each animal) pointed out that there were statistically significant differences in the % ID/g values for all of the tissues except

lodobenzoyl Antibody Fab Conjugate Evaluations

muscle, liver, and neck at 4 h and tumor, muscle, lung, stomach, and neck at 20 h. These very small, but significant, differences appeared to be due to the fact that animals injected with the m- [1251]iodobenzoylFab conjugate had slightly lower concentrations in tissues than those injected with the p-[1311]iodobenzoyllabeled Fab. However, it cannot be ruled out that the (consistent) small differences may have been brought about by radioisotope counting errors. Repeat of the biodistribution with a reversal of isotopes on the m- and p-iodobenzoyl conjugates would have answered this question, but the differences were so small as to not warrant such an undertaking. While the data suggests a significant difference in isotopic ratio within an individual animal, the isotopic ratio differences were smaller, in general, than the differences noted from animal-to-animal variation. The high lung activity observed in the first animal biodistribution study (Table I) was not observed in the second or third biodistribution studies (Tables I1 and 111). The reason for the higher than normal lung concentrations is not readily apparent. If one isotope had been observed to be high in the lung while the other normal, an explanation might have been that the antibody had been damaged in the radiolabeling process. However, both iodobenzoyl-conjugated Fab preparations gave similar localization in the individual animals. Further, analysis of the HPLC chromatograms did not indicate that there was anything unusual about the preparations. As expected, the stomach and neck radioisotopic localization was appreciably different for the m- and p-iodobenzoyl conjugates from that of the directly labeled Fab, denoting the differences in the in vivo deiodination for the labeled preparations. The values obtained were consistent with those previously observed for radioiodinelabeled NR-ML-05 Fab, an antimelanoma antibody (26). Also, consistent with the initial comparative biodistribution, p-iodobenzoyl-labeled Fab ( [1251]-5) had considerably higher quantities of radioactivity in the kidney than the chloramine-T-labeled Fab ( [1311]ChT)with isotopic ratios of 2.2 at 4 h and 3.1 at 16 h (Table 11). In contrast, only small differences were noted for the kidneys when m-iodobenzoyl-labeled Fab ([1251]-6) was compared with chloramine-T-labeled Fab (Table 111). From our studies, and other reported results on labeled Fab fragments, it is evident that Fab conjugates are rapidly accumulated in the kidneys. A large difference in the kidney localization or retention of the p-iodobenzoyl conjugate of NR-LU-10 Fab was observed in this study. Similar differences in kidney radioactivity had been observed previously when an antimelanoma antibody, NRML-05 Fab, labeled as the p-iodobebenzoyl conjugate was compared with the same antibody labeled with radioiodide/ChT (26). This same observation was made when iodovinyl-Fab conjugates (27) and an N-(p-iodopheny1)maleimide-Fab conjugate4 were compared with p-iodobenzoyl-labeled Fab. Indeed, the coinjected, dual-labeled Fab biodistribution studies presented herein demonstrate a reproducible difference in mice for the p-iodobenzoyl conjugate’s accumulation or retention in the kidney from that of the m-iodobenzoyl conjugate of the same Fab. Presumably antibodies are metabolized in the same manner as other circulating proteins in renal cells (28), most likely resulting in complete hydrolysis to amino acids leading to either the free acid or lysine adducts of the iodobenzoates. Data obtained in these laboratories from metabolites in urine samples of patients suggests that the degradation of the p-iodobenzoyl conjugate yields principally the lysine adduct, with a minor amount of glycine

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adduct (hippuric acid) being present (29). Contrary to this, Zalutsky has reported (22) that free m-iodobenzoic acid is obtained from degradation of the intact antibodies in his studies and has observed principally the glycine adduct being present in studies of the m-iodobenzoic acid (30). Such a difference in the metabolism could readily explain the differences in kidney activity noted. However, other factors such as alteration of metabolite binding in the renal transport system and/or the rate of transport (31) may be involved. Delineation of the mechanism of the observed kidney radioactivity differences is beyond the scope of this investigation. Unlike the results obtained in the radioiodinated Fab, other investigations of p-iodobenzoyl antibody conjugates involving intact antibodies or F(ab’)2 fragments duallabeled and coinjected with the same antibody radioiodinated by using chloramine-T have not shown the appreciable differences observed in kidney radioactivity concentrations (16, 32, 33). Additional information is needed as to whether the observed kidney differences for the p- and m-iodobenzoyl conjugated NR-LU-10 Fab are unique to mice or common in other species of animal. The fact that the differences in most tissues are minor, with the exception of the kidneys and intestines, has led us to the conclusion that no difference would be expected to be seen in a clinical application of antibodies radioiodinated as either the m-iodobenzoyl conjugate or the p-iodobenzoyl conjugate due to patient-to-patient variability. This statement is made with the caveat that determination of variance in the kidney activity between the two radiolabeling agents could be important as a substantial difference in the radiation dose to that organ may ultimately favor the use of the m-iodobenzoyl conjugate. ACKNOWLEDGMENT

We thank Dr. Mary Ann Gray, Karen Poole, Denise DuPont, and Joanne Stevens for their efforts in obtaining the biodistribution data presented herein. We also thank Dr. Alan R. Fritzberg, Dr. A. Charles Morgan, and Dr. Paul L. Beaumier for their discussions on the experiments and manuscript. LITERATURE CITED

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Registry No. 1,107759-58-0; 2,112725-22-1; [1261]-3,12521572-7; [1311]-3,126296-26-2;['251]-4, 125215-73-8; ["11]-4, 12773321-5.