Conjugation of antibodies with bifunctional chelating agents: Isothiocyanate and bromoacetamide reagents, methods of analysis, and subsequent addition of metal ions

ANALYTICAL

BIOCHEMISTRY

142,68-78

(1984)

Conjugation of Antibodies with Bifunctional Chelating Agents: lsothiocyanate and Bromoacetamide Reagents, Methods of Analysis, and Subsequent Addition of Metal Ions’ CLAUDE F. MEARES,* MICHAEL J. MCCALL,* DAYTON T. REARDAN,* DAVID A. GOODWIN,~ CAROL I. DIAMANTI,~ AND MAUREEN MCTIGUE~ *Department of Chemistry, University of California, Davis, California 95616, and TDepartment of Nuclear Medicine, Veterans Administration Medical Center, Palo Alto. California 94304. and Stanford University School of Medicine, Stanford, California 94305 Received March 20, 1984 Preparation of the chelating agent (St4-[2,3-bis[bis(carboxymethyl)amino]propyl]phenyl isothiocyanate is reported. Procedures for conjugation of this and (S)-N-4-[2,3-bis[bis (carboxymethyl)amino]propyl]phenyl bromoacetamide to monoclonal antibodies and other proteins are described. The conjugates may be purified quickly by centrifugation through Sephadex G-50. The number of protein-bound chelating groups may be measured by titration with standard “Co’+, using thin-layer chromatography to monitor binding. The labeled products retain their immunoreactivity, as illustrated by experiments in vivo with chelate-conjugated antibody to mouse I-AK antigen. @1984 Academic PRS, Inc. KEY WORDS: immunoglobulins; organic synthesis; radioactive; thin-layer chromatography; gel filtration; radioactivity measurement; luminescence: metal ions.

Recently there has been a great deal of interest in the attachment of metal chelates to proteins, particularly monoclonal antibodies, for use as radiopharmaceuticals in vivo or as fluorescent tracers in vitro (l- 12). Because they form stable chelates with a large number of different metallic elements, reagents incorporating EDTA or DTPA* groups have been used most frequently. Previously published reactions for coupling chelating agents to proteins have included diazonium

coupling (I 3,14) and acylation with activated carboxyl groups (l-4,12,15,16). As part of our research on chelate-tagged radiopharmaceuticals, we have prepared EDTA analogs bearing isothiocyanate or bromoacetamide as the reactive groups for conjugation to antibodies or other biological molecules. Here we describe straightforward procedures for attaching these reagents to mouse monoclonal antibodies, quickly purifying the products, analyzing the number of attached chelating groups, and labeling with “‘In or various other metal ions.

’ Supported by Research Grants CA16861 (C.F.M.), CA18343 (D.A.G.), and Research Career Development Award CA00462 (C.F.M.) from the National Cancer Institute, National Institutes of Health, and a Veterans Administration research grant (D.A.G.). ’ Abbreviations: DTPA, diethylenetriaminepentaacetic acid, ABE, (Q-4-[2,3-bis[bis(carboxymethyl)amino]propyl]aniline; BABE, (S>N-4-[2,3-bis[bis(carboxymethyl) amino]propyl]phenyl bromoacetamide (see Fig. I); CITC, (S) - 4 - [2,3 - bis[bis(carboxymethyI)amino]propyl]phenyl isothiocyanate (see Fig. I); DDW, deionized distilled water; Ab, antibody; MIgG, mouse immunoglobin G. 0003-2697184 $3.00 Copyright 0 1984 by Academic F~cs.s, Inc. All rights of reproduction in any fom reserved.

MATERIALS

AND

METHODS

General Reagents. Mouse monoclonal antibody to the human transferrin receptor was obtained from Dr. Dennis Carlo, Hybritech, Inc., San Diego, California. Mouse monoclonal antibody to BALB/K B lymphocytes (anti I-AK) was obtained from Dr. Hugh McDevitt, 68

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AND

Stanford University. Mouse immunoglobin (MIgG) was purchased from Sigma Chemical Company (whole MIgG) or was from Hybritech (monoclonal MIgG). No difference in properties, with respect to labeling, was observed. Human serum transferrin was purchased from Behring Diagnostics, Somerville, New Jersey. Cobalt powder (99.999%) was obtained from Aldrich. Indium foil (99.999%) was purchased from Alpha. Carrier-free 57CoC12 was purchased from ICN. The water used throughout was fresh deionized distilled water (DDW). Citrate buffer was prepared by dissolving the appropriate amount of citric acid in DDW and adjusting the pH to the desired value with either saturated sodium hydroxide or gaseous ammonia filtered through glass wool. All buffers used with antibodies were filtered through sterile 0.45 km nitrocellulose (Millipore) filters prior to use. All other reagents were the purest commercially available. All glassware was mixed-acid washed (17) and liberally rinsed with DDW. All plasticware was soaked overnight in 3 M HCl and rinsed liberally with DDW. Volumes under 1 ml were measured with Eppendorftype pipets. Metal-free disposable pipet tips were purchased from Bio-Rad. Standard cellulose dialysis tubing with a molecular weight cutoff of 12,000 to 14,000 g/mol was used. Dialysis tubing was prepared for use by soaking 1 h in 1% aqueous acetic acid; rinsing with DDW; boiling for 30 min in 1% aqueous Na2C03, 10 InM H.+EDTA, 10 mM ascorbic acid; heating to 75°C for a few minutes in fresh 1% Na2COs, 10 mM H,EDTA; and five iterations of heating to 75°C for a few minutes in fresh portions of DDW. Dialysis tubing was stored at 4°C in plastic bottles in 0.02% aqueous NaN3, 1 mM Na2EDTA. Before use, it was rinsed thoroughly with DDW. Instrumentation High-pressure liquid chromatography. HPLC was performed on the chelating agents at room temperature using Waters 6000A

CHELATES

69

and M-45G solvent delivery systems controlled by a Waters Model 660 solvent programmer and employing an Alltech 10 X 250mm Cl8 column. Compounds were detected by absorbance at 254 nm using an ISCO UA-5 absorbance monitor. Twenty-minute linear gradients were used, beginning at 100% 0.05 M triethylammonium acetate, pH 6, and stopping at 100% methanol, with a flow rate of 3 ml/min. Ultraviolet spectroscopy. Optical density measurements were made at 280 nm on either a Gilford Model 250 spectrophotometer or a Hewlett-Packard Model 8450A uv/vis spectrophotometer using a 1-cm-path-length microcell masked with black photographic tape to give reproducible results on volumes of 300 ~1. Antibody concentrations were determined using E& = 14 ( 18) and a molecular weight of 155,000 g/mol. Optical densities were measured on a suitable dilution (usually lOO-fold) to give absorbance readings of 0.1-0.3. NMR. NMR spectra were run on a Varian EM 360 NMR spectrometer. Samples were dissolved in DzO and the pH was adjusted with NaOD prior to obtaining the spectra. The HDO peak was set at 4.7 ppm and used as the reference. Infrared spectroscopy. Infrared spectra of solid samples in KBr pellets were obtained using a Perkin-Elmer 237B grating infrared spectrophotometer. Fluorescence measurements were performed on a Perkin-Elmer Model MPF-44B fluorescence spectrophotometer, at room temperature, in the ratio mode, uncorrected for instrumental response. The instrument was very stable over the 2 h required for a fluorescence titration. Radiation counting. Gamma counting was done in a Beckman Model 3 10 counter with the appropriate energy windows for the isotope being counted (O-170 keV for 57Co and O-300 keV for ” ‘In). Chelate Synthesis (S)-4-[2,3-Bis[bis(carboxymethylamino)]propyllphenyl isothiocyanate (CITC, Fig. 1).

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MEARES

H,\

,NKH,CO,-1,

HZ H, “C R~CHZ~

‘NKH,C02-),

CITC:

R = SCN-

BABE:

R = BrCH,C

e

NH-

FIG. 1. Structures of the bifunctional chelating agents used in this work. Both were prepared starting from naturally occurring phenylalanine, and have the S configuration as shown. ABE, the immediate precursor of BABE and CITC, has R = HrN-. The precursor of ABE has R = OrN-.

(S)-pAminobenzyl-EDTA3 (ABE) was prepared by the catalytic reduction of (S)-p nitrobenzyl-EDTA. p-Nitrobenzyl-EDTA (507 mg, 1.2 mmol), prepared according to DeRiemer et al. (19), was dissolved in about 50 ml cold HrO, with the careful addition of NaOH, and finally adjusted to pH 11.5 with 0.1 M NaOH. A 92-mg amount of 10% Pd/ charcoal was added to the cold solution, and the reaction was stirred under 1 atm H2 for 3-5 h in an ice bath. After Hz uptake ceased, the reaction mixture was neutralized with 1 N HCl, titered through 0.45-pm nitrocellulose (Millipore) to remove the catalyst, and iyophilized. The lyophilized residue, containing the product and .sodium chloride, was used without further purification. Lyophilization residue (1.066 g), containing 405 mg of (S)paminobenzyl-EDTA (as determined by s7Co TLC assay described below), was dissolved in 10 ml of 3 N HCl and 1.O ml of thiophosgene (85% in CCb, Aldrich) was added with 3 There are many ways to name multifunctional compounds such as these. Previously (16,19) we chose to name them as derivatives of EDTA. But in this work it seems clearer to emphasize the different protein-labeling groups by naming BABE and CITC as derivatives of bromoacetamide and phenyl isothiocyanate, respectively. In keeping with this, aminobenzyl-EDTA can be named as a derivative of aniline (see ABE, footnote 2) and nitrobenxyl-EDTA can be named (S)4[2,3-bis[bis(carboxymethyl)amino]propyl]nitrobenzene. See Fig I for structures. Note that BABE can also be called bromoacetamidobenzyl-EDTA ( 19).

ET AL.

stirring. Since thiophosgene is toxic, this reaction was carried out in a good fume hood. The reaction mixture was stirred at room temperature for 6 h, until the aqueous phase was negative to fluorescamine (20). Excess thiophosgene and carbon tetrachloride were removed by extraction with ethyl ether (care was taken to avoid spilling), and the acidic aqueous layer was lyophilized using apparatus in the hood. The product, CITC, was characterized on the basis of ir (SCN- stretch at 2100 cm-‘), NMR (the aromatic aa’bb’ pattern of ABE, centered at 6.8 ppm (pH lo1 I), changed to an unresolved multiplet at 7.3 ppm; aliphatic to aromatic proton ratio 3.22 vs 3.25 theoretical), and HPLC retention time (15.5 vs 10 min for ABE). Addition of ammonia to a solution of CITC caused the HPLC peak at 15.5 min to disappear and a new peak to appear at 10 min retention time, consistent with formation of a thiourea from the isothiocyanate. The yield was 1.100 g of residue containing 4 18 mg (93% theoretical) of CITC as determined by 57Co TLC assay (described below). (S)-N-4-[2,3-Bi~bis(carboxymethyl)amino]propyljphenyl bromoacetamide (BABE, Fig. 1). This was prepared as described earlier (19). The reaction mixture was extracted with ethyl ether to remove the excess bromoacetylbromide, and the acidic aqueous layer was lyophilized. The BABE-salt residue was used without further purification. The percentage of BABE in the residue was determined by the 57Co TLC assay outlined below; the yield was practically quantitative. Purification of “‘InCl~. This was performed behind 2-in. lead shielding in a fume hood. Four vials, each containing 3 mCi of “‘InC13 in 1.5 ml aqueous NaCl solution (Medi + Physics), were treated with 0.4-ml aliquots of 12 M HCl (final concentration 2.5 M HCI) and applied to a l-cm-diameter column containing 15 g of Bio-Rad AGl-X4 anionexchange resin (200-400 mesh) equilibrated with 2 M HCl. Each vial was washed with an additional l-ml aliquot of 2 M HCl, the washings were added to the column, and the

ANTIBODIES

AND CHELATES

liquid was allowed to run into the column. After the column was washed with an additional 17.5 ml of 2 M HCl, the ” ‘InCla was eluted with 0.2 M HCl. Fractions were collected in acid-washed polypropylene vials and dried in a block heater (90°C) under a gentle stream of air. The vials were capped and stored in lead pigs until needed for radiolabeling. They were generally used within 1 week after column purification. Preparation of standardized 57CoCl~ and “‘fnClj. Nonradioactive samples of each pure metal were weighed (four significant figures) and dissolved in 6 M HCl at 60°C and then diluted with DDW to the mark in volumetric flasks. Another volumetric dilution was performed to yield solutions of each which were about 0.06 M in HCl and about 400 PM in metal (exact value accurately known in each case). A 500-~1 amount of each solution was added to about 0.1 mCi of the appropriate carrier-free radioisotope to yield a solution with a radioactivity concentration greater than 50,000 cpm/pl. Thin-layer chromatography. As shown in Fig. 2, every TLC plate was prepared as follows: 2 cm from the bottom of a IO-cm plate, a very light pencil line was drawn to indicate the origin; 5 cm up from the origin a 2-mm-wide path was scraped free of silica gel to stop the solvent. Each labeled lane was spotted with 0.5 to 2.0 ~1 of sample and dried with a stream of air. A hole was punched in the top of the TLC to allow for easy removal from the solvent tank. The TLC system consisted of O.Zmm-thick silica gel 60 FzS4 on plastic backing (E. Merck, Cat. No. 5775), with 10% (w/v) ammonium acetate in DDW:methanol (1: 1, v/v) as the developing solvent. Fresh solvent was used for each TLC, and the TLC was removed promptly after developing and allowed to air-dry at room temperature. To count the separated components in the gamma counter, each lane of the TLC plate was cut with scissors at approximately Rf = 0.2 and 0.3. The resulting three sections contained protein-bound radiometal (RJ O.O-0.2), back-

71

FIG. 2. Autoradiogram (25 h with an intensifying screen) of a standard TLC showing the TLC characteristics. Lane 1: 10 ~1 0. I M sodium citrate, pH 6.5, + 10 gl bifunctional chelate-tagged mouse IgG + 2 pl standardized “CoC12 solution. Lane 2: 10 pl 0.1 M sodium citrate, pH 6.5, + 10 ~1 unmodified mouse IgG + 2 al standardized “CoC12 solution. Lane 3: 10 ~1 0.1 M sodium citrate, pH 6.5, + 2 ~1 standardized %JC& solution.

ground (R/0.2-0.3), and free radiometal chelates (RJ 0.3- 1.O). TLC analysis of chelator concentration in stock solutions. Exactly 5 ~1 of the standard 400 PM cobalt solution was added to 10 ~1 of 0.1 M sodium phosphate at pH 6.5 (it is important that phosphate buffer be used in this case, so that unchelated cobalt will remain near the origin). To this was added an accurately measured volume of a stock solution containing one of the chelating agents (sufficient to chelate about 50% of the cobalt). This solution was vortex mixed and allowed to stand for 5 min at room temperature and then 1 ~1 was applied to a standard silica TLC plate and developed. Cobalt chelates migrated with R/B 0.3, while unchelated cobalt remained near the origin. The concentration of chelator was calculated using the equation

72

MEARES

moles (Co2+) X rwm (RJ> 0.3)1 .

I

L wm (toW

J

= moles (chelator).

[l]

Calibration of this assay with known concentrations of EDTA gave agreement within 5%, with a coefficient of variance (SD X lOO/ mean) of 3.5%.

TLC method for determining the extent of the antibody-chelator coupling reaction. The reaction mixture could be tested by removing a 2-~1 aliquot and adding it to 10 ~1 of 0.1 M ammonium citrate buffer at pH 6.5 (it is important that citrate buffer be used in this case, so that unchelated cobalt will move near the front) and then adding 1 ~1 of carrier-free 57CoC12. The resulting solution was vortexed and allowed to stand for 10 min at room temperature and then analyzed by TLC. The antibody-bound 57Co chelates remained at the origin while the free chelates moved with RJ 0.8-0.95. By knowing the initial ratio of bifunctional chelating agent to protein and measuring the radioactivity at the origin versus the radioactivity at RJ> 0.3 as described above, an approximate coupling yield could be measured. It was determined with a series of carrier-added cobalt solutions (up to a stoichiometric ratio of cobalt to total chelator) that this procedure was not biased in favor of free chelator.

TLC method for ~uantitating the number of protein-bound chelating groups after isolation of antibody-chelator conjugate from nonbound chelator. First, the protein concentration was determined by measuring the absorbance of an appropriately diluted sample at 280 nm. Then exactly 10 ~1 of the conjugate solution was added to 10 ~1 of 0.1 M ammonium citrate buffer (at pH 6.5 for assay with standardized “CoC12, at pH 5.0 for assay with standardized ” ‘InClJ. An amount of standardized metal solution sufficient to give about a twofold molar excess of metal relative to the estimated amount of chelator in the lo-p1 aliquot of antibody-chelator conjugate was added to the above solution of protein and buffer (the pH of the solution

ET AL.

did not change significantly). The assay was not attempted unless the concentration of antibody-bound chelator was greater than 10 PM, because trace-metal contamination could interfere with it. The solution was vortexed and then allowed to stand for 20 min. If standardized “‘1nCl~ was used to assay the chelate/protein ratio, or if there was a question of nonspecific binding of metal to the protein, as determined by the control lane (see lane 2, Fig. 2), the solution was challenged with EDTA prior to spotting the TLC. The EDTA challenge was performed as follows: after the above solution had stood for 20 min, 2 ~1 of 0.05 M EDTA, pH 6.5 (enough to scavenge all of the added metal), was added to it. The resulting solution was vortexed and allowed to stand for 5 min. For analysis, 1 ~1 was then applied to the TLC and dried, developed, and counted as described above. After development of the TLC a fluorescamine test (20) for protein could be performed on the TLC plate to check for overloading and subsequent “smearing” of the protein at the origin. The concentration of antibody-bound chelator was calculated using the equation moles (Co’+ or In3+) X

cpm (RJ< 0.2) [ cpm (total)

= moles (Ab chelator).

1 (21

The TLC assay was corroborated by fluorescence titration using terbium(III), which has a narrow bandwidth emission peak at 546 nm. To do this it was convenient to prepare a MIgG solution with a high chelator/ protein molar ratio, because BABE and CITC are not as effective at stimulating lanthanide luminescence as the azo chelator used by Leung and Meares (21). BABE was used for the reaction; the bound chelator/protein ratio of the conjugate was determined to be 10.2 by TLC assay. This same solution was then titrated with TbC13; the result was the expected titration curve (21) whose endpoint gave a value of 10.6 chelators/protein. The 4% difference between the TLC chelator/ protein ratio (10.2) and the fluorescence ti-

ANTIBODIES

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AND CHELATES

tration (10.6) is within the experimental error of the two systems. The TLC assay was also found to work well with antibodies conjugated to DTPA anhydride (4,22). Centrifuged column procedure. For the antibody coupling reactions below, buffer changes and removal of unbound chelator were carried out by a centrifuged column procedure, previously reported by Penefsky (23), which we adapted to our use (see Fig. 3). The procedure used the barrel of a disposable, 1-ml plastic tuberculin syringe (Becton-Dickinson No. 5625) fitted with a porous polyethylene disk cut from sheets (l/l 6-m thick, 70-pm pore size, Bolab, Inc., Cat. No. BB2062-70L) using a No. 1 cork borer. The syringe was filled to the l-ml mark with Sephadex G-50-80 (Sigma) previously allowed to swell in either a 0.15 M sodium phosphate buffer (pH 8) or a 0.1 M ammonium citrate buffer (pH 6) depending on the desired final buffer for the antibody. The column was allowed to stand until no further liquid drained from it; then it was transferred to a 15 X 125-mm test tube containing a 0.5-ml polypropylene microcentrifuge tube (VWR) with the cap removed, resting on a No. 2 cork (Fig. 3). The centrifuge tube acted as the collection vessel for the column. The test-tube assembly was placed in a four-place swinging-bucket rotor (No. 2 13) of a simple tabletop centrifuge (Model CL, International Equipment Co., Needham Heights, Mass.) and centrifuged at approximately half speed (setting - 3 out of 7, or -1OOg) for 2 min. This caused some buffer to run into the collection vessel, and the gel column length decreased approximately 30%. Then a fresh microcentrifuge tube was put in place, and 100 ~1 of an antibody solution was applied to the top of the gel column. The apparatus was then recentrifuged as above, and the antibody in the desired final buffer was collected in the microcentrifuge tube. This was used twice in the antibody-chelator coupling procedures below, the first time to prepare the antibody for coupling by transferring it to the reaction buffer (0.15 M sodium phos-

i-ml Plastic Tuberculin Syringe Packed with Sephadex O-50-80

Polyethylene

Frit

OS-ml Poly ropylene Micro Test 7 ube, Cap Removed 15xi25mm Pyrex Test +2

Tube

Cork

FIG. 3. Schematic drawing of the centrifuged column used to separate antibodies from small molecules (see Materials and Methods).

phate, pH 8) and the second time to remove unreacted chelator and to place the antibodychelator conjugate in a buffer suitable for radioactive metal labeling (0.1 M ammonium citrate, pH 6). The volume of effluent collected was approximately the volume applied (k 10%). The amount of antibody recovered was approximately 90% of that applied, based on its absorbance at 280 nm. There was no evidence, as shown by “Co TLC assay, of any free chelator in the effluent. Similar results were achieved using the protein transferrin instead of an antibody. Alternate procedures to achieve the same ends, notably dialysis with several changes of buffer, required several days, were not completely effective in removing free chelator, and were accompanied by antibody losses of similar or greater magnitude, especially when handling small amounts (~100 ~1). The centrifuged column procedure reduced the total elapsed time needed for preparation, purification, and TLC analysis of an antibodychelator conjugate to approximately 6 h. Antibody-Chelator Coupling General procedures. The antibody solution (-25 mg/ml) was first prepared for conjugation by the Sephadex G-50-80 centrifuged column procedure above, using 0.15 M so-

74

MEARES

dium phosphate, pH 8, as the gel buffer. The collected effluent was transferred to a polypropylene vial and an excess of chelator was added (BABE- 1.5 mM final concentration; CITC-0.5 mM). The added chelator could be in solution (0.15 M sodium phosphate, pH 8) or in solid form. The concentration of antibody in the final solution was at least 15 mg/ml. The pH of the antibody-chelator solution was adjusted to 9.0-9.5 with saturated aqueous trisodium phosphate. The solution was incubated at 37°C for 2 h. An aliquot of reaction mixture was removed and a TLC assay to estimate the coupling yield was carried out (see above). If the chelator:antibody conjugation ratio was less than 1, the reaction mixture pH was readjusted if necessary, and incubation and assay procedures were repeated. Otherwise, excess chelator was removed and the antibody-chelator conjugate placed in a 0.1 M ammonium citrate buffer, pH 6, using the centrifuged column described above. Quantitative determinations of the antibody and bound-chelate concentrations were made using absorbance and the 57Co TLC assay described above. The absence of free, unreacted chelator in the centrifuged column effluent was shown by repeating the carrier-free 57Co TLC analysis of coupling yield and observing that the area of the TLC plate at R,-> 0.3 was nonradioactive. Coupling of BABE to mouse monoclonal antibody: A specific example. An 85-~1 aliquot of mouse monoclonal anti-transfenin receptor solution (36.6 m&/ml, 2.4 X 10T4 M) was applied to a Sephadex G-50-80 column which had been previously prepared with 0.15 M sodium phosphate solution (pH 8). The column was centrifuged as described above and 95 ~1 of effluent was collected. Absorbance measurement of the effluent at 280 nm showed an antibody concentration of 29 mg/ ml (88% recovery, 1.9 X 10m4 M). To 90 ~1 of antibody solution was added 25 ~1 of 6.9 mM BABE in 0.15 M sodium phosphate, pH 8 (final concentration 1.5 mM BABE, ca. 1O:l ratio of BABE to antibody), and the

ET AL.

reaction mixture was adjusted to pH 9 with 8 ~1 of saturated aqueous trisodium phosphate. The reaction was incubated at 37°C for 2 h. A 2-~1 ahquot of the reaction mixture was removed and a carrier-free 57Co TLC assay run to estimate the coupling yield. Of the total radioactivity, 35% was present at Rf < 0.2, indicating that an average of 3.5 molecules of BABE had coupled to each molecule of the antibody. The reaction mixture (- 120 ~1) was applied to a Sephadex G-50-80 centrifuge column, which had been prepared with 0.1 M ammonium citrate (pH 6). Centrifugation produced - 110 ~1 of effluent. The absorbance of the effluent indicated an antibody concentration of 25.5 mg/ml (81% recovery, 1.6 X 10e4 M). Overall, the amount of antibody recovered was approximately 70% of the initial amount. A carrier-free 57Co TLC assay showed no unconjugated chelator in the effluent. A 57Co assay for quantitating the number of chelators on the antibody gave a chelator concentration of 3.8 X 10e4 M, therefore giving a BABE-to-antibody ratio of 2.4: 1 in the final 0.1 M ammonium citrate solution (pH 6). The apparent decrease in the chelator:antibody ratio from 3.5:l to 2.4:l may be due to traces of metal ions in the Sephadex G-50-80 column. “‘In incorporation experiments. Five carrier-free ’ “In stock solutions in 0.1 M ammonium citrate buffers were prepared by adding 3 ~1 of carrier-free “‘1nCl~ solution (in 1 mM HCl) to 22-~.d aliquots of 0.1 M ammonium citrate buffer, pH 5, 4, 3, and 2.1. To an 8-~1 aliquot of each of the five “‘In-citrate solutions was added 8 ~1 of antitransferrin receptor-BABE conjugate in 0.1 M ammonium citrate solution, pH 6 (antibody concentration: -95 pM, BABE/antibody ratio: 1.3). As control experiments, 8 ~1 of each of the five “‘In-citrate solutions was mixed with 8 ~1 of unconjugated antitransferrin receptor antibody in 0.1 M ammonium citrate solution, pH 6 (antibody concentration: 125 pm). The 10 solutions were mixed thoroughly and allowed to stand for -15 min before 16 ~1 of 10 mM

ANTIBODIES

AND CHELATES

NazEDTA solution, pH 5, was added to each and the solutions were mixed again. After 5 min, 1.5~~1 aliquots of each solution were spotted on a silica gel TLC plate and developed. At 10 and 30 min after the addition of the EDTA challenge solution, the TLC procedure was repeated. Radioactivity distributions in the TLCs were visualized by autoradiography and quantitatively determined by sectioning the plate and counting each section for gamma radioactivity. The data showed that the extent of “‘In incorporation in the antibody-chelate conjugate was not sensitive to the pH of the “‘In-citrate solution. In addition the EDTA challenge procedure did not result in a detectable increase of radioactivity at Rf > 0.3 over a period of 30 min. In the case of the unconjugated antibody, there was no evidence to suggest that “‘In was nonspecifically bound to the antibody (after the introduction of the EDTA). A second experiment was undertaken along the same lines, except that only the antibodychelator conjugate and the “‘In-citrate, pH 5, solutions were used. Two solutions, each containing 10 ~1 of the antibody-chelator conjugate solution and 5 ~1 of the “‘Incitrate solution, were prepared. One solution was kept at room temperature and the other placed in a 37°C water bath. At time intervals of 5, 10, 20, 40, and 80 min after initial mixing, a l-r1 aliquot of each solution was removed and mixed with 5 ~1 of a 10 mM EDTA solution, pH 5. Five minutes later l~1 aliquots of the solutions containing the EDTA challenge were spotted on a silica gel TLC plate and developed. The amount of unbound “‘In, moving with Rf > 0.2, was only 3-5% of the total “‘In. There was no significant change observed with respect to time nor was there any significant difference between the room temperature solution and the 37°C solution. In vivo distribution. Mouse monoclonal antibody to BALB/k B lymphocytes (anti-IAK), conjugated with 3.3 chelators per antibody molecule (using the method described

75

above for BABE), was radiolabeled with “‘In. Uptake in the lymph nodes of BALB/k (IAK positive), BALB/c (I-AK negative), and C3H (I-AK positive) mice was measured (19) 24 h after subcutaneous (both hindfoot pads) or intravenous (tail vein) injection of the “‘In-labeled anti-I-AK conjugate. For comparison, an ’ ” In-labeled human antibody conjugate (prepared using the method described above for BABE, approximately one chelator per antibody molecule) and ‘*‘Ianti-I-AK (prepared according to Ref. (24), approximately 0.04 ‘*‘I atoms per antibody molecule) were also studied. Results are given in Fig. 4. RESULTS AND DISCUSSION

The procedures described above provide a practical way to attach strong metal-chelating groups to mouse monoclonal antibodies, and other proteins as well. The methods used to prepare BABE and CITC from phenylalanine are lengthy (seven steps), but straightforward. To assure stability, both reagents should be stored dry; we keep ours in the freezer over &SO, desiccant. Isothiocyanate and haloacetamide reagents are well known in protein chemistry (25,26). Isothiocyanates are usually considered to be specific for amino groups and have found wide use in the attachment of fluorescent dyes to antibodies (25). Haloacetamides are known to react readily with sulfhydryl groups on proteins, but will also react with amino groups and other nucleophiles (26). At pH 9 to 9.5, reaction of haloacetamides with lysine c-amino groups is expected to occur. Since antibodies seldom contain free sulthydryl groups, lysine and histidine residues and Nterminal amino groups may be the dominant sites of reaction of antibodies with BABE. We have not attempted to verify the residues labeled by BABE and CITC. The cyclic anhydride of DTPA (22) is expected to react with protein amino groups (4); the product of this reaction is a strong chelator, but it releases In3+ ions at a higher rate than do chelators like BABE and CITC (27).

76

MEARES

SALSR “‘ho0

sAlB/c “‘has

M “‘InuS

SALWC ~“lnlHllgG1251~S

CM

FIG. 4. Demonstration of in viva immunoreactivity of “‘IrmB (“‘In chelate-labeled antibody to B lymphocytes bearing the I-AK antigen) by comparing uptake of radioactivity in the lymph nodes of animals whose B lymphocytes express the antigen (BALB/k and C3H) to those which do not (BALB/c). 24 h after subcutaneous (SC) injection in the rear footpads, lymph node uptake of “‘IncuB in antigen-bearing mice was about an order of magnitude higher than in BALB/c mice. Lymph node uptake of ” ‘ItmB in BALB/c mice was comparable to uptake of a nonspecific human immunoglobulin (“‘ln(H)IgG). All the mice took up “‘In(H)IgG to a similar degree. Included for comparison is the lymph node uptake of ‘*%xB in antigen-bearing C3H mice. This is significantly lower than “‘IncrB uptake, possibly due to loss of the lzsI radiolabel (3,12). Data are also included to show that intravenous (IV) injection of “‘InaB into the tail vein led to much lower uptake in the lymph nodes, but still there was a significant d8emnce between antigen-positive and antigen-negative animals.

It is important to measure the average number of chelators per antibody, both dur-

ing conjugation and after purification of the conjugate. Simple TLC methods can do this easily and with reasonable accuracy. We have found that 0.1 M ammonium citrate buffers are useful in preventing tight binding of Co*+ In3+, or Tb3+ ions to unmodified antiboiies, but permit quantitative delivery of these metal ions to protein-bound chelating groups. For some other metals, it may be necessary to use other buffers with weak

ET AL.

metal-binding properties such as acetate, lactate, Tris, or various amino acids. When using short-lived radionuclides such as “‘In (T,,* 2.8 days), it is desirable to store the purified antibody-chelator conjugate in nonradioactive form until just before it is needed. Then the radiometal can be bound and the product used immediately, providing maximum specific radioactivity and minimum degradation of the radioimmunoglobulin. On the other hand, when it is desired to attach a stable metal ion (such as fluorescent Eu3+ or Tb3+) to an antibody, there are certain advantages to adding the metal ion before the conjugation reaction. For example, if a CITC-Eu chelate is prepared and then conjugated to an antibody, this would remove the need for a metal-transfer buffer such as citrate and it would prevent contaminating metal ions from binding to the chelating groups. Some adjustment of the conjugation conditions might be necessary to quantitatively retain the chelated europium, however. The conjugation conditions reported here were chosen to make it convenient to add the metal ion last, after preparation and analysis of the antibody-chelator conjugate. This practically requires that the final concentration of protein-bound chelating groups be relatively high (>10m5 M) so that added metal ions may be bound quickly and quantitatively. By starting with a concentrated solution of antibody (> 15 mg/ml), the desired final conditions are easily achieved. Because BABE and CITC do not hydrolyze significantly in 2’h at pH 9.5, 37°C the conjugation reaction conditions used here ( 1.5 tnM BABE or 0.5 t’nM CITC) probably represent reasonable concentrations of reagents for use with more dilute solutions of antibody. For lower antibody concentrations the ratio of bound chelator to antibody should change little, though the fraction of the total chelator bound to antibody will be smaller. This result would be quite acceptable if the metal ion were added first, since the centrifuged column cleanly removes unbound chelate. The antibody-chelate conjugates retain

ANTIBODIES

their immunoreactivity. As shown in Fig. 4, very high lymph node concentrations of ’ ’ ‘Inanti-I-AK were found in BALB/k and C3H mice (antigen positive) as compared to BALB/ c mice (antigen negative). It is interesting to note that the chelate label was superior to “‘1 (3,12). Further experiments with “‘Inanti-I-AK showed striking in vivo visualization of the spleen of a BALB/k mouse (% dose per gram spleen = 116) but not of a BALB/ c mouse (% dose per gram spleen = 10) (2829). Conjugation of CITC to monoclonal anti-Raji lymphoma antibodies (0.4-2.3 chelators/antibody), followed by labeling with 57C02+, led to 77-84s retention of immunoreactivity in vitro relative to minimally radioiodinated antibody (W. C. Cole, S. .I. DeNardo, C. F. Meares, G. L. DeNardo, A. L. Epstein, and H. O’Brien, unpublished results). Conjugation of BABE to anti-human transferrin receptor antibodies, followed by labeling with ’ ’ ‘In, led to in vivo visualization of the human hematopoietic system; in contrast, conjugation of BABE to human transferrin, followed by labeling of the chelating groups with “‘In, led to in vivo visualization of the human circulatory system (30). So far our experience has been that labeling with up to 3-4 chelators per antibody molecule does not seriously degrade immunoreactivity. Of course this may not be true for every antibody to be studied in the future. On the other hand, it may be practical in some cases to attach many more chelators per antibody while maintaining adequate immunoreactivity. The labeling of antibodies with bifunctional chelating agents has a number of potential applications, including in vivo diagnosis and therapy (l-5,1 1,12,28-30) and in vitro immunoassay (6- 10). These involve different metal ions with different physical properties, but practically the same conjugation chemistry. Because EDTA and related chelating agents form stable complexes with more than 50 metallic elements, it is likely that other potential

the future.

applications

will

come

to light

77

AND CHELATES

in

ACKNOWLEDGMENTS We thank Dr. Hugh McDevitt of Stanford University for providing the anti-I-AK antibody and Dr. Dennis Carlo of Hybritech Inc. for providing the anti-human transferrin receptor antibody.

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