Bioconjugate Chem. 1991, 2, 67-70
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TECHNICAL NOTE Characterization of Antibody-Chelator Conjugates: Determination of Chelator Content by Terbium Fluorescence Titration Kimberly D. Brandt, Karen E. Schnobrich, and David K. Johnson* Abbott Laboratories, Department 90M, Abbott Park, Illinois 60064. Received September 17, 1990
Fluorescence titrations were performed by adding varying mole ratios of terbium(II1) to antibody conjugates formed by benzyl isothiocyanate derivatives of three different polyaminopolycarboxylate chelators (NTA, EDTA, and DTPA) and the results compared to values for average chelator content obtained by cobalt-57 binding assays. For two different murine monoclonal antibodies, the average chelator content obtained by terbium fluorescence titration correlated closely with that measured by the cobalt47 binding assay. It is concluded that lanthanide fluorescence titrations provide a useful alternative to radiometal binding assays for the determination of chelator content in protein-chelator conjugates.
Monoclonal antibodies derivatized with polyaminopolycarboxylate chelates of a variety of radiometals are finding increasing use in the detection (1-5) and potential treatment (6-8) of a number of cancers. Among many parameters that influence the behavior of such chelate immunoconjugates, the average level of substitution (i.e. the number of chelating groups incorporated into each antibody molecule) is a fundamental characteristic that has nevertheless been poorly defined in many of the preparations used clinically. In our laboratory, the 57C0 binding assay (9) has been the method of choice for measuring the average chelator substitution of EDTA' and DTPA immunoconjugates. In contrast to the use of 14C-labeled chelators (10, I I ) , the 57C0 binding assay provides functional information as to the available metal binding capacity of a conjugate and is sufficiently simple that it can be performed routinely on every preparation that is produced. However, as with any assay, equivocal results are sometimes obtained and, in the course of efforts to develop an independent technique by which to confirm chelator substitution levels measured by the 57Coassay, we evaluated the use of terbium fluorescence titrations. Binding of lanthanide metals such as terbium(II1) and europium(II1) to chelating agents that contain an aromatic moiety held close to the coordination sphere can lead to "sensitized" fluorescence, in which light is absorbed through the aromatic system and the energy transferred to the metal, which then produces emissions characterized by a very large Stokes shift and fluorescence lifetimes of up to several seconds (12). Earlier studies (13) of an albumin conjugate of a bifunctional EDTA derivative (1, Chart I) had shown that the phenyl moiety present in that chelator was able to sensitize europium and terbium fluorescence a t micromolar concentrations. As each chelating site in the conjugate bound one lanthanide ion, and as a lanthanide ion had to be in a chelate site if energy transfer was to occur, this technique could be used to quantitate the number of chelating groups present (13). 'The following abbreviations are used: CEA, carcinoembryonic antigen;DTPA, diethylenetriaminepentaaceticacid;EDTA, ethylenediaminetetraacetic acid; NTA, nitrilotriacetic acid. 1043-180219 1/29O2-O067$O2.5OIO
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Similarly, we observed that the phenyl group in the bifunctional EDTA derivative 3 (Chart I) produced sensitized terbium fluorescence (Figure 1). Conjugates of an anti-CEA monoclonal antibody (C110) with a homologous series of bifunctional polyaminopolycarboxylates of varying denticity (2-4, Chart I) were therefore prepared and terbium fluorescence titrations performed, in order to assess the extent of correlation with 57C0binding assay data and the applicability of this technique to different chelate structures. Chelating agents 3 and 4 were synthesized as previously reported (14). The synthesis of compound 2 will be presented elsewhere. The generation and characterization of the anti-CEA antibody used in these studies has been previously described (15). CllO is an IgGl murine monoclonal antibody that is presently in clinical trials in the form of an indium-lll-labeled conjugate of compound 4 (16). All glassware used in these studies was washed sequentially with 3 M HC1, deionized water, and methanol. Deionized water was obtained from a MILLI-Q system (Millipore Corp., Bedford, MA), and all buffer salts were reagent grade or better. Antibody concentrations were determined from the absorbance a t 280 nm, using a 0 1991 American Chemical Society
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Figure 1. Excitation (A) and emission (B) scans, obtained as described in the text, for an antibody conjugate of compound 3 that had been exposed to a 6-fold molar excess of terbium(II1) then subjected to size-exclusion chromatography by spin column to remove unbound terbium. correlation coefficient previously established for that particular antibody from plots of A280 vs antibody concentration as measured by the Bradford dye binding assay (Bio-Rad Laboratories, Richmond, CAI. Conjugates of C110 with each of the chelators were prepared as previously described (1.9, using a 40:l input mole ratio of chelator: antibody at an antibody concentration of 10 mg/mL. Each conjugate was diluted into 0.05 M citrate buffer, pH 6.0, to a final concentration of 2.8-4.2 X M. To aliquots of this stock solution (100 pL) were then added terbium(II1) citrate solutions (10 pL per aliquot) obtained by dissolvingTbCly6HzO (99.999%, Aldrich Chemical Co., Milwaukee, WI) in 0.05 M citrate, pH 6.0. Terbium solutions of varying molarity were used so as to span a range of Tb:antibody input mole ratios. After incubation in the presence of terbium for 30 min a t room temperature, each aliquot was centrifuged through a Sephadex G-25 spin column to remove non-antibody-bound terbium. The eluate (100 pL) was then diluted into 0.05 M Tris/ 0.9 50 NaC1/5 % ethylene glycol, pH 7.5, to a final volume of 2.0 mL and transferred to a cuvette. Fluorescence measurements were made with a Perkin-Elmer LS-5 fluorimeter operating in time-resolved mode, exciting a t 268, 271, and 283 nm, respectively, for the NTA, DTPA, and EDTA conjugates and monitoring emission a t 548 nm. A
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Figure 2. Fluorescence intensity at 548 nm as a function of terbium:antibody input mole ratio for CllO conjugates of (A) compound 2, (B) compound 3, and (C)compound 4. Each data point shown is the mean of 10 fluorimeter readings, the standard deviation from the mean being less than 1% in all cases. Underivatized CllO antibody at the same concentration as the conjugates gave fluorescence intensities of less than 20 units (same scale) when exposed to terbium under the same conditions at Tb:antibody input mole ratios up to 101. 200-ps delay time was employed, with a 1.0-s gate and excitation and emission slit widths of 15 and 20 nm, respectively. Fluorescence titration curves for the three C110-chelator conjugates are shown in Figure 2. In each case there was a distinct break in the curve a t a terbium:antibody ratio of ca. 6, with no further increase in fluorescence intensity observed beyond that ratio. This indicated that each conjugate contained an average of six chelators per antibody molecule, a value identical with that previously obtained from 57C0binding assays performed on CllO conjugates of 4 ( 1 5 ) . The similarity in results obtained with three different chelators suggests that this method may also be applicable to other bifunctional ligands that contain phenyl groups (4-6, IO).
Technical Note
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Bioconjugate Chem., Vol. 2, A
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Figure 3. Fluorescence intensity at 548 nm as a function of terbium:antibody input mole ratio for a B72.3 conjugate of compound 4. Each data point shown is the mean of 10 fluorimeter readings, the standard deviation from the mean being less than 1% in all cases.
To further confirm the correspondence between substitution levels measured by 57Cobinding assays and those obtained by terbium fluorescence titration, the latter method was applied to a conjugate of 4 with a second IgGl murine monoclonal antibody, B72.3. This antibody is directed against a high molecular weight tumor-associated mucin (17) and its production and conjugation to 4 have been described elsewhere (14,18). The conjugate used in the fluorescence study was obtained at an input mole ratio of 4:B72.3 of 3:l and contained approximately two chelators per antibody by 57Cobinding assay (18). The corresponding fluorescence titration curve (Figure 3) showed a plateau in intensity a t a terbium:antibody mole ratio of ca. 2, again demonstrating good correlation between the two methods. The primary constraints on configuring fluorescence assays of this type appear to be related to adventitious metal contamination of samples and materials used in the assay. The practical limit of detection of terbium in these systems is well below nanomolar and the Tris/ethylene glycol/salt buffer minimizes physical trapping and adsorption of proteins (to columns, cuvettes, etc.) at such low conjugate concentrations. Thus, in principle, an assay could be performed a t antibody concentrations some 4-5 log units lower than are used here, making it a sensitive procedure requiring very small sample sizes. However, in practice, conjugate concentrations of a t least micromolar are needed to obtain reasonable assay precision in the face of ambient levels of metal contaminants. When replicate titrations were performed on a single CllO conjugate of 4 under the conditions described earlier, the break point in the titration curve was reasonably reproducible, but given samples a t a particular (but apparently random) mole ratio sometimes gave intensities that fell substantially below the curve. We attribute this to contamination of individual spin columns, sample vials, or cuvettes with metals that were able to compete with terbium for protein-bound chelating sites. This level of variability in the assay, plus the cumbersome nature of a procedure requiring separate processing of samples a t each mole ratio, led us to investigate an alternative, simplified configuration that employed a single aliquot of conjugate, a single buffer, and a single cuvette. A 1.0 X lo4 M solution of a CllO conjugate of 4 in 0.05 M Tris, pH 7.5, was prepared and a 2.5-mL aliquot
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Figure 4. Fluorescence intens'lty at 548 nm as a function of terbium:antibody input mole ratio for a CllO conjugate of compound 4. Each data point shown is the mean of 10 fluorimeter readings, the standard deviation from the mean being less than 1% in all cases. Each panel (A-E) represents a single titration experiment performed on an aliquot of a single conjugate stock solution.
transferred to a 5-mL cuvette. A 2 X M stock solution of terbium in 0.05 M Tris, pH 7.5, was prepared by dissolving TbClr6HzO in 0.1 M HC1 to give a 0.01 M stock, which was then diluted with 0.05 M Tris, pH 7.5. A 100pL aliquot of the 2 X M terbium solution was added to the cuvette containing the conjugate and the resulting solution stirred a t room temperature for 10 min, using a small magnetic stir bar placed in the cuvette. Fluorescence measurements were then performed with instrument settings identical with those described earlier. This procedure was repeated until a total of 15 100-pL aliquots of the terbium solution had been added serially to the
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Bioconjugate Chem., Vol. 2, No. 1, 1991
cuvette, fluorescence readings being made after each addition. A dilution correction was applied to the fluorescence intensity measured at each mole ratio, to compensate for the change in volume of the test solution. Replicate fluorescence titrations of this type were performed on five aliquots of the 1.0 X 104M conjugate stock, the results of these assays appearing in Figure 4. The scatter in the data in the plateau region of the curve was consistently less than that evident in Figure 2. However, although four of the five replicates agreed very closely (panels A, C, D, E, Figure 41, the fifth (panel B, Figure 4) gave a completely disparate substitution level (2.5 chelators/antibody vs. 5). Again, the most likely explanation for this variability in the data is metal contamination of either the cuvette, the stir bar, or the pipet used to transfer aliquot B. In this assay configuration, such contamination affects all data points, as opposed to the individual data points that are affected when the spin column procedure is used. Thus, the choice of a particular configuration depends upon the type of variability that is easiest to deal with in a given application. For routine analysis of DTPA immunoconjugates that are available in quantities permitting the generation of micromolar stock solutions, the single cuvette titration in Tris buffer is the preferred procedure. However, given the data in Figure 4, it is recommended that assays be run in duplicate and that, if duplicate measured substitution levels differ by more than 2096, the titration should be repeated. With these precautions, the procedure is relatively straightforward to perform, given access to a fluorimeter with time-resolution capabilities. Attempts to measure terbium fluorescence without time gating are generally confounded by interfering short-lived fluorescent signals from buffers, etc. However, any fluorimeter with a ratio mode that permits intensities to be compared at at least two different time points after exciting the sample can be used to perform this assay. T h e average substitution level of a chelator immuno conjugate affects performance characteristics, such as theease of labeling with a radiometal (19),and may also influence the disposition of such radiometal labeled conjugates in vivo. We conclude that lanthanide fluorescence titrations offer an independent, nonisotopic method for quantitating this basic parameter in conjugates that incorporate a sensitizing moiety. LITERATURE CITED (1) Siccardi, A. G., Buraggi, G. L., Callegaro, L., Colella, A. C.,
De Filippi, P. G., Galli, G., Mariani, G., Masi, R., Palumbo, R., Riva, P., Salvatore, M., Scassellati, G. A., Scheidhauer, K., Turco, G. L., Zaniol, P., Benini, S., Deleide, G., Gasparini, M., Lastoria,S., Mansi, L., Paganelli, G., Salvischiani,E., Seregni, E., Viale, G., and Natali, P. G. (1989) Immunoscintigraphy of adenocarcinomas by means of radiolabeled F(ab')z fragments of an anti-carcinoembryonic antigen monoclonal antibody: A multicenter study. Cancer Res. 49, 3095-3103. (2) Chatal, J-F., Saccavani,J-C., Gestin,J-F.,Th&lrez, P.,Curtet, C., Kremer, M., Guerreau, D., Nolib6, D., Fumoleau, P., and Guillard, Y. (1989)Biodistribution of indium-111 labeled OC 125 monoclonal antibody intraperitoneally injected into patients operated on for ovarian carcinomas. Cancer Res. 49, 3087-3094. (3) Beatty, J. D., Hyams, D. M., Morton, B. A., Beatty, B. G., Williams, L. E., Yamauchi, D., Merchant, B., Paxton, R. J., and Shively, J. E. (1989) Impact of radiolabeled antibody imaging on management of colon cancer. Am. J . Surg. 157, 13-19. (4) Craig, A. S., Helps, I. M., Jankowski, K. J., Parker, D., Beeley, N. R. A,, Boyce, B. A., Eaton, M. A. W., Millican, A. T., Millar, K., Phipps, A,, Rhind, S. K., Harrison, A., and Walker, C. (1989) Towards tumor imaging with indium-111 labelled
Brandl et al. macrocycle-antibody conjugates. J . Chem. SOC.Chem. Commun. 794-796. (5) Mathias, C. J., Sun, Y., Welch, M. J., Connett, J. M., Philpott, G. W., and Martell, A. E. (1989)BraHBED: an improved bifunctionalchelatefor radiolabeling antibodies. J . Nucl. Med. 30, 763. (6) Deshpande, S. V., DeNardo, S. J., Kukis, D. L., Moi, M. K., McCall, M. J., DeNardo, G. L., and Meares, C. F. (1990) Yttrium-90 labeled monoclonal antibody for therapy: labeling by a new macrocyclic bifunctional chelating agent. J . Nucl. Med. 31, 473-479. (7) Macklis, R. M., Kinsey, B. M., Kassis,A. I., Ferrara, J. L. M., Atcher, R. W., Hines, J. J., Coleman, C. N., Adelstein, S. J., and Burakoff, S. J. (1988) Radioimmunotherapy with alphaparticle-emittingimmunoconjugates. Science 240,1024-1026. (8) Boniface, G. R., Izard, M. E., Walker, K. Z., McKay, D. R., Sorby, P. J., Turner, J. H., and Morris, J. G. (1989) Labeling of monoclonal antibodies with samarium-153 for combined radioimmunoscintigraphyand radioimmunotherapy. J . Nucl. Med. 30,683-691. (9) Meares, C. F., McCall, M. J., Reardon, D. T., Goodwin, D. A., Diamanti, C. I., and McTigue, M. (1984) Conjugation of antibodies with bifunctional chelating agents: isothiocyanate and bromoacetamide reagents, methods of analysis .and subsequent addition of metal ions. Anal. Biochem. 142,6878. (10)Brechbiel, M. W., Gansow, 0. A., Atcher, R. W., Schlom, J., Esteban, J., Simpson, D. E., and Colcher, D. (1986) "Synthesis of 1-(p-isothiocyanatobenzyl) derivativesof DTPA and EDTA. Antibody labeling and tumor imaging studies. Inorg. Chem. 25,2772-2781. (11) Mirzadeh, S., Brechbiel, M. W., Atcher, R. W., and Gansow, 0. A. (1990) Radiometal labeling of immunoproteins: Covalent linkage of 2-(4-isothiocyanatobenzyl)diethylenetriaminepentaacetic acid ligands to immunoglobulin. Bioconjate Chem. I, 59-65. (12) Richardson, F. S. (1982) Terbium(II1) and europium(II1) as luminescent probes and stains for biomolecular systems. Chem. Rev. 82,541-552. (13) Leung, C. S-H., and Meares, C. F. (1977) Attachment of fluorescent metal chelates to macromolecules using "bifunctional" chelating agents. Biochem. Biophys. Res. Commun. 75, 149-155. (14) Westerberg, D. A., Carney, P. L., Rogers, P. E., Kline, S. J., and Johnson, D. K. (1989)Synthesis of novel bifunctional chelators and their use in preparing monoclonal antibody conjugates for tumor targeting. J . Med. Chem. 32, 236-243. (15) Sumerdon, G. A., Rogers, P. E., Lombardo, C. M., Schnobrich, K. E., Melvin, S. L., Hobart, E. D., Tribby, I. I. E., Stroupe, S. D., and Johnson, D. K. (1990) An optimized antibody-chelator conjugate for imaging of carcinoembryonic antigen with indium-111. Nucl. Med. Biol. 17, 247-254. (16) Griffin, T., Brill, B., Bokhari, F., Collins, J., Stochl, M., Gionet, M., Ruschowski, M., Stroupe, S., Kiefer, H., Sumerdon, G., Johnson, D., and Hnatowich, D. (1989) Radioimmunodetection of recurrent colorectalcancer with an 111Inlabeled anti-CEA antibody. Fourth International Conferenceon Monoclonal Antibody Immunoconjugates for Cancer, San Diego, CA, March 30-April 1, Abstract 23. (17) Nuti, M., Teramoto, Y. A., Mariani-Costantini, R., Horan Hand, P., Colcher, D., and Schlom, J. (1982) A monoclonal antibody (B72.3) defines patterns of distribution of a novel tumor-associated antigen in human mammary carcinoma cell populations. Znt. J . Cancer 29, 539-545. (18) Carney, P. L., Rogers, P. E., and Johnson, D. K. (1989)Dual isotope study of iodine-125 and indium-111 labeled antibody in athymic mice. J . Nucl. Med. 30, 374-384. (19) Rogers, P. E., Schnobrich, K. E., and Johnson, D. K. (1988) Correlation of radiochemicalyield with chelator concentration in indium-111labeled antibody-chelator conjugates. J . Nucl. Med. 29, 924. Registry No. 1, 53641-65-9; 2, 131322-70-8;3, 117499-22-6; 4,117499-23-7;terbium, 7440-27-9.
