Bioconjugate Chem. 1990, 1, 59-65
principle of protein-dye binding. Anal. Biochem. 72, 248255. (25) Laemmli, V. K. (1970)Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 222,680-685. (26) Roholt, O., Onoue, K., and Pressman, D. (1964) Specific combination of H and L chains of rabbit gamma-globulins. Proc. Natl. Acad. Sci. U.S.A. 51, 173-178. (27) Sutton, J., Alden, J. R., and Easterbrook-Smith, S.B. (1984) The effects of cleavage of the inter-chain disulfide bonds of rabbit IgG on its ability to bind Clq. Biochem. Biophys. Acta 787, 39-44. (28) Wahl, R. L., Liebert, M., Fisher, S., Sherman, P., Jackson, G., Laino, L., and Wissing, J. (1987) Radioimmunotherapy of human ovarian carcinoma xenografts: preliminary evaluation. Proc. AACR 28, 384 (abstr.). (29) Amzel, L. M., and Poljak (1979).Threedimensional structure of immunoglobulins. In Annual Review of Biochemistry (E. E. Snell, P. D. Boyer, A. Meister, and C. C. Richardson, Eds.) Vol. 48, pp 961-967, Annual Reviews, Inc., Palo Alto, CA.
59
(30) Davies, D. R., and Metzger, H. (1983) Structural basis of antibody function. In Annual Review of Immunology (W. E. Paul, C. G. Fathman, H. Metzer, Eds.) Vol. I, pp 87-117, Annual Reviews, Inc., Palo Alto, CA. (31) Brennan, M., Davison, P. F. and Paulus, H. (1985) Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments. Science 229,
--.
R1-9.2
(32) Husain, S.S.and Lowe, G. J. (1968) Evidence for histidine in the active site of papain. Biochem. J. 108, 855-859. (33) Paik, C. H., Ebbert, M. A., Murphy, P. R., Lassman, C. R., Reba, R. C., Eckelman, W. C., Pak, K. Y., Powe, J., Steplewski, Z., and Koprowski, H. (1983) Factors influencing DTPA conjugation with antibodiesby cyclic DTPA anhydride. J . Nucl. Med. 24, 1158-1163. (34) Sears, D. W., Mohrer, J., and Beychok, S. (1975) A kinetic study of the reoxidation of interchain disulfide bonds in a human immunoglobulin IgGlk. Correlation between sulfhydry1 dissappearance and intermediates in covalent assembly of H,L,. Proc. Natl. Acad. Sci. U.S.A. 72, 353-357.
Radiometal Labeling of Immunoproteins: Covalent Linkage of 2-(4-Isothiocyanatobenzyl)diethylenetriaminepentaacetic Acid Ligands to Immunoglobulin Saed Mirzadeh,* Martin W. Brechbiel, Robert W. Atcher,+ and Otto A. Gansow Inorganic and Radioimmune Chemistry Section, Radiation Oncology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892. Received July 27, 1989
A study was made of the covalent attachment of the bifunctional 2-(4-isothiocyanatobenzyl)diethylenetriaminetetraacetic acid family of chelate ligands to proteins for the purpose of labeling monoclonal antibodies with radiometals. The parameters and the chemical variables examined included pH, reaction period, temperature, and ligand and protein concentrations. It is shown that these variables, with the exception of protein concentration, have significant effects on the rate of protein conjugation. Conjugation of three monoclonal antibodies and human IgG under identical conditions showed only 17% variation. Finally, the effect of the concentration of conjugated IgG on radiolabeling yield was studied.
Attachment of radioactive metals to proteins by use of bifunctional chelate ligands (ligands) has expanded in use as an alternative to radiohalogenation (1-3) as the availability of these ligands has increased (4-7). The variety of metal isotopes with useful nuclear properties exceeds that of the halogen isotopes, giving the investigator greater flexiblity in choosing a diagnostic or therapeutic agent. Further, the development of monoclonal antibodies (MoAb) has yielded an ideal vehicle for transporting radioactivity to living cells, perhaps the magic bullet long sought for use in medicine (8). Derivatives of diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) have been extensively used as ligands in recent years (9-15). The cyclic and mixed anhydrides DTPA (CA- and MA-
* To
whom all correspondence should be addressed:
NIH, 10/B3B69,9000 Rockville Pike, Bethesda, MD 20892. Chemistry Division, Argonne National Laboratory, Argonne, IL 60439.
DTPA, respectively) react with immunoglobulins (predominately with €-amino groups of lysine side chains) through one of the carboxylate groups of the ligand (57, 14), resulting in a net loss in the metal-binding capability of the ligand molecule. This problem is resolved in bifunctional ligands such as DTTA-azo-imidate [N’[4-[ [2-hydroxy-5-(iminomethoxymethyl)phenyl]azo] benzylldiethylenetriaminetetraacetic acid] (15) and 2-(4isothiocyanatobenzy1)-EDTA(SCN-Bzl-EDTA) or -DTPA (9-11), which contain a protein-binding group that is distinct from the metal-chelating group. In the latter cases the SCN group on these ligands reacts with €-aminogroups with formation of a thiourea group (9-12, 16, 17). As demonstrated in Figure l a , the ligand portion of the molecule remains unchanged upon protein attachment, thus ensuring the expected stability of metal-ligand complex (metal chelate). The CA- and MA-DTPA also have the potential for cross-linking of protein inter- and intramolecularly. In the case of the isothiocyanate linkage, no potential for
1043-1802/90/2901-0059$02.50/00 1990 American Chemical Society
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Mirzadeh et ai.
EXPERIMENTAL PROCEDURES
NCS I
f"l i ("I
CO2H C02H C02H C02H CO2H
Figure 1. (a) Attachment of isothiocyanate group to protein via formation of a thiourea group with the c-amino group of lysine side chain; (b) structure of 1M3B-DTPA. cross-linking exists, thus the integrity of the protein molecules is more likely to be conserved. In addition, the coupling reaction does not involve the release of 4 equiv of H+ per mole of ligand as does the hydrolysis of the dianhydride. Furthermore, the SCN-Bzl derivatives of EDTA or DTPA are relatively stable in aqueous solutions yet are adequately reactive to proteins. Importantly, they can be stored for long periods without noticeable degradation (9, 10). Recently, we have reported a series of biodistribution studies of radiolabeled MoAb employing SCN-BzlEDTA, SCN-Bzl-DTPA,and various alkyl derivatives of SCN-Bzl-DTPA (12,13). In this paper, a systematic study of the chemical factors which influence binding of the SCN-Bzl-DTPA family of ligands [specifically, 14Clabeled 3-(4-isothiocyanatobenzyl)-6-methyldiethylenetriaminepentaacetic acid (1MSB-DTPA), Figure l b ] to proteins is presented. Variables explored included pH, reaction period, temperature, and ligand and protein concentrations. In addition, the effect of the concentration of chelate ligand conjugated IgG (conjugated IgG) on the "'In-radiolabeling yield was also examined. Human IgG, a polyclonal protein with an average molecular weight of 150 kDa, was used as the protein model for these investigations. This choice was primarily based on the ready availability of this protein in high purity and high concentration. Finally, the direct applicability of the results and methodology presented in this work was demonstrated by results from conjugations of three clinically used MoAb's (B72.3, anti-Tac, UPC10) with the 1M3BDTPA ligand. The immunoreactivity or the binding affinity of the chelate ligand modified MoAb to its antigenic target is an important criterion which deserves systematic study and discussion which are beyond the scope of the present work. In brief, we have shown that antiTac with two or less ligands per antibody retained its full binding affinity (12) and B72.3 retained its immunoreactivity when the ligand to protein ratio was less than one (9,13). This compromise between the immunoreactivity and the number of ligands bonded to immunoproteins let us limit our study over a region of the ligand concentration which produced a practical ligand to protein ratio 54. All measurements of ligand binding are presented in terms of a final chelate ligand to protein molar ratio, (CL/ P ) , moles of ligands bound per mole of protein as determined by use of carbon-14 labeled ligands.
Materials, Reagents, and Instrumentation. T o reduce the metal ion contamination, specifically the common metals [e.g. Fe(III), Zn(II), Cu(II)], plasticware (metalfree low protein binding polypropylene) was used in almost all protein work. All reagents used were of analytical grade or better. In general, all buffers were prepared in 1OX concentration in 2-L volumes. They were first filtered through 0.45-pm cellulose nitrate filter papers (e.g. Whatman aD552), then passed through a column of Chelex-100 resin (2.5 X 10 cm, preequilibrated with the same buffer, see below for specifications), and finally, prior to storage at 4 "C, they were filtered through a 0.22-pm sterile cellulose acetate membrane filtration unit (e.g. Corning 25942, Corning, NY). The composition of the buffers and reagents were as follows: HEPES [N-(2-Hydroxyethyl)piperazineN'-ethanesulfonic acid] buffer, 50 mM in HEPES, 150 mM in NaC1, pH = 8.6; MES [2-(N-morpholino)ethanesulfonic acid] buffer, 20 mM in MES, 150 mM in NaCl, 0.05% NaN,, pH = 6.2; citrate buffer, 50 mM in sodium citrate, 150 mM in NaC1, 0.05% NaN,, pH = 5.5; phosphate buffer, 50 mM in KH,P04, the pH (6.7-8.3) was adjusted with the addition of 0.1 M NaOH; borate buffer, 50 mM with respect to both and KC1, the pH (8.0-10.3) was adjusted with the addition of 0.1 M of NaOH; NaCl reagent, 150 mM in NaCI, 0.05% NaN,; Human IgG, GAMMAGARD, obtained from Travenol Laboratory, Inc. (Glendale, CA). The lyophilized IgG was rehydrated to produce a preparation 150 mM in NaCl (pH = 6.8) containing -50 mg/mL of protein (-94% IgG and -6% human albumin). Bovine plasma yglobulin (BSgG) as protein standard was obtained from BioRad Laboratories (Richmond, CA, catalogue 51500-0005). Monoclonal antibodies anti-Tac and UPClO were furnished by Dr. Thomas Waldmann (18) (Metabolism Branch, NCI, NIH), and B72.3 and BL3 were furnished by Dr. David Colcher (19) (Laboratory for Tumor Immunology and Biology, NCI, NIH). Tubular membrane for dialysis made of regenerated cellulose with a molecular weight cutoff (MWCO) of 12-14 kDa (Spectra/Por2,10mm flat width) was obtained from Spectrum Medical Industries (Los Angeles, CA). The dry membrane after treatment with EDTA to remove heavy metals was stored a t 4 "C in a 0.05% solution of sodium azide. Chelex-100 resin was the Na+ form, 100-200 mesh (Bio-Rad Laboratories). Resin was exhaustively washed with and stored in deionized H,O. Size-exclusion columns for HPLC were Bio-Si1 TSK-125, 250, and 400 series (Bio-Rad Laboratories). The protein microconcentrator used was a Centricon 30, MWCO = 30 kDa, Amicon Corp. (Danvers, MA). The HPLC used was a Model 2150/2152, LKB (Bromma, Sweden). Ligand. 14C-labeled 3-(4-isothiocyanatobenzyl)-6methyldiethylenetriaminepentaacetic acid (1M3BDTPA) was used as the ligand. A detailed description of synthetic methods of this compound and a procedure for 14C synthesis is given by Brechbiel et al. (9, 10). The 14Clabel was introduced by use of Br14CH,C0,H in the alkylation step of the synthesis. The tricesium salt of 14C-1M3B-DTPA was stored over Drierite in a vacuum desiccator over a period of 1year without any noticeable degradation in reactivity. As needed, 1.4 mg was disM solusolved in 100 pL of H,O to produce a 1.0 x tion of ligand. The specific activity of this batch (used throughout these studies) was 1.807 X lo6 dpm/pmol, which was measured by correlating the 14Ccounting rate (dpm) of a known volume of a ligand solution with ligand
Covalent Attachment of Ligands to Proteins
concentration, as measured by UV absorption at 280 nm
(9). Radioactivity and Counting Methods. No-carrieradded '"In (specific activity of about 50 mCi/pg) was obtained from New England Nuclear/Du Pont (Boston, MA). The activity concentration was approximately 50 mCi/mL in 0.05 M HC1. The '"In activity was quantitated by measuring its 171.3 (90.3%) and 245.4 (94.0%) KeV y-rays in a NaI (Tl) well type scintillation detector coupled to a multichannel analyzer (MCA). Samples of constant liquid geometry were counted for a duration sufficient to provide counting statistics of 12%. A gas ionization chamber (CRC-7, Capintec Instrument Inc., Ramsey, NJ) was used for gross activity measurements. A 1000-channel scintillation counter (LS5801, Beckman, Fullerton, CA) was used for quantitative determination of 14C activity. Generally, a 50-pL aliquot of a sample containing 14Cwas mixed with 10 mL of scintillation cocktail (Biofluor, NEN/Du Pont). The absolute disintegration of 14C was determined by appropriate corrections for background and quench factor. Protein Preparation and Measurement. The protein solutions were dialyzed against a t least a 500-fold volume excess of HEPES buffer (or other buffers as needed) for 24 h. T o deplete metal ion contaminants, Chelex-100 resin was added to the dialysates (-1 g/L). The protein concentrations were determined from UV absorption, at 280 nm. A value of 1.35 absorbance units per mg/mL was used for the extinction coefficient for IgG and the four monoclonal antibodies anti-Tac, UPC10, B72.3, and BL3. The SCN-Bzl-DTPA family of ligands also exhibits some absorption a t this wavelength (due to the aromatic ring). Depending on the value of (CL/P),, some corrections of the protein concentration were necessary (about 3% for every ligand per protein molecule of 150 kDa molecular weight). The UV absorption method was compared with the bicinchoninic acid (BCA) colorimetric determination (20). The BCA assay is more sensitive than the UV method and can be used for determination of protein concentrations as low as a few pg/mL. This procedure is sensitive to temperature and reaction time and does not result in a linear Beer's law plot. The most convenient and reproducible conditions were found to be a 30-min incubation at 60 "C. Typically, 100 pL of a protein solution (100 pg/mL) was added to 2 mL of a BCA working solution in a standard 1.3 X 10 cm Pyrex tube. The BCA working solution was freshly prepared by mixing 600 pL of solution A (4% CuS04.5H,0) with 30 mL of solution B (1% BCA-Na,, 2% Na,CO,.H,O, 1.6% sodium tartrate, 0.4% NaOH, 0.95% NaHCO,, pH = 11.25). After a 30min incubation a t 60 "C, the samples were quickly cooled to room temperature and their absorbance was measured vs a reagent blank a t 562 nm. The protein standard bovine plasma y-globulin in concentrations ranging from 20 to 200 pg/mL in the same buffer as the other proteins, was used to construct the Beer's law plot. Reaction of Isothiocyanatobenzyl-DTPA with IgG. In general, 2-4 mg of IgG in a concentration of about 10 mg/mL (in HEPES buffer, pH = 8.62,200-400 pL in volume) was mixed with 2-10 p L of 1M3B-DTPA ligand solution (1 X lo-, M in H,O). The pH of the reaction mixture was measured by a semimicro glass electrode. The initial ligand-to-protein molar ratio, (CL/P),, was determined by measuring the concentration of ligand in the reaction mixture. Typically, 20 pL of the reaction mixture was taken for ligand determination by 14Ccounting. The protein concentration in the reaction mixture
Bioconjugate Chem., Vol. 1, No. 1, 1990 61
was based on the protein measurement of an external standard which was prepared in parallel and was identical with the reaction mixture, but excluded the ligand. The reaction mixture was allowed to stand a t room temperature for the desired period of time. The unconjugated or free ligand was separated from protein by serial dialysis. Typically, 2-4 mg of protein was dialyzed against 2 X 500 mL of citrate buffer for at least 12 h each time and then against NaCl reagent for a few hours. The (CL/ P)f of the conjugated IgG was then determined. In all experiments duplicate samples were used. The following describes the specific modifications to the above general procedure. Effect of pH. The following reaction mixture was prepared. To 80 p L of protein stock solution (in 150 mM NaC1) containing 4 mg of protein was added 320 p L of the appropriate buffer of various pH values and 10.0 pL of the ligand solution. The pH of the reaction mixtures ranged from 6.75 to 10.1. Effect of Reaction Period and Temperature. Reaction mixtures were prepared in a cold room a t 5 "C. At time zero, duplicate samples were placed in various temperature baths. At various time points, 300 pL of each reaction mixture was taken for the (CL/P), determination as described in the general procedure. Effect of Concentration of Ligand. To 400 pL of IgG solution (10.0 mg/mL in HEPES buffer, pH = 8.62) were added various amounts of a ligand solution (3-50 pL), for (CL/P), ranging from 0.73 to 10.3. Then the total volume of the reaction mixture was adjusted to 460 pL with HEPES buffer. After this point the general assay procedure was followed. Effect of Protein Concentration. By using sequential dilution, six IgG samples in concentrations ranging from 3.1 to 25.0 mg/mL in HEPES buffer were prepared from a stock solution of IgG (-50 mg/mL). Appropriate amounts of ligand solution were then added to each sample in order to keep the (CL/P), constant at 4.0 f 0.2. Then the volumes of the reaction mixtures were adjusted to 440 pL by the addition of HEPES buffer. Although in these experiments membrane dialysis principally was used to isolate the free ligand from the conjugated IgG, two other methods, ultracentrifugation and size-exclusion HPLC, were also tested. Ultracentrifugation was performed by using microconcentrators with MWCO of 30 kDa and an active membrane surface area of 0.92 cm2. In this case the reaction was quenched by the addition of 1 mL of MES buffer followed by 20 min of centrifugation a t 4200g a t 4 "C. To remove 99.9% of the unconjugated ligand the above step was repeated twice. Purification of conjugated IgG from free ligand by sizeexclusion HPLC was also explored. Excellent separation was achieved on a TSK-125 column. The eluent was MES buffer, pumped a t a flow rate of 1mL/min a t room temperature. Chromatography was monitored a t 280 nm. Under the above conditions, IgG eluted with a retention time of 6 min (almost at the column void volume) and the free ligand eluted at 11.2 min with FWHM = 2.0 min. Better than 85% of IgG was recovered in 4-mL volume when the initial reaction mixture contained about 1 mg of protein. Procedure for '"In Labeling of Conjugated IgG. An [lllIn]Indium acetate solution was prepared a t pH = 3.8-4.0 by addition of appropriate amounts of 2 M NaOAc to a '"InCl,/HCl mixture. Typically, 15 pL of the above mixture containing about 350 pCi of "'In was added to a reaction vial containing 135 pL of a mixture of ligand-
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Table 11. Effect of Concentration of Chelate Ligand. [ligand], M 3.88 x 10-5 6.23 x 10-5 1.17 x 10-4 1.82 x 10-4 2.42 x 10-4 5.50 x 10-4
0
2
4
6
8 10 12 14 16 18 Time, min.
Figure 2. A typical HPLC chromatogram of "'In-labeled [ 1M3BDPTAI-IgG [conjugated IgG = 50 pg [(CL/P), = 0.231, unconjugated IgG = 450 pg; '"In activity = 350 pCi; buffer = MESC1, p H = 6.2; flow rate = 1 mL/min; chart speed = 1 cm/min; pressure = 20 bar; TSK-400 size-exclusion column].
uv
BCA colorimetricb 9i protein (280 nm) (562 nm) difference" B72.3 242.5 245.9 1.4 229.5 3.2 BL3 222.2 anti-Tac 215.8 221.0 2.4 UPClO 234.3 238.1 1.6 IgG 238.1 264.9 10.1 " Percent difference = (BCA - UV) X 100/BCA. Vs primary external protein standards (see the Experimental Procedures).
conjugated and unconjugated IgG (total of 1 mg of protein in MES buffer). The pH of the reaction mixture was kept between 4.0 and 4.2. After 30 min, the reaction was quenched by raising the pH to about 6 by the addition of 10 pL of 2 M NaOAc followed by 3 pL of lo-* M Na,EDTA to scavenge any free In-111. Subsequently, the radiolabeled protein was isolated from unreacted or free indium by HPLC employing a TSK-400 sizeexclusion column. The HPLC was operated isocratically at pH = 6.2 (MES buffer) with 1.0 mL/min flow rate and was equipped with dual on-line UV and radioactivity detectors. Typically, 24 0.5-mL fractions were collected and the radiolabeling yield was determined as the ratio of the radioactivity of the protein peak to the total activity in all tubes. A representative chromatogram is shown in Figure 2. Procedure for Purification of "'In. '"In activity was retained by cation-exchange resin from 8 M HBr (AG50Wx4, 2 X 20 mm, 100-200 mesh, preequilibrated with 8 M HBr). Under these conditions, excellent separations could be achieved from Co, Ni, Cu, and Zn. Subsequently, "'In was eluted with 8 M HC1 while a trace amount of Fe was strongly retained (21). The eluent was evaporated to dryness under an IR heat lamp and the lllIn activity was leached from the surface of the glass with 0.1 M HC1. RESULTS AND DISCUSSION
As discussed in the introduction, the chelate ligand to protein molar ratio, (CL/P), was used as a measure of efficiency of ligand-protein binding. Obviously, determination of (CL/P) depends on the protein concentration and an accurate assay of protein is therefore necessary. The results of the protein assay by UV absorption and BCA calorimetric method are summarized in Table I. In addition to IgG, four monoclonal antibodies (anti-
(CL/P), 0.33 0.55 0.93 1.42 1.84 3.71
(CL/P),,, 0.45 0.47 0.43 0.42 0.41 0.36
M (6.9 f 0.3 mg/mL); reaction " [IgG] = (4.6 f 0.2) X period = 17.3 h; p H = 8.62; temperature = 27 "C; volume of reaction = 460 pL. Table 111. Effect of Protein Concentration. IkGI 1.62 x 3.99 x 6.18 x 7.60 x 1.46 x 1.56 x
2.43 5.99 9.27 11.4 21.9 23.4
Table I. Comparison of Protein Concentration Measurements via UV Absorption at 280 nm and BCA Colorimetric Assav [protein], pg/mL
(CL/P), 0.73 1.17 2.19 3.41 4.54 10.3
1.12 1.29 1.43 1.45 1.52 1.58
10-5 10-5 10-5 10-5 10-4 10-4
*
" (CL/P)i = 4.0 0.2; reaction period = 17.0 h; pH = 8.62; volume of reaction mixture = 440 pL; temperature = 27 "C. Table IV. Conjugation of Various Proteins. [proteinlrxn,M 5.19 x 10-5 anti-Tac 4.79 X upcio 5.21 x 10-5 ~ 7 2 . 3 5.39 x 10-5 average (5.15 f 0.23) X protein
(CL/P), 4.26 4.82 4.39 4.75 4.56 f 0.29
(CL/P), 1.48 1.04 1.16 1.52 1.30 f 0.15
(CL/P),,, 0.34 0.23 0.26 0.32 0.29 f 0.05
a Reaction period = 16.1 h; temperature = 27 "C; volume of reaction mixture = 204 ILL.
Table V. '"In Labeling of Conjugated IgG: Effect of the Concentration of Conjugated IgG on Radiolabeling Yield at Constant "'In Activity. ratio of % fraction of % '"In to available sites [conj IgGl, radiolabeling conj IgG, occupied by M (CL/P), yield' pCi/pg "'In atomsd O.Ob
6.7 X 1.3 X 1.7 X 3.3 X 6.7 X 1.3 X 1.3 X
lo*
lo* 10* lo*
0.0 0.23 0.23 0.23 0.23 0.23 0.23 0.35
0.0
69 72 89 92 93 90 92
0.0 4.8 2.5 2.5 1.3 0.65 0.32 0.32
0.0 31.0 16.0 16.0 8.2 4.1 2.0 1.4
Volume of reaction mixture = 150 pL; the concentration of protein in the reaction mixture was adjusted to 1.3 X M by the addition of unconjugated IgG; reaction period = 30 min; temperature = 27 "C; activity of "'In = 350 pCi; pH = 4.C-4.2. 1.0 mg of unconjugated IgG. See the text for definition. Based on the specific activity of commercially available "'In, see the Experimental Procedures.
'
Tac, UPC10, B72.3, and BL3) were also used in these comparative studies. The UV assays were only 2-3% lower than results obtained by BCA colorimetric assays for all four monoclonal proteins of 150-kDa molecular weight. However, in the case of the polyclonal human IgG (with the same average molecular weight) the difference was about 10%. The UV absorption bands are primarily due to the aromatic amino acids (e.g. tryptophan and tyrosine, e = 5700 and 1300 M-' cm-l, respectively, at 280 nm). Proteins do not all have the same content of aromatic amino acid; consequently, two proteins at the same concentration may absorb to a different degree. As indi-
Covalent Attachment of Ligands to Proteins 2.0
I
I
I
I
I
Bioconjugate Chem., Vol. 1, No. 1, 1990 I
I
2.0
1.5
-e
1.5 +
c 1.0
I
u
63
6 -
1.0
0.5
0
Phosphate
A
Borate
-
0.5
h 7.0
8.0
9.0
10.0
PH
Figure 3. Variation of the final ligand-to-protein molar ratio, (CL/P),, as a function of pH [(CL/P), = 3.9 0.4; [IgGl,,, = 6.1 x loq5M, 8.9 mg/mL; reaction period = 16.6 h, temperature = 27 "C].
5
10 15 20 25 30 35
Reaction Temperature, O C Figure 5. Effect of temperature on (CL/P), for various reaction periods [(CL/P), = 3.7; [IgG],,, = 5.7 X lo-' M; pH = 8.421, 4.0
1.0
15T
0.5
0.2
5oc
-
0.4
0.6
[Ligand], mM
Figure 6. Variation of (CL P), as a function of the concen-
10
20
30
Reaction Period, h Figure 4. Effect of reaction period on (CL/P) for various temperatures [(CL/P), = 3.7; [IgG],,, = 5.7 x lo-%M; pH = 8.421. cated above, no significant variations in UV assay of the four MoAb's were observed by using 1.35 absorbance units per mg/mL as the extinction coefficient. As far as conjugation was concerned, no significant differences were observed among three methods, dialysis, ultracentrifugation, and size-exclusion HPLC, for isolation of free ligand from conjugated IgG. Inherently, dialysis is a slow process and requires a few days to obtain the final product. Both centrifugation and HPLC methods are faster techniques and separation could be completed within a few hours. However, in subsequent radiolabeling of the conjugated-IgG samples prepared by these three methods, appreciable higher radiolabeling yield was obtained for the dialysis sample. Possibly, this is an indication of the effectiveness of serial dialysis in the removal of metal ion contaminants. The results of binding of the SCN-Bzl-DTPA to the proteins illustrating the effect of pH, reaction period, temperature, and ligand and protein concentrations are presented in Tables 11-V and Figures 3-6. Effect of pH and Buffers. The results of these experiments, plotted in Figure 3, showed a simple and interM, the (CL/P), pretable pattern. At [IgG] = 6.1 X increased sharply from 0.29 to 1.96, as the pH of the reaction mixture increased from 6.71 to about 9. Above pH 9 (9.0-10.0), the (CL/P), leveled off a t 1.96 and remained rather independent of pH. The increase in (CL/P), as a
i
tration of ligand (see Table I for conditions).
function of pH is likely due to the greater deprotonation of the €-amino group of the lysines a t higher pH values providing additional NH, sites for reaction with the isothiocyanate group. Degradation of isothiocyanate to thiourea at pH 9 and above (22, 23) could account for the pH-independent behavior of (CL/P),. The use of different buffers of equal ionic strength did not seem to have any significant effect on the rate of conjugation, as indicated in Figure 3. Effects of Reaction Period and Temperature. In a series of parallel experiments, the effect of the reaction period was studied a t 5, 15, 25, and 35 "C. Results are shown in Figure 4. All data points (except 17-h time points) represent the average of duplicate measurements. The data for the 17-h time points are the average of data from two independent experiments. At 25 "C, (CL/P), increased rapidly to 0.94 within the first 8 h. Thereafter, from 8 to 32 h, (CL/P), increased only 0.86 unit to 1.8. At 35 "C, the initial rate of the reaction was quite fast and (CL/P), reached about 1.5 during the first 8 h. The effects of varying the temperature for different reaction periods are illustrated in Figure 5. The exponential dependence of the yield on temperature, which is expected for biomolecular reactions, is clearly suggested for reaction periods up to 17 h. However, for 17, 24, and 32 h this exponential dependence is less clear and a decreasing trend in (CL/P), even can be seen at 35 "C. The data describing the variation in yield as a function of temperature is important for two reasons. First, it markedly improves the kinetics of the reaction. Second, most antibodies are best maintained at lower tem-
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Bioconjugate Chem., Vol. 1, No. 1, 1990
perature until use, and these results could be used to optimize the conjugation temperature of various antibodies. Effect of Concentration of Chelate Ligand. These data are summarized in Table 11, and a plot of (CL/P), vs [ligand],,, is shown in Figure 6. A t [IgG] = 6.9 f 0.3 mg/mL, pH = 8.62, and reaction period of 17.3 h, a 14fold increase in concentration of ligand from 3.9 X low5 to 5.5 X M resulted in a 12-fold increase in (CL/P), from 0.33 to 3.71. The ratio of (CL/P), to (CL/P), ((CL/ PIf ,,a measure of the linearity of the relationship) remains ratker constant a t 0.44 f 0.03 when the (CL/P), increased from 0.73 to 4.54 (Table 11, column 4), indicating firstorder dependence of the reaction rate on the ligand concentration over this concentration range. A t (CL/P), = 10.3, the corresponding ratio dropped to 0.36; however, under the above conditions the saturation point of the lysines has not yet been observed. These data indicate that the reaction between IgG and 1M3B-SCN-BzlDTPA proceeds well a t low initial ligand-to-protein ratio, in contrast to the reaction between protein and DTPA dianhydride or mixed anhydrides of DTPA, where a minimum of a 50-fold excess of ligand to protein is required to produce comparable results (6,7). Moreover, the reaction between SCN-Bzl-DTPA and IgG can be allowed to proceed over a greater range of ligand concentrations since there is no possibility of cross-linkage. Effect of Protein Concentration. At (CL/P), = 4.0 f 0.2, pH = 8.62, and reaction period = 17.0 h, a 10-fold increase in the concentration of IgG from 2.4 to 23.4 mg/ mL resulted in only a 1.4-fold increase in the (CL/P),, from 1.12 to 1.58 (Table 111). These results do not represent the first-order dependence of the reaction rate on the protein concentration. Conjugation of Various Proteins. When IgG and three other monoclonal antibodies (anti-Tac, UPC10, and B72.3) were simultaneously conjugated under identical experimental conditions, no significant variation was observed in conjugation efficiencies (Table IV). An average value of 1.2 0.3 for (CL/P), was obtained for the above proteins when concentrations of proteins in the reaction mixture were (5.2 f 0.2) X M, with (CL/ P)i = 4.6 f 0.3, and when the reactions were allowed to proceed for 17.0 h. This would indicate that it is possible to define a set of standard conditions for the reaction of SCN ligand with MoAb's. Radiolabeling: Effect of t h e Concentration of 1MSB-DTPA-IgG on t h e Radiolabeling Yield a t Cons t a n t '"In Activity. Results of these experiments are summarized in Table V. When 1.0 mg of unconjugated IgG was used in the reaction, the radiolabeling yield was 0 (Table V, first row), indicative of the absence of nonspecific binding of "'In under our experimental conditions. The radiolabeling yield remained about 90% when the concentration of conjugated IgG [with (CL/P), = 0.231 in the reaction mixture decreased over 1 order of magnitude from 1.3 X low5to 1.7 X lo4 M. Further decrease in concentration of conjugated IgG resulted in a decrease in radiolabeling yield. When the concentration of conjugated IgG was 6.7 X lo4 M, the yield dropped to 69%. The ratio of activity (pCi) incorporated per microgram of protein also decreased from 4.8 to 0.32 as the concentration of conjugated protein increased from 6.7 X to 1.3 X M (Table V, column 4). It was not possible to attain a 100% radiolabeling yield under any conditions. In addition to acetate, two other weakly complexing ligands, iminodiacetic acid and citrate, were tested with no significant improvement of the labeling yield.
*
Use of a conjugated-IgG preparation with a 52% higher (CL/P), also did not result in a 100% yield (Table V, last row). An extensive purification of lllIn (by column chromatography, see the Experimental Procedures) resulted in a negligible increase in radiolabeling yield. Similar behavior has also been observed in the radiolabeling of various MoAb's. SUMMARY
The foregoing results demonstrate the chemical factors which influenced binding of the SCN-Bzl-DTPA family of ligands (specifically, 1M3B-DTPA) to immunoglobulins. Variables explored included pH, reaction period, temperature, and ligand and protein concentration. These variables, with the exception of protein concentration, have significant effects on the rate of protein conjugation. The effect of pH was demonstrated by a 7-fold increase in the labeling efficiency when pH was increased from 6.7 to 9.0. Above pH 9.0 the reaction rate was rather independent of pH. The kinetics of the reaction were studied at 5, 15, 25, and 35 "C. For reaction periods up to 17 h an exponential dependence of yield on temperature was obtained which was expected for a bimolecular reaction. This exponential dependence decreased over longer reaction periods and a t higher temperatures. Firstorder dependence of the reaction rate on ligand concentration was obtained over a range from 3.0 X to 5.5 X M. A 10-fold increase in [IgG], from 2.4 to 23.4 mg/mL, resulted in only a 1.4-fold increase in the (CL/ P ) , from 1.12 to 1.58. When IgG and three other monoclonal antibodies were simultaneously conjugated under identical experimental conditions, no significant variation was observed in conjugation efficiencies. ACKNOWLEDGMENT
We wish to acknowledge Pierre St. Raymond and Jane Fitzgerald for their preliminary work in this field (unpublished data) and Drs. Tom McMurry and Robert Kozak for their critical review of the manuscript. LITERATURE CITED
(1) Goldenberg, D. M., Edmund Kim, E., Deland, F. H., Bennett, S., and James Primus, F. (1980) Radioimmunodetection of cancer with radioactive antibodies t o carbinoembryonic antigen. Cancer Res. 40, 2984-2992. (2) Scheinberg, D. A., Strand, M., and Gansow, 0. A. (1982) Tumor imaging with radioactive metal chelates conjugated t o monoclonal antibodies. Science 215,1511-1513. (3) Scheinberg, D. A. and Strand, M. (1982) Leukemic cell targeting and therapy by monoclonal antibody in mouse model system. Cancer Res. 42,44-49. (4) Yeh, S. M., Sherman, D. G., and Meares, C. F. (1979) A new route to bifunctional chelating agents: Conversion of amino acids to analogs of ethylenedinitrilotetraacetic acid. Anal. Biochem. 100, 152-159. ( 5 ) Krejcarek, G. E. and Tucker, K. L. (1977) Covalent attachment of chelating groups to macromolecules. Biochem. Biophys. Res. Commun. 7 7 , 581-585. (6) Gansow, 0. A,, Atcher, R. W., Link, D. C., et al. (1984) Generator-produced Bi-212; chelated to chemically modified monoclonal antibody for use in radiotherapy. .. Amer. Chem. SOC. Symp. Ser. 241,215-227. ( 7 ) Hnatowich, D. J., Layne, W. W., and Childs, R. L. (1982) PreDaration and labeline of DTPA-couded albumin. Znt. J . Appl. Radiat. h o t . 33, g26-332. (8) Kohler, C. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495-499.
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(9) 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)derivatives of DTPA and EDTA. Antibody labeling and tumor-imaging studies. Inorg. Chem. 25, 2772, 2781. (10) Brechbiel, M. W. (1988) New Bifunctional Ligands f o r Radioimmunoimaging and Radioimmunotherapy Ph. D. Thesis, The American University, Washington, D. C. (11) 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. (12) Kozak, R. W.; Raubitschek, A., Mirzadeh, S., Brechbiel, M. W., Junghaus, R., Gansow, 0. A., and Waldmann, T. A. (1989) Nature of the bifunctional chelating agent used for radioimmunotherapy with yttrium-90 monoclonal antibodies: Critical factors in determining in vivo survival and organ toxicity. Cancer Res. 49, 2639-2644. (13) Roselli, M., Schlom, J., Gansow, 0. A., Raubitschek, A., Mirzadeh, S., Brechbiel, M. W., and Colcher, D. (1989)Comparative biodistributions of yttrium- and indium-labeledmonoclonal antibody B72.3 in athymic mice bearing human colon carcinoma xenografts. J. Nucl. Med. 30,672-682. (14) Paik, C. H., Murphy, P. R., Eckelman, W. C., Volkert, W. A., and Reba, R. C. (1983) Optimization of DTPA mixed-anhydride reaction with antibodies at low concentration. J. Nucl. Med. 24, 932-936. (15) Paik, C. H., Herman, E., Eckelman, W. C., and Reba, R. C. (1980) Synthesis, plasma clearance, and in vitro stability of protein containing a conjugated indium-111 chelate. J. Radioanal. Chem. 57,553-564. (16) Kawamura, A. (1969) in Fluorescent Antibody Tech-
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ter 3, University of Tokoyo, Tokyo. (17) Mizusawa, E. A,, Thompson, M. R., and Hawthorne, M. F. (1985) Synthesis and antibody-labeling studies with the p-isothiocyanatobenzene derivatives of 1,2-dicarba-closododecaborane and the dodecahydro-7,8-dicarba-nido-undecaborate(I-) ion for neutron-capture therapy of human cancer. Znorg. Chem. 24, 1911-1916. (18) Leonard, W. J., Depper, J. M., Robb, R. J., Waldmann, T. A., and Green, W. C. (1983) Characterization of the human receptor for T-cell growth factor. Proc. Natl. Acad. Sci. U.S.A. 80, 6957-6961. (19) Colcher,D., Horan Hand, P., Nuti, M., and Schlom,J. (1981) A spectrum of monoclonal antibodies reactive with mammary tumor cells. Proc. Natl. Acad. Sci. U.S.A. 78, 31993203. (20) Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fugimoto,E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C. (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150, 7685. (21) Nelson, F., Murase, F., and Kraus, K. A. (1964) I. Cation exchange in concentrated HC1 and HClO,. J. Chromatog. 13, 503. (22) Assony, S. J. (1961) The Chemistry of Isothiocyanates. In The Chemistry of Organic Sulfur Compounds (N. Kharasch and C. Y. Meyers, Eds.) pp 326-338, Pergamon Press, New York. (23) Satchell, D. P. N. and Satchell, R. S. (1975) Acylation by ketenes and isothiocyanates. A mechanistic comparison. Chem. SOC.Rev. 4, 231-250. Registry No. lM3B-DTPA, 121806-84-6; l"In, 15750-15-9.
Preparation and Characterization of Paramagnetic Polychelates and Their Protein Conjugates Paul F. Sieving, Alan D. Watson, and Scott M. Rocklage* Salutar, Incorporated, 428 Oakmead Parkway, Sunnyvale, California 94086. Received July 31, 1989
The gadolinium complexes of poly-L-lysine-poly(diethylenetriamine-N,N~,N",N"-pentaacetic acid) (Gd-PL-DTPA) and poly-L-lysine-poly(1 ,4,7,1O-tetraazacyclododecane-~,N'JV",N"'-tetraaceticacid) (Gd-PL-DOTA) and their conjugates with human serum albumin (HSA) have been prepared and characterized. Poly-L-lysine (PL, degree of polymerization = 100) was N-acylated with a mixed anhydride of the chelating ligand (DTPA or DOTA). Sixty to ninety chelating groups per molecule of PL could be attached in this way. Following purification of the polychelate by size-exclusion chromatography, the gadolinium complexes were prepared by standard methods and conjugated to HSA with heterobifunctional cross-linking reagents. The molar relaxivities of these macromolecular species were 2-3-fold higher than those of the corresponding monomeric metal complexes ([Gd(DTPA)] and [Gd(DOTA)]). The conjugation conditions were optimized to produce conjugates containing 60-90 metal centers per molecule of HSA (ca. one polychelate per protein).
We are conducting a research program to investigate the feasibility of preparing derivatives of biologically active macromolecules containing a large number of covalently bound metal chelates. The utility of such species for producing target-specific contrast enhancement in mag1043-1802/90/2901-0065$02.50/0
netic resonance imaging (MRI), as well as for applications in nuclear medicine, is anticipated. The present system was proposed as a model of the physical and chemical properties of such systems in general. The polychelate approach was developed as a means of preserving 0 1990 American Chemical Society
