Extended Length Heterobifunctional Coupling Agents for Protein

Daiki Tomita , Takuya Kimura , Hitomi Hosaka , Yuta Daijima , Risa Haruki , Kai Ludwig , Christoph Böttcher , and Teruyuki Komatsu. Biomacromolecules...
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Bioconjugate Chem. 1996, 7, 88−95

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Extended Length Heterobifunctional Coupling Agents for Protein Conjugations Christopher Bieniarz,* Mazhar Husain, Grady Barnes, Carol A. King,† and Christopher J. Welch‡ Department of Immunochemistry, Diagnostics Division, Abbott Laboratories, Abbott Park, North Chicago, Illinois 60064. Received May 24, 1995X

A series of extended length heterobifunctional coupling agents is described. The successive aminocaproic acid homologation of succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, a known 9-atom long maleimide active ester linker, yielded 16-, 23-, and 30-atom long maleimide active ester homologues. The performance study of these coupling agents in automated microparticle enzyme immunoassays showed that, in the R fetoprotein assay, in which the linkers were employed in the construction of the alkaline phosphatase-antibody conjugates, the signal increased 64% when the length of the linker was incremented from 9 atoms to 23 atoms and 82% for the 30-atom long linker as compared with the 9-atom homologue. Similar improvements were observed in the performance of carbohydrate antigen, marker of ovarian cancer (CA-125), immunoassay where the linkers were used for conjugation of the capture antibody anti-CA-125 to the microparticle. Thus, a 300% signal improvement resulted when a 30-atom linker was used instead of the 9-atom homologue. The observed differences in the performance of the conjugates are interpreted as resulting from improved antibody binding and lowering of the steric hindrance of the complementarity-determined region of the antibody when longer coupling agents were used.

INTRODUCTION

The cross-linking agents currently in use for bioconjugations exploit a wide variety of functionalities and reactivities. The chemistry of the bioconjugation reagents has been extensively reviewed (1-5). Heterobifunctional reagents, especially maleimide active ester linkers, are particularly attractive, in that they may be reacted chemoselectively with amines on the first molecule and then conjugated to a thiol-containing second molecule in a highly controlled manner, at physiological pH (6-8), and they are quite more selective than iodoacetyl groups in that they do not react with histidine and methionine or thionucleotides (10, 11). Steric effects are among the most important factors influencing the reactivity of organic molecules. In particular, steric hindrance exerts very large influence over the rates of reactions in organic and aqueous media (12). An essential aspect in the construction of a bioconjugate is the preservation of the optimal biological and chemical characteristics of the elements in the conjugate. Although it is generally assumed that the length of the spacer arm is important, with longer spacer arms presumably more effective in coupling large proteins, there is a relative dearth of studies relating to the effects of the length of cross-linking agents on the performance of the conjugates. We have synthesized a series of heterobifunctional coupling reagents of various lengths useful for covalent coupling of proteins through active ester carbonyl-amine and maleimide-thiol bonds (13-16). Successive elongation of the commercially available 9-atom linker SMCC1 * Current address: Christopher Bieniarz, Advanced Drug Delivery (D-97D) AP4, Hospital Product Division, Abbott Laboratories, 1 Abbott Park Road, Abbott Park, North Chicago, IL 60064-3500. † Ne ´ e Carol A. Schlesinger. ‡ Present address: Regis Chemical Co., 8210 Austin Avenue, P.O. Box 519, Morton Grove, IL 60053. X Abstract published in Advance ACS Abstracts, December 1, 1995.

1043-1802/96/2907-0088$12.00/0

(8), compound 1 of Figure 1, with aminocaproic acid yielded a series of 16-, 23-, and 30-atom long heterobifunctional maleimide active esters, compounds 2-4 shown in Figure 2. In the work presented here, these linkers were then used to construct two types of conjugates: (a) alkaline phosphatase-monoclonal antibody (mAb) conjugates and (b) microparticle-polyclonal antibody conjugates. We studied the effect of these linkers on the performance of the microparticle capture enzyme immunoassay (MEIA) (17). In this work, we show that the length of the cross-linking reagent in the conjugate is directly related to the performance of these conjugates in the MEIA format. We observed this effect both for antibody-enzyme label conjugates as in the case of alkaline phosphatase-anti-AFP mIgG (a) and where the capture antibody anti-CA-125 IgG was immobilized through a series of increasing length linkers to aminated microparticles (b). EXPERIMENTAL SECTION

Materials. Except as noted, reagents were obtained commercially and used without further purification. All solvents were high-performance liquid chromatography (HPLC) grade. Anhydrous DMF, N-hydroxysuccinimide, dicyclohexylcarbodiimide, Sephadex G-25 chromatographic packing, and silica gel 60 Merck 70-230 mesh were purchased from Aldrich Chemical Co. SMCC and EDC were purchased from Pierce, Rockford, IL. 5,5′Dithiobis(2-nitrobenzoic acid), (2-mercaptoethyl)amine, 4-methyl umbelliferyl phosphate, and p-nitrophenyl phosphate were from Sigma Chemical Co. Bovine intestinal alkaline phosphatase (EC 3.2.1.23, immunoassay grade) was purchased from Boehringer Mannheim Co. as a 10 1 Abbreviations: SMCC, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; AFP, R fetoprotein; CA-125, carbohydrate antigen, marker of ovarian cancer; DMF, dimethylformamide; EDC, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride; EDTA, ethylenediaminetetraacetic acid; TRIS, tris(hydroxymethyl)aminomethane; DTT, dithiothreitol; SPDP, N-succinimidyl 3-(2-pyridyldithio)propionate.

© 1996 American Chemical Society

Coupling Agents for Protein Conjugations

mg/mL solution in triethanolamine, NaCl, MgCl2, and ZnCl2. Monoclonal anti-AFP IgG was produced at Abbott Laboratories, North Chicago, IL. Monoclonal anti-CA125 IgG was from Centocor Co. Polyclonal IgGs were produced at Abbott Laboratories. Amine and carboxyl microparticles were from Seradyn or Interfacial Dynamics Corp., Portland, OR. All detergents and buffer components were from Sigma and Aldrich. General Methods. Immunoassays were performed on an Abbott Laboratory automated fluorescence analyser IMx. Electronic spectra were recorded on a HewlettPackard 8452A diode array spectrophotometer. Nuclear magnetic resonance spectra were obtained on a Varian Unity 300 MHz instrument. HPLC analyses of the conjugates were done on a Perkin-Elmer Series 4 analytical HPLC equipped with a 7125-075 Rheodyne injector or Waters 600 equipped with a U6K injector and a Waters 484 tunable absorbance detector. Elemental analyses were by Oneida Research Services Inc., Whitesboro, NY. Synthesis of the Extended Heterobifunctional Linker Arms. (a) 4-[(2,5-Dihydro-2,5-dioxo-1H-pyrrol1-yl)methyl]-N-[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]cyclohexanecarboxamide (2). To a 1.0 mL dry DMF stirred solution of 1-[[4-[[(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl]cyclohexyl]methyl]-1H-pyrrole-2,5-dione (SMCC, 9-atom linker 1) (3.34 g, 0.01 mol) was added 6-aminocaproic acid (2.51 g, 0.01 mol) in 1.0 mL of DMF, and stirring under a nitrogen atmosphere was continued at room temperature for 12 h. A solution of dicyclohexylcarbodiimide (2.06 g, 0.01 mol) in 1.0 mL of DMF was added, followed by N-hydroxysuccinimide (1.15 g, 0.01 mol), and the solution was stirred at room temperature for an additional 6 h. Precipitated dicyclohexylurea was removed by filtration, and the filtrate was evaporated under reduced pressure to give a tacky solid, which was purified by flash chromatography on silica gel using as eluent 5% CH3OH/95% CHCl3 to give 2.46 g of the 16atom linker, compound 2. Yield: 2.46 g, 55%. Mp: 147149 °C. Analytical HPLC on Waters Associates system using a µ Porosil silica gel column, 4% CH3OH/CHCl3, a 20 µL injection, a flow of 20 mL/min, and a Waters tunable detector at 300 nm showed a single peak (peak area > 98%) at 1.61 min. 1H NMR (300 MHz, 1:1 CD3OD/CDCl3): δ 6.78 (s, 2H), 3.35 (d, 2H, J ) 7.2 Hz), 3.33 (m, 1H), 3.18 (m, 2H, J ) 7.3 Hz), 2.88 (s, 4H), 2.63 (t, 2H, J ) 7.2 Hz), 2.09 (tt, 1H, J ) 14.4, 4.3 Hz), 1.920.93 (m, 16H). FAB m/z 448 (M + H)+. Anal. Calcd for C22H29O7N3‚0.5H2O: C, 57.88; H, 6.62; N, 9.20. Found: C, 58.09; H, 6.63; N, 9.54. (b) 4-[(2,5-Dihydro-2,5-dioxo-1H-pyrrol-1-yl)methyl]-N[6-[[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]-6oxohexyl]cyclohexanecarboxamide (3). This compound was prepared in a manner analogous to the preparation of 2. With 2.00 g of 2 as starting material, 1.25 g or 50% of 3 was obtained after flash chromatography using 10% CH3OH/90% CHCl3 as eluent. Mp: 144-145 °C. Analytical HPLC performed under conditions identical to those for compound 2 showed a single peak (peak area > 98%) at 2.16 min. 1H NMR (300 MHz, 1:1 CD3OD/ CDCl3): δ 6.78 (s, 2H), 3.47 (tt, 1H, J ) 12.0, 3.6 Hz), 3.35 (d, 2H, J ) 7.2 Hz), 3.33 (m, 2H), 3.19 (t, 2H, J( ) 7.2 Hz), 3.16 (t, 2H, J ) 7.2 Hz), 2.88 (s, 4H), 2.64 (t, 2H, J ) 7.2 Hz), 2.18 (t, 2H, J ) 8.4 Hz), 2.09 (tt, 1H, J ) 13.2, 4.3 Hz), 1.93-0.92 (m, 20H). FAB m/z 561 (M + H)+. Anal. Calcd for C28H40O8N4‚0.5H2O: C, 59.02; H, 7.26; N, 9.84. Found: C, 58.92; H, 7.54; N, 10.00. (c) 4-[(2,5-Dihydro-2,5-dioxo-1H-pyrrol-1-yl)methyl]-N[6-[[6-[[6-[(2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]6-oxohexyl]amino]-6-oxohexyl]cyclohexanecarboxamide (4).

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This compound was prepared as 2 and 3 above. With 1.00 g of 3 as starting material, 0.60 g or 50% of 4 was obtained after flash chromatography using 10% CH3OH/ 90% CHCl3 as eluent. Mp: 170-173 °C. Analytical HPLC performed under conditions identical to those for compounds 2 and 3 showed a single peak (peak area > 98%) at 4.01 min. 1H NMR (300 MHz, 1:1 CD3OD/ CDCl3): δ 6.78 (s, 2H), 3.55, (bm, 1H), 3.37 (d, 2H, J ) 7.2 Hz), 3.35 (m, 3H), 3.17 (m, 6H, J ) 7.2 Hz), 2.88 (s, 4H), 2.63 (t, 2H, J ) 7.2 Hz), 2.18 (t, 2H, J ) 8.4 Hz), 2.16 (t, 2H, J ) 8.4 Hz), 2.09 (tt, 1H, J ) 13.2, 4.3 Hz), 1.98-0.92 (m, 26H). FAB m/z 674 (M + H)+. Anal. Calcd for C34H50O9N5: C, 60.70; H, 7.49; N, 10.41. Found: C, 60.43; H, 7.46; N, 10.24. Conjugation of Monoclonal Anti-AFP IgG to Calf Intestinal Alkaline Phosphatase Using 4. (a) Derivatization of Alkaline Phosphatase. A 250 µL sample of a 10 mg/mL, 7.4 × 10-5 M, original solution of calf intestinal alkaline phosphatase was placed in a vial. A 100 µL volume of a 5.0 × 10-3 M DMF solution of compound 4 was added (27 equiv of 4/enzyme), and the solution was agitated on a rotary agitator for 30 min at room temperature. The crude reaction solution was chromatographed on a pre-equilibrated Sephadex G-25 (coarse) column with 0.1 M sodium phosphate buffer (pH 7.0), 0.1 M NaCl, 1.0 mM MgCl2, and 0.1 mM ZnCl2 as eluent. Enzyme-containing fractions were detected by examination of their catalytic activity of inducing color in the p-nitrophenyl phosphate solutions (18). Enzyme fractions were pooled, and protein concentration was estimated by measuring absorbance at 280 nm using the equation protein concentration (mg/mL) ) A280/1 cm × 1.05 mL mg-1 cm-1. (b) Monoclonal Anti-AFP IgG Derivatization. A 6.4 mg/ mL, 4.7 × 10-5 M, solution of monoclonal anti-AFP was incubated for 20 min in a pH 7.0 sodium phosphate buffer at room temperature with 25 mM dithiothreitol (DTT), stirring on a rotary agitator. The solution of partially reduced antibody was then chromatographed on a preequilibrated Sephadex G-25 (coarse) column with 0.1 M sodium phosphate buffer (pH 7.0), 0.1 M NaCl, and 5 mM EDTA. Fractions from the column were collected, proteincontaining fractions were pooled, and protein concentration of the pooled solutions was estimated by measuring absorbance at 280 nm using the equation protein concentration (mg/mL) ) A280/1 cm × 1.39 mL mg-1 cm-1. (c) Conjugation of Partially Reduced Antibody with Derivatized Alkaline Phosphatase. A 1.00 mL solution of the derivatized alkaline phosphatase (0.48 mg/mL, pH 7.0) from (a) was combined with an equal volume of a 0.40 mg/mL solution of the partially reduced antibody from (b) (molar ratio of 1.2:1 enzyme to antibody known through preceding experimentation to yield the best conjugates). This solution was made 0.1 M in NaCl and 0.1 M in TRIS, 3% Triton X-100, and 1 mM MgCl2 and 0.1 mM ZnCl2 and 10% mannitol, yielding the final conjugation solution (pH 7.0). The solution was stirred for 10 h at 2-8 °C on a rotary agitator. After that, unreacted thiols were capped with 100 µL of 5 mM DMF solution of N-ethylmaleimide. It was determined earlier that up to 30% DMF in the buffered conjugation solutions was not detrimental either to the enzyme or to the antibody activities (13, 19). The conjugate concentrate thus obtained could be diluted as necessary for use in sandwich-automated IMx assays or stored in a refrigerator at 2-5 °C for extended periods of time. HPLC gel filtration analysis of this conjugate was performed as described before (19, 20) and revealed mostly a monomeric 1:1 conjugate of enzyme to antibody, although a significant amount of material, 20-25%, had a higher

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molecular weight. Conjugations using shorter heterobifunctional linkers 1-3 were done in a manner analogous to the procedure described above. Conjugation of the Polyclonal Anti-AFP IgG to the Carboxylated Microparticle. The capture antibody in the AFP immunoassay was attached to carboxylated microparticles by incubation of a 1.00 mL volume of microparticles (0.39 µm, 1.25% solids) with 0.18 M 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC) and 0.76 mg, 5.2 × 10-9 mol, of polyclonal anti-AFP IgG in 3.9 mM 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 6.5). After being stirred at room temperature for 2 h, the microparticles were filtered through a medium sintered glass filter and washed with a 0.05 M TRIS, 0.1 M NaCl, and 0.1% Tween detergent (pH 8.0) buffer. The derivatized microparticles were centrifuged at 15 000 rpm and diluted in storage buffer 0.05 M TRIS, 0.1 M NaCl, 14% sucrose, and 0.1% sodium azide (pH 8.0). Conjugation of Polyclonal Anti-CA-125 IgG to Amine Microparticle Using 4. (a) Pretreatment of Amine Microparticles. A 1.00 mL volume of amine microparticles (0.164 µm, 2.5% solids) was mixed with 0.5 g of Biorex ion exchange resin (20-50 mesh, Bio-Rad catalog no. 142-7425). The mixture was rotated endover-end for 1 h at room temperature and then vacuumfiltered through a coarse sintered glass funnel with washing. The filtrate was collected and centrifuged at 15 000 rpm for 30 min. The supernatant was discarded, and the microparticle pellet was resuspended in distilled water with vortexing and adjusted to 2.5% solids by addition of distilled water. (b) Derivatization of the Microparticles. Resuspended, pretreated microparticles from part (a) were mixed with an equal volume of a 3 mM DMF solution of 30-atom linker compound 4 and allowed to react at room temperature for 1 h with end-over-end rotation. The reaction mixture was then diluted 10-fold with phosphate-buffered saline (PBS) (pH 7.2) and centrifuged at 15 000 rpm for 30 min. The resulting supernatant was discarded, and the microparticle pellet was resuspended with PBS. The centrifugation-resuspension sequence was repeated twice, the solution was again centrifuged, the supernatant was discarded, and the pellet was resuspended to a concentration of 2.5% solids with 0.05 M TRIS buffer (pH) (8.0) and 0.1 M NaCl. (c) Reduction of the Anti-CA-125 IgG. A 7.4 mg/mL, 4.9 × 10-5 M, solution of sheep anti-CA-125 IgG in PBS buffer was incubated with 25 mM DTT for 20 min at room temperature with stirring on a rotary agitator. The solution of partially reduced antibody was then chromatographed on a pre-equilibrated Sephadex G-25 (coarse) column with pH 7.0 phosphate buffer, 0.1 M phosphate, 0.1 M NaCl, and 5 mM EDTA as eluent. Fractions were collected, protein-containing fractions were pooled, and the protein concentration of the pooled solution was estimated by measuring absorbance at 280 nm. (d) Conjugation of Partially Reduced Antibody to Derivatized Microparticles. A 1 mL volume of a 1.0 mg/ mL, 6.7 × 10-6 M, solution of the partially reduced antibody from part (c) was combined with 1.0 mL of the 2.5% solids maleimide-derivatized microparticles from part (b). The mixture was rotated end-over-end at room temperature for 12 h. The reaction mixture was diluted 10-fold with wash buffer (pH 7.2), 0.01 M sodium phosphate, and 1% Tween detergent and then centrifuged at 15 000 rpm for 30 min. The resulting supernatant was discarded, and the microparticle pellet was washed twice, by vortexing in 1.0 mL of wash buffer, diluting 10-fold with wash buffer, and then centrifuging at 15 000 rpm

Bieniarz et al.

for 30 min followed by discarding of the supernatant. The washed pellet was resuspended to a final concentration of 0.125% solids in storage buffer (pH 8.1), 0.01 M TRIS, 0.1 M NaCl, 0.1% sodium azide, and 13.6% sucrose. The final microparticle suspension was first passed through a 23- and then a 25-gauge needle. These microparticles were stored in storage buffer until further use. Amine microparticle derivatizations with shorter heterobifunctional linkers 1-3 were done in a manner analogous to the procedure described above. Conjugation of the Monoclonal Anti-CA-125 IgG to Bovine Alkaline Phosphatase. The other half of the sandwich in the CA-125 immunoassay, alkaline phosphatase-labeled monoclonal anti-CA-125, was prepared by a method entirely analogous to the one employed in the conjugation of monoclonal anti-AFP IgG to calf intestinal alkaline phosphatase described above. This conjugate was made using compound 4. Immunoassay Performance of the Conjugates. The assay performance of the conjugates was evaluated with an Abbott IMx automated immunoassay analyzer on the basis of the microparticle capture enzyme immunoassay technology (MEIA) (17). In MEIA technology, antibody-derivatized microparticles capture the analyte and are then reacted with an alkaline phosphate-labeled antibody conjugate, which completes the sandwich. The nonfluorescent substrate, 4-methylumbelliferyl phosphate, is added, and the rate of the appearance of the fluorescent product, 4-methylumbelliferone, is measured (21). A 500 ng/mL AFP standard was used to compare the performance of the conjugates by examination of the rate of fluorescence increase expressed in arbitrary units of counts per second per second at the given concentrations. AFP (R fetoprotein) is an oncofetal antigen expressed in hepatic carcinoma cells (22), and CA-125 is an epithelial ovarian cancer marker used in detection and quantitation of the ovarian tumor-associated antigens (23). Control Experiments. Derivatization of Alkaline Phosphatase and Amine Microparticles with Heterobifunctional Linkers. Aliquots of alkaline phosphatase were treated with 27 equiv of SMCC and heterobifunctional linkers 2-4 in 1 mL of 0.1 M sodium phosphate, 0.1 M NaCl, 1 mM MgCl2, and 0.1 mM ZnCl2 as described before for the preparation of anti-AFP-alkaline phosphatase conjugates. Microparticles used in the following experiments did not contain any detergent and therefore did not require any pretreatment. Aliquots (1 mL) of the particles (0.18 µm, 1.2% solids) containing 2.16 µ equiv of amine were washed twice by mixing with an excess of 0.1 M sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl, vortexing the mixture vigorously, and then recovering the particles by centrifugation at 15 000 rpm for 30 min. The washed particles were suspended in 1 mL of 0.1 M sodium phosphate and 0.1 M NaCl (pH 7.0) and allowed to react with the linkers at linker to amine molar ratios of 1:1 and 3:1. The linkers were dissolved in 0.5 mL of DMF and slowly added to the particles. After incubation for 1 h at ambient temperature on a rotator, the particles were washed three times with 0.1 M sodium phosphate and 0.1 M NaCl (pH 7.0) to remove unreacted linkers. The derivatized particles were suspended in a known volume of 0.1 M sodium phosphate and 0.1 M NaCl (pH 7.0) and stored on ice until quantitation of maleimide groups, usually within 30-60 min. Quantitation of Maleimide Groups Introduced into Alkaline Phosphatase and Amine Microparticles. The determinations of maleimide groups incorporated into alkaline phosphatase upon linker derivatization and the

Coupling Agents for Protein Conjugations

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Figure 1. Syntheses of the extended heterobifunctional linkers.

phosphatase activity were carried out as already published (19). This method was also used with minor modifications to quantitate maleimide groups introduced into amine microparticles upon linker derivatization. Samples of derivatized microparticles were incubated with 100 µM (2-mercaptoethyl)amine in a final volume of 1 mL of 0.1 M sodium phosphate buffer (pH 7.0) containing 0.1 M NaCl and 2 mM EDTA. After incubation for 15 min at ambient temperature, particles were removed by filtration through a 0.2 µm filter, and the remaining thiol content in the filtrate was quantitated by the method of Ellman (24). As a control, amine microparticles treated with just DMF were also reacted with (2-mercaptoethyl)amine to determine if there was any nonspecific consumption of thiols. In another control, carboxyl microparticles (1 mL, 1.2% solids) were treated with an excess of SMCC (7 µmol) for 1 h at ambient temperature, washed as amine microparticles, and then reacted with (2-mercaptoethyl)amine. RESULTS

The syntheses of the extended heterobifunctional linkers 2-4 are shown in Figure 1. Successive homologation of SMCC with aminocaproic acid yielded the indicated coupling agents. These reagents remain stable for periods of several years when stored as solids in the desiccator at 2-8 °C. Millimolar solutions of these reagents in dry DMF (25) in brown glass ampules were stable for several months at 2-8 °C. We examined the rates of aqueous hydrolysis of the active ester functionality in these reagents by UV spectroscopy by following the time-based increase of the absorbance at 259 nm ( ) 8600 M-1 cm-1) which is due to the release of the N-hydroxysuccinimidyl anion at pH 7.0, with 0.1 M sodium phosphate buffer, at 25 °C, and with 15% DMF (26). Under those conditions, half-life times for hydrolysis of SMCC, 2, 3, and 4 are 3.0, 2.2, 2.5, and 2.5 h, respectively. We studied the effect of the length of the coupling agents on the performance of the conjugates in the automated immunoassays in two different settings. The conjugates of bovine intestinal alkaline phosphatase and anti-AFP mIgG were constructed as depicted in Figure 2a, while derivatization of the aminated microparticle with a polyclonal anti-CA-125 was prepared as shown in Figure 2b.

Anti-AFP mIgG-Alkaline Phosphatase Conjugates. Figure 3 shows the rate of appearance of the MEIA fluorescence signal as the function of the length of the linker arms used in the antibody-enzyme conjugates. The signal of the 500 ng/mL calibrator increased as the function of the length of the heterobifunctional linker. The increase in signal paralleled the increase of the linker arm length at all concentrations of the AFP standards (data not shown). The solid bars show the values of the blank background signal (noise) corresponding to 0 ng/mL of AFP antigen. The signal of the high standard increased 64% when the length of the spacer arm was increased from 9-atom SMCC to 23-atom linker 3 and increased 82% when the spacer arm was increased to 30-atom linker 4. The performance of the conjugates was optimized by three-dimensional plots correlating signal/noise as the function of the equivalents of linker used and Ab/enzyme ratios. All conjugates were compared under identical values of these last two independent variables. In order to keep constant the distance between the capture antibody and the microparticle, in all the runs, the covalent conjugation of the capture antibody to the carboxylated microparticle was done using a soluble carbodiimide EDC (27), a zero-length conjugation reagent. Therefore, it appears that the enhancement of the performance of the assay is directly related to the length of the linker arm in the antibodyenzyme conjugate. The conjugates made with increasing lengths of spacer arms also showed increased thermal stabilities. Thus, conjugates made with 23-atom linker showed 65% less degradation than those made with SMCC when stressed at 45 °C for 7 days. Although the background signal was identical, 2.9 counts per second per second for the shortest and the longest linkers, signal/ noise almost doubled when the 30-atom linker was compared with 9-atom SMCC, 250 and 138, respectively, although these ratios for the two intermediate length linkers were markedly lower, 65 and 92 for the 16- and 23-atom linkers, respectively. Anti-CA-125-Aminated Microparticle Conjugates. In order to examine the effect of the different length spacers between the capture antibody and the microparticles, polyclonal anti-CA-125 IgG was covalently conjugated to the aminated microparticles using 9-, 16-, 23-, and 30-atom heterobifunctional coupling agents as shown

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Figure 2. (a) Conjugation of alkaline phosphatase to antibodies using extended length heterobifunctional linkers. (b) Derivatization of the aminated microparticle with capture antibody using extended linkers.

Figure 4. Rates of appearance of the CA-125 MEIA fluorescence signal (counts per second per second) as a function of CA125 concentration for conjugates of varying lengths: (O) EDC, (0) linker 1, (]) linker 2, × linker 3, and (b) linker 4.

Figure 3. Rate of AFP MEIA fluorescence signal (counts per second per second) as a function of the length of the linker arm used in the antibody-enzyme conjugate. Dark bars represent the background signal at 0 ng/mL AFP, and light bars show the signal at 500 ng/mL AFP.

in Figure 2b. Incubation with 35, 65, 165, 325, and 650 units/mL CA-125 standards followed by addition of the anti-CA-125 mIgG-alkaline phosphatase conjugate and

MUP fluorogenic enzyme substrate yielded graphs shown in Figure 4. In these experiments, the antibody-enzyme conjugates were all prepared using the 9-atom linker SMCC. The optimized ratios of linkers per microparticle, molarity of the capture polyclonal IgG, and antibodyenzyme conjugates were kept constant throughout all the experiments. Therefore, the observed effects may be confidently attributed to the effect of the varying length of the spacer between the microparticle and the capture antibody. As shown in Figure 4, there is a 3-fold increase

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Coupling Agents for Protein Conjugations Table 1. Number of Maleimide Groups Introduced into Calf Intestinal Alkaline Phosphatase upon Activation with SMCC and Heterobifunctional Linkers 2-4 linker used

maleimide groups introduced

% starting activity retained

SMCC linker 2 linker 3 linker 4

4.0 4.3 4.6 4.2

92 94 90 92

of the signal at the 650 and 325 units/mL standards going from the 9- to the 30-atom linker in capture antibodymicroparticle conjugates. Interestingly, at the reagent stoichiometries used in these experiments, the 16- and 23-atom linkers yielded conjugates with very similar performance, with the 16-atom linker yielding slightly better conjugates than the 23-atom homologue. Signal/ noise ratios of 650 units/0 unit standards were 20.5, 10.6, 10.4, and 12.6 for SMCC, 16-, 23-, and 30-atom linkers, respectively, showing relative invariance at higher linker lengths. The higher value of signal/noise for the SMCC conjugate is due to the lowest value of the blank, 20 counts per second per second, while the blank values at 0 unit/mL for microparticles derivatized with 16-, 23-, and 30-atom linkers were 88, 92, and 95 counts per second per second, respectively. Figure 4 also shows that the performance of the best optimized conjugate based on EDC-mediated coupling of the antibody to a carboxylated microparticle was markedly inferior. The stability of the aminated microparticles derivatized with the polyclonal anti-CA-125 IgG was studied by comparison of the performance of the capture antibody-microparticle conjugates incubated at 4 and 45 °C for 7 days. The standard curves of the 30-atom conjugates incubated at those two temperatures were virtually superimposable and identical to the standard curve at time zero. Unlike the anti-AFP-alkaline phosphatase conjugates, little difference in thermal stabilities was observed between the microparticle conjugates made with different length linkers. The Effect of Introduction of Maleimide Groups into Alkaline Phosphatase and Amine Microparticles. The relative reactivity of the linkers with alkaline phosphatase was determined by quantitation of maleimide groups introduced by treatment with the linkers under identical conditions. The number of maleimide groups introduced was determined by treatment of the derivatized enzyme with excess of (2-mercaptoethyl)amine, followed by titration of the unreacted thiol groups with DTNB (19). The effect of linker derivatization on the enzyme activity was also examined. It can be seen from the results presented in Table 1 that the extent of the modification of alkaline phosphatase upon treatment with the linkers, as judged by the number of maleimide groups incorporated, was essentially constant for all the linkers. Treatment of the enzyme with a 27-fold molar excess of the linkers resulted in the incorporation of four to five maleimide groups per enzyme molecule. The derivatized enzyme retained 90-94% of the starting activity. These results are consistent with those reported earlier (19). The relative reactivity of the linkers with amine microparticles was also determined by quantitation of maleimide groups incorporated upon treatment with the linkers at linker to amine molar ratios of 1:1 and 3:1. As seen from the data presented in Table 2, the extent of derivatization by the various linkers was essentially constant for all the linkers, ranging from 2.3-3.0% for 1:1 and 25.0-31.9% for 3:1 linker to amine molar ratios. Carboxyl microparticles treated with excess SMCC as

Table 2. Maleimide Modification of Amine Microparticles by Treatment with SMCC and Heterobifunctional Linkers 2-4 % fraction of amine sites derivatized with linker maleimide linker

1:1 linker to amine

3:1 linker to amine

SMCC linker 2 linker 3 linker 4

2.7 2.3 2.7 3.0

25.0 29.6 26.9 31.9

well as amine microparticles treated only with DMF showed no incorporation of maleimide groups. DISCUSSION

Although it is generally accepted that the length and the flexibility of the coupling agent are important in the design of a bioconjugate, few systematic studies on this subject appeared in the literature. Some literature reported enhanced binding of the antibody covalently conjugated to the solid phase through an extended, flexible coupling agent (28, 29). The enhancement in the antibody ability to recognize antigens attached through linkers when compared to passive adsorptions has also been described (30). The present work shows that the length of a heterobifunctional coupling agent in an antibody-enzyme conjugate is a crucial factor in the performance of the latter in an automated MEIA. Similarly, the performance of the conjugates of the capture antibodies and the microparticles was enhanced by employing successively longer and more flexible coupling agents. Since the performance of the bioconjugate in the MEIA setting is the composite of the performances of the antibody and the signal-generating enzyme, the question arises whether the observed enhancement of the signal with longer coupling agents is the consequence of improved immunoglobulin binding of the antigen or improved enzyme activity. Our data in Figure 3 show that the blank signal at the 0 ng/mL AFP standard, which originates from nonspecific interaction of the anti-AFP IgG-alkaline phosphatase conjugate with the matrix, and incomplete removal of the unbound conjugate, is relatively constant for all the cojugates, regardless of the length of the linker. Thus, the alkaline phosphatase activity in the unbound conjugates is constant, regardless of the length of the linker. The data in Table 1 show that the activity of the alkaline phosphatase retained after the modification with different length linkers is essentially the same for all the linkers. Moreover, the data show a virtually identical number of maleimides introduced into the alkaline phosphatase, regardless of the length of the linker used. Thus, the differences in performance of the conjugates may be confidently attributed to the length of the linkers. Spurious factors, i.e., differential reactivity of the linkers and differences in the remaining enzymatic activity, may be discounted. Similarly, Table 2 shows that the degree of amine microparticle derivatization is essentially the same for all the linkers, regardless of their length. As expected, lower ratios of the linker/amine in the microparticle resulted in lower levels of microparticle derivatization. We have shown previously that the functionalization of the alkaline phosphatase with a 30-atom heterobifunctional reagent only minimally affects the enzymatic activity (31). We have also found that, in many antibodyalkaline phosphatase conjugations, enzyme activity can be uncompromised in conjugates that have lost most of their antibody activity. Moreover, conjugation, even with lower linker arm concentrations, can cause considerable

94 Bioconjugate Chem., Vol. 7, No. 1, 1996

loss (>95%) in antibody binding (32). Our data in Figure 4 show that successive elongation of the distance between the microparticle and the capture antibody in the CA125 assay also results in increases in signal. Since the antibody-alkaline phosphatase conjugate was held constant throughout these experiments, the signal increase may be attributed to the successively longer distance between the capture antibody and the microparticle. We therefore conclude that the signal enhancement is the result of the alleviation of the steric crowding between the antibody and the entity conjugated to it, i.e., enzyme or solid phase in this work. The complementarity-defined region (CDR) of the immunoglobulins is most severely affected in the case of shorter linkers, i.e., SMCC, and the least when longer linkers are employed. Thus, the effect of the longer linkers on the enzyme activity appears to be smaller than the effect of alleviating the steric hindrance on the antibody. We have recently shown that the construction of the Fc site-specific conjugates based on the extended linkers resulted in 200-400% improvement in signal as compared to the best non-Fc conjugates (19). Apparently, factors relieving steric crowding of an antibody CDR may lead to conjugates of enhanced activity. Figure 4 patently illustrates the last point by showing the zero-length EDC-mediated conjugates’ markedly inferior performance as compared with that of even the shortest linker SMCC. Although the chemistries of these two conjugates are different, we believe that the steric hindrance of the immunoglobulin’s CDR is the principal reason for the difference in the conjugates’ performance. The findings of the enhancement of signal as a result of the increasing distance between the two entities in a bioconjugate are general. We confirmed the above in many different conjugates of monoclonal and polyclonal antibodies modified not only by DTT disulfide reduction but also by Traut iminothiolane derivatization (33) or SPDP-mediated thiolation (34). The design of the coupling agents providing appropriate chemical and geometric environment for the conjugates will no doubt continue to be a crucially important endeavor in bioconjugate chemistry. Thus, the design of coupling agents possessing several advantageous characteristics, i.e., length, flexibility, hydrophilicity, and ability to introduce selective functionalities in site-specific regions of the biomolecules, will be a challenging objective of bioconjugate chemistry. ACKNOWLEDGMENT

The authors thank Ms. Caroline S. Miceli for her contributions in assay development and microparticle conjugations. LITERATURE CITED (1) Means, G. E., and Feeney, R. E. (1990) Chemical Modification of Proteins: History and Applications. Bioconjugate Chem. 1, 2. (2) Maggio, E. T. (1985) Enzyme Immunoassay, CRC Press, Boca Raton, FL. (3) Wong, S. H. (1991) Chemistry of Protein Conjugation and Cross-linking, CRC Press, Boca Raton, FL. (4) Brinkley, M. (1992) A Brief Survey of Methods for Preparing Protein Conjugates with Dyes, Haptens, and Cross-Linking Reagents. Bioconjugate Chem. 3, 2. (5) Feeney, R. E. (1987) Chemical Modification of Proteins: Comments and Perspectives. Int. J. Pept. Protein Res. 29, 145. (6) Kitagawa, T., and Aikawa, T. (1976) Enzyme Coupled Immunoassay of Insulin Using a Novel Coupling Reagent. J. Biochem. (Tokyo) 79, 233.

Bieniarz et al. (7) Youle, R. J., and Neville, D. M. (1980) Anti-thy 1.2 Monoclonal Antibody Linked to Ricin is a Potent Cell-Type-Specific Toxin. Proc. Natl. Acad. Sci. U.S.A. 77, 5483. (8) Yoshitake, S., Yamada, Y., Ishikawa, E., and Masseyeff, R. (1979) Conjugation of Glucose Oxidase from Aspergillus niger and Rabbit Antibodies using N-Hydroxysuccinimide ester of N-(4-carboxycyclohexyl-methyl) maleimide. Eur. J. Biochem. 101, 395. (9) Gregory, J. D. (1955) The Stability of N-Ethylmaleimide and Its Reaction with Sulfhydryl Groups. J. Am. Chem. Soc. 77, 3922. (10) Smyth, D. G., Blumenfeld, O. O., and Konigsberg, W. (1964) Reaction of N-Ethylmaleimide with Peptides and Aminoacids. Biochem. J. 91, 589. (11) Brown, R. D., and Matthews, K. S. (1979) Chemical Modification of Lactose Repressor Proteins Using N-Substituted Maleimides. J. Biol. Chem. 254, 5128. (12) March, J. (1992) Advanced Organic Chemistry, pp 275278, John Wiley & Sons, Inc., New York. (13) Bieniarz, C., Welch, C. J., and Barnes, G. (1991) Heterobifunctional Couplings Agents. U.S. Patent 4,994,385. (14) Bieniarz, C., Welch, C. J., Barnes, G., and Schlesinger, C. A. (1991) Covalent Attachment of Antibodies and Antigens to Solid Phases Using Extended Length Heterobifunctional Coupling Agents. U.S. Patent 5,002,883. (15) Bieniarz, C., Welch, C. J., and Barnes, G. (1991) Heterobifunctional Maleimide Containing Coupling Agents. U.S. Patent 5,053,520. (16) Bieniarz, C., Welch, C. J., Barnes, G., and Schlesinger, C. A. (1991) Covalent Attachment of Antibodies and Antigens to Solid Phases Using Extended Length Heterobifunctional Coupling Agents. U.S. Patent 5,063,109. (17) Fiore, M., Mitchell, J., Doan, T., Nelson, R., Winter, G., Grandone, C., Zeng, K., Haraden, R., Smith, J., Harris, K., Leszczynski, J., Berry, D., Stafford, S., Barnes, G., Scholnick, A., and Ludington, K. (1988) The Abbott IMx Automated Benchtop Immunochemistry Analyzer System. Clin. Chem. (Washington, D.C.) 34, 1726. (18) Boehringer Mannheim Biochemicals (1993/4) Biochemicals for the Diagnostic Industry Clinical Chemistry, Indianapolis, IN. (19) Husain, M., and Bieniarz, C. (1994) Fc Site-Specific Labeling of Immunoglobulins with Calf Intestinal Alkaline Phosphatase. Bioconjugate Chem. 5, 482. (20) Yoshitake, S., Imagawa, M., and Ishikawa, E. (1982) Efficient Preparation of Rabbitt Fab′-Horseradish Peroxidase Conjugates Using Maleimide Compounds and its Use for Enzyme Immunoassay. Anal. Lett. 15 (B2), 147. (21) McComb, R. B., Bowers, G. N., Jr., and Posen, S. (1979) Alkaline Phosphatase, Plenum Press, New York and London. (22) Roitt, I. (1991) Essential Immunology, p 297, Blackwell Scientific Publication, Oxford. (23) Bast, R. C., Jr. (1993) Perspective on the Future of Cancer Markers. Clin. Chem. (Washington, D.C.) 39, 2444. (24) Ellman, G. L. (1959) Tissue Sulphydryl Groups. Arch. Biochem. Biophys. 82, 70-77. (25) Perrin, D. D., Armarego, W. L. F., and Perrin, D. R. (1980) Purification of Laboratory Chemicals, 2nd ed., Pergamon Press, Oxford. (26) Pollak, A., Blumenfeld, H., Wax, M., Baughn, R. L., and Whitesides, G. M. (1980) Enzyme Immobilization by Condensation Copolymerization into Cross-Linked Polyacrylamide Gels. J. Am. Chem. Soc. 102, 6324. (27) Hoare, D. G., and Koshland, D. E. (1967) A Method for the Quantitative Modification and Estimation of Carboxylic Acid Groups in Proteins. J. Biol. Chem. 242, 2447. (28) Suter, M., and Butler, J. E. (1986) The Immunoassay of Sandwich ELISAs. II. A Novel System Prevents the Denaturation of Capture Antibodies. Immunol. Lett. 13, 313. (29) Peterman, J. H., Tarcha, P. J., Chu, V. P., and Butler, J. E. (1988) The Immunochemistry of Sandwich ELISAs. IV. The Antigen Capture Capacity of Antibody Covalently Attached to Bromoacetyl Surface-Functionalized Polystyrene. J. Immunol. Methods 55, 271. (30) Kennel, S. J. (1982) Binding of Monoclonal Antibody to Protein Antigen in Fluid Phase or Bound to Solid Supports. J. Immunol. Methods 55, 1.

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Coupling Agents for Protein Conjugations (31) Bieniarz, C., Young, D. F., and Cornwell, M. J. (1994) Thiolate and Phosphorothioate Functionalized Fluoresceins and Their Use as Fluorescent Labels. Bioconjugate Chem. 5, 31. (32) Rohr, T., Young, D. F., and Bieniarz, C. Unpublished results. (33) Jue, R., Lambert, J. M., Pierce, L. R., and Traut, R. R. (1978) Addition of Sulfhydryl Groups to Escherichia coli

Ribosomes by Protein Modification with 2-Iminothiolane(methyl 4-mercaptobutyrimidate). Biochemistry 17, 5399. (34) Carlsson, J., Drevin, H., and Axen, R. (1978). Protein Thiolation and Reversible Protein-Protein Conjugation. NSuccinimidyl 3-(2-pyridyldithio)propionate, a New Heterobifunctional Reagent. Biochemistry J. 173, 723.

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