Bioconjugate Chem. 1998, 9, 399−402
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TECHNICAL NOTES Thermally Stabilized Immunoconjugates: Conjugation of Antibodies to Alkaline Phosphatase Stabilized with Polymeric Cross-Linkers Christopher Bieniarz,*,† Douglas F. Young,‡ and Michael J. Cornwell‡ Advanced Drug Delivery, Hospital Products Division, and Diagnostics Division, Abbott Laboratories, Abbott Park, Illinois 60064-3500. Received February 9, 1998
A method of conjugating poly(glutamic acid) poly(phosphorothioate)-cross-linked alkaline phosphatase to maleimide-derivatized immunoglobulin is described. Intramolecular autocatalyzed cross-linking of alkaline phosphatase at 2:1 to 4:1 polymer:enzyme ratios introduced 32-68 thiolates on the surface of the enzyme. Depending on the stoichiometry of polymer to enzyme, the cross-linked alkaline phosphatase retained 75-90% of its native catalytic activity. The cross-linked thiolate-functionalized alkaline phosphatase was conjugated to maleimide-derivatized immunoglobulin. Compared to a control prepared using non-cross-linked alkaline phosphatase, these conjugates were smaller in size and more stable to heat. The enzymatic activity of the cross-linked conjugates after incubation at 45 °C and pH 7.5 for 25 days was 35% higher than those of the highest-activity control conjugates. The conjugation process could be controlled by varying the stoichiometries of poly(glutamic acid) poly(phosphorothioate), alkaline phosphatase, and immunoglobulin.
INTRODUCTION
Alkaline phosphatase is one of the enzymes most frequently employed in ELISA1 (1, 2). ALP popularity can be attributed to its relatively high stability, its high turnover number, the sensitivity of its enzyme assays (3), and the existence of excellent fluorogenic and chromogenic substrates (4, 5). Although many bioconjugation methods have been developed for efficient conjugation of ALP and other enzymes to antibodies (6-9), there is a continuing need for development of conjugation methods which preserve the chemical and biological properties of those biomolecules in the conjugate. Although ALP is considered to have good thermal stability, in our experience, prolonged storage of ALP-IgG conjugates leads to gradual loss of the enzymatic signal. In the preceding paper (10), we describe stabilization of proteins via novel polymeric cross-linkers, poly(phosphorothioate) polymers, activated by alkaline phosphatase. The protein is first derivatized with thiolreactive electrophiles e.g., maleimides. The poly(phosphorothioate) polymer is then exposed to the action of ALP which very efficiently catalyzes the hydrolysis of the phosphates to unmask thiolates on the polymeric back* To whom correspondence should be addressed: Abbott Laboratories, Hospital Products Division D-97D, Abbott Park, North Chicago, IL 60064-3500. Phone: (847) 937-2239. Fax: (847) 938-3645. E-mail:
[email protected]. † Hospital Products Division. ‡ Diagnostics Division. 1Abbreviations: ELISA, enzyme-linked immunosorbent assay; ALP, alkaline phosphatase; TSH, thyroid-stimulating hormone; DMF, dimethylformamide; PGA, poly(L-glutamic acid); STMCC, succinimidyl tris(caproamido) 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; SMCC, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate.
bone. The thiolates on the polymer then react with the electrophiles on the surface of the protein, resulting in predominantly intramolecular cross-linking and concomitant stabilization of the protein. When the protein is ALP, the process is self-catalyzed in that the very enzyme to be cross-linked catalyzes the hydrolysis of the phosphorothioate bonds and the cross-linking of the protein surface. Here we describe the application of the ALP-catalyzed auto-cross-linking to the construction of stabilized alkaline phosphatase-anti-TSH conjugates. These conjugates are crucially important in enzyme immunoassays for the immunodiagnostic assessment of the function of the thyroid gland. Stabilization of these immunoconjugates against thermal denaturation is of great practical interest because of the prolonged storage requirements for such immunoreagents. EXPERIMENTAL SECTION
Materials and General Procedures. The synthesis and analysis of poly(glutamic acid) poly(phosphorothioate) PGA(SPO3)24, materials, and other reagents were as described in the preceding paper (10). Monoclonal antiTSH was from Abbott Laboratories (Abbott Park, IL). STMCC 30-atom heterobifunctional maleimide-active ester linker was synthesized as described previously (11). Conjugation of Anti-TSH IgG to PGA(SPO3)24 Cross-Linked Bovine Alkaline Phosphatase. (a) Derivatization of Anti-TSH IgG. To 1 mL of 6.6 mg/mL anti-TSH IgG (44.0 nmol) was added 1 mL of buffer composed of 0.1 M sodium phosphate, 0.1 M NaCl, 1.0 mM MgCl2, and 0.1 mM ZnCl2 at pH 7.0 (buffer B). The antibody was concentrated to approximately 0.2 mL using a Centricon-30 concentrator with a MW cutoff of 30 000. The concentrate was rediluted to 2 mL using
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Scheme 1. Conjugation of Poly(phosphorothioate)-Cross-Linked ALP to IgG
buffer B and then reconcentrated to 0.2 mL. This concentration-dilution cycle was repeated three times. The volume of the antibody solution was increased to 1 mL with buffer B and the mixture placed in a vial. The concentration of the protein was 5.3 mg/mL (35 mM by A280). To 50 mL of DMF was added 0.56 mg (831 nmol) of succinimidyl tris(caproamido) 4-(N-maleimidoethyl)cyclohexane-1-carboxylate (STMCC), a 30-atom heterobifunctional maleimide active ester cross-linking agent (11). This solution was added to 0.47 mL of the 35 mM solution of IgG (16.6 nmol) with a 50-fold molar excess of STMCC linker to protein. The solution was reacted for 1 h at room temperature while being rotated at 100 rpm on a rotary agitator. The reaction solution was passed through a Sephadex G-25 column which was eluted with buffer B. Fractions with an A280 of >0.5 were pooled. The concentration of IgG in the pooled fractions was 0.83 mg/mL (5.5 nM). The antibody pool was stored on ice until it was conjugated. (b) Conjugation of STCM-Derivatized Anti-TSH IgG to PGA(SPO3)24 Cross-Linked Bovine ALP. To a 0.72 mL aliquot of 0.83 mg/mL (4 nmol) STMCC linker-derivatized anti-TSH IgG was added 0.69 mL of 1.30 mg/mL (3.3 nmol) PGA(SPO3)24 cross-linked ALP prepared as described in the preceding paper (10). The resulting solution was allowed to react overnight at 5 °C while being rotated at 100 rpm on a rotary agitator. The progress of the conjugation was followed by gel permeation HPLC using a Bio-Rad Bio-Sil SEC-400 column and pH 7.0 buffer containing 0.1 M sodium phosphate and
0.1 M NaCl (buffer C) at a flow rate of 1.0 mL/min (detector at A280). After 12 h, the reaction was essentially complete with >80% of the protein conjugated. Conjugates were concentrated to a volume of 200 mL with 30 000 MW cutoff Amicon membranes. The process was repeated three times. The concentration of the protein was 3.2 mg/mL as determined by A280. As a control experiment, the thermal stability and GPC profile of the PGA(SPO3)24-cross-linked ALP-anti-TSH conjugate was compared to that of the highest-activity and stability un-cross-linked ALP-anti-TSH control conjugate. Derivatization of ALP with iminothiolane hydrochloride and its conjugation to STCM-derivatized anti-TSH IgG were carried out as described previously (11). Thermal Stability Evaluation of the Conjugates. Stock solutions of PGA(SPO3)24-cross-linked ALP-antiTSH IgG conjugates from above were diluted with pH 7.5 buffer containing 0.1 M Tris, 1.0 mM MgCl2, and 0.1 mM ZnCl2 to a concentration of 10 mg/mL. These dilutions were stored at 45 °C in an incubator for the duration of the study. The values of the enzyme activity of all the conjugates were normalized to 100% at the start of the incubation. Progressive changes in the enzyme activity of the conjugates were monitored as described in the preceding paper (10). RESULTS AND DISCUSSION
Scheme 1 shows the conjugation of PGA(SPO3)24-crosslinked, stabilized ALP to IgG derivatized with the 30-
Technical Notes
Bioconjugate Chem., Vol. 9, No. 3, 1998 401
Figure 2. GP chromatographs of the conjugation: (A) native IgG, (B) native ALP, (C) unfractionated cross-linked ALP, (D) the unfractionated cross-linked ALP-IgG conjugate, with 1:1 enzyme:IgG stoichiometry, and (E) unfractionated Traut reagentderivatized ALP conjugated to STMCC-derivatized IgG. Figure 1. Thermal stability of PGA(SPO3)24-cross-linked ALPanti-TSH IgG conjugates at 45 °C in pH 7.5 buffer containing 0.1 M Tris, 1.0 mM MgCl2, and 0.1 mM ZnCl2. Decay of enzyme activity in the best conjugate based on Traut reagent-derivatized ALP and conjugated to STMCC-derivatized IgG, compared with conjugates of ALP cross-linked at 2:1 and 4:1 stoichiometries with PGA(SPO3)24.
atom heterobifunctional coupling agent STMCC. We have shown before that the performance of this longer maleimide active ester is significantly better than the performance of the shorter analogues (11). Self-catalyzed cross-linking of ALP by PGA(SPO3)24 is shown in Scheme 1A. Poly(glutamic acid) poly(phosphorothioate) is incubated in the presence of SMCC-derivatized ALP. This enzyme catalyzes the hydrolysis of phosphate and unmasks thiols in the polymer, which react with the maleimides on the surface of ALP. Between 32 and 68 thiolates are thus introduced into the ALP, lowering the activity of the enzyme by 10-25%. In the preceding paper, we have presented data which show that PGA-cross-linked ALP is considerably stabilized against thermal denaturation (10). The stoichiometries of PGA(SPO3)24:ALP may be varied from 1:1 to 6:1. As shown in the preceding paper (10), higher PGA(SPO3)24: ALP ratios result in predominantly intramolecular crosslinking of ALP. The cross-linking process generates a multiplicity of thiols in the cross-linked protein. These unreacted thiols are used to further conjugate the thermally stabilized enzyme to anti-TSH IgG. Scheme 1B shows derivatization of the antibody with the 30-atom heterobifunctional coupling agent SMCC. The maleimides introduced into the antibody molecule react in the final step with the thiolates on the PGA-cross-linked ALP. The thermal stability of the conjugates is shown in Figure 1. After 8 days at 45 °C in a pH 7.5 buffer containing 0.1 M Tris, 1.0 mM MgCl2, and 0.1 mM ZnCl2, the best cross-linked ALP-anti-TSH conjugates (2:1 and 4:1 PGA(SPO3)24:ALP) had a normalized enzyme activity 15% higher than that of the unmodified native ALP and 30% higher than that of the most active control conjugates based on un-cross-linked ALP, made using the Traut method (12). Notably, the conjugates prepared with the cross-linked ALP also had better thermal stability than the native unconjugated ALP. In our experience, the enzyme thermal stability of ALP conjugates is generally lower than that of the unconjugated ALP. Unlike PGA-cross-linked ALP, in which the cross-
linked enzyme experiences a dramatic loss of activity followed by a quick recovery within the first 72 h of incubation at 45 °C (10), the cross-linked ALP conjugates do not show similar behavior. If the loss of activity followed by the recovery, observed in the first 72 h in the cross-linked ALP, is due to the reversibility of the changes in the quaternary structure, the conjugated IgG molecule may have a stabilizing effect which prevents such transient changes in the quaternary structure possibly because of the presence of covalent cross-links to IgG and the contiguity of the IgG and ALP molecules. All conjugates were prepared from antibodies derivatized with a 50-fold molar excess of STMCC linkers and ALP cross-linked with 2 or 4 equiv of PGA(SPO3)24. As described in the Experimental Section, the ratio of IgG to cross-linked enzyme was 1:1. The data in Figure 1 are normalized to 100% at day 0 to visualize the effect of thermal stress on various preparations. N-Ethylmaleimide-capped conjugates underwent thermal denaturation at a faster rate than the uncapped conjugates (data not shown). The progress of the conjugations was followed by HPLC using a Bio-Sil SEC-400 column. The ALP enzyme activity of 1:1 ALP-IgG conjugates at day 0 was between 65 and 80% of that of the native enzyme. Panels A-E of Figure 2 show GPC profiles of IgG, native ALP, unfractioned 4:1 PGA-cross-linked ALP, the crude unfractionated IgG-cross-linked ALP conjugate, and the best crude unfractionated conjugate based on the Traut method. Chromatogram D appears to consist predominantly of the conjugated IgG. The crude conjugates at 10.11 min and higher molecular weights all show enzymatic activity, as demonstrated by careful fractionation of this material. The shoulder peak at 11.15 min shows ALP activity and has retention times virtually identical to that of PGA-cross-linked ALP. As seen in Figures 2E and 1, the most active unfractionated Traut ALP-IgG conjugate yields material with a much higher molecular weight whose thermal stability is considerably lower than that of the cross-linked material. Free thiolates on the enzyme do not lead to aggregation of the conjugates as demonstrated by GPC analysis of the incubated materials. In view of the decrease in the number of titratable thiolates in the cross-linked ALP (10), the constant size of the conjugates can possibly be attributed to the exhaustion of the thiolates through some oxidative process, i.e. intramolecular disulfide bond formation.
402 Bioconjugate Chem., Vol. 9, No. 3, 1998 CONCLUSIONS
Bieniarz et al. (4) Maggio, E. T. (1980) Enzyme-Immunoassay, p 174, CRC Press, Boca Raton, FL.
Auto-catalyzed cross-linking of maleimide-derivatized alkaline phosphatase with poly(glutamic acid) poly(phosphorothioate) stabilizes the protein against the thermal denaturation and introduces multiple thiolates on the surface of the enzyme. The stabilized enzyme is then conjugated to an the immunoglobulin molecule through the reaction of thiolates with maleimides in the IgG. The conjugation process is highly controllable; conjugate size may be adjusted by selection of appropriate stoichiometries of the cross-linking polymer, enzyme, and immunoglobulin. The method leads to thermally stabilized conjugates.
(8) Brinkley, M. (1992) A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents. Bioconjugate Chem. 3, 2.
ACKNOWLEDGMENT
(9) Feeney, R. E. (1987) Chemical modification of proteins: comments and perspectives. Int. J. Pept. Protein Res. 29, 145.
We thank Dr. Eric Ginsburg of the Abbott Laboratories Advanced Drug Delivery Department for his critical reading of the manuscript.
(10) Bieniarz, C., Cornwell, M. J., and Young, D. F. (1997) Alkaline phosphatase activatable polymeric cross-linkers and their use in the stabilization of proteins. Bioconjugate Chem. 9, 390-398.
LITERATURE CITED
(11) Bieniarz, C., Husain, M., Barnes, G., King, C. A., and Welch, C. J. (1996) Extended length heterobifunctional coupling agents for protein conjugation. Bioconjugate Chem. 7, 88-95.
(1) Butt, W. R. (1984) Practical Immunoassay, pp 47-50, Marcel Dekker, New York. (2) Maggio, E. T. (1980) Enzyme-Immunoassay, pp 64-65, CRC Press, Boca Raton, FL. (3) Ishikawa, E., Imagawa, M., Hashida, S., Yoshitake, S., Hamaguchi, Y., and Ueno, T. (1983) Enzyme-labeling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining. J. Immunoassay 4 (3), 209327.
(5) McComb, R. B., Bowers, G. N., and Posen, S. (1979) Alkaline Phosphatase, pp 304-318, Plenum Press, New York. (6) Means, G. E., and Feeney, R. E. (1990) Chemical modification of proteins: history and applications. Bioconjugate Chem. 1, 2. (7) Wong, S. H. (1991) Chemistry of Protein Conjugation and Cross-linking, CRC Press, Boca Raton, FL.
(12) 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, 5399-5406.
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