Bioconjugate
Chemhw JULY/AUGUST 1992 Volume 3, Number 4 0 Copyright 1992 by the American Chemical Society
REVIEWS Purifying Antibody-Plasminogen Activator Conjugates? Christoph Bode,’ Marschall S. Runge,t and Edgar Haber* Medical Clinic I11 (Cardiology), University of Heidelberg, 6900 Heidelberg, Germany, Division of Cardiology, Emory University School of Medicine, Atlanta, Georgia 30322, and Harvard School of Public Health, Boston, Massachusetts 02115. Received December 2,1991 INTRODUCTION
Conjugates of monoclonal antibodies and plasminogen activators have been constructed in an effort to target these therapeutically useful enzymes to thrombi. Because thrombi may contain fibrin-richand platelet-rich material, antifibrin as well as antiplatelet antibodies have been investigated as targeting devices. To reduce the antigenicity and alter the pharmacokinetic properties of these potential therapeutic agents, efforts were made to reduce the molecular size of the conjugate’s antibody portion by using the antigen-binding fragment (Fab) rather than the whole antibody. To obtain a homogeneous preparation of conjugate and to facilitate production, recombinant antibody-plasminogen activator constructs have been produced. Bispecificmonoclonal antibodiescontaining one binding site that recognizes a thrombus and one binding site that recognizes a plasminogen activator have been shown to concentrate plasminogen activator at the surface of a thrombus. Theoretically,such bispecific antibodies could be used to enhance the potency of endogenous plasminogen activators. Alternatively, bispecific antibodies or bispecific (Fab’)o could be combined with a plasminogen activator and used as immunoconjugates. The potential therapeutic use of these constructs has been reviewed previously (I,2). The present review focuses on the purification of antibody-plasminogen activator conjugates and bispecific antibodies.
* Address for correspondence: Dr. Christoph Bode, Medizinische Klinik I11 (Kardiologie), Universitiit Heidelberg, Bergheimerstrasse 58, 6900 Heidelberg, Germany. ‘This research was supported in part by a grant from the Deutache Forschungsgemeinschaft (Bo 726/3-2). Emory University School of Medicine. 8 Harvard School of Public Health.
*
1043-1802/92/2903-0269$03.00/0
PURIFICATION OF CHEMICAL AND RECOMBINANT ANTIBODY-PLASMINOGEN ACTIVATOR CONJUGATES
Purification by Gel Filtration. The chemistry used to synthesizethe antibody-plasminogen activatormolecule can be controlled to the extent that predominantly 1:l conjugates of whole antibody or Fab’ and plasminogen activator are formed. In addition to the desired conjugate the reaction mixture always contains uncoupled antibody and uncoupled plasminogen activator. For example, with a conjugation mixture of antifibrin IgG (150 kDa) and the B-chain of urokinase (30 m a ) , the uncoupled B-chain of urokinase could be effectively separated from the antifibrin antibody-urokinase conjugate (180 kDa) by gel filtration on Sephacryl2-200(3). However, the conjugate could not be completely separated by uncoupled antibody. Resolution on Sephacryl2-300did produce better results, but the 5-300 column was still unsuitable for the evaluation of conjugates with antibodies that exhibit functional properties of their own (e.g. platelet inhibition). These antibodies had to be separated quantitatively from the conjugate so that they would not interfere with assays comparing the functional properties of conjugated versus unconjugated enzyme. Although it is possible to separate 104 kDa conjugates of antifibrin Fab’ (50 kDa) and urokinase (54 kDa) by gel filtration, this method has not been extensively applied because other purification procedures are superior (see below). Affinity Purification Based on Functional Properties. The use of two sequential affinity procedures, one selecting for active enzyme and the other for functionally intact antibody, has proved to be the method of choice for purifying active conjugate. This method is schematically presented in Figure 1. For the plasminogen activators urokinase (4, 5) and tissue-type plasminogen activator (t-PA) (6,7),which are 0 1992 American Chemical Soclety
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Bloconlugte Chem.. Vol. 3, No. 4, 1992
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Figure 1. The principle of sequential affinity chromatography based on functional properties. This is the method of choice for purifying highly active conjugates of plasminogen activators and antifibrin antibodies or antifibrin Fab.
both serine proteases, the specific inhibitor benzamidine was used as an immobilized ligand (8). Only enzyme antibody conjugates in which the enzymatic center remained intact during the coupling procedure (and uncoupled intact enzyme from the reaction mixture) could bind and be retained by the benzamidine-Sepharose affinity column. Uncoupled whole antibody or uncoupled antibody fragments such as Fab have no affinity for benzamidine and were thus not retained on the column. To reduce nonspecific binding on the affinity column to a minimum, a high-salt washing step was performed prior to elution (Figure 2). Free antibody in the nonbinding fraction from the column could then be collected and reused for conjugation in order to enhance yield. The desired conjugate (and uncoupled enzyme) was eluted by a mild change in pH. Application of an elution buffer containing 0.1 M sodium acetate and 0.1 M sodium chloride, pH 4.0, regularly resulted in complete elution without measurable loss of enzyme activity. The pH of the eluate was adjusted to 7.4 with a 3.0 M Tris-HC1 solution, pH 8.6. In the case of antifibrin antibody-plasminogen activator conjugates, the eluate was then applied to a (Gly-HisArg-Pro-Leu-Asp-Lys-Cys)-MB-lysine-Sepharose column (peptide-Sepharose). The peptide, which was used as an antigen to raise monoclonal antifibrin antibodies, represents the seven amino-terminal residues of the fibrin Bchain (BO15-22) (9). The cysteine residue was added to allow for predefined coupling to lysine immobilized on cyanogen bromide-activated Sepharose C1-4B using the heterobifunctionalcross-linkingreagent p-maleimidobenzoic acid succinimido ester (MBS). Peptide-Sepharose has been used as an efficient purification matrix for antifibrin antibody, antifibrin Fab’, and their respective conjugates. Because this matrix was used for the eluate of the benzamidine-Sepharose purification step, which contained only uncoupled enzyme and conjugate and no free antibody, only the conjugate was retained. Elution was effected again through pH change (0.2 M glycine, pH 2.8). As the capacity of the peptide-Sepharose matrix was very high (usually in excess of 20 mg of antibody/mL of affinity gel) the column could be used to concentrate conjugate, making concentration by filtration in pressure cells (Amicon) unnecessary. Figure 2 shows a representative elution profile. Purificationresults were confirmed by SDS-PAGE (IO,11) and Western blotting (11). Sequential affinity chromatography as described above has yielded conjugates with an active enzyme portion and an antibody portion capable of binding to the intended target. Because the conjugationchemistry was controlled so that predominantly 1:l conjugates were synthesized, further purification by gel filtration to eliminate active aggregates has usually been unnecessary. The purification of chemical conjugates by sequential affinity chromatography yields active, predominantly 1:l
FUACTION NUAylER
Figure 2. Purification of antibody-urokinase conjugates by affinity chromatography based on functional properties. Antibody-urokinase conjugates were first purified by passage over a benzamidine-Sepharosecolumn. (A) the reaction mixture (6mL at 2 mg/mL) was loaded onto a 4-mL benzamidine-Sepharose column pre-equilibrated with PBSA (0.1 M NaHZPOd, 0.1 M NaC1,0.2% sodium azide, pH 6.6). After a washing step with PBSA and then 0.1 M sodium phosphate, 0.5 M NaCl, 0.02% sodium azide, pH 7.4, the bound material was eluted with 0.1 M sodium acetate, 0.1 M NaC1, pH 4.0. The arrow marks the beginning of the elution. The eluted material was neutralized with 3 M Tris-HC1, pH 8.6. (B) the material eluted from the benzamidine-Sepharose column was loaded onto a peptideSepharose column pre-equilibrated with PBSA. This column was also washed with 0.1 M sodium phosphate, 0.5 M NaC1, 0.02% sodium azide, and the bound material was eluted with 0.2 M glycine, pH 2.8. The arrow marks the beginning of the elution (from ref 4).
conjugatesthat are, however, not completelyhomogeneous because the heterobifunctional cross-linking reagent Nsuccinimidyl3-(2-pyridy1thio)propionate(SPDP)can react with different amino groups on the plasminogen activator. Using this purification procedure for recombinant conjugates yields a uniform, homogeneous preparation (12). Purification by Immunoaffinity. In moat cases affinity purification can be performed by immobilizing an antibody to one of the conjugate components on a cyanogen bromide-activatedSepharosematrix. For example, a goat antibody directed against mouse Fab (goat antimouse Fab) has been used to select for antiplatelet Fab’, antiplateletIgG, and their respective conjugates (13).Using benzamidine-Sepharose first and then goat anti-mouse Fab-Sepharose we were able to efficientlypurify antiplatelet-urokinase conjugates. However, this procedure does not select for antibody (or Fab) in the conjugate that has retained its capacity to bind to the antigen. Therefore,
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we suggest that affinity chromatography based on functional criteria be used whenever such an approach is feasible. Immunoaffinity chromatography (on an anti-plasminogen activator column) has also been combined with affinity chromatography based on functional properties (on a fibrin fragmentD-dimer column) to purify antibodyplasminogen activator conjugates (14,15).Because binding with the immunoaffinity column is based on antigen recognition rather than functional activity, this approach may in theory result in conjugates with diminishedspecific activity because the final preparation may contain a mixture of active and inactive components. The reported conjugates (14,15),however, showed specific activities on fibrin plate assays or toward chromogenic substrate that were comparable to those of uncoupled activator. Thus the theoretical limitation of this approach may not always be of practical importance. Another method for selecting intact IgG that might be contained within a conjugate is the use of protein A immobilized on Sepharose. Protein A is a streptococcal protein that binds to the constant domains of immunoglobulins with high affinity. As our goal has been to develop strategies that eliminate the constant domains from conjugatesby using Fab fragments, this approach has not been widely practiced in our laboratories. PURIFICATION OF BISPECIFIC ANTIBODIES AND IMMUNOCONJUGATES
Purification by Gel Filtration. Bispecific antibodies have been constructed by chemically couplingIgG (or Fab’) molecules (16, 17) of the same isotype but different specificity. The example discussed below was synthesized from a monoclonal antifibrin antibody and a monoclonal anti-tPA antibody. Bispecific IgG molecules (300kDa) have been purified by gel filtration from reaction mixtures containing the two antibodies (each 150 kDa). Usually 9 mL of reaction mixture was applied to a Sephacryl S-300column (2.5X 85cm). Fractions from the first peak contained the desired bispecific antibody. Fractions from the second peak (at about 150 kDa) presumably contained both antibody monomers. An immunochemicalcomplex (immunoconjugate)made up of tPA and bispecific antibody was formed by mixing a 3:l molar excess (tPAbispecific antibody) of tPA (3.5 mg) with bispecific antibody (5 mg). This mixture was again applied in a 9-mL volume to the Sephacryl S-300 column. Chromatography revealed a peak at approximately 400 kDa, which was interpreted as tPA bound to the bispecific antibody, and a second peak of approximately 70 kDa, representing unbound tPA. On the basis of enzymatic activity in a chromogenic substrate assay (S-2288), approximately 1.5 mol of tPA appeared to bind per mole of bispecific antibody. Purification by gel filtration is an effectiveway to obtain bispecific antibodies in high yield. Harsh elution conditions can be avoided. However, antibodies that have lost function during the coupling procedure are not removed. In addition, the resolving power of most gel filtration resins is insufficient to discriminate effectively between bispecific antibody-tPA complexes (370 kDa) and bispecific antibody alone (300m a ) . Therefore,the preparation may contain functionally inactive molecules. Affinity PurificationBased on Functional Groups. The method of choice for purifying bispecific antibodies is affinity purification based on functional criteria. Bispecific (Fab’)2 molecules made from the Fab’ of an an-
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