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Carbohydrate-Directed Conjugation of Cobra Venom Factor to Antibody by Selective Derivatization of the Terminal Galactose Residues Qinglan Fu and D. Channe Gowda* Department of Biochemistry and Molecular Biology, 3900 Reservoir Road, Georgetown University Medical Center, Washington, DC 20007. Received August 14, 2000; Revised Manuscript Received January 12, 2001
Cobra venom factor (CVF) can cause cell death by complement-mediated bystander cell lysis. Several studies have investigated CVF for application in cancer therapy by conjugating CVF to antibodies against tumor cell surface-specific antigens via the side-chain amino acid residues. In most cases, the activity of CVF was markedly impaired, presumably by modification of the factor B binding domain due to random derivatization. Since CVF is a glycoprotein and its oligosaccharide chains are distal to the factor B binding domain, coupling of CVF to antibodies through its oligosaccharide chains is expected to yield immunoconjugates with retention of CVF activity and elimination of the immunoreactivity of the terminal R-galactosyl residues. In this study, we investigated the carbohydrate sitedirected conjugation of CVF to a monoclonal IgG specific to a cell-surface antigen of human ovarian cancer cells. The terminal galactosyl residues of CVF were selectively modified at C-6 by treatment with galactose oxidase, and the generated aldehyde groups were derivatized in situ with hydrazides containing either protected thiol or maleimide functional groups. The CVF derivatives were allowed to react with thiol groups introduced to the antibody by derivatization with 2-iminothiolane to yield carbohydrate site-directed CVF-antibody conjugates. In both cases, 30-40% of the antibody crosslinked to CVF to yield predominantly monovalent CVF-antibody conjugates. The purified immunoconjugates retained 70-75% of CVF activity and significant level of antigen-binding capacity. This is the first study to exploit the oligosaccharide chains of CVF for the preparation of active immunoconjugates.
INTRODUCTION
(CVF),1
Cobra venom factor a nontoxic complementactivating glycoprotein in cobra venom, is a functional analogue of mammalian complement component C3b. When added to mammalian serum, CVF forms CVF,Bb, a C3/C5 convertase of the alternative pathway of the complement (1, 2). CVF,Bb can activate the complement in a manner similar to C3b,Bb, the physiologic C3/C5 convertase. However, CVF,Bb is completely resistant to the action of complement regulatory factors H and I. Therefore, when administered to animals, CVF can deplete the complement and build up membrane-attaching complexes; the latter can cause bystander lysis of cells (1). This property of CVF has been used for selective killing of tumor cells by conjugating to antibodies against cancer-specific cell surface antigens (1, 3-10). CVF consists of three polypeptide chains, designated R, β, and γ, and three N-linked oligosaccharides; two in * To whom correspondence should be addressed. Phone: (202) 687-3840. Fax: (202) 687-7186. E-mail: gowda@ bc.georgetown.edu. 1 Abbreviations: CVF, cobra venom factor; PDPH, 3-(2pyridyldithio)propionic acid hydrazide; MPBH, 4-(4-N-maleimidopheny)butyric acid hydrazide; M2C2H, 4-(maleimidomethyl)cyclohexane-1-carboxylic acid hydrazide; PDPH-CVF, PDPH derivative of CVF; M2C2H-CVF, M2C2H derivative of CVF; MPBH-CVF, MPBH derivative of CVF; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); SDS-PAGE, sodium dodecyl sulfatepolyacrylamide electrophoresis; HRP, horseradish peroxidase; FITC, fluorescein isothocyanate; DTT, dithiothreitol; OVB-3, mouse monoclonal IgG2b directed against a cell surface antigen of human ovarian cancer cells.
the R-chain, one in the β-chain, and no carbohydrates in the γ-chain (11, 12). More than 80% of the oligosaccharide chains of CVF terminate with the unusual R-galactosylated Lewis X structures (12-14). CVF is immunoreactive to natural human anti-R-Gal antibody because of the presence of terminal GalR1-3Gal residues in its oligosaccharide chains (12, 15). Therefore, therapeutic application of CVF requires the abolition of this immunoreactivity. Although the anti-R-Gal antibody immunoreactivity of CVF can be abolished by the removal of terminal galactosyl residues, the exposed Lewis X structures are recognized by liver cell lectins, leading to rapid clearance of CVF from the circulation (16). The complement-activating activity of CVF is not affected by either the removal of its oligosaccharide chains or the binding of the terminal R-galactosyl residues with lectin or antibodies (12). This implies that the oligosaccharide chains of CVF are distal to the factor B binding domain. Therefore, coupling of CVF through the terminal galactosyl residues to antibodies should yield immunoconjugates with the retention of the CVF activity and simultaneously eliminating the anti-R-Gal immunoreactivity due to masking of the terminal R-galactosyl residues. In addition, this mode of cross-linking of CVF to antibodies should avoid the rapid, asialoglycoprotein receptor-dependent, hepatic clearance of CVF immunoconjugates from the circulation. We have recently shown that the terminal galactosyl residues of CVF can be modified conveniently at C-6 by galactose oxidase treatment and in situ derivatization with hydrazides (17) and that this modification completely abolishes the anti-RGal immunoreactivity without affecting CVF activity (17).
10.1021/bc000100u CCC: $20.00 © 2001 American Chemical Society Published on Web 02/16/2001
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In this study, we used this site-specific derivatization procedure to conjugate CVF to a monoclonal antibody (OVB-3) directed against a cell surface antigen of human ovarian cancer cells and assessed the purified immunoconjugates for CVF and antibody activities. EXPERIMENTAL PROCEDURES
Materials. CVF was isolated from Naja naja kaouthia venom as described previously (18). 3-(2-Pyridyldithio)propionic acid hydrazide (PDPH) and 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH) were synthesized as reported previously by Zara et al. and Chamow et al., respectively (19, 20). 4-(Maleimidomethyl)-cyclohexane-1-carboxylic acid hydrazide (M2C2H), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and 2-iminothiolane hydrochloride were purchased from Pierce Chemical Co. (Rockford, IL). Galactose oxidase (25 units/mg of protein) and coffee bean R-galactosidase (10 units/mg of protein) were from Boehringer Mannheim (Indianapolis, IN). HRP (type VIA, 1000 units/mg) was from Sigma Chemical Co. (St. Louis, MO). Centricon 30 tubes were from Amicon (Danvers, MA). FITC-conjugated goat anti-mouse IgG was from Southern Biotechnology Associates, Inc. (Birmingham, AL). RPMI 1640 medium, insulin, penicillin/ streptomycin were from Life Technologies (Gaithersburg, MD). Guinea pig erythrocytes were obtained by femoral vein puncture. OVB-3, a mouse monoclonal antibody (IgG2b, ref 21) was a generous gift from Dr. Ira Pastan, National Cancer Institute, NIH. Carbohydrate Analysis. OVB-3 antibody (50 µg) was hydrolyzed with 400 µL of 2.5 M trifluoroacetic acid at 100 °C for 5 h. The hydrolysates were evaporated to dryness in a Speed Vac, dissolved in water and then analyzed for neutral sugars and hexosamines using a Dionex HPLC (Dionex Corp., Sunnyvale, CA) with pulsed amperometric detection on a CarboPac PA1 column (4 × 250 mm) (22). Elution was with 20 mM NaOH at a flow rate of 0.9 mL/min. Sialic acids from OVB-3 antibody (20 µg) were quantitatively released either by hydrolysis with 0.1 M sulfuric acid (40 µL) at 80 °C for 50 min or by treatment with Clostridium perfringens sialidase (20 milliunits) in 50 µL of 50 mM sodium acetate, pH 5.5, at 37 °C for 24 h. The released sialic acids were analyzed on a CarboPac PA1 high-pH anion-exchange column (4 × 250 mm) with 100 mM NaOH/150 mM NaOAc at a flow rate of 0.8 mL/min (22). Derivatization of the Terminal Galactosyl Residues of CVF by Acid Hydrazides. Derivatization of the primary hydroxyl groups of the terminal galactosyl residues of CVF was carried out by a modification of the published procedures (23, 24). CVF (2-2.5 mg) in 1 mL of 100 mM sodium phosphate, pH 6.5, was incubated with galactose oxidase (10 units, added in two equal aliquots at 0 and 8 h), HRP (30 units/mL) and PDPH (50-100 mol/mol CVF) at 37 °C for 30 h (17). To minimize the hydrolysis of reactive maleimide groups to maleic acid, derivatization of the enzyme-oxidized CVF with MPBH or M2C2H was performed at the end of enzyme incubation for 2 h at room temperature. Uncoupled hydrazides were removed by filtration using Centricon 30 tubes or by gel filtration on Sephadex G-25 columns (0.9 × 15 cm) in 50 mM sodium phosphate, 150 mM NaCl, 2 mM EDTA, pH 6.5, 7.2 or 8.0. To determine the extent of derivatization, an aliquot of PDPH-CVF (150 µg in PBS, pH 7.2) was reduced with 50 mM DTT at room temperature for 20 min (25). The released pyridine-2-thione was determined by measuring
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the absorbance of the solution at 343 nm and by using the molar extinction coefficient of 8.08 × 103 M-1 cm-1 (25). To determine the amount of M2C2H or MPBH coupling to CVF, M2C2H-CVF, and MPBH-CVF were reduced with sodium borohydride and treated with coffee bean R-galactosidase. The released galactose was measured by HPLC using high-pH anion-exchange column (see below). Stability of M2C2H and MPBH in Buffer Solutions. Stock solutions of M2C2H or MPBH (28 mM in N,N-dimethylformamide) were diluted to 350 nM with 100 mM NaOAc (pH 5.5) or 100 mM sodium phosphate (pH 6.5, 7.0, 7.5, or 8.0). The hydrolysis of the maleimide moiety of M2C2H and MPBH was monitored at intervals by measuring the increase in absorbance at 320 nm for M2C2H or the decrease in absorbance at 300 nm for MPBH (20). Derivatization of OVB-3 with 2-Iminothiolane. OVB-3 (1.2 mg) in 800 µL of 50 mM sodium phosphate, 150 mM NaCl, 2 mM EDTA, pH 8.0, was incubated in a nitrogen atmosphere with 2-iminothiolane (104 µg) at room temperature for 1 h (26). The unreacted 2-iminothiolane was removed by gel filtration on Sephadex G-25 columns (0.9 × 15 cm) in 150 mM NaCl, 2 mM EDTA, pH 8.0. The amount of 2-iminothiolane that coupled to OVB-3 was determined by treating the derivatized antibody with DTNB and measuring spectrophotometrically the released 2-nitro-5-thiol-benzoic acid (26). Coupling of Hydrazide-Derivatized CVF to 2-Iminothiolane Derivative of OVB-3. PDPH-CVF, M2C2HCVF and MPBH-CVF (0.5 mg each) were separately mixed with 2-iminothiolane-derivatized OVB-3 (0.5 mg) in 400 µL of 150 mM NaCl, 2 mM EDTA, pH 6.5, 7.2, or 8.0, and incubated overnight at 25 °C with gentle rocking in a nitrogen atmosphere. Unreacted thiol groups in the derivatized OVB-3 and maleimide groups in M2C2H-CVF and MPBH-CVF were blocked with cysteine at 25 °C for 1 h. Aliquots of the solutions were analyzed by SDSPAGE under nonreducing conditions (27). The reaction mixtures (200-250 µg protein) were chromatographed on Bio-Silect SEC 400-5 gel filtration HPLC columns (300 × 7.8 mm) (Bio-Rad, Hercules, CA) using a Dionex Bio-LC HPLC in PBS, pH 7.2. The elution was with 50 mM sodium phosphate, 150 mM NaCl, pH 7.2, at a flow rate of 0.9 mL/min. Proteins were monitored by on-line UV detection at 280 nm using a Dionex variable wavelength detector. Fractions corresponding to CVF-IgG conjugates were pooled and analyzed for CVF activity. Derivatization of the Oligosaccharide Chains of OVB-3 with PDPH. OVB-3 (2 mg/mL) in 50 mM sodium phosphate, 150 mM NaCl, pH 6.5, was treated with 5 mM sodium periodate (1 mL) in the above buffer at 4 °C for 10 min. Excess periodate was allowed to react with glycerol (25 µL) at 4 °C for 30 min. The solution was dialyzed against 50 mM sodium phosphate, 150 mM NaCl, pH 6.5, and then incubated with 50-fold molar excess PDPH at 25 °C for 5 h. The amount of PDPH coupled to the antibody was determined as described above (25). Coupling of CVF to OVB-3 via the Carbohydrate Moieties of Both Proteins. The PDPH-derivatized OVB-3 (0.5 mg) was treated with 20 mM DTT in 200 µL of 100 mM NaOAc, pH 4.5, at 25 °C for 20 min. The solution was chromatographed on Sephadex G-25 (0.9 × 15 cm) in 50 mM sodium phosphate, 150 mM NaCl, 2 mM EDTA, pH 8.0. The void volume fractions were concentrated in Centricon 30 tubes and allowed to react with PDPH-CVF (0.5 mg) in 250 µL of 50 mM sodium
Site-Directed Conjugation of CVF and Antibody
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Figure 1. Scheme for the derivatization of the terminal galactosyl residues of the oligosaccharide chains (partial structure is shown) of CVF with heterobifunctional hydrazides. Oxidation of CVF with galactose oxidase in the presence of HRP generates aldehyde groups at the C-6 positions of the terminal galactose residues. The derivatization of the aldhedyde groups with protected thiol groupor maleimide group-containing hydrazides yielding stable hydrazones offers a convenient approach to couple CVF to antibodies.
phosphate, 150 mM NaCl, 2 mM EDTA, pH 8.0, overnight at 25 °C in a nitrogen atmosphere. Unreacted thiol groups were blocked with cysteine at 25 °C for 1 h. Aliquots of the solutions were analyzed on 5% SDSpolyacrylamide gel under nonreducing conditions. Hemolytic Assay for CVF Activity. CVF, hydrazidederivatized CVF, or CVF-OVB-3 conjugates (8-2000 ng CVF protein in each case) in PBS, 1 mM CaCl2, 0.5 mM MgCl2, and 0.1% (w/v) gelatin, pH 7.2 (20 µL) were mixed with guinea pig serum (20 µL) and guinea pig erythrocytes (10 × 106 in 20 µL of the above buffer) and incubated at 37 °C for 40 min (12). Cold PBS, pH 7.2 (1 mL), was added, the unlysed erythrocytes were removed by centrifugation and the released hemoglobin was measured at 412 nm. OVB-3 Binding to Ovarian Cancer Cell Surface Antigen. Human ovarian cancer cells (NIH:OVCAR-3 cells obtained from ATCC, Rockville, MD) were cultured as monolayers in RPMI 1640 medium with 10% fetal calf serum, insulin (10 µg/mL), penicillin (100 units/mL), and streptomycin (100 µg/mL), according to Pirker et al. (28). Cells were detached by gentle scraping, washed with serum-free medium and suspended in PBS, pH 7.2, containing 2% bovine serum albumin. The cells were treated with OVB-3, 2-iminothiolane-derivatized OVB-3 or CVF-OVB-3 conjugates (5 µg antibody/mL in PBS, pH
7.2) at 4 °C for 2 h. After washing with PBS, pH 7.2, the cells were treated with FITC-labeled goat anti-mouse IgG at 4 °C for 1 h, washed, fixed with 2% paraformaldehyde, and surface fluorescence was measured using a Becton Dickinson FACS II analyzer at the Georgetown University Lombardi Cancer Center core facility. RESULTS
Derivatization of Terminal Galactosyl Residues of CVF with Hydrazides. CVF contains approximately 5.6 terminal galactosyl residues (∼4.1 R-linked and ∼1.5 β-linked) (17). The C-6 hydroxyl groups of the terminal galactosyl residues were oxidized with galactose oxidase in the presence of HRP and the generated aldehyde groups were derivatized with three different hydrazides, which contain electrophilic centers for cross-linking of CVF to antibodies as depicted in Figure 1 (17). Treatment of PDPH-CVF with DTT released about 5.5 mol of pyridyl-2-thione, indicating that >95% of the terminal galactosyl residues of CVF were derivatized. In agreement with this result, treatment of sodium borohydridereduced PDPH-CVF, M2C2H-CVF, or MPBH-CVF with coffee bean R-galactosidase did not release detectable amounts of galactose. The hemolytic activity of CVF derivatized with all three hydrazides, as measured by the
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Figure 2. Scheme for the conjugation of OVB-3 to the oligosaccharide chains of CVF. The derivatization of the OVB-3 with 2-iminothiolane introduces thiol groups to the antibody. When the derivatized antibody allowed to react with PDPH-CVF (A) or M2C2H-CVF (B) under nitrogen atmosphere yields disulfide- or thioether-linked CVF-OVB-3 conjugates.
Figure 3. SDS-polyacrylamide gel electrophoresis of CVFOVB-3 conjugates. The hydrazide-derivatized CVF (5-10 µg), 2-iminothiolane derivatized OVB-3 (5-10 µg) and CVF-OVB-3 conjugates (20-30 µg), prepared as outlined in Figure 2, were electrophoresed on 5% (panel A) and 6% (panel B) SDSpolyacrylamide gel under nonreducing conditions. The gels were stained with Coomassie blue. (A) Lane 1, PDPH-CVF; lane 2, 2-iminothiolane derivative of OVB-3; lane 3, CVF-OVB-3 conjugates prepared from PDPH-CVF. Panel B: Lane 1, CVFOVB-3 prepared form MPBH-CVF; lane 2, CVF-OVB-3 prepared from M2C2H-CVF, lane 3, MPBH-CVF; lane 4, 2-iminothiolane derivatized OVB-3.
bystander lysis of guinea pig erythrocytes in guinea pig serum, was similar to that of untreated CVF (see Figure 5). Carbohydrate Moieties of OVB-3. In view of coupling hydrazide-derivatized CVF to carbohydrate moieties of the monoclonal antibody OVB-3, the nature of the carbohydrate moieties of the antibody was examined. More than 90% of the carbohydrate moiety of OVB-3 was released by N-glycanase, an enzyme that removes asparagine-linked oligosaccharides in glycoproteins (29). Carbohydrate compositional analysis of the enzymereleased carbohydrates revealed sialic acid, galactose,
Figure 4. HPLC fractionation of CVF-OVB-3 conjugates. The CVF-OVB-3 conjugates prepared as shown in Figure 2 were fractionated by HPLC gel filtration. Shown is the separation of CVF-OVB-3 prepared from the conjugation of 2-iminothiolanederivatized OVB-3 to PDPH-CVF. PDPH-CVF monovalent conjugates (fraction III, Mr ≈ 300 kDa) and multivalent conjugates (fractions II and I, Mr ≈ 450 and 600 kDa, respectively) were pooled as indicated. The immunoconjugates prepared by cross-linking 2-iminothiolane derivatized OVB-3 to MPBH-CVF and M2C2H-CVF were also fractionated by HPLC (not shown).
N-acetylglucosamine, mannose, fucose, N-acetylgalactosamine in the approximate molar ratios of 2.1:2.8:4.4: 3.0:0.9:0.3. Sialic acid was found to be a mixture of 60% N-acetylneuraminic acid and 40% N-glycolylneuraminic acid. The results suggest that the carbohydrate moieties of OVB-3 are predominantly complex type N-linked oligosaccharides, present most likely in the Fc region of
Site-Directed Conjugation of CVF and Antibody
Figure 5. Effect of hydrazide derivatization and conjugation of the antibody on CVF activity. CVF activity was measured by complement-mediated lysis of unsensitized guinea pig erythrocytes. The indicated amounts of the hydrazide derivatives of CVF and CVF-antibody conjugates were incubated with unsensitized guinea pig erythrocytes in guinea pig serum. The absorbance of the released hemoglobin was measured at 412 nm. (b) CVF; (O) PDPH-CVF; (2) CVF-OVB-3 conjugate fractions III in Figure 4; (4) CVF-OVB-3 conjugate fraction II in Figure 4; (9) CVF-OVB-3 conjugate fraction I in Figure 4.
the antibody. IgGs contain two N-linked oligosaccharides in their Fc regions; one oligosaccharide per polypeptide chain. Derivatization of OVB-3 with 2-Iminothiolane and Conjugation to Hydrazide Derivatives of CVF. The OVB-3 antibody was derivatized with 2-iminothiolane, a widely used reagent for the introduction of thiol groups to proteins for cross-linking (26, 30-33). Treatment of OVB-3 with ∼90-fold molar excess of 2-iminothiolane introduced 4-5 thiol groups/molecule of the antibody. The derivatization procedure did not significantly affect the antigen-binding activity of the antibody (see legend to Figure 6). OVB-3 derivatized with 2-iminothiolane was allowed to react separately with the PDPH-, M2C2H-, and MPBHderivatives of CVF at various pH according to the scheme outlined in Figure 2. SDS-PAGE analysis of the reaction mixtures showed that the derivatized OVB-3 efficiently conjugated to PDPH-CVF at pH 8.0, yielding a major product at ∼300 kDa and minor products at >450 kDa; approximately 40% of OVB-3 was coupled to CVF (Figure 3). The cross-linking of OVB-3 to PDPH-CVF was 5-10% at pH 6.5 or 7.2 (not shown). In the case of M2C2H-CVF and MPBH-CVF, the coupling efficiency at pH 8.0 was
