Synthesis and Characterization of Carbohydrate-Linked Murine

Nov 1, 1994 - ... human serum albumin–sialoglycoside conjugates containing O-acetylated sialic acids. Hai Yu , Harshal A. Chokhawala , Ajit Varki , ...
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Bioconjugate Chem. 1994, 5, 602-61 1

602

Synthesis and Characterization of Carbohydrate-Linked Murine Monoclonal Antibody K20-Human Serum Albumin Conjugates L. H. Kondejewski, J. A. Kralovec, A. H. Blair, and T. Ghose* Departments of Biochemistry and Pathology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7. Received April 22, 1994@

Efficacy of antibody mediated targeting depends on retention of immunoreactivity in conjugates. Retention can be improved by site-specific linkage of drugs or drug-loaded carriers to residues that are located well away from the antigen-binding sites. In this study we describe the site-specificlinkage of a potential drug carrier, human serum albumin (HSA), t o the carbohydrate residues in Dal K20, a murine IgGl monoclonal antibody (mAb) against human renal cell carcinoma, using disulfide exchange between 3-(2-pyridyldithio)propionicacid succinimidyl ester (SPDP)-derivatized HSA and 11-[[3-(2pyridyldithio)propionyllaminolundecanoicacid hydrazide (AUPDP)-derivatized mAb Dal K 20. AUPDP gave a higher yield of the conjugate than a functionally analogous 3-(2-pyridyldithio)propionic acid hydrazide (HPDP), suggesting that the extra length of the former facilitated the linkage. The conjugates were found to be unstable without reduction of the hydrazone linkage using sodium cyanoborohydride. Stabilized 1:1HSA:K20 carbohydrate-linked conjugates were isolated and compared with non-site-specific 1:l conjugates in which HSA was conjugated to amino groups in mAb Dal K20. The yield and stability of the two conjugates were comparable, but the site-specific conjugate was found to retain three times more antibody activity than the non-site-specific conjugate.

INTRODUCTION

Cancer chemotherapeutic agents have been conjugated to antibodies in an effort to make these agents more specific for cancer cells and to reduce their systemic toxicity (1-3). Both high drug loadings as well as the retention of immunoreactivity are important for the synthesis of potent and tumor-specific conjugates. A number of anticancer drugs have been conjugated directly to amino groups of immunoglobulins, but the general finding has been that in these conjugates immunoreactivity of the antibody is reduced, probably due to the direct modification of residues in the antigen binding site and/or changes in tertiary structure following derivatization of the antibody (3). In order to achieve higher drug loadings while retaining immunoreactivity, carriers have been loaded with drug molecules and then linked to antibodies (4-7). The linkage of drug-loaded carriers to a single or a very small number of sites in an antibody molecule may allow higher molar incorporation of the drug (e.g., 210) with retention of antibody activity, but linkage to residues such as amino groups that are present throughout the antibody molecule, including the antigen binding sites, has the inherent risk of interference with antibody activity. In recent years, the carbohydrate Abstract published in Advance ACS Abstracts, September 1, 1994. Abbreviations: AUPDP, 1l-[[3-(2-pyridyldithio)propionyllaminolundecanoic acid hydrazide; DMF, dimethylformamide; DNP-AU-hydrazide, ll-[(2,4-dinitrophenyl)aminolundecanoic acid hydrazide; DNP-AU-OMe, 11-[(2,4-dinitrophenyl)amino]undecanoic acid methyl ester; DNP-IgG, 11-[(2,4-dinitrophenyl)aminolundecanoic acid hydrazide-modified IgG; DTT, dithiothreitol; GMBS, y-maleimidobutyric acid N-hydroxysuccinimide ester; HPDP, 3-(2-pyridyldithio)propionicacid hydrazide; HSA, human serum albumin; HSA-K20, conjugate consisting of HSA coupled to mAb Dal K20; IgG, immunoglobulin G; mAb, monoclonal antibody; MTX, methotrexate; NRG, normal rabbit globulin; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate buffered saline (0.01 M phosphate in 0.15 M sodium chloride, pH 7.2);SPDP, 3-(2-pyridyldithio)propionicacid succinimidyl ester; SDS, sodium dodecyl sulfate.

residues of IgGl antibodies have become the preferred site for IgG modification because the majority of the carbohydrate residues in IgG are located in the hinge region of the molecule, well away from the antigenbinding site. Indeed, antibodies have been modified at their carbohydrate residues with low molecular weight drugs (2,8-10), liposomes (111,toxin molecules (121, and radionuclides (13) with full retention of antibody activity in many cases. The specific modification of carbohydrate residues involves the oxidation of immunoglobulin carbohydrates with sodium periodate to generate aldehyde groups, followed by reaction with suitable hydrazides, resulting in the formation of a hydrazone bond. We have used HSA as a model carrier protein and conjugated it to the carbohydrate moiety of a murine monoclonal antibody against a human renal cell carcinoma antigen, i.e., mAb Dal K20, using two different cross-linkers. HSA was selected primarily because of its well elucidated structure and anticipated lack of significant immunogenicity in patients. We also investigated the stability of the hydrazone bonds in these conjugates. In this study we report on the synthesis, stability, and immunoreactivity of carbohydrate-linked HSA-Dal K20 conjugates and compare them to a non-site-specific conjugate in which HSA was linked to amino groups in mAb Dal K20.

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1043-1802/94/2905-0602$04.50/0

MATERIALS AND METHODS

The antibody used in these studies was mAb Dal K20, a murine IgGIK monoclonal antibody directed against a human renal cell carcinoma associated cell surface antigen (14). mAb Dal K20 was produced by standard hybridoma methods and purified from mouse ascites by protein A chromatography (Bio-Rad, Richmond, CA) following the instructions of the manufacturer. NRG was used to standardize some conjugation procedures and was isolated from rabbit serum using caprylic acid (15). HSA, 2,2’-dipyridyl disulfide, 3-mercaptopropionic acid, 5,5‘dithiobis(2-nitrobenzoicacid), Extravidin-peroxidase, biotin hydrazide, dicyclohexylcarbodiimide, and hydrazine were from Sigma (St. Louis, MO). 11-Aminoundecanoic 0 1994 American Chemical Society

Carbohydrate-Linked Antibody-Albumin Conjugates

acid and N-hydroxysuccinimide were from Aldrich Chemical Co. (Milwaukee, WI). PDG desalting columns, BioGel P10, Bio-Gel P300, and goat anti-mouse IgGperoxidase were from Bio-Rad (Richmond, CA). Sulfosuccinimidyl 6-(biotinamid0)hexanoate was from Pierce (Rockford, IL). Thin layer chromatography was performed on silica gel 60 F 254 plates (Merck, Darmstadt, FRG) using 9:l chlorofordmethanol (system A) or 19:l chloroform/ methanol (system B). IH NMR spectra were recorded in CDCl3 on a Nicolet NT360NB spectrometer with shifts reported relative to tetramethylsilane. Melting points were determined with an Electrothermal 9100 melting point apparatus. Buffer A, 0.1 M sodium acetate, pH 4.0; buffer B, 0.1 M sodium phosphate, pH 7.2, containing 1 mM ethylenediaminetetraacetic acid. 3-(2-Pyridyldithio)propionicAcid Succinimidyl Ester (la). SPDP was synthesized by the method of Carlsson (16): yield 1.5 g, 56%; Rf 0.84 (A); lH NMR 6 2.83 (s, 4H), 3.13 (m, 4H), 7.13 (m, lH), 7.69 (m, 2H), 8.50 (m, 1H). 3-(2-Pyridyldithio)propionic Acid Hydrazide (lb). To SPDP (1.0 g, 3.2 mmol) dissolved in 20 mL of methanol was added hydrazine (160 pL, 4.8 mmol) in 5 mL of methanol. The solution was stirred at room temperature for 15 min, methanol removed under reduced pressure, the oil taken up in methylene chloride, washed with saturated sodium bicarbonate and then water, and dried with sodium sulfate, and the methylene chloride evaporated. The product was crystallized from diethyl ether to give a fine white powder: yield 0.63 g, 85%; Rf 0.41 (A); mp 91.5-92.5 "C; 'H NMR (CDC13) 6 2.62 (t, 2H), 3.10 (t, 2H), 3.98 (s, 2H), 7.14 (m, lH), 7.62 (m, 2H), 8.24 (s, lH), 8.52 (m, 1H). 1 l-[[3-(2-Pyridyldithio)propionyllaminolundecanoic Acid (2a). To 1l-aminoundecanoic acid (129 mg, 0.64 mmol) and sodium bicarbonate (108 mg, 1.28 mmol) dissolved in 2 mL of a water-ethanol mixture (2:l) was added SPDP (200 mg, 0.64 mmol) dissolved in 4 mL of ethanol. The solution was stirred at room temperature for 1h, adjusted to pH 7.0 with 1.0 M HCl, and the solvent removed under reduced pressure. The residue was distributed between water and chloroform, extracted with water, and dried over sodium sulfate. Chloroform was evaporated to give a white powder: yield 232 mg, 91%; R f 0.46 (A). 1 l-[[3-(2-Pyridyldithio)propionyllaminolundecanoic Acid Hydrazide (2c). To compound 2a (232 mg, 0.58 mmol) dissolved in 5 mL of methylene chloride was added N-hydroxysuccinimide (73 mg, 0.64 mmol) and dicyclohexylcarbodiimide (131mg, 0.64 mmol). The mixture was stirred for 2 h at room temperature and cooled to -20 "C, the dicyclohexylurea removed by filtration, and methylene chloride evaporated. The obtained 11-[[3-(2-pyridyldithio)propionyl]amino]udecanoic acid succinimidyl ester (2b),Rf 0.64 (A) (284 mg, 0.58 mmol), was dissolved in 5 mL of methanol and treated with hydrazine (37 pL, 1.16 mmol) dissolved in a small amount of methanol for 1 h at room temperature. Methanol was removed by evaporation, the residue taken up in methylene chloride, washed with saturated sodium bicarbonate and with saturated NaC1, dried over sodium sulfate, and evaporated, and the solid washed with ether to give a white powder: yield 160 mg, 61%; mp 105 "C; Rf 0.32 (A); 'H NMR 6 1.22 (s, 12H), 1.51 (p, 2H), 1.62 (p, 2H), 2.50 (t, 2H), 2.60 (t, 2H), 3.08 (t, 2H), 3.26 (q, 2H), 3.96 (bs, 2H), 6.50 (s, lH), 7.13 (q, lH), 7.20 (s, lH), 7.66 (m, 2H), 8.45 (m, 1H). 11-[(2,4-Dinitrophenyl) amino]undecanoic Acid

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Methyl Ester (3a). To 4 mL of methanol at -10 "C was added thionyl chloride (1040 pL, 14 mmol) followed by the slow addition of ll-aminoundecanoic acid (800 mg, 4 mmol) while stirring. The mixture was stirred at room temperature for 1 h, and then two volumes of diethyl ether were added and the precipitated 1l-aminoundecanoic acid methyl ester hydrochloride was washed with ether and collected by filtration. To ll-aminoundecanoic acid methyl ester hydrochloride (150 mg, 600 pmol) and triethylamine (167 pL, 1.2 mmol) in 10 mL of DMF was added l-fluoro-2,4-dinitrobenzene (68 pL, 540 pmol) and the solution stirred a t room temperature for 1 h. DMF was removed by evaporation, the residue taken up in chloroform, extracted with 0.1 M HC1, washed with saturated NaC1, and dried with sodium sulfate, and the solvent evaporated. The product was azeotroped with ether to give a yellow powder: yield 155 mg, 75%; Rf 0.96 (B); 'H NMR 6 1.29 (s, 12H), 1.46 (m, 2H), 1.78 (p, 2H), 2.33 (t, 2H), 3.44 (9, lH), 3.68 (s, 3H), 6.94 (d, lH), 8.28 (d, lH), 8.58 (bs, lH), 9.15 (t, 1H). ll-[(2,4-Dinitrophenyl)aminolundecanoic Acid Hydrazide (3b). To compound 3a (130 mg, 341 pmol) in 50 mL of methanol was added hydrazine (5.2 mL, 166 mmol) and the reaction mixture stirred for 16 h at room temperature. Methanol was removed by evaporation and the residue taken up in chloroform, extracted with saturated sodium bicarbonate, dried with sodium sulfate, and evaporated to give a yellow powder: yield 117 mg, 90%; R f 0.17 (B); mp 119-121 "C; ,Y = 360 nm, 6 = 17 000 M-l cm-l; lH NMR 6 1.29 (s, lOH), 1.46 (m, 2H), 1.60 (m, 2H), 1.78 (p, 2H), 2.33 (t, 2H), 3.44 (9,2H), 3.68 (s, 3H), 6.94 (d, lH), 8.28 (d, lH), 8.58 (bs, lH), 9.15 (t, 1H). Oxidation of IgG and Reaction with HPDP or ATJPDP. To mAb Dal K20 (10 mg/mL, 1.0 mL) in 0.1 M acetate buffer, pH 5.5, containing 0.15 M NaCl at 0 "C was added sodium periodate (23.4 mg in 0.1 mL) dropwise while stirring to give a final periodate concentration of 100 mM. After 20 min at 0 "C in the dark, the solution was desalted into 0.1 M sodium acetate, pH 4.0 (buffer A) and DMF added slowly while stirring to a concentration of 15%(v/v). HPDP or AUPDP dissolved in DMF was added to give a 100-fold molar excess of hydrazide spacer over mAb K20 and a final DMF concentration of 25% (v/v). After 2 h at room temperature the solutions were desalted into 0.1 M sodium phosphate, 1mM EDTA, pH 7.2 (buffer B). The number of pyridyldithio groups incorporated into mAb Dal K20 was determined in the presence of 0.1 M DTT, using E = 8080 M-l cm-l at 343 nm for released pyridine-2-thione (16). Protein concentration was determined by absorbance at 280 nm (1.0 mg/ mL = 1.4) with correction for pyridyldithio contribution using the fOI-mula A280 due t o I& - A280 measured - (5100 [PDT]), where [PDT] is the molar concentration of pyridine-2-thione. Pilot Studies with NRG To Determine Conditions for Spacer Incorporation and Hydrazone Bond Stabilization. (i) Oxidation Conditions. To NRG (8.7 mg/mL, 0.45 mL) in 0.1 M acetate buffer, pH 5.5, containing 0.15 M NaCl a t 0 "C was added a 0.1%-1.0% aqueous solution of sodium periodate (0.55 mL) dropwise while stirring to give periodate concentrations in the range of 0.26-260 mM. After 20 min, reaction mixtures were desalted into buffer A and reacted with AUPDP, and the number of pyridyldithio groups incorporated was determined as described above. (ii) Incorporation of DNP-AU-hydrazide. To NRG (25 mg, 1.47 mL) at 0 "C in 0.1 M sodium acetate buffer, pH 5.5, containing 0.15 M NaC1, was added sodium periodate (100 mg in 0.8 mL dHzO)with stirring. After 20 min in the dark at

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0 "C the solution was desalted into 3.2 mL of buffer A and 0.56 mL of DMF added followed by DNP-AUhydrazide (6.4 mg in 0.63 mL of DMF). After 16 h a t room temperature the solution was desalted into buffer A. The number of DNP-AU-hydrazide groups incorporated into the protein was determined using E = 17 000 M-' cm-' at 360 nm (18)and protein concentration was measured by the Lowry assay using NRG as the standard. Control reactions were carried out in which (i) a 100-fold molar excess of DNP-AU-OMe was added to oxidized NRG in buffer A, and (ii) a 100-fold molar excess of DNP-AU-hydrazide was added to nonoxidized NRG in Buffer A. After 4 h at room temperature the mixtures were desalted into buffer A and the incorporation of DNP groups/NRG determined. (iii) Stabilization of Hydrazone Bonds between IgG and DW-AU-hydrazide with Sodium Cyanoborohydride. NRG derivatized with DNP-AU-hydrazide in buffer A prepared as described above, at a concentration of 1.2 mg/mL, was incubated in the presence of 15 mM sodium cyanoborohydride or 15 mM sodium borohydride. After 48 h, the solutions were desalted into buffer A. A third sample was treated in the same way but received no reducing agent. After desalting, aliquots of cyanoborohydridetreated, borohydride-treated, and untreated protein were dialyzed against buffer A at 37 "C for 11 days. Periodic samples were taken, and the number of DNP groups per NRG was determined. A second group of aliquots of DNP-AU-hydrazide-derivatized NRG in buffer A were incubated with a 10 000-fold molar excess of propanal (80 mM), and after 48 h, the solutions were desalted into buffer A and the incorporation of DNP-AU-hydrazide in NRG was determined. To determine the effect of cyanoborohydride treatment prior to reaction with DNPAU-hydrazide, oxidized NRG at a concentration of 4.1 mg/ mL in buffer A was incubated with a molar excess of sodium cyanoborohydride ranging from 0 to 4000 (0-110 mM). The reaction mixtures were left at room temperature for 45 h after which time they were desalted into buffer A and reacted with DNP-AU-hydrazide, and the incorporation of DNP-AU-hydrazide into NRG was determined as described above. Coupling of SPDP and GMBS to IgG. mAb Dal K20 was derivatized with SPDP or GMBS by reaction of mAb K20 at 10 mg/mL in PBS with a 5-fold molar excess of either SPDP or GMBS dissolved in a small volume of DMF for 30 min. The solutions were then desalted into buffer B. Incorporation of pyridyldithio groups was determined as described above, and incorporation of maleimide groups was determined using N-(2,4-dinitrophenyl)-L-cysteine (17). The latter compound was prepared from N,N-bis(2,4-dinitrophenyl)-~-cystine by reduction with an excess of /3-mercaptoethanol. Labeling of mAb Dal B O with Biotin Hydrazide or Biotin Succinimidyl Ester. mAb Dal K20 was labeled with either sulfosuccinimidyl 6-(biotinamid0)hexanoate or biotin hydrazide. Non-site-specificmAb Dal JS2O-biotin was prepared by treating mAb Dal K20 (2.4 mg, 0.6 mL) in PBS with a 10-fold molar excess of sulfosuccinimidyl6-(biotinamido)hexanoate (90 pg in 25 pL of DMF) and desalting the solution into PBS after 1 h. For the preparation of site-specifically modified mAb Dal K20-biotin, oxidized mAb K20 in buffer A was reacted with a 100-fold molar excess of biotin hydrazide dissolved in a small amount of DMF. The solution was left for 4 h a t room temperature and then sodium cyanoborohydride added to a concentration of 13 mM and the solution desalted into PBS after 1 h. Digestion of Biotin-Labeled mAb Dal K20 with Pepsin. To mAb K20 (10 pL, 25 pg) in PBS modified

Kondejewski et al.

either site-specifically with biotin hydrazide or non-sitespecifically with sulfosuccinimidyl 6-(biotinamido)hexanoate was added 30 p L of 0.1 M sodium citrate buffer, pH 3.5, and 5 pL of pepsin (0.25 pg, 10 pglmg IgG) in citrate buffer. The digestion mixture was incubated at 37 "C for 30 min, at which time 4 pL of 2.0 M Tris/HCl, pH 9.0, was added, and aliquots mixed with equal volumes of SDS-PAGE sample buffer and samples were analyzed by SDS-PAGE. Localization of Sites of Biotin Reactivity by Western Blotting. Site-specific Dal K20-biotiq nonsite-specific Dal K20-biotin, and their pepsin digests prepared as above were separated on SDS-PAGE in duplicate. Gels were transferred to nitrocellulose and either stained for total protein using India ink or probed for biotin using Extravidin-peroxidase. The composition of the transfer buffer was 50 mM Tris, 380 mM glycine, 0.1% SDS, and 20%methanol, and the transfer conditions were 85 V (constant) for 1.5 h at 4 "C. For total protein staining, blots were washed in PBS containing 0.4% Tween 20 (PBS/Tween) with two changes of 5 min each, stained with 0.1% (v/v) India ink in PBSmween for 30 min, and destained in PBS. For biotin-specific staining, blots were rinsed with PBS, blocked with 2% BSA in PBS (PBSBSA) for 1 h, and incubated with Extravidinperoxidase diluted to 1pg/mL in PBS/BSA for 1h. Blots were washed with PBS/BSA and peroxidase detected with substrate solution consisting of 7 mg/mL of 4-chloro1-naphthol in PBS containing 20% methanol and 0.03% (v/v) HzOz. Activation of HSA for Reaction with mAb Dal BO-Spacer. 1. HSA-SPDP-SH. To HSA at 10 mg/ mL in PBS was added a &fold molar excess of SPDP with stirring at room temperature. The SPDP had been dissolved in an amount of DMF that did not exceed 20% of the volume of the HSA solution. After 30 min, the solution was desalted into 0.1 M acetate buffer, pH 4.5, containing 0.1 M NaC1, and DTT added to give a concentration of 10 mM. After 20 min at room temperature, the DTT-treated mixture was desalted into buffer B. The number of pyridyldithio groups incorporated into HSA was determined as described for IgG above with the exception that protein was measured by the Lowry assay. 2. HSA-SH. HSA in PBS at a concentration of 10 mg/ mL was reduced by the addition of a 20-fold molar excess of DTT (3 mM) for 20 min and desalted into buffer B. The number of thiol groups generated was determined with 5,5'-dithiobis(2-nitrobenzoicacid) (18). Conjugation of HSA to Spacer-ModifiedmAb Dal K20. For the preparation of site-specificconjugates, mAb Dal K20 in buffer B (2.0 mg/mL, 1.0 mL) derivatized with HPDP (3.8 HPDP/mAb Dal K20) or AUPDP (4.6 AUPDP/ mAb Dal K20) was mixed with HSA-SPDP-SH or HSASH in buffer B to give a 4: 1molar ratio of HSA over mAb Dal K20 in a final volume of 1.5 mL. For the preparation of non-site-specificconjugates, mAb Dal K20 (3.6 mg/mL, 3.0 mL) in buffer B derivatized with SPDP or GMBS (approximately 2.5 SPDP or GMBS/mAb Dal K20) was mixed with HSA-SPDP-SH in buffer B at a 4:l molar ratio of HSA over mAb Dal K20 in a final volume of 6.2 mL. All reaction mixtures were stirred briefly and left a t room temperature for 16 h a t which time thiols were blocked by the addition of a 20-fold molar excess of N-ethylmaleimide over mAb Dal K20. Some site-specific conjugates were prepared on a 20 times larger scale, desalted into buffer A, and treated with 15 mM sodium cyanoborohydride for 90 min to reduce hydrazone bonds. The reactions were monitored by both SDS and native PAGE under nonreducing conditions (7% gels). Purification of Dal K20-HSA Conjugates. Dal

Carbohydrate-Linked Antibody-Albumin Conjugates

K20-HSA conjugates were purified by gel filtration chromatography on Bio-Gel P 300 (2.5 cm x 90 cm) equilibrated with PBS using an upward flow system with a flow rate of 7.5 m u . Fractions containing purified 1:l HSA-Dal K20 conjugates were pooled, concentrated by precipitation with ammonium sulfate (60% saturation), desalted into PBS, and stored a t 4 "C. In certain experiments, Dal K20-HSA conjugates were isolated from native PAGE gels in which SDS was omitted. Conjugates were separated by native PAGE on 5%gels in a Bio Rad Mini-Protean I1 apparatus with gels of 0.75" thickness. Samples containing between 150 and 260 pg of mAb Dal K20 were mixed with an equal volume of sample buffer and loaded onto gels using a preparative comb. After electrophoresis, a narrow strip from the edge of the gel was cut out, stained, and used as a reference to cut out the remaining conjugatecontaining band from the gel. The electroelution apparatus consisted of two reservoirs connected by dialysis bags in each reservoir and a bridge between the two bags. The reservoir, dialysis bags, and bridge were filled with 50 mM TrisMC1, pH 8.6, with the gel-containing bag in the cathode compartment. Elution conditions were 100 V for 7 h, after which the collection bags were removed and eluted conjugates dialyzed against 50 mM NH4C03, lyophillized, and analyzed by SDS-PAGE. Immunoreactivity of mAb Dal K20 and Its Conjugates. lZ5I-mAbK20 (3.38 pCi/pg, 0.52 pg) in PBS containing 0.1% (w/v) BSA (PBSBSA) was mixed in duplicate with dilutions of either native mAb Dal K20 or conjugated mAb Dal K20 in a volume of 0.3 mL in PBS/ BSA. Caki-1 cells (8) were removed from flasks by treatment with 0.02% (w/v) EDTA in Hank's buffered saline solution, washed with PBS, and resuspended in PBSBSA and 5.0 x lo5 cells in 0.1 mL added per tube. Cells were incubated at 4 "C with occasional shaking and after 2 h were washed three times with PBSBSA and the tubes counted for radioactivity. The percent inhibition of lZ5I-DalK20 binding was determined using the formula [l - (cpm bound in presence of noniodinated mAb Dal K20/cpm bound in absence of noniodinated mAb Dal K20)llOO. Percent activity of mAb Dal K20 conjugates was determined using the formula (IC50 native mAb Dal K2O/IC50 conjugated mAb Dal K20)100, where IC50 is the concentration of noniodinated Dal K20 required to give 50% inhibition of iodinated Dal K20 binding. Other Methods. Concentrations of unconjugated proteins were determined using the Lowry assay with HSA and NRG as protein standards (19)or by absorbance at 280 nm using = 1.4 for 1.0 mg/mL of IgG. For purified 1:l HSA-Dal K20 conjugates, the protein concentration was determined by the Lowry assay using a 1:l molar mixture of IgG and HSA as the standard. mAb Dal K20 was radiolabeled with 1251using the chloramine-T method (20). SDS-PAGE was carried out according to the method of Laemmli (21). Samples were desalted using either disposable PDG columns or columns containing Bio-Gel P 10. RESULTS

Synthesis of HSA-Dal K20 Conjugates. Synthesis Site-Specific Heterobifunctional Cross-Linkers. The heterobifunctional spacer AUPDP was designed to contain an aldehyde-reactive hydrazide group and a thiolreactive pyridyl disulfide group. In our first attempt to synthesize AUPDP 2c, the methyl ester of ll-aminoundecanoic acid was reacted with SPDP la (Figure 1). Treatment of the resulting methyl ester of 2a with a 20fold excess of hydrazine led to release of pyridine-2thione. However, when SPDP was reacted with 11of

Bioconjugate Chem., Vol. 5, No. 6, 1994 605

la: R = 0 - N I

5

l b : R=NHNH2

2 28: R = OH

2b: R = 0 - N

3

2c : R = " H l

3a: R =WH3 3b:R="H2

4

Figure 1. Structures of the cross-linkers 3-(2-pyridyldithio)propionic acid succinimidyl ester (SPDP, la),3-(2-pyridyldithio)propionic acid hydrazide (HPDP, lb), 11-[[3-(2-pyridyldithio)propionyllaminolundecanoic acid hydrazide (AUPDP, 2c), the hydrazone stability probe 1l-[(2,4-dinitrophenyl)aminolundecanoic acid hydrazide (DNP-AU-hydrazide,3b),and y-maleimidobutyric acid sucinimidyl ester (GMBS, 4).

aminoundecanoic acid, the product 2a was obtained which was then converted to the succinimidyl ester 2b. Active ester 2b was then treated with hydrazine to smoothly give the hydrazide derivative 2c. A 1.5-2.0fold excess of hydrazine over the succinimidyl ester was important in preventing the formation of side products. A similar sensitivity toward hydrazine was observed when defining optimal conditions for SPDP conversion to HPDP. Introduction of Pyridyldithio Spacers into mAb Dal K20. mAb Dal K20 was oxidized with sodium periodate to render it reactive toward the hydrazide group of HPDP or AUPDP. IgG is typically oxidized with periodate using concentrations between 1 and 10 mM at pH 5.5 for 20 min at 0 "C for subsequent reaction with hydrazidecontaining spacers (12,22-24). However, we found that under these conditions few cross-linkers were incorporated into mAb Dal K20. We therefore investigated the effect of the NRG oxidation level on subsequent reactivity toward AUPDP. As shown in Figure 2, the use of 1 or

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606 Bioconjugate Chem., Vol. 5, No. 6,1994

"1

I

+mAbK20

+conjugate

+HSA 0e 0

, 50

I

I

100

150

200

I

I

250

300

sodium periodate concentration (mM) Figure 2. Effect of oxidation level on incorporation of AUPDP into NRG. NRG in 0.1 M sodium acetate, pH 5.5,0.15 M NaC1, was oxidized with an excess of sodium periodate indicated. After 20 min at 0 "C, samples were desalted into 0.1 M sodium acetate, pH 4.0, DMF added to a concentration of 15%, and a 50-fold molar excess of AUPDP dissolved in DMF added to give a final DMF concentration of 25%. Samples were desalted into 0.1 M sodium acetate, pH 4.0, after 3 h and the incorporation of AUPDP into NRG determined as described in the Materials and Methods.

10 mM periodate resulted in the incorporation of 0.6 or 1.4 AUPDPDgG, respectively. By increasing the excess of periodate over IgG, we were able to incorporate up to five spacers, i.e., when IgG was oxidized with 260 mM periodate. Approximately four pyridyldithio groups were routinely introduced into NRG or mAb Dal K20 using a periodate concentration of 100-150 mM. An average of three to four cross-linkers per IgG was found to be best for the subsequent reaction with thiol-containing HSA. It was also necessary to include DMF at a concentration of 25%during coupling to prevent cross-linker precipitation. Neither oxidation with periodate nor incubation in 25% DMF impaired antibody-binding activity. However, the incorporation of four AUPDP/mAb Dal K20 did result in a loss of 35% activity. For the synthesis of non-site-specificconjugates, mAb Dal K20 was derivatized with the heterobifunctional cross-linkers SPDP (la)or GMBS (4). Typically, a &fold molar excess of SPDP or GMBS over IgG resulted in the incorporation of 2.5 cross-linkers/&$. mAb Dal K20 was found to be sensitive to amino group modification as the incorporation of 2.5 SPDP/mAb Dal K20 resulted in the loss of 60% binding activity. The incorporation of GMBS into DalK20 resulted in comparable loss of binding activity. Conjugation of HSA with mAb Dal D O . In order to compare the two site-specific cross-linkers, we examined their ability to form conjugates between HSA and mAb Dal K20. mAb Dal K20 which had been derivatized with either HPDP (3.8 HPDP/Dal K20) or AUPDP (4.6 AUPDP/ Dal K20) was reacted with either HSA-SH which was obtained by reduction of HSA directly with DTT (2 thiols/ HSA) or with HSA-SPDP-SH, obtained from HSA which had been modified with SPDP and then reduced with DTT (2.5 thiols/HSA). The HSA-SPDP-SH gives an increase in the total spacer length between Dal K20 and HSA. Conjugate formation was monitored by native PAGE, and the yield of the conjugate with HSA-SH was found to be much greater with AUPDP-derivatized DalK20 than with HPDP-derivatized DalK20 (lanes 6 and 4, respectively, Figure 3). Irrespective of whether HSA-SPDP or DTT-reduced HSA was used for conjugation, Dal K20-AUPDP gave a higher yield of the conju-

1

2

3

4

5

6

Figure 3. Analysis of site-specific conjugation yields by native PAGE. HSA or SPDP-HSA were reduced with DTT and reacted with mAb K20 derivatized with AUPDP or HPDP as described in the text. Following conjugation, an aliquot of each reaction mixture was subjected to native PAGE; lane 1, HPDP-Dal K20; lane 2, AUPDP-Dal K20; lane 3, HPDP-Dal K20 HSASPDP-SH; lane 4, HPDP-Dal K20 HSA-SH; lane 5, AUPDPDal K20 HSA-SPDP-SH; lane 6, AUPDP-Dal K20 HSASH.

+ +

+

+

+conjugate +mAb

K20

4- HSA

1

2

3

4

5

6

7

Figure 4. SDS-PAGE analysis of conjugates eluted from native PAGE gels. HSA-Dal K20 site-specific conjugates were prepared with either HPDP or AUPDP and non-site-specific conjugates with SPDP or GMBS as described in the Materials and Methods. Each conjugation mixture was subjected to preparative native PAGE, and conjugates were eluted from the gels and analyzed by SDS PAGE: lanes 1,4,and 7, mAb Dal K20; lane 2, AUPDP conjugate; lane 3, HPDP conjugate; lane 5, SPDP conjugate; lane 6, GMBS conjugate. gate than did Dal K20-HPDP (lanes 5 and 6 vs lanes 3 and 4). These results suggest that the ability to link HSA to the carbohydrate of mAb Dal K20 is dependent on the length of spacer or spacers between the two proteins (Figure 3). Non-site-specific Dal K20-HSA conjugates synthesized using SPDP were isolated in pure form by gel filtration with no contamination by free HSA or mAb Dal K20. In contrast, gel filtration of site-specificconjugates synthesized using HPDP or AUPDP showed free HSA and mAb Dal K20 at the elution position for the conjugate, indicating partial breakdown of these conjugates. Figure 4 shows an experiment in which the site-specificconjugates were purified from reaction mixtures by native PAGE and bands corresponding to Dal K20-HSA conjugates eluted from the gels. When material from these bands was subjected to SDS-PAGE, there were substantial amounts of material that migrated to positions expected for free mAb Dal K20 and HSA (Figure 4, lanes 2 and 3), indicating instability of the hydrazone bond in the sitespecific conjugate. In contrast, non-site-specific conjugates of HSA and Dal K20 prepared using SPDP or GMBS as the bifunctional linking reagent showed very little of the unbound components (lanes 5 and 6).

Bioconjugale Chem., Vol. 5, No. 6,1994 607

Carbohydrate-Linked Antibody-Albumin Conjugates Table 1. Reduction of IgG Aldehydes by Sodium Csanoborohsdridea

molar molar excess of DNP groups excess of DNP groups NaCNBH3 (mol DNP/mol IgG) NaCNBH3 (mol DNP/mol IgG) 0 6.2 2000 0.8 500 1.0 4000 1.2 1000 1.0 a NRG was oxidized with sodium periodate and treated with a molar excess of sodium cyanoborohydride (0-110 mM) for 48 h a t pH 4.0. Samples were desalted into 0.1 M sodium acetate, pH 4.0, and reacted with a 100-fold molar excess of DNP-AUhydrazide for 4 h. Unreacted DNP-AU-hydrazide was removed by gel filtration and the incorporation of DNP groups into IgG determined as described in the Materials and Methods.

Stabilization and Isolation of Conjugates. Model Stabilization Studies with DNP-AU-hydrazide. King et al. (25)demonstrated that hydrazone bonds in conjugates could be stabilized by cyanoborohydride reduction. To assist in defining conditions for stabilization, a chromophoric DNP-hydrazide was synthesized (3b,Figure 1). DNP-AU-hydrazide was expected to display similar reactivity toward oxidized IgG as AUPDP because both compounds possess an identical undecanoic acid hydrazide moiety. Retention of DNP groups in the protein was used to monitor stability of the hydrazone bonds instead of monitoring the pyridyldithio groups donated by AUPDP because the latter were susceptible to disulfide reduction by cyanoborohydride. DNP-AU-hydrazide was incorporated into NRG following the oxidation. Control reactions in which oxidized NRG was treated with DNP-AU-OMe or where nonoxidized NRG was treated with DNP-AU-hydrazide showed no incorporation of DNP into NRG, indicating the specificity for reaction between oxidized NRG and hydrazide. We first examined whether we could prevent incorporation of DNP-AU-hydrazide into oxidized IgG by reduction of IgG aldehydes with cyanoborohydride prior to reaction with the DNP-probe. Table 1 shows that IgG which had been oxidized and reacted with DNP-AUhydrazide incorporated 6.2 DNP groups/IgG. IgG which was treated with an excess of cyanoborohydride prior to reaction with probe showed that approximately 1 DNP group/IgG was introduced, demonstrating that the majority of aldehydes generated by periodate oxidation of IgG carbohydrate were also sterically accessible to reduction by cyanoborohydride. In order to evaluate the stability of the hydrazone bonds formed between IgG aldehydes and the hydrazide 3b, DNP-IgG was dialyzed at 37 "C against 0.1 M acetate buffer, pH 4.0. Figure 5 shows that approximately 60% of probe in both untreated and borohydride-treated DNPIgG was lost after 11days. In contrast, DNP-IgG which had been treated with cyanoborohydride showed retention of 80% of the DNP moiety after the same period of time, indicating that a proportion of the hydrazone bonds had been stabilized by cyanoborohydride. To further confirm the stabilization of hydrazone bonds by cyanoborohydride reduction, we treated DNP-IgG with an excess of propanal. As shown in Table 2, IgG which was initially derivatized with 7.9 DNP groups retained only 1.8 DNP/IgG after treatment with propanal for 48 h. DNP-IgG which had been treated with cyanoborohydride prior t o aldehyde treatment retained substantially more DNP residues, Le., 4.5 DNPAgG. Data obtained from this competition system suggested that treatment of DNP-IgG with 15 mM cyanoborohydride at pH 4.0 for 1.5 h is sufficient to reduce the majority of hydrazone bonds.

2ol O

0

2

4

8

6

10

l

dialysis time (days)

Figure 5. Stabilization of hydrazone-linked DNP reporter groups by sodium cyanoborohydride. DNP-NRG was treated with 15 mM sodium cyanoborohydride (open circles), 15 mM sodium borohydride (open squares), or not treated (filled circles) as described in the Materials and Methods. Samples were dialyzed against 0.1 M sodium acetate, pH 4.0, at 37 "C,aliquots were taken at the times indicated, and the number of DNP groups bound to NRG was determined. Table 2. Competition of Hydrazone-Linked DNP ReDorter GrouDs with an Excess of ProDanal" ~

~

treatment of DNP-IgG start after NaCNBH3 control NaCNBH3 aldehyde control aldehyde

+

+

~~

~~

DNP groups (mol DNP/mol IgG) 7.9 5.1 7.0 4.5 1.8

~

~

% DNP

remaining 100

65 89 57 23

a Periodate oxidized NRG in 0.1 M sodium acetate, pH 4.0, was reacted with a 100-fold molar exess of DNP-AU-hydrazide for 4 h and desalted into the same buffer. Samples were either treated with 15 mM sodium cyanoborohydride (NaCNBH3) or received no treatment (control). After 48 h, samples were desalted into 0.1 M sodium acetate, pH 4.0, and treated with a 10 000-fold molar excess of propanal (80 mM) for 48 h at room temperature, desalted, and the number of DNF' reporter groups bound to NRG determined as described in the Materials and Methods.

Stabilization and Isolation of Carbohydrate-Linked HSA-Dal K20 Conjugates. Conjugates between mAb Dal K20 and HSA were synthesized as shown in Scheme 1. AUPDP was incorporated into mAb Dal K20 by reaction with oxidized carbohydrate residues, and AUPDPderivatized mAb K20 reacted with HSA-SPDP-SH. Following conjugation, reaction mixtures were treated with cyanoborohydride under the conditions determined with the DNP-probe in order to stabilize hydrazone bonds, and the conjugate was purified from the reaction mixture by gel filtration chromatography. As shown in Figure 6A (lane 4), the carbohydrate-linked conjugate could now be isolated without contamination by free HSA or mAb K20, indicating that the hydrazone bonds in the conjugate had also been reduced by cyanoborohydride. The yield of mAb K20 in conjugate form was on average 15%. A non-sitespecific mAb K20-HSA conjugate prepared by reaction of SPDP-derivatized mAb K20 with HSA-SPDP-SH was also synthesized and purified for subsequent comparison with the site-specific conjugate (lane 3, Figure 6A). Recovery of mAb K20 in the non-site-specific conjugate was comparable to that of the site-specificconjugate, with 20% average recovery. These numbers represent yield of the two conjugates from the clean fractions only and do not include conjugates in unresolved fractions which were not rechromatographed. Rechromatography is likely to improve the yield.

Kondejewski et al.

608 Bioconjugafe Chem., Vol. 5, No. 6,1994

Scheme 1. Synthesis of Carbohydrate-Linked mAb K20-HSA Conjugates

A.

+-conjugate 4- mAb K20

SPDP

1 +HSA

4

AUPDP

1

2

3

4

5

B. 4- HSA

I

NaCNBH3

"r Characterization of HSA-Dal K20 Conjugates. The conjugates shown in Figure 6A were analyzed by SDS-PAGE under reducing conditions. It can be seen that they were reduced to the components expected from disulfide-linked conjugates (Figure 6B, lanes 3 and 4), i.e., both contain IgG heavy and light chains as well as HSA. The migration position for 1:l HSA-IgG conjugates was confirmed by a dual labeling experiment in which HSA was labeled with 1251 and an NRG preparation with 1311. Conjugation was carried out by incorporating GMBS into the IgG followed by reaction with directly reduced HSA-MTX as described in the Materials and Methods. The conjugate preparation was subjected to SDS-PAGE and stained with Coomassie Blue. The band migrating directly above IgG itself contained 1251 and 1311in a ratio of 0.88 f 0.10. To further investigate the site-specific nature of the linkage, we designed an experiment in which a low molecular weight probe, biotin hydrazide, was reacted with periodate-treated mAb Dal K20. We also reacted a sample of mAb Dal K20 non-site-specifically with a succinimidyl ester derivative of biotin, and each conjugate was digested with pepsin. Analysis under nonreducing conditions (Figure 7A) showed that the samples were completely digested and the labels were incorporated into both samples; however, only the non-site-specifically labeled mAb Dal K20 (lane 6) contained the biotin label in the F(ab')2 portion (lanes 5 and 6). The cleavage site for pepsin is below the interchain disulfide bonds joining the two heavy chains (26)and gives rise to F(ab')2 and a number of small fragments since the Fc portion is degraded by pepsin. The lack of staining of the sitespecific F(ab')Z is consistent with labeling of the carbohydrate moiety, the location of which is known to be below the pepsin cleavage site (27). Analysis under

1

2

3

4

5

4-

heavy chain

4-

light chain

6

Figure 6. SDS-PAGE comparison of purified HSA-Dal K20 site-specific and non-site-specific conjugates. HSA-Dal K20 conjugates were synthesized using the non-site-specific crosslinker SPDP or with the site-specific cross-linker AUPDP as described in the Materials and Methods and the 1:l HSAmAb Dal K20 conjugates purified by gel filtration chromatography. Purified conjugates were analyzed by SDS-PAGE under nonreducing (A) or reducing (B) conditions. A lanes 1and 5, mAb Dal K20; lane 2, HSA, lane 3, purified non-site-specific HSADal K20 conjugate; lane 4,purified site-specificHSA-Dal K20 conjugate. B: lanes 1and 5, HSA, lanes 2 and 6, mAb Dal K20; lane 3, purified site-specific HSA-Dal K20 conjugate; lane 4, purified non-site-specificHSA-Dal K20 conjugate. reducing conditions showed that the label was present on both heavy and light chains in the non-site-specifically modified sample, this being consistent with a random modification of amino groups by succinimidyl esters. Selective heavy chain reactivity was exhibited by biotin hydrazide, again indicating selective carbohydrate labelling by hydrazide compounds (Figure 7B). The retention of antigen-binding activity in the two types of HSA-Dal K20 conjugates was assayed as shown in Figure 8. The site-specific HSA-Dal K20 conjugate and the non-site-specific HSA-Dal K20 conjugate retained 30%and 10% activity, respectively, of the parent mAb. DISCUSSION Our results provide a method for the synthesis of structurally well-defined conjugates of antibodies with other macromolecules. We used HSA as a prototype multivalent carrier that may furnish the basis for production of active drug-HSA-mAb ternary conjugates. Although there are reports of site-specific conjugation of various unloaded (e.g., poly-g-glutamyl hydrazide (30)) or drug-loaded macromolecules (e.g., methotrexate-HSA (4),bleomycin-dextran (31),methotrexate-dextran (32)) to IgG, a successful site-specific linkage of HSA to IgG has not been reported. An advantage of HSA is that its discrete molecular weight helped significantly in the key purification step, i.e., fractionation of the reaction mix-

Carbohydrate-Linked Antibody-Albumin Conjugates

Bioconjugate Chem., Vol. 5, No. 6, 1994 609

A.

pepsin

------

pepsin

-----4- mAbK20

4- F(ab’),

perception was that insufficiency in the length of that spacer was responsible, and indeed, its replacement with AUPDP, carrying an extra 12 atoms of chain length, led to much better conversions. These findings can be attributed to steric restriction arising from the attachment of the carbohydrate at conserved Asn 297 residues of both heavy chains (28), lying between the two C H ~ domains of IgG (33,34). A similar dependence on chain length has been observed in conjugating toxins to IgM (12).

S

n s n

S

n

s n

B.

4- heavy chain

+ light chain n s Figure 7. Localization of site-specific and non-site-specific biotin labels on mAb Dal K20. mAb Dal K20 was labeled sitespecificallywith biotin hydrazide (s) or non-site-specificallywith sulfosuccinimidyl 6-(biotinamido)hexanoate (n), digested with pepsin, and separated by SDS PAGE under nonreducing (7% gels, A) or under reducing conditions (12%gels, B). Gels were run in duplicate,transferred to nitrocellulose, and either stained for total protein with India ink (left panels) or for biotin with Extravidin-peroxidase (right panels). For the lane number in the text the lanes are counted from left to right.

1

10

concentration of mAb Dal K20 (pg/mL) Figure 8. Retention of antigen binding activity in HSA-Dal K20 conjugates. 1251-mAbDal K20 was mixed with native mAb Dal K20 (open squares), site-specific HSA-Dal K20 conjugate (filled circles),or non-site-specificHSA-Dal K20 conjugate (open circles) and the inhibition of 1251-mAbK20 binding to Caki-1 cells determined as described in the Materials and Methods. Each value is the mean and range from duplicate samples.

ture on Bio-Gel P-300, leading to the isolation of the 1:l HSA’K20 conjugate. Our method of conjugation is based on hydrazone bond formation between the oxidized carbohydrate moiety of IgG and HSA via a tandem of two heterobifunctional cross-linking spacers (AUPDP and SPDP). HPDP was selected originally as the site-specific linker bearing the hydrazide group, but the yield of conjugate was low. Our

To prevept denaturation of IgG but at the same time promote the coupling reaction, it was important to restrict the incorporation ratio of AUPDP to a relatively narrow range, established in preliminary tests. Parallel considerations applied to introduction of the thiol into HSA. Defining these incorporation ratios served to favor the production of 1:l conjugates over conjugates with multiple IgG or HSA units, thus making it possible to carry out purification by gel filtration. Synthesizing 1:1conjugates by these spacer-containing crosslinkers entailed limiting the number of hydrazone bonds between HSA and IgG. A potential consequence of this limitation is that the conjugate will be less stable than one linked by multiple hydrazones, and such instability was observed in our site-specific conjugates a t the stage of hydrazone bond formation. However, cyanoborohydride reduction resulted in the stabilization of a significant proportion of hydrazone bonds, and this made it possible to isolate the conjugates. It is interesting that, in the stability evaluation experiment in which DNP-AU-hydrazide was used instead of AUPDP to act as a probe, we were unable to remove all DNP reporter groups either by dialysis or by competition with propanal. This indicates that a fraction of the hydrazone bonds formed between oxidized IgG and DNP-AU-hydrazide were stable without reduction. Formation of stable hydrazone bonds under comparable conditions has also been observed by others (2,24) and could be attributed to the microenvironment of individual bonds. The disulfide bond between HSA and mAb may not be adequately stable in vivo because disulfide bonds are subject to reduction by various serum and tissue components, e.g., glutathione. A thioether bond between HSA and mAb may be more appropriate for the construction of drug-HSA-mAb ternary conjugates. One of the ways to introduce a thioether linkage would be to replace SPDP, the agent modiwng 11-aminoundecanoicacid, by a maleiimido group-containing spacer, e.g., by succinimidyl y-maleiimidobutyrate. On the other hand a number of disulfide-linked immunotoxins have been found to be several times more potent then their thioether-linked counterparts (28) because dissociation of the toxin is essential for its translocation to intracytoplasmic target sites. If necessary, the disulfide bond in site-specific immunotoxins produced by our method can be rendered more stable by the introduction of “hindered disulfide bonds” using crosslinking agents such as 4-[(succinimidyloxy)carbonyl]-a-methyl-a-(2-pyridyldithio)toluene (29) or derivatives of iminothiolane in which methyl groups are incorporated on the carbon atom adjacent to the disulfide bond (28). The isolation of well-defined 1:lHSA-K20 conjugates allowed comparison of the immunoreactivity of the sitespecific and non-site-specific forms that were free of unreacted mAb and other high molecular weight conjugates. The site-specific HSA-K20 conjugate retained three times the antibody activity compared to corresponding non-site-specificconjugates, showing that the greater retention of activity was due to specific attachment of HSA to the carbohydrate of mAb K20. Non-site-

610 Bioconjugate Chem., Vol. 5, No. 6, 1994

specific HSA-mAb conjugates obtained by others ( 4 , 6 ) have been reported to retain 30% immunoreactivity but the preparations tested might have contained unreacted mAb which would be predicted to increase their apparent immunoreactivity. In our study, site-specific derivatization of mAb Dal K20 with AUPDP led to 35%loss of its antibody activity. In synthesizing non-site-specific counterparts, incorporation of 2.5 thiol groups into mAb Dal K20 using SPDP led to 60% loss. The further losses in antibody activity in both the site-specific and non-site-specific forms were most likely due to steric factors imposed by the relatively large HSA molecule. The presence of HSA linked to the C H domain ~ of mAb K20 may sterically interfere with the flexibility of the antigen binding sites and antigenbinding capacity (35, 36). Increasing the chain length in the linking spacer improved conjugation, but further studies will be required to define the optimal length. Also, other factors, e.g., lipophilicity, could have played a significant role, influencing steric orientation of the spacer and therefore its ability to react with thiols introduced into HSA with SPDP. A problem encountered with AUPDP was its limited solubility. This could have led to the tendency toward precipitation observed after the incorporation of AUPDP in IgG (Scheme 1) and therefore contribute to low yield of the conjugate. A more appropriate crosslinker of the required length may be one which is derived from an oligopeptide into which hydrophilic groups are introduced to confer increased solubility. This study has shown that site-specific synthesis in conjugation with reductive stabilization can produce a stable, well-defined HSA-mAb conjugate with greater retention of antibody activity than that synthesized nonsite-specifically. This site-specific approach utilizing carbohydrate residues in the immunoglobulin should be applicable to conjugation of other proteins including toxins and enzymes. LITERATURE CITED (1) Kulkarni, P. N., Blair, A. H., and Ghose, T. (1981) Covalent binding of methotrexate to immunoglobulins and the effect of antibody-linked drug on tumor growth in vivo. Cancer Res. 41, 2700-2706. (2) Greenfield, R. S., Kaneko, T., Daues, A., Edson, M. A., Fitzgerald, D. A., Olech, L. J., Grattan, J . A., Spitalny, G. L., and Braslawsky, G. R. (1990) Evaluation in vitro of adriamycin immunoconjugates synthesized using an acid-sensitive hydrazone linker. Cancer Res. 50, 6600-6607. (3) Ghose, T., and Blair, A. H. (1987) The design of cytotoxicagent-antibody conjugates. CRC Crit. Rev. Ther. Drug Carrier Syst. 3 , 263-359. (4) Endo, N., Kato, Y., Takeda, Y., Saito, M., Umemoto, N., Kishida, K., and Hara, T. (1987) I n vitro cytotoxicity of a human serum albumin-mediated conjugate of methotrexate with anti-MM46 monoclonal antibody. Cancer Res. 47,10761080. (5) Garnett, M. C., Embleton, M. J., Jacobs, E., and Baldwin, R. W. (1983) Preparation and properties of a drug-carrierantibody conjugate showing selective antibody-directed cytotoxicity in vitro. Znt. J . Cancer 31, 661-670. (6) Garnett, M. C., and Baldwin, R. W. (1986) An improved synthesis of a methotrexate-albumin 79IT/36 monoclonal antibody conjugate cytotoxic to human osteogenic sarcoma cell lines. Cancer Res. 46, 2407-2421. (7) Amon, R., and Sela, M. (1982) I n vitro and in vivo efficacy of conjugates of daunomycin with anti-tumor antibodies. Zmmunol. Rev. 62, 5-27. ( 8 ) Kralovec, J., Singh, M., Mammen, M., Blair, A. H., and Ghose, T. (1989) Synthesis of site-specific methotrexate-IgG

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