Bioconjugafe Chem. 1990, 1, 212-221
212
Preparation and Characterization of Immunoconjugates for Antibody -Target ed Photolysis Scott L. Rakestraw,+.$Ronald G. Tompkins,+ and Martin L. Yarmush*,+,§ Department of Chemical and Biochemical Engineering and the Center for Advanced Biotechnology and Medicine, Rutgers University, Piscataway, New Jersey 08855, and Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114. Received January 17, 1990 Monoclonal antibody (MAb)-dextran-tin(1V) chlorin e6 (SnCe6) immunoconjugates were prepared by a new technique involving the use of reducing, terminal-modified dextran carriers and site-specific modification of the Fc oligosaccharide moiety on the antibodies. Dextran carriers were synthesized to increase the number of SnCe6 molecules attached to a MAb. The dextran carriers were coupled to the MAb via a single, chain-terminal hydrazide group to prevent aggregation of MAbs. Conjugates were prepared with antimelanoma MAb 2.1 containing up to 18.9 SnCe6 molecules per MAb. Under neutral conditions, no hydrolysis of the hydrazone bond between the MAb and the dextran carrier could be detected, and the hydrazone was not stabilized by reduction with NaCNBH3 or NaBH4. Analysis of the purified immunoconjugates showed that approximately two dextran carrier chains were attached to a MAb regardless of the number of SnCe6 molecules linked to a dextran carrier. Site-specific covalent attachment of the SnCe6-dextran chains to the MAb was confirmed by SDSPAGE. HPLC analysis of the conjugates gave a single species eluting in the range of 200-240 kDa. As determined by a competitive inhibition radioimmunoassay using viable SK-MEL-2 human malignant melanoma cells, the conjugates showed excellent retention of antigen-binding activity relative to unconjugated MAb. INTRODUCTION Methods for coupling active agents to antibodies can be grouped into two categories: (1) those which attach the agent directly to the antibody, and (2) those which rely on an intermediary for attachment. To date, most of the direct coupling procedures involve modification of amino acid side chains (1,2). These approaches generally lack specificity of the coupling reaction in that the active agent cannot be directed to a particular site on the antibody. T o exert control in this regard, direct methods which target the Fc oligosaccharide moiety of the antibody for coupling have been developed ( 3 ) . While site-directed coupling to the heavy-chain carbohydrate has served to produce more well-defined immunoconjugates (4-61, the ratio of active agent to antibody is limited by the amount of periodate-oxidizable carbohydrate expressed by the immunoglobulin. T o increase the number of active agents coupled to each molecule of antibody, indirect attachment approaches have been developed. Most reported methods have linked the active agent of interest to a carrier biopolymer, followed by attachment of the resulting macromolecule to the immunoglobulin in a manner which does not offer site-directed control. Immunoconjugates prepared with carrier polymers such as poly amino acids (71, serum albumins ( 8 ) , and dextran derivatives (9, I O ) have been described. Recently, methods which attempted to combine the indirect carrier approach with site-directed control of the coupling reaction between carrier and antibody have been reported ( I I , I 2 ) . Attachment of a agent-
* Address correspondence
and reprint requests to Dr. Martin L. Yarmush, Rutgers University, P.O. Box 909, Piscataway, N J 08854. +
Harvard Medical School.
* Present address: E. I. du Pont de Nemours, Experimental
Station, Wilmington, DE 19880-0304. 5 Rutgers University.
1043-1802/90/2901-0212$02.50/0
substituted dextran to the antibody was accomplished via Schiff base formation between t h e periodateoxidized Fc oligosaccharide and an excess of free amino groups on the carrier dextran. No attempt, however, was made to limit the number of free amino groups on the carrier, raising the possibility of carrier-mediated crosslinking of antibodies to form high molecular weight aggregates. In addition, conjugation via Schiff base formation requires stabilization of the bond with reducing agents such as NaCNBH3 or NaBH4. Reductive stabilization of Schiff base bonds has been reported to often result in undesirable modification of some amino acids in the immunoglobulin ( 3 ) . The present work was undertaken to synthesize a monoclonal antibody (MAb)l immunoconjugate which (1)possessed a favorable active agent to antibody ratio by using the carrier approach, (2) demonstrated a site-directed coupling of the carrier by a single, chain-terminal group to the Fc carbohydrate moiety of the immunoglobulin, and (3) could be reproducibly prepared with retention of both active agent and antibody activity. By limiting the linkage of the antibody and carrier to a single area in each molecule, the possibility of carrier-mediated aggregate formation is reduced. EXPERIMENTAL PROCEDURES Chemicals. Unless noted otherwise, all organic chemicals were obtained from Aldrich (Milwaukee) and were used without further purification. Chlorin e6 was obtained Abbreviations: MAb, monoclonal antibody; SnCe6, tin(1V) chlorin e6; DCC, dicyclohexylcarbodiimide; HOBt, N-hydroxybenzotriazole; EDAC, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; Trt, triphenylmethyl (trityl) group, DPBS, DulbecCO’S phosphate-buffered saline; DMSO, dimethyl sulfoxide;DMF, dimethylformamide; BSA, bovine serum albumin; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. C 1990 American Chemical Society
Bioconjugafe Chem., Vol. 1, No. 3, 1990
Antibody-Targeted Photolysis
213
Scheme I. Synthetic Scheme for the Preparation of the Dextran Carrier Containing SnCe6 and a Chain-Terminal Hydrazide Group for Attachment to the MAba
-co7
1
(V)
Excess Adipic Dihydrazide NaCNElH,
DMSO
- h W i H - & d
(I)
H
O
W
0
bH
1
-Lo,
(VI111
OH
--
0
6H
I (XI)
--
DMSO Excess Acetaldehyde
-To7
I
1
5% Tifluomacetic acid
OH
R = SnCe6 or NHNH,
(XIV)
Roman numerals refer to the structures of comDounds described in this paper and shown in the schemes. Trt designates the triphenylmethyl (trityl) protection group. 0
from Porphyrin Products (Logan, UT). The organic solvents were purchased from EM Science (Omnisolv grade) and were stored over molecular sieves to remove water. MAb Purification. An antimelanoma MAb 2.1 (a kind gift of R. E. Saxton, UCLA Medical Center) was purified from ascites by affinity chromatography on a protein G column (13). A purified antilymphoma MAb 2.130 was obtained from Damon Biotechnology (Needham, MA). The purity of all of the IgG antibodies was checked by HPLC on a Du Pont Zorbax GF-250 column eluted with 0.20 M phosphate, pH 7.2, and by both SDS-PAGE and isoelectric focusing electrophoresis on a PHAST system (Pharmacia, Piscataway, NJ). I. Synthesis of the Dextran Carrier. A dextran carrier was synthesized to attach the photosensitizer tin(1V) chlorin e6 (SnCe6) to the MAbs. The primary aim of the synthesis was to generate a single group on the dextran carrier for attachment to the Fc carbohydride moiety of the MAb. A chain-terminal hydrazide group was inserted at the reducing terminus of the dextran carrier and then protected by a triphenylmethyl (Trt) group while subsequent chemistry was performed on the carrier. After coupling of multiple SnCe6 molecules and capping of excess SnCe6 linkage moieties on the carrier chain, the Trt group was removed to expose the terminal hydrazide group. The deprotected carrier was then attached to the periodateoxidized oligosaccharide group on the MAb via formation of an intermolecular hydrazone bond. The synthetic schemes used in the present work are outlined in Schemes 1-111. Roman numerals correspond to the com-
pounds whose structures are illustrated in these schemes. ( A ) Attachment of a Terminal Group onto Dextran uia Reductiue Amination. Dextran T40 (Pharmacia, 50 g, -1.43 mmol, M , = 35 kDa) and adipic acid dihydrazide (41.1 g, 236 mmol) were dissolved in 0.2 M acetate buffer pH 4.75 (500 mL). The resulting solution was heated to 40 " C at which point NaCNBH3 (12.5 g, 200 mmol) was added. The reaction mixture was stirred at 40 "C for 48 h with maintenance of the pH a t 4.75 f 0.2 by addition of concentrated HCl as needed. Hydrazinodextran I1 was precipitated by portionwise addition of the reaction mixture to 4 L of vigorously stirred methanol. The crude precipitate was dissolved in 300 mL of deionized water and extensively dialyzed (tubing MWCO 12-14 kDa) against several changes of deionized water for 2 days. Lyophilization of the dialysate yielded the product as a flaky, white powder (46.2 g, 92.4% yield). The extent of reducing-end modification was estimated by dinitrosalicylate titration (14) versus standards of the unmodified starting material dextran T40. ( B ) Preparation of a n N-Trityl Active Ester (V)f o r Protection of the Terminal Hydrazide i n Hydrazinodextran I I . Adipic acid monethyl ester was converted into the sodium salt of its monohydrazide by using the method of Hurd and co-workers (15). Briefly, monoethyl adipate (15 g, 86.1 mmol) was neutralized with 1 equiv of sodium ethoxide in ethanol. The salt was recovered by rotary vacuum evaporation followed by trituration of the residue with a mixture of ether and ethanol (yield 91%,). Sodium ethyl adipate (10 g, 51 mmol) was
Rakestraw et al. Scheme 111. Synthetic Scheme for the Preparation of the in Situ Active Ester of SnCe6 Used To Couple the Photosensitizer to Carbazate Groups on the Dextran Carrier (Scheme 1)a CH 5 CH2 H3C H
\ C NHH
N 2L
C
H
3
NaOEt
(IX)
ElOH
COOH
N2H4
1. MqSiCI, Et3N
I
SnCI, Acetic Acid 1% Sodium Acetate
I
H3C
CH2CH3 \
.'
(XI
COOH N-Hydrox yphthalimide DMFAcetonitrile
DCC
a:- 0
I
HOBt EDAC
H \ ,3 c CH2CH3 m
0
a Roman numerals refer to the structures of compounds described in this paper and shown in the schemes.
refluxed in hydrazine hydrate (85% ' ) for 1 h, poured into ethanol/ether (l:l), and evaporated to dryness in vacuo. The residue was treated with ethanol and vacuum evaporated until the odor of hydrazine could no longer be detected. The residue (111) was triturated with a mixture of ethanol and ether and collected (8.56 g, 92 %, mp 149-151 "C dec). Tritylhydrazo diformate IV was prepared by using a previously reported procedure with appropriate modifications (16). T o a suspension of finely ground I11 (1.82 g, 10 mmol) in 20 mL of chloroform/acetonitrile (5:l) was added trimethylsilyl chloride (1.27 mL, 10 mmol). The mixture was refluxed for 2 h and cooled to room temperature, and triethylamine (2.79 mL, 20 mmol) and a solution of trityl chloride (2.79 g, 10 mmol) in 20 mL of chloroform were sequentially added. After stirring for 1 h, methanol ( 5 mL) was added with an additional 15 min of stirring to effect the hydrolysis of the trimethylsilyl ester. The light yellow solution was poured into ether and quickly washed with chilled 5 % citric acid. The aqueous phase was discarded, and the organic phase was extracted twice with 1 N NaOH (50 mL). The combined alkaline extracts were cooled overnight a t 4 "C and shaken with fresh ether (50 mL). Upon cooling of the separated aqueous layer to 0 "C and neutralization with glacial acetic acid, a dense white precipitate was formed.
V N - O A c ) NSN
The axial ligands associated with the central Sn ion have been omitted for clarity. The axial ligands are chloride anions for the anhydrous preparation of X, but are thought to be (OH-)* when SnCe6 is dissolved in water. Roman numerals refer to the structures of compounds described in this paper and shown in the schemes. a
The solid was resuspended in ethyl acetate (2 X 150 mL), washed with water, and dried over anhydrous MgS04. Rotary evaporation of the solvent yielded a gummy residue, which was dissolved in T H F and slowly poured into rapidly stirred hexane to yield 1.75 g (44%) of a white solid (IV), which was used without further characterization. T o a solution of IV (1.75 g, 4.35 mmol) in dry T H F (100 mL) was added N-hydroxyphthalimide (0.71 g, 4.36 mmol) followed by a solution of dicyclohexylcarbodiimide (896 mg, 4.35 mmol) in 20 mL of THF. The reaction contents were stirred for 20 h a t room temperature, and the urea was removed by filtration. The filter cake was washed extensively with THF. Evaporation in vacuo of the combined filtrate and washes left a residue which was immediately taken up in chloroform (150 mL) and washed sequentially with saturated sodium bicarbonate
Antibody-Targeted Photolysis
(2 x 100 mL), saturated NaCl (100 mL), and water (2 X 100 mL). The organic layer was dried over MgS04 and evaporated to a syrup which was slowly run into vigorously stirred hexane to yield V: 1.73 g; 73% yield; mp 168-170 O C ; IR 1813,1788,1749,1618 (C=O), 1534 (NH), 1490 (aryl) cm-1; TLC on silica gel with a mobile phase of CH2Clz/MeOH/acetic acid (9:l:trace) showed a single band a t R f 0.92. Theory: C, 72.4; H, 5.3; N, 7.7. Found: C, 71.8; H, 5.9; N, 7.8. A [3H]glycine analogue of V was prepared as described above by substituting tritiated glycine for the monohydrazide of adipic acid (111). The [SHIglycine analogue was used to label the position of the terminal hydrazide for strutural studies. (C) Terminal Blocking of Hydrazinodextran ZZ with the Protected Active Ester V. The trityl-protected active ester V (530 mg, 0.97 mmol) was added to a solution of I1 (5.0 g, 0.14 mmol) in 25 mL of dry DMSO. The mixture was stirred for 3.5 h a t room temperature during which time a slight, pale yellow tint developed due to release of N-hydroxyphthalimide upon reaction of the terminal hydrazide in I1 with the active ester. The reaction was terminated by pouring the contents into 500 mL of methanol, which caused precipitation of tritylhydrazinodextran VI. The product was collected by filtration and washed extensively with methanol. The filter cake was dissolved in deionized water (100 mL) and the solution was filtered to remove a small amount of insoluble material. The filtrate was dialyzed (tubing MWCO 12-14 kDa) versus several changes of deionized water for 2 days and lyophilized to yield 4.32 g (86.4 9; ) of VI. Digestion of purified VI with dextranase (17) for 30 min in 0.1 phosphate buffer, pH 6.0, a t room temperature followed by TLC of the sugar fragments on silica gel plates (Analtech, Newark, DE) with ethyl acetate/ methanol/water (4:2:1) produced three bands with anthrone detection (18): R f 0.06 (smear), 0.20 (isomaltose), and 0.39 (glucose). A preparative plate was run and bands corresponding to the R f values above were scraped from the plate. The compounds were eluted with excess mobile phase, and the solvent was evaporated to yield residues, which were dissolved in 60% perchloric acid. The R f 0.06 band was the only fraction that exhibited the intense yellow color of the T r t cation. T r t content of the R f 0.06 band (60% HC104; 430 nm, € = 32 000 M-' cm-' for triphenylmethyl cation) was determined spectrophotometrically, and total carbohydrate content for each band was determined by using the anthrone assay (18).
To confirm the position of the hydrazide group in the dextran chain, a sample of I1 was reacted with the trityl-protected [3H]glycine active ester described previously. The resulting tritiated hydrazinodextran was digested for 30 min with 400 mM NaI04 a t pH 6.5, and the digestion mixture was applied to a Sephadex G-25 column (2.5 X 40 cm) equilibrated with deionized water. The collected fractions were assayed for 3H content by liquid-scintillation counting and trityl content by acidification with 60% HC104 and spectrophotometry, and totalcarbohydrate content was determined by the anthrone method (18). ( D ) Preparation of (Trity1hydrazino)dextrantransCarbonate (VZZ). The cyclic trans-carbonate was prepared according to the general procedure of Doane et al. (19). A portion of VI (4.0 g) was dissolved in dry DMSO (40 mL) to which dioxane (4.8 mL) and triethylamine (20 mL) were subsequently added. The mixture was cooled in an ice bath under vigorous stirring while ethyl chloroformate (12 mL) was added dropwise over 4 min. The turbid mixture was stirred an additional 5 min, and the
Bioconjugafe Chem., Vol. 1, No. 3, 1990 215
product was precipitated by pouring into 1 L of rapidly stirred methanol. The solid was collected by filtration followed by extensive washing with fresh methanol. The product (VII) was air-dried on the filter after washing with ether: yield 3.92 g; IR 1810 (cyclic carbonate); 1752 (acyclic carbonate) cm-'. The product was slightly soluble in water, but readily soluble in DMSO. The degree of substitution of the carbonate groups on the dextran chain was determined by acidic titration of VI1 that had been digested with barium hydroxide (19). ( E ) Conversion of VZI to (Trity1hydrazino)dextran Carbazate (VZZZ) by Hydrazinolysis. Dextran transcarbonate VI1 (3.50 g) was ground to a fine powder and shaken with 30 mL of hydrazine hydrate (85%). The initial powder dissolved over the course of about 5 min, yielding a homogeneous solution, which was shaken for an additional minute, and poured into 1 L of methanol to effect precipitation of carbazate VIII. The solid was collected by filtration, washed with methanol, and airdried on the filter. The white powder was dissolved in 100 mL of deionized water, dialyzed overnight versus several changes of deionized water, and lyophilized to yield 3.38 g. The infrared spectrum of the product showed a single strong carbonyl band at 1717 cm-' and the appearance of a new NH band at 1513 cm-'. Addition of a drop of benzaldehyde to a solution of the product in 0.2 M acetate buffer, pH 4.75, containing 10% ethanol led to rapid formation of a precipitate, which was found to contain aromatic components by UV spectroscopy. 11. In Situ Preparation of the Tin(1V) Chlorin e6 Active Ester. The photosensitizer chlorin e6 was converted to its tin(1V) metallochlorin analogue and then activated for coupling to the side-chain carbazate groups on dextran carrier VIII. Activation of the SnCe6 was accomplished by in situ generation of an active ester by reaction with a carbodiimide and N-hydroxybenzotriazole in molar amounts equivalent to the amount of SnCe6 used. The in situ active ester (probable structure XI) was not isolated and characterized but used immediately to couple SnCe6 to the carrier. ( A )Preparation of Tin(ZV) Chlorin e6. Chlorin e6 (IX; 500 mg, 0.837 mmol) was dissolved in 100 mL of 1%(w/w) anhydrous sodium acetate in glacial acetic acid and heated to 60 "C under nitrogen in darkness. Finely powdered SnC12 (1.0 g, 5.26 mmol) was charged into the reaction vessel with stirring. The incorporation of tin into the chlorin was followed by vis spectroscopy over the course of 1.5 h, during which time the red peak underwent a hypsochromic shift to 640 nm (pyridine) from the metal-free value of 664 nm (pyridine). Upon complete disappearance of the 664-nm peak, the reaction was cooled to room temperature with exposure to air (oxidation of SnI1 to SnIV) and poured into 1 L of chloroform. The dark green solution was extracted three times with 3 N HCl(300 mL) and twice with water (300 mL). The organic layer was evaporated in vacuo to dryness. The residue was dissolved in absolute ethanol (300 mL) and evaporated again in vacuo. The ethanol evaporation procedure was repeated twice more to strip any remaining water. The residue was triturated in hexane to produce dark blue-green plates, which were recrystallized from ethyl acetate to yield tin(1V) chlorin e6 dichloride (SnCe6; X): 391 mg; 59.5% yield; vis (pyridine) A,, 415 nm; €415 = 1.50 X 105; 640 nm €640 = 6.00 X lo4. TLC of the product on silica gel plates with 2.6-lutidine/water (6:4 v/v) in an atmosphere saturated with NH3 vapor gave a single band at R f 0.59. To facilitate quantitation, X was also prepared with a trace l13Sn y-radiolabel in the manner described above using l13SnC16*- (New England
216
Rakestraw et at.
Bioconjugate Chem., Vol. 1, No. 3, 1990
Nuclear, Boston) prereduced with a small amount of magnesium metal in glacial acetic acid. ( B )I n Situ Generation of the SnCe6 Active Ester. In order to produce the SnCe6 active ester, N-hydroxybenzotriazole (HOBt; 14 mg, 0.104 mmol) was initially added to a solution of SnCe6 (X; 30 mg, 0.042 mmol) in 1.0 mL of DMF and 700 pL of acetonitrile. After 5 min, l-ethyl3- [ 3- (dimethylamino)propyl]carbodiimide(EDAC; 8 mg, 0.042 mmol) was added to initiate the reaction. The reaction was continued for 3 h in the dark at room temperature to generate XI, which was not isolated but used in situ in the next step to couple to dextran carbazate VIII. 111. Coupling of In Situ SnCe6 Active Ester XI to Dextran Carbazate VIII. In a separate vessel, a solution of the terminally protected carbazate VI11 (120 mg, -3.43 pmol) in 7.0 mL of dry DMSO was prepared just prior to the completion of the 3-h reaction time required for in situ generation of the chlorin active ester XI. Upon complete dissolution of VIII, the entire in situ active ester solution from above was added. The dark green mixture was allowed to react for 2 h in darkness at room temperature under tumbling agitation. Triethylamine (500 pL) was then added, and the filtered reaction mixture was applied to a Sephadex G-50 column (2.5 X 45 cm) and eluted with deionized water. Two peaks showing strong absorbance at 634 nm (water) were observed. The first peak (XII), which eluted at the void volume and contained the majority of the green pigment, was collected for use in further reactions. The low molecular weight peak, which contained unbound X, was discarded. To render excess side chain hydrazide groups on XI1 unreactive after coupling of the SnCe6, the remaining hydrazides were capped by reactions with excess acetaldehyde under reduction trapping conditions. To the pooled high molecular weight SnCe6-dextran fractions from above was added enough sodium acetate to bring the concentration to 300 mM. Cold acetaldehyde (2.0 mL, 35.8 mmol) was added, and the reaction mixture was adjusted to pH 5.0 with glacial acetic acid. Sodium cyanoborohydride was then added to a final concentration of 200 mM. The reaction was stirred at room temperature in the dark for 3 h. The pH, which changed rapidly at first due to consumption of protons by the reductive amination capping, was maintained at 5.0 by addition of acetic acid. Excess capping reagents were removed by extensive dialysis (tubing MWCO 12-14 kD) of the reaction solution against deionized water. The dialyzed material was lyophilized to yield 106 mg of a fluffy, green solid (XIII) readily soluble in water, DMSO, and neutral or alkaline buffers, but only slightly soluble in acidic buffers. The product was stored in darkness at -20 "C until further use. IV. Removal of the Chain-Terminal Trityl Blocking Group to Expose the Hydrazide Group. The lyophilized product from above (XIII; 100 mg) was dissolved in dry DMSO (5.0 mL), and trifluoroacetic acid (250 pL) was added to cleave the chain-terminal trityl protection group. After 5 min, the reaction was terminated by addition of triethylamine (500 KL),and the filtered mixture was eluted from a Sephadex G-50 column (2.5 x 45 cm) with deionized water. A single strong green peak (A, 405,634 nm; XIV) eluted at the void volume followed by an intense yellow peak (A, 430 nm in 60%) HC104; trityl cation) which eluted at the low molecular weight retention limit. The high molecular weight green peak was collected and lyophilized to yield 93 mg of SnCe6dextran XIV, which was stored in the dark at -20 "C until needed.
V. Coupling of SnCe6-Dextran to the Fc Carbohydrate Moiety in Antibodies. In preparation for coupling, the MAb (10 mg) was brought to a concentration of 5 mg/mL in 0.15 M acetate buffer, pH 4.75, by passage through a PD-10 desalting column (Pharmacia) followed by ultrafiltration as needed. Oxidation of the Fc carbohydrate moiety was performed as previously described with some modifications ( 3 , 4 ) . Sodium metaperiodate was added to the MAb solution to a final concentration of 20 mM. The oxidation was allowed to proceed in darkness for 15 min at room temperature, followed by the addition of ethylene glycol (50 pL/mL MAb solution) with 15 min of further incubation to scavenge excess periodate. The oxidized MAb was purified by passage through PD-10 gel filtration column (Pharmacia) eluted with acetate buffer. Protein-containing fractions (determined by appreciable A2801 were pooled. In a separate vessel, a solution of XIV (40 mg, -1.04 pmol) in 1.0 mL of deionized water was prepared. This solution was added immediately to the oxidized MAb in acetate buffer and incubated for 48 h at 4 "C in darkness. Following completion of the coupling, the reaction mixture was brought to pH 7.5 by dropwise addition of 1.0 M K2HP04. The immunoconjugates were purified by a two-step procedure. First, the coupling mixture was applied to a protein G-Sepharose column (Pharmacia) to remove unbound SnCe6-dextran XIV. After elution of the bound material with 0.15 M glycine pH 2.5 and immediate neutralization with 1.0 M K2HPO4, unconjugated MAb was separated from the immunoconjugate by passage through a Sephacryl S-300 (1.5 X 25 cm) column. The high molecular weight conjugate fractions were pooled and dialyzed against PBS overnight at 4 "C. The immunoconjugates were stable at 4 "C for up to 1 week, and they could be stored frozen indefinitely at -70 "C. VI. Analysis of Immunoconjugates. Substitution ratios of SnCe6 per MAb were determined by y-counting (l13Sn radiolabeled SnCe6) and A280 (MAb content). The measurement of MAb concentration was corrected for SnCe6-dextran absorbance at 280 nm with a standard curve produced from known concentrations of l13Snlabeled SnCe6-dextran. Conjugate structures were analyzed with SDS-PAGE (8-25 % continuous gradient, mercaptoethanol) on a PHAST electrophoresis system (Pharmacia) with silver staining for protein visualization. Conjugate isoelectric points were determined with the same system using PI 3-9 gradient gels. HPLC of the purified conjugates was performed with a Du Pont Zorbax GF-250 column with 0.2 M phosphate buffer, pH 7.2 (1.0 mL/min). The binding activity of the antimelanoma MAb 2.1 for its cell surface antigen (20) was measured by radioimmunoassay (RIA) (21)using 1251-labeledMAb and a suspension of SK-MEL-2 human malignant melanoma cells (22). Previous results indicated that saturation of the available cell surface antigens occurred at a total MAb concentration of 20 nM for a suspension of 250 000 SK-MEL-2 cells. The binding activities of both the specific conjugate (antimelanoma 2.1-dextran-SnCe6) and the control conjugate (antilymphoma 2.130-dextran-SnCe6) were assessed by a competitive inhibition radioimmunoassay (21) in which increasing concentrations of the conjugates were incubated simultaneously with a nonsaturating amount (total MAb concentration of 10 nm) of unconjugated 1251-labeledantimelanoma 2.1 and a suspension of 265 000 SK-MEL-2 melanoma cells. RIA data was plotted according to the following equation:
A= I
1
+~/KA
Bioconjugate Chem., Vol. 1, No. 3, 1990
Antibody-Targeted Photolysis
where I = percentage of inhibition expressed as a decimal, A = total molar concentration of the unlabeled inhibitor MAb, and K is the equilibrium affinity constant of the unlabeled MAb. Under conditions in which the total antibody concentration is sufficient to occupy all cell surface antigen, the slope of the inhibition curve 1 / K is proportional to the average affinity constant of the inhibitor MAb relative to the 1251-labeled reference MAb antimelanoma 2.1.
OH NalO,
RESULTS
Synthesis of the SnCe6-Dextran Carrier. The synthetic schemes used in the present work are detailed in Schemes 1-111. A reactive hydrazide group was added to the reducing terminus of dextran via reductive amination with a large excess of adipic acid dihydrazide in the presence of NaCNBH3. Titration of the unmodified reducing end in I1 using the dinitrosalicylate method (14) showed a 92%' modification of the terminal aldehyde. Presumably, a hydrazone is formed by condensation of the dihydrazide with the small equilibrium amount of terminal acyclic aldehyde in dextran. The hydrazone is trapped to a hydrazide by reduction with NaCNBH3 (23). The hydrazide group functions as the point of attachment of the dextran chain to the MAb. To prevent modification of the chain-terminal hydrazide in I1 during the subsequent synthetic steps, a triphenylmethyl (Trt) protection group was introduced via an active ester of carboxypentanoic hydrazide (V, Scheme 11). Digestion of purified VI with dextranase (17) followed by TLC of the sugar fragments on silica gel plates produced three bands of Rf 0.06 (smear, minor), 0.20 (isomaltose, major), and 0.39 (glucose, minor). The R f 0.06 band was the only fraction that exhibited the intense yellow color of the T r t cation. Spectrophotometric measurement of the T r t content of the Rf 0.06 band combined with total carbohydrate content for each band yielded an average ratio of 125 mol of glucose per mol of Trt. This value was in good agreement with the number average molecular weight of 21.2 kDa (degree of polymerization = 131) previously determined by gel-permeation chromatography against dextran standards, suggesting that only one molecule of T r t was attached to each dextran chain. To confirm the position of the hydrazide group in the dextran chain, a [3H]glycine analogue of t h e T r t protected active ester was reacted with 11. Periodate digestion of the tritiated analogue followed by chromatography of the fragments on Sephadex G-25 showed virtually all of the 3H activity in the low molecular weight peak, while the bulk of the carbohydrate content was found in the void fractions (Figure 1). The observed chromatographic behavior can be explained by release of a low molecular weight tritiated fragment from the terminal glucose residue. Because the hydrazide group has been attached to the chain by reductive amination, the terminal glucose residue can no longer cyclize. Treatment of the chain with periodate cleaves the glucose subunit ring between the vicinal diols releasing C-3 as formic acid, and C-1 and C-2 as part of a tritiated low molecular weight fragment containing the trityl group. Although the remaining glucose subunits are also cleaved between the vicinal diol positions, the presence of the high molecular weight peak in the chromatogram of the digestion mixture (Figure 1)indicates that the dextran backbone remains intact. Trt-protected hydrazinodextran VI was reacted with ethyl chloroformate in DMSO by using the general method of Doane and co-workers (19). The reaction yielded a mixture of cyclic and acyclic carbonates, which were evident from the IR spectra (carbonyl bands at 1810, 1752
217
1
0
10
20
10
20
39
40
50
60
70
3Q
40
50
60
70
Fraction Number
1.0
4 9
0.8
'3
8
2
0.6
w 0.4
'3
2
2
0.2 0.0 0
Fraction Number Figure 1. Chromatography of the mixture resulting from digestion of an 3H-labeled analogue of the trityl-protected hydrazinodextran VI (Scheme I) with 400 mM NaI04 in pH 6.5 acetate buffer (reaction shown). The chromatograms were generated by passage of the digestion mixture through a Sephadex G-25 column (2.5 X 38 cm) equilibrated with deionized water. Fractions (5 mL) were collected, and each fraction was divided into three equal aliquots (1.67 mL). The refractive index of one aliquot was determined by an Abbe refractometer. The second aliquot was acidified with 60% perchloric acid and its absorbance a t 430 nm was determined. The final aliquot was combined with 19 mL of liquid-scintillation cocktail (Hydrofluor, National Diagnostics) and 3H content was determined. (A) A chromatogram of the refractive index ( 0 )and 3H content (0) of each fraction is shown. Results are expressed relative to the maximum signal observed for each assay. (B) A chromatogram of the refractive index ( 0 )and asorbance at 430 nm (0, in 60% HC104) of each fraction is shown. Results are expressed relative to the maximum signal observed for each assay.
cm-l). In the present work, the dextran carbonate was found to have a total degree of substitution of 0.54 as determined by acidic back-titration of the sugar following barium hydroxide digestion (19). Treatment of dextran carbonate VI1 with hydrazine hydrate led to an immediate solvolysis of the carbonate groups with quantitative formation of a dextran carbazate (VIII). Evidence for the transformation was detected in the complete disappearance of the cyclic (1810 cm-l) and acyclic (1752 cm-l) carbonyl bands in the IR spectrum of the carbon-
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ate with concurrent appearance of a single carbonyl band at 1717 cm-1 and a NH bending band at 1513 cm-' in the spectrum of the carbazate. SnCe6 was coupled to VI11 by reaction with a SnCe6 active ester (XI) generated in situ in a separate vessel (Scheme 111). The protocol reported in the current work gave five chlorins per chain based on an average molecular weight of the dextran chains of -35 kDa. By varying the amount of in situ SnCe6 active ester XI reacted with each dextran chain in the coupling reaction, the ratio of SnCe6 per chain could be varied between 0.5 and 12.3 based on y-counting of 113Sn-labeled SnCe6. Introduction of Sn4+ into the chlorin macrocycle was observed to substantially increase the solubility of the SnCe6dextran chains in aqueous buffers at modest loadings of SnCe6 per dextran chain. At ratios of SnCeGldextran exceeding 10, the lyophilized material required dissolution in 0.10 M NaOH followed by dialysis into the working buffer of choice. Coupling of SnCe6 to dextran carbazate VI11 resulted in only partial coverage of the available hydrazide functions. Since the chain-terminal hydrazide was designed to be the sole point of linkage to MAbs, excess hydrazide groups in the remainder of the chain were capped to render them unreactive. Capping was accomplished by a reductive ethylation of the spare hydrazides with excess acetaldehyde in the presence of NaCNBH3. The outcome of this procedure was measured by incubation of the purified XI11 with an excess of benzaldehyde in 0.15 M acetate buffer, pH 4.75, containing 10% (v/v) ethanol. Prior to the reductive alkylation, rapid precipitation of the SnCe6-dextran chains was observed following benzaldehyde addition due to formation of hydrophobic phenyl hydrazones along the chain backbone. However, after the capping reaction, no precipitate was formed upon addition of benzaldehyde. Finally, the T r t protecting group was removed from the terminal hydrazide group on the capped SnCe6dextran chain by treatment with trifluoroacetic acid in dry DMSO. Elution of the reaction mixture on Sephadex G-50 with deionized water initially showed two bands. The rapidly eluting band contained all of the dark bluegreen pigment present, whereas, the slower eluting band faded from deep yellow to colorless as it progresed down the column. The disappearance of the trailing yellow band can be explained by retardation of the trityl group due to hydrophobic interaction with the column packing. Initially, the cleaved T r t group comigrated with hydrophilic trifluoroacetic acid and thus displayed the characteristic yellow pigment of the T r t cation. As elution progressed, separation of the faster moving trifluoroacetic acid caused a shift of the equilibrium between T r t and its cation toward the uncharged species, which is colorless. The appearance of the slowly eluting yellow band provided evidence of deprotection of the chain-terminal hydrazide to yield XIV. Preparation of Immunoconjugates. The MAbs used in the present work (antimelanoma MAb 2.1 and antilymphoma 2.130) were stable to oxidation with 20 mM NaI04. No evidence of precipitation of the MAbs was ever observed in multiple preparations of immunoconjugates. Table I displays the various conjugates assembled from the MAb. Comparison of the SnCe6/MAb ratio with the SnCeGldextran ratio showed that approximately two dextran carrier chains were able to be coupled to each antibody molecule. Coupling of the SnCe6dextran to the MAb was always performed with the SnCe6dextran present in at least 20-fold molar excess relative to the MAb. No attempts were made to prepare conju-
Table I. Conjugation Characteristics of MAbs immunocarrier conjugate SnCe6/ SnCe6/ dextran sample dextran MAb chains per MAb no. ratio" ratiob antibody 2.1
1 2 3 4 1
2.130d
0.9 3.5 5.5 9.9 3.5
1.7 6.8 11.2 18.9 7.2
1.89 1.94 2.04 1.91 2.25
%
conjugate yieldc 31 28 27 24
56
Determined by using lWnCe6 to prepare the SnCeG-dextran (dextran average MW -35 kDa). Determined by using l13SnCe6dextran and A m (MAb content) corrected for SnCeG-dextran absorbance. Based on the amount of oxidized MAb used in the coupling reaction with 113SnCe6-dextran. MAb 2.130 is an antilymphoma MAb used as a nonbinding control antibody. MAb 2.1 is an antimelanoma MAb.
200
300
400
500
600
700
Wavelength, nm
8
1.0
-
0.5
-
mE
%
2
0.0 400 500 600 700 Wavelength, nm F i g u r e 2. Optical absorption spectra of compounds used in the preparation of the immunoconjugates described in this paper: (A) A spectrum of unconjugated SnCe6 (14.7 pM) in DPBS, (B) a spectrum of an immunoconjugate (SnCeG/MAb molar ratio 11.2) in DPBS. The light path of the cuvette was 10 mm for both samples. Both the Soret band (409 nm) and the red band (634 nm) become broader with less pronounced absorbance as a result of conjugation. The Soret band also develops a shoulder a t 378 nm and an isosbestic point a t 390 nm.
200
300
gates with less of an excess of SnCe6-dextran in the coupling reaction mixture. Even at a 20-fold excess of SnCe6dextran not all of the MAb was observed to form conjugate. However, for a particular MAb, the yield of conjugate did not appear to be significantly effected by the SnCe6/ dextran molar ratio of the SnCe6-dextran used in the coupling (Table I). Instead, the yield of conjugate appears to be governed by the hydrazone equilibrium established between the hydrazide group on the carrier and the reactive aldehyde groups on the particular MAb. Analysis of the Immunoconjugates. The spectrum of a typical conjugate is shown in Figure 2b. For comparison, the spectrum of free SnCe6 is illustrated in Figure 2a. Both the Soret and red bands showed were broad-
Bioconjugate Chem., Vol. 1, No. 3, 1990
Antibody-Targeted Photolysis
219
I
t
I
J I I I I I I I I I ( I I ( I J I (0 45
W E fminutes 1
Figure 3. HPLC of antimelanoma 2.1 MAb and a purified antimelanoma MAb 2.1 immunoconjugate (SnCe6/MAb molar ratio 6.8:l). The chromatograms were produced by elution of each sample from a Du Pont GF-250 column (0.8 X 25 cm) with 0.2 M phosphate buffer, pH 7.2, a t 1.0 mL/min. (A) Unmodified MAb detected by A2m and (B) purified immunoconjugate detected by A280 (solid line) and A634 (dotted line) are shown. kla
200 11694 67 -
43 -
21 1
U
Standards
Figure 4. SDS-PAGE of antimelanoma MAb 2.1 and a purified antimelanoma MAb 2.1 immunoconjugate (SnCe6/MAb molar ratio 6.8:l). A continuous gradient gel (8-2576) was used with silver staining for visualization of protein bands. Samples were prereduced with mercaptoethanol prior to migration on the gel. Lane 1 is the unmodified MAb 2.1 showing the characteristic light and heavy chain bands for IgG. Lane 2 shows the migration pattern for the purified immunoconjugate. The modified immunoglobulin heavy chain in the conjugate (lane 2) was observed to be green prior to silver staining.
ened as a result of conjugation to the dextran carrier and MAb. In addition, the Soret band displayed a shoulder peak at 378 nm and an isosbestic point at 390 nm. Purified conjugates analyzed by HPLC were found to elute in a single peak corresponding to a molecular weight range of 200-240 kDa (Figure 3). Small amounts (
