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Bioconjugate Chem. 1990, 1, 51-59
Sulfhydryl Site-Specific Cross-Linking and Labeling of Monoclonal Antibodies by a Fluorescent Equilibrium Transfer Alkylation Cross-Link Reagent Renato B. del Rosario,t Richard L. Wahl,*lt Stephen J. Brocchini,$ Richard G. Lawton,t and Richard H. Smitht Division of Nuclear Medicine, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0028, and Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109. Received July 20, 1989
The site-specific intramolecular cross-linking of sulfhydryls of monoclonal antibodies via a new class of “equilibrium transfer alkylation cross-link (ETAC) reagents” is described. Following complete or partial reduction of interchain disulfides with dithiothreitol (DTT), two murine IgG2a monoclonal antibodies, 225.288 and 5G6.4, were reacted with a,a-bis[(p-tolylsulfonyl)methyl]-m-aminoacetophenone (ETAC la) and a fluorescent conjugated derivative, sulforhodamine B m-(a,a-bis(p-tolylsulfonylmethy1)acetyl)anilide derivative (ETAC lb). Reducing SDS-polyacrylamide gel electrophoresis analysis of the products from lb indicated the formation of S-ETAC-S interchain heavy and light chain cross-links (-23-34% overall yield by video-camera densitometry) which do not undergo disulfide-thiol exchange with DTT at 100 OC. In contrast, no interchain cross-links were observed upon reaction of unreduced or reduced antibody wherein the thiols have been previously alkylated with iodoacetamide. These results indicated site-specific cross-linking of interchain sulfhydryls and places their distance within 3-4 A. Flow cytometry of the ETAC lb 5G6.4 cross-linked product using 77 IP3 human ovarian carcinoma target cells showed positive binding and retention of immunoreactivity. The in vivo biodistributions of 1311-labeledintact 5G6.4 and 1251-labeledreduced 5616.4 ETAC la product in rats were essentially identical over a period of 24 h. The present study illustrates the potential applications of labelable ETAC reagents as thiol-specific probes for a wide variety of immunological studies.
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The search for site-specific methods of introducing labels into monoclonal antibodies is currently an area of intense interest in immunochemical research. While traditional “random” methods of labeling are commonly achieved by conjugation to protein lysyl €-aminogroups (1-4) and radioiodination of tyrosines (5-7), more recent work has targeted carbohydrate (aldehyde) and sulfhydryl (cysteine) functionalities (8-10). A primary impetus for targeting these latter residues is that “tagging” is directed regiospecifically and away from the antigen combining regions. This may avoid unwanted chemical reactions which can arise from random labeling procedures and potentially may yield labeled antibodies with higher immunoreactivities. This feature is especially important in biochemical applications of immunoconjugates and radioimaging where diminishment of immunoreactivity can lead to poor target localization. Alkylation of reduced disulfides is particularly attractive because of the greater nucleophilicity of thiols over amines and the possibility for selective alkylation of different types of interchain disulfides (11-14). A major limiting factor in the development of thiolspecific labeling techniques is the scarcity of thiol-reactive reagents. The recent synthesis of new N-substituted aromatic maleimide derivatives (9,lO) and crabescein (15) represent a promising class of organic compounds. We have recently reported our success with the sulfhydryl site-specific labeling of partially reduced monoclonal antibodies with biotin (16,17). An additional caveat in thiol-directed labeling is that alkylation occurs with
* To whom reprint requests should be addressed. University of Michigan Medical Center. University of Michigan. 1043-1802/90/2901-0051$02.50/0
concomitant and irreversible cleavage of interchain disulfide bonds. Cleavage of these covalent bonds may adversely affect not only the overall stability of the natural conformation of the antibody but can also diminish immunoreactivity if substantial dissociation of complementary heavy and light chains ensues. A plausible solution to this problem is design of reagents which carry the desired label and have thiol-specific cross-linking properties. In this paper we describe the reactions of reduced disulfides of two murine monoclonal antibodies, 225.288 and 506.4, with a new class of cross-linking reagents characterized as “equilibrium transfer alkylation cross-link” (ETAC) reagents (18-20). The compounds in the present study are structurally and mechanistically similar to the earlier prototypes (18) and are designated in Figure 1. The insertion of ETAC 1 type compounds into reduced disulfide bonds of interchain IgG heavy chains is depicted in Scheme I. Compounds of type 1 are unique in that they possess an amine functionality on the aromatic ring attached to the bridge which serves as a chemical carrier for introduction of a desired label. We illustrate the potential of this structural feature with the synthesis of its rhodamine conjugate, lb. Details of the syntheses and chemistry of the other structurally related extended ETAC compounds are described elsewhere (19). EXPERIMENTAL PROCEDURES
a,a-Bis[(tolylsulfonyl)methyl]-m-aminoacetophenone (ETACla). Bis[(p-tolylsulfonyl)methyl]-m-nitroacetophenone (20)(0.5 g, 1.0 mmol) was finely ground in a mortar and this fine powder was then dispersed in a solution of 25 mL of absolute ethanol and 1 mL of glacial acetic acid, forming a white suspension. This sus0 1990 American Chemical Society
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Bioconjugafe Chem., Vol. 1, No. 1, 1990
O l S I O
I NH
Figure 1. Chemical structures of ETAC compounds:a,a-bis[( p tolylsulfonyl)methyl]-m-aminoacetophenone(la) and tetraethylrhodamine derivative lb.
pension was added to a stirring suspension of 5% Pd/C (0.05 g) in 15 mL of absolute ethanol under a hydrogen atmosphere a t ambient temperature. The reaction mixture was maintained under a slight positive pressure of hydrogen for 24 h as determined by a differential mineral oil manometer. The Pd/C catalyst was then filtered by gravity through Celite and the filtrate was rotoevaporated. Absolute ethanol (10-20 mL) was used to wash the filter cake, and the washings were added to the residue and again evaporated in vacuo, leaving 0.18 g (38%) of solid product (mp 143-144 "C). Less pure material was obtained if the Pd/C catalyst was allowed to soak in chloroform and then again filtered by gravity. The chloroform filtrate yielded material with very little difference by spectroscopic analyses and crude yields were increased to 70-7570. ETAC la: IR (Nicolet 5DX and 5DX-B FT-IR) (KBr, cm-') 3473,3378,1683,1596,1291, 1149,1085; 'H NMR (Bruker WM 360 and AM 300 MHz, CDC1,) 6 2.46 (s, 6 H), 3.54 (AB q, 4 H, JAB= 6.31, 6.28 Hz), 4.26 (t, 1 H, J = 6.27 Hz), 6.82 (d, 1 H), 6.92 (d, 1 H), 7.00 (s, 1 H), 7.07 (t, 1 H, J = 7.79 Hz), 7.52 (AB q, 8 H, JAB= 8.02 Hz); 13C NMR (CDC1,) 6 195.3, 147.1, 145.3,135.4,130.1,129.1,128.44,128.38, 120.6,118.2,114.2, 55.3, 35.6, 20.7; MS (Finnigan 4021 GC-MS system) m / e (relative intensity, EI) 471 (Me+,0.071, 278 (18.4), 246 (20.9), 160 (38.4), 155 (16.9), 139 (70.3), 120 (47.2), 91 (100.0). Sulforhodamine B m-[a,cr-Bis[(ptolylsulfonyl)methyl]acetyl]anilide Derivatives (ETAC lb). a,aBis[ (p-tolylsulfonyl)methyl]-m-aminoacetophenone (la; 100 mg, 0.21 mmol) was dissolved in 2 mL of pyridine and to this solution was added 120 mg (0.20 mmol) of the sulfonyl chloride derivative of sulforhodamine B (Kodak). A trace of 4-(dimethylamino)pyridinewas added and the mixture was stirred overnight at room temperature. A few drops of water was added and the mixture continued to stir for 2 h. The intensely colored solution was diluted with chloroform and treated with 10% HC1. The chloroform layer was washed with 10% HC1 several times and then, after drying over anhydrous sodium sulfate, evaporated to give 130 mg of a dark purple foam after vacuum drying (0.01 mm) for 6 h. This material was essentially homogeneous by TLC in CHC1,/ methanol ( l o / l ) , showing an intense orange-red fluorescent spot at R, 0.43 and a trace of an orange fluorescent spot at R, 0.64 believed to be the Michael elimination product from the bis-sulfone moiety. ETAC lb: 'H NMR (CDCl,) 6 1.28 (br t, 1 2 H), 2.48 (s, 6 H), 3.50 (m, 8 H) overlaps 3.54 (m, 4 H), 4.20 (p, 1 H), peaks a t 6.62, 6.92, 7.00, 7.16 superimposed on 7.35 (A of AB q, 4 H), 7.65
del Rosario et al.
(B of AB q, 4 H); FAB-MS (VG Instruments 702-508) m / e (relative intensity, FAB) 1012 (MH+, 13.0), 855 (MH+ - S02CGH4CH3,1.75). Isolation and Purification of 225.289 and 5G6.4. 225.288 is an IgG2ak murine monoclonal antibody reactive with the high molecular weight antigen of melanoma (21). Hybridoma cells producing this reagent were grown in pristane-primed (Aldrich) Balb/c mice as ascites and then purified by staphylococcal protein A chromatography (22). The antibody was eluted with 0.1 M citrate buffer (pH = 5) and directly reduced with DTT. 5G6.4 is an IgG2a murine monoclonal antibody with preferential reactivity with ovarian and other epithelial cancers and was similarly grown, purified, dialyzed, and concentrated against 0.1 M sodium phosphate buffer (pH = 7) (23). Protein concentrations were measured by using the method of Bradford with bovine serum albumin or bovine IgG (Bio-Rad) as reference standards (24). The purity and immunoreactivity of these preparations were regularly monitored by SDS-polyacrylamide gel electrophoresis conducted under reducing conditions and flow cytometry analysis using a Coulter Epics instrument. Reductions and Reactions of Reduced Monoclonal Antibody with ETAC 1. The procedure is illustrated for the reactions of 225.283 and ETAC lb. To M) was added by syringe 100 pL of 225.288 (-3.3 X 2 pL of DTT (Aldrich or Sigma, 1.71 M). The mixture was allowed to incubate at 37 "C for 2-3 h and chromatographed through a 53/4 -in. glass pipet column containing -2 mL of Sephadex G-25-150 which had been previously allowed to equilibrate in 0.05 M Tris-HC1 (pH = 8). The reaction mixture was collected in -200-250-1L fractions into tubes each containing 15 pL of ETAC lb (0.026 M) dissolved in DMSO (Sigma). The tubes were allowed to incubate a t 37 "C for -3 h after vortexing and were subsequently examined for protein content qualitatively with Bradford's reagent (Bio-Rad). Fractions (normally 1 or 2) which gave a blue color were rechromatographed separately with a similar desalting column as above. The ETAC lb labeled 225.288 fraction eluted as a blue-violet band, leaving excess reagent a t the origin of the column. Reactions of partially reduced 5G6.4 (-50-7O:l molar ratio of DTT to IgG2a, -5 h) antibody with l b were similarly performed with the omission of the desalting step. Upon addition of an equimolar quantity of lb (to DTT), the mixture was vortexed and placed on a nutator for overnight incubation a t room temperature. The reaction mixtures were then spun in a microfuge for 15 min to remove insoluble material prior to gel filtration chromatography. Yields of cross-linked products were estimated by videocamera densitometry at the computer image analysis facility of the University of Michigan Medical Center. With this technique, the Coomassie blue content of each fragment (e.g. H2L2) in lane A of Figure 3 was measured as a function of optical density and obtained as integrated areas with a program which allows for variable background subtraction arising from nonuniform gel destaining. With use of the total integrated areas of heavy and light chains from a completely reduced sample of antibody, a correction factor to incorporate the difference of Coomassie blue staining for a heavy (H) chain and a light (L) chain was calculated for each gel in determining H and L contributions on areas of whole fragments. FPLC (fast protein-liquid chromatography) analysis of chromatographed product mixtures were run on a Pharmacia P-500 system equipped with a single or a series of
Bioconjugate Chem., Vol. 1, No. 1, 1990
Sulfhydryl Site-Specific Cross-Linking of Antibodies
53
Scheme I. Intramolecular Cross-Linkingof Interchain IgG Heavy Chain Thiols NHR
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two Superose 12 sizing columns with PBS (pH = 7.2) as eluting buffer and a flow rate of 24 mL/h. SDS-Polyacrylamide Gel Electrophoresis (SDSPAGE) Analysis (25). SDS-polyacrylamide gel electrophoresis was routinely performed with commercially available Pharmacia 8-25% polyacrylamide gradient gels on a Pharmacia Phast System. Sample preparation typically involved mixing 1 volume of protein sample with at least twice the volume of a Tris-HC1 (pH 6.8) buffer solution which contained 8% SDS (by weight), 20% glycerol (by weight), and 10% 2-mercaptoethanol (by volume) or DTT (0.67 M) for reducing SDS-PAGE. These mixtures were heated for 2-3 min in a boiling-water bath or incubated at 37 "C for 24 h prior to electrophoresis. (Both reaction conditions led to complete reduction and dissociation of heavy and light chains of intact monoclonal antibody.) Gels were stained with Coomassie blue. Biodistribution of 1311-Labeled5G6.4 and 12'I-Labeled Reduced 506.4 ETAC l a in Rats. Approximately 40 pg (-3 X pmol) of 5G6.4 (Figure 4, lane B, E) and reduced 506.4 + ETAC l a (Figure 4, lane B, F) were radioiodinated via the Iodogen (-20 pg; -4.5 X pmol, -20 min) method (6)with carrier free iodide (ICN Biomedicals), purified by anion-exchange chromatography (Bio-Rad AG-l-X8,200-400 mesh), to give 1311labeled 5G6.4 (-8 X lo6 mCi/pmol) and '251-labeled laalkylated 5G6.4 (-1.2 X lo' pCi/pmol) which contained 50 000 (heavy chain, second band from the bottom of the gel) represented the quantity of cross-linked product of mass = the HL, H2, H2L, and H2L2 (very faint by visual inspection) mass with respect to the entire mixture of fragments. For lane A on Figure 3, this method gave -1.3% (H2L2), -3.9% (H2L), -9.2% (H2), and -8.8% (HL), which corresponded to a total yield of -23% cross-linked products. The relative H and L chain content in each cross-linked species is proportional to percent area of cross-linked fragment X fraction of H (or L) chains in a given fragment {e.g. 2H/[2 X H/(area of 1 heavy chain/area of 1 light chain) + 2H] for H2L2 as corrected for the higher absorbance of H vs L; see the Experimental Procedures). Summation of each contribution from H2L2, H2L, H2, and HL bands gives a -30% yield for heavy and 15% for light chains with respect to the total quantity of heavy and light chains in the product mixture (%H/ %L = 2). Similarly, the proportion of heavy-heavy (H-H) and heavy-light (H-L) linkages can be estimated from the ratio of total area for H-H fragments/total area for HL fragments. This calculation gives an H-H/H-L ratio = 1. The above ratios suggest that there is no large preference for heavy-heavy vs heavy-light cross-linking in reactions with completely reduced 225.288 although the number of cross-linked heavy chains is significantly higher than that of light chains. Although the above experiments successfully demonstrated the thiol site specificity of ETAC 1 type com-
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Sulfhydryl Site-Specific Cross-Unking of Antibodies
Bioconlugate Chem., Vol. 1, No. 1, 1990 55 A. [I-1311506.4 D
A B C D E F G H
I
Figure 3. Lane designations: A and B, reducing (DTT) SDS-
PAGE analysis of completely reduced 225.288 + ETAC l b product mixture; C, intact 225.288 in the presence of an equivalent quantity of DTT and SDS (control); D, reducing (DTT) SDSPAGE of unreduced 225.283 + ETAC l b product mixture; E, control reduction of 225.28s for comparison to D; F and G, reducing (DTT) SDS-PAGE of reaction product mixtures from iodoacetamide-alkylatedreduced 225.283 + ETAC lb; H, control reduction of 225.283 for comparison to F and G; I, intact 225.283.
1
a,
0
10
0
”
-
40
(0
B. [I-125]reduci 506.4 + la
1OOOOO
40
(0
8)
fraction numbcr (min.)
Figure 5. Size-exclusion radio-FPLC profile of radioiodinated 5G6.4 (A) and reduced 5G6.4
A B C D E F G H I Figure 4. Lane designations: A, Pharmacia molecular mass
standards were used (from top to bottom) were phosphorylase b (94 OOO), bovine serum albumin (67 OOO), ovalbumin (43 OOO), carbonic anhydrase (30 OOO), soybean trypsin inhibitor (20 loo), a-lactalbumin (14 400); B, nonreducing SDS-PAGE of intact 5G6.4; C, nonreducing SDS-PAGE of reduced 5G6.4 + ETAC l a product mixture; D, reducing SDS-PAGE of intact 5G6.4; E, reducing SDS-PAGE of reduced 5G6.4 + ETAC l a product mixture; F-I, exact corresponding SDS-PAGEanalysis for 5G6.4 and reduced 5G6.4 + ETAC lb.
pounds, there were two practical limitations of the labeling procedure. First, the preparation and isolation of the cross-linked antibodies required chromatography at least twice and the quantity of ETAC reagent used always exceeded the actual amount needed for cross-linking. This was necessary to ensure that all reduced species from sizing chromatography were trapped by ETAC. The desalting chromatography step after DTT reduction also led to variable protein dilution and loss. It was also desirable to have a procedure that could be more easily adapted for scale up involving larger columns and longer periods of chromatography. To address three issues we simplified the procedure by employing a strategy which involved a longer reduction period (-5 h) and lower DTT concentrations (-5070 molar excess over IgG2a). The major difference was that a stoichiometric quantity of ETAC reagent (with respect to DTT concentration)was added after the reduction period and the mixture was allowed to incubate overnight at 37 “C prior to chromatography. Since la,b were only fairly soluble in aqueous DMSO solutions, the use of lower ETAC concentrations also minimized unwanted precipitation during the reaction. Figure 4 depicts the nonreducing and reducing SDSPAGE analysis of the product mixtures upon the application of this technique to the antiovarian IgG2a antibody 566.4 + ETAC l a and lb. Following reduction with a 70-fold molar excess of DTT, video-camera densitometry and image analysis of the product mixture from
+ ETAC l a product B.
l b gave a -34% yield of cross-linked products distributed among H2L2 (
