Bioconjugate Chem. 1992, 3, 104-107
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ARTICLES Photoactivation of Toxin Conjugates' Victor S. Goldmacher,' Peter D. Senter,+John M. Lambert, and Walter A. Blattler ImmunoGen, Inc., 148 Sidney Street, Cambridge, Massachusetts 02139. Received July 1, 1991
A novel photocleavable protein cross-linking reagent has been used for conjugation of the ribosomeinactivating protein pokeweed antiviral protein from seeds of Phytolacca americana (PAP-S),with either the monoclonal antibody 5E9 directed against the human transferrin-receptor or the B-chain of ricin that binds to cell-surface oligosaccharides bearing terminal D-galactose residues. When irradiated with near-UV light (350 nm), the linker of these conjugates undergoes photolytic degradation, resulting in the release of native toxin that is fully functional. The cytotoxicities of these 5E9-PAP-S and ricin B-chain-PAP-S conjugates for HeLa cells could be enhanced by irradiating the cells with light after they had internalized the conjugates.
INTRODUCTION
We have previously described the synthesis of novel photocleavable cross-linking reagents which can be used for constructing conjugates between ribosome-inactivating proteins and targeting molecules such as monoclonal antibodies or lectins (1). UV irradiation (350 nm) of such conjugates results in the photolytic fragmentation of the linker and release of nonmodified toxin that is fully active in a cell-freetranslation system (Scheme I). Here we report the cytotoxic effects on cultured human cells of conjugates of the ribosome-inactivating protein PAP-S1 to two different targeting molecules made with photocleavable cross-linking reagents. One targeting molecule, the monoclonal antibody 5E9, binds to the human transferrin receptor, but does not compete with transferrin for binding, and is not cytotoxic (2). The other targeting molecule, ricin B-chain, binds to terminal D-galactose residues of oligosaccharide components of cell-surface molecules (3).
Scheme I. Release of the Ribosome-Inactivating-Protein (RIP) Moiety from Its Conjugate with a Targeting Molecule upon Irradiation with Light 0
OCONH
"-..-./
TARGETING MOLECULE
I
I
t H,N-
h'
RIP]
pound by refluxing in a mixture of tetrahydrofuran and 3 74 aqueous perchloricacid (2:l,v/v) under an atmosphere of nitrogen for 20 h. After chromatography on silica, 1[5-(N-maleimidomethyl)-2-nitrophenyllethanol was obtained as a light yellowish solid, which was converted to the corresponding chloroformate as described in ref 1. EXPERIMENTAL PROCEDURES Preparation of Conjugates. The conjugate between the monoclonal antibody 5E9 [IgGl, anti-human transSynthesis of Photocleavable Cross-Linking Reaferrin receptor (21,purified as described previously ( 4 ) ] gents. The chloroformate derivative of S-[4-nitro-3-(1and PAP@ was prepared as illustrated in Scheme I1 (left hydroxyethy1)phenyllmethylthioacetate (see Scheme 11, column) using procedures described previously (I). Briefly, top of left column) was synthesized as described previously PAP-S was reacted with the chloroformate of S-[Cnitro(1). The chloroformate of 1-[5-(N-maleimidomethyl)-2-ni- 3-(1-hydroxyethyl)phenyllmethylthioacetate using conditions described previously (11,and free sulfhydryl groups trophenyllethanol (see Scheme 11, top of right column) was synthesized as follows. 1-[5-(Bromomethyl)-2-nitro- were generated by treating the modified PAP-S with 50 mM hydroxylamine at pH 7.3 for 30 min at 25 "C followed phenyllethanol (see ref 1) was converted with aqueous by gel filtration through Sephadex G 25 equilibrated with ammonia in methanol to the corresponding 1-[5-(aminom100mM potassium phosphate buffer (pH 7.0). An average ethyl)-2-nitrophenyllethanol. The amine was reacted with of 0.7 sulfhydryl groups per molecule of PAP-S were 1equiv of maleic anhydride in methanol, thus forming the introduced in this way, as measured by Ellman's reagent corresponding amido carboxylicacid. Ring closure to form (5). The modified PAP-S was mixed at pH 7.0 with 5E9 a maleimido group was effected with anhydrous sodium antibody which had been functionalized with succinimacetate in acetic anhydride. The resulting 0-acetyl so derivative of 1-[5-(N-maleimidomethyl)-2-nitrophenyl]- idyl 4-(N-maleimidomethyl)cyclohexane-l-carboxylate as to introduce an average of 1.5 maleimido groups per ethanol was then hydrolyzed to the final hydroxy comantibody molecule using procedures for antibody modification described previously (1,5). The incorporation of This work was done during the authors' appointment in the maleimido groups was assayed with [l4C1cysteineusing Division of Tumor Immunology of the Dana-Farber Cancer the method described previously (5). The conjugation Institute and Harvard Medical School. f
Present address: Oncogen, Inc., Seattle, WA 98121. PAP-S, pokeweed antiviral protein from seeds of Phytolacca americana. +
The source and purification of PAP-S has been reported elsewhere (5).
1043-1802/92/2903-0104$03.00/00 1992 American Chemical Soclety
Bloconjugate Chem., Voi. 3, No. 2, 1992 105
Photoactlvatlon of Toxin Conjugates
Scheme 11. Construction of Light-Sensitive Conjugates of PAP-S with either 5E9 Antibody (left) or Ricin B-Chain (right)
II
0
~ C O N PAP-s H ~
I
O C O N H * F i
reaction mixture was kept at 4 "C for 4 hand the conjugate was then purified by ion-exchange chromatography followed by gel filtration as described previously (I),yielding conjugates of similar purity to those described previously as shown by polyacrylamide/sodium dodecyl sulfate gel electrophoresis ( I ) . PAP-Swas conjugated to ricin B-chain (Scheme 11, right column) by first reacting PAP-S (1mg/mL) with the chloroformate derivative of 1-[5-(N-maleimidomethy1)-2-nitrophenyllethanol (20-fold molar excess) in 100 mM NaHC03 at 0 "C for 30 min followed by gel filtration through a column of Sephadex G-25 equilibrated in 100 mM sodium phosphate buffer (pH 7.0). The modified PAP-S was then reacted with 1mol equiv of ricin B-chain (Worthington, Freehold, NJ). After a 3-h incubation, the reaction mixture was applied to a column (2 X 90 cm) of Sephadex G-100 equilibrated in 5 mM bistris/acetate (pH 5.8) containing lactose (50 mM), NaCl (50 mM), EDTA (1 mM), and dithioerythritol (1 mM). Fractions were analyzed by polyacrylamide/sodium dodecyl sulfate gel electrophoresis, and those fractions containing the conjugate (1:lratio of ricin B-chain and PAP-S)were dialyzed against 10 mM potassium phosphate buffer (pH 7.2) containing NaCl(145 mM) and stored in aliquots (90 pL) at -80 "C. Irradiation of Cells. Samples were irradiated with near-UV light for 7 min at room temperature. A Rayonet fluorescenceUV lamp (Southern New England Ultraviolet Co., Middletown, CT) with an emission peak at 350 nm was used as a source of UV light. The lamp was placed at a 15-cm distance from the Petri dishes with samples. SDS gel electrophoresis showed that 185% of the conjugate was cleaved under these conditions. Cells and Cell Culture Maintenance. HeLa cells (ATCC CCL 2) were grown as monolayer cultures in a growth medium composed of RPMI-1640 medium supplemented with 10% heat-treated (30 min at 56 "C) fetal calf serum and 2 mM L-glutamine (all three from GIBCO). Cells were passaged twice a week, using the standard trypsin-Versen mixture (Whittaker M.A. Bioproducts, Walkersville, MD) to suspend the cells. Cytotoxicity Studies. Cytotoxicities of toxin conjugates were determined by a clonogenic assay. Cells were plated onto 20 cm2 polystyrene tissue culture grade Petri
dishes in 10 mL of growth medium at a density of 5-1000 cells/cm2 and left for 24 h at 37 "C in a humidified atmosphere containing 5 % C02. Within this interval of time, the cells adhered to the substratum and resumed exponential growth, and the expression of cell-surface transferrin receptors and binding sites for ricin returned to normal after being decreased during the trypsinization procedure (data not shown). Medium was then replaced with 10 mL of fresh growth medium containing a toxin conjugate. After a desired time period of exposure to the toxin at 37 "C, Petri dishes (with the lid on) were irradiated for 7 min with near-UV light at room temperature. Following the irradiation, the cells were rinsed with warm growth medium and incubated in 10 mL of fresh medium for 8 days. Colonies of 20 or more cells were enumerated as described previously (6). RESULTS
We have previously established that irradiation of photocleavable conjugates with UV light under conditions used in experiments with cells induced dissociation of more than 85% of the toxin molecules, as determined by polyacrylamide/SDS gel electrophoresis ( I ) . The 5E9-PAPS and the ricin B-chain-PAP-S conjugates were cleaved using identical conditions (results not shown). As described previously (I),wealso further confirmed the release of fully active PAP-S from the photocleavable conjugates by measuring its ability to inhibit protein synthesis in a cell-free translation system ( I , 7). PAP-S released from these conjugates was as efficient at inactivating protein synthesis as native PAP-S. The cytotoxicity of the toxin conjugates was examined by measuring the colony-forming ability of HeLa cells which had been exposed to the reagents for a desired period of time under normal growth conditions. Plating efficiency of control HeLa cells in these experiments was 0.4-0.5. UV irradiation of cells in the absence of toxins under conditions described in Experimental Procedures was nontoxic (surviving fractions L 0.9). Photocleavable conjugates had a significant cytotoxicity even in the dark (Figure l ) , which was consistent with our observation ( I ) that PAP-S conjugated with an antibody via a photocleavable linker could inactivate ribosomes in a cell-free translation system. Irradiation of
OoMmacher et el.
108 Bloconjugete Chem., Vol. 3, No. 2. 1992 5E9 - PAP-S
BEFORE UV-irradiation C a
AFTER UV-irradiation
\.Light
Antibody-Toxin outside the cell
0
outside the cell
sfeadystate IS quickly established
Light
Antibody-Toxin in a primary compartment 0
0.2
0.4
0
2
Toxin IS liberated
Event
released
4
Concentration (nM)
Figure 1. Cytotoxicity of photocleavable conjugates 5E9-PAP-
S and ricin B-chain-PAP-S. Cells were incubated with a conjugate for 24 h under normal growth conditions, and then the cells were either light irradiated (empty symbols) or not (filled symbols). The dishes with the cells were then rinsed and placed into fresh medium, and the surviving fractions of cells were determined by their ability to form colonies.
cells that had been exposed for 24 h to ricin B-PAP-S or 5E9-PAP-S with UV light caused a further profound decrease in the surviving fraction (Figure 1). The following controls have been done in order to confirm the specificity of the cytotoxic effects. Photocleavable conjugates 5E9-PAP-S (0.4 nM) mixed with 1 pM 5E9 antibody or ricin B-chain-PAP-S (2 nM) mixed with 30 mM a-lactose were essentially not cytotoxic (surviving fractions I 0.9)whether followed or not by UV irradiation of the cells. PAP-S, 5E9, or ricin B-chain alone, with or without the UV-irradiation step, were not toxic to the cells at concentrations up to 50 nM. A short (1 h) exposure of cells to photocleavable ricin B-PAP-S (20 nM), or to 5E9-PAP-S (1 nM) with or without subsequent UV irradiation, was not toxic for HeLa cells (surviving fractions I 0.95). DISCUSSION
It has been reported previously that immunotoxins in which antibody and single-chain ribosome-inactivating protein moieties are linked via a disulfide bond are more cytotoxic than similar immunotoxins linked in a noncleavable manner, presumably because the disulfide bond is reductively cleaved in the cells ( 5 , 9 1 2 ) . The data reported here indicate that the disulfide linkage is not unique in this respect and suggest that other linkers which allow release of the single-chain ribosome-inactivating protein inside cells will permit the formation of a cytotoxic immunotoxin. In this respect photocleavable conjugates of PAP-S were similar to conjugates of antibodies or ricin B-chain with PAP-S or with gelonin linked via a disulfide bond (5). Gelonin is a single-chain ribosome-inactivating protein that is similar to PAP-S in its enzymatic activity and specificity and its cytotoxicity properties (48).Cytotoxicities of ricin B-gelonin and of 5E9-gelonin conjugates linked via a disulfide bond for HeLa cells after a 24-h exposure [with the IC37 being 0.12 nM for the both conjugates (1311 are very similar to those of the photolabile conjugates described in this report (IC3, = 0.05 and 1.3 nM for photocleavable conjugates of 5E9-PAP-S and ricin B-chain-PAP-S after a 24-h exposure followed by UV irradiation, respectively). It is well-established (14-16) that endocytosis is an obligatory step in the process of intoxication of cells by antibody conjugated to single-chainribosome-inactivating proteins or ricin A-chain. In particular, experiments with a large number of disulfide-linked conjugates of gelonin with antibodies showed that, invariably, those conjugates
Antibody-Toxin in a secondary compartment
retained inside the cell and eventually enters the cytoplasm
1
COMMIllED TO DIE
that were not endocytosed exhibited low cytotoxicity similar to that of free gelonin, while those conjugates that were endocytosed demonstrated enhanced cytotoxicity (14). Similar results were obtained in experiments with disulfide-linked PAP-S conjugates (Goldmacher, V. S., Lambert, J. M., and Blattler, W. A., unpublished results). The observation that free PAP-S at 50 nM did not have a cytotoxic effect on HeLa cells also suggested that the PAP-S moieties of the conjugates which were located outside the cell at the time of the irradiation were not toxic to the cells. These results imply that only the internalized PAP-Smolecules were involved in killing cells. Endocytosis delivers proteins into endosomes and lysosomes that are acidified to pH 4.5-5.5 (17). The photolysis of o-nitrobenzyl alcohol derivatives is thought to involve an aci-nitro intermediate that decomposes to the final product (18). This decomposition is catalyzed by protons. Therefore, the acidified Compartments create a favorable environment for the reaction. Thus, photolytic cleavage of the photocleavable conjugates is possible in any intracellular compartments, including the acidified ones. The fact that a short (1h) incubation of cells with photocleavable PAP-S conjugates followed by UV irradiation of the cells does not induce any noticeable cytotoxic effect is of interest. It suggests that a single round of endocytosis is not sufficient for achieving an intracellular concentration of PAP-S conjugate that is high enough to kill the cell upon the release of free toxin by UVirradiation. Endocytosis for a 24-h period appears to be necessary to accumulate sufficient amounts of the conjugates to kill the cells once the PAP-S can be released. The compartment where this accumulation of the conjugate takes place is probably a compartment that is distinct from the primary endosome. Previously we have shown that most molecules of ricin B-chain and 5E9 antibody that had been internalized by cells via endocytosis are quickly transported back to the cell surface via exocytosis (14). PAP-S molecules liberated from the conjugate in endosomes might therefore be quickly excreted into the external medium and thus would not cause any cytotoxic effect. The tentative pathway of intoxication of cells by photocleavable conjugates is depicted in Scheme 111. The intracellular site of cleavage of disulfide-linked conjugates is also unknown. It seems likely that such conjugates would be cleaved under reducing conditions in the cell. Such conditions are not found in endosomes or lysosomes (19),but rather in the cytoplasm that contains 1-10 mM glutathione (20),or in the endoplasmic reticulum or the Golgi complex that contain protein disulfide isomerase (19).
Photoactlvation of Toxin Conjugates
We have demonstrated that the cytotoxicity of conjugates prepared with photocleavable linkers can be induced by photolysis. Such conjugates may be of interest as a research tool because their cytotoxicity can be increased by an external signal at the desired site and/or at the desired moment in time. LITERATURE CITED (1) Senter, P. D., Tansey, M. J., Lambert, J. M., and Blattler, W. A. (1985) Novel photocleavable protein crosslinking reagents and their use in the preparation of antibody-toxin conjugates. Photochem. Photobiol. 42, 231-237. (2) Haynes, B. F., Hemler, M., Cotner, T., Mann, D. L., Eisenbarth, G. S., Strominger, J. L., and Fauci, A. S. (1981) Characterization of a monoclonal antibody (5E9) that defines a human cell surface antigen of cell activation. J. Immunol. 127, 347-351. (3) Olsnes, S., and Pihl, A. (1982) Toxic lectins and related proteins. In Molecular Action of Toxins and Viruses (P.Cohen, and S. van Heyningen, Eds.) pp 195-234, Elsevier, Amsterdam. (4) Scott, C. F., Goldmacher, V. S., Lambert, J. M., Jackson, J. V., and McIntyre, G. D. (1987) An immunotoxin composed of a monoclonal anti-transferrin receptor antibody linked by a disulfide bond to the ribosome-inactivating protein gelonin: potent in vitro and in vivo effects against human tumors. J . Natl. Cancer Inst. 79, 1163-1172. (5) Lambert, J. M., Senter, P. D., Yau-Young, A., Blattler, W. A., Goldmacher, V. S. (1985) Purified immunotoxins that are reactive with human lymphoid cells. Monoclonal antibodies conjugated to the ribosome-inactivating proteins gelonin and the pokeweed antiviral proteins. J. Biol. Chem. 160, 1203512041. (6) Goldmacher, V. S., Tinnel, N. L., and Nelson, B. C. (1986) Evidence that pinocytosis in lymphoid cells has a low capacity. J . Cell Biol. 102, 1312-1319. (7) Pelham, H. R., and Jackson, R. J. (1976)An efficient mRNAdependent translation system from reticulocyte lysates. Eur. J. Biochem. 67,247-256. (8) Endo, Y., Tsurugi, K., and Lambert, J. M. (1988) The site of action of six different ribosome-inactivating proteins from plants on eukariotic ribosomes: The RNA N-glycosidase activity of the proteins. Biochem. Biophys. Res. Commun. 150,1032-1036. (9) Oeltman, T. N., and Forbes, J. T. (1981) Inhibition of mouse spleen cell function by diphtheria toxin fragment A coupled to anti-mouse Thy-1.2 and by ricin A chain coupled to antimouse IgM. Arch. Biochem. Biophys. 209, 362-370. (10) Masuho, Y., Kishida, K., Saito, M., Umemoto, N., and Hara, T. (1982) Importance of the antigen-binding valency and the nature of the crosslinking bond in ricin A-chain conjugates with antibody. J . Biochem. 91, 1583-1591.
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(11) Blythman, H. E., Bord, A., Buisson, I., Jansen, F. K., Richer, G., and Thurneyssen, 0. (1984) Antitumor effect of immunotoxin in mouse and human tumor models. Protides 32,421424. (12) Ramakrishnan, S., and Houston, L. L. (1984) Comparison of the selective cytotoxic effects of immunotoxins containing ricin A chain or pokeweed antiviral protein and anti-Thy 1.1 monoclonal antibodies. Cancer Res. 44, 201-208. (13) Goldmacher, V. S., Blattler, W. A., Lambert, J. M., McIntyre, G., and Stewart, J. (1990) Cytotoxicity of gelonin conjugated to targeting molecules: effects of weak amines, monensin, adenovirus, and adenoviral capsid proteins penton, hexon, and fiber. Mol. Pharmacol. 36, 818-822. (14) Goldmacher, V. S., Scott, C. F., Lambert, J. M., McIntyre, G. D., Blattler, W. A., Collinson, A. R., Stewart, J. K., Chong, L. D., Cook, S., Slayter, H. S., Beaumont, E., and Watkins, S. (1989) Cytotoxicity of gelonin and its conjugates with antibodies is determined by the extent of their endocytosis. J. Cell. Physiol. 141, 222-234. (15) Preijers, F. W. M. B., Tax, W. J. M., De Witte, T., Janssen, A., Heijden, H. V. D., Vidal, H., Wessels, J. M. C., and Capel, P. J. A. (1988) Relationship between internalization and cytotoxicity of ricin A-chain immunotoxins. Br. J. Haematol. 70, 289-294. (16) Wargalla, U. C., and Reisfeld, R. A. (1989) Rate of internalization of an immunotoxin correlates with cytotoxic activity against human tumor cells. Proc. Natl. Acad. Sci. U.S.A. 86, 5146-5150. (17) Yamashiro, D. J., and Maxfield, F. R. (1984) Acidification of endocytic compartments and the intracellular pathways of ligands and receptors. J . Cell. Biochem. 26, 231-246. (18) Walker, J. W., Reid, G. P., McGray, J. A,, and Trentham, D. R. (1988) Photolabile 1-(2-nitrophenyl)ethyl phosphate esters of adenine nucleotide analogues. Synthesis and mechanism of photolysis. J . Am. Chem. Soc. 110, 7170-7177. (19) Feener, E. P., Shen, W.-C., and Ryser, H. J.-P. (1990) Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes. J. Biol. Chem. 265, 18780-18785. (20) Meister, A. (1988) Glutathione metabolism and its selective modification. J . Biol. Chem. 263, 17205-17208. Registry No. S-[4-Nitro-3-(1-hydroxyethyl)phenyl]methyl thioacetate chloroformate derivative, 99821-62-2; 1-[5-(N-maleimidomethyl)-2-nitrophenyl]ethanol chloroformate derivative, 99821114317-16-7;l-[5-(bromomethyl)-2-nitrophenyl]ethanol, 60-0; 1-[5-(aminomethyl)-2-nitrophenyl]ethanol, 138957-91-2;1[5-(aminomethyl)-2-nitrophenyl]ethanol maleic acid derivative, 138957-92-3; 1-[5-(N-maleimidomethyl)-2-nitrophenyl]ethanol 0-acetyl derivative, 138957-93-4; 1-[5-(N-maleimidomethyl)-2nitrophenyllethanol, 138957-94-5.
