Eur. J. Biochem. 196,203-209 (1991)
0FEBS 1991 0014295691001324
Biochemical, cytotoxic and pharmacokinetic properties of an immunotoxin composed of a mouse monoclonal antibody Fib75 and the ribosome-inactivating protein a-sarcin from Aspergihs giganteus Edward J. WAWRZYNCZAK', Raymond V. HENRY', Alan J. CUMBER', Geoffrey D. PARNELL', Elaine J. DERBYSHIRE' and Norbert ULBRICH2 Institute of Cancer Research, Sutton, England Department of Biochemistry, Free University of Berlin, Federal Republic of Germany (Received August 22/0ctober 31, 1990) - EJB 90 1012
An immunotoxin was synthesized by the attachment of a-sarcin, the ribosome-inactivating protein derived from the mould Aspergillus giganteus, to a monoclonal mouse IgG2a antibody Fib75. The a-sarcin immunotoxin exerted toxic effects in tissue culture against the EJ human bladder carcinoma cell line, expressing the antigen recognised by the Fib75 antibody, inhibiting the incorporation of [3H]leucine by 50% at a concentration of 0.46 nM. The cytotoxic effects of the a-sarcin immunotoxin were indistinguishable from those of a Fib75 immunotoxin made with rich A chain. Fib75-a-sarcin was cleared from the circulation of the rat with biphasic kinetics following intravenous administration. The a- and p-phase half-lives were 0.8 h and 6 h, respectively, similar to the serum half-lives of analogous Fib75 immunotoxins made with ribosome-inactivating proteins derived from plants. a-Sarcin was completely stable in physiological saline buffer at 37 "C, whereas the ribosomeinactivating activity of rich A chain was gradually lost under identical conditions. a-Sarcin may be a valuable alternative to ricin A chain for the construction of therapeutic immunotoxins because of its smaller size and greater thermostability.
Immunotoxins are hybrid protein molecules generated by the covalent linkage of antibodies and toxins such as the A chain of the plant toxin ricin. Rich A chain is a ribosomeinactivating protein (RIP) that modifies eukaryotic ribosomes by a unique catalytic mechanism, cleavage of the N-glycosidic bond at A4324 in 28s rRNA, thereby inactivating cellular protein synthesis irreversibly (Olsnes and Pihl, 1982; Endo and Tsurugi, 1987). Immunotoxins constructed with ricin A chain have been shown to exert selective toxic effects against animal and human cells bearing the target antigens recognised by the antibody component and several of these agents are being evaluated in patients with malignant disease (Blakey et al., 1988). Cytotoxic immunotoxins have also been made with single-chain plant-derived RIP which resemble ricin A chain in size (about 30 kDa), structure, and mechanism of catalytic action (Stirpe and Barbieri, 1986; Stirpe et al., 1988; Endo et al., 1988). a-Sarcin is a RIP, first isolated from the mould Aspergillus giganteus, and was shown to be capable of inhibiting the growth of a number of different animal tumours (Olson and Goerner, 1965; Olson et al., 1965). This basic, nonCorrespondence to E. J. Wawrzynczak, Drug Targeting Laboratory, Section of Medicine, Institute of Cancer Research, Cotswold Rd., Sutton, Surrey SM2 SNG, England Abbreviations. ICs0, concentration at which the incorporation of [3H]leucine is inhibited by 50% ; RIP, ribosome-inactivating protein(s) ; SPDP, N-succinimidy1-3-(2-pyridyldithio)propionate. Enzymes. a-Sarcin (EC 3.1.27. -); ricin A chain (EC 3.2.2. -).
glycosylated protein has properties that differ significantly from those of the plant-derived RIP (Wool, 1984). Firstly, its size (17 kDa) is about half that of the plant RIP (Sacco et al., 1983). Secondly, it bears no similarities to plant RIP at the level of the primary structure (Sacco et al., 1983). Thirdly, its enzymic action upon eukaryotic ribosomes proceeds by a different mechanism, hydrolysis of the phosphodiester linkage between G4325 and A4326 in 28s rRNA (Endo and Wool, 1982). Two other RIP of this type, restrictocin and mitogillin, have been isolated from the mould Aspergillus restrictus. These RIP closely resemble one another, are homologous with asarcin in primary structure and have a similar catalytic activity (Lopez-Otin et al., 1984; Fernandez-Luna et al., 1985; Fando et al., 1985). In this study, we have compared the properties of an immunotoxin consisting of a-sarcin linked to a monoclonal mouse IgG2a antibody Fib75, which recognises a human antigen, with those of a Fib75 immunotoxin made with ricin A chain. We have previously shown that Fib75 -ricin-A-chain exerted selective toxic effects in tissue culture against a human tumour cell line (Forrester et al., 1984). Fib75 - a-sarcin formed a stable immunotoxin with cytotoxic potency indistinguishable from that of Fib75 - ricin-A-chain, and pharmacokinetic properties similar to those of analogous Fib75 immunotoxins made with single-chain plant RIP. The findings presented suggest that a-sarcin may be a valuable alternative to ricin A chain and other plant-derived RIP for the construction of immunotoxins intended for therapy.
204 MATERIALS AND METHODS
Cell-free assays of ribosome-inactivating activity
Materials
The ribosome-inactivating activity of a-sarcin and ricin A chain was determined by measuring the inhibition of [3H]leucine incorporation into newly synthesized protein using a rabbit reticulocyte lysate assay similar to that described by Forrester et al. (1984). Triplicate samples were first treated with 2-mercaptoethanol at a final concentration of 0.2 M for 1 h at 37°C. The effect of incubation at 37°C upon ribosome-inactivating activity was measured using samples of RIP or immunotoxin prepared in Dulbecco's phosphate-buffered saline solution A containing 1 mM disodium EDTA and 0.1 mg/ml S-carboxymethylated bovine serum albumin as the carrier protein, at a final RIP concentration of 6 nM. The solutions were filter sterilized, distributed into sterile plastic tubes fitted with O-ring seals, and incubated for different lengths of time up to 96 h at 37°C. A control sample of each preparation was kept at 4°C for the duration of the experiment. The samples were diluted to final concentrations in the rabbit reticulocyte lysate assay giving 60-80% inhibition of protein synthesis, i. e. 50 pM ricin A chain, or 500 pM a-sarcin.
The monoclonal mouse IgG2a antibody Fib75 was purified from the ascitic fluid of mice bearing the hybridoma cell line LICR-LOND Fib75 by ammonium sulphate precipitation followed by chromatography on immobilised Staphylococcus protein A. Ricin A chain was purified from an aqueous extract of delipidated castor bean cake of Sri Lankan origin, as described by Forrester et al. (1984). Sephadex G-25 (M), Sephadex G-25 (SF), Sephadex G-50 (F), Sephacryl S-200 (SF), Blue Sepharose CL-6B and CNBractivated Sepharose 4B were purchased from Pharmacia Ltd., Milton Keynes, UK. CM52-cellulose was from Whatman Ltd, Maidstone, UK. TSK-G3000SWXL columns were purchased from Anachem Ltd, Luton, UK. Sheep anti-(mouse 1g)-antiserum linked to horseradish peroxidase (NA.931), sodium ['251]iodide (IMS.30) and L[4,5-3H]leucine(TRK.170) were purchased from Amersham International plc., Amersham, UK. Iodo-gen was from Pierce and Warriner Ltd, Chester, UK. S-Carboxymethylated bovine serum albumin was obtained from Sigma Chemical Co. Ltd, Poole, UK. N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) was obtained from Pharmacia Ltd. All other reagents were of analytical grade or of the highest purity available. Cytotoxicity experiments in tissue culture Normal male albino rats of the Wistar/CBI strain, each of Cytotoxicity experiments using the EJ human bladder mass approximately 250 g, were supplied by the MRC Animal carcinoma cell line were carried out essentially as described Breeding Unit, National Institute of Medical Research, London, England, and allowed free access to food and water. by Forrester et al. (1984). Briefly, dilutions of RIP or immunotoxin solution were added to subconfluent monolayer cultures of EJ cells in triplicate and incubated for 24 h. Preparation of a-sarcin [3H]Leucine (1 pCi) was added to each culture followed by incubation for a further 24 h. The incorporation ~f[~H]Ieucine A . giganteus MDH 18894 was grown at 30°C for 96 h in was then determined by liquid scintillation counting. a rotary shaker at 200 rpm in 2% (mass/vol.) soluble corn starch, 1.5% (massivol.) beef extract, 2% (massjvol.) peptone, 0.5% (massivol.) sodium chloride inoculated with approxi- Measurement of immunotoxin concentration by ELISA mately 6 x lo7 spores/400 ml medium in 1-1 culture flasks. A rabbit antiserum to a-sarcin was raised by the procedure After removal of mycelia by filtration and low-speed centrifugation (10000 x g for 10 min), the medium was adjust- described by Worrell et al. (1986 b). a-Sarcin-specific antibody ed to neutral pH and loaded on to a Whatman CM52 cation- was isolated from the antiserum by affinity chromatography exchange column. Bound protein was eluted with 20 mM Tris/ on a column of a-sarcin linked to CNBr-activated Sepharose HCI containing 1.5 M NaCl, pH 7.2. The protein was then 4B. The solid-phase ELISA procedure used was a modification subjected to gel filtration on a column (150 cm x 4 cm diameter) of Sephadex G-50 (F),equilibrated with 20 mM Tris/HCl, of the method originally described by Worrell et al. (1986a) for the detection of intact immunotoxins made with ricin A pH 7.2. chain. Briefly, affinity-purified anti-(a-sarcin) antibody was first adsorbed to 96-well microELISA plates. Serum samples Synthesis, purification and characterization of Fib75 - a-sarcin containing Fib75 - a-sarcin were added to the plates and, The procedures used for the synthesis of Fib75 - a-sarcin following incubation and washing, the bound immunotoxin were similar in outline to those described by Cumber et al. was detected using sheep anti-(mouse Ig) antiserum linked to horseradish peroxidase in combination with an O-phenyl( 1985) for the preparation of disulphide-linked immunotoxins made with single-chain RIP. a-Sarcin, in 0.1 M sodium phos- enediamine substrate solution developing colour at 492 nm. phate containing 0.14 M sodium chloride (NaC1/Pi), pH 7.0, The mean absorbance at 492 nm of triplicate serum samples was reacted with SPDP to introduce an average of 0.2 was used to calculate immunotoxin concentration at each 2-pyridyldisulphide group/molecule, then treated with a time point by reference to a standard curve spanning the 200-fold molar excess of dithiothreitol in NaC1/Pi, pH 5.5, for concentration range 0.25 - 20 ng/ml. 30min at 25°C to remove S-pyridyl groups. The modified a-sarcin was incubated with Fib75 substituted with an average Blood clearance measurements of 1.O 2-pyridyldisulphide groups in NaC1/Pi, pH 7.0, for 20 h at 25 "C. The a-sarcin immunotoxin was purified by gel-perFib75 - a-sarcin was prepared in sterile solution at a conmeation chromatography on Sephacryl S-200 (SF) and affin- centration of 14 pg conjugated RIP/ml. Clearance studies were ity chromatography on Blue Sepharose CL-6B (Knowles and performed in triplicate as described by Worrell et al. (1986b) Thorpe, 1987). Fib75 - ricin-A-chain was prepared as de- following a single intravenous injection of 1 - 2 pg conjugated scribed previously by Forrester et al. (1984) and was used a-sarcin. The concentration of intact immunotoxin in serum without further purification. samples was determined using the ELISA procedure described
205 above. A blood clearance curve was fitted to the determined serum concentrations of the immunotoxin by a computerized non-linear least-squares regression algorithm (Jennrich and Sampson, 1968). The experimental data were most consistent with a two-compartment open pharinacokinetic model described by the biexponential equation c = Ae-'* Be-O', where c is the concentration at time t , and A , B and a, p are the concentration and _rate constants, respectively. The weighting function l/(Y+ q2was applied to all measurements (Ottaway, 1973). a-Sarcin was radiolabelled to a specific activity of approximately 250 pCi 12sI/mgprotein using the Iodo-gen method (Fraker and Speck, 1978). Rats received a single intravenous injection of '"1-a-sarcin in sterile solution containing a total of approximately 1.2 x lo6 cpm. Serum samples isolated between 2 min and 1 h after injection were treated with trichloroacetic acid at a final concentration of 20% (mass/ vol.) for 1 h at 4°C to precipitate protein. The radioactivity present in the precipitate was measured using a Packard 5266 y counter.
+
0.27
0
a,
c
0
40 80 Fraction number
Fraction number
0.003
670 158
The modified a-sarcin used for conjugation contained an average of 0.2 2-pyridyldisulphide group/RlP molecule. At this level of modification, greater than 90% of the derivatized RIP molecules would have been substituted with a single 2pyridyldisulphide group, assuming a distribution governed by the Poisson ratio. The reaction mixture of reduced, derivatized a-sarcin and Fib75 substituted with SPDP was subjected to gel-permeation chromatography on Sephacryl S-200 to separate immunotoxins and unconjugated antibody (molecular mass = 120 - 200 kDa), from unconjugated a-sarcin (molecular mass = 17 kDa) and the low-molecular-mass by-products of the reaction (Fig. 1A). The immunotoxin was further purified by affinity chromatography on Blue Sepharose CL-6B. Fractions 55-61 from the Sephacryl S-200 column were pooled and dialysed into the starting buffer for chromatography on the Blue Sepharose column (Fig. 1 B). Material which eluted from the affinity column unretarded (fractions 8 - 15) was found by SDSjPAGE to consist entirely of unconjugated antibody (not shown). Material that bound to the column and was eluted with 0.5 M NaCl buffer (fractions 33 - 34) represented the final product, Fib75 - a-sarcin at a concentration of 13.4 pg conjugated RIP/ml solution. Analysis of a sample of the final immunotoxin preparation by gel-permeation HPLC revealed that the immunotoxin eluted as a single peak with an elution position corresponding closely to that of Fib75 run under identical conditions, and that no aggregated protein was present (Fig. 1 C). The Fib75 - a-sarcin preparation was also analysed by SDS/PAGE in direct comparison with Fib75 - ricin-A-chain and the unconjugated antibody and RIP components (Fig. 2). Ricin A chain appeared as two bands, corresponding to differently glycosylated forms, with apparent molecular masses of 32 kDa and 34 kDa (lane 1). Fib75 - ricin-A-chain consisted of several bands corresponding to unconjugated antibody and to antibody linked to one, two or three molecules of A chain in order of decreasing mobility (lane 2). Fib75 - a-sarcin consisted predominantly of a single band which had a mobility intermediate between that of unconjugated Fib75 and the monosubstituted ricin A chain immunotoxin species (lane 3) and probably represents a monosubstituted a-sarcin imniuno-
17
1.4
A
RESULTS Synthesis, purification and characterisation ofFib75 - a-sarcin
120
0
4
8
12
16
Elution volume (mi)
Fig. 1. Chromatographic purification and analysis of Fib75 - a-sarcin. (A) Sephacryl S-200 gel-permeation chromatography. The reaction mixture was applied to a column (85 cm x 1.6 cm diameter) of Sephacryl S-200 equilibrated with NaC1/Pi, pH 7.0, and eluted at a flow rate of 15 ml/h. Fractions of 1.5 ml were collected. (B) Blue Sepharose CL-6B affinity chromatography. The mixture of immunotoxin and unconjugated antibody from gel-permeation chromatography was applied to a column (17 cm x 1.5 cm diameter) of Blue Sepharose CL-6B equilibrated with 50mM sodium phosphate, pH 7.0, and eluted at a flow rate of 0.4 ml/miu. Fractions of 2.4 ml were collected. The arrow denotes the start of elution with the same buffer containing 0.5 M NaCl. (C) Gel-permeation HPLC. A sample (0.1 ml) of the Fib75 - x-sarcin preparation purified by affinity chromatography was applied to a TSK-G3000SWXL column (30 cm x 0.78 cm diameter) equilibrated with 20 mM sodium phosphate containing 0.1 M sodium sulphate, pH 6.8, and eluted at a flow rate of 0.4 ml/min. The arrows indicate the elution positions of molecular mass standards (kDa)
toxin. In this preparation, there was no evidence for the presence of unconjugated a-sarcin, which ran as a single main band with a mobility consistent with its expected molecular mass, 17 kDa (lane 4). The preparation did contain a faint band which co-migrated with unconjugated Fib75 (lane 5). The presence of unconjugated antibody may be attributed, at least in part, to a proportion of the Fib75 preparation which was found by subsequent experiments to bind to Blue Sepharose CL-6B and elute under the conditions used for immunotoxin purification. The Fib75 - a-sarcin preparation could be stored frozen at - 70 "C for over one year or kept at 4°C for at least three months without breakdown, loss of material, or formation of aggregates, and retained full cytotoxic activity (see below). Ribosome-inactivating activity of a-sarcin and Fib75 - a-sarcin
The ribosome-inactivating activity of a-sarcin and Fib75 a-sarcin was determined by measuring the inhibition of de
206
100
1
0
Fig. 2. SDSjPAGE analysis of Fib7.5, Fib75 immunotoxins and unconjugated RIP. The samples were run on a 4-12.5% gradient polyacrylamide gel under non-reducing conditions and protein bands were visualized using Coomassie brilliant blue staining. Lane 1, unconjugated ricin A chain; lane 2, Fib75 -rich-A-chain; lane 3, Fib75 - u-sarcin; lane 4, unconjugated a-sarcin; lane 5, unconjugated Fib75. The running positions of protein molecular mass standards (kDa) are shown 120 1 100 -
0
24
48
72
96
Time of incubation (h)
Fig. 4. Effect of incubation upon the ribosome-inactivating activity of u-sarcin, Fib75- u-sarcin, ricin A chain and Fib75 - ricin-A-chain. The ribosome-inactivating activity of samples of unconjugated RIP or immunotoxin, incubated at 37°C for different lengths of time, was measured by the rabbit reticulocyte lysate assay. (A) a-sarcin ( 0 )and Fib75-u-sarcin (0).(B) Ricin A chain ( W ) and Fib75-ricin-Achain (El). Each point represents the mean value of triplicate assays of [3H]leucine incorporation expressed as a percentage of control incubations. The error bars denote the standard deviations from the mean values unless smaller than the symbol used
80 -
ribosome-inactivating activity of the conjugated a-sarcin was consistently lower than that of the unconjugated RIP in several experiments but the difference was twofold or less, and 40 the statistical significance of the difference was low, P > 0.1 by Student’s t-test. Thus, the ribosome-inactivating activity 20 of a-sarcin was largely or completely retained throughout the various steps involved in the synthesis and purification of the 01 Fib75 immunotoxin. In the same assay, ricin had an ICs0 of 1o-1310-1210-11 1 o - l 01 o - 1 ~ o . ~10.’ 7.3 f 3.8 pM (not shown). The ribosome-inactivating activity Concentration of alpha sarcin (M) of ricin A chain was unimpaired following conjugation to Fib75 (Forrester et al., 1984). Fig. 3. Inhibition of cell-free protein synthesis by a-sarcin and Fib75In the cell-free assay, immunotoxin samples were reduced a-sarcin. Protein synthesis by a rabbit reticulocyte lysate mixture was measured in the presence of different concentrations of a-sarcin ( 0 ) with 2-mercaptoethanol before assay to ensure complete reand Fib75-a-sarcin (0).Each point represents the mean value of lease of RIP from the Fib75 antibody and were compared [3H]leucine incorporation into protein determined in three separate with the unconjugated RIP similarly treated with reducing experiments and expressed as a percentage of the incorporation in the agent. In the absence of reduction, Fib75-a-sarcin had an absence of RIP ( > 10000 cpm). The error bars denote the standard 1Cs0 greater than 50 nM, indicating that the ribosome-indeviations from the mean values activating activity of the RIP was blocked by attachment to the antibody, but was recovered following separation of the two components by reduction. The inhibitory activity of n o w protein synthesis in a cell-free rabbit reticulocyte lysate unconjugated a-sarcin was not significantly diminished by (Fig. 3). a-Sarcin gave a dose-dependent inhibition of protein exposure to reducing agent (not shown). synthesis, inhibiting the incorporation of [3H]leucineinto protein at a concentration (IC,o) in the assay mixture of Heat stability of a-sarcin, ricin A chain and immunotoxins The heat stability of a-sarcin and ricin A chain, in conju55 23 pM. The 1Cs0 of the Fib75-a-sarcin preparation tested under identical assay conditions was 110 & 50 pM. The gated and unconjugated forms, was compared by measuring 60 -
I
207 1201
E
2
v)
I'
I
0 Concentration of RIP (M)
Fig. 5. Cytotoxic Cflects oja-sarcin, Fib75-aa-sarcin, ricin A chain and Fib75 - ricin- A-chain axainst the EJ human bladder carcinoma cell line in tissue culture. EJ cells were incubated for 48 h in the presence of different concentrations of a-sarcin ( O ) , Fib75 - a-sarcin (0),ricin A chain ( M ) or Fib75 - ricin-A-chain (n).Each point represents the mean value of ['Hlleucine incorporated by the cells during the final 24 h of incubation, determined from three separate experiments, and exprcssed as a percentage of the incorporation by untreated cultures ( > 40000 cpm). The error bars denote the standard deviations from the mean values unless smaller than the symbols used
the effect of incubating the RIP or immunotoxin under physiological conditions of ionic strength, pH and temperature upon ribosome-inactivating activity. The samples were prepared at the same concentration in buffer containing carrier protein, incubated for different lengths of time under sterile conditions, then diluted in the rabbit reticulocyte assay to give a final concentration of RIP predicted to give between 60% and 80% inhibition of cell-free protein synthesis in each case. Over the 4-day period of incubation, there was no significant diminution in the degree of protein synthesis inhibition given by either unconjugated a-sarcin or Fib75 - a-sarcin (Fig. 4A). In contrast, the ability of ricin A chain to inhibit protein synthesis was lost progressively with incubation, declining from approximately 80% inhibition at the start of the experiment to about 20% inhibition after 4 days of incubation (Fig. 4B). Following conjugation to Fib75, the activity of ricin A chain was completely preserved (Fig. 4 B) suggesting that the attachment of the ricin A chain to the antibody protected it against denaturation. Contparative cytotoxic ej'ects and Fib75 - ricin-A-chain
of Fib75 - a-sarcin
The cytotoxic activities of a-sarcin and Fib75 - a-sarcin were assessed in direct comparison with those of rich A chain and Fib75 - ricin-A-chain by measuring their ability to inhibit the incorporation of [jH]leucine by the EJ human bladder carcinoma cell line which expresses the antigen recognised by Fib75. Fib75 - cc-sarcin and Fib75 - ricin-A-chain caused a dose-dependent inhibition of [3H]leucineincorporation upon continuous incubation with the EJ cells in tissue culture (Fig. 5). The ICSOof Fib75 - ricin-A-chain in these assays was 310 & 220 pM consistent with the ICsOpreviously reported (Forrester et al., 1984). The IC,o of Fib75-a-sarcin, 460 & 360 pM was not significantly different from that of the ricin A chain immunotoxin ( P > 0.5) and both immunotoxins caused greater than 90% inhibition of [3H]leucineincorporation at the highest concentration tested, 10 nM (Fig. 5). The two Fib75 immunotoxins were between approximately 500-
8
16
24
Time after injection (h)
Fig. 6. Pharmacokinetics of a-sarcin and Fib75-a-sarcin in the rat. Normal male Wistar rats received a single intravenous injection of '251-a-sarcin ( 0 )or unlabelled Fib75 -a-sarcin (0).Each point represents the mean value of serum concentration, determined as described in Materials and Methods, expressed as a percentage of the serum concentration measured 2 min after injection. The error bars denote the standard deviations from the mean values unless smaller than the symbols used
fold and 800-fold more cytotoxic than the unconjugated RIP. Rich A chain had an ICSoof 180 40 nM compared with an IC50of 360 150 nM for a-sarcin, a difference that was only weakly significant (P>0.1) although the inhibition of [3H]leucine incorporation at a concentration of 1 pM was significantly less ( P < 0.001) with a-sarcin than with ricin A chain (Fig. 5). Therefore, attachment of either ct-sarcin or ricin A chain to the Fib75 antibody enhanced the cytotoxicity of the RIP to the target cell. The cytotoxic activities of both Fib75 - a-sarcin and Fib75 - ricin-A-chain were completely preserved after incubation in physiological saline buffer at 37°C for up to 4 days (not shown). Pharrnacokinetics of a-sarcin and Fib75 -a-sarcin The pharmacokinetics of Fib75 - a-sarcin in the normal Wistar rat were determined following intravenous administration of a single bolus of immunotoxin. The concentration of the a-sarcin immunotoxin present in serum samples isolated up to 24 h after injection was determined using a solid-phase ELISA detecting intact immunotoxin molecules. The a-sarcin immunotoxin was cleared from the circulation with biphasic kinetics (Fig. 6) that were best described by a two-compartment open pharmacokinetic model. The calculated a- and pphase half-lives were0.80 & 0.11 hand 6.0 & 0.1 h, respectively. The serum concentration of the a-sarcin immunotoxin measured 2 min after injection was not significantly different from the concentration of immunotoxin predicted to be present on the basis of the injected dose. 24 h after the injection, about 4% of the injected dose of immunotoxin remained in the circulation. The blood clearance of unconjugated a-sarcin labelled with ''1 was considerably more rapid than that of the immunotoxin (Fig. 6). The initial half-life of the RIP was less than 5 min. 1 h after injection, less than 2 % of the RIP remained in the bloodstream compared with greater than 80% in the case of the immunotoxin. DISCUSSION In the present study, we have demonstrated the synthesis of an immunotoxin consisting of a-sarcin and a mouse mono-
208 clonal antibody Fib75, and have analysed its important properties. The a-sarcin was stable to the procedures commonly employed in the synthesis and purification of immunotoxins made with plant-derived RIP. The affinity purified a-sarcin immunotoxin was completely stable to storage conditions and to incubation under physiological conditions, had identical cytotoxic activity to a Fib75 immunotoxin made with ricin A chain, and had similar pharmacokinetic properties to analogous Fib75 immunotoxins made with single-chain plant RIP. The ribosome-inactivating activity of the a-sarcin, which was better than or similar to those reported for other preparations of a-sarcin using a variety of cell-free assays of protein synthesis inhibition (Conde et al., 1978, 1989; Fando et al., 1985), was largely or completely preserved during the preparation of the Fib75 immunotoxin. These results are in accord with the findings of Conde et al. (1989) who demonstrated that the modification of restrictocin with SPDP had only a small effect on the ability of the RIP to inhibit protein synthesis in a cell-free assay. The enzymic activity of a-sarcin was completely preserved after incubation in physiological saline buffer at 37°C for up to 4 days in both unconjugated form and as part of the Fib75 immunotoxin. This result confirmed that the activity of a-sarcin was completely stable to physiological conditions, in agreement with its previously reported thermostability (Olson and Goerner, 1965), and that its stability was not lessened following attachment to antibody. In contrast, unconjugated ricin A chain was found to undergo inactivation during the course of incubation. This is consistent with the demonstration that the isolated A and B chains of ricin are conformationally less stable than the intact toxin (Olsnes et al., 1975; Wawrzynczak et al., 1988). Following attachment to the Fib75 antibody, the ricin A chain was stabilised against denaturation. The protective effect of the antibody may have been due to conformational stabilisation of the A chain present in the immunotoxin, as a result of a close molecular association between the two components. The cytotoxic activity of Fib75 -a-sarcin was identical to that of a Fib75 immunotoxin made with ricin A chain in parallel assays and was about 800-fold greater than that of unconjugated a-sarcin against the EJ human bladder carcinoma cell line in tissue culture. Restrictocin has previously been reported to form immunotoxins with cytotoxic activity (Orlandi et al., 1988; Conde et al., 1989). A restrictocin immunotoxin made with a mouse monoclonal IgM antibody MBrl, recognising an antigen associated with human breast carcinoma, had a cytotoxic potency similar to that reported for an MBrl - ricin-A-chain immunotoxin (Canevari et al., 1985) although the cytotoxic activities of these two immunotoxins, and of the unconjugated restrictocin and ricin A chain, were not directly compared in the same assay. In the experiments presented here, a-sarcin was only slightly less toxic to the EJ human bladder carcinoma cell line than ricin A chain. In contrast, Conde et al. (1989) found that restrictocin was consistently less toxic than ricin A chain to a number of human breast carcinoma cell lines in tissue culture. This discrepancy could reflect differences in the susceptibility of different cell lines to intoxication by these RIP or may indicate a real difference between a-sarcin and restrictocin. The relatively weak cytotoxicity of unconjugated a-sarcin reflects the low efficiency with which RIP present in the extracellular milieu can enter intact cells and gain access to the ribosomes. Experiments in which a-sarcin was directly introduced into Xenopus oocytes by microinjection showed that protein synthesis in intact oocytes was rapidly inactivated as
a result of ribosomal modification occurring by the highly specific mechanism of rRNA cleavage demonstrated in vitro (Ackerman et al., 1988). It is reasonable to presume, therefore, that the enhancement of cytotoxic potency upon conjugation to the Fib75 antibody was due to the more efficient delivery of the a-sarcin to the cytosolic compartment following binding of the immunotoxin to the target antigen at the cell surface. Although the cell-free ribosome-inactivating activity of the a-sarcin immunotoxin was less than that of the ricin A chain immunotoxin, the toxic effects of the immunotoxins upon the EJ human bladder carcinoma cell line were identical. This suggests that the rate-limiting step in the action of the immunotoxins was not ribosome inactivation. Preliminary kinetic experiments have shown that Fib75 - a-sarcin and Fib75 - ricin-A-chain inhibited the incorporation of [3H]leucine by EJ cells at an identical rate and did so following a similar lag period after exposure of the cells to the immunotoxins (unpublished results). It is likely that, in this system, the rate-limiting step was the antibody-dependent delivery of RIP to an intracellular compartment favouring entry of the RIP to the cytosol. The mechanism of transmembrane penetration into the cytosol is obscure, but is clearly not a property that is restricted to molecules with the structure of the plant RIP. The clearance of unconjugated 251-a-sarcin from the bloodstream of the rat occurred rapidly (half-life < 5 min) following intravenous administration. Similarly, rapid elimination was reported for restrictocin (Conde et al., 1989), ricin A chain (Worrell et al., 1986b) and other plant RIP (Wawrzynczak et al., 1990) consistent with the expected renal filtration of macromolecules of this size. In contrast, the conjugated a-sarcin persisted in the bloodstream at much higher levels following its attachment to the Fib75 antibody. Fib75 a-sarcin exhibited biphasic clearance kinetics characteristic of the pharmacokinetic behaviour of Fib75 immunotoxins made with ricin A chain and other RIP in comparable experiments (Worrell et al., 1986a; Wawrzynczak et al., 1990). There was no significant loss of Fib75 - a-sarcin from the circulation before the first sample of blood was taken just 2 min after injection. In contrast, Fib75 - ricin-A-chain was previously shown to suffer an early rapid disappearance from the bloodstream because of mannose-dependent receptor-mediated hepatic recognition (Worrell et al., 1986b, c). The aand 6-phase half-lives of the a-sarcin immunotoxin, about 0.8 h and 6 h, respectively, were comparable with the halflives determined in the rat of Fib75 immunotoxins made by similar methodology with the plant single-chain RIP gelonin and momordin: a-phase, 0.4-0.7 h ; P-phase, 8-9 h. The reasons why immunotoxins exhibit biphasic pharmacokinetics, during the first 24 h following administration, are not understood. This phenomenon could be related to the structural heterogeneity inherent in immunotoxin preparations synthesized by chemical procedures with regard to the point of attachment of the RIP to the antibody, but cannot, in this case, be related to differences in the level of substitution of the antibody because the cc-sarcin immunotoxin preparation contained the monosubstituted immunotoxin species almost exclusively. In conclusion, we have demonstrated that a-sarcin is RIP useful for the construction of immunotoxins with intact antibody. The Fib75 -a-sarcin immunotoxin had equal stability and cytotoxic potency, and similar pharmacokinetics, to analogous Fib75 immunotoxins made with plant-derived RIP. YSarcin may prove advantageous over ricin A chain in the context of the future design of immunotoxins intended for
209 cancer therapy because of two useful properties: firstly, its smaller size, and secondly, its greater thermostability. One approach to overcoming the problem of limited penetration of solid tumour masses by large macromolecules such as immunotoxins is to attempt the miniaturisation of the component parts of the immunotoxin. The smaller size of asarcin compared with that of the plant RIP should prove useful in making the smallest possible immunotoxin molecule which retains its component functions. A risk associated with reducing the size of the antibody component is the potential loss of the protective effect that stabilises conjugated ricin A chain. This would not prove a drawback in the case of asarcin which is highly stable in both the conjugated and the unconjugated form. The findings presented in this study indicate that further studies are warranted to determine the antitumour efficacy of a-sarcin immunotoxins in vivo and to explore the construction of immunotoxins with an cc-mcin produced in Escherichin coli by recombinant DNA techniques (Henze et al., 1990). This work was supported by funds from the Cancer Research Campaign and thc Medical Research Council, UK. The financial support to N. U. of Hocchst AG, Frankfurt am Main, FRG, is gratefully acknowlcdged.
REFERENCES Ackerman, E. J., Saxena, S. K. & Ulbrich, N . (1988) J . B i d . Chem. 263, 17076 - 17083. Blakey, D. C., Wawrzynczak, E. J., Wallace, P. M. & Thorpe, P. E. (1988) in Monoclonal antihodj therapy (Waldmann, H., ed.) pp. 50-90, Karger, Basel. Canevari, S., Orlandi, R., Ripamonti, M., Tagliabue, E., Aguanno, S., Miotti, S., Menard, S. & Colnaghi, M. I. (1985) J . Nut1 Cuncer inst. 75, 831 - 839. Conde, F. P., Fernandez-Puentes, C., Montcro, M. T. V. & Vazquez, D. (1978) FEMS illicvohiol. Lett. 4, 349-355. Conde, F. P., Orlandi, R., Canevari, S., Mazzanzanica, D., Ripamonti, M., Munoz, S. M., Jorge, P. & Colnaghi, M. I. (1 989) C t r . J . Biochem. 178, 795 - 802. Cumber, A. J., Forrester, J. A , , Foxwell, B. M . J., Ross, W. C. J. & Thorpe, P. E. (1985) Methods Enzymol. 112, 207-225. Endo, Y. & Tsurugi, K . (1987) J . Biol. Chem. 262, 8128-8130. Endo. Y.. Tsurugi, K . & Lambert, J . M. (1988) Biochem. Biophys. RPS.Commun. 150, 1032-1036.
Endo, Y. & Wool, 1. G. (1982) J . B i d . Chem. 257,9054-9060. Fando, J. L., Alaba, l., Escarmis, C., Fernandez-Luna, J. L., Mendez, E. & Salinas, M. (1985) Eur. J . Biochem. 149, 29 - 34. Fernandez-Luna, J. L., Lopez-Otin, C., Soriano, F. & Mendez, E. (1985) Biochemistry 24, 861 -867. Forrester, J. A,, McIntosh, D. P., Cumber, A. J., Parneli, G. D. & Ross, W. C. J. (1984) Cancer Drug Delivery 1, 283-292. Fraker, P. J . & Speck, J . C. (1978) Biochem. Biophys. Res. Commun. 80, 849- 857. Henze, P.-P. C., Hahn, U., Erdmann, V. A. & Ulbrich, N . (1990) Eur. J . Biochem. 192, 127-131. Jcnnrich, R. I. & Sampson, P. F. (1968) Technometrim 10,63-72. Knowles, P. P. & Thorpe, P. E. (1987) Anal. Biochem. 160,440-443. Lopez-Oh, C., Barber, D., Fernandez-Luna, J. L., Soriano, F. & Mendez, E. (1984) Eur. J . Biochem. 143, 621 -634. Olsnes, S. & Pihl, A . (1982) in Molecufar action uftoxins and viruses (Cohen, P. & van Heyningen, S., eds) pp. 51 -105, Elsevier, Amsterdam. Olsnes, S., Refsnes, K., Christensen, T. B. & Pihl, A. (1975) Biochim. Biophys. Acta 405, 1 - 10. Olson, B. H. & Goerner, G. L. (1965) Appl. Microhiol. 13, 314-321. Olson, B. H., Jennings, J. C., Roga, V., Junek, A. J. & Schuurmans, D. M. (1965) Appl. Microbiol. 13, 322-326. Orlandi, R., Canevari, S., Conde, F. P., Leoni, F., Mezzanzanica, D., Ripamonti, M. & Colnaghi, M. I. (1988) Cancer Zmmunol. Immunother. 26, 114- 120. Ottaway, J. H. (1973) Biochem. J . 134, 729-732. Sacco, G., Drickamer, K. & Wool, I. G. (1983) J . B i d . Chetn. 258, 5811 -5818. Stirpe, F., Bailey, S., Miller, S. P. & Bodley, J. W. (1988) Nuckic Acids Res. 16, 1348-1357. Stirpe, F. & Barbieri, L. (1986) FEES Lett. 195, 1-8. Wawrzynczak, E. J., Cumber, A. J., Henry, R. V., May, J., Newell, D. R., Parnell, G. D., Worrell, N. R . & Forrester, J. A . (1990) Cancer Res. 50, 7519 - 7526. Wawrzynczak, E. J., Drake, A. F., Watson, G. J., Thorpe, P. E. & Vitetta, E. S. (1988) Biochim. Biophys Acta 971, 55-62. Wool, I. G. (1984) Trends Biochem. Sci. 9, 14-17. Worrell, N. R., Cumber, A. J., Parnell, G. D., Mirza, A., Forrester, J . A. & Ross, W. C. J. (1986a) Anti-Cancer Drug Design 1, 179- 188. Worrcll, N. R., Cumber, A. J., Parnell, G . D., Ross, W. C. J. & Forrester, J. A. (1986b) Biochem. Phannacol. 35, 417-423. Worrell, N . R., Skilleter, D. N., Cumber, A. J. & Price, R. J. (1986~) Biochem. Biuphys. Res. Commun. 137, 892 - 896.
0FEBS 1991 0014295691001324
Biochemical, cytotoxic and pharmacokinetic properties of an immunotoxin composed of a mouse monoclonal antibody Fib75 and the ribosome-inactivating protein a-sarcin from Aspergihs giganteus Edward J. WAWRZYNCZAK', Raymond V. HENRY', Alan J. CUMBER', Geoffrey D. PARNELL', Elaine J. DERBYSHIRE' and Norbert ULBRICH2 Institute of Cancer Research, Sutton, England Department of Biochemistry, Free University of Berlin, Federal Republic of Germany (Received August 22/0ctober 31, 1990) - EJB 90 1012
An immunotoxin was synthesized by the attachment of a-sarcin, the ribosome-inactivating protein derived from the mould Aspergillus giganteus, to a monoclonal mouse IgG2a antibody Fib75. The a-sarcin immunotoxin exerted toxic effects in tissue culture against the EJ human bladder carcinoma cell line, expressing the antigen recognised by the Fib75 antibody, inhibiting the incorporation of [3H]leucine by 50% at a concentration of 0.46 nM. The cytotoxic effects of the a-sarcin immunotoxin were indistinguishable from those of a Fib75 immunotoxin made with rich A chain. Fib75-a-sarcin was cleared from the circulation of the rat with biphasic kinetics following intravenous administration. The a- and p-phase half-lives were 0.8 h and 6 h, respectively, similar to the serum half-lives of analogous Fib75 immunotoxins made with ribosome-inactivating proteins derived from plants. a-Sarcin was completely stable in physiological saline buffer at 37 "C, whereas the ribosomeinactivating activity of rich A chain was gradually lost under identical conditions. a-Sarcin may be a valuable alternative to ricin A chain for the construction of therapeutic immunotoxins because of its smaller size and greater thermostability.
Immunotoxins are hybrid protein molecules generated by the covalent linkage of antibodies and toxins such as the A chain of the plant toxin ricin. Rich A chain is a ribosomeinactivating protein (RIP) that modifies eukaryotic ribosomes by a unique catalytic mechanism, cleavage of the N-glycosidic bond at A4324 in 28s rRNA, thereby inactivating cellular protein synthesis irreversibly (Olsnes and Pihl, 1982; Endo and Tsurugi, 1987). Immunotoxins constructed with ricin A chain have been shown to exert selective toxic effects against animal and human cells bearing the target antigens recognised by the antibody component and several of these agents are being evaluated in patients with malignant disease (Blakey et al., 1988). Cytotoxic immunotoxins have also been made with single-chain plant-derived RIP which resemble ricin A chain in size (about 30 kDa), structure, and mechanism of catalytic action (Stirpe and Barbieri, 1986; Stirpe et al., 1988; Endo et al., 1988). a-Sarcin is a RIP, first isolated from the mould Aspergillus giganteus, and was shown to be capable of inhibiting the growth of a number of different animal tumours (Olson and Goerner, 1965; Olson et al., 1965). This basic, nonCorrespondence to E. J. Wawrzynczak, Drug Targeting Laboratory, Section of Medicine, Institute of Cancer Research, Cotswold Rd., Sutton, Surrey SM2 SNG, England Abbreviations. ICs0, concentration at which the incorporation of [3H]leucine is inhibited by 50% ; RIP, ribosome-inactivating protein(s) ; SPDP, N-succinimidy1-3-(2-pyridyldithio)propionate. Enzymes. a-Sarcin (EC 3.1.27. -); ricin A chain (EC 3.2.2. -).
glycosylated protein has properties that differ significantly from those of the plant-derived RIP (Wool, 1984). Firstly, its size (17 kDa) is about half that of the plant RIP (Sacco et al., 1983). Secondly, it bears no similarities to plant RIP at the level of the primary structure (Sacco et al., 1983). Thirdly, its enzymic action upon eukaryotic ribosomes proceeds by a different mechanism, hydrolysis of the phosphodiester linkage between G4325 and A4326 in 28s rRNA (Endo and Wool, 1982). Two other RIP of this type, restrictocin and mitogillin, have been isolated from the mould Aspergillus restrictus. These RIP closely resemble one another, are homologous with asarcin in primary structure and have a similar catalytic activity (Lopez-Otin et al., 1984; Fernandez-Luna et al., 1985; Fando et al., 1985). In this study, we have compared the properties of an immunotoxin consisting of a-sarcin linked to a monoclonal mouse IgG2a antibody Fib75, which recognises a human antigen, with those of a Fib75 immunotoxin made with ricin A chain. We have previously shown that Fib75 -ricin-A-chain exerted selective toxic effects in tissue culture against a human tumour cell line (Forrester et al., 1984). Fib75 - a-sarcin formed a stable immunotoxin with cytotoxic potency indistinguishable from that of Fib75 - ricin-A-chain, and pharmacokinetic properties similar to those of analogous Fib75 immunotoxins made with single-chain plant RIP. The findings presented suggest that a-sarcin may be a valuable alternative to ricin A chain and other plant-derived RIP for the construction of immunotoxins intended for therapy.
204 MATERIALS AND METHODS
Cell-free assays of ribosome-inactivating activity
Materials
The ribosome-inactivating activity of a-sarcin and ricin A chain was determined by measuring the inhibition of [3H]leucine incorporation into newly synthesized protein using a rabbit reticulocyte lysate assay similar to that described by Forrester et al. (1984). Triplicate samples were first treated with 2-mercaptoethanol at a final concentration of 0.2 M for 1 h at 37°C. The effect of incubation at 37°C upon ribosome-inactivating activity was measured using samples of RIP or immunotoxin prepared in Dulbecco's phosphate-buffered saline solution A containing 1 mM disodium EDTA and 0.1 mg/ml S-carboxymethylated bovine serum albumin as the carrier protein, at a final RIP concentration of 6 nM. The solutions were filter sterilized, distributed into sterile plastic tubes fitted with O-ring seals, and incubated for different lengths of time up to 96 h at 37°C. A control sample of each preparation was kept at 4°C for the duration of the experiment. The samples were diluted to final concentrations in the rabbit reticulocyte lysate assay giving 60-80% inhibition of protein synthesis, i. e. 50 pM ricin A chain, or 500 pM a-sarcin.
The monoclonal mouse IgG2a antibody Fib75 was purified from the ascitic fluid of mice bearing the hybridoma cell line LICR-LOND Fib75 by ammonium sulphate precipitation followed by chromatography on immobilised Staphylococcus protein A. Ricin A chain was purified from an aqueous extract of delipidated castor bean cake of Sri Lankan origin, as described by Forrester et al. (1984). Sephadex G-25 (M), Sephadex G-25 (SF), Sephadex G-50 (F), Sephacryl S-200 (SF), Blue Sepharose CL-6B and CNBractivated Sepharose 4B were purchased from Pharmacia Ltd., Milton Keynes, UK. CM52-cellulose was from Whatman Ltd, Maidstone, UK. TSK-G3000SWXL columns were purchased from Anachem Ltd, Luton, UK. Sheep anti-(mouse 1g)-antiserum linked to horseradish peroxidase (NA.931), sodium ['251]iodide (IMS.30) and L[4,5-3H]leucine(TRK.170) were purchased from Amersham International plc., Amersham, UK. Iodo-gen was from Pierce and Warriner Ltd, Chester, UK. S-Carboxymethylated bovine serum albumin was obtained from Sigma Chemical Co. Ltd, Poole, UK. N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP) was obtained from Pharmacia Ltd. All other reagents were of analytical grade or of the highest purity available. Cytotoxicity experiments in tissue culture Normal male albino rats of the Wistar/CBI strain, each of Cytotoxicity experiments using the EJ human bladder mass approximately 250 g, were supplied by the MRC Animal carcinoma cell line were carried out essentially as described Breeding Unit, National Institute of Medical Research, London, England, and allowed free access to food and water. by Forrester et al. (1984). Briefly, dilutions of RIP or immunotoxin solution were added to subconfluent monolayer cultures of EJ cells in triplicate and incubated for 24 h. Preparation of a-sarcin [3H]Leucine (1 pCi) was added to each culture followed by incubation for a further 24 h. The incorporation ~f[~H]Ieucine A . giganteus MDH 18894 was grown at 30°C for 96 h in was then determined by liquid scintillation counting. a rotary shaker at 200 rpm in 2% (mass/vol.) soluble corn starch, 1.5% (massivol.) beef extract, 2% (massjvol.) peptone, 0.5% (massivol.) sodium chloride inoculated with approxi- Measurement of immunotoxin concentration by ELISA mately 6 x lo7 spores/400 ml medium in 1-1 culture flasks. A rabbit antiserum to a-sarcin was raised by the procedure After removal of mycelia by filtration and low-speed centrifugation (10000 x g for 10 min), the medium was adjust- described by Worrell et al. (1986 b). a-Sarcin-specific antibody ed to neutral pH and loaded on to a Whatman CM52 cation- was isolated from the antiserum by affinity chromatography exchange column. Bound protein was eluted with 20 mM Tris/ on a column of a-sarcin linked to CNBr-activated Sepharose HCI containing 1.5 M NaCl, pH 7.2. The protein was then 4B. The solid-phase ELISA procedure used was a modification subjected to gel filtration on a column (150 cm x 4 cm diameter) of Sephadex G-50 (F),equilibrated with 20 mM Tris/HCl, of the method originally described by Worrell et al. (1986a) for the detection of intact immunotoxins made with ricin A pH 7.2. chain. Briefly, affinity-purified anti-(a-sarcin) antibody was first adsorbed to 96-well microELISA plates. Serum samples Synthesis, purification and characterization of Fib75 - a-sarcin containing Fib75 - a-sarcin were added to the plates and, The procedures used for the synthesis of Fib75 - a-sarcin following incubation and washing, the bound immunotoxin were similar in outline to those described by Cumber et al. was detected using sheep anti-(mouse Ig) antiserum linked to horseradish peroxidase in combination with an O-phenyl( 1985) for the preparation of disulphide-linked immunotoxins made with single-chain RIP. a-Sarcin, in 0.1 M sodium phos- enediamine substrate solution developing colour at 492 nm. phate containing 0.14 M sodium chloride (NaC1/Pi), pH 7.0, The mean absorbance at 492 nm of triplicate serum samples was reacted with SPDP to introduce an average of 0.2 was used to calculate immunotoxin concentration at each 2-pyridyldisulphide group/molecule, then treated with a time point by reference to a standard curve spanning the 200-fold molar excess of dithiothreitol in NaC1/Pi, pH 5.5, for concentration range 0.25 - 20 ng/ml. 30min at 25°C to remove S-pyridyl groups. The modified a-sarcin was incubated with Fib75 substituted with an average Blood clearance measurements of 1.O 2-pyridyldisulphide groups in NaC1/Pi, pH 7.0, for 20 h at 25 "C. The a-sarcin immunotoxin was purified by gel-perFib75 - a-sarcin was prepared in sterile solution at a conmeation chromatography on Sephacryl S-200 (SF) and affin- centration of 14 pg conjugated RIP/ml. Clearance studies were ity chromatography on Blue Sepharose CL-6B (Knowles and performed in triplicate as described by Worrell et al. (1986b) Thorpe, 1987). Fib75 - ricin-A-chain was prepared as de- following a single intravenous injection of 1 - 2 pg conjugated scribed previously by Forrester et al. (1984) and was used a-sarcin. The concentration of intact immunotoxin in serum without further purification. samples was determined using the ELISA procedure described
205 above. A blood clearance curve was fitted to the determined serum concentrations of the immunotoxin by a computerized non-linear least-squares regression algorithm (Jennrich and Sampson, 1968). The experimental data were most consistent with a two-compartment open pharinacokinetic model described by the biexponential equation c = Ae-'* Be-O', where c is the concentration at time t , and A , B and a, p are the concentration and _rate constants, respectively. The weighting function l/(Y+ q2was applied to all measurements (Ottaway, 1973). a-Sarcin was radiolabelled to a specific activity of approximately 250 pCi 12sI/mgprotein using the Iodo-gen method (Fraker and Speck, 1978). Rats received a single intravenous injection of '"1-a-sarcin in sterile solution containing a total of approximately 1.2 x lo6 cpm. Serum samples isolated between 2 min and 1 h after injection were treated with trichloroacetic acid at a final concentration of 20% (mass/ vol.) for 1 h at 4°C to precipitate protein. The radioactivity present in the precipitate was measured using a Packard 5266 y counter.
+
0.27
0
a,
c
0
40 80 Fraction number
Fraction number
0.003
670 158
The modified a-sarcin used for conjugation contained an average of 0.2 2-pyridyldisulphide group/RlP molecule. At this level of modification, greater than 90% of the derivatized RIP molecules would have been substituted with a single 2pyridyldisulphide group, assuming a distribution governed by the Poisson ratio. The reaction mixture of reduced, derivatized a-sarcin and Fib75 substituted with SPDP was subjected to gel-permeation chromatography on Sephacryl S-200 to separate immunotoxins and unconjugated antibody (molecular mass = 120 - 200 kDa), from unconjugated a-sarcin (molecular mass = 17 kDa) and the low-molecular-mass by-products of the reaction (Fig. 1A). The immunotoxin was further purified by affinity chromatography on Blue Sepharose CL-6B. Fractions 55-61 from the Sephacryl S-200 column were pooled and dialysed into the starting buffer for chromatography on the Blue Sepharose column (Fig. 1 B). Material which eluted from the affinity column unretarded (fractions 8 - 15) was found by SDSjPAGE to consist entirely of unconjugated antibody (not shown). Material that bound to the column and was eluted with 0.5 M NaCl buffer (fractions 33 - 34) represented the final product, Fib75 - a-sarcin at a concentration of 13.4 pg conjugated RIP/ml solution. Analysis of a sample of the final immunotoxin preparation by gel-permeation HPLC revealed that the immunotoxin eluted as a single peak with an elution position corresponding closely to that of Fib75 run under identical conditions, and that no aggregated protein was present (Fig. 1 C). The Fib75 - a-sarcin preparation was also analysed by SDS/PAGE in direct comparison with Fib75 - ricin-A-chain and the unconjugated antibody and RIP components (Fig. 2). Ricin A chain appeared as two bands, corresponding to differently glycosylated forms, with apparent molecular masses of 32 kDa and 34 kDa (lane 1). Fib75 - ricin-A-chain consisted of several bands corresponding to unconjugated antibody and to antibody linked to one, two or three molecules of A chain in order of decreasing mobility (lane 2). Fib75 - a-sarcin consisted predominantly of a single band which had a mobility intermediate between that of unconjugated Fib75 and the monosubstituted ricin A chain immunotoxin species (lane 3) and probably represents a monosubstituted a-sarcin imniuno-
17
1.4
A
RESULTS Synthesis, purification and characterisation ofFib75 - a-sarcin
120
0
4
8
12
16
Elution volume (mi)
Fig. 1. Chromatographic purification and analysis of Fib75 - a-sarcin. (A) Sephacryl S-200 gel-permeation chromatography. The reaction mixture was applied to a column (85 cm x 1.6 cm diameter) of Sephacryl S-200 equilibrated with NaC1/Pi, pH 7.0, and eluted at a flow rate of 15 ml/h. Fractions of 1.5 ml were collected. (B) Blue Sepharose CL-6B affinity chromatography. The mixture of immunotoxin and unconjugated antibody from gel-permeation chromatography was applied to a column (17 cm x 1.5 cm diameter) of Blue Sepharose CL-6B equilibrated with 50mM sodium phosphate, pH 7.0, and eluted at a flow rate of 0.4 ml/miu. Fractions of 2.4 ml were collected. The arrow denotes the start of elution with the same buffer containing 0.5 M NaCl. (C) Gel-permeation HPLC. A sample (0.1 ml) of the Fib75 - x-sarcin preparation purified by affinity chromatography was applied to a TSK-G3000SWXL column (30 cm x 0.78 cm diameter) equilibrated with 20 mM sodium phosphate containing 0.1 M sodium sulphate, pH 6.8, and eluted at a flow rate of 0.4 ml/min. The arrows indicate the elution positions of molecular mass standards (kDa)
toxin. In this preparation, there was no evidence for the presence of unconjugated a-sarcin, which ran as a single main band with a mobility consistent with its expected molecular mass, 17 kDa (lane 4). The preparation did contain a faint band which co-migrated with unconjugated Fib75 (lane 5). The presence of unconjugated antibody may be attributed, at least in part, to a proportion of the Fib75 preparation which was found by subsequent experiments to bind to Blue Sepharose CL-6B and elute under the conditions used for immunotoxin purification. The Fib75 - a-sarcin preparation could be stored frozen at - 70 "C for over one year or kept at 4°C for at least three months without breakdown, loss of material, or formation of aggregates, and retained full cytotoxic activity (see below). Ribosome-inactivating activity of a-sarcin and Fib75 - a-sarcin
The ribosome-inactivating activity of a-sarcin and Fib75 a-sarcin was determined by measuring the inhibition of de
206
100
1
0
Fig. 2. SDSjPAGE analysis of Fib7.5, Fib75 immunotoxins and unconjugated RIP. The samples were run on a 4-12.5% gradient polyacrylamide gel under non-reducing conditions and protein bands were visualized using Coomassie brilliant blue staining. Lane 1, unconjugated ricin A chain; lane 2, Fib75 -rich-A-chain; lane 3, Fib75 - u-sarcin; lane 4, unconjugated a-sarcin; lane 5, unconjugated Fib75. The running positions of protein molecular mass standards (kDa) are shown 120 1 100 -
0
24
48
72
96
Time of incubation (h)
Fig. 4. Effect of incubation upon the ribosome-inactivating activity of u-sarcin, Fib75- u-sarcin, ricin A chain and Fib75 - ricin-A-chain. The ribosome-inactivating activity of samples of unconjugated RIP or immunotoxin, incubated at 37°C for different lengths of time, was measured by the rabbit reticulocyte lysate assay. (A) a-sarcin ( 0 )and Fib75-u-sarcin (0).(B) Ricin A chain ( W ) and Fib75-ricin-Achain (El). Each point represents the mean value of triplicate assays of [3H]leucine incorporation expressed as a percentage of control incubations. The error bars denote the standard deviations from the mean values unless smaller than the symbol used
80 -
ribosome-inactivating activity of the conjugated a-sarcin was consistently lower than that of the unconjugated RIP in several experiments but the difference was twofold or less, and 40 the statistical significance of the difference was low, P > 0.1 by Student’s t-test. Thus, the ribosome-inactivating activity 20 of a-sarcin was largely or completely retained throughout the various steps involved in the synthesis and purification of the 01 Fib75 immunotoxin. In the same assay, ricin had an ICs0 of 1o-1310-1210-11 1 o - l 01 o - 1 ~ o . ~10.’ 7.3 f 3.8 pM (not shown). The ribosome-inactivating activity Concentration of alpha sarcin (M) of ricin A chain was unimpaired following conjugation to Fib75 (Forrester et al., 1984). Fig. 3. Inhibition of cell-free protein synthesis by a-sarcin and Fib75In the cell-free assay, immunotoxin samples were reduced a-sarcin. Protein synthesis by a rabbit reticulocyte lysate mixture was measured in the presence of different concentrations of a-sarcin ( 0 ) with 2-mercaptoethanol before assay to ensure complete reand Fib75-a-sarcin (0).Each point represents the mean value of lease of RIP from the Fib75 antibody and were compared [3H]leucine incorporation into protein determined in three separate with the unconjugated RIP similarly treated with reducing experiments and expressed as a percentage of the incorporation in the agent. In the absence of reduction, Fib75-a-sarcin had an absence of RIP ( > 10000 cpm). The error bars denote the standard 1Cs0 greater than 50 nM, indicating that the ribosome-indeviations from the mean values activating activity of the RIP was blocked by attachment to the antibody, but was recovered following separation of the two components by reduction. The inhibitory activity of n o w protein synthesis in a cell-free rabbit reticulocyte lysate unconjugated a-sarcin was not significantly diminished by (Fig. 3). a-Sarcin gave a dose-dependent inhibition of protein exposure to reducing agent (not shown). synthesis, inhibiting the incorporation of [3H]leucineinto protein at a concentration (IC,o) in the assay mixture of Heat stability of a-sarcin, ricin A chain and immunotoxins The heat stability of a-sarcin and ricin A chain, in conju55 23 pM. The 1Cs0 of the Fib75-a-sarcin preparation tested under identical assay conditions was 110 & 50 pM. The gated and unconjugated forms, was compared by measuring 60 -
I
207 1201
E
2
v)
I'
I
0 Concentration of RIP (M)
Fig. 5. Cytotoxic Cflects oja-sarcin, Fib75-aa-sarcin, ricin A chain and Fib75 - ricin- A-chain axainst the EJ human bladder carcinoma cell line in tissue culture. EJ cells were incubated for 48 h in the presence of different concentrations of a-sarcin ( O ) , Fib75 - a-sarcin (0),ricin A chain ( M ) or Fib75 - ricin-A-chain (n).Each point represents the mean value of ['Hlleucine incorporated by the cells during the final 24 h of incubation, determined from three separate experiments, and exprcssed as a percentage of the incorporation by untreated cultures ( > 40000 cpm). The error bars denote the standard deviations from the mean values unless smaller than the symbols used
the effect of incubating the RIP or immunotoxin under physiological conditions of ionic strength, pH and temperature upon ribosome-inactivating activity. The samples were prepared at the same concentration in buffer containing carrier protein, incubated for different lengths of time under sterile conditions, then diluted in the rabbit reticulocyte assay to give a final concentration of RIP predicted to give between 60% and 80% inhibition of cell-free protein synthesis in each case. Over the 4-day period of incubation, there was no significant diminution in the degree of protein synthesis inhibition given by either unconjugated a-sarcin or Fib75 - a-sarcin (Fig. 4A). In contrast, the ability of ricin A chain to inhibit protein synthesis was lost progressively with incubation, declining from approximately 80% inhibition at the start of the experiment to about 20% inhibition after 4 days of incubation (Fig. 4B). Following conjugation to Fib75, the activity of ricin A chain was completely preserved (Fig. 4 B) suggesting that the attachment of the ricin A chain to the antibody protected it against denaturation. Contparative cytotoxic ej'ects and Fib75 - ricin-A-chain
of Fib75 - a-sarcin
The cytotoxic activities of a-sarcin and Fib75 - a-sarcin were assessed in direct comparison with those of rich A chain and Fib75 - ricin-A-chain by measuring their ability to inhibit the incorporation of [jH]leucine by the EJ human bladder carcinoma cell line which expresses the antigen recognised by Fib75. Fib75 - cc-sarcin and Fib75 - ricin-A-chain caused a dose-dependent inhibition of [3H]leucineincorporation upon continuous incubation with the EJ cells in tissue culture (Fig. 5). The ICSOof Fib75 - ricin-A-chain in these assays was 310 & 220 pM consistent with the ICsOpreviously reported (Forrester et al., 1984). The IC,o of Fib75-a-sarcin, 460 & 360 pM was not significantly different from that of the ricin A chain immunotoxin ( P > 0.5) and both immunotoxins caused greater than 90% inhibition of [3H]leucineincorporation at the highest concentration tested, 10 nM (Fig. 5). The two Fib75 immunotoxins were between approximately 500-
8
16
24
Time after injection (h)
Fig. 6. Pharmacokinetics of a-sarcin and Fib75-a-sarcin in the rat. Normal male Wistar rats received a single intravenous injection of '251-a-sarcin ( 0 )or unlabelled Fib75 -a-sarcin (0).Each point represents the mean value of serum concentration, determined as described in Materials and Methods, expressed as a percentage of the serum concentration measured 2 min after injection. The error bars denote the standard deviations from the mean values unless smaller than the symbols used
fold and 800-fold more cytotoxic than the unconjugated RIP. Rich A chain had an ICSoof 180 40 nM compared with an IC50of 360 150 nM for a-sarcin, a difference that was only weakly significant (P>0.1) although the inhibition of [3H]leucine incorporation at a concentration of 1 pM was significantly less ( P < 0.001) with a-sarcin than with ricin A chain (Fig. 5). Therefore, attachment of either ct-sarcin or ricin A chain to the Fib75 antibody enhanced the cytotoxicity of the RIP to the target cell. The cytotoxic activities of both Fib75 - a-sarcin and Fib75 - ricin-A-chain were completely preserved after incubation in physiological saline buffer at 37°C for up to 4 days (not shown). Pharrnacokinetics of a-sarcin and Fib75 -a-sarcin The pharmacokinetics of Fib75 - a-sarcin in the normal Wistar rat were determined following intravenous administration of a single bolus of immunotoxin. The concentration of the a-sarcin immunotoxin present in serum samples isolated up to 24 h after injection was determined using a solid-phase ELISA detecting intact immunotoxin molecules. The a-sarcin immunotoxin was cleared from the circulation with biphasic kinetics (Fig. 6) that were best described by a two-compartment open pharmacokinetic model. The calculated a- and pphase half-lives were0.80 & 0.11 hand 6.0 & 0.1 h, respectively. The serum concentration of the a-sarcin immunotoxin measured 2 min after injection was not significantly different from the concentration of immunotoxin predicted to be present on the basis of the injected dose. 24 h after the injection, about 4% of the injected dose of immunotoxin remained in the circulation. The blood clearance of unconjugated a-sarcin labelled with ''1 was considerably more rapid than that of the immunotoxin (Fig. 6). The initial half-life of the RIP was less than 5 min. 1 h after injection, less than 2 % of the RIP remained in the bloodstream compared with greater than 80% in the case of the immunotoxin. DISCUSSION In the present study, we have demonstrated the synthesis of an immunotoxin consisting of a-sarcin and a mouse mono-
208 clonal antibody Fib75, and have analysed its important properties. The a-sarcin was stable to the procedures commonly employed in the synthesis and purification of immunotoxins made with plant-derived RIP. The affinity purified a-sarcin immunotoxin was completely stable to storage conditions and to incubation under physiological conditions, had identical cytotoxic activity to a Fib75 immunotoxin made with ricin A chain, and had similar pharmacokinetic properties to analogous Fib75 immunotoxins made with single-chain plant RIP. The ribosome-inactivating activity of the a-sarcin, which was better than or similar to those reported for other preparations of a-sarcin using a variety of cell-free assays of protein synthesis inhibition (Conde et al., 1978, 1989; Fando et al., 1985), was largely or completely preserved during the preparation of the Fib75 immunotoxin. These results are in accord with the findings of Conde et al. (1989) who demonstrated that the modification of restrictocin with SPDP had only a small effect on the ability of the RIP to inhibit protein synthesis in a cell-free assay. The enzymic activity of a-sarcin was completely preserved after incubation in physiological saline buffer at 37°C for up to 4 days in both unconjugated form and as part of the Fib75 immunotoxin. This result confirmed that the activity of a-sarcin was completely stable to physiological conditions, in agreement with its previously reported thermostability (Olson and Goerner, 1965), and that its stability was not lessened following attachment to antibody. In contrast, unconjugated ricin A chain was found to undergo inactivation during the course of incubation. This is consistent with the demonstration that the isolated A and B chains of ricin are conformationally less stable than the intact toxin (Olsnes et al., 1975; Wawrzynczak et al., 1988). Following attachment to the Fib75 antibody, the ricin A chain was stabilised against denaturation. The protective effect of the antibody may have been due to conformational stabilisation of the A chain present in the immunotoxin, as a result of a close molecular association between the two components. The cytotoxic activity of Fib75 -a-sarcin was identical to that of a Fib75 immunotoxin made with ricin A chain in parallel assays and was about 800-fold greater than that of unconjugated a-sarcin against the EJ human bladder carcinoma cell line in tissue culture. Restrictocin has previously been reported to form immunotoxins with cytotoxic activity (Orlandi et al., 1988; Conde et al., 1989). A restrictocin immunotoxin made with a mouse monoclonal IgM antibody MBrl, recognising an antigen associated with human breast carcinoma, had a cytotoxic potency similar to that reported for an MBrl - ricin-A-chain immunotoxin (Canevari et al., 1985) although the cytotoxic activities of these two immunotoxins, and of the unconjugated restrictocin and ricin A chain, were not directly compared in the same assay. In the experiments presented here, a-sarcin was only slightly less toxic to the EJ human bladder carcinoma cell line than ricin A chain. In contrast, Conde et al. (1989) found that restrictocin was consistently less toxic than ricin A chain to a number of human breast carcinoma cell lines in tissue culture. This discrepancy could reflect differences in the susceptibility of different cell lines to intoxication by these RIP or may indicate a real difference between a-sarcin and restrictocin. The relatively weak cytotoxicity of unconjugated a-sarcin reflects the low efficiency with which RIP present in the extracellular milieu can enter intact cells and gain access to the ribosomes. Experiments in which a-sarcin was directly introduced into Xenopus oocytes by microinjection showed that protein synthesis in intact oocytes was rapidly inactivated as
a result of ribosomal modification occurring by the highly specific mechanism of rRNA cleavage demonstrated in vitro (Ackerman et al., 1988). It is reasonable to presume, therefore, that the enhancement of cytotoxic potency upon conjugation to the Fib75 antibody was due to the more efficient delivery of the a-sarcin to the cytosolic compartment following binding of the immunotoxin to the target antigen at the cell surface. Although the cell-free ribosome-inactivating activity of the a-sarcin immunotoxin was less than that of the ricin A chain immunotoxin, the toxic effects of the immunotoxins upon the EJ human bladder carcinoma cell line were identical. This suggests that the rate-limiting step in the action of the immunotoxins was not ribosome inactivation. Preliminary kinetic experiments have shown that Fib75 - a-sarcin and Fib75 - ricin-A-chain inhibited the incorporation of [3H]leucine by EJ cells at an identical rate and did so following a similar lag period after exposure of the cells to the immunotoxins (unpublished results). It is likely that, in this system, the rate-limiting step was the antibody-dependent delivery of RIP to an intracellular compartment favouring entry of the RIP to the cytosol. The mechanism of transmembrane penetration into the cytosol is obscure, but is clearly not a property that is restricted to molecules with the structure of the plant RIP. The clearance of unconjugated 251-a-sarcin from the bloodstream of the rat occurred rapidly (half-life < 5 min) following intravenous administration. Similarly, rapid elimination was reported for restrictocin (Conde et al., 1989), ricin A chain (Worrell et al., 1986b) and other plant RIP (Wawrzynczak et al., 1990) consistent with the expected renal filtration of macromolecules of this size. In contrast, the conjugated a-sarcin persisted in the bloodstream at much higher levels following its attachment to the Fib75 antibody. Fib75 a-sarcin exhibited biphasic clearance kinetics characteristic of the pharmacokinetic behaviour of Fib75 immunotoxins made with ricin A chain and other RIP in comparable experiments (Worrell et al., 1986a; Wawrzynczak et al., 1990). There was no significant loss of Fib75 - a-sarcin from the circulation before the first sample of blood was taken just 2 min after injection. In contrast, Fib75 - ricin-A-chain was previously shown to suffer an early rapid disappearance from the bloodstream because of mannose-dependent receptor-mediated hepatic recognition (Worrell et al., 1986b, c). The aand 6-phase half-lives of the a-sarcin immunotoxin, about 0.8 h and 6 h, respectively, were comparable with the halflives determined in the rat of Fib75 immunotoxins made by similar methodology with the plant single-chain RIP gelonin and momordin: a-phase, 0.4-0.7 h ; P-phase, 8-9 h. The reasons why immunotoxins exhibit biphasic pharmacokinetics, during the first 24 h following administration, are not understood. This phenomenon could be related to the structural heterogeneity inherent in immunotoxin preparations synthesized by chemical procedures with regard to the point of attachment of the RIP to the antibody, but cannot, in this case, be related to differences in the level of substitution of the antibody because the cc-sarcin immunotoxin preparation contained the monosubstituted immunotoxin species almost exclusively. In conclusion, we have demonstrated that a-sarcin is RIP useful for the construction of immunotoxins with intact antibody. The Fib75 -a-sarcin immunotoxin had equal stability and cytotoxic potency, and similar pharmacokinetics, to analogous Fib75 immunotoxins made with plant-derived RIP. YSarcin may prove advantageous over ricin A chain in the context of the future design of immunotoxins intended for
209 cancer therapy because of two useful properties: firstly, its smaller size, and secondly, its greater thermostability. One approach to overcoming the problem of limited penetration of solid tumour masses by large macromolecules such as immunotoxins is to attempt the miniaturisation of the component parts of the immunotoxin. The smaller size of asarcin compared with that of the plant RIP should prove useful in making the smallest possible immunotoxin molecule which retains its component functions. A risk associated with reducing the size of the antibody component is the potential loss of the protective effect that stabilises conjugated ricin A chain. This would not prove a drawback in the case of asarcin which is highly stable in both the conjugated and the unconjugated form. The findings presented in this study indicate that further studies are warranted to determine the antitumour efficacy of a-sarcin immunotoxins in vivo and to explore the construction of immunotoxins with an cc-mcin produced in Escherichin coli by recombinant DNA techniques (Henze et al., 1990). This work was supported by funds from the Cancer Research Campaign and thc Medical Research Council, UK. The financial support to N. U. of Hocchst AG, Frankfurt am Main, FRG, is gratefully acknowlcdged.
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