Pharmacokinetics in the Rat of a Panel of Immunotoxins Made with Abrin a Chain, Ricin a Chain, Gelonin, and Momordin1

[CANCER RESEARCH 50, 7519-7526. December 1, 1990] Pharmacokinetics in the Rat of a Panel of Immunotoxins Made with Abrin A Chain, Ricin A Chain, Gelonin, and Momordin1

Edward J. Wawrzynczak,2 Alan J. Cumber, Raymond V. Henry, Julie May, David R. Newell,3 Geoffrey D. Parnell, Nan R. Worrell, and J. Anthony Forrester Drug Targeting Laboratory, Section of Medicine Â¡E.J. W., A. J. C., R. V. H., J. M., G. D. P.. N. R. W.. J. A. F.], and Section of Drug Development [D. R. N.J. Institute of Cancer Research, Cotswold Road, Sulton, Surrey, SM2 5A/G, England

ABSTRACT patients (reviewed in Refs. 1-4). Ricin A chain, a RIP isolated from the plant toxin ricin, has A panel of immunotoxins was constructed by chemically attaching the most frequently been used in the synthesis of ITs. The first ribosome-inactivating proteins abrin A chain, ricin A chain, gelonin, and experimental studies in which ricin A chain ITs were adminis momordin to the monoclonal mouse IgG2a antibody Fib75 by means of a disulfide linkage. All the immunotoxins were toxic in tissue culture to tered i.v. to rabbits and rats revealed that the IT was rapidly the I.I human bladder carcinoma cell line expressing the antigen recog lost from the circulation (5-7). Subsequent studies clearly dem nized by Fib75, inhibiting the incorporation of |'U|lcmine by 50% at onstrated that the cause of the initial rapid clearance after concentrations between 1 x IO"10M and 8 x 10"'Â°M.The pharmacoki- injection of the IT was specific receptor-mediated hepatic rec netics of the immunotoxins in the normal Wistar rat was determined ognition of an oligomannose side chain present exclusively on following i.v. administration. The concentrations of intact immunotoxin the A2 form of native ricin A chain (8-10). Three strategies in serum samples taken at various intervals after injection for up to 24 h have been shown to minimize uptake of ricin A chain ITs by were measured by solid-phase enzyme-linked immunosorbent assays the liver in vivo: (a) saturation of the hepatic receptor system specific for each of the four different ribosome-inactivating proteins. The by coadministration of the IT and an excess of mannose- Fib75 immunotoxins were cleared from the circulation with comparable, but not identical, biphasic kinetics best described by a two compartment containing protein or carbohydrate (8, 9, 11); (b) chemical open pharmacokinetic model. The a-phase half-lives of the panel, be treatment of the native ricin to destroy the mannose residues tween 0.35 and 0.71 h, were similar. The #-phase half-life of Fib75-abrin (10); and (c) the use of the A, form of native ricin A chain A chain, 13.3 h, was significantly longer than the Â¿f-phasehalf-lives of which lacks the oligomannose side chain (12). Fib75-ricin A chain, Fib75-gelonin, and Fib75-momordin, between 7.5 An alternative approach to bypassing the problem of hepatic and 8.6 h. Fib75-abrin A chain was found to be about 3- to 4-fold more clearance is to construct ITs with RIPs other than ricin A chain. resistant than the other immunotoxins to breakdown by reduction of the A large family of RIPs of diverse plant origin has now been disulfide linkage between the A chain and the antibody with glutathione identified (13). These RIPs resemble ricin A chain in size and in vitro. This suggests that the longer serum half-life of Fib75-abrin A mode of action but differ from ricin A chain and from one chain may have been due to greater stability against reduction in vivo. Analysis of serum samples obtained up to 24 h after injection of Fib75- another in primary structure, pi, and the degree and type of abrin A chain revealed that the chemically intact immunotoxin present glycosylation. Pharmacokinetic studies have been reported for in the circulation retained full cytotoxic activity. An abrin A chain ITs made with abrin A chain (14), pokeweed antiviral protein immunotoxin made with a different monoclonal mouse IgG2a antibody (15, 16), gelonin (16-21), saporin (17, 18, 22-25), and tricho- was also found to be more stable against reduction by glutathione in vitro kirin (26). A common feature of the majority of these studies than an analogous ricin A chain immunotoxin. Thus, abrin A chain may is the finding that all types of IT have a much shorter half-life possess unique molecular properties that endow immunotoxins made with in the bloodstream than the parent antibody. However, it is this A chain with greater stability in vivo than immunotoxins made with difficult to make direct comparisons between the results of the ricin A chain or other ribosome-inactivating proteins. various studies because of major differences in experimental detail. These differences include: (a) the nature of the antibod INTRODUCTION ies; (b) the methods of IT preparation and purification; (c) the ITs4 are hybrid protein molecules consisting of a toxic protein animal species; (d) the times of the sampling points; (e) the procedures for IT quantification; and (/) the methods of phar linked to an antibody. Many agents of this type can bind macokinetic analysis. selectively to cells that express the target antigen recognized by In this study, we have measured the blood clearance of a the antibody component and kill the cells by the intracellular panel of ITs prepared using a single monoclonal antibody, action of the toxic protein. Immunotoxins made using antibod ies recognizing tumor-associated antigens have been shown to Fib75, which recognizes an integral membrane glycoprotein (M, 19,000) present on most differentiated normal and neo- exert potent and selective cytotoxic effects in animal tumour plastic human cells and not expressed by rodent tissues. Cyto models and are currently undergoing clinical trials in cancer toxic ITs were prepared by similar chemical procedures using Received 6/12/90; accepted 8/30/90. abrin A chain, native ricin A chain, gelonin, and momordin. The costs of publication of this article were defrayed in part by the payment Samples of IT were injected into normal rats i.v. and blood of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. samples were taken at identical times after injection. The con ' This work was supported by funds from the Medical Research Council and centration of IT in serum was determined using RIP-specific the Cancer Research Campaign, United Kingdom. ! To whom requests for reprints should be addressed. ELISAs detecting only intact IT molecules. All the experimen 3Present address: University of Newcastle-upon-Tyne, Department of Clinical tal data were analyzed by the same mathematical procedures. Oncology, Cancer Research Unit, Medical School, Framlington Place, Newcastle- This study showed that the Fib75-abrin A chain IT had the upon-Tyne, NE2 4HH. England. *The abbreviations used are: IT, immunotoxin; ELISA, enzyme-linked im longest blood half-life of the ITs in the panel. The disulfide munosorbent assay: HPLC, high performance liquid chromatography; PAGE, bond linking abrin A chain to the Fib75 antibody was found to polyacry'amide gel electrophoresis; PBS. phosphate-buffered saline; RIP. ribo some-inactivating protein; SDS. sodium dodecyl sulfate; SPDP, /V-succinimidyl- be less susceptible to reduction by glutathione in vitro than the 3-(2-pyridyldithio)propionate. disulfide linkages in the ITs made with the other RIPs suggest- 7519

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. PHARMACOKINETICS OF IMMUNOTOXINS IN THE RAT ing that the longer persistence of the abrin A chain IT may be gelonin and momordin both appeared as single bands with apparent attributed, in whole or in part, to a slower rate of breakdown molecular weights of about 30,500 and 31,000, respectively, by SDS- in vivo. PAGE. Preparation and Characterization of Immunotoxins MATERIALS AND METHODS Abrin A chain and ricin A chain were attached to the Fib75 antibody Materials using the methodology described in detail by Cumber et al. (28). Briefly, 2-pyridyl disulfide groups were first introduced into Fib75 at an average Castor bean cake derived from seeds of Ricinus communis of Kenyan derivatization level of about 1.8 groups/antibody by reaction with origin was obtained from Croda Premier Oils, Hull, Humberside, SPDP. The derivatized antibody was then reacted with an excess of England. Seeds of Abrus precatorius were obtained from the Department freshly reduced toxin A chain. The reaction mixture was applied to a of Botany, University of Ghana, Accra, Ghana. Seeds of Gelonium column of Sephacryl S-200 and the material which eluted at a position multiflorum and Momordica charantia were from United Chemicals corresponding to IT (M, 180,000-210,000) was collected. Immunotox and Allied Products, Calcutta, India. ins made with the 2AL-1 antibody were prepared by the same procedure. The murine hybridoma LICR-LOND Fib75 secreting a mouse mono Gelonin and momordin were modified by reaction with SPDP to clonal antibody of the IgG2a isotype was provided by Dr. R. A. J. introduce an average of 1.0 2-pyridyl disulfide groups/RIP molecule Mcllhinney. The antibody Fib75 was purified from the ascitic fluid of and were reduced with dithiothreitol before mixing with derivatized hybridoma-bearing mice by ammonium sulfate precipitation followed antibody as above. The gelonin and momordin ITs were purified free by chromatography on immobilized staphylococcal protein A using of unconjugated RIP by gel permeation chromatography on Sephacryl stepwise elution. The mouse monoclonal antibody 2AL-1, raised S-200 as for the toxin A chain ITs. against vesicular stomatitis virus and also of the lgG2a isotype, was The IT preparations were analyzed by SDS-PAGE and gel permea purified by a similar procedure. tion HPLC. Electrophoresis was performed on 4.0-12.5% gradient Chromatography media, Sephadex G-25 (F), Sephacryl S-200, Seph- polyacrylamide gels in the presence of 0.2 g SDS/100 ml solution (32). arose 4B, protein A-Sepharose 4B, and CNBr-activated Sepharose 4B, Samples were prepared for electrophoresis in the absence of reducing were purchased from Pharmacia, Ltd., Milton Keynes, Buckingham agent to preserve the disulfide linkage between the antibody and RIP. shire, England. TSK-G3000 HPLC columns were purchased from Gel permeation HPLC was performed on a TSK-G3000SW column Anachem, Ltd., Luton, Bedfordshire, England. (7.5 mm inside diameter x 60 cm). Samples (0.1 ml) were applied to Sheep anti-mouse immunoglobulin-horseradish peroxidase (NA.931 ), the column and eluted at a flow rate of 0.03 ml/min. The running sodium ['"IJiodide (IMS.30), and L-[4,5-'H]leucine (TRK.170) were buffer was 20 mivi sodium phosphate-0. l M sodium sulfate, pH 6.8, purchased from Amersham International pic., Amersham, Buckingh containing 0.05 g NaNi/100 ml solution. The column was calibrated amshire, England. Emulsifier-Safe liquid scintillation cocktail was ob using protein standards of known molecular weight. tained from Canberra Packard Ltd., Pangbourne, Berkshire, England. The RIP content of the final IT preparations was determined as lodo-Gen was purchased from Pierce (UK), Ltd., Chester, England. described by Cumber et al. (28). The content of unconjugated antibody lodoacetamide was obtained from BDH, Ltd., Poole, Dorset, Eng and different IT species in the final preparations was estimated follow land. Glutathione and o-phenylenediamine were purchased from Sigma ing SDS-PAGE by densitometry of Coomassie blue-stained protein Chemical Co., Ltd., Poole, Dorset, England. SPDP was from Phar bands using a Bio-Rad Model 620 video densitometer. macia. All other reagents were of the highest purity available. Tissue culture media, RPMI 1640 and Dulbecco's modified Eagle's Preparation of Affinity-purified Anti-RIP Antibody medium, and fetal calf serum were purchased from Gibco, Ltd., Paisley, Rabbit antisera to abrin A chain, ricin A chain, gelonin, and mo Scotland. Flat-bottomed 96-well micro-ELISA plates (Immulon 2) were ob mordin were produced by the procedure described by Worrell et al. (17). Abrin A chain- and ricin A chain-specific antibody was isolated tained from Dynatech Laboratories, Ltd., Billingshurst, Sussex, Eng from serum by affinity chromatography on columns of A. precatorius land. Sterile tissue culture plates (24 wells) were from Nunclon, Ltd., agglutinin and R. communis agglutinin, respectively, linked to CNBr- Uxbridge, Middlesex, England. activated Sepharose 4B. Gelonin- and momordin-specific antibody was Normal male albino Wistar/CBI rats, 8-16 weeks old, were supplied isolated from antiserum by affinity chromatography on a column of the by the M.R.C. Animal Breeding Unit, National Institute of Medical appropriate RIP linked to CNBr-activated Sepharose 4B. Research, London, England, and allowed free access to food and water. Male New Zealand White rabbits were obtained from J. & L. G. Measurement of Immunotoxin Concentration in Serum Samples by En Phillips, Ltd., Petersfield, Hampshire, England. zyme-linked Immunosorbent Assay

Purification of Ribosome-inactivating Proteins The solid-phase ELISA procedure used was the method originally described for detecting intact ITs made with ricin A chain (33). Briefly, Ricin was purified from an aqueous extract of defatted castor bean affinity-purified anti-RIP antibody was first immobilized on micro- cake by ammonium sulfate precipitation, affinity chromatography on ELISA plates. Serum samples containing IT were added to the plates acid-treated Sepharose 4B, and gel permeation chromatography on and, following incubation and washing, the bound IT was detected Sephacryl S-200 essentially as described by Nicolson and Blaustein (27) using anti-mouse immunoglobulin-horseradish peroxidase in combi with the modifications previously described by Cumber et al. (28). Ricin nation with an o-phenylenediamine substrate solution developing color A chain was isolated from the toxin by reductive cleavage and further at 492 nm. The mean absorbance at 492 nm of triplicate serum samples purified by the method described by Forrester et al. (29). This prepa was used to calculate IT concentration at each time point by reference ration gave two bands with apparent molecular weights of about 30,000 to a standard curve spanning a concentration range of 0.125-15 ng/ and 32,000 when examined by SDS-PAGE corresponding to the two ml. The maximum standard deviation from the determined mean values differently glycosylated forms of the A chain. ranged between 5 and 9% in individual experiments. Abrin was purified from defatted meal of A. precatorius seeds and abrin A chain was isolated from the toxin and purified by procedures Measurement of IT Breakdown in Vitro identical with those described for ricin A chain (28, 29). The preparation gave a single band on SDS-PAGE with an apparent molecular weight ELISA. A stock solution of glutathione was prepared in PBS at a of about 29,000. concentration of 10 mivi and sterilized by passage through a 0.22-Â¿

sulfhydryl groups in dilutions of the stock solution using Ellman's were weighed and the radioactivity present in the samples was deter reagent (34). Sterile solutions of IT containing between 0.3 and 0.5 ug mined in a Packard 5266 gamma counter. of conjugated RIP per ml were prepared in PBS containing 4% (w/w) alkylated rat serum to provide carrier proteins. Each IT solution (0.5 Pharmacokinetic Analysis ml) was mixed with an equal volume of 10 BIM glutathione solution and incubated at 37Â°Cunder sterile conditions. Control samples were Blood clearance curves were fitted to the determined serum levels of IT by a computerized nonlinear least-squares regression algorithm (36). treated in identical fashion except that the glutathione solution was The experimental data were most consistent with a two compartment replaced by PBS. Samples were removed after different times of incu bation up to 8 h and mixed with an equal volume of 20 mM iodoacet- open pharmacokinetic model described by the biexponential equation amide solution in PBS to block free sulfhydryl groups. The concentra C = Ac'"' + Be'1" tion of intact IT in each sample was determined by a solid-phase ELISA similar to the method described above. Rate constants for the slower where C is the concentration at time / and A, B and Â«,ÃŸare the phase of the reaction were calculated assuming pseudo-first order concentration and rate constants, respectively. The weighting function reaction kinetics. \/(Y + Y)2 was applied to all measurements (37). The C0 values were Gel Permeation HPLC. Solutions of glutathione were prepared in calculated from the fitted curves by back extrapolation, i.e., Co = A + PBS at concentrations of 0.1 M, 10 mM, and 1 mM and sterilized by B. The Â«-and /3-phase half-lives were calculated as rw= 0.6931 /a or ÃŸ. filtration. Sterile IT solutions containing between 100 and 170 Â¿igof The area under the serum concentration versus time curve at infinite conjugated RIP per ml were prepared in PBS without the addition of time after injection (ADC) was calculated as the integral of the curve carrier protein. Each IT solution (135 pi) was mixed with 15 Â¿ilof using the formula glutathione solution at the three concentrations. After incubation for 1 h at 37Â°Cunder sterile conditions, iodoacetamide was added to a final ADC = A/a + B/ÃŸ concentration of 13 mM. Control samples were treated in identical fashion except that the glutathione was omitted. Samples (0.1 ml) of the IT solutions were analyzed by gel permeation HPLC on a TSK- RESULTS G3000SWXL column (7.8 mm inside diameter x 30 cm) equilibrated with 20 mM sodium phosphate-0. l Msodium sulfate, pH 6.8, containing Characterization of the Fib75 Immunotoxins. Fib75 ITs made 0.05 g of NaN VI 00 ml of solution at a flow rate of 0.4 ml/min. The with abrin A chain, ricin A chain, gelonin, and momordin were absolute amounts of ricin A chain or abrin A chain released from intact synthesized using closely matched preparative conditions. In IT by coincubation with glutathione were calculated by comparing the each case, the IT preparation was completely separated from peak heights of material eluting with the retention time of the uncon- unconjugated RIP and partially resolved from unmodified an jugated A chains and the peak heights from linear standard curves tibody by a single step of gel permeation chromatography on generated with samples of the appropriate A chain at concentrations of Sephacryl S-200. The four IT preparations all had a similar 1-15 MgA chain/ml. The total A chain content of the ITs was deter composition as judged by SDS-PAGE (Fig. 1). Densitometry mined from the A chain released by incubation of the IT in the presence of 10 mM glutathione for 5 h at 37Â°C. was used to calculate the mean percentage content of the different molecular species and the standard deviations from the mean values. The predominant species present was the Cytotoxicity Experiments in Tissue Culture singly substituted antibody-RIP conjugate [51 Â±3% (SD)J, Cytotoxicity experiments using the EJ human bladder carcinoma containing one RIP molecule linked to one antibody molecule, with lesser amounts of multiply substituted antibody-RIP con cell line were carried out essentially as described by Forrester et al. (29). Briefly, dilutions of IT solution were added to subconfluent jugates [23 Â±12%] and of unconjugated Fib75 antibody [26 Â± monolayer cultures of EJ cells in triplicate and incubated for 24 h. 11%]. All the IT preparations were further analyzed by gel [3H]Leucine (1 jjCi) was added to each culture followed by incubation permeation HPLC. Fig. 2 shows the elution profile of Fib75- for a further 24 h. At the end of this period, the incorporation of abrin A chain which is representative of the elution profiles ['Hjleucine was determined by liquid scintillation counting. The results seen for all the ITs in the figure. This HPLC analysis showed were expressed as a percentage of the ['Hjleucine incorporated by control cultures not receiving IT. The incorporation of [â€¢'Hjleucineby control cultures was greater than 80,000 cpm. 1 Blood Clearance Measurements

Immunotoxins. ITs were prepared in sterile solution at concentrations of conjugated RIP between 109 and 187 Â¿ig/ml.These preparations were checked by gel permeation HPLC shortly before administration to animals in order to ensure the absence of aggregated protein. Clear ance studies Â»ereperformed as described by Worrell et al. (7) following a single i.v. injection of 11-22 ^g of conjugated RIP. The concentration of intact IT in serum samples was determined using the ELISA proce dure described above. The experimental data were adjusted according to the amount of conjugated RIP injected and animal weight in each experiment and are expressed as the serum concentration based on the injection of 10 /jg of conjugated RIP/300 g of rat weight. Ribosome-inactivating Proteins. RIPs were radiolabeled to a specific activity of 200-400 ^Ci '"I/mg protein using the lodo-Gen method (35). In the case of abrin A chain and ricin A chain, the intact toxins were labeled and the A chains were isolated subsequently using the Fig. 1. SDS-PAGE analysis of Fib75 antibody and ITs. Samples were run on a 4-12.5% gradient polyacrylamide gel under nonreducing conditions. Protein usual procedure. Groups of rats, treated as above, received injections bands were visualized by Coomassie Brilliant Blue staining. Lane I, unconjugated of I25I-RIP solution in PBS containing between 1.0 and 1.3 x 106cpm. Fib75; Lane 2, Fib75-abrin A chain; Lane 3, Fib75-ricin A chain: Lane 4. Fib75- Samples of blood taken at intervals between 2 and 30 min after injection gelonin; Lane 5, Fib75-momordin. 7521

the presence of multiply substituted IT species (Fig. 2, Peak A), singly substituted IT (Fig. 2, Peak B), and unconjugated anti body (Fig. 2, Peak C) in proportions consistent with the analysis by SDS-PAGE and of minimal amounts of aggregated protein or unconjugated RIP. 0.1 The Fib75 ITs were all toxic to the EJ human bladder carcinoma cell line in tissue culture. The concentrations at which ['H]leucine incorporation by cells was reduced by 50% for the panel of ITs ranged between approximately 1 x 10 '" and 8 x 10"'Â°M.In direct comparisons of cytotoxicity in vitro, 0.01 J such as in the representative experiment shown in Fig. 3, the 8 12 16 20 24 ITs differed consistently in potency. The order of potency was: Time after injection (h) Fib75-abrin A chain > Fib75-ricin A chain > Fib75-momordin Fig. 4. Blood clearance rates of Fib75 ITs in the normal rat. Normal male > Fib75-gelonin. The difference in cytotoxicity between Fib75- Wistar rats received a single i.v. injection of Fib75-abrin A chain (â€¢).Fib75-ricin abrin A chain and Fib75-ricin A chain was statistically signifi A chain (O). Fib75-gelonin (â€¢).or Fib75-momordin (D). Blood samples were removed at various intervals after injection, and the serum was isolated and the cant: P < 0.02 by Student's / test. The unconjugated RIPs or concentration of intact IT measured using solid-phase ELISA as described under "Materials and Methods." The serum concentration of IT is expressed in terms unconjugated Fib75 antibody exerted no significant cytotoxic effects upon EJ cells at concentrations of 1 x 10~" M (not of tig of conjugated RIP per ml of scrum. Points, mean values of serum concen trations determined on samples from groups of two to three animals per IT. shown). Pharmacokinetics of the Fib75 Immunotoxins in the Rat. The serum concentrations of the four Fib75 IT preparations in In the case of Fib75-ricin A chain, a substantial amount of normal Wistar rats were determined at various intervals up to the IT had disappeared from the bloodstream by the time the 24 h after i.v. injection. All four ITs were cleared from the first sample was taken at 2 min after the injection of the IT circulation with biphasic kinetics (Fig. 4) that were best de solution. This rapid loss was excluded from the analysis in scribed by a two compartment open pharmacokinetic model. order to allow fitting of the two compartment pharmacokinetic The pharmacokinetic data are shown in Table 1. model to the data and afford a direct comparison with the other ITs in the panel (see "Discussion"). The calculated Â«-phase half-lives of the abrin A chain, ricin A chain, and momordin 158 17 0.04 680 l44 I 1.4 ITs, 0.50, 0.70, and 0.71 h, respectively, were not significantly different. The gelonin IT had the shortest Â«-phasehalf-life of 0.35 h. This value was significantly shorter than the half-lives of the ricin A chain and momordin ITs (0.1 > P > 0.05). The 0-phase half-lives of the ricin A chain, gelonin, and momordin c 0.02 ITs, 7.5, 8.0, and 8.6 h, respectively, were similar. In contrast, the /i-phase half-life of Fib75-abrin A chain was 13.3 h. The longer half-life of the abrin A chain IT in the tf-phase of clearance compared with those of the other ITs was highly 0.00 significant (0.01 > P> 0.001). 8 12 1 6 Fib75-gelonin did not suffer the early rapid clearance shown Retention time (h) by Fib75-ricin A chain and thus was present at higher concen Fig. 2. Gel permeation HPLC of Fib75-abrin A chain. Fib75-abrin A chain was applied to a column of TSK-G3000SW and cluted as described under tration in the bloodstream than the ricin A chain IT. However, "Materials and Methods. "Arrows, elution positions of molecular weight standards when compared as a percentage of the calculated C(1values after with values in thousands. 4 and 24 h, the clearance of these two ITs was very similar. The serum concentration of Fib75-gelonin was lower than that of Fib75-momordin from 1 h after injection onwards because the 100 gelonin IT had a shorter Â«-phase half-life and because its Â«- phase was prolonged compared with that of the momordin IT ' o (see Table 1). This indicates that the principal difference in the Ã 60 clearance of the gelonin and momordin ITs occurred shortly after injection since the /i-phase half-lives of the two ITs did not differ significantly. Fib75-abrin A chain persisted in the circulation longer than Fib75-momordin and was present in the highest amount after 24 h, at 18.3% of the calculated Câ€ž.The concentration of the abrin A chain IT was comparable with that of the momordin IT at 4 h after injection but was about 1.5- IO'13 10'12 10" 101Â° 10 10" fold higher at 24 h after injection indicating that the difference Concentration of RIP (M) in the clearance of these two ITs occurred predominantly in the Fig. 3. Cytotoxic effects of Fib75 ITs upon FJ human bladder carcinoma cells 0-phase. As a result, the area under the serum concentration in tissue culture. Monolayer cultures were incubated for 48 h in the presence of different concentrations of Fib75-abrin A chain (â€¢).Fib75-ricin A chain (O). versus time curve for the abrin A chain IT was calculated to be Fib75-gelonin (â€¢),or Fib75-momordin (D). Points, mean values of triplicate 1.5-fold greater than that for the momordin IT. measurements of [3H]leucine incorporated by the cells during the final 24 h of The blood clearance of unconjugated abrin A chain, ricin A culture expressed as a percentage of the incorporation in untreated cultures. The standard deviations from the mean values were <8% and have been omitted for chain, gelonin, and momordin was also measured following i.v. the sake of clarity. administration of '"I-labeled RIPs. In contrast with the rates 7522

Table 1 Pharmacokinetic data for the blood clearance of the Fib75 immunotoxins ofCO4h51.8level (% d)A38.3 intercepts (% of

ITFib75-abrin (h)0.50 (h)13.3 h18.3 kg/ml xh)12.1 A Â±0.08 Â±0.5 Â±3.2 Â±1.7 Fib75-ricin A 0.70 Â±0.16 7.5 Â±0.4 50.6 Â±6.0 49.0 Â±3.5 35.6 5.5 2.9 Fib75-gelonin 0.35 Â±0.08 8.0 Â±0.4 55.8 Â±8.1 46.4 Â±2.8 33.4 5.912.4AUC 5.7 Fib75-momordinIÂ»*' 0.71 Â±0.191*0 8.6 Â±0.2/ero-lime 21.0 Â±2.7B63.6 80.3 Â±2.0Blood 64.324 7.9 * tv,a and /ufi, a- and tf-phase half-lives in blood; A and B, concentration constants in the biexponential equation

C = Be~" for the a- and ff-phases of clearance, respectively: Al'C. area under the serum concentration versus time curve. of clearance of the ITs, all the RIPs cleared very rapidly from chain broke down with a 3-4-fold lower rate constant of 0.043 the bloodstream with an initial half-life of less than 5 min (not h~'. The amount of Fib75-abrin A chain remaining was signif shown). icantly greater than that of the other ITs at all time points and Breakdown of Fib75 and Control Immunotoxins in Vitro. The more than 50% of the starting amount of the abrin A chain IT susceptibility of the panel of Fib75 ITs to breakdown in the was left intact after 8 h of incubation. A similar difference in presence of reducing agent was assessed by incubating a solution the rates of breakdown of the abrin A chain and ricin A chain of each IT in the presence of 5 mivi glutathione at 37Â°Cin vitro. ITs could be detected in the presence of glutathione concentra The amount of intact IT remaining after different times of tions as low as 0.2 mM (not shown). exposure to the glutathione was measured using solid-phase To confirm that the decrease in the amount of intact IT ELISA. In each case, coincubation with glutathione led to a measured by the ELISA was, in fact, due to the cleavage of the progressive decline in the amount of intact IT (Fig. 5) whereas disulfide bond, the release of the A chains from Fib75-abrin A there was no significant loss of IT as measured by the ELISA chain and Fib75-ricin A chain upon treatment with glutathione when glutathione was omitted from the incubation (not shown). was measured directly. Gel permeation HPLC was used to The breakdown of the ITs in the presence of the large (approx measure the absolute amount of A chain released upon incu imately 500,000-fold) molar excess of glutathione used in the bation of the ITs in the presence of different concentrations of experiment would have been predicted to follow pseudo-first glutathione for 1 h at 37Â°Cin vitro (Fig. 6A). The amount of A order reaction kinetics giving a linear relationship between the chain released by glutathione was about 2- to 3-fold lower in logarithm of the IT concentration and time. The biphasic the case of Fib75-abrin A chain consistent with the greater kinetics actually observed indicated that the IT preparations stability of the abrin A chain IT measured by ELISA. The might be heterogeneous with respect to the rate of cleavage of release of A chain by glutathione was also measured directly the disulfide bond. In the more rapid phase of breakdown, the decrease in the amount of intact IT was most pronounced in the case of Fib75-gelonin, greater than 50% loss occurring within 1 h of incubation, compared with the loss of only about 20% of Fib75-abrin A chain (Fig. 5). Fib75-ricin A chain and Fib75-momordin broke down in similar amounts intermediate between those of the abrin A chain and gelonin ITs. In the slower phase of breakdown, the Fib75 ITs made with ricin A 40 chain, gelonin, and momordin all broke down with similar rate constants of between 0.14 and 0.17 h"1 whereas Fib75-abrin A

0 100 n 100

10 0 J 02468 .1 1 10 Time of incubation (h) Concentration of glutathione (mM) Fig. 5. Instability of Fib75 ITs in the presence of glutathione. Solutions of Fib75-abrin A chain (â€¢).Fib75-ricin A chain (O), Fib75-gelonin (â€¢).and Fib75- Fig. 6. Release of A chain from Fib75 and 2AL-1 ITs made with abrin A momordin (O) were each incubated in the presence of 5 mM glutathione at 37Â°C chain and ricin A chain by glutathione. ITs made with abrin A chain (â€¢)orricin in vitro. The concentration of intact IT was determined after various times of A chain (O) linked to the antibodies Fib75 (A) or 2AL-1 (A) were incubated in incubation using solid-phase ELISA as described under "Materials and Methods." the presence of different concentrations of glutathione for I h at 37"C in vitro. Points, mean values of triplicate determinations expressed as a percentage of the The amount of A chain released was measured using gel permeation HPLC as amount of intact IT at the start of the incubation; bars, SD from the mean values described under "Materials and Methods." Points are expressed as the percentage unless smaller than the points as plotted. of A chain released relative to the total A chain content. 7523

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. PHARMACOKINETICS OF 1MMUNOTOXINS IN THE RAT using abrin A chain and ricin A chain ITs made with a control erties, abrin A chain, ricin A chain, gelonin, and momordin, monoclonal antibody of the same isotype, 2AL-1 (Fig. 6B). As were cleared from the bloodstream of the normal rat with with the Fib75 ITs, abrin A chain was released less rapidly from comparable, although not identical, biphasic clearance kinetics; the 2AL-1 antibody than ricin A chain indicating that the (b) Fib75-abrin A chain was eliminated from the bloodstream relative resistance of Fib75-abrin A chain to reduction was not less rapidly in the ÃŸ-phaseofclearance than the Fib75 ITs made an idiosyncracy of the Fib75 antibody. No release of A chains with ricin A chain, gelonin, and momordin; (c) the abrin A was detected in control experiments in which the ITs were chain IT was less susceptible to cleavage via reduction of the incubated under the same conditions but in the absence of (lisiillide bond by glutathione in vitro than the other ITs; (d) glutathione (not shown). Moreover, a Fib75-ricin A chain IT the Fib75-abrin A chain persisting in the circulation of the rat made with a nonreducible thioether linkage did not break down 24 h after injection retained the full cytotoxic activity of the when coincubated with glutathione (not shown). injected IT. Cytotoxic Activity of Fib75-Abrin A Chain in the Circulation. The pharmacokinetic properties of a panel of ITs made with The cytotoxic activity of Fib75-abrin A chain remaining in the abrin A chain, ricin A chain, gelonin, and momordin were circulation after i.v. injection of IT into the normal Wistar rat measured using procedures matched as closely as possible for was determined from serum samples isolated 2 min, 4 h, and each IT. The ITs were prepared using a single mouse monoclo 24 h after injection. The concentration of intact IT was meas nal antibody, Fib75, derivatized with the same cross-linking ured by ELISA. The ability of the serum samples to inhibit the agent, SPDP. The IT preparations used in the pharmacokinetic incorporation of [3H]leucine by EJ human bladder carcinoma experiments were purified using similar procedures, had similar cells in tissue culture was measured. In the case of serial serum composition with respect to the content of singly and multiply samples isolated from two different animals, no significant substituted conjugate species, and exerted cytotoxic effects difference could be detected between the cytotoxic activities of upon the EJ human bladder carcinoma cell line bearing the the serum samples isolated at the different times (Fig. 7). The target antigen recognized by Fib75. The blood clearance rates IC50 values, between 1.5 x 10"'Â°and 2.5 x 10"I0 M, were in of the ITs were determined by administering the ITs to the accord with the determined potency of the abrin A chain IT same strain of rat, sampling blood at identical intervals, and before injection indicating that the activity of the IT was com determining the concentration of IT present in isolated serum pletely preserved throughout the entire period of the clearance using analogous solid-phase ELISAs. The experimental data experiment. were analyzed using the same pharmacokinetic model and mathematical curve fitting procedures. The early rapid clearance of a proportion of Fib75-ricin A DISCUSSION chain within 2 min of injection was consistent with similar The major findings of the present study are as follows: (a) results reported previously by this laboratory (7, 12) and by ITs made using four RIPs with different physicochemical prop- other workers (5, 6). The preparation of native ricin A chain used in this study consisted of a mixture of the two differently

120 glycosylated forms. A, and A2, in a molar ratio of approximately 2:1. The A, chain contains a single complex-type oligosaccha- 100 ride side chain whereas the A2 chain carries an additional oligomannose side chain (38). We have shown previously that 80 the rapid clearance of Fib75-ricin A chain was due to mannose- 60 dependent receptor-mediated hepatic recognition (8). Fib75 IT molecules containing the A2 chain were eliminated virtually 40 completely from the circulation of the rat within l h and those 20 persisting in the circulation after this time were cleared with the same kinetics as a Fib75 IT made with purified ricin A, 0 chain (12). Fib75-gelonin did not show an early loss comparable 120 with the ricin A chain IT preparation despite the fact that gelonin has been reported to contain a single oligomannose side 100 chain (30, 39). This finding is consistent with previous studies 80 of the blood clearance of ITs made with gelonin (16-21) and suggests that the oligosaccharide side chain of gelonin was not 60 recognized avidly by the hepatic mannose receptor system in 40 the rat. Nevertheless, the more rapid elimination of the gelonin IT in the Â«-phasecompared with the other ITs in the panel 20 could indicate that a proportion of the gelonin IT preparation was eliminated by a similar process. However, all the Fib75 10" 10' ITs, including the IT made with abrin A chain, which is not Concentration of abrin A chain (Mi glycosylated (40), had similar Â«-phasehalf-lives. This phase of Fig. 7. Cytotoxic activity of serum samples from rats given injections of Fib75- clearance cannot, therefore, be adequately explained in terms abrin A chain. The cytotoxic effects of serum samples isolated from two rats at 2 of receptor-mediated uptake via carbohydrate recognition. In min (â€¢),4h (O), and 24 h (â€¢)afterthe i.v. injection of Fib75-abrin A chain were deed, other studies have shown that Fib75-abrin A chain is measured against the EJ human bladder carcinoma cell line as described in the legend to Fig. 3. The test samples were prepared to give the concentrations stated taken up by hepatic parenchyma! and nonparenchymal cells in by making the appropriate dilutions of the serum samples based on the concen only very low amounts both in vitro and in vivo (41). tration of intact IT present as measured by ELISA. Points, mean values of triplicate measurements of [3Hjleucine incorporation: bars, SD from the mean The biphasic pharmacokinetics observed with the Fib75 ITs values unless smaller than the points as plotted. in the panel could have been the result of the structural heter- 7524

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. PHARMACOKINETICS OF IMMUNOTOXINS IN THE RAT ogeneity inherent in IT preparations synthesized by chemical measuring the actual release of A chain by glutathione using a procedures. First, the experiments measuring the rate of break quantitative HPLC method. Since glutathione is the most abun down of the Fib75 ITs in vitro indicated the presence of IT dant reducing molecule in the circulation (43), this finding molecules with different susceptibilities to reduction by gluta- strongly suggests that the longer half-life of Fib75-abrin A chain thione. Thus, the a-phase of clearance could reflect the prefer in the 0-phase of clearance compared with the other Fib75 ITs ential elimination of the fraction of IT molecules in which the may be due, either in part or in whole, to a slower rate of disulfide bond is most readily cleaved. However, in a different cleavage of the disulfide bond in vivo. In our experiments, a study, Fib75-ricin A chain ITs made with a disulfide linkage or high concentration of glutathione was used to give readily with a nonreducible thioether linkage had comparable Â«-phase measurable rates of breakdown for comparison. The concentra half-lives (33). Second, IT molecules within a single preparation tion of glutathione in peripheral blood is relatively low but it is differ with regard to the position of attachment of the RIP present at higher levels in the liver, the site of glutathione molecule to the antibody and may differ with respect to the synthesis (43). It is therefore possible that the major site of IT relative configuration of the two components even when linked breakdown by reduction is within the hepatic circulation. via the same position. The more rapid clearance of a proportion The slower rate of breakdown of the IT made with abrin A of IT molecules could have been due to an impairment to the chain in vitro could be associated with the physicochemical ability of the antibody component to persist in the circulation. properties of this RIP. Abrin A chain has a considerably more This might occur either by enhancement of interactions between acidic isoelectric point, pi 4.6 (40), than ricin A chain, gelonin, the conjugated antibody and normal tissues mediated via nat and momordin, pis 7.5, 8.15, and 8.6, respectively (39, 40). It ural mechanisms such as Fc receptor recognition or by novel is possible that a localized electrostatic interaction between interactions made possible by structural changes to the antibody abrin A chain and the Fib75 antibody could induce the hybrid as a result of conjugation. molecule to adopt a more compact conformation in which the The clearance rates of Fib75-ricin A chain, Fib75-gelonin, disulfide bond is partially shielded from attack by reducing and Fib75-momordin were very similar in the /3-phase of clear agents. A similar explanation was suggested previously for the ance suggesting that the different modes of construction nec apparently slower breakdown of an IT made with saporin (44) essarily used in the synthesis of ITs with toxin A chains on the although subsequent pharmacokinetic analyses claimed that one hand, compared with single-chain RIPs on the other, did there was no difference in the rate of splitting compared with not have a major influence upon the /3-phase half-life. In con an analogous ricin A chain IT (24). An alternative explanation trast, the j3-phase half-life of Fib75-abrin A chain was signifi is that the proximity of negatively charged side chains of amino cantly longer than that of the other ITs in the panel. A possible acid residues of abrin A chain in the vicinity of the disulfide explanation for the shorter 0-phase half-life of ITs made with bond causes electrostatic repulsion of glutathione which is ricin A chain, gelonin, and momordin is that these RIPs may negatively charged at physiological pH. Such a mechanism of all contain a complex-type oligosaccharide side chain (38, 39) protection would be a unique feature of abrin A chain since all and that the presence of this structural feature somehow en the other RIPs that have been used for IT construction carry a hances their clearance. One way to test this hypothesis would net positive charge at physiological pH. The relative resistance be to determine the effect of removing the oligosaccharide side to reduction of the abrin A chain IT made with Fib75 was not chain from the RIPs upon the rate of clearance of IT. However, a peculiarity of the Fib75 antibody because an abrin A chain IT it has been reported that the complex-type oligosaccharide side made with another mouse monoclonal antibody of the same chain of ricin A chain cannot be cleaved enzymically without isotype (IgG2a) was also cleaved less readily in vitro than the loss of ribosome-inactivating activity (38). Similarly, we were corresponding ricin A chain IT. It remains to be determined unable to remove the oligosaccharide side-chains from gelonin whether this phenomenon is restricted to antibodies of the and momordin without first denaturing the RIPs.5 We have IgG2a isotype. The ability of an IT made with abrin A chain to persist longer recently found that a Fib75 IT made with an aglycosyl recom binant ricin A chain had a .; phase half-life similar to that of in the circulation than ITs made with ricin A chain, gelonin, Fib75-ricin Ai-chain suggesting that the presence of the com and momordin would be of no advantage were IT molecules plex-type oligosaccharide side chain had no influence in this that are reduced less readily in vitro to possess a diminished phase of clearance.6 cytotoxic potency. The analysis of samples obtained from rats up to 24 h after injection of Fib75-abrin A chain clearly showed The rate at which the disulfide bond linking the antibody and that the IT present in the circulation remained fully active. The RIP can be cleaved by reduction has been shown previously to influence IT half-life in the /3-phase. Thus, ITs made with cross- cytotoxic activity of ITs during the course of blood clearance linking agents such as SPDB (33) and 4-succinimidyloxycar- experiments has rarely been reported. In one study, it was found bonyl-Â«-methyl-Â«-(2-pyridyldithio)toluene (42), which contain that the rate of disappearance of a ricin A chain IT was mirrored exactly by the rate of loss of cytotoxic activity (9). In contrast, hindered disulfide bonds that are less susceptible to chemical reduction, have significantly longer ÃŸ-phasehalf-lives than the clearance studies of a gelonin IT revealed that the cytotoxic activity of the IT remaining in the bloodstream was substan corresponding ITs made with nonhindered linkages. In this study, we found that Fib75-abrin A chain was cleaved less tially diminished (20). rapidly (by 3-4-fold) by glutathione in vitro than the Fib75 ITs In conclusion, we have demonstrated that an IT made with abrin A chain had the highest cytotoxic potency and the longest made with ricin A chain, gelonin, or momordin. Direct evidence serum half-life of a panel of ITs made with different RIPs and for the slower cleavage of the abrin A chain IT was obtained by that the IT retained full cytotoxic activity in the circulation. ' Unpublished results. Our results suggest that the slower clearance of the abrin A 6 E. J. Wawrzynczak, A. J. Cumber, R. V. Henry, and G. D. Parncll. Compar chain IT may have been due to its greater resistance to reduction ative biochemical, cytotoxic and pharmacokinetic properties of immunotoxins made with native ricin A chain, ricin A, chain and recombinant ricin A chain, by glutathione. These findings indicate that abrin A chain can submitted for publication. form ITs with greater stability than ITs made with other RIPs 7525

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. Pharmacokinetics in the Rat of a Panel of Immunotoxins Made with Abrin A Chain, Ricin A Chain, Gelonin, and Momordin

Edward J. Wawrzynczak, Alan J. Cumber, Raymond V. Henry, et al.

Cancer Res 1990;50:7519-7526.

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