Comparison of the Pharmacokinetics and Hepatotoxic Effects of Saporin and Ricin A-Chain Immunotoxins on Murine Liver Parenchymal Cells David C

(CANCER RESEARCH 48. 7072-7078. December 15. 1988] Comparison of the Pharmacokinetics and Hepatotoxic Effects of Saporin and Ricin A-Chain Immunotoxins on Murine Liver Parenchymal Cells David C. Blakey,1 David N. Skilleter, Roger J. Price, Graham J. Watson, Leigh I. Hart, David R. Newell, and Philip E. Thorpe2 Drug Targeting Laboratory, Imperial Cancer Research Fund, Lincoln 's Inn Fields, London WC2A 3PX [D. C. B., G. J. W., P. E. TJ; MRC Toxicology Unit, Medical Research Council Laboratories, Woodmansterne Road, Carshalton, Surrey SMS 4EF [D. N. S., R. J. P.]; and Drug Development Section, Institute of Cancer Research, Clifton Avenue, Sulton, Surrey SM2 5PX fL. I. H., D. R. N.], England

ABSTRACT of Thy-1.1-expressing AKR-A lymphoma cells (22). A single i.v. injection of IT-sap eliminated approximately 99.999% of Immunotoxins containing the ribosome-inactivating protein, saporin, the lymphoma cells growing in the peritoneal cavity of the mice, are very effective antitumor agents but are highly toxic to mice. They whereas an equivalent dose of IT-A eliminated only 99% of the induce severe necrotic lesions in the liver parenchyma of the recipients. cells. Unfortunately, the IT-sap was 5 times more toxic to mice. Such extensive damage to the liver parenchyma is not observed with ricin A-chain immunotoxins even at 5-fold higher dosage. The hepatotoxicity It produced necrotic lesions in the liver parenchymal cells of of the saporin immunotoxins was found in the present study to arise from the mice which were severe enough to account for death (22, a combination of two effects. First, saporin and saporin immunotoxins 23). In contrast, the IT-A caused only minor damage to the were 30- and 6-fold more toxic to primary cultures of mouse liver liver parenchymal cells, even at doses approaching the median parenchymal cells than were ricin A-chain and ricin A-chain Â¡mmunotox- lethal dose (Table 1). The hepatotoxic effects of the IT-sap were ins, respectively. This was despite the fact that the cells bound 4- to 5- not due to its binding to liver cells via the antigen binding sites fold less saporin or saporin immunotoxins than ricin A-chain or ricin A- or the Fc portion of the antibody or to hepatic recognition of chain immunotoxins. The binding of ricin A-chain and its immunotoxin the carbohydrate in the antibody component (22, 23). to the cells was mediated through the carbohydrate residues present on In the present study, we investigated the cause of the hepa the A-chain whereas saporin is not glycosylated and thus must bind to totoxicity of IT-sap. We found that the toxicity appears to arise other sites on the cell surface which result in transport of saporin from a combination of two effects, (a) Isolated mouse liver relatively efficiently to the cytosol. The second reason for the hepatotoxic action of the saporin immunotoxin was that it had a longer blood half- parenchymal cells are about 30-fold more sensitive to saporin life (t:,n = 1.1 h; tifÃŸ=17.1 h) than the ricin A-chain immunotoxin (Iv, and 6-fold more sensitive to IT-sap than to ricin A-chain and = 0.52 h; r.,/i = 9.7 h). Analyses using a two-compartment pharmacoki- IT-A, respectively, (b) IT-sap is not subject to clearance by the netic model showed that the two immunotoxins broke down in vivo to carbohydrate recognition systems of the liver. It therefore per give free antibody at a similar rate (r... = 10-12 h) but that the ricin A- sists for longer periods in the mice giving it more time to chain immunotoxin was eliminated 11 times more rapidly than the saporin interact with the parenchymal cells and kill them. The slower immunotoxin by routes other than breakdown. It was calculated that, in clearance of the IT-sap in vivo may also explain its better mice given a median lethal dose of saporin immunotoxin, the blood levels antitumor activity in the AKR-A tumor model. of immunotoxin remained above the concentration that killed 50% of parenchymal cells in vitro for more than 48 h. In mice given a median lethal dose of ricin A-chain immunotoxin, the blood levels fell below the MATERIALS AND METHODS concentration that was toxic to parenchymal cells in vitro within 4 h. The longer blood half-life of the saporin immunotoxin may also explain Materials. Saporin was kindly supplied by Drs. F. Stirpe and L. our previous finding that it had antitumor activity superior to that of a Barbieri (Istituto di Patologia Generale, UniversitÃ degli Studi di Bo ricin A-chain immunotoxin in mice. logna, Bologna, Italy). It had been isolated from an aqueous extract of the seeds of S. officinalis by ion exchange chromatography on carbox- ymethylcellulose (1) and further purified on a column of Sephacryl S- INTRODUCTION 200(10). Ricin was purified from an aqueous extract of defatted castor bean Saporin is a protein from the seeds of Saponaria officinalis cake as described by Nicolson and Blaustein (24). Highly purified ricin (soapwort) which inactivates eukaryotic ribosomes in a manner A-chain was prepared from the ricin as described previously (25). similar to that of the A-chain of ricin and other plant toxins The hybridoma cell line, MRC OX7, secreting a mouse IgGl anti- (1, 2). Novel antitumor agents (ITs)3 have been synthesized by Thy 1.1 antibody, was kindly provided by Dr. A. F. Williams (MRC attaching these and other ribosome-inactivating proteins to Cellular Immunology Unit, University of Oxford). The antibody was monoclonal antibodies directed against tumor-associated anti purified from the blood and ascitic fluid of hybridoma-bearing mice as gens (3-21). We previously showed that an IT prepared from described by Mason and Williams (26). The hybridoma cell line, LICR- LON-R10, secreting a mouse IgGl subclass antibody to human glyco- saporin and monoclonal anti-Thy-1.1 antibody (OX7) produced phorin, was kindly supplied by Dr. P. A. W. Edwards (Ludwig Institute, an antitumor effect superior to that of an analogous IT prepared Sutton, England). from ricin A-chain when administered to mice bearing allografts Williams E tissue culture medium and fetal calf serum were pur chased from Gibco-Biocult, Ltd. (Paisley, Scotland). SPDP was pur Received 6/2/88; revised 8/31/88; accepted 9/16/88. chased from Pharmacia (Uppsala, Sweden). Na'"I (IMS 30), L-[4,5- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in 3H]leucine (TRK 170) and L-[{/-14C]leucine (CFB 67) were obtained accordance with 18 U.S.C. Section 1734 solely to indicate this fact. from Amersham International (Amersham, England). The lodo-Gen 1Present address: ICI Pharmaceuticals, CTL Building, Alderley Park, Mac- reagent for protein iodination was from Pierce, Ltd., (Chester, Eng clesfield, Cheshire SK10 4TJ, England. 2To whom requests for reprints should be addressed. land). Collagenase type 1A was from Sigma (Poole, England). All other ' The abbreviations used are; IT, immunotoxin; SPDP, A'-succinimidyl-3-(2- reagents were of analytical grade. pyridyldithio)propionate; SDS, sodium dodecyl sulfate; K ',,,. 50% inhibitory Preparation of ITs. Saporin (M, 30,000) and ricin A-chain (M, 30,000 concentration; OX7, monoclonal antibody against Thy-1.1; IT-A, immunotoxin containing ricin A-chain; IT-sap, immunotoxin containing saporin; AB, antibody and 32,000) were coupled to OX7 antibody (M, 150,000). Where released from IT by breakdown in vivo; AUC, area under plasma concentration specified in the text, analogous ITs prepared with RIO antibody, a versus time curve. monoclonal antibody against human glycophorin were used. The cou- 7072

Table 1 Toxic lesions caused by saporiÂ«,ricin A-chain, and ITs and oxidative phosphorylation prevent endocytosis (33). As shown LDso" Oig ricin A or previously, they reduce the uptake of ricin by 85% in rat liver cells and lesions'SaporinIT-sapRicinsaporin/25 g mouse) Major histolÃ³gica! the toxin associated with the cells can be almost completely removed 70 Damagetubulesof to convoluted with galactose (0.1 M) showing that it is attached to the cell surface kidney.25 (32). Severe necrosis of hepatic pa For toxicity measurements, the hepatocyte cultures were incubated renchyma!tored cells. Damage with saporin, ricin A-chain, or ITs for 24 h in serum free medium or spleen.710 pulp of AIT-A1 Destructionnon-parenchymal of hepatic in medium containing 5% (v/v) autologous mouse serum. The cells cells and ger were then washed twice with serum-free medium and 1 Â¿Â¿CiofL-[4,5- Lie-berkuhn.1 minal cells in crypts of 3H]leucine was added to each culture. They were then incubated for a further 2-h period after which the radioactivity incorporated by the cells 20 Destructionnon-parenchyma) of hepatic cells and ger was measured as described by Villa et al. (34). Lie-berkuhnminal cells in crypts of Blood Clearance Measurements. Groups of 3 or 4 adult male HAI .H/ c mice weighing about 25 g each were given i.v. injections of radioio * The median lethal doses (LDsos) for the immunotoxins refer to the quantities dinated saporin, ricin A-chain, ITs, or antibody (10 ng of protein of ricin A-chain or saporin that they contained to facilitate comparison with containing I x 10* cpm). Samples of blood were drawn from the tail unconjugated ricin A-chain or saporin. The LDwS of the immunotoxins in terms of total protein were approximately 6-fold greater. The route of administration vein of mice after various time intervals. The radioactivity in the blood was i.p. samples (50

IT at 1=0 same rate as native OX7. This assumption is validated by the fact that OX7 derivatized with SPDP followed by reduction with dithiothreitol is cleared from the bloodstream at a rate identical to that for native OX7 and by the fact that the values of A",0for AB released from ITs 1 are identical to that of the /Go for native OX7 (results not shown). The IT infâ€”bloodX.*sÂ¿IT in tissues second assumption is that conversion of IT to AB is a first order or pseudo-first order process. This would be true, for example, for gluta- IT Kspl thione-mediated splitting since the plasma levels of glutathione are in splitting large excess over those of the IT (40). The finding that IT-As are stable in whole blood in vitro (28) does not conflict with this possibility because, in vitro, glutathione is very rapidly eliminated by processes not K,0(AbjAB involving oxidation by molecular oxygen (40). Conversion of IT to AB in bloodN.enzyme mediated, provided that the enzyme obeyed Michaelis-Menton Fig. l. Two compartment pharmacokinetic model. kinetics and that the levels of IT were well below the Km. The third assumption is that the elimination of IT and the breakdown of IT to (39), i.e. AB occurs in the central compartment (i.e., the blood, or in tissues in immediate contact with the blood). AUC (AB) Toxicity to Mice. Graded doses of saporin, ricin A-chain, or ITs were AUC (OX7) injected i.p. into groups of 3 BALB/c mice weighing about 25 g each. The median lethal dose was calculated according to the method of Weil The AUC (OX7) was calculated from the values generated by the nonlinear least-squares regression analysis of the plasma concentration (41). versus time data after injection of OX7. The AUC (OX7) at infinite time is given by RESULTS

AUC (OX7) = - + - Inhibition of Protein Synthesis in Reticulocyte Lysates a ÃŸ Native saporin reduced the rate of incorporation of [3H] The AUC<_(AB) was calculated using the trapezoidal rule (39) and leucine into protein in a reticulocyte lysate by 50% at a concen corrected to infinity by adding tration of 8.6 x 10"" M. Seventy-two % of the inhibitory activity was lost when saporin was derivatized with one or two mole AUC,.^ (AB) = â€”¿ cules of SPDP, coupled to OX7 antibody, and released from P the antibody with dithiothreitol (Table 2). By contrast, native where C, is the plasma concentration of AB at the final time point t ricin A-chain was about 4-fold more active than native saporin and ÃŸisthe /3-phase rate constant for OX7. at inhibiting protein synthesis in the reticulocyte lysate assay The microscopic rate constant, ATio,isthe sum of all the rate constants and no loss in inhibitory activity was seen when it was coupled for processes which result in irreversible loss of IT from the animal. to OX7 antibody with SPDP and released with dithiothreitol Thus (Table 2). As a result, the IT-A was 14-fold more effective at inhibiting protein synthesis in reticulocyte lysates than the IT- sap. Release of the saporin or ricin A-chain moiety from the where Kv>is the rate constant for the splitting of IT to AB and Kfl is antibody by reduction of the disii Itide linkage with dithiothreitol the sum of the rate constants for elimination of IT by processes other was essential for potent inhibitory activity to be expressed. than splitting. The values of A,,,,,and K,\ were calculated as follows: Unreduced ITs did not inhibit protein synthesis in the lysates Since at concentrations up to 3.3 x 10~9M.

Toxicity to Hepatocyte Cultures AI Saporin was about 30-fold more toxic than ricin A-chain to mouse liver parenchymal cells in vitro. Saporin reduced their [3H]leucine incorporation by 50% at a concentration of 2.1 x AUC (AB) 10~9M, as compared with 5.6 x 10~" M for ricin A-chain (Fig. AUC (OX7) 2). Attachment of saporin or ricin A-chain to OX7 antibody reduced their toxicity to parenchymal cells 13- and 3-fold, respectively (Fig. 2). The IT-sap had an IC50 of 2.8 x 10~8M whereas the IT-A had an IC50 of 1.6 x IO"7M. Analogous ITs The half-life with which the IT split up to AB is given by

log,2 0.693 Table 2 Inhibition of protein synthesis in reticulocyte lysate A",,, The concentration of reduced material required to inhibit [3H]leucine incor Â»â€¢pi poration by 50% in rabbit reticulocyte lysates was determined as described previously (31). Both ITs were prepared with OX7 antibody. The results shown The half-life with which the IT is eliminated by processes other than are the arithmetic mean values of two K

IT-A Table 3 Uptake and binding of saporin, ricin A-chain, and ITs by mouse liver 100 parenchymal cells in vitro Parenchymal cells (1.5 x 10') were incubated at 37'C for 60 min with 3 x 10"' M radioiodinated saporin, ricin A, or ITs. The uptake of radioactivity by the 80 cells was then measured and is expressed below relative to the amount of cellular protein found in the cultures or on a molecules per cell basis. One mg of cellular ricin A protein is equivalent to approximately 0.75 x 10*parenchymal cells. For binding S-o 60 measurements, cells were preincubated with deoxyglucose and azide to inhibit 11 endocytosis; the radioactive reagents were then added and the radioactivity associated with the cells was measured 60 min later as above. The values shown co 40 are the geometric mean Â±SD of 3 determinations. ND, not determined. Uptake Binding 20 IT-sap Approximate Approximate fmol/mg cell molecules/ fmol/mg cell molecules/ protein cell protein cell OLI 10' 10'' Saporii!OX7-sapOX7-sap122 118Â± 10 Â±121 117 +0X7Â°RIO-sapRicin Concentration (M) Â±218 Â±115 Â±178 Â±162 Fig. 2. Cytotoxicity of saporii), ricin A-chain, and ITs to mouse liver paren AOX7-AOX717Â± +595 Â±666 chyma! cells in vitro. Monolayer cultures of mouse liver parenchyma! cells (2 X Â±52Â± Â±2ND11,30014,50013,70012,10049,80053,100ND 1(1"cells) were incubated at 37'C in the presence of the indicated concentrations 113,70017,70016,90014,50062,70076,4001,60014Â± of saporin. IT-sap, ricin A-chain or IT-A. The ITs were prepared with OX7 " 100-fold excess of unlabeled OX7 antibody. antibody which does not bind to the cells. Cellular protein synthesis was deter mined 24 h later by pulsing cells for 2 h with [3H]leucine and measuring the radioactivity incorporated as described in "Materials and Methods." The radio binding is due to recognition of carbohydrate on the A-chain activity incorporated by treated cultures is expressed as a percentage of that in untreated cultures (mean incorporation, 39,230 cpm). Each point represents the by receptors on parenchymal cells (42). However, saporin is geometric mean of triplicate measurements; bars, SD. free from carbohydrate and thus must bind to other sites on the cell surface. prepared from an antibody of different specificity (RIO) had Incubation of saporin or ricin A-chain with parenchymal cells toxicity similar to that of the OX7 ITs showing that the toxicity for a shorter period of time (20 min) resulted in the uptake of to parenchyma! cells was not due to antigen binding (results 4,000 and 22,000 molecules/cell, respectively. The uptake of not shown). saporin and ricin A-chain by parenchymal cells therefore ap Saporin, ricin A-chain, and the ITs had the same toxicity pears to be linear with time up to 60 min. when applied to the cells in the presence of 5% autologous mouse serum as they did in its absence showing that the greater Pharmacokinetics toxicity of saporin and the IT-saps was not due to their inter Blood Half Lives. Fig. 3 shows the amounts of IT-sap and action with a plasma component to form a toxic complex which IT-A that remained in intact (M, 180,000) form in the blood was then taken up by the cells. plasma of mice at various times after injection. The clearance curves were biphasic having an initial rapid a-phase followed Uptake in Hepatocyte Cultures by a slower /8-phase. The a- and /3-phase half-lives and various We explored the possibility that the toxicity of IT-saps to other pharmacokinetic parameters were determined by analyz parenchyma! cells exceeded that of IT-As because the cells were ing the plasma levels of ITs using a two-compartment open able to bind and endocytose saporin and saporin containing pharmacokinetic model (Fig. 1). These values are listed in Table molecules in greater amounts. This was done by incubating 4. parenchymal cells with 3 x IO"9M radioiodinated saporin, ricin The IT-A disappeared from the bloodstream about twice as A-chain, or ITs for 60 min in the presence or absence of rapidly as the IT-sap during the a- and ^-phases of clearance. deoxyglucose and azide and measuring the radioactivity asso Also, more IT-A than IT-sap was lost from the plasma during ciated with the cells. The agents were used under conditions the Â«-phase.The percentage of drop in the blood level of IT-A which we previously established stopped endocytosis of ricin during the a-phase was 91% as compared with 67% of the IT- (32). It was found that the cells bound and took up about one- sap. Twenty-four h after injection, 11% of the IT-sap remained fourth as much saporin and the IT-saps as they did ricin A- in the bloodstream as compared with only 1% of the IT-A. The chain and the IT-A (Table 3). In the presence of inhibitors of disappearance of the IT-sap (f%a = 1.1 h; tv,ÃŸ=17.1 h) was, endocytosis, the cells each bound 11,000-14,000 molecules of however, still faster than that of the native antibody (tv,a = 3.0 saporin and IT-saps as compared with about 50,000 molecules h;fv,/3= 114h). of ricin A-chain and IT-A. In the absence of inhibitors of The AUC for the IT-A was 17.9 /ig/ml x h as compared with endocytosis, the numbers of molecules bound and taken up by 83.7 tig/ml x h for the IT-sap. The AUC of the IT-sap was, the cells were only slightly (1.2- to 1.4-fold) greater than in therefore, 4.7-fold greater than that of the IT-A. their presence. Control experiments showed that the uptake of In accordance with our previous studies (22, 43), free ricin radioiodinated OX7 antibody alone was small (1,600 mole A-chain and saporin were very rapidly cleared from the blood cules/cell) compared with that of the ITs and that addition of stream. The clearance of free saporin was faster than that of a 100-fold excess of un labeled OX7 antibody had no effect on free ricin A-chain. Ten min after injection of ricin A-chain, the uptake of OX7-sap (Table 3). Thus the binding and uptake 15% of the radioactivity remained in the bloodstream as com of the ITs appear to be mediated primarily through the inter pared with only 4% of the free saporin. The ce- and /3-phase action of the saporin and ricin A-chain moieties with the half-lives for ricin A-chain were 0.08 and 2.1 h, respectively. parenchymal cell surface. We have shown previously that bind Too few useful data points were gathered for saporin to permit ing of ricin A-chain and IT-A is reduced by more than 50% by pharmacokinetic analysis. chemical deglycosylation of the toxin or by competition with Stability. In accordance with previous results (15,17, 28, 30), 0.1 M mannose or fucose showing that the major portion of autoradiographs of SDS-gels of blood samples from mice given 7075

100-1 IT-A r 50 the bloodstream by routes other than breakdown to free anti body was about 11-fold faster than that of the IT-sap (Table 5). The half-life (i^el) for the elimination of the IT-A was 1.5 h as

-6 compared with 16.1 h for the IT-sap. This difference was expected from our earlier finding that, in mice, IT-As are o trapped by the parenchymal and nonparenchymal cells of the liver which have receptors that recognize the oligosaccharides -0.5 present on ricin A-chain (28, 42). Saporin is free from carbo C o hydrate (1) and thus homes to the liver in smaller amounts and to different receptors (23).

0.1 -1 0.05 In support of this, the K^s at steady state were 98 and 104 O) o ml/kg for OX7 and IT-sap, respectively. In contrast the Vafor C 100-1 b) IT-sap â€¢¿SO o the IT-A was 211 ml/kg suggesting that the IT-A is trapped by o tissues whereas the distribution of IT-sap is similar to that of co OX7 and is mainly restricted to the blood and extracellular (O fluids. 10- -5 JD a. DISCUSSION

-0.5 1- t One of the major findings of this study was that saporin was approximately 30-fold more cytotoxic to mouse liver parenchy mal cells in vitro than was ricin A-chain. The high sensitivity of parenchymal cells to saporin is unusual (IC5o 2.1 x 10~9M). 0.1 -J "T" â€”¿r~ â€”¿Iâ€” 1-0.05 Other cells such as the lymphoma cell lines AKR-A and EL4 24 48 72 96 and mouse splenic T- and B-cells have the same low sensitivity to saporin as they do to ricin A-chain, with ICso values in the Time after injection (hours) range 1 x 10~8-2 x 10"7M (22). Fig. 3. Blood clearance rates of ITs and release of free antibody. Mice were A possible explanation for the high sensitivity of parenchymal given i.v. injections of 10 Â«igofradioiodinated IT-A or IT-sap. Both ITs were prepared with OX7 antibody. Blood samples were removed various periods of cells to saporin is that they have a cell surface molecule to time later and the plasma was electrophoresed on polyacrylamide gels in SDS. which saporin binds and which delivers the protein with high Autoradiographs of these gels were then scanned by densitometry to determine efficiency to the cytosol. This is suggested by the binding studies the percentage of radioactivity in each blood sample that corresponded to intact which showed that, at a concentration of 3 x 10~9 M, the IT (M, \ 80,000) ( ) orto released antibody (M, 150,000) ( ). Each point represents the geometric mean of triplicate measurements on each of 3 or 4 mice/ parenchymal cells bind about 11,000 molecules of saporin per group; bars, SD. cell in l h as compared with fewer than 2,000 molecules of immunoglobulin under the same experimental conditions. injections of radioiodinated IT-A or IT-sap revealed that both However, in these studies we did not distinguish between spe ITs were unstable in vivo and broke down progressively to give cific and nonspecific binding. More detailed experiments are a long lived product (M, 150,000) corresponding to free anti required to demonstrate conclusively that saporin binds to a body. We have shown previously that the breakdown product specific receptor molecule on the parenchymal cell surface. from an IT-A is reactive with anti-mouse immunoglobulin Ricin A-chain bound to the cells in greater number (50,000 antibody but not with anti-ricin antibody indicating that it is molecules/cell) than saporin yet had low toxicity. The binding indeed free antibody (28). Free saporin and free ricin A-chain of ricin A-chain was expected from our previous studies (42, (M, 30,000) were not seen at any time point on the gels probably 43) which showed that the carbohydrate (probably the fucose because they were very rapidly cleared and were lightly labeled. residue) of the A-chain was recognized by mouse parenchymal The rate at which IT-sap broke down to release free antibody cells in vivo and that chemical modification of the carbohydrate was the same as that of IT-A. Both ITs split up with a half-life prevents this recognition. It is therefore likely that binding to (fviSpl) of 10-12 h (Table 5). However, because of the lesser carbohydrate receptors is not a route through which ricin A- tissue entrapment of IT-sap (see below), a greater fraction of chain can express potent cytotoxicity. Saporin is free from the injected dose of IT-sap was available for splitting than of carbohydrate (1) and thus must bind to other sites on the surface IT-A. The fraction of injected IT-sap that split up was 5.5-fold of parenchymal cells and ones which relatively efficiently trans greater than that of IT-A (Table 5). port saporin to the cytosol. It is possible that, after binding to Elimination. The rate at which the IT-A was eliminated from these sites, saporin is efficiently delivered to the trans-Golgi

Table 4 Pharmacokinetic data for ITs, native OX7, and A-chain valueB32.6 level Material residence 0

Table 5 Splitting and elimination rates oflTs In conclusion the liver toxicity of IT-sap in vivo can be explained as: (a) parenchymal cells are unusually sensitive to IT (h) (h) saporin and the IT-sap; (b) the lack of carbohydrate-mediated IT-sap 0.61 Â±0.02 10.3 16.1 clearance of the IT-sap gives it a long blood half-life and more IT-A 0.11 Â±0.01 12.3 1.5 Â°fid, fraction of IT that splits to give free antibody; fuspl, half-life with which time for toxic amounts to accumulate in the liver parenchyma. IT splits to give free antibody; r el. half-life of elimination of IT by routes other The slower clearance of the IT-sap in vivo may also explain than splitting. why it had antitumor activity superior to that of an analogous IT-A in the AKR-A tumor model (22). network which has been suggested by the studies of Van Deurs et al. (44) to be the site of ricin A-chain translocation to the ACKNOWLEDGMENTS cytosol. Alternatively, saporin may resist catabolism better than ricin A-chain, leaving more material in active form to enter the We thank Dr. A. Creighton and Dr. E. Vitella for Iheir helpful cytosol. commenis on ihis manuscripi and Audrey Beckel for excellent typing Attachment of saporin to antibody decreased its toxicity to and secretan al assistance. parenchymal cells by 13-fold. This can partly be explained by the fact that its inhibitory effect on cell-free protein synthesis was reduced by 4-fold after conjugation. The remainder of the REFERENCES decrease in cytotoxicity, which is also seen when ricin A-chain 1. Stirpe, F., Gasperi-Campani, A., Barbieri, L., Patasca, A., Abbondanza, A., is attached to antibody, may be because the antibody portion of and Stevens, W. A. Ribosome-inactivating proteins from the seeds of Sapon the IT changes the fate of the saporin and ricin A-chain after aria officinali! L. (soapwort), oÃAgrostemma githago L. (corn cockle) and of Asparagus officinalis L. 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S., and Uhr, J. W. Immunotoxins. Annu. Rev. Immunol., 3:197- which have receptors that recognize the oligosaccharides 212, 1985. present on ricin A-chain (28, 42). The longer blood half-life of 6. Vitetta, E. S., Fulton. R. J., May, R. D., Till, M., and Uhr, J. W. Redesigning the IT-sap compared with the IT-A may explain why it gave a nature's poisons to create anti-tumor reagents. Science (Wash. DC), 238: much better antitumor effect in the AKR-A lymphoma model 1098-1104, 1987. 7. Blakey, D. C., Wawrzyncazak, E. J., Wallace, P. M., and Thorpe, P. E. (22) at equivalent dosage. In support of this, chemical degly- Antibody toxin conjugates: a perspective. In: H. Waldmann (ed.), Progress cosylation of ricin A-chain has, in the same model, been found in Allergy, Basel: S. Karger AG, in press, 1988. 8. Jansen, F. K., Blythman, H. E., Carriere, D., Casellas, P., Gros, O., Gros, to extend the in vivo half-life of an IT-A and improve its P., Laurent, J. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1988 American Association for Cancer Research. Comparison of the Pharmacokinetics and Hepatotoxic Effects of Saporin and Ricin A-Chain Immunotoxins on Murine Liver Parenchymal Cells

David C. Blakey, David N. Skilleter, Roger J. Price, et al.

Cancer Res 1988;48:7072-7078.

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