[CANCER RESEARCH 54, 2650-2653, May 15, 19941 Effects of Sonicated Eosinophils on the in Vitro Sensitivity of Human Lymphoma Cells to Glucose Oxidase'

Michael K. Samoszuk,2 Vincent Nguyen, Cherry T. Thomas, and Dawn M. Jacobson

Pathology Department, University of California, Irvine, California 92717

ABSTRACT cokinetic properties in vivo compared to native enzyme, as is the case for ricin A chain (11, 12)? What concentration of glucose oxidase can We report here that cultured human lymphoma cells in the absence of be attained within lymphoma implants in vivo? sonicated eosinophils are sensitive to killing by glucose oxldase (@3-D- Perhaps the most interesting biological issue that needs to be glucose:oxygen..oxido reductase; EC 1.1.3.4) at concentrations as low as 0.025pg/mI,a levelthat canberapidlyattainedin s.c.tumorimplantsin addressed was raised by our recent immunoscintigraphic fmding that mice that receive a single nonlethal injection of enzyme. Multiple clono certain human lymphomas contain extensive extracellular deposits of genie assays were used to measure the survival of human lymphoma cell eosinophil peroxidase (13). Because eosinophil peroxidase increases lines (H9 and ARH-77) cultured for 14 days in complete RPMI 1640 the cytotoxic activity of hydrogen peroxide in the presence of Br, 1, supplemented with exogenous glucose oxidase (0.025—23@igJnil)oran or SCN (14, 15), we hypothesized that sonicated human eosinophils Immunoconjugate containing glucose oxidase (0.25—25pg/mI) in the pres could be used to increase the tumoricidal activity of glucose oxidase ence or absence of catalase (10 @igJml)oran equal number of sonicated as measured in a clonogenic assay. This report describes the results of human eosinophils with or without supplemental 100 pas Br, 1, or SCN. our experiments that were intended to explore these issues prior to the In addition, we used an immunoassay to measure the concentration of start of clinical safety studies using glucose oxidase in lymphoma glucose oxidase in s.c. implants of the Sp 2/0 myeloma tumor at 0—30mm patients. after an l.v. Injection of 50 @agofenzyme into 21 BALB/c mice. Doses of glucose oxidase as small as 0.025 @aaJmlkilled more than 3 logs of tumor cells. Catalase completely inhibited, and sonicated human eosinophfls MATERIALS AND METhODS partially inhibited, the killing by glucose oxidase or immunoconjugate, whereas supplemental halides had no effect. Glucose oxldase i.v. produced Glucose Oxidase and Immunoconjugate. Glucose oxidase was purified, levels >0.04 pg/g of tumor for 30 mm after injection with a peak concen processed, and analyzed for enzymatic activity as described previously (10). tratlon of 0.079 pg/g of tumor within 5 mEnofinjection. These results are An immunoconjugate directed against human eosinophil peroxidase was crc important because certain human lymphomas contain extensive extracel ated by cross-linking glucose oxidase with glutaraldehyde to a murine mono lular deposits of eoslnophll peroxidase, thereby making these tumors clonal antibody called EOS (10, 13). potentially less susceptible to killing by otherwise therapeutic doses of In order to quantify the immunoreactivity of the immunoconjugate,we used glucose oxidase. a Lineweaver-Burke approach as described by Schuhmacher et aL (16). In brief, serial 2-fold dilutions of sonicated eosinophils (starting at 2 X i0@ cells/mi) or a negative control cell line were incubated for 60 mm with a fixed INTRODUCTION concentration (30 @.iWml)of immunoconjugate (approximately 50 units/mg). Glucose oxidase is a Mr 160,000 flavoenzyme that is produced by After the cells were washed, the bound conjugate was measured spectropho tometrically by its glucose oxidase activity (10). The immunoreactivity data Aspergillus niger. The enzyme is not present in mammalian cells. were then analyzed using a double inverse plot of immunoconjugate binding to Under physiological conditions, it catalyzes the following reactions: target cells over the range of cell concentrations. The immunoreactive fraction at infmite antigen excess was then extrapolated by calculating the reciprocal of @3-n-Glucose+ enzyme-FAD .-* enzyme-FADH2 + D-glucono-6-lactone the intercept on the total/bound (Y) axis. Pharmacokinetics of Deglycosylated Glucose Oxidase. We attempted to Enzyme-FADH2 + 02 -@enzyme-FAD + H202 reduce the clearance of glucose oxidase by carbohydrate receptors in the mouse liver by deglycosylating the enzyme with a-mannosidase and endo Some lymphomas and carcinomas are sensitive to killing by H202 glycosidase H (Boehringer-Mannheim, Indianapolis, IN) using a method de (1—9).Consequently, in vitro and in vivo studies have demonstrated scribed by Kalisz et aL (17). Following overnight digestion of the glucose that in the presence of adequate oxygen concentrations, glucose oxi oxidase with the deglycosylating enzymes, the deglycosylated glucose oxidase dase has potent tumoricidal activity (4—8)mediated in part by hydro was purified by low pressure liquid chromatography over a hydroxylapatite gen peroxide. Lymphomas in particular have an intrinsic sensitivity to column (Bio-Rad Laboratories, Richmond, CA) using a linear gradient of the toxic effects of hydrogen peroxide (5—8).The mechanisms of phosphate buffer from 10 to 400 mMat pH 6.8. The deglycosylated glucose cytotoxicity of hydrogen peroxide are incompletely understood at this oxidase eluted as a single sharp peak at a phosphate concentration of approx time but are believed to be mediated by free radical production. imately 40 mM. In recent preclinical studies, we have demonstrated that glucose After a buffer exchange into phosphate-buffered saline (jH 7.4) using a oxidase has relatively predictable toxicities and is probably safe for Sephadex PD-b column (Pharmacia, Piscataway, NJ), the enzyme was asia lyzed for purity by silver-stained sodium dodecyl sulfate-polyacrylamide gel initiating Phase I human studies (10). Before such clinical trials can electrophoresis (Bio-Rad), which showed a single broad band at approximately begin, however, it will be necessary to answer some important ques M, 75,000. Purified enzyme was then filter sterilized and stored refrigerated up tions. For example, what effect does glucose oxidase have on the to 14 days before use. The enzymatic activity of the deglycosylated enzyme survival of cultured lymphoma cells as measured in a clonogenic averaged 105 units/mg of protein (compared to approximately 200 units/mg for assay? Does deglycosylated glucose oxidase have superior pharma the native enzyme). In order to measure the blood half-life of the deglycosylated enzyme, 18 Received 1/12/94; accepted 3/16/94. adult BALB/c mice (20—22gbody weight) were given i.v. injections of 100 The costs of publication of this article were defrayed in part by the payment of page ,Lgof enzyme in a volume of 200 @.tlofphosphate-buffered saline. At intervals charges. This article must therefore be hereby marked advertisement in accordance with of 0, 15, 30, 45, 60, and 120 mm after injection, groups of three mice were 18 U.S.C. Section 1734 solely to indicate this fact. phlebotomized, and the serum concentration of glucose oxidase was measured 1 This work was supported by USPHS Grant NIH R29 CA 48713. 2 To whom requests for reprints should be addressed, at Medical Sciences D-440, with a capture enzyme-linked immunosorbent assay (10). The serum half-life Pathology Department, University of California, Irvine, CA 92717. was extrapolated from a plot of mean serum concentration versus mm after 2650

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sonication procedure was intended to mimic the in vivo degranulation of eosinophils. Pilot clonogenic assays performed with glucose oxidasc and intact eosinophils demonstrated no significant effect on survival. Catalase was tested because of its ability to neutralize hydrogen peroxide. Sonication was performed for 10 s at medium power (setting, 50) using a Vibra Cell ultrasonicator(Sonics and Materials,Inc., Danbury,CT) and 3.6 X 106 eosinophils suspended in 1.5 ml of ice-cold complete cell culture medium. Microscopic examination of the sonicate revealed amorphous cellular debris and intact granules which could be precipitated by centrifugation. When measured with a colorimetric assay, greater than 90% ofthe peroxidase activity could be removed by centrifugation, suggesting that less than 10% of the .• pesoxidase was liberated by somcation into the fluid phase. In a subsequent set of experiments, the lymphoma cells were cocultured with glucose oxidase plus sonicated eosinophils in the presence of supplemen ta.l1, Br, and SCN (100 p.Meach). For negative control purposes, we also performedclonogenic assays consistingof untreatedlymphomacells only or lymphomacells plus somcatedeosinophilsonly. Fig. 1. Cytopreparation of cosinophils purified from human blood by hypotonic lysis The lymphomacells from each experimentwere plated at serial 10-fold method.The cytopreparationwasincubatedfor3 mm in a chromogenicsubstratefor dilutions (beginning at 2500 cells/ed) into 24 wells of 96-well microtiter eosinophil peroxidase (aminoethyl carbazole plus hydrogen peroxide; 0.01%) and then plates. After 14 days of growth in a humidified CO2 incubator, the number of counterstained with hematoxylin. Note that the morphologically intact eosinophils are free wells showing growth at each concentrationwas counted by microscopic of contaminatingbloodcellsandretaincytoplasmicperoxidaseactivity.Eosinophilsare recognizablebytheirtypicalbibbedor unilobednucleus,incontrasttoneutrophilswhich examination, and the following formula (Spearman estimator) was used to contain a multilobed nucleus. The purity ofthe cosinophils was also confirmed by Wright calculate the number of surviving clonogenic units (9) at each dose of glucose stainingofcytopreparation.X400. oxidase (18): 0 = (e')(e@)

injection. For comparative purposes, a similar experiment was also performed where .y = 0.57722 (Euler's constant) and @iiscalculated as: using 100 ,.ig of native (glycosylated) enzyme. Purification of Eosinophll& For the studies described in this report, a — x,+d @@ novel andsimple methodwas used to purifyeosinophilsfromhumanperiph ,@—2 —d cml blood samples. The method exploited the relative resistance of human eosinophils to hypotomc lysis. In brief, fresh, EDTA-anticoagulated blood In the latter formula, x@= ln z, where z is the highest concentration of the samples (3-10 ml) from patients with eosinophil counts >500/pJ were centri original suspension used; d in a, where a = 10 is the serial dilution factor; @ fuged at 250 X g to sediment the blood cells. After removal of the plasma and p1is the proportion of wells showing growth at dilution z@(where = the layer, the cell sediment was transferred to 50-nil conical centrifuge tubes. number of serial dilutions tested). Under the conditions used in these experi Foreach 1 ml of sediment,44 ml of sterile,distilledwaterwere added,and ments (four serial 10-fold dilutions into 24 wells at each dilution), the maxi the tubeswere immediatelycapped.For the next 60 s, the tubeswere gently mum numberof clonogemc units that could be estimated if growth were inverted and reinverted without producing foaming. At the end of 60 s, 5 ml of observed in all wells at all dilutions was 1760 UnitS/mi. lox phosphate-buffered saline were added to each tube to restore the solution We also attemptedto study a model of tumor cell killing in which the to isotonicity, and the semipurified eosinophils were pelleted by centrifuging lymphoma cells were first pelleted with sonicated eosinophils and then inca the mixture at 600 X g for 7 miii. At this speed, the intact eosinophils settled bated for 1 h in the presence of varying concentrations of glucose oxidase to the bottomof the tubes,while most of the cellulardebrisfrom lysis of the immunoconjugate directed against cosinophil peroxidase. This model was other cells remained in suspension. intended to mimic an in vivo situation in which the lymphoma cells are The wine-red supernatant was then removed, and the cell pellet was resus intimately admixed with degranulated eosinophils that can bind the peroxide pended in 1 ml of phosphate-buffered saline, transferred to a 1.5-ml micro generating immunoconjugate. Following the incubation with immunoconju centrifuge tube, and centrifuged at 4000 rpm for 2 missin an Eppendorf Model gate, the lymphomacells were washed, and the clonogenic assay was per 5415 centrifuge. The thin red layer of debris was carefully removed by formed as described above. aspiration, and the small beige pellet of eosinophils was then washed in saline Accumulation of Glucose Oxidase in Tumor Implants. Adult BALB/c and counted after staining with phloxine dye. mice received s.c. inoculations into the flank of approximately 10@Sp 2/0 This simple method routinely recovered >80% of the eosinophils in a blood murine myeloma cells Obtainedfrom ascites passage. When the tumors reached specimen in <45 lull) with >85% viability by trypan-blue exclusion. The approximately 10 mm in diameter, the mice received a single i.v. injection of preparations typically contained >95% pure eosinophils, with only rare con 50 g@gofnative glucose oxidase. At 5-mistintervals from 0—30mm,groups of taminating mononuclear and R.BCs as assessed by Wright staining of cyto three mice were sacrificed, the tumors were removed, and the concentrations preparations. Moreover, the eosinophils purified by this method were morpho of glucose oxidase were measured in the tumor homogenates by a capture logically Intact and retained their pemxithsc activity (Fig. 1). For use in the enzyme-linked immunosorbent assay (10). subsequent clonogenic assays, the purified eosinophils were cryopreserved in 90% fetal calf serum-10% dimethyl sulfoxide and then rapidly thawed before RESULTS sonication. Clonogenic Assays. In order to characterize the tumoricidal properties of Glucose Oxidase Immunocoi@jugate. The double inverse Liz glucose oxidase and EOS-glucose oxidase in greater detail, we performed eweaver-Burke plot of the binding of the immunoconjugate to som clonogenic assays with two human lymphoma cell lines, AR.H-77 (a B-cell line cated eosinophils is illustrated in Fig. 2. On the basis of the Y-axis infected with Epstein-Barr virus) and H9 (a 1-cell lymphoma). Both cell lines intercept, the immunoreactivity of the immunoconjugate was extrap were obtainedfrom the AmericanType CultureCollection (Rockville, MD) olated to be 65%. By contrast, intact eosinophils as the target yielded and cultured in RPMI 1644)with 10-20% fetal calf serum (complete medium). an immunoreactivity of less than 8%. In the initial series of experiments (performed in duplicate), the lymphoma cells were suspended in complete medium containing varying concentrations Pharmacokinefics of Deglycosylated Glucose Oxidase. The so of unconjugatedglucoseoxidase (0.025—25g.ig/ml)inthe presenceor absence rum half-life of deglycosylated glucose oxidase was determined to be of an equal number of purified, sonicated human eosinophils or catalase approximately 48 min, compared to a serum half-life of45 mm for the (Sigma Chemical Co., St. Louis, MO) at a concentration of 10 @ig/ml.The native enzyme. Because there was no substantial difference in the 2651

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half-life between native and deglycosylated forms of glucose oxidase, we elected to use only native enzyme in the subsequent studies. Clonogenic Assays. The two cell lines that were tested yielded essentially identical results in duplicate assays, and representative data from a clonogenic assay of the ARH-77 cell line plus glucose oxidase I are presented in Fig. 3. In the absence of eosinophils, glucose oxidase at concentrations as low as 0.25 p@g/mlcompletely sterilized the cell cultures (no detectable growth). Eosinophils consistently produced I partial neutralization of the killing by glucose oxidase, and supple mental F, Br, and SCN (not plotted) did not reverse the neutral ization. The Spearman estimate of the number of clonogenic units in the negative controls (tumor cells only and tumor cells plus sonicated 0.1 0.01 eosinophils only) was 1760 clonogenic units/mi. Catalase (10 @g/ml) Immunoconjugate (units/mi) completely neutralized the killing by glucose oxidase, also yielding an Fig. 4. Clonogenic assay using H9 cell line incubated with varying concentrations of estimate of 1760 clonogenic units/ml at all concentrations of glucose immunoconjugate (approximately 50 units/mg) in the presence (0) or absence (•)of sonicated eosinophils. In the absence of sonicated eosinophils, there was a significant oxidase tested. (P < 0.05) decrease in the number of clonogenic units at the two highest concentrations Representative data from the studies with the immunoconjugate and of immunoconjugate.Similarresultswere obtainedwith the ARH-77cell line, and the H9 cell line pelleted with sonicated eosinophils are shown in Fig. addition of supplemental halides did not alter the apparent protective effect of the sonicated eosinophils. Note that the sensitivity of the target cells to killing by the 4. In this model, the sonicated eosinophils again seemed to provide immunoconjugate in this assay was less than the sensitivity to killing by tinconjugated protection from killing by the immunoconjugate because tumoricidal glucose oxidase (Fig. 3) because the conditions of the assay were slightly different, as activity was observed only in the absence of eosinophils. Studies with described in the text. the ARH-77 cell line produced similar results and are not shown. Accumulation ofGlucose Oxidase in Tumor Implants The time Table 1 Concentration ofglucose oxidase in s.c. implants of course of glucose oxidase accumulation in s.c. tumor implants is sp2/0myelomatumorcells mminBALB/c mice having s.c. implants of Sp 2/0 myeloma tumor (approximately 10 tabulated in Table 1. Notably, there was significant accumulation of timeintervalsdiameter) received a single i.v. injection of 50 @igofglucose oxidase. At varying theamountafter injection, the tumors were removed, weighed, homogenized, and enzyme-linkedimmunosorbentof glucose oxidase in the homogenate measured by a capture assay.Mean concentrationMinutes injectedafter ±1 SD % of injectiontumor0 (@.tgIgoftumor) dose/g 05 not detectable 0.1610 0.079±0.28(n3) 0.1615 0.079 ±0.01(n 3) 0.1620 0.078 ±0.06(n 3) 0.0825 0.041 ±0.016(n 3) 0.1230 0.061 ±0.027(n 3) 0.046 ±0.015(n 3) 0.10

@ 0@ , , , , , II I I I 1 I I 1 3 5 7 9 11 13 15 l/CeUDMuflon glucose oxidase (0.16% of the injected dose/g of tumor) only 5 mm Fig. 2. Immunoreactivity plot of the EOS monoclonal antibody conjugated to glucose after injection, and levels in excess of0.04 p@g/goftumor persisted for oxidase using sonicated eosinophils as the target. Extrapolation of the curve to the Y-axis at least 30 mm after injection. intercept yields an immunoreactivity estimate of 65%. DISCUSSION

Various forms of glucose oxidase (native enzyme, enzyme conju gated to monoclonal antibodies, and enzyme adsorbed to polystyrene microspheres) have been proposed as potential antineoplastic agents (1—8).The data from our studies indicate that relatively low concen trations of native glucose oxidase (0.025—0.25 @ig/ml)are highly I —.— No Eosinophils tumoricidal to cultured lymphoma cells in a clonogenic assay, pro -0 Eo@noph@ vided that sonicated (degranulated) eosinophils are not present. This finding is significant because many human lymphomas (particularly Hodgkin's disease) contain extensive deposits of eosinophil peroxi dase, as recently demonstrated by our in vivo immunoscintigraphic studies with radiolabeled EOS antibody (13). Thus, eosinophil de 0.5 0.05 0.005 0.0005 Gkicose oxidase (units/mi) granulation within tumors represents a previously unrecognized and Fig. 3. Results of clonogenic assay using ARH-77 cells incubated with varying potentially significant problem that may interfere with antineoplastic concentrations of glucose oxidase (approximately 200 units/mg) in the presence (0) or therapy using glucose oxidase. absence (•)of sonicated eosinophiis. At glucose oxidase concentrations of 0.005 and In our experiments, the neutralization of the tumoricidal activity of 0.0005 units/ml, there were significantly fewer clonogenic units when sonicated eosi nophils were excluded from the clonogenic assay. @,P< 0.05 using Student's unpaired glucose oxidase by degranulated eosinophils was somewhat unex test and Spearman estimate for the SD of the clonogenic unit estimate. Addition of pected, particularly in view of the numerous previous reports that supplemental halides did not increase the killing by sonicated eosinophils. Similar results hydrogen peroxide augments the cytotoxicity of eosinophil peroxidase were obtained with the H9 cell line. The untreated negative controls and clonogenic assays with catalase yielded an estimate of 1760 clonogenic units/mI. (8, 14, 15, 19—21).We speculate that the reason for this apparent 2652

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1994 American Association for Cancer Research. GLUCOSE OXIDASE PULLING OF LYMPHOMA CELLS discrepancy is that eosinophil peroxidase seems to be cytotoxic only from peripheral blood specimens. This simple method will undoubt when directly adherent to target cell membranes. In the previous edly aid other investigators who wish to explore the biology of studies, purified eosinophil peroxidase was used to coat target cells eosinophils and human cancers in the future. directly prior to administration of hydrogen peroxide in short-term cytotoxicity assays, whereas the current study used sonicated (de REFERENCES granulated) eosinophils mixed with tumor cells in a much more 1. Stanislawski, M., Rousseau, V., Goavec, M., and Ito, H. Immunotoxins containing sensitive clonogenic assay. glucoseoxidaseand lactoperoxidasewithtumoricidalproperties:in vitro killing Thus, in our clonogenic assay, the peroxidase in the sonicated effectiveness in a mouse plasmacytoma model. Cancer Res., 49: 5497—5504, 1989. 2. Philpott, G. W., Bower, R. i., and Parker, C. W. Selective iodination and cytotoxicity eosinophils probably functioned in essentially the same manner as the of tumorcellswithan antibody-enzymeconjugate.Surgery,74:51—58,1973. exogenous catalase to eliminate hydrogen peroxide rapidly before it 3. Philpou, G. W., Shearer, W. T., Bower, R. J., and Parker, C. W. Selective cytotoxicity of hapten-substituted cells with an antibody-enzyme conjugate. i. Immunol., 111: could kill the uncoated tumor cells. Accordingly, the highly reactive 921—925,1973. haloacids generated by eosinophil peroxidase from the hydrogen 4. Higuchi, Y., Shoin, S., and Matsukawa, S. Enhancement of the antitumor effect of peroxide and supplemental halides probably halogenated proteins in glucose oxidase by combined administration of decomposition inhibitors together with an oxygenated fluorocarbon. Jpn. J. Cancer Rca., 82: 942—949,1991. the eosinophil granules and cell culture medium rather than killing the 5. Nathan, C. F., and Cohn, Z. A. Antitumor effects of hydrogen peroxide in vivo. tumor cells. Consequently, most of the tumor cells could survive and J. Exp. Med., 154: 1539—1552,1981. proliferate because the sonicated eosinophils had neutralized the hy 6. Farber, C. M., Liebes, L F., Kanganis, D. N., and Silber, R. Human B-lymphocytes shown greater susceptibility to hydrogen peroxide toxicity than T-lymphocytes. drogen peroxide produced by glucose oxidase. J. Immunol.,132:2543—2545,1984. It is important to note, however, that it is still uncertain whether our 7. O'Donnell-Tormey, i., DeBoer, C. i., and Nathan, C. F. Resistance of human tumor clonogenic assay model truly approximates the in vivo situation in cells in vitro to oxidative cytolysis. J. Clin. Invest., 76: 80—86,1985. 8. Samoszuk, M. K., Petersen, A., Lo-Hsueh, M., and Rietveld, C. A peroxide-gener human lymphomas that are characterized by degranulation of eosi ating immunoconjugate directed to eosinophil peroxidase is cyotoxic to Hodgkin's nophils. If, for example, eosinophil peroxidase from degranulated diseasecellsin vitro.Antibodylmmunoconj.Radiopharm.,2:37-46, 1989. 9. Nathan, C. F., Silverstein, S. C., Brukner, L H., and Cohn, Z. A. Extracellular eosinophils directly coated tumor cells or vasculature in vivo, then cytolysis by activated macrophages and granulocytes. I. Exp. Med., 148: 100—113, exogenous glucose oxidase could still be expected to have potent 1979. tumoricidal activity because the peroxidase-coated cells would be 10. Samoszuk,M., Ehrlich,D., and Ramzi,D. Preclinicalsafetystudiesof glucose oxidase. J. Pharmacol. Exp. Ther., 266: 1643—1648,1993. exceptionally sensitive to killing by hydrogen peroxide. 11. Amlot, P. L, Stone, M. i., Cunningham, i., Fay, i., Newman, i., Collins, R., May, R., Indeed, our study clearly demonstrated that tumoricidal levels of McCarthy, M., Richardson, i., Ghetie, V., Ramilo, 0., Thorpe, P. E., Uhr, i. W., and glucose oxidase can accumulate rapidly in s.c. tumor implants even in Vitetta, E. S. A Phase I study of an anti-CD22-deglycosylated ricin A chain immu notoxin in the treatment of B-cell lymphomas resistant to conventional therapy. the absence of eosinophils. Specifically, a single nonlethal (10) dose Blood, 82: 2624—2633, 1993. of glucose oxidase (50 @g)rapidlyproduced levels of enzyme within 12. Thorpe, P. E., Wallace, P. M., Knowles, P. P., ReIf, M. G., Brown, A. N., Watson, G. J., Blakey, D. C., and Newell, D. R. Improved anti-tumor effects of immunotoxins the tumor (0.079 @ig/goftumor) that greatly exceeded the concentra prepared with degiycosylated ricin A chain and hindered disulfide linkages Cancer tion (0.025 ,A.g/ml) needed to kill more than 99% of the cells in a Rca., 48: 6396-6402, 1988. clonogenic assay (Fig. 3). 13. Samoszuk, M., Anderson, A. L, Ramzi, E., Wang, F., Braunstein, P., Lutsky, i., Majmundar, H., and Slater, L. Radioimmunodetection of Hodgkin's disease and Moreover, it is particularly significant that a relatively high non-Hodgkin's lymphomas with monoclonal antibody to eosinophil peroxidase. proportion of the injected glucose oxidase accumulated in the i. NucLMed.,34: 1246—1253,1993. tumor implants only 5 mm after injection (0.16% of the injected 14. Slungaard, A., and Mahoney, i. R. Bromide-dependent toxicity of eosinophil perox idase for endothelium and isolated working rat hearts: a model for eosinophiiic dose/g of tumor). By contrast, most monoclonal antibodies require endocarditis. i. Exp. Med., 173: 117—126,1991. many h to achieve tumor concentrations of only 0.01—0.03%of the 15. Slungaard, A., and Mahoney, i. R. Thiocyanate is the major substrate for eosinophil peroxidase in physiologic fluids. i. Biol. Chem., 266: 4903—4910,1991. injected dose (22). One possible explanation for this difference is 16. Schuhmacher, i., Klivenyi, G., Matys, R, Kirchgebner, H., Hauser, H., Maier-Borst, that the hydrogen peroxide produced by glucose oxidase is known W., and Matzku, S. Uptake of indium-ill in the liver of mice following administra to initiate hydrolysis of endothelial cell phospholipids, thereby tion of indium-111-DTPA-labeled monoclonal antibodies: influence of labeling pa rameters, physiologic parameters, and antibody dose. i. NucI. Med., 31: 1084—1093, leading to reversible increases in vascular permeability (23). Thus, 1990. our data suggest that it is theoretically possible that intravascular 17. Kalisz, H. M., Hecht, H. J., Schomburg, D., and Schmid, R. D. Effects of carbohy glucose oxidase can promote its own passage across endothelial drate depletion on the structure, stability, and activity of glucose oxidase from Aspergillus niger. Biochim. Biophys. Acts, 1080: 138—142,1991. barriers into tumor tissue. 18. iohnson, E. A., and Brown, B. W. The Spearman estimator for serial dilution assays. In addition to improving our understanding of the potential use of Biometrics, 97: 79-88, 1961. 19. iong, E. C., and Klebanoff, S. J. Eosinophil-mediated mammalian tumor cell cyto glucose oxidase as an antineoplastic therapeutic agent, this study also toxicity: role of the peroxidase system. i. Immunol., 124: 1949—1954,1980. generated some other interesting findings. On the basis of our data, for 20. Nathan, C. F., and Kiebanoff, S. i. Augmentation of spontaneous macrophage example, it appears that there is no significant difference in the mediated cytolysis by eosinophil peroxidase. i. Exp. Med., 155: 1291—1308,1982. 21. Sainoszuk, M. IC, Petersen, A., Gidanian, F., and Rietveld, C. Cytophilic and sensitivity of a B-cell line (ARH-77) and a T-cell line (H9) to killing cytotoxic properties of human eosinophil peroxidase plus major basic protein. Am. i. by glucose oxidase. Moreover, there does not appear to be any Pathol., 132: 455—460,1988. 22. Carrasquillo,i.A.,Buns,P.A.,Keenan,A.M,Reynolds,i.C.,Schroff,R.W.,Foon, pharmacokinetic advantage to using deglycosylated glucose oxidase, K. A., Ming-Hsu,S.,Gazdar,A. F., Mulshine,i. L., Oldham,R. K.,Perentesis,P, and there was no detectable increase in tumoricidal activity when Horowitz, M., Eddy, i., iames, P., and Larson, S. M. Radioimmunodetection of using a peroxide-generating immunoconjugate targeted against human cutaneousT-celilymphomawithWIn-labeledTiOlmonoclonalantibody.N.EngI. i. Med., 315: 673—680,1986. eosinophil peroxidase. Finally, our study documented the use of a 23. Shasby,D.M.,Yorek,M.,andShasby,S.S. Exogenousoxidantsinitiatehydrolysis novel method for rapidly and efficiently purifying human eosinophils of endothelial cell inositol phospholipids. Blood, 72: 491—499,1988.

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Michael K. Samoszuk, Vincent Nguyen, Cherry T. Thomas, et al.

Cancer Res 1994;54:2650-2653.

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