[CANCER RESEARCH 39, 1927-i 933, June 1979]0008-5472/79/0039-0000$02.00

Improvement in the Therapeutic, Immunological, and Clearance Propertiesof Escherichia coil and Erwinla carotovora L-Asparaginases byAttachment of Poly-DL-alanylPeptides'

Jack R. Uren2 and Richard C. Ragin

Division of Medicinal Chemistry and Pharmacology, Sidney Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT

The chemical modification of both EscheriChia coli and Erwinia Carotovora asparaginases by a DL-alanine-N-carboxyanhydnide polymerization technique produced modified enzymes which had greater protease stability, retained most oftheir catalytic activity, and demonstrated a 7- to 10-fold prolongation in plasma clearance properties in normal mice andrats. Concomitantly, plasma substrate depletion was also extended 5 to 13 days longer for the modified as compared withthe native enzymes. For preparations of modified enzymes withplasma half-lives longer than 24 hr, the therapeutic activity wassuperior to that of the native enzymes. In addition, the modifiedE. coli preparations were less immunogenic in mice than wasthe native enzyme, and they cross-reacted with antibodiesdeveloped to the native enzyme to a 300-fold lesser degree,such that the modified enzyme still showed prolonged clearance in an animal which had been immunized previously to thenative enzyme. The native enzyme was immediately clearedfrom the plasma of such immune animals, although hypenimmune animals would rapidly clear both the native and modifiedenzymes. Similarly, the modified E. Carotovora enzyme wouldcross-react to a 500-fold lesser degree with antibodies developed against the native E. carotovora enzyme.

INTRODUCTION

Therapy of acute lymphocytic leukemia with the enzyme Lasparaginase is accompanied by immunological toxicity in approximately 25% of the patients, ranging in severity from mildallergic reactions to anaphylactic shock (22). Not only theseverity of the allergic reaction but also the presence of cmculating antiasparaginase antibody (which greatly acceleratesthe clearance of the enzyme) restricts subsequent therapy withthe enzyme (9). Consequently, therapy with the enzyme ismanaged in such a way as to minimize these immunologicalcomplications: (a) patients are given short courses of enzymefor remission induction or consolidation as opposed to continued administration for maintenance therapy; (b) they aretreated with combination chemotherapeutic agents, many ofwhich are immunosuppressive; and (c) when allergic reactionsdo occur, therapy is changed to an enzyme isolated fromanother source which does not share cross-reacting antigenicdeterminants, i.e. , from the E. Coli enzyme to the E. Carotovora

enzyme. Therefore, any procedure which greatly decreasesthe immunogenicity of asparaginase can not only decrease theallergic reactions associated with the use of the enzyme butalso broaden the ways and expand the time over which theenzyme can be effectively given. This report describes such aprocedure.

The immunological consequences of modifying proteins withDL-alanyl polymers have been extensively studied by Sela etal. (2, 4, 18). These investigators found that such modifiedproteins were much less able to cross-react with antibodiesdirected against the native protein (2, 4, 18) and would elicitmuch lower antisera titers when animals were immunized withthe modified, as opposed to the native, protein (4, 18). Theantisera derived from animals immunized with the richly alanylated preparation contained antibodies almost exclusivelyspecific toward poly-DL-alanine (i 8). In addition, it has beenpossible to induce tolerance to the poly-DL-alanine determinantby injecting poly-DL-alanine into newborn rabbits (i 7, 18). TheE. coli poly-DL-alanylated asparaginase preparations in thiswork were also less reactive with the antiserum elicited by thenative enzyme and would elicit less precipitating antibodieswhen used to immunize mice, as compared with the amountelicited by the native enzyme. Unlike the previous work, however, the antibodies obtained from immunization with the modified enzyme would not cross-react with poly-DL-alanine, butthey would cross-react with the native enzyme.

In addition to the immunological difficulties associated withenzyme therapy, the rapid plasma clearance of a foreign protein is another general problem facing the therapeutic use ofenzymes. Factors such as isoelectnic point (i 6), state of aggregation (21), and sialic acid content (13) have been shown toinfluence the plasma clearance rate of specific proteins, but nogeneral properties will adequately predict the clearance rate offoreign proteins. In mice, the presence of the lactic dehydrogenase-elevating virus will cause a prolongation in the plasmahalf-life of many enzymes (i 5). Whether this is true for otherspecies has not been demonstrated. Poly-DL-alanylation ofboth the E. coli and the E. carotovora L-asparaginases in thiswork has increased the plasma half-lives of both of theseenzymes 7- to 10-fold. Possibly, these procedures may proveto be a general technique for overcoming the limitations oftherapy with rapidly cleared enzymes.

MATERIALS AND METHODS

L-Asparaginase from E. coli was obtained from the Merck,Sharp and Dohme Research Laboratories, West Point, Pa. (LotC-E603), and the enzyme from E. carotovora was obtainedfrom the Microbiological Research Establishment, Salisbury,England (Lot MREi 6). Both trypsin and a-chymotrypsin were

1 Supported by Research Grants CA 0651 6, CA 1 9589, and CA 1 891 7 from

NIH, Bethesda, Md.2 To whom requests for reprints should be addressed, at the Sidney Farber

Cancer Institute, Division of Medicinal Chemistry and Pharmacology, 44 BinneyStreet, Boston, Mass. 02115.

Received August 2i , 1978; accepted February 16, i 979.

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J, R. Uren and R. C. Ragin

purchased from the Worthington Biochemical Corporation,Freehold, N. J. Rabbit anti-mouse y-globulin was purchasedfrom Cappel Laboratories, Cochranville, Pa. DL-Alanine-N-carboxyanhydnide was purchased from Miles Research Products,Elkhart, Ind., and Sephadex G-25 came from Pharmacia FineChemicals, Piscataway, N. J. 5-Dizao-4-oxo-L-norvaline was agift from Dr. Robert Handschumacher, Vale University. Corn

plete Freund's adjuvant came from Difco Laboratories, Detroit,Mich., and Na'25l was purchased from the Amersham Corponation, Arlington Heights, Ill. Male C57BL/6 x DBA/2 (hereafter called BD2F1) mice came from the Jackson Laboratory,Bar Harbor, Maine, and the L5i 78V tumor line was obtainedfrom I. Wodinsky at Arthur D. Little, Inc., Cambridge, Mass.Rats were obtained from Charles River Breeding Laboratories,Wilmington, Mass.

Preparation and Purification of PoIy-DL-alanyl Asparaginases. After preliminary experiments which evaluated the effects of the nature of the organic solvent (dimethyl sulfoxide,dioxane, glycerol) and its percentage (40 to 90%), buffer pH(6.8 or 8.0), and ratio of DL-alanine-N-carboxyanhydride toprotein (1 to 4 mg/mg) on retention of enzyme activity, alanineincorporation, and precipitation with antiasparaginase antibodies, the following preparation procedure was developed. Onevial (10,000 lU) of asparaginase from either E. Cob(40 mg) orE. Carotovora (10 mg) was dissolved in 5 ml of 0.1 M K2HPO4,pH 6.8. The enzymes (with or without prior dialysis) werecooled to 0°in ice, and 5 ml of dimethyl sulfoxide were addedgradually. To the cooled reaction mixture, 150 mg of DL-alanine-N-carboxyanhydnide were added. After 1 hr the reactionmixture was purified by gel filtration on Sephadex G-25 in 0.05M K2HP04 buffer, pH 6.8. Fractions containing enzyme activity

were pooled and further purified by ammonium sulfate precipitation between 45 and 60% saturation. An overall recovery of20% of the initial enzyme activity was observed.

Immunological Procedures. An injection of asparaginase inexperimental animals suppresses the animal's immunologicalresponsiveness (22). In order to remove the immunosuppressive activity of the enzyme, both native and modified E. coliasparaginases were inactivated with a site-specific reagent,DONV,3at room temperature for 30 mm at a final concentrationof 0.5 mg per ml of protein, 50% dimethyl sulfoxide, and 2 m@DONV in 0.05 M K2HPO4,pH 6.8 (12). If at the end of this time>1 0% enzyme activity still remained, a further addition ofDONV was made. The inactivated proteins were dialyzed overnight versus 0.01 M K2HP04, pH 7.8. Equal volumes of theinactivated proteins or native E. Carotovora or E. coli enzymeand complete Freund's adjuvant were homogenized in a VirTis45 homogenizer at top speed for 5 mm. Twenty @.tgof protein(0.1 ml) were injected weekly i.p. into BD2F1 male mice.Antibody-antigen precipitin reactions were visualized on Milesimmunodiffusion plates in 0.8% agarose in PBS with 5 @tlofapproximately 1 mg per ml of antigen.

A radioimmunoassay for asparaginase was established bythe following procedures. Dialyzed native E. coli or E. carotovora asparaginase (20 @tIof 0.8 mg/mI) was radiolabeled byincubating with 20 jsl of chloramine-T (5 mg/mI) and 10 @.dofNa1251(100 mCi/mI) for 30 sec, followed by the addition of 0.1ml of 5 mg per ml of sodium metabisulfite and 0. 1 ml of KI (1

3 The abbreviations used are: DONV, 5-diazo-4-oxo-L-norvaline; PBS, 0.9%

NaCl-9 mM NaHPO4,pH 7.2; TCA, trichloroacetic acid.

mg/mI). The reaction mixture was then purified by gel filtrationon a 10-mI Sephadex G-25 column in 0.01 M K2HPO4,1 mgper ml of bovine serum albumin buffer, pH 7.5. The radioactiveprotein fractions (approximately 1O@Ci/mol, 4 iodines permolecule for the E. coli and 2 x 106 Ci/mol, 1 iodine permolecule for the E. Carotovora-enzyme) were pooled and frozen. Seventy % of the radioactivity could be precipitated with10% TCA. Competitive binding assays were performed in microtiter plates containing 100 j@lof PBS, 5 mt@iEDTA (pH 7.2),10,000 cpm 1251-labeledasparaginase, and 5-fold dilutions ofantigen (25-@iICook microdilutors were used), and followed by5 @lof antisera diluted 1000-fold with normal mouse sera foranti-E. COlisera and 50-fold for anti-E. carotovora sera. Incubations for 30 mm at 37°were performed, followed by theaddition of 20 @lof rabbit anti-mouse y-globulin and an additional 1-hr incubation at 37°.Antigen in the immune precipitatewas separated from unbound antigen by layering the reactionmixture on top of 120 @Iof 10% glycerol in PBS and centnifuging for 1 mm at 8000 x g. The precipitate was cut from thebottom of the tube and counted in a Beckman ‘y4000 counter.Only 4% of the counts (relative to those precipitated by TCA)were in the pellet when nonimmune mouse sera were used.One hundred % of the TCA-precipitable counts were precipitated by immune sera and 1000-fold diluted immune E. colisera and 50-fold diluted immune E. Carotovora sera wouldprecipitate 20 to 50% of the counts such that the competitivebinding results are more indicative of the tighter-binding antibody population.

RESULTS

Electrophoretic and Chemical Characterization of Poly-iLalanyl Asparaginase. Sodium dodecyl sulfate electrophoresis(23) of modified E. Cobenzyme gave a single band of increasedmolecular weight consistent with the degree of alanine incorporation (see below). A single band of slightly differing mobilityfrom the native enzyme was observed with agarose gel stripelectrophoresis at pH 8.6. With polyacrylamide gel electrophoresis at pH 8.3 (Chart 1), 2 bands of differing mobility and ofapproximately 40 to 60% intensity were observed. Attempts atisolating these forms by ion-exchange chromatography and gelfiltration have not been successful. The modified E. carotovoraenzyme, on the other hand, showed primarily a single bandwith polyacrylamide electrophoresis differing in mobility fromthat of the native enzyme (Chart 1).

Since the N-carboxyanhydnide copolymerization terminateswith a free amino-terminal amino acid, the number of chains ofalanine polymer was determined by the amino-terminal proceduneof Stark and Smyth (20). The results of these experimentsand the increase in alanine composition are presented in Table1. It is apparent from this table that a variable amount of DLalanine had reacted with both enzymes from preparation topreparation even though the conditions were kept constant.Trace contaminations in the reagents, DL-alanine-N-carboxyanhydride instability, and the rate of dissolving of the solid DLalanine-N-carboxyanhydride in the reaction mixture may account for this variability. The range in alanine incorporationwas between 200 and 900 residues/mol protein. Only 2 preparations for the E. coli enzyme, characteristic of each end ofthe range, and Preparation 3 for the E. Carotovora enzyme willbe discussed. Consequently, the further characterization of the

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Enzymaticpropertiesof nativeandpoly-ot.-alanylasparaginasefromE. coli and E. carotovorasourcesEnzyme

activity was determinedby the coupledenzymeassayofCooneyet al. (6). Km'Sweredeterminedby Lineweaver-Burkplots ofenzymeactivityversussubstrateconcentrationbetween5 and 50 @a.iasparaginein 0.1 PAK2HPO4,pH8.0. Proteinconcentrationwasdeterminedbyabsorbanceat 280 nm.Extinctioncoefficientsusedwere7.1for a 1% solution of the E. coli enzyme(10) and 6.1 for the E.carotovoraenzyme(11).Specific

activity(IU/ K@(x i0°

Enzym? Source mg) % nativeM)Native

E. co/i 124 100 1Preparation 1b E. co/i 25 20 1Preparation 2 E. coli 59.3 48Native Erwinia 259 100 3Preparation 3 Erwinia 169 65 4

Table1Chemicalcharacterizationof poly-DL-alanyl-modifiedasparaginasePrepa

rationSourceIncreaseIn al

anine/mol'No.of chains/molbAlanine/chain1

234E.

coliE.coliE.carotovoraE.carotovora805

243685

292±i3c23.5

17.031.5

24.65±1534.3

14.321.7

ii.8±.6

75‘°@fed50-\

xEco/i25-

Biological Properties of Poly-DL-alanyl L-AsparaginaSeS

sitive measure of enzyme homogeneity, we chose to measurethe heat inactivation rate of the E. carotovora and E. coil nativeand modified (Preparation 1) asparaginases. As depicted inChart 3, 40% of modified E. coil enzyme shows relative heatstability similar to the native enzyme, whereas 60% of thematerial demonstrated a heat inactivation half-time of i 8 mm.The inactivated enzyme upon cooling would recover its lostactivity in concentrated solutions. The modified E. carotovoraenzyme, on the other hand, did not show a biphasic heatinactivation rate, and the modified enzyme /2 = 22 mm) wasmore heat stable than was the native enzyme /2 = 7 mm).

The stability of the native and modified (Preparation 1) E. coilasparaginases to proteolysis was also investigated (Chart 4).The modified enzyme was totally resistant to trypsin at 100 @g/ml over 1 hr, whereas the native enzyme was 50% destroyedby a 30-mm incubation with trypsin (1 @g/ml).In the presenceof a-chymotrypsmn,the modified enzyme was slightly more than

Table2

E

cmChart i . Electrophoretic mobility of native and modified (Preparation 2) E. co/i

asparaginase and native and modified E. carotovora asparaginase at pH 8.3 on7.5% polyacrylamlde gels (7). Gels were stained with amido black and scannedwith a Gilford model 2520 gel scanner.

a@ Tat@e 1 for chemical characterization.

b This preparation was not further purified by ammonium sulfate precipitation.a Amino acid analysis with a Beckman 1 21 amino acid analyzer following 24

hr of hydrolysis with 6 N HCI at 110°.Calculations were based on 132 residuesof alanine and 140 residues of valine per mol of native E. coli asparaginase (10)and i 16 alanine and 119 vallne residues per mol of native E. carotovoraasparaginase (5).

b Determined by the amino-terminal procedure of Stark and Smyth (20).C Mean ±S.D.

E. coli preparations will be identified as to which preparationwas utilized.

Enzymatic Properties of Poly-oL-alanyl Asparaginase. Thespecific activity and the Kmfor both the native and polymermodified E. coil and E. carotovora enzymes are given in Table2. The modification procedure caused no change in the Km,and the specific activity of the modified enzyme was about50% of the native enzyme in both cases. The polymer modification procedure did not appear to reduce greatly the substrateaccessibility or turnover for either of these enzymes.

The pH optima for aspanagine hydrolysis by both the nativeand modified E. coli and E. carotovora enzymes are shown inChart 2. The modified enzymes demonstrated slightly morealkaline pH optima than did the native enzymes. At physiological pH (7.2), both the modified and native E. coil enzymeswere close to their optimal values, whereas the E. carotovoramodified enzyme differed by only about 10% from the nativeenzyme.

Stability of PoIy-ou.alanyl, E. coil, and E. carotovora Asparaginases. Since temperature stability is sometimes a sen

Native

.\

A@XV

Erw@rncCoroFovoro

. I • I

75 -

50 -

25 -

‘4.o 6.0 80pH

Chart 2. pH rate profile of native and modified (Preparation 2) E. coil andnative and modified E. carotovora asparaginase. Enzyme activity was measuredby the loss of absorbance at 215 nm (1 1) with 5 m@.iasparagine in 0.1 M Triscitrate buffers. An extinction coefficient was calculated for each pH.

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J. R. Uren and R. C. Ragin

presented in Chart 5. A log-linear decay of all 4 enzymeactivities was observed in the plasma of mice, with the modifiedenzymes exhibiting approximately a 7-fold-longer plasma halflife than the native enzymes. The consequences of theseinjections on plasma asparagine concentration were that it tookabout 5 days longer for the asparagine to return to the plasmawhen the modified E. coil enzyme was injected and 12 dayslonger when the modified E. carotovora enzyme was injected,as compared to the time with unmodified enzymes. These datawere consistent with the plasma half-lives of each enzyme, i.e.,modified E. carotovora > modified E. co/i > native E. carotovora

@ native E. coil.The plasma half-life of native and polymer-modified (Prepa

ration 2) E. coil asparaginase in a rat following an i.v. injectionin the femoral vein is shown in Chart 6. A biphasic clearancerate was observed with a short-lived component (66%) havinga half-life of 4 hr and a longer-lived component (34%) with ahalf-life of 13 hr, both of which are longer than the nativeenzyme /2 = 1.5 hr). These data again may reflect the 2components observed with polyacrylamide gel electrophoresisand by heat inactivation rates. These results also demonstratedthat the prolonged plasma half-life of the polymer-modifiedenzyme was not totally species specific.

TherapeutIc Activity of Poly-DL-alanyl Asparaglnases. Theimproved plasma clearance properties and extent of substratedepletion presented in the previous section should predict animproved therapeutic response for the modified as opposed tothe native aspanaginasesat equivalent dosage. When the standard National Cancer Institute survival test was used with theasparaginase-sensitive L5178 leukemia in mice (8), this resultwas not always observed. A possible reason was that tumorbearing animals exhibited a much longer plasma half-life forthe native enzyme (24 as opposed to 3 hr), probably due to thepresence of the lactic dehydnogenase-elevating virus in ourtumor line (14). This extension of the plasma half-life for the

Time (Mm)

.@

Chart 3. Heat stability of native and modified (Preparation 1) E. co/i asparaginase at 50°and native and modified E. carotovora asparaginase at 65°.Aliquots were removed, and enzyme activity was determined by the loss ofabsorbance at 215 nm (1 i ) with 5 mp.iasparagine in 0.05 Tris, pH 7.3.

I0C• AT:@ °ModIf,edJr •. 05y/mITrypsin50f:@ •@“NIt@%,,@£I00y/mI Trypsln

,. NotIve―@'@.,@

Iy/mI Trypsln

‘@‘

.@ @I00 I I I

50

\\1q@@@ModifiedIy/mI

Modified 5y/mIa- Chymotrypsin

Native Iy'/mIa - Chymotrypsin

I_ I I._.o 20 40 60 80

Time(mmChart 4. StabilIty of native and modified (Preparation i ) E. coil asparaginase

in the presence of trypsin (1, 5, and 100 g@g/ml)and a-chymotrypsln (1 or 5 @ag/ml). Asparaginase (0.6 mg/mI) was incubated with the proteins in 0.i M K2HPO4buffer, pH 7.8 at 37°,and aliquots were removed and assayed for DONVhydrolyzing ability (12).

5-fold more resistant to proteolysis. Since lysine residues aremodified by the polymerization procedure and unmodified lysines are one of the primary substrate requirements for trypsinhydrolysis, it is not surprising that the modified enzyme is moreresistant to trypsin than to a-chymotrypsin hydrolysis. Theresistance of the modified protein to a-chymotrypsin digestionmay reflect a steric barrier imposed by the polymer to theapproach of the a-chymotrypsin macnomolecule.

Plasma Clearance and Substrate Depletion of Poly-oLalanyl Asparaglnases In Rodents. The in vivo effect of theinjection of 10 IU i.p. of both E. coli native and modified(Preparation 2) and the E. carotovora native and modifiedenzymes on plasma clearance rate and substrate levels is

4

@‘1

Chart 5. Effects of 10 lU of native and modified (Preparation 2) E. coilasparaginase and native and modified E. carotovora asparaginase on plasmaenzyme activity and asparagine concentration in BD2F, mice. Following the i.p.injection of the enzymes, 0.25 ml of blood was collected by orbital bleeding, andthe plasma was removed following centrifugation for 1 mm at 8000 x g. Analiquot of the plasma(10 to 50 @tI)was assayed for enzyme activity by the coupledenzyme assay described by Cooney et al. (6), and 100 @lof the remaining plasmawere deproteinized with 66 @lof 10% sulfosalicylic acid. The precipitated proteinwas removed by centrifugation at 130,000 x g for 30 mm in a Beckman airfuge,and I 10 @tlof the supematant were analyzed for the amino acid content with aBeckman Model 121 M amino acid analyzer. Asparagine concentrations werenormalized to the threonine content to correct for handling losses.

8

Time ( Days)

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One millionL5178Y cells per mousewere implantedi.p. intomaleBD2F1miceon Day0 and treatedby i.p. boluson Day1. Eachgroupcontained

5 mice except for 10 control mice. Initial mouseweightsaveraged25 g each.%

ofincreaseinlifespanDosage

(lU/mouse) NativeModifieda10

54±9c1i6±350±70.1—2±0.315±20.01

2±0.3 —5±0.9

Percentage of increase in life span when mice bearing theasparaginase-sensitive L5 178Y leukemia are treated with various

native and modifiedasparaginasesEarlytreatmenta LatetreatmentbE.

caroto- E. carobEnzyme E. coil vora E. coilvoraNative

22±2― 12±2 45±5Modified 39±4 56±4 42±4

Immunogenicity of native and modified (Preparations 1 and 2) E. coilasparaginase in complete Freund's adjuvant with BD2F1miceMice

were immunizedi.p. weeklywith 20 @sgof DONV-inactivatedenzyme, and Ouchterlony precipitin reactions were measured 7 dayslater (See “MaterialsandMethods―).Native

enzyme Modified Preparation 1 Mod:@@nP@ePa@

No.Time Precipitin No. posi- No. posi- Precipi- posi(wk) line tive Precipitin line tive tin linefive1

None 0/25 None 0/19 None 0/52 Weak 25/25 None 0/19 None 0/53 Strong 25/25 None O/i9 None 0/54 Strong 25/25 Weak i5/i9 None 0/55 Strong 25/25 Weak-strong i9/19 Weak 2/5

Biological Properties of Poly-DL-alanyl L-Asparaginases

native enzyme was not observed with the modified enzymes.Consequently, only modified enzyme preparations whichshowed plasma half-lives in excess of 24 hr demonstratedsuperior therapeutic activity over the native enzyme; otherwise,activities similar to the native enzymes were observed. Thedata in Table 3 support these conclusions. If therapy wasinitiated early before the virus could exert its plasma life-extending ability, both modified enzymes were superior to theirnative counterparts. If therapy was delayed until the virus hadestablished itself, only the modified E. carotovora enzyme /2= 36 hr) was superior to its native counterpart. The modified(Preparation 2) E. coil enzyme /2 = 20 hr) was equivalent toits native counterpart. When the more extensively modified(Preparation 1) E. coil enzyme /2 = 36 hr) was used fortherapy, a greatly improved therapeutic activity was observedeven with late therapy (Table 4). This preparation appeared tobe about 10-fold better than the native enzyme and was capable of curing mice with single injections of 400 lU/kg.

Immunological Properties of Poly-oi.-alanyl Asparaginase.When both native and polymer-modified E. coil asparaginase(both preparations) were inactivated with the site-specific reagent DONV to remove the immunosuppressive activity of theenzymes and were injected in complete Freund's adjuvant intomice, it took a much longer time (4 + weeks as opposed to 2weeks) for precipitating antibodies to be elicited by the modified

Chart 6. Plasma clearance of native and modified (Preparation 2) E. coilasparaginase following an i.v. Injection of 50 lU in the femoral vein of a rat. Bloodsamples were obtained by tail bleeds, and enzyme activity In the plasma sampleswere determined as In Chart 5.

as opposed to the native enzyme, as judged by Ouchterlonyprecipitin reactions in agarose gels (Table 5). The fact thatprecipitating antibodies eventually were formed suggests thatthe modified protein was capable of diffusing through the geland precipitating with the antibody, validating the assay. Theseobservations suggest that under these experimental conditionsthe modified enzyme is much less immunogenic than the nativeprotein. Antisera derived from mice immunized with the modifled enzyme displayed a reaction of identity between nativeand modified proteins but gave no precipitin line with poly-DLalanine in an Ouchtenlony immunodiffusion assay.

A quantitative measure of the degree of cross-reaction between the native and modified enzyme was determined by a

radioimmunocompetition assay (Chart 7). It took 300-fold moremodified E. coil asparaginase than native enzyme to displacethe nadiolabeled native enzyme from the antisera derived fromanimals immunized with the native enzyme and 100-fold moremodified enzyme when the antisera derived from the miceimmunized with the modified enzyme were utilized. The significance of this difference is unknown because only 2 antiserawere evaluated. Native E. carotovora asparaginase could notdisplace the radiolabeled E. coil enzyme from either antiserumwhen assayed up to 30,000 times the concentration of nativeenzyme which would cause 50% displacement, confirming thepreviously reported lack of cross-reactivity between these 2enzymes (22). Similarly, it took 500-fold more modified E.carotovora enzyme than native to displace the radiolabeled E.carotovora enzyme from antisera derived from mice immunized

Table 4

Effect of E. coli asparaginase modification on therapy of murineleukemia L51 78V

Modified

a Modified E. coil asparaginase Preparation 1 (see Table 1 ) was used (plasma

t,/2@ 36 hr).b Three of five 60-day survivors.C Mean ±S.D.

Table 5

Time(Hr)

Table3

28 ±476 ±6

aOnemillionL5178YcellsimplantedIntoeachof5 maleBD2F,miceonDay0 and treated 1 hr post-implantation with 10 lU of each enzyme.

b Treatment as above but 24 hr post-tumor implantation.CModified E. coil asparaginase Preparation 2 (see Table i ) was used (plasma

b,,2 —20 hr).d Mean ±S.D.

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useful in the treatment of animals (or patients) who have a lowantibody titer directed against the native enzyme but not inanimals with a high antiasparaginase titer.

DISCUSSION

The polyacrylamide gel electrophoresis, heat stability, andintravascular plasma clearance rates have all indicated that,for as yet unknown reasons, DL-alanine-N-carboxyanhydnidepolymerization of E. coli asparaginase has more heterogeneitywithin a given preparation than that exhibited by the E. carotovora enzyme. Nevertheless, all of the modified enzymesexamined have shown reasonable retention of catalytic activitywith improved plasma clearance properties, protease stability,substrate depletion in vivo, therapeutic activity, and decreasedimmunogenicity and cross-reacting antigenicity compared totheir native counterparts. Chart 9 depicts a possible model toexplain this collection of results. The polymers act as a stericbarrier which retards or prevents the interaction of macnomolecules (proteases, antibodies, immunogenic, and/or clearancerecognition receptors) with the native enzyme while micromolecular substances (substrates, products, hydrogen ions, coenzymes, etc.) can still interact with the enzyme with littledifficulty. Needless to say, the nature as well as the extent ofpolymerization are important factors. Sela and Arnon (19) havedocumented the fact that the addition of poly-L-tyrosine, phenylalanine, or tryptophan peptides has increased rather thandecreased immunogenicity of gelatin, and others have shownthat amino acid-copolymenized enzymes are in many casesmuch more water insoluble (3). In general, we have observedthat the greater the extent of modification, the longer theplasma half-lives the poorer is the ability to precipitate withanti-native asparaginase antibodies and the greater is the lossof catalytic activity. All of the properties need to be balanced

Chart 8. Plasma clearance of native and modified (Preparation 1) E. coliasparaginase in normal BD2F, mice @D,0) and BD2F mice immunized with thenative enzyme (U, C). Ten lU were injected p., and enzyme activity wasmeasured as in Chart 5. MIce were immunized with 2 injections of 20 @igofinactivated native enzyme 1 week apart, as described in •Materials and Methods.―A 25-fold dilution of serum would still bind >20% radiolabeled enzyme.

Log Conc.(nanograms)

Chart 7. Effects of native (0, L@)and modified ce, A, X, ) asparaginase oncompetitive displacement of ‘25l-labelednative enzyme from antisera derivedfrom immunization with the native (—) and modified (————)enzymes. Preparation 2-modified E. coil enzyme was used in the top experiments, and Preparation 3- (X) and 4- (L modified E. carotovora enzyme was used in the bottomexperiments (see Table 1). Procedures are described in the text.

with the native E. carotovora enzyme.If the modification procedure totally masked only some of the

antigenic determinants, we would expect the modified enzymenot to displace the radiolabeled native enzyme from the nativeimmunized antisera directed against these masked determinants. In contrast, 100% of the counts was displaceable by thenative on modified enzymes when either antiserum was used.In addition, immunization with the modified enzyme would beexpected to elicit antibodies only to the unmasked immunogenic sites such that the native and modified proteins shouldbe equivalent at displacing the radiolabeled antigen. This isunlike the 100-fold difference observed. If all of the antigenicdeterminants were masked and a small amount of contamination of the modified enzyme with the native enzyme caused theobserved competitive binding, then we would expect only 0.3to 1% precipitation of the E. coil enzyme activity by the antiserum. In contrast, between 96 and 99% of both native and

modified enzyme activity was precipitated by the double antibody technique when either antiserum was utilized. One possible interpretation of these results suggests that most of theantigenic determinants still exist in the modified enzyme preparation, but their ability to bind to the antisera is retarded bythe stenic effects of the polymer.

A previous section has demonstrated the prolonged plasmaclearance properties of modified E. coli asparaginase. Whenthe plasma clearance rate was reexamined in mice immunizedwith the native enzyme, the results in Chart 8 were observed.If early immune mice were used (25-fold diluted serum wouldstill bind >20% radiolabeled enzyme), the native enzyme wasimmediately cleared from the animal and no detectable circulating enzyme activity could be observed. With the modified(Preparation 1) enzyme, however, a prolonged plasma cleanance was observed in both the normal and immune animals. Ifhighly immune animals were used (625-fold diluted serum

would still bind >20% nadiolabeled enzyme), activity of neitherthe native nor the modified enzyme could be detected in theplasma following an i.p. injection of i 0 lU of the enzymes.These results suggest that the modified enzyme would be

Time(days)

CANCER RESEARCH VOL. 391932

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Antibody,Protease,Clearance,orImmunogenicRecognitionSites

JUNE 1979 1933

Biological Properties of Poly-DL-alanyl L-Asparaginases

REFERENCES

1. Abuchowski,A., van Es, T., Palczuk, N. C., and Davis, F. F. Alterationofimmunologic properties of bovine serum albumin by covalent attachment ofpolyethylene glycol. J. Blol. Chem., 252: 3578—3581, 1977.

2. Arnon,R., and Neurath, H. lmmunochemlcalstudieson bovinetrypslnandtrypsinogen derivatives. Immunochemistry, 7: 241—250,1970.

3, Bar-Eli, A., and Katchalski, E. Preparation and properties of water-Insolublederivatives of trypsin. J. Blol. Chem., 238: 1690—1698, 1963.

4. Brown, R. K., TapEs, M. A., Sela, M., and Anfinsen,C. B. Studieson theantigenic structure of nlbonuclease. IV. Polyalanyl ribonuclease. J. Blol.Chem., 238: 3876-3883, 1963.

5, Cammack, K. A., Marlborough, D. I., and Miller, D. S. Physical propertiesand subunit structure of L-asparaginase Isolated from Erwinia carotovora.Biochem. J., 126: 361 -379, 1972.

6. 000ney, D. A., Capizzi, R. L, and Handschumacher,R. E. Evaluationof Lasparaglnase metabolism In animals and man. Cancer Res., 30: 929—935,1970.

7. GabrIel, 0. Analytical disc gel electrophoresis. Methods Enzymol. 22: 565—578. 1971.

8. Geran, R. I., Greenberg,N. H., MacDonald,M. M., Schumacher,A. M., andAbbott, B. J. Protocols for screening chemical agents and natural productsagainst animal tumors and other biological systems. Cancer Chemother.Rep., 3 (Part 2): 96-97, 1972.

9, Goldberg, A. I., Conney, D. A., Glynn, J. P., Homan, E. R., Gaston, M. R.,and Milman, H. A. The effects of Immunization to L-aSparaglnaseon antitumar and enzymatic activity. Cancer Res., 33: 256—261, 1973.

10. Ho, P. K., Milikin, E. B., Bobbltt, J. L., Grinnan, E. L., Burck, P. J., Frank, B.H., Boeck, L. D., and Squires,R. W. CrystallineL-asparaglnasefromEscherichia coil B. Purification and chemical characterization. J. Blol. Chem.,245:3708—3715, 1970.

11. Howard,J.B.,andCarpenter,F.H.L-AsparaginasefromErwiniacarotovora.Substrate specificity and enzymatic properties. J. Biol. Chem., 24 7: 1020—1030, 1972.

12. Jackson, R. C., and Handschumacher, R. E. Escherichia coil L-Asparaglnase. Catalytic activity and subunit nature. Biochemistry, 9: 3585—3590,1970.

13. MoreIl, A. G., Gregorladis, G., Scheinberg, I. H., Hickman, J., and Ashwell,G. The role of sialicacid In determiningthe survivalof glycoprotelnsin thecirculation. J. Blol. Chem., 246: 1461—1467,1971.

14. Riley, V. Role of the LDH-elevatlng virus in leukemia therapy by asparaginase. Nature (Lond.), 220: 1245—i246, 1968.

15. RIley, V., Campbell, H. A., and Stock, C. C. Asparaginase clearance:influence of the LDH-elevatlng virus. Proc. Soc. Exp. Biol. Med., 133: 38—42, 1970.

16. Rutter, D. A., and Wade, H. E. The influence of the iso-electric point of Lasparaginase upon Its persistence In the blood. Br. J. Exp. Pathol., 52:610-614, i97i.

17. Schechter, I., Bauminger, S., and Sela, M. Induction of Immunologicaltolerance towards a peptide determinant with a non-immunogenic polypeptide. Biochim. Blophys. Acta, 93: 686-687, 1964.

18. SeIa, M. Immunological studies with synthetic polypeptides. Adv. Immunol.5: 29-129, 1966.

19. Sela, M., and Amon, R. Studies on the chemical basis of the antigenicity ofproteins. 1. Antlgenlcity of polypeptidyl gelatins. Blochem. J., 75: 91—102.1960.

20. Stark, G. R., and Smyth,D. G. The use of cyanate for the determinationofNH@-terminalresidues in proteins. J. Blol. Chem., 238: 214—225,1963.

21. Thorbecke, G. J., Maurer, P. H., and Benacerraf, B. The affinity of thereticulo-endothellal system for various modified serum proteins. Br. J. Exp.Pathol., 41: i90—197,1960.

22. Uren, J. R., and Handschumacher,R. E. Enzymetherapy. In: F. F. Becker(ed), Cancer: A comprehensivetreatise, Vol. 5, pp. 457-487. New York:Plenum Press, 1977.

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Chart 9. Proposed model to explain the enzymatic and biological propertiesof poly-Di-alanylasparaglnases. Polymer modification may: (a) mask antigenicdeterminants; (b) mask Immunogenic recognition sites; (c) mask protease-susceptible sites; (d) mask clearance recognition signals; (a) allow free access tolow-molecular-weight substrates; ( 1)maintain systemic injectabillty; and ( g) alterpH optimum by changing microenvironment.

in order to achieve the most optimal preparations. Further workin our laboratory is being done to identify other amino acid-N-carboxyanhydnides for enzyme attachment, which may alsoimprove their biological activity possibly beyond that presentedhere. A recent report by Abuchowski et a/. (1) has showndecreased immunological properties by the attachment of polyethylene glycol to bovine serum albumin.

A truly general procedure which can decrease the immunogenicity while increasing the plasma clearance properties ofproteins with retention of catalytic activity can be of immensevalue in the development of proteins and enzymes as therapeutic agents. The improved therapeutic activity and decreased immunogenicity of asparaginase reported here, aswell as the decreased immunogenicity of proteins previouslyreported by Arnon and Neurath (2), Brown et a!. (4), and Sela(1 8), suggest that amino acid-N-carboxyanhydnide polymeri

zation may be one such general procedure. The specific thenapeutic and immunological improvements of poly-DL-alanyl asparaginases require further immunological and toxicologicalcharacterization as a prerequisite for human trials as agentsfor therapy of acute lymphocytic leukemia.

ACKNOWLEDGMENTS

The very fine technical assistance of Frank Chin and Michelle Gorman isgratefully acknowledged. Thanks are also given to Dr. victor Raso for advicewith respect to immunochemistry and to Dr. Emil Frel, III, and Dr. Edward Modestfor their continued interest and input in this research program.

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1979;39:1927-1933. Cancer Res   Jack R. Uren and Richard C. Ragin  l-Asparaginases by Attachment of Poly-dl-alanyl Peptides

Erwinia carotovora and Escherichia coliProperties of Improvement in the Therapeutic, Immunological, and Clearance

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