The Effects of Immunization to L-Asparaginase on Antitumor ......L-asparaginase therapy that has been linked to the development of resistance (10-12, 14, 15). Immunization of animals

(CANCER RESEARCH 33, 256-261, February 1973)

The Effects of Immunization to L-Asparaginase onAntitumor and Enzymatic Activity

Arthur I. Goldberg, David A. Cooney, John P. Glynn,1 Elton R. Homan, Marilyn R. Gaston,

and Harry A. Milman

Drug Evaluation Branch, Drug Research ami Development ¡A.1. G., J. P. G., M. R. G.], and Laboratory of Toxicology, Experimental Therapeutics¡D.A. C., E. R. H., H. A. M.]. Chemotherapy, National Cancer Institute, Bethesda. Maryland 20014

SUMMARY

BALB/c mice were immunized with Escherichia coliL-asparaginase and tested for the presence of hemagglutinationantibody (HA) to the enzyme. Mice were then groupedaccording to whether they were immune to L-asparaginase(HA titer, >4) or were non-HA responders (negative HA titer).In vivo antitumor activity of L-asparaginase against P-1798 (anL-asparaginase-sensitive lymphoma) was tested in both groups,as well as in control mice (never before exposed toL-asparaginase). L-Asparaginase was inactive against the tumorin immune mice but was equally active in non-HA respondersand control animals. Another experiment comparedL-asparaginase derived from E. coli with enzyme derived fromErwinia carotovora (which does not cross-reactimmunologically with E. coli L-asparaginase). Mice immune toE. coli L-asparaginase received about the same benefit fromthe antitumor activity of E. carotovora L-asparaginase as didcontrol mice. In separate studies, blood samples from immune,non-HA responder, and control mice were tested by in vitroassays for levels of L-asparaginase and L-asparagine at varioustime intervals up to 120 hr following a single i.v. injection ofL-asparaginase. Most immune mice cleared the plasma ofen/.yme 30 min after injection and consequently had a rapidreturn to the circulation of L-asparagine. In non-HAresponders and control animals, L-asparaginase was detectablefor over 48 hr after injection, and L-asparagine remainedabsent from the circulation. These studies relate the presenceof humoral antibody to L-asparaginase with loss of antitumorand enzymatic activity and suggest that, in immunized animalswithout detectable HA, the antitumor and enzymatic activityof L-asparaginase is intact.

INTRODUCTION

Of all the chemotherapeutic agents that are active intreating human neoplasia, L-asparaginase is the only drug witha mechanism of action that is thought to depend on a specificmetabolic defect in the malignant cell (7, 9). The basis of thetherapeutic activity of L-asparaginase is assumed to be a resultof the depletion of L-asparagine from the circulation, thus

'Deceased (May 7, 1971).Received February 4, 1972; accepted October 20, 1972.

depriving sensitive tumor cells of their sole source of theamino acid (2,9). Most normal and resistant cells are thoughtto escape the cytotoxic effects of the L-asparaginase becauseof their ability to synthesize L-asparagine (18). However,although the antitumor activity of L-asparaginase has beendescribed as an "... all-or-none-response .. . (depending uponthe susceptibility or resistance of a particular leukemia) .. ."

(1), it is clear that resistance to the activity of the drug willeventually occur, in most cases, even against tumors that wereinitially sensitive. Thus, although L-asparaginase will inducecomplete remission in over 60% of patients with acutelymphocyte leukemia (3, 4), the duration of remission isusually short (18). The development of resistance to theantitumor activity of the enzyme may involve several differentmechanisms. Resistance of tumor cells correlates well with thelevel of L-asparagine synthetase (1, 6). If tumor cells cansynthesize their own L-asparagine, they are independent ofcirculating L-asparagine and therefore are resistant toL-asparaginase-induced cytotoxicity. The enhanced ability oftumor cells to extract L-asparagine from the plasma, RBC's, or

other adjacent cells also has been suggested as a mechanismthat may explain tumor cell resistance to L-asparaginase (1).

Immunological response is another consequence ofL-asparaginase therapy that has been linked to thedevelopment of resistance (10-12, 14, 15). Immunization ofanimals with L-asparaginase has been reported to nullify itsantitumor activity (10, 11, 14, 15).

Incubation of L-asparaginase with immune serum that isspecific for the enzyme has been demonstrated to decreaseenzymatic activity up to 50% (10). These observations suggestthat the production of antibody against L-asparaginase mayplay a role in the development of resistance. However,Peterson et al. (10) reported that the enzymatic activity ofL-asparaginase could be demonstrated in an enzyme-antibodycomplex. Moreover, sustained depression of circulatingL-asparagine has been observed in a small number of patients,even after L-asparaginase could not longer be detected in theplasma (10). These observations suggested that, while antibodyto L-asparaginase may develop, binding the L-asparaginase andcausing extravascular sequestration of the enzyme-antibodycomplex, enzymatic and therefore presumably antitumoractivity would still be exerted (10, 11).

This study was conducted to clarify the role that thehumoral response to L-asparaginase may play in thedevelopment of resistance to this chemotherapeutic agent. Theantitumor activity of L-asparaginase was evaluated in immune

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mice (previously sensitized to L-asparaginase and with HA2

titers of 8 or higher), in non-HA responder mice (alsopretreated with L-asparaginase, but with negative HA antibodytiters), and in control mice (never before exposed toL-asparaginase).

Enzymatic activity also was studied in these groups of micein separate experiments, and correlations were made betweenthe presence of measurable HA titers, the loss of antitumoractivity, and the loss of enzymatic activity.

MATERIALS AND METHODS

Sensitization of Animals to L-Asparaginase

L-Asparaginase, derived from E. coli with a specific activityof ~300 i.u./mg protein, was a gift to the National Cancer

Institute from Merck, Sharp and Dohme (West Point, Pa.).Both male and female BALB/c mice received i.p. injections of

2The abbreviations used are: HA, hcmagglutinating antibody; MST,median survival time.

Asparagi/tose Activity after Immunization

E. coli L-asparaginase weekly or biweekly, for periods of up to2 years beginning at either the 1st day or the 8th week of life.The dosage per mouse was constant and ranged from 0.10 to200 i.u. (Tables 1 and 2). All injections were given at 0.10ml/mouse.

Passive HA Titers

Passive HA titers were determined by the method ofStavitsky (14) as modified by Peterson et al. (10). The endpoint chosen was the highest dilution with a definite mat ofagglutinated cells and was expressed as the reciprocal of thedilution of the serum at the end point. Blood samples for theHA determination were obtained by retroorbital bleeding atvarious time intervals over a 2-year period.

In Vivo Antitumor Activity of L-Asparaginase in Immune,Non-HA Responder, and Control Mice

Mice with HA titers that were negative following injections

Table 1Hemagglutination titers to L-asparaginasefollowing weekly immunization with L-asparaginase beginning at 24 hr oj age

All mice alive at each time interval were bled and all serum samples were then tested for antibody titer.

HA titers ofL-asparaginase"L-Asparaginase

dosei(i.u./mouse)20020ID10.10Totalno. of

nicebeginningtreatment2743383119Micebled at 18-59 days Mice bled at 112 155daysNegative23/2740/4331/3823/3110/192-2564/273/437/388/319/19512-4096

Negative12/1611/332/368/242/112-2564/1618/3330/3610/246/11512-40964/334/366/243/11Mice

bled at 366-41 2daysNegative1/137/171/84/91/32-25611/139/174/83/9512-40961/131/173/82/92/3

1The HA titers are tabulated as the number of mice with particular range of titers over the total number of mice tested on the particular day.

Table 2Hemagglutination liters to L-asparaginasefollowing weekly immunization with L-asparaginase hi'ginning at fi weeks of age

No. of mice with following HA tilers to L-asparaginasebonL-Asparaginasedose(i.u./mouse)020010020Day

13Nega-

2-tive853

25Nega

tive323Day

272-812116-128\c\t1'Nega

tive222Day

562_812216-4096T*I'1'Nega

tive12Day

1392_8316-5123e\'Day158Nega-

2-tive8223161024\ri*

0 1-ive mice per group began the study and all mice alive at each time interval were bled and all serum samples were then tested for antibody

titers.6 The HA titers are tabulated as the number of mice with the particular range of liters over the total number of mice tested on the particular

day.c Tiler of 16.d Titer of 32.e Titers of 32, 16, and 16, respectively.f Titer of 128.«Titerof512.h Tiler of 1024.'Titer of 4096.

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Goldberg, Cooney, Glynn, Roman, Gaston, Milman

of E. coli L-asparaginase from birth were designated as non-HAresponders. "Immune mice" was the designation given animals

that received L-asparaginase injections from birth and that hadHA titers of 8 or higher. BALB/c males and females ofapproximately the same age as the non-HA responder andimmune aimais, but never before exposed to L-asparaginase,were designated as controls.

P-1798 is a lymphosarcoma that is sensitive toL-asparaginase (8, 11) and is carried as a transplant in BALB/cX DBA/2 F, (hereafter called CDF. female mice. On the 1stexperimental day (Day 0), CDF, mice with large, palpabletumors were killed, and a brei was prepared that consisted of

0.10 g of tumor per ml of Hanks' balanced salt solution.Five-tenths ml of this brei was then injected s.c. into the righthind leg of the immune, non-HA responder, and controlBALB/c mice to be tested. L-Asparaginase therapy was givenas described in Tables 3 and 4, and mice were then examineddaily for 30 days and weekly thereafter to observe tumorsize. Mortality was recorded daily. MST was measured for alltest groups. L-Asparaginase, derived from E. carotovora with aspecific activity of ~500 i.u./mg protein, was obtained fromthe British Microbiological Research Establishment, PortónDown, England. Following the P-1798 inoculation on Day 0,E. carotovora L-asparaginase was injected i.p. on Days 3 and

Table 3Therapeutic efficacy of L-asparaginaseagainst P-l 798 (inoculated s.c. on Day 0) in immune, nonimmune, and control BAI.B/c mice

Group1.

Control62. Control63.Nonimmune''4

Immune^S

Immune^No.

ofmice4

4119gL-Asparaginase

(i.u./mouse) i.p. onDays 1 and100

505050100Days

ofdeath20,

24, 27, 27105+, 105+, 105+, 105+41,70,77, 105+(X8)16,

18.21.24.25,27,29,105+,105+

2,23,24,27,27,28,40MST

(days)25.5

105+''105+c2527No.

of survivorson Day1050

482e0Pa0.05d0.05d

Asparaginase Activity after Immunization

10 (Table 4). This enzyme was tested because animalssensitized to E. coli L-asparaginase do not cross-reactimmunologically with E. carotovora L-asparaginase (11).Because of the distribution of survival time, statistical analysiswas performed with a nonparametric method, the Wilcoxonrank sum test (13).

In Vitro Assays of L-Asparaginase Clearance and Activity inImmune, Non-HA Responder, and Control Mice

Procurement of Blood. Mice were bled from the ophthalmicplexus with disposable capillary pipets. An aliquot of blood(usually 20 n\} was added immediately to 100 ¡Aof ice-cold0.9% NaCl solution and centrifuged for 1 sec at 12,000 rpm inthe Eppendorf Zentrifuge. One aliquot of the supernatant, forthe measurement of L-asparaginase, was immediately iced.L-Asparaginase was quantitated by the method of Cooney etal. (5). Another aliquot for the measurement of L-asparaginewas dispensed into plastic 1.5-ml Eppendorf conical vesselsmaintained at 95°on an Eppendorf thermoregulated hotplate

and heated for 5 min. This operation was completed within 30sec of venipuncture and served to destroy any L-asparaginasein the sample.

Measurement of L-Asparagine. L-Asparagine in the sampleswas measured by a radiometrie technique to be publishedelsewhere. Briefly, duplicate samples of mouse plasma ( 1.6 jul)were treated with bacterial decarboxylases to removeL-aspartic and L-glutamic acids. Then, L-asparagine in thesample was hydrolyzed to L-aspartic acid with L-asparaginase.The L-aspartic acid that was generated was transaminated withradioactive a-ketoglutaric acid-1-14C to yield radioactiveL-glutamic acid. Unreacted a-ketoglutaric acid-l-14C was

removed with acid hydrogen peroxide; the residualradioactivity was counted in a scintillation spectrometer. Withthis technique as little as 25 pmoles of L-asparagine can bedetected in 1.6 /Lilof blood. Statistical analysis of the enzymeconcentration at 1 hr was carried out with the least significantdifference test (13).

RESULTS

Development of HA Titer to E. coli L-Asparaginasefollowing Regular Injections with the Enzyme. Tables 1 and 2give the HA titer to L-asparaginase in BALB/c mice, receivingweekly injections of L-asparaginase from 24 hr or 8 weeks ofage, respectively. The titers, determined at various timeintervals over a 2-year period (Tables 1 and 2), include theresults of single serum samples taken from all mice surviving ateach of the time intervals. Occasionally, for testing purposes,mice were bled 2 or 3 times during a time interval. In thesecases the titer of only the 1st sample is reported. In the groupreceiving L-asparaginase from 1 day of age (Table 1), thegeneral pattern of antibody response was a consistent increasein titer between the 1- and 2-month sample (Bleeding 1) andthe 3.5- to 5-month sample (Bleeding 2), with a plateau in titerover the next 1.5 years. There were fewer mice with negativetiters among the groups receiving the lower dose levels ofL-asparaginase. The most effective dose in producingsensitization was 10 i.u./mouse, with 34 of 36 positive titers at

the 2nd bleeding period and 7 of 8 positive titers at the finalbleeding. A varying number of mice with negative antibodytiters was observed for all the doses of L-asparaginase studied.Table 2 presents the titers for mice that received weekly dosesof L-asparaginase beginning at 8 weeks of age. In general,lower titers were observed in this group than in mice in whichsensitization began at 1 day of age. However, in theexperimental groups that received L-asparaginase, 20i.u./mouse, from 8 weeks of age, there were no negative titersafter 139 days. In contrast 33% of the mice that begansensitization to L-asparaginase, 20 i.u./mouse from theneonatal period, had negative HA titers.

Analysis of the Antitumor Activity of L-Asparaginase inControl Mice and Mice Either Immune to L-Asparaginase orwith Negative HA Titers following Sensitization. From themice that were receiving weekly injections of L-asparaginasefrom birth, groups of non-HA responders and groups of immune mice were formed. Two groups of mice never before exposed to L-asparaginase (control mice) were also followed. Allmice were challenged with P-1798 on Day 0. The MST in thegroup given tumor and left untreated was 25.5 days with therange of death from Day 20 to Day 27 (Table 3). Thesensitivity of the tumor to L-asparaginase was demonstrated inthe 2nd control group, which received L-asparaginase, 50i.u./mouse, on Days 1 and lO.Of 4 animals, 4 were alive without tumor at Day 105 (Table 3). Non-HA responders (i.e., negative HA titers) were also treated with L-asparaginase, 50i.u./mouse, on Days 1 and 10. The survival pattern was verysimilar to that of control mice with 8 of 11 mice alive at Day105 without palpable tumor. The 3 deaths occurred on Days41, 70, and 77 and the animals died with either large tumorsor ascites (Table 3). L-Asparaginase had a reduced antitumoreffect in the immune group. When L-asparaginase, 50i.u./mouse, was injected on Days 1 and 10, the MST was 26days, with 6 of 8 animals dead by Day 105. A 100-i.u./mousedose produced a MST of 27 days, with all mice dead by Day41 (Table 3). The survival times for immune mice treated with50 or 100 i.u./mouse were significantly less than the survivaltimes of the control and nonimmune treated animals (p <0.01).

A Comparative Study of the Therapeutic Effect of E. coliand E. carotovora L-Asparaginase in Control and Immune Miceand E. coli L-asparaginase. From the group of mice treatedweekly from birth with E. coli L-asparaginase, 3 groups ofimmune mice, with HA titers of 128 to 512, were formed.Three control groups (no prior exposure to L-asparaginase)were treated in a manner identical to that for the immunegroups. All the mice were inoculated with P-1798 on Day 0.One of the immune and one of the control groups were leftuntreated. The 2nd immune and control groups received E.coli L-asparaginase, 40 i.u./mouse, on Days 3 and 10. The final2 groups were given injections of E. carotovora L-asparaginase,35 i.u./mouse, on Days 3 and 10. Without L-asparaginasetherapy immune and control mice all died with large tumors30 days after tumor inoculation, with MST's of 24.5 and 22

days, respectively (Table 4). Treatment with E. coliL-asparaginase produced no increase in survival for theimmune mice over the untreated animals with a MST of 23days and with all 10 animals dying with tumor 28 days aftertumor inoculation (Table 4). All control animals treated with

FEBRUARY 1973 259

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Goldberg, Cooney, Glynn, Homan, Gaston, Milman

E. coli L-asparaginase survived to Day 30 and 5 of 10 werealive and tumor free on Day 150. In contrast E. carotovoraL-asparaginase was effective in both immune and controlgroups with MST's of 36 and 41 days, respectively. The

survival times for the immune animals treated with E.carotovora L-asparaginase were significantly higher thanuntreated animals (p < 0.01), while immune mice given E. coliL-asparaginase died with a pattern that did not differsignificantly from that of untreated mice (p > 0.05).

A Comparison of L-Asparaginase Clearance andL-Asparagine Recovery following a Single Injection ofL-Asparaginase in Immune, Non-HA Responder, and ControlMice. From the animals treated from birth with E. coliL-asparaginase, 6 immune and 6 non-HA responder mice werechosen. The immune group had HA titers of between 128 and512. The non-HA responders had negative HA titers. A 3rdgroup of 6 mice never before exposed to L-asparaginase served

as controls.Two mice each, from the immune, non-HA responders, and

control groups were given L-asparaginase, 50, 5, or 0i.u./mouse, i.v. at Time 0. Nine blood samples were obtainedover the next 120 hr at time intervals indicated in Chart 1. Acontrol sample was obtained from all mice 10 min prior toinjections of L-asparaginase. In all samples both L-asparaginaseand L-asparagine were measured. Two basic observationsemerged from this in vitro analysis. First, L-asparagine wasdetectable in a plasma sample only when L-asparaginase wasnot present. Even the smallest amount of L-asparaginasedetectable in this study, 0.10 i.u./ml of blood, correlated withan L-asparagine level of

Asparaginase Activity after Immunization

biologically active enzyme might have been sequestered in thereticuloendothelial system.

Our study indicated that the development of passive HAtiters to L-asparaginase correlated with a loss of antitumoractivity. The experiment with E. carotovora L-asparaginasedemonstrated that, while immune mice were resistant to theantitumor effects of E. coli L-asparaginase, the enzyme derivedfrom E. carotovora, which does not cross-reactimmunologically with E. coli L-asparaginase, was still effective.Furthermore, the in vitro measurement of L-asparaginase andL-asparagine plasma levels in immune, non-HA responder, andcontrol mice demonstrated that the presence of passive HAtiters is associated with and may actually result in a very rapidclearance of L-asparaginase from the circulation. Moreover, theelimination of the L-asparaginase from the circulation did notappear to be a sequestration of a still active enzyme-antibodycomplex, because if the enzyme were still active theL-asparagine level presumably would not have returned topretreatment levels as rapidly as was observed in the immunemice. The in vivo and in vitro studies, therefore, indicate thatthe development of humoral antibody to L-asparaginase isdirectly correlated with loss of therapeutic effectiveness ofL-asparaginase. The observation that mice pretreated withL-asparaginase but with no measurable HA titer demonstratedthe same antitumor effects from the drug as control mice,never previously exposed to the enzyme, warrants furtherstudy in terms of how they may contribute to more effectiveuse of L-asparaginase.

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1973;33:256-261. Cancer Res Arthur I. Goldberg, David A. Cooney, John P. Glynn, et al. Enzymatic ActivityThe Effects of Immunization of l-Asparaginase on Antitumor and

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