a Murine Thymoma and Colon Carcinoma Xenografts

Antitumor Effect of 2′-Deoxy-5-fluorouridine Conjugates against a Murine Thymoma and Colon Carcinoma Xenografts Kenia G. Krauer, Ian F. C. McKenzie and Geoffrey A. Pietersz Cancer Res 1992;52:132-137.

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ICANCERRESEARCH52. 132-137.January 1, 19921

Antitumor Effect of 2'-Deoxy-5-fluorouridine Thymoma and Colon Carcinoma Xenografts Kenia G. Krauer, Ian F. C. McKenzie,

Conjugates against a Murine

and Geoffrey A. Pietersz'

The Austin Research institute, Kronheimer Building, Austin Hospital, Heidelberg, Victoria 3084, Australia

inhibiting the enzyme thymidylate synthetase and as a result inhibits DNA synthesis. 2'-Deoxy-S-fluorouridine was successfully conjugated to

relatively smaller doses inhibit tumor growth. 5FdUrd, a more potent derivative of 5FUra, an agent pres ently used for the treatment of adenocarcinoma of the breast, colon, and ovary, was therefore examined. This highly cytotoxic drug can exert its action in two ways; either by incorporation into RNA after metabolism into 5-fluorouridine diphosphate or by inhibition of DNA synthesis due to 2-deoxy-5-fluorouri dine monophosphate inhibition ofthe enzyme thymidylate syn

anti-Ly-2.l reactive with the murine thymoma ITF(I)7SNS E3, I-i, and

thetase. In a previous paper we reported the conjugation of

250-30.6 reactive with human colon cancer cells using the active ester of

5FdUrd to monoclonal antibodies, via the active ester of the 5FdUrdsucc derivative (I 2). Initial reactivity of 5FdUrdsucc immunoconjugates was in vestigated by using anti-Ly-2.I conjugates within a tumor

ABSTRACT

The conjugation of antineoplastic drugs to monoclonal antibodies reactive

with tumor

associated

antigens

conveys selective

cytotoxicity,

overcoming the systemic toxicities caused by drugs during standard chemotherapy. 2'-Deoxy-5-fluorouridine, a more potent derivative of 5fluorouracil, is an antimetabolite which exerts its cytotoxic action by

2'-deoxy-5-fluoro-3'-O-succinoyluridine

(SFdUrdsucc).

In vitro,

5Fd

Urdsucc-anti-Ly-2.l was selectively cytotoxic against I1T(1)7SNS E3 murine thymoma cells at nanomolar concentrations. The human colon carcinoma

cell L1M1899

was inhibited

by SFdUrdsucc-I-I

conjugates

in

the range of 10-@10-@M, as were Cob 205 cells by SFdUrdsucc-25030.6 conjugates. In vivo, SFdUrdsucc conjugates were more effective than equivalent amounts of free SFdUrdsucc. Against the ITF(1)75NS

E3 murine thymoma, a single dose of 100 isg (SFdUrdsucc equivalents)

SFdUrdsucc-anti-Ly-2.1resulted in 85% tumor inhibition compared to meantumor size of control mice.Irrelevant SFdUrdsuccconjugatesfailed to inhibit tumor growth. Multiple doses of SFdUrdsucc-I-1 conjugate produced

50% growth inhibition

of the moderately

differentiated

tumor

INTRODUCTION Conventional chemotherapy, possibly effective in preventing tumor growth and occasionally eradicating tumors, can be accompanied by severe side effects and a dose limiting toxicity (1). Targeting of drugs, toxins, and radioisotopes, directly to tumor associated antigens by using MoAbs2 is now under examination as an alternative to overcome these problems (2â€” 6). The decrease in systemic drug toxicity noted with conjuga tion to antibodies, and the greater drug uptake by tumors using drug conjugates in comparison to free drug, make drug-antibody conjugates an attractive alternative for cancer therapy (7). Chemical coupling methods have been established for a number of drugs (8) and some drug-immunoconjugates have been used data indicate

that this

in combination

with the partial

loss in both

monoclonal

antibody(ies);

CEA,

Australia

and were maintained

in patho

Tumor Cells. The clonal variant E3 of a murine thymoma (Ly-2.1@) (14) was maintained in vitro in Dulbecco's modified Eagle's medium supplemented with 10% heat inactivated newborn calf serum (Flow

Laboratories, Sydney, Australia) and 2 mM glutamine (CSL), 100 @g/ ml streptomycin (Glaxo, Melbourne) and 100 lU/mb penicillin (CSL). The E3 cell line was maintained by serial passage in CBF1mice. For in vivo studies, cells from ascites fluid were washed and centrifuged (1000 rpm, 5 mm) twice in PBS (pH 7.3), and resuspended in PBS before s.c.

using

carci

noembryonic antigen; E3, ITT(1)75NS E3; 5FUra, 5-fluorouracil; FUrd, 5fluorouridine; 5FdUrd, 2'-deoxy-S-fluorouridine; SFdUrdsucc, 2'-deoxy-S-flu oro-3'-O-succinoyluridine; PBS, phosphate buffered saline; TFR, transferrin re

of 3-5 x 106 cells into CBF

mice.

Cob

205 (16) was maintained

in vitro under

the same

protein

A-Sepharose

(Pharmacia,

Inc.,

Piscataway,

NJ)

(18).

Anti-transferrin receptor (IgGl) (19), reactive with human transfemn receptor, I-i (IgGl) (20), reactive with CEA and 250-30.6 (IgG2b), reactive

ceptor, CSL, CommonwealthSerum Laboratories,Melbourne,Australia DMF, dimethylformamide;

in Western

Monoclonal Antibodies. The anti-Ly-2.l (IgG2a) (17) reactive with the murine thymoma E3 was isolated from ascitic fluid and purified by

Building, Austin Hospital, Studley Road, Heidelberg,

Victoria 3084, Australia. MoAb,

Centre

in nude mice by injecting cells (5 x l0@)directly from tissue culture, followingwashing and centrifugation (1000 rpm, 5 mm).

bTo whom requests for reprints should be addressed, at The Austin Research

are:

Resource

gen-free conditions.

conditions as LIM1899. Cob 205 was established as a s.c. xenograft

accordancewith 18 U.S.C. Section 1734solelyto indicatethis fact.

used

Mice. CBF mice were bred in the Department of Pathology, Uni versity of Melbourne. Swiss nude mice were obtained from the Animal

carcinoma

Received7/24/91; accepted 10/16/91.

abbreviations

AND METHODS

mM glutamine (CSL), 100 @g/mlstreptomycin (Glaxo, Melbourne), and 100 lU/mb penicillin (CSL). For in vivo studies, L1M1899 was serially transplanted as a s.c. tumor xenograft. The human colon

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

2 The

were tested against

LIM1899 (15), a human colon carcinoma cell line, obtained from Dr. R. H. Whitehead (Ludwig Institute for Cancer Research, Mel bourne), was maintained in vitro in RPMI 1640 supplemented with 10% heat inactivated fetal calf serum (Flow Laboratories, Sydney), 2

drug and antibody activity, resulting from the conjugation pro cedure, has prompted the use of more potent drugs where

Institute, Kronheimer

MATERIALS

injection

mode of treatment has therapeutic potential, however, some limitations are also evident. Restricted tumor access and poor blood perfusion results in relatively inefficient uptake of drug antibody conjugates into tumors (1 1). The inefficient permea tion into tumors

and 5FdUrdsucc-250-30.6

colon carcinoma xenografts grown in nude mice, LIM1899 and Cob 205, respectively. We now report the efficacy of 5FdUrdsucc conjugates in vivo.

tively resistant to free SFdUrdsucc and SFdUrdsucc-250-30.6 conjugates.

trials (9, 10). The resulting

BALB/c F1 (hereafter called CBF) mice which are Ly-2.1 negative. This model has been used in a number of studies to characterize the in vivo antitumor activity of a variety of drugs and toxin immunoconjugates (13). Using this model, initial dose response and optimal dose scheduling studies were per formed. In light of the activity of 5FUra in colon cancer, SFdUrdsucc-I-1

LIM1899. In contrast, the human colon carcinoma Cob 205 was rela

in clinical

specific model, E3 thymoma (Ly-2.1 positive) growing in B6 x

with an antigen

present

on normal

and malignant

gastrointestinal epithelium, were also used is this study (21).

IC,,, 50% inhibition of@3HJdeoxyuridine incorporation. 132


human

IN Jâ€•IVO ACFIVITY OF 5FdUrdsucc-MoAbCONJUGATES

Preparation

and Quantitation

of Conjugates.

The active ester deriva

RESULTS

tive of 5FdUrdsucc (10) was prepared by dissolving 1.73 mg of 5FdUrdsucc

in 70

@l of dry DMF.

N-Hydroxysuccinimide

17 @tl dry DMF) and dicyclohexylcarbodiimide DMF) were added and the reaction

mixture

In Vitro Antibody Activity

(0.59 mg in

(618 mg in 50 @tl dry

was kept at room temper

ature for 3 h, then at 4'C overnight. An aliquot of active ester was added to MoAb while vortexing and the mixture was maintained at room temperature for 3 h. Following centrifugation (400 x g for 5 mm) to remove precipitate, the solution was passed through a PD-10 column Sephadex G-25 (Pharmacia) equilibrated in PBS. Antibody concentra tion was determined

as described

previously

(22), and the amount

of

5FdUrdsucc bound was measured by absorbance spectrophotometry at @

260 nm (t2,o FdUrdsucc = 7600 M1 cm' M'

cm').

The addition

of 20-fold

and

MoAb = 129,300

molar excess of active ester led to

an incorporation of 7â€”9 molecules of SFdUrdsucc/antibody molecule with 70â€”80%protein recovery. All in vitro and in vivo experiments were carried out by using 5FdUrdsucc conjugates with a similar incor poration ratio.

The binding activity of MoAbs and MoAb drug-conjugates was measured by using flow cytometry. A saturating amount of anti-Ly-2.1 MoAb (5 @g) on E3 cells had a mean fluorescence of 118, whereas 5 jsg of 5FdUrdsucc-anti-Ly-2.1 (5.8 residues) had a fluorescence of 106 on the same cells, indicating an 11.0%

decrease in binding. Equivalent saturating quantities (8 @sg) of I-i MoAb and 5FdUrdsucc-I-1 conjugate (8.0 residues) had mean fluorescence values of 29 and 21 , respectively, demon strating a 24.0% decrease in antibody binding on LIM 1899 cells. A 15.5% decrease in binding was observed for 5FdUrdsucc-30.6

comparison

(8.2 residues),

mean

fluorescence,

133, in

to the same quantity of 250-30.6 MoAb, mean

fluorescence,

154, against

Cob

205 cells. This

decrease

in

by flow cytometry with the use of a FACScan (Becton Dickinson,

MoAb binding displayed by all conjugates is expected since the addition of a number of drug residues to an antibody may

Mountain View, CA) (15). Briefly, indirect binding assays were per

interfere

In Vitro Antibody Activity. Binding activity of MoAb was measured

with its binding to its epitope.

formed where 3 x 10' cells were incubated with MoAb or conjugate for

In Vitro Cytotoxicity

1 h at 4C. Cells were washed and centrifuged 3 times with buffer (PBS containing 2% newborn calf serum and 0.025% sodium azide) and

The cytotoxicity of 5FdUrd, 5FdUrdsucc, and 5FdUrdsucc immunoconjugates was tested on the cell lines (Table 1). 5FdUrdsucc and 5FdUrdsucc-anti-Ly-2.1 conjugate showed

resuspended in buffer containing 10 @gfluorescein isothiocyanate labeled rabbit anti-mouse immunogbobulin F(ab)'2 fragment (Silenus

Laboratories Pty Ltd., Hawthorn, Australia). After a 1-h incubation on ice, the cells were washed and centrifuged

similar growth inhibition

twice with buffer, and finally

resuspended in the same buffer, and fluorescence was measured by using a FACScan.

on E3 in a 24-h assay with IC50 values

of 5 x l0-@ M and 3.0 x i0@ M, respectively, whereas 5FdUrd showed 10-fold greater activity (ICso 4.0 X 10@'Â° M). In a 30-

In Vitro Drug Activity. The activity of the drug before and after conjugation was determined by a 24-h cytotoxicity assay. Briefly,sterile, filtered dilutions of drug or conjugate (100 @l in PBS) were added to 1-

Ly-2.1) was 4 times more active than the nonspecific conjugate (5FdUrdsucc-anti-TFR) on E3 cells (Ly-2.1 positive, TFR neg

2 x 10' cells in 100

ative), indicating

@lmedium

and incubated

in 96-well plates for 24

mm exposure

assay the specific conjugate

(5FdUrdsucc-anti

the selective toxicity of the anti-Ly-2.1

MoAb

h at 3'lT and 7% CO2. Following this incubation period, 1 @iCi 16-3H1 conjugate. On the LIM1899 cells, a 6-fold difference was oh deoxyuridine

(Amersham

International,

Buckinghamshire,

England)

in

50 @l medium was added and the cells were incubated for a further 4â€”

served between 5FdUrdsucc (IC50 6.25 x 10_a M) and

5FdUrdsucc-I-1 conjugate (ICso 4.0 X i0@ M), 5FdUrdsucc was also 10-fold more active than 5FdUrd (ICso 7.5 X iO@ M) after 24 h of exposure. The L1M1899 reactive antibody conju lation System, Packard Instrument Co., Downers Grove, IL) in 5 ml gate 5FdUrdsucc I-I showed 10-fold greater activity than the scintillation fluid. The specificity of conjugates was assessed by com LIM1899 nonreactive 5FdUrdsucc-anti-Ly-2.1 conjugate, paring conjugates made with MoAbs reactive and nonreactive with a again illustrating the specificity of 5FdUrdsucc conjugates. The given cell line. Dilutions of each conjugate (200 Ml in PBS) were cytotoxicity of 5FdUrdsucc (IC50 6.0 x 10_6 M) on Cob 205 incubated with the cells (2 x 10' cells in 200 @il medium) for 30 mm at cells was 2.5 times greater than SFdUrdsucc-30.6 conjugate room temperature. To remove unbound conjugate the cells werewashed (IC50 I .5 x i0-@ M), and 4.0 times less than 5FdUrd (ICso 1.5 and centrifuged (1000 rpm, 5 mm) three times using fresh medium, x 10_6 M) during the 24-h drug exposure assay. Using Cob then resuspended in 200 medium before 100 MIaliquots were added 205, selective cytotoxicity could not be demonstrated as suffi to microtiter plates. Following incubation for 24 h at 37T and 7% C02, the cells were pulsed and harvested as describedabove.The results cient concentrations ofconjugate could not be used which would were expressed as a percentage of inhibition of [6-3Hjdeoxyuridine produce cell killing. 6 h. Cells were harvested, using a microtiter plate harvestor (Dynatech, England), onto glass fiber filters (Enzo Biochem., Inc., New York, NY) and counted in a fi scintillation counter (Tri-Carb 460C Liquid Scintil

@

incorporation compared to control wells. In Vivo Use of SFdUrdsucc-MoAb Immunoconjugates.

the in vivo potency of 5FdUrdsucc-MoAb

To examine

Table I In vitrocytotoxicitiesof5FdUrd, 5FdUrdsucc,and 5FdUrdsucc

conjugates three tumor

conjugateson cell lines

models were used; the E3 tumor (Ly-2.l positive, Ly-2.2 negative) grown in CBF mice (Ly-2.1 negative and Ly-2.2 positive), which is a congenic

tumor

model, and Cob

205 and LIM1899

(M)ITT(l)7SNSâ€¢E3L1M1899Cob IC,@,

which are human

tumor xenografts grown in nude mice.

205Assay24

E3 (5 )10' NDSFdUrdsucc-l-1ND1.2xl07NDSFdUrdsucc-30.6NDND>10'

were recorded as mean tumor size (cm2)Â±SE. Groups of 6â€”8 mice, of the same age and sex, were used for each experimental group. In therapy studies the dose injected

is expressed

as 5FdUrd

equivalents

(jig) in the

conjugate.

a IC,@

value

b ND,

not

represents

50%

inhibition

of l3Hjdeoxyuridine

done.

133


incorporation.

IN J'IVO ACTIVITY OF 5FdUrdsucc-MoAb CONJUGATES 6 @

SFdUrdsucc could be safely administered on 4 alternative days without any toxicity (Fig. 3). To establish the specificity of SFdUrdsucc conjugates and the contribution unconjugated antibody or free drug had on tumor inhibition, the effects of (a) FdUrdsucc conjugated to anti-TFR (E3 nonreactive); (b) a mixture of unconjugated SFdUrdsucc and anti-Ly-2.l; and (c) SFdUrdsucc conjugated to anti-Ly-2. 1 (E3 reactive) were compared on the E3 thymoma. Four days after tumor inoculation, individual treatments con

A

5.

@

4,

Cl) @

3,

E I-.

@

2'

C C C

1'

sisting of a total of 80 jsg of SFdUrdsucc, in either the conju

70

gated or unconjugated form, were administered on 4 alternate days (Fig. 3). The SFdUrdsucc-anti-TFR conjugate, which is unreactive with the E3 thymoma showed 7.0% tumor inhibition on Day 13, while the specific conjugate, SFdUrdsucc-anti-Ly 2.1, showed 60% tumor inhibition, thus demonstrating the specificity of 5FdUrd MoAb conjugates. The mixture of free drug and MoAb (with doses of each equivalent to the anti-Ly

60

at these low doses has been shown to have no antitumor effect

10

20

15

Days After Tumor Inoculation

B 0 Câ€• OC

80

2.1 conjugate) Ec

Free SFdUrdsucc

(data not shown). In vivo growth inhibition studies using 0

@

showed 25% tumor inhibition.

50

5FdUrd, in the range 50 @igâ€”1 mg/dose for 5 days, showed no

40

40

.

.

.

.

.

.

50

60

70

80

90

100

5FUclrsucc

dose

significant

of the E3 thymoma

growing s.c. (data not

may therefore be able to participate in antibody dependent cellular cytotoxicity to inhibit tumor growth; previous experi

(ug)

Fig. 1. A, growth inhibitory effects of one dose of SFdUrdsucc-anti-Ly-2.1

inhibition

shown). The anti-Ly-2.1 MoAb is of the IgG2a subclass and

110

on

the E3 thymoma. Groups of 8 CBF, mice 6 days after tumor inoculation (5 x

ments using this antibody have shown similar results (13, 23).

lO@cells s.c.); mice were given injections of PBS J); 48 @tg 5FdUrdsucc-anti-Ly 2.1 (Li); 70 @gSFdUrdsucc-anti-Ly-2.1 (A); 85 @g5FdUrdsucc-anti-Ly-2.l (@J); and 100 @igSFdUrdsucc-anti-Ly-2.l (â€¢).Mean tumor size Â±SE was plotted against days after tumor inoculation. B, tumor inhibition of E3 thymoma growing s.c. induced by various doses of 5FdUrdsucc (5FUdrsucc) conjugated to anti-Ly 2.1 administered i.p. seen 15 days after tumor inoculation.

The in vivo efficacy and specificity of SFdUrdsucc conjugates illustrated in this model prompted further studies using colon

C,'

In Vivo Activity

HT(1)75NS E3 Tumor Model. Groups of 8 CBF1 mice given

w5 N

injections s.c. of 5 x 106 E3 cells, developed tumors 7 days after inoculation, and were then given a single i.p. injection of 48, 70, 85, or 100 @ig of SFdUrdsucc conjugated to anti-Ly-2. 1 (Fig. 1A). Marked tumor inhibition was observed at the highest dose administered (100 pg), where tumors were 15.0% of con trol mean tumor size on day 15 and 100% survival of mice was seen within this group. Minor weight loss, however, was oh served, with some diarrhea. By Day 15, 50% tumor inhibition (Fig. 1B), compared with control tumors, was produced by the lowest dose of conjugated SFdUrdsucc (48 @g)administered, thus demonstrating the tumor inhibiting potential of these conjugates at low doses. Initial in vivo experiments, in which 7 sequential doses of SFdUrdsucc conjugates were administered (data not shown), resulted in death of some mice at a total dose of 120 @tgof SFdUrdsucc. It was necessary, therefore, to establish the appro priate dose schedule for the most effective treatment without toxicity. Treatment was initiated where mice received 4 or 5 doses of the low or high dose protocol; (a) low dose (2.4, 10.6, 10.9, and 8.6 @g: total dose administered, 32.5 zg); and (b) high dose (5.9, 21.3, 21.9, 17.3, and 5.4 @tg: total dose administered, 7 1.8 zg) of SFdUrdsucc conjugated to anti-Ly-2. 1. Five days after tumor inoculation of 5 x 106 E3 cells s.c., mice were given injections i.v. of either 4 or 5 doses of conjugate (Fig. 2). By day 13, tumors were inhibited by 41.0 and 57.0% compared to mean control tumors, with low and high dosages, respectively, and no signs ofsystemic toxicity or death was observed. Further dose scheduling experiments showed 20 @zgof conjugated

Cl) 0 IU) 0 0

5

10

15

25

20

DAYS AFTER TUMOR INOCULATION Fig. 2. Growth of the E3 thymoma s.c. in groups of 8 CBF, mice treated i.p. with PBS (N), SFdUrdsucc-anti-Ly-2.1 conjugate; high dose total 71.8 @g(â€¢), and low dose total 32.5

@g(A). Treatments

denoted ( A ) began 5 days after

inoculationof 5 x 10@E3 cells s.c. Bars, SE.

C,'

E U

UI

N Cl) 0

z

4 UI 4

6

8

10

12

14

DAYS AFTER TUMOR INOCULATION

Fig. 3. Comparativeeffectson the E3 thymomain CBF, miceofvarious forms of 5FdUrdsucc. Groups of 8 mice were given injections i.p. (denoted ) of 4 doses of 20 @zg SFdUrdsucc:conjugatedto anti-Ly-2.1(0); or anti-TFR (Lx),and free SFdUrdsucc with anti-Ly-2.l PBS alone (U). Bars, SE.

MoAb (â€¢);control mice given injections of

134


IN VWO AC1IVITY OF SFdUrdsucc-MoAb CONJUGATES 2

received four doses of SFdUrdsucc

A

total dose, 122 @sg,(Day 7,

31 gig; Day 10, 30 @Lg; Day 13, 25 @g;Day 16, 36 gig). No significant differences in tumor growth among the various groups were observed (Fig. 5).

C,' E U

C (81 0

DISCUSSION

E 3 I. C a

Colorectal cancer is the second most common cause of can cer-related deaths. One in 20 people will develop colorectal cancer and the majority of these people ultimately die of their disease. 5FUra, an antimetabolite which has an objective re sponse rate of 15â€”20%,is the most active single agent against the disease. However, most remissions are only partial and little impact on survival is noted. Gastrointestinal and dermatobogi cal involvement has been noted with 5-FUra chemotherapy, while the major dose-limiting toxicity is hematological. More effective therapies with fewer side effects are clearly needed. The linkage ofantineoplastic drugs to monoclonal antibodies has led to a new method of treatment, where drug is targeted directly to tumors, thus overcoming the side effects of com monly used drugs. The delivery of large quantities of drug to tumors still poses a problem but this can be partly overcome by coupling more toxic drugs to the monoclonal antibodies.

C

A

A

A

A

Days

After

Tumor

20

40

30 Inoculation

C,'

E U

U)

N U) 0

z 4

U)

30

SFdUrdsucc,


C

3

E .2.2

of the highly cytotoxic

5FdUrd,

was

SFdUrdsucc

and SFdUrdsucc-anti-Ly-2.1

were cytotoxic

to

ward E3 thymoma cells at nanomolar concentrations in vitro

U)

N

(Table 1). Once 5FdUrdsucc and SFdUrdsucc conjugates enter the cell they must be cleaved by esterases or proteolytic diges tion to release their active species, possibly 5FdUrd. The finding that the drug conjugate was slightly more active than free

U)

0 I-

z 4

U)

5FdUrdsucc 20

30


mice were given injections i.p. of either PBS () or 5FdUrdsucc conjugated to I1 (0) on the days denoted g. Bars, SE. Individual tumor growth curves for control mice and conjugate treated mice are shown in Fig. 4B and Fig. 4C, respectively.

carcinoma tumor models, since 5FUra and its derivatives have been shown to be effective against colon carcinomas (24).

Effect of 5FdUrdsucc-MoAb on Colon Tumor Xenografts. Nude mice bearing the given injections ofeither days after implantation SFdUrdsucc conjugated

IC50 5.0 x 10@ M, 5FdUrdsucc-anti

arising from the increased hydrophiicity of SFdUrdsucc, as a consequence

LIM1899 tumor as a xenograft were SFdUrdsucc-I-1 conjugate or PBS, 21 of tumor pieces. A total of 66 ig of to I-i MoAb was administered i.p.

of the introduction

of the carboxyl group. Differ

ence in 5FdUrd and 5FdUrdsucc

cytotoxicity was seen on

2.0

every third day (Day 21, 5 @g; Day 24, 20 @tg; Day 27, 29 @zg; Day 30, 22 sg). The administration of SFdUrdsucc-I-1 conju gates produced 46.7% tumor inhibition on Day 38 compared to the mean tumor size of control animals (Fig. 4A). The individual tumor growth curves for control mice (Fig. 4B) and those receiving conjugate (Fig. 4C) demonstrated the in vivo

E

U

1.5

U)

N U)

1.0'

0

efficacy of SFdUrdsucc conjugates against the LIM1899 colon carcinoma. As an alternative, human colon xenograft, Cob 205, was chosen. Groups of 6 Swiss nude mice were given injections s.c. of 5 x 106 Cob 205 cells. Seven days later i.p. treatment was initiated where mice received either (a) PBS, (b) 5FdUrdsucc 250-30.6 (Cob 205 reactive), or (c) 5FdUrdsucc-anti-Ly-2.1 (Cob 205 nonreactive), every third day. Conjugate groups

(5FdUrdsucc

Ly-2.1 ICso 3.0 x 10-a M) suggests that drug conjugate uptake and subsequent digestion within the cell is very effective. This also implies that the introduction of the succinyl group onto 5FdUrd, for conjugation purposes, and the conjugation of antibody via this group did not interfere with the cytotoxic activity of 5FdUrd. The 10-fold loss in activity of SFdUrdsuce (IC50 5.0 x 10-p M) compared to 5FdUrd (IC50 4.0 x 10â€•@ M) on E3 cells, may possibly be due to difficulty in its entry into cells via the nucleoside transport system, or diffusion problems

40

Fig. 4. A, tumor growth of LIM1899 xenograft in nude mice. Groups of 6

@

a derivative

coupled to MoAb and these conjugates were highly cytotoxic and had specific activity in vitro (12). These studies have now been extended to establish the in vivo efficacy of 5-dUrdsucc immunoconjugates.

I-

z

0.5

4

U)

L 0

5

L

I

10

DAYS AFTER

I 15

20

25

TUMOR INOCULATION

Fig. 5. GrowthofColo 205 s.c. xenograftsin nude micetreated i.p. (total dose, 122 @zg 5FdUrdsuccequivalents)with PBS (), 5FdUrdsucc-anti-Ly-2.1 conjugate (Li), and 5FdUrdsucc-30.6 conjugate (0) on days denoted A.Bars, SE.

135


IN VIVO ACTIVITY OF SFdUrdsucc-MoAb CONJUGATES

LIM1899 and Cob 205 cells, suggesting that these problems may also influence killing of these cell lines. A 6-fold decrease in activity was observed,

however,

when SFdUrdsucc-I-1

con

jugate was compared to free SFdUrdsucc on LIM1899 cells, and a 2.5-fold decrease in drug activity was demonstrated for free SFdUrdsucc and SFdUrdsucc-30.6 conjugate on Cob 205 cells. This loss in activity may have resulted

due to inefficient

processing of the drug conjugates into 5FdUrd by these cell lines once they have entered the cell. It is also possible that because LIM1899 secretes CEA, and since the I-i antibody reacts with CEA, some of the drug conjugates may have bound to the free antigen in addition to CEA found on the cell surface, or possibly shed as an immune complex. Selective cytotoxicity of antigen reactive and nonreactive drug conjugates was dem onstrated on both E3 and L1M1899 cell lines. SFdUrdsucc anti-Ly-2.1 was 4 times more active than they nonspecific 5FdUrdsucc-anti-TFR on E3 cells, and SFdUrdsucc-I-1 was 10 times

more

active

than

SFdUrdsucc-anti-Ly-2.

1 (LIM 1899

nonreactive) on L1M1899 cells. SFdUrdsucc and SFdUrdsucc conjugates were more toxic toward mouse E3 cells than the human LIM 1899 cells. Evans et a!. (25) previously demon strated that most mouse cells were more sensitive than most human cells to growth inhibition by 5-FUra due to a difference in activities of several enzymes responsible for the initial me tabolism of the drugs. Single dose response studies of SFdUrdsucc conjugates in vivo (Fig. 1) revealed them to be a potent inhibitor of tumor growth at a dose of SFdUrdsucc, which in an unconjugated form would be ineffective. A single injection of 48 @tg SFdUrdsucc-anti-Ly-2.1 (SFdUrdsucc equivalent) was required to produce a 50% inhibition ofmean tumor size. In comparison, previous studies using idarubicin, a more toxic analogue of doxorubicin, upon conjugation to antibody required 135 @igof idarubicin

(2.8 times more drug than in SFdUrdsucc

conjugates)

that the coupling procedure used was effective in retaining both the drug and antibody activity. Furthermore, it also suggests that the tumor inhibition was not due to slow release of free drug from the immunoconjugate. Human colon carcinomas grown in nude mice were used to further assess the antitumor activity of the drug conjugates in vivo. These models are considered by some to be representative oftumors in humans (27). In the L1M1899 model, a moderately differentiated tumor, SFdUrdsucc-I-1 conjugate therapy was able to retard tumor growth 48% compared to mean control tumor size (Fig. 4). This tumor inhibition was produced by multiple dose administration of only 66 @igof SFdUrdsucc conjugated to MoAb. Unlike LIM 1899, Cob 205 cells were relatively resistant to SFdUrdsucc. No significant differences in tumor growth were observed when animals treated with either reactive or nonreac tive conjugates were compared to those given PBS (Fig. 5). FACSCan

analysis

of the

250-30.6

MoAb,

and

SFdUrdsucc

250-30.6 conjugate, showed both were capable of binding to Cob 205 cells, therefore the drug-immunoconjugate should have had the binding capacity to kill its target cells. This in vivo finding reflects the in vitro results, where high concentrations of SFdUrdsucc were required to produce any killing of Cob 205 cells; neither free nor conjugated drug was effective in vitro on this tumor. The relative resistance of Cob 205 is probably not surprising, as the Cob 205 cell line originated from the ascites fluid of a patient with adenocarcinoma of the colon, where prior to culture the patient received 5-fluorouracil ther apy (16). The inability of SFdUrdsucc and its conjugate to inhibit Cob 205 growth in vitro and in vivo may therefore reflect an acquired resistance of the tumor or a natural intrinsic resistance to this drug. More importantly, it is apparent that this form of resistance,

unlike multidrug

resistance,

cannot

be

overcome by using immunoconjugates.3 to inhibit growth of s.c. E3 tumors by 50% compared to control From the above results it has been clearly shown that tumor size. Few drug conjugates have shown such tumor inhib SFdUrdsucc conjugates are highly efficacious in in vitro and in iting potential as 5FdUrdsucc conjugates at such low doses. vivo. In previous studies Hurwitz et a!. (28) linked 5-fluorouri Multiple dose administration of SFdUrdsucc immunoconju dine to antibodies via a dextran bridge, and demonstrated gates produced some signs of systemic toxicity such as weight selective cytotoxicity on leukemic B-cells. Various preparations loss, diarrhea, and some deaths. Toxicity associated with im had between 7 and 24 molecules of fluorouridine per immuno munoconjugate administration is fairly uncommon. However, methotrexate (26) and aminopterin3 conjugates when given globulin molecule, and showed killing similar to that of free FUrd. Even though a greater number of residues of drug were sequentially or at high doses have also been shown to produce coupled to antibody using the dextran bridge, our SFdUrdsucc toxic effects in animals. The increased half-life of toxic drug, MoAb conjugates showed enhanced killing, comparable to due to the presence of antibody molecule in drug-immunocon SFdUrdsucc, a more toxic drug than FUrd. 5-Fluorouridine jugates or their continual administration, which would maintain high concentrations of drug within the circulation, may be was also recently coupled to a carbohydrate moiety of an anti CEA monoclonal antibody by using aminodextran as the inter possible causes. Even though SFdUrdsucc conjugate associated systemic toxicity was unexpected, dose scheduling proved to be mediate carrier (29). The drug conjugates contained 30â€”35 molecules of FUrd per molecule of antibody and exhibited effective, where toxicity could be abolished and considerable an IC50 of 1.0 sg/ml. In comparison to the drug conjugate tumor growth inhibitory activity retained (Figs. 2 and 3). The conjugation of cytotoxic drugs to MoAb requires the prepared within this report these FUrd conjugates were 7 times production of a conjugate which is cytotoxic to tumor cells but less active when tested against colon carcinomas, even though retains its selective action in vivo; this has been demonstrated there are 4 times as many residues of drug per antibody mole for SFdUrdsucc conjugates (Fig. 3). SFdUrdsucc-anti-Ly-2. 1 cule. The more potent cytotoxicity of the SFdUrdsucc drug conjugates inhibited growth of E3 tumor (60% in comparison conjugates prepared by using a succinyl linkage implies that to mean control tumor size, on Day 13), whereas SFdUrdsucc these conjugates upon entry to the cell, are able to be readily anti-TFR conjugates, which are unreactive to E3 tumor cells, cleaved into 5FdUrd and/or 5FUra. Both these active species were unable to produce any significant tumor inhibition (7% can inhibit growth, 5FdUrd being the more potent. Due to on Day 13). SFdUrdsucc-anti-Ly-2. 1 conjugates successfully the conjugation procedure used, the drug conjugate described inhibit growth at microgram amounts and exhibit their action by Shih et a!. (29) may only be cleaved into 5FUra, so a less predominantly on antigen positive cell lines. This illustrates active drug immunoconjugate results. The SFdUrdsucc drug conjugates produced within this study therefore appear to 3 Unpublished results. 136


IN VWO ACFIVITY OF 5FdUrdsucc-MoAb CONJUGATES

be more

active

than

previously

reported

12. Goerlach, A., Krauer, K. G., McKenzie, I. F. C., and Pietersz, G. A. In vitro antitumor activity of 2'-deoxy-S-fluorouridine-monoclonal antibody conju gates. Bioconjugate Chem., 2: 96â€”101,1991.

5-fluorouridine

immunoconjugates.

Although we have demonstrated that SFdUrdsucc conjugates are extremely potent and selective in inhibiting tumor growth in vivo, tumor eradication was not observed at any of the doses used. To further improve chemotherapy it may be necessary to use compounds that enhance the activity of the fluoropyrimi dines. Leucovorin, a folinic acid, when used in combination with 5FUra and 5FdUrd has shown to potentiate their cytotox

13. Pietersz, G. A., Smyth, M. J., Kanellos,J., Cunningham, Z., Sacks, N. P.

icities

16. Semple,T. U., Quinn, L. A., Woods, L K., and Moore, G. E. Tumor and lymphoidcell linesfrom a patient with carcinomaof the colon for a cytotox icitymodel.Cancer Res., 38: 1345â€”1355, 1978.

in vitro and in vivo (30, 3 1). The

presence

M.,and McKenzie,I. F. C. PreClinical andclinicalstudieswitha varietyof immunoconjugates. Antibody Immunocon. Radiopharm., 1: 79â€”103,1988.

14. Hogarth, P. M., Henning, M. M., and McKenzie, I. F. C. Alloantigenic phenotype of radiation induced thymomas in the mouse. J. NatI. Cancer Inst., 69:619â€”625, 1982. 15. Andrew, S. A., Teh, iC., Johnstone, R., Russell, S. M., Whitehead, R. H., McKenzie, I. F. C., and Pietersz, G. A. Tumor localization by combinations

of monoclonalantibodiesin a new human colon carcinomacell line (LIMI899). Cancer Res., SO:4423â€”4428,1990.

of high

quantities of intracellular reduced folates, induced by leucovo rin, stabilizes the ternary complex offluorodeoxyuridylate mon ophosphate and thymidylate synthetase, resulting in an in creased cytotoxicity of fluoropyridines (32). Studies are there fore under way to incorporate this potentiation observed with leucovorin into a combination therapy with SFdUrdsucc con jugates, to further increase the potency ofthese drug conjugates.

17. Hogarth, P. M., Edwards, J., McKenzie, I. F. C., Goding, J. W., and Liew, F. Y. Monoclonal antibodies to murine Ly-2.l cell surface antigen. Immu nology, 46: 135â€”144, 1982.

18. Ey, P. L., Prowse,S. J., and Jenkin, C. R. Isolationofpure IgGl, IgG2a and lgG2b immunoglobulins from mouse serum using protein A Sepharose. Immunochemistry, 15: 429â€”434,1978. 19. Panaccio, M., Zalcberg, J. R., Thompson, C. H., Leyden, M. J., Sullivan, J. R., Lichenstein, M., and McKenzie, I. F. C. Heterogeneity of human trans

ferrinreceptorantibodies:heterogeneityofthe TFR and useto detecttumours in vivo.ImmunoL Cell BioL, 65: 461â€”472, 1988.

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