in Vivo and in Vitro Mitotoxin Antitumor Activity of ...

Antitumor Activity of Basic Fibroblast Growth Factor-Saporin Mitotoxin in Vitro and in Vivo Julie G. Beitz, Pamela Davol, Jeffrey W. Clark, et al. Cancer Res 1992;52:227-230. Published online January 1, 1992.

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(CANCERRESEARCH52, 227-230, JanuaryI, 19921

Advances in Brief

Antitumor Activity of Basic Fibroblast Growth Factor-Saporin and in Vivo' Julie G. Beitz,2 Pamela Davol, Jeffrey W. Clark, Junji Kato, Maria Medina, Douglas A. Lappi, Andrew Baird, and Paul Calabresi

Mitotoxin in Vitro

A. Raymond Frackelton,

Jr.,

Roger Williams Cancer Center and Brown University, Providence,RhodeIsland 02908/i. G. B., P. D., J. W. C., J. K., M. M., A. R. F., P. C.J,and The Whittier institute for Diabetesand Endocrinology, La Jolla, California 92037 (D. A. L., A. B.]

Materials

Abstract

Many cancer cell lines express basic fibroblast growth factor (FGF) receptors,

cytotoxic

making

them

compounds.

potential

targets

for the delivery

To this end, we have investigated

and Methods

Mitotoxin. Conjugation of saporin to basic FGF was accomplished

of FGF-based

after derivatization

the antitumor

proprionate as described (10, 13). The conjugate was purified by hepa

of saporin

with N-succinimidyl-3(2-pyridyldithio)-

rin-Sepharose (Pharmacia) column chromatography, followed by di activity of a novel mitotoxin, Fibroblast Growth Factor-saporin (FGF on a Mono 5/5 NaCI cation SAP), a conjugateofFGF and the ribosome-inactivatingprotein, saporin. alysis against water and chromatography In vitro, FGF-SAP is cytotoxic for human melanoma, teratocarcinoma, exchange column (Pharmacia). Fractions containing the conjugate were and neuroblastoma cells expressing FGF-receptors. Mice treated with detected by silver staining after sodium dodecyl sulfate-polyacrylamide FGF-SAP i.v., on a variety of schedules, showed dramatic tumor growth gel electrophoresis. inhibition with minimal toxicity. Thus, FGF-SAP appears to be a well tolerated and potent antitumor agent. The potential of FGF-targeted cytotoxicity is discussed.

Cell Lines.

SK-Mel-1,

a human

melanoma,

SK-N-MC,

a human

neuroblastoma, and PA-i, a human ovary teratocarcinoma cell line, were obtained from the American Type Culture Collection (Rockville, MD) and were grown in modified Eagle's medium supplemented with

10% fetal calf serum. A431, a human epidermoid carcinoma, was also

Introduction The advantage ofconjugating toxins from bacteria and plants to cell-targeting antibodies and ligands is well recognized (1). The conjugation of a toxin to a cytokine such as FGF3 to achieve local antitumor effects has been utilized less frequently, although this approach is effective with transforming growth factor a (2), interleukin 4 (3), interleukin 6 (4), and acidic fibroblast growth factor (5). There is evidence implicating the involvement of FGF in the growth of a variety of neoplastic cell lines, including melanoma (6), astrocytoma (7), and ovarian carcinoma (8). For example, the growth of certain melanoma cell lines is stimulated by FGF (6) and can be inhibited by antagonistic FGF peptides (6). Thus, the development of ther apeutic strategies utilizing FGF to deliver toxin into FGF responsive cancer cells might prove effective against a number of malignancies. SAP, derived from the seeds of the plant Saponaria

officin

alis, irreversibly inhibits the protein synthesis of eukaryotic cells by rendering the 605 subunits of ribosomes unable to bind elongation factor 2 (9). Accordingly, the conjugate of FGF to saporin (FGF-SAP) is a ligand toxin which has cellular target ing specificity

and potent cytotoxicity

in vitro for cells express

obtained from the American Type Culture Collection and was grown in RPMI 1640 supplemented with 10% fetal calf serum. FSaIIC, a

murine fibrosarcoma, was a gift of Dr. Beverly Teicher (Dana Farber Cancer Institute, Boston, MA) and was grown in a modified Eagle's

medium supplemented with 5% fetal calf serum. â€œ@I-FGF Receptor BindingAssay. Cells were seeded in 12-welltissue culture plates (Costar) at iO@cells/well and grown until confluent in their respective medium. â€˜25I-FGF binding was performed using a radioreceptor assay as described (14). Briefly, cell monolayers were

incubated with fresh, unsupplemented medium containing 0.2% gelatin and 3 ig/ml heparin (Sigma) for 1 h at 37C and 5% CO2 and then washed with ice-cold medium and allowed to cool for 1 mm. Cells were

incubated with various concentrations of â€˜25I-FGF in 250 @il ofthe same medium for 2 h on ice. The cells were then gently washed twice with ice-cold 0.9% phosphate-buffered

saline to remove

unbound

wI@FGF,

and remaining cell-associated radioactivity was extracted with 1% Tri

ton X-lOOand quantitated using a Beckman gamma counter. Nonspe cific binding was determined

by inhibiting

specific binding

using a 200-

fold excess of nonradiolabeled FGF. In Vitro Survival Studies. Cells were plated in 96-well tissue culture plates (Costar) at iO@cells/well in their respective medium. One day later, the medium was removed, and medium containing 1 PMto 1 @iM of the conjugate FGF-SAP or free SAP was added. Cells were treated in triplicate and maintained at 37T and 5% CO2. Seventy-two h after

the treatment was initiated, the medium was removed and the cells ing FGF receptors, including baby hamster kidney cells, corneal were trypsinized and counted using a Coulter counter (Coulter Elec and aortic arch endothelial cells, and AIDS-KS-3 cells (10â€”12). tronics, Inc., Hialeah, FL). Results are expressed as the mean cell Because of the reported successful in vivo targeting efficacy of number from treated wells, normalized to media controls, as a function other saporin conjugates (13), we investigated the cytotoxic of the FGF-SAP or SAP concentration. The 50% inhibition values effects in vitro and in vivo of a saporin-based conjugate directed werecalculated from dose-responsecurves and represent the concentra against the FGF-receptor found on FGF-receptor-bearing can tion of FGF-SAP or SAP which resulted in a 50% reduction in cell number. cer cell lines. In Vivo Antitumor Studies. Experiments with SK-Mel-i, SK-N-MC, Received 8/9/91; accepted 11/8/91. 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This

work

was

supported

in

part

by

a

grant

from

Farmitalia

Carlo

Erba,

Milan, Italy. 2 To

whom

requests

for

reprints

should

be

addressed,

at

Roger

Williams

Hospital, 825 Chalkstone Avenue, Providence, RI 02908. 3 The

abbreviations

bFGF, basic FGF.

used are:

FGF,

fibroblast

growth

factor,

SAP,

saporin;

and A43l cell lines were performed in 8â€”10-week-oldmale nu/nu mice,

while those with PA-i cells used 8â€”10-week-old female nu/nu mice. Nude mice were bred and maintained by the Roger Williams Hospital Animal Care Facility. FSaIIC cells were carried in 8â€”10-week-old male C3H/HeN mice (Taconic Laboratories, Germantown, NY). The 50% lethal dose of FGF-SAP in BALB/c mice was found to be 500 pig/kg, with toxicity manifested

as extensive

hemorrhage,

often in the intestinal

tract (1 1). For in vivo studies, groups of five mice were inoculated with 227


ANTITUMOR ACflVITY OF FGF-SAP 2 x 106 tumor cells s.c. in the right rear flank. In the initial studies, 125 ag/kg FGF-SAP or 85 big/kg SAP (that is, equivalent molecular

A , vu

concentrations of saporin in each treatment group) were dissolved in sterile phosphate-buffered saline in the absence of heparin and were administered as a single injection via tail vein, 0, 1, 5, 10, or 15 days

80 C

after tumor implantation. In subsequent studies, mice received a course of i.v. injections

of 0.5 @zg/kgFGF-SAP

administered

0

C)

at weekly inter

at least twice weekly, beginning 5 days after tumor implantation, and

0

tumor volumes

C 0

were calculated

60

a, C)

vals, for a total of four doses. The progress of each tumor was measured as (15)

40

C.)

(Minimum measurement)2 (maximum measurement)

20

2

a

Results are expressed as mean tumor volumes of treated groups, nor malized to untreated controls, as a function of time. Errors are SEM.

0.01

0.1

1

10

100

1000

Concentration(nM)

Statistical comparisons of mean tumor volumes for the various treat

ment groups were made using the Mann-Whitney rank-sum test.

B Results Inhibition of Tumor Cell Proliferation. The FGF-SAP conju gate has been shown to interact with the high-affinity FGF

(2 (2

receptor and to be cytotoxic to cells expressing this receptor (10â€”12). Furthermore, FGF-SAP cytotoxic activity is inhibited

a, E

0.1

in a dose-dependent fashion by exogenous FGF (10, 12). To assess the potential cytotoxicity of FGF-SAP against different tumor cell lines, we performed 125I-FGF binding studies to demonstrate

the presence

of high-affinity

0

> 0

E I-

binding sites for FGF

on these cells. Scatchard analysis showed that SK-Mel-l, PA 1, SK-N-MC, and FSaIIC cells expressed high-affinity FGF receptors,

whereas

(Table I). Next, we determined cell survival after treatment with various concentrations of FGF-SAP or SAP for 72 h in vitro. FGF SAP is a potent inhibitor of cell growth for each of the cell lines expressing FGF receptors (Table 1 and Fig. 1A). FGF SAP demonstrated minimal cytotoxic effects in A431 cells. SAP-associated growth inhibition was observed for PA-i and SK-N-MC cells, but only after exposure to SAP concentrations that were 2â€”6orders of magnitude greater than that of the conjugate.

0.01

A43 1 cells were devoid of FGF receptors

The addition

of FGF

and SAP in a noncovalent

mixture had no cytotoxic effects. Inhibition of Tumor Growth in Vivo. In subsequent studies we explored the FGF-SAP mitotoxin's efficacy in vivo for each cell

Day Fig. 1. A, inhibition of PA-i cell proliferation after treatment with various concentrations of FGF-SAP (0), unconjugated FGF (â€¢),unconjugated SAP (0), or a noncovalent mixture of FGF plus SAP (U) in vitro. Cells were seeded into 96-well plates, treated in triplicate for 72 h with concentrations of FGF-SAP or controls as indicated, trypsinized, and counted. Results are expressed as the mean cell number from treated wells, normalized to media controls, as a function of concentration. Nonnormalized values with SD were: control (no addition), 15.72 x l0@Â±2.46; 10 nM FGF-SAP, 3.23 x 10' Â±0.32; I MMsaporin, 5.85 x 10' Â± 0.1 1. B, Tumor growth inhibition in nude mice bearing PA-I xenografts treated with FGF-SAP or free SAP administered 5 days after tumor implantation. Mean tumor volumes are expressed as a function of time for mice receiving a single dose of FGF-SAP 125 ag/kg (O), FGF-SAP 0.5 @zg/kgweekly for four doses (0), SAP 85 pg/kg (0), or no treatment (U). n = 10 mice/treatment group. Bars, SE.

tation. FGF-SAP caused marked inhibition of tumor growth,

line grown s.c. as solid tumor xenografts in mice. Preliminary and in some animals, complete tumor regression was observed toxicological evaluation of FGF-SAP showed a dose of 500 @tg/ (Fig. 1B, data not shown). Tumor inhibition was observed even

kg to be lethal in BALB/c mice (1 1) and 250 @zg/kgto be nonlethal. Accordingly, we chose 125 jzg/kg as an initial dose of FGF-SAP. We performed pilot studies in nude mice bearing human tumor xenografts in which a single i.v. dose of FGF SAP was administered 1, 5, 10, or 15 days after tumor implan Table 1 High-affinity FGF receptor number, FGF dissociation constant, and growth inhibition oftumor SAPFGF-R cell lines in thepresenceoJFGF-SAP or

bFGF-SAP IC,oâ€•SK-Mel-l Cell Line K0@ 167 PA-I SK-N-MC FSaIIC A43l

41 NAC

Number

IC,oâ€•

19,000 33,000 45,000 7,000 0

0.1 1.0 0.01 2.5 No effect

SAP No effect 500 1000 No effect No effect

aKd,in pM. b Concentration

(in

nM)

calculated

from

dose-response

curves

which

resulted

in a 50% reduction in cell number. Each value is the mean of at least three determinations. C NA,

not

applicable.

when treatment was delayed until day 15, when tumor volumes are approximately 50â€”100mm3. By day 30, mean volumes of tumors in treated mice measured only 5â€”33%ofthose of control tumors. Since FGF-SAP at this dose level appeared equally efficacious when administered on day 1, 5, or 10 (data not shown), we chose day 5 as the treatment day for further inves tigations.

At this time, tumors

are approximately

40â€”50 mm3

in volume. The next series of studies compared a broad range of FGF SAP doses with equivalent doses of SAP to determine in vivo dose responses.

Mean tumor volumes on day 30 for FGF-SAP

or SAP-treated xenografts compared to those of untreated controls are displayed in Table 2. Studies performed in nude mice bearing SK-Mel-i, PA-i, or SK-N-MC xenografts dem onstrated growth inhibition with FGF-SAP and lack of efficacy with free SAP (Table 2 and Fig. 1B). Complete tumor regres sion was observed on day 30 in 16% of SK-N-MC xenografts

treated with a single dose of FGF-SAP (125 .@g/kg)on day 5. 228


ANTITUMOR ACFIVITY OF FGF-SAP Table 2 Dose efficacy oIFGF-SAP versus SAP administered as a single i.v. injection 5 days after tumor implantation in mice'

deaths were observed either in mice receiving free SAP in the doses used or in untreated control mice.

Mean tumor volumeon day 30 (% of control)b (pg/kg)SK-Mel-lPA-lSK-N-MCFSaIICA43lbFGF-SAP

Discussion 128Â±5bFGF-SAP 90Â±783Â±13 105Â±0150Â±26 118Â±2232Â±8c 103Â±53lÂ±I5c SAP125.0 85.039Â±l4c

The ribosome-inactivating protein, saporin, covalently linked to FGF exerts potent cytotoxic effects in vitro against a variety of tumor cell types expressing cell surface receptors for FGF Â±15 (Table 1). The in vitro data accurately predict the superior 100Â±3a 0.01771 SAP0.025 cytotoxicity of FGF-SAP as compared with free SAP for FGF Tumor celIs (2 Xl0@) were inoculated s.c. into the right rear fiank of host receptor-bearing SK-Mel-I, PA-i, and SK-N-MC xenografts mice. b Mean tumor volumes for treated xenografts were calculated using 2â€”1 1 mice/ (Table 2) and the absence of significant antitumor effects on treatment group. There were 2â€”24mice in the control groups. Errors are SEM. A43i xenografts. The extent to which FGF-SAP cytotoxicity directly resulted in tumor growth inhibition was expected, since 0.01. A43l cells did not express cell surface receptors for FGF. The efficacy of FGF-SAP administered as multiple low doses in vivo is particularly impressive. This regimen affords the Antitumor responses to FGF-SAP were also observed in im munocompetent host mice bearing FSaIIC tumors (Table 2), advantage of delivering repeated, relatively nontoxic FGF-SAP although the effects of FGF-SAP were characteristically short doses to tumor cells that survive the initial treatment. Certainly, lived in this single-dose regimen (Table 3). No growth inhibition from the studies presented here, some tumor cells survive FGF was observed in mice bearing A43l xenografts following treat SAP treatment, be it after a 72-h exposure in vitro, or after a ment with FGF-SAP or SAP. single, i.v. high dose in vivo. Resistant tumor subpopulations In conjunction with our evaluation of lower FGF-SAP doses may thus be capable of continued proliferation in vivo. Poor in vivo, we performed studies in which multiple doses of FGF vascular access of the conjugate; alterations in FGF-receptor SAP were administered i.v. Because we observed only modest number, occupancy, or internalization in resistant cells; or, antitumor effects with FGF-SAP at a single dose of 0.5 @sg/kg perhaps, the stage in cell cycle of tumor cells at the time of (Table 2; data not shown), we treated groups of mice bearing treatment must all be considered. Thus, from a therapeutic SK@-Mel-l,PA-i, SK-N-MC, or FSaIIC tumors with FGF-SAP standpoint, it is of great practical importance that more than 0.5 big/kg beginning on day 5, and then once a week for a total one dose of conjugate be administered with safety. of four doses. Table 3 compares mean tumor volumes for the Formal pharmacokinetic studies of FGF-SAP, including multiple low-dose FGF-SAP regimen to volumes for the single blood half-life, stability, and elimination of the conjugate, have high-dose regimen. Significant tumor inhibition was seen on not yet been performed. In the absence of these studies, how day 35 for each of the tumor types examined under the multiple ever, we speculate that FGF-SAP persists in vivo long enough low-dose treatment, and complete tumor regression was oh to exert both its antitumor effects and its toxic effects. Since served in 20% of SK-N-MC xenografts. This potent antitumor saporin is not glycosylated, saporin conjugates may have an effect was not consistently observed with the single high-dose enhanced ability to escape hepatic clearance from the circula treatment. tion (16). Basic FGF has a high affinity for natural glycosami FGF-SAP Toxicity. Preliminary evaluation of hematological noglycans in vitro and in vivo, which is also conferred to SAP and hepatic parameters has been conducted in adult rats 7 days when conjugated. Studies in which FGF was injected locally in after a single i.v. dose of FGF-SAP (50 @g/kg).4A modest rise the rat brain have shown that FGF binds to the extracellular in WBC, without change in hematocrit, and modest elevations matrix initially and is subsequently transferred and internalized of serum transaminase levels (serum glutamic-oxaloacetic within target tissues, in this case, the vasculature, 2 days later Â±19

92Â±9bFGF-SAP SAP0.5 0.361

C Significant

difference

transaminase

between

treatment

and

control

and serum glutamic-pyruvic

tumor

volumes,

transaminase)

P

s

were

observed. These findings are believed to represent mild hepa tocellular injury. In general, FGF-SAP or SAP treatment was well tolerated in the majority of mice evaluated in the above-mentioned stud ies. Subcutaneous hemorrhage and edema, accompanied by weight loss and ultimately death, occurred between 10 and 14 days with the highest dose of FGF-SAP (125 pg/kg) in I 0% of mice bearing SK-Mel-i, PA-i, SK-N-MC, or FSaIIC tumors. Premature death also occurred in nearly 60% of mice bearing A43l xenografts receiving this highest FGF-SAP dose, in spite ofthe fact that autopsies failed to reveal any gross abnormalities in vital organs and no animals died of metastatic disease. In contrast, single lower doses of FGF-SAP were well tolerated and were not associated with any deaths. Furthermore, no cumulative toxicities were noted in mice receiving the multiple low-dose regimen of FGF-SAP. Thus, chronic treatment of tumors in vivo with low doses of FGF-SAP appeared to be both efficacious and nontoxic. No toxic side effects or premature

Tablemice'Cell 3 Comparison ofFGF-SAP treatment regimensin tumor-bearing control)bSingle tumor volume (% of lineDayMean dosesdSK-Mel-i12

68Â±12 72Â±13 l9Â±6@SK-N-MC12 3554Â±10' 56Â±11 84Â±5 18Â±7'FSaIIC12 3558Â±11 30Â±Ile 51Â±3' 42Â±4'

3534Â±12' 92Â±18

a Tumor cells (2 x 10') were inoculated s.c. into the right rear flank of host mice. b Mean tumor volumes were calculated using 2â€”14mice/treatment There were 2â€”24mice in the control groups. Errors are SEM. C A

single

d A dose

dose

of

FGF-SAP

ofFGF-SAP

125

0.5

pg/kg

@&g/kg was

was

administered

administered

on day

on

day

group.

5.

5, followed

by weekly

injections for a total of four doses. â€˜ Statistical

personal

Multiple low

iIÂ±2'PA-I12 3538Â±12' 26Â±7'

@ 4 W. Casscells,

high dosec

difference

between

treatment

0.01.

communication.

229


and

control

tumor

volumes,

P

ANTITUMOR ACTIVITY OF FGF-SAP

(I7).@Thus, it is plausible that the single FGF-SAP dose exerts its effects on target tissues over a relatively long time, perhaps days. In-depth toxicological studies need to be performed, includ ing evaluations of bone marrow, hepatic, and renal function, in mice receiving each ofthe various schedules

ofFGF-SAP.

With

the exception of the highest FGF-SAP dose, the conjugate was well tolerated in vivo, even when multiple low doses were administered. In addition, autoradiographic or immunohisto chemical studies designed to localize FGF-SAP binding within tumor xenografts and their supporting vasculature are being planned since they will be crucial for a complete understanding of the mechanism of action of FGF-SAP. Particularly critical to the delineation ofFGF-SAP-induced toxicities is the binding of FGF-SAP within normal tissues expressing FGF receptors. Along these lines, a number of equally important references can be extrapolated from the data presented here. Like Lindner et a!. (18), we find that there is minimal toxicity of FGF-SAP for normal tissues. We thus conclude that, under normal con ditions, the basic FGF receptor is not expressed at high enough levels to mediate the internalization effects of the conjugate. This proposal is compatible with our unpublished finding and the results ofWhalen et aL (19) that the systemic administration of basic FGF has little or no effect. Accordingly and surpris ingly, the selective expression of the FGF receptor in tumors makes them exquisitely susceptible to FGF-SAP action, almost as if they were exposing a unique antigen to immunotoxins. No detectable toxicity was observed in vivo with free SAP. Others have shown that SAP is rapidly cleared from the circu lation, with only 4% of free SAP detectable in the blood 10 mm after i.v. injection (16). In addition, the doses of SAP used in our studies were several orders of magnitude lower than the reported 50% lethal dose for SAP of 6.8 mg/kg mouse (16). Not surprisingly, we failed to observe significant antitumor effects with SAP administration in vivo. In conclusion, we believe that therapies utilizing mitotoxins designed to target cells expressing cell surface receptors for a particular growth factor or cytokine will provide viable alter native approaches to conventional cancer treatments, including immunotherapy. Toxins coupled to epidermal growth factor and to interleukin 2 are already the focus of clinical cancer trials, and some excellent clinical responses have been reported with the latter (20). In light of these recent successes using other targeted agents, the antitumor activity of FGF-SAP against FGF-receptor-bearing tumors is immensely exciting and is potentially immediately applicable to cancer therapy. 5 Unpublished

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