antitumor activity of tp3(anti-p80)-pokeweed antiviral protein … · vol. 4, 1641-1647, july 1998...

Vol. 4, 1641-1647, July 1998 Clinical Cancer Research 1641

Antitumor Activity of TP3(anti-p80)-Pokeweed Antiviral Protein

Immunotoxin in Hamster Cheek Pouch and Severe Combined

Immunodeficient Mouse Xenograft Models of

Human Osteosarcoma

Onur Ek, Barbara Waurzyniak,Dorothea E. Myers, and Fatth M. Uckun’Departments of Biotherapy [0. E., D. E. M., F. M. U.], Experimental

Oncology [0. E.}, Experimental Pathology [0. E., B. W.], and DrugDiscovery Program [0. E., D. E. M., F. M. U.], Wayne Hughes

Institute, St. Paul, Minnesota 55 1 13, and Department of TherapeuticRadiology, University of Minnesota, Minneapolis, Minnesota 55455

[O.E.]

ABSTRACT

TP3-pokeweed antiviral protein (PAP) immunotoxin is

directed against the p80 antigen on osteosarcoma cells. Pre-

vious studies have demonstrated that TP3-PAP kills clone-

genic human osteosarcoma cells in vitro and shows signifi-

cant antitumor activity in a murine soft tissue sarcomamodel (P. M. Anderson, et a!., Cancer Res., 55: 1321-1327,

1995.) In this study, we demonstrate that TP3-PAP elicitspotent in vivo antitumor activity in a hamster cheek pouch

model of human osteosarcoma. Furthermore, treatment

with TP3-PAP at nontoxic dose levels significantly delayed

the emergence and progression of leg tumors and markedlyimproved tumor-free survival in severe combined immuno-

deficient mice challenged with OHS human osteosarcoma

cells. Thus, TP3-PAP may be useful in the treatment of poorrisk osteosarcoma.

INTRODUCTION

Osteosarcoma accounts for approximately 60% of primary

bone neoplasms in the first two decades of life (1-5). The

long-term survival of pediatric patients with osteosarcoma has

dramatically improved as a result of contemporary multimodal-

ity treatment programs (1-5). However, osteosarcoma patients

who present with metastatic disease or develop pulmonary me-

tastases as well as a subgroup of patients whose tumors show an

inadequate necrotic response to neoadjuvant chemotherapy con-

tinue to have a very poor prognosis (4-8). Presently, the major

challenge in the treatment of pediatric osteosarcoma is to cure

such poor risk patients who require more effective chemother-

apy. Therefore, the development of new potent anti-osteosar-

Received 3/1 1/98; revised 4/28/98; accepted 4/29/98.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

� To whom requests for reprints should be addressed, at Wayne HughesInstitute, 2665 Long Lake Road, St. Paul, MN 55 1 13. Phone: (612)697-9228; Fax: (612) 697-1042.

coma drugs has become one of the focal points in translational

cancer research.

TP3-PAP2 is a potent immunotoxin directed against a Mr

80,000 surface antigen on osteosarcoma cells (9). TP3-PAP was

shown to selectively kill >99.9% of human osteosarcoma cells

at picomolar concentrations in vitro (10). The purpose of the

present study was to further evaluate the clinical potential of this

immunotoxin by examining its in vivo toxicity profile and its

efficacy in preclinical animal model systems.

MATERIALS AND METHODS

TP3(anti-pSO)-PAP Immunotoxin. TP3-PAP was con-

structed by covalent conjugation of the TP3(IgG2b) murine

monoclonal antibody (Refs. 1 1, 12; ATCC accession number

HB-12340) to the plant-derived ribosome inhibitory hemitoxin

PAP. The procedures used for the production and purification of

TP3-PAP immunotoxin have been described previously in detail

(10). B43-PAP, an anti-CD19 immunotoxin directed against

human leukemia cells (13, 14), was used as a control agent.

Cells. The OHS human osteosarcoma cell line (15) was

maintained by serial passages in RPMI 1640 (Life Technologies,

Grand Island, NY) supplemented with 10% (vol/vol) heat-macti-

vated fetal bovine serum (Hyclone Laboratories, Logan, UT) and

1% (vol/vol) penicillin-streptomycin (Life Technologies), as re-

ported previously (10). Cells were cultured in tissue culture flasks

at 37#{176}Cin a humidified 5% CO2 atmosphere. Before s.c. injection

into the right hind legs of SCID mice, cells were washed twice in

PBS. SCID mice were then inoculated s.c. with 0.2 ml of the cell

suspension containing a total of 1 X 106 OHS cells.

HCP Tumor Assay. The effects of in vivo exposure to

TP3-PAP on tumor development and angiogenesis were exam-

med in the HCP assay system using 2-month-old female Golden

Syrian hamsters (Harlan, Indianapolis, IN), as described previ-

ously in detail (16, 17). Hamsters with established OHS cheek

tumors were treated systemically with 1 mg/kg TP3-PAP ad-

ministered in daily i.p. boluses for a total of 5 treatment days.

Toxicity Studies in BALB/c Mice. All of the BALB/c

mice used in the toxicity studies were obtained from the SPF

breeding facilities of NIH (Bethesda, MD) at 6-8 weeks of age.

The mice were housed in the American Association for the

Assessment and Accreditation of Laboratory Animal Care

(AAALAC)-approved and -accredited Research Animal Re-

sources Mouse Facility of the University of Minnesota (Minne-

2 The abbreviations used are: PAP, pokeweed antiviral protein; SCID,

severe combined immunodeficient; SPF, specific pathogen-free; HCP,

hamster cheek pouch.

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�- .

1642 Antitumor Activity of TP3(anti-p80)-PAP Immunotoxin

Fig. 1 Anti-osteosarcoma ac-tivity of TP3-PAP in HCP tu-

mor assays. Innoculations of5 x 106 OHS cells in 100 jj.l

PBS were given, and the cellswere allowed to grow in the

HCP for 7 days. OHS cellsformed highly vascularized tu-

mors in all of the four hamsters

challenged (see A and B).Treatment of OHS cheek-tu-mor-bearing hamsters with i.p.

injections ofTP3-PAP (0.2 mg/kg/day for 5 days) resulted in

tumor shrinkage. C, OHScheek tumor before initiation ofTP3-PAP therapy in a repre-sentative hamster; D, OHScheek tumor of reduced size in

the same hamster after 5 days

of TP3-PAP therapy.

apolis, MN). All of the husbandry and experimental contact

made with the mice maintained SPF conditions. The mice were

kept in Micro-Isolator cages (Lab Products, Inc., Maywood,

NY) containing autoclaved food, water, and bedding. In each of

the toxicity studies, female BALB/c mice were administered an

i.p. bolus injection of TP3-PAP in 0.2 ml PBS or of 0.2 ml PBS

alone (control mice). In the first study, groups of four to six

mice received a single treatment of one of five different dose

levels of TP3-PAP ranging from 1 mg/kg to 5 mg/kg. In the

second study, groups of four to six mice received five consec-

utive daily treatments of TP3-PAP at five different cumulative

dose levels ranging from 1 mg/kg to 5 mg/kg. In each study,

four control mice were administered 0.2 ml PBS i.p. in accord-

ance with the experimental protocol. No sedation or anesthesia

was used throughout the treatment period. Mice were monitored

daily for mortality in order to determine the day-30 LD5O

values. Multiple organs were collected within 4 h after death,

grossly examined, and processed for histopathological exami-

nation, as reported previously (14). Mice who survived 30 days

after the treatment were killed; the tissues were immediately

collected from randomly selected mice and preserved in 10%

neutral buffered formalin.

Efficacy Studies in SCifi Mice. All of the SCID mice

used in the efficacy study were produced by SPF CB-17 SCID/

SCID breeders (originally obtained from Dr. Melvin Bosma,

Fox Chase Cancer Center, Philadelphia, PA) in the AAALAC-

approved and -accredited Research Animal Resources SCID

Mouse Facility of the University of Minnesota (Minneapolis,

MN). All of the husbandry and experimental contact made with

the mice maintained SPF conditions. The mice were housed in

Micro-Isolator cages containing autoclaved food, water and

bedding. Trimethoprim sulfamethoxidole (Bactrim) was added

to the drinking water of the mice three times a week. Female

SCID mice (ages 4-6 weeks) were inoculated s.c. with 1 X 106

OHS cells followed by i.p. administration of the TP3-PAP

immunotoxin at one of the three cumulative dose levels of 0.25

mg/kg, 0.50 mg/kg, or 1.0 mg/kg. Throughout the experimental

period, they were monitored daily for overall health and sur-

vival. At the time of death, necropsies were performed, and the

osteosarcoma burden of the mice was assessed as described

previously (18-23). For histopathological studies, tissues were

fixed in 10% neutral buffered formalin, dehydrated, and embed-

ded in paraffin by routine methods. Glass slides with affixed

6-p.m tissue sections were prepared, stained with H&E, and

submitted to the veterinarian pathologist for examination. The

control group, comprising 78 female SCID mice, was treated

with three daily i.p. injections of 0.2 ml PBS (n = 53), TP3

monoclonal antibody (1 mg/kg cumulative dose) + unconju-

gated PAP (0.25 mg/kg cumulative dose; n = 5), the control

immunotoxin B43-PAP (n = 5) at a total cumulative dose of 1

mg/kg, adriamycin (25 mg/m2/day for 3 days; n = 5), a single

dose of methotrexate (12.5 g/m2/day for 1 day; n = 5), or

cyclophosphamide (50 mg/kg/day for 2 days; n = 5). The

primary end points of interest were tumor growth and tumor-

free survival outcome. Estimation of life table outcome and

comparisons of outcome between groups were done with the log

rank test. The efficacy of TP3-PAP against established tumors

was examined by treating SCID mice who had s.c. OHS xc-

nografts of 0.5-cm or 1 .0-cm diameter with a cumulative dose of

1 mg/kg TP3-PAP given in three i.p. injections (injection vol-

ume, 0.2 ml) 24 h apart from each other. Control mice were

treated with 0.2 ml PBS/day for 3 days.

RESULTS

Anti-Osteosarcoma Activity of TP3-PAP Immunotoxin

in the HCP Model. Within 7 days after transplantation to the

cheek pouches of female Golden Syrian hamsters (cell dose, 5 X



Clinical Cancer Research 1643

Table 1 Toxicity of TP3-PAP in BALB/c mice

Treatmentschedule

Total dose ofTP3-PAP

(mg/kg)

Bed y weight (g) Survival a fter TP3-PAP

Pretreatment Day 30 or at death

Median survival (days)”

Proportion surviving (%)

Day 15 Day 30

A. Single dose” 0 (n = 4)C

1(n4)2 (n 4)

3 (n 6)4 (n 6)5 (n 6)

24.2 ± 1.4

25.5±1.124.9 ± 0.2

25.7 ± 0.424.7 ± 0.526.1 ± 0.6

25.8 ± 0.624.2±0.624.2 ± 0.122.1 ± 0.916.9 ± I .1

17.9 ± 1.2

>30>30>30>301 1 .5

9.5

100 ± 0 100 ± 0100±0 100±0

100 ± 0 100 ± 083 ± 15 67 ± 19

0 ± 0 0 ± 00 ± 0 0 ± 0

B. Five daily doses” 0 (n = 4)

1 (n 4)

2(n4)

3 (n 6)4(n6)5(n6)

22.4 ± 0.5

25.7 ± 0.7

24.1 ±0.3

25.6 ± 0.625.0±0.326.1±0.6

25.4 ± 0.524.9 ± 0.3

23.1 ±0.4

16.8 ± 0.918.3±0.717.9±1.2

>30>30

>30

131313

100 ± 0 100 ± 0100 ± 0 100 ± 0100±0 100±0

17 ± 15 0 ± 00±0 0±00±0 0±0

a All of the mice were electively killed on day 30. See “Materials and Methods” for experimental details.b Mice were treated with either a single i.p. bolus dose (A) or five daily i.p. bolus doses (B).

C � number of mice.

106 cells/hamster; volume of inoculated cell suspension = 0.1

ml), proliferating OHS cells formed highly vascularized >10-

mm2 tumors in four of four hamsters (Fig. 1 , A and B). Impor-

tantly, when hamsters carrying established 9-16 mm2 cheek

pouch OHS tumors received a 5-day (i.e., days 8-12) systemic

therapy with i.p. injections of TP3-PAP, there was a 57 ± 19%

tumor shrinkage in three hamsters, as determined by tumor

measurements 48 h after completion of the treatment (i.e. , on

day 14) without any obvious side effects (Fig. 1, C and D).

Thus, TP3-PAP showed significant in vivo antitumor activity in

our initial pilot study that used the HCP model, which provided

the rationale for detailed toxicity and efficacy studies in mice.

Mouse Toxicity of TP3-PAP Immunotoxin. In the first

study, groups of four to six female BALB/c mice treated with a

single i.p. bolus dose of TP3-PAP at one of five different dose

levels (I, 2, 3, 4, and 5 mg/kg) developed signs of toxicity at

doses of 3 mg/kg and higher (Table lA). At these high-dose

levels of immunotoxin, the mice became weak and lethargic,

had decreased levels of activity, lost weight, and developed

scruffy skin. All of the 12 mice treated with 4 mg/kg or 5 mg/kg

TP3-PAP died within 15 days with median survival times of

1 1.5 days and 9.5 days, respectively (Table 1). The day-30

LD5O was approximately 3 mg/kg with 67 ± 19% of the mice

remaining alive on day 30. The histopathological lesions in the

TP3-PAP-treated mice were dose-dependent and were observed

at �2 mg/kg dose levels. The lesions attributed to TP3-PAP

toxicity included: (a) acute to subacute degeneration and mild to

severe necrosis of cardiac muscle fibers with variable mineral-

ization and satellitosis of necrotic muscle fibers at dose levels �

2 mg/kg; (b) mild to moderate necrosis of skeletal muscle fibers

with variable degrees of mineralization at dose levels � 4

mg/kg; (c) mild periacinal vacuolization of hepatocytes and

mild scattered pinpoint hepatocellular necrosis at dose levels �

4 mg/kg; and (d) mild and subtle kidney damage with widely

scattered necrotic tubules and/or regenerative tubules that con-

tamed variable amounts of intraluminal granular cellular debris

at dose levels � 3 mg/kg. Control mice that were given 0.2 ml

PBS i.v. developed no clinical signs of toxicity and had no gross

or histological lesions.

In the second study, groups of four to six female BALB/c

mice were treated with a single daily i.p bolus injection of

TP3-PAP for 5 consecutive days at one of five cumulative dose

levels ( 1 , 2, 3, 4, and 5 mg/kg). Similar to the mice who received

single doses of TP3-PAP, these mice developed signs of toxicity

at cumulative doses of 3 mg/kg and higher. All of the 18 mice

treated at �3 mg/kg of TP3-PAP died with a median survival of

13 days (Table lB). The histopathological lesions in the TP3-

PAP-treated mice were dose-dependent and were observed at

�3 mg/kg dose levels. The lesions attributed to TP3-PAP tox-

icity were virtually identical to those observed in mice treated

with single-dose TP3-PAP injections and included: (a) acute to

subacute degeneration and mild to severe necrosis of cardiac

muscle fibers with variable mineralization and satellitosis of

necrotic muscle fibers at dose levels �3 mg/kg; (b) mild to

moderate necrosis of skeletal muscle fibers with variable de-

grees of mineralization at dose levels � 4 mg/kg; (c) mild

periacinal vacuolization of hepatocytes at dose levels �3 mg/

kg; and (‘0 mild and subtle kidney damage with widely scattered

necrotic tubules and/or regenerative tubules that contained vari-

able amounts of intraluminal granular cellular debris at dose

levels �3 mg/kg. Control mice that were given 0.2 ml of PBS

i.v. developed no clinical signs of toxicity and had no gross or

histological lesions.

In Vivo Antitumor Activity of TP3-PAP Immunotoxin

in SCID Mice Xenografted with OHS Human OsteosarcomaCells. All of the 25 CB-17-SCID mice that were inoculated

s.c. with 1 X 106 OHS osteosarcoma cells in their hind legs

developed tumors at a median of 12 days. These tumors rapidly

progressed to reach a mean (± SE) volume of 3.3 ± 2.2 cm3 on

day 52 (n = 25) and 4.9 ± 0.6 cm3 on day 77 (n 10). The

tumors were poorly circumscribed, fingering into the adjacent

skeletal muscle, and invading the femur. The tumor masses were

highly cellular and were divided into incomplete lobules by thin

fibrous septa. There were large, often confluent, areas of necro-



8

6

4

2

0

-a.- PBS (N=53)

.-*- TP3-PAP, 0.25 mg/kg (N=6)

-0- TP3-PAP, 0.50 mg/kg (N=6)

.-1�- TP3-PAP, 1 mg/lCg (N=6)

-.-. TP3-Ab, 1 mg/kg+PAP, 0.25 mgflg (N=5)

-A- B43-PAF� 1 mgflcg (N=5)

-.- CHEMO (N=15)

C’)

E0

U)

E

0E

�0.8

0E

I- 0.60)C>

�0.4

C0

00.�0

1.0

0 30 60 90 120 150

-0- PBS (N=53)

-0’- TP3-Ab+PAP (N=5)

-0- B43-PAP (N=5)

-.- CHEMO (N=15)

-&. TP3-PAl� I mg/kg (N=6)

0 30 60 90 120 150


Time After Inoculation of OHS Osteosarcoma Cells (Days)

sis in the centers of many lobules. The cells in the viable regions

were closely packed into sheets with little intercellular stroma.

The cells were large with abundant, clearly outlined, ampho-

philic to slightly eosinophilic, finely vacuolated cytoplasm. The

large, vesicular, round to ovoid nuclei were often eccentrically

located and had one to two large eosinophilic nucleoli. Mitotic

figures were numerous. Microscopic metastases were found in

the lung and spleen.

We examined the in vivo antitumor activity of TP3-PAP in

our SCID mouse xenograft model of human osteosarcoma de-

scribed above. We first sought to determine whether TP3-PAP

could delay the emergence and progression of tumors when

administered according to a nontoxic 3-day treatment program

at cumulative dose levels of 0.25 mg/kg, 0.50 mg/kg, or 1.00

mg/kg commencing 2 h after the s.c. tumor cell inoculation.

Control mice were treated with PBS, unconjugated TP3 anti-

body (1 mg/kg cumulative dose) + unconjugated PAP (1 mg/kg

cumulative dose), or B43-PAP (1 mg/kg cumulative dose), a

control immunotoxin directed against the CD19 surface antigen

on human leukemia cells. All of the 63 control mice treated with

PBS (n = 53), TP3 antibody + PAP (n 5), or B43-PAP (n

Fig. 2 Anti-osteosarcoma activity of TP3-PAP

in SCID mice. A, the tumor volume of s.c. OHSxenografts according to the amount of time (days)after the inoculation of OHS cells and the specifictreatment program applied to prevent tumorgrowth. B, tumor-free survival curves of SCIDmice inoculated with OHS cells and treated with

TP3-PAP, TP3 antibody (Ab) plus PAP, B43-

PAP, PBS, or chemotherapy. For details of thetreatment programs, see the “Materials and Meth-ods” section.

5) developed tumors within 45 days with a median tumor-free

survival of only 19 days (Fig. 2). Tumors rapidly progressed and

reached a size of 4.0 cm3 by 53.7 ± 2.6 days in PBS-treated

mice, by 43.6 ± 2.9 days in mice treated with TP3 + PAP, and

by 46.2 ± 3.3 days in B43-PAP-treated mice. At the 0.25- and

0.50-mg/kg dose levels, TP3-PAP delayed the emergence of

tumors (mean tumor-free survival = 26.5 days at 0.25 mg/kg

and 32.0 days at 0.50 mg/kg). On day 30, only 15.9 ± 4.6% of

the control mice were tumor-free, whereas no tumors were

detected in 33.3 ± 19.2% of mice treated with 0.25 mg/kg

TP3-PAP and 50 ± 20.4% of mice treated with 0.50 mg/kg

TP3-PAP (Table 2). However, once developed, OHS tumors in

TP3-PAP-treated mice progressed as rapidly as in control mice

(Fig. 2). TP3-PAP showed remarkably potent antitumor activity

at the 1.0-mg/kg cumulative dose level; none of the SCID mice

treated with TP3-PAP at this dose level developed a tumor for

up to 1 10 days after inoculation (Fig. 2), and 67 ± 19% were

still alive and tumor-free at 150 days (Table 2). In contrast, all

of the 15 mice that were treated with the chemotherapeutic

agents adriamycin, methotrexate, or cyclophosphamide devel-

oped tumors within 45 days with a median tumor-free survival



A.

-0-- PBS (N=20)

-0- Adriamycin (N=5)

-.-- Methotrexate (N=5)

-0- TP3-PAP, 1 mg/kg (N=16)

4’

3’

2

0

�1�

B.

6

5,

4,

3,

2’

PBS(N=20)

0 10 20 30

Time After Initiation of Therapy (Days)


Table 2 In vivo anti-osteosarcoma activity of TP3-PAP immunotoxin

Female SCID mice were inoculated s.c. with 1 X 106 OHS human osteosarcoma cells. Two hours later, mice were started on treatment programsemploying TP3-PAP immunotoxin (0.25 mg/kg, 0.5 mg/kg, 1.0 mg/kg in daily i.p. injections over 3 days) or chemotherapeutic agents (adriamycin,methotrexate, cyclophosphamide) as described in “Materials and Methods.” Control mice were treated with PBS, TP3 antibody + unconjugated PAP,or B43-PAP immunotoxin. Results are expressed as the cumulative proportion of mice surviving tumor-free according to the time after the inoculationof OHS cells. Life table analysis used the log-rank statistics.

Proportion surviving tumor-free (%)Median tumor-

Treatment group No. of mice 30 days 60 days 90 days 120 days free survival (days) P (log rank)

Control 63 16±5 0±0 0±0 0±0 19TP3-PAP, 0.25mg/kg 6 33 ± 19 0 ± 0 0 ± 0 0 ± 0 26.5 0.04TP3-PAP, 0.5mg/kg 6 50 ± 20 0 ± 0 0 ± 0 0 ± 0 32 0.04TP3-PAP, 1mg/kg 6 100 ± 0 100 ± 0 100 ± 0 83 ± 15 >150 <0.000001Chemotherapeutic agents 15 0 ± 0 0 ± 0 0 ± 0 0 ± 0 15 0.35

time of only 14.8 days (Fig. 2; Table 2). The inability of

unconjugated TP3 antibody + unconjugated PAP to elicit sig-

nificant antitumor activity in this SCID mouse model of human

osteosarcoma demonstrated that the antitumor activity of TP3-

PAP cannot be explained by its antibody moiety or PAP moiety

alone. However, the targeting TP3 antibody moiety was essen-

tial for the observed antitumor activity of TP3-PAP inasmuch as

no inhibition was observed with the control immunotoxin B43- �

PAP.

We next examined the effects of TP3-PAP on established

0.5-cm diameter OHS xenografts in SCID mice. Control mice

were treated with PBS, (n 20) or with the standard chemo- E

therapeutic drugs (n 10) adriamycin (25 mg/m2/d for 3 days; i�

n 5) or methotrexate (12.5 g/m2/day for 1 day; n = 5),

whereas the immunotoxin treatment group (n 16) received a

cumulative 1 mg/kg TP3-PAP over 3 days. Tumor progression

was monitored for 30 days. Tumors of the PBS-treated mice

showed rapid progression after the first week, whereas TP3-

PAP-treated mice showed no significant tumor progression for 3

weeks. Chemotherapy regimens did not seem to be as effective

as TP3-PAP. Consequently, on day 30, the mean tumor volume

of TP3-PAP-treated mice was substantially smaller than the

mean tumor volume of PBS-treated control mice (1.1 ± 0.1

versus 3.9 ± 0.4 cm3, P < 0.001) as well as the mean tumor �

volumes of mice treated with methotrexate (1 .1 ± 0. 1 versus

2.5 ± 0.4 cm3, P < 0.0001) or adriamycin (1.1 ± 0.1 versus

2.6 ± 0.2 cm3, P < 0.001; Fig. 3A). E

We also examined the antitumor activity of TP3-PAP (1- �

mg/kg cumulative dose level) in SCID mice with very large �

1.0-cm diameter OHS tumors. As shown in Fig. 3B, tumor

progression was significantly slower in the TP3-PAP-treated

mice (n = 9) than in the PBS-treated control mice (n = 20).

However, the magnitude of the antitumor effect seemed less

than in mice with smaller OHS tumors. Thus, TP3-PAP is likely

to have optimal utility only at a very early disease stage or in 0

adjuvant settings after surgical resection and/or chemotherapy

of the tumor mass.

Fig. 3 Anti-osteosarcoma activity of TP3-PAP in SCID mice with

DISCUSSION established xenograft tumors. A, SCID mice with 0.5-cm diameter OHS

Immunotoxins have been prepared by covalently linking a tumors were treated with PBS, TP3-PAP, or chemotherapeutic agents,. . . . as described in the “Materials and Methods” section. Tumor progression

cell type-specific monoclonal antibody to a variety of catalytic over time is shown for each of the treatment groups. B, SCID mice with

ribosome inhibitory protein toxins (24-27). For an immuno- 1.0-cm diameter OHS tumors were treated with TP3-PAP (1 mg/kg) or

toxin to be optimally effective against osteosarcoma, the target PBS as described in the “Materials and Methods” section.




antigen recognized by its monoclonal antibody moiety has to

fulfill at minimum the following essential requirements: (a) it

has to be expressed on clonogenic cells from the majority of

patients; (b) it has to be expressed on the self-renewing clono-

genic osteosarcoma cell populations; (c) it has to undergo anti-

body-induced internalization and thus allow the toxin moiety to

be transported into the targeted osteosarcoma cells; (d) it has to

be absent from the peripheral blood myeloid/erythroid elements

and thus allow immunotoxin to reach and effectively kill the

small number of clonogenic osteosarcoma cells; (e) it should not

be shed from the surface nor circulate in blood in soluble form

competing with surface-bound antigen for the administered im-

munotoxin molecules; and (J) it has to be absent from the

parenchymal cells of the life-maintaining nonhematopoietic or-

gans.

The tumor-associated antigen p80 is expressed at very high

levels on the surface membrane of human osteosarcoma cells

(9). The very limited normal-tissue distribution of p80 antigen

(9, 1 1, 12, 27, 28) makes it an attractive target for biotherapy of

osteosarcomas. The murine IgG2b anti-p80 antibody TP3 reacts

with mesenchymal tumors including osteosarcomas, hemangio-

pericytomas, chondrosarcomas, malignant fibrous histiocyto-

mas, and synovial cell sarcomas (11). In a previous study, we

found that the PAP immunoconjugate of the TP3 antibody (i.e.,

TP3-PAP imniunotoxin) kills >99.99% of clonogenic human

osteosarcoma cells within 18 h, suggesting that this biothera-

peutic agent may be useful in the treatment of therapy-refractory

osteosarcoma (10). This report provides a detailed characteriza-

tion of the pharmacokinetics, toxicity, and antitumor activity of

TP3-PAP immunotoxin directed against osteosarcoma cells.

The in vivo toxicity profile as well as pharmacokinetics of

TP3-PAP in mice were identical to the previously reported

toxicity profiles and pharmacokinetic features of other PAP

immunotoxins that are presently under clinical investigation (14,

26, 29). In efficacy studies, TP3-PAP elicited potent in vitro and

in vivo antitumor activity in a HCP model of human osteosar-

coma. Treatment with TP3-PAP at nontoxic systemic exposure

levels significantly delayed the emergence and progression of

leg tumors and markedly improved tumor-free survival in SCID

mice challenged with OHS human osteosarcoma cells.

Several investigators have evaluated the toxicities associ-

ated with the administration of immunotoxins (24-27). There is

limited information about the toxicity profiles of immunotoxins

containing the plant hemitoxin PAP (14, 29). B43-PAP, an

anti-CD19 immunotoxin directed against B-lineage leukemia

cells, was reported previously to cause dose-limiting renal and

cardiac toxicities in mice (30) and dose-limiting renal toxicity

due to severe and diffuse renal tubular necrosis in cynomolgus

monkeys (14). In addition, B43-PAP caused a mild hepatic

injury in cynomolgus monkeys, characterized by transient dc-

vation of transaminases and a mild multifocal hepatitis detected

histopathologically and a transient episode of proteinuria and

hypoalbuminemia, which could be due to a mild capillary leak.

The toxicities associated with TXU-PAP immunotoxin detailed

in a recent report were reminiscent of those associated with

B43-PAP immunotoxin (30). In mouse toxicity studies, TP3-

PAP immunotoxin caused compound-related histological Ic-

sions. TP3-PAP-related histological lesions in BALB/c mice

included mild to severe myocardial necrosis, characterized by

the presence of occasional necrotic myofiber segments in one or

both ventricles, and mild or moderate skeletal muscle necrosis,

characterized by occasional to many necrotic myofiber seg-

ments. There was also mild acute multifocal hepatocellular

necrosis, characterized by the presence of a small number of

individual necrotic hepatocytes in the livers of some mice. This

toxicity profile is identical to that seen in BALB/c mice treated

with B43-PAP immunotoxin (30), which is in clinical trials

under BB-IND-3864, or with TXU-PAP immunotoxin (14),

which is in clinical trials under BB-IND-6982. Lesions that

might be expected to cause morbidity are myocardial necrosis

and skeletal muscle necrosis. The hepatocellular necrosis may

have been a direct toxic effect of TP3-PAP inasmuch as it was

seen only at higher dose levels, but it may also have been a

consequence of impaired cardiac function.

Humoral immune responses to the monoclonal antibody as

well as toxin portions of immmunotoxins have contributed to

their limited clinical utility (13). Immunogenicity of TP3-PAP

might be reduced by replacing the mouse antibody with a

chimeric or humanized version of TP3 as well as by attaching

allergens, haptens, or chemical agents, such as polyethylene

glycol, that suppress immune responses.

On the basis of the encouraging anti-osteosarcoma activity

of TP3-PAP presented herein, we postulate that the incorpora-

tion of this immunotoxin into clinical treatment protocols may

improve the prognosis for therapy-refractory osteosarcoma pa-

tients. TP3 antibody also reacts with budding capillaries of a

wide variety of tumors (9, 10). Therefore, toxin conjugates of

the TP3 antibody can inhibit the growth of tumors not only by

selectively destroying p80 antigen positive cancer cells but also

by damaging the tumor neovasculature. Such damage to the

tumor neovasculature (3 1) may also facilitate the tumor entry of

antineoplastic agents by providing “windows” in the vasculature

through which the antineoplastic agents may easily enter the

tumor.

REFERENCES

1. Meyers, P. A., Heller, G., Healey J., Huvos, A., Lane, J., Marcove,R., Applewhite, A., Vlamis, A., and Rosen, G. Chemotherapy fornonmetastatic osteogenic sarcoma: the Memorial Sloan-Kettering expe-rience. J. Clin. Oncol., 10: 5-15, 1992.

2. Hudson, M., Jaffe, M. R., Jaffe, N., Ayala, A., Raymond, A. K.,Carrasco, H., Wallace, S., Murray, J., and Robertson, R. Pediatricosteosarcoma: therapeutic strategies, results, and prognostic factors de-

rived from a 10-year experience. J. Clin. Oncol., 8: 1988-1997, 1990.

3. Frei, E., III, and Goorin, A. Osteogenic sarcoma: the development of

curative treatment. ln: J. F. Novak and J. H. McMaster (eds.), Frontiersof Osteosarcoma Research, pp 5-13. Bern, Switzerland: Hogrefe &Huber Publishers, 1993.

4. Ward, W. G., Mikaelian, K., Dorey, F., Mirra, J. M., Sassoon, A.,

Holmes, E. C., Eilber, F. R., and Eckhardt, J. J. Pulmonary metastasesof stage IIB osteosarcoma and subsequent pulmonary metastases.J. Clin. Oncol., 12: 1849-1858, 1994.

5. Meyers, P. A., Heller, G., Healey, J., Huvos, A., Applewhite, A.,Sun, M., and LaQuaglia, M. Osteogenic sarcoma with clinically detect-able metastasis at initial presentation. J. Clin. Oncol., 11: 449-453,

1993.

6. Raymond, A. K., Chawla, S. P., Carrasco, C. H., Ayala, A. G.,Fanning, C. V., Grice, B., Arman, T., Plager, C., Papadopoupolos,N. E. J., Edeiken, J., Wallace, S., Jaffe, N., Murray, 1. A., and Benjamin,R. S. Osteosarcoma chemotherapy effect: a prognostic factor. Semin.Diagn. Pathol., 4: 212-236, 1987.




7. Winkler, K., Beron, G., and Detting, G. Neoadjuvant chemotherapyof osteosarcoma: results of a randomized cooperative trial (COSS-82)with salvage chemotherapy based on histologic tumor response. J. Clin.Oncol., 6: 329-339, 1988.

8. Provisor, A. J., Ettinger, L. J., Nachman, J. B., Krailo, M. D.,Makley, J. T., Yunis, E. J., Huvos, A. G., Betcher, D. L., Baum, E. S.,Kisker, T., and Miser, J. S. Treatment of nonmetastatic osteosarcoma ofthe extremity with preoperative and postoperative chemotherapy: areport from the Children’s Cancer Group. J. Clin. Oncol., 15: 76-84,

1997.

9. Bruland, 0. S., Fodstad, 0., Stenwig, A. E., and Pihl, A. Expressionand characteristics of a novel human osteosarcoma-associated cell sur-face antigen. Cancer Res., 48: 5302-5309, 1988.

10. Anderson, P. M., Meyers, D. E., Hasz, D. E., Covalciuc, K.,Saltzman, D., Khanna, C., and Uckun, F. M. In vitro and in vivocytotoxicity of an anti-osteosarcoma immunotoxin containing pokeweed

antiviral protein. Cancer Res., 55: 1321-1327, 1995.

11. Bruland, 0., Fodstad, 0., Funderud, S., and Pihl, A. New mono-clonal antibodies specific for human sarcomas. mt. J. Cancer, 38:

27-31, 1986.

12. Bruland, 0., Fodstad, 0., Skretting, A., and Pihi, A. Selective

localisation of two radiolabelled anti-sarcoma monoclonal antibodies inhuman osteosarcoma xenografts. Br. J. Cancer, 56: 21-25, 1987.

13. Uckun, F. M., and Reaman, G. H. Immunotoxins for treatment of

leukemia and lymphoma. Leuk. Lymphoma, 18: 195-201, 1995.

14. Uckun, F. M., Yanishevski, Y., Waurzyniak, B., Messinger, Y.,

Chelstrom, L. M. Schneider, E. A., Ek, 0., Zeren, T., Haissig, S.,Langlie, M., Irvin, J. D., Myers, D. E., Evans, W., and Gunther, R.

Pharmacokinetic features, immunogenicity, and toxicity of B43(anti-

CD19)-pokeweed antiviral protein immunotoxin in cynomolgus mon-keys. Clin. Cancer Res., 3: 325-337, 1997.

15. Fodstad, 0., Brogger, A., Bruland, 0., Soiheim, 0., Nesland, J., andPihl, A. Characteristics of a cell line established from a patient withmultiple osteosarcoma, appearing 13 years after treatment for bilateralretinoblastoma. Int. J. Cancer, 38: 33-40, 1986.

16. Wallace Toolan, H. Growth of human tumors in cortisone-treatedlaboratory animals: the possibility of obtaining permanently transplant-able human tumors. Cancer Res., 13: 389-402, 1953.

17. Blumenthal, R. D., Kashi, R., Stephens, R., Sharkey, R. M., and

Goldenberg, D. M. Improved radioimmunotherapy of colorectal cancerxenografts using antibody mixtures against carcinoembryonic antigenand colon-specific antigen-p. Cancer Immunol. Immunother., 32: 303-

310, 1991.

18. Gerlowski, L. E., and Jam, R. K. Physiologically based pharmaco-

kinetic modelling: principles and applications. J. Pharm. Sci., 72: 1 103-

1126, 1983.

19. Uckun, F. M., Evans, W. E., Forsyth, C. J., Waddick, K. G.,Ahlgren, L. T., Chelstrom, L. M., Burkhardt, A., Bolen, J., and Myers,D. E. Biotherapy of B-cell precursor leukemia by targeting genistein toCD19-associated tyrosine kinases. Science (Washington DC), 267:

886-891, 1995.

20. Messinger, Y., Yanishevski, Y., Ek, 0., Zeren, T., Waurzyniak, B.,

Gunther, R., Chelstrom, L., Chandan-Langlie, M., Schneider, E., Myers,D. E., Evans, W., and Uckun, F. M. In vivo toxicity and pharmacokinetic

features of B43 (anti-CD19)-genistein immunoconjugate in nonhuman

primates. Clin. Cancer Res., 4: 165-170, 1998.

21. Uckun, F. M., Narla, R., Zeren, T., Yanishevski, Y., Myers, D. E.,

Waurzyniak, B., Ek, 0., Schneider, E., Messinger, Y., Chelstrom, L. M.,Gunther, R., and Evans, W. In vivo toxicity, pharmacokinetics, and

anticancer activity of genistein conjugated to human epidermal growth

factor. Clin. Cancer Res., 4: 1 125-1 134, 1998.

22. Perentesis, J. P., Gunther, R., Waurzyniak, B., Yanishevski, Y.,Myers, D. E., Ek, 0., Messinger, Y., Shao, Y., Chelstrom, L. M.,

Schneider, E., Evans, W. E., and Uckun, F. M. In vivo biotherapy of

HL-60 myeloid leukemia with a genetically engineered recombinantfusion toxin directed against the human granulocyte macrophage cob-

ny-stimulating factor receptor. Clin. Cancer Res., 3: 2217-2227, 1997.

23. Uckun, F. M., Chelstrom, L. M., Irvin, J. D., Finnegan, D., Gunther,R., Young, J., Kuebelbeck, V., Myers, D. E., and Houston, L. L. ln vivo

efficacy of B43 (anti-CD19)-pokeweed antiviral protein immunotoxin

against BCL-1 murine B-cell leukemia. Blood, 79: 2649-2661, 1992.

24. Vitetta, E. S., Fulton, R. J., May, R. D., Till, M., and Uhr, J. W.

Redesigning nature’s poisons to create antitumor reagents. Science

(Washington DC), 238: 1098-1 101, 1988.

25. Neville, D. M., and Youle, R. J. Monoclonal antibody-ricin or ricinA chain hybrids: kinetic analysis of cell killing for tumor therapy.Immunol. Rev., 62: 75-91, 1982.

26. Irvin, J. D., and Uckun, F. M. Pokeweed antiviral protein: ribosome

inactivation and therapeutic applications. Pharmacol. Ther., 55: 279-

302, 1993.

27. Uckun, F. M. Immunotoxins for the treatment of leukemia. Br. J.

Haematol., 85: 435-438, 1993.

28. Bruland, 0. S., Fodstad, 0., Aas, M., Soiheim, 0. P., Hoie, J.,Skretting, A., Winderen, M., Michaelsen, T., and Pihl, A. Immunoscin-

tography of bone sarcomas-results in S patients. Eur. J. Cancer, 30A:

1484-1489, 1994.

29. Waurzyniak, B., Schneider, E. A., Turner, N., Yanishevski, Y.,

Gunther, R., Chebstrom, L. M., Wendorf, H., Myers, D. E., Irvin, J. D.,Messinger, Y., Ek, 0., Zeren, T., Chandan-Langlie, M., Evans, W. E.,

and Uckun, F. M. In vivo toxicity, pharmacokinetics, and antileukemic

activity of TXU (anti-CD7)-pokeweed antiviral protein immunotoxin.Clin. Cancer Res., 3: 881-890, 1997.

30. Gunther, R., Chelstrom, L. M., Wendorf, H. R., Schneider, E. A.,Covalciuc, K., Johnson, B., Cbementson, D., Irvin, J. D., Myers, D. E.,

and Uckun, F. M. Toxicity profile of the investigational biotherapeutic

agent, B43 (anti-CD19)-pokeweed antiviral protein immunotoxin. Leuk.

Lymphoma, 22: 61-70, 1996.

31. Folkman, M. J. Antiangiogenesis. In: V. T. DeVita, Jr., S. Heilman,and S. A. Rosenberg (eds.), Biologic Therapy of Cancer, pp. 743-753.

Philadelphia: J. B. Lippincott Co., 1991.



1998;4:1641-1647. Clin Cancer Res O Ek, B Waurzyniak, D E Myers, et al. osteosarcoma.immunodeficient mouse xenograft models of humanimmunotoxin in hamster cheek pouch and severe combined Antitumor activity of TP3(anti-p80)-pokeweed antiviral protein

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