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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
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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-
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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
1644 Antitumor Activity of TP3(anti-p80)-PAP Immunotoxin
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
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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)
Clinical Cancer Research 1645
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.
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1646 Antitumor Activity of TP3(anti-p80)-PAP Immunotoxin
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.
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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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