1. Healthcare

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RAPID COMMUNICATION
Eradication of Minimal Disease in Severe Combined Immunodeficient Mice
With Disseminated Daudi Lymphoma Using Chemotherapy and an
Immunotoxin Cocktail
By M.-A. Ghetie, K. Tucker, J. Richardson, J.W. Uhr, and E.S. Vitetta
Severe combined immunodeficient (SCID) mice injected intravenously with ahumanBurkitt’slymphoma
cell line
(Daudi)
develop
disseminated
lymphoma
(SCID/Daudi),
which is fatal in 100% of the mice. Earlytreatment of these
mice with either an immunotoxin (IT) cocktail (consisting
of anti-CD19-ricin A chain plus anti-CD=-ricin A chain) or
chemotherapy significantly prolonged survival
but was not
curative. Combination therapywith the IT cocktail and any
one of three chemotherapeutic drugs (doxorubicin, cytoxan,
or camptothecin) cured the mice. Cure was demonstrated
by both histopathologic examination of treated mice and,
more importantly, by adoptive transferof cells from organs
of the cured miceto naive SCID micewhere 100 tumor cells
would have caused disease in the recipients. These results
provide a strong rationale for combining IT therapy with
conventional chemotherapyin the treatment of B-cell neoplasia.
0 1994 by The American Societyof Hematology.
C
phosphamide), doxorubicin, and methotrexate, as well as a
less commonly used drug, camptothecin, which has recently
been re-evaluated for the therapy of human carcinoma^.^"^
Neither doxorubicin nor methotrexate has beenusedin
combination with ITS.The most significant therapeutic effect
of either drug alone was achieved using an immunoconjugate
containing doxorubicin and the BR96 antibody. This conjugate cured xenografted human lung, breast, and colon carcinomas grown subcutaneously in athymic mice. This conjugate also cured 70%of mice with metastatic human lung
carcinoma.I’ Liposome-encapsulated doxorubicin has also
been used to eradicate lung cancer in
The toxicity
of doxorubicin to the gastrointestinal tract and myocardium
is reduced whenthe drug is administered in encapsulated
form. In a phase I clinical trial, TLCD-99 (doxorubicin encapsulated in liposomes) was well-tolerated andproduced
fewer side effects than would be expected with nonencapsulated drug at equal doses.“
Clinical trials with camptothecin in its water-soluble form
were discontinued because of severe
However,
new derivatives of camptothecin have been used with some
success in phase I1 clinical studies in patients with refractory
leukemia and lymphomaI7 and in phase I and I1 studies in
patients with advanced non-small cell lung carcinoma” or
with gynecologic cancers.” Complete inhibition of the
growth of human malignant melanoma cells in vitro and
regression of human melanoma xenografts,’ breast xenografts,” and ovarian carcinomas” in nude mice have also
been induced with camptothecin. These studies suggested
that a combination of ITS and different chemotherapeutic
regimens might improve the efficacy of ITS reactive with
human B lymphoma.
This study shows that therapy with a cocktail of two ITS
(anti-CD22-dgA and anti-CD19-dgA) plus a single chemotherapeutic agent cures human Burkitt’s lymphoma (Daudi
cells) in SCID mice with no evidence of minimal residual
disease or dormant tumor cells.
ONVENTIONAL APPROACHES to the treatment of
B-cell lymphoma have emphasized the use of cytotoxic agents that cancure 30%to 50% of high-grade lymphomas, but not low-grade lymphomas.’,2We have used our
severe combined immunodeficient (SCID)/Daudi model of
disseminated human Burkitt’s B lymphoma3 for preclinical
evaluation of different immunotoxin (IT) constructs containing either anti-CD22 or anti-CD19 antibodies and deglycosylated ricin A chain (dgA).4,5These ITS have also been
used in over 100 patients in phase VI1 clinical trials in patients with refractory non-Hodgkin’s lymphoma (NHL)6,7
and (Stone et al, unpublished data; Sausville et al, unpublished data, Kaplan et al, unpublished data, 1994).
Our previous studies have shown that neither Fab’ nor
IgG anti-CD22 (RFB4)-dgA4 or IgG anti-CD19 (HD37)dgA5 cured SCIDDaudi mice with early disease, although
survival was significantly prolonged. A combination of the
two ITS or a combination of anti-CD22-dgA and anti-CD19
antibody (without dgA) extended survival of SCID/Daudi
mice significantly longer than either treatment alone. Thus
far, the only combination treatment that has been curative
in SCID mice was a large amount of anti-CD19 antibody and
the anti-CD22-immunotoxin, RFB4-dgA. The mice treated
with this combination survived more than 1 year, at which
timethey appeared tumor free.8Inthe present study we
compare the therapeutic effect of combination treatment with
chemotherapy and ITS. We selected three chemotherapeutic
drugs that are commonly used in humans: cytoxan (cyclo-
From the Cancer Immunobiology Center, the Department of Pathology, and the Department of Microbiology, University of Texas
Southwestern Medical Center at Dallas.
Submitted April 4, 1994; accepted May 17, 1994.
Supported by National Institutes of Health Grants No. CA-28149
and CA-41087.
Address reprint requests to E.S. Vitetta, PhD, Cancer Immunobiology Center and Department of Microbiology, 6000 Harry Hines
Blvd, Dallas, lX 75235-8576.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“adveaisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 I994 by The American Society of Hematology.
0006-497//94/8403-0038$3.00/0
702
MATERIALS AND METHODS
Cells. The Burkitt’s lymphoma cell line Daudi was maintained
in culture by serial passage in RMPI 1640 medium containing 25
mmol/L HEPES, 10% heat-inactivated fetal bovine serum, 100 U/
mL penicillin, 100 pg/mL streptomycin, and 100 mmoVL L-glutamine (complete medium). The cells were grown in a humidified
Blood, VOI 84, NO 3 (August l), 1994: PP 702-707
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IMMUNOTOXINS IN SCID/LYMPHOMA
703
atmosphere of 5% CO2and air. Cells were used for intravenous (IV)
inoculation of SCID mice. Cell viability was determined by trypan
blue exclusion.
Animals. Female SCID mice (CB-l7 SCIDISCID) were purchased from the Harlan Sprague Dawley Laboratory (Madison, WI).
They were housed and maintained in a specific pathogen-free (SPF)
facility. Animals were fed autoclaved food and sterile water ad libitum, and all manipulations were performed in a laminar flow hood.
Induction ofhumantumor in SCID mice. Six- to10-week old
SCIDs were inoculated IVwith 5 X lo6 Daudi cells in 0.1 mL
phosphate-buffered saline (PBS). Mice were monitored daily and
killed at the onset of paralysis of both hind legs, a clinical symptom
that preceeds death.3 (The mean paralysis time [MPT] has been
established as the end point in this animal model.*) Complete necropsy and histopathologic examination were performed on several
animals in each experimental group. Tissue from lung, heart, liver,
spleen, kidney, ovary, and vertebrae were fixed in 10% buffered
formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.
Therapy of SCIDDaudi mice. SCID mice were inoculated with
5 x lo6 Daudi cells 24 hours before treatment. In each experiment,
four groups of 5 to 10 mice with an average weight of 20 g were
used and treatment was administered on days 1 through 4 after tumor
injection as follows: (1) control mice were injected retroorbitally
(RO) with PBS or saline; (2) mice were injected R 0 with an IT
cocktail (WB4-dgA [60 pg] + HD37-dgA [60 pg]); (3) mice received either one of the following chemotherapeutic drugs: methotrexate (1 12 pg), doxorubicin (80 pg), cytoxan (1.6 mg), or camptothecin (640 pg) (all chemotherapeutics but camptothecin were
administered IV on days 1 through 4 divided into four equal amounts;
camptothecin was injected intramuscular (IM) in an emulsion in
intralipid two times per week for 4 weeks); (4) mice were administered a combination of treatments (2) and (3) as described above.
When therapy was initiated, the SCID mice had 1 X lo7disseminated
Daudi cells as described el~ewhere.~
Adoptive transfer. Mice surviving for 150 to 200 days were
killed and cells from ovaries and spinal cords' were injected IV into
healthy SCID mice. Adoptive recipients were followed-up for 200
days. At this time, the transfer of 100 tumor cells should have caused
death in the recipients.'
Cytotoxic effect of different chemotherapeutic drugs alone or in
combination with ITS. A Daudi killing assay for each drug (methotrexate, doxorubicin, cytoxan, and camptothecin) was performed as
described else~here.*~
The inhibition of 'H-thymidine incorporation
was used to determine individual ICs0s (M). The cytotoxic effect of
a cocktail containing different amounts of anti-CD22-dgA and a
constant amount of anti-CD19-dgA (eg, the ICso dose) andvice
versa was also determined and compared with combination treatment
using each IT plus either one of the chemotherapeutic drugs at a
Table 1. Cytotoxic Effect of Dierent Chemotherapeutics on Daudi
Cells In Vitro and on SCID Mice
Chemotherapeutic
Methotrexate
Category
Antimetabolite
ICw (mollL)
LDm Dose
(mg/kg)*
Not toxic u p to 14
2.2
x
mol/L
Doxorubicin
Antibiotic
4.2 ? 1.3 X lo-' (3)t
10
200
Alkylating agent 8.8 t 1.6 x 10" (3)t
Cytoxan
1.1 ? 0.2 X 10-9 (3)t
ND
Camptothecin Alkaloid
Abbreviation: ND, not determined.
Two mice were used to determine the LDm.
t The number in parentheses is the number of experiments performed.
Table 2. Antitumor Effect of DHerent Chemotherapeutic
Drugs in SCID/Daudi Mice
Drug
Treatment (dose)*
MW 2 SD (d)
Control
Methotrexate
Cytoxan
No treatment
40% LDW ( 1 12 pg)
20% LD5o (0.8mg)
40% LD50 (1.6 mg)
20% LDso (40 pg)
40% LDso (80/le)
40 pg X 8 = 320 pg
80 pg X 8 = 640 pg
31.7 t 2.6(10)t
31.6 ? 2.4 (5)
51.8 2 4.1 (5)
63.4 t 12.7 (11)
31.2 C 1.2 ( 1 1 )
62.2 ? 13.6 (27)
44.4 t 9.7 (7)
74.7 ? 17.5 (12)
~
Doxorubicin
Camptothecin
~~~
*All drugs but camptothecin were injected IV in four equal doses
on days 1-4 after injection with tumor cells; camptothecin (sonicated
in intralipid)was administered IM 2 times per week for 4 weeks.
t The number in parentheses is the number of mice in each group.
certain concentration (eg, the ICs0 dose). To determine whether the
cytotoxic effect of combination treatment was additive or synergistic,
an analysis using an algebraic method was performed and the combination index (CI) was calculated using the equation: C1 = A/Ae +
BlBe < 1 (synergistic); = 1 (additive), where A and B are the
concentrations of the two drugs that, in combination, kill 90% of
the Daudi cells, and Ae and Be are the concentrations of the two
drugs extrapolated from their individual killing curves and corresponding to 90% killing."
Immunojuorescence assay. The phenotype of Daudi tumor
grown in SCID mice has been previously de~cribed.~
To determine
whether treatment with drugs generated CD22- or CD19- Daudi
cells, we harvested tumors from doxorubicin-treated mice (ovary
and spine) and as well as from untreated SCIDDaudi mice (ovary).
Single-cell suspensions were prepared and cells were analyzed by
indirect immunofluorescence, using the parental Daudi cells as a
control." Cells were stained with the primary antibodies anti-CD22
and anti-CD19 followed by a fluorescein isothiocyanate (FITC)labeled goat-antimouse Ig (GAMIg). The cells were analyzed on a
fluorescence-activated cell sorter (Becton-Dickinson, Oxnard, CA).
RESULTS
Cytotoxic effect of different Chemotherapeutic agents on
Daudi cells. The chemotherapeutic drugs used
in
the
SCJDDaudi model are described in Table 1. These drugs
(methotrexate, cytoxan, doxorubicin, and camptothecin) induce antitumor effects by interacting with cellular DNA. As
shown in the table, methotrexate didnot kill Daudi cells
whereas doxorubicin and camptothecin had comparable
IC50s
m o m ) and cytoxan was less potent (ICso =
mom).
Antitumor effect of difSerent drugs in SCID/Daudi mice.
Two different doses (20% and 40% of the LD50 dose) were
administered to separate groups of SCIDDaudi mice.As
shown in Table 2, methotrexate did not prolong survival of
SCIDDaudi mice at the 40% LDS0 dose whereas the other
three drugs gave dose-dependent prolongation of survival.
Therefore, 40% of the LD50 dose of cytoxan or doxorubicin
or 640 pg campothecin was used insubsequent experiments,
because this dose induced a significant prolongation of MPT
in SCIDDaudi mice. Furthermore, at this dose mice did not
show toxic effects as determined by histopathologic examination.
Phenotype of Daudi tumor inSCID mice treatedwith
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704
GHETIE ET AL
Table 3. Effect of Treatment With Doxorubicin on the Phenotype of
Residual Daudi Tumor in Treated SCID Mice
% Positive Cells
Untreated
Daudi Staining
Mice
Mice Treated With
Doxorubicin
Antibody
Parental
Used
for
Anti-CD22
Anti-CD19
4.1
MOPC-21
83.8
87.6
4.3
64.2
91.7
2.1
79.9
97.1
2.8
86.4
93.8
doxorubicin. Daudi cells from SCID mice treatedwith
40% of the LDSodose of doxorubicin were analyzed for their
CD19/CD22 phenotype. As shown in Table 3, residual tumor
cells from treated mice had the same phenotype as parental
Daudi cells andtumor cells from untreated mice. Hence,
chemotherapy did not change the expression of the target
antigens for the two ITS.
Effect of combination therapy of SCID/Daudi mice. Chemotherapeutic drugs (doxorubicin, cytoxan, or camptothecin) significantly extended the MPT of treated mice as
did treatment with a cocktail of the two ITS (anti-CD22-dgA
+ anti-CD19-dgA).' However, at the time of killing these
mice showed the same pattern of tumor growth as control
mice. The mice treated with any one of the chemotherapeutic
drugs in combination with the cocktail of the two ITS survived over 150 days with no sign of paralysis (Fig 1). When
gross and histopathologic examination was performed, none
of these mice showed any sign of tumor. At least lo7 cells
from ovary and spine were adoptively transferred into naive
SCID mice. There was no evidence of tumor growth in recipients after 7 months, at whichtime the experiments were
terminated. Because 100 Daudi cells are sufficient to cause
progressive tumor in all the adoptive recipients at 5 months,'
we considered the original donor mice to be cured.
Synergistic effect of treatment with a cocktail of ITS plus
chemotherapeutic drugs in Daudi cells. Both ITS have a
significant cytotoxic effect on Daudi cells with ICsos of 5.0
X lo"* mol/L (RFBCdgA) and 4.5 X lo-" moVL (HD37dgA).' The combination of the two ITS (Fig 2A) or each of
the two ITS together with a single chemotherapeutic drug
had an enhanced cytotoxic effect, which is shown inFig
2, B and C. Figure 2D shows the cytotoxic effect of each
chemotherapeutic drug. The analysis (algebraic method)" of
the cytotoxic effect of combination treatment shows that the
addition of chemotherapeutic drugs to the two ITS induced
a synergistic cytotoxic effect on Daudi cells because the C1
was < 1 for 90% killing. The data in Table 4 show that the
combination of the two ITS or one IT plus one chemotherapeutic drug increases the cytotoxic effect on Daudi cells and
that the cytotoxic effect is synergistic.
DISCUSSION
The objective of the present study was to compare the
antitumor activity of different chemotherapeutic drugs alone
or in combination with the two ITS used in other studies in
SCID/Daudi mice4,5,8 as well
as in clinical trials6.' (and Stone
so
2
100
150
PARALYSIS T I M t ( d a y s )
"
.
50
.
.
1 00
l50
PARALYSIS TIME (days)
C
100
-
80
>
W
2
60
k
z
40
W
W
a
20
0
100
PARALYSIS TIME ( d a y s )
50
0
150
Fig 1. A combination of a cocktail of two ITS (anti-CD22-dgA +
anti-CD19-dgA) and chemotherapeutic drugs in SCID/Daudi mice induces complete inhibition of tumor growth: (A) cocktail
of ITS +
doxorubicin (40% of theLDw administered in 4 equal doses); (B) cocktail of ITS cytoxan (40% of LDw administered in 4 equal doses); (C)
cocktail of ITS camptothecin (totaldose = 640 p g administered in
8 equal doses [2 times per week for 4 weeks]). SClDmice wereinoculated IV with Daudi cells (5 x lo6 per mouse) and were treated as
follows: (0)
saline (control) (10 mice); (A)chemotherapeutic drugs
only: doxorubicin, cytoxan, or camptothecin (10 mice); (0)cocktail
of two I T S (RFB4-dgA + HD37-dgA) (20% of LDS each, totaling 120
p g administered in 4 equal doses) (10 mice); ( ) chemotherapeutic
drug (any one above) plus cocktail oftwo ITS (10 mice). The percent
of survivors is plotted
against the paralysis time. Each panel shows
a representative experiment of two t o four performed.
+
+
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L
0
U
I 0-1'
1 o+
10"'
IT concentrotion (M)
1 o-'L
1 o"6
IO"'
1 0-12
IT concentration (M)
10"'
\
10 - I S
1 0-11
IT concentration (M)
1 0-g
10"O
1 0-B
10"
1 0"
Chernotheropeuticconcentrotion
(M)
1 o-2
Fig 2. Cytotoxic effect of combination treatment was analyzed on Daudi cells(1 x 105/we11/200pLl as follows: (A) anti-CD22-dgA (0);antiCDl9-dgA (0);anti-CDP2-dgA (2.8 x lo-' to 2.8 x 10"' mol/L) plus anti-CD19-dgA at 2.8 x 10"' mollL (A); anti-CD19-dgA (2.8 x lo-' to 2.8
x 10"' mollLI plus anti-CD22-dgA at 2.8 x 10"' mol/L (AI; (B) anti-CD22-dgA alone (0)or anti-CD22-dgA (2.8 x 10"' t o 2.8 x lo-" mollLI
plus doxorubicin at 9.2 x 10" mol/L (A);or plus cytoxan at 9.0 x 10" mollL (M); or plus camptothecin at 1.4 x 10"' mollL (0);(Cl anti-CD19dgA alone (0)or anti-CD19-dgA (2.8 x lo-' t o 2.8 x 10"' mol/L) plus doxorubicin at 9.2 x lo-' mol/L (AI; or plus cytoxan at 9.0 x 10" moll
L (M); or plus camptothecin at 1.4 x 10"' mol/L (0);(D) doxorubicin (01;camptothecin (Dl; cytoxan (A).The plates with cells were incubated
for 24 hours, pulsed with IaHl-thymidinefor 18 hours, harvested, and counted. The
reduction of incorporation of cells treated with ITS (percent
of control) was plotted against the concentration.
et al, unpublished data; Sausville et al, unpublished data,
Kaplan et al, unpublished data, 1994). In a previous study,'
we showed that SCIDDaudi mice treated with a combination
of anti-CD22-dgA (20% of LD50dose) and large amounts
of F(ab'), fragments of anti-CD19 antibodies were tumorfree 1 year after treatment. The present study was undertaken
inan attempt tofind less expensive but equally effective
curative therapies for minimal disease in SCIDDaudi mice.
Our major finding is that chemotherapy and immunotoxins
have synergistic antitumor activity in vitro and cure SCID
mice with disseminated B lymphoma.
The in vitro studies of Daudi cells treated with methotrexate, doxorubicin, cytoxan, or camptothecin showed that: (1)
methotrexate was not effective at killing Daudi cells either
because it must be metabolized by the liver to be active or
the Daudi cells are naturally resistant to this drug; (2) Daudi
cells were very sensitive to both doxorubicin and carnptothecin, with
an IC5o of
m o m ; (3) cytoxan was less
cytotoxic for Daudi cells and its lower toxic effect (IC50 =
m o m ) was consistent with its higher LDso in mice.
The doses chosen for in vivo studies (20% and 40% of
the LD50doses) for methotrexate, doxorubicin, and cytoxan,
and 640 pg for camptothecingwere administered safely with
no signs of toxicity. Because methotrexate did not extend
the MPT at 40% of the LD5,,, it was not used in combination
with the ITS in vivo.
In a previous studywe have shown that a cocktail of
two ITS (anti-CD22-dgA and anti-CD19-dgA) extended
survival of SCIDDaudi mice in a manner consistent with
the killing of 6 logs of tumor cells (50% of mice survived
over 100 days).' In this study, we combined the two therapies
known to be effective individually and show that the combination of the two ITS and one of the above chemotherapeutic
agents had a synergistic effect in vitro and eradicated disseminated minimal disease in vivo.Our studies confirmand
extend earlier reports that ITS and mafosfamid eliminated
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706
GHETIE ET AL
Table 4. Increase of IT'SCytotoxic Effect When Used in
Combination With Each Other or With Chemotherapeutics
Drug
None
Doxorubicin§
Cytoxanll
Camptothecinf
Anti-CDZZ-dgA
Anti-CD19dgA
+*
-
-
+*
+*
+t
0.08
+S
+*
0.03
+*
-
0.005
-
+*
+*
ICso
rnol/L
X 10."
1.2
22
-
0.08
0.00004
-
+*
0.0007
+*
-
0.004
-
+*
0.32
~~~~
~
For each combination above, a Cl was calculated using the formula
Cl = NAe + B/Be (see Materials and Methods)21;for 90%killing of
Daudi cells, the Cl was < l for all the combinations in the abovetable
meaning that the effect was synergistic.
* Each IT was titrated from2.8 x IO-' to 2.8 x
mol/L.
t Anti-CD19-dgAwas added at 2.8 x IO"' mol/L.
Anti-CD22-dgAwas added at 2.8 x IO'" mol/L.
§ Doxorubicin was added at 9.2 x IO-' mol/L.
11 Cytoxan was added at 9.0 x
mol/L.
f Camptothecin was added at 1.4 x IO"' mol/L.
*
neoplastic T cells from autologous marrow grafts before
bone marrow transplantation" and induced a greater antitumor effect than either therapy alone in human solid tumors
(T-ALL) grown in nude mice.23In addition, an anti-CD19pokeweed antiviral protein (PAP) IT in combination with
cyclophosphamide was highly effective against human
t(4; 1I ) leukemia and CALLA' Cp' human pre-B ALL in
SCID mi~e.'~.'~
The antitumor activity of two ITS (SNI-RA
and SNZRA) directed against human T leukemia in nude
mice was potentiated by recombinant a-interferon and daunorubicin."
Doxorubicin has not been used previously in combination
therapy with ITS, but it has been shown that immunoconjugates containing doxorubicin and a tumor-reactive antibody
cured several xenografted human tumors in athymic mice.13
The toxicity of doxorubicin in SCID miceI5 and humans"
has been circumvented by multiple injections at lower doses.
The fact that mice treated with combination therapy (cocktail
of ITS and doxorubicin) were tumor free at 200 days was
supported by the adoptive transfer experiment which gave
no evidence for the presence of residual tumor cells.
In summary, our results support the rationale of using
chemotherapy together with an immunotoxin cocktail to treat
early and/or minimal disease.
ACKNOWLEDGMENT
We thank Cindy Patterson for expert secretarial assistance. We
are indebted to Dr P. Pantazis for providing campothecin and Dr V.
Ghetie for preparing the immunotoxins.
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From www.bloodjournal.org by guest on February 6, 2015. For personal use only.
1994 84: 702-707
Eradication of minimal disease in severe combined immunodeficient
mice with disseminated Daudi lymphoma using chemotherapy and an
immunotoxin cocktail
MA Ghetie, K Tucker, J Richardson, JW Uhr and ES Vitetta
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