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Experimental Therapeutics, Preclinical Pharmacology |
The Immunology Graduate Program [B-R. W., E. S. V.], the Cancer Immunobiology Center [B-R. W., M-A. G., E. S. V.], and the Department of Microbiology [M-A. G. , E. S. V.], The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-8576
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since 1997, two "naked" mAbs have been approved by the Federal Drug Administration for the treatment of lymphoma (chimerized Rituxan) (4 , 5) and breast cancer (humanized Herceptin) (6 , 7) . These mAbs have the advantage of low immunogenicity, few side effects, and significant efficacy. ICs consisting of mAbs coupled to toxins, radionuclides, enzymes, or drugs are often more potent but are more difficult to develop clinically because they have significant side effects at high doses. Therefore, lower doses must be administered and clinical trials are proceeding more cautiously.
Radiolabeled mAbs and ITs are among the ICs currently in clinical trials. The former are usually mAbs labeled with ß emitters, such as 131I and 90Y. The latter are mAbs coupled to a variety of native, modified, or genetically engineered plant or bacterial toxins or RIPs. Both modalities have advanced well into clinical trials (8 , 9) , and thus far, the most impressive clinical results have been observed in the treatment of lymphomas (10, 11, 12, 13) . Lymphomas are excellent targets for ICs, because, in comparison to solid tumors, cells are relatively accessible to systemically administered therapeutic agents (14) . There are also numerous well-characterized, lineage-restricted antigens on lymphoma cells, and the normal lymphocytes killed during therapy are replaced. Furthermore, the disease itself is immunosuppressive (resulting in neutralizing antibody responses in only about 30% of the patients) (15 , 16) .
Clearly, future studies will involve combining various traditional and targeted therapies in an effort to eliminate large tumor masses, metastatic disease, dormant cells, and antigen-negative variants. For this reason, it is important to develop combinatorial regimens that are safe and have significant efficacy. This can be accomplished to some extent by studying human tumor xenografts in immunodeficient mice (17) .
Because each targeted therapy has different advantages as well as dose-limiting toxicities, it is important to consider these in developing combinatorial regimens. For example, RIT is advantageous in treating large tumors because the radiation emitted from a few mAb-coated tumor cells can penetrate several cell diameters and kill surrounding mAb-inaccessible tumor cells [even when they lack the targeted antigen (18) ]. In contrast, ITs must bind to every tumor cell and be appropriately internalized. Although, there is no bystander effect in IT therapy, ITs are highly potent (19) . When ITs are used as a "cocktail," they are effective in eliminating MRD (20) . Because of this, it would be attractive to combine ITs and RIT to treat advanced metastatic tumors. Indeed, these therapies could even follow more traditional chemotherapy regimens or surgery.
The primary goal of this study was to test the effectiveness of RIT plus IT therapy to treat disseminated human B-cell lymphomas in SCID and nude mice. Using highly sensitive methods for detecting MRD, we compared a variety of dose regimens for both efficacy and toxicity. Our studies suggest that although each modality is effective when administered alone, the combination of ITs followed by RIT is most effective and indeed curative in advanced, disseminated disease. In contrast, RIT followed by IT therapy is highly toxic and often fatal. In the latter instance, toxicity appears to be related to the fact that both therapies damage the vasculature but with different kinetics. In mice, RIT induces late PVL, whereas IT therapy induces early PVL. Thus, when RIT is given prior to IT therapy, the PVL induced by the two agents is cumulative, resulting in fatalities. In contrast, if the IT is administered first, PVL induced by the IT resolves before RIT is administered. These studies underscore the importance of scheduling in combining targeted therapy regimens and emphasize that there is increased efficacy when therapies are combined in the appropriate temporal order.
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MATERIALS AND METHODS |
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Mice.
Female outbred ICR SCID mice (Taconic Farms, Germantown,
NY ) and athymic nude mice (Harlan) were housed in
sterile cages with paper filter covers. Sterilized cages, covers,
bedding, food, and drinking water were changed weekly. In the case of
mice treated with RIT, the cages and bedding were changed twice a week.
Mice were 6–7 weeks old at the time of tumor cell inoculation. All
procedures were performed in a laminar flow hood.
Tumor Xenografts and Experimental Models.
Two tumor models, including localized s.c. and disseminated tumors,
were established in SCID and nude mice.
Localized s.c. Tumors.
Nude (but not SCID) mice received 500 rads of whole body 
irradiation from a 137Cs source 24 h before
tumor cell inoculation. Daudi cells, 2 x
107, in 0.2 ml RPMI medium were inoculated s.c.
into normal SCID and preirradiated nude mice. To follow tumor growth,
three diameters of the tumor nodule were measured twice a week using
calipers. Tumor volumes (V) were calculated using the
following equation: V = 4/3
x
r1 x
r2 x
r3, where r is the radius.
Therapy was initiated when tumor volumes reached 300–500
mm3 (approximately 30–35 days after tumor cell
injection). Mice were sacrificed when tumor volumes exceeded 1500
mm3 (
1.2 g).
Disseminated Tumors in SCID Mice.
A model of disseminated tumors was established in SCID mice as
previously described (21)
. Briefly, 5 x
106 Daudi cells in 150 µl RPMI medium were
injected into SCID mice via the lateral tail vein. Tumors
grew systemically and mice became paralyzed when tumor cells
infiltrated the spinal canal, resulting in hind-leg paralysis. For
humane reasons, hind-leg paralysis was used as the end point in these
studies, because 100% of mice died about 7–10 days later.
Disseminated Tumors in Nude Mice.
Because of the low tumor take (16%) in nonirradiated nude mice, all
nude mice received 400–500 rad whole body irradiation 1 day prior
to tumor cell inoculation. Optimal tumor growth was achieved by
injecting i.v. 2 x 107 Daudi cells into the
tail vein. Body weights were followed weekly. Mice were sacrificed when
hind-leg paralysis was observed or when animals lost 30% of their body
weight.
mAbs and IT.
Murine antihuman CD22 (RFB4, a gift from Dr. G. Janossy, Royal Free
Hospital, London, United Kingdom), CD19 (HD37, a gift from Dr.
D. Dorken, Heidelberg, Germany), and anti-CD20 (2H7;
Bristol-Myers, New York, NY) mAbs were used as the targeting mAbs in
this study. All mAbs were of IgG1-
isotype. An isotype-matched
irrelevant mAb, 3F12 (a gift from Dr. E. Hansen at UTSW, Medical
school, Dallas, TX), was used as a control. The RFB4-dgRTA IT was
prepared as described previously using purified RFB4, the SMPT
cross-linker (22)
, and dgRTA (23)
. The
immunoreactivity of the IT was confirmed by fluorescence-activated cell
sorter analysis. The cytotoxicity of each batch of IT was determined
both in in vitro and in vivo. Forty percent of
the LD50 was used as the therapeutic dose as
previously described (24)
.
Radioiodination.
Radioiodination of mAbs with Na131I was performed
using the Iodogen method. Briefly, 100 µg of each mAb was mixed with
2 mCi Na131I (NEN, Boston, MA) in 50 µl PBS, pH
7.0, in a glass tube coated with 20 µg Iodogen (Pierce, Rockville,
IL). The tube was then incubated in ice for 7 min, and the mixture was
chromatographed on a PG-10 column (Pharmacia Biotech, Piscataway, NJ)
to remove unbound Na131I. The
131I-mAb was eluted with 2 ml of PBS. The amounts
of free radioiodine and the specific activities of the
131I-mAbs were determined by precipitation in
10% trichloroacetic acid. In all experiments, less than 5% of the
total radioactivity was trichloroacetic acid-soluble, and the specific
activity of the 131I-mAb was between 5 and 15
µCi/µg. Human serum albumin (Sigma, St. Louis, MO) was added as a
carrier protein to a final concentration of 0.5% to prevent
radiolysis, aggregation, and denaturation. The immunoreactivity of
131I-mAbs was unchanged, based on the fact that:
(1)
the binding of 131I-mAbs to Daudi cells was
completely inhibited by adding cold-specific mAb but not by adding an
irrelevant mAb and (2)
Scatchard analyses confirmed that the
affinity was unchanged.
Therapy Protocols for s.c. Tumors.
When tumor volumes exceeded 300 mm3, mice were
treated with 131I-mAbs; SCID mice received 3
µCi/g and nude mice received 15 µCi/g by i.p. injection.
131I-mAb was mixed with unlabeled mAb and PBS so
that each injection contained 100 µg mAb in 500 µl. Control animals
received equal amounts of unlabeled mAbs (100 µg) or an equal volume
of PBS (500 µl). Tumor volumes were measured twice a week. On the day
of injection and twice a week thereafter, mice were weighed. Blood was
collected weekly. Each blood sample was mixed with a 20-fold volume of
2.5% acetic acid to lyse RBCs and the numbers of WBCs were counted
under the light microscope. Decreases in body weight and peripheral WBC
counts were used to evaluate the systemic toxicity of the RIT. The
observation period was 80 days. A PR was defined as a temporary
decrease in tumor volume during the 80-day observation period. A CR was
defined as a complete disappearance of tumor nodules for 30 days
followed by reappearance during the 80-day observation period. A cure
was defined as failure of the tumor to reappear either at the original
inoculation site or elsewhere for 80 days. Mice from all groups were
selected at random for detection of tumor cells in tissues outside the
original inoculation sites, using human cell-specific dot blots.
Therapy Protocols for Disseminated Tumors.
RIT (131I-HD37) and/or IT therapy (RFB4-dgRTA)
were used to treat disseminated tumors. The reasons that we chose
RFB4-dgA and 131I-HD37 as the representative
reagents for IT therapy and RIT, respectively, were as follows: (1)
To
avoid antigenic modulation, we targeted different antigens with the two
therapies. (2)
RFB4-dgA is 10-fold more potent than HD37-dgA, so we
chose this as our IT. On day 0, 5 x 106 or
2 x 107 Daudi cells were injected i.v. into
SCID and preirradiated nude mice, respectively. Therapy was initiated
with the first treatment 2 weeks later followed by a second treatment
on week 4 (Table 1)
. It required
approximately 2 weeks for WBCs to recover to normal levels in mice
undergoing RIT. Therefore, we used a 2-week interval between the two
therapies. Single treatments at 2 or 4 weeks were performed for
comparison. For RIT, 3 and 10 µCi/g were administered i.p. to SCID
and nude mice, respectively (10 µCi per gram was the maximum
tolerated dose for nude mice with disseminated Daudi tumors). For IT
therapy, 40% of the LD50 (2.8–4.8 µg/g) was
divided into four equal doses and injected i.v. on 4 consecutive days
(i.e., 10% of the LD50/day). Mice
were weighed twice a week to monitor systemic toxicity.
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were sacrificed on
week 8; (2)
mice receiving regimens 1 or 2 were randomly divided into
two groups: one group was sacrificed on week 8 and the other on week
12. Gross examination, the dot blot assay, and the Daudi cell-specific,
nested PCR (in order of increasing of sensitivity) were used to
evaluate tumor burdens in treated mice. Gross examination was first
performed in all nude mice. Dot blot analysis and/or PCR were
subsequently performed on DNA extracts from organs of those mice
showing no evidence of tumors by gross examination. A "cure" was
operationally defined as no detectable tumor cells in evaluated organs
by gross examination, dot blot, and the Daudi cell-specific, nested
PCR.
Preparation of Genomic DNA.
Cells or organs were first incubated in 0.5 ml digestion buffer (100
mM NaCl, 10 mM Tris-HCl, 25 mM
EDTA, 0.5% SDS, and 0.1 mg/ml proteinase K) at 50°C overnight.
Following incubation, RNA was digested by the addition of 50 µl (1
mg/ml) of RNase A. One-tenth volume of 3 M sodium acetate
and 2 volumes of isopropanol were then added, and the genomic DNA was
precipitated by a 20-min centrifugation at 10,000 rpm. The DNA pellet
was dissolved in 200 µl sterile water.
Dot Blots.
Five hundred nanograms of genomic DNA was applied to a nylon
transfer membrane (Amersham Life Science Inc., Arlington Heights, IL),
which was then baked in an 85°C vacuum oven for 2 h to fix the
DNA onto the membrane. Prehybridization was performed by incubating the
membrane with the prehybridization buffer [50% formamide, 5x SSC
solution (0.75 M NaCl and 0.075 M citric acid),
5% Denhardt’s solution, 0.1% SDS, and 0.1 mg/ml denatured salmon
sperm DNA] at 42°C for 2 h. Human DNA was detected by adding a
32P[dCTP]-labeled human Cot-1 DNA (GIBCO BRL,
Gaithersburg, MD) followed by incubation at 42°C for 8 h. The
membrane was washed with 2x SSC and 0.1% SDS at 42°C for 10 min,
twice, and then with 0.1x SSC and 0.1% SDS at 65°C for 30 min.
Autoradiography was used to detect the binding of the probe to the
human DNA.
Daudi Cell-specific, Nested PCR.
A Daudi cell-specific, nested PCR was designed to detect Daudi
cells remaining in the organs of treated mice. Two pairs of primers
were used to amplify a 405-bp DNA fragment from the immunoglobulin
kappa light chain variable region of the Daudi cell. The
oligonucleotide primers (Daudi2: 5'-GCTCTGTGGAAGTGACCTAA-3', L1:
5'-CTGGCTCCGACGTAAGGA-3', L2: 5'-GACGTAAGGAGGGAG-AGAAC-3', and R1:
5'-GTTGACAGTAGTAGGTTG-3') were synthesized at the Ruybern Cardiology
Center facility at UTSW. The sensitivity of the PCR assay was
determined by amplifying the target gene from the genomic DNA from
106 mouse cells and varying numbers of Daudi
cells. To detect tumor cells in mice with disseminated Daudi tumors,
500 ng of genomic DNA extracted from organs of treated mice was used as
the template, and the same amplification procedures were carried out.
PVL.
PVL was evaluated by measuring the amount of fluid accumulated in the
lungs of treated mice (25
, 26)
. In these experiments, the
same agents (131I-HD37 and RFB4-dgRTA), doses,
and regimens used in therapy experiments were administered to
tumor-free SCID and nude mice. Beginning 1 week after the completion of
therapy and for 4 consecutive weeks, animals were sacrificed weekly,
and lungs were excised from one group of 5–10 treated animals. Because
of severe weight loss that occurred in animals receiving RIT prior to
IT therapy (RIT 
IT), SCID mice treated with combination therapies
were sacrificed 1 week after the completion of therapy. The lungs were
weighed before and after lyophilization and the wet:dry weight ratios
were calculated. This value was used as a measurement of PVL.
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RESULTS |
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300 µCi/injection).
The therapy of mice with s.c. tumors was initiated once the tumor
volume exceeded 300 mm3. SCID mice received one
i.p. injection of 131I-RFB4, whereas nude mice
received 131I-RFB4, -HD37, -2H7, or -3F12 (Table 2)
. Mice in the control groups received
either equal amounts of unlabeled mAb or PBS. Tumor volumes were
measured twice a week for 80 days to determine efficacy.
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. In summary, in these experiments,
131I-labeled tumor-reactive mAbs were highly
effective in reducing large s.c. tumor burdens, and cures could be
induced when large doses were used.
Combinations of RIT and IT Therapy in SCID Mice with Advanced,
Disseminated Tumors.
We next tested the combination of RIT and IT therapy in mice with more
clinically relevant disseminated tumors. Growth of disseminated Daudi
cells in SCID mice has been described previously (21)
.
Briefly, 5 x 106 Daudi cells were injected
i.v. into SCID mice and tumor cells grew systemically. Therapy was
initiated 2 weeks after tumor cell inoculation, and 2 weeks later (week
4) the second treatment was administered (Table 1)
.
131I-HD37 and RFB4-dgRTA were used as the
therapeutic agents for RIT and IT therapy, respectively. SCID mice,
which are very radiosensitive, were used in this study with the intent
of developing effective and safe therapy regimens with low doses of
RIT. Because we found that 131I-3F12 had only a
modest and transient effect on tumor growth,
131I-3F12 was not included in subsequent
experiments.
Individual treatments (131I-HD37 or RFB4-dgRTA)
administered after either 2 or 4 weeks significantly delayed paralysis
(Fig. 1)
. When IT was administered prior
to RIT, the mean survival time was further extended (Fig. 1)
.
Unexpectedly, Regimen 1 (RIT IT) was fatal in 100% of the mice,
with a mean survival time of 44.6 days. Thus, there were
schedule-related toxicities when RIT and IT were combined, although
both treatments were safe when used individually.
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When therapy was performed in nude mice with disseminated tumors,
animals were sacrificed when any one of the following occurred: (1)
hind-leg paralysis, (2)
30% weight loss, or (3)
when mice remained
healthy for 8 or 12 weeks. Gross examinations were first performed on
all organs. For those mice without visible tumor nodules, DNA was
extracted from various organs, and dot blot assays were performed. When
dot blot assays were negative, the Daudi cell-specific, nested PCR was
subsequently performed. A cure was operationally defined as failure to
detect tumor cells by gross examination, dot blot assays, and the Daudi
cell-specific, nested PCR.
Combination RIT and IT Therapy in Nude Mice with Advanced,
Disseminated Tumors.
Nude mice were treated as described in Table 1
. Fifty percent of the 26
PBS-treated mice became paralyzed, and tumors were observed in all of
the mice (Table 3
, Regimen 7). Single
treatments reduced paralysis to 30% (Table 3
, Regimen 3) or 0% (Table 3
, Regimens 4–6) of the mice. However, tumors were detected in all
animals receiving single treatments by either gross examination or dot
blot assays. Hence, single treatments were effective, but not curative.
The antitumor effects of RIT and IT therapy, administered at either
week 2 or week 4, were comparable in the treatment of advanced,
disseminated disease.
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RIT), paralysis did not
occur (Table 3)
. Thus, on week 8, the 20 mice were divided into two
groups and sacrificed either at that time (week 8) or 4 weeks later
(week 12). At week 8, gross examination revealed no tumors in 10 of 10
mice. According to the protocol described above, dot blots and nested
PCRs were then performed to determine whether residual tumor cells were
present. Tumor cells were detected in 6 of 10 mice (Table 3)
using the
dot blot assay (data not shown). Of the remaining 4 mice, tumors were
detected in 3 of 4 mice using the nested PCR (Table 3)
. On week 12, the
remaining 10 mice were sacrificed. By gross examination, there were no
visible tumors in the 8 mice in which dot blots or PCRs were performed.
Four cures were documented by nested PCR (data not shown).
Regimen 1 (RIT IT) was highly toxic; 9 of 19 mice died before week
8 (Table 3)
. The remaining 10 mice were divided into two groups with 5
mice per group. On week 8, no tumors were observed by gross
examination, but residual tumor cells were detected in 4 of 5 mice. On
week 12, tumor cells were detected in all 5 mice by either gross
examination or dot blots (data not shown).
In summary, individual treatments were effective but not curative.
Regimen 1 (RIT IT) was highly toxic and often fatal, whereas
Regimen 2 (IT RIT) was curative, as determined at week 12. On week
8, the tumor burdens were comparable in surviving mice receiving either
Regimen 1 or 2. However, on week 12, tumor relapses occurred in mice
treated with Regimen 1 (RIT IT) while cures were achieved in 40%
of the mice receiving Regimen 2 (IT RIT).
Toxicity of RIT and/or IT Therapy.
To evaluate the toxicity of RIT in mice with s.c. tumors, decreases in
body weight and peripheral WBCs were determined. In SCID mice, both
weights and WBCs reached their nadirs 2–3 weeks after treatment; mice
then recovered (data not shown). Weight losses were not observed in
nude mice. WBCs declined and recovered in a manner similar to those
observed in SCID mice (data not shown). The toxicity of RIT appeared to
be transient and reversible.
Weights were followed during the therapy of disseminated tumors. The
dose of RIT used in this study was well tolerated; no severe weight
losses (20% of body weight) were observed (data not shown). Minor
weight losses (5–20% of body weight) were observed in both SCID and
nude mice after the administration of the IT (data not shown), but
these were transient and the mice recovered after 1 week. Approximately
5% of the SCID mice showed a >20% loss in weight prior to the onset
of hind-leg paralysis, because of the large tumor burdens. Overall, the
doses of RIT and IT therapy used in this study were well tolerated, and
life-threatening toxicities were not observed.
When RIT and IT therapy were combined, mice treated with Regimen 2 (IT
RIT) maintained their weights (Fig. 2, C and D)
.
However, mice treated with Regimen 1 (RIT IT) lost weight
immediately after IT treatment (Fig. 2, A and B)
,
and the weight losses were followed by deaths in all of the SCID mice
and 47% of the nude mice. Overall, Regimen 1 (RIT IT) caused
severe and, for some mice, irreversible toxicities.
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. At
weekly intervals, mice were sacrificed and lungs were excised. The
wet:dry lung weight ratios were calculated to determine the amount of
fluid accumulated in the lungs. In IT-treated mice, fluid accumulation
in the lungs increased 1 week after treatment and declined to baseline
2 weeks later, whereas in mice treated with RIT, the ratios did not
increase until 3 weeks after treatment (Fig. 3)
. Using Regimen 2 (IT RIT), PVL was
moderately increased 1 week after therapy. However, during the same
time interval, there was severe PVL in the mice treated with Regimen 1
(RIT IT).
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IT), the late PVL induced by
the RIT was exacerbated by the early PVL induced by the IT. In
contrast, when Regimen 2 (IT RIT) was used, IT-mediated PVL
occurred early and resolved before RIT-induced PVL was initiated.
Roles of RIT and IT in the Toxicity of Regimen 1 (RIT IT).
We next explored the individual roles of RIT and IT in the toxicity
induced by Regimen 1. We administered 50% of the dose of either RIT or
IT. All mice survived and weight losses became less severe when the
dose of RIT was reduced by 50%. All nude mice and 2 of 5 SCID mice
survived (Fig. 4, A and B)
, that is, the survival rates were increased. In contrast,
reducing the dose of the IT did not improve survival or prevent weight
loss (Fig. 4, C and D)
. In addition, RIT mediated
the toxicity of Regimen 1 in a dose-dependent manner (data not shown),
that is, the weight losses, which occurred after the administration of
IT, decreased as the RIT doses were reduced. Thus, RIT played the
dominant role in the toxicity of Regimen 1 (RIT IT).
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. With Regimen 1 (RIT IT), 8 weeks
after tumor cell inoculation, tumors were detected by dot blot, and, by
week 12, tumor nodules were detected in all mice (60% detectable by
dot blotting, and the remaining 40% detectable by PCR). There were no
cures. With Regimen 2 (IT RIT), 8 weeks after tumor cell
inoculation, tumor cells were detected in 60% of the mice by dot blots
and in 40% of the mice by nested PCR. At week 12, one cure (20%) was
achieved, and the remaining 80% of the mice had tumors that were
detectable by dot blots or nested PCRs, but not by gross examination.
Using Regimen 2 (IT RIT), the outcome of therapy was comparable
when either 100% or 50% of RIT was administered. Overall, as
determined by the tumor burdens on week 12, Regimen 2 (IT RIT) had
better antitumor activity than Regimen 1 (RIT IT).
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DISCUSSION |
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IT) and was dose-dependent. (7)
The efficacy of
Regimen 2 (IT RIT) was higher than that of Regimen 1 (RIT IT).
Thus, in contrast to our initial prediction that RIT followed by IT
therapy would be the better regimen, the reverse was true. Whether or
not this will be the case in humans remains to be determined. In humans, RIT has been more successful in treating bulky lymphomas than IT therapy. Using the 131I-B1 anti-CD20 mAb, myeloablative doses of RIT have been evaluated in treating advanced, relapsed B-cell lymphoma (10) . Very high overall and progression-free survival rates have been reported, but autologous bone marrow or peripheral blood stem cell transplantation is required. Nonmyeloablative doses of the 131I-B1 also induced excellent responses, but fewer durable remissions or cures have been achieved (11) . In contrast to the prevalent strategy of using ITs to treat MRD in mice, RIT has not been used in this setting. Thus, the efficacy of RIT on MRD in humans is unclear.
Therapy with dg-RTA-containing ITs has been more difficult to develop in humans because of VLS and hence the need to give smaller doses that cannot eliminate large tumor burdens. Nevertheless, Phase I trials with ITs have shown antitumor activity in patients with lymphoma (31 , 32) . Because of their dose-limiting toxicity, ITs should perform best in MRD. Indeed, in mice, IT therapy has been more successful in treating MRD than in treating large s.c. tumors (20 , 33) .
Although RIT and IT therapy have not been combined previously, Buchsbaum et al. (34) used a radiolabeled IT to reduce s.c. tumors. From their in vitro study (34 , 35) , the IT itself appeared to be responsible for the majority of the antitumor activity, because radiolabeling the IT did not increase the potency of the IT. However, no additional toxicity was reported after administrating the radiolabeled IT.
RIT and IT therapy have both been used in combination with other
agents. ITs have been combined with chemotherapeutic agents and
additive or synergistic effects were observed (20
, 36)
.
RIT has been combined with cytotoxic agents that also function as
radiosensitizers [e.g., 5-fluorouracil (37)
and SR4233 (38)
], agents that can alter the permeability
of vasculature [e.g., tumor necrosis factor
(39)
or IL-2 (40)
], or hyperthermia
(41)
to increase efficacy. Experiments have also been
carried out using agents that elevate the expression of target antigens
[e.g., IFN stimulates carcinoembryonic antigen
expression on colon carcinoma cells (42
, 43)
].
The importance of temporal order in administering different agents has been demonstrated previously. For example, DeNardo et al. (44 , 45) combined 90Y-chL6 (a chimeric antibody reacting with an integral membrane glycoprotein that is expressed at high levels on human breast, colon, ovary, and lung carcinomas) with either taxol or cold ch225 (an anti-EGFR mAb) to treat breast carcinoma xenografts. Synergistic effects were observed when taxol was administered after the 90Y-mAb or when ch225 was given before 90Y-mAb. However, high mortality rates were observed when ch225 was combined with 90Y-chL6 in either temporal order. Ghetie et al. (46) have also combined ITs with various chemotherapeutic agents. Significant therapeutic benefits were achieved when the IT therapy was administered before or during chemotherapy but not after chemotherapy.
In this study, we found that, when RIT and IT therapy were combined, therapeutic efficacy was increased when ITs were administered prior to RIT. In contrast to the report by DeNardo et al. (44) , in which greater toxicity occurred in either temporal order, in our study, toxicity only occurred when RIT was administered prior to IT therapy. This suggested that either RIT predisposed mice to the toxicity of the IT or that the kinetics of toxicities caused by RIT versus IT therapy were different and became cumulative only when RIT was administered first.
Changes in vascular permeability (VP) following RIT have been studied in mice with s.c. tumors (47) . Radiolabeled tracers were injected into mice after the completion of RIT. The amounts of the tracer accumulating in the s.c. tumors and normal organs (liver and lung) were measured to determine changes in VP. No changes in the VP of normal organs were observed. However, RIT-induced VP changes in tumor sites varied and were idiosyncratic for the tumor, that is, VP increased in some tumors and decreased in others after RIT. In this study, instead of measuring tracer accumulation in organs of tumor-bearing mice, we measured the accumulation of pulmonary fluid, that is, PVL, in normal mice after RIT, IT therapy or both. PVL was used to measure damage to the vasculature. The kinetics of PVL were studied to further explore the differences between Regimens 1 and 2. In normal mice, PVL was induced by both RIT and IT, but with different kinetics. Hence, RIT induced late and long-lasting vascular toxicity that was exacerbated when ITs were administered. In contrast, ITs induced early PVL, which rapidly resolved so that when RIT was administered, there was no exacerbation of toxicity.
Toxicity could be avoided in some animals treated with RIT followed by IT therapy by reducing the dose of RIT by 50–75%; it could not be improved by delaying the time interval between the two therapies (data not shown) or by giving less IT. This implies that the RIT-induced damage predisposes mice to the toxicity of ITs. Thus, in this animal model, the most effective regimen is IT therapy followed by RIT.
It has been suggested that ITs can sensitize tumor cells to
chemotherapeutic agents (36
, 46
, 48)
. In our
experiments, it is possible that in Regimen 2 (IT RIT), that the IT
did indeed sensitize tumor cells to RIT. It is also possible that the
increase in vascular permeability facilitated the penetration of
radiolabeled mAbs and that the efficacy of Regimen 2 reflects the
cumulative effect of two events. Although a small amount of vascular
leak is probably advantageous for the extravazation of mAbs and ICs
(49)
, severe VLS can be fatal in patients. Clearly, our
results suggest that for the two therapies to be combined safely and
effectively, IT therapy should be given prior to RIT. Whether this will
be the case in treating advanced disseminated disease in humans remains
to be determined in clinical trials.
We have also demonstrated that both cold mAbs and an irrelevant 131I-mAb were much less effective in treating large s.c. tumors. Thus, in the case of the mAbs tested in this study, both specificity and radiolabeling were optimal for their antitumor activity. However, it has been reported that some mAbs (lacking radionuclides) have good antitumor activity. Moreover, in one instance, an unlabeled anti-CD20 mAb (B1) had better efficacy than the 131I-labeled mAb in an animal model (28) , although the reasons for this were not investigated. In such case, it is possible, for example, that radiolabeling some mAbs damages portions of the mAb that are responsible for binding, signaling, or effector functions. Whatever the explanation, taken together with the results of others, our results underscore the idiosyncratic behavior of different mAbs, even against the same molecule.
In conclusion, the results of our studies demonstrate the excellent efficacy of both RIT and IT therapy in local and disseminated lymphoma in mice. They also underscore the increased efficacy achieved by combining the two therapies to treat disseminated disease. Finally, in this experimental model, the necessity of using ITs before RIT (unless the dose of the latter is reduced) can be attributed at least in part to the different kinetics of vascular toxicity induced by the two agents.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by NIH Grant CA28149 and a grant from
Meadows Foundation. 
2 To whom requests for reprints should be
addressed, at The Cancer Immunobiology Center, University of Texas
Southwestern Medical Center at Dallas, 6000 Harry Hines Boulevard,
Dallas, TX 75235-8576. Fax: (214) 648-1204; E-mail: evitet{at}mednet.swmed.edu
3 The abbreviations used are: mAb, monoclonal
antibody; IC, immunoconjugate; IT, immunotoxin; RIT,
radioimmunotherapy; RIP, ribosome inactivating protein; MRD, minimal
residual disease; SCID, severe combined immunodeficiency disease; PVL,
pulmonary vascular leak; dgRTA, deglycosylated ricin A chain; SMPT,
N-succinimidyl-oxycarbonyl-
-mehty--(2-pyridyldithio)
toluene; PR, partial response; CR, complete response; VP, vascular
permeability.
Received 8/16/99; revised 11/11/99; accepted 11/12/99.
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