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Blood, Vol. 94 No. 1 (July 1), 1999:
pp. 233-243
The Importance of Antibody-Specificity in Determining Successful
Radioimmunotherapy of B-Cell Lymphoma
By
Timothy M. Illidge,
Mark S. Cragg,
Harry M. McBride,
Ruth R. French, and
Martin J. Glennie
From the Tenovus Research Laboratory, Southampton University
Hospitals, Southampton, UK.
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ABSTRACT |
We report the radioimmunotherapy of mouse B-cell lymphoma,
BCL1, using a panel of anti-B-cell monoclonal antibodies
(MoAb) (anti-CD19, anti-CD22, anti-major histocompatibility complex
(MHC) II, and anti-idiotype (Id) radiolabeled with 131-iodine. When administered early in disease (day 4), the 131I-anti-MHCII
MoAb cured tumors as a result of targeted irradiation alone, the
unlabeled MoAb being nontherapeutic. In contrast,
131I-anti-Id, despite targeting irradiation and having
therapeutic activity as an unconjugated antibody, protected mice for
only 30 days; 131I-anti-CD19 and anti-CD22 were
therapeutically inactive. Binding and biodistribution studies showed
that the anti-Id, unlike anti-MHCII, MoAb was cleared from target cells
in vivo and delivered 4 times less irradiation to splenic tumor.
Treating later in the disease (day 14) increased tumor load and
produced the expected reduction in therapeutic activity with the
anti-MHCII, but surprisingly, allowed 131I-anti-Id to cure
most mice. This unexpected potency of 131I-anti-Id late in
the disease appeared to result from the direct cytotoxicity of the
anti-Id MoAb, which was more active in established disease, in
combination with targeted irradiation. We believe the ability of
targeted irradiation and certain cytotoxic MoAb to work cooperatively
against tumor in this way has important implications for the selection
of reagents in radioimmunotherapy of B-cell lymphoma.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
RADIOIMMUNOTHERAPY (RIT) is an expanding
field and over the last few years some of the most compelling clinical
data in the treatment of advanced non-Hodgkin's lymphoma (NHL) have derived from this approach. Impressive durable partial and complete responses have been reported using a variety of antibodies, delivery schedules, radioisotopes, and doses of radioactivity.1-3
Press et al4,5 have adopted a myeloablative strategy with
peripheral blood stem cell transplantation, whereas other groups,
including Kaminski et al, using lower doses of nonmyeloblative
fractionated treatments, have reported similarly impressive response
rates.6-10 One feature, which appears common to all of
these studies, is the exquisite sensitivity of low-grade NHL to RIT.
However, the mechanisms behind these encouraging clinical responses
remain largely undefined. This has meant that the optimal treatment
approach for achieving remissions, including the required dose of
radioactivity and the importance of the monoclonal antibody (MoAb)
specificity, remains unknown.
To date, target antigens which have been used for RIT clinical trials
include the major histocompatibility complex (MHC) class II allele
HLA-DR10,11-13 Ig idiotype (Id),14
CD20,2-10 CD22,15,16 and
CD37.3,5,17,18 The main criteria in the selection of MoAb
for RIT has been that the target antigen is well expressed on the
tumor, but not on critical, nonrenewable, normal tissue such as the
nervous system. Recently, much of the clinical work has focused on the
B-cell specific antigen, CD20. Indeed, anti-CD20 MoAb appear extremely
promising for targeting B-cell lymphomas. The CD20 molecule is a plasma
membrane protein that is well expressed on most B-cell tumors, it is
not expressed on hematopoietic stem cells, does not appear to undergo
antibody-induced endocytosis,19 and is not shed from the
plasma membrane before or after treatment.20-22 In vitro
studies have shown the advantages of targeting CD20 in RIT over a range
of other potential B-cell targets.20-22 However, one of the
difficulties of evaluating clinical RIT data with anti-CD20 MoAb is
that these MoAb are therapeutic in their own right,23,24 either through recruitment of natural effectors, such as complement, or
direct cytotoxic signaling through the CD20 antigen.25
Maloney et al,23,24 have recently reported that 50% of
patients achieve partial or complete remission following treatment with
a human/mouse chimeric anti-CD20 MoAb. Therefore, in any RIT study it
is difficult to establish which part of the clinical success is
dependent on antibody effectors, antigen specificity, targeted
radiation, or whole-body irradiation.
Answering such questions in a clinical setting is difficult and ideally
requires good preclinical animal testing. Unfortunately most animal
work to date has been restricted to human lymphoma xenografts, which
suffer from the lack of cross-reactivity of the treatment MoAb with
normal mouse tissue and the unusual distribution of human tumors in
immunodeficient animals.26 Ideally, syngeneic animal models
are needed in which the tumor develops in the presence of an intact
immune system and with the targeting MoAb able to cross-react with
appropriate normal tissue. RIT and biodistribution work in syngeneic
lymphoma models has mainly been limited to observations of
radioiodinated anti-Id MoAb in the Rauscher murine T-cell
erythroleukemia27-29 and the B-cell lymphoma
38C13.30,31 Such systems suffer a number of limitations.
In this present study we report the biodistribution, in vivo
internalization, and RIT of a range of B-cell specific MoAb (anti-CD19, anti-CD22, anti-MHCII, and anti-Id) in the BCL1 B-cell
lymphoma of BALB/c mice. For three of these reagents, namely anti-CD22, anti-MHCII, and anti-Id, there are similar clinical
131I-labeled MoAb either currently undergoing clinical
evaluation,13,15 or that have formed the basis of recent
trials.14 Currently, no anti-mouse CD20 MoAb has been
developed for such work. Our results show the relative contribution of
the MoAb and the targeted irradiation to the therapeutic effects in
vivo. For the first time in animals, we show at least an additive
therapeutic effect between the activity of anti-Id MoAb and that of
targeted irradiation, which allows the eradication of advanced tumor
without toxic side effects. These results have important implications
for clinical work. They suggest that the success of recent RIT in NHL
may not derive solely from the targeting activity of radio-conjugated MoAb and that dose of radioactivity delivered to tumor does not correlate with tumor response. Instead the eradication of tumor appears
to stem from a combination of the effect of radio-targeting activity
and that of the endogenous cytotoxic activity of the carrier MoAb.
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MATERIALS AND METHODS |
Animals and cell lines.
Ten- to 12-week-old female BALB/c mice (mean weight, 27 g; range, 25 to
30 g) were supplied by Harlan UK Limited (Blackthorn, Oxon, UK), and
maintained in local animal facilities.
BCL1 is a B-cell leukemia that arose spontaneously in a
2-year-old BALB/c mouse and is transplanted in syngeneic recipients by
injection of spleen lymphocytes from leukemic animals.32,33 Unlike most human lymphomas, which are disseminated, BCL1
and most other animal lymphomas develop predominantly in the spleen and
then the liver with a leukemic spill in the terminal stages of the
disease. Experimental results show that the BCL1 is a
highly malignant tumor and as few as 10 cells can transfer the
BCL1 tumor to unirradiated BALB/c mice.34 The
BCL1-3B3 cell line is a variant of the BCL1
tumor,35 which is phenotypically similar but can be
maintained in culture.  BCL1 is a recently derived
variant of BCL1 that arose in this laboratory from the
wild-type BCL1 and is capable of growth both in culture and
also in BALB/c mice (T. Illidge, unpublished results, September
1996). Cell culture of BCL1-3B3 was performed
in supplemented RPMI (RPMI containing 2-mercaptoethanol [50
µmol/L], glutamine [2 mmol/L], pyruvate [1 mmol/L], penicillin
and streptomycin [100 IU/mL], fungizone [2 mg/mL], and 10% fetal
calf serum [FCS] [Myoclone]) (GIBCO-BRL, Paisley, Scotland).
BCL1 was maintained in supplemented RPMI and 20% FCS.
Antibody production and purification.
A list of the MoAb used in this report and their source is given in
Table 1.36-39 The MoAb TI2-3 is an anti-MHC
class II reagent that was produced in house. Its specificity was
confirmed by sequential immunoprecipitation using a well recognized
anti-MHC class II MoAb, N22 American Type Culture Collection (ATCC).
MoAb-secreting hybridoma cells were expanded in culture using
supplemented Dulbecco's Modified Eagle's Medium (containing the same
supplements as supplemented RPMI [above], but without
2-mercaptoethanol). To purify the IgG MoAb, the culture supernatants
were concentrated 20 times by membrane filtration (Millipore, Bedford,
MA), precipitated with saturated ammonium sulphate, and
then dialyzed and fractionated on protein G (1D3, NIMR6, and TI2-3) or
protein A (N22) (Amersham Pharmacia Biotech UK Ltd, Little Chalfont,
Bucks, UK) according to the manufacturer's instructions. Two of the
rat MoAb (Mc39-16 and Mc10-6A5) were prepared by ion-exchange
chromatography on DEAE (Whatman, Maidstone, UK) as described by
Elliott et al.40 The purity of all IgG preparations was checked by electrophoresis (Beckman EP system; Beckman
Instruments, Irvine, CA).
Iodination, labeling efficiency, and specific activity of MoAb.
Iodine-125 and iodine-131 were supplied as sodium iodide in dilute
sodium hydroxide solution at pH 10 and free from reducing agent
(Amersham Pharmacia Biotech UK Ltd). Purified antibodies were iodinated (in phosphate-buffered saline) using Iodobeads (Pierce
Chemicals Co, Rockford, IL) according to the manufacturers' instructions.
The labeled protein was separated from unbound iodine by gel filtration
on a Sephadex G-50 column (Amersham Pharmacia Biotech UK Ltd). Between
80% and 90% of the counts of labeled antibody were precipitable with
trichloroacetic acid. Labeling efficiency was determined as the amount
of radioactive iodine incorporated into the recovered product as
compared with the amount added to the reaction mixture. This varied
between 43.5% to 56.5% for both the Iodine-125 and the Iodine-131.
The specific activity, which was expressed as the amount of
radionuclide attached per mg of MoAb in the final product, varied from
8.1 to 15.0 MBq/mg (0.22 to 0.40 mCi/mg), but was routinely 10 to 11.33 MBq/mg for all of the Iodine-131 therapies.
Flow cytometry.
Splenic BCL1 cells were prepared and analyzed by direct
immunofluorescence36 using each of the MoAb (fluorescein
isothiocyanate-IgG) under investigation (Table 1).
Binding of iodinated MoAb to the surface of BCL1.
The binding of radiolabeled MoAb to cells was determined as described
previously.36 Briefly, 125I-MoAb were serially
diluted and incubated with fresh BCL1 cells for 2 hours at
37°C in the presence of NaN3 and 2-deoxyglucose to
prevent endocytosis. The cell-bound and free 125I-MoAb were
then separated by centrifugation through a mixture of dibutyl
phthalate:dioctyl phthalate oils (1.1:1, vol:vol). This allowed rapid
separation of bound and free antibody without disturbing the binding
equilibrium. The cell pellets with bound radiolabeled MoAb were counted
on a gamma counter (Wallac UK Ltd, Milton Keynes, UK).
Clearance of surface bound MoAb in vivo.
To determine the level of MoAb remaining bound to the surface of the
tumor cells after treatment, surface MoAb was assessed by two-color
flow cytometry using a FACS Vantage (Becton Dickinson, Mountain View,
CA). Mice were treated with 0.5 mg of the appropriate MoAb or a
control, nonbinding isotype-match, MoAb by tail-vein injection, 4 days
after they had received 5 × 107 BCL1 cells by
intravenous (IV) injection. Sixteen hours later mice were killed, their
spleens homogenized to give a single cell suspension, and the
homogenate washed twice with PBS. Surface-bound MoAb remaining after
treatment was detected using the appropriate concentration of
FITC-mouse anti-rat IgG (Jackson ImmunoResearch Laboratories Inc, West
Grove, PA). To allow gating on only the BCL1 cells and
exclude normal spleen cells, phycoerythrin (PE)-labeled rat
anti-BCL1 Id (PE-Mc10-6A5; Serotec, Oxford, UK) MoAb (25 µg/mL) was added after the cells had been labeled for rat IgG. The
results are displayed in histograms which show FITC-anti-rat IgG
binding to Id positive cells. In the case of BCL1 taken
from mice treated with anti-Id MoAb, surface Id was significantly
blocked or internalized by the in vivo treatment, but we were still
able to achieve sufficient PE-anti-Id MoAb-staining to allow gating on
the tumor cells.
Measurement of apoptosis in vitro.
Radiation and MoAb-induced apoptosis was analyzed by the method of
Nicoletti et al.41 Briefly, treated samples (approximately 5 × 105 cells) were washed in PBS, resuspended in
hypotonic fluorochrome solution (50 µg/mL propidium iodide (PI),
0.1% (wt/vol) sodium citrate, 0.1% (vol/vol) Triton X-100) and then
stored overnight in the dark at 4°C to allow staining of DNA.
Analysis was performed by flow cytometry on a FACScan (Becton
Dickinson). PI-stained nuclei fluoresce in the red wavelength, which
was analysed using a 488-nm argon laser for excitation and a 560-nm
dichroic mirror and 600-nm band pass filter (bandwidth 35 nm) for
detection. For comparison of results, the G1 peak in each sample was
adjusted to around channel 250 on a logarithmic scale.
In vitro irradiation of cells was achieved using a Caesium source (AECL
Gammacell 1,000; Toronto, Canada) at a dose rate of 2.18 Gy/min. Culture plates (24-well multi-plates; Life Technology, Paisley,
UK) were coated with MoAb (5 µg/mL) in PBS for 2 hours at 37°C
before addition of cells (2.5 to 5 × 105/mL). In
combination experiments, cells were added immediately postirradiation.
Biodistribution studies.
Groups of BALB/c mice were injected through the tail vein with
106 fresh BCL1 cells. They were given Lugol's
solution (5.0 mL Lugol's stock/400 mL H2O; Lugol's stock:
10 g KI, 5 g elemental iodine in 100 mL H2O) in their
drinking water 3 days before initiation of the biodistribution or RIT.
On day 14 post tumor inoculation animals received 500 µg of
trace-labeled 125I-MoAb by tail-vein injection.
Biodistribution of each radioactive MoAb (anti-Id, anti-CD22, and
anti-MHCII) was compared with that of an irrelevant antibody, which
failed to bind to the tumor cells. Animals were killed 1, 24, 48, 96, and 120 hours after receiving the radioactive MoAb. The weight and
radioactive counts of the dissected organs (spleen, liver, kidneys,
heart, lungs, and thymus) were measured, and the percentage of the
injected dose/g of tissue (% ID/g) was calculated as described by
Badger et al.27 In addition, blood samples were obtained by
cardiac puncture immediately postmortem. Marrow samples were obtained
by removing both ends of one femur and passing a 21-guage needle
attached to a 2-mL syringe through the marrow cavity. The marrow sample
was expressed into a preweighed test tube containing 1 mL 10%
formalin. Data were not adjusted for minor differences in individual
mouse weights. The calculation of the area under the curve (AUC), used
as a measure of the total delivered dose, was performed using Fig. P
Scientific processor (Software Corporation, Durham, NC).
RIT with 131I-labeled MoAb.
For RIT experiments, groups of mice were inoculated with freshly
prepared BCL1 tumor cells. The number of tumor cells given, by tail vein injection, varied according to individual experiment but
was usually 106/mouse. Radiolabeled MoAb was given by IV
injection on day 4 or 14 after tumor inoculation. For the initial
therapies, approximately 500 µg (490 to 520 µg) with 5 MBq of
131I were given by tail-vein injection. In subsequent
therapies, approximately 750 µg (730 to 800 µg) of MoAb with
between 7.5 and 8.5 MBq of 131I was injected per mouse,
although the amount of antibody injected was adjusted to keep the dose
of radioactivity the same within an experiment. In each individual
therapy the quantity of antibody injected varied by less than 10%, to
achieve a constant radioactive dose. At this level of irradiation no
animals were lost because of irradiation toxicity, however, a
consistent toxicity was patchy hair loss over the backs of animals in
20% to 30% of cases, which occurred from around 3 weeks after
treatment and did not always recover in the long-term survivors. To
assess the relative contributions of the irradiation and the MoAb to
the therapeutic activity of iodinated MoAb, parallel groups of mice
were always treated with "naked" IgG.
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RESULTS |
Binding activity of MoAb with BCL1 mouse lymphoma cells.
A panel of anti-mouse B-cell specific MoAb has been characterized for
this study (Table 1). Although these reagents, other than the anti-Id
MoAb, are not tumor-specific, they have all been considered at one time
or another for treatment of human B-cell malignancy either as
"naked" MoAb or conjugated to toxic substances.36 Figure 1 shows the levels of binding
achieved with each MoAb against the mouse B-cell lymphoma
BCL1 using flow cytometric analysis. They can be divided
into two groups: anti-Id and anti-MHCII MoAb, which bind at
comparatively high levels; and anti-CD19 and anti-CD22 MoAb, which bind
at a much lower level. Detailed analysis of binding activity was also
performed using radioiodinated MoAb. Binding curves (not shown) were
established for 125I-MoAb and, once at equilibrium, free-
and cell-bound radioactivity separated rapidly by spinning through
phthalate oil. The average number of MoAb molecules bound to each
BCL1 cell and their affinities were estimated (Table 1).
These results confirm the flow cytometry and show the apparent Ka
values in the range of 108 to 109
mmol/L 1.
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| Fig 1.
Flow cytometric analysis of MoAb binding to
BCL1 cells using direct immunofluorescence staining. Fresh
BCL1 cells were stained with FITC-labeled MoAb
(specificities shown) at 10 µg/mL. The specificity of the MoAb used
(see Table 1) is as indicated.
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RIT with 131I-labeled and unlabeled MoAb.
We next tested the same panel of reagents in RIT against
BCL1 lymphoma (Fig 2).
Age-matched groups of mice received 106 BCL1
cells IV followed 4 days later by various unlabeled and radioiodinated
MoAb preparations. Our most striking observation was the therapeutic
efficacy of TI2-3, an anti-class II MoAb (Fig 2A). While this MoAb was
completely nontherapeutic when unlabeled, used as a
131I-conjugate it "cured" 85% of animals (34 out of
40). These results where achieved with around 500 µg of radiolabeled
antibody, carrying approximately 5 MBq per mouse (Fig 2A). At this
level no animals were lost due to irradiation. RIT experiments were
generally terminated after 100 days and at this time, none of the
surviving animals showed any signs of tumor, even when spleen cells
were examined by flow cytometry with anti-Id MoAb. Interestingly, 16 of
18 animals from the initial 131I-anti-MHCII MoAb (TI2-3)
experiment survived for more than 350 days and never developed
BCL1. The lack of efficacy of the "naked" anti-MHCII
MoAb (TI2-3) and the marginal therapeutic effects of nonspecific
irradiation delivered by control antibody strongly suggest that the
success of 131I-anti-MHCII MoAb depends on its ability to
"target" irradiation to the tumor-bearing organs.
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| Fig 2.
RIT of BCL1 using radioiodinated and
unlabeled anti-B cell MoAb. (A) Composite graph showing results from
three individual RIT (minimum of 40 animals per group) performed under
the same experimental conditions. Mice were given 106 fresh
BCL1 cells IV on day 0 and then treated with MoAb (500 µg
with or without approximately 5 MBq 131I) 4 days later. (B)
Groups of 10 BCL1-bearing mice were treated as in Fig 2A,
but with 750 µg of MoAb (with or without approximately 7.5 MBq of
131I) on day 4. Treatments included: Control IgG ( );
131I-control IgG ( ); anti-MHCII (TI2-3;  ,  );
anti-Id ( ,  ); anti-CD19 ( ;
); anti-CD22 ( ,  ); and
anti-MHCII MoAb (N22;  ,  ). The solid and open symbols represent
MoAb labeled with and without radioactive iodine, respectively. Results
with the anti-MHCII MoAb (, ) are shown in both (A) and (B).
Animal survival was monitored daily. Control 131I-IgG1 MoAb
had a very small, but statistically significant therapeutic effect
(P < .01).
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Treatment with anti-Id MoAb produced markedly different therapeutic
results. The survival curves in Fig 2A show that unconjugated anti-Id
MoAb extended survival over controls by approximately 10 days,
confirming earlier work from this laboratory.36 Iodination of the MoAb increased survival to around 20 days but none of the animals treated (42 mice) achieved long-term survival. Thus, unlike the
anti-MHCII MoAb, the therapeutic activity of 131I-anti-Id
MoAb may have two components, one resulting directly from the MoAb, and
a second from its targeted irradiation. It is important to note that
tumors emerging after anti-Id treatment were phenotypically unchanged
and were still bound by treatment MoAb. Thus, it would appear that
despite binding at a similar level to the BCL1 tumor, and
being more active as an unconjugated reagent, the anti-Id MoAb is less
therapeutic in RIT than the anti-MHCII MoAb.
We next extended this work to include the remainder of our panel of
MoAb. Figure 2B shows that neither anti-CD19 nor anti-CD22 MoAb were
appreciably active in therapy, regardless of whether or not they were
radiolabeled. Previous work from this laboratory36 has
shown that anti-CD19 MoAb can be therapeutic in BCL1 but
only when given in high amounts and over an extended period. A second anti-MHCII MoAb, N22, behaved exactly like TI2-3 and showed that, although inactive as an unconjugated reagent, it was highly effective once radioiodinated.
Thus from these RIT experiments both the anti-MHCII MoAb (N22 and
TI2-3), which bound at high levels (Fig 1), were active against
BCL1 tumor. The reason for the poorer efficacy of anti-Id MoAb was not clear and did not seem to result from any marked difference in its binding characteristics (Table 1). We decided, therefore, to look for possible explanations in vivo.
Biodistribution of 125I-MoAb in tumor-bearing animals.
Initial dosimetry work with BCL1 showed that 14 days
postinoculation (106 cells), spleens were macroscopically
enlarged with disrupted architecture when examined by immunohistology
(data not shown). Spleens from such animals weighed 0.522 ± 0.23 g
(mean ± 2 SEM), compared with only 0.15 ± 0.04 g for those from
normal mice. These mice also show slight liver enlargement because of
developing tumor.
To investigate the biodistribution of iodinated MoAb in such mice and
estimate the total amount of irradiation delivered to the splenic
tumor, we injected 500 µg of 125I-anti-CD22 (low
binding), -anti-MHCII (TI2-3, high binding), or -anti-Id (high,
tumor-specific binding) MoAb into 14-day tumor-bearing mice. At given
times over the next 5 days the animals were killed and various organs
removed to determine radioactive content. The results showed (Fig
3) that the different MoAb all accumulated rapidly in the spleen where the majority of the tumor was localized. Even just 1 hour after MoAb injection the spleens contained around 30%
ID/g of tissue (anti-Id and anti-MHCII). The corresponding amount for
the anti-CD22 MoAb was around 15%. Other organs, such as the liver,
kidney, and lungs, carried between 10% and 15% ID/g of tissue at the
same time point. Furthermore, unlike the results from these organs, the
results for the spleen showed a large differential between uptake of
the specific MoAb and uptake of the control IgG (approximately 5%),
consistent with specific targeting to the tumor.
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| Fig 3.
Biodistribution of trace-labeled 125I-MoAb in
BCL1-carrying mice. Matched groups of mice, which had been
injected IV with BCL1 cells (106) 14 days
previously, were injected IV with 500 µg of 125I-MoAb.
The MoAb under investigation included: , anti-Id; , anti-MHCII;
, anti-CD22; and , control IgG2a. At the intervals shown, the
animals were killed, blood samples obtained immediately postmortem, and
the various organs (as shown) removed for weighing and estimation of
radioactive content. Results are expressed as the percentage of the
injected dose of 125I-MoAb per gram of tissue recovered (% ID/g; Materials and Methods). For reasons of clarity, the anti-CD22
MoAb results are shown for the spleen only. Each point shows the mean
and range for four or five animals investigated. These results
represent one of two similar experiments.
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Figure 3 also shows the rate at which MoAb, once accumulated in the
spleen, were cleared. This was particularly interesting with regard to
the RIT. Thus, while the half-life for 125I-anti-Id MoAb
was about 9 hours, that for 125I-anti-MHCII MoAb was
almost 24 hours. As a result of such a large difference, the AUC for
these two reagents, which gives a measure of total dose of irradiation
delivered to the organ during RIT, differ by close to 4:1.
Interestingly, the anti-CD22 MoAb, which accumulates at relatively low
levels in the spleen, presumably because of the relatively low
expression of CD22, also has a short half-life resulting in a very
small AUC and probably explaining its lack of RIT.
The total dose of radiolabeled anti-Id, anti-MHCII, and control IgG
delivered to organs other than the spleen is quite similar over the
5-day period with little evidence of specific uptake. In the blood,
however, we did find an extended half-life for anti-MHCII MoAb. This
probably results from binding to MHCII-expressing cells, particularly B
cells, in the circulation, which extends the half-life beyond that of
the other MoAb.
Clearance of tumor surface antigen by MoAb in vivo.
We next investigated whether surface antigens, once bound by treatment
MoAb in vivo, are likely to remain at the surface or be endocytozed
along with the treatment MoAb inside the cell. The results of such work
might explain why anti-Id and anti-MHCII MoAb differed in their splenic
survival and consequent AUC (Fig 3) and could have important
implications for selecting target antigens in RIT.
For these experiments, BCL1-bearing mice were treated with
the same amount of MoAb that was used in RIT (0.5 mg/animal) of anti-Id, anti-CD19, anti-CD22, or anti-MHCII MoAb (TI2-3), and then 24 hours later splenic tumor cells were investigated for the presence of
surface antibody. The level of the therapeutic MoAb on the
surface of recovered tumor cells was detected by adding FITC-mouse
anti-rat IgG, and then PE-anti-BCL1 Id MoAb used to allow
analysis (gating) of just the tumor cell population. The results (Fig
4) showed that in vivo
exposure to anti-MHC II MoAb (TI2-3) did little or nothing to the
expression of MHC II by BCL1, and that the recovered tumor
cells remained coated with MoAb. In contrast, those tumors that had
been exposed to anti-Id, anti-CD19, or anti-CD22 MoAb showed clear
evidence of MoAb-clearing in vivo, and carried less MoAb/cell than that
on control cells stained with these reagents. For example, fresh tumor
cells stained with anti-Id MoAb gave a mean fluorescence intensity that
was more that 10 times higher than obtained on cells recovered after in vivo treatment. However, despite such losses, the cells treated with
the three clearing MoAb were not completely negative for treatment
MoAb, because they all stained more strongly than untreated cells
stained with control MoAb (dotted histogram). These data confirm and
extend previous work showing that MoAb that are not cleared from the
targets, such as anti-MHCII MoAb, are likely to be more active in RIT,
than those that tend to modulate.20-22 These results are
also consistent with the biodistribution studies showing the smaller
AUC for the internalizing anti-CD22 and anti-Id MoAb.
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| Fig 4.
Changes in the level of surface MoAb after treatment in
vivo. BCL1-bearing mice were treated with 0.5 mg of
anti-CD19, anti-CD22, anti-Id, anti-MHCII (TI2-3), or control IgG
(Materials and Methods). Sixteen hours later spleen cells were taken
and any MoAb remaining at the tumor surface detected by adding
FITC-mouse anti-rat IgG polyclonal Ab. PE-anti-Id MoAb was also added
to the staining mixture to allow gating on the tumor cells. The dotted
histogram in the anti-CD22 MoAb box shows background staining with
FITC-anti-rat IgG. These cells were taken from mice treated with
control IgG. The solid histogram shows the maximum level of
FITC-staining obtained when cells from mice treated with control IgG
were stained in vitro with a saturating level of MoAb (25 µg/mL
anti-CD22, anti-CD19, anti-Id, or anti-MHCII as indicated in each box).
The open histograms (solid lines) show the level of MoAb remaining on
the surface of tumor cells when they were recovered from mice treated
with MoAb. Mice treated with anti-CD22, anti-CD19, and anti-Id MoAb
show evidence of clearing in vivo; anti-MHCII MoAb does not.
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RIT of advanced BCL1 lymphoma.
In an attempt to increase tumor burden and extend the tumor model more
in line with human disease, in the next set of RIT experiments
treatment was delayed until day 14 of tumor development when the
spleens were enlarged and tumor was beginning to appear in the liver.
These experiments were confined to the anti-Id and the anti-MHCII MoAb
that had been successful when used in RIT early in the disease. For
this work we increased the amount of MoAb administered to 750 µg/mouse, which was conjugated to approximately 7.5 MBq to 8.5 MBq
131I. The results with unconjugated MoAb were as expected,
with only the anti-Id showing any therapeutic activity (Fig
5). However, somewhat surprisingly we found
that when treating at this late time point, the anti-Id MoAb gave, if
anything, a more effective therapy (13 days) than had been achieved
when used on day 4 of the disease (Fig 2A). The radioiodinated
anti-MHCII MoAb, rather than curing mice as had been seen earlier, only
extended animal survival moderately (by approximately 12 to 15 days
over those in control groups), little better than unlabeled anti-Id
MoAb under these conditions. However, by far the most impressive result was achieved with 131I-anti-Id MoAb, which extended the
survival of all the animals to beyond 100 days with no signs of tumor.
This observation was confirmed in two subsequent experiments. Thus by
delaying the treatment and increasing the tumor burden it appears that
the anti-Id therapy had become more effective, a result which is in direct contrast to that seen using the anti-MHC II MoAb.
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| Fig 5.
Treatment of the BCL1 lymphoma with RIT late
in the disease. Groups of 10 mice were given 106 cells on
day 0 as usual and then left until day 14 before treating IV with
radioiodinated MoAb (750 µg/animal [approximately 7.5 MBq]). The
treatments included: Control IgG (); control IgG2a (); anti-Id
(, ); anti-MHC II (TI2-3; , ); and anti-MHCII (N22; ,
). The solid and open symbols represent MoAb labeled with and
without radioactive iodine, respectively. Surprisingly the
131I-anti-Id MoAb provided long-term protection to all the
mice.
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To investigate the relative contribution of the anti-Id MoAb and the
radiation on the improved efficacy of 131I-anti-Id when
treating later in the disease, an investigation was set up using
unlabeled anti-Id MoAb to directly compare the efficacy of treating on
days 4 and 14 after tumor inoculation. The results from this work are
shown in Fig 6 and confirm an improved survival with anti-Id MoAb when treating later in the disease. It would
appear, therefore, that by delaying treatment with anti-Id MoAb for 10 days we have been able to increase survival by between 8 and 9 days. No
improvement in efficacy was seen by delaying treatment with any of the
other MoAb under investigation (data not shown).
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| Fig 6.
The effect of delaying anti-Id MoAb-treatment in
BCL1 lymphoma. Groups of 10 mice were given 106
BCL1 on day 0 and then treated with MoAb (1 mg/mouse) 4 or
14 days later as shown. For comparison, an additional group of mice was
treated with 131I-anti-Id MoAb (750 µg/animal
[approximately 7.5 MBq]) on day 14. Treatment groups include: Control
IgG (); anti-Id on day 4 and 14 (); 131I-anti-Id
(); and anti-MHCII () on day 14.
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Anti-Id MoAb and irradiation provide additive cytotoxicity.
It is possible that the unexpected increase in therapeutic activity
seen when delaying anti-Id MoAb treatment might relate to the growth
status of the tumor cells at different times during its development.
Preliminary data suggest that during the first 10 days after
inoculation of a small dose of BCL1 cells
(105/mouse) there is very little, if any, proliferation
that can be detected (not shown). After this period, tumor cells are
detectable in the log phase of an exponential growth curve. One
possible explanation for the current results is that anti-Id MoAb is
therapeutically more effective in rapidly growing cells. We next
measured the sensitivity of rapidly growing BCL1 cells
lines (BCL1-3B3 and BCL1) to anti-Id MoAb
and external beam irradiation. Cells were exposed to MoAb, irradiation,
or both, and then the level of growth arrest and DNA fragmentation
assessed by flow cytometry following nuclear staining with PI. Figure
7A shows the DNA cell-cycle profiles obtained when BCL1 cells (log phase) were exposed to
MoAb for 72 hours. During log-phase growth, the DNA in cells treated
with control IgG was distributed with 51% to 53% in G1/G0 phase, 16% to 18% in S phase, and 12% to 13% in G2/M phase. In addition, approximately 13.5% to 14.4% of the DNA was in the sub-G1/G0
position, which represents fragmented DNA derived from cells undergoing apoptosis.41 Most MoAb under investigation
(anti-CD19, anti-CD22, and anti-MHCII) had no effect on cell growth or
DNA profiles. However, as expected,42 anti-Id and
anti-Fcµ MoAb caused profound cell-cycle arrest (not shown), which
was followed by an increase in DNA fragmentation (Fig 7A), indicative
of apoptosis.
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| Fig 7.
Growth arrest and apoptosis of BCL1 cells
treated with external beam irradiation and MoAb. BCL1
cells were either treated with 2 Gy external beam irradiation, or
remained untreated, before being cultured for 72 hours in 24-well
plates, which were either coated (or uncoated) with MoAb as indicated.
The cells were then harvested after 72 hours, stained with PI to detect
DNA content, and analyzed by flow cytometry. (A) Flow cytometry
histograms for one typical experiment. The horizontal bars show the
gated areas representing fragmented DNA (indicative of apoptosis). The
percentage of DNA within each gate is shown. (B) Average (+SD) of
three similar experiments to that in Fig 7A. The * indicates
statistical significance (P < .01) over other combinations
of MoAb and irradiation.
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Cells treated with external beam irradiation underwent a number of
changes in their DNA composition, including the well-described G2
cell-cycle arrest and marked DNA fragmentation (37.7% to 41.5%). However, the most interesting results were obtained when cells were
irradiated and then cultured in anti-Id or anti-Fcµ MoAb. The
combination of cytotoxic MoAb and irradiation resulted in greatly
increased DNA fragmentation (61% to 67%), suggesting the two
modalities had an additive cytotoxic activity. None of the other MoAb
under study (Table 1) increased the toxic effect of irradiation. The
potency of irradiation and MoAb binding to the surface Ig of
BCL1 was confirmed in three separate experiments (Fig 7B)
and on BCL1-3B3 cells (not shown). Finally, we asked the
question whether the combination of anti-Id MoAb and external beam
irradiation showed the same additive effect in vivo. Cultured BCL1 cells were treated with 2 Gy external beam
irradiation and then injected (106/mouse) with or without
0.5 µg/mouse anti-Id MoAb. Figure 8 shows a significant benefit (P < .01) of treating with both MoAb
and irradiation over either treatment alone and suggests that this cooperative effect may play an important role in the therapeutic activity of radiolabeled anti-Id MoAb.
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| Fig 8.
Anti-Id and external beam irradiation has a significant
benefit over either treatment alone in vivo. BCL1 are
treated in vitro followed by growth in mice. Cells (106/mL)
were either treated with 2 Gy of external beam irradiation or remained
untreated before being mixed with MoAb (0.5 µg/mL) as described,
before being transferred into BALB/c (5/group). Each mouse received
106 cells and 0.5 µg of MoAb as indicated in the key.
Survival was monitored daily.
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DISCUSSION |
In this study we have investigated RIT in an animal model in an attempt
to define the relative contributions of "targeted" low-dose
irradiation and MoAb to the therapeutic effect. Dosimetry and treatment
planning for therapeutic infusions of radiolabeled MoAb in lymphoma are
usually performed by extrapolation from biodistribution studies with
trace-labeled MoAb.3,6,8,27 This extrapolation assumes that
the biodistribution of a therapeutic conjugate will be similar to that
seen with trace-labeled MoAb.27 A primary aim of this
investigation was to provide a model of targeted, nonmyeloablative
irradiation that would accurately predict organ dosimetry for
subsequent low dose RIT. We believe this is the first time a range of
anti-B-cell MoAb have been compared in such a well-defined syngeneic
lymphoma model using both radioiodinated and "naked" MoAb.
A number of observations have been made with regard to RIT. First,
predictions initially made in vitro20-22 that strongly
expressed surface B-cell antigens (MHCII and Id) are more suitable
targets for RIT than weakly expressed molecules (CD19, CD22), have been confirmed in vivo. Thus tumor-bearing animals, if treated early in the
disease with 131I-anti-MHCII MoAb (TI2-3 or N22), were
mainly cured, while those given 131I-anti-CD19 or
131I-anti-CD22 MoAb were given very little protection,
despite similar binding constants. These results appear to reflect
levels of antigen expression, but are also influenced by the tendency
of MoAb to clear in vivo (see below). Interestingly, under these
conditions, 131I-anti-Id MoAb, which bound to
BCL1 at high levels, gave only partial protection. Second,
although the anti-MHCII and anti-Id MoAb bound at similar levels to
BCL1, the anti-MHCII (TI2-3) delivered about four times
more irradiation to tumor-bearing spleens. This result reflected its
much longer half-life and may be explained by the observation that
TI2-3 remains on the surface of tumor cells after MoAb treatment in
vivo while Id was cleared, probably by endocytosis. Third,
radioiodinated anti-MHCII MoAb, which were highly effective when used
early in the disease (day 4), were much less effective when used
against advanced tumor on day 14 and no longer provided any cures.
Fourth, our most unexpected observation was that under these demanding
conditions, 131I-anti-Id MoAb, which had given about 30 days protection when used on day 4, was able to cure (as determined by
survival >100 days after inoculation) almost all animals when given
on day 14. It would thus appear that with this particular MoAb, which
was the only one investigated that had intrinsic therapeutic activity, treating later in the disease was highly beneficial. Biodistribution studies performed also on day 14, indicated that this tumor response did not correlate with the dose of radioactivity delivered to the tumor
and indeed that the anti-Id MoAb delivered four times less irradiation
to the tumor-bearing organs. Finally, in vitro experiments indicated an
interesting additive effect between the therapeutic capacity of
irradiation and the cytotoxic activity of anti-Id MoAb, so that when
used in combination, these two treatments induced significantly more
apoptosis than either alone. Importantly, of the panel of MoAb, only
those reacting with the B-cell receptor (anti-Id and anti-Fcµ) were
capable of inducing this effect, supporting the idea that
radioiodinated anti-Id MoAb achieves its therapeutic effects through a
coupling of the cytotoxic potential of irradiation and MoAb.
The growth and organ distribution of the BCL1 lymphoma has
been very well characterized.33 Knapp et al34
showed that the tumor is confined mainly to the spleen until the
terminal stages of the disease, when large numbers of tumor cells
appear in the peripheral blood. These workers also showed that 14 days
postinjection of 106 cells, the spleen contained
approximately 30%  - and Id-positive cells with little or no
-positive cells in the blood. We conducted biodistribution studies
on tumor at this stage of development and showed high-level uptake of
trace-labeled anti-MHCII and anti-Id MoAb in the spleen, which was
about six times that of a control IgG, and twice that of anti-CD22 MAb.
Thus as expected, uptake was MoAb-dependent and probably related
directly to binding to tumor cells and, in the case of anti-CD22 and
anti-MHCII MoAb, antigen-positive normal cells. However, although the
initial uptake of anti-MHCII and anti-Id MoAb was similar,
biodistribution studies over the next 5 days showed that they had quite
different half-lives of 24 hours and 8 to 9 hours, respectively.
Integrating the AUC gives an approximation of the total dose of
irradiation delivered to an organ and is commonly used to estimate this
parameter.30,43 The AUC calculation using our data showed
that the 125I-conjugated anti-MHCII MoAb (TI2-3) delivered
a total dose of irradiation that was at least four times that delivered
by the anti-Id MoAb. The explanation for the difference in half-life between anti-Id and anti-MHCII MoAb (TI2-3) is most likely related to
the rate at which anti-Id MoAb was internalized leading to rapid
dehalogenation.20 The rapid internalization,
dehalogenation, and excretion of radiolabeled MoAb against the B-cell
receptor and tumor Id compared with MoAb against MHCII have been
well-documented in vitro.19,20 However, other factors may
also contribute and it is possible that low levels of secreted
BCL1 Id could form immune complexes with the MoAb and
thereby promote degradation in FcR-bearing cells.44 Our
biodistribution data for 125I-anti-MHCII MoAb confirm
previous reports from similar models30 and show that the
125I-control IgG has an increased half-life over the
anti-Id MoAb. Also of note was the observation that at these modest
doses of radioactivity, there was no detectable bone marrow
accumulation. This was in keeping with the report by Badger et
al,45 which suggested that, unless larger doses, greater
than 250 µCi (9.4 MBq) of radioactive iodine were used,
myelosuppression is not an expected treatment related toxicity in these
animal models.
The results from the initial RIT with 131I-MoAb strongly
implicate targeted irradiation to the tumor-bearing organs by the MHCII MoAb (TI2-3 or N22) as the major cause of the tumoricidal effects. The
lack of effects seen with "naked" anti-MHCII MoAb is in agreement with previous work,36 and the negligible effects of the
radiolabeled control IgG appear to exclude either a direct therapeutic
effect from the MoAb or a nonspecific "total body" irradiation
effect. Importantly, when interpreting these results in animal models and attempting to extrapolate to clinical RIT of B-cell lymphomas, the
effects observed here with the anti-MHCII MoAb appear entirely compatible with those seen in the clinic using 131I-Lym-1
directed against the HLA-DR10 antigen.13 Like the
anti-MHCII MoAb used here, the Lym-1 MoAb appears to have little or no
therapeutic efficacy as a naked antibody in vivo and appears to be an
inactive delivery vehicle for the targeting of what DeNardo et
al13 describe as "systemic radiotherapy." To increase
the efficacy of this approach, DeNardo et al12,13 have used
fractionation of RIT, which has in turn enabled an increase in the
maximum-tolerated dose and response rates.
Thus our initial data underline what has been shown by others mainly
from in vitro data,20-22 that a highly expressed,
nonendocytosed surface target is likely to be the most successful for
RIT. The survival advantage seen here with 131I-anti-MHCII
MoAb is more marked than that previously reported in this model using
other delivery systems,46 or than those seen in the murine
T-cell lymphoma EL4 with Yttrium90-labeled
MoAb.47 Knox et al30,31 have performed
extensive investigations in the 38C13 murine B-cell subcutaneous
lymphoma model showing the radiobiological effect of
131I-anti-Id MoAb and comparing its activity with
dose-equivalent external beam irradiation. This study showed that,
although there was a statistically significant difference between
specific (131I-anti-Id MoAb) and nonspecific irrelevant
(131I-control MoAb) MoAb on tumor response, the relative
efficacy of the 131I-anti-Id was low, indicative of poor
tumor targeting. A difficulty in interpreting such results is the lack
of another anti-B cell MoAb to define the contribution that anti-Id
MoAb may have had in these tumor responses.
The most unexpected observation to emerge from the current study was
the capacity of 131I-anti-Id MoAb to cure animals when
given as a single dose on day 14 of the BCL1 tumor (Figs 5
and 6). Part of the therapeutic activity of this derivative comes from
the anti-Id MoAb, which as an unconjugated reagent gave partial
protection to tumor-bearing mice and was also more active when
administered as a naked antibody later in the disease (Fig 6).
Considerable evidence now shows that anti-Id MoAb can be directly
cytotoxic to human and mouse lymphoma cells and that this effect seems
to depend on the ability of the MoAb to cross-link the surface Ig and
thereby deliver transmembrane signals to the cells (Fig
7).42 Racila et al,48 working in the
BCL1 model, have presented strong evidence that anti-Id
MoAb can provoke intracellular signals in tumor cells, which regulate their growth and can leave them in a state of dormancy for extended periods. Our own work36 shows that in a similar mouse
B-cell lymphoma model, anti-Id MoAb, when administered in vivo, causes an abrupt growth arrest of tumor without immediate eradication. The
reason that anti-Id MoAb might be more therapeutic when used on day 14 rather than day 4 is not clear. However, it is possible that the growth
rate of the tumor may play some role in this phenomenon. For example,
if BCL1 cells are cycling faster on day 14 than on day 4, then it does not seem unreasonable to expect changes in their
sensitivity to signaling through the B-cell receptor. The c-myc
oncogene protein is expressed throughout the G1, S, and G2M stages of
the cell cycle and is one of a number of genes responsible for
regulating the linked pathways of proliferation and
apoptosis.49 The Myc protein is not expressed in resting
(G0) cells. In cycling cells, perturbations in myc levels are known to
induce apoptosis.50 Signaling through the B-cell receptor
is one such way of perturbating myc, and for some time it has been
known that such signaling in vitro can result in apoptosis. Therefore,
if in established disease (day 14) more cells are in cell cycle, then
anti-Id might be expected to be more effective.
It is also quite clear that in addition to the MoAb effect, target
irradiation plays an important role in providing tumor control. Our in
vitro data show that anti-Id, unlike the anti-MHCII, MoAb were able to
give at least an additive effect with external beam irradiation
resulting in unexpected levels of apoptosis in cultured
BCL1-3B3 and BCL1 cells. Cells treated with
a combination of irradiation and anti-Id MoAb in vitro were also less
viable than cells treated with either moiety alone when inoculated into animals. This observation offers a potential explanation for the immunotherapy results. By treating later in the disease, we have increased the activity of the MoAb, which together with the effect of
the targeted irradiation, can eradicate all tumor cells. The improvement in therapeutic efficacy that is provided by combined irradiation and anti-Id MoAb treatment may arise from events that occur
at the transmembrane signaling stage. Recently, it has been discovered
that, at least for lymphoblasts, apoptosis can be induced by
irradiation with release of the intracellular messenger
ceramide.51 When elevated, this molecule can induce a
variety of cellular effects, including growth arrest and apoptosis.
Intriguingly, signaling through the B-cell receptor, through MoAb such
as anti-µ and presumably anti-Id, can also yield increases in
ceramide. Therefore, it may be that the improved potency of the
radiolabeled anti-Id MoAb is governed by an enhanced ceramide response.
An alternate explanation may center on the fact that both irradiation and anti-Id MoAb treatments are capable of modulating the expression of
c-myc. These two explanations may not be mutually exclusive, indeed, it may be that elevated levels of ceramide themselves translate
to a more potent perturbation of myc and hence, apoptotic signaling.
We believe these results may also help to explain some of the excellent
therapeutic effects seen in the clinic with radiolabeled anti-CD20
MoAb. In contrast to the anti-MHCII MoAb, recent evidence suggests that
anti-CD20 MoAb may be directly cytotoxic to cells and operate at least
in part through a signaling pathway that induces apoptosis in target
cells.25 Therefore, it seems plausible that together,
targeted irradiation and anti-CD20 MoAb, may have a combined
therapeutic efficacy that is greater than either treatment alone.
Studies are underway to discover if any other radioconjugated MoAb
benefit from the additive effects of targeted irradiation and direct Ab
cytotoxicity. For example, anti-CD79 MoAb that bind to the invariant
signaling chains associated with the B-cell receptor may provide an
interesting target.52 These MoAb may have similar signaling
properties to anti-Id MoAb, but will not suffer from the inherent
problems of genetic instability and the requiremen |
TOP
ABSTRACT
INTRODUCTION