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Experimental Therapeutics, Preclinical Pharmacology |
Garden State Cancer Center, Belleville, New Jersey 07109
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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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The current study was intended to extend these results in two ways: to
test other cell lines expressing Ii, including adherent target cells;
and to test other Abs reacting with B-cell lymphomas. Melanoma cells
and many carcinoma cells can be induced to express high levels of both
mature MHC class II antigen and the invariant chain by IFN-
.
Anti-CD74 uptake by induced SK-MEL-37 melanoma cells was comparable
with the uptake by Raji cells (2)
. However, the very
different cellular morphology is likely to have a significant impact on
killing by internalized radionuclides. More specifically, the spreading
of the cells on plastic would tend to decrease the proximity of nucleus
and cytoplasm. Therefore, we have tested cytotoxicity for these
adherent cell lines, as well as for other B-cell lymphomas, to
establish the generality of the results.
Whereas the massive intracellular uptake of anti-CD74 is not matched by any other Ab to our knowledge, some other Abs can bind to the cell surface in similar amounts. For example, the number of mature MHC class II molecules on Raji B-lymphoma cells is approximately 3 x 106 (2) . (Note that the mature MHC class II molecules lack the invariant chain, which must be removed before binding of peptide antigens.) This level of expression suggests that some cytotoxicity might be obtained with anti-MHC class II, although perhaps not as strong as with anti-CD74. A basic difference between anti-CD74 and antimature MHC class II is in their subcellular localization. Thus, whereas the former is delivered to lysosomes, the latter remains primarily on the cell surface. According to dosimetry calculations, isotopes in the cytoplasm are expected to be more potent than isotopes on the cell surface, but the difference is relatively small for the radionuclides used in these experiments. For example, the advantage of internalization for 125I and 111In is 53 and 67%, respectively (3) , as calculated for a cell with the dimensions of Raji (cell diameter, 16 µm; nuclear diameter, 12 µm; Ref. 1 ), which suggests that isotopes localized to the cell surface would still be quite active. It should also be noted that considerable evidence suggests a recycling pathway for mature MHC class II, in which the molecules enter an intracellular compartment where peptide antigens are exchanged (4 , 5) . This factor, of course, would further enhance the cytotoxic capacity of the Ab, but the effect would be small because most of the antigen is on the cell surface at any one time (4) .
Another Ab was also tested, namely anti-CD20. With approximately 3 x 105 sites/cells (6 , 7) , this Ab was not expected to provide an effective target for killing in this experimental system, but it seemed possible that some cytotoxicity would be observed. Unexpectedly, radiolabeled anti-CD20 killed cells very effectively, and the explanation for such killing is partially elucidated below.
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MATERIALS AND METHODS |
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Antibodies and Radiolabeling.
Anti-CD74 (LL1) was supplied by the Antibody Production facility at
Immunomedics, Inc. (Morris Plains, NJ). The source of L243 (anti-MHC
class II antigen) was described previously (5
, 8)
. The
hybridoma producing 1F5 was obtained from the American Type Culture
Collection (Rockville, MD). Ab-producing cells were found to constitute
<10% of the cell population, so the Ab-producing cells were cloned by
limiting dilution before expansion of the cells. Ab purification was on
an affinity column of Sepharose linked to protein A or protein G
(Amersham Pharmacia, Piscataway, NJ), as appropriate for the Ab
isotype. Abs were labeled with 125I and
131I by the chloramine-T method and with
111In with the chelator
isothiocyanate-benzyl-DTPA, as described previously in detail
(1)
. Labeled preparations were analyzed by instant TLC,
gel filtration high-performance liquid chromatography, or both by
methods that have been described (9)
to determine the
level of radioactivity not bound to the Ab, which was always <10% and
usually <5%. Representative preparations of radiolabeled Abs, with
each radiolabel, were tested for immunoreactivity (percentage bindable)
by incubating with a large excess of cells. Control tubes had excess
unlabeled Ab added to block specific binding and therefore to indicate
the level of nonspecific binding; specific binding was calculated by
subtraction. The range of specific binding was as follows: LL1,
53.0–71.7%; LL2, 42.1–60.1%; 1F5, 50.6–74.8%; and L243,
56.3–58.6%. Such variation in immunoreactivity could have some effect
on the data presented, but this would be at most a small effect, and it
would not significantly alter any of the conclusions.
Cytotoxicity of Nonadherent Cells.
All of the methods were described in detail previously
(1)
. Briefly, cells were incubated for 2 days with varying
concentrations of radiolabeled Abs and then diluted 14.3-fold. Cell
counts were obtained every 3–5 days until the cells had multiplied
16-fold or until day 21. The fraction of cells killed was calculated
from the growth curves, without considering possible division delay
induced by irradiation.
Cytotoxicity of Adherent Cells.
SK-MEL-37 melanoma cells were preincubated for 2 days with human
IFN- (IFN-1b; Actimmune; Genentech, South San Francisco, CA) at
250 units/ml, and IFN- at this concentration was included in the
medium used throughout the assay. After trypsinization, 0.15 ml of a
0.1% suspension (packed cell v/v) was plated in wells of 96-well
plates and incubated overnight. After aspiration of the medium, 0.2 ml
of medium containing radiolabeled Ab was added. Serial dilutions of the
Abs were used, each tested in triplicate, and the incubation was for
48 h. The control wells contained excess unlabeled Ab at 62.5
µg/ml to inhibit specific Ab binding and thereby to indicate the
level of nonspecific cytotoxicity. Some wells were used to measure
uptake of radioactivity, some were used for cell counts after
trypsinization, and some were used for a clonogenic assay. To measure
uptake, cells were washed three times with tissue culture medium and
then harvested with 50 µl 2.0 M NaOH, which was collected
with a cotton swab. For the clonogenic assay, cells from a single well
were trypsinized and suspended in 6.7 ml medium. Serial 4-fold
dilutions were prepared, and 5 ml of each dilution were plated in T30
flasks. After 12–15 days of growth, colonies of >50 cells were
counted in those flasks that contained 50–200 colonies. Before
counting, colonies were fixed for 10 min with 5.0% glutaraldehyde in
0.1 M sodium cacodylate HCl (pH 7.2), stained for 10 min
with 0.5% methylene blue in 25% ethanol, and washed thoroughly with
water. Irradiated cells grew more slowly than control cells, which can
be attributed to radiation-induced growth delay (10)
or to other toxic effects of the radiation. Therefore, compared
with the control cells, the treated cells were allowed 2–3 more
days to grow before counting colonies. In practice, this allowed all of
the healthy colonies to reach the countable size of 
50 cells. The
cloning efficiency of control cells was approximately 60%.
Monitoring Uptake of Radioactivity.
Uptake of cpm was determined in experiments identical to the
cytotoxicity experiments. At various times, cells were collected,
pelleted, and washed three times with 5 ml tissue culture medium, and
the radioactivity was determined. Routinely, 1/20 of the total sample
was counted because otherwise the cpm were too high to be counted
accurately (>106
cpm). Cell counts were also
determined at each time point, although, for reasons discussed below,
the data are generally expressed in terms of the initial cell count.
The number of Ab molecules bound/cell was calculated from the cpm bound
and the specific activity and was corrected for the fraction of cpm in
the stock Ab preparation that was not associated with intact IgG
(always <10%). For radiation dosimetry, the area under the curve of
bound cpm versus time was calculated, which provided the
cumulative counts. Cumulative disintegrations were calculated from the
gamma counter efficiencies, which were 70.9% for
111In and 76.5% for 125I.
Cellular S-values for Raji-sized cells (diameter, 16 µm; nuclear
diameter, 12 µm) were from Goddu et al. (3)
.
Elution of Bound Radiolabeled Ab by Excess Unlabeled Ab.
Experiments were set up like the cytotoxicity experiments, with cells
in 24-well plates. 125I-labeled L243 was included
at 5 µCi/ml, which kills approximately 95% of the cells after a
standard 2-day incubation. After 2, 22, or 44 h, cells from three
wells were collected, pooled, pelleted, washed three times, and
suspended in 2.5 ml. Aliquots of 0.5 ml were replated in four wells of
a 24-well plate, and 0.5 ml of L243 hybridoma ascites diluted 1:25 was
added to two of the wells, whereas the other two wells received control
medium. The principle of this assay is that excess unlabeled Ab
replaces the labeled Ab on the cell surface, because of the fact that
the bound labeled Ab, although predominantly bivalently bound,
frequently wobbles on the cell surface (11)
. After 24-h
incubation under tissue culture conditions, the cells and supernatant
were analyzed for remaining cpm, as described previously in detail
(8)
. The supernatant was also tested by precipitation with
cold 10% TCA to distinguish between intact and degraded Ab.
Large fragments of Ab would also be TCA precipitable, but such
fragments are not generated from L243 in significant amounts in these
experiments, as determined previously (5)
, and, in any
case, would not affect the interpretation of the results in this study.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, were generally similar to the results described with Raji cells, with
high levels of antigen-specific cytotoxicity with all of the cell
lines. With three of the four cell lines, 100% killing was obtained
(> approximately 6 logs) at the highest Ab concentration tested,
whereas the killing of RL cells was slightly less. Daudi was markedly
more susceptible to killing than the other three cell lines, with both
antigen-specific and nonspecific killing. In the assay of nonspecific
killing, a nonreactive Ab at 90 µCi/ml killed 85–96% of Daudi
cells, whereas killing of the other three cell lines was only 30–50%.
By comparison with previous experiments in which higher µCi
concentrations were tested in the killing of Raji (15)
,
Daudi was approximately 3-fold more sensitive to nonspecific killing.
In specific killing with 111In-labeled LL1, Daudi
was also 2–3-fold more sensitive, as shown in Fig. 1
. Also, unlike the
other target cells, Daudi cells did not gradually become very large
before lysis, as described previously for Raji (1)
, but
instead lysed more rapidly. Both the lack of cell enlargement and the
susceptibility to killing of Daudi cells are likely to be related to
the presence of wild-type p53, which plays a role in the induction of
apoptosis (16)
, but additional studies are required to
confirm this possibility.
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, strong, antigen-specific killing was observed. With the highest Ab
concentration tested, 10 µg/ml, which had 159 µCi/ml, the
colony-forming units were reduced by 97.4%. A low level of nonspecific
cytotoxicity was observed; this was attributable to true nonspecific
cytotoxicity and not to residual specific binding that was not
inhibited by excess unlabeled Ab, because essentially the same results
were obtained with a nonreactive Ab, labeled similarly (data not
shown). The uptake of radioactivity, determined at day 2 (the time of
cloning) in the same experiment, was 3.1 x
106
cpm/8.0 x 104
cells/well or 38.8 cpm/cell. At least 88% of this uptake was antigen
specific because it was inhibited by excess unlabeled Ab. However,
100% killing, as observed with lymphoma target cells, was not obtained
with the melanoma targets, despite comparable levels of cpm
uptake/cell.
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. In comparison with the previous CD74 experiments, L243 was comparable
in potency, whereas CD20 was somewhat less potent on the basis of the
dose-response curves. At the highest concentration tested, CD20 killed
slightly <100% of the target cells. For example, in the
experiment shown in Fig. 3
, the fraction surviving with 1F5 at 90
µCi/ml was 2.99 x 10-6, which
corresponds to approximately 1–2 cells surviving of the initial 5 x 105
cells. Nonreactive Abs labeled in the same
way had no significant cytotoxic effect at the concentrations used, as
shown in Fig. 3
C, demonstrating that the cytotoxicity was
antigen-specific.
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A. This Ab appeared to
have a transient effect at day 2, which was probably because of the
fact that the Ab causes marked clumping of the cells, but by day 5 the
cells had multiplied essentially as much as untreated cells. In a
similar experiment with unlabeled 1F5, the Ab had no detectable
effect on cell growth (Fig. 3
B).
Uptake of cpm was determined in similar experiments. As shown in Fig. 4
, the uptake of both Abs was very high. The peak uptake with L243 was 47
cpm/cell, and the peak uptake with 1F5 was 29 cpm/cell. Although we
determined the cell count at each time point in all of the experiments,
the data are presented in terms of the initial number of cells
plated/well. The reason for this is that the cells start to die as
early as day 2, and therefore use of the actual viable cell number
results in artifactual high values of cpm/cell at later time points.
For example, in the experiment shown in Fig. 4
, L243 killed
approximately 70% of the initial cells at day 2, which resulted in a
value of >150 cpm/viable cell, which is clearly misleading. This issue
was discussed previously in greater detail (1)
. Fig. 5
shows typical growth curves in experiments of this type. Note that
cells were killed much more rapidly with L243 than with 1F5, but even
with 1F5, cell division was blocked rapidly, and there was very little
increase in cell number after time 0. This was a consistent finding in
experiments in which a large fraction of cells were killed and supports
the validity of using the initial cell count to calculate the cpm/cell.
The ability of radiation to rapidly block cell division is well known
(10)
.
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also shows the number of Ab molecule-equivalents bound/cell at
various times. These are referred to in this way because some of the
isotope retained by the cells is no longer on intact Ab but rather in
the form of catabolic products. The uptake of L243 by the cells peaked
at 7.2 x 106
Ab molecules/cell at 48 h
but was already at a high level at 1 h. This pattern of binding
kinetics is markedly different from that described previously with LL1,
where binding was very low at 1 h. This difference can be
attributed to the fact that the number of antigenic sites/cell on the
cell surface is much higher with L243 than with LL1. The substantial
increase in L243 binding with time that does occur can be attributed to
two factors. First, although L243 does not display rapid
internalization, it will be gradually internalized and degraded because
of normal turnover of cell surface constituents. Although not a major
factor, this will contribute to an increase of cpm/cell over time when
residualizing labels are used. More specifically, we determined
previously that, of the total L243 counts bound initially to Raji
cells, 21% were catabolized within 2 days (5)
. Second,
the irradiated cells grow substantially in size as an effect of lethal
radiation. As described previously (1)
, by day 2 the mean
cell diameter increased from 15.4 µm to 20.9 µm, corresponding to
an increase in surface area of 84% (assuming that the abundance of
microvilli and other surface conformations do not change). These two
factors can readily explain the uptake of 7.2 x
106
Ab molecule-equivalents/cell at 48 h,
given 3 x 106
sites/cell normally.
With 1F5, however, the high level of Ab uptake/cell, peaking at
approximately 5 x 106
Ab molecules/cell at
day 2, is not so readily explained. The number of CD20 sites/cell on
Raji cells was reported to be 3.6 x 105
(7)
, which is similar to the value reported for another
B-lymphoma cell line, Daudi (6)
. We redetermined the
sites/cell at saturation and found that there were 2.4 x
105
sites/cell4
, which is consistent with the previous reports. Thus, the number of
111In-labeled Ab molecules bound/cell at 48 h, as shown in Fig. 4
, was approximately 20-fold higher than the number
of sites/cell. The increase in cell size because of lethal irradiation,
as noted above, can account for only a 2–3-fold increase, so a 10-fold
increase remains to be accounted for. Another factor, as with L243, is
the turnover of the antigen (and any Ab bound to it). However, CD20 is
considered to turn over slowly, as indicated by low rates of catabolism
of bound Ab. Press et al. (6)
reported that
there was no significant catabolism of a CD20 Ab by Daudi cells. We
described significant levels of catabolism using the Ramos cell line,
but this was still at a relatively low level (17)
. The
impact of this factor can be readily investigated by comparing
residualizing with nonresidualizing radiolabels. A convenient
nonresidualizing label is a conventional chloramine-T iodine label,
because iodotyrosine rapidly leaves the cell after it is generated in
lysosomes (discussed in Ref. 18
). Results of such a
comparison with anti-CD20 demonstrated that the advantage of a
residualizing label is modest,
<2-fold.5
Therefore, the high level of binding of 1F5, much higher than the
number of sites/cell, remains to be explained and is discussed further
below.
Killing Cells with L243 or 1F5 Conjugated with 131I or
125I, and Uptake of 125I.
Because both of these Abs are internalized at relatively low rates, the
majority of bound Ab is expected to be at the cell surface, at least
for the first 2–3 days of incubation with Ab. The implication of this
statement is that the cells are killed primarily by radiation emitted
from the cell surface. This represents a major difference from the
situation with LL1, because this Ab is localized primarily to
lysosomes. Because cytoplasmic radionuclides are expected to be
somewhat more cytotoxic than radionuclides on the cell surface, as
noted in the "Introduction," it seemed important to confirm further
that radionuclides on the cell surface were responsible for cell
killing. Accordingly, experiments were performed with conventional
iodine labels, both 131I and
125I. These labels, as noted above, rapidly leave
the cell after catabolism, so are not trapped to a significant extent
in lysosomes. Therefore, they remain localized in target cells
primarily at the cell surface.
Fig. 6
demonstrates effective cytotoxicity obtained with both
131I and 125I labels. Both
radiolabels were tested at the highest concentration that produces
little if any nonspecific killing, as determined previously (1
, 15)
. The 131I-labeled conjugate was tested
at a starting concentration of 16 µCi/ml, whereas the
125I-labeled conjugate was tested at
concentrations up to 138 µCi/ml. With both isotopes, L243 was
considerably more potent than 1F5, but 1F5 still had high levels of
antigen-specific cytotoxicity. For example, in Fig. 6
B, the
cytotoxicity from 131I-labeled 1F5 may seem weak
in comparison with L243, yet it produced a cell kill of >3 logs. Fig. 6
also includes data with LL1 for comparison, as well as data with a
nonreactive control Ab. With LL1, the labeled conjugate is rapidly
catabolized and released from the cells, resulting in little
accumulation and consequently little cytotoxicity, as shown.
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. The uptake of 131I-labeled Abs is expected to be
very similar, because the specific activities and the
immunoreactivities with both isotopes were similar. These experiments
were set up exactly like the cytotoxicity experiments, using the
minimum Ab concentration that kills nearly 100% of the cells. This
concentration was 16 µCi/ml for L243 and 138 µCi/ml for 1F5, so
8.6-fold higher for 1F5. As shown, high levels of uptake were observed
with both Abs, with a peak uptake of approximately 20 cpm/cell at 2
days.
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, which also lists the calculated cGy dose delivered in 2 days. We have
not determined the time of functional cell death, but it is likely that
much of the dose delivered at later time points was redundant, because
the cells were already dead. Similar values are also shown for
111In-labeled conjugates. With the
111In label, the situation is complicated by the
fact that some of the bound Ab is internalized and catabolized, and the
catabolites are retained within lysosomes. Thus, the label is not
entirely on the cell surface, and it may be appropriate to use the
cytoplasmic S-value for some fraction of the radiolabel. However, we
know from previous studies that the great majority of the bound Ab
remains on the cell surface with these two Abs (see below, also), so
use of the cell surface S-value is appropriate to estimate the
radiation dose delivered. We have not attempted to correct these values
of radiation dose for the gradual increase in the size of the cells
resulting from lethal irradiation, which was described above. This
Table
is intended only to provide an estimate of the radiation dose
delivered in these experiments and does not allow a precise comparison
between Abs and radionuclides, because the uptake naturally depends on
the Ab concentration used. For 131I, assuming the
same uptake as with 125I, the dose delivered
would be only slightly lower than with 125I,
because the S-value is 1.06 x 10-4
Gy/Bq·s. Radiobiological parameters for Raji, using external
X-irradiation, was determined previously (1)
. The
D0 was 90 cGy, and the extrapolation
number ñ (10)
was 1.31. Therefore, the expected killing is
consistent with the cytotoxicity observed. The expected dose for a kill
of 6 logs is 1268 cGy.
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, the great majority of the bound L243 Ab (80–90%) was eluted with
excess unlabeled Ab, and this fraction was the same whether the initial
Ab incubation was for 2, 22, or 47 h. These data demonstrate that
the Ab remains on the cell surface and does not accumulate in any
intracellular compartment to a substantial level. Because elution in
these experiments requires 24 h for completion, the possibility
remains that the bound Ab recycles between the cell surface and an
intracellular compartment, such as early endosomes, but we can conclude
that the bound Ab spends at least some of the time at the cell surface.
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DISCUSSION |
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Earlier work suggested that radiolabeled Abs would not effectively kill single cells, but this conclusion was based on a limited body of experimental data, much of which we discussed previously (1) . Warters et al. (19) first attempted to kill cells with 125I bound to the cell surface. They used 125I-labeled con A, which is expected to bind to a very large number of sites/cell, but only weak killing was observed. The maximum killing obtained was approximately 90%. But there were several problems associated with the use of con A. First, the bound con A was very rapidly catabolized by the cells, which made it necessary to perform the binding incubation for 36 h at 4°C to maintain the con A on the cell surface. Second, the unlabeled con A was cytotoxic for the cells, necessitating the use of trypsinized, monovalent con A. The avidity of this monovalent ligand is unlikely to be as high as that of bivalent Abs, which bind essentially irreversibly (11 , 20) . In any case, it should be noted that the D0 (dose required for 63% kill in the linear part of the semilog dose-response curve) for 125I-labeled con A was 12,600 disintegrations/cell with CHO cells, which is in the same range as the values we found with Ab LL1. The D0 for 125I-labeled IMP-R2-LL1 was approximately 24,900 with Raji cells (15) . The CHO cells appear to be approximately 2-fold more resistant to radiation than Raji cells, as judged by results of external beam irradiation (1 , 19) . One important variable in the method of calculating D0 must be recognized. The total cumulative disintegrations used by Warters et al. (19) was over a 2-day period, whereas we used a period of 4–6 days because the isotopes were strongly retained by the cells, primarily within lysosomes. It is quite possible that, in our experiments, the cells were already killed by day 2 and that radiation delivered after that time was redundant. If this were the case, the true D0 would be much less than our estimate. In general, we consider the results of Warters et al. (19) to be consistent with our data.
Another key study was by Lindmo et al. (21)
,
who attempted to kill melanoma cells with an
131I-labeled Ab that reacts with a high density
antigen on the cell surface. These authors showed that strong killing
was obtained only if Ab-coated cells were stored frozen for many weeks
to allow radiation damage to accumulate. This result, then, emphasizes
the low level of cytotoxicity obtained. In general, the relatively weak
cytotoxicity that has rarely been reported with radiolabeled Abs has
been attributed to unusual properties of the particular Abs, such as
transport to the nucleus (22
, 23)
or accumulation in
macropinosomes (24)
. Cytotoxicity with
131I-labeled anti-CD20 was recently reported by
Johnson and Press (25)
. These investigators intentionally
pelleted the target cells to increase crossfire from adjacent cells, so
their results may or may not demonstrate single-cell kill. We note that
-particle emitters are exceptions to many of the above statements.
This type of radiation can efficiently kill single cells
(26)
; however, the available isotopes have short
half-lives of 
7 h, which seem unsuitable for treatment of solid
tumors where substantial time is required for tumor penetration.
-Particle emitters also appear to be very toxic, considering that
only 20 µCi of 212Pb-labeled Ab can be
administered to a mouse (27)
.
From the cellular S-values published by Goddu et al. (3) , we can calculate the number of disintegrations on the cell surface required to produce a defined level of cell kill. For Raji cells, the radiation parameters, determined from Cs137 irradiation experiments, were D0 = 90 and ñ = 1.31 (1) , so the dose required for a kill of 6 logs is approximately 1270 cGy. For a cell the size of Raji (RC = 8 µm; RN = 6 µm), this would require approximately 110,000 disintegrations of 125I, 120,000 disintegrations of 131I, and 164,000 disintegrations of 111In. From our data, the amount of radioactivity delivered/cell reached these levels, and the cytotoxicity observed was generally consistent with the calculated radiation dose. However, as noted above, the time course of irradiation is an important factor that has not been incorporated into the calculations.
Although the ability to kill single cells is clearly critical for therapy of micrometastases, its importance for therapy of solid tumors is uncertain. Indeed, radiolabeled Abs used for radioimmunotherapy, both experimentally and clinically, are very rarely even tested for in vitro cytotoxicity. This is partly because crossfire is a major factor in the killing of cells within solid tumors, whereas it does not apply in the killing of single cells (28) . In vivo experiments, both in animal models and in patients, are required to ascertain the relevance of single-cell kill to therapy. The results described herein do demonstrate, however, that two of the Abs that have been most effective in clinical studies of radioimmunotherapy, anti-CD20 (29) and anti-MHC class II (30) , are both in fact able to effectively kill single cells. This suggests that the importance of single-cell kill may be underestimated. At the doses used in patients, initial Ab concentrations are high enough to produce substantial single-cell kill, according to our in vitro data. For example, with a dose of approximately 60 mCi of 131I-labeled Lym-1, as used by DeNardo et al. (30) , the peak concentration in interstitial fluid will be approximately 6 µCi/ml, which from our in vitro data would produce a high level of cell kill. This is also true for the myeloablative dose of up to 800 mCi of 131I-labeled anti-CD20 used by Press et al. (31) . We note that the anti-MHC class II Ab used herein, L243, is not allele specific, unlike Lym-1 (32) , so would be expected to have more consistent reactivity with diverse B-cell lymphomas.
An enigma regarding anti-CD20 binding is that the uptake of Ab is much higher than the number of sites/cell by a factor of approximately 20. There are two known factors that contribute to increasing the Ab uptake. First, there is some turnover of the antigen, which provides some increase in the Ab molecule-equivalents binding/cell. Second, once radiation damage occurs, the cells increase markedly in size, which naturally results in an increase in the Ab molecules bound/cell. However, these factors can explain only a 3–4-fold increase in Ab sites/cell, so a further explanation is required. Two possibilities are apparent. First, the level of antigen may be up-regulated as a result of Ab binding. Secondly, there may be a pool of intracellular antigen that equilibrates with the cell surface and is gradually bound by Ab. Which of these possibilities is correct, if either, is currently under investigation.
Although the 98% kill of the adherent target cells, SK-MEL-37, was substantial, it was not as complete as the killing of B-lymphoma cells, which was 100%. This is likely to be attributable at least partially to the shape of the cell. Although we have not attempted to perform dosimetry calculations for cells adherent to plastic, which would be quite complex, it is clear that the flattened shape of the cell will tend to separate radionuclides in the cytoplasm from the nucleus. Moreover, the rim of cytoplasm will be quite thin above and below the nucleus. Thus, the radiation dose delivered to the nucleus will be considerably less than with round lymphoma cells. In addition, the SK-MEL-37 cells may be less sensitive to radiation than B-lymphoma cells. We would also like to suggest another possible contributory factor, though speculative. When the SK-MEL-37 cells form a monolayer, the cells are always most dense along the edges of the wells, and it seems possible that a few cells may be buried beneath other cells at the edges and therefore inaccessible to Ab.
The major advantage of Auger electron emitters over ß-particle emitters appears to be their much lower level of nonspecific cytotoxicity (15) . This was demonstrated in vitro (15) and has also recently been shown in vivo in a SCID mouse xenograft model.6 In regard to single-cell kill alone, 131I was more potent than either 111In or 125I, on the basis of the µCi/ml required for a particular level of kill, and this was true for all of the three Abs tested. The potency of 131I can be at least partially attributed to its significant nonspecific toxicity, which will enhance the effect of specific Ab binding. The higher energy ß-particles of 90Y (relative to 131I) are expected to be somewhat less potent at single-cell kill, and this was demonstrated in vitro with Ab LL1 (15) .
The three antigens targeted in this study are clinically important. However, the significance of these results will be amplified if the approach can be applied to other antigens that are not present at such a high density. This can potentially be achieved by using more potent radionuclides or higher specific activity. It should be emphasized that the radionuclides we have tested were selected only because of availability and that many more potent radionuclides (based on theoretical S-values) could be selected. There are at least four radionuclides that are expected to be at least 3-fold more potent than 111In when delivered to the cell surface, having suitable half-lives of 1.7–15.4 days. These are 153Sm, 191Os, 195mPt, and 195mHg. 191mOs would also be potent, because it decays rapidly to 191Os. On the basis of the analysis of Sastry et al. (33) , 119Sb and 119mTe are also expected to be potent (although cellular S-values have not been published for these isotopes). 195mPt, the most potent of the group, is expected to be 9.9-fold stronger than 111In. An isotope like 153Sm is attractive because it emits both abundant conversion electrons and moderate-energy ß-particles. None of these radionuclides are currently available carrier-free, and only 153Sm has been conjugated to Abs with high stability (34) . Large increases in specific activity, using currently available isotopes, are also possible. Conjugation of a single 111In atom/Ab would yield a specific activity of approximately 240 mCi/mg, which is 6-fold higher than the highest specific activity we have used in these experiments. Furthermore, it has been demonstrated that some Abs can be conjugated with five chelates without impairing immunoreactivity (35) . In summary, there are realistic approaches by which much lower-density antigens might provide effective targets.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by NIH Grants CA79620 (to
M. J. M.) and CA39841 (to D. M. G.). S. E. E. was supported by
the Undergraduate Projects in Technology and Medicine
program of the Stevens Institute of Technology. 
2 To whom requests for reprints should be
addressed, at Garden State Cancer Center, 520 Belleville Avenue,
Belleville, NJ 07109. Phone: (973) 844-7013; Fax: (973) 844-7020;
E-mail: mjmattes.gscancer{at}worldnet.att.net
3 The abbreviations used are: MHC, major
histocompatibility complex; Ab, antibody; DTPA,
diethylenetriaminepentaacetic acid; con A, concanavalin A; TCA,
trichloroacetic acid.
6 R. Ochakovskaya, L. Osorio, D. M.
Goldenberg, and M. J. Mattes. Therapy of disseminated B-cell
lymphoma xenografts in SCID mice with an anti-CD74 antibody conjugated
with 111In, 67Ga, or 90Y, submitted
for publication.
Received 6/ 6/00; revised 10/ 4/00; accepted 10/ 5/00.
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-particle-emitting radionuclides as modalities for radioimmunotherapy. Proc. Natl. Acad. Sci. USA, 83: 474-478, 1986.This article has been cited by other articles:
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 R. B. Michel, A. V. Rosario, P. M. Andrews, D. M. Goldenberg, and M. J. Mattes Therapy of Small Subcutaneous B-Lymphoma Xenografts with Antibodies Conjugated to Radionuclides Emitting Low-Energy Electrons Clin. Cancer Res., January 15, 2005; 11(2): 777 - 786. [Abstract] [Full Text] [PDF]
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J. D. Burton, S. Ely, P. K. Reddy, R. Stein, D. V. Gold, T. M. Cardillo, and D. M. Goldenberg CD74 Is Expressed by Multiple Myeloma and Is a Promising Target for Therapy Clin. Cancer Res., October 1, 2004; 10(19): 6606 - 6611. [Abstract] [Full Text] [PDF]
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R. B. Michel, M. E. Castillo, P. M. Andrews, and M. J. Mattes In vitro Toxicity of A-431 Carcinoma Cells with Antibodies to Epidermal Growth Factor Receptor and Epithelial Glycoprotein-1 Conjugated to Radionuclides Emitting Low-Energy Electrons Clin. Cancer Res., September 1, 2004; 10(17): 5957 - 5966. [Abstract] [Full Text] [PDF]
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R. B. Michel, M. W. Brechbiel, and M. J. Mattes A Comparison of 4 Radionuclides Conjugated to Antibodies for Single-Cell Kill J. Nucl. Med., April 1, 2003; 44(4): 632 - 640. [Abstract] [Full Text] [PDF]
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R. B. Michel, R. Ochakovskaya, and M. J. Mattes Antibody Localization to B-Cell Lymphoma Xenografts in Immunodeficient Mice: Importance of Using Residualizing Radiolabels Clin. Cancer Res., August 1, 2002; 8(8): 2632 - 2639. [Abstract] [Full Text] [PDF]
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R. B. Michel and M. J. Mattes Intracellular Accumulation of the Anti-CD20 Antibody 1F5 in B-Lymphoma Cells Clin. Cancer Res., August 1, 2002; 8(8): 2701 - 2713. [Abstract] [Full Text] [PDF]
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R. Ochakovskaya, L. Osorio, D. M. Goldenberg, and M. J. Mattes Therapy of Disseminated B-Cell Lymphoma Xenografts in Severe Combined Immunodeficient Mice with an Anti-CD74 Antibody Conjugated with 111Indium, 67Gallium, or 90Yttrium Clin. Cancer Res., June 1, 2001; 7(6): 1505 - 1510. [Abstract] [Full Text] [PDF]
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, 
, and 
) in comparison
with a control nonreactive Ab labeled similarly (
, 
, 
, and
). Cells were incubated for 2 days with the labeled Ab at the
indicated concentration and then diluted 14.3-fold, and cell growth was
monitored for a total of 21 days. Results are shown for Raji (
