Evaluation of the Binding of Radiolabeled Rituximab to CD20



Evaluation of the Binding of Radiolabeled Rituximab to CD20
Volume 22, Number 4, 2007
© Mary Ann Liebert, Inc.
DOI: 10.1089/cbr.2007.371
Evaluation of the Binding of Radiolabeled Rituximab
to CD20-Positive Lymphoma Cells: An In Vitro
Feasibility Study Concerning Low-Dose-Rate
Radioimmunotherapy with the -Emitter 227Th
Katrine B. Melhus, Roy H. Larsen, Trond Stokke, Olav Kaalhus, Pål K. Selbo,
and Jostein Dahle
Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital,
Montebello, Oslo, Norway
Radioimmunotherapy (RIT) with the alpha-emitter 227Th is currently under evaluation. 227Th is conjugated to
the chimeric anti-CD20 monoclonal antibody rituximab, using the chelator p-isothiocyanato-benzyl-DOTA.
In this study, the binding of 227Th-DOTA-p-benzyl-rituximab to three different CD-20-positive lymphoma cell
lines, Raji, Rael, and Daudi, were evaluated. Equilibrium and kinetic binding experiments were used to determine binding parameters, including the association and dissociation rate constants, the equilibrium dissociation constants, and the total number of antigens for Raji, Rael, and Daudi cells. There were significant differences between the cell lines with respect to both Kd and the total number of antigens. Rael cells had more
than three times as many antigens as the other two cell lines, and the functional Kd found for Rael cells was
significantly higher than that found for Raji and Daudi cells. These results were confirmed using flow cytometry. Rituximab was found to be localized in patches on the cell membrane. The findings indicated that
227Th-labeled rituximab has relevant antigen-targeting properties for radioimmunotherapy.
Key words: -particle-radioimmunotherapy, antibody-binding parameters, antibody affinity,
Monoclonal antibodies (MAbs) labeled with alpha-emitting radionuclides are a new potential
treatment modality of disseminated and metastatic cancer.1–3 Alpha particles have a short track
length of typically less than 100 m, high relaAddress reprint requests to: Jostein Dahle; Department of
Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital; Building J, Montebello, 0310 Oslo,
Norway; Tel.: 47 22 78 12 03; Fax: 47 22 78 12 07
E-mail: [email protected]
tive biological effectiveness, and a low oxygen
enhancement ratio, which may give therapeutic
advantages over radiation qualities of lower linear energy transfer (LET).4 However, the alphaemitting radionuclides tested so far are challenging to apply in clinical settings because of their
short half-lives, low production capability, and/or
limited supply of source material.2,5 Cationic radium-223 (t1/2 11.4 d) has shown considerable
promise in clinical trials in patients with skeletal
metastases.6,7 Although 223Ra can be stably retained in liposomes, it cannot currently be stably
conjugated to mAbs.8,9 We are now testing
whether 227Th, the parent of 223Ra, is suitable for
radioimmunotherapy (RIT). Thorium-227 has a
half-life of 18.7 days and can be conjugated to
rituximab by using the bifunctional chelator pisothiocyanato-benzyl-DOTA. Long-term operating generators for 227Th can be prepared. 227ThDOTA-p-benzyl-rituximab is stable both in vitro
and in vivo.10 Low initial activity levels of 227ThDOTA-p-benzyl-rituximab inhibit growth of
CD20 positive cells in vitro.10 The long half-life
of 227Th may permit the administration and targeting of a 227Th-labeled radioimmunoconjugate
(RIC) to occur before a significant amount of
223Ra is generated. The relatively long half-life
of 223Ra, in turn, assures that this nuclide would
be largely excreted or trapped in skeletal hydroxyapatite before decay occurs.6 Although a
considerable amount of 223Ra is taken up in
bone,10 this will probably not cause bone marrow
toxicity because of the short range of the alphaparticles.11 Thus, a therapeutic window may exist, which permits therapy with 227Th.
To assess the potential of 227Th in -RIT, the
binding to antigen-positive cells is important to
evaluate. Because of the relatively long half-life
of 227Th, the dose rate would probably have to be
lower than for other -emitters considered for therapy. It is, therefore, necessary to have a high affinity RIC and a specific activity that ensures that
enough 227Th-atoms decay at the cells. This paper
refers to CD20-positive lymphoma cells targeted
with rituximab as the experimental system.
Cell Lines
Three B-cell lymphoma lines, Raji, Rael, and
Daudi, and one B-cell leukemia line, Reh, were
used. The B-cell lymphoma cell lines express the
CD20 antigen, whereas the Reh cells are CD20
negative and were used as control cells. Single
cell suspensions were grown in RPMI 1640
medium (PAA; Linz, Austria), supplemented
with 13% heat-inactivated fetal calf serum (FCS)
(PAA), 1% L-glutamine (PAA), and 1% penicillin-streptomycin (PAA) in a humid atmosphere
with 95% air/5% CO2.
and the conjugation of 227Th-p-isothiocyanato-benzyl-DOTA to rituximab has been described before.10 Briefly, 227Ac was produced through the
thermal neutron irradiation of 226Ra followed by decay of 227Ra (t1/2 42.2 m) to 227Ac.3 227Th was
selectively retained from a 227Ac decay mixture in
7 M of HNO3 solution by anion exchange chromatography.8 After 227Ac, 223Ra and its daughters
had eluted from the column, and 227Th was extracted from the column with 12 M of HCl. The
227Th, prepared this way, was more than 99.93%
pure. The 227Th-DOTA complex was prepared by
adding 20 L of a 10-mg/mL solution of p-SCNbenzyl-DOTA (Macrocyclics Inc.; Dallas, TX) to a
solution containing 20 L (150 mg/mL) of L-ascorbic acid (Sigma-Aldrich Co. Ltd; Gillingham, UK)
and 20–150 L of 100–300 MBq/mL of 227Th in
HCl in a 2-mL glass vial. Then, 1–2 mg of rituximab in 100–200 L was added to the 227Th and
p-SCN-benzyl-DOTA reaction. Thereafter, the reaction mixture was incubated for 40 minutes at approximately 55°C by using a Thermomixer Comfort (Eppendorf AG; Hamburg, Germany). The
solution was cooled down to 37°C, and the pH was
adjusted to 8–9 by adding 1 M of NaCO3/NaHCO3
buffer. Then, the rituximab was added. After 45
minutes of reaction, 20 L of saturated diethylene
triamine pentaacetic acid (DTPA, Fluka Chemie,
Buchs, Switzerland) was added and the mixture further incubated for 5 minutes. Thereafter, the reaction mixture was purified by gel filtration (EconoPac10 DG; Bio-Rad, Hercules, CA) and eluted with
1% bovine serum albumin (BSA; Sigma Chemical
Co., St Louis, MO) in phosphate-buffered saline
PBS; pH, 7.4. Finally, the purified product was sterile filtered (Millex GV-13; Millipore Co., Bedford,
MA) into a sterile 10-mL glass vial (Wheaton; Millville, NJ), which was subsequently capped with a
sterile rubber cap (Sigma; St. Louis, MO). The specific activity of the RIC was typically 650–5300
Quantification of
Quantification of 227Th can be problematic in the
presence of 223Ra. Therefore, the RIC was always
purified by gel filtration, as described above, before the binding experiments. The activity was
counted in a gamma counter (Cobra AutoGamma; Packard; Downers Grove, IL).
Radiolabeling of Rituximab
The mAb rituximab (MabThera®; Roche, Basel
Switzerland) targeting CD20 was labeled with
227Th. The preparation of 227Th-DOTA complexes
Measurement of Immunoreactive Fraction
The quality of the RIC was measured using lymphoma cells and cell concentrations up to 108 Raji
cells/ml to compensate for the modest specific
activity and counting efficacy of the RIC.10,13
The immunoreactive fraction (IRF) of 227Thp-isothiocyanato-benzyl-DOTA-rituximab was
between 50 and 70%.
Equilibrium Binding Experiments
Specific binding was measured at seven different concentrations of RIC to determine the total number of antigens, Bmax, and the equilibrium dissociation constant, Kd, by Scatchard
analysis.14,15 Two (2) million cells in 0.4 mL
of PBS were incubated with 10–50,000 ng/mL
of RIC for 2 hours in a shaker at 4°C. Then, the
cells were washed twice with PBS containing
1% FCS, and the activity was counted in a
gamma counter (Packard). Both cells and supernatant were counted. Nonspecific binding
was measured by blocking with cold rituximab.
The analysis of equilibrium binding experiments by the Scatchard method has been explained before.14,15 Briefly, specific binding, B,
divided by the concentration of free antibody,
[Ab], is plotted versus specific binding. The
slope of this plot equals 1/Kd and the intercept with the x-axis gives the total number of
antigens, Bmax, directly:
B [Ab]
Binding of Cold Rituximab
Cells were incubated with unlabeled rituximab
(Roche, Basel, Switzerland) or herceptin (Roche)
(irrelevant mAb) for 1 hour in PBS at 4°C, and
washed twice with PBS. Then, the cells were incubated with fluorescein isothiocyanate (FITC)labeled mouse-anti-human Ab (BD Biosciences;
Erembodegen, Belgium) for 0.5 hours in PBS at
4°C. Subsequently, the cells were washed twice,
and the fluorescence was measured on viable
cells in a FACSCalibur flowcytometer (BD Biosciences). Dead cells were excluded by adding
propidium iodide (PI; Calbiochem, La Jolla, CA)
to the cells directly before flow cytometry and
gating on the PI-negative cells.
Fluorescence Labeling of Rituximab
Rituximab was labeled with the fluorescing dye,
Alexa Fluor 488 (hereby named Alexa-rituximab), according to the instructions of the manufacturer (Sigma).
Measurements of Binding Using
Raji and Rael cells were incubated with 0.1
g/mL of Alexa-rituximab for 24 hours at 37°C.
The cells were then washed twice with PBS and
studied by using 100 magnification and a fluorescence microscope (AX70; Olympus, Hamburg, Germany) equipped with a charge-coupled
device (CCD) camera. PI was used to exclude
dead cells.
In another experiment, 5 million Rael cells
were stained with 2 g/mL of Hoechst 33342 for
0.5 hours at 37°C, then washed and mixed with
5 million unstained Raji cells. Subsequently, the
cell suspension was incubated with 0.01–100
g/mL of Alexa-rituximab for 1 hour at 4°C, then
washed and analyzed by flow cytometry by gating on the Hoechst 33342 fluorescence (Stokke,
unpublished date). The experiment was performed three times and each time the cell line
stained with Hoechst 33342 was switched.
Dissociation Rate Constant
The dissociation rate constant kd was found by
allowing RIC to bind to cells for 1.5 hours at 4°C,
and then further binding was blocked by adding
unlabeled rituximab in excess. Five (5) million
cells per mL of PBS with 50–300 ng/mL of RIC
were incubated for 1.5 hours and washed three
times with PBS and then resuspended in fresh
medium with 100 g/mL of unlabeled rituximab.
Activity was counted in a gamma counter
(Packard) before the addition of unlabeled rituximab. Subsequently, three 0.4-mL aliquots were
subtracted at several time points, and cell-bound
activity was measured. Nonspecific binding was
measured, using the same cells blocked with 25
g/mL of rituximab. The dissociation rate constant was determined by plotting total binding
versus time and fitting the data to Equation 2 using Sigma plot (version 7; SPSS; San Jose, CA).
Btot Bunspecific a ekd t b ekd2 t
Association Rate Constant
Two (2) million cells in 0.4 mL of PBS were incubated from 5 minutes to 3.5 hours at 4°C with
50–300 ng/mL of RIC, washed twice with PBS
as for the Scatchard analysis, and binding was
measured in a gamma counter (Cobra AutoGamma, Packard). Nonspecific binding was measured by using the same cells blocked with 25
g/mL of rituximab. The observed rate of asso471
ciation, kobs, was determined by plotting specific
binding versus time and fitting the data to Equation 3, using the Sigma plot (version 7, SPSS):
B Ymax (1 ekobs t )
Here, kobs is a measure of how long it takes to
reach equilibrium between association and dissociation. Thus, it depends on the association
rate, ka, the dissociation rate, kd, and the start concentrations of Ab and Ag, [Ab]0 and Bmax, respectively. That is, kobs ka([Ab]0 Bmax) kd. Consequently, an expression for ka can be
kobs kd
ka (4)
[Ab]0 Bmax
Equations 3 and 4 can also be found as approximations of the solution of the differential equation describing the net rate of formation of the
antigen-antibody complex.16
mean number of antigens, Bmax, and the mean
equilibrium dissociation constant, Kd, for the
three different cell lines.
The specific activity obtained for 227ThDOTA-p-benzyl-rituximab was typically approximately 1 kBq/g, which was low, compared
with most radioimmunoconjugates.1–3 However,
in the prescribing information for rituximab, the
affinity is 8 nM, which is similar to our data for
Raji and Daudi cells, indicating that the low specific activity and the labeling procedure did not
alter the binding properties of the antibody. Furthermore, results from the Scatchard experiments
with 125I-rituximab did not differ considerably
from the results for 227Th-DOTA-p-benzyl-rituximab (not shown). Thus, the values found for
227Th-DOTA-p-benzyl-rituximab seem to be of
the right order of magnitude.
Significant differences were found between the
Rael cell line and the two other cell lines. There
were 2.5 times more available antigens on the
Rael cells than on the Raji and Daudi cells, and
the Kd was 2.3 times higher for Rael cells than
for Raji and Daudi cells.
Scatchard Analysis
The specific activity of a RIC affects the measurements of IRF and may also affect the measurements of affinity and number of binding sites.
Affinity and number of binding sites were measured using the Scatchard analysis and three different CD20-positive cell lines: Raji, Rael, and
Daudi. Figure 1 shows representative Scatchard
experiments, and Table 1 shows the resulting
Binding of Unlabeled Rituximab and
Alexa-Rituximab to CD20-Positive
Lymphoma Cells
The binding of unlabeled rituximab and AlexaRituximab was assessed using flow cytometry
and fluorescence microscopy. Figure 2 shows the
binding of 0.1 g/mL of unlabeled rituximab to
Raji and Rael lymphoma cells. There was no difference in fluorescence signal from herceptin,
rituximab, or secondary antibody-labeled CD20-
Figure 1. Scatchard analysis. Representative plots of specific binding/free antibody (Ab) concentration versus number of antigens per cell for (A) Raji, (B) Rael, and (C) Daudi cells labeled with 227Th-DOTA-p-benzyl-rituximab.
Table 1. Number of Available Antigens (Bmax, Mean Standard Error [SE]) and the Equilibrium Dissociation Constant
(Functional Kd, Mean SE) of Raji, Rael, and Daudi Cells Measured Using 227Th-DOTA-p-Benzyl-Rituximab and the
Scatchard Method
Bmax (antigens/cell)
Functional Kd (nM)
248.000 34.000
8.1 1.9
637.000 73.000
18.7 2.5
211.000 49.000
6.9 1.3
and Kd for Rael cells was significantly higher than for Raji cells and Daudi cells (p 0.01; Tukey test).
Figure 2. Specific binding of rituximab to lymphoma cells (Raji and Rael) and not to leukemia cells (Reh). (A) Reh cells, (B)
Raji cells, or (C) Rael cells were incubated with no primary antibody (grey fill), 0.1 g/mL of cold rituximab (solid black line),
or 0.1 mg/mL of cold herceptin (solid green line), washed and costained with fluorescein isothiocyanate (FITC)-conjugated mouseanti-human secondary antibody. Fluorescence was measured using flow cytometry. (D) Mean values of fluorescence from five
experiments. * significantly different form Rael (t test, p 0.05). Error bars standard deviation.
negative Reh cells (Fig. 2A). Raji and Rael cells
labeled with Rituximab fluoresced significantly
stronger than cells labeled with herceptin or the
secondary antibody only (Fig. 2B and 2C, t test,
p 0.05). The fluorescence signal from Raji
cells was significantly stronger than from Rael
cells. Similar results as for Raji cells were found
for Daudi cells (not shown). The antigen expression for all three cell lines resembled log normal
distributions and varied with at least a factor of
50 between the cells with the lowest and the cells
with the highest antigen expression. The antigen
expression of rituximab-labeled cells overlapped
with the herceptin background distribution for the
lower fluorescence intensities.
Figure 2D shows that the mean numbers of
bound antibodies was higher for Raji cells than
for Rael cells at 0.1 g/mL of rituximab. This
was not in agreement with the data found by the
Scatchard analysis (Table 1), where Rael cells
were shown to have the largest number of antigens. To investigate the difference between the
Scatchard analysis and the flow cytometry data
with regard to the number of antigen sites, Alexaconjugated rituximab was used. The concentration of Alexa-rituximab was varied over 5
decades, and Rael and Raji cells were stained simultaneously for 1 hour to avoid any differences
in staining conditions (Fig. 3). One of the cell
lines was labeled with Hoechst 33342 to distinguish the two cell lines by gating on DNA fluorescence. Figure 3 shows that for low concentrations of rituximab (below 1 g/mL), the
fluorescence intensity (e.g., the number of bound
antibodies) was higher for Raji cells than for Rael
cells, while opposite for concentrations above 1
g/mL. For the experiments with cold rituximab
(Fig. 2), a concentration of 0.1 g/mL of rituximab was used. For the Scatchard analysis, the
highest concentrations are the most important for
the determination of the maximum number of
antigens. Thus, the data in Figure 3 reproduce the
difference between Figure 2 and Table 1. A
Scatchard analysis of the Alexa-rituximab experiments was performed (Fig. 3B) and resulted in
Kds of a similar order of magnitude as with radioactive rituximab: Kd was 13 nM for Raji cells
and 45 nM for Rael cells, and Bmax was more
than twice as large for Rael cells than for Raji
Binding was also studied at 37°C by using
Alexa-conjugated rituximab and fluorescence microscopy (Fig. 4). Neither of the cell lines internalized Alexa-rituximab, which was localized to
Figure 3. Binding of rituximab to Raji and Rael cells as
a function of concentration of Alexa-rituximab. (A) Rael
cells were stained with Hoechst 33342, mixed with unstained Raji cells, and then the different cells were incubated
with Alexa-rituximab in the same vial. Propidium iodide was
used to gate away the dead cells. There was no unspecific
binding to Reh cells (not shown). For Alexa-rituximab concentrations above 1 g/ml, Raji cells fluoresced with a lower
intensity than Rael cells (p 0.01), whereas it was the other
way around for lower concentrations (p 0.03). A Student
t test was used to test for significance. (B) A Scatchard plot
of the data in (A). Kd 13 and 45 nM for Raji and Rael
cells, respectively. Bmax 32.400 and 75.600 (arbitrary
units) for Raji and Rael cells, respectively.
patches on the plasma membrane (results from
Rael and Daudi cells not shown).
Kinetic Binding Experiments
To further characterize, 227Th-DOTA-p-benzylrituximab, we also measured the dissociation rate
constant (Fig. 5; Table 2) and the association rate
constant (Fig. 6; Table 2) and calculated the Kd kd/ka (Table 2) for viable cells. The dissociation
seemed to be biexponential; in the first hours after the addition of cold antibody, the hot antibody
dissociated relatively faster than for later time
Figure 4. Subcellular localization of rituximab. Raji cells were incubated with 0.1 g/mL of Alexa-rituximab for 24 hours at
37°C. (A) Phase contrast image of four Raji cells shows a difference in cell size. (B–D) Fluorescence images from the lower to
upper focal plane shows that the rituximab is located heterogeneously in the plasma membrane.
points (Fig. 5). The half-life for the first phase of
dissociation was 1.8 hours and 46 minutes,
whereas it was 10.3 hours and 28.7 hours for the
second phase, for Raji and Rael cells, respectively. Notably, Figure 5 shows that even 2 days
after adding cold rituximab, in excess of
10%–20% of the RIC was still binding to both
types of cells. This retention might have been the
result of the internalization of at least a portion
of the antibody. Furthermore, the dissociation
rate constants determined in this paper were
found by adding a large amount of unlabeled an-
tibody to inhibit further binding. Thus, the dissociation might be even slower, as an eventual
fraction of monovalently bound hot antibody
could be competed out by the cold antibody. Additionally, a bivalently bound antibody is probably continuously wobbling, such that one arm
becomes transiently detached. Therefore, the dissociation rate constant might depend on the
amount of unlabeled antibody added. To prevent
this artifact, the dissociation rate constant might
instead be measured after a large dilution of the
reaction mixture.
diffusion of antibody might still have limited the
association of antibody with antigen.
Figure 5. Dissociation rate constant. Decreasing number
of counts per minute (cpm) as a function of time after the
addition of 100 g/mL of cold rituximab to (A) Raji or (B)
Rael cells in equilibrium with three different concentrations
of 227Th-p-benzyl-DOTA-rituximab. The cells were incubated with 227Th-p-benzyl-DOTA-rituximab for 1.5 hours
to reach equilibrium. Then, the cells were washed and 100
g/mL cold rituximab was added. The cells were washed
before each measurement. Mean unspecific binding to Reh
cells for all time points were 25 cpm for 50 ng/mL, 24 cpm
for 100 ng/mL, and 36 cpm for 300 ng/mL. Unspecific binding did not vary significantly with time. Error bars standard deviation.
Equilibrium was assumed to be reached in the
Scatchard experiments after 2 hours. Therefore,
the dissociation rate constant for the first phase
of dissociation (kd in Equation 2) was used to calculate Kd. The Kd found by the Scatchard analysis has been termed functional Kd, and it was 41
times larger than Kd for Raji cells and 6 times for
Rael cells (Table 2).
The association rate constants observed were
slower than one might expect at the relatively
high concentrations used. The cells were incubated during continuous shaking, but the slow
In this study, we measured the binding parameters of 227Th-DOTA-p-benzyl-rituximab, a new
potential RIC against CD20-positive lymphomas.
We have found similar affinities using the
Scatchard analysis, as given in the prescribing information for rituximab and reported by others,17
which shows that labeling with 227Th does not
significantly damage the antibody. The measured
affinity was confirmed by using Alexa-labeled
rituximab and flow cytometry. Fluorescence microscopy of rituximab bound to lymphoma cells
showed no internalization of antibody during the
first 24 hours of incubation, but indicated a heterogeneous distribution of antigens on the cell
surface. Flow cytometry showed a wide range of
antigen distribution between the cells.
The mean number of available binding sites
was at least twice as high for Rael cells as for
Raji and Daudi cells (Table 1), and the distribution of available antigens resembled a log normal
distribution (Fig. 2). The cells in Figure 3 were
DNA stained, which made information about cell
cycle available. However, the variation in the
number of antigens was not related to position in
the cell cycle (not shown). For therapy, the cells
with the lowest number of available binding sites
are of particular interest, as they may result in tumor regrowth. For low specific activity, antigen
saturation may occur, preventing a sufficient
number of radioactive molecules to attach to the
cell. Microbeam experiments have shown that to
Table 2. Dissociation Rate Constant (Kd), Association
Rate Constant (Ka), Kinetic Dissociation Constant (Kd),
and Functional Equilibrium Dissociation Constant
(Functional Kd) for Raji and Rael Cells Labeled with
Cell line
0.4 0.3 1.9 0.5
0.9 0.8 0.3 0.1
Functional kd
8.1 1.9
18.7 2.5
0.2 0.2
3.3 3.3
calculated using kd and ka.
of Rael cells was significantly higher than the Kd of
Raji cells.
Figure 6. Association rate constant. Increasing number of
counts per minute (cpm) as a function of time after the addition of three different concentrations of 227Th-p-benzylDOTA-rituximab to (A) Raji or (B) Rael cells. The cells
were washed before measurement and unspecific binding to
Reh cells was subtracted before plotting. Error bars standard deviation.
obtain a 1% cell survival probability, 21 alpha
particles on average have to hit the nucleus,18
which fits well with microdosimetry calculations
of the alpha-RIT of lymphoma cells11 and with
simulations of tumor-control probability.19 Lymphoma cells have a relatively large nucleus,
which ensures that approximately one third of the
alpha-particles emitted from the cell surface will
hit the nucleus.20 Thus, at least 63 227Th-rituximab conjugates have to decay at the cell surface
to get a cell kill probability of 99%. Hence, assuming log normal distributions of available antigens, the number of antigens in the 0.14% (mean3) of cells with the lowest number of antigens
was calculated. For these cells, a specific activity of 2375 Bq/g for Raji cells, 1275 Bq/g for
Rael cells, and 6000 Bq/g for Daudi cells was
necessary to obtain 63 227Th-rituximab conju-
gates on the cell surface (Table 3). We have previously shown that the incubation of cells with
only 200 Bq/mL of 227Th-rituximab significantly
inhibited the growth of Raji cells.10 This corresponded to only 0.2 g/mL of 227Th-rituximab
(specific activity, 1000 Bq/g), which means
that, on average, approximately Bmax/7 antigens
had bound antibody (Fig. 3). For the 0.14% cells
with the lowest number of antigens, this means
approximately four 227Th atoms per Raji cell. In
addition, daughter cells get half of the 227Th-rituximab of the mother, so for the calculations
above to be valid, we have to assume no tumor
growth occurred during the decay of 227Th. However, because 227Th-rituximab has a long biological half-life in vivo (5.3 days in mice10), tumor
cells undergoing division may later be irradiated
owing to a continuous association of RIC from
the pool of circulating compound.
For low concentrations of rituximab, a larger
amount of the antigens on Raji cells versus Rael
cells had bound rituximab (Fig. 2D) and vice
versa at concentrations above 1 g/mL (Fig. 3).
This result may be explained by differences in
the association rate for the two cell lines. For low
concentrations of antibody, only a small fraction
of the available antigens is occupied by antibody
and it takes a longer time to reach equilibrium.
Thus, if the association constant is higher on the
Raji cells than on the Rael cells, the Raji cells
will bind antibody faster and thus appear to have
Table 3. The 0.14% Limit of Number of Antigens and
the Specific Activity Necessary to Get 63227Th Decays
Per Cell
Number of antigensa
Specific activity (Bq/g)b
aAssuming a log normal distribution with a standard deviation of FWHM/2(2ln2), the percentage of cells with
the lower number of available binding sites than
2 is 0.14%.
e 3 2
bSpecific activity [#227Th] T1/2
2 ,
e 3 NA
where T1/2 is the half-life of 227Th, Mw, is the molecular
weight of rituximab, and NA is Avogadro’s number.
more antigen sites than the Rael cells. For higher
concentrations of antibody, it takes a shorter time
to reach equilibrium and then kinetics have less
impact. We have found that the association rate
was seven times higher for Raji cells than for Rael
cells (Table 2), so this explanation might be the
answer. Furthermore, it is tempting to speculate
that the reason for the lower ka and higher kd of
the CD20 antigen on Rael cells than on Raji cells
might be related to differences in organization of
the antigens on the cell surface. Other explanations may be mutation in the CD20 gene or posttranslational modifications of the CD20 protein
in either of the cell lines.
There was a large difference between the
functional Kd and Kd determined by the kinetic
experiments, which might indicate that the radioimmunoconjugate bound irreversibly, and
consequently, had an almost infinite affinity or
Kd almost equal to zero. If kd2 (Equation 2) was
used, the values of Kd were approximately
1 1015 M for both Raji and Rael cells. Thus,
the difference between functional Kd and Kd
were certainly larger than an order of magnitude. The reason for this difference might be
the disagreement between the assumption about
equilibrium that is the basis for the Scatchard
analysis and the two-phase dissociation of the
antibody conjugate. These results imply that the
Scatchard analysis does not always give reliable estimates of antibody affinity.
The findings of this study show that 227ThDOTA-p-benzyl-rituximab has relevant antigenbinding properties for use in targeted -immunotherapy against CD20-positive lymphomas
and that the association and dissociation properties of the antigen-antibody complex is compatible with the relatively long half-life of 227Th.
Thus, low-dose-rate -RIT might be a promising
strategy against CD20-positive tumors.
We are grateful to Jørgen Borrebæk of Algeta
ASA, Oslo, Norway, for the production of 227ThDOTA-p-benzyl-rituximab. This study was financed by the MEDKAP program of The Norwegian Research Council.
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