ANTICANCER RESEARCH 33: 85-96 (2013)
Evaluating Antigen Targeting and Anti-tumor Activity
of a New Anti-CD37 Radioimmunoconjugate
Against Non-Hodgkin’s Lymphoma
JOSTEIN DAHLE1, ADA H.V. REPETTO-LLAMAZARES1, CAMILLA S. MOLLATT2,
KATRINE B. MELHUS2, ØYVIND S. BRULAND3,4, ARNE KOLSTAD4 and ROY H. LARSEN5
1Nordic
Nanovector AS, Oslo, Norway;
of Radiation Biology, Institute for Cancer Research, and
3Oncology, The Norwegian Radium Hospital, Oslo, Norway;
4Insititute for Clinical Medicine, University of Oslo and 5Sciencons Ltd., Oslo, Norway
2Departments
Abstract. The monoclonal antibody against CD20, rituximab,
alone, or as part of combination therapies, is standard therapy
for non-Hodgkin’s B-cell lymphoma. Despite significantly better
clinical results obtained for beta-emitting radioimmunoconjugates (RICs), RICs targeting CD20 are not commonly used
in medical practice, partly because of competition for the CD20
target. Therefore, novel therapeutic approaches against other
antigens are intriguing. Here, the binding properties of a novel
antibody against CD37 (tetulomab) were compared with those
of rituximab. The therapeutic effect of 177Lu-tetulomab was
compared with 177Lu-rituximab on Daudi cells in vitro. The
biodistribution, therapeutic and toxic effects of 177Lu-tetulomab
and unlabeled tetulomab were determined in SCID mice injected
with Daudi cells. The affinity of tetulomab to CD37 was similar
to the affinity of rituximab to CD20, but the CD37-tetulomab
complex was internalized 10-times faster than the CD20rituximab complex. At the same concentration of antibody,
177Lu-tetulomab was significantly more efficient in inhibiting cell
growth than was 177Lu-rituximab, even though the cell-bound
activity of 177Lu-rituximab was higher. Treatment with 50 and
100 MBq/kg 177Lu-tetulomab resulted in significantly increased
survival of mice, compared with control groups treated with
tetulomab or saline. The CD37 epitope recognized by tetulomab
was highly expressed in 216 out of 217 tumor biopsies from
patients with B-cell lymphoma. This work warrants further preclinical and clinical studies of 177Lu-tetulomab.
This article is freely accessible online.
Correspondence to: Jostein Dahle, Nordic Nanovector AS,
Kjelsåsveien 168 B, 0884 Oslo, Norway. Tel: +47 98458850, Fax:
+47 22580007, E-mail: [email protected]
Key Words: Radioimmunotherapy,
lymphoma, 177Lu.
0250-7005/2013 $2.00+.40
CD37,
non-Hodgkin’s
Beta-emitting radioimmunoconjugates have significant
antitumor activity in patients with relapsed or refractory B-cell
lymphoma (1-4), including those refractory to rituximab, a
monoclonal antibody to CD20 (5, 6) and chemotherapy (7).
Radioimmunotherapy (RIT) is administered with large
quantities of unlabeled “cold” antibody to CD20 one week
and 4 h prior to radiolabeled antibodies to CD20. Such a
priming dose is necessary to reduce antibody binding to
normal B-cells by depleting peripheral blood B-cells and
lymph node B-cells (8, 9). Thus, sufficient amounts of
radiolabeled antibody can bypass these sites, penetrate lessaccessible compartments, such as the lymph nodes, and
target tumor cells. However, both clinical and experimental
studies in mice have shown that in some circumstances, even
quite low rituximab concentrations in the blood can reduce
tumor cell targeting and thus impair the clinical efficacy of
CD20-directed RIT (10). Furthermore, the majority of
patients selected for CD20-based RIT have received several
cycles of “cold” rituximab, which may result in selection of
tumor cells with low CD20 expression and thus could lower
the effect of subsequent anti-CD20 treatments (11-13).
By targeting B-cell antigens, other than CD20, a better
response might be achieved. CD37 is a heavily glycosylated
40- to 52-kDA glycoprotein and a member of the tetraspan
transmembrane family of proteins (14, 15). CD37
internalizes and has modest shedding in transformed B-cells
expressing the antigen (16, 17). During B-cell development,
CD37 is expressed in cells progressing from pre-B to
peripheral mature B-cell stages and is absent on terminal
differentiation to plasma cells (18). Given its relative B-cell
selectivity, CD37 thus represents a valuable target for
therapies in chronic lymphocytic leukemia (CLL), hairy-cell
leukemia (HCL), B-cell non-Hodgkin’s lymphoma (NHL)
and other B-cell malignancies.
CD37 has been previously studied as a target for RIT
using 131I-labeled MB-1 (16). Recently, there has been a
85
ANTICANCER RESEARCH 33: 85-96 (2013)
revival in interest in the targeting of CD37. Both an Fcengineered monoclonal antibody and a small modular
immuno-pharmaceutical have been developed for the
targeting of CD37 in CLL (19, 20).
At the Norwegian Radium Hospital a murine IgG1
antibody, HH1 (tetulomab, according to the nomenclature for
naming of antibodies), was developed against CD37 in the
1980s (21). In the present study we investigated binding to
different subtypes of NHL and compared binding properties
of tetulomab with the chimeric IgG1 antibody rituximab.
Furthermore, the therapeutic effects of 177Lu-tetulomab,
177Lu-rituximab, “cold” tetulomab and “cold” rituximab was
compared in vitro. Increasing concentrations of 177Lutetulomab were used to treat SCID mice injected intravenously
with human Daudi lymphoma cells. This animal model (22), is
a worst-case scenario model of NHL because the tumor cells
will, to a large degree, end up in the bone marrow. This
localization will result in irradiation of bone marrow cells and
corresponding hematological toxicity. The biodistribution of
antibodies in SCID mice may be different from that in other
mouse strains because of low concentrations of endogeneous
antibodies in the blood (23, 24). In addition, because of the
SCID mutation this mouse strain cannot repair DNA doublestrand breaks and is thus very sensitive to ionizing radiation
(25). Regardless of the limitations of this model, a significant
therapeutic effect of 177Lu-tetulomab was shown at a dosage
level resulting in tolerable toxicity.
Materials and Methods
Cell lines. The CD20- and CD37-expressing B-cell lymphoma cell
lines Raji, Rael and Daudi were used in the current study (LGC
Standards, Boras, Sweden). Single-cell suspensions were grown in
RPMI 1640 medium supplemented with 10 % heat-inactivated FCS,
1% L-glutamine and 1% penicillin-streptomycin (all from PAA,
Linz, Austria), in a humid atmosphere with 95% air/5% CO2.
Labeling of antibodies with 177Lu. Tetulomab and rituximab were first
labeled with a chelator, 2-(4-isothiocyanatobenzyl)-1,4,7,10
tetraazacycododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA,
DOTA) (Macrocyclics, Dallas, TX, USA). DOTA was dissolved in
0.05 M HCl, added to the antibody in a 5:1 ratio and the pH adjusted
to 8.5-9 by washing with carbonate buffer using AMICON-30
centrifuge tubes (Millipore, Cork, Ireland). The solution was shaken
for 60 min at room temperature, and the reaction was terminated by
adding 50 μl of 200 mM glycine solution (per mg antibody). To
remove free chelator, the conjugated antibody was centrifuged 4-5times with phosphate buffered saline (PBS) (PAA) at 1:10 dilution
using AMICON-30 centrifuge tubes (Millipore). Before labeling with
177Lu (Perkin Elmer, Boston, MA, USA) the PBS buffer was changed
to ammonium acetate buffer, pH 5.4, using the same centrifuge tubes.
177Lu was then added to 0.5 mg DOTA-Ab, and shaken for 45 min at
37ºC. Specific activity was between 50 and 150 MBq/kg.
Immunoreactivity. The quality of the radioimmunoconjugates was
measured using lymphoma cells and a modified Lindmo method
86
(26). Cell densities of up to 3×108 cells/ml were used. All conjugates
used in experiments had immunoreactivity greater than 50 %.
Binding parameters. The association rate constant, ka, and the
equilibrium dissociation constant, Kd, were measured for tetulomab
and rituximab, and the mean number of binding sites, Bmax, was
determined for Raji, Rael and Daudi cells using a one-step curve
fitting method (27). Specific binding was measured as a function of
time and antibody concentration, and the solution of the differential
equation describing the net rate of formation of the antigen-antibody
complex was fitted to the experimental data points using ka, Kd and
Bmax as parameters. Five million cells/ml were used, with four
concentrations of “hot” antibody (100 ng/ml, 1000 ng/ml, 5000 ng/ml
and 10000 ng/ml) and seven incubation time points (5 min, 10 min,
20 min, 30 min, 1 h, 1.5 h and 2 h). After incubation, cells were
washed twice with PBS, and then counted in a gamma counter (Cobra
II; Packard Instrument Company, Meriden, CT, USA).
Internalization and retention of 177Lu-tetulomab and 177Lurituximab. Daudi cells were harvested, counted and diluted to a
density of 2×106 cells/ml and incubated with 1 μg/ml 177Lutetulomab, 177Lu-rituximab, 125I-tetulomab or 125I-rituximab. Some
parallels were pre-blocked with the corresponding “cold” antibody
(100 μg/ml). The cells were incubated for 0, 10, 20, 30, 60, 120 and
180 min. Subsequently, the cells were centrifuged and washed twice
with medium, and the supernatant and the wash medium were
collected for counting. Cells were incubated with 1 ml stripping
buffer (150 mM NaCl and 50 mM glycine, pH 2.6) for 10 min, at
room temperature. The cells were washed twice and the
supernatants and the cells were counted using a calibrated gamma
detector (Cobra II).
In another experiment, 106 Daudi cells/ml medium were
incubated with 1 μg/ml 125I- or 111In-labeled tetulomab or rituximab
for one hour, washed twice with medium and incubated further for
four days. The cell-bound activity was determined immediately after
washing and after four days of incubation by measuring the number
of cells (Vi Cell Viability Analyzer, Beckman Coulter, Fullerton,
CA, USA) and the amount of radioactivity with the gamma detector
(Cobra II).
Effect of 177Lu-tetulomab and 177Lu-rituximab on growth of Daudi
cells in vitro. One milliliter of Daudi cell suspension (1×106
cells/ml) was pipetted into each of 24 tubes. Six tubes each were
blocked with 100 μg/ml of either tetulomab or rituximab and
incubated for 30 min at 37˚C. Subsequently, either 177Lu-tetulomab
or 177Lu-rituximab was added to a final concentration of 0, 1, 2.5, 5,
10 or 20 μg/ml and the cells incubated further at 37˚C. The specific
activity was 91.6 kBq/μg for 177Lu-tetulomab and 136.6 kBq/μg for
177Lu-rituximab. The amount of added activity was measured during
the incubation period with a gamma detector (Cobra II). After two
hours, half of the cells were washed and the cell-bound activity was
measured, while the other half of the cells was incubated overnight
before washing and measurement of cell-bound activity. For
measurement of cell growth, 50,000 cells from each tube were
seeded in three wells in a 12-well plate and the number of cells was
measured at several time points over the next 14 days using an
automatic imaging system (Clone Select Imager, Molecular Devices
Ltd., New Milton, Hampshire, UK) that captured images of each
well, analyzed the images and calculated the number of cells in each
well. Growth delay was estimated as the difference in time between
Dahle et al: New Anti-CD37 Radioimmunoconjugate for NHL
exponential growth of the control cells and that of the treated cells.
Survival was estimated by extrapolating the exponential part of the
growth curve to the y-axis (28). Growth delay factor was calculated
by dividing the delay in growth to 100,000 cells/ml for cells treated
with 177Lu-tetulomab with the corresponding delay in growth for
cells treated with 177Lu-rituximab.
Biodistribution of 177Lu-tetulomab in severe combined immune
deficient (SCID) mice with intravenously injected Daudi cells.
Biodistribution of 177Lu-tetulomab was determined in female SCID
mice (NOD.CB17/Prkdc scid JHsd; Harlan Laboratories, An Venray,
The Netherlands) with intravenously-injected Daudi tumor cells.
The preparation was administered by tail vein injection of 260-1400
kBq in 100 μl solution in each animal, one week after an injection
of 10×106 Daudi cells. Four to five animals were used per time
point. Autopsies were performed after cervical dislocation at various
time points after injection. The weight of each tissue sample (blood,
lung, heart, liver, spleen, kidneys, stomach, small intestine, large
intestine, femur, muscle, brain, skull, lymph node and neck) was
determined, and 177Lu was measured by a calibrated gamma
detector (Cobra II). Samples of the injectate were used as references
in the measurement procedures. All procedures and experiments
involving animals in this study were approved by the National
Animal Research Authority and carried out according to the
European Convention for the Protection of Vertebrates used for
Scientific Purposes.
Therapeutic and toxic effect of 177Lu-tetulomab in SCID mice
intravenously injected with Daudi cells. Female, 4- to 8-week-old,
SCID mice with body weights in the range of 16- 23 g at the start of
the experiment, were used. The animals were maintained under
pathogen-free conditions, and food and water were supplied ad
libitum. All mice were ear-tagged and followed individually
throughout the study. Mice were injected intravenously with 10×106
Daudi cells in 0.1 ml PBS without Ca2+ and Mg2+ (PAA, Paasching,
Austria) one week before treatment with NaCl (N=23), 50 μg
tetulomab (N=14), 50 (N=10), 100 (N=10) and 200 (N=10) MBq/kg
177Lu-tetulomab. A pilot study was performed to validate 100 %
tumor formation following injection of 10×106 Daudi cells per
mouse. Mice were killed by cervical dislocation, if suffering from
hind leg paralysis (primary end point), body weight decreased by
20% from baseline, or if they otherwise showed symptoms of illness
and discomfort. The mice were dissected and histological sections
were stained with antibody against CD20 (EP459Y, Novus
Biologicals, Littleton, CO, USA) to locate tumor cells. The mice
were checked for hind leg paralysis every day and weighed two to
three times per week. The different treatment groups were compared
by Kaplan-Meier survival analysis using SPSS version 13.0 (SPSS,
Chicago, IL, USA).
Toxicity measurements. Prior to the study start and every two weeks
thereafter for up to 12 weeks, 50-75 μl blood was collected from
the vena saphena lateralis in 0.5 ml EDTA-coated tubes (BD
Microtainer K2E tubes; Becton, Dickinson and Company, Franklin
Lakes, NJ, USA). White blood cells, red blood cells and platelets
were counted using an automated hematology analyzer (Scil Vet abc
animal blood counter, ABX Diagnostics, Montpellier, France).
Patient samples. A total of 217 B-cell lymphoma biopsies from
patients treated at the Norwegian Radium Hospital were stained
Table I. The mean number of antigens (Bmax) on Raji, Rael and Daudi
cells, the equilibrium dissociation constant (Kd) and the association rate
constant (ka) for tetulomab and rituximab.
Antibody
Cell line
Tetulomab
Tetulomab
Tetulomab
Rituximab
Rituximab
Rituximab
Raji
Rael
Daudi
Raji
Rael
Daudi
aTetulomab
Bmax (Ag/cell)a
Kd (nM)
ka (nM–1h–1)
146000±7600
263000±27000
340000±5000
272000±69000
626000±36000
307000±12000
6.3±1.7
12.7±5.5
2.7±0.3
4.8±0.9
12.0±2.0
5.8±1.5
0.36±0.14
0.07±0.01
0.72±0.07
0.08±0.006
0.08±0.007
0.14±0.01
and rituximab bind to CD37 and CD20, respectively.
with the following antibodies (Dako, Glostrup, Denmark): anti-CD3
(UCHT-1), anti-IgGl [polyclonal rabbit anti-human lambda light
chains, rabbit F(ab’)2], anti-IgGk [polyclonal rabbit anti-human
kappa light chains, rabbit F(ab’)2], and anti-CD37 (in-house
tetulomab antibody). The expression of the different antigens was
measured using light microscopy after horseradish peroxidase
visualization (Dako).
Results
Binding properties of tetulomab and rituximab. The
association rate constant, ka, the equilibrium dissociation
constant, Kd, and the mean number of binding sites, Bmax,
were determined for three different cell lines, Raji, Rael and
Daudi, and for the tetulomab and rituximab antibodies (Table
I). For Raji and Rael cells, the number of CD37 antigens was
approximately half the number of CD20 antigens, while for
Daudi cells the number of antigens was similar. The Kd was
higher for Rael than for Raji and Daudi cells for both
antibodies. The Kd was similar for the two antibodies for
Raji and Rael cells, while for Daudi cells it was significantly
lower for tetulomab than for rituximab (t-test, p<0.05),
indicating that tetulomab had better affinity for the CD37
antigen than rituximab had for the CD20 antigen, in this cell
line. For Rael cells, ka was similar for tetulomab and
rituximab, while it was significantly higher for tetulomab
than for rituximab for Raji and Daudi cells, indicating faster
binding of tetulomab to CD37 than of rituximab to CD20 in
these two cell lines. The Daudi cells were chosen for further
study since they had the highest amount of CD37 antigens:
an average of 340,000 CD37 antigens and 307,000 CD20
antigens per cell.
Internalization of 177Lu-tetulomab and 177Lu-rituximab.
Tetulomab was internalized faster and to a greater extent than
rituximab (Figure 1). For the first half hour of incubation the
internalization speed was 0.19 fg/min/cell for tetulomab and
0.02 fg/min/cell for rituximab. There was no significant
difference between experiments performed with 125I and 177Lu.
Therefore, the results were pooled. In another experiment,
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ANTICANCER RESEARCH 33: 85-96 (2013)
Table II. Number of 177Lu atoms bound per Daudi cell after two and 18 h of incubation.
Incubation time
Antibody dosage
(μg/ml)
2 Hours
177Lu-tetulomab
Unblocked
0a
1
2.5
5
10
20
aThe
12
8318
9105
10025
13646
16290
Blocked
7
449
720
1837
3521
8473
18 Hours
177Lu-rituximab
Unblocked
8
8554
11629
13658
17344
30095
177Lu-tetulomab
Blocked
53
372
885
2019
2769
9709
177Lu-rituximab
Unblocked
Blocked
Unblocked
12
10327
11757
12123
11548
15233
5
301
787
1857
3205
5445
10
12831
18836
24097
24249
26639
Blocked
53
356
1385
1871
2860
5824
different counts for the control samples is indicative of the variation in background radiation of the counter.
however, of longer duration, Daudi cells were incubated with
125I- and 111In-tetulomab for 1 h, washed and cell-bound
activity was measured after four days of incubation (Figure 1,
insert). The amount of antibody bound to cells was higher for
111In-tetulomab than for 125I-tetulomab.
Cell-bound activity of 177Lu-tetulomab and 177Lu-rituximab.
Daudi cells were incubated with increasing concentrations of
177Lu-tetulomab or 177Lu-rituximab for two hours and 18 h
before washing. Cell-bound activity was measured and the
cells were seeded for further growth. The cell bound activity
was lower for cells incubated with tetulomab than for cells
incubated with rituximab after two-hour incubation and 18 h
incubation (Table II). However, radiolabeled tetulomab
saturated the antigen quicker and at lower antibody
concentration than did rituximab. The specific activity was
92 MBq/mg for 177Lu-tetulomab and 137 MBq/mg for
177Lu-rituximab, which explains the difference in the number
of 177Lu atoms attached to the cells for the two antibodies.
Non-specific binding (blocked) was similar for the two RICs.
At the 1 μg/ml dosage, there were almost no differences in
the number of specifically bound radioactive atoms for both
incubation times.
Cell growth experiments. Growth of Daudi cells incubated
with RICs for two hours before washing, was followed for
14 days (Figure 2 A and B). There was no effect of unlabeled
antibodies alone on cell growth (data not shown). The
blocked cells treated with the 177Lu-antibody clearly did not
grow as fast as the untreated control cells, indicating that
there was an effect of unbound 177Lu-antibody or a nonspecific bound 177Lu-antibody on the cells. Treatment of
unblocked cells with 177Lu-antibody resulted in an increase
in growth delay of 44% for cells treated with 10 μg/ml of
177Lu- tetulomab (Figure 2A) and of 31% for cells treated
with 10 μg/ml 177Lu-rituximab (Figure 2B), as compared
with blocked cells. For treatment with 20 μg/ml of 177Lu-
88
Figure 1. Internalization of tetulomab and rituximab in Daudi cells. The
insert figure shows cell-bound tetulomab 96 h after washing for
incubation of Daudi cells with 1 μg/ml 111In-tetulomab or 125Itetulomab. N=3. Error bars=standard deviation.
antibody, the difference between tetulomab and rituximab
was even larger, since there was no re-growth of the cells
treated with 177Lu- tetulomab.
Growth of Daudi cells incubated with RICs for 18 h
before washing was also followed for 14 days (Figure 2C
and D). There was no effect of unlabeled antibody alone on
cell growth (data not shown). The inhibition of cell growth of
the blocked cells treated with 177Lu-antibody was more
pronounced than when the cells were incubated for two
hours before washing, probably because of the 16-h
increased incubation time with RIC in the medium.
Treatment of unblocked cells with 177Lu-antibody resulted
Dahle et al: New Anti-CD37 Radioimmunoconjugate for NHL
Figure 2. Growth of Daudi cells incubated with 177Lu-tetulomab (A, C) or 177Lu-rituximab (B, D) for 2 h (A, B) or 18 h (C, D) before washing. Open
symbols: unblocked cells; closed symbols: blocked cells.
in a growth delay of 107% for cells treated with 2.5 μg/ml
177Lu-tetulomab (Figure 2C) and of 52% for cells treated
with 2.5 μg/ml 177Lu-rituximab (Figure 2D), even though
cells labeled with 177Lu-rituximab had 1.6-times as much
cell-bound activity than did the cells labeled with 177Lutetulomab (Table II). The growth delay factor was 1.4 for
cells incubated for two hours with 10 μg/ml 177Lutetulomab, as compared with the same concentration of
177Lu-rituximab and greater than 1.6 for cells treated with
20 μg/ml of the RICs. For 18-h incubation, the growth delay
factor was 1.6 for cells incubated with 1 μg/ml of the RICs.
Biodistribution of 177Lu-tetulomab in SCID mice. Uptake and
retention data of 177Lu-tetulomab in normal tissues of female
SCID mice intravenously injected with Daudi cells are
presented in Figure 3. There was no apparent re-distribution
of nuclide from or to any organs after the initial uptake of
RICs, which indicates the in vivo stability of 177Lu-tetulomab
since free 177Lu tends to relocate to the bone (29). However,
the uptake and retention of 177Lu-tetulomab in blood, liver,
spleen and kidney was significantly higher than in nude mice
(data not shown). There was no significant difference
between the biodistribution in SCID mice with and without
intravenously injected Daudi cells (data not shown). The
biodistribution of 177Lu-rituximab was also investigated and
the uptake in the spleen was extremely high (data not
shown). Although the biodistribution was not ideal, we
performed therapy experiments with 177Lu-tetulomab.
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ANTICANCER RESEARCH 33: 85-96 (2013)
Table III. Distribution of Daudi cells in organs of mice with hind leg
paralysis.
Organ
Lymph nodes
Kidney
Fat tissue
Ovaries
Liver
Spleen
Lungs
Spine
No. of mice with Daudi cells in organ (%)
8 of 28 (28.6)
14 of 31 (45.2)
12 of 30 (40)
17 of 30 (56.7)
1 of 30 (3.2)
1 of 30 (3.2)
22 of 31 (71)
30 of 31 (96)
Table IV. Lymphoma biopsies tested for CD37 expression.
Diagnosis
B-Lymphoblastic lymphoma
Burkitt’s lymphoma
Diffuse large B-cell lymphoma (DLBCL)
DLBCL transformed from low-grade
Follicular lymphoma
Low-grade, unspecified
Marginal zone lymphoma
Mantle cell lymphoma
Small lymphocytic lymphoma
Total
No. of
samples
CD37-positive
(%)
1
4
25
19
92
2
9
28
37
100.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
97.3
217
99.5
Toxic and therapeutic effects of 177Lu-tetulomab in SCID
mice with intravenously injected Daudi cells. SCID mice
were injected intravenously with 10×106 Daudi cells one
week before administration of 50, 100 or 200 MBq/kg 177Lutetulomab, 50 μg tetulomab, or NaCl. The Daudi cells
migrated to lymph nodes, kidney, fat tissue, ovaries, lungs
and to the spine (Table III and Figure 4). The mice were
monitored for hind leg paralysis, radiation toxicity and
bodyweight every other day and blood counts every other
week (Figures 5 and 6).
In Figure 5A, data are analyzed with deaths due to
radiotoxicity as end-point. Radiotoxicity was defined as a
white blood cell count (WBC) lower than 1.5×109 cells/l, no
hind leg paralysis and decreased body weight. Treatment
with 200 MBq/kg of 177Lu-tetulomab was too toxic for the
mice and they died two to three weeks after injection (Figure
5A). The WBC decreased below 1×109 cells/l for these mice
(Figure 6A) and the platelets decreased below 300×109
cells/l (Figure 6B). The bodyweight of these mice also
decreased significantly. There were no apparent treatmentinduced changes in bodyweight in the other treatment
90
Figure 3. Biodistribution (% injected dose/gram (ID/g)) of 177Lutetulomab. The mice were injected with Daudi cells one week before
injection of RIC. N=5. Data are mean±standard deviation.
groups, except for the 100-MBq/kg 177Lu-tetulomab-treated
group, where the two mice that died from radiation toxicity
had significantly decreased body weight. There were no
deaths due to the 50-MBq/kg 177Lu-tetulomab treatment.
Treated mice died from radiation toxicity before any mice
died in the control group, where the first mouse died at 21
days. Treatments did not have a significant effect on red
blood cell counts (Figure 6C).
The 50 and 100 MBq/kg 177Lu-tetulomab treatments
resulted in significantly improved survival as compared with
NaCl, with respect to the hind leg paralysis end-point
(p<0.01, Mantel-Cox log-rank test) (Figure 5B). Treatment
with 50 and 100 MBq/kg 177Lu-tetuomab was also
significantly more effective than treatment with tetulomab
(p<0.05), while the tetulomab group was not significantly
different from the NaCl group (p>0.05).
Expression of the tetulomab CD37 epitope in B-cell
lymphoma biopsies. Expression of CD37 by binding of
tetulomab monoclonal antibody was tested in 217 lymphoma
biopsies from different subtypes of NHL. All samples were
also stained for CD3, κ and λ light chain expression. In 216
out of the 217 samples, more than 50% of the κ- and/or
λ-positive cells (B-cells) expressed CD37 (Table IV).
Tetulomab did not show binding to the CD3-positive
population (T-cells).
Dahle et al: New Anti-CD37 Radioimmunoconjugate for NHL
Figure 4. Microscopy images of sections of organs of SCID mice i.v. injected with Daudi cells. The sections were stained with an antibody against
CD20 in order to locate the Daudi cells (Left panels). A: Spinal cord. B: Kidney. C: Ovary. Magnification=10 times. Right panels show corresponding
sections stained with hematoxylin and eosin.
Discussion
Treatment of patients with lymphoma with current CD20directed RIT is challenging in those previously treated with
rituximab, due to antigenic drift and possible blockage of the
CD20 antigen. Therefore, RIT targeting other antigens could
have some advantages. We have shown that there was a
significantly enhanced inhibition of cell growth after
91
ANTICANCER RESEARCH 33: 85-96 (2013)
Figure 5. Survival analysis of SCID mice i.v. injected with Daudi cells
and then treated with 50, 100 or 200 MBq/kg 177Lu-tetulomab, 50 μg
tetulomab, or NaCl. A: Survival with radiation toxicity as end-point, i.e.
white blood cell count <1.5×109 cells/l and no hind leg paralysis. B:
Survival with hind leg paralysis as end point. In panel A no mice in the
NaCl–, 50 μg tetulomab- or the 50 MBq/kg 177Lu-tetulomab-treated
groups died of radiation toxicity, so these lines overlap. The “cold”
tetulomab-treated group was omitted from this panel. The crosses in
panel A represent animals that were censored because of hind leg
paralysis. Similarly, the crosses in panel B represent animals that were
censored because they died of causes other than hind leg paralysis.
92
treatment with CD37-directed 177Lu-tetulomab as compared
with CD20-directed 177Lu-rituximab. The growth delay
factor of 177Lu-tetulomab vs. 177Lu-rituximab was 1.6. The
uptake of 177Lu-tetulomab in lymphoma cells was lower or
similar to the uptake of 177Lu-rituximab directly after
labeling. Furthermore, 177Lu-tetulomab was significantly
more effective than unlabeled tetulomab in treating SCID
mice intravenously injected with Daudi cells. The Kd of
tetulomab was similar to that for rituximab. Only one out of
217 clinical biopsies from nine different types of NHL did
not express the CD37 antigen epitope targeted by tetulomab.
RIT with CD37 as the target has previously been explored
using a 131I-labeled murine monoclonal antibody (MB-1),
both in a mouse model and in humans (30-35). CD37
antibodies were compared with CD20 antibodies and a
higher grade of internalization and de-halogenation of 131Ilabeled RIC was found for CD37 than for CD20 (35).
Despite clinical responses observed in that study, CD20 was
chosen for further development. No subsequent efforts have
been made to target CD37 with RICs.
In the early studies of CD37 RIT described above, the
chloramine-T method of 131I-labeling, was used (35). 131I
labeled to antibodies with the iodogen or the chloramine-T
method will not be well-retained in the cells if the antigenantibody complex is internalized (16, 17). The same pattern
of de-halogenation has been shown with CD22 antibodies,
which also are internalized (36). However, metallic
radionuclides labeled to antibodies with chelators were better
retained intracellularly when internalized (37).
There are several metallic nuclides that can be used for
RIT against CD37. Although we demonstrated results in a
mouse model with the alpha emitting nuclide 227Th (38), a
β-emitting nuclide may be more suitable as therapy for bulky
lymphoma. Clinical data indicate that NHL is responsive to
low Linear Energy Transfer (LET) β-emitters (1-4), thus in
the current work, 177Lu was chosen because of its
availability, suitable radiochemistry and half-life, and
promising radiation properties.
The inhibition of cell growth after treatment with CD37directed 177Lu-tetulomab was significantly better than that
for CD20-directed 177Lu-rituximab, when compared at the
same antibody concentration. The differences in cell growth
inhibition were higher for 18-h than for two-hour incubation
with the RICs and thus might, to some extent, be explained
by the higher internalization of tetulomab than of rituximab.
The internalization of tetulomab and thus retention of 177Lu
will probably result in a higher absorbed radiation dose to
the cells treated with tetulomab than with rituximab, even
though the initial binding was similar or higher for
rituximab. The 1.5- to 2-fold higher binding for rituximab
than for tetulomab can be explained by the 1.5-fold higher
specific activity for rituximab than for tetulomab in this
experiment. The growth inhibition induced by 177Lu-
Dahle et al: New Anti-CD37 Radioimmunoconjugate for NHL
Figure 6. Hematology of SCID mice i.v. injected with Daudi cells and then treated with 50, 100 or 200 MBq/kg 177Lu-tetulomab, 50 μg tetulomab
or NaCl. A: White blood cell counts (WBC). B: Platelet counts. C: Red blood cell counts (RBC).
tetulomab and 177Lu-rituximab was related to selective
targeting of the CD37 and CD20 antigens, respectively, since
blocking with “cold” tetulomab and rituximab significantly
reduced the inhibition of cell growth.
The cell growth experiments were carried out with Daudi
cells, which had the highest expression of the CD37 antigen,
more than two-fold that for Raji cells. Therefore, one might
wonder if the treatment would also be effective for Raji cells.
One would expect, however, that it would be possible to
attain a similar effect on Raji cells, as on Daudi by
increasing the concentration of the antibody by the same
factor as the difference in the number of antigens they carry.
The unusually high retention of 177Lu-tetulomab in blood,
liver, spleen and kidneys and the extremely high uptake of
177Lu-rituximab in the spleen in SCID mice cannot be
explained by binding to Daudi cells located in the respective
93
ANTICANCER RESEARCH 33: 85-96 (2013)
organs, since similar biodistribution without Daudi cells
being injected was found (results not shown). The reason for
the unusual biodistribution might be the low production of
endogeneous antibodies in SCID mice. This effect on
biodistribution has been previously described (39).
We report on a similar migration pattern of Daudi cells as
the one presented by Gethie et al. in (22). The relatively high
toxicity of 177Lu-tetulomab in this model was probably
related to both the unusual biodistribution and the high
radiosensitvity of these DNA double-strand repair-defective
mice (due to the SCID mutation). Nevertheless, the
therapeutic effect of 177Lu-tetulomab was significantly better
than that for the unlabeled antibody.
The binding data for 125I-labeled tetulomab and rituximab
showed that the antigen-binding properties of tetulomab and
rituximab were similar, with a Kd between 2.7 and 12.7 for
tetulomab and between 4.8 and 12 for rituximab, depending
on the cell line used. The reason for the variability in Kd
between cell lines might be that the three parameters
estimated by the curve-fitting method, to some extent may
influence each other, or there could be differences in the
antigen from cell line to cell line due to mutations or posttranslational changes. The data for the number of antigens
on the Daudi cells fitted well with the measured binding after
two hours in the growth inhibition experiment if the
difference in specific activity between 177Lu-tetulomab and
177Lu-rituximab is taken into consideration.
In conclusion, the data presented here indicate that the
tetulomab antibody is well-suited for RIT of CD37expressing lymphoma cells. The study of binding of
tetulomab to patient biopsies indicates that targeting of the
CD37 epitope by tetulomab could be clinically relevant and
warrants for future pre-clinical and clinical investigations.
Declaration of Interest
JD and AHVRL are employed by and have stock options in Nordic
Nanovector AS. RHL owns Nordic Nanovector shares.
Acknowledgements
The Authors are grateful to Steinar Funderud and Erlend Smeland
who developed the tetulomab monoclonal antibody, to Bjørn
Erikstein, Erlend Smeland and Harald Holte for collecting the
lymphoma biopsies, and to the Histology Research Laboratory at
the Norwegian Radium Hospital for preparing the lymphoma slides.
This study was supported by a grant from Innovation Norway.
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Received October 18 2012
Revised November 7, 2012
Accepted November 8, 2012
95