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 CD20-rituximab 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 pre-clinical and clinical studies of 177Lu-tetulomab.
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 less-accessible 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 revival in interest in the targeting of CD37. Both an Fc-engineered 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 177Lu-tetulomab 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 double-strand 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 4-5-times 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 37°C. 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 (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 177Lu-rituximab. Daudi cells were harvested, counted and diluted to a density of 2×106 cells/ml and incubated with 1 μg/ml 177Lu-tetulomab, 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 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 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, 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 non-specific 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-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 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 177Lu-tetulomab (Table II). The growth delay factor was 1.4 for cells incubated for two hours with 10 μg/ml 177Lu-tetulomab, 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.
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 177Lu-tetulomab, 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 treatment-induced changes in bodyweight in the other treatment 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).
Discussion
Treatment of patients with lymphoma with current CD20-directed 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 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 131I-labeled 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 antigen-antibody 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 CD37-directed 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-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 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 post-translational 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 CD37-expressing 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.
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.
Footnotes
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This article is freely accessible online.
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Declaration of Interest
JD and AHVRL are employed by and have stock options in Nordic Nanovector AS. RHL owns Nordic Nanovector shares.
- Received October 18, 2012.
- Revision received November 7, 2012.
- Accepted November 8, 2012.
- Copyright© 2013 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved