Evaluating Antigen Targeting and Anti-Tumor Activity of a New Anti-CD37 Radioimmunoconjugate Against Non-Hodgkin’S Lymphoma
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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; 2Departments 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 Abstract. The monoclonal antibody against CD20, rituximab, Beta-emitting radioimmunoconjugates have significant alone, or as part of combination therapies, is standard therapy antitumor activity in patients with relapsed or refractory B-cell for non-Hodgkin’s B-cell lymphoma. Despite significantly better lymphoma (1-4), including those refractory to rituximab, a clinical results obtained for beta-emitting radioimmuno- monoclonal antibody to CD20 (5, 6) and chemotherapy (7). conjugates (RICs), RICs targeting CD20 are not commonly used Radioimmunotherapy (RIT) is administered with large in medical practice, partly because of competition for the CD20 quantities of unlabeled “cold” antibody to CD20 one week target. Therefore, novel therapeutic approaches against other and 4 h prior to radiolabeled antibodies to CD20. Such a antigens are intriguing. Here, the binding properties of a novel priming dose is necessary to reduce antibody binding to antibody against CD37 (tetulomab) were compared with those normal B-cells by depleting peripheral blood B-cells and of rituximab. The therapeutic effect of 177Lu-tetulomab was lymph node B-cells (8, 9). Thus, sufficient amounts of compared with 177Lu-rituximab on Daudi cells in vitro. The radiolabeled antibody can bypass these sites, penetrate less- biodistribution, therapeutic and toxic effects of 177Lu-tetulomab accessible compartments, such as the lymph nodes, and and unlabeled tetulomab were determined in SCID mice injected target tumor cells. However, both clinical and experimental with Daudi cells. The affinity of tetulomab to CD37 was similar studies in mice have shown that in some circumstances, even to the affinity of rituximab to CD20, but the CD37-tetulomab quite low rituximab concentrations in the blood can reduce complex was internalized 10-times faster than the CD20- tumor cell targeting and thus impair the clinical efficacy of rituximab complex. At the same concentration of antibody, CD20-directed RIT (10). Furthermore, the majority of 177Lu-tetulomab was significantly more efficient in inhibiting cell patients selected for CD20-based RIT have received several growth than was 177Lu-rituximab, even though the cell-bound cycles of “cold” rituximab, which may result in selection of activity of 177Lu-rituximab was higher. Treatment with 50 and tumor cells with low CD20 expression and thus could lower 100 MBq/kg 177Lu-tetulomab resulted in significantly increased the effect of subsequent anti-CD20 treatments (11-13). survival of mice, compared with control groups treated with By targeting B-cell antigens, other than CD20, a better tetulomab or saline. The CD37 epitope recognized by tetulomab response might be achieved. CD37 is a heavily glycosylated was highly expressed in 216 out of 217 tumor biopsies from 40- to 52-kDA glycoprotein and a member of the tetraspan patients with B-cell lymphoma. This work warrants further pre- transmembrane family of proteins (14, 15). CD37 clinical and clinical studies of 177Lu-tetulomab. 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 This article is freely accessible online. differentiation to plasma cells (18). Given its relative B-cell selectivity, CD37 thus represents a valuable target for Correspondence to: Jostein Dahle, Nordic Nanovector AS, therapies in chronic lymphocytic leukemia (CLL), hairy-cell Kjelsåsveien 168 B, 0884 Oslo, Norway. Tel: +47 98458850, Fax: +47 22580007, E-mail: [email protected] leukemia (HCL), B-cell non-Hodgkin’s lymphoma (NHL) and other B-cell malignancies. Key Words: Radioimmunotherapy, CD37, non-Hodgkin’s CD37 has been previously studied as a target for RIT lymphoma, 177Lu. using 131I-labeled MB-1 (16). Recently, there has been a 0250-7005/2013 $2.00+.40 85 ANTICANCER RESEARCH 33: 85-96 (2013) revival in interest in the targeting of CD37. Both an Fc- (26). Cell densities of up to 3×108 cells/ml were used. All conjugates engineered monoclonal antibody and a small modular used in experiments had immunoreactivity greater than 50 %. immuno-pharmaceutical have been developed for the targeting of CD37 in CLL (19, 20). Binding parameters. The association rate constant, ka, and the equilibrium dissociation constant, K , were measured for tetulomab At the Norwegian Radium Hospital a murine IgG1 d and rituximab, and the mean number of binding sites, Bmax, was antibody, HH1 (tetulomab, according to the nomenclature for determined for Raji, Rael and Daudi cells using a one-step curve naming of antibodies), was developed against CD37 in the fitting method (27). Specific binding was measured as a function of 1980s (21). In the present study we investigated binding to time and antibody concentration, and the solution of the differential different subtypes of NHL and compared binding properties equation describing the net rate of formation of the antigen-antibody of tetulomab with the chimeric IgG1 antibody rituximab. complex was fitted to the experimental data points using ka, Kd and Furthermore, the therapeutic effects of 177Lu-tetulomab, Bmax as parameters. Five million cells/ml were used, with four concentrations of “hot” antibody (100 ng/ml, 1000 ng/ml, 5000 ng/ml 177Lu-rituximab, “cold” tetulomab and “cold” rituximab was 177 and 10000 ng/ml) and seven incubation time points (5 min, 10 min, compared in vitro. Increasing concentrations of Lu- 20 min, 30 min, 1 h, 1.5 h and 2 h). After incubation, cells were tetulomab were used to treat SCID mice injected intravenously washed twice with PBS, and then counted in a gamma counter (Cobra with human Daudi lymphoma cells. This animal model (22), is II; Packard Instrument Company, Meriden, CT, USA). a worst-case scenario model of NHL because the tumor cells will, to a large degree, end up in the bone marrow. This Internalization and retention of 177Lu-tetulomab and 177Lu- localization will result in irradiation of bone marrow cells and rituximab. Daudi cells were harvested, counted and diluted to a 6 177 corresponding hematological toxicity. The biodistribution of density of 2×10 cells/ml and incubated with 1 μg/ml Lu- tetulomab, 177Lu-rituximab, 125I-tetulomab or 125I-rituximab. Some antibodies in SCID mice may be different from that in other parallels were pre-blocked with the corresponding “cold” antibody mouse strains because of low concentrations of endogeneous (100 μg/ml). The cells were incubated for 0, 10, 20, 30, 60, 120 and antibodies in the blood (23, 24). In addition, because of the 180 min. Subsequently, the cells were centrifuged and washed twice SCID mutation this mouse strain cannot repair DNA double- with medium, and the supernatant and the wash medium were strand breaks and is thus very sensitive to ionizing radiation collected for counting. Cells were incubated with 1 ml stripping (25). Regardless of the limitations of this model, a significant buffer (150 mM NaCl and 50 mM glycine, pH 2.6) for 10 min, at therapeutic effect of 177Lu-tetulomab was shown at a dosage room temperature. The cells were washed twice and the supernatants and the cells were counted using a calibrated gamma level resulting in tolerable toxicity. detector (Cobra II). In another experiment, 106 Daudi cells/ml medium were Materials and Methods incubated with 1 μg/ml 125I- or 111In-labeled tetulomab or rituximab for one hour, washed twice with medium and incubated further for Cell lines. The CD20- and CD37-expressing B-cell lymphoma cell four days. The cell-bound activity was determined immediately after lines Raji, Rael and Daudi were used in the current study (LGC washing and after four days of incubation by measuring the number Standards, Boras, Sweden). Single-cell suspensions were grown in of cells (Vi Cell Viability Analyzer, Beckman Coulter, Fullerton, RPMI 1640 medium supplemented with 10 % heat-inactivated FCS, CA, USA) and the amount of radioactivity with the gamma detector 1% L-glutamine and 1% penicillin-streptomycin (all from PAA, (Cobra II). Linz, Austria), in a humid atmosphere with 95% air/5% CO2. Effect of 177Lu-tetulomab and 177Lu-rituximab on growth of Daudi Labeling of antibodies with 177Lu. Tetulomab and rituximab were first cells in vitro. One milliliter of Daudi cell suspension (1×106 labeled with a chelator, 2-(4-isothiocyanatobenzyl)-1,4,7,10 cells/ml) was pipetted into each of 24 tubes. Six tubes each were tetraazacycododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA, blocked with 100 μg/ml of either tetulomab or rituximab and DOTA) (Macrocyclics, Dallas, TX, USA). DOTA was dissolved in incubated for 30 min at 37˚C. Subsequently, either 177Lu-tetulomab 0.05 M HCl, added to the antibody in a 5:1 ratio and the pH adjusted or 177Lu-rituximab was added to a final concentration of 0, 1, 2.5, 5, to 8.5-9 by washing with carbonate buffer using AMICON-30 10 or 20 μg/ml and the cells incubated further at 37˚C. The specific centrifuge tubes (Millipore, Cork, Ireland). The solution was shaken activity was 91.6 kBq/μg for 177Lu-tetulomab and 136.6 kBq/μg for for 60 min at room temperature, and the reaction was terminated by 177Lu-rituximab. The amount of added activity was measured during adding 50 μl of 200 mM glycine solution (per mg antibody). To the incubation period with a gamma detector (Cobra II).