Abstract. The monoclonal antibody against CD20, rituximab,alone, or as part of combination therapies, is standard therapyfor non-Hodgkin’s B-cell lymphoma. Despite significantly betterclinical results obtained for beta-emitting radioimmuno-conjugates (RICs), RICs targeting CD20 are not commonly usedin medical practice, partly because of competition for the CD20target. Therefore, novel therapeutic approaches against otherantigens are intriguing. Here, the binding properties of a novelantibody against CD37 (tetulomab) were compared with thoseof rituximab. The therapeutic effect of 177Lu-tetulomab wascompared with 177Lu-rituximab on Daudi cells in vitro. Thebiodistribution, therapeutic and toxic effects of 177Lu-tetulomaband unlabeled tetulomab were determined in SCID mice injectedwith Daudi cells. The affinity of tetulomab to CD37 was similarto the affinity of rituximab to CD20, but the CD37-tetulomabcomplex was internalized 10-times faster than the CD20-rituximab complex. At the same concentration of antibody,177Lu-tetulomab was significantly more efficient in inhibiting cellgrowth than was 177Lu-rituximab, even though the cell-boundactivity of 177Lu-rituximab was higher. Treatment with 50 and100 MBq/kg 177Lu-tetulomab resulted in significantly increasedsurvival of mice, compared with control groups treated withtetulomab or saline. The CD37 epitope recognized by tetulomabwas highly expressed in 216 out of 217 tumor biopsies frompatients with B-cell lymphoma. This work warrants further pre-clinical and clinical studies of 177Lu-tetulomab.

Beta-emitting radioimmunoconjugates have significantantitumor activity in patients with relapsed or refractory B-celllymphoma (1-4), including those refractory to rituximab, amonoclonal antibody to CD20 (5, 6) and chemotherapy (7).

Radioimmunotherapy (RIT) is administered with largequantities of unlabeled “cold” antibody to CD20 one weekand 4 h prior to radiolabeled antibodies to CD20. Such apriming dose is necessary to reduce antibody binding tonormal B-cells by depleting peripheral blood B-cells andlymph node B-cells (8, 9). Thus, sufficient amounts ofradiolabeled antibody can bypass these sites, penetrate less-accessible compartments, such as the lymph nodes, andtarget tumor cells. However, both clinical and experimentalstudies in mice have shown that in some circumstances, evenquite low rituximab concentrations in the blood can reducetumor cell targeting and thus impair the clinical efficacy ofCD20-directed RIT (10). Furthermore, the majority ofpatients selected for CD20-based RIT have received severalcycles of “cold” rituximab, which may result in selection oftumor cells with low CD20 expression and thus could lowerthe effect of subsequent anti-CD20 treatments (11-13).

By targeting B-cell antigens, other than CD20, a betterresponse might be achieved. CD37 is a heavily glycosylated40- to 52-kDA glycoprotein and a member of the tetraspantransmembrane family of proteins (14, 15). CD37internalizes and has modest shedding in transformed B-cellsexpressing the antigen (16, 17). During B-cell development,CD37 is expressed in cells progressing from pre-B toperipheral mature B-cell stages and is absent on terminaldifferentiation to plasma cells (18). Given its relative B-cellselectivity, CD37 thus represents a valuable target fortherapies in chronic lymphocytic leukemia (CLL), hairy-cellleukemia (HCL), B-cell non-Hodgkin’s lymphoma (NHL)and other B-cell malignancies.

CD37 has been previously studied as a target for RITusing 131I-labeled MB-1 (16). Recently, there has been a

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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: jd@nordicnanovector.no

Key Words: Radioimmunotherapy, CD37, non-Hodgkin’slymphoma, 177Lu.

ANTICANCER RESEARCH 33: 85-96 (2013)

Evaluating Antigen Targeting and Anti-tumor Activity of a New Anti-CD37 Radioimmunoconjugate

Against Non-Hodgkin’s LymphomaJOSTEIN 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

0250-7005/2013 $2.00+.40

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revival in interest in the targeting of CD37. Both an Fc-engineered monoclonal antibody and a small modularimmuno-pharmaceutical have been developed for thetargeting of CD37 in CLL (19, 20).

At the Norwegian Radium Hospital a murine IgG1antibody, HH1 (tetulomab, according to the nomenclature fornaming of antibodies), was developed against CD37 in the1980s (21). In the present study we investigated binding todifferent subtypes of NHL and compared binding propertiesof tetulomab with the chimeric IgG1 antibody rituximab.Furthermore, the therapeutic effects of 177Lu-tetulomab,177Lu-rituximab, “cold” tetulomab and “cold” rituximab wascompared in vitro. Increasing concentrations of 177Lu-tetulomab were used to treat SCID mice injected intravenouslywith human Daudi lymphoma cells. This animal model (22), isa worst-case scenario model of NHL because the tumor cellswill, to a large degree, end up in the bone marrow. Thislocalization will result in irradiation of bone marrow cells andcorresponding hematological toxicity. The biodistribution ofantibodies in SCID mice may be different from that in othermouse strains because of low concentrations of endogeneousantibodies in the blood (23, 24). In addition, because of theSCID 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 significanttherapeutic effect of 177Lu-tetulomab was shown at a dosagelevel resulting in tolerable toxicity.

Materials and Methods

Cell lines. The CD20- and CD37-expressing B-cell lymphoma celllines Raji, Rael and Daudi were used in the current study (LGCStandards, Boras, Sweden). Single-cell suspensions were grown inRPMI 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 firstlabeled with a chelator, 2-(4-isothiocyanatobenzyl)-1,4,7,10tetraazacycododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA,DOTA) (Macrocyclics, Dallas, TX, USA). DOTA was dissolved in0.05 M HCl, added to the antibody in a 5:1 ratio and the pH adjustedto 8.5-9 by washing with carbonate buffer using AMICON-30centrifuge tubes (Millipore, Cork, Ireland). The solution was shakenfor 60 min at room temperature, and the reaction was terminated byadding 50 μl of 200 mM glycine solution (per mg antibody). Toremove free chelator, the conjugated antibody was centrifuged 4-5-times with phosphate buffered saline (PBS) (PAA) at 1:10 dilutionusing AMICON-30 centrifuge tubes (Millipore). Before labeling with177Lu (Perkin Elmer, Boston, MA, USA) the PBS buffer was changedto 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 at37ºC. Specific activity was between 50 and 150 MBq/kg.

Immunoreactivity. The quality of the radioimmunoconjugates wasmeasured using lymphoma cells and a modified Lindmo method

(26). Cell densities of up to 3×108 cells/ml were used. All conjugatesused in experiments had immunoreactivity greater than 50 %.

Binding parameters. The association rate constant, ka, and theequilibrium dissociation constant, Kd, were measured for tetulomaband rituximab, and the mean number of binding sites, Bmax, wasdetermined for Raji, Rael and Daudi cells using a one-step curvefitting method (27). Specific binding was measured as a function oftime and antibody concentration, and the solution of the differentialequation describing the net rate of formation of the antigen-antibodycomplex was fitted to the experimental data points using ka, Kd andBmax as parameters. Five million cells/ml were used, with fourconcentrations of “hot” antibody (100 ng/ml, 1000 ng/ml, 5000 ng/mland 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 werewashed twice with PBS, and then counted in a gamma counter (CobraII; Packard Instrument Company, Meriden, CT, USA).

Internalization and retention of 177Lu-tetulomab and 177Lu-rituximab. Daudi cells were harvested, counted and diluted to adensity of 2×106 cells/ml and incubated with 1 μg/ml 177Lu-tetulomab, 177Lu-rituximab, 125I-tetulomab or 125I-rituximab. Someparallels were pre-blocked with the corresponding “cold” antibody(100 μg/ml). The cells were incubated for 0, 10, 20, 30, 60, 120 and180 min. Subsequently, the cells were centrifuged and washed twicewith medium, and the supernatant and the wash medium werecollected for counting. Cells were incubated with 1 ml strippingbuffer (150 mM NaCl and 50 mM glycine, pH 2.6) for 10 min, atroom temperature. The cells were washed twice and thesupernatants and the cells were counted using a calibrated gammadetector (Cobra II).

In another experiment, 106 Daudi cells/ml medium wereincubated with 1 μg/ml 125I- or 111In-labeled tetulomab or rituximabfor one hour, washed twice with medium and incubated further forfour days. The cell-bound activity was determined immediately afterwashing and after four days of incubation by measuring the numberof 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 Daudicells in vitro. One milliliter of Daudi cell suspension (1×106

cells/ml) was pipetted into each of 24 tubes. Six tubes each wereblocked with 100 μg/ml of either tetulomab or rituximab andincubated for 30 min at 37˚C. Subsequently, either 177Lu-tetulomabor 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 specificactivity was 91.6 kBq/μg for 177Lu-tetulomab and 136.6 kBq/μg for177Lu-rituximab. The amount of added activity was measured duringthe incubation period with a gamma detector (Cobra II). After twohours, half of the cells were washed and the cell-bound activity wasmeasured, while the other half of the cells was incubated overnightbefore washing and measurement of cell-bound activity. Formeasurement of cell growth, 50,000 cells from each tube wereseeded in three wells in a 12-well plate and the number of cells wasmeasured at several time points over the next 14 days using anautomatic imaging system (Clone Select Imager, Molecular DevicesLtd., New Milton, Hampshire, UK) that captured images of eachwell, analyzed the images and calculated the number of cells in eachwell. 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 thegrowth curve to the y-axis (28). Growth delay factor was calculatedby dividing the delay in growth to 100,000 cells/ml for cells treatedwith 177Lu-tetulomab with the corresponding delay in growth forcells treated with 177Lu-rituximab.

Biodistribution of 177Lu-tetulomab in severe combined immunedeficient (SCID) mice with intravenously injected Daudi cells.Biodistribution of 177Lu-tetulomab was determined in female SCIDmice (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 260-1400kBq in 100 μl solution in each animal, one week after an injectionof 10×106 Daudi cells. Four to five animals were used per timepoint. Autopsies were performed after cervical dislocation at varioustime points after injection. The weight of each tissue sample (blood,lung, heart, liver, spleen, kidneys, stomach, small intestine, largeintestine, femur, muscle, brain, skull, lymph node and neck) wasdetermined, and 177Lu was measured by a calibrated gammadetector (Cobra II). Samples of the injectate were used as referencesin the measurement procedures. All procedures and experimentsinvolving animals in this study were approved by the NationalAnimal Research Authority and carried out according to theEuropean Convention for the Protection of Vertebrates used forScientific Purposes.

Therapeutic and toxic effect of 177Lu-tetulomab in SCID miceintravenously 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 ofthe experiment, were used. The animals were maintained underpathogen-free conditions, and food and water were supplied adlibitum. All mice were ear-tagged and followed individuallythroughout 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 μgtetulomab (N=14), 50 (N=10), 100 (N=10) and 200 (N=10) MBq/kg177Lu-tetulomab. A pilot study was performed to validate 100 %tumor formation following injection of 10×106 Daudi cells permouse. Mice were killed by cervical dislocation, if suffering fromhind leg paralysis (primary end point), body weight decreased by20% from baseline, or if they otherwise showed symptoms of illnessand discomfort. The mice were dissected and histological sectionswere stained with antibody against CD20 (EP459Y, NovusBiologicals, Littleton, CO, USA) to locate tumor cells. The micewere checked for hind leg paralysis every day and weighed two tothree times per week. The different treatment groups were comparedby Kaplan-Meier survival analysis using SPSS version 13.0 (SPSS,Chicago, IL, USA).

Toxicity measurements. Prior to the study start and every two weeksthereafter for up to 12 weeks, 50-75 μl blood was collected fromthe vena saphena lateralis in 0.5 ml EDTA-coated tubes (BDMicrotainer K2E tubes; Becton, Dickinson and Company, FranklinLakes, NJ, USA). White blood cells, red blood cells and plateletswere counted using an automated hematology analyzer (Scil Vet abcanimal blood counter, ABX Diagnostics, Montpellier, France).

Patient samples. A total of 217 B-cell lymphoma biopsies frompatients 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 lightchains, rabbit F(ab’)2], anti-IgGk [polyclonal rabbit anti-humankappa light chains, rabbit F(ab’)2], and anti-CD37 (in-housetetulomab antibody). The expression of the different antigens wasmeasured using light microscopy after horseradish peroxidasevisualization (Dako).

Results

Binding properties of tetulomab and rituximab. Theassociation rate constant, ka, the equilibrium dissociationconstant, Kd, and the mean number of binding sites, Bmax,were determined for three different cell lines, Raji, Rael andDaudi, and for the tetulomab and rituximab antibodies (TableI). For Raji and Rael cells, the number of CD37 antigens wasapproximately half the number of CD20 antigens, while forDaudi cells the number of antigens was similar. The Kd washigher for Rael than for Raji and Daudi cells for bothantibodies. The Kd was similar for the two antibodies forRaji and Rael cells, while for Daudi cells it was significantlylower for tetulomab than for rituximab (t-test, p<0.05),indicating that tetulomab had better affinity for the CD37antigen than rituximab had for the CD20 antigen, in this cellline. For Rael cells, ka was similar for tetulomab andrituximab, while it was significantly higher for tetulomabthan for rituximab for Raji and Daudi cells, indicating fasterbinding of tetulomab to CD37 than of rituximab to CD20 inthese two cell lines. The Daudi cells were chosen for furtherstudy since they had the highest amount of CD37 antigens:an average of 340,000 CD37 antigens and 307,000 CD20antigens per cell.

Internalization of 177Lu-tetulomab and 177Lu-rituximab.Tetulomab was internalized faster and to a greater extent thanrituximab (Figure 1). For the first half hour of incubation theinternalization speed was 0.19 fg/min/cell for tetulomab and0.02 fg/min/cell for rituximab. There was no significantdifference between experiments performed with 125I and 177Lu.Therefore, the results were pooled. In another experiment,

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Table I. The mean number of antigens (Bmax) on Raji, Rael and Daudicells, the equilibrium dissociation constant (Kd) and the association rateconstant (ka) for tetulomab and rituximab.

Antibody Cell line Bmax (Ag/cell)a Kd (nM) ka (nM–1h–1)

Tetulomab Raji 146000±7600 6.3±1.7 0.36±0.14Tetulomab Rael 263000±27000 12.7±5.5 0.07±0.01Tetulomab Daudi 340000±5000 2.7±0.3 0.72±0.07Rituximab Raji 272000±69000 4.8±0.9 0.08±0.006Rituximab Rael 626000±36000 12.0±2.0 0.08±0.007Rituximab Daudi 307000±12000 5.8±1.5 0.14±0.01

aTetulomab and rituximab bind to CD37 and CD20, respectively.

however, of longer duration, Daudi cells were incubated with125I- and 111In-tetulomab for 1 h, washed and cell-boundactivity was measured after four days of incubation (Figure 1,insert). The amount of antibody bound to cells was higher for111In-tetulomab than for 125I-tetulomab.

Cell-bound activity of 177Lu-tetulomab and 177Lu-rituximab.Daudi cells were incubated with increasing concentrations of177Lu-tetulomab or 177Lu-rituximab for two hours and 18 hbefore washing. Cell-bound activity was measured and thecells were seeded for further growth. The cell bound activitywas lower for cells incubated with tetulomab than for cellsincubated with rituximab after two-hour incubation and 18 hincubation (Table II). However, radiolabeled tetulomabsaturated the antigen quicker and at lower antibodyconcentration than did rituximab. The specific activity was92 MBq/mg for 177Lu-tetulomab and 137 MBq/mg for177Lu-rituximab, which explains the difference in the numberof 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 inthe number of specifically bound radioactive atoms for bothincubation times.

Cell growth experiments. Growth of Daudi cells incubatedwith RICs for two hours before washing, was followed for14 days (Figure 2 A and B). There was no effect of unlabeledantibodies alone on cell growth (data not shown). Theblocked cells treated with the 177Lu-antibody clearly did notgrow as fast as the untreated control cells, indicating thatthere was an effect of unbound 177Lu-antibody or a non-specific bound 177Lu-antibody on the cells. Treatment ofunblocked cells with 177Lu-antibody resulted in an increasein growth delay of 44% for cells treated with 10 μg/ml of177Lu- tetulomab (Figure 2A) and of 31% for cells treatedwith 10 μg/ml 177Lu-rituximab (Figure 2B), as comparedwith blocked cells. For treatment with 20 μg/ml of 177Lu-

antibody, the difference between tetulomab and rituximabwas even larger, since there was no re-growth of the cellstreated with 177Lu- tetulomab.

Growth of Daudi cells incubated with RICs for 18 hbefore washing was also followed for 14 days (Figure 2Cand D). There was no effect of unlabeled antibody alone oncell growth (data not shown). The inhibition of cell growth ofthe blocked cells treated with 177Lu-antibody was morepronounced than when the cells were incubated for twohours before washing, probably because of the 16-hincreased incubation time with RIC in the medium.Treatment of unblocked cells with 177Lu-antibody resulted

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Figure 1. Internalization of tetulomab and rituximab in Daudi cells. Theinsert figure shows cell-bound tetulomab 96 h after washing forincubation of Daudi cells with 1 μg/ml 111In-tetulomab or 125I-tetulomab. N=3. Error bars=standard deviation.

Table II. Number of 177Lu atoms bound per Daudi cell after two and 18 h of incubation.

Incubation time 2 Hours 18 HoursAntibody dosage (μg/ml) 177Lu-tetulomab 177Lu-rituximab 177Lu-tetulomab 177Lu-rituximab

Unblocked Blocked Unblocked Blocked Unblocked Blocked Unblocked Blocked

0a 12 7 8 53 12 5 10 531 8318 449 8554 372 10327 301 12831 3562.5 9105 720 11629 885 11757 787 18836 13855 10025 1837 13658 2019 12123 1857 24097 187110 13646 3521 17344 2769 11548 3205 24249 286020 16290 8473 30095 9709 15233 5445 26639 5824

aThe different counts for the control samples is indicative of the variation in background radiation of the counter.

in a growth delay of 107% for cells treated with 2.5 μg/ml177Lu-tetulomab (Figure 2C) and of 52% for cells treatedwith 2.5 μg/ml 177Lu-rituximab (Figure 2D), even thoughcells labeled with 177Lu-rituximab had 1.6-times as muchcell-bound activity than did the cells labeled with 177Lu-tetulomab (Table II). The growth delay factor was 1.4 forcells incubated for two hours with 10 μg/ml 177Lu-tetulomab, as compared with the same concentration of177Lu-rituximab and greater than 1.6 for cells treated with20 μg/ml of the RICs. For 18-h incubation, the growth delayfactor was 1.6 for cells incubated with 1 μg/ml of the RICs.

Biodistribution of 177Lu-tetulomab in SCID mice. Uptake andretention data of 177Lu-tetulomab in normal tissues of female

SCID mice intravenously injected with Daudi cells arepresented in Figure 3. There was no apparent re-distributionof nuclide from or to any organs after the initial uptake ofRICs, which indicates the in vivo stability of 177Lu-tetulomabsince 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 differencebetween the biodistribution in SCID mice with and withoutintravenously injected Daudi cells (data not shown). Thebiodistribution of 177Lu-rituximab was also investigated andthe uptake in the spleen was extremely high (data notshown). Although the biodistribution was not ideal, weperformed therapy experiments with 177Lu-tetulomab.

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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. Opensymbols: unblocked cells; closed symbols: blocked cells.

Toxic and therapeutic effects of 177Lu-tetulomab in SCIDmice with intravenously injected Daudi cells. SCID micewere injected intravenously with 10×106 Daudi cells oneweek before administration of 50, 100 or 200 MBq/kg 177Lu-tetulomab, 50 μg tetulomab, or NaCl. The Daudi cellsmigrated to lymph nodes, kidney, fat tissue, ovaries, lungsand to the spine (Table III and Figure 4). The mice weremonitored for hind leg paralysis, radiation toxicity andbodyweight every other day and blood counts every otherweek (Figures 5 and 6).

In Figure 5A, data are analyzed with deaths due toradiotoxicity as end-point. Radiotoxicity was defined as awhite blood cell count (WBC) lower than 1.5×109 cells/l, nohind leg paralysis and decreased body weight. Treatmentwith 200 MBq/kg of 177Lu-tetulomab was too toxic for themice and they died two to three weeks after injection (Figure5A). 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 alsodecreased significantly. There were no apparent treatment-induced changes in bodyweight in the other treatment

groups, except for the 100-MBq/kg 177Lu-tetulomab-treatedgroup, where the two mice that died from radiation toxicityhad significantly decreased body weight. There were nodeaths due to the 50-MBq/kg 177Lu-tetulomab treatment.Treated mice died from radiation toxicity before any micedied in the control group, where the first mouse died at 21days. Treatments did not have a significant effect on redblood cell counts (Figure 6C).

The 50 and 100 MBq/kg 177Lu-tetulomab treatmentsresulted in significantly improved survival as compared withNaCl, with respect to the hind leg paralysis end-point(p<0.01, Mantel-Cox log-rank test) (Figure 5B). Treatmentwith 50 and 100 MBq/kg 177Lu-tetuomab was alsosignificantly more effective than treatment with tetulomab(p<0.05), while the tetulomab group was not significantlydifferent from the NaCl group (p>0.05).

Expression of the tetulomab CD37 epitope in B-celllymphoma biopsies. Expression of CD37 by binding oftetulomab monoclonal antibody was tested in 217 lymphomabiopsies from different subtypes of NHL. All samples werealso stained for CD3, κ and λ light chain expression. In 216out 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-positivepopulation (T-cells).

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Figure 3. Biodistribution (% injected dose/gram (ID/g)) of 177Lu-tetulomab. The mice were injected with Daudi cells one week beforeinjection of RIC. N=5. Data are mean±standard deviation.

Table III. Distribution of Daudi cells in organs of mice with hind legparalysis.

Organ No. of mice with Daudi cells in organ (%)

Lymph nodes 8 of 28 (28.6)Kidney 14 of 31 (45.2)Fat tissue 12 of 30 (40)Ovaries 17 of 30 (56.7)Liver 1 of 30 (3.2)Spleen 1 of 30 (3.2)Lungs 22 of 31 (71)Spine 30 of 31 (96)

Table IV. Lymphoma biopsies tested for CD37 expression.

Diagnosis No. of CD37-positivesamples (%)

B-Lymphoblastic lymphoma 1 100.0Burkitt’s lymphoma 4 100.0Diffuse large B-cell lymphoma (DLBCL) 25 100.0DLBCL transformed from low-grade 19 100.0Follicular lymphoma 92 100.0Low-grade, unspecified 2 100.0Marginal zone lymphoma 9 100.0Mantle cell lymphoma 28 100.0Small lymphocytic lymphoma 37 97.3

Total 217 99.5

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 theCD20 antigen. Therefore, RIT targeting other antigens couldhave some advantages. We have shown that there was asignificantly enhanced inhibition of cell growth after

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Figure 4. Microscopy images of sections of organs of SCID mice i.v. injected with Daudi cells. The sections were stained with an antibody againstCD20 in order to locate the Daudi cells (Left panels). A: Spinal cord. B: Kidney. C: Ovary. Magnification=10 times. Right panels show correspondingsections stained with hematoxylin and eosin.

treatment with CD37-directed 177Lu-tetulomab as comparedwith CD20-directed 177Lu-rituximab. The growth delayfactor of 177Lu-tetulomab vs. 177Lu-rituximab was 1.6. Theuptake of 177Lu-tetulomab in lymphoma cells was lower orsimilar to the uptake of 177Lu-rituximab directly afterlabeling. Furthermore, 177Lu-tetulomab was significantlymore effective than unlabeled tetulomab in treating SCIDmice intravenously injected with Daudi cells. The Kd oftetulomab was similar to that for rituximab. Only one out of217 clinical biopsies from nine different types of NHL didnot express the CD37 antigen epitope targeted by tetulomab.RIT with CD37 as the target has previously been exploredusing a 131I-labeled murine monoclonal antibody (MB-1),both in a mouse model and in humans (30-35). CD37antibodies were compared with CD20 antibodies and ahigher 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 waschosen for further development. No subsequent efforts havebeen made to target CD37 with RICs.

In the early studies of CD37 RIT described above, thechloramine-T method of 131I-labeling, was used (35). 131Ilabeled to antibodies with the iodogen or the chloramine-Tmethod will not be well-retained in the cells if the antigen-antibody complex is internalized (16, 17). The same patternof de-halogenation has been shown with CD22 antibodies,which also are internalized (36). However, metallicradionuclides labeled to antibodies with chelators were betterretained intracellularly when internalized (37).

There are several metallic nuclides that can be used forRIT against CD37. Although we demonstrated results in amouse model with the alpha emitting nuclide 227Th (38), aβ-emitting nuclide may be more suitable as therapy for bulkylymphoma. Clinical data indicate that NHL is responsive tolow Linear Energy Transfer (LET) β-emitters (1-4), thus inthe current work, 177Lu was chosen because of itsavailability, suitable radiochemistry and half-life, andpromising radiation properties.

The inhibition of cell growth after treatment with CD37-directed 177Lu-tetulomab was significantly better than thatfor CD20-directed 177Lu-rituximab, when compared at thesame antibody concentration. The differences in cell growthinhibition were higher for 18-h than for two-hour incubationwith the RICs and thus might, to some extent, be explainedby the higher internalization of tetulomab than of rituximab.The internalization of tetulomab and thus retention of 177Luwill probably result in a higher absorbed radiation dose tothe cells treated with tetulomab than with rituximab, eventhough the initial binding was similar or higher forrituximab. The 1.5- to 2-fold higher binding for rituximabthan for tetulomab can be explained by the 1.5-fold higherspecific activity for rituximab than for tetulomab in thisexperiment. The growth inhibition induced by 177Lu-

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Figure 5. Survival analysis of SCID mice i.v. injected with Daudi cellsand then treated with 50, 100 or 200 MBq/kg 177Lu-tetulomab, 50 μgtetulomab, 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 theNaCl–, 50 μg tetulomab- or the 50 MBq/kg 177Lu-tetulomab-treatedgroups died of radiation toxicity, so these lines overlap. The “cold”tetulomab-treated group was omitted from this panel. The crosses inpanel A represent animals that were censored because of hind legparalysis. Similarly, the crosses in panel B represent animals that werecensored because they died of causes other than hind leg paralysis.

tetulomab and 177Lu-rituximab was related to selectivetargeting of the CD37 and CD20 antigens, respectively, sinceblocking with “cold” tetulomab and rituximab significantlyreduced the inhibition of cell growth.

The cell growth experiments were carried out with Daudicells, which had the highest expression of the CD37 antigen,more than two-fold that for Raji cells. Therefore, one mightwonder if the treatment would also be effective for Raji cells.

One would expect, however, that it would be possible toattain a similar effect on Raji cells, as on Daudi byincreasing the concentration of the antibody by the samefactor 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 of177Lu-rituximab in the spleen in SCID mice cannot beexplained by binding to Daudi cells located in the respective

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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 tetulomabor NaCl. A: White blood cell counts (WBC). B: Platelet counts. C: Red blood cell counts (RBC).

organs, since similar biodistribution without Daudi cellsbeing injected was found (results not shown). The reason forthe unusual biodistribution might be the low production ofendogeneous antibodies in SCID mice. This effect onbiodistribution has been previously described (39).

We report on a similar migration pattern of Daudi cells asthe one presented by Gethie et al. in (22). The relatively hightoxicity of 177Lu-tetulomab in this model was probablyrelated to both the unusual biodistribution and the highradiosensitvity of these DNA double-strand repair-defectivemice (due to the SCID mutation). Nevertheless, thetherapeutic effect of 177Lu-tetulomab was significantly betterthan that for the unlabeled antibody.

The binding data for 125I-labeled tetulomab and rituximabshowed that the antigen-binding properties of tetulomab andrituximab were similar, with a Kd between 2.7 and 12.7 fortetulomab and between 4.8 and 12 for rituximab, dependingon the cell line used. The reason for the variability in Kdbetween cell lines might be that the three parametersestimated by the curve-fitting method, to some extent mayinfluence each other, or there could be differences in theantigen from cell line to cell line due to mutations or post-translational changes. The data for the number of antigenson the Daudi cells fitted well with the measured binding aftertwo hours in the growth inhibition experiment if thedifference in specific activity between 177Lu-tetulomab and177Lu-rituximab is taken into consideration.

In conclusion, the data presented here indicate that thetetulomab antibody is well-suited for RIT of CD37-expressing lymphoma cells. The study of binding oftetulomab to patient biopsies indicates that targeting of theCD37 epitope by tetulomab could be clinically relevant andwarrants for future pre-clinical and clinical investigations.

Declaration of Interest

JD and AHVRL are employed by and have stock options in NordicNanovector AS. RHL owns Nordic Nanovector shares.

Acknowledgements

The Authors are grateful to Steinar Funderud and Erlend Smelandwho developed the tetulomab monoclonal antibody, to BjørnErikstein, Erlend Smeland and Harald Holte for collecting thelymphoma biopsies, and to the Histology Research Laboratory atthe 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 2012Revised November 7, 2012

Accepted November 8, 2012

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