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A. Vidal-Crespo et al.
(I vs. R: **P<0.01), where 70% of the mice were alive at day 107 (Figure 4). Mean survival was 73 days for the con- trol group, 97 for rituximab and 103 for daratumumab groups. On autopsy, the disease had spread to the brain, BM, spleen and lungs (Online Supplementary Figure S4A). Daratumumab treatment reduced the disease burden in the brain, BM, and spleen by 93%, 63%, and 48%, respec- tively (Online Supplementary Figure S4B). Rituximab-treat- ed mice showed a similar reduction in tumor load to that achieved by daratumumab in the brain (73%) and BM (69%) compared to that observed with the isotype con- trol, while no effect was observed in spleen infiltration. Finally, daratumumab did not diminish lung infiltration, while a moderate reduction was seen in rituximab-treated mice (18%) compared to isotype control mice (Online Supplementary Figure S4B), suggesting an organ-dependent differential immunotherapeutic response.
In the FL systemic model, CD20low WSU-FSCCL cells (Table 1) were intravenously inoculated in SCID mice and one week later mice were treated weekly with daratu- mumab, rituximab or isotype control (20/10/10/10 mg/kg). All control-treated and rituximab-treated mice rapidly succumbed to the disease and died or had to be euthanized due to severe disease signs by day 40 (control group) and day 62 (rituximab group), respectively (Figure 4). In contrast, in the daratumumab-treated group, OS was significantly prolonged (I vs. D: ***P<0.001; I vs. R: **P<0.01; D vs. R *P< 0.05). Mean survival was 35 days for the control group, 41 for rituximab and 60 for daratumum-
ab. By the end of the experiment (day 90), 30% of mice treated with daratumumab were cured (Figure 4). On autopsy, WSU-FSCCL cells showed systemic dissemina- tion of disease in the brain, BM and spleen (identified as huCD45+/CD19+CD10+; Online Supplementary Figure S4C). Daratumumab robustly decreased dissemination to the brain (90%, P< 0.01) compared to the control group, while rituximab was not effective (Online Supplementary Figure S4D). Homing of WSU-FSCCL cells to the spleen and BM was less prominent. There were no significant differences in the reduction of dissemination to the spleen and BM by daratumumab compared to the rituximab-treated group (Online Supplementary Figure S4C).
Altogether, these data confirm that daratumumab shows comparable to superior activity with respect to rit- uximab in systemic models of MCL and FL.
Daratumumab synergizes with R-CHOP to reduce tumor burden in pre-clinical models of MCL and FL
The synergy between daratumumab and rituximab and/or chemotherapy regimens was evaluated in MCL (REC-1) and tFL (RL) xenograft heterotopic models. SCID mice were inoculated subcutaneously with NHL cells and when tumors became palpable, mice were randomly assigned into the following groups: i) Isotype ii) daratu- mumab iii) CHOP iv) D-CHOP v) R-CHOP and vi) R-D- CHOP. Tumor volume was assessed twice a week and tumor weights at sacrifice. In the MCL model (Figure 5A), daratumumab induced significant anti-tumor activity
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Figure 2. Daratumumab effect in a three-dimensional model of follicular lymphoma. Three-dimesional (3D) spheroids of RL-GFP cells were obtained after three days of culture in hanging drop plates or 96-well ultra-low attachment plates, and then treated with 10 μg/mL of Isotype control (IgG1), or 1 or 10 μg/mL of daratumumab at different times. (A) 3D reconstruction images produced by SPIM; fluorescence was measured at a λex of 488nm (GFP) and 561nm (monoclonal antibody [mAb] labeling). (B) Percentage of mAb diffusion, representing quantity of the mAb in the spheroid. (C) Percentage of mAb penetration, representing maximum depth of the mAb in the spheroid. (D) Spheroid volume (mm3) time course (n=number of spheroids per treatment). Statistical differences between groups were assessed by unpaired t-test (**P<0.01; ***P<0.001).
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