Enhanced in vivo antimyeloma effects induced by 2 different mAb/B-BiTE complexes. (A) Three million MM1S/CD38^lowSLAMF7^high cells were injected subcutaneously into NOD/Shi-scid IL-2rγ(null) mice, then 1.0 × 10⁷ freshly isolated PBMCs obtained from the donor were injected IV on day 4. Daratumumab (30 μg) or daratumumab/B-BiTE complex (daratumumab, 30 μg; B-BiTE, 10 μg) was administered IV on day 5. On day 12, 30 μg elotuzumab was administered IV to the mice treated with daratumumab alone. On the other hand, daratumumab/B-BiTE or elotuzumab/B-BiTE (mAb, 30 μg; B-BiTE, 10 μg) was administered to the mice treated with daratumumab/B-BiTE on the same day (top left). Tumor sizes after treatment are depicted (bottom left), and the overall survival of each murine group (n = 6) is summarized in the form of Kaplan-Meier curves (right). Overall survival was compared among the groups using a log-rank (Mantel-Cox) test. ∗P  < .05; ∗∗∗P  < .001. (B) To perform histological analyses, the experiments in panel A were repeated, and subcutaneous tumors were resected from each murine group on day 17. Representative hematoxylin and eosin staining and fluorescence immunohistochemistry for human CD138 at the tumor sites are shown (left). The images in white rectangles are magnified, and the scale bars in the pictures represent 600 μm (black) and 100 μm (yellow). In addition, tumor tissues were homogenized, and the phenotypes of human cells that accumulated at the tumor sites were also analyzed by flow cytometry. Representative dot plots of human CD45^–CD138⁺ residual MM1S myeloma cells (labeled red), human CD45⁺CD3⁺CD8⁺ T cells (labeled blue), human CD45⁺CD3⁺CD4⁺ T cells (labeled green), as well as human CD45⁺CD3^–CD16⁺CD56⁺ NK cells (labeled orange) obtained from each murine group (n = 3) are shown (right). (C) Three hundred thousand MM1S/CD38^lowSLAMF7^high/SLR⁺ cells were injected into the right tibia of irradiated NOD/Shi-scid IL-2rγ(null) mice. After confirming tumor engraftment by bioluminescence imaging assays on day 7, a total of 3.0 × 10⁶ human PBMCs were injected IV on day 8. Daratumumab (30 μg) or daratumumab/B-BiTE (daratumumab, 30 μg; B-BiTE, 10 μg) was similarly administered on day 9, followed by administration of elotuzumab (30 μg), daratumumab/B-BiTE, or elotuzumab/B-BiTE (mAb, 30 μg; B-BiTE, 10 μg) on day 13. Total photon counts for each mouse were acquired, and luminescence intensity was calculated as arbitrary units (n = 4 mice per group). Obtained whole images are shown in supplemental Figure 10B. Magnified images of the right tibia from each mouse are displayed on the left, and luminescence data for each murine group are summarized on the right. SLR, stable luciferase red.