DLL3 expression analysis in B-hDLL3 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hDLL3 MC38 cultures were stained with anti-human DLL3 antibody AMG-757 (in house). Human DLL3 was detected on the surface of B-hDLL3 MC38 cells but not wild-type MC38 cells. The 3-G07 clone of B-hDLL3 MC38 cells was used for in vivo experiments. 

Subcutaneous homograft tumor growth of B-hDLL3 MC38 cells. B-hDLL3 MC38 cells (5x105) and wild-type MC38 cells (5x105) were subcutaneously implanted into C57BL/6 mice (female, 7-week-old). Tumor volume and body weight were measured twice a week. (A) Average tumor volume ± SEM. (B) Body weight (Mean± SEM). Volume was expressed in mm3 using the formula: V=0.5 X long diameter X short diameter2. As shown in panel A, B-hDLL3 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Subcutaneous homograft tumor growth of B-hDLL3 MC38 cells. B-hDLL3 MC38 cells (1x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into B-hCD3E mice (female, 7-week-old, n=6). Tumor volume and body weight were measured twice a week. (A) Average tumor volume ± SEM. (B) Body weight (Mean± SEM). Volume was expressed in mm³ using the formula: V=0.5 X long diameter X short diameter². As shown in panel A, B-hDLL3 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Note：

Due to the presence of immunogenicity, the expression level of hDLL3 in tumor tissue has been slightly reduced. We suggest enrolling 8-10 mice per group, or inoculating the DLL3 cell line in DLL3-related humanized mice, to evaluate tumor efficacy.

B-hDLL3 MC38 cells were subcutaneously transplanted into B-hCD3E mice (n=6), and on 28 days post inoculation, tumor cells were harvested and assessed for human DLL3 expression by flow cytometry using anti-human DLL3 antibody AMG-757 (in house). As shown, human DLL3 was expressed on the surface of tumor cells. Therefore, B-hDLL3 MC38 cells can be used for in vivo efficacy studies of novel DLL3 therapeutics.

Antitumor activity of bispecific antibody (BsAb) X in B-hCD3E mice. B-hDLL3 MC38 cells were subcutaneously implanted into B-hCD3E mice (female, 8 week-old, n=6). Mice were grouped when the tumor size was approximately 100 mm³, at which time they were treated with BsAb X provided by the client with doses and schedules indicated in panel. (A) Tumor volume changes during treatment. (B) Body weight changes during treatment. As shown in panel A, BsAb X was efficacious in controlling tumor growth in B-hCD3E mice, demonstrating that the B-hCD3E mouse model is a powerful tool for in vivo efficacy study of T cell bispecific antibody. Values are expressed as mean ± SEM.