The mouse Hvem gene was replaced by human HVEM coding sequence in B-hHVEM MC38 cells. Human HVEM is highly expressed on the surface of B-hHVEM MC38 cells.

Gene targeting strategy for B-hHVEM MC38 cells. The exogenous promoter and human HVEM coding sequence were inserted to replace part of murine exon 3 and all of exon 4. The insertion disrupts the endogenous murine Hvem gene, resulting in a non-functional transcript.

Subcutaneous homograft tumor growth of B-hHVEM MC38 cells. B-hHVEM MC38 cells (5x10⁵ and 1x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6N mice (female, 5-8-week-old, n=5). 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-hHVEM MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Subcutaneous homograft tumor growth of B-hHVEM MC38 cells. B-hHVEM MC38 cells (1x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into homozygous B-hBTLA/hHVEM mice (female, 6-week-old, n=7). 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-hHVEM MC38 cells were able to form tumors in vivo and can be used for efficacy studies.

B-hHVEM MC38 cells were subcutaneously transplanted into homozygous B-hBTLA/hHVEM mice (n=7), and on 28 days post inoculation, tumor cells were harvested and assessed for human HVEM expression by flow cytometry. As shown, human HVEM was highly expressed on the surface of tumor cells. Therefore, B-hHVEM MC38 cells can be used for in vivo efficacy studies of HVEM therapeutics.