The mouse Gprc5d gene was replaced by human GPRC5D coding sequence in B-hGPRC5D MC38 cells. Human GPRC5D is highly expressed on the surface of B-hGPRC5D MC38 cells.

Gene targeting strategy for B-hGPRC5D MC38 cells. The exogenous promoter and human GPRC5D coding sequence were inserted to replace part of murine exon 2 and all of exons 3 and 4. The insertion disrupts the endogenous murine Gprc5d gene, resulting in a non-functional transcript.

GPRC5D expression analysis in B-hGPRC5D MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hGPRC5D MC38 cultures were stained with species-specific anti-GPRC5D antibody. Human GPRC5D was detected on the surface of B-hGPRC5D MC38 cells, but not on the surface of wild-type MC38 cells. The 3-D10 clone of B-hGPRC5D MC38 cells was used for in vivo tumor growth assays.

B-hGPRC5D MC38 cells were subcutaneously transplanted into C57BL/6 mice (n=6). At the end of the experiment, tumor cells were harvested and assessed for human GPRC5D expression by flow cytometry. As shown, human GPRC5D was highly expressed on the surface of tumor cells. Therefore, B-hGPRC5D MC38 cells can be used for in vivo efficacy studies of GPRC5D therapeutics.

Subcutaneous homograft tumor growth of B-hGPRC5D MC38 cells. B-hGPRC5D MC38 cells (1x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6 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-hGPRC5D MC38 cells were able to form tumors in vivo and can be used for efficacy studies.

Subcutaneous homograft tumor growth of B-hGPRC5D MC38 cells. B-hGPRC5D MC38 cells and wild-type MC38 cells were subcutaneously implanted into B-h4-1BB mice (female, 7-week-old). Tumor volume and body weight were measured twice a week. (A) Average tumor volume. (B) Body weight. Volume was expressed in mm³ using the formula: V=0.5 X long diameter X short diameter². As shown in panel A, B-hGPRC5D MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

B-hGPRC5D MC38 tumor growth of individual mice. B-hGPRC5D MC38 cells (1x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6 mice (female, 7-week-old, n=6). As shown in panel, B-hGPRC5D MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hGPRC5D MC38 tumor growth of individual mice. B-hGPRC5D MC38 cells and wild-type MC38 cells were subcutaneously implanted into B-h4-1BB mice (female, 7-week-old). As shown in panel, B-hGPRC5D MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.