The human CLDN6 coding sequence was inserted to mouse Cldn6 exon 2. Human CLDN6 is highly expressed on the surface of B-hCLDN6 MC38 cells.

Gene targeting strategy for B-hCLDN6 MC38 cells. The exogenous promoter and human CLDN6 coding sequence were inserted to mouse Cldn6 exon 2 gene locus site. The insertion disrupts the endogenous murine Cldn6 gene, resulting in a non-functional transcript.

CLDN6 expression analysis in B-hCLDN6 MC38 cells by flow cytometry. Single cell suspensions from B-hCLDN6 MC38 cultures were stained with species-specific anti-CLDN6 antibody. Human CLDN6 was detected on the surface of B-hCLDN6 MC38 cells, but not on the surface of wild-type MC38 cells. The 2-B06 clone of B-hCLDN6 MC38 cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-hCLDN6 MC38 cells. B-hCLDN6 MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6 mice (female, 7-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 × long diameter × short diameter². As shown in panel A, B-hCLDN6 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-hCLDN6 MC38 cells were subcutaneously transplanted into C57BL/6 mice (n=5) . At the end of the experiment, tumor cells were harvested and assessed for human CLDN6 expression by flow cytometry. As shown, human CLDN6 was highly expressed on the surface of tumor cells. Therefore, B-hCLDN6 MC38 cells can be used for in vivo efficacy studies of novel CLDN6 therapeutics.

B-hCLDN6 MC38 tumor growth of individual mice. B-hCLDN6 MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6 mice (female, 7-week-old, n=5). As shown in panel, B-hCLDN6 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Subcutaneous homograft tumor growth of B-hCLDN6 MC38 cells. B-hCLDN6 MC38（2-B06 clone） cells and wild-type MC38 cells were subcutaneously implanted into B-h4-1BB mice (female, 6-week-old, n=6). 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 × long diameter × short diameter². As shown in panel A, B-hCLDN6 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

B-hCLDN6 MC38 tumor growth of individual mice. B-hCLDN6 MC38 cells and wild-type MC38 cells were subcutaneously implanted into B-h4-1BB mice (female, 6-week-old, n=6). As shown in panel, B-hCLDN6 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.