Gene targeting strategy for B-hHLA-E MC38 plus cells. The exogenous promoter and human HLA-E coding sequence was inserted to replace part of murine exon 3 and all of exons 4~7. The insertion disrupts the endogenous murine H2-T23 gene, resulting in a non-functional transcript.

HLA-E expression analysis in B-HLA-E MC38 plus cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-HLA-E MC38 plus cultures were stained with species-specific anti-HLA-E antibody. Human HLA-E was detected on the surface of B-HLA-E MC38 plus cells but not wild-type MC38 cells. The 1-A07 clone of B-HLA-E MC38 plus cells was used for in vivo experiments.

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

B-HLA-E MC38 plus cells were subcutaneously transplanted into C57BL/6 mice (n=5). At the end of the experiment, tumor cells were harvested and assessed for human HLA-E expression by flow cytometry. As shown, human HLA-E was highly expressed on the surface of tumor cells. Therefore, B-HLA-E MC38 plus cells can be used for in vivo efficacy studies of novel NKG2A or CD94 therapeutics.