The coding sequence of human B2M gene and the whole genomic sequence of human HLA-A2.1 gene were randomly inserted into the genome of B-Tg(WT1 peptide-HLA-A2.1) MC38. Human HLA-A2.1 is highly expressed on the surface of B-Tg(WT1 peptide-HLA-A2.1) MC38.

Gene targeting strategy for B-Tg(WT1 peptide-HLA-A2.1) MC38. The coding sequence of human B2M gene and the whole genomic sequence of human HLA-A2.1 gene were randomly inserted into the genome of B-Tg(WT1 peptide-HLA-A2.1) MC38. Human HLA-A2.1 is highly expressed on the surface of B-Tg(WT1 peptide-HLA-A2.1) MC38.

HLA-A2.1 expression analysis in B-Tg(WT1 peptide-HLA-A2.1) MC38 by flow cytometry. Single cell suspensions from wild-type MC38 and B-Tg(WT1 peptide-HLA-A2.1) MC38 cultures were stained with species-specific anti-HLA-A2.1 antibody. Human HLA-A2.1 was detected on the surface of B-Tg(WT1 peptide-HLA-A2.1) MC38 cells but not wild-type MC38 cells. The 1-G03 clone of B-Tg(WT1 peptide-HLA-A2.1) MC38 cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-Tg(WT1 peptide-HLA-A2.1) MC38 cells. B-Tg(WT1 peptide-HLA-A2.1) MC38  cells (5x10⁵) were subcutaneously implanted into B-HLA-A2.1 mice (female, 7-week-old, n=8). Tumor volume and body weight were measured three times 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-Tg(WT1 peptide-HLA-A2.1) MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.