The B2M gene (Exon2 to Exon3) of mouse were replaced by the sequence encompassing the human B2M CDS, HLA-A*0201 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the murine H-2D^b gene containing the α3, transmembrane and cytoplasmic domains and NY-ESO-1 CDS. Human HLA-A2.1 is highly expressed on the surface of B-HLA-A2.1/hNY-ESO-1 MC38.

Gene targeting strategy for B-HLA-A2.1/hNY-ESO-1 MC38. The B2M gene (Exon2 to Exon3) of mouse were replaced by the sequence encompassing the human B2M CDS, HLA-A*0201 gene that included leader sequence, α1 and α2 domains ligated to a fragment of the murine H-2Db gene containing the α3, transmembrane and cytoplasmic domains and NY-ESO-1 CDS. Human HLA-A2.1 is highly expressed on the surface of B-HLA-A2.1/hNY-ESO-1 MC38.

HLA-A2.1 expression analysis in B-HLA-A2.1/NY-ESO-1 MC38 by flow cytometry. Single cell suspensions from wild-type MC38 and B-HLA-A2.1/NY-ESO-1 MC38 cultures were stained with species-specific anti-HLA-A2.1 antibody. Human HLA-A2.1 was detected on the surface of B-HLA-A2.1/NY-ESO-1 MC38 cells but not wild-type MC38 cells. The 3-D04 clone of B-HLA-A2.1/NY-ESO-1 MC38 cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-HLA-A2.1/hNY-ESO-1 MC38 cells. B-HLA-A2.1/hNY-ESO-1 MC38 cells (5x10⁵) were subcutaneously implanted into B-HLA-A2.1 mice (male, 7-week-old, n=6). 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 AB-HLA-A2.1/hNY-ESO-1 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

B-HLA-A2.1/hNY-ESO-1 MC38 cells were subcutaneously transplanted into B-HLA-A2.1 mice (n=6), and on 24 days post inoculation, tumor cells were harvested and assessed for human HLA-A2.1 expression by flow cytometry and NY-ESO-1 expression by western blot, respectively. As shown, human HLA-A2.1 was highly expressed on the surface of tumor cells(A). Human NY-ESO-1 was highly expressed(B). Therefore, B-HLA-A2.1/hNY-ESO-1 MC38 cells can be used for in vivo efficacy studies of novel NY-ESO-1 therapeutics.