The mouse Cd19 gene was replaced by human CD19 coding sequence in B-hCD19 MC38 cells. Human CD19 is highly expressed on the surface of B-hCD19 MC38 cells.

Gene targeting strategy for B-hCD19 MC38 cells. The exogenous promoter and human CD19 coding sequence were inserted to replace part of murine exon 2 and all of exons 3~12. The insertion disrupts the endogenous murine Cd19 gene, resulting in a non-functional transcript.

CD19 expression analysis in B-hCD19 MC38 cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hCD19 MC38 cultures were stained with species-specific anti-CD19 antibody. Human CD19 was detected on the surface of B-hCD19 MC38 cells but not wild-type MC38 cells. The 2-G1 clone of B-hCD19 MC38 cells was used for in vivo experiments.

Subcutaneous homograft tumor growth of B-hCD19 MC38 cells. B-hCD19 MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6 mice (female, 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-hCD19 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Subcutaneous tumor growth of B-hCD19 MC38 cells.
B-hCD19 MC38 cells (1x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into B-hCD3E mice (female, 6-8-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. Results indicate that B-hCD19 MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

CD19 expression evaluated on B-hCD19 MC38 tumor cells by flow cytometry.
B-hCD19 MC38 cells were subcutaneously transplanted into B-hCD3E mice (n=6). Upon conclusion of the experiment, tumor cells were harvested and assessed for human CD19 expression by flow cytometry. As shown, human CD19 was highly expressed on the surface of tumor cells. Therefore, B-hCD19 MC38 cells can be used for in vivo efficacy studies evaluating novel CD19 therapeutics.

B-hCD19 MC38 cell line was subcutaneously implanted into B-hCD3E mice. Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with Blinatumomab with doses and schedules indicated in panel (B) Body weight changes during treatment. As shown in panel A, high dose of CD3e Bispecific antibody (Blinatumomab) was efficacious in controlling tumor growth in B-hCD3E mice, demonstrating that the B-hCD19 MC38 were able to establish tumor in vivo and can be used for efficacy study. Values are expressed as mean ± SEM.