Gene targeting strategy for B-CAG-hCD19 MC38. The exon 2-12 of mouse CD19 gene that encode the extracellular domain were replaced by human CD19 CDS region in B-CAG-hCD19 MC38.
Protein expression analysis
The CD19 expression analysis in B-CAG-hCD19 MC38 cells by flow cytometry. Single cells were collected from wild-type MC38 and B-CAG-hCD19 MC38 cells, and analyzed by flow cytometry with species-specific anti-CD19 antibody. Human CD19 was detectable in B-CAG-hCD19 MC38 cells but not wild-type MC38 cells.
Tumor Growth Curve & Body Weight Changes
Subcutaneous xenograft tumor growth of B-CAG-hCD19 MC38 cells. B-CAG-hCD19 MC38 cells and wild-type MC38 cells (5×105) were subcutaneously implanted into the C57BL/6 mice (female, 5-8 week-old, n=5).Tumor size and mice body weight were measured twice a week. (A) Tumor average volume ± SEM, (B) Mice body weight (Mean± SEM). Volume was expressed in mm3 using the formula: V=0.5a X b2, where a and b were the long and short diameters of the tumor, respectively. As shown in panel A, B-CAG-hCD19 MC38 were able to establish tumor in vivo and can be used for efficacy study.
Protein expression analysis in tumor cells
1. pre-vaccination cell detection:
Conclusion: Enzyme digestion did not affect the expression of hCD19 in modified cell lines
2. Cell detection during tumor growth
Conclusion: The expression of hCD19 is gradually lost during the tumor growth.
3. Post-tumor cell detection
Conclusion: After digestion, the expression of hCD19 in B-CAG-hCD19 MC38 cells can be restored.
B-CAG-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-CAG-hCD19 MC38 were able to establish tumor in vivo and can be used for efficacy study.Values are expressed as mean ± SEM