Basic Information
Description
The chimeric CDS composed of mouse signal peptide, human IgV region, mouse extracellular region except IgV domain, moue transmembrane region and mouse intracellular region of PD-L1 gene was inserted to replace the IgV domain of mouse Pdl1 gene. Human PD-L1 is highly expressed on the surface of B-hPD-L1 low MC38 plus cells.
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Targeting strategy
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Gene targeting strategy for B-hPD-L1 low MC38 plus cells. The chimeric CDS composed of mouse signal peptide, human IgV region, mouse extracellular region except IgV domain, moue transmembrane region and mouse intracellular region of PD-L1 gene was inserted to replace the IgV domain of mouse Pdl1 gene. The insertion disrupts the endogenous mouse Pdl1 gene, resulting in a non-functional transcript.
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Protein expression analysis
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PD-L1 expression analysis in B-hPD-L1 low MC38 plus cells by flow cytometry. Single cell suspensions from wild-type MC38 and B-hPD-L1 low MC38 plus cultures were stained with species-specific anti-PD-L1 antibody. Human PD-L1 was detected on the surface of B-hPD-L1 low MC38 plus cells but not wild-type MC38 cells. The 2-D10 clone of B-hPD-L1 low MC38 plus cells was used for in vivo experiments.
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Tumor growth curve & Body weight changes
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Subcutaneous homograft tumor growth of B-hPD-L1 low MC38 plus cells. B-hPD-L1 low MC38 plus cells (1×106), B-hPD-L1 low MC38 plus cells (3×106) and wild-type MC38 cells (5×105) were subcutaneously implanted into C57BL/6 mice (female, 9-week-old, n=5). 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 X long diameter X short diameter2. As shown in panel A, B-hPD-L1 low MC38 plus cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.
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Protein expression analysis of tumor cells
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B-hPD-L1 low MC38 plus cells were subcutaneously transplanted into C57BL/6 mice (n=5), and on 29 days post inoculation, tumor cells were harvested and assessed for human PD-L1 expression by flow cytometry. As shown, human PD-L1 was highly expressed on the surface of tumor cells. Therefore, B-hPD-L1 low MC38 plus cells can be used for in vivo efficacy studies of novel PD-L1 therapeutics.
