Expression cassettes containing the exogenous promoter and human TROP2 CDS were randomly inserted into the genome of B-hTROP2 low MC38 cells. Human TROP2 was detected to be expressed on the surface of B-hTROP2 low MC38 cells.

Gene targeting strategy for B-hTROP2 low MC38 cells. Expression cassettes containing the exogenous promoter and human TROP2 CDS were randomly inserted into the genome of MC38 cells that do not express the mouse TROP2.

Human TROP2 expression analysis in B-hTROP2 low MC38 cells by flow cytometry. Single cell suspensions of wild-type MC38 and B-hTROP2 low MC38 cultures were stained with species-specific anti-TROP2 antibody. Human TROP2 was detected on the surface of B-hTROP2 low MC38 cells but not wild-type MC38 cells. The 3-B10 clone of B-hTROP2 low MC38 cells was used for in vivo tumor growth assays.

Subcutaneous homograft tumor growth of B-hTROP2 low MC38 cells. B-hTROP2 low MC38 cells (5x10⁵) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into C57BL/6 mice (female, 7-week-old, 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 X long diameter X short diameter². As shown in panel A, B-hTROP2 low MC38 cells were able to form tumors in vivo and can be used for efficacy studies.

B-hTROP2 low MC38 cells were subcutaneously transplanted into C57BL/6 mice (n=5), and on 28 days post inoculation, tumor cells were harvested and assessed for human TROP2 expression by flow cytometry. As shown, human TROP2 was highly expressed on the surface of tumor cells. Therefore, B-hTROP2 low MC38 cells can be used for in vivo efficacy studies of TROP2 therapeutics.

Subcutaneous homograft tumor growth of B-hTROP2 low MC38 cells. B-hTROP2 low MC38 cells (5x10⁵, 1x10⁶, 5x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into B-hCD3E mice (female, 7-week-old, n=6). 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 X long diameter X short diameter². As shown in panel A, B-hTROP2 low MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Subcutaneous homograft tumor growth of B-hTROP2 low MC38 cells. B-hTROP2 low MC38 cells (5x10⁵, 1x10⁶, 5x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into B-h4-1BB mice (female, 9-week-old, n=6). 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 X long diameter X short diameter². As shown in panel A, B-hTROP2 low MC38 cells were able to establish tumors in vivo and can be used for efficacy studies.

Subcutaneous tumor growth of B-hTROP2 low MC38 cells. B-hTROP2 low MC38 cells (5x10⁵, 1x10⁶) and wild-type MC38 cells (5x10⁵) were subcutaneously implanted into B-hCTLA4 mice (female, 6-9-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 X long diameter X short diameter². Results indicate that B-hTROP2 low MC38 cells were able to establish tumors in vivo and can be used for efficacy studies. Values are expressed as mean ± SEM.

Antitumor activity of TROP2 antibody conjugated TLR8 agonists in B-hTLR8 mice. (A) Anti-human TROP2 antibody conjugated TLR8 agonists inhibited MC38 tumor growth in B-hTLR8 mice. Murine colon cancer B-hTROP2 low MC38 cells were subcutaneously implanted into homozygous B-hTLR8 mice (female, 9 week-old, n=8). Mice were grouped according to body weight differences, at which time they were treated antibody conjugates with different doses. (B) Body weight changes during treatment. As shown in panel A, anti-human TROP2 antibody conjugated TLR8 agonists were efficacious in controlling tumor growth in B-hTLR8 mice, demonstrating that B-hTLR8 mouse model is a promising model for preclinical in vivo studies to evaluate antibodies conjugated TLR8 agonists. Values are expressed as mean ± SEM.