B-hCD16A mice(CB-17 SCID)

Basic Information

Targeting Strategy

Gene targeting strategy for B-hCD16A mice(CB-17 SCID).

The regulatory region and exons 1~5 of mouse Fcgr4 gene that encode the full-length protein were replaced by human FCGR3A exons 1~4 and regulatory region in B-hCD16A mice(CB-17 SCID).

Surface Expression in NK Cells

Species-specific FCGR3A expression analysis in wild-type CB-17 SCID mice and homozygous B-hFCGR3A(CB-17 SCID) by flow cytometry.  Splenocytes were collected from wild-type CB-17 SCID mice(+/+)  and homozygous B-hFCGR3A mice(CB-17 SCID) (H/H). Mouse FCGRIV was not detectable in NK cells of wild-type CB-17 SCID mice. Human FCGR3A was detectable in NK cells of B-hFCGR3A mice(CB-17 SCID). Note: the clone of anti-human CD16A antibody is 3G8.

Surface Expression in Granulocytes

Species-specific FCGR3A expression analysis in wild-type CB-17 SCID mice and homozygous B-hFCGR3A(CB-17 SCID)  by flow cytometry.  Splenocytes were collected from wild-type CB-17 SCID mice(+/+)  and homozygous B-hFCGR3A mice (CB-17 SCID) (H/H). Mouse FCGR3A was exclusively detectable in granulocytes of wild-type CB-17 SCID. Human FCGR3A was not detectable in granulocytes of wild-type CB-17 SCID mice and homozygous B-hFCGR3A mice (CB-17 SCID). Note: the clone of anti-human CD16A antibody is 3G8.

Surface Expression in Macrophages

Strain specific FCGR3A expression analysis in wild-type CB-17 SCID mice and homozygous B-hFCGR3A(CB-17 SCID)  by flow cytometry. Peritoneal exudative macrophages(PEMs) were collected from wild-type CB-17 SCID mice(+/+)  and homozygous B-hFCGR3A mice (CB-17 SCID) (H/H). Mouse FCGRIV was exclusively detectable in PEMs of wild-type C57BL/6 mice. Human FCGR3A was exclusively detectable in PEMs of homozygous B-hFCGR3A mice (CB-17 SCID). Note: the clone of anti-human CD16A antibody is 3G8.

Surface Expression in Monocytes

Strain specific CD16A expression analysis in wild-type CB-17 SCID mice and homozygous B-hCD16A (CB-17 SCID) by flow cytometry.  Splenocytes were collected from wild-type CB-17 SCID mice(+/+)  and homozygous B-hCD16A mice(CB-17 SCID) (H/H). Mouse CD16A was exclusively detectable in monocytes of wild-type CB-17 SCID mice. Human CD16A was exclusively detectable in monocytes of B-hCD16A mice(CB-17 SCID). Note: the clone of anti-human CD16A antibody is 3G8.

Strain specific CD16A expression analysis in wild-type CB-17 SCID mice and homozygous B-hCD16A (CB-17 SCID) by flow cytometry. Blood cells were collected from wild-type CB-17 SCID mice(+/+)  and homozygous B-hCD16A mice(CB-17 SCID) (H/H). Mouse CD16A was exclusively detectable in monocytes of wild-type CB-17 SCID mice. Human CD16A was exclusively detectable in monocytes of B-hCD16A mice(CB-17 SCID). Note: the clone of anti-human CD16A antibody is 3G8.

Analysis of Leukocyte Cell Populations

Analysis of spleen leukocyte subpopulations by FACS. Splenocytes were isolated from female wild-type CB-17 SCID and B-hCD16A mice(CB-17 SCID) (n=3, 8-week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hCD16A mice(CB-17 SCID) were similar to those in the CB-17 SCID mice, demonstrating that CD16A humanization does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Analysis of blood leukocyte subpopulations by FACS. Splenocytes were isolated from female wild-type CB-17 SCID and B-hCD16A mice(CB-17 SCID) (n=3, 8-week-old). Flow cytometry analysis of the blood leukocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hCD16A mice(CB-17 SCID) were similar to those in the CB-17 SCID mice, demonstrating that CD16A humanization does not change the overall development, differentiation or distribution of these cell types in spleen. Values are expressed as mean ± SEM.

Tumor Growth Curves

Subcutaneous CDX tumor growth of B-hHep3B-Luc cells in B-hCD16A mice(CB-17 SCID).

B-hHep3B-Luc cells(8×10⁶) were subcutaneously implanted into B-hCD16A mice(CB-17 SCID) (female, 8-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-hHep3B-Luc cells were able to establish tumors in B-hCD16A mice(CB-17 SCID) and can be used for efficacy studies.

Subcutaneous CDX tumor growth in B-hCD16A mice(CB-17 SCID).

HC116 (5×10⁶), K-562 (1×10⁶)  and NCI-N87 (1×10⁷) were subcutaneously implanted into B-hCD16A mice(CB-17 SCID) (female, 8-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, HCT116, K-562 and NCI-N87 were able to establish tumors in B-hCD16A mice(CB-17 SCID) and can be used for efficacy studies.

In vivo Efficacy of Anti-human CLDN18.2 Antibody

Antitumor activity of anti-human CLDN18.2 antibody in B-hCD16A mice (CB-17 SCID) bearing B-hCLDN18.2 BxPC-3 cells. (A) Anti-hCLDN18.2 antibody inhibited tumor growth. Human pancreas B-hCLDN18.2 BxPC-3 cells were subcutaneously implanted into homozygous B-hCD16A mice (CB-17 SCID) (female, 10 week-old, n=6). Mice were grouped when tumor volume reached approximately 100 mm³, at which time they were treated with antibodies in panel A. (B) Body weight changes during treatment. As shown in panel, B-hCD16A mice (CB-17 SCID) bearing B-hCLDN18.2 BxPC-3 cells provide a powerful preclinical model for in vivo evaluation of anti-CLDN18.2 antibodies. Values are expressed as mean ± SEM. Last dosing was on day 56.