B-hCD28 mice

Basic Information

Gene targeting strategy

Exons 2~3 of mouse Cd28 gene that encode the extracellular domain were replaced by human CD28 exons 2~3 in B-hCD28 mice.

Protein expression analysis

Strain specific CD28 expression analysis in homozygous B-hCD28 mice by flow cytometry. Splenocytes were collected from WT and homozygous B-hCD28 (H/H) mice stimulated with anti-CD3ε in vivo, and analyzed by flow cytometry with species-specific anti-CD28 antibody. Mouse CD28 was detectable in WT mice. Human CD28 was exclusively detectable in homozygous B-hCD28 but not WT mice.

Analysis of spleen leukocytes cell subpopulations in B-hCD28 mice

Analysis of spleen leukocyte subpopulations by FACS.

Splenocytes were isolated from female C57BL/6 and B-hCD28 mice (n=3, 6 week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for CD45 population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T, B, NK, Monocyte, DC and macrophage cells in homozygous B-hCD28 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types in spleen.

Analysis of spleen T cell subpopulations in B-hCD28 mice

Analysis of spleen T cell subpopulations by FACS.

Splenocytes were isolated from female C57BL/6 and B-hCD28 mice (n=3, 6 week-old). Flow cytometry analysis of the splenocytes was performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live CD45+ cells were gated for CD3 T cell population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of CD8, CD4, and Treg cells in homozygous B-hCD28 mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hCD28 in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell sub types in spleen. Values are expressed as mean ± SEM.

Analysis of T cell proliferation stimulated with anti-CD3 and anti-CD28 antibody in vitro

Analysis of T cell proliferaion stimulated with anti-CD3 and anti-CD28 antibody in vitro. Homozygous B-hCD28 mice were treated with anti-mCD3e antibody and anti-mCD28 antibody or anti-hCD28 antibody as indicated on the graph (female, 6 week-old, n=3). T cells were analyzed at 48 and 72 hours after treatment. Anti-mCD28 antibody resulted in an increased proliferation of T cells in WT mice, and anti-hCD28 antibody resulted in an increased proliferation of T cells in B-hCD28 mice. Values are expressed as mean ± SEM.

Analysis of T cell activation stimulated with anti-CD3 and anti-CD28 antibody in vitro

Analysis of T cell activation stimulated with anti-CD3 and anti-CD28 antibody in vitro.

Homozygous B-hCD28 mice were treated with anti-mCD3e antibody and anti-mCD28 antibody or anti-hCD28 antibody as indicated on the graph (female, 6 week-old, n=3). T cells were analyzed at 24, 48 and 72 hours after treatment. Anti-mCD28 antibody resulted in an increased activation of T cells in WT mice, and anti-hCD28 antibody resulted in an increased activation of T cells in B-hCD28 mice. Values are expressed as mean ± SEM.

In Vivo Efficacy Evaluation of Anti-Human CD28 Antibodies

Anti-tumor activity of anti-human CD28 antibodies in humanized B-hCD28 mice. Murine colon cancer MC38 cells were subcutaneously implanted into homozygous B-hCD28 mice (female, 6-7-week-old, n=5). (A) Anti-human CD28 antibodies inhibited tumor growth in humanized B-hCD28 mice, (B) without negatively impacting body weight changes. Values are expressed as mean ± SEM.

In vivo efficacy of anti-human PD-L1xCD28 bispecific antibody

Antitumor activity of anti-PD-L1 and anti-CD28 (PD-L1xCD28) bispecific antibody (BsAb) in B-hCD28 mice.Murine colon cancer B-hPD-L1 MC38 plus cells were subcutaneously implanted into homozygous B-hCD28 mice (female, 7-8 week-old, n=10). Mice were grouped when tumor volume reached approximately 200 mm³, at which time they were treated with anti-human PD-L1xCD28 BsAbs provided by the client. (A) anti-human PD-L1xCD28 BsAbs inhibited B-hPD-L1 MC38 plus cells tumor growth in B-hCD28 mice. (B) Body weight changes during treatment. As shown in panel A, anti-human PD-L1xCD28 BsAbs were efficacious in controlling tumor growth in B-hCD28 mice, demonstrating that the B-hCD28 mice provide a powerful preclinical model for in vivo evaluation of anti-human PD-L1xCD28 BsAbs. Values are expressed as mean ± SEM.

References

1. FEBS J. 2016 Sep;283(18):3325-34. doi: 10.1111/febs.13754. Epub 2016 Jun 6.
2. Cytokine Growth Factor Rev. 2016 Apr;28:11-9. doi: 10.1016/j.cytogfr.2016.02.004. Epub 2016 Mar 4.
3. Proc Natl Acad Sci U S A. 2016 Oct 18;113(42):E6437-E6446. Epub 2016 Oct 5.