Description

• PD-1 (PDCD1, programmed cell death 1) is primarily expressed on activated T cells (including CD4+, CD8+, and exhausted T cells), B cells, and NKT cells. Its ligands, PD-L1 and PD-L2, are found on antigen-presenting cells and tumor cells. The PD-1 signaling pathway delivers inhibitory signals by recruiting SHP2 phosphatase upon ligand binding, which dampens TCR/CD28-mediated activation, promotes T cell exhaustion, and maintains peripheral tolerance to prevent autoimmunity and facilitate tumor immune evasion.
• The CDS that encodes human PD-1 signal peptide, extracellular domain, transmembrane and cytoplasmic domain is inserted right after mouse PD-1 ATG to replace the mouse PD-1 gene in B-hPD-1 mice ad.
• Mouse PD-1 was only detectable in wild-type C57BL/6 mice. Human PD-1 was exclusively detectable in homozygous B-hPD-1 mice ad.
• The anti-human PD-1 antibodies (pembrolizumab analog or nivolumab analog, in-house) effectively inhibited the tumor growth in B-hPD-1 mice ad.
• Application: B-hPD-1 mice ad can be used as a mouse model to validate the pharmacodynamics and safety of drugs for treating tumors or autoimmune diseases.

Targeting Strategy

Gene targeting strategy for B-hPD-1 mice ad. The CDS that encode human PD-1 signal peptide, extracellular domain, transmembrane and cytoplasmic domain is inserted right after mouse PD-1 ATG to replace the mouse PD-1 gene. The human PD-1 protein expression will be driven by endogenous mouse PD-1 promoter, while mouse PD-1 gene transcription and translation will be disrupted.

Protein Expression Analysis in Spleen

Strain specific PD-1 ad expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hPD-1 mice ad by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hPD-1 mice ad (H/H) stimulated with or without anti-mouse CD3ε antibody (7.5 μg/mice, i.p.) in vivo for 24 hrs (female, 10-week-old, n=1). Protein expression was analyzed with anti-mouse PD-1 antibody (Biolegend, 329904) and anti-human PD-1 antibody (Biolegend, 109104) by flow cytometry. Mouse PD-1 was only detectable in wild-type C57BL/6 mice. Human PD-1 was exclusively detectable in homozygous B-hPD-1 mice ad, but not in wild-type C57BL/6 mice.

In Vivo Efficacy of Anti-human PD-1 Antibodies

Antitumor activity of anti-human PD-1 antibody (pembrolizumab analog, in-house) in B-hPD-1 mice ad. (A) Anti-human PD-1 antibody inhibited MC38 tumor growth in B-hPD-1 mice ad. Murine colon cancer MC38 cells (5×10⁵) were subcutaneously implanted into homozygous B-hPD-1 mice ad (male, 7-week-old, n=7). Mice were grouped when tumor volume reached approximately 100-150 mm³, at which time they were intraperitoneally injected with anti-human PD-1 antibodies indicated in panel. (B) Body weight changes during treatment. As shown in panel A, anti-human PD-1 antibody was efficacious in controlling tumor growth in B-hPD-1 mice ad, demonstrating that the B-hPD-1 mice ad provides a powerful preclinical model for in vivo evaluation of anti-human PD-1 antibodies. Values are expressed as mean ± SEM.

Antitumor activity of anti-human PD-1 antibody (nivolumab analog, in-house) in B-hPD-1 mice ad. (A) Anti-human PD-1 antibody inhibited MC38 tumor growth in B-hPD-1 mice ad. Murine colon cancer MC38 cells (5×10⁵) were subcutaneously implanted into homozygous B-hPD-1 mice ad (male, 7-week-old, n=7). Mice were grouped when tumor volume reached approximately 100-150 mm³, at which time they were intraperitoneally injected with anti-human PD-1 antibodies indicated in panel. (B) Body weight changes during treatment. As shown in panel A, anti-human PD-1 antibody was efficacious in controlling tumor growth in B-hPD-1 mice ad, demonstrating that the B-hPD-1 mice ad provides a powerful preclinical model for in vivo evaluation of anti-human PD-1 antibodies. Values are expressed as mean ± SEM.