PD-1/hPD-L1: An immune-checkpoint and tumor-associated target for therapeutic intervention

•Gene Information: PD-1 (PDCD1/CD279) is an inhibitory immune-checkpoint receptor. PD-L1 (CD274/B7-H1) is a key PD-1 ligand that mediates immune suppression in tumors and antigen-presenting-cell contexts.

•Protein Expression: PD-1 is induced after antigen-specific T-cell stimulation and is often sustained during chronic activation. PD-L1 can be constitutively expressed by tumor cells or induced by IFN-γ and other inflammatory cues.

•Signaling Pathway: PD-L1/PD-1 interaction initiates inhibitory signaling, including dephosphorylation events that suppress TCR downstream signaling. This reduces T-cell activation, cytotoxicity, and tumor-cell killing.

•Therapeutic Inhibition: Anti-PD-1 or anti-PD-L1 monoclonal antibodies block the inhibitory interaction and restore cytotoxic T-cell activity. B-hPD-1/hPD-L1 mice provide an in vivo platform for evaluating PD-1/PD-L1 checkpoint blockade and combination therapies.

PD-1

•Exon 2 of the mouse PD-1 gene, which encode the IgV domain, was replaced with human PD-1 exon 2 in B-hPD-1/hPD-L1 mice.

•The endogenous mouse promoter, 5′ UTR, 3′ UTR, and genomic regions encoding the signal peptide, non-IgV domain of the Extracellular domain, transmembrane and cytoplasmic domains are retained. The chimeric PD-1 expression is driven by the endogenous mouse PD-1 promoter, while endogenous mouse PD-1 transcription and translation are disrupted.

PD-L1

•Exon 3 of the mouse PD-L1 gene, which encode the IgV domain, was replaced with human PD-L1 exon 3 in B-hPD-1/hPD-L1 mice.

•The endogenous mouse promoter, 5′ UTR, 3′ UTR, and genomic regions encoding the signal peptide, non-IgV domain of the Extracellular domain, transmembrane and cytoplasmic domains are retained. The chimeric PD-L1 expression is driven by the endogenous mouse PD-L1 promoter, while endogenous mouse PD-L1 transcription and translation are disrupted.

Strain-specific analysis of PD-1 and PD-L1 gene expression in wild-type C57BL/6 mice and homozygouse B-hPD-1/hPD-L1 mice by RT-PCR. Splenocytes were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hPD-1/hPD-L1 mice (H/H). Mouse PD-1 and PD-L1 mRNA was detected in wild-type mice, while human PD-1 and PD-L1 mRNA was detected in homozygous B-hPD-1/hPD-L1 mice but not in wild-type mice.

Strain-specific analysis of PD-1 and PD-L1 protein expression in wild-type C57BL/6 mice and homozygous B-hPD-1/hPD-L1 mice by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice (+/+) and homozygous B-hPD-1/hPD-L1 mice (H/H) and stimulated with anti-CD3ε antibody. Mouse PD-1 and PD-L1 were detected in wild-type mice, while human PD-1 and PD-L1 were detected in B-hPD-1/hPD-L1 mice but not in wild-type mice.

Complete blood count (CBC) analysis of B-hPD-1/hPD-L1 mice. Blood was collected from female C57BL/6 and B-hPD-1/hPD-L1 mice (6-week-old, n = 5) and analyzed for CBC parameters. B-hPD-1/hPD-L1 mice showed blood cell composition and morphology comparable to C57BL/6 controls, indicating no obvious hematological abnormalities associated with PD-1/hPD-L1 humanization. Values are expressed as mean ± SEM.

Blood chemistry analysis of B-hPD-1/hPD-L1 mice. Serum was collected from female C57BL/6 and B-hPD-1/hPD-L1 mice (6-week-old, n = 5) and analyzed for AST, ALB, GLU, CHOL, and CREA. No obvious differences were observed between B-hPD-1/hPD-L1 mice and C57BL/6 controls, indicating no apparent clinical chemistry abnormalities associated with PD-1/PD-L1 humanization. Values are expressed as mean ± SEM.

Antitumor activity of pembrolizumab analog in the B-hPD-L1-luc MC38 plus syngeneic tumor model. B-hPD-L1-luc MC38 plus cells (5×105) were subcutaneously implanted into homozygous B-hPD-1/hPD-L1 mice (male, 8 weeks old, n = 5). Mice were randomized when tumor volume reached approximately 80 mm³ and treated with pembrolizumab analog (in-house). (A) Tumor volume. (B) Body weight. Pembrolizumab analog inhibited tumor growth, supporting B-hPD-1/hPD-L1 mice as an in vivo model for evaluating anti-human PD-1 antibodies. Values are expressed as mean ± SEM.

Antitumor activity of atezolizumab analog in the B-hPD-L1-luc MC38 plus syngeneic tumor model. B-hPD-L1-luc MC38 plus cells (5×105) were subcutaneously implanted into homozygous B-hPD-1/hPD-L1 mice (male, 5 weeks old, n = 5). Mice were randomized when tumor volume reached approximately 100 mm³ and treated with atezolizumab analog (in-house). (A) Tumor volume. (B) Body weight. Atezolizumab analog inhibited tumor growth, supporting this model for anti-human PD-L1 antibody efficacy evaluation. Values are expressed as mean ± SEM.