PD-1 (programmed cell death protein 1, PDCD1) and its ligand PD-L1 (CD274, also known as B7-H1) are pivotal immune checkpoint molecules that regulate T cell activation, tolerance, and peripheral immune homeostasis. While PD-1 is primarily expressed on activated T cells, B cells, and myeloid cells, PD-L1 is broadly expressed not only on antigen-presenting cells (APCs) but also on non-hematopoietic tissues and many tumor cells.

Through PD-1/PD-L1 interactions, tumors exploit this pathway to suppress T cell function, leading to immune evasion. Overexpression of PD-L1 is strongly associated with poor prognosis in cancers such as melanoma, lung cancer, and renal cell carcinoma, making PD-L1 a critical biomarker and therapeutic target in oncology. The success of anti-PD-L1 antibodies (e.g., atezolizumab, durvalumab, avelumab) highlights its importance in clinical immunotherapy.

In PD-1/PD-L1 humanized mice, the murine Pdcd1 and Cd274 genes are replaced with the corresponding human PDCD1 and CD274 sequences, enabling the expression of human PD-1 and PD-L1 under physiological promoters. This design maintains normal immune cell development and immune homeostasis while allowing for specific evaluation of human PD-1/PD-L1 interactions in vivo.

This PD-1/PD-L1 humanized mouse model provides a robust and translationally relevant platform for preclinical efficacy testing, antibody validation, biomarker discovery, and combination therapy development targeting the PD-1/PD-L1 axis.

Key Advantages

• Humanized expression of both PD-1 and PD-L1 under native promoters.
• Enables evaluation of anti-PD-1, anti-PD-L1, and combination checkpoint inhibitor therapies.
• Maintains normal immune system development for reliable translational studies.
• Provides a robust platform for immuno-oncology drug discovery.
• Suitable for studying tumor immune evasion and autoimmune disease mechanisms.
• Bridges the translational gap for antibody validation in vivo.

Validation

• Flow cytometry confirmed species-specific expression:
  human PD-1 and PD-L1 exclusively detected in homozygous PD-1/PD-L1 humanized mice, absent in WT controls.
• In vivo validation with checkpoint inhibitors:
  pembrolizumab (anti-PD-1) and atezolizumab (anti-PD-L1) inhibited tumor growth in a dose-dependent manner.
• Stable immune function:
  blood routine and biochemistry tests indicated largely normal immune and metabolic profiles, ensuring translational relevance.
• Demonstrated compatibility with syngeneic tumor models (e.g., MC38), enabling accurate evaluation of drug response.

Application

• Preclinical efficacy testing of anti-PD-1 and anti-PD-L1 antibodies.
• In vivo antibody validation for checkpoint inhibitors, including clinically approved drugs.
• Combination therapy studies (anti-PD-1 + anti-PD-L1, or with chemotherapy, radiotherapy, targeted therapies, novel immunotherapies).
• Biomarker discovery and resistance mechanism studies, especially PD-L1 expression as a predictive marker.
• Autoimmunity and chronic infection models, where PD-1/PD-L1 dysregulation contributes to disease.
• Immuno-oncology drug development, bridging the gap between basic research and clinical translation.

PD-1

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

•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 PD-1/PD-L1 humanized mice (B-hPD-1/hPD-L1).

•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 mouse and human PD-1 and PD-L1 mRNA expression in wild-type C57BL/6 mice and PD-1/PD-L1 humanized mice by RT-PCR. Splenocytes were isolated from wild-type C57BL/6 mice (+/+) and homozygous PD-1/PD-L1 humanized mice (H/H). Mouse Pd-1 and Pd-l1 mRNA were detected in wild-type C57BL/6 mice, whereas human PD-1 and PD-L1 mRNA were detected in homozygous PD-1/PD-L1 humanized mice but not in wild-type C57BL/6 mice.

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

Complete blood count (CBC) analysis of PD-1/PD-L1 humanized mice. Blood samples were collected from female wild-type C57BL/6 mice and PD-1/PD-L1 humanized mice (6 weeks old, n = 5) and analyzed for complete blood count (CBC) parameters. PD-1/PD-L1 humanized mice exhibited blood cell composition and morphology comparable to those of wild-type C57BL/6 controls, indicating no obvious hematological abnormalities associated with PD-1 and PD-L1 humanization. Values are expressed as mean ± SEM.

Blood biochemical analysis of PD-1/PD-L1 humanized mice. Serum samples were collected from female wild-type C57BL/6 mice and PD-1/PD-L1 humanized mice (6 weeks old, n = 5) and analyzed for aspartate aminotransferase (AST), albumin (ALB), glucose (GLU), cholesterol (CHOL), and creatinine (CREA) levels. No obvious differences in these blood biochemical parameters were observed between PD-1/PD-L1 humanized mice and wild-type C57BL/6 controls, indicating no apparent clinical chemistry abnormalities associated with PD-1 and PD-L1 humanization. Values are expressed as mean ± SEM.

Antitumor efficacy of a pembrolizumab analog in a human PD-L1-expressing, luciferase-labeled MC38 syngeneic tumor model using PD-1/PD-L1 humanized mice. MC38 cells (B-hPD-L1-luc MC38 plus, 5 × 10⁵) were subcutaneously implanted into homozygous PD-1/PD-L1 humanized mice (male, 8 weeks old, n = 5). When tumor volumes reached approximately 80 mm³, mice were randomized into treatment groups and administered an in-house pembrolizumab analog. (A) Tumor volume. (B) Body weight. Treatment with the pembrolizumab analog inhibited tumor growth, demonstrating the utility of PD-1/PD-L1 humanized mice as a preclinical model for evaluating the in vivo antitumor efficacy of anti-human PD-1 antibodies. Values are expressed as mean ± SEM.

Antitumor efficacy of an atezolizumab analog in a human PD-L1-expressing, luciferase-labeled MC38 syngeneic tumor model using PD-1/PD-L1 humanized mice. MC38 cells (B-hPD-L1-luc MC38 plus, 5 × 10⁵) were subcutaneously implanted into homozygous PD-1/PD-L1 humanized mice (male, 5 weeks old, n = 5). When tumor volumes reached approximately 100 mm³, mice were randomized into treatment groups and administered an in-house atezolizumab analog. (A) Tumor volume. (B) Body weight. Treatment with the atezolizumab analog inhibited tumor growth, demonstrating the utility of PD-1/PD-L1 humanized mice as a preclinical model for evaluating the in vivo antitumor efficacy of anti-human PD-L1 antibodies. Values are expressed as mean ± SEM.