Gene Information: PD‑1 is encoded by the PDCD1 gene on chromosome 2q37.3, while PD‑L1 is encoded by CD274 on chromosome 9p24.1. Both are type I transmembrane proteins; PD‑1 belongs to the immunoglobulin superfamily and functions as an inhibitory receptor, whereas PD‑L1 is a member of the B7 family of co‑stimulatory molecules.
Protein Expression: PD-1 is primarily expressed on activated T cells, B cells, and NK cells, whereas PD-L1 is expressed on antigen-presenting cells and many tumor cells, with expression often induced by IFN-γ.
Signaling Pathway: Binding of PD-L1 to PD-1 recruits SHP2 phosphatase and suppresses TCR/CD28-mediated PI3K–AKT signaling, resulting in reduced T-cell proliferation, cytokine production, and cytotoxic activity.
Therapeutic Inhibition: Anti-PD-1 and anti-PD-L1 antibodies block this inhibitory pathway, restoring T-cell function and enhancing anti-tumor immune responses.
VEGFA: a master regulator of angiogenesis and therapeutic intervention
Gene Information: VEGFA is a protein‑coding gene located on chromosome 6p21.1. It encodes a potent angiogenic growth factor that belongs to the PDGF/VEGF family, with multiple isoforms generated by alternative splicing.
Protein Expression: VEGFA is secreted by tumor cells, cancer‑associated fibroblasts, tumor‑infiltrating macrophages, and endothelial cells themselves. Its expression is strongly upregulated by hypoxia, oncogenic mutations, and inflammatory mediators such as IL‑6 and TGF‑β, creating a pro‑angiogenic gradient within the tumor microenvironment.
Signaling Pathway: VEGFA binds with high affinity to receptor tyrosine kinases VEGFR‑1 and VEGFR‑2 on endothelial cells. This drives endothelial cell proliferation, migration, tube formation, vascular permeability, and survival. Additionally, VEGFA promotes the recruitment of immunosuppressive cells and upregulates PD‑L1 on endothelial and tumour cells, directly linking angiogenesis to immune evasion.
Therapeutic Inhibition: Anti‑VEGFA monoclonal antibodies and small‑molecule multikinase inhibitors targeting VEGFRs block ligand‑receptor binding or downstream kinase activity.
VEGFR2: A central regulator of angiogenesis and its therapeutic intervention
Gene Information: Vascular endothelial growth factor receptor 2 (VEGFR2), also known as KDR or FLK1, is a protein-coding gene located on chromosome 4q12. It encodes a receptor tyrosine kinase belonging to the VEGFR family.
Protein Expression: VEGFR2 is primarily expressed on vascular endothelial cells, lymphatic endothelial cells, and hematopoietic cells. It predominantly exists as a full-length transmembrane receptor, serving as the main transducer of angiogenic signals.
Signaling Pathway: VEGFR2 binds VEGFA, VEGFC, and VEGFD, utilizing NP1/2 as co-receptors. It possesses strong tyrosine kinase activity and plays a primary role in mediating vascular permeability, endothelial cell proliferation, migration, and survival..
Therapeutic Inhibition: By blocking VEGFR2 binding, dimerization, or kinase activity with monoclonal antibodies or tyrosine kinase inhibitors, downstream signaling and cell migration are inhibited, offering a major strategy for anti-angiogenic therapy.
Complete Human Pathway: Quadruple humanization of PD-1, PD-L1, VEGFA, and VEGFR2 preserves native human signaling, enabling precise in vivo evaluation.
IND-Enabling Data Package: Provides a robust platform for generating preclinical safety and efficacy data to support IND submissions for combination therapeutics.
Versatile Disease Modeling: Enables robust in vivo modeling of immune evasion and pathological angiogenesis within the tumor microenvironment.
Target & Pathway Validation: Enables study of the PD-1/PD-L1 axis and its regulatory crosstalk with VEGFA/VEGFR2 signaling pathways.
Efficacy & Safety Evaluation: Supports efficacy and safety assessment of anti-hPD-1/PD-L1, anti-hVEGFA/VEGFR2 antibodies, bispecifics, and combination therapies.
Novel Therapeutic Screening: Facilitates the discovery and screening of novel immuno-oncology and anti-angiogenic combination strategies for translational research.
PD-1
The exon 2 of mouse Pdcd1 gene that encodes the IgV domain was replaced by human PDCD1 exon 2 in B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice.
PD-L1
The exon 3 of mouse Cd274 gene that encodes the IgV domain was replaced by human CD274 exon 3 in B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice. Sequences of other regions still belonged to mice.
VEGFA
The exons 1-8 of mouse Vegfa gene that encode the full-length protein were replaced by human VEGFA exons 1-8 in B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice.
VEGFR2
The exons 2-15 of mouse Kdr gene that encodes the extracellular region coding sequences were replaced by human KDR exons 2-15 in B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice.
Strain specific PD-1 expression analysis in wild-type C57BL/6 mice and homozygous B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) (male, 17-week-old, n=2) and homozygous B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice (H/H) (male, 17-week-old, n=2). Cells were stimulated in vivo via intraperitoneal injection of 7.5 μg/mouse anti-CD3ε antibodies (clone 145-2C11, BioXCell: BE0001-1) for 24 hours. Protein expression was analyzed with anti-mouse PD-1 antibody (Biolegend, 109104) and anti-human PD-1 antibody (Biolegend, 329908) by flow cytometry.
Strain specific PD-L1 expression analysis in wild-type C57BL/6 mice and homozygous B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+)(male, 17-week-old, n=2). and homozygous B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice (H/H)(male, 17-week-old, n=2). Cells were stimulated in vivo via intraperitoneal injection of 7.5 μg/mouse anti-CD3ε antibodies (clone 145-2C11, BioXCell: BE0001-1) for 24 hours. Protein expression was analyzed with anti-mouse PD-L1 antibody (Biolegend, 124312) and anti-human PD-L1 antibody (Biolegend, 329706) by flow cytometry.
VEGFR2 expression analysis in homozygous B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice by flow cytometry. Fetal mouse lung endothelial cells were collected from homozygous B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice (H/H). Protein expression was analyzed with anti-human VEGFR2 antibody (Biolegend, 359904) by flow cytometry.
Strain specific VEGFA expression analysis in wild-type C57BL/6 mice and homozygous humanized B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice by ELISA. Lung tissue homogenate was collected from wild-type C57BL/6 mice (+/+) (male, n=3, 10-week-old) and homozygous B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice (H/H) (male, n=3, 10-week-old). Expression level of mouse and human VEGFA were analyzed by ELISA (anti-mouse VEGFA antibody: R&D, MMV00; anti-human VEGFA antibody: R&D, DVE00). Values are expressed as mean ± SEM.
