• PD-1 functions as a critical "immune checkpoint" that negatively regulates adaptive immunity. Upon binding to its ligands—programmed death-ligand 1 (PD-L1) or PD-L2—PD-1 initiates inhibitory signaling cascades, resulting in reduced T cell proliferation, cytokine secretion (e.g., IFN-γ, IL-2), and survival. This mechanism limits excessive immune activation and maintains self-tolerance. VEGFA is the primary driver of angiogenesis (new blood vessel formation). It binds to endothelial cell receptors VEGFR-1, VEGFR-2 (predominant), and VEGFR-3, activating signaling pathways (e.g., PI3K/AKT, MAPK) that promote endothelial cell proliferation, migration, and tube formation. Tumor cells upregulate VEGFA to induce tumor angiogenesis, securing nutrient/oxygen supply and facilitating metastasis. Bispecific antibodies (BsAbs) are engineered as dual inhibitors, integrating immune activation (via PD-1 blockade) with vascular normalization (via VEGFA inhibition) to enhance cytotoxic T lymphocyte (CTL) recruitment and function within the tumor microenvironment (TME), thereby overcoming resistance to single-agent checkpoint inhibitors.
• The B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 knock-in mouse model was generated by intercrossing B-hPD-1, B-hPD-L1, B-hVEGFA, and B-hVEGFR2 mice. In this model, the IgV domains of mouse Pdcd1 and Cd274 were replaced with the corresponding human PD-1 and CD274 sequences. Additionally, mouse Vegfa exons 1–8, encoding the full-length protein, were substituted with human VEGFA exons 1–8, while mouse Vegfr2 exons 2–15, encoding the extracellular region, were replaced with human VEGFR2 exons 2–15.
• In homozygous B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice, human VEGFA was detected in lung tissue homogenates, and human VEGFR2 was expressed on lung endothelial cells in fetal mice. Furthermore, human PD-1 and PD-L1 were detectable in splenic T cells from these mice.
• This product is applicable to PD-1/VEGFA bispecific antibody research, thereby accelerating the progression of drug development

Gene targeting strategy for B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice.

The exon 2 of mouse Pdcd1 gene that encodes the IgV domain was replaced by human PD-1 exon 2.

The exon 3 of mouse Cd274 gene that encodes the IgV domain was replaced by human CD274 exon 3.

The exons 1-8 of mouse Vegfa gene that encode the full-length protein were replaced by human VEGFA exons 1-8.

The exons 2-15 of mouse Vegfr2 gene that encode the extracellular region were replaced by human VEGFR2 exons 2-15.

The B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice knock-in model was developed by breeding the B-hPD-1 mice, the B-hPD-L1 mice, B-hVEGFA mice and the B-hVEGFR2 mice .

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 ELISA kit: RD, MVR100; anti-human VEGFA ELISA kit: RD, DVR100C). Human VEGFA was only detectable in homozygous B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice but not in wild-type C57BL/6 mice, with an expression level of approximately 150 pg per milligram of total protein. Mouse VEGFA was only detectable in wild-type C57BL/6 mice. Values are expressed as mean ± SEM.

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. Human VEGFR2 was exclusively detectable in homozygous 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. Human PD-1 was exclusively detectable in homozygous B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice, but not in wild-type C57BL/6 mice.

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. Human PD-L1 was exclusively detectable in homozygous B-hPD-1/hPD-L1/hVEGFA/hVEGFR2 mice, but not in wild-type C57BL/6 mice.