• 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

• 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 tumour cells, cancer‑associated fibroblasts, tumour‑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 tumour 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. 

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 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 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 mice.

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

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

Strain specific PD-1 expression analysis in wild-type C57BL/6 and homozygous B-hPD-1/hPD-L1/hVEGFA mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hPD-1/hPD-L1/hVEGFA mice (H/H) after stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs (female, 6-week-old, n=1) or not. Protein expression was analyzed with anti-mouse PD-1 antibody (Biolegend, 109104) and anti-human PD-1 antibody (Biolegend, 329904) by flow cytometry.

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

Strain specific PD-L1 expression analysis in wild-type C57BL/6 and homozygous B-hPD-1/hPD-L1/hVEGFA mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6 mice (+/+) and homozygous B-hPD-1/hPD-L1/hVEGFA mice (H/H) after stimulated with anti-mouse CD3ε antibody (7.5 μg, i.p.) in vivo for 24 hrs (female, 6-week-old, n=1) or not. Protein expression was analyzed with anti-mouse PD-L1 antibody (Biolegend, 124312) and anti-human PD-L1 antibody (Biolegend, 329706) by flow cytometry.

• Mouse VEGFA was detected exclusively in wild-type C57BL/6 mice
• Human VEGFA was detected in homozygous B-hPD-1/hPD-L1/hVEGFA mice, but not in wild-type mice.

Strain specific VEGFA expression analysis in wild-type C57BL/6 mice and homozygous B-hPD-1/hPD-L1/hVEGFA mice by ELISA. Lung homogenates were collected from wild-type C57BL/6 mice (+/+) (male, n=3, 8-week-old) and homozygous B-hPD-1/hPD-L1/hVEGFA mice (H/H; H/H; H/H) (male, n=3, 8-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).

• The frequencies of T cells, B cells, NK cells, DCs, granulocyte, monocytes, macrophages, CD4+ T cells, CD8+ T cells and Tregs in homozygous B-hPD-1/hPD-L1/hVEGFA mice were similar to those in C57BL/6 mice
• Humanization of PD-1, PD-L1 and VEGFA does not change the frequency or distribution of these cell types in spleen.

Frequency of leukocyte subpopulations in spleen by flow cytometry. Splenocytes were isolated from wild-type C57BL/6 mice and homozygous B-hPD-1/hPD-L1/hVEGFA mice (n=3, 10-week-old). A. Flow cytometry analysis of the splenocytes was performed to assess the frequency of leukocyte subpopulations. B. Frequency of T cell subpopulations. Values are expressed as mean ± SEM. Significance was determined by two-way ANOVA test.  *P < 0.05, **P < 0.01, ***p < 0.001.

Establishment of a B-hVEGFA MC38 model and in vivo efficacy study of Ivonescimab antibody. B-hVEGFA MC38 cells were implanted subcutaneously into homozygous B-hPD-1/hPD-L1/hVEGFA mice (female, 8 weeks old, n=6). When the average tumor volume reached approximately 70~90 mm³, mice were randomized and subsequently administered the Ivonescimab analog via intraperitoneal injection.

Antitumor activity of Ivonescimab analog (in-house) in B-hPD-1/hPD-L1/hVEGFA mice. (A) Tumor growth curves. (B) Body weight changes during treatment. As shown in panel A, Ivonescimab was efficacious in controlling tumor growth in B-hPD-1/hPD-L1/hVEGFA mice. Values are expressed as mean ± SEM. The overage of this tumor model is 50%.

Establishment of a B-hVEGFA MC38 model and in vivo efficacy study of Bevacizumab & Keytruda combination antibody. B-hVEGFA MC38 cells were implanted subcutaneously into homozygous B-hPD-1/hPD-L1/hVEGFA mice (female, 8 weeks old, n=6). When the average tumor volume reached approximately 70~90 mm³, mice were randomized and subsequently administered with anti-PD-1 & anti-VEGFA antibody via intraperitoneal injection.

Antitumor activity of Bevacizumab & Keytruda in B-hPD-1/hPD-L1/hVEGFA mice. (A) Tumor growth curves. (B) Body weight changes during treatment. As shown in panel A, Bevacizumab was efficacious in controlling tumor growth in B-hPD-1/hPD-L1/hVEGFA mice. The combination of these two antibodies is not more effective than monotherapy. Values are expressed as mean ± SEM.

The overage of this tumor model is 40%.   

Establishment of a B-hVEGFA/hPD-L1 plus MC38 model and in vivo efficacy study of Ivonescimab antibody. B-hVEGFA/hPD-L1 plus MC38 cells were implanted subcutaneously into homozygous B-hPD-1/hPD-L1/hVEGFA mice (male, 8 weeks old, n=6). When the average tumor volume reached approximately 300 mm³, mice were randomized and subsequently administered with anti-PD-1 & anti-VEGFA antibody via intraperitoneal injection.

Antitumor activity of Ivonescimab (also known as AK112) in B-hPD-1/hPD-L1/hVEGFA mice. (A) Tumor growth curves. (B) Body weight changes during treatment. As shown in panel A, Ivonescimab was efficacious in controlling tumor growth in B-hPD-1/hPD-L1/hVEGFA mice. Values are expressed as mean ± SEM. The overage of this tumor model is 60%.

Establishment of a B-hVEGFA/hPD-L1 B16-F10 model and in vivo efficacy study of Ivonescimab antibody. B-hVEGFA/hPD-L1 B16-F10 cells were implanted subcutaneously into homozygous B-hPD-1/hPD-L1/hVEGFA mice (female, 8 weeks old, n=6). When the average tumor volume reached approximately 150 mm³, mice were randomized and subsequently administered with anti-PD-1 & anti-VEGFA antibody via intraperitoneal injection.

Antitumor activity of Ivonescimab (also known as AK112) in B-hPD-1/hPD-L1/hVEGFA mice. (A) Tumor growth curves. (B) Body weight changes during treatment. As shown in panel A, Ivonescimab was efficacious in controlling tumor growth in B-hPD-1/hPD-L1/hVEGFA mice. Values are expressed as mean ± SEM. The overage of this tumor model is 60%. Note: one mouse dead in G3 on day12.