• PD-1 (PDCD1) and its ligand PD-L1 (CD274) are key immune checkpoint molecules that negatively regulate T-cell activation in the tumor microenvironment. VEGFA is a major pro-angiogenic factor that promotes tumor vascularization and immune suppression. Co-targeting PD-1/PD-L1 and VEGFA has become an important strategy in oncology, with several anti-PD-1/VEGF or anti-PD-L1/VEGF combination therapies and bispecific antibodies currently under clinical investigation.
• In B-hPD-1/hPD-L1/hVEGFA humanized mice, the endogenous mouse Pdcd1, Cd274 and Vegfa genes are replaced by their human counterparts on a C57BL/6 background. Homozygous animals express human PD-1, human PD-L1 and human VEGFA while maintaining normal immune cell composition in spleen, providing a physiologically relevant triple humanized PD-1/PD-L1/VEGFA mouse model. This allows direct in vivo evaluation of human-specific PD-1/PD-L1 checkpoint inhibitors, anti-VEGFA antibodies, and PD-1 x VEGF bispecific antibodies.
• When implanted with B-hVEGFA MC38 or B-hVEGFA/hPD-L1 plus MC38 syngeneic tumors, B-hPD-1/hPD-L1/hVEGFA mice support robust antitumor responses to anti-PD-1/VEGFA bispecific antibodies, bevacizumab & Keytruda combination therapy, and ivonescimab (AK112), enabling translational assessment of tumor growth inhibition, body weight change, and dosing regimens.

Key Advantages

• Triple humanized checkpoint/angiogenesis axis: Simultaneous humanization of PD-1, PD-L1, and VEGFA on a C57BL/6 background provides a unique humanized PD-1/PD-L1/VEGFA mouse model to evaluate immuno-oncology and anti-angiogenic therapies within a single system.
• Validated expression of human PD-1, PD-L1 and VEGFA: Flow cytometry and ELISA confirm the loss of mouse PD-1, PD-L1, and VEGFA and exclusive expression of human PD-1, human PD-L1, and human VEGFA in the spleen and lung.
• Intact immune cell composition: Comparable frequencies of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes, macrophages, CD4⁺ and CD8⁺ T cells, and Tregs relative to wild-type mice demonstrate that triple humanization does not disrupt immune homeostasis.
• Ready-to-use syngeneic tumor models: Compatible with B-hVEGFA MC38 and B-hVEGFA/hPD-L1 MC38 cell lines, enabling fully human ligand–receptor interactions and robust antitumor responses for checkpoint inhibitor and anti-VEGFA drug evaluation.
• Demonstrated efficacy of clinical-relevant agents: The model supports in vivo efficacy testing of anti-PD-1/VEGF bispecific antibodies (ivonescimab analog, ivonescimab/AK112) and bevacizumab + Keytruda combination therapy, aligning preclinical findings with ongoing clinical strategies.
• Ideal for antibody validation and combination-strategy optimization: Suitable for dose finding, sequence and combination optimization, biomarker exploration and comparative studies versus other humanized PD-1 or PD-L1 mouse models.

Strain-specific PD-1 and PD-L1 expression was analyzed in wild-type C57BL/6 mice (+/+) and homozygous PD-1/PD-L1/VEGFA humanized mice (H/H) by flow cytometry. Splenocytes were collected from both strains with or without in vivo anti-CD3ε stimulation.
Mouse PD-1 and mouse PD-L1 were detectable only in wild-type C57BL/6 mice, whereas human PD-1 and human PD-L1 proteins were exclusively detected in the PD-1/PD-L1/VEGFA humanized mouse model, confirming successful replacement of the endogenous mouse genes.

Strain-specific VEGFA expression was evaluated in lung homogenates from wild-type C57BL/6 mice (+/+) and homozygous PD-1/PD-L1/VEGFA humanized mice (H/H; H/H; H/H) using ELISA. Mouse VEGFA protein was detectable only in wild-type mice, while human VEGFA was exclusively expressed in PD-1/PD-L1/VEGFA humanized mice, demonstrating complete humanization of the Vegfa locus suitable for anti-VEGFA drug evaluation.

Splenocytes were isolated from wild-type C57BL/6 mice and homozygous PD-1/PD-L1/VEGFA humanized mice (n=3, 10-week-old) and analyzed by flow cytometry to determine leukocyte and T cell subset frequencies. The percentages of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes, macrophages, CD4⁺ T cells, CD8⁺ T cells and Tregs in humanized PD-1/PD-L1/VEGFA mice were comparable to those in wild-type controls. These results indicate that humanization of PD-1, PD-L1 and VEGFA does not alter immune cell distribution in the spleen. Values are expressed as mean ± SEM. Significance was determined using a two-way ANOVA test (*P < 0.05, **P < 0.01, ***P < 0.001).

The antitumor activity of an PD-1 x VEGFA bispecific antibody (Ivonescimab analog, in-house) was evaluated in PD-1/PD-L1/VEGFA humanized mice. Murine colon cancer B-hVEGFA MC38 cells were subcutaneously implanted into homozygous mice (female, 8-week-old, n=6). When tumor volume reached approximately 70–90 mm³, animals were grouped and intraperitoneally injected with the bispecific antibody as indicated. The bispecific antibody significantly inhibited tumor growth in the humanized PD-1/PD-L1/VEGFA mouse model, demonstrating its suitability for evaluating PD-1 x VEGF dual-targeting agents. Body weight changes were monitored throughout the study. Values are presented as mean ± SEM, and significance was assessed by two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001).
The overage of this tumor model is 50%.

Antitumor activity of bevacizumab and Keytruda (pembrolizumab) was assessed in PD-1/PD-L1/VEGFA humanized mice implanted with murine colon cancer B-hVEGFA MC38 cells (female, 8-week-old, n=6). When tumors reached 70–90 mm³, mice were assigned to treatment groups and intraperitoneally injected with anti-PD-1 and/or anti-VEGFA antibodies. Bevacizumab monotherapy effectively controlled tumor growth in the humanized model. However, the combination of bevacizumab with Keytruda did not yield enhanced efficacy compared to monotherapy.Values are presented as mean ± SEM.

The overage of this tumor model is 40%.

The antitumor activity of Ivonescimab (AK112), an PD-1 x VEGF bispecific antibody, was evaluated in PD-1/PD-L1/VEGFA humanized mice engrafted with B-hVEGFA/hPD-L1 MC38 cells (male, 8-week-old, n=5). Mice were grouped when tumor volume reached approximately 300 mm³ and received intraperitoneal injections of ivonescimab as indicated.
Ivonescimab treatment effectively inhibited tumor growth in the triple humanized PD-1/PD-L1/VEGFA model. Values are expressed as mean ± SEM.

The overage of this tumor model is 60%.

Q1. What are B-hPD-1/hPD-L1/hVEGFA mice mainly used for?

B-hPD-1/hPD-L1/hVEGFA mice are triple humanized PD-1/PD-L1/VEGFA mice designed for in vivo efficacy studies of human-specific PD-1/PD-L1 checkpoint inhibitors, anti-VEGFA antibodies and anti-PD-1/VEGF bispecific antibodies, including ivonescimab and bevacizumab/Keytruda combinations in B-hVEGFA MC38 syngeneic tumor models.

Q2. How do these humanized PD-1/PD-L1/VEGFA mice differ from single humanized PD-1 mice?

Compared with single-humanized PD-1 mice, this triple-humanized PD-1/PD-L1/VEGFA model simultaneously expresses human PD-1, PD-L1, and VEGFA. It better recapitulates the clinical setting in which anti–PD-1/PD-L1 and anti-VEGF/VEGFA agents act on multiple human targets, making it more predictive for combination therapies and bispecific antibody development.

Q3. Can I use B-hPD-1/hPD-L1/hVEGFA mice for ivonescimab (AK112) preclinical studies?

Yes. Ivonescimab (AK112) has been tested in B-hPD-1/hPD-L1/hVEGFA mice bearing B-hVEGFA/hPD-L1 MC38 tumors, where it showed significant antitumor activity, demonstrating that this model is suitable for ivonescimab and other anti-PD-1/VEGF bispecific antibody evaluation. 

Q4. What tumor models are recommended with B-hPD-1/hPD-L1/hVEGFA mice?

B-hVEGFA MC38 and B-hVEGFA/hPD-L1 MC38 tumor models are recommended, as they express human VEGFA and human PD-L1, ensuring fully human ligand–receptor engagement with human PD-1/PD-L1/VEGFA in the host mice.