• Gene Information: PD-1 (PDCD1) and PD-L1 (CD274) are key immune checkpoint molecules regulating T-cell activity, whereas PTK7 is a tumor-associated cell-surface glycoprotein. Together, these targets link tumor-targeted therapy with cancer immunotherapy.

• Protein Expression: PD-1 is primarily expressed on activated T cells, while PD-L1 is expressed on tumor cells and various immune cells. PTK7 is frequently overexpressed in multiple solid tumors, including colorectal, breast, ovarian, and lung cancers, and is associated with tumor progression.

• Signaling Pathway: PD-1 binding to PD-L1 inhibits T-cell activation and promotes tumor immune evasion. PTK7 primarily regulates non-canonical Wnt/planar cell polarity (PCP) signaling and interacts with Wnt pathway components to control cell polarity, migration, and invasion.

• Model application: PD-1/PD-L1 blockade restores anti-tumor T-cell activity, while PTK7-targeted ADCs selectively eliminate PTK7-positive tumor cells. Their combination provides a promising strategy for enhancing anti-tumor efficacy and overcoming immune suppression.

PD-1

•  A chimeric CDS including human PD-1 gene encoding the signal peptide and extracellular region, mouse PD-1 gene encoding the transmembrane and cytoplasmic region, a stop sequence WPRE were inserted after the initiation codon ATG of mouse PD-1 gene in B-hPD-1 plus/hPD-L1/hPTK7 mice. 
• The endogenous mouse promoter and 5′ UTR 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.

•  Exon 3 of the mouse PD-L1 gene, which encodes the IgV domain, was replaced with human PD-L1 exon 3 in B-hPD-1 plus/hPD-L1/hPTK7 Mice. 
• 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.

• The exons 2–14 of the mouse Ptk7 gene, which encode the extracellular domain, were replaced by human counterparts in B-hPD-1 plus/hPD-L1/hPTK7 mice. The genomic region of the mouse Ptk7 gene encoding the signal peptide, transmembrane domain, and cytoplasmic portion was retained.

PD-1 and PD-L1 expression analysis in wild-type C57BL/6JNifdc mice and homozygous humanized B-hPD-1 plus/hPD-L1/hPTK7 mice by flow cytometry. Splenocytes were collected from wild-type C57BL/6JNifdc mice (+/+) and homozygous B-hPD-1 plus/hPD-L1/hPTK7 mice (H/H, H/H, H/H) (Female, 6-8-week-old, n=1) stimulated with anti-CD3ε in vivo (7.5 μg/mice, stimulation for 24 hours, i.p.). Protein expression was analyzed with anti-human PD-1 antibody (Biolegend, 329908), anti-mouse PD-1 antibody (Biolegend, 109104), anti-human PD-L1 antibody (Biolegend, 329706) and anti-mouse PD-L1 antibody (Biolegend, 124312) by flow cytometry.

Immunohistochemical (IHC) analysis of PTK7 protein expression in wild-type C57BL/6JNifdc mice and B-hPD-1 plus/hPD-L1/hPTK7 mice. Major tissues were collected from wild-type C57BL/6JNifdc mice and homozygous B-hPD-1 plus/hPD-L1/hPTK7 mice and analyzed by IHC with anti-PTK7 antibody (Abcam, 325170).