Cancer therapy often faces challenges such as drug resistance and immune evasion. Conventional therapeutic antibodies for immune anti-cancer therapy have shown shortcomings like high immunogenicity and lacking capability of enabling efficient intracellular delivery.

PD-L1, an immune checkpoint inhibitor, and VEGFR2, which is essential for cancer metastasis, play pivotal roles in tumorigenesis. However, their miniature bispecific intracellular nanobodies for combining check-point blockade and anti-metastasis anticancer therapy is underexplored.

In a study published in Molecular Cancer, Prof. ZHANG Lei and Prof. CHEN Zhuo from the Fujian Institute of Research on the Structure of Matter of the Chinese Academy of Sciences, and the collaborators, developed a novel bispecific nanobody, FAP1V2, and explored the mechanisms involved in how transient intracellular expression of PD-L1 and VEGFR2 bispecific nanobody in cancer cells inspires long-term T cell activation and infiltration to combat tumor and inhibit cancer metastasis.

The FAP1V2 is designed to simultaneously target two critical pathways: immune checkpoint blockade and metastasis suppression. Researchers found that the FAP1V2 nanobody contains the heavy chain variable regions V_H of antibodies against PD-L1 and VEGFR2. Lacking the Fc fragment, FAP1V2 has reduced immunogenicity and enables efficient intracellular delivery. It keeps highly specific and efficient ability to bind and block the antigens PD-L1 and VEGFR2.

Researchers found that two rounds of transient FAP1V2 expression in Lewis lung carcinoma cells resulted in remarkable tumor suppression in mice models, and one out of six treated mice exhibited complete eradication of secondary tumors. The therapy enhanced T cell infiltration into tumors, reduced PD-1^high immune cells, and boosted CD25^high activated T cells in the spleen, indicating robust systemic immune activation. FAP1V2 significantly inhibited cancer cell migration by blocking VEGFR2 signaling, preventing metastasis to the lung and liver.

Transcriptome analysis revealed that FAP1V2 modulates critical pathways including Wnt/β-catenin and PI3K/AKT, while downregulating transcription of metastasis-promoting genes. This dual-action mechanism, which enhances immune response while stifling tumor spread, made FAP1V2 as a transformative candidate for combination cancer therapies. With high biocompatibility and targeted approach, FAP1V2 offers hope for more effective and personalized therapies.

This study presents a potential strategy for enhancing immune activation while inhibiting metastasis in tumor therapy. It introduces a new framework for investigating the intracellular regulation of signaling pathways through the application of V_H intrabodies.