Background

Anti-PD-1 antibodies have transformed cancer immunotherapy by inducing long-term complete remissions in a subset of patients. However, studies have shown that a significant proportion of patients remain unresponsive to immune checkpoint blockade (ICB) therapy. Antibody-cytokine fusions are a class of drugs that show potential to enhance the anticancer properties of other immunotherapies. EDA-FN is expressed in most solid and hematologic tumors but is virtually undetectable in healthy adult tissues, making it an attractive target for cytokine delivery at the site of disease.

On August 13, 2024, researchers at Philogen published a study titled "An IL-7 fusion protein targeting EDA fibronectin upregulates TCF1 on CD8+ T-cells, preferentially accumulates to neoplastic lesions, and boosts PD-1 blockade" in the Journal for immunotherapy of cancer. The study describes the generation and characterization of a novel interleukin-7 fusion protein, F8(scDb)-IL7, that targets EDA-FN. The product consists of an F8 antibody in a single-chain, bivalent antibody (scDb) format that is specific for the alternatively spliced EDA of FN and fused to IL-7. F8(scDb)-IL7 significantly increased the expression of T cell factor 1 (TCF-1) on CD8+ T cells. In preclinical cancer models, F8(scDb)-IL7 exhibited potent single-agent activity and eliminated sarcoma lesions in combination with anti-PD-1.

IL-7 fusion protein preparation

The alternative splicing domains of FN's EDA and EDB are recognized by F8 and L19 antibodies, respectively, and are believed to be used to generate tumor-homing fusion proteins. The researchers designed three IL-7 fusion proteins with L19, namely L19 (scDb)-IL7, L19 (IgG4)-IL7, KIH-IL7, L19(IgG4)-IL7. Quantitative biodistribution experiments in mice revealed that the single-chain form possessed superior biodistribution characteristics. Subsequently, F8 (scDb)-IL7 was generated by using the F8 portion of the same single-chain diabody format as L19(scDb) -IL7, which exhibited significant tumor uptake.

Antigen expression analysis in tumor sections

The expression of EDA and EDB in F9, WEHI-164, MC38, and GL-261 tumor tissues was examined and it was found that all tumor models expressed comparable levels of these two antigens.

Extensive analysis of EDA and EDB expression using F8(scDb)-IL7 and L19(scDb)-IL7 revealed high levels of EDA and EDB expression in all analyzed malignancies, whereas these two antigens were barely detectable in most normal adult tissues.

F8(scDb)-IL7 bioactivity

F8(scDb)-IL7 effectively stimulated human peripheral blood mononuclear cells (hPBMCs) in vitro and significantly increased T cell factor 1 (TCF-1) expression on CD8+ T cells compared with IL2 fusion protein.

F8 (scDb)-IL7 subcutaneous tumor model treatment experiment

F8(scDb)-IL7 was found to exhibit potent single-agent activity in clinical cancer models and was found to induce complete tumor regression or growth inhibition in combination with PD-1 blockade.

The increased spleen weight and elevated IFNγ levels in the blood of treated mice indicated that F8 (scDb)-IL7 may have promoted the activation and proliferation of immune cells.

Immunofluorescence analysis showed that CD8+ T cells infiltrated the tumors of all F8(scDb)-IL7 treatment groups, and enhanced CD8+ T cell density was observed in the combination group.

Summarize

This study describes the generation and characterization of a novel interleukin-7 fusion protein, F8(scDb)-IL7, that targets EDA-FN. It significantly increases TCF1 expression and promotes PD-1 blockade. F8(scDb)-IL7 represents a promising candidate for combination with anti-PD-1 antibodies to enhance tumor-specific memory T cells. Because EDA-FN is expressed in most human solid malignancies, this product may have broad applicability. Based on the results, combining an ICB with EDA-FN-targeted IL-7 represents a promising strategy worthy of clinical exploration.