Studies on single domain antibodies VHH against FAPα as potential cancer therapeutics

Fibroblast activation protein-α (FAPα) is a serine protease highly expressed on cancer-associated fibroblasts (CAFs) within the tumor microenvironment (TME), where it contributes to immunosuppression and correlates with poor patient prognosis. In this study we report the identification and characterization of high affinity single-domain antibodies (VHH) against FAPα, discovered using a naïve llama yeast display library and optimized through structure guided modeling. Docking and Dynamic simulations revealed a stable FAPα-VHH complex, with the antibody. Multiple VHH clones were identified, including variants exhibiting limited enzymatic inhibition but highly specific binding. Lead VHH clones A01 and A02 demonstrated sub-nanomolar binding affinity to FAPα and negligible cross-reactivity with homologous proteins (CD26/DPP4), as confirmed by ELISA and BLI. These ∼15 kDaVHH antibodies retain the advantages of nanobodies, including efficient tissue penetration and modularity. Fc-fusion with human IgG1 domains, including wild-type (WT) and ADCC-enhanced DLE (S239D/I332E) Fc variants, conferred bivalent engagement and enhanced FcγRIIIa-dependent effector recruitment. The resulting lead candidates demonstrate therapeutic potential for selectively targeting FAPα-expressing stromal cells to remodel the tumor microenvironment (TME) in solid tumors. Functionally, VHH-Fc constructs promote antibody-dependent cellular cytotoxicity (ADCC), supporting selective targeting of FAPα⁺ stromal cells. Collectively, this work establishes a structurally validated and functionally optimized anti-FAPα VHH-Fc candidates for stromal-targeted immunotherapy in solid tumors.