Targeting the Complement C1 Proteases Using Camelid-Derived Single Domain Antibodies: Production, Characterization and Possible Application for Tissue/Fluid Analysis

Background. The complement C1r and C1s proteases initiate the classical complement pathway within the C1 complex, but emerging evidence points to additional roles in tissues, including cell-intrinsic functions linked to cancer and connective tissue disorders. Few tools are available to explore these plasma and tissue-relevant functions. We report the first development and characterization of anti-C1r and anti-C1s VHH single-domain antibodies (nanobodies). Methods. A lama was immunized with the purified human C1r2/C1s2 tetramer. After phage display selection, two anti-C1r VHHs (C1rCl1, C1rCl6) and one anti-C1s VHH (C1sCl7) were produced in E. coli and characterized by ELISA, Western blot, SPR and size-exclusion chromatography using recombinant protease fragments and proenzyme forms. Functional impact on C1s activation was assessed by cleavage assay, and tissue applicability by immunohistochemistry on human skin sections. Results. All three VHHs bound with high affinity to both active and proenzyme forms. Epitope mapping localized C1rCl1 and C1rCl6 to the CCP2 module of C1r, and C1sCl7 to the CCP1 module of C1s. The two anti-C1r VHHs recognize distinct, non-overlapping epitopes and can bind simultaneously to C1r. C1rCl1 mildly inhibited C1s activation by C1r. The VHH pair C1rCl1/C1rCl6 enabled sandwich ELISA detection of C1r in serum, plasma, and cell culture supernatant. Immunohistochemistry on human skin confirmed clean C1r staining in keratinocytes and fibroblasts, with a dissociated tissue distribution compared to C1s. Conclusions. These first anti-C1r and anti-C1s nanobodies constitute useful tools for detection in biological fluids and tissue staining, with potential applications in diseases where these proteases play a pathological role.