A potent complement factor C3–specific nanobody inhibiting multiple functions in the alternative pathway of human and murine complement

Abstract

The complement system is a complex, carefully regulated proteolytic cascade for which suppression of aberrant activation is of increasing clinical relevance, and inhibition of the complement alternative pathway is a subject of intense research. Here, we describe the nanobody hC3Nb1 that binds to multiple functional states of C3 with subnanomolar affinity. The nanobody causes a complete shutdown of alternative pathway activity in human and murine serum when present in concentrations comparable with that of C3, and hC3Nb1 is shown to prevent proconvertase assembly, as well as binding of the C3 substrate to C3 convertases. Our crystal structure of the C3b-hC3Nb1 complex and functional experiments demonstrate that proconvertase formation is blocked by steric hindrance between the nanobody and an Asn-linked glycan on complement factor B. In addition, hC3Nb1 is shown to prevent factor H binding to C3b, rationalizing its inhibition of factor I activity. Our results identify hC3Nb1 as a versatile, inexpensive, and powerful inhibitor of the alternative pathway in both human and murine model systems of complement activation.

Functional and Structural Characterization of a Potent C1q Inhibitor Targeting the Classical Pathway of the Complement System

“…Clear electron density was observed for the vast majority of residues in gC1q and C1qNb75 ( Figure 4B ) which allowed us to model the intermolecular interface with high confidence. PISA analysis ( 33 ) revealed an epitope with an area of 740 Å 2 on C1q, which is in the low end for such complexes, whereas the observed shape complementarity of 0.74 is in the average range ( 34 ). In the crystal structure, there are two complexes in the asymmetric unit with the two gC1q copies being almost identical.…”

“…Immunization, library generation and selection was performed essentially as described in ( 34 ). A llama was immunized four times each with a 100 μg mixture of purified C1q and gC1q, the latter generated by collagenase digestion of C1q ( 3 ).…”

“…Following this round of selection, single colonies from 2xYT agar plates were inoculated into a 96-well plate containing 200 µL LB media with 100 µg/mL ampicillin. The plate was incubated at 37 • C for 8 h, added IPTG to a final concentration of 1 mM and incubated ON at 30 • C. The following day the plate was centrifuged for 10 min at 450 g and the supernatant was used in an ELISA as described (34). Phagemids from ELISA positive clones were isolated and sequenced.…”

Complement activation by human IgG antibodies to galactose‐α‐1,3‐galactose

“…26 Nanobodies were derived from heavy-chain antibodies from llamas, selected by phage display, expressed in bacteria and purified as described previously. 27 Four nanobodies were used: (i) C1qNb75 binds to the globular head of C1q, blocking C1q docking to immunoglobulin Fc domains; 28 (ii) hC3Nb1 binds C3, selectively blocking the C3 convertase of the alternative pathway (AP); ,27 (iii) hC3Nb2 binds C3, blocking both the CP and AP C3 convertases; 29 and (iv) KRA152, which was used as control.…”

“…Hence, it was important to examine this possibility. We challenged the complement deposition of IgG anti-αGal on pRBCs using four different inhibitors: C1qNb75, hC3Nb2 (nanobody that binds C3, blocking C3 cleavage by both CP and AP 27 ), eculizumab (monoclonal IgG that binds C5, blocking C5 cleavage 26 ) and EDTA (chelates divalent cations, inhibiting complement activation in general). These experiments were conducted in 10% HHS and IgG anti-αGal at 20 mg/l.…”