Structure-Guided Engineering of a Complement Component C3-Binding Nanobody Improves Specificity and Adds Cofactor Activity

Abstract

The complement system is a part of the innate immune system, where it labels intruding pathogens as well as dying host cells for clearance. If complement regulation is compromised, the system may contribute to pathogenesis. The proteolytic fragment C3b of complement component C3, is the pivot point of the complement system and provides a scaffold for the assembly of the alternative pathway C3 convertase that greatly amplifies the initial complement activation. This makes C3b an attractive therapeutic target. We previously described a nanobody, hC3Nb1 binding to C3 and its degradation products. Here we show, that extending the N-terminus of hC3Nb1 by a Glu-Trp-Glu motif renders the resulting EWE-hC3Nb1 (EWE) nanobody specific for C3 degradation products. By fusing EWE to N-terminal CCP domains from complement Factor H (FH), we generated the fusion proteins EWEnH and EWEµH. In contrast to EWE, these fusion proteins supported Factor I (FI)-mediated cleavage of human and rat C3b. The EWE, EWEµH, and EWEnH proteins bound C3b and iC3b with low nanomolar dissociation constants and exerted strong inhibition of alternative pathway-mediated deposition of complement. Interestingly, EWEnH remained soluble above 20 mg/mL. Combined with the observed reactivity with both human and rat C3b as well as the ability to support FI-mediated cleavage of C3b, this features EWEnH as a promising candidate for in vivo studies in rodent models of complement driven pathogenesis.

Keywords: Factor H; alternative pathway; complement system; inhibitor; single-domain antibody.

Figure 1

Design and validation of EWE. (A, B) Structural comparison of the hC3Nb1 (grey) in complex with native C3 (red) and C3b (green). C3 and C3b are aligned on their MG-ring. (C, D) Comparison of the binding interface of hC3Nb1 in complex with C3 and C3b. The structures in panels (C, D) are aligned on the hC3Nb1 molecule. PDB entries are 6RU5 for the C3:hC3Nb1 complex and 6EHG for the C3b:hC3Nb1 complex. (E, F) SEC-based analyses of the interaction of EWE with C3b and native C3. (G) SDS-PAGE analysis of peak fractions from panels (E, F), indicated by bars. (H–J) SEC-based analyses of the complex-formation between native C3 and WE-hC3Nb1, E-hC3Nb1, and hC3Nb1. (K) SEC-based analysis of the interaction between hC3Nb1 and C3b. (L, M) Cleavage of C3 by CVFBb was monitored in presence or absence of EWE or hC3Nb1. mAU, milli absorbance units. M, molecular weight marker.

Figure 2

Binding kinetics of EWE. Surface plasmon resonance based analyses of the interaction between EWE and (A) C3b, (B) C3MA, or (C) native C3. (D) Table summarizing the binding kinetics of EWE. The averages of rate constants for n=3 experiments are shown. The interaction with native C3 is possibly a result of C3 tick-over during the experiment. RU, response units. a.u., arbitrary units. nM, nanomolar.

Figure 3

Design and characterization of EWEµH and EWEnH. (A) Model of the EWEµH and EWEnH fusion proteins. The crystal structure of hC3Nb1 [PDB entry 6EHG] was superimposed onto the structure of C3b:mini-FH [PDB entry 5O35]. The dashed line illustrates a linker joining the C-terminus of the EWE moiety and CCP2. The lower panel illustrates the domain structure of EWEµH and EWEnH. (B, C) Effect of EWE and hC3Nb1 on C3b cleavage. C3b was incubated with FH, FI as well as a 1.2-fold molar excess of either (B) EWE or (C) hC3Nb1 and the cleavage was monitored by SDS PAGE. (D) EWEµH cleavage assay. C3b was incubated with a two-fold molar excess of mini-FH, EWEµH or mini-FH as well as EWE in the presence of FI. The cleavage of C3b was monitored by SDS-PAGE analysis upon the indicated incubation at 37°C. (E, F) Comparison of EWEµH and EWEnH. C3b was incubated with FI and EWEµH or EWEnH in molar C3b:EWEµH/EWEnH ratios of (E) 1:0.5 and (F) 1:2. (G) Cleavage assay of rat C3b performed using a molar C3b:EWEµH/EWEnH ratio of 1:2. The C3b was generated from rat C3 using CVFBb and was immediately used for the cleavage assay, explaining the presence of C3a in the reactions. M, molecular weight marker.

Figure 4

Bio-layer interferometry-based analyses of EWE fusion proteins. (A) EWE, (B) EWEµH or (C) EWEnH was immobilized on a biosensor via the C-terminal 6xHis-tag and transferred into dilution series of (A, B) 1.5625-50 nM C3b, and (C) 3.125-100 nM C3b. Similarly, (D) EWE, (E) EWEµH or (F) EWEnH was immobilized on biosensors and transferred into dilution series of (D, E) 3.125-50 nM iC3b or (F) 6.25-100 nM iC3b. (G) The table summarizes the rate constants of association and dissociation from n=3 experiments for C3b kinetics and n=2 experiments for iC3b kinetics. nm, nanometer.

Figure 5

Functional comparison of EWEµH and EWEnH in serum conditions. The effects of EWE, EWEµH, and EWEnH on (A) AP mediated deposition of C3 fragments onto a surface of zymosan or (B) AP mediated hemolysis of rabbit erythrocytes. In panels A and B, the effects were assayed in 11% human serum and data were normalized to 100% in serum without nanobodies, and 0% without serum. The effects were compared to the parental AP inhibitor hC3Nb1 (16), the broad inhibitor hC3Nb2 (22), and the inactive hC3Nb1 (W102A) mutant (16). Dashed lines indicate putative C3 concentrations assuming a C3 concentration of 5.4 µM in undiluted human serum (17). Average and S.D. (error bars) are shown for n=3 experiments in both panels. (C) Ultrafiltration assay. The EWEnH was concentrated to 22 mg/mL and was subjected to SEC. (D) SDS-PAGE analysis of peak fractions from panel (C) mAU, milli absorbance units. M, molecular weight marker.