Bispecific Complement Engagers for Targeted Complement Activation.

Activation of the complement system represents an important effector mechanism of endogenous and therapeutic Abs. However, efficient complement activation is restricted to a subset of Abs due to the requirement of multivalent interactions between the Ab Fc regions and the C1 complex. In the present study, we demonstrate that Fc-independent recruitment of C1 by modular bispecific single-domain Abs that simultaneously bind C1q and a surface Ag can potently activate the complement system. Using Ags from hematological and solid tumors, we show that these bispecific Abs are cytotoxic to human tumor cell lines that express the Ag and that the modular design allows a functional exchange of the targeting moiety. Direct comparison with clinically approved Abs demonstrates a superior ability of the bispecific Abs to induce complement-dependent cytotoxicity. The efficacy of the bispecific Abs to activate complement strongly depends on the epitope of the C1q binding Ab, demonstrating that the spatial orientation of the C1 complex upon Ag engagement is a critical factor for efficient complement activation. Collectively, our data provide insight into the mechanism of complement activation and provide a new platform for the development of immunotherapies.

A Complement C3-Specific Nanobody for Modulation of the Alternative Cascade Identifies the C-Terminal Domain of C3b as Functional in C5 Convertase Activity.

The complement system is an intricate cascade of the innate immune system and plays a key role in microbial defense, inflammation, organ development, and tissue regeneration. There is increasing interest in developing complement regulatory and inhibitory agents to treat complement dysfunction. In this study, we describe the nanobody hC3Nb3, which is specific for the C-terminal C345c domain of human and mouse complement component C3/C3b/C3c and potently inhibits C3 cleavage by the alternative pathway. A high-resolution structure of the hC3Nb3-C345c complex explains how the nanobody blocks proconvertase assembly. Surprisingly, although the nanobody does not affect classical pathway-mediated C3 cleavage, hC3Nb3 inhibits classical pathway-driven hemolysis, suggesting that the C-terminal domain of C3b has an important function in classical pathway C5 convertase activity. The hC3Nb3 nanobody binds C3 with low nanomolar affinity in an SDS-resistant complex, and the nanobody is demonstrated to be a powerful reagent for C3 detection in immunohistochemistry and flow cytometry. Overall, the hC3Nb3 nanobody represents a potent inhibitor of both the alternative pathway and the terminal pathway, with possible applications in complement research, diagnostics, and therapeutics.

Functional and structural insight into properdin control of complement alternative pathway amplification.

Properdin (FP) is an essential positive regulator of the complement alternative pathway (AP) providing stabilization of the C3 and C5 convertases, but its oligomeric nature challenges structural analysis. We describe here a novel FP deficiency (E244K) caused by a single point mutation which results in a very low level of AP activity. Recombinant FP E244K is monomeric, fails to support bacteriolysis, and binds weakly to C3 products. We compare this to a monomeric unit excised from oligomeric FP, which is also dysfunctional in bacteriolysis but binds the AP proconvertase, C3 convertase, C3 products and partially stabilizes the convertase. The crystal structure of such a FP-convertase complex suggests that the major contact between FP and the AP convertase is mediated by a single FP thrombospondin repeat and a small region in C3b. Small angle X-ray scattering indicates that FP E244K is trapped in a compact conformation preventing its oligomerization. Our studies demonstrate an essential role of FP oligomerization in vivo while our monomers enable detailed structural insight paving the way for novel modulators of complement.

Structural insight into proteolytic activation and regulation of the complement system.

The complement system is a highly complex and carefully regulated proteolytic cascade activated through three different pathways depending on the activator recognized. The structural knowledge regarding the intricate proteolytic enzymes that activate and control complement has increased dramatically over the last decade. This development has been pivotal for understanding how mutations within complement proteins might contribute to pathogenesis and has spurred new strategies for development of complement therapeutics. Here we describe and discuss the complement system from a structural perspective and integrate the most recent findings obtained by crystallography, small-angle X-ray scattering, and electron microscopy. In particular, we focus on the proteolytic enzymes governing activation and their products carrying the biological effector functions. Additionally, we present the structural basis for some of the best known complement inhibitors. The large number of accumulated molecular structures enables us to visualize the relative size, position, and overall orientation of many of the most interesting complement proteins and assembled complexes on activator surfaces and in membranes.

Structure and function of a family of tick-derived complement inhibitors targeting properdin.

Activation of the serum-resident complement system begins a cascade that leads to activation of membrane-resident complement receptors on immune cells, thus coordinating serum and cellular immune responses. Whilst many molecules act to control inappropriate activation, Properdin is the only known positive regulator of the human complement system. By stabilising the alternative pathway C3 convertase it promotes complement self-amplification and persistent activation boosting the magnitude of the serum complement response by all triggers. In this work, we identify a family of tick-derived alternative pathway complement inhibitors, hereafter termed CirpA. Functional and structural characterisation reveals that members of the CirpA family directly bind to properdin, inhibiting its ability to promote complement activation, and leading to potent inhibition of the complement response in a species specific manner. We provide a full functional and structural characterisation of a properdin inhibitor, opening avenues for future therapeutic approaches.

Properdin oligomers adopt rigid extended conformations supporting function.

Properdin stabilizes convertases formed upon activation of the complement cascade within the immune system. The biological activity of properdin depends on the oligomerization state, but whether properdin oligomers are rigid and how their structure links to function remains unknown. We show by combining electron microscopy and solution scattering, that properdin oligomers adopt extended rigid and well-defined conformations which are well approximated by single models of apparent n-fold rotational symmetry with dimensions of 230-360 Å. Properdin monomers are pretzel-shaped molecules with limited flexibility. In solution, properdin dimers are curved molecules, whereas trimers and tetramers are close to being planar molecules. Structural analysis indicates that simultaneous binding through all binding sites to surface-linked convertases is unlikely for properdin trimer and tetramers. We show that multivalency alone is insufficient for full activity in a cell lysis assay. Hence, the observed rigid extended oligomer structure is an integral component of properdin function.

Recruitment of properdin by bi-specific nanobodies activates the alternative pathway of complement.

The complement system represents a powerful part of the innate immune system capable of removing pathogens and damaged host cells. Nevertheless, only a subset of therapeutic antibodies are capable of inducing complement dependent cytotoxicity, which has fuelled the search for new strategies to potentiate complement activation. Properdin (FP) functions as a positive complement regulator by stabilizing the alternative pathway C3 convertase. Here, we explore a novel strategy for direct activation of the alternative pathway of complement using bi-specific single domain antibodies (nanobodies) that recruit endogenous FP to a cell surface. As a proof-of-principle, we generated bi-specific nanobodies with specificity toward FP and the validated cancer antigen epidermal growth factor receptor (EGFR) and tested their ability to activate complement onto cancer cell lines expressing EGFR. Treatment led to recruitment of FP, complement activation and significant deposition of C3 fragments on the cells in a manner sensitive to the geometry of FP recruitment. The bi-specific nanobodies induced complement dependent lysis of baby hamster kidney cells expressing human EGFR but were unable to lyse human tumour cells due to the presence of complement regulators. Our results confirm that FP can function as a surface bound focal point for initiation of complement activation independent of prior C3b deposition. However, recruitment of FP by bi-specific nanobodies appears insufficient for overcoming the inhibitory action of the negative complement regulators overexpressed by many human tumour cell lines. Our data provide general information on the efficacy of properdin as an initiator of complement but suggest that properdin recruitment on its own may have limited utility as a platform for potent complement activation on regulated cell surfaces.

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

The classical pathway of complement is important for protection against pathogens and in maintaining tissue homeostasis, but excessive or aberrant activation is directly linked to numerous pathologies. We describe the development and in vitro characterization of C1qNb75, a single domain antibody (nanobody) specific for C1q, the pattern recognition molecule of the classical pathway. C1qNb75 binds to the globular head modules of human C1q with sub-nanomolar affinity and impedes classical pathway mediated hemolysis by IgG and IgM. Crystal structure analysis revealed that C1qNb75 recognizes an epitope primarily located in the C1q B-chain that overlaps with the binding sites of IgG and IgM. Thus, C1qNb75 competitively prevents C1q from binding to IgG and IgM causing blockade of complement activation by the classical pathway. Overall, C1qNb75 represents a high-affinity nanobody-based inhibitor of IgG- and IgM-mediated activation of the classical pathway and may serve as a valuable reagent in mechanistic and functional studies of complement, and as an efficient inhibitor of complement under conditions of excessive CP activation.

Soluble collectin-12 mediates C3-independent docking of properdin that activates the alternative pathway of complement.

Properdin stabilizes the alternative C3 convertase (C3bBb), whereas its role as pattern-recognition molecule mediating complement activation is disputed for decades. Previously, we have found that soluble collectin-12 (sCL-12) synergizes complement alternative pathway (AP) activation. However, whether this observation is C3 dependent is unknown. By application of the C3-inhibitor Cp40, we found that properdin in normal human serum bound to Aspergillus fumigatus solely in a C3b-dependent manner. Cp40 also prevented properdin binding when properdin-depleted serum reconstituted with purified properdin was applied, in analogy with the findings achieved by C3-depleted serum. However, when opsonized with sCL-12, properdin bound in a C3-independent manner exclusively via its tetrameric structure and directed in situ C3bBb assembly. In conclusion, a prerequisite for properdin binding and in situ C3bBb assembly was the initial docking of sCL-12. This implies a new important function of properdin in host defense bridging pattern recognition and specific AP activation.

Structural Basis for Properdin Oligomerization and Convertase Stimulation in the Human Complement System.

Properdin (FP) is a positive regulator of the immune system stimulating the activity of the proteolytically active C3 convertase C3bBb in the alternative pathway of the complement system. Here we present two crystal structures of FP and two structures of convertase bound FP. A structural core formed by three thrombospondin repeats (TSRs) and a TB domain harbors the convertase binding site in FP that mainly interacts with C3b. Stabilization of the interaction between the C3b C-terminus and the MIDAS bound Mg2+ in the Bb protease by FP TSR5 is proposed to underlie FP convertase stabilization. Intermolecular contacts between FP and the convertase subunits suggested by the structure were confirmed by binding experiments. FP is shown to inhibit C3b degradation by FI due to a direct competition for a common binding site on C3b. FP oligomers are held together by two sets of intermolecular contacts, where the first is formed by the TB domain from one FP molecule and TSR4 from another. The second and largest interface is formed by TSR1 and TSR6 from the same two FP molecules. Flexibility at four hinges between thrombospondin repeats is suggested to enable the oligomeric, polydisperse, and extended architecture of FP. Our structures rationalize the effects of mutations associated with FP deficiencies and provide a structural basis for the analysis of FP function in convertases and its possible role in pattern recognition.

Microbial growth studies in biodiesel blends.

Introduction of biofuels to the fuel matrix poses new questions and challenges. The present study investigates the microbiological stability of biodiesel blends in small scale microcosms. The study presents results from incubations of diesel-biodiesel blends with contaminated inoculation water collected from diesel storage tanks to ensure the presence of relevant fuel degrading bacteria. DAPI and qPCR analyses has subsequently shown an increased bacterial growth and activity in the microcosms containing biodiesel blends as the carbon source compared to those microcosms where neat fossil diesel made up the carbon source. Several anaerobic microorganisms have been identified after incubation. Presence of methanogens, sulfate-reducing bacteria and nitrate reducing bacteria has furthermore been confirmed by chemical analyses, supplemented by observations of methane formation in biodiesel incubations. The findings will contribute to the knowledge base for a safer introduction of biodiesel in the fuel matrix by employment of proper house-keeping and monitoring methods.