Characterization of the bispecific VHH antibody gefurulimab (ALXN1720) targeting complement component 5, and designed for low volume subcutaneous administration.

The bispecific antibody gefurulimab (also known as ALXN1720) was developed to provide patients with a subcutaneous treatment option for chronic disorders involving activation of the terminal complement pathway. Gefurulimab blocks the enzymatic cleavage of complement component 5 (C5) into the biologically active C5a and C5b fragments, which triggers activation of the terminal complement cascade. Heavy-chain variable region antigen-binding fragment (VHH) antibodies targeting C5 and human serum albumin (HSA) were isolated from llama immune-based libraries and humanized. Gefurulimab comprises an N-terminal albumin-binding VHH connected to a C-terminal C5-binding VHH via a flexible linker. The purified bispecific VHH antibody has the expected exact size by mass spectrometry and can be formulated at greater than 100 mg/mL. Gefurulimab binds tightly to human C5 and HSA with dissociation rate constants at pH 7.4 of 54 pM and 0.9 nM, respectively, and cross-reacts with C5 and serum albumin from cynomolgus monkeys. Gefurulimab can associate with C5 and albumin simultaneously, and potently inhibits the terminal complement activity from human serum initiated by any of the three complement pathways in Wieslab assays. Electron microscopy and X-ray crystallography revealed that the isolated C5-binding VHH recognizes the macroglobulin (MG) 4 and MG5 domains of the antigen and thereby is suggested to sterically prevent C5 binding to its activating convertase. Gefurulimab also inhibits complement activity supported by the rare C5 allelic variant featuring an R885H substitution in the MG7 domain. Taken together, these data suggest that gefurulimab may be a promising candidate for the potential treatment of complement-mediated disorders.

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.

Nanobody-mediated complement activation to kill HIV-infected cells.

The complement system which is part of the innate immune response against invading pathogens represents a powerful mechanism for killing of infected cells. Utilizing direct complement recruitment for complement-mediated elimination of HIV-1-infected cells is underexplored. We developed a novel therapeutic modality to direct complement activity to the surface of HIV-1-infected cells. This bispecific complement engager (BiCE) is comprised of a nanobody recruiting the complement-initiating protein C1q, and single-chain variable fragments of broadly neutralizing antibodies (bNAbs) targeting the HIV-1 envelope (Env) protein. Here, we show that two anti-HIV BiCEs targeting the V3 loop and the CD4 binding site, respectively, increase C3 deposition and mediate complement-dependent cytotoxicity (CDC) of HIV-1 Env-expressing Raji cells. Furthermore, anti-HIV BiCEs trigger complement activation on primary CD4 T cells infected with laboratory-adapted HIV-1 strain and facilitates elimination of HIV-1-infected cells over time. In summary, we present a novel approach to direct complement deposition to the surface of HIV-1-infected cells leading to complement-mediated killing of these cells.

Structural studies offer a framework for understanding the role of properdin in the alternative pathway and beyond.

Structures of alternative pathway proteins have offered a comprehensive structural basis for understanding the molecular mechanisms governing activation and regulation of the amplification pathway of the complement cascade. Although properdin (FP) is required in vivo to sustain a functional alternative pathway, structural studies have been lagging behind due to the extended structure and polydisperse nature of FP. We review recent progress with respect to structure determination of FP and its proconvertase/convertase complexes. These structures identify in detail regions in C3b, factor B and FP involved in their mutual interactions. Structures of FP oligomers obtained by integrative studies have shed light on how FP activity depends on its oligomerization state. The accumulated structural knowledge allows us to rationalize the effect of point mutations causing FP deficiency. The structural basis for FP inhibition by the tick CirpA proteins is reviewed and the potential of alphafold2 predictions for understanding the interaction of FP with other tick proteins and the NKp46 receptor on host immune cells is discussed. The accumulated structural knowledge forms a comprehensive basis for understanding molecular interactions involving FP, pathological conditions arising from low levels of FP, and the molecular strategies used by ticks to suppress the alternative pathway.

Structure determination of an unstable macromolecular complex enabled by nanobody-peptide bridging.

Structure determination of macromolecular complexes is challenging if subunits can dissociate during crystallization or preparation of electron microscopy grids. We present an approach where a labile complex is stabilized by linking subunits though introduction of a peptide tag in one subunit that is recognized by a nanobody tethered to a second subunit. This allowed crystal structure determination at 3.9 Å resolution of the highly non-globular 320 kDa proconvertase formed by complement components C3b, factor B, and properdin. Whereas the binding mode of properdin to C3b is preserved, an internal rearrangement occurs in the zymogen factor B von Willebrand domain type A domain compared to the proconvertase not bound to properdin. The structure emphasizes the role of two noncanonical loops in thrombospondin repeats 5 and 6 of properdin in augmenting the activity of the C3 convertase. We suggest that linking of subunits through peptide specific tethered nanobodies represents a simple alternative to approaches like affinity maturation and chemical cross-linking for the stabilization of large macromolecular complexes. Besides applications for structural biology, nanobody bridging may become a new tool for biochemical analysis of unstable macromolecular complexes and in vitro selection of highly specific binders for such complexes.

Methodology to identify optimal subject-specific laxity tests to stretch individual parts of knee ligaments.

Laxity tests are performed to diagnose ligament injuries or to estimate subject-specific ligament properties. While the current laxity tests are easy to perform, they are not optimized to isolate specific ligament bundles. Therefore, we developed a methodology to identify optimal laxity tests to either stretch specific ligaments relatively more than other ligaments or maximally stretch a specific ligament within the boundaries of maximally applied loads to the knee. The method was applied to a subject-specific knee model and the identified optimal laxity tests compared against standard tests. For the laxity tests isolating the stretch in a specific ligament, we found laxity tests that performed better for all ligament bundles except a few where neither the optimal nor standard laxity tests could isolate the ligament. We found force ratios, between the force change in the ligament of interest compared to the maximal force change in the other ligaments, of over 2.0 for six ligament bundles with the optimized load cases whereas the standard laxity tests only resulted in one bundle over 2.0. For the tests to maximally load the ligament of interest, increased force changes were seen for all optimal load cases and force changes of over 200 N were seen for six ligament bundles whereas only one bundle had a force change over 200N with the standard laxity tests. Our results show that there is a potential to develop better laxity tests than those performed today and emerging laxity test equipment enable implementation of such tests.

Quantification of the pro-form of human complement component factor D (adipsin).

Factor D (also known as adipsin) is a serine protease and part of the complement system, involved in innate immune responses and effector functions of antibodies. Factor D cleaves factor B complexed with C3b, leading to the C3 convertase C3bBb. This C3 convertase is central in the alternative activation pathway and the amplification loop, which amplifies the two other complement activation pathways: the classical pathway and the lectin pathway. Adipocytes synthesize factor D as a pro-form comprising 6 additional residues that must be cleaved off to generate a mature form. The MBL-associated serine protease 3 (MASP-3), found in complex with the pattern recognition molecules of lectin activation pathway, converts the pro-form to mature factor D, which reportedly is the most abundant form found in the circulation at concentrations of 1-2 µg/ml among healthy individuals. The mature factor D is rate-limiting for complement activation, but little is known about the distribution of pro vs. mature factor D in the circulation, the regulation hereof and the potential activation stimuli of the lectin pathway, responsible for activation of MASP-3 and subsequent conversion of pro-form of factor D. In this light we established and validated an ELISA specific for measuring the pro-form of complement factor D. With a working range of 0.82-25 ng/ml, acceptable intra and inter assay CVs, and a relative recovery rate above 90%, we found that the median plasma concentration in Danish blood donors was 134 ng/ml; corresponding to that 8-15% factor D circulates as pro-form. We also found that blood sampling procedures affect conversion and hence the levels measured in serum and plasma.

Measuring Knee Joint Laxity in Three Degrees-of-Freedom In Vivo Using a Robotics- and Image-Based Technology.

Accurate and reliable information about three-dimensional (3D) knee joint laxity can prevent misdiagnosis and avoid incorrect treatments. Nevertheless, knee laxity assessments presented in the literature suffer from significant drawbacks such as soft tissue artifacts, restricting the knee within the measurement, and the absence of quantitative knee ligament property information. In this study, we demonstrated the applicability of a novel methodology for measuring 3D knee laxity, combining robotics- and image-based technology. As such technology has never been applied to healthy living subjects, the aims of this study were to develop novel technology to measure 3D knee laxity in vivo and to provide proof-of-concept 3D knee laxity measurements. To measure tibiofemoral movements, four healthy subjects were placed on a custom-built arthrometer located inside a low dose biplanar X-ray system with an approximately 60 deg knee flexion angle. Anteroposterior and mediolateral translation as well as internal and external rotation loads were subsequently applied to the unconstrained leg, which was placed inside a pneumatic cast boot. Bone contours were segmented in the obtained X-rays, to which subject-specific bone geometries from magnetic resonance imaging (MRI) scans were registered. Afterward, tibiofemoral poses were computed. Measurements of primary and secondary laxity revealed considerable interpersonal differences. The method differs from those available by the ability to accurately track secondary laxity of the unrestricted knee and to apply coupled forces in multiple planes. Our methodology can provide reliable information for academic knee ligament research as well as for clinical diagnostics in the future.

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.

A Methodology to Evaluate the Effects of Kinematic Measurement Uncertainties on Knee Ligament Properties Estimated From Laxity Measurements.

Ligaments are important joint stabilizers but assessing their mechanical properties remain challenging. We developed a methodology to investigate the effects of kinematic measurement uncertainty during laxity tests on optimization-based estimation of ligament properties. We applied this methodology to a subject-specific knee model with known ligament properties as inputs and compared the estimated to the known knee ligament properties under the influence of noise. Four different sets of laxity tests were simulated with an increasing number of load cases, capturing anterior/posterior, varus/valgus, and internal/external rotation loads at 0 deg and 30 deg of knee flexion. 20 samples of uniform random noise ([-0.5,0.5] mm and degrees) were added to each set and fed into an optimization routine that subsequently estimated the ligament properties based on the noise targets. We found a large range of estimated ligament properties (stiffness ranges of 5.97 kN, 7.64 kN, 8.72 kN, and 3.86 kN; reference strain ranges of 3.11%, 2.53%, 1.88%, and 1.58% for anterior cruciate ligament (ACL), posterior cruciate ligament (PCL), medical collateral ligament (MCL), and lateral collateral ligament (LCL), respectively) for three sets of laxity tests, including up to 22 load cases. A set of laxity tests with 60 load cases kept the stiffness and reference strain ranges below 470 N per unit strain and 0.85%, respectively. These results illustrate that kinematic measurement noise have a large impact on estimated ligament properties and we recommend that future studies assess and report both the estimated ligament properties and the associated uncertainties due to kinematic measurement noise.

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.

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.

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.

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.

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.

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.

Crystallization and X-ray analysis of monodisperse human properdin.

The 54 kDa protein properdin, also known as factor P (FP), plays a major role in the complement system through the stabilization of the alternative pathway convertases. FP circulates in the blood as cyclic dimers, trimers and tetramers, and this heterogeneity challenges detailed structural insight into the mechanism of convertase stabilization by FP. Here, the generation of an intact FP monomer and a variant monomer with the third thrombospondin repeat liberated is described. Both FP monomers were excised from recombinant full-length FP containing internal cleavage sites for TEV protease. These FP monomers could be crystallized, and complete data sets extending to 2.8 Šresolution for the intact FP monomer and to 3.5 Šresolution for the truncated variant were collected. The principle of specific monomer excision and domain removal by the insertion of a protease cleavage site may be broadly applicable to structural studies of oligomeric, flexible and modular proteins.

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.

Tropical rainforest palm communities in Madre de Dios in Amazonian Peru / Comunidades de palmas en los bosques tropicales de Madre de Dios de la Amazonía Peruana

We studied palm communities, in particular species-richness and abundance, in the tropical rainforests in southeastern Peru in 54 transects (5×500m) covering an area of 13.5 hectares in flood plain, terra firme, terrace and premontane hills. We found 42 palm species in 18 genera in the transects. Terra firme forest had the highest species richness (38 species) followed by floodplain and premontane hills with 27 species and terrace forests with 26 species. The highest palm abundances were found in premontane hill forest which had 3243 palms per hectare and terra firme forest which had 2968 palms per hectare. The floodplain forests were intermediate in palm abundance with 2647 and the terrace forests had the lowest abundance with 1709 palms per hectare. Intermediate sized palms were the most common being represented by 18 species, while large palms were represented with 16 species. There were only eight species of small palms of which one was acaulescent. Only one species of liana palm was registered. Of the 42 species observed in the 54 transects, 20 were cespitose, 21 solitary and two had colonial growth. Seven species were found 40-320 km outside of their previously known range.

Estudiamos las comunidades de palmas de los bosques pluviales tropicales del sur de Perú, con especial énfasis en su riqueza de especies y abundancia, utilizando 54 transectas (5×500m), que cubrieron un área de 13.5 hectáreas en planicie inundable, terra firme, terraza y colinas premontanas. Encontramos 42 especies de palmas en las transectas. En el bosque de terra firme se encontró la mayor riqueza de especies (38 especies), seguido por la planicie inundable y las colinas premontanas con 27 especies y los bosques de terraza con 26 especies. Las mayores abundancias se encontraron en el bosque de colinas premontanas, con 3243 palmas por hectárea, y en el bosque de terra firme con 2968 palmas por hectárea. Los bosques de la planicie inundable presentaron una abundancia intermedia con 2647 palmas por hectárea y los bosques de terraza presentaron la menor abundancia con 1709. Las palmas de tamaño intermedio fueron las más comunes, estando representadas por 18 especies, mientras que las palmas grandes estuvieron representadas por 16 especies. Se encontraron solamente ocho especies de palmas pequeñas, una de las cuales era acaulescente. Sólo se registró una especie de palma trepadora. De las 42 especies observadas en las 54 transectas, 20 fueron cespitosas, 21 solitarias y dos presentaron crecimiento colonial. Siete especies se encontraron 40-320 km fuera del rango de distribución conocido previamente.