IgG antibodies can organize into ordered hexamers on cell surfaces after binding their antigen. These hexamers bind the first component of complement C1 inducing complement-dependent target cell ...killing. Here, we translated this natural concept into a novel technology platform (HexaBody technology) for therapeutic antibody potentiation. We identified mutations that enhanced hexamer formation and complement activation by IgG1 antibodies against a range of targets on cells from hematological and solid tumor indications. IgG1 backbones with preferred mutations E345K or E430G conveyed a strong ability to induce conditional complement-dependent cytotoxicity (CDC) of cell lines and chronic lymphocytic leukemia (CLL) patient tumor cells, while retaining regular pharmacokinetics and biopharmaceutical developability. Both mutations potently enhanced CDC- and antibody-dependent cellular cytotoxicity (ADCC) of a type II CD20 antibody that was ineffective in complement activation, while retaining its ability to induce apoptosis. The identified IgG1 Fc backbones provide a novel platform for the generation of therapeutics with enhanced effector functions that only become activated upon binding to target cell-expressed antigen.
The classical complement pathway contributes to the natural immune defense against pathogens and tumors. IgG antibodies can assemble at the cell surface into hexamers via Fc:Fc interactions, which ...recruit complement component C1q and induce complement activation. Biophysical characterization of the C1:IgG complex has remained elusive primarily due to the low affinity of IgG-C1q binding. Using IgG variants that dynamically form hexamers efficient in C1q binding and complement activation, we could assess C1q binding in solution by native mass spectrometry and size-exclusion chromatography. Fc-domain deglycosylation, described to abrogate complement activation, affected IgG hexamerization and C1q binding. Strikingly, antigen binding by IgG hexamers or deletion of the Fab arms substantially potentiated complement initiation, suggesting that Fab-mediated effects impact downstream Fc-mediated events. Finally, we characterized a reconstituted 2,045.3 ± 0.4-kDa complex of intact C1 bound to antigen-saturated IgG hexamer by native mass spectrometry, providing a clear visualization of a complete complement initiation complex.
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•Complement components C1q and C1 bind efficiently to soluble IgG hexamers•IgG glycosylation contributes to both the hexamer stability and C1q binding•A complement initiation complex is reconstituted and visualized by mass spectrometry
Through certain mutations, Wang et al. stabilize IgG hexamers and demonstrate, by native mass spectrometry, how they bind efficiently to C1q/C1. Molecular features of these IgG hexamers, such as glycosylation, are assessed for their specific role in complement activation.
Complement activation by antibodies bound to pathogens, tumors, and self antigens is a critical feature of natural immune defense, a number of disease processes, and immunotherapies. How antibodies ...activate the complement cascade, however, is poorly understood. We found that specific noncovalent interactions between Fc segments of immunoglobulin G (IgG) antibodies resulted in the formation of ordered antibody hexamers after antigen binding on cells. These hexamers recruited and activated C1, the first component of complement, thereby triggering the complement cascade. The interactions between neighboring Fc segments could be manipulated to block, reconstitute, and enhance complement activation and killing of target cells, using all four human IgG subclasses. We offer a general model for understanding antibody-mediated complement activation and the design of antibody therapeutics with enhanced efficacy.
Danger patterns on microbes or damaged host cells bind and activate C1, inducing innate immune responses and clearance through the complement cascade. How these patterns trigger complement initiation ...remains elusive. Here, we present cryo-electron microscopy analyses of C1 bound to monoclonal antibodies in which we observed heterogeneous structures of single and clustered C1-immunoglobulin G1 (IgG1) hexamer complexes. Distinct C1q binding sites are observed on the two Fc-CH2 domains of each IgG molecule. These are consistent with known interactions and also reveal additional interactions, which are supported by functional IgG1-mutant analysis. Upon antibody binding, the C1q arms condense, inducing rearrangements of the C1r
s
proteases and tilting C1q's cone-shaped stalk. The data suggest that C1r may activate C1s within single, strained C1 complexes or between neighboring C1 complexes on surfaces.
IgG antibodies play a central role in protection against pathogens by their ability to alert and activate the innate immune system. Here, we show that IgGs assemble into oligomers on antigenic ...surfaces through an ordered, Fc domain-mediated process that can be modulated by protein engineering. Using high-speed atomic force microscopy, we unraveled the molecular events of IgG oligomer formation on surfaces. IgG molecules were recruited from solution although assembly of monovalently binding molecules also occurred through lateral diffusion. Monomers were observed to assemble into hexamers with all intermediates detected, but in which only hexamers bound C1. Functional characterization of oligomers on cells also demonstrated that C1 binding to IgG hexamers was a prerequisite for maximal activation, whereas tetramers, trimers, and dimers were mostly inactive. We present a dynamic IgG oligomerization model, which provides a framework for exploiting the macromolecular assembly of IgGs on surfaces for tool, immunotherapy, and vaccine design.
Activation of membrane receptors through clustering is a common mechanism found in various biological systems. Spatial proximity of receptors may be transduced across the membrane to initiate ...signaling pathways or alternatively be recognized by peripheral proteins or immune cells to trigger external effector functions. Here we show how specific immunoglobulin G (IgG) binding induces clustering of monomeric target molecules in lipid membranes through Fc–Fc interactions. We visualize and characterize the dynamic IgG oligomerization process and the molecular interactions involved using high-speed atomic force microscopy, single-molecule force spectroscopy, and quartz crystal microbalance experiments. We found that the Fc–Fc interaction strength is precisely tuned to be weak enough to prevent IgG oligomerization in solution at physiological titers, but enabling IgG oligomerization when Fabs additionally bind to their cognate surface epitopes, a mechanism that ultimately targets IgG-mediated effector functions such as classical complement activation to antigenic membranes.
•Transient expression of antibodies up to 400mg/L.•Experimental design techniques lead to an optimal process.•A combination of SV40 large T antigen, p21 and p27 increases expression levels.•Fully ...scalable from 0.1 up to 1200ml.
Transient expression systems in mammalian cells have become the method of choice for producing research quantities of antibodies. Both the speed and yield of the available transient systems and the natural posttranslational modifications favor these systems above expression in lower eukaryotes, prokaryotes or stable cell lines.
We describe an optimized mammalian transient expression system, capable of producing up to 400mg/L of native secreted antibodies in less than a week. The system is composed of commercially available components and is based on expression in the fast growing suspension cell line, FreeStyle™ 293-F (HEK-293F).
The method depends on an optimal combination of a gene transfer method, an expression vector and cotransfection with expression enhancing plasmids, encoding the large T antigen of the SV40 virus and the cell cycle inhibitors p21 and p27.
Optimization of all components of the expression system, by experimental design techniques, yielded maximal expression levels (including antibody isotypes IgG1, 2, 3, 4 and Fab fragments of various species).
Expression volumes were scalable from 0.1ml up to 1.2L in a simple shaker flask system in animal component free, low protein medium, enabling consistent production of relatively high amounts of a large number of native antibodies.
The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. ...Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and size-exclusion chromatography (SEC) analysis. Standard electrospray ionization (ESI)-mass spectrometry does not provide a direct solution as this approach is hindered by extensive interference of ion signals caused by closely spaced charge states of broadly distributed glycoforms. Here, we introduce a native tandem MS-based approach, enabling charge-state resolution and charge assignment of protein ions including those that escape mass analysis under standard MS conditions. Using this method, we determined the MW of two model glycoproteins, the extra-cellular domains of the highly and heterogeneously glycosylated proteins CD38 and epidermal growth factor receptor (EGFR), as well as the overall MW and binding stoichiometries of these proteins in complex with a specific antibody.
CD20 monoclonal antibody therapies have significantly improved the outlook for patients with B-cell malignancies. However, many patients acquire resistance, demonstrating the need for new and ...improved drugs. We previously demonstrated that the natural process of antibody hexamer formation on targeted cells allows for optimal induction of complement-dependent cytotoxicity. Complement-dependent cytotoxicity can be potentiated by introducing a single point mutation such as E430G in the IgG Fc domain that enhances intermolecular Fc-Fc interactions between cell-bound IgG molecules, thereby facilitating IgG hexamer formation. Antibodies specific for CD37, a target that is abundantly expressed on healthy and malignant B cells, are generally poor inducers of complement-dependent cytotoxicity. Here we demonstrate that introduction of the hexamerization-enhancing mutation E430G in CD37-specific antibodies facilitates highly potent complement-dependent cytotoxicity in chronic lymphocytic leukemia cells
Strikingly, we observed that combinations of hexamerization-enhanced CD20 and CD37 antibodies cooperated in C1q binding and induced superior and synergistic complement-dependent cytotoxicity in patient-derived cancer cells compared to the single agents. Furthermore, CD20 and CD37 antibodies colocalized on the cell membrane, an effect that was potentiated by the hexamerization-enhancing mutation. Moreover, upon cell surface binding, CD20 and CD37 antibodies were shown to form mixed hexameric antibody complexes consisting of both antibodies each bound to their own cognate target, so-called hetero-hexamers. These findings provide novel insights into the mechanisms of synergy in antibody-mediated complement-dependent cytotoxicity and provide a rationale to explore Fc-engineering and antibody hetero-hexamerization as a tool to enhance the cooperativity and therapeutic efficacy of antibody combinations.
Immunoglobulin (Ig) G molecules are essential players in the human immune response against bacterial infections. An important effector of IgG-dependent immunity is the induction of complement ...activation, a reaction that triggers a variety of responses that help kill bacteria. Antibody-dependent complement activation is promoted by the organization of target-bound IgGs into hexamers that are held together via noncovalent Fc-Fc interactions. Here we show that staphylococcal protein A (SpA), an important virulence factor and vaccine candidate of
, effectively blocks IgG hexamerization and subsequent complement activation. Using native mass spectrometry and high-speed atomic force microscopy, we demonstrate that SpA blocks IgG hexamerization through competitive binding to the Fc-Fc interaction interface on IgG monomers. In concordance, we show that SpA interferes with the formation of (IgG)
:C1q complexes and prevents downstream complement activation on the surface of
Finally, we demonstrate that IgG3 antibodies against
can potently induce complement activation and opsonophagocytic killing even in the presence of SpA. Together, our findings identify SpA as an immune evasion protein that specifically blocks IgG hexamerization.
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