The Making of Pieces of the Puzzle: Reflections on a Collaborative Ethnographic Filmmaking Process

This is a companion piece to the “Pieces of the Puzzle” film. &nbsp

Hexamerization-enhanced CD20 antibody mediates complement-dependent cytotoxicity in serum genetically deficient in C9

We examined complement-dependent cytotoxicity (CDC) by hexamer formation-enhanced CD20 mAb Hx-7D8 of patient-derived chronic lymphocytic leukemia (CLL) cells that are relatively resistant to CDC. CDC was analyzed in normal human serum (NHS) and serum from an individual genetically deficient for C9. Hx-7D8 was able to kill up to 80% of CLL cells in complete absence of C9. We conclude that the narrow C5b-8 pores formed without C9 are sufficient for CDC due to efficient antibody-mediated hexamer formation. In the absence of C9, we observed transient intracellular increases of Ca2 + during CDC (as assessed with FLUO-4) that were extended in time. This suggests that small C5b-8 pores allow Ca2 + to enter the cell, while dissipation of the fluorescent signal accompanying cell disintegration is delayed. The Ca2 + signal is retained concomitantly with TOPRO-3 (viability dye) staining, thereby confirming that Ca2 + influx represents the most proximate mediator of cell death by CDC

Promoting Fc-Fc interactions between anti-capsular antibodies provides strong immune protection against Streptococcus pneumoniae.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia and an important cause of childhood mortality. Despite the introduction of successful vaccines, the global spread of both non-vaccine serotypes and antibiotic-resistant strains reinforces the development of alternative therapies against this pathogen. One possible route is the development of monoclonal antibodies (mAbs) that induce killing of bacteria via the immune system. Here, we investigate whether mAbs can be used to induce killing of pneumococcal serotypes for which the current vaccines show unsuccessful protection. Our study demonstrates that when human mAbs against pneumococcal capsule polysaccharides (CPS) have a poor capacity to induce complement activation, a critical process for immune protection against pneumococci, their activity can be strongly improved by hexamerization-enhancing mutations. Our data indicate that anti-capsular antibodies may have a low capacity to form higher-order oligomers (IgG hexamers) that are needed to recruit complement component C1. Indeed, specific point mutations in the IgG-Fc domain that strengthen hexamerization strongly enhance C1 recruitment and downstream complement activation on encapsulated pneumococci. Specifically, hexamerization-enhancing mutations E430G or E345K in CPS6-IgG strongly potentiate complement activation on S. pneumoniae strains that express capsular serotype 6 (CPS6), and the highly invasive serotype 19A strain. Furthermore, these mutations improve complement activation via mAbs recognizing CPS3 and CPS8 strains. Importantly, hexamer-enhancing mutations enable mAbs to induce strong opsonophagocytic killing by human neutrophils. Finally, passive immunization with CPS6-IgG1-E345K protected mice from developing severe pneumonia. Altogether, this work provides an important proof of concept for future optimization of antibody therapies against encapsulated bacteria

Antibody Engineering & Therapeutics 2016: The Antibody Society's annual meeting, December 11–15, 2016, San Diego, CA

ABSTRACT Antibody Engineering & Therapeutics, the largest meeting devoted to antibody science and technology and the annual meeting of The Antibody Society, will be held in San Diego, CA on December 11-15, 2016. Each of 14 sessions will include six presentations by leading industry and academic experts. In this meeting preview, the session chairs discuss the relevance of their topics to current and future antibody therapeutics development. Session topics include bispecifics and designer polyclonal antibodies; antibodies for neurodegenerative diseases; the interface between passive and active immunotherapy; antibodies for non-cancer indications; novel antibody display, selection and screening technologies; novel checkpoint modulators / immuno-oncology; engineering antibodies for T-cell therapy; novel engineering strategies to enhance antibody functions; and the biological Impact of Fc receptor engagement. The meeting will open with keynote speakers Dennis R. Burton (The Scripps Research Institute), who will review progress toward a neutralizing antibody-based HIV vaccine; Olivera J. Finn, (University of Pittsburgh School of Medicine), who will discuss prophylactic cancer vaccines as a source of therapeutic antibodies; and Paul Richardson (Dana-Farber Cancer Institute), who will provide a clinical update on daratumumab for multiple myeloma. In a featured presentation, a representative of the World Health Organization's INN expert group will provide a perspective on antibody naming. “Antibodies to watch in 2017” and progress on The Antibody Society's 2016 initiatives will be presented during the Society's special session. In addition, two pre-conference workshops covering ways to accelerate antibody drugs to the clinic and the applications of next-generation sequencing in antibody discovery and engineering will be held on Sunday December 11, 2016

C1q binding to surface-bound IgG is stabilized by C1r(2)s(2) proteases

Complement is an important effector mechanism for antibodymediated clearance of infections and tumor cells. Upon binding to target cells, the antibody's constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr2s2) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r2s2). While interactions between C1 and IgG-Fc are believed to be mediated by the globular heads of C1q, we here find that C1r2s2 proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various 2,4-dinitrophenol [DNP]-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r2s2 contributes to C1q-IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG, we reveal that hexamer-enhancing mutations improve C1q-IgG stability, both in the absence and presence of C1r2s2. In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.Transplantation and autoimmunit

IgG4 breaking the rules

Immunoglobulin G4 (IgG4) antibodies have been known for some time to be functionally monovalent. Recently, the structural basis for this monovalency has been elucidated: the in vivo exchange of IgG half-molecules (one H-plus one L-chain) among IgG4. This process results in bispecific antibodies that in most situations will behave as functionally monovalent antibodies. The structural basis for the abnormal behaviour of IgG4 seems to be largely the result of a single amino acid change relative to human IgG1: the change of a proline in core hinge of IgG1 to serine. This results in a marked shift in the equilibrium between interchain disulphide bridges and intrachain disulphide bridges, which for IgG4 results in 25-75% absence of a covalent interaction between the H-chains. Because of strong non-covalent interactions between the CH3 domains (and possibly also between the CH1 domain and the trans-CH2 domain) IgG4 is a stable four-chain molecule and does not easily exchange half-molecules under standard physiological conditions in vitro. We postulate that the exchange is catalysed in vivo by protein disulphide isomerase (PDI) and/or FcRn (the major histocompatibility complex (MHC)-related Fc receptor) during transit of IgG4 in the endosomal pathway in endothelial cells. Because IgG4 is predominantly expressed under conditions of chronic antigen exposure, the biological relevance of this exchange of half-molecules is that it generates antibodies that are unable to form large immune complexes and therefore have a low potential for inducing immune inflammation. In contrast to monovalent immunoglobulin fragments, these scrambled immunoglobulins have a normal half-life. The significance of the ensuing bispecificity needs further evaluation, because this will be relevant only in situations where high IgG4 responses are found to two unrelated antigens that happen to be present in the body at the same time and place. In this context the significance of IgG4 autoreactivity might have to be re-evaluated. The main function of IgG4, however, is presumably to interfere with immune inflammation induced by complement-fixing antibodies, or, in the case of helminth infection or allergy, by IgE antibodie

IgG4 breaking the rules

Immunoglobulin G4 (IgG4) antibodies have been known for some time to be functionally monovalent. Recently, the structural basis for this monovalency has been elucidated: the in vivo exchange of IgG half-molecules (one H-plus one L-chain) among IgG4. This process results in bispecific antibodies that in most situations will behave as functionally monovalent antibodies. The structural basis for the abnormal behaviour of IgG4 seems to be largely the result of a single amino acid change relative to human IgG1: the change of a proline in core hinge of IgG1 to serine. This results in a marked shift in the equilibrium between interchain disulphide bridges and intrachain disulphide bridges, which for IgG4 results in 25–75% absence of a covalent interaction between the H-chains. Because of strong non-covalent interactions between the CH3 domains (and possibly also between the CH1 domain and the trans-CH2 domain) IgG4 is a stable four-chain molecule and does not easily exchange half-molecules under standard physiological conditions in vitro. We postulate that the exchange is catalysed in vivo by protein disulphide isomerase (PDI) and/or FcRn (the major histocompatibility complex (MHC)-related Fc receptor) during transit of IgG4 in the endosomal pathway in endothelial cells. Because IgG4 is predominantly expressed under conditions of chronic antigen exposure, the biological relevance of this exchange of half-molecules is that it generates antibodies that are unable to form large immune complexes and therefore have a low potential for inducing immune inflammation. In contrast to monovalent immunoglobulin fragments, these scrambled immunoglobulins have a normal half-life. The significance of the ensuing bispecificity needs further evaluation, because this will be relevant only in situations where high IgG4 responses are found to two unrelated antigens that happen to be present in the body at the same time and place. In this context the significance of IgG4 autoreactivity might have to be re-evaluated. The main function of IgG4, however, is presumably to interfere with immune inflammation induced by complement-fixing antibodies, or, in the case of helminth infection or allergy, by IgE antibodies

When binding is enough: nonactivating antibody formats

Most therapeutic antibodies currently used in the clinic are based on the human IgG1 format, which is a bivalent molecule that efficiently interacts with the immune system's effector functions. In clinical applications where binding to the target alone is sufficient for therapeutic efficacy; however, engagement of the immune system is not required and may even cause unwanted side-effects. Likewise, bivalent binding to the target may negatively influence the therapeutic efficacy of an antibody. Here we discuss the state of the art for antibody-based therapeutics, designed to be nonactivating (i.e. do not engage the innate immune system's effector functions), in both monovalent and bivalent format

Human IgG4 binds to IgG4 and conformationally altered IgG1 via Fc-Fc interactions

The Fc fragment of IgG4 can interact with the Fc fragment of another IgG molecule. This interaction is a confounding factor when measuring IgG4 rheumatoid factor levels. Recently, we demonstrated that half-molecules of IgG4 can exchange to form a bispecific Ab. We expected these two phenomena to be related and investigated the physicochemical aspects of IgG4 Fc-Fc interactions. We found that IgG4 is >99% monomeric by size-exclusion chromatography; therefore, IgG4 Fc-Fc interactions in the fluid phase (if any) would be short-lived. However, (125)I-labeled IgG4 does bind to IgG1 and IgG4 coupled to a solid phase. By contrast, IgG1 does not bind to coupled IgG4. Furthermore, conditions that induce partial unfolding/dissociation of the CH3 domains enhance IgG4 Fc binding, suggesting that Fc binding is primarily CH3 mediated. IgG4 slowly associates with both IgG4 and IgG1 coupled to a biosensor chip. Remarkably, subsequent dissociation was much faster for IgG4 than for IgG1. Moreover, after binding of an IgG4 mAb to Sepharose-coupled Ag, we observed additional binding of IgG4 with irrelevant specificity, whereas similar binding was not observed with Ag-bound IgG1. We propose that the IgG4-IgG4 Fc interaction resembles an intermediate of the Fab-arm (half-molecule) exchange reaction that is stabilized because one of the IgG4 molecules is coupled to a solid phase. By contrast, IgG4 Fc recognizes IgG1 only after a conformational change that renders CH3(IgG1) accessible to an interaction with the CH3(IgG4). Such Fc interactions may enhance Ag binding of IgG4 in viv