A. Roberto Temming

Objectives: Listeria monocytogenes is an opportunistic pathogen, associated with foodborne infections that disproportionately affect newborns, elderly and immunocompromised patients. L. monocytogenes can be classified on the antigenic and related structural variation of cell-associated wall teichoic acid (WTA) molecules through conventional serotyping techniques. The WTA structure of serovars (SV) 1/2, 1/2*, 3 and 7 consists of a linear poly-ribitolphosphate (RboP) polymer either with or without decoration with rhamnose (Rha) and/or N-acetylglucosamine (GlcNAc). Of these four SVs, SV1/2 (WTA with GlcNAc and Rha) causes ~ 99% of all listeriosis cases. However, conventional serotyping cannot accurately discriminate between these four SVs, particularly SVs1/2* (WTA with Rha). Methods: Here we applied two identified monoclonal antibodies (mAb), with specificity for the RboP backbone or GlcNAc modification to develop a discriminatory serotyping scheme for SV1/2, 1/2*, 3 and 7. Isogenic mutants for the different SVs were created in L. monocytogenes SV1/2 strain EGD-e. The typing scheme was then adapted to an immnoblot assay and applied to a collection of 317 previously classified listeriosis isolates from the Netherlands Reference Laboratory for Bacterial Meningitis. Results: Binding of the RboP-specific mAb was limited to EGD-e wild type (SV1/2), but increased significantly for isogenic EGD-e mutants representing SV1/2*, 3 and 7. In contrast, the GlcNAc-specific mAb only recognized EGD-e mutants representing SVs 1/2 and 3. The combined staining profiles of the two mAbs allowed accurate discrimination of the four SVs as verified on clinical isolates. Applying this typing scheme to 317 listeriosis isolates previously typed as SV1/2, we confirmed SV designation in >90% of isolates, but also identified SV1/2* (5.4%), SV3 (0.6%) and SV7 (0.3%) isolates. SV1/2* isolates were also identified among meningitis patients. Conclusion: The increased discriminatory capacity of L. monocytogenes serotyping provides a more detailed insight of the epidemiological landscape and the critical factors for L. monocytogenes infections.

Bacterial infections represent a substantial global health challenge, impacting both human and veterinary health. The ongoing evolution of antibiotic-resistant pathogens, coupled with limited new antibiotic discoveries, urges the need for alternative strategies to treat and prevent these infections. Passive immunization with monoclonal antibodies (mAbs) is gaining interest as a promising alternative. Here, we report an experimental pipeline for generating human mAbs from healthy donor B cells using synthetic mimics of complex bacterial glycans. We identified functional mAbs recognizing discrete and unique epitopes on the surface glycans of two bacterial priority pathogens; Staphylococcus aureus and Streptococcus pyogenes. The use of chemically-defined synthetic glycans was critical for the discovery and systematic characterization of mAbs. From a heterogeneous mix of B cell specificities, antibody sequences were identified, leading to the production of mAbs with distinct reactivities against immunodominant but also to less common or even masked epitopes. The pipeline can be adapted to different glycan targets, donor material or specific antibody isotypes. This work thereby paves the way for the discovery of glycan-specific mAbs with clinical relevance to treat, prevent or diagnose infections with S. aureus, S. pyogenes or other bacterial pathogens.

Bacterial infections represent a substantial global health challenge, impacting both human and veterinary health. The ongoing evolution of antibiotic-resistant pathogens, coupled with limited new antibiotic discoveries, urges the need for alternative strategies to treat and prevent these infections. Passive immunization with monoclonal antibodies (mAbs) is gaining interest as a promising alternative. Here, we report an experimental pipeline for generating human mAbs from healthy donor B cells using synthetic mimics of complex bacterial glycans. We identified functional mAbs recognizing discrete and unique epitopes on the surface glycans of two bacterial priority pathogens; Staphylococcus aureus and Streptococcus pyogenes . The use of chemically-defined synthetic glycans was critical for the discovery and systematic characterization of mAbs. From a heterogeneous mix of B cell specificities, mAbs were isolated with reactivities against immunodominant but also to less common or even masked epitopes. The pipeline can be adapted to different glycan targets, donor material or specific antibody isotypes. This work thereby paves the way for the discovery of glycan-specific mAbs with clinical relevance to treat, prevent or diagnose infections with S. aureus , S. pyogenes or other bacterial pathogens.

Most clinically used anticancer mAbs are of the IgG isotype, which can eliminate tumor cells through NK cell–mediated antibody-dependent cellular cytotoxicity and macrophage-mediated antibody-dependent phagocytosis. IgG, however, ineffectively recruits neutrophils as effector cells. IgA mAbs induce migration and activation of neutrophils through the IgA Fc receptor (FcαRI) but are unable to activate NK cells and have poorer half-life. Here, we combined the agonistic activity of IgG mAbs and FcαRI targeting in a therapeutic bispecific antibody format. The resulting TrisomAb molecules recruited NK cells, macrophages, and neutrophils as effector cells for eradication of tumor cells in vitro and in vivo. Moreover, TrisomAb had long in vivo half-life and strongly decreased B16F10gp75 tumor outgrowth in mice. Importantly, neutrophils of colorectal cancer patients effectively eliminated tumor cells in the presence of anti-EGFR TrisomAb but were less efficient in mediating killing in the presence of IgG anti-EGFR mAb (cetuximab). The clinical application of TrisomAb may provide potential alternatives for cancer patients who do not benefit from current IgG mAb therapy.

Abs of the IgG isotype mediate effector functions like Ab-dependent cellular cytotoxicity and Ab-dependent cellular phagocytosis by Fc interactions with FcγRs and complement-dependent cytotoxicity upon IgG-Fc binding to C1q. In this study, we describe the crucial role of the highly conserved dual glycines at position 236–237 in the lower hinge region of human IgG, including the lack of one glycine as found in IgG2. We found several permutations in this region that either silence or largely abrogate FcγR binding and downstream FcγR effector functions, as demonstrated by surface plasmon resonance, Ab-dependent cellular phagocytosis, and Ab-dependent cellular cytotoxicity assays. Although the binding regions of FcγRs and C1q on the IgG-Fc largely overlap, IgG1 with a deletion of G236 only silences FcγR-mediated effector functions without affecting C1q-binding or activation. Several mutations resulted in only residual FcγRI binding with differing affinities that are either complement competent or silenced. Interestingly, we also found that IgG2, naturally only binding FcγRIIa, gains binding to FcγRI and FcγRIIIa after insertion of G236, highlighting the crucial importance of G236 in IgG for FcγR interaction. These mutants may become invaluable tools for FcγR-related research as well as for therapeutic purposes in which only complement-mediated functions are required without the involvement of FcγR.

Ab-dependent cellular cytotoxicity (ADCC) is one of the most important effector mechanisms of tumor-targeting Abs in current immunotherapies. In ADCC and other Ab-dependent activation of myeloid effector cells, close cell–cell contact (between effector and target cell) and formation of immunological synapses are required. However, we still lack basic knowledge on the principal factors influencing ADCC potential by therapeutic Abs. In this study we investigated the combined roles of five factors affecting human NK cell–mediated ADCC, namely: 1) Ag density, 2) target cell membrane composition, 3) IgG FcγR polymorphism, 4) FcγR-blocking cytophilic Abs, and 5) Ab fucosylation. We demonstrate that the magnitude of NK cell–mediated ADCC responses is predominantly influenced by Ag density and Ab fucosylation. Afucosylation consistently induced efficient ADCC, even at very low Ag density, where fucosylated target Abs did not elicit ADCC. On the side of the effector cell, the FcγRIIIa–Val/Phe158 polymorphism influenced ADCC potency, with NK cells expressing the Val158 variant showing more potent ADCC. In addition, we identified the sialic acid content of the target cell membrane as an important inhibitory factor for ADCC. Furthermore, we found that the presence and glycosylation status of aspecific endogenous Abs bound to NK cell FcγRIIIa (cytophilic Abs) determine the blocking effect on ADCC. These five parameters affect the potency of Abs in vitro and should be further tested as predictors of in vivo capacity.

The precise mechanisms underlying anti-inflammatory effects of intravenous immunoglobulin (IVIg) therapies remain elusive. The sialylated IgG fraction within IVIg has been shown to be therapeutically more active in mouse models. Functionally, it has been suggested that IgG undergoes conformational changes upon Fc-sialylation which sterically impede binding to conventional FcγRs, but simultaneously allow binding to human DC-SIGN (SIGN-R1 in mice) and also CD23. These latter C-type lectins have been proposed responsible for the immunomodulatory effects in mouse models. However, there is conflicting evidence supporting direct interactions between sialylated human IgG and CD23/DC-SIGN. While cells expressing human CD23 and DC-SIGN in their native configuration bound their natural ligands IgE and ICAM-3, respectively, no IgG binding was observed, regardless of Fc-glycan sialylation in any context (with or without bisection and/or fucosylation) or presence of sialylated Fab-glycans. This was tested by both by FACS and a novel cellular Surface Plasmon Resonance imaging (cSPRi) approach allowing for monitoring low-affinity but high-avidity interactions. In summary, we find no evidence for human CD23 or DC-SIGN being bona fide receptors to human IgG, regardless of IgG Fc- or Fab-glycosylation status. However, these results do not exclude the possibility that either IgG glycosylation or C-type lectins affect IVIg therapies.

Neonatal Fc-receptor (FcRn), the major histocompatibility complex (MHC) class I-like Fc-receptor, transports immunoglobuline G (IgG) across cell layers, extending IgG half-life in circulation and providing newborns with humoral immunity. IgG1 and IgG2 have similar half-lives, yet IgG2 displays lower foetal than maternal concentration at term, despite all known FcRn binding residues being preserved between IgG1 and IgG2. We investigated FcRn mediated transcytosis of V_H-matched IgG1 and IgG2 and mutated variants thereof lacking Fc-gamma receptor (FcγR) binding in human cells expressing FcRn. We observed that FcγR binding was not required for transport and that FcRn transported less IgG2 than IgG1. Transport of IgG1 with a shortened lower hinge (ΔGly236, absent in germline IgG2), was reduced to levels equivalent to IgG2. Conversely, transport of IgG2 + Gly236 was increased to IgG1 levels. Gly236 is not a contact residue between IgG and FcRn, suggesting that its absence leads to an altered conformation of IgG, possibly due to a less flexible Fab, positioned closer to the Fc portion. This may sterically hinder FcRn binding and transport. We conclude that the lack of Gly236 is sufficient to explain the reduced FcRn-mediated IgG2 transcytosis and accounts for the low maternal/fetal IgG2 ratio at term.