Biology subjects

Delidakis, G.

Publications and source records attributed to Delidakis, G..

4 recordsLinked to original sources

bioRxiv2026

Antibodies with Engineered Fc Domains Having Absolute Binding Selectivity to either FcγRIIa or FcγRI Delineate the Respective Effector Phenotypes by Human Monocytes and Macrophages

IgG1 immune complexes bind to all the Fc{gamma} receptors (Fc{gamma}R) expressed on myeloid cells, making it challenging to determine the precise role of each Fc{gamma}R on Fc effector phenotypes. Here we report the engineering of Fc2KG, an aglycosylated human IgG1 Fc domain that binds with near physiological affinity to Fc{gamma}RIIa/b with no detectable binding to any other Fc{gamma}Rs. Crystallographic analysis elucidated the structural basis of how mutations in the Fc domain compensate for the absence of the N297 glycan and enable selective binding. Using single-cell phagocytosis assays, we show that particles opsonized with Fc-engineered antibodies formatted with Fc2KG or with an Fc domain that binds only Fc{gamma}RI (Fc5), are ingested by THP-1 cells with near identical kinetics. We find that with CD16+ primary human monocytes, selective Fc{gamma}RII engagement is a major contributor to the ADCP mediated by wild-type IgG1. Additionally, we showed that Fc2KG formatted antibodies induce high levels of GM-CSF. With M1-like monocyte-derived human macrophages, trastuzumab formatted with wild-type IgG1 Fc, with Fc2KG or Fc5 were all equally proficient in the trogocytotic killing of opsonized SK-BR-3 HER2+ cells but only Fc{gamma}RI engagement led to secretion of proinflammatory cytokines. Collectively, our results highlight how precisely tuned, Fc-engineered antibodies can be deployed to answer long-standing questions regarding the precise effector functions mediated by human Fc{gamma}Rs, information which is key for the optimization of therapeutic antibodies.

immunology↗

bioRxiv2026

Structure-Function Analysis of the FCRL5-IgG1 Fc Complex Reveals an Unappreciated Effect of Fc-Silent Antibodies on B cells

Human Fc receptor-like 5 (FCRL5) is a low-affinity IgG Fc receptor expressed on various B cell subsets and a potential therapeutic target. We discovered that commonly used Fc-silencing mutations, designed to prevent interactions between the Fc{gamma} receptors on immune cells and the Fc domain of therapeutic IgG, do not prevent binding to FCRL5. As a result, unintended interactions between Fc-silent therapeutic IgG and human B cells may occur. We isolated a well-expressed variant of the Fc-binding portion of human FCRL5 by directed evolution and used structural modeling to guide the engineering of a human IgG1 Fc variant with approximately 100-fold higher affinity for FCRL5, enabling us to produce FCRL5:Fc complexes in solution. Native mass spectrometry, size exclusion chromatography, and the crystal structure of the FCRL5- IgG1 Fc complex solved at 3.4 [A] indicate that the two proteins bind in a 1:1 stoichiometry. Furthermore, the structure revealed that FCRL5 binds to IgG1 Fc in a manner completely distinct from that of previously characterized Fc-binding proteins, such as Fc{gamma} receptors, explaining why most Fc-silencing mutations do not disrupt FCRL5 binding. We demonstrate that selective cross-linking of FCRL5 with the B cell receptor (BCR) in cis, using Fc-engineered antibodies with either physiological or enhanced FCRL5 affinity, inhibits Ca2+ flux in FCRL5-expressing B cells. We compare this effect with the selective co-ligation of Fc{gamma}RIIb with the BCR. Our work demonstrates that FCRL5 interacts with human IgG Fc in a distinctive manner and that engagement of FCRL5 by Fc-silent therapeutic IgG could influence B cell function.

bioengineering↗

bioRxiv2025

Selective decoupling of IgG1 binding to viral Fc receptorsrestores antibody-mediated NK cell activation against HCMV-infected cells

A key mechanism of antiviral antibodies is to bind cell-surface viral antigens and activate cellular immunity to clear infected cells, yet antibodies targeting human cytomegalovirus (HCMV) have exhibited limited efficacy. This appears due to HCMVs multiple immune evasion mechanisms, including viral receptors (vFc{gamma}Rs) which bind human IgG Fc domains to co-operatively inhibit Fc activation of host Fc{gamma} receptors and impair Fc-mediated effector functions. We biochemically characterized and evaluated the functions of two highly conserved vFc{gamma}Rs, gp34 and gp68, and mapped their binding epitopes on the Fc domain. Based on this information, we then engineered Fc variants that retain binding to CD16A, which is essential for NK activation, and to FcRn but have markedly attenuated binding to gp34 and gp68. IgG1 antibodies targeting the gB fusogen with engineered Fc domains were not internalized by infected cells, mediated enhanced CD16A activation and limited viral spread in HCMV-infected fibroblasts more effectively than wild-type Fc. Together, this work demonstrates a strategy to enhance the efficacy of antibody therapies to clear HCMV infections. HighlightsO_LIHost and HCMV FcR compete for IgG1 binding but engage different residues. C_LIO_LIFc-engineering abrogates viral FcR antagonism while retaining CD16A activation. C_LIO_LIAntibodies that resist vFcR capture promote superior ADCC against infected cells. C_LIO_LIDesigner Fc domains complement Fabs to create enhanced disease-specific therapies. C_LI

immunology↗

bioRxiv2020

Prevalent, protective, and convergent IgG recognition of SARS-CoV-2 non-RBD spike epitopes in COVID-19 convalescent plasma

Although humoral immunity is essential for control of SARS-CoV-2, the molecular composition, binding epitopes and effector functions of the immunoglobulin G (IgG) antibodies that circulate in blood plasma following infection are unknown. Proteomic deconvolution of the circulating IgG repertoire (Ig-Seq1) to the spike ectodomain (S-ECD2) in four convalescent study subjects revealed that the plasma response is oligoclonal and directed predominantly (>80%) to S-ECD epitopes that lie outside the receptor binding domain (RBD). When comparing antibodies directed to either the RBD, the N-terminal domain (NTD) or the S2 subunit (S2) in one subject, just four IgG lineages (1 anti-S2, 2 anti-NTD and 1 anti-RBD) accounted for 93.5% of the repertoire. Although the anti-RBD and one of the anti-NTD antibodies were equally potently neutralizing in vitro, we nonetheless found that the anti-NTD antibody was sufficient for protection to lethal viral challenge, either alone or in combination as a cocktail where it dominated the effect of the other plasma antibodies. We identified in vivo protective plasma anti-NTD antibodies in 3/4 subjects analyzed and discovered a shared class of antibodies targeting the NTD that utilize unmutated or near-germline IGHV1-24, the most electronegative IGHV gene in the human genome. Structural analysis revealed that binding to NTD is dominated by interactions with the heavy chain, accounting for 89% of the entire interfacial area, with germline residues uniquely encoded by IGHV1-24 contributing 20% (149 [A]2). Together with recent reports of germline IGHV1-24 antibodies isolated by B-cell cloning3,4 our data reveal a class of shared IgG antibodies that are readily observed in convalescent plasma and underscore the role of NTD-directed antibodies in protection against SARS-CoV-2 infection.

immunology↗