Biology subjects

Ravetch, J. V.

Publications and source records attributed to Ravetch, J. V..

9 recordsLinked to original sources

bioRxiv2024

The anti-inflammatory activity of IgG requires the co-engagement of types I and II Fc receptors

Intravenous immunoglobulin administered at high doses has been used for decades as an effective anti-inflammatory preparation to treat a wide array of autoimmune diseases. Studies in murine models have found this activity to be dependent on 1) sialylation of the N-linked glycan on the CH2 domain of IgG, 2) the type I IgG inhibitory Fc receptor Fc{gamma}RIIB, and 3) the type II Fc receptor DC-SIGN. Here, we demonstrate that the ectodomain glycan on Fc{gamma}RIIB interacts with the lectin domain of DC-SIGN to form a cell surface complex, thereby enabling the binding of sialylated IgG1. We have exploited this observation to Fc-engineer sialylated IgG1 to enhance its affinity for Fc{gamma}RIIB and demonstrate that the resulting molecule is at least 100-fold more potent in reducing the inflammatory sequelae of antibody or T cell mediated autoimmune diseases, providing the basis for a new class of anti-inflammatory therapeutics.

immunology↗

bioRxiv2023

IL-15 synergizes with CD40 agonist antibodies to induce durable immunity against bladder cancer

CD40 is a central co-stimulatory receptor implicated in the development of productive anti-tumor immune responses across multiple cancers, including bladder cancer. Despite strong preclinical rationale, systemic administration of therapeutic agonistic antibodies targeting the CD40 pathway have demonstrated dose limiting toxicities with minimal clinical activity to date, emphasizing an important need for optimized CD40-targeted approaches, including rational combination therapy strategies. Here, we describe an important role for the endogenous IL-15 pathway in contributing to the therapeutic activity of CD40 agonism in orthotopic bladder tumors, with upregulation of trans-presented IL-15/IL-15R surface complexes, particularly by cross-presenting cDC1s, and associated enrichment of activated CD8 T cells within the bladder tumor microenvironment. In bladder cancer patient samples, we identify DCs as the primary source of IL-15, however, they lack high levels of IL-15R at baseline. Using humanized immunocompetent orthotopic bladder tumor models, we demonstrate the ability to therapeutically augment this interaction through combined treatment with anti-CD40 agonist antibodies and exogenous IL-15, including the fully-human Fc-optimized antibody 2141-V11 currently in clinical development for the treatment of bladder cancer. Combination therapy enhances the crosstalk between Batf3-dependent cDC1s and CD8 T cells, driving robust primary anti-tumor activity and further stimulating long-term systemic anti-tumor memory responses associated with circulating memory-phenotype T and NK cell populations. Collectively, these data reveal an important role for IL-15 in mediating anti-tumor CD40 agonist responses in bladder cancer and provide key proof-of-concept for combined use of Fc-optimized anti-CD40 agonist antibodies and agents targeting the IL-15 pathway. These data support expansion of ongoing clinical studies evaluating anti-CD40 agonist antibodies and IL-15-based approaches to evaluate combinations of these promising therapeutics for the treatment of patients with bladder cancer.

immunology↗

bioRxiv2023

Mechanism of glycoform specificity and protection against antibody dependent enhancement by an anti-afucosylated IgG nanobody

Immunoglobulin G (IgG) antibodies contain a single, complex N-glycan on each IgG heavy chain protomer embedded in the hydrophobic pocket between its C{gamma}2 domains. The presence of this glycan contributes to the structural organization of the Fc domain and determines its specificity for Fc{gamma} receptors, thereby determining distinct cellular responses. On the Fc, the variable construction of this glycan structure leads to a family of highly-related, but non-equivalent glycoproteins known as glycoforms. We previously reported the development of synthetic nanobodies that distinguish IgG glycoforms without cross-reactivity to off-target glycoproteins or free glycans. Here, we present the X-ray crystal structure of one such nanobody, X0, in complex with its specific binding partner, the Fc fragment of afucosylated IgG1. Two X0 nanobodies bind a single afucosylated Fc homodimer at the upper C{gamma}2 domain, making both protein-protein and protein-carbohydrate contacts and overlapping the binding site for Fc{gamma} receptors. Upon binding, the elongated CDR3 loop of X0 undergoes a conformational shift to access the buried N-glycan and acts as a glycan sensor, forming hydrogen bonds with the afucosylated IgG N-glycan that would otherwise be sterically hindered by the presence of a core fucose residue. Based on this structure, we designed X0 fusion constructs that disrupt pathogenic afucosylated IgG1-Fc{gamma}RIIIa interactions and rescue mice in a model of dengue virus infection.

immunology↗

bioRxiv2022

Human FcγRIIIa activation on splenic macrophages drives the in vivo pathogenesis of dengue disease

Although dengue virus (DENV) infection typically causes asymptomatic disease, DENV-infected patients can experience severe complications. A risk factor for symptomatic disease is pre-existing anti-DENV IgG antibodies. Cellular assays suggested that these antibodies can enhance viral infection of Fc{gamma} receptor (Fc{gamma}R)-expressing myeloid cells. Recent studies, however, revealed more complex interactions between anti-DENV antibodies and specific Fc{gamma}Rs by demonstrating that modulation of the IgG Fc glycan correlates with disease severity. To investigate the in vivo mechanisms of antibody-mediated dengue pathogenesis, we developed a mouse model for dengue disease that recapitulates the unique complexity of human Fc{gamma}Rs. Our studies reveal that the in vivo pathogenic activity of anti-DENV IgG antibodies is exclusively mediated through engagement of Fc{gamma}RIIIa expressed on splenic macrophages, resulting in inflammatory sequelae and mortality. These findings highlight the importance of IgG-Fc{gamma}RIIIa interactions in dengue disease, with important implications in the design of safer vaccination approaches and effective therapeutic strategies.

immunology↗

bioRxiv2022

Antibody-lectin chimeras for glyco-immune checkpoint blockade

Despite the curative potential of checkpoint blockade immunotherapy, most patients remain unresponsive to existing treatments. Glyco-immune checkpoints - interactions of cell-surface glycans with lectin, or glycan-binding, immunoreceptors - have emerged as prominent mechanisms of immune evasion and therapeutic resistance in cancer. Here, we describe antibody-lectin chimeras (AbLecs), a modular platform for glyco-immune checkpoint blockade. AbLecs are bispecific antibody-like molecules comprising a cell-targeting antibody domain and a lectin "decoy receptor" domain that directly binds glycans and blocks their ability to engage inhibitory lectin receptors. AbLecs potentiate anticancer immune responses including phagocytosis and cytotoxicity, outperforming most existing therapies and combinations tested. By targeting a distinct axis of immunological regulation, AbLecs synergize with blockade of established immune checkpoints. AbLecs can be readily designed to target numerous tumor and immune cell subsets as well as glyco-immune checkpoints, and therefore represent a new modality for cancer immunotherapy.

biochemistry↗

bioRxiv2020

Antibody potency, effector function and combinations in protection from SARS-CoV-2 infection in vivo

SARS-CoV-2, the causative agent of COVID-19, is responsible for over 24 million infections and 800,000 deaths since its emergence in December 2019. There are few therapeutic options and no approved vaccines. Here we examine the properties of highly potent human monoclonal antibodies (hu-mAbs) in a mouse adapted model of SARS-CoV-2 infection (SARS-CoV-2 MA). In vitro antibody neutralization potency did not uniformly correlate with in vivo activity, and some hu-mAbs were more potent in combination in vivo. Analysis of antibody Fc regions revealed that binding to activating Fc receptors is essential for optimal protection against SARS-CoV-2 MA. The data indicate that hu-mAb protective activity is dependent on intact effector function and that in vivo testing is required to establish optimal hu-mAb combinations for COVID-19 prevention.

microbiology↗

bioRxiv2020

Engineered ACE2 receptor traps potently neutralize SARS-CoV-2

An essential mechanism for SARS-CoV-1 and -2 infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human Fc domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2 pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50) in the 10-100 ng/ml range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-utilizing coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be pre-designed for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated or generated from convalescent patients.

bioengineering↗

bioRxiv2020

FcRn, but not FcgRs, drives maternal-fetal transplacental transport of human IgGantibodies

The IgG Fc domain has the capacity to interact with diverse types of receptors, including FcRn and Fc{gamma}Rs, which confer pleiotropic biological activities. Whereas FcRn regulates IgG epithelial transport and recycling, Fc effector activities, such as ADCC and phagocytosis are mediated by Fc{gamma}Rs, which upon crosslinking transduce signals that modulate the function of effector leukocytes. Despite the well-defined and non-overlapping functional properties of FcRn and Fc{gamma}Rs, recent studies have suggested that Fc{gamma}Rs mediate transplacental IgG transport, as certain Fc glycoforms were reported to be enriched in fetal circulation. To determine the contribution of Fc{gamma}Rs and FcRn to the maternal-fetal transport of IgG, we characterized the IgG Fc glycosylation in paired maternal-fetal samples from patient cohorts from Uganda and Nicaragua. No differences in IgG1 Fc glycan profiles and minimal differences in IgG2 Fc glycans were noted, whereas the presence or absence of galactose on the Fc glycan of IgG1 did not alter Fc{gamma}RIIIA or FcRn binding, half-life, or their ability to deplete target cells in Fc{gamma}R/FcRn humanized mice. Modeling maternal/fetal transport in Fc{gamma}R/FcRn humanized mice confirmed that only FcRn contributed to transplacental transport of IgG; IgG selectively enhanced for FcRn binding resulted in enhanced accumulation of maternal antibody in the fetus. In contrast, enhancing Fc{gamma}RIIIA binding did not result in enhanced maternal/fetal transport. These results argue against a role for Fc{gamma}Rs in IgG transplacental transport, suggesting Fc engineering of maternally administered antibody to only enhance FcRn binding as a means to improve maternal/fetal transport of IgG. Significance StatementTransport of IgG antibodies from the maternal to the fetal circulation is a key process for neonatal immunity, as neonates cannot sufficiently generate IgG antibodies to reach protective levels during the first months after birth. In humans and other primates, maternal to fetal transport of IgG antibodies is largely mediated through the placental tissue. FcRn has been previously identified as the major driver of IgG transplacental transport. Here we examined whether other receptors, such as Fc{gamma}Rs, also contribute to the maternal-fetal IgG transfer. By characterizing the Fc domain structure of paired maternal-fetal IgG samples and modeling transplacental IgG transport in genetically engineered mouse strains, we determined that FcRn, but not Fc{gamma}Rs, is the major receptor that mediates transplacental IgG transport.

immunology↗

bioRxiv2020

A Combination of Two Human Monoclonal Antibodies Limits Fetal Damage by Zika Virus in Macaques

Human infection by Zika virus (ZIKV) during pregnancy can lead to vertical transmission and fetal aberrations, including microcephaly. Prophylactic administration of antibodies can diminish or prevent ZIKV infection in animal models, but whether passive immunization can protect nonhuman primates and their fetuses during pregnancy has not been determined. Z004 and Z021 are neutralizing monoclonal antibodies to domain III of the envelope (EDIII) of ZIKV. Together the two antibodies protect nonpregnant macaques against infection even after Fc modifications to prevent antibody-dependent enhancement in vitro (ADE) and extend their half-lives. Here we report on prophylactic co-administration of the Fc-modified antibodies to pregnant rhesus macaques challenged 3 times with ZIKV during first and second trimester. The two antibodies did not entirely eliminate maternal viremia but limited vertical transmission protecting the fetus from neurologic damage. Thus, maternal passive immunization with two antibodies to EDIII can shield primate fetuses from the harmful effects of ZIKV. Significance statementZika virus (ZIKV) infection during pregnancy can cause fetal abnormalities. Vaccines against ZIKV are under development, but because of potential safety concerns due to disease enhancing antibodies, and the time required by active immunization to induce protective antibodies, there is a need to explore alternative strategies. Recombinant monoclonal antibodies can be modified to prevent enhancement of infection, and thus could be an efficacious and safe alternative to vaccines to confer rapid protection. We show that prophylactic administration of two engineered antibodies, Z004 and Z021, to pregnant macaques partially protects against fetal neurologic damage and limits vertical transmission of ZIKV.

immunology↗