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Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities.

ABSTRACT: Soluble Angiotensin-Converting Enzyme 2 (ACE2) constitutes an attractive antiviral capable of targeting a wide range of coronaviruses utilizing ACE2 as their receptor. Here, using structure-guided approaches, we developed divalent ACE2 molecules by grafting the extracellular ACE2-domain onto a human IgG1 or IgG3 (ACE2-Fc). These ACE2-Fcs harbor structurally validated mutations that enhance spike (S) binding and remove angiotensin enzymatic activity. The lead variant bound tightly to S, mediated in vitro neutralization of SARS-CoV-2 variants of concern (VOCs) with sub-nanomolar IC ₅₀ and was capable of robust Fc-effector functions, including antibody-dependent-cellular cytotoxicity, phagocytosis and complement deposition. When tested in a stringent K18-hACE2 mouse model, it delayed death or effectively resolved lethal SARS-CoV-2 infection in a prophylactic or therapeutic setting utilizing the combined effect of neutralization and Fc-effector functions. These data confirm the utility of ACE2-Fcs as valuable agents in preventing and eliminating SARS-CoV-2 infection and demonstrate that ACE2-Fc therapeutic activity require Fc-effector functions.

SUBMITTER: Chen Y

PROVIDER: S-EPMC8629194 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Engineered ACE2-Fc counters murine lethal SARS-CoV-2 infection through direct neutralization and Fc-effector activities.

Project description:Soluble angiotensin-converting enzyme 2 (ACE2) constitutes an attractive antiviral capable of targeting a wide range of coronaviruses using ACE2 as their receptor. Using structure-guided approaches, we developed a series of bivalent ACE2-Fcs harboring functionally and structurally validated mutations that enhance severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) receptor binding domain recognition by up to ~12-fold and remove angiotensin enzymatic activity. The lead variant M81 potently cross-neutralized SARS-CoV-2 variants of concern (VOCs), including Omicron, at subnanomolar half-maximal inhibitory concentration and was capable of robust Fc-effector functions, including antibody-dependent cellular cytotoxicity, phagocytosis, and complement deposition. When tested in a stringent K18-hACE2 mouse model, Fc-enhanced ACE2-Fc delayed death by 3 to 5 days or effectively resolved lethal SARS-CoV-2 infection in both prophylactic and therapeutic settings via the combined effects of neutralization and Fc-effector functions. These data add to the demonstrated utility of soluble ACE2 as a valuable SARS-CoV-2 antiviral and indicate that Fc-effector functions may constitute an important component of ACE2-Fc therapeutic activity.

| S-EPMC9278865 | biostudies-literature

Mutations from bat ACE2 orthologs markedly enhance ACE2-Fc neutralization of SARS-CoV-2.

Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates infection of cells expressing angiotensin-converting enzyme 2 (ACE2). ACE2 is also the viral receptor of SARS-CoV (SARS-CoV-1), a related coronavirus that emerged in 2002-2003. Horseshoe bats (genus Rhinolophus ) are presumed to be the original reservoir of both viruses, and a SARS-like coronavirus, RaTG13, closely related SARS-CoV-2, has been isolated from one horseshoe-bat species. Here we characterize the ability of S-protein receptor-binding domains (RBDs) of SARS-CoV-1, SARS-CoV-2, and RaTG13 to bind a range of ACE2 orthologs. We observed that the SARS-CoV-2 RBD bound human, pangolin, and horseshoe bat ( R. macrotis) ACE2 more efficiently than the SARS-CoV-1 or RaTG13 RBD. Only the RaTG13 RBD bound rodent ACE2 orthologs efficiently. Five mutations drawn from ACE2 orthologs of nine Rhinolophus species enhanced human ACE2 binding to the SARS-CoV-2 RBD and neutralization of SARS-CoV-2 by an immunoadhesin form of human ACE2 (ACE2-Fc). Two of these mutations impaired neutralization of SARS-CoV-1. An ACE2-Fc variant bearing all five mutations neutralized SARS-CoV-2 five-fold more efficiently than human ACE2-Fc. These data narrow the potential SARS-CoV-2 reservoir, suggest that SARS-CoV-1 and -2 originate from distinct bat species, and identify a more potently neutralizing form of ACE2-Fc.

| S-EPMC7337384 | biostudies-literature

Engineered antibody Fc variants with enhanced effector function.

Project description:Antibody-dependent cell-mediated cytotoxicity, a key effector function for the clinical efficacy of monoclonal antibodies, is mediated primarily through a set of closely related Fcgamma receptors with both activating and inhibitory activities. By using computational design algorithms and high-throughput screening, we have engineered a series of Fc variants with optimized Fcgamma receptor affinity and specificity. The designed variants display >2 orders of magnitude enhancement of in vitro effector function, enable efficacy against cells expressing low levels of target antigen, and result in increased cytotoxicity in an in vivo preclinical model. Our engineered Fc regions offer a means for improving the next generation of therapeutic antibodies and have the potential to broaden the diversity of antigens that can be targeted for antibody-based tumor therapy.

| S-EPMC1389705 | biostudies-literature

Engineered Fc-glycosylation switch to eliminate antibody effector function.

Project description:Antibodies mediate effector functions through Fcγ receptor (FcγR) interactions and complement activation, causing cytokine release, degranulation, phagocytosis, and cell death. They are often undesired for development of therapeutic antibodies where only antigen binding or neutralization would be ideal. Effector elimination has been successful with extensive mutagenesis, but these approaches can potentially lead to manufacturability and immunogenicity issues. By switching the native glycosylation site from position 297 to 298, we created alternative antibody glycosylation variants in the receptor interaction interface as a novel strategy to eliminate the effector functions. The engineered glycosylation site at Asn298 was confirmed by SDS-PAGE, mass spectrometry, and X-ray crystallography (PDB code 6X3I). The lead NNAS mutant (S298N/T299A/Y300S) shows no detectable binding to mouse or human FcγRs by surface plasmon resonance analyses. The effector functions of the mutant are completely eliminated when measured in antibody-dependent cell-meditated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays. In vivo, the NNAS mutant made on an antibody against a human lymphocyte antigen does not deplete T cells or B cells in transgenic mice, in contrast to wild-type antibody. Structural study confirms the successful glycosylation switch to the engineered Asn298 site. The engineered glycosylation would clash with approaching FcγRs based on reported Fc-FcγR co-crystal structures. In addition, the NNAS mutants of multiple antibodies retain binding to antigens and neonatal Fc receptor, exhibit comparable purification yields and thermal stability, and display normal circulation half-life in mice and non-human primate. Our work provides a novel approach for generating therapeutic antibodies devoid of any ADCC and CDC activities with potentially lower immunogenicity.

| S-EPMC7531572 | biostudies-literature

Fc-engineered antibodies with immune effector functions completely abolished.

Project description:Elimination of the binding of immunoglobulin Fc to Fc gamma receptors (FcγR) is highly desirable for the avoidance of unwanted inflammatory responses to therapeutic antibodies and fusion proteins. Many different approaches have been described in the literature but none of them completely eliminates binding to all of the Fcγ receptors. Here we describe a set of novel variants having specific amino acid substitutions in the Fc region at L234 and L235 combined with the substitution G236R. They show no detectable binding to Fcγ receptors or to C1q, are inactive in functional cell-based assays and do not elicit inflammatory cytokine responses. Meanwhile, binding to FcRn, manufacturability, stability and potential for immunogenicity are unaffected. These variants have the potential to improve the safety and efficacy of therapeutic antibodies and Fc fusion proteins.

| S-EPMC8691596 | biostudies-literature

Neutralization of SARS-CoV-2 pseudovirus using ACE2-engineered extracellular vesicles.

Project description:The spread of coronavirus disease 2019 (COVID-19) throughout the world has resulted in stressful healthcare burdens and global health crises. Developing an effective measure to protect people from infection is an urgent need. The blockage of interaction between angiotensin-converting enzyme 2 (ACE2) and S protein is considered an essential target for anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) drugs. A full-length ACE2 protein could be a potential drug to block early entry of SARS-CoV-2 into host cells. In this study, a therapeutic strategy was developed by using extracellular vesicles (EVs) with decoy receptor ACE2 for neutralization of SARS-CoV-2. The EVs embedded with engineered ACE2 (EVs-ACE2) were prepared; the EVs-ACE2 were derived from an engineered cell line with stable ACE2 expression. The potential effect of the EVs-ACE2 on anti-SARS-CoV-2 was demonstrated by both in vitro and in vivo neutralization experiments using the pseudovirus with the S protein (S-pseudovirus). EVs-ACE2 can inhibit the infection of S-pseudovirus in various cells, and importantly, the mice treated with intranasal administration of EVs-ACE2 can suppress the entry of S-pseudovirus into the mucosal epithelium. Therefore, the intranasal EVs-ACE2 could be a preventive medicine to protect from SARS-CoV-2 infection. This EVs-based strategy offers a potential route to COVID-19 drug development.

| S-EPMC8427979 | biostudies-literature

In planta deglycosylation improves the SARS-CoV-2 neutralization activity of recombinant ACE2-Fc

Project description:The ectodomain of human ACE2-Fc was transiently expressed in leaves of Nicotiana benthamiana plants and protein A and SEC purified. SEC-purified ACE2-Fc was sequentially digested with chymotrypsin and trypsin and the glycopeptides were subjected to LC-ESI-MS analysis using the LC-OTMS, Orbitrap Exploris 480 from Thermo Scientific.

2023-04-18 | PXD041320 | Pride

Fc-Engineered Antibodies with Enhanced Fc-Effector Function for the Treatment of B-Cell Malignancies.

Project description:Monoclonal antibody (mAb) therapy has rapidly changed the field of cancer therapy. In 1997, the CD20-targeting mAb rituximab was the first mAb to be approved by the U.S. Food and Drug Administration (FDA) for treatment of cancer. Within two decades, dozens of mAbs entered the clinic for treatment of several hematological cancers and solid tumors, and numerous more are under clinical investigation. The success of mAbs as cancer therapeutics lies in their ability to induce various cytotoxic machineries against specific targets. These cytotoxic machineries include antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC), which are all mediated via the fragment crystallizable (Fc) domain of mAbs. In this review article, we will outline the novel approaches of engineering these Fc domains of mAbs to enhance their Fc-effector function and thereby their anti-tumor potency, with specific focus to summarize their (pre-) clinical status for the treatment of B-cell malignancies, including chronic lymphocytic leukemia (CLL), B-cell non-Hodgkin lymphoma (B-NHL), and multiple myeloma (MM).

| S-EPMC7603375 | biostudies-literature

Structural characterization of a human Fc fragment engineered for lack of effector functions.

Project description:The first three-dimensional structure of a human Fc fragment genetically engineered for the elimination of its ability to mediate antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity is reported. When introduced into the lower hinge and CH2 domain of human IgG1 molecules, the triple mutation L234F/L235E/P331S ('TM') causes a profound decrease in their binding to human CD64, CD32A, CD16 and C1q. Enzymatically produced Fc/TM fragment was crystallized and its structure was solved at a resolution of 2.3 A using molecular replacement. This study revealed that the three-dimensional structure of Fc/TM is very similar to those of other human Fc fragments in the experimentally visible region spanning residues 236-445. Thus, the dramatic broad-ranging effects of TM on IgG binding to several effector molecules cannot be explained in terms of major structural rearrangements in this portion of the Fc.

| S-EPMC2467532 | biostudies-literature

IgG3 enhances neutralization potency and Fc effector function of an HIV V2-specific broadly neutralizing antibody.

Project description:Broadly neutralizing antibodies (bNAbs) protect against HIV infection in non-human primates and their efficacy may be enhanced through interaction with Fc receptors on immune cells. Antibody isotype is a modulator of this binding with the IgG3 subclass mediating potent Fc effector function and is associated with HIV vaccine efficacy and HIV control. BNAb functions are typically assessed independently of the constant region with which they are naturally expressed. To examine the role of natural isotype in the context of a bNAb lineage we studied CAP256, an HIV-infected individual that mounted a potent V2-specific bNAb response. CAP256 expressed persistently high levels of plasma IgG3 which we found mediated both broad neutralizing activity and potent Fc function. Sequencing of germline DNA and the constant regions of V2-directed bNAbs from this donor revealed the expression of a novel IGHG3 allele as well as IGHG3*17, an allele that produces IgG3 antibodies with increased plasma half-life. Both allelic variants were used to generate CAP256-VRC26.25 and CAP256-VRC26.29 IgG3 bNAbs and these were compared to IgG1 versions. IgG3 variants were shown to have significantly higher phagocytosis and trogocytosis compared to IgG1 versions, which corresponded to increased affinity for FcγRIIa. Neutralization potency was also significantly higher for IgG3 bNAbs, particularly against viruses lacking the N160 glycan. By exchanging hinge regions between subclass variants, we showed that hinge length modulated both neutralization potency and Fc function. This study showed that co-operation between the variable and natural IgG3 constant regions enhanced the polyfunctionality of antibodies, indicating the value of leveraging genetic variation which could be exploited for passive immunity.

| S-EPMC6936867 | biostudies-literature

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