Granzyme B Deficiency Protects against Angiotensin II-Induced Cardiac Fibrosis.

Cardiac fibrosis is observed across diverse etiologies of heart failure. Granzyme B (GzmB) is a serine protease involved in cell-mediated cytotoxicity in conjunction with the pore-forming protein, perforin. Recent evidence suggests that GzmB also contributes to matrix remodeling and fibrosis through an extracellular, perforin-independent process. However, the role of GzmB in the onset and progression of cardiac fibrosis remains elusive. The present study investigated the role of GzmB in the pathogenesis of cardiac fibrosis. GzmB was elevated in fibrotic human hearts and in angiotensin II-induced murine cardiac fibrosis. Genetic deficiency of GzmB reduced angiotensin II-induced cardiac hypertrophy and fibrosis, independently of perforin. GzmB deficiency also reduced microhemorrhage, inflammation, and fibroblast accumulation in vivo. In vitro, GzmB cleaved the endothelial junction protein, vascular endothelial (VE)-cadherin, resulting in the disruption of endothelial barrier function. Together, these results suggest a perforin-independent, extracellular role for GzmB in the pathogenesis of cardiac fibrosis.

Granzyme B inhibits keratinocyte migration by disrupting epidermal growth factor receptor (EGFR)-mediated signaling.

Chronic non-healing wounds including diabetic, venous, and decubitus skin ulcers are currently lacking effective therapies. Non-healing diabetic ulcers can lead to amputations as progress into a highly chronic state before detection and existing treatments for these wounds often fail. Granzyme B (GzmB) is a serine protease that was, until recently, believed to function exclusively in cytotoxic lymphocyte-mediated apoptosis. However, during excessive or chronic inflammation, GzmB can accumulate in the extracellular milieu, retain its activity, and cleave a number of important extracellular proteins. Epidermal growth factor receptor (EGFR) is a transmembrane receptor involved in cellular processes such as proliferation and migration. EGFR signaling is integral to the wound healing process. The present study investigated the effects of GzmB on keratinocyte cell migration using HaCaT cell line. Using electric cell-substrate impedance sensing and scratch assays, the present study demonstrates that GzmB inhibits keratinocyte migration by interfering with the EGFR pathway. GzmB limited cell transition into a migratory morphology and was found to reduce ligand-induced EGFR phosphorylation. Inhibition of GzmB reversed the aforementioned effects. In summary, data from the present study suggest key role for GzmB in the pathogenesis of impaired wound healing through the impairment of EGFR signaling and cell migration.

Olfactory ensheathing cells promote corticospinal axonal outgrowth by a L1 CAM-dependent mechanism.

Olfactory ensheathing cells (OECs) support the ability of the olfactory neuraxis to continually retarget within the mature central nervous system. This has led many groups to transplant OECS into the lesioned rodent spinal cord (SCd) in vivo, with variable degrees of anatomical, physiological, and behavioral success. Some of the most conflicting results in OEC transplantation have come from the corticospinal tract (CST) which has shown a relatively poor regeneration response. Although spinal neurite sprouting occurs in response to OECs in vivo and in vitro, we do not know if OECs possess the molecular machinery to stimulate outgrowth of functionally important motor tracts like the CST. Here, we assay cultured postnatal day 8 mouse CST neurons expressing yellow fluorescent protein (YFP) for their ability to extend axons and dendrites in response to different glia, and show that CST axons elongate in response to proteins in OEC plasma membrane (PM). In contrast, CST dendritic branching preferentially occurs in response to factors secreted by both OECs and astrocytes. We identify the L1-neural cell adhesion molecule (L1-NCAM) as a major component of OEC-induced corticospinal axon elongation, and have determined that OEC PM factors (including L1), can stimulate CST outgrowth even when inhibition is induced by myelin associated glycoprotein. Together, these results suggest that in the right context, OEC-derived PM factors could enhance CST axonal regeneration, and potentially contribute to approaches to ameliorate recovery from SCd injury.

LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization from coronavirus disease 2019 (COVID-19) when administered early. However, SARS-CoV-2 variants of concern (VOCs) have negatively affected therapeutic use of some authorized mAbs. Using a high-throughput B cell screening pipeline, we isolated LY-CoV1404 (bebtelovimab), a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody. LY-CoV1404 potently neutralizes authentic SARS-CoV-2, B.1.1.7, B.1.351, and B.1.617.2. In pseudovirus neutralization studies, LY-CoV1404 potently neutralizes variants, including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved, except for N439 and N501. The binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The broad and potent neutralization activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants.

LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants.

SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization when administered early during COVID-19 disease. However, the emergence of variants of concern has negatively impacted the therapeutic use of some authorized mAbs. Using a high throughput B-cell screening pipeline, we isolated a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody called LY-CoV1404 (also known as bebtelovimab). LY-CoV1404 potently neutralizes authentic SARS-CoV-2 virus, including the prototype, B.1.1.7, B.1.351 and B.1.617.2). In pseudovirus neutralization studies, LY-CoV1404 retains potent neutralizing activity against numerous variants including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant and retains binding to spike proteins with a variety of underlying RBD mutations including K417N, L452R, E484K, and N501Y. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved with the exception of N439 and N501. Notably, the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The breadth of reactivity to amino acid substitutions present among current VOC together with broad and potent neutralizing activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic agent to treat all known variants causing COVID-19. In Brief: LY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated. Highlights: LY-CoV1404 potently neutralizes SARS-CoV-2 authentic virus and known variants of concern including the B.1.1.529 (Omicron), the BA.2 Omicron subvariant, and B.1.617.2 (Delta) variantsNo loss of potency against currently circulating variantsBinding epitope on RBD of SARS-CoV-2 is rarely mutated in GISAID databaseBreadth of neutralizing activity and potency supports clinical development.

Stabilized coronavirus spike stem elicits a broadly protective antibody.

Current coronavirus (CoV) vaccines primarily target immunodominant epitopes in the S1 subunit, which are poorly conserved and susceptible to escape mutations, thus threatening vaccine efficacy. Here, we use structure-guided protein engineering to remove the S1 subunit from the Middle East respiratory syndrome (MERS)-CoV spike (S) glycoprotein and develop stabilized stem (SS) antigens. Vaccination with MERS SS elicits cross-reactive ß-CoV antibody responses and protects mice against lethal MERS-CoV challenge. High-throughput screening of antibody-secreting cells from MERS SS-immunized mice led to the discovery of a panel of cross-reactive monoclonal antibodies. Among them, antibody IgG22 binds with high affinity to both MERS-CoV and severe acute respiratory syndrome (SARS)-CoV-2 S proteins, and a combination of electron microscopy and crystal structures localizes the epitope to a conserved coiled-coil region in the S2 subunit. Passive transfer of IgG22 protects mice against both MERS-CoV and SARS-CoV-2 challenge. Collectively, these results provide a proof of principle for cross-reactive CoV antibodies and inform the development of pan-CoV vaccines and therapeutic antibodies.

The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) poses a public health threat for which preventive and therapeutic agents are urgently needed. Neutralizing antibodies are a key class of therapeutics that may bridge widespread vaccination campaigns and offer a treatment solution in populations less responsive to vaccination. Here, we report that high-throughput microfluidic screening of antigen-specific B cells led to the identification of LY-CoV555 (also known as bamlanivimab), a potent anti-spike neutralizing antibody from a hospitalized, convalescent patient with coronavirus disease 2019 (COVID-19). Biochemical, structural, and functional characterization of LY-CoV555 revealed high-affinity binding to the receptor-binding domain, angiotensin-converting enzyme 2 binding inhibition, and potent neutralizing activity. A pharmacokinetic study of LY-CoV555 conducted in cynomolgus monkeys demonstrated a mean half-life of 13 days and a clearance of 0.22 ml hour-1 kg-1, consistent with a typical human therapeutic antibody. In a rhesus macaque challenge model, prophylactic doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract in samples collected through study day 6 after viral inoculation. This antibody has entered clinical testing and is being evaluated across a spectrum of COVID-19 indications, including prevention and treatment.

LY-CoV555, a rapidly isolated potent neutralizing antibody, provides protection in a non-human primate model of SARS-CoV-2 infection.

SARS-CoV-2 poses a public health threat for which therapeutic agents are urgently needed. Herein, we report that high-throughput microfluidic screening of antigen-specific B-cells led to the identification of LY-CoV555, a potent anti-spike neutralizing antibody from a convalescent COVID-19 patient. Biochemical, structural, and functional characterization revealed high-affinity binding to the receptor-binding domain, ACE2 binding inhibition, and potent neutralizing activity. In a rhesus macaque challenge model, prophylaxis doses as low as 2.5 mg/kg reduced viral replication in the upper and lower respiratory tract. These data demonstrate that high-throughput screening can lead to the identification of a potent antiviral antibody that protects against SARS-CoV-2 infection. ONE SENTENCE SUMMARY: LY-CoV555, an anti-spike antibody derived from a convalescent COVID-19 patient, potently neutralizes SARS-CoV-2 and protects the upper and lower airways of non-human primates against SARS-CoV-2 infection.

Culturing olfactory ensheathing cells from the mouse olfactory epithelium.

Olfactory ensheathing cells (OECs) are not a class of stem cell, but they are a specialized and highly plastic glial cell that can continuously support the neurogenesis and axonal regeneration of olfactory receptor neurons. Because of this, they have been transplanted into sites of spinal cord injury to test their efficacy in promoting repair. They also have been demonstrated to have some ability to support the remyelination of demyelinated axons. Although the majority of these transplantation studies have used OECs prepared from the olfactory bulb (OB-OECs), OECs also can be prepared from the olfactory mucosa, and they are thus a candidate peripherally accessible population of glia that may be effective in promoting repair in a variety of central nervous system lesions. This protocol is designed to produce a highly enriched population of OECs from the lamina propria (LP) of the olfactory mucosa (LP-OECs), which are antigenically similar to OB-OECs and bear some phenotypic similarities to embryonic Schwann cells, but may demonstrate some distinct functional differences.

Topical small molecule granzyme B inhibitor improves remodeling in a murine model of impaired burn wound healing.

Granzyme B (GzmB) is a serine protease that has long been thought to function exclusively in lymphocyte-mediated apoptosis. In recent years, this paradigm has been revisited due to the recognition that GzmB accumulates in the extracellular milieu in many autoimmune and chronic inflammatory disorders, and contributes to impaired tissue remodeling due to the cleavage of extracellular matrix proteins. Knockout studies suggest that GzmB-mediated cleavage of decorin (DCN) contributes to impaired collagen fibrillogenesis and remodeling. As DCN is anti-fibrotic and contributes to reduced hypertrophic scarring, GzmB-induced DCN cleavage could play a role in wound healing following burn injury. In the present study, a novel, gel-formulated, first-in-class small-molecule inhibitor of GzmB, VTI-1002, was assessed in a murine model of impaired, diabetic burn wound healing. VTI-1002 exhibited high specificity, potency, and target selectivity. Gel-formulated VTI-1002 was able to penetrate the stratum corneum and was retained in the skin with minimal systemic absorption. Daily topical administration of VTI-1002 gel for 30 days following thermal injury showed significantly accelerated wound closure, increased DCN protein levels, and collagen organization that was translated into significantly increased wound tensile strength compared to controls. Overall, VTI-1002 gel was well-tolerated in vivo and no adverse events were observed. Topical application of VTI-1002 represents a novel therapeutic approach for the treatment of cutaneous burn wounds.