Rapid discovery of diverse neutralizing SARS-CoV-2 antibodies from large-scale synthetic phage libraries.

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.

Functional GLP-1R antibodies identified from a synthetic GPCR-focused library demonstrate potent blood glucose control.

G protein-coupled receptors (GPCRs) are a group of seven-transmembrane receptor proteins that have proven to be successful drug targets. Antibodies are becoming an increasingly promising modality to target these receptors due to their unique properties, such as exquisite specificity, long half-life, and fewer side effects, and their improved pharmacokinetic and pharmacodynamic profiles compared to peptides and small molecules, which results from their more favorable biodistribution. To date, there are only two US Food and Drug Administration-approved GPCR antibody drugs, namely erenumab and mogamulizumab, and this highlights the challenges encountered in identifying functional antibodies against GPCRs. Utilizing Twist's precision DNA writing technologies, we have created a GPCR-focused phage display library with 1 × 1010 diversity. Specifically, we mined endogenous GPCR binding ligand and peptide sequences and incorporated these binding motifs into the heavy chain complementarity-determining region 3 in a synthetic antibody library. Glucagon-like peptide-1 receptor (GLP-1 R) is a class B GPCR that acts as the receptor for the incretin GLP-1, which is released to regulate insulin levels in response to food intake. GLP-1 R agonists have been widely used to increase insulin secretion to lower blood glucose levels for the treatment of type 1 and type 2 diabetes, whereas GLP-1 R antagonists have applications in the treatment of severe hypoglycemia associated with bariatric surgery and hyperinsulinomic hypoglycemia. Here we present the discovery and creation of both antagonistic and agonistic GLP-1 R antibodies by panning this GPCR-focused phage display library on a GLP-1 R-overexpressing Chinese hamster ovary cell line and demonstrate their in vitro and in vivo functional activity.

Anti-TNF modulation reduces myocardial inflammation and improves cardiovascular function in systemic rheumatic diseases.

BACKGROUND: Rheumatoid arthritis (RA), ankylosing spondylitis (AS) and psoriatic arthritis (PsA) are common disorders associated with increased rates of cardiovascular disease (CVD), but the contribution of cytokine-induced inflammation to impaired cardiovascular function in these conditions remains poorly understood. OBJECTIVES: We assessed the effect of anti-TNF therapy on myocardial and vascular function, myocardial tissue characteristics and perfusion in inflammatory arthropathy and systemic rheumatic disease (IASRD) patients, using cardiovascular magnetic resonance (CMR). METHODS: 20 RA patients, 7 AS patients, 5 PsA patients without previously known CVD scheduled to commence anti-TNF therapy and 8 RA patients on standard disease modifying antirheumatic drugs underwent CMR at 1.5 T, including cine, tagging, pulse wave velocity (PWV), T2-weighted, native and postcontrast T1 mapping, ECV quantification, rest and stress perfusion and late gadolinium enhancement (LGE) imaging. RESULTS: Following anti-TNF therapy, there was significant reversal of baseline subclinical cardiovascular dysfunction, as evidenced by improvement in peak systolic circumferential strain (p < 0.001), peak diastolic circumferential strain rate (p < 0.001), and total aortic PWV, (p < 0.001). This was accompanied by a reduction in myocardial inflammation, as assessed by T2-weighted imaging (p = 0.005), native T1 mapping (p = 0.009) and ECV quantification (p = 0.001), as well as in serum inflammatory markers like CRP (p < 0.001) and ESR (p < 0.001), and clinical measures of disease activity (DAS28-CRP, p = 0.001; BASDAI, p < 0.001). A trend towards improvement in myocardial perfusion was observed (p = 0.07). Focal myocardial fibrosis, as detected by LGE CMR was not altered by anti-TNF therapy (p = 0.92). CONCLUSIONS: Anti-TNF therapy reduces subclinical myocardial inflammation and improves cardiovascular function in RA, AS and PsA. CMR may be used to track disease progression and response to therapy. Future CMR-based studies to demonstrate effect of anti-TNF therapy modulation of vascular structure and function on hard clinical events and outcomes would be useful.

No Evidence of Myocardial Oxygen Deprivation in Nonischemic Heart Failure.

BACKGROUND: Whether the myocardium in nonischemic heart failure experiences oxygen limitation remains a long-standing controversy. We addressed this question in patients with dilated cardiomyopathy (DCM) using a dual approach. First, we tested the changes in myocardial oxygenation between rest and stress states, using oxygenation-sensitive cardiovascular magnetic resonance. Second, we sought to assess the functional consequences of oxygen limitation at rest by measuring myocardial energetics before and after short-term oxygen supplementation. METHODS AND RESULTS: Twenty-six subjects (14 DCM and 12 normal) underwent cardiac magnetic resonance imaging at 3 Tesla to assess cardiac volumes, function, oxygenation, and first-pass perfusion (0.03 mmol/kg Gd-DTPA bolus) at stress and rest (4-6 minutes IV adenosine, 140 µg/kg per minute). Signal intensity change (SIΔ) and myocardial perfusion reserve index (MPRI) were measured from oxygenation and perfusion images, respectively. Furthermore, the effect of oxygen supplementation on resting myocardial energy metabolism was tested using (31)P MR spectroscopy, measuring PCr/ATP ratios in both groups at baseline and after 4 hours of oxygen via facemask in the DCM group. During stress, there were equivalent rises in rate pressure product in both groups (DCM, 76±15% and normal, 79±9%; P=0.84). MPRI was significantly reduced in DCM (1.51±0.11 versus normal 1.86±0.10; P=0.03). However, there was no difference in oxygenation between groups: SIΔ in DCM 17±3% versus normal 20±2% (P=0.38). Furthermore, at a left ventricular segmental level, there was no correlation between oxygenation-sensitive SIΔ and MPRI (R=0.06; P=0.43). Resting PCr/ATP was reduced in DCM (1.66±0.07 versus normal 2.12±0.06; P=0.002). With oxygen supplementation, there was no change in PCr/ATP (1.61±0.08; P=0.58; Δ=0.04±0.05). There was also no effect of oxygen on systolic function (ejection fraction pre oxygen, 34±1%; post oxygen, 36±2%; P=0.46; Δ 2±1%). CONCLUSIONS: Our results demonstrate dissociation between microvascular dysfunction and oxygenation in DCM, suggesting that the impairment of perfusion is not sufficient to cause deoxygenation during stress. Cardiac energetics are unaffected by oxygen supplementation, indicating the absence of relevant myocardial hypoxia at rest. Our study suggests that novel treatments for nonischemic heart failure should focus on efforts to directly target cardiomyocyte function and metabolism rather than oxygen delivery and microvascular function.

Blunted myocardial oxygenation response during vasodilator stress in patients with hypertrophic cardiomyopathy.

OBJECTIVES: This study sought to assess myocardial perfusion and tissue oxygenation during vasodilator stress in patients with overt hypertrophic cardiomyopathy (HCM), as well as in HCM mutation carriers without left ventricular (LV) hypertrophy, and to compare findings to those in athletes with comparable hypertrophy and normal controls. BACKGROUND: Myocardial perfusion under vasodilator stress is impaired in patients with HCM. Whether this is associated with impaired myocardial oxygenation and tissue ischemia is unknown. Furthermore, it is not known whether perfusion and oxygenation are impaired in HCM mutation carriers without left ventricular hypertrophy (LVH). METHODS: A total of 27 patients with overt HCM, 10 HCM mutation carriers without LVH, 11 athletes, and 20 healthy controls underwent cardiovascular magnetic resonance (CMR) scanning at 3-T. Myocardial function, perfusion (perfusion reserve index [MPRI]), and oxygenation (blood-oxygen level dependent signal intensity [SI] change) under adenosine stress were assessed. RESULTS: MPRI was significantly reduced in HCM (1.3 ± 0.1) compared to controls (1.8 ± 0.1, p < 0.001) and athletes (2.0 ± 0.1, p < 0.001), but remained normal in HCM mutation carriers without LVH (1.7 ± 0.1; p = 0.61 vs. controls, p = 0.02 vs. overt HCM). Oxygenation response was attenuated in overt HCM (SI change 6.9 ± 1.4%) compared to controls (18.9 ± 1.4%, p < 0.0001) and athletes (18.7 ± 2.0%, p < 0.001). Interestingly, HCM mutation carriers without LVH also showed an impaired oxygenation response to adenosine (10.4 ± 2.0%; p = 0.001 vs. controls, p = 0.16 vs. overt HCM, p = 0.003 vs. athletes). CONCLUSIONS: In overt HCM, both perfusion and oxygenation are impaired during vasodilator stress. However, in HCM mutation carriers without LVH, only oxygenation is impaired. In athletes, stress perfusion and oxygenation are normal. CMR assessment of myocardial oxygenation has the potential to become a novel risk factor in HCM.