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The coronavirus disease 2019 (COVID-19) pandemic is the result of widespread infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Compared to other highly transmissible viruses, SARS-CoV-2 is associated with high rates of morbidity and mortality, and it represents an unprecedented challenge to global public health [1]. Most people infected with SARS-CoV-2 experience mild to moderate respiratory illness similar to influenza or other virus infections, with symptoms such as fever, dry cough, and dyspnea. However, a considerable number of infected people develop pneumonia and acute lung injury or acute respiratory distress syndrome (ARDS); these conditions are closely associated with the relatively high mortality rate of COVID-19 [2]. Some patients also exhibit pulmonary alveolitis, bronchiolitis, accumulation of mucus and edema fluid, and different degrees of inflammation marked by infiltration of various immune cells into the pulmonary interstitium [3, 4].
In order to screen Ab candidates for neutralizing capability in vitro, most groups test Abs against authentic living SARS-CoV-2, while some use pseudovirus with reporter readouts. A few methods have been used to quantify inhibitory concentrations, such as plaque reduction neutralization test (PRNT), focus reduction neutralization assay (FRNT), cytopathic effect (CPE), luciferase luminescence quantification, immunofluorescence assay (IFA), and virus mRNA quantification by quantitative polymerase change reaction (qPCR). The use of such a wide variety of in vitro assay methods makes it difficult to directly compare Abs from different publications (Table 1). To bring nAbs one step closer to clinical trials, a handful of publications also include data from in vivo animal models, which demonstrate the efficacy of the Ab as a treatment or prophylactic agent. Mice are not affected by SARS-CoV-2, presumably due to differences in ACE2 amino acid sequence compared to humans. Hence, mouse models for testing SARS-CoV-2 neutralizing capability must be generated by introducing human ACE2 into the lung cells of mice, either by the use of transgenic methods or by infecting normal mice with adenovirus encoding the human ACE2 gene for transient expression. As an alternative to mice, Shi et al. performed animal experiments in a rhesus macaque model; in this model, nAbs administered in both protection and treatment contexts caused clear reductions in viral load and lung damage [51]. Moreover, hamsters develop severe and easily observed signs of illness after infection with SARS-CoV-2, including rapid weight loss, a very high viral load in the lungs, and severe lung pathology [52]. Therefore, hamsters have become a commonly used model to evaluate the prophylactic and therapeutic efficacy of Abs (Table 1).
REGN-COV2 is a cocktail of the human Abs, casirivimab and imdevimab (formerly known as REGN10933 and REGN10987, respectively), which both target the S protein RBD but were identified by different methods [41]. Casirivimab was identified from VelocImmune hAb transgenic mice immunized with a DNA plasmid encoding SARS-CoV-2 S protein, followed by a booster of injected recombinant S protein. Meanwhile, imdevimab was identified from isolated PBMCs of three human donors previously infected with SARS-CoV-2. In both cases, the murine or human single B cells bound to S protein were sorted by FACS. The Kd values of casirivimab and imdevimab for S protein are both about 0.04 nM by measurement with Biacore T200. The PRNT50 of casirivimab and imdevimab are 0.0374 and 0.0421 nM, respectively. Novel S gene mutants rapidly appeared when virus was passaged in the presence of individual Abs, resulting in loss of neutralization. However, treatment of casirivimab and imdevimab together can prevent the selection of escape mutants in vitro since they comprise a non-competing Ab cocktail [81]. In vivo efficacy of this Ab cocktail has been evaluated in both rhesus macaques (used to model mild disease) and golden hamsters (model for more severe disease) [82]. In the rhesus macaques, REGN-COV2 greatly reduced virus load in the lower and upper airways and decreased virus-induced pathological sequelae when administered prophylactically (50 mg/kg dosage) or therapeutically (25 mg/kg dosage). Administration in hamsters (5 mg/kg dosage) inhibited weight loss and reduced viral titers in the lung.
Etesevimab (CB6, JS016, LY-CoV016) was identified by screening single B cells from a convalescent patient [51]. X-ray crystallography revealed that its epitope on SARS-CoV-2 RBD largely overlaps with ACE2 binding residues. To reduce the potential risk of an Ab-dependent enhancement (ADE) [93] and effector functions, the Fc of etesevimab was modified by two leucine-to-alanine substitutions at residues 234 and 235 (known as the LALA mutation), which abolished its affinity for the Fcγ receptor. In rhesus monkey models, treatment with etesevimab inhibited viral titers and reduced lung damage under both prophylactic and therapeutic usages. Etesevimab has been evaluated in a completed phase I clinical trial (NCT04441931) and a phase II/III study in combination with bamlanivimab (NCT04427501).
CTCs can also be used in vivo for the generation of patient-derived tumor models that assist in treatment. It has been shown that BC xenografts composed of luminal BC CTCs contain MICs that are shown to induce metastasis of bone, liver, and lungs in mice [83]. The study revealed a correlation between CTC surface markers like EPCAM+, CD44+, CD47+and MET+ve, with an increase in the number of metastatic sites and reduced survival rates, thus providing data for the development of better diagnostic tools for the treatment of metastatic BC [83]. Similar studies on LC CTC-derived xenografts have provided better insights into therapeutic drug trials, disease prognosis, and resistance mechanisms [84]. To overcome the problem of low numbers of CTCs, studies have also focused on generating continuous cell lines from CTCs. For example, the CL cell line CTC-MCC-41 established from patients has been shown to have a stable phenotype sharing properties with its primary tumors, thus allowing functional studies and both in vivo and in vitro drug therapeutic trials to be carried out [85]. 2ff7e9595c
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