Although SARS-CoV-2-neutralizing antibodies are promising therapeutics against COVID-19, little is known about their mechanism(s) of action or effective dosing windows. We report the generation and development of SC31, a potent SARS-CoV-2 neutralizing antibody, isolated from a convalescent patient. Antibody-mediated neutralization occurs via an epitope within the receptor-binding domain of the SARS-CoV-2 Spike protein. SC31 exhibited potent anti-SARS-CoV-2 activities in multiple animal models. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, treatment with SC31 greatly reduced viral loads and attenuated pro-inflammatory responses linked to the severity of COVID-19. Importantly, a comparison of the efficacies of SC31 and its Fc-null LALA variant revealed that the optimal therapeutic efficacy of SC31 requires Fc-mediated effector functions that promote IFNγ-driven anti-viral immune responses, in addition to its neutralization ability. A dose-dependent efficacy of SC31 was observed down to 5mg/kg when administered before viral-induced lung inflammatory responses. In addition, antibody-dependent enhancement was not observed even when infected mice were treated with SC31 at sub-therapeutic doses. In SARS-CoV-2-infected hamsters, SC31 treatment significantly prevented weight loss, reduced viral loads, and attenuated the histopathology of the lungs. In rhesus macaques, the therapeutic potential of SC31 was evidenced through the reduction of viral loads in both upper and lower respiratory tracts to undetectable levels. Together, the results of our preclinical studies demonstrated the therapeutic efficacy of SC31 in three different models and its potential as a COVID-19 therapeutic candidate.

SARS-CoV-2-neutralizing antibodies are promising therapeutics for COVID-19. However, little is known about the mechanisms of action of these antibodies or their effective dosing windows. We report the discovery and development of SC31, a potent SARS-CoV-2 neutralizing IgG1 antibody, originally isolated from a convalescent patient at day 27 after the onset of symptoms. Neutralization occurs via a binding epitope that maps within the ACE2 interface of the SARS-CoV-2 Spike protein, conserved across all common circulating SARS-CoV-2 mutants. In SARS-CoV-2 infected K18-human ACE2 transgenic mice, SC31 demonstrated potent survival benefit by dramatically reducing viral load concomitant with attenuated pro-inflammatory responses linked to severe systemic disease, such as IL-6. Comparison with a Fc-null LALA variant of SC31 demonstrated that optimal therapeutic efficacy of SC31 requires intact Fc-mediated effector functions that can further induce an IFNγ-driven anti-viral immune response. Dose-dependent efficacy for SC31 was observed down to 5mg/kg when dosed before the activation of lung inflammatory responses. Importantly, despite FcγR binding, no evidence of antibody dependent enhancement was observed with the Fc-competent SC31 even at sub-therapeutic doses. Therapeutic efficacy was confirmed in SARS-CoV-2-infected hamsters, where SC31 again significantly reduced viral load, decreased lung lesions and inhibited progression to severe disease manifestations. This study underlines the potential for significant COVID-19 patient benefit for the SC31 antibody that justifies rapid advancement to the clinic, as well as highlighting the importance of appropriate mechanistic and functional studies during development.

BackgroundDespite significant progress in cancer immunotherapy in recent years, resistance to existing immune checkpoint therapies (ICT) is common. V-domain Ig suppressor of T cell activation (VISTA), a predominantly myeloid immune checkpoint regulator, represents a promising therapeutic target due to its role in suppressing proinflammatory antitumor responses in myeloid-enriched tumor microenvironments. However, uncertainty around the cognate VISTA ligand has made the development of effective anti-VISTA antibodies challenging. The expression of VISTA on normal immune cell subtypes argues for a neutralizing non-depleting antibody, however, previous reported anti-VISTA antibodies use IgG1 Fc isotypes that deplete VISTA+ cells by antibody dependent cellular cytotoxicity/complement dependent cytotoxicity and these antibodies have shown fast serum clearance and immune toxicities.MethodHere we used a rational antibody discovery approach to develop the first Fc-independent anti-VISTA antibody, HMBD-002, that binds a computationally predicted functional epitope within the C-C-loop, distinct from other known anti-VISTA antibodies. This epitope is species-conserved allowing robust in vitro and in vivo testing of HMBD-002 in human and murine models of immune activation and cancer including humanized mouse models.ResultsWe demonstrate here that blockade by HMBD-002 inhibits VISTA binding to potential partners, including V-Set and Immunoglobulin domain containing 3, to reduce myeloid-derived suppression of T cell activity and prevent neutrophil migration. Analysis of immune cell milieu suggests that HMBD-002 treatment stimulates a proinflammatory phenotype characterized by a Th1/Th17 response, recapitulating a phenotype previously noted in VISTA knockout models. This mechanism of action is further supported by immune-competent syngenic and humanized mouse models of colorectal, breast and lung cancer where neutralizing VISTA, without depleting VISTA expressing cells, significantly inhibited tumor growth while decreasing infiltration of suppressive myeloid cells and increasing T cell activity. Finally, we did not observe either the fast serum clearance or immune toxicities that have been reported for IgG1 antibodies.ConclusionIn conclusion, we have shown that VISTA-induced immune suppression can be reversed by blockade of the functional C-C’ loop region of VISTA with a first-in-class rationally targeted and non-depleting IgG4 isotype anti-VISTA antibody, HMBD-002. This antibody represents a highly promising novel therapy in the VISTA-suppressed ICT non-responder population.

Obesity is a global chronic disease epidemic that is attributed to the abnormal accumulation of lipids in adipose tissue. Astaxanthin (AST) from Haematococcus pluvialis, a natural carotenoid, exhibited antioxidant, anti-lipogenic,...

Focal adhesion (FA) proteins, kindlin-2 and integrin-linked kinase (ILK), regulate cell adhesion and migration. ILK interacts with and promotes kindlin-2 targeting to FAs. Leu353 and Leu357 in kindlin-2 have been reported to be important for the interaction between kindlin-2 and ILK. However, the binding interface between kindlin-2 and ILK remains unclear. Using molecular modeling and molecular dynamics simulations, we show that Asp344, Asp352, and Thr356 in kindlin-2 and Arg243 and Arg334 in ILK kinase domain (KD) are important in kindlin-2/ILK complex formation. Mutations that disrupt these interactions abrogate kindlin-2 and ILK colocalization in HeLa cells. The interactions are direct based on data from pull-down assays using purified recombinant kindlin-2 F2-pleckstrin homology and ILK KDs. These data provide additional insights into the binding interface between kindlin-2 and ILK.