Low-dose in vivo protection and neutralization across SARS-CoV-2 variants by monoclonal antibody combinations

Prevention of viral escape and increased coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern require therapeutic monoclonal antibodies (mAbs) targeting multiple sites of vulnerability on the coronavirus spike glycoprotein. Here we identify several potent neutralizing antibodies directed against either the N-terminal domain (NTD) or the receptor-binding domain (RBD) of the spike protein. Administered in combinations, these mAbs provided low-dose protection against SARS-CoV-2 infection in the K18-human angiotensin-converting enzyme 2 mouse model, using both neutralization and Fc effector antibody functions. The RBD mAb WRAIR-2125, which targets residue F486 through a unique heavy-chain and light-chain pairing, demonstrated potent neutralizing activity against all major SARS-CoV-2 variants of concern. In combination with NTD and other RBD mAbs, WRAIR-2125 also prevented viral escape. These data demonstrate that NTD/RBD mAb combinations confer potent protection, likely leveraging complementary mechanisms of viral inactivation and clearance.

Protective mitochondrial fission induced by stress-responsive protein GJA1-20k

The Connexin43 gap junction gene GJA1 has one coding exon, but its mRNA undergoes internal translation to generate N-terminal truncated isoforms of Connexin43 with the predominant isoform being only 20 kDa in size (GJA1-20k). Endogenous GJA1-20k protein is not membrane bound and has been found to increase in response to ischemic stress, localize to mitochondria, and mimic ischemic preconditioning protection in the heart. However, it is not known how GJA1-20k benefits mitochondria to provide this protection. Here, using human cells and mice, we identify that GJA1-20k polymerizes actin around mitochondria which induces focal constriction sites. Mitochondrial fission events occur within about 45 s of GJA1-20k recruitment of actin. Interestingly, GJA1-20k mediated fission is independent of canonical Dynamin-Related Protein 1 (DRP1). We find that GJA1-20k-induced smaller mitochondria have decreased reactive oxygen species (ROS) generation and, in hearts, provide potent protection against ischemia-reperfusion injury. The results indicate that stress responsive internally translated GJA1-20k stabilizes polymerized actin filaments to stimulate non-canonical mitochondrial fission which limits ischemic-reperfusion induced myocardial infarction.

Multi-specific DARPin® therapeutics demonstrate very high potency against mutated SARS-CoV-2 variants in vitro

The SARS-CoV-2 virus responsible for the COVID-19 pandemic has so far infected more than 100 million people globally, and continues to undergo genomic evolution. Emerging SARS-CoV-2 variants show increased infectivity and may lead to resistance against immune responses of previously immunized individuals or existing therapeutics, especially antibody-based therapies.Several monoclonal antibody therapeutics authorized for emergency use or in development start to lose potency against various SARS-CoV-2 variants. Cocktails of two different monoclonal antibodies constitute a promising approach to protect against such variants as long as both antibodies are potent, but come with increased development complexity and therefore cost. As an alternative, we developed two multi-specific DARPin® therapeutics, each combining three independent DARPin® domains binding the SARS-CoV-2 spike protein in one molecule, to potently neutralize the virus and overcome virus escape.Here, we show in a panel of in vitro studies that both multi-specific DARPin® therapeutics, ensovibep (MP0420) and MP0423, are highly potent against the new circulating SARS-CoV-2 variants B.1.1.7 (UK variant) and B.1.351 (South African variant) and the most frequent emerging mutations in the spike protein. Additionally, viral passaging experiments show potent protection by ensovibep and MP0423 against development of escape mutations. Furthermore, we demonstrate that the cooperative binding of the individual modules in a multi-specific DARPin® antiviral is key for potent virus inhibition and protection from escape variants. These results, combined with the relatively small size and high production yields of DARPin® molecules, suggests ensovibep and MP0423 as superior alternatives to monoclonal antibody cocktails for global supply and demonstrate the strength of the DARPin® platform for achieving potent and lasting virus inhibition for SARS-CoV-2 and possibly other viruses.

Preventing cancer by long-term partial Myc suppression

Myc+/− haploinsufficient mice and mice with deleted Myc enhancers express reduced levels of the pleiotropic transcription factor Myc. Such mice are viable, with relatively mild pathologies, but show delay in onset of certain cancers. However, many phenotypes arising from germline gene perturbation are indirect consequences of adaptive developmental compensation and so do not translate to equivalent phenotypes when applied to adults. To ascertain whether systemic Myc hypomorphism also conferred cancer protection when acutely imposed in adults, and what the side-effects of this might be, we constructed a genetically engineered mouse model in which Myc expression may be systemically and reversibly hypomorphed at will. Acute imposition of Myc hypomorphism in adult mice conferred potent protection against both KRasG12D-driven lung and pancreatic cancers yet elicited only mild haematopoietic side effects. These side effects were completely suppressed by imposing Myc hypomorphism metronomically – a regimen that nonetheless retained potent cancer prophylaxis. Our data identify a key bottleneck at the transition from pre-malignant hyperplasia to overt tumour that is peculiarly reliant on levels of Myc that are higher than those required for most adult physiology, offering a possible window of opportunity for Myc inhibition in cancer prophylaxis.

Discovery and Development of Human SARS-CoV-2 Neutralizing Antibodies using an Unbiased Phage Display Library Approach

ABSTRACTSARS-CoV-2 neutralizing antibodies represent an important component of the ongoing search for effective treatment of and protection against COVID-19. We report here on the use of a naïve phage display antibody library to identify a panel of fully human SARS-CoV-2 neutralizing antibodies. Following functional profiling in vitro against an early pandemic isolate as well as a recently emerged isolate bearing the D614G Spike mutation, the clinical candidate antibody, STI-1499, and the affinity-engineered variant, STI-2020, were evaluated for in vivo efficacy in the Syrian golden hamster model of COVID-19. Both antibodies demonstrated potent protection against the pathogenic effects of the disease and a dose-dependent reduction of virus load in the lungs, reaching undetectable levels following a single dose of 500 micrograms of STI-2020. These data support continued development of these antibodies as therapeutics against COVID-19 and future use of this approach to address novel emerging pandemic disease threats.

Early precursors and molecular determinants of tissue-resident memory CD8+ T lymphocytes revealed by single-cell RNA sequencing

During an immune response to microbial infection, CD8+ T cells give rise to distinct classes of cellular progeny that coordinately mediate clearance of the pathogen and provide long-lasting protection against reinfection, including a subset of noncirculating tissue-resident memory (TRM) cells that mediate potent protection within nonlymphoid tissues. Here, we used single-cell RNA sequencing to examine the gene expression patterns of individual CD8+ T cells in the spleen and small intestine intraepithelial lymphocyte (siIEL) compartment throughout the course of their differentiation in response to viral infection. These analyses revealed previously unknown transcriptional heterogeneity within the siIEL CD8+ T cell population at several stages of differentiation, representing functionally distinct TRM cell subsets and a subset of TRM cell precursors within the tissue early in infection. Together, these findings may inform strategies to optimize CD8+ T cell responses to protect against microbial infection and cancer.

Neutralizing Antibodies Isolated by a site-directed Screening have Potent Protection on SARS-CoV-2 Infection

Neutralizing antibody is one of the most effective interventions for acute pathogenic infection. Currently, over three million people have been identified for SARS-CoV-2 infection but SARS-CoV-2-specific vaccines and neutralizing antibodies are still lacking. SARS-CoV-2 infects host cells by interacting with angiotensin converting enzyme-2 (ACE2) via the S1 receptor-binding domain (RBD) of its surface spike glycoprotein. Therefore, blocking the interaction of SARS-CoV-2-RBD and ACE2 by antibody would cause a directly neutralizing effect against virus. In the current study, we selected the ACE2 interface of SARS-CoV-2-RBD as the targeting epitope for neutralizing antibody screening. We performed site-directed screening by phage display and finally obtained one IgG antibody (4A3) and several domain antibodies. Among them, 4A3 and three domain antibodies (4A12, 4D5, and 4A10) were identified to act as neutralizing antibodies due to their capabilities to block the interaction between SARS-CoV-2-RBD and ACE2-positive cells. The domain antibody 4A12 was predicted to have the best accessibility to all three ACE2-interfaces on the spike homotrimer. Pseudovirus and authentic SARS-CoV-2 neutralization assays showed that all four antibodies could potently protect host cells from virus infection. Overall, we isolated multiple formats of SARS-CoV-2-neutralizing antibodies via site-directed antibody screening, which could be promising candidate drugs for the prevention and treatment of COVID-19.