Intracellular domain of epithelial cell adhesion molecule induces Wnt receptor transcription to promote colorectal cancer progression.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) has been widely studied as a tumor antigen due to its expression in varieties of solid tumors. Moreover, the glycoprotein contributes to critical cancer-associated cellular functionalities via its extracellular (EpEX) and intracellular (EpICD) domains. In colorectal cancer (CRC), EpCAM has been implicated in the Wnt signaling pathway, as EpICD and ß-Catenin are coordinately translocated to the nucleus. Once in the nucleus, EpICD transcriptionally regulates EpCAM target genes that; however, remains unclear whether Wnt signaling is modulated by EpICD activity. METHODS: Patient-derived organoids (PDOs), patient-derived xenografts (PDXs), and various CRC cell lines were used to study the roles of EpCAM and EpICD in Wnt receptor expression. Fluorescence and confocal microscopy were used to analyze tumors isolated from PDX and other xenograft models as well as CRC cell lines. EpCAM signaling was intervened with our humanized form of EpCAM neutralizing antibody, hEpAb2-6. Wnt receptor promoters under luciferase reporters were constructed to examine the effects of EpICD. Luciferase reporter assays were performed to evaluate promoter, γ-secretase and Wnt activity. Functional assays including in vivo tumor formation, organoid formation, spheroid and colony formation experiments were performed to study Wnt related phenomena. The therapeutic potential of EpCAM suppression by hEpAb2-6 was evaluated in xenograft and orthotopic models of human CRC. RESULTS: EpICD interacted with the promoters of Wnt receptors (FZD6 and LRP5/6) thus upregulated their transcriptional activity inducing Wnt signaling. Furthermore, activation of Wnt-pathway-associated kinases in the ß-Catenin destruction complex (GSK3ß and CK1) induced γ-secretase activity to augment EpICD shedding, establishing a positive-feedback loop. Our hEpAb2-6 antibody blocked EpICD-mediated upregulation of Wnt receptor expressions and conferred therapeutic benefits in both PDX and orthotopic models of human CRC. CONCLUSIONS: This study uncovers relevant functions of EpCAM where Wnt receptors are upregulated via the transcriptional co-factor activity of EpICD. The resultant enhancement of Wnt signaling induces γ-secretase activity further stimulating EpICD cleavage and its nuclear translocation. Our humanized anti-EpCAM antibody hEpAb2-6 blocks these mechanisms and may thereby provide therapeutic benefit in CRC.

Current landscape of mRNA technologies and delivery systems for new modality therapeutics.

Realizing the immense clinical potential of mRNA-based drugs will require continued development of methods to safely deliver the bioactive agents with high efficiency and without triggering side effects. In this regard, lipid nanoparticles have been successfully utilized to improve mRNA delivery and protect the cargo from extracellular degradation. Encapsulation in lipid nanoparticles was an essential factor in the successful clinical application of mRNA vaccines, which conclusively demonstrated the technology's potential to yield approved medicines. In this review, we begin by describing current advances in mRNA modifications, design of novel lipids and development of lipid nanoparticle components for mRNA-based drugs. Then, we summarize key points pertaining to preclinical and clinical development of mRNA therapeutics. Finally, we cover topics related to targeted delivery systems, including endosomal escape and targeting of immune cells, tumors and organs for use with mRNA vaccines and new treatment modalities for human diseases.

Development of AAV-delivered broadly neutralizing anti-human ACE2 antibodies against SARS-CoV-2 variants.

The ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in the emergence of new variants that are resistant to existing vaccines and therapeutic antibodies, has raised the need for novel strategies to combat the persistent global COVID-19 epidemic. In this study, a monoclonal anti-human angiotensin-converting enzyme 2 (hACE2) antibody, ch2H2, was isolated and humanized to block the viral receptor-binding domain (RBD) binding to hACE2, the major entry receptor of SARS-CoV-2. This antibody targets the RBD-binding site on the N terminus of hACE2 and has a high binding affinity to outcompete the RBD. In vitro, ch2H2 antibody showed potent inhibitory activity against multiple SARS-CoV-2 variants, including the most antigenically drifted and immune-evading variant Omicron. In vivo, adeno-associated virus (AAV)-mediated delivery enabled a sustained expression of monoclonal antibody (mAb) ch2H2, generating a high concentration of antibodies in mice. A single administration of AAV-delivered mAb ch2H2 significantly reduced viral RNA load and infectious virions and mitigated pulmonary pathological changes in mice challenged with SARS-CoV-2 Omicron BA.5 subvariant. Collectively, the results suggest that AAV-delivered hACE2-blocking antibody provides a promising approach for developing broad-spectrum antivirals against SARS-CoV-2 and potentially other hACE2-dependent pathogens that may emerge in the future.

Homogeneous antibody and CAR-T cells with improved effector functions targeting SSEA-4 glycan on pancreatic cancer.

Pancreatic cancer is usually asymptomatic in the early stages; the 5-y survival rate is around 9%; and there is a lack of effective treatment. Here we show that SSEA-4 is more expressed in all pancreatic cancer cell lines examined but not detectable in normal pancreatic cells; and high expression of SSEA-4 or the key enzymes B3GALT5 + ST3GAL2 associated with SSEA-4 biosynthesis significantly lowers the overall survival rate. To evaluate potential new treatments for pancreatic cancer, homogeneous antibodies with a well-defined Fc glycan for optimal effector functions and CAR-T cells with scFv construct designed to target SSEA-4 were shown highly effective against pancreatic cancer in vitro and in vivo. This was further supported by the finding that a subpopulation of natural killer (NK) cells isolated by the homogeneous antibody exhibited enhancement in cancer-cell killing activity compared to the unseparated NK cells. These results indicate that targeting SSEA-4 by homologous antibodies or CAR-T strategies can effectively inhibit cancer growth, suggesting SSEA-4 as a potential immunotherapy target for treating pancreatic disease.

Epithelial cell adhesion molecule (EpCAM) regulates HGFR signaling to promote colon cancer progression and metastasis.

BACKGROUND: Epithelial cell adhesion molecule (EpCAM) is known to highly expression and promotes cancer progression in many cancer types, including colorectal cancer. While metastasis is one of the main causes of cancer treatment failure, the involvement of EpCAM signaling in metastatic processes is unclear. We propose the potential crosstalk of EpCAM signaling with the HGFR signaling in order to govern metastatic activity in colorectal cancer. METHODS: Immunoprecipitation (IP), enzyme-linked immunosorbent assay (ELISA), and fluorescence resonance energy transfer (FRET) was conducted to explore the extracellular domain of EpCAM (EpEX) and HGFR interaction. Western blotting was taken to determine the expression of proteins in colorectal cancer (CRC) cell lines. The functions of EpEX in CRC were investigated by proliferation, migration, and invasion analysis. The combined therapy was validated via a tail vein injection method for the metastasis and orthotopic colon cancer models. RESULTS: This study demonstrates that the EpEX binds to HGFR and induces downstream signaling in colon cancer cells. Moreover, EpEX and HGF cooperatively mediate HGFR signaling. Furthermore, EpEX enhances the epithelial-to-mesenchymal transition and metastatic potential of colon cancer cells by activating ERK and FAK-AKT signaling pathways, and it further stabilizes active ß-catenin and Snail proteins by decreasing GSK3ß activity. Finally, we show that the combined treatment of an anti-EpCAM neutralizing antibody (EpAb2-6) and an HGFR inhibitor (crizotinib) significantly inhibits tumor progression and prolongs survival in metastatic and orthotopic animal models of colon cancer. CONCLUSION: Our findings illuminate the molecular mechanisms underlying EpCAM signaling promotion of colon cancer metastasis, further suggesting that the combination of EpAb2-6 and crizotinib may be an effective strategy for treating cancer patients with high EpCAM expression.

Structure-guided antibody cocktail for prevention and treatment of COVID-19.

Development of effective therapeutics for mitigating the COVID-19 pandemic is a pressing global need. Neutralizing antibodies are known to be effective antivirals, as they can be rapidly deployed to prevent disease progression and can accelerate patient recovery without the need for fully developed host immunity. Here, we report the generation and characterization of a series of chimeric antibodies against the receptor-binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Some of these antibodies exhibit exceptionally potent neutralization activities in vitro and in vivo, and the most potent of our antibodies target three distinct non-overlapping epitopes within the RBD. Cryo-electron microscopy analyses of two highly potent antibodies in complex with the SARS-CoV-2 spike protein suggested they may be particularly useful when combined in a cocktail therapy. The efficacy of this antibody cocktail was confirmed in SARS-CoV-2-infected mouse and hamster models as prophylactic and post-infection treatments. With the emergence of more contagious variants of SARS-CoV-2, cocktail antibody therapies hold great promise to control disease and prevent drug resistance.

Rapid generation of mouse model for emerging infectious disease with the case of severe COVID-19.

Since the pandemic of COVID-19 has intensely struck human society, small animal model for this infectious disease is in urgent need for basic and pharmaceutical research. Although several COVID-19 animal models have been identified, many of them show either minimal or inadequate pathophysiology after SARS-CoV-2 challenge. Here, we describe a new and versatile strategy to rapidly establish a mouse model for emerging infectious diseases in one month by multi-route, multi-serotype transduction with recombinant adeno-associated virus (AAV) vectors expressing viral receptor. In this study, the proposed approach enables profound and enduring systemic expression of SARS-CoV-2-receptor hACE2 in wild-type mice and renders them vulnerable to SARS-CoV-2 infection. Upon virus challenge, generated AAV/hACE2 mice showed pathophysiology closely mimicking the patients with severe COVID-19. The efficacy of a novel therapeutic antibody cocktail RBD-chAbs for COVID-19 was tested and confirmed by using this AAV/hACE2 mouse model, further demonstrating its successful application in drug development.

Bivalent mRNA vaccine effectiveness against SARS-CoV-2 variants of concern.

BACKGROUND: Sequential infections with SARS-CoV-2 variants such as Alpha, Delta, Omicron and its sublineages may cause high morbidity, so it is necessary to develop vaccines that can protect against both wild-type (WT) virus and its variants. Mutations in SARS-CoV-2's spike protein can easily alter viral transmission and vaccination effectiveness. METHODS: In this study, we designed full-length spike mRNAs for WT, Alpha, Delta, and BA.5 variants and integrated each into monovalent or bivalent mRNA-lipid nanoparticle vaccines. A pseudovirus neutralization assay was conducted on immunized mouse sera in order to examine the neutralizing potential of each vaccine. RESULTS: Monovalent mRNA vaccines were only effective against the same type of virus. Interestingly, monovalent BA.5 vaccination could neutralize BF.7 and BQ.1.1. Moreover, WT, Alpha, Delta, BA.5, and BF.7 pseudoviruses were broadly neutralized by bivalent mRNA vaccinations, such as BA.5 + WT, BA.5 + Alpha, and BA.5 + Delta. In particular, BA.5 + WT exhibited high neutralization against most variants of concern (VOCs) in a pseudovirus neutralization assay. CONCLUSIONS: Our results show that combining two mRNA sequences may be an effective way to develop a broadly protective SARS-CoV-2 vaccine against a wide range of variant types. Importantly, we provide the optimal combination regimen and propose a strategy that may prove useful in combating future VOCs.

mRNA-based vaccines and therapeutics: an in-depth survey of current and upcoming clinical applications.

mRNA-based drugs have tremendous potential as clinical treatments, however, a major challenge in realizing this drug class will promise to develop methods for safely delivering the bioactive agents with high efficiency and without activating the immune system. With regard to mRNA vaccines, researchers have modified the mRNA structure to enhance its stability and promote systemic tolerance of antigenic presentation in non-inflammatory contexts. Still, delivery of naked modified mRNAs is inefficient and results in low levels of antigen protein production. As such, lipid nanoparticles have been utilized to improve delivery and protect the mRNA cargo from extracellular degradation. This advance was a major milestone in the development of mRNA vaccines and dispelled skepticism about the potential of this technology to yield clinically approved medicines. Following the resounding success of mRNA vaccines for COVID-19, many other mRNA-based drugs have been proposed for the treatment of a variety of diseases. This review begins with a discussion of mRNA modifications and delivery vehicles, as well as the factors that influence administration routes. Then, we summarize the potential applications of mRNA-based drugs and discuss further key points pertaining to preclinical and clinical development of mRNA drugs targeting a wide range of diseases. Finally, we discuss the latest market trends and future applications of mRNA-based drugs.

Broadly neutralizing human antibodies against Omicron subvariants of SARS-CoV-2.

BACKGROUND: The COVID-19 pandemic continues to pose a significant worldwide threat to human health, as emerging SARS-CoV-2 Omicron variants exhibit resistance to therapeutic antibodies and the ability to evade vaccination-induced antibodies. Here, we aimed to identify human antibodies (hAbs) from convalescent patients that are potent and broadly neutralizing toward Omicron sublineages. METHODS: Using a single B-cell cloning approach, we isolated BA.5 specific human antibodies. We further examined the neutralizing activities of the most promising neutralizing hAbs toward different variants of concern (VOCs) with pseudotyped virus. RESULTS: Sixteen hAbs showed strong neutralizing activities against Omicron BA.5 with low IC50 values (IC50 < 20 ng/mL). Among four of the most promising neutralizing hAbs (RBD-hAb-B22, -B23, -B25 and -B34), RBD-hAb-B22 exhibited the most potent and broad neutralization profiles across Omicron subvariant pseudoviruses, with low IC50 values (7.7-41.6 ng/mL) and a low PRNT50 value (3.8 ng/mL) in plaque assays with authentic BA.5. It also showed potent therapeutic effects in BA.5-infected K18-hACE2 mice. CONCLUSIONS: Thus, our efficient screening of BA.5-specific neutralizing hAbs from breakthrough infectious convalescent donors successfully yielded hAbs with potent therapeutic potential against multiple SARS-CoV-2 variants.

Reversal of pancreatic desmoplasia by a tumour stroma-targeted nitric oxide nanogel overcomes TRAIL resistance in pancreatic tumours.

OBJECTIVE: Stromal barriers, such as the abundant desmoplastic stroma that is characteristic of pancreatic ductal adenocarcinoma (PDAC), can block the delivery and decrease the tumour-penetrating ability of therapeutics such as tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), which can selectively induce cancer cell apoptosis. This study aimed to develop a TRAIL-based nanotherapy that not only eliminated the extracellular matrix barrier to increase TRAIL delivery into tumours but also blocked antiapoptotic mechanisms to overcome TRAIL resistance in PDAC. DESIGN: Nitric oxide (NO) plays a role in preventing tissue desmoplasia and could thus be delivered to disrupt the stromal barrier and improve TRAIL delivery in PDAC. We applied an in vitro-in vivo combinatorial phage display technique to identify novel peptide ligands to target the desmoplastic stroma in both murine and human orthotopic PDAC. We then constructed a stroma-targeted nanogel modified with phage display-identified tumour stroma-targeting peptides to co-deliver NO and TRAIL to PDAC and examined the anticancer effect in three-dimensional spheroid cultures in vitro and in orthotopic PDAC models in vivo. RESULTS: The delivery of NO to the PDAC tumour stroma resulted in reprogramming of activated pancreatic stellate cells, alleviation of tumour desmoplasia and downregulation of antiapoptotic BCL-2 protein expression, thereby facilitating tumour penetration by TRAIL and substantially enhancing the antitumour efficacy of TRAIL therapy. CONCLUSION: The co-delivery of TRAIL and NO by a stroma-targeted nanogel that remodels the fibrotic tumour microenvironment and suppresses tumour growth has the potential to be translated into a safe and promising treatment for PDAC.

Monoclonal antibodies against S2 subunit of spike protein exhibit broad reactivity toward SARS-CoV-2 variants.

BACKGROUND: The variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) harbor diverse spike (S) protein sequences, which can greatly influence the efficacies of therapeutics. Therefore, it would be of great value to develop neutralizing monoclonal antibodies (mAbs) that can broadly recognize multiple variants. METHODS: Using an mRNA-LNP immunization strategy, we generated several mAbs that specifically target the conserved S2 subunit of SARS-CoV-2 (B-S2-mAbs). These mAbs were assessed for their neutralizing activity with pseudotyped viruses and binding ability for SARS-CoV-2 variants. RESULTS: Among these mAbs, five exhibited strong neutralizing ability toward the Gamma variant and also recognized viral S proteins from the Wuhan, Alpha, Beta, Gamma, Delta and Omicron (BA.1, BA.2 and BA.5) variants. Furthermore, we demonstrated the broad reactivities of these B-S2-mAbs in several different applications, including immunosorbent, immunofluorescence and immunoblotting assays. In particular, B-S2-mAb-2 exhibited potent neutralization of Gamma variant (IC50 = 0.048 µg/ml) in a pseudovirus neutralization assay. The neutralizing epitope of B-S2-mAb-2 was identified by phage display as amino acid residues 1146-1152 (DSFKEEL) in the S2 subunit HR2 domain of SARS-CoV-2. CONCLUSION: Since there are not many mAbs that can bind the S2 subunit of SARS-CoV-2 variants, our set of B-S2-mAbs may provide important materials for basic research and potential clinical applications. Importantly, our study results demonstrate that the viral S2 subunit can be targeted for the production of cross-reactive antibodies, which may be used for coronavirus detection and neutralization.

Monoclonal antibodies for COVID-19 therapy and SARS-CoV-2 detection.

The coronavirus disease 2019 (COVID-19) pandemic is an exceptional public health crisis that demands the timely creation of new therapeutics and viral detection. Owing to their high specificity and reliability, monoclonal antibodies (mAbs) have emerged as powerful tools to treat and detect numerous diseases. Hence, many researchers have begun to urgently develop Ab-based kits for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ab drugs for use as COVID-19 therapeutic agents. The detailed structure of the SARS-CoV-2 spike protein is known, and since this protein is key for viral infection, its receptor-binding domain (RBD) has become a major target for therapeutic Ab development. Because SARS-CoV-2 is an RNA virus with a high mutation rate, especially under the selective pressure of aggressively deployed prophylactic vaccines and neutralizing Abs, the use of Ab cocktails is expected to be an important strategy for effective COVID-19 treatment. Moreover, SARS-CoV-2 infection may stimulate an overactive immune response, resulting in a cytokine storm that drives severe disease progression. Abs to combat cytokine storms have also been under intense development as treatments for COVID-19. In addition to their use as drugs, Abs are currently being utilized in SARS-CoV-2 detection tests, including antigen and immunoglobulin tests. Such Ab-based detection tests are crucial surveillance tools that can be used to prevent the spread of COVID-19. Herein, we highlight some key points regarding mAb-based detection tests and treatments for the COVID-19 pandemic.

A critical overview of current progress for COVID-19: development of vaccines, antiviral drugs, and therapeutic antibodies.

The novel coronavirus disease (COVID-19) pandemic remains a global public health crisis, presenting a broad range of challenges. To help address some of the main problems, the scientific community has designed vaccines, diagnostic tools and therapeutics for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The rapid pace of technology development, especially with regard to vaccines, represents a stunning and historic scientific achievement. Nevertheless, many challenges remain to be overcome, such as improving vaccine and drug treatment efficacies for emergent mutant strains of SARS-CoV-2. Outbreaks of more infectious variants continue to diminish the utility of available vaccines and drugs. Thus, the effectiveness of vaccines and drugs against the most current variants is a primary consideration in the continual analyses of clinical data that supports updated regulatory decisions. The first two vaccines granted Emergency Use Authorizations (EUAs), BNT162b2 and mRNA-1273, still show more than 60% protection efficacy against the most widespread current SARS-CoV-2 variant, Omicron. This variant carries more than 30 mutations in the spike protein, which has largely abrogated the neutralizing effects of therapeutic antibodies. Fortunately, some neutralizing antibodies and antiviral COVID-19 drugs treatments have shown continued clinical benefits. In this review, we provide a framework for understanding the ongoing development efforts for different types of vaccines and therapeutics, including small molecule and antibody drugs. The ripple effects of newly emergent variants, including updates to vaccines and drug repurposing efforts, are summarized. In addition, we summarize the clinical trials supporting the development and distribution of vaccines, small molecule drugs, and therapeutic antibodies with broad-spectrum activity against SARS-CoV-2 strains.

A booster dose of Delta × Omicron hybrid mRNA vaccine produced broadly neutralizing antibody against Omicron and other SARS-CoV-2 variants.

BACKGROUND: With the continuous emergence of new SARS-CoV-2 variants that feature increased transmission and immune escape, there is an urgent demand for a better vaccine design that will provide broader neutralizing efficacy. METHODS: We report an mRNA-based vaccine using an engineered "hybrid" receptor binding domain (RBD) that contains all 16 point-mutations shown in the currently prevailing Omicron and Delta variants. RESULTS: A booster dose of hybrid vaccine in mice previously immunized with wild-type RBD vaccine induced high titers of broadly neutralizing antibodies against all tested SARS-CoV-2 variants of concern (VOCs). In naïve mice, hybrid vaccine generated strong Omicron-specific neutralizing antibodies as well as low but significant titers against other VOCs. Hybrid vaccine also elicited CD8+/IFN-γ+ T cell responses against a conserved T cell epitope present in wild type and all VOCs. CONCLUSIONS: These results demonstrate that inclusion of different antigenic mutations from various SARS-CoV-2 variants is a feasible approach to develop cross-protective vaccines.

α-2,6-sialic acid/IgG anti-dsDNA ratios correlate with human lupus disease activity and possible mechanisms: A pilot study.

OBJECTIVE: To study the association of α2,6-sialic acid (SIA) content in serum IgG anti-dsDNA with human systemic lupus erythematosus disease activity index (SLEDAI) and the effect of sialylated and desialylated (deSIA) IgG anti-dsDNA on lupus B cells. METHODS: Blood from lupus patients was collected to determine the ratio of SIA in isolated IgG anti-dsDNA over serum IgG anti-dsDNA (SIA/IgG anti-dsDNA) ratios, which were plotted against SLEDAI using a receiver-operating-characteristics curve. Lupus B cells were cultured in vitro with chimeric sialylated IgG anti-dsDNA and its deSIA form. Culture supernatants were assayed for anti-inflammatory IL-10 and SIA/IgG anti-dsDNA ratios, which were compared among different pre-treatment groups using t-tests. RESULTS: The area-under-the-curve (AUC) for anti-dsDNA levels against SLEDAI was 0.791 positively (95% confidence interval [C.I.]: 0.699-0.884) and SIA/IgG anti-dsDNA ratios against SLEDAI yielded an AUC of 0.705 inversely (95% C.I: 0.601-0.809): not significantly different. SIA/IgG anti-dsDNA ratios discriminated significantly between patients without and patients with proteinuria (p = .046). SIA/IgG anti-dsDNA ratios correlated significantly and positively with serum C3c and C4 levels. Pre-treatment with IgG anti-dsDNA and its immune complexes (dsDNA/IgG anti-dsDNA IC) induced higher IL-10 from lupus B cells than medium pre-treatment (most p < .01 from day 2 to day 5 culture). DeSIA IgG anti-dsDNA IC induced lower IL-10 (p < .05) and lower SIA/IgG anti-dsDNA ratios (p < .001) from lupus B cells than medium and dsDNA pre-treatment. CONCLUSION: α2,6-SIA/IgG anti-dsDNA ratios inversely forecasted SLEDAI scores. Possible mechanisms may be due to the different effects of sialylated and deSIA IgG anti-dsDNA on lupus B cells in terms of IL-10 secretion and SIA/IgG anti-dsDNA ratios.

Antibody cocktail effective against variants of SARS-CoV-2.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an RNA virus with a high mutation rate. Importantly, several currently circulating SARS-CoV-2 variants are associated with loss of efficacy for both vaccines and neutralizing antibodies. METHODS: We analyzed the binding activity of six highly potent antibodies to the spike proteins of SARS-CoV-2 variants, assessed their neutralizing abilities with pseudovirus and authentic SARS-CoV-2 variants and evaluate efficacy of antibody cocktail in Delta SARS-CoV-2-infected hamster models as prophylactic and post-infection treatments. RESULTS: The tested RBD-chAbs, except RBD-chAb-25, maintained binding ability to spike proteins from SARS-CoV-2 variants. However, only RBD-chAb-45 and -51 retained neutralizing activities; RBD-chAb-1, -15, -25 and -28 exhibited diminished neutralization for all SARS-CoV-2 variants. Notably, several cocktails of our antibodies showed low IC50 values (3.35-27.06 ng/ml) against the SARS-CoV-2 variant pseudoviruses including United Kingdom variant B.1.1.7 (Alpha), South Africa variant B.1.351 (Beta), Brazil variant P1 (Gamma), California variant B.1.429 (Epsilon), New York variant B.1.526 (Iota), and India variants, B.1.617.1 (Kappa) and B.1.617.2 (Delta). RBD-chAb-45, and -51 showed PRNT50 values 4.93-37.54 ng/ml when used as single treatments or in combination with RBD-chAb-15 or -28, according to plaque assays with authentic Alpha, Gamma and Delta SARS-CoV-2 variants. Furthermore, the antibody cocktail of RBD-chAb-15 and -45 exhibited potent prophylactic and therapeutic effects in Delta SARS-CoV-2 variant-infected hamsters. CONCLUSIONS: The cocktail of RBD-chAbs exhibited potent neutralizing activities against SARS-CoV-2 variants. These antibody cocktails are highly promising candidate tools for controlling new SARS-CoV-2 variants, including Delta.

Identification of COVID-19 B-cell epitopes with phage-displayed peptide library.

BACKGROUND: Coronavirus disease 19 (COVID-19) first appeared in the city of Wuhan, in the Hubei province of China. Since its emergence, the COVID-19-causing virus, SARS-CoV-2, has been rapidly transmitted around the globe, overwhelming the medical care systems in many countries and leading to more than 3.3 million deaths. Identification of immunological epitopes on the virus would be highly useful for the development of diagnostic tools and vaccines that will be critical to limiting further spread of COVID-19. METHODS: To find disease-specific B-cell epitopes that correspond to or mimic natural epitopes, we used phage display technology to determine the targets of specific antibodies present in the sera of immune-responsive COVID-19 patients. Enzyme-linked immunosorbent assays were further applied to assess competitive antibody binding and serological detection. VaxiJen, BepiPred-2.0 and DiscoTope 2.0 were utilized for B-cell epitope prediction. PyMOL was used for protein structural analysis. RESULTS: 36 enriched peptides were identified by biopanning with antibodies from two COVID-19 patients; the peptides 4 motifs with consensus residues corresponding to two potential B-cell epitopes on SARS-CoV-2 viral proteins. The putative epitopes and hit peptides were then synthesized for validation by competitive antibody binding and serological detection. CONCLUSIONS: The identified B-cell epitopes on SARS-CoV-2 may aid investigations into COVID-19 pathogenesis and facilitate the development of epitope-based serological diagnostics and vaccines.

Monoclonal Antibodies against Nucleocapsid Protein of SARS-CoV-2 Variants for Detection of COVID-19.

Mitigation strategies of the coronavirus disease 2019 (COVID-19) pandemic have been greatly hindered by the continuous emergence of SARS-CoV-2 variants. New sensitive, rapid diagnostic tests for the wide-spectrum detection of viral variants are needed. We generated a panel of 41 monoclonal antibodies against the SARS-CoV-2 nucleocapsid protein (NP) by using mice hybridoma techniques. Of these mAbs, nine exhibited high binding activities and were applied in latex-based lateral flow immunoassays (LFIAs). The LFIAs utilizing NP-mAb-7 and -40 had the best sensitivity and lowest limit of detection: 8 pg for purified NP and 625 TCID50/mL for the authentic virus (hCoV-19/Taiwan/4/2020). The specificity tests showed that the NP-mAb-40/7 LFIA strips did not cross-react with five human coronavirus strains or 20 other common respiratory pathogens. Importantly, we found that 10 NP mutants, including alpha (B.1.1.7), beta (B.1.351), gamma (P.1), and delta (B.1.617.2) variants, could be detected by NP-mAb-40/7 LFIA strips. A clinical study (n = 60) of the NP-mAb-40/7 LFIA strips demonstrated a specificity of 100% and sensitivity of 90% in infected individuals with cycle threshold (Ct) values < 29.5. These anti-NP mAbs have strong potential for use in the clinical detection of SARS-CoV-2 infection, whether the virus is wild-type or a variant of concern.

The extracellular domain of epithelial cell adhesion molecule (EpCAM) enhances multipotency of mesenchymal stem cells through EGFR-LIN28-LET7 signaling.

Mesenchymal stem cells (MSCs) are widely considered to be an attractive cell source for regenerative therapies, but maintaining multipotency and self-renewal in cultured MSCs is especially challenging. Hence, the development and mechanistic description of strategies that help promote multipotency in MSCs will be vital to future clinical use. Here, using an array of techniques and approaches, including cell biology, RT-quantitative PCR, immunoblotting, immunofluorescence, flow cytometry, and ChIP assays, we show that the extracellular domain of epithelial cell adhesion molecule (EpCAM) (EpEX) significantly increases the levels of pluripotency factors through a signaling cascade that includes epidermal growth factor receptor (EGFR), signal transducer and activator of transcription 3 (STAT3), and Lin-28 homolog A (LIN28) and enhances the proliferation of human bone marrow MSCs. Moreover, we found that EpEX-induced LIN28 expression reduces the expression of the microRNA LET7 and up-regulates that of the transcription factor high-mobility group AT-hook 2 (HMGA2), which activates the transcription of pluripotency factors. Surprisingly, we found that EpEX treatment also enhances osteogenesis of MSCs under differentiation conditions, as evidenced by increases in osteogenic markers, including Runt-related transcription factor 2 (RUNX2). Taken together, our results indicate that EpEX stimulates EGFR signaling and thereby context-dependently controls MSC states and activities, promoting cell proliferation and multipotency under maintenance conditions and osteogenesis under differentiation conditions.