Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody.

The effectiveness of SARS-CoV-2 vaccines and therapeutic antibodies have been limited by the continuous emergence of viral variants and by the restricted diffusion of antibodies from circulation into the sites of respiratory virus infection. Here, we report the identification of two highly conserved regions on the Omicron variant receptor-binding domain recognized by broadly neutralizing antibodies. Furthermore, we generated a bispecific single-domain antibody that was able to simultaneously and synergistically bind these two regions on a single Omicron variant receptor-binding domain as revealed by cryo-EM structures. We demonstrated that this bispecific antibody can be effectively delivered to lung via inhalation administration and exhibits exquisite neutralization breadth and therapeutic efficacy in mouse models of SARS-CoV-2 infections. Importantly, this study also deciphered an uncommon and highly conserved cryptic epitope within the spike trimeric interface that may have implications for the design of broadly protective SARS-CoV-2 vaccines and therapeutics.

Structural analysis of the dual agonism at GLP-1R and GCGR.

Glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR), two members of class B1 G protein-coupled receptors, play important roles in glucose homeostasis and energy metabolism. They share a high degree of sequence homology but have different functionalities. Unimolecular dual agonists of both receptors developed recently displayed better clinical efficacies than that of monotherapy. To study the underlying molecular mechanisms, we determined high-resolution cryo-electron microscopy structures of GLP-1R or GCGR in complex with heterotrimeric Gs protein and three GLP-1R/GCGR dual agonists including peptide 15, MEDI0382 (cotadutide) and SAR425899 with variable activating profiles at GLP-1R versus GCGR. Compared with related structures reported previously and supported by our published pharmacological data, key residues responsible for ligand recognition and dual agonism were identified. Analyses of peptide conformational features revealed a difference in side chain orientations within the first three residues, indicating that distinct engagements in the deep binding pocket are required to achieve receptor selectivity. The middle region recognizes extracellular loop 1 (ECL1), ECL2, and the top of transmembrane helix 1 (TM1) resulting in specific conformational changes of both ligand and receptor, especially the dual agonists reshaped ECL1 conformation of GLP-1R relative to that of GCGR, suggesting an important role of ECL1 interaction in executing dual agonism. Structural investigation of lipid modification showed a better interaction between lipid moiety of MEDI0382 and TM1-TM2 cleft, in line with its increased potency at GCGR than SAR425899. Together, the results provide insightful information for the design and development of improved therapeutics targeting these two receptors simultaneously.

Antibody signatures in hospitalized hand, foot and mouth disease patients with acute enterovirus A71 infection.

Enterovirus A71 (EV-A71) infection is a major cause of severe hand, foot and mouth disease (HFMD) in young children. The characteristics of EV-A71 neutralizing antibodies in HFMD patients are not well understood. In this study, we identified and cloned EV-A71-neutralizing antibodies by single cell RNA and B cell receptor sequencing of peripheral blood mononuclear cells. From 145 plasmablasts, we identified two IgG1 monoclonal antibodies (mAbs) and six IgM mAbs that neutralized EV-A71. Four of the IgM mAbs harbor germline variable sequences and neutralize EV-A71 potently. Two genetically similar IgM antibodies from two patients have recurrent heavy chain variable domain gene usage and similar complementarity-determining region 3 sequences. We mapped the residues of EV-A71 critical for neutralization through selection of virus variants resistant to antibody neutralization in the presence of neutralizing mAbs. The residues critical for neutralization are conserved among EV-A71 genotypes. Epitopes for the two genetically similar antibodies overlap with the SCARB2 binding site of EV-A71. We used escape variants to measure the epitope-specific antibody response in acute phase serum samples from EV-A71 infected HFMD patients. We found that these epitopes are immunogenic and contributed to the neutralizing antibody response against the virus. Our findings advance understanding of antibody response to EV-A71 infection in young children and have translational potential: the IgM mAbs could potentially be used for prevention or treatment of EV-A71 infections.

Antibody Nanotweezer Constructing Bivalent Transistor-Biomolecule Interface with Spatial Tolerance.

Establishing a multivalent interface between the biointerface of a living system and electronic device is vital to building intelligent bioelectronic systems. How to achieve multivalent binding with spatial tolerance at the nanoscale remains challenging. Here, we report an antibody nanotweezer that is a self-adaptive bivalent nanobody enabling strong and resilient binding between transistor and envelope proteins at biointerfaces. The antibody nanotweezer is constructed by a DNA framework, where the nanoscale patterning of nanobodies along with their local spatial adaptivity enables simultaneous recognition of target epitopes without binding stress. As such, effective binding affinity increases by 1 order of magnitude compared with monovalent antibody. The antibody nanotweezer operating on transistor offers enhanced signal transduction, which is implemented to detect clinical pathogens, showing ∼100% overall agreement with PCR results. This work provides a perspective of engineering multivalent interfaces between biosystems with solid-state devices, holding great potential for organoid intelligence on a chip.

Artificial intelligence in cancer immunotherapy: Applications in neoantigen recognition, antibody design and immunotherapy response prediction.

Cancer immunotherapy is a method of controlling and eliminating tumors by reactivating the body's cancer-immunity cycle and restoring its antitumor immune response. The increased availability of data, combined with advancements in high-performance computing and innovative artificial intelligence (AI) technology, has resulted in a rise in the use of AI in oncology research. State-of-the-art AI models for functional classification and prediction in immunotherapy research are increasingly used to support laboratory-based experiments. This review offers a glimpse of the current AI applications in immunotherapy, including neoantigen recognition, antibody design, and prediction of immunotherapy response. Advancing in this direction will result in more robust predictive models for developing better targets, drugs, and treatments, and these advancements will eventually make their way into the clinical setting, pushing AI forward in the field of precision oncology.

Identification and validation of anti-protein arginine methyltransferase 5 (PRMT5) antibody as a novel biomarker for systemic sclerosis (SSc).

OBJECTIVES: In the complex panorama of autoimmune diseases, the characterisation of pivotal contributing autoantibodies that are involved in disease progression remains challenging. This study aimed to employ a global antibody profiling strategy to identify novel antibodies and investigate their association with systemic sclerosis (SSc). METHODS: We implemented this strategy by conducting immunoprecipitation (IP) following on-bead digestion with the sera of patients with SSc or healthy donors, using antigen pools derived from cell lysates. The enriched antigen-antibody complex was proceeded with mass spectrometry (MS)-based quantitative proteomics and over-represented by bioinformatics analysis. The candidate antibodies were then orthogonally validated in two independent groups of patients with SSc. Mice were immunised with the target antigen, which was subsequently evaluated by histological examination and RNA sequencing. RESULTS: The IP-MS analysis, followed by validation in patients with SSc, revealed a significant elevation in anti-PRMT5 antibodies among patients with SSc. These antibodies exhibited robust diagnostic accuracy in distinguishing SSc from healthy controls and other autoimmune conditions, including systemic lupus erythematosus and Sjögren's syndrome, with an area under the curve ranging from 0.900 to 0.988. The elevation of anti-PRMT5 antibodies was verified in a subsequent independent group with SSc using an additional method, microarray. Notably, 31.11% of patients with SSc exhibited seropositivity for anti-PRMT5 antibodies. Furthermore, the titres of anti-PRMT5 antibodies demonstrated a correlation with the progression or regression trajectory in SSc. PRMT5 immunisation displayed significant inflammation and fibrosis in both the skin and lungs of mice. This was concomitant with the upregulation of multiple proinflammatory and profibrotic pathways, thereby underscoring a potentially pivotal role of anti-PRMT5 antibodies in SSc. CONCLUSIONS: This study has identified anti-PRMT5 antibodies as a novel biomarker for SSc.

A novel human single-domain antibody-drug conjugate targeting CEACAM5 exhibits potent in vitro and in vivo antitumor activity.

Leveraging the specificity of antibody to deliver cytotoxic agent into tumor, antibody-drug conjugates (ADCs) have become one of the hotspots in the development of anticancer therapies. Although significant progress has been achieved, there remain challenges to overcome, including limited penetration into solid tumors and potential immunogenicity. Fully human single-domain antibodies (UdAbs), with their small size and human nature, represent a promising approach for addressing these challenges. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is a glycosylated cell surface protein that rarely expressed in normal adult tissues but overexpressed in diverse cancers, taking part in tumorigenesis, progression, and metastasis. In this study, we investigated the therapeutic potential of UdADC targeting CEACAM5. We performed biopanning in our library and obtained an antibody candidate B9, which bound potently and specifically to CEACAM5 protein (KD = 4.84 nM) and possessed excellent biophysical properties (low aggregation tendency, high homogeneity, and thermal stability). The conjugation of B9 with a potent cytotoxic agent, monomethyl auristatin E (MMAE), exhibited superior antitumor efficacy against CEACAM5-expressing human gastric cancer cell line MKN-45, human pancreatic carcinoma cell line BxPC-3 and human colorectal cancer cell line LS174T with IC50 values of 38.14, 25.60, and 101.4 nM, respectively. In BxPC-3 and MKN-45 xenograft mice, administration of UdADC B9-MMAE (5 mg/kg, i.v.) every 2 days for 4 times markedly inhibited the tumor growth without significant change in body weight. This study may have significant implications for the design of next-generation ADCs.

Cross-reactive antibody response to Monkeypox virus surface proteins in a small proportion of individuals with and without Chinese smallpox vaccination history.

BACKGROUND: After the eradication of smallpox in China in 1979, vaccination with the vaccinia virus (VACV) Tiantan strain for the general population was stopped in 1980. As the monkeypox virus (MPXV) is rapidly spreading in the world, we would like to investigate whether the individuals with historic VACV Tiantan strain vaccination, even after more than 40 years, could still provide ELISA reactivity and neutralizing protection; and whether the unvaccinated individuals have no antibody reactivity against MPXV at all. RESULTS: We established serologic ELISA to measure the serum anti-MPXV titer by using immunodominant MPXV surface proteins, A35R, B6R, A29L, and M1R. A small proportion of individuals (born before 1980) with historic VACV Tiantan strain vaccination exhibited serum ELISA cross-reactivity against these MPXV surface proteins. Consistently, these donors also showed ELISA seropositivity and serum neutralization against VACV Tiantan strain. However, surprisingly, some unvaccinated young adults (born after 1980) also showed potent serum ELISA activity against MPXV proteins, possibly due to their past infection by some self-limiting Orthopoxvirus (OPXV). CONCLUSIONS: We report the serum ELISA cross-reactivity against MPXV surface protein in a small proportion of individuals both with and without VACV Tiantan strain vaccination history. Combined with our serum neutralization assay against VACV and the recent literature about mice vaccinated with VACV Tiantan strain, our study confirmed the anti-MPXV cross-reactivity and cross-neutralization of smallpox vaccine using VACV Tiantan strain. Therefore, it is necessary to restart the smallpox vaccination program in high risk populations.

Structural Study of SARS-CoV-2 Antibodies Identifies a Broad-Spectrum Antibody That Neutralizes the Omicron Variant by Disassembling the Spike Trimer.

The continuous emergence of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants poses new challenges in the fight against the coronavirus disease 2019 (COVID-19) pandemic. The newly emerging Omicron strain caused serious immune escape and raised unprecedented concern all over the world. The development of an antibody targeting a conserved and universal epitope is urgently needed. A subset of neutralizing antibodies (NAbs) against COVID-19 from convalescent patients were isolated in our previous study. In this study, we investigated the accommodation of these NAbs to SARS-CoV-2 variants of concern (VOCs), revealing that IgG 553-49 neutralizes pseudovirus of the SARS-CoV-2 Omicron variant. In addition, we determined the cryo-electron microscopy (cryo-EM) structure of the SARS-CoV-2 spike (S) protein complexed with three monoclonal antibodies targeting different epitopes, including 553-49, 553-15, and 553-60. Notably, 553-49 targets a novel conserved epitope and neutralizes the virus by disassembling S trimers. IgG 553-15, an antibody that neutralizes all of the VOCs except Omicron, cross-links two S trimers to form a trimer dimer, demonstrating that 553-15 neutralizes the virus by steric hindrance and virion aggregation. These findings suggest the potential to develop 553-49 and other antibodies targeting this highly conserved epitope as promising therapeutic reagents for COVID-19. IMPORTANCE The emergence of the Omicron strain of SARS-CoV-2 caused higher immune escape, raising unprecedented concerns about the effectiveness of antibody therapies and vaccines. In this study, we identified a SARS-CoV-2 neutralizing antibody, 553-49, which neutralizes all variants by targeting a completely conserved novel epitope. In addition, we revealed that IgG 553-15 neutralizes SARS-CoV-2 by cross-linking virions and that 553-60 functions by blocking receptor binding. Comparison of different receptor binding domain (RBD) epitopes revealed that the 553-49 epitope is hidden in the S trimer and keeps a high degree of conservation during SARS-CoV-2 evolution, making 553-49 a promising therapeutic reagent against the emerging Omicron and future variants of SARS-CoV-2.

Vaccinia virus induces EMT-like transformation and RhoA-mediated mesenchymal migration.

The emerging outbreak of monkeypox is closely associated with the viral infection and spreading, threatening global public health. Virus-induced cell migration facilitates viral transmission. However, the mechanism underlying this type of cell migration remains unclear. Here we investigate the motility of cells infected by vaccinia virus (VACV), a close relative of monkeypox, through combining multi-omics analyses and high-resolution live-cell imaging. We find that, upon VACV infection, the epithelial cells undergo epithelial-mesenchymal transition-like transformation, during which they lose intercellular junctions and acquire the migratory capacity to promote viral spreading. After transformation, VACV-hijacked RhoA signaling significantly alters cellular morphology and rearranges the actin cytoskeleton involving the depolymerization of robust actin stress fibers, leading-edge protrusion formation, and the rear-edge recontraction, which coordinates VACV-induced cell migration. Our study reveals how poxviruses alter the epithelial phenotype and regulate RhoA signaling to induce fast migration, providing a unique perspective to understand the pathogenesis of poxviruses.

A highly stable human single-domain antibody-drug conjugate exhibits superior penetration and treatment of solid tumors.

The inefficient tumor penetration of therapeutic antibodies has hampered their effective use in treating solid tumors. Here, we report the identification of a fully human single-domain antibody (UdAb), designated as n501, targeting the oncofetal antigen 5T4. The high-resolution crystal structure indicates that n501 adopts a compact structure very similar to that of camelid nanobodies, and binds tightly to all eight leucine-rich repeats of 5T4. Furthermore, the UdAb n501 exhibits exceptionally high stability, with no apparent activity changes over 4 weeks of storage at various temperatures. Importantly, the UdAb-based antibody-drug conjugate (n501-SN38) showed much deeper tumor penetration, significantly higher tumor uptake, and faster accumulation at tumor sites than conventional IgG1-based antibody-drug conjugate (m603-SN38), resulting in improved tumor inhibition. These results highlight the potential of UdAb-based antibody-drug conjugates as a potential class of antitumor therapeutics with characteristics of high stability and strong tumor penetration for the effective treatment of solid tumors.

Potent Therapeutic Strategies for COVID-19 with Single-Domain Antibody Immunoliposomes Neutralizing SARS-CoV-2 and Lip/cGAMP Enhancing Protective Immunity.

The worldwide spread of COVID-19 continues to impact our lives and has led to unprecedented damage to global health and the economy. This highlights the need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. We modified a single-domain antibody, SARS-CoV-2 VHH, to the surface of the liposomes. These immunoliposomes demonstrated a good neutralizing ability, but could also carry therapeutic compounds. Furthermore, we used the 2019-nCoV RBD-SD1 protein as an antigen with Lip/cGAMP as the adjuvant to immunize mice. Lip/cGAMP enhanced the immunity well. It was demonstrated that the combination of RBD-SD1 and Lip/cGAMP was an effective preventive vaccine. This work presented potent therapeutic anti-SARS-CoV-2 drugs and an effective vaccine to prevent the spread of COVID-19.

A novel and effective approach to generate germline-like monoclonal antibodies by integration of phage and mammalian cell display platforms.

Phage display technology allows for rapid selection of antibodies from the large repertoire of human antibody fragments displayed on phages. However, antibody fragments should be converted to IgG for biological characterizations and affinity of antibodies obtained from phage display library is frequently not sufficient for efficient use in clinical settings. Here, we describe a new approach that combines phage and mammalian cell display, enabling simultaneous affinity screening of full-length IgG antibodies. Using this strategy, we successfully obtained a novel germline-like anti-TIM-3 monoclonal antibody named m101, which was revealed to be a potent anti-TIM-3 therapeutic monoclonal antibody via in vitro and in vivo experiments, indicating its effectiveness and power. Thus, this platform can help develop new monoclonal antibody therapeutics with high affinity and low immunogenicity.

Facile Separation of PEGylated Liposomes Enabled by Anti-PEG scFv.

PEGylated nanocarriers have gained increasing attention due to reduced toxicity and enhanced circulation compared with free drugs. According to guidances of drug regulatory departments worldwide, it is crucial to determine free and liposomal drug concentrations; however, the conventional used separation methods including dialysis, ultrafiltration, and solid-phase extraction (SPE) have drawbacks of time-consuming, drug leakage, environmental pollution or error bias of trace level drug. Here we developed a facile PEG-scFv-based separation method combined with HPLC to quantify free doxorubicin (DOX) and liposomal DOX in plasma. Anti-PEG single chain variable fragment antibody (PEG-scFv) was adopted to sediment PEGylated liposomes by simple incubation and low speed centrifugation. Compared to SPE, it demonstrated sufficient accuracy and sensitivity to evaluate free and liposomal DOX with intact liposomes. Therefore, it can serve as an alternative approach of SPE, which is suitable for quality assessment and pharmacokinetics evaluation of PEGylated liposomal drugs and possible other PEGylated nanocarriers.

Deciphering Protein Corona by scFv-Based Affinity Chromatography.

It remains challenging to precisely decipher the structural and functional characteristics of protein coronas. To overcome the drawbacks frequently occurring in the traditional separation methods, an anti-PEG single-chain variable fragment (PEG-scFv) based affinity chromatography (AfC) was developed to achieve precise and efficient separation of protein coronas on PEGylated liposomes (sLip). His-tagged PEG-scFv could readily capture sLip without affecting protein corona compositions, and separate sLip/protein complex from plasma protein aggregates and endogenous vesicles through the Ni-NTA column. AfC demonstrated 43-fold higher protein corona collecting efficiency than centrifugation, which was extremely crucial for separation of in vivo protein coronas due to the limitation of sample size. AfC evaded contamination by endogenous vesicles and protein aggregates occurring in centrifugation, and reserved the loosely bound proteins, providing an unprecedented approach to deeply decipher protein coronas. The scFv-based AfC also paves new avenues for the separation of protein coronas formed on other nanomedicines.

Ultrasensitive Detection of SARS-CoV-2 Antibody by Graphene Field-Effect Transistors.

The fast spread of SARS-CoV-2 has severely threatened the public health. Establishing a sensitive method for SARS-CoV-2 detection is of great significance to contain the worldwide pandemic. Here, we develop a graphene field-effect transistor (g-FET) biosensor and realize ultrasensitive SARS-CoV-2 antibody detection with a limit of detection (LoD) down to 10-18 M (equivalent to 10-16 g mL-1) level. The g-FETs are modified with spike S1 proteins, and the SARS-CoV-2 antibody biorecognition events occur in the vicinity of the graphene surface, yielding an LoD of ∼150 antibodies in 100 µL full serum, which is the lowest LoD value of antibody detection. The diagnoses time is down to 2 min for detecting clinical serum samples. As such, the g-FETs leverage rapid and precise SARS-CoV-2 screening and also hold great promise in prevention and control of other epidemic outbreaks in the future.

Ultraprecise Antigen 10-in-1 Pool Testing by Multiantibodies Transistor Assay.

Effective screening of infectious diseases requires a fast, cheap, and population-scale testing. Antigen pool testing can increase the test rate and shorten the screening time, thus being a valuable approach for epidemic prevention and control. However, the overall percent agreement (OPA) with polymerase chain reaction (PCR) is one-half to three-quarters, hampering it from being a comprehensive method, especially pool testing, beyond the gold-standard PCR. Here, a multiantibodies transistor assay is developed for sensitive and highly precise antigen pool testing. The multiantibodies capture SARS-CoV-2 spike S1 proteins with different configurations, resulting in an antigen-binding affinity down to 0.34 fM. The limit of detection reaches 3.5 × 10-17 g mL-1SARS-CoV-2 spike S1 protein in artificial saliva, 4-5 orders of magnitude lower than existing transistor sensors. The testing of 60 nasopharyngeal swabs exhibits ∼100% OPA with PCR within an average diagnoses time of 38.9 s. Owing to its highly precise feature, a portable integrated platform is fabricated, which achieves 10-in-1 pooled screening for high testing throughput. This work solves the long-standing problem of antigen pool testing, enabling it to be a valuable tool in precise diagnoses and population-wide screening of COVID-19 or other epidemics in the future.

A broadly neutralizing germline-like human monoclonal antibody against dengue virus envelope domain III.

Dengue is the most widespread vector-borne viral disease caused by dengue virus (DENV) for which there are no safe, effective drugs approved for clinical use. Here, by using sequential antigen panning of a yeast antibody library derived from healthy donors against the DENV envelop protein domain III (DIII) combined with depletion by an entry defective DIII mutant, we identified a cross-reactive human monoclonal antibody (mAb), m366.6, which bound with high affinity to DENV DIII from all four DENV serotypes. Immunogenetic analysis indicated that m366.6 is a germline-like mAb with very few somatic mutations from the closest VH and Vλ germline genes. Importantly, we demonstrated that it potently neutralized DENV both in vitro and in the mouse models of DENV infection without detectable antibody-dependent enhancement (ADE) effect. The epitope of m366.6 was mapped to the highly conserved regions on DIII, which may guide the design of effective dengue vaccine immunogens. Furthermore, as the first germline-like mAb derived from a naïve antibody library that could neutralize all four DENV serotypes, the m366.6 can be a tool for exploring mechanisms of DENV infection, and is a promising therapeutic candidate.

Synergistic Effect by Combining a gp120-Binding Protein and a gp41-Binding Antibody to Inactivate HIV-1 Virions and Inhibit HIV-1 Infection.

Acquired immune deficiency syndrome (AIDS) has prevailed over the last 30 years. Although highly active antiretroviral therapy (HAART) has decreased mortality and efficiently controlled the progression of disease, no vaccine or curative drugs have been approved until now. A viral inactivator is expected to inactivate cell-free virions in the absence of target cells. Previously, we identified a gp120-binding protein, mD1.22, which can inactivate laboratory-adapted HIV-1. In this study, we have found that the gp41 N-terminal heptad repeat (NHR)-binding antibody D5 single-chain variable fragment (scFv) alone cannot inactivate HIV-1 at the high concentration tested. However, D5 scFv in the combination could enhance inactivation activity of mD1.22 against divergent HIV-1 strains, including HIV-1 laboratory-adapted strains, primary HIV-1 isolates, T20- and AZT-resistant strains, and LRA-reactivated virions. Combining mD1.22 and D5 scFv exhibited synergistic effect on inhibition of infection by divergent HIV-1 strains. These results suggest good potential to develop the strategy of combining a gp120-binding protein and a gp41-binding antibody for the treatment of HIV-1 infection.

Synthetic Homogeneous Glycoforms of the SARS-CoV-2 Spike Receptor-Binding Domain Reveals Different Binding Profiles of Monoclonal Antibodies.

SARS-CoV-2 attaches to its host receptor, angiotensin-converting enzyme 2 (ACE2), via the receptor-binding domain (RBD) of the spike protein. The RBD glycoprotein is a critical target for the development of neutralizing antibodies and vaccines against SARS-CoV-2. However, the high heterogeneity of RBD glycoforms may lead to an incomplete neutralization effect and impact the immunogenic integrity of RBD-based vaccines. Investigating the role of different carbohydrate domains is of paramount importance. Unfortunately, there is no viable method for preparing RBD glycoproteins with structurally defined glycans. Herein we describe a highly efficient and scalable strategy for the preparation of six glycosylated RBDs bearing defined structure glycoforms at T323, N331, and N343. A combination of modern oligosaccharide, peptide synthesis and recombinant protein engineering provides a robust route to decipher carbohydrate structure-function relationships.