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

The effectiveness of SARS-CoV-2 vaccines and therapeutic antibodies has 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 Omicron variant RBD recognized by broadly neutralizing antibodies. Based on this finding, we generated a bispecific single-domain antibody that was able to simultaneously and synergistically bind these two regions on a single Omicron variant RBD as revealed by Cryo-EM structures. This inhalable antibody exhibited exquisite neutralization breadth and therapeutic efficacy in mouse models of SARS-CoV-2 infections. The structures also deciphered an uncommon cryptic epitope within the spike trimeric interface that may have implications for the design of broadly protective SARS-CoV-2 vaccines and therapeutics.

COVID 19 breakthrough infection risk: a simple physical model describing the dependence on antibody concentration

The empirically-observed dependence on blood IgG anti-receptor binding domain antibody concentration of SARS-CoV-2 vaccine efficacy against infection has a rational explanation in the statistics of binding of antibody to spike proteins on the virus surface: namely that the probability of protection is the probability of antibody binding to more than a critical number of the spike proteins protruding from the virus. The model is consistent with the observed antibody concentrations required to induce immunity and with the observed dependence of vaccine efficacy on antibody concentration and thus is a useful tool in the development of models to relate, for an individual person, risk of breakthrough infection given measured antibody concentration

Correlation of the Commercial Anti-SARS-CoV-2 Receptor Binding Domain Antibody Test with the Chemiluminescent Reduction Neutralizing Test and Possible Detection of Antibodies to Emerging Variants

This study provides a diagnostic evidence of test validity, which can lead to vaccine efficacy and proof of recovery after COVID-19. It is not easy to know neutralization against SARS-CoV-2 in the clinical laboratory because of technical and biohazard issues.

COVID 19 breakthrough infection risk: a simple physical model describing the dependence on antibody concentration

The empirically-observed dependence on blood IgG anti-receptor binding domain antibody concentration of SARS-CoV-2 vaccine efficacy against infection has a rational explanation in the statistics of binding of antibody to spike proteins on the virus surface: namely that the probability of protection is the probability of antibody binding to more than a critical number of the spike proteins protruding from the virus. The model is consistent with the observed antibody concentrations required to induce immunity and with the observed dependence of vaccine efficacy on antibody concentration and thus is a useful tool in the development of models to relate, for an individual person, risk of breakthrough infection given measured antibody concentration

A non-ACE2 competing human single-domain antibody confers broad neutralization against SARS-CoV-2 and circulating variants

The current COVID-19 pandemic has heavily burdened the global public health system and may keep simmering for years. The frequent emergence of immune escape variants have spurred the search for prophylactic vaccines and therapeutic antibodies that confer broad protection against SARS-CoV-2 variants. Here we show that the bivalency of an affinity maturated fully human single-domain antibody (n3113.1-Fc) exhibits exquisite neutralizing potency against SARS-CoV-2 pseudovirus, and confers effective prophylactic and therapeutic protection against authentic SARS-CoV-2 in the host cell receptor angiotensin-converting enzyme 2 (ACE2) humanized mice. The crystal structure of n3113 in complex with the receptor-binding domain (RBD) of SARS-CoV-2, combined with the cryo-EM structures of n3113 and spike ecto-domain, reveals that n3113 binds to the side surface of up-state RBD with no competition with ACE2. The binding of n3113 to this novel epitope stabilizes spike in up-state conformations but inhibits SARS-CoV-2 S mediated membrane fusion, expanding our recognition of neutralization by antibodies against SARS-CoV-2. Binding assay and pseudovirus neutralization assay show no evasion of recently prevalent SARS-CoV-2 lineages, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) for n3113.1-Fc with Y58L mutation, demonstrating the potential of n3113.1-Fc (Y58L) as a promising candidate for clinical development to treat COVID-19.

Enhancement of NK Cell Antitumor Effector Functions Using a Bispecific Single Domain Antibody Targeting CD16 and the Epidermal Growth Factor Receptor

The ability to kill tumor cells while maintaining an acceptable safety profile makes Natural Killer (NK) cells promising assets for cancer therapy. Strategies to enhance the preferential accumulation and activation of NK cells in the tumor microenvironment can be expected to increase the efficacy of NK cell-based therapies. In this study, we show binding of a novel bispecific single domain antibody (VHH) to both CD16 (FcRγIII) on NK cells and the epidermal growth factor receptor (EGFR) on tumor cells of epithelial origin. The bispecific VHH triggered CD16- and EGFR-dependent activation of NK cells and subsequent lysis of tumor cells, regardless of the KRAS mutational status of the tumor. Enhancement of NK cell activation by the bispecific VHH was also observed when NK cells of colorectal cancer (CRC) patients were co-cultured with EGFR expressing tumor cells. Finally, higher levels of cytotoxicity were found against patient-derived metastatic CRC cells in the presence of the bispecific VHH and autologous peripheral blood mononuclear cells or allogeneic CD16 expressing NK cells. The anticancer activity of CD16-EGFR bispecific VHHs reported here merits further exploration to assess its potential therapeutic activity either alone or in combination with adoptive NK cell-based therapeutic approaches.

Expression, purification and characterization of anti-FGF7 domain antibody identified using phage display technique

Background: Fibroblast growth factors (FGFs) are involved in angiogenesis, wound healing and embryonic development. However, one of the causes of cancer cell growth in fibroblast-dependent cancers is FGF7 secreted by fibroblasts. Therefore, antibodies against FGF7 can be used for treatment of these types of cancers. Methods: In previous studies, a phage displaying single domain antibody, D53, against human FGF7 has been identified using the phage display technique. In the present study, D53 was produced and purified in its isolated form. ELISA experiment was performed to evaluate the binding of D53 to FGF7. The mode of interaction of D53-FGF7 was explored using docking study and molecular dynamics (MD) simulations. Results: The expression and purification processes were verified using western blotting and SDS-PAGE analyses. ELISA experiment showed that D53 is able to recognize and bind FGF7. Docking study and MD simulations indicated that compared to dummy VH, D53 has more affinity towards FGF7. Conclusion: The findings in the current study can be useful for generation and development of FGF7 inhibitors with potential use in fibroblast-dependent cancers.

Single-domain antibody screening by isPLA-seq

Single-domain antibody (sdAb) holds the promising strategies for diverse research and translational applications. Here, we describe a method for the adaptation of the in situ proximity ligation assay (isPLA) followed by sequencing (isPLA-seq) to facilitate screening of a high-sensitive, high-throughput sdAb library for a given protein at subcellular and single-cell resolution. Based on the sequence of complementarity-determining region 3 (CDR3), the recombinant sdAb can be produced for in vitro and in vivo utilities. This method provides a general means to identify the functional measure of sdAb and its complementary epitopes and its potential applications to investigate cellular processes.