Dr. Tianlei Ying’s group developed a broad-spectrum inhalable bispecific single-domain antibody against SARS-CoV-2
Source：Tianlei Ying
2022-06-13
Dr. Tianlei Ying’s group from MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, Shanghai Institute of Infectious Disease and Biosecurity and School of Basic Medical Sciences of Fudan University, in collaboration with Lei Sun’s team from Institutes of Biomedical Sciences of Fudan University and Yanling Wu’s team from School of Basic Medical Sciences of Fudan University published a research article entitled “Broad neutralization of SARS-CoV-2 variants by an inhalable bispecific single-domain antibody” on Cell on April 14th, 2022. The research reported a broad-spectrum inhalable bispecific single-domain antibody bn03, revealing two conserved non-ACE2-competing epitopes on RBD. This antibody exhibits potent neutralizing efficacy against all known SARS-CoV-2 variants of concern (VOC) and displayed significant anti-viral effects in both mildly and severely infected hACE2 mice.
Since the outbreak of the COVID-19 pandemic, over 533 million confirmed cases and 6.3 million death have been reported. The highly mutational rate enables the virus to frequently overcome immune defense, bringing challenges for the development of broad antibodies and vaccines. The newly emerging variant Omicron and its sublineages possessed over 15 mutational sites within RBD, far more than previously reported variants of concern, which with no more than 3 mutations in RBD. A number of studies have reported the escape of Omicron to vaccinated serum and the majority of existing SARS-CoV-2 neutralizing antibodies, especially to those targeting the receptor-binding motif (RBM). Antibodies that received emergency use authorizations (EUA) from the food and drug administration earlier have been suspended due to the occurrence of Omicron and the sublineage BA.2. To conquer the threat of emerging SARS-CoV-2 variants, development of broad-spectrum antibodies is of urgent need.
Based on the analysis of mutations in Omicron, the research team discovered that more mutations are located at the RBM, further determination of antibodies’ neutralizing abilities against Omicron discovered that those targeting RBM were the most obviously escaped by the virus. For vaccinated sera with a higher level of non-RBM antibodies, they maintained higher neutralizing activity to Omicron. These results suggested that Omicron variant may be less susceptible to antibodies of RBM. However, there are still two regions found conserved in RBD, one located in the cryptic interface of the trimeric spike, and the other lies in the outer lateral surface.
Dr. Tianlei Ying’s team previously developed a versatile platform for rapid isolation of fully human single-domain antibodies and identified a series of antibodies targeting five types of epitopes on RBD. Through the screening of the combination of antibodies, a broad-spectrum bispecific single-domain antibody with superior properties and high yield stands out from other combinations, named bn03. The antibody consisted of two non-ACE2-competing single-domain antibodies connected through a linker, with one arm recognizing a highly conserved cryptic epitope inside the spike trimer (n3130v), the other targeting the outer lateral surface of RBD (n3113v). According to the result of cryo-EM, two arms of bn03 were able to bind simultaneously and synergistically to one RBD of spike trimer. Specifically, binding of n3130v can pull the adjacent “down” RBD inward to the central axis, so that the outer lateral surface was fully exposed from the N-terminal domain (NTD), making space for further binding of n3113v.
Targeting two conserved epitopes of RBD confers bn03 the ability to potently neutralize all known VOCs including Omicron. Based on the deep mutational analysis, no mutation was found accumulated in n3130v epitope (frequency>0.1%). Antibodies targeting this site are uncommon and rarely reported, indicating the epitope was under less immune selection pressure, which can result in the antigenic shift. Besides, the extreme conservative of the cryptic n3130v epitope allows it a potential target for broad antibody and vaccine design.
Since SARS-CoV-2 infection mainly causes respiratory illness, in order to improve the efficiency of drug delivery to lungs and reduce the side effects of systemic administration, bn03 was delivered through inhaled route. Based on the in vivo pharmacokinetic and pharmacodynamic study, aerosol delivery of single-domain antibodies can significantly increase the drug accumulation in lungs and airway and improve the therapeutic efficacy in SARS-CoV-2 infected mice. The excellent property of bn03 enables it to be aerosolized to uniform particles under diagrams of 5 μm, determined by next-generation impactor (NGI). In addition, bn03 (27 kDa) possesses higher aerosol efficacy than conventional IgG antibodies (150 kDa). These findings showed the virtue of single-domain antibodies as an inhaled drug for respiratory disease.
In the end, inhalation of bn03 displayed remarkable therapeutic efficacy in SARS-CoV-2 challenged mice with severe or mild symptoms. The viral RNA copies in lung were largely reduced in the treatment group with ameliorated lung pathology. In mild symptom mice, the viral copies were close to the minimum detection limit, and in severely infected mice, the live virus in lung was undetectable.
Altogether, this article reported a broad-spectrum inhalable bispecific single-domain antibody against SARS-CoV-2, with superior properties, and potent anti-viral effects in vivo. Currently, bn03 has entered the pilot production stage and is accelerating its advance to clinical trials. Furthermore, a highly conserved cryptic epitope hidden inside the trimeric interface was identified, providing insights for the design of broad-spectrum antibodies and vaccines.
Dr. Tianlei Ying, Dr. Lei Sun, and Dr. Yanling Wu are the co-corresponding authors of the paper. Ph.D. candidates Cheng Li, Wuqiang Zhan, Dr. Zhenlin Yang, Dr. Chao Tu, and Dr. Gaowei Hu are the co-first authors of the paper. The research was supported by grants from the National Key R&D Program of China, National Natural Science Foundation of China, etc.
In recent years, the research team of Professor Tianlei Ying of Fudan University has carried out systematic research on the theoretical system of fully human germline-like monoclonal antibody, design of novel antibody fragments, and antibody repertoire research. These studies have revealed the molecular characteristics of the antibody repertoire of the human immune system, and also provides an important theoretical basis for exploring safer, more economical, and more effective antibody drugs and immunotherapy strategies (https://csi.fudan.edu.cn). The team is now recruiting postdoctoral fellows with research directions in bioinformatics, synthetic immunology or antibodyomics. Applications should be sent to kongyu@fudan.edu.cn with the resume attached.
Since the outbreak of the COVID-19 pandemic, over 533 million confirmed cases and 6.3 million death have been reported. The highly mutational rate enables the virus to frequently overcome immune defense, bringing challenges for the development of broad antibodies and vaccines. The newly emerging variant Omicron and its sublineages possessed over 15 mutational sites within RBD, far more than previously reported variants of concern, which with no more than 3 mutations in RBD. A number of studies have reported the escape of Omicron to vaccinated serum and the majority of existing SARS-CoV-2 neutralizing antibodies, especially to those targeting the receptor-binding motif (RBM). Antibodies that received emergency use authorizations (EUA) from the food and drug administration earlier have been suspended due to the occurrence of Omicron and the sublineage BA.2. To conquer the threat of emerging SARS-CoV-2 variants, development of broad-spectrum antibodies is of urgent need.
Based on the analysis of mutations in Omicron, the research team discovered that more mutations are located at the RBM, further determination of antibodies’ neutralizing abilities against Omicron discovered that those targeting RBM were the most obviously escaped by the virus. For vaccinated sera with a higher level of non-RBM antibodies, they maintained higher neutralizing activity to Omicron. These results suggested that Omicron variant may be less susceptible to antibodies of RBM. However, there are still two regions found conserved in RBD, one located in the cryptic interface of the trimeric spike, and the other lies in the outer lateral surface.
Dr. Tianlei Ying’s team previously developed a versatile platform for rapid isolation of fully human single-domain antibodies and identified a series of antibodies targeting five types of epitopes on RBD. Through the screening of the combination of antibodies, a broad-spectrum bispecific single-domain antibody with superior properties and high yield stands out from other combinations, named bn03. The antibody consisted of two non-ACE2-competing single-domain antibodies connected through a linker, with one arm recognizing a highly conserved cryptic epitope inside the spike trimer (n3130v), the other targeting the outer lateral surface of RBD (n3113v). According to the result of cryo-EM, two arms of bn03 were able to bind simultaneously and synergistically to one RBD of spike trimer. Specifically, binding of n3130v can pull the adjacent “down” RBD inward to the central axis, so that the outer lateral surface was fully exposed from the N-terminal domain (NTD), making space for further binding of n3113v.
Targeting two conserved epitopes of RBD confers bn03 the ability to potently neutralize all known VOCs including Omicron. Based on the deep mutational analysis, no mutation was found accumulated in n3130v epitope (frequency>0.1%). Antibodies targeting this site are uncommon and rarely reported, indicating the epitope was under less immune selection pressure, which can result in the antigenic shift. Besides, the extreme conservative of the cryptic n3130v epitope allows it a potential target for broad antibody and vaccine design.
Since SARS-CoV-2 infection mainly causes respiratory illness, in order to improve the efficiency of drug delivery to lungs and reduce the side effects of systemic administration, bn03 was delivered through inhaled route. Based on the in vivo pharmacokinetic and pharmacodynamic study, aerosol delivery of single-domain antibodies can significantly increase the drug accumulation in lungs and airway and improve the therapeutic efficacy in SARS-CoV-2 infected mice. The excellent property of bn03 enables it to be aerosolized to uniform particles under diagrams of 5 μm, determined by next-generation impactor (NGI). In addition, bn03 (27 kDa) possesses higher aerosol efficacy than conventional IgG antibodies (150 kDa). These findings showed the virtue of single-domain antibodies as an inhaled drug for respiratory disease.
In the end, inhalation of bn03 displayed remarkable therapeutic efficacy in SARS-CoV-2 challenged mice with severe or mild symptoms. The viral RNA copies in lung were largely reduced in the treatment group with ameliorated lung pathology. In mild symptom mice, the viral copies were close to the minimum detection limit, and in severely infected mice, the live virus in lung was undetectable.
Altogether, this article reported a broad-spectrum inhalable bispecific single-domain antibody against SARS-CoV-2, with superior properties, and potent anti-viral effects in vivo. Currently, bn03 has entered the pilot production stage and is accelerating its advance to clinical trials. Furthermore, a highly conserved cryptic epitope hidden inside the trimeric interface was identified, providing insights for the design of broad-spectrum antibodies and vaccines.
Dr. Tianlei Ying, Dr. Lei Sun, and Dr. Yanling Wu are the co-corresponding authors of the paper. Ph.D. candidates Cheng Li, Wuqiang Zhan, Dr. Zhenlin Yang, Dr. Chao Tu, and Dr. Gaowei Hu are the co-first authors of the paper. The research was supported by grants from the National Key R&D Program of China, National Natural Science Foundation of China, etc.
In recent years, the research team of Professor Tianlei Ying of Fudan University has carried out systematic research on the theoretical system of fully human germline-like monoclonal antibody, design of novel antibody fragments, and antibody repertoire research. These studies have revealed the molecular characteristics of the antibody repertoire of the human immune system, and also provides an important theoretical basis for exploring safer, more economical, and more effective antibody drugs and immunotherapy strategies (https://csi.fudan.edu.cn). The team is now recruiting postdoctoral fellows with research directions in bioinformatics, synthetic immunology or antibodyomics. Applications should be sent to kongyu@fudan.edu.cn with the resume attached.
