scholarly journals Affinity maturation of cross-reactive CR3022 antibody against the receptor binding domain of SARS-CoV-2 via in silico site-directed mutagenesis

2021 ◽  
Author(s):  
Samvedna Saini ◽  
Manusmriti Agarwal ◽  
Amartya Pradhan ◽  
Savitha Pareek ◽  
Ashish K Singh ◽  
...  
Keyword(s):  
Binding Affinity ◽  
In Silico ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Affinity Maturation ◽  
Antibody Engineering ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
Directed Mutagenesis ◽  
Human Antibody

Abstract Introduction: Computational antibody engineering, affinity maturation, and screening greatly aid in vaccine and therapeutic antibody development by increasing the speed and accuracy of predictions. This study presents a protocol for designing affinity enhancing mutants of antibodies through in silico mutagenesis. A SARS-CoV-2 cross-reactive neutralizing antibody, CR3022, is considered as a case study.Methods: Our study aimed at generating antibody candidates from the human antibody CR3022 (derived from convalescent SARS patient) against the RBD of SARS-CoV-2 via in silico affinity maturation using site-directed mutagenesis in mutation hotspots. We optimized the paratope of the CR3022 antibody towards the RBD of SARS-CoV-2 for better binding affinity and stability, employing molecular modeling, docking, dynamics simulations, and molecular mechanics energies combined with generalized Born and surface area (MM-GBSA). Results: Nine antibody candidates were generated post in silico site-directed mutagenesis followed by preliminary screening. Molecular dynamics simulation of 100 nanoseconds and MM-GBSA analysis confirmed L-K45S as a lead antibody with the highest binding affinity against the RBD compared to wild-type and mutant counterparts. Three out of the remaining mutants were also found to have distinct epitopes and binding, possessing a potential to be developed against emerging SARS-CoV-2 variants of concern. Conclusion: The study demonstrates the use of an integrative antibody engineering protocol for enhancing affinity and neutralization potential through mutagenesis using robust open-source computational tools and predictors. This study highlights unique scoring and ranking methods for evaluating docking efficiency. It also underscores the importance of framework mutations for developing broadly neutralizing antibodies.

scholarly journals Affinity maturation of cross-reactive CR3022 antibody against the receptor binding domain of SARS-CoV-2 via in silico site-directed mutagenesis

2020 ◽  
Author(s):  
Amartya Pradhan ◽  
Samvedna Saini ◽  
Manusmriti Agarwal ◽  
Yatender Kumar
Keyword(s):  
Binding Affinity ◽  
Receptor Binding ◽  
In Silico ◽  
Affinity Maturation ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
Directed Mutagenesis ◽  
Human Antibody ◽  
Receptor Binding Domain

Abstract Background: The coronavirus disease 2019 (COVID-19) has unequivocally affected the lives of people across the planet and has imposed an unprecedented burden on our healthcare systems. With no potent regimen for treatment, there is a dire need for finding promising candidates. Receptor binding domain (RBD) of the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has proven to be a promising target owing to its role in viral invasion. Methods: Our study aimed at generating antibody candidates from the human antibody CR3022 (derived from convalescent SARS patient) against the RBD of SARS-CoV-2 via in silico affinity maturation. We optimized the paratope of the CR3022 antibody towards the RBD of SARS-CoV-2 for better binding affinity and stability, employing molecular modeling, docking, and dynamics simulations. Results: Out of seven antibody leads generated post in silico site-directed mutagenesis followed by preliminary screening, antibody named SAM3 was predicted to have the highest binding affinity towards RBD. However, molecular dynamics simulation of fifty nanoseconds set the seal on SAM1 and SAM2. Both demonstrated a higher binding affinity and stability compared to other counterparts and CR3022. Conclusion: We hypothesize that SAM1, SAM2, and SAM3 antibody candidates can bind to the RBD and potentially disrupt the viral invasion. All three antibody candidates to bind residues on the human ACE-2 binding site of SARS-CoV-2 which were not conserved from SARS-CoV. Our study calls for further in vitro and in vivo testing of SAM1, SAM2, and SAM3 candidates for COVID-19 treatment.

scholarly journals Enhancement of SARS-CoV-2 Receptor Binding Domain -CR3022 Human Antibody Binding Affinity via in Silico Engineering Approach

2020 ◽  
Author(s):  
Fateme Sefid ◽  
Zahra Payandeh ◽  
Ghasem Azamirad ◽  
Behzad Mansoori ◽  
Behzad Baradaran ◽  
...  
Keyword(s):  
Amino Acids ◽  
Binding Affinity ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
Affinity Maturation ◽  
Binding Domain ◽  
Human Antibody ◽  
Receptor Binding Domain ◽  
Receptor Binding Site ◽  
Specificity And Sensitivity

Abstract Background: The nCoV-2019 is a cause of COVID-19 disease. The surface spike glycoprotein (S), which is necessary for virus entry through the intervention of the host receptor and it mediates virus-host membrane fusion, is the primary coronavirus antigen (Ag). The angiotensin-converting enzyme 2 (ACE2) is reported to be the effective human receptor for SARS-CoVs 2. ACE2 receptor can be prevented by neutralizing antibodies (nAbs) such as CR3022 targeting the virus receptor-binding site. Considering the importance of computational docking, and affinity maturation we aimed to find the important amino acids of the CR3022 antibody (Ab). These amino acids were then replaced by other amino acids to improve Ab-binding affinity to a receptor-binding domain (RBD) of the 2019-nCoV spike protein. Finally, we measured the binding affinity of Ab variants to the Ag. Result: Our findings disclosed that several variant mutations could successfully improve the characteristics of the Ab binding compared to the normal antibodies. Conclusion: The modified antibodies may be possible candidates for stronger affinity binding to Ags which in turn can affect the specificity and sensitivity of antibodies.

scholarly journals Application of an integrated computational antibody engineering platform to design SARS-CoV-2 neutralizers

2021 ◽  
Author(s):  
Saleh Riahi ◽  
Jae Hyeon Lee ◽  
Shuai Wei ◽  
Robert Cost ◽  
Alessandro Masiero ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Receptor Binding ◽  
In Silico ◽  
Half Life ◽  
Neutralizing Antibodies ◽  
Binding Domain ◽  
Antibody Engineering ◽  
Receptor Binding Domain ◽  
Effector Functions

Abstract As the COVID-19 pandemic continues to spread, hundreds of new initiatives including studies on existing medicines are running to fight the disease. To deliver a potentially immediate and lasting treatment to current and emerging SARS-CoV-2 variants, new collaborations and ways of sharing are required to create as many paths forward as possible. Here we leverage our expertise in computational antibody engineering to rationally design/engineer three previously reported SARS-CoV neutralizing antibodies and share our proposal towards anti-SARS-CoV-2 biologics therapeutics. SARS-CoV neutralizing antibodies, m396, 80R, and CR-3022 were chosen as templates due to their diversified epitopes and confirmed neutralization potency against SARS-CoV (but not SARS-CoV-2 except for CR3022). Structures of variable fragment (Fv) in complex with receptor binding domain (RBD) from SARS-CoV or SARS-CoV-2 were subjected to our established in silico antibody engineering platform to improve their binding affinity to SARS-CoV-2 and developability profiles. The selected top mutations were ensembled into a focused library for each antibody for further screening. In addition, we convert the selected binders with different epitopes into the trispecific format, aiming to increase potency and to prevent mutational escape. Lastly, to avoid antibody induced virus activation or enhancement, we suggest application of NNAS and DQ mutations to the Fc region to eliminate effector functions and extend half-life.

scholarly journals Application of an integrated computational antibody engineering platform to design SARS-CoV-2 neutralizers

2021 ◽  
Author(s):  
Saleh Riahi ◽  
Jae Hyeon Lee ◽  
Shuai Wei ◽  
Robert Cost ◽  
Alessandro Masiero ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Receptor Binding ◽  
In Silico ◽  
Half Life ◽  
Neutralizing Antibodies ◽  
Binding Domain ◽  
Antibody Engineering ◽  
Receptor Binding Domain ◽  
Effector Functions

As the COVID-19 pandemic continues to spread, hundreds of new initiatives including studies on existing medicines are running to fight the disease. To deliver a potentially immediate and lasting treatment to current and emerging SARS-CoV-2 variants, new collaborations and ways of sharing are required to create as many paths forward as possible. Here we leverage our expertise in computational antibody engineering to rationally design/optimize three previously reported SARS-CoV neutralizing antibodies and share our proposal towards anti-SARS-CoV-2 biologics therapeutics. SARS-CoV neutralizing antibodies, m396, 80R, and CR-3022 were chosen as templates due to their diversified epitopes and confirmed neutralization potency against SARS. Structures of variable fragment (Fv) in complex with receptor binding domain (RBD) from SARS-CoV or SARS-CoV2 were subjected to our established in silico antibody engineering platform to improve their binding affinity to SARS-CoV2 and developability profiles. The selected top mutations were ensembled into a focused library for each antibody for further screening. In addition, we convert the selected binders with different epitopes into the trispecific format, aiming to increase potency and to prevent mutational escape. Lastly, to avoid antibody induced virus activation or enhancement, we applied NNAS and DQ mutations to the Fc region to eliminate effector functions and extend half-life.

Affinity enhancement of nanobody binding to EGFR: in silico site-directed mutagenesis and molecular dynamics simulation approaches

2016 ◽  
Vol 35 (8) ◽  
pp. 1710-1728 ◽  
Author(s):  
Alireza Farasat ◽  
Fatemeh Rahbarizadeh ◽  
Ghader Hosseinzadeh ◽  
Sharareh Sajjadi ◽  
Mehdi Kamali ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Molecular Dynamics Simulation ◽  
In Silico ◽  
Site Directed Mutagenesis ◽  
Dynamics Simulation ◽  
Directed Mutagenesis

scholarly journals High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis

2018 ◽  
Author(s):  
Derek M Mason ◽  
Cédric R Weber ◽  
Cristina Parola ◽  
Simon M Meng ◽  
Victor Greiff ◽  
...  
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Mammalian Cells ◽  
Affinity Maturation ◽  
Antibody Engineering ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Variant Discovery ◽  
Novel Variant

ABSTRACTAntibody engineering is performed to improve therapeutic properties by directed evolution, usually by high-throughput screening of phage or yeast display libraries. Engineering antibodies in mammalian cells offers advantages associated with expression in their final therapeutic format (full-length glycosylated IgG), however, the inability to express large and diverse libraries severely limits their potential throughput. To address this limitation, we have developed homology-directed mutagenesis (HDM), a novel method which extends the concept of CRISPR/Cas9-mediated homology-directed repair (HDR). HDM leverages oligonucleotides with degenerate codons to generate site-directed mutagenesis libraries in mammalian cells. By improving HDM efficiency (>35-fold) and combining mammalian display screening with next-generation sequencing (NGS), we validated this approach can be used for key applications in antibody engineering at high-throughput: rational library construction, novel variant discovery, affinity maturation, and deep mutational scanning (DMS). We anticipate that HDM will be a valuable tool for engineering and optimizing antibodies in mammalian cells, and eventually enable directed evolution of other complex proteins and cellular therapeutics.

scholarly journals A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

1994 ◽  
Vol 180 (6) ◽  
pp. 2147-2153 ◽  
Author(s):  
M Pizza ◽  
M R Fontana ◽  
M M Giuliani ◽  
M Domenighini ◽  
C Magagnoli ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cholera Toxin ◽  
Neutralizing Antibodies ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
E Coli ◽  
Heat Labile ◽  
A Subunit ◽  
Adp Ribosyltransferase ◽  
Derivatives Of

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.

scholarly journals Dissecting Human Antibody Responses: Useful, Basic, and Surprising Findings

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. SCI-49-SCI-49
Author(s):  
Antonio Lanzavecchia
Keyword(s):  
B Cell ◽  
Dna Sequences ◽  
Neutralizing Antibodies ◽  
Influenza A ◽  
Somatic Mutations ◽  
Vaccine Design ◽  
Human Memory ◽  
Affinity Maturation ◽  
Effective Vaccine ◽  
Human Antibody

Abstract We use cell culture-based high-throughput methods to interrogate human memory B cell and plasma cell repertoires and to isolate antibodies selected on the basis of their neutralizing potency and breadth. Relevant examples are antibodies that neutralize all influenza A viruses or even four paramyxoviruses. By targeting conserved structures, these broadly neutralizing antibodies are less prone to select escape mutants and are promising candidates for prophylaxis and therapy of infections, as well as tools for vaccine design. The value of a target-agnostic approach to vaccine design is illustrated by our discovery of extremely potent antibodies that neutralize human cytomegalovirus, which led to the identification of their viral ligand, a pentameric complex that was then produced and tested as an effective vaccine. By reconstructing the genealogy trees of specific B cell clones, we investigate the role of somatic mutations in affinity maturation and in generation of antibody variants with broader or different specificity. Somatic mutations can also generate autoantibodies, as found in patients with pemphigus and autoimmune pulmonary alveolar proteinosis. Recently, while searching for antibodies that broadly react with malaria variant antigens, we discovered a new mechanism of antibody diversification, which relies on templated insertions of genomic DNA sequences into immunoglobulin genes, followed by somatic mutations. Disclosures Lanzavecchia: Humabs SA: Equity Ownership, Research Funding.

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