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2022 ◽  
Author(s):  
Fangzhu Zhao ◽  
Celina Keating ◽  
Gabriel Ozorowski ◽  
Namir Shaabani ◽  
Irene M. Francino-Urdaniz ◽  
...  

The rapid spread of SARS-CoV-2 variants poses a constant threat of escape from monoclonal antibody and vaccine countermeasures. Mutations in the ACE2 receptor binding site on the surface S protein have been shown to disrupt antibody binding and prevent viral neutralization. Here, we use a directed evolution-based approach to engineer three neutralizing antibodies for enhanced binding to S protein. The engineered antibodies showed increased in vitro functional activity in terms of neutralization potency and/or breadth of neutralization against viral variants. Deep mutational scanning revealed that higher binding affinity reduced the total number of viral escape mutations. Studies in the Syrian hamster model showed two examples where the affinity matured antibody provided superior protection compared to the parental antibody. These data suggest that monoclonal antibodies for anti-viral indications could benefit from in vitro affinity maturation to reduce viral escape pathways and appropriate affinity maturation in vaccine immunization could help resist viral variation.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Christopher R. Corbeil ◽  
Mahder Seifu Manenda ◽  
Traian Sulea ◽  
Jason Baardsnes ◽  
Marie-Ève Picard ◽  
...  

AbstractThe design of superior biologic therapeutics, including antibodies and engineered proteins, involves optimizing their specific ability to bind to disease-related molecular targets. Previously, we developed and applied the Assisted Design of Antibody and Protein Therapeutics (ADAPT) platform for virtual affinity maturation of antibodies (Vivcharuk et al. in PLoS One 12(7):e0181490, 10.1371/journal.pone.0181490, 2017). However, ADAPT is limited to point mutations of hot-spot residues in existing CDR loops. In this study, we explore the possibility of wholesale replacement of the entire H3 loop with no restriction to maintain the parental loop length. This complements other currently published studies that sample replacements for the CDR loops L1, L2, L3, H1 and H2. Given the immense sequence space theoretically available to H3, we focused on the virtual grafting of over 5000 human germline-derived H3 sequences from the IGMT/LIGM database increasing the diversity of the sequence space when compared to using crystalized H3 loop sequences. H3 loop conformations are generated and scored to identify optimized H3 sequences. Experimental testing of high-ranking H3 sequences grafted into the framework of the bH1 antibody against human VEGF-A led to the discovery of multiple hits, some of which had similar or better affinities relative to the parental antibody. In over 75% of the tested designs, the re-designed H3 loop contributed favorably to overall binding affinity. The hits also demonstrated good developability attributes such as high thermal stability and no aggregation. Crystal structures of select re-designed H3 variants were solved and indicated that although some deviations from predicted structures were seen in the more solvent accessible regions of the H3 loop, they did not significantly affect predicted affinity scores.


2021 ◽  
Author(s):  
Line Ledsgaard ◽  
Andreas H. Laustsen ◽  
Urska Pus ◽  
Jack Wade ◽  
Pedro Villar ◽  
...  

AbstractThe monocled cobra (Naja kaouthia) is one of the most feared snakes in Southeast Asia. It is a highly dangerous species with a potent venom deriving its toxicity predominantly from abundant long-chain α-neurotoxins. The only specific treatment for snakebite envenoming is antivenom, which is based on animal-derived polyclonal antibodies. Despite the lifesaving importance of these medicines over the past 120 years, and their ongoing role in combating snakebite disease, major limitations in safety, supply consistency, and efficacy creates a need for a new generation of improved treatments based on modern biotechnological techniques. Here, we describe the initial discovery and subsequent optimization of a recombinant human monoclonal immunoglobin G (IgG) antibody against α-cobratoxin using phage display technology. Affinity maturation of the parental antibody by light chain-shuffling resulted in an 8-fold increase in affinity, translating to a significant increase in in vitro neutralization potency and in vivo efficacy. While the parental antibody prolonged survival of mice challenged with purified α-cobratoxin, the optimized antibody prevented lethality when incubated with N. kaouthia whole venom prior to intravenous injection. This study is the first to demonstrate neutralization of whole snake venom by a single recombinant monoclonal antibody. Importantly, this suggests that for venoms whose toxicity relies on a single predominant toxin group, such as that of N. kaouthia, as little as one monoclonal antibody may be sufficient to prevent lethality, thus providing a tantalizing prospect of bringing recombinant antivenoms based on human monoclonal or oligoclonal antibodies to the clinic.One Sentence SummaryA recombinant human monoclonal immunoglobulin G antibody, discovered and optimized using in vitro methods, was demonstrated to neutralize the lethal effect of whole venom from the monocled cobra in mice via abrogation of α-neurotoxin-mediated neurotoxicity.


2021 ◽  
Author(s):  
Tyler N Starr ◽  
Nadine Czudnochowski ◽  
Fabrizia Zatta ◽  
Young-Jun Park ◽  
Zhuoming Liu ◽  
...  

An ideal anti-SARS-CoV-2 antibody would resist viral escape, have activity against diverse SARS-related coronaviruses, and be highly protective through viral neutralization and effector functions. Understanding how these properties relate to each other and vary across epitopes would aid development of antibody therapeutics and guide vaccine design. Here, we comprehensively characterize escape, breadth, and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD), including S309, the parental antibody of the late-stage clinical antibody VIR-7831. We observe a tradeoff between SARS-CoV-2 in vitro neutralization potency and breadth of binding across SARS-related coronaviruses. Nevertheless, we identify several neutralizing antibodies with exceptional breadth and resistance to escape, including a new antibody (S2H97) that binds with high affinity to all SARS-related coronavirus clades via a unique RBD epitope centered on residue E516. S2H97 and other escape-resistant antibodies have high binding affinity and target functionally constrained RBD residues. We find that antibodies targeting the ACE2 receptor binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency, but we identify one potent RBM antibody (S2E12) with breadth across sarbecoviruses closely related to SARS-CoV-2 and with a high barrier to viral escape. These data highlight functional diversity among antibodies targeting the RBD and identify epitopes and features to prioritize for antibody and vaccine development against the current and potential future pandemics.


2020 ◽  
Author(s):  
Romain Rouet ◽  
Ohan Mazigi ◽  
Gregory J Walker ◽  
David B Langley ◽  
Meghna Sobti ◽  
...  

Antibodies against coronavirus spike protein potently protect against infection and disease, however it remains unclear if such protection can be extended to variant coronaviruses. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here we explore antibody maturation strategies to change and broaden their specificity, enabling potent binding and neutralization of SARS-CoV-2. Using targeted mutagenesis as well as light chain shuffling on phage, we identified variants with considerably increased affinity and neutralization potential. The most potent antibody, derived from the NIH-developed mAb m396, neutralized live SARS-CoV-2 virus with a half-maximal inhibitory concentration (IC50) of 160 ng/ml. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo- electron microscopy, indicating that a limited set of antibodies can give rise to variants targeting diverse epitopes. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.


Biomolecules ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 919 ◽  
Author(s):  
Jong-Hee Ko ◽  
Hyuk-Sang Kwon ◽  
Bomin Kim ◽  
Gihong Min ◽  
Chorong Shin ◽  
...  

Although bevacizumab (Avastin®) has been approved as an antiangiogenic agent against some cancers, the efficacy is transient and unsatisfactory in other cancers most likely owing to the presence of alternative proangiogenic factors. Therefore, simultaneous blocking of several proangiogenic factors may be a promising strategy for antiangiogenic cancer therapeutics. Accordingly, neuropilin-1 (NRP1) is an attractive target because it serves as a multifunctional receptor for the vascular endothelial growth factor (VEGF) family. Here, we aimed to generate and test an anti-VEGFA and anti-NRP1 dual-targeting bispecific antibody (named as IDB0076) by genetic fusion of an NRP1-targeting peptide to the C-terminus of the bevacizumab heavy chain. Similar to the parental antibody (bevacizumab), IDB0076 suppressed VEGFA-induced migration of human endothelial cells. In contrast, IDB0076 inhibited endothelial-cell migration induced by other angiogenesis growth factors and manifested a more potent antitumor activity than that of bevacizumab in a murine tumor xenograft model. When toxicity was preliminarily evaluated in cynomolgus monkeys, IDB0076 showed no substantial adverse effects, e.g., the absence of noticeable nephrotoxicity, which has previously been documented for the combination therapy of bevacizumab and an anti-NRP1 antibody. Thus, VEGFA-and-NRP1 dual-targeting bispecific antibody IDB0076 may be a potent and safe anticancer agent worthy of further preclinical and clinical studies.


2020 ◽  
Vol 48 (10) ◽  
pp. 5281-5293 ◽  
Author(s):  
Alex R Nanna ◽  
Alexander V Kel’in ◽  
Christopher Theile ◽  
Justin M Pierson ◽  
Zhi Xiang Voo ◽  
...  

Abstract Gene silencing by RNA interference (RNAi) has emerged as a powerful treatment strategy across a potentially broad range of diseases. Tailoring siRNAs to silence genes vital for cancer cell growth and function could be an effective treatment, but there are several challenges which must be overcome to enable their use as a therapeutic modality, among which efficient and selective delivery to cancer cells remains paramount. Attempts to use antibodies for siRNA delivery have been reported but these strategies use either nonspecific conjugation resulting in mixtures, or site-specific methods that require multiple steps, introduction of mutations, or use of enzymes. Here, we report a method to generate antibody–siRNA (1:2) conjugates (ARCs) that are structurally defined and easy to assemble. This ARC platform is based on engineered dual variable domain (DVD) antibodies containing a natural uniquely reactive lysine residue for site-specific conjugation to β-lactam linker-functionalized siRNA. The conjugation is efficient, does not compromise the affinity of the parental antibody, and utilizes chemically stabilized siRNA. For proof-of-concept, we generated DVD-ARCs targeting various cell surface antigens on multiple myeloma cells for the selective delivery of siRNA targeting β-catenin (CTNNB1). A set of BCMA-targeting DVD-ARCs at concentrations as low as 10 nM revealed significant CTNNB1 mRNA and protein knockdown.


Retrovirology ◽  
2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Tumelo Moshoette ◽  
Stuart Alvaro Ali ◽  
Maria Antonia Papathanasopoulos ◽  
Mark Andrew Killick

Abstract The existing repertoire of HIV-1 patient derived broadly neutralising antibodies (bNAbs) that target the HIV-1 envelope glycoprotein (Env) present numerous and exciting opportunities for immune-based therapeutic and preventative strategies against HIV-1. Combination antibody therapy is required to ensure greater neutralization coverage and limit Env mediated escape mutations following treatment pressure. Engineered bispecific bNAbs (bibNAbs) assimilate the advantages of combination therapy into a single antibody molecule with several configurations reporting potency enhancement as a result of the increased avidity and simultaneous engagement of targeted epitopes. We report the engineering of a novel bibNAb (iMab-CAP256) comprising the highly potent, CAP256.VRC26.25 bNAb with anticipated extension in neutralization coverage through pairing with the host directed, anti-CD4 antibody, ibalizumab (iMab). Recombinant expression of parental monoclonal antibodies and the iMab-CAP256 bibNAb was performed in HEK293T (Human embryonic kidney 293 T antigen) cells, purified to homogeneity by Protein-A affinity chromatography followed by size exclusion chromatography. Antibody assembly and binding functionality of Fab moieties was confirmed by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and ELISA, respectively. Breadth and potency were evaluated against a geographical diverse HIV-1 pseudovirus panel (n = 20). Overall, iMab-CAP256 demonstrated an expanded neutralizing coverage, neutralizing single, parental antibody resistant pseudovirus strains and an enhanced neutralization potency against all dual sensitive strains (average fold increase over the more potent parental antibody of 11.4 (range 2 to 31.8). Potency enhancement was not observed for the parental antibody combination treatment (iMab + CAP256) suggesting the presence of a synergistic relationship between the CAP256 and iMab paratope combination in this bibNAb configuration. In addition, iMab-CAP256 bibNAbs exhibited comparable efficacy to other bibNAbs PG9-iMab and 10E08-iMab previously reported in the literature. The enhanced neutralization coverage and potency of iMAb-CAP256 over the parental bNAbs should facilitate superior clinical performance as a therapeutic or preventative strategy against HIV-1.


2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Aimin Tang ◽  
Zhifeng Chen ◽  
Kara S. Cox ◽  
Hua-Poo Su ◽  
Cheryl Callahan ◽  
...  

Abstract Respiratory syncytial virus (RSV) infection is the leading cause of hospitalization and infant mortality under six months of age worldwide; therefore, the prevention of RSV infection in all infants represents a significant unmet medical need. Here we report the isolation of a potent and broadly neutralizing RSV monoclonal antibody derived from a human memory B-cell. This antibody, RB1, is equipotent on RSV A and B subtypes, potently neutralizes a diverse panel of clinical isolates in vitro and demonstrates in vivo protection. It binds to a highly conserved epitope in antigenic site IV of the RSV fusion glycoprotein. RB1 is the parental antibody to MK-1654 which is currently in clinical development for the prevention of RSV infection in infants.


2019 ◽  
Vol 116 (32) ◽  
pp. 15889-15894 ◽  
Author(s):  
Yuhan Zhang ◽  
Changming Fang ◽  
Rongsheng E. Wang ◽  
Ying Wang ◽  
Hui Guo ◽  
...  

To direct checkpoint inhibition to the tumor microenvironment, while avoiding systemic immune activation, we have synthesized a bispecific antibody [norleucine4, d-Phe7]-melanocyte stimulating hormone (NDP-MSH)-antiprogrammed cell death-ligand 1 antibody (αPD-L1) by conjugating a melanocyte stimulating hormone (α-MSH) analog to the antiprogrammed cell death-ligand 1 to (αPD-L1) antibody avelumab. This bispecific antibody can bind to both the melanocortin-1 receptor (MC1R) and to PD-L1 expressed on melanoma cells and shows enhanced specific antitumor efficacy in a syngeneic B16-SIY melanoma mouse model compared with the parental antibody at a 5 mg/kg dose. Moreover, the bispecific antibody showed increased infiltrated T cells in the tumor microenvironment. These results suggest that a tumor-targeted PD-L1-blocking bispecific antibody could have a therapeutic advantage in vivo, especially when used in combination with other checkpoint inhibitors.


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