single domain antibodies
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2022 ◽  
Vol 18 (1) ◽  
pp. e1010169
Author(s):  
Kwok-ho Lam ◽  
Jacqueline M. Tremblay ◽  
Kay Perry ◽  
Konstantin Ichtchenko ◽  
Charles B. Shoemaker ◽  
...  

Botulinum neurotoxins (BoNTs) are among the deadliest of bacterial toxins. BoNT serotype A and B in particular pose the most serious threat to humans because of their high potency and persistence. To date, there is no effective treatment for late post-exposure therapy of botulism patients. Here, we aim to develop single-domain variable heavy-chain (VHH) antibodies targeting the protease domains (also known as the light chain, LC) of BoNT/A and BoNT/B as antidotes for post-intoxication treatments. Using a combination of X-ray crystallography and biochemical assays, we investigated the structures and inhibition mechanisms of a dozen unique VHHs that recognize four and three non-overlapping epitopes on the LC of BoNT/A and BoNT/B, respectively. We show that the VHHs that inhibit the LC activity occupy the extended substrate-recognition exosites or the cleavage pocket of LC/A or LC/B and thus block substrate binding. Notably, we identified several VHHs that recognize highly conserved epitopes across BoNT/A or BoNT/B subtypes, suggesting that these VHHs exhibit broad subtype efficacy. Further, we identify two novel conformations of the full-length LC/A, that could aid future development of inhibitors against BoNT/A. Our studies lay the foundation for structure-based engineering of protein- or peptide-based BoNT inhibitors with enhanced potencies and cross-subtypes properties.


Author(s):  
Paula Segovia-de los Santos ◽  
Pedro Quintero-Campos ◽  
Sergi Morais ◽  
Cesar Echaides ◽  
Ángel Maquieira ◽  
...  

2021 ◽  
Author(s):  
Jessica Krakow ◽  
Michal Hammel ◽  
Ying Zhu ◽  
Brian J Hillier ◽  
Bryce Paolella ◽  
...  

Abstract Background COBRA™ (COnditional Bispecific Redirected Activation) T-cell engagers are designed to target solid tumors as a single polypeptide chain prodrug that becomes activated by proteolysis in the tumor microenvironment. One COBRA molecule comprises seven Ig domains: three single-domain antibodies (sdAbs) recognizing a tumor target or human serum albumin (HSA), and CD3ε-binding VH and VL and their inactivated counterparts, VHi and VLi. Pairing of VH and VL, and VLi and VHi, into scFvs is prevented by shortened inter-domain linkers. Instead, VH and VL are expected to interact with VLi and VHi, respectively, thus making a diabody whose binding to CD3ε on the T-cells is impaired. Methods We analyzed the structure of an EGFR COBRA in solution using negative stain electron microscopy (EM) and small-angle X-ray scattering (SAXS). Results We found that this EGFR COBRA forms stable monomers with a very dynamic interdomain arrangement. At most, only five domains at a time appeared ordered, and only one VH-VL pair was found in the Fv orientation. Non-enzymatic post-translational modifications suggest that the CDR3 loops in the VL-VHi pair are exposed but are buried in the VH-VLi pair. The MMP9 cleavage rate of the prodrug when bound to recombinant EGFR or HSA is not affected, indicating positioning of the MMP9-cleavable linker away from the EGFR and HSA binding sites. Conclusion Here we propose a model for EGFR COBRA where VH and VLi form an Fv, and VL and VHi do not, possibly interacting with other Ig domains. SAXS and MMP9 cleavage analyses suggest that all COBRA molecules tested have a similar structural architecture.


2021 ◽  
Author(s):  
Hari P. Dulal ◽  
David J. Vance ◽  
Durga P. Neupane ◽  
Xiangcheng Chen ◽  
Jacqueline M. Tremblay ◽  
...  

Typhoid toxin is secreted by the typhoid fever-causing bacterial pathogen Salmonella Typhi and has tropism for immune cells and brain endothelial cells. Here, we generated a camelid single domain antibody (VHH) library from typhoid toxoid-immunized alpacas and identified 41 VHHs selected on the glycan-receptor binding PltB and nuclease CdtB. VHHs exhibiting potent in vitro neutralizing activities from each sequence-based family were epitope binned via competition ELISAs, leading to 6 distinct VHHs, two anti-PltB (T2E7 and T2G9) and four anti-CdtB VHHs (T4C4, T4C12, T4E5, and T4E8), whose in vivo neutralizing activities and associated toxin neutralizing mechanisms were investigated. We found that T2E7, T2G9, and T4E5 effectively neutralized typhoid toxin in vivo , as demonstrated by 100% survival of mice administered a lethal-dose of typhoid toxin and with little to no typhoid toxin-mediated upper motor function defect. Cumulatively, these results highlight the potential of the compact antibodies to neutralize typhoid toxin by targeting the glycan-binding and/or nuclease subunits.


2021 ◽  
Vol 12 ◽  
Author(s):  
Bjoern Traenkle ◽  
Philipp D. Kaiser ◽  
Stefania Pezzana ◽  
Jennifer Richardson ◽  
Marius Gramlich ◽  
...  

The advancement of new immunotherapies necessitates appropriate probes to monitor the presence and distribution of distinct immune cell populations. Considering the key role of CD4+ cells in regulating immunological processes, we generated novel single-domain antibodies [nanobodies (Nbs)] that specifically recognize human CD4. After in-depth analysis of their binding properties, recognized epitopes, and effects on T-cell proliferation, activation, and cytokine release, we selected CD4-specific Nbs that did not interfere with crucial T-cell processes in vitro and converted them into immune tracers for noninvasive molecular imaging. By optical imaging, we demonstrated the ability of a high-affinity CD4-Nb to specifically visualize CD4+ cells in vivo using a xenograft model. Furthermore, quantitative high-resolution immune positron emission tomography (immunoPET)/MR of a human CD4 knock-in mouse model showed rapid accumulation of 64Cu-radiolabeled CD4-Nb1 in CD4+ T cell-rich tissues. We propose that the CD4-Nbs presented here could serve as versatile probes for stratifying patients and monitoring individual immune responses during personalized immunotherapy in both cancer and inflammatory diseases.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Obinna C. Ubah ◽  
Eric W. Lake ◽  
Gihan S. Gunaratne ◽  
Joseph P. Gallant ◽  
Marie Fernie ◽  
...  

AbstractSingle-domain Variable New Antigen Receptors (VNARs) from the immune system of sharks are the smallest naturally occurring binding domains found in nature. Possessing flexible paratopes that can recognize protein motifs inaccessible to classical antibodies, VNARs have yet to be exploited for the development of SARS-CoV-2 therapeutics. Here, we detail the identification of a series of VNARs from a VNAR phage display library screened against the SARS-CoV-2 receptor binding domain (RBD). The ability of the VNARs to neutralize pseudotype and authentic live SARS-CoV-2 virus rivalled or exceeded that of full-length immunoglobulins and other single-domain antibodies. Crystallographic analysis of two VNARs found that they recognized separate epitopes on the RBD and had distinctly different mechanisms of virus neutralization unique to VNARs. Structural and biochemical data suggest that VNARs would be effective therapeutic agents against emerging SARS-CoV-2 mutants, including the Delta variant, and coronaviruses across multiple phylogenetic lineages. This study highlights the utility of VNARs as effective therapeutics against coronaviruses and may serve as a critical milestone for nearing a paradigm shift of the greater biologic landscape.


Antibodies ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 47
Author(s):  
Arghavan Soleimanizadeh ◽  
Heiko Dinter ◽  
Katharina Schindowski

IgG antibodies are some of the most important biopharmaceutical molecules with a high market volume. In spite of the fact that clinical therapies with antibodies are broadly utilized in oncology, immunology and hematology, their delivery strategies and biodistribution need improvement, their limitations being due to their size and poor ability to penetrate into tissues. In view of their small size, there is a rising interest in derivatives, such as single-domain antibodies and single-chain variable fragments, for clinical diagnostic but also therapeutic applications. Smaller antibody formats combine several benefits for clinical applications and can be manufactured at reduced production costs compared with full-length IgGs. Moreover, such formats have a relevant potential for targeted drug delivery that directs drug cargo to a specific tissue or across the blood–brain barrier. In this review, we give an overview of the challenges for antibody drug delivery in general and focus on intranasal delivery to the central nervous system with antibody formats of different sizes.


2021 ◽  
Author(s):  
Irina A Favorskaya ◽  
Dmitry V Shcheblyakov ◽  
Ilias B Esmagambetov ◽  
Inna V Dolzhikova ◽  
Irina A Alekseeva ◽  
...  

Virus-neutralizing antibodies are one of the few treatment options for COVID-19. The evolution of SARS-CoV-2 virus has led to the emergence of virus variants with reduced sensitivity to some antibody-based therapies. The development of potent antibodies with a broad spectrum of neutralizing activity is urgently needed. Here we isolated a panel of single-domain antibodies that specifically bind to the receptor-binding domain of SARS-CoV-2 S glycoprotein. Three of the selected antibodies exhibiting most robust neutralization potency were used to generate dimeric molecules. We observed that these modifications resulted in up to a 200-fold increase in neutralizing activity. The most potent heterodimeric molecule efficiently neutralized each of SARS-CoV-2 variant of concern, including Alpha, Beta, Gamma and Delta variants. This heterodimeric molecule could be a promising drug candidate for a treatment for COVID-19 caused by virus variants of concern.


Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2214
Author(s):  
Qilong Tang ◽  
Raymond J. Owens ◽  
James H. Naismith

Nanobodies are 130 amino acid single‑domain antibodies (VHH) derived from the unique heavy-chain-only subclass of Camelid immunogloblins. Their small molecular size, facile expression, high affinity and stability have combined to make them unique targeting reagents with numerous applications in the biomedical sciences. The first nanobody agent has now entered the clinic as a treatment against a blood disorder. The spread of the SARS-CoV-2 virus has seen the global scientific endeavour work to accelerate the development of technologies to try to defeat a pandemic that has now killed over four million people. In a remarkably short period of time, multiple studies have reported nanobodies directed against the viral Spike protein. Several agents have been tested in culture and demonstrate potent neutralisation of the virus or pseudovirus. A few agents have completed animal trials with very encouraging results showing their potential for treating infection. Here, we discuss the structural features that guide the nanobody recognition of the receptor binding domain of the Spike protein of SARS-CoV-2.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Q. Lecocq ◽  
P. Debie ◽  
J. Puttemans ◽  
R. M. Awad ◽  
L. De Beck ◽  
...  

AbstractRecent advancements in the field of immune-oncology have led to a significant increase in life expectancy of patients with diverse forms of cancer, such as hematologic malignancies, melanoma and lung cancer. Unfortunately, these encouraging results are not observed in the majority of patients, who remain unresponsive and/or encounter adverse events. Currently, researchers are collecting more insight into the cellular and molecular mechanisms that underlie these variable responses. As an example, the human lymphocyte activation gene-3 (huLAG-3), an inhibitory immune checkpoint receptor, is increasingly studied as a therapeutic target in immune-oncology. Noninvasive molecular imaging of the immune checkpoint programmed death protein-1 (PD-1) or its ligand PD-L1 has shown its value as a strategy to guide and monitor PD-1/PD-L1-targeted immune checkpoint therapy. Yet, radiotracers that allow dynamic, whole body imaging of huLAG-3 expression are not yet described. We here developed single-domain antibodies (sdAbs) that bind huLAG-3 and showed that these sdAbs can image huLAG-3 in tumors, therefore representing promising tools for further development into clinically applicable radiotracers.


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