Neutralization of typhoid toxin by alpaca-derived, single-domain antibodies targeting the PltB and CdtB subunits

2021 ◽  
Author(s):  
Hari P. Dulal ◽  
David J. Vance ◽  
Durga P. Neupane ◽  
Xiangcheng Chen ◽  
Jacqueline M. Tremblay ◽  
...  
Keyword(s):  
Lethal Dose ◽  
Bacterial Pathogen ◽  
Salmonella Typhi ◽  
Single Domain ◽  
Single Domain Antibody ◽  
Function Defect ◽  
Single Domain Antibodies ◽  
Typhoid Toxin

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.

scholarly journals Mechanisms of substrate recognition by a typhoid toxin secretion-associated muramidase

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Tobias Geiger ◽  
Maria Lara-Tejero ◽  
Yong Xiong ◽  
Jorge E Galán
Keyword(s):  
Substrate Recognition ◽  
Bacterial Pathogen ◽  
Salmonella Typhi ◽  
Intracellular Bacteria ◽  
Atomic Structures ◽  
Toxin Secretion ◽  
Typhoid Toxin ◽  
Close Homolog

Typhoid toxin is a virulence factor for the bacterial pathogen Salmonella Typhi, which causes typhoid fever in humans. After its synthesis by intracellular bacteria, typhoid toxin is secreted into the lumen of the Salmonella-containing vacuole by a secretion mechanism strictly dependent on TtsA, a specific muramidase that facilitates toxin transport through the peptidoglycan layer. Here we show that substrate recognition by TtsA depends on a discrete domain within its carboxy terminus, which targets the enzyme to the bacterial poles to recognize YcbB-edited peptidoglycan. Comparison of the atomic structures of TtsA bound to its substrate and that of a close homolog with different specificity identified specific determinants involved in substrate recognition. Combined with structure-guided mutagenesis and in vitro and in vivo crosslinking experiments, this study provides an unprecedented view of the mechanisms by which a muramidase recognizes its peptidoglycan substrate to facilitate protein secretion.

scholarly journals Single Domain Antibodies as Carriers for Intracellular Drug Delivery: A Proof of Principle Study

Biomolecules ◽  
2021 ◽  
Vol 11 (7) ◽  
pp. 927
Author(s):  
Sebas D. Pronk ◽  
Erik Schooten ◽  
Jurgen Heinen ◽  
Esra Helfrich ◽  
Sabrina Oliveira ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Targeted Delivery ◽  
Single Domain ◽  
Drug Conjugates ◽  
Intracellular Drug Delivery ◽  
Internalization Rate ◽  
Single Domain Antibodies ◽  
Different Characteristics

Antibody-drug conjugates (ADCs) are currently used for the targeted delivery of drugs to diseased cells, but intracellular drug delivery and therefore efficacy may be suboptimal because of the large size, slow internalization and ineffective intracellular trafficking of the antibody. Using a phage display method selecting internalizing phages only, we developed internalizing single domain antibodies (sdAbs) with high binding affinity to rat PDGFRβ, a receptor involved in different types of diseases. We demonstrate that these constructs have different characteristics with respect to internalization rates but all traffic to lysosomes. To compare their efficacy in targeted drug delivery, we conjugated the sdAbs to a cytotoxic drug. The conjugates showed improved cytotoxicity correlating to their internalization speed. The efficacy of the conjugates was inhibited in the presence of vacuolin-1, an inhibitor of lysosomal maturation, suggesting lysosomal trafficking is needed for efficient drug release. In conclusion, sdAb constructs with different internalization rates can be designed against the same target, and sdAbs with a high internalization rate induce more cell killing than sdAbs with a lower internalization rate in vitro. Even though the overall efficacy should also be tested in vivo, sdAbs are particularly interesting formats to be explored to obtain different internalization rates.

scholarly journals Single-Domain Antibodies for Targeting, Detection, and In Vivo Imaging of Human CD4+ Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Bjoern Traenkle ◽  
Philipp D. Kaiser ◽  
Stefania Pezzana ◽  
Jennifer Richardson ◽  
Marius Gramlich ◽  
...  
Keyword(s):  
T Cell ◽  
Immune Cell ◽  
Inflammatory Diseases ◽  
Xenograft Model ◽  
Single Domain ◽  
Positron Emission ◽  
Depth Analysis ◽  
Single Domain Antibodies

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.

scholarly journals Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71

2020 ◽  
Vol 64 (5) ◽  
Author(s):  
Peng-Nien Huang ◽  
Hsiang-Ching Wang ◽  
Hui-Chen Hung ◽  
Sung-Nien Tseng ◽  
Teng-Yuan Chang ◽  
...  
Keyword(s):  
Emerging Infectious Diseases ◽  
Conformational Epitope ◽  
Single Domain ◽  
Therapeutic Interventions ◽  
Asia Pacific ◽  
Single Domain Antibody ◽  
Domain Antibody ◽  
Enterovirus A71

ABSTRACT In the past few decades, enterovirus A71 (EVA71) has caused devastating outbreaks in the Asia-Pacific region, resulting in serious sequelae in infected young children. No preventive or therapeutic interventions are currently available for curing EVA71 infection, highlighting a great unmet medical need for this disease. Here, we showed that one novel single-domain antibody (sdAb), F1, isolated from an immunized llama, could alleviate EVA71 infection both in vitro and in vivo. We also confirmed that the sdAb clone F1 recognizes EVA71 through a novel conformational epitope comprising the highly conserved region of VP3 capsid protein by using competitive-binding and overlapping-peptide enzyme-linked immunosorbent assays (ELISAs). Because of the virion’s icosahedral structure, we reasoned that adjacent epitopes must be clustered within molecular ranges that may be simultaneously bound by an engineered antibody with multiple valency. Therefore, two single-domain binding modules (F1) were fused to generate an sdAb-in-tandem design so that the capture of viral antigens could be further increased by valency effects. We showed that the tetravalent construct F1×F1-hFc, containing two sdAb-in-tandem on a fragment crystallizable (Fc) scaffold, exhibits more potent neutralization activity against EVA71 than does the bivalent sdAb F1-hFc by at least 5.8-fold. We also demonstrated that, using a human scavenger receptor class B member 2 (hSCARB2) transgenic mouse model, a half dose of the F1×F1-hFc provided better protection against EVA71 infection than did the F1-hFc. Thus, our study furnishes important insights into multivalent sdAb engineering against viral infection and provides a novel strategic deployment approach for preparedness of emerging infectious diseases such as EVA71.

scholarly journals Single-domain antibodies for targeting, detection and in vivo imaging of human CD4+ cells

2021 ◽  
Author(s):  
Bjoern Traenkle ◽  
Philipp D. Kaiser ◽  
Stefania Pezzana ◽  
Jennifer Richardson ◽  
Marius Gramlich ◽  
...  
Keyword(s):  
In Vivo Imaging ◽  
Immune Cell ◽  
Inflammatory Diseases ◽  
Xenograft Model ◽  
Single Domain ◽  
High Signal ◽  
Cd4 Cells ◽  
Single Domain Antibodies

The advancement of new immunotherapies for the treatment of cancers, infections, immune-mediated inflammatory diseases, and autoimmune diseases necessitates the co-development of appropriate probes to detect and monitor the distribution and infiltration of distinct immune cell populations. Considering the key role of CD4+ T cells in regulating immunological processes, we have developed a set of novel single-domain antibodies (nanobodies, Nbs) that specifically recognize the human CD4 co-receptor in its native state on various CD4+ cells. Following detailed characterization of binding properties, epitope mapping, and site-directed functionalization, we selected biologically inert Nbs that do not affect T cell proliferation or cytokine expression in vitro. We used fluorescently labeled Nbs to track the presence and location of CD4+ cells in a xenograft model, demonstrating a high signal-to-background ratio by in vivo optical imaging. In summary, this study reports for the first time the generation and application of human CD4-specific Nbs for the detection and in vivo imaging of CD4+ cells in a preclinical animal model. We anticipate that the Nbs presented in this study will be versatile probes, e.g. in immunoPET imaging for patient stratification and for monitoring individual immune responses during personalized immunotherapy.

scholarly journals Generation of a safe and efficacious llama single-domain antibody fragment (vHH) targeting the membrane-proximal region of 4-1BB for engineering therapeutic bispecific antibodies for cancer

2021 ◽  
Vol 9 (6) ◽  
pp. e002131
Author(s):  
Tianhang Zhai ◽  
Chao Wang ◽  
Yifeng Xu ◽  
Weifeng Huang ◽  
Zhijun Yuan ◽  
...  
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Single Domain ◽  
Single Domain Antibody ◽  
X Ray ◽  
X Ray Crystallography ◽  
Domain Antibody ◽  
Cell Assays

BackgroundThe discovery of checkpoint inhibitors towards cytotoxic T-lymphocyte protein 4 (CTLA-4) and programmed cell death protein 1 (PD-1) has been revolutionary for the treatment of cancers. These therapies have only offered an average of 20%–30% response rates across the tumor spectrum and the combination of agonists towards the tumor-necrosis superfamily members, such as 4-1BB and CD40, has shown potent efficacy in preclinical studies; however, these agonists have exhibited high degrees of toxicity with limited efficacy in human trials. In this study, we have generated a single-domain antibody towards a unique epitope of 4-1BB that limits its potential on-target toxicity while maintaining sufficient potency. This 4-1BB binder is ideal for use in the engineering of multispecific antibodies to localize 4-1BB activation within the tumor microenvironment, as shown here by a anti-PD-L1/4-1BB bispecific candidate (PM1003).MethodsTo determine the functional activity of the 4-1BB- and PD-L1-binding elements of PM1003, in vitro luciferase reporter and primary cell assays were used to test the potency of programmed cell death 1 ligand 1 (PD-L1) blockade and PD-L1-mediated 4-1BB activation via cross-bridging. X-ray crystallography was conducted to resolve the binding epitopes of the respective binding arms, and accurate binding kinetics were determined using standard affinity measurement techniques. Human 4-1BB and/or PD-L1 knock-in mice were used in cancer models for testing the in vivo antitumor efficacy of PM1003, and safety was evaluated further.ResultsPM1003 shows potent activation of 4-1BB and blockade of PD-L1 in cell-based assays. 4-1BB activation was exerted through the bridging of PD-L1 on target cells and 4-1BB on effector cells. No PD-L1-independent activation of 4-1BB was observed. Through X-ray crystallography, a unique binding epitope in the cysteine-rich domain 4 (CRD4) region was resolved that provides high potency and potentially low on-target toxicity as determined by primary immune cell assays and toxicity evaluation in vivo.ConclusionsA unique single-domain antibody was discovered that binds to the CRD4 domain of 4-1BB. When incorporated into a 4-1BB/PD-L1 bispecific (PM1003), we have shown the potent inhibition of PD-L1 activity with 4-1BB agonism upon cross-bridging with PD-L1 in vitro. Antitumor activity with minimal toxicity was found in vivo. Thus, PM1003 is a uniquely differentiating and next generation therapeutic agent for cancer therapy.

scholarly journals Comparative studies on Salmonella typhi grown in vivo and in vitro I. Virulence, toxicity, production of infection-promoting substances and DPN-ase activity

1963 ◽  
Vol 61 (1) ◽  
pp. 1-20 ◽  
Author(s):  
A. L. Olitzki ◽  
Dina Godinger
Keyword(s):  
Antibody Production ◽  
Comparative Studies ◽  
Lethal Dose ◽  
Sodium Taurocholate ◽  
Salmonella Typhi ◽  
Agar Gel ◽  
Bacterial Antigens ◽  
Lethal Doses

1. The virulence of S. typhi strains Ty 2 and O 901 injected intra-abdominally into white mice was examined. The lethal dose of strain Ty 2 grown in vivo was lower than that of the corresponding culture grown in vitro, while the lethal doses of the in vivo- and in vitro-grown strain O 901 were almost identical.2. The extracts of infected livers and spleens were not toxic, but acted as infection-promoting substances. Extracts from normal organs exerted similar but weaker effects. Glycogen together with sodium taurocholate were powerful infection-promoting substances. The highest increases of virulence were observed when spleen-grown bacteria together with extracts from infected organs were injected.3. Extracts from infected spleen, pancreas, stomach and lower intestines, spleen-grown S. typhi, other spleen-grown Enterobacteriaceae, and bacteria taken from the intestines of normal and diseased animals, exhibited high DPN-ase activity.4. The extracts from infected organs contained soluble bacterial antigens. Their presence was demonstrated by tube precipitation, agar gel precipitation and antibody production after immunization of rabbits with extracts from infected organs with addition of adjuvants.5. Spleen-grown bacteria were able to absorb host antigens and to produce, in addition to the known agglutinating antibodies, anti-spleen precipitins.

scholarly journals Single-domain antibody screening by isPLA-seq

2021 ◽  
Vol 5 (1) ◽  
pp. e202101115
Author(s):  
Yueyuan Yin ◽  
Fei Yan ◽  
Ruimin Zhou ◽  
Mingchen Li ◽  
Jinyi Ma ◽  
...  
Keyword(s):  
Single Domain ◽  
Proximity Ligation Assay ◽  
Single Domain Antibody ◽  
Cellular Processes ◽  
Proximity Ligation ◽  
Domain Antibody ◽  
Functional Measure ◽  
Potential Applications

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.

scholarly journals Diagnostic nanoparticle targeting of the EGF-receptor in complex biological conditions using single-domain antibodies

Nanoscale ◽  
2014 ◽  
Vol 6 (11) ◽  
pp. 6046-6056 ◽  
Author(s):  
K. Zarschler ◽  
K. Prapainop ◽  
E. Mahon ◽  
L. Rocks ◽  
M. Bramini ◽  
...  
Keyword(s):  
Egf Receptor ◽  
Single Domain ◽  
Single Domain Antibodies

Nanoparticles functionalized with single domain antibodies are shown to specifically target the EGF receptor in vitro. We investigate the effects on uptake and specificity when increasing the environmental serum toward more in vivo "realistic" concentrations.

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