Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering

Lüdel, Frank; Bufe, Sandra; Bleymüller, Willem M.; Jonge, Hugo de; Iamele, Luisa; Niemann, Hartmut H.; Hellweg, Thomas

doi:10.3390/antib8040048

Cited by 7 publications

(3 citation statements)

References 60 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The greatest density occurred at low molecular weights, suggesting that these scaffolds offered enhanced flexibility around the other design parameters. Named scaffolds were plotted as follows: nanobody (Nb – 15 kDa), scFv (27 kDa), Fab (50 kDa), F(ab’) 2 (100 kDa), IgG (150 kDa), nanobody dimer (Nb-Nb – 30 kDa), Diabody (scFv dimer - 54 kDa (Lüdel et al, 2019)).…”

Section: Resultsmentioning

confidence: 99%

Global parameter optimisation and sensitivity analysis of antivenom pharmacokinetics and pharmacodynamics

Morris

Blee

Hauert

2023

Preprint

View full text Add to dashboard Cite

In recent years it has become possible to design snakebite antivenoms with diverse pharmacokinetic properties. Owing to the pharmacokinetic variability of venoms, the choice of antivenom scaffold may influence a treatments neutralisation coverage. Computation offers a useful medium through which to assess the pharmacokinetics and pharmacodynamics of envenomation-treatment systems, as antivenoms with identical neutralising capacities can be simulated. In this study, we simulate envenomation and treatment with a variety of antivenoms, to define the properties of effective antivenoms. Systemic envenomation and treatment were described using a two-compartment pharmacokinetic model. Treatment of Naja sumatrana and Cryptelytrops purpureomaculatus envenomation was simulated with a set of 200,000 theoretical antivenoms across 10 treatment time delays. These two venoms are well-characterised and have differing pharmacokinetic properties. The theoretical antivenom set varied across molecular weight, dose, kon, koff, and valency. The best and worst treatments were identified using an area under the curve metric, and a global sensitivity analysis was performed to quantify the influence of the input parameters on treatment outcome. The simulations show that scaffolds of diverse molecular formats can be effective. Molecular weight and valency have a negligible direct impact on treatment outcome, however low molecular weight scaffolds offer more flexibility across the other design parameters, particularly when treatment is delayed. The simulations show kon to primarily mediate treatment efficacy, with rates above 10*5 M-1s-1 required for the most effective treatments. koff has the greatest impact on the performance of less effective scaffolds. While the same scaffold preferences for improved treatment are seen for both model snakes, the parameter bounds for C. purpureomaculatus envenomation are more constrained. This paper establishes a computational framework for the optimisation of antivenom design.

show abstract

Section: Resultsmentioning

confidence: 99%

Global parameter optimisation and sensitivity analysis of antivenom pharmacokinetics and pharmacodynamics

Morris

Blee

Hauert

2023

Preprint

View full text Add to dashboard Cite

show abstract

“… 36 They show a higher binding affinity and structural stability compared to single chain F v (scFvs). 37 Another strength of diabodies is their bivalency, which can be used to induce dimerization of target proteins 38 , 39 and allows for the development of bispecific diabodies 40 , 41 , 42 , 43 , 44 , 45 , 46 : Several formats have been developed, including Tandem Diabodies, 47 BiTE, 48 and DARTs. 49 , 50 Among these, the DART Blinatumomab by Amgen was approved in 2014 by the FDA.…”

Section: Diabody Structurementioning

confidence: 99%

Cross‐linking disulfide bonds govern solution structures of diabodies

et al. 2023

View full text Add to dashboard Cite

In the last years, antibodies have emerged as a promising new class of therapeutics, due to their combination of high specificity with long serum half‐life and low risk of side‐effects. Diabodies are a popular novel antibody format, consisting of two Fv domains connected with short linkers. Like IgG antibodies, they simultaneously bind two target proteins. However, they offer altered properties, given their smaller size and higher rigidity. In this study, we conducted the—to our knowledge—first molecular dynamics (MD) simulations of diabodies and find a surprisingly high conformational flexibility in the relative orientation of the two Fv domains. We observe rigidifying effects through the introduction of disulfide bonds in the Fv–Fv interface and characterize the effect of different disulfide bond locations on the conformation. Additionally, we compare VH–VL orientations and paratope dynamics between diabodies and an antigen binding fragment (Fab) of the same sequence. We find mostly consistent structures and dynamics, indicating similar antigen binding properties. The most significant differences can be found within the CDR‐H2 loop dynamics. Of all CDR loops, the CDR‐H2 is located closest to the artificial Fv–Fv interface. All examined diabodies show similar VH–VL orientations, Fv–Fv packing and CDR loop conformations. However, the variant with a P14C‐K64C disulfide bond differs most from the Fab in our measures, including the CDR‐H3 loop conformational ensemble. This suggests altered antigen binding properties and underlines the need for careful validation of the disulfide bond locations in diabodies.

show abstract

“…ScFvs are composed of the variable regions of the antibody heavy and light chains (V H and V L domains) connected by a flexible peptide linker, and they represent the smallest engineered antibody fragment containing the parental specificity. ScFvs can display significant inter-domain flexibility between V H and V L domains 13 and have a tendency to form dimers and higher order oligomers depending on the length of the linker peptide 14 – 16 . However, the identification and characterization of a metal-dependent tetramerization motif utilizing a polyhistidine sequence in scFvs represents a novel and unexplored area of research.…”

Section: Introductionmentioning

confidence: 99%

Structure, dynamics and transferability of the metal-dependent polyhistidine tetramerization motif TetrHis for single-chain Fv antibodies

et al. 2023

View full text Add to dashboard Cite

The polyhistidine (6XHis) motif is one of the most ubiquitous protein purification tags. The 6XHis motif enables the binding of tagged proteins to various metals, which can be advantageously used for purification with immobilized metal affinity chromatography. Despite its popularity, protein structures encompassing metal-bound 6XHis are rare. Here, we obtained a 2.5 Å resolution crystal structure of a single chain Fv antibody (scFv) bearing a C-terminal sortase motif, 6XHis and TwinStrep tags (LPETGHHHHHHWSHPQFEK[G3S]3WSHPQFEK). The structure, obtained in the presence of cobalt, reveals a unique tetramerization motif (TetrHis) stabilized by 8 Co2+ ions. The TetrHis motif contains four 6 residues-long β-strands, and each metal center coordinates 3 to 5 residues, including all 6XHis histidines. By combining dynamic light scattering, small angle x-ray scattering and molecular dynamics simulations, We investigated the influence of Co2+ on the conformational dynamics of scFv 2A2, observing an open/close equilibrium of the monomer and the formation of cobalt-stabilized tetramers. By using a similar scFv design, we demonstrate the transferability of the tetramerization property. This novel metal-dependent tetramerization motif might be used as a fiducial marker for cryoelectron microscopy of scFv complexes, or even provide a starting point for designing metal-loaded biomaterials.

show abstract

Distinguishing Between Monomeric scFv and Diabody in Solution Using Light and Small Angle X-ray Scattering

Cited by 7 publications

References 60 publications

Global parameter optimisation and sensitivity analysis of antivenom pharmacokinetics and pharmacodynamics

Global parameter optimisation and sensitivity analysis of antivenom pharmacokinetics and pharmacodynamics

Cross‐linking disulfide bonds govern solution structures of diabodies

Structure, dynamics and transferability of the metal-dependent polyhistidine tetramerization motif TetrHis for single-chain Fv antibodies

Contact Info

Product

Resources

About