The structure of dual-variable-domain immunoglobulin molecules alone and bound to antigen

Correia, Ivan; Sung, Joyce; Burton, Randall E.; Jakob, C.G.; Carragher, Bridget; Ghayur, Tariq; Radziejewski, Czeslaw

doi:10.4161/mabs.24258

Cited by 40 publications

(25 citation statements)

References 50 publications

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“…2). 26,27 The crystal structures of CODV-Fab IL4 x IL13 evince reasons for maintenance of antigen affinities and independence of antigen binding in CODV format. The binding sites of Fv IL4 and Fv IL13 are about 65 A apart, oriented in opposing directions, and very few interactions are observed between these domains or with Fc1 (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…24,25 Structural studies suggest that Fv1 may hinder antigen access by swinging "like a bucket" over the antigen-binding region of Fv2. 26,27 Biotherapeutic agents require a proper balance of target tissue penetration and maintenance of therapeutic concentrations at the site of action. Low molecular weight bispecific Igs devoid of Fc domains aim at superior pharmacodynamics by increased target tissue penetration.…”

Section: Introductionmentioning

confidence: 99%

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CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

Steinmetz

Vallée

Beil

et al. 2016

mAbs

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Bispecific immunoglobulins (Igs) typically contain at least two distinct variable domains (Fv) that bind to two different target proteins. They are conceived to facilitate clinical development of biotherapeutic agents for diseases where improved clinical outcome is obtained or expected by combination therapy compared to treatment by single agents. Almost all existing formats are linear in their concept and differ widely in drug-like and manufacture-related properties. To overcome their major limitations, we designed cross-over dual variable Ig-like proteins (CODV-Ig). Their design is akin to the design of circularly closed repeat architectures. Indeed, initial results showed that the traditional approach of utilizing (G4S) x linkers for biotherapeutics design does not identify functional CODV-Igs. Therefore, we applied an unprecedented molecular modeling strategy for linker design that consistently results in CODV-Igs with excellent biochemical and biophysical properties. CODV architecture results in a circular self-contained structure functioning as a self-supporting truss that maintains the parental antibody affinities for both antigens without positional effects. The format is universally suitable for therapeutic applications targeting both circulating and membrane-localized proteins. Due to the full functionality of the Fc domains, serum half-life extension as well as antibody-or complement-dependent cytotoxicity may support biological efficiency of CODV-Igs. We show that judicious choice in combination of epitopes and paratope orientations of bispecific biotherapeutics is anticipated to be critical for clinical outcome. Uniting the major advantages of alternative bispecific biotherapeutics, CODV-Igs are applicable in a wide range of disease areas for fast-track multi-parametric drug optimization.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

Steinmetz

Vallée

Beil

et al. 2016

mAbs

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“…Electron microscopy with its unique capability for providing direct visual information of size, shape, and aggregation extent of a sample is a powerful tool in the arsenal of characterization techniques applied to protein therapeutics . Molecular electron microscopy uses advanced specimen preparation and imaging methods designed specifically to visualize complex biological samples under conditions close to their native hydration state.…”

Section: Introductionmentioning

confidence: 99%

Transmission Electron Microscopy as an Orthogonal Method to Characterize Protein Aggregates

Sung

Pardeshi

Mulder

et al. 2015

Journal of Pharmaceutical Sciences

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Aggregation of protein-based therapeutics is a challenging problem in the biopharmaceutical industry. Of particular concern are implications for product efficacy and clinical safety due to potentially increased immunogenicity of the aggregates. We used transmission electron microscopy (TEM) to characterize biophysical and morphological features of antibody aggregates formed upon controlled environmental stresses. TEM results were contrasted with results obtained in parallel by independent methods, including size exclusion chromatography, dynamic light scattering, microflow imaging and nanoparticle tracking. For TEM, stressed samples were imaged by negative staining and in the frozen-hydrated state. In both cases, aggregates appeared amorphous but differed in fine structural detail. Specifically, negatively stained aggregates were compact and consisted of smaller globular structures that had a notable three dimensional character. Elements of the native IgG structure were retained, suggesting that the aggregates were not assembled from denatured protein. In contrast, aggregates in frozen-hydrated samples appeared as extended, branched protein networks with large surface area. Using multiple scales of magnification, a wide range of particle sizes was observed and semi-quantitatively characterized. The detailed information provided by TEM extended observations obtained with the independent methods, demonstrating the suitability of TEM as a complementary approach to submicron particle analysis.

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“…Averaging is based on the assumption that protein particles are identical in structure and conformation but different in orientations, however many proteins are known to undergo thermodynamic fluctuation in solution. By applying the averaging method without previously knowing the protein thermodynamic fluctuation fluctuations, the averaged structure can often cause missing domains 10,80 or local variation in resolution 81 in cryo-EM reconstructions.…”

Section: Discussionmentioning

confidence: 99%

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

Rames

Ren

2014

JoVE

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Structural determination of proteins is rather challenging for proteins with molecular masses between 40 – 200 kDa. Considering that more than half of natural proteins have a molecular mass between 40 – 200 kDa1,2, a robust and high-throughput method with a nanometer resolution capability is needed. Negative staining (NS) electron microscopy (EM) is an easy, rapid, and qualitative approach which has frequently been used in research laboratories to examine protein structure and protein-protein interactions. Unfortunately, conventional NS protocols often generate structural artifacts on proteins, especially with lipoproteins that usually form presenting rouleaux artifacts. By using images of lipoproteins from cryo-electron microscopy (cryo-EM) as a standard, the key parameters in NS specimen preparation conditions were recently screened and reported as the optimized NS protocol (OpNS), a modified conventional NS protocol 3. Artifacts like rouleaux can be greatly limited by OpNS, additionally providing high contrast along with reasonably high#resolution (near 1 nm) images of small and asymmetric proteins. These high-resolution and high contrast images are even favorable for an individual protein (a single object, no average) 3D reconstruction, such as a 160 kDa antibody, through the method of electron tomography4,5. Moreover, OpNS can be a high#throughput tool to examine hundreds of samples of small proteins. For example, the previously published mechanism of 53 kDa cholesteryl ester transfer protein (CETP) involved the screening and imaging of hundreds of samples 6. Considering cryo-EM rarely successfully images proteins less than 200 kDa has yet to publish any study involving screening over one hundred sample conditions, it is fair to call OpNS a high-throughput method for studying small proteins. Hopefully the OpNS protocol presented here can be a useful tool to push the boundaries of EM and accelerate EM studies into small protein structure, dynamics and mechanisms.

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The structure of dual-variable-domain immunoglobulin molecules alone and bound to antigen

Cited by 40 publications

References 50 publications

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

CODV-Ig, a universal bispecific tetravalent and multifunctional immunoglobulin format for medical applications

Transmission Electron Microscopy as an Orthogonal Method to Characterize Protein Aggregates

Optimized Negative Staining: a High-throughput Protocol for Examining Small and Asymmetric Protein Structure by Electron Microscopy

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