Biophysical Properties of Human Antibody Variable Domains

Ewert, Stefan; Huber, Thomas; Honegger, Annemarie; Plückthun, Andreas

doi:10.1016/s0022-2836(02)01237-8

Cited by 308 publications

(304 citation statements)

References 55 publications

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“…Such individual stabilizing mutations likely occur in response to specific "affinity mutations" during the affinity maturation process, complicating an analysis of their overall additivity in the context of only the germ-line and affinity-matured antibody. Previous directed evolution experiments have shown that mutations in antibody variable regions, particularly those in turns at the surface or residues at the variable-constant interface (21,22), can significantly increase the stability of antibodies or antibody fragments (23)(24)(25)(26). Here we show that the clonal selection process is likely more sophisticated than previously realized-it efficiently selects such stabilizing mutations to compensate for somatic mutations that mature affinity but are deleterious to stability.…”

Section: Effects Of Somatic Mutations On Affinity and Thermodynamic Smentioning

confidence: 59%

RETRACTED: Somatic hypermutation maintains antibody thermodynamic stability during affinity maturation

Wang

Sen

Zhang

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Somatic hypermutation and clonal selection lead to B cells expressing high-affinity antibodies. Here we show that somatic mutations not only play a critical role in antigen binding, they also affect the thermodynamic stability of the antibody molecule. Somatic mutations directly involved in antigen recognition by antibody 93F3, which binds a relatively small hapten, reduce the melting temperature compared with its germ-line precursor by up to 9 °C. The destabilizing effects of these mutations are compensated by additional somatic mutations located on surface loops distal to the antigen binding site. Similarly, somatic mutations enhance both the affinity and thermodynamic stability of antibody OKT3, which binds the large protein antigen CD3. Analysis of the crystal structures of 93F3 and OKT3 indicates that these somatic mutations modulate antibody stability primarily through the interface of the heavy and light chain variable domains. The historical view of antibody maturation has been that somatic hypermutation and subsequent clonal selection increase antigen–antibody specificity and binding energy. Our results suggest that this process also optimizes protein stability, and that many peripheral mutations that were considered to be neutral are required to offset deleterious effects of mutations that increase affinity. Thus, the immunological evolution of antibodies recapitulates on a much shorter timescale the natural evolution of enzymes in which function and thermodynamic stability are simultaneously enhanced through mutation and selection.

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Section: Effects Of Somatic Mutations On Affinity and Thermodynamic Smentioning

confidence: 59%

RETRACTED: Somatic hypermutation maintains antibody thermodynamic stability during affinity maturation

Wang

Sen

Zhang

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…V-genes from the VH1, VH3 and VH5 families are found to be better biotherapeutic candidates than those from other families. 26 However, CDR grafting onto a subset of frameworks does not always result in stable antibodies because there may be some incompatibility between the CDR and the framework residues. This is often compensated for by mutations in the framework region to recover the native mouse structure pair.…”

Section: Introductionmentioning

confidence: 99%

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

Apgar

Mader

Agostinelli

et al. 2016

mAbs

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Antibodies are an important class of biotherapeutics that offer specificity to their antigen, long half-life, effector function interaction and good manufacturability. The immunogenicity of non-human-derived antibodies, which can be a major limitation to development, has been partially overcome by humanization through complementarity-determining region (CDR) grafting onto human acceptor frameworks. The retention of foreign content in the CDR regions, however, is still a potential immunogenic liability. Here, we describe the humanization of an anti-myostatin antibody utilizing a 2-step process of traditional CDR-grafting onto a human acceptor framework, followed by a structure-guided approach to further reduce the murine content of CDR-grafted antibodies. To accomplish this, we solved the co-crystal structures of myostatin with the chimeric (Protein Databank (PDB) id 5F3B) and CDR-grafted antimyostatin antibody (PDB id 5F3H), allowing us to computationally predict the structurally important CDR residues as well as those making significant contacts with the antigen. Structure-based rational design enabled further germlining of the CDR-grafted antibody, reducing the murine content of the antibody without affecting antigen binding. The overall "humanness" was increased for both the light and heavy chain variable regions.

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“…An antibody library constructed based on different frameworks would introduce distinct biophysical and biochemical characteristics to its members, so it is expedient to construct a library based on frameworks with similar expression traits. It was found that a scFv derived from V H 3 (DP47) and V k 3 (DPK-22) combination possessed relative good expression pattern (12,21,22). In this study, soluble portion could be detected in most cases; however, the products were mainly expressed in inclusion body forms in a T7 promoter-based expression vector, which makes it difficult to be purified by simple affinity chromatography method.…”

Section: Discussionmentioning

confidence: 71%

Construction of a Fully Synthetic Human scFv Antibody Library with CDR3 Regions Randomized by a Split-Mix-Split Method and Its Application

Yin

Ren

Zhu

et al. 2008

Journal of Biochemistry

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The randomization scheme of hypervariable region takes crucial role in construction of a synthetic antibody library. The codon bias and inevitable 'stop' codon of conventional 'NNK' and 'NNS' codons limit their applications. Here we report a splitmix-split DNA synthesis method that can control over the amino acid composition and distribution of randomized sequences effectually. A fully synthetic human antibody library with a diversity of 1.56 3 10 9 was successfully generated with complementarity determining region 3 (CDR3) randomized by this strategy. Sequencing analysis indicated that >60% of colonies had completely correct scFv genes and the amino acid composition and distribution were designed well in accordance. The utility was demonstrated by screening of scFv clones against BHL (anti-CD3 3 anti-ovarian carcinoma bispecific antibody). These results proved the feasibility of the split-mix-split DNA randomization strategy in library construction and site-directed mutagenesis.

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Biophysical Properties of Human Antibody Variable Domains

Cited by 308 publications

References 55 publications

RETRACTED: Somatic hypermutation maintains antibody thermodynamic stability during affinity maturation

RETRACTED: Somatic hypermutation maintains antibody thermodynamic stability during affinity maturation

Beyond CDR-grafting: Structure-guided humanization of framework and CDR regions of an anti-myostatin antibody

Construction of a Fully Synthetic Human scFv Antibody Library with CDR3 Regions Randomized by a Split-Mix-Split Method and Its Application

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