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Abstract
Monoclonal antibodies (mAbs) have proven to be useful for development of new therapeutic drugs and diagnostic techniques. To overcome the difficulties posed by their complex structure and folding, reduce undesired immunogenicity, and improve pharmacokinetic properties, a plethora of different Ab fragments have been developed. These include recombinant Fab and Fv segments that can display improved properties over those of the original mAbs upon which they are based. Antibody (Ab) fragments such as Fabs, scFvs, diabodies, and nanobodies, all contain the variable Ig domains responsible for binding to specific antigenic epitopes, allowing for specific targeting of pathological cells and/or molecules. These fragments can be easier to produce, purify and refold than a full Ab, and due to their smaller size they can be well absorbed and distributed into target tissues. However, the physicochemical and structural properties of the immunoglobulin (Ig) domain, upon which the folding and conformation of all these Ab fragments is based, can limit the stability of Ab-based drugs. The Ig domain is fairly sensitive to unfolding and aggregation when produced out of the structural context of an intact Ab molecule. When unfolded, Ab fragments may lose their specificity as well as establish non-native interactions leading to protein aggregation. Aggregated antibody fragments display altered pharmacokinetic and immunogenic properties that can augment their toxicity. Therefore, much effort has been placed in understanding the factors impacting the stability of Ig folding at two different levels: 1) intrinsically, by studying the effects of the amino acid sequence on Ig folding; 2) extrinsically, by determining the environmental conditions that may influence the stability of Ig folding. In this review we will describe the structure of the Ig domain, and the factors that impact its stability, to set the context for the different approaches currently used to achieve stable recombinant Ig domains when pursuing the development of Ab fragment-based biotechnologies.
Highlights
Abs are secreted glycoproteins of ~150 kDa that represent the soluble form of the antigen receptor of B cells [1]
The multitude of studies describing the stability of different Ab fragments, and their response to changes in their environment, allows the establishment of common strategies at three levels: 1) Engineering of covalent bonds to stabilize the interaction of H and L chains in the Ab fragment; 2) sequence mutagenesis to increase stability and solubility of the Ig domains composing the Ab fragment; 3) buffering the environmental conditions that cause Ig instability during production, storage and utilization of Ab fragments
We have recently described the case of four unrelated anti-TCR/CD3 Fab fragments that dimerize at low protein concentrations (0.2 mg/ml) while preserving their Ag binding specificity [67]
Summary
Abs are secreted glycoproteins of ~150 kDa that represent the soluble form of the antigen receptor of B cells [1] Their function is to bind to specific antigens (Ag) from pathogens, mediating Ag neutralization and clearance in cooperation with other components of the immune system [2]. Binding of Abs to specific proteins at the cell surface allows the targeting of pathologic tissues like tumoral masses. While the size and valency of Ab fragments is tailored to suit best the application of choice (tissue imaging, tumor targeting, etc.), in every case the Ig domain(s) that structure and provide functionality to these molecules may unfold during production, storage and/or administration. The multitude of studies describing the stability of different Ab fragments, and their response to changes in their environment, allows the establishment of common strategies at three levels: 1) Engineering of covalent bonds to stabilize the interaction of H and L chains in the Ab fragment; 2) sequence mutagenesis to increase stability and solubility of the Ig domains composing the Ab fragment; 3) buffering the environmental conditions that cause Ig instability during production, storage and utilization of Ab fragments
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