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Strategies to stabilize compact folding and minimize aggregation of antibody-based fragments.

Gil D, Schrum AG - Adv Biosci Biotechnol (2013)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Mayo Clinic College of Medicine, Rochester, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Diagrams depicting the structure of (A) an scFv fragment (IgG); and the derived (B) diabody; (C) triabody; and (D) tetrabody. The circles in each panel represent the specific Ag of the depicted Fv.
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Figure 3: Diagrams depicting the structure of (A) an scFv fragment (IgG); and the derived (B) diabody; (C) triabody; and (D) tetrabody. The circles in each panel represent the specific Ag of the depicted Fv.

Mentions: Given the interest in developing Ab fragments smaller than a Fab, stabilization of Ig folding in Fv fragments has been pursued by different approaches. The design of a covalent disulfide bond between VH and VL domains of the Fv (dsFv) was one of the first strategies employed to reinforce the V interface [28,29]. Another way to stabilize Fv fragments is to link the VH and VL chains with a flexible peptide sequence resistant to endopeptidases, generating single chain Fv (scFv) fragments (Figure 3A) [30,31]. Additionally sc-dsFv fragments have been developed by combining both disulfide bonding and peptide linking of VH and VL domains [32]. From these fragments, the most employed scFv still displays a tendency to unfold at the V interface, resulting in sub-optimal stability of the Ig domains [33]. When this happens, a phenomenon known as “protein domain swapping” can occur, wherein complementary Ig domains from adjacent scFv molecules interact with each other to result in scFv oligomerization [26]. This protein domain swapping has been described in different types of proteins other than Igs [34–36]. Indeed, Ig domain swapping can be engineered and optimized as a mechanism for controlling the precise oligomerization of scFv molecules [37,38]. Depending on the length of the peptide linker and the amino acid sequence of the Ab, short linkers that impede the proper rotation of the complementary Ig domains covalently linked to establish an interface, promote the swap of the same Ig domain between two, three or even four molecules of scFv (Figure 3A). The resulting diabodies, triabodies and tetrabodies (Figures 3B and D) display stable Ig folding and functional Ag recognition. On the other hand, they present different pharmacokinetic properties than the parental scFvs, such as stronger multivalent binding to the targeted epitope and prolonged retention in tissue [13,39]. An interesting application of the Ig domain swapping in scFv molecules is the development of bispecific diabodies [26]. A dimer of Fabs of different specificities can be formed when the VHA is linked to VLB in the scFv (A and B referring to different epitope specificities recognized by different FvA and FvB fragments) [40]. Bispecific diabodies are designed to either recognize epitopes located in the same antigenic structure, to increase binding to the antigen, or to recognize epitopes belonging to different Ags, in which case the diabody will crosslink/juxtapose otherwise separate Ags. The crosslinking properties of bispecific diabodies have been specially applied in the field of cancer therapeutics [13,41]. In some cases the diabodies are designed to promote the contact between tumor cells and cytotoxic effector cells of the immune system, such as cytotoxic T lymphocytes (CTLs) or Natural Killer (NK) cells, In other strategies, diabodies have been utilized to deliver specific toxins, radioactive haptens, or adenoviral gene delivery vehicles as a payload to tumor cells, resulting in their destruction.


Strategies to stabilize compact folding and minimize aggregation of antibody-based fragments.

Gil D, Schrum AG - Adv Biosci Biotechnol (2013)

Diagrams depicting the structure of (A) an scFv fragment (IgG); and the derived (B) diabody; (C) triabody; and (D) tetrabody. The circles in each panel represent the specific Ag of the depicted Fv.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307952&req=5

Figure 3: Diagrams depicting the structure of (A) an scFv fragment (IgG); and the derived (B) diabody; (C) triabody; and (D) tetrabody. The circles in each panel represent the specific Ag of the depicted Fv.
Mentions: Given the interest in developing Ab fragments smaller than a Fab, stabilization of Ig folding in Fv fragments has been pursued by different approaches. The design of a covalent disulfide bond between VH and VL domains of the Fv (dsFv) was one of the first strategies employed to reinforce the V interface [28,29]. Another way to stabilize Fv fragments is to link the VH and VL chains with a flexible peptide sequence resistant to endopeptidases, generating single chain Fv (scFv) fragments (Figure 3A) [30,31]. Additionally sc-dsFv fragments have been developed by combining both disulfide bonding and peptide linking of VH and VL domains [32]. From these fragments, the most employed scFv still displays a tendency to unfold at the V interface, resulting in sub-optimal stability of the Ig domains [33]. When this happens, a phenomenon known as “protein domain swapping” can occur, wherein complementary Ig domains from adjacent scFv molecules interact with each other to result in scFv oligomerization [26]. This protein domain swapping has been described in different types of proteins other than Igs [34–36]. Indeed, Ig domain swapping can be engineered and optimized as a mechanism for controlling the precise oligomerization of scFv molecules [37,38]. Depending on the length of the peptide linker and the amino acid sequence of the Ab, short linkers that impede the proper rotation of the complementary Ig domains covalently linked to establish an interface, promote the swap of the same Ig domain between two, three or even four molecules of scFv (Figure 3A). The resulting diabodies, triabodies and tetrabodies (Figures 3B and D) display stable Ig folding and functional Ag recognition. On the other hand, they present different pharmacokinetic properties than the parental scFvs, such as stronger multivalent binding to the targeted epitope and prolonged retention in tissue [13,39]. An interesting application of the Ig domain swapping in scFv molecules is the development of bispecific diabodies [26]. A dimer of Fabs of different specificities can be formed when the VHA is linked to VLB in the scFv (A and B referring to different epitope specificities recognized by different FvA and FvB fragments) [40]. Bispecific diabodies are designed to either recognize epitopes located in the same antigenic structure, to increase binding to the antigen, or to recognize epitopes belonging to different Ags, in which case the diabody will crosslink/juxtapose otherwise separate Ags. The crosslinking properties of bispecific diabodies have been specially applied in the field of cancer therapeutics [13,41]. In some cases the diabodies are designed to promote the contact between tumor cells and cytotoxic effector cells of the immune system, such as cytotoxic T lymphocytes (CTLs) or Natural Killer (NK) cells, In other strategies, diabodies have been utilized to deliver specific toxins, radioactive haptens, or adenoviral gene delivery vehicles as a payload to tumor cells, resulting in their destruction.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Mayo Clinic College of Medicine, Rochester, USA.

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.

No MeSH data available.


Related in: MedlinePlus