High‐Affinity Antibody Detection with a Bivalent Circularized Peptide Containing Antibody‐Binding Domains

Abstract

Direct chemical labeling of antibody produces molecules with poorly defined modifications. Use of a small antibody-binding protein as an adapter can simplify antibody functionalization by forming a specific antibody-bound complex and introducing site-specific modifications. To stabilize a noncovalent antibody complex that may be used without chemical crosslinking, a bivalent antibody-binding protein is engineered with an improved affinity of interaction by joining two Z domains with a conformationally flexible linker. The linker is essential for the increase in affinity because it allows simultaneous binding of both domains. The molecule is further circularized using a split intein, creating a novel adapter protein ("lasso"), which binds human immunoglobulin G1 (IgG1) with K D = 0.53 nM and a dissociation rate that is 55to 84-fold slower than Z. The lasso contains a unique cysteine for conjugation with a reporter and may be engineered to introduce other functional groups, including a biotin tag and protease recognition sequences. When used in enzyme-linked immunosorbent assay (ELISA), the lasso generates a stronger reporter signal compared to a secondary antibody and lowers the limit of detection by 12-fold. The small size of the lasso and a long half-life of dissociation make the peptide a useful tool in antibody detection and immobilization.

Stability Enhancement of a Dimeric HER2-Specific Affibody Molecule through Sortase A-Catalyzed Head-to-Tail Cyclization

“…Upon treatment with sortase A, a native peptide bond is formed between Thr 146 and Gly 1 , and the cyclic dimer, (Z HER2 ) 2:C , is formed ( Figure 2 B). For reference, the designs of three cyclic Affibody variants described earlier in the literature [ 24 , 25 , 26 ] are schematically shown ( Figure 2 C). It can be noted that the two previously described cyclic HER2-binding Affibody molecules are monomeric or truncated versions of the protein, not taking advantage of the possible avidity effects that are expected from dimeric constructs.…”

“…Zhou and coworkers used split-intein technology to produce a dimeric and backbone-cyclized Z-domain capable of binding to human IgG1 with increased affinity compared to the original monomeric protein [ 24 ], suggesting that the two domains can bind simultaneously on opposite sides of the symmetrical IgG1 dimer. A linear precursor protein, with a 17-residue linker separating the two Z-domains, showed a 10–12-fold higher affinity for human IgG1 compared to monomeric Z, and importantly, the subsequent backbone cyclization reaction, in which a significantly longer second 45–54 residue linker was formed, did not have a detrimental effect on the antibody binding affinity according to SPR and flow cytometry data [ 26 ].…”

“…Lasso is a recombinantly expressed Z-domain dimer. The two IgG-binding Z-domains are joined by flexible linkers and the construct is backbone-cyclized using split-intein technology [ 26 ]. The schematic drawings of Z HER2:CL , Z min and lasso are based on information found in references [ 24 , 25 , 26 ].…”

“…Intramolecular crosslinking and backbone cyclization of Affibody molecules and Z-domain variants have previously been investigated by our group and others [ 24 , 25 , 26 ]. An intramolecular thioether bond was, for example, shown to thermally stabilize a solid-phase synthesized and monomeric HER2-binding Affibody by 10 °C [ 24 ], and in a chemically synthesized 2-helix variant of the Z-domain, the introduction of a native peptide bond connecting the N-terminus to the C-terminus improved the ability of the protein to refold following thermal denaturation [ 25 ].…”

Affinity-Based Methods for Site-Specific Conjugation of Antibodies

“…It was also equipped with an N-terminal biotinylation tag as well as a cysteine for additional reporter conjugation. The lasso was used as a capturing agent in an ELISA and shown to lower the limit of detection compared to an immobilized secondary Fab fragment by 12-fold and also, when conjugated to a fluorophore, used as a detecting agent in a confocal microscopy cell detection assay …”