A single‐chain variable fragment intrabody prevents intracellular polymerization of Z α ₁ ‐antitrypsin while allowing its antiproteinase activity

Abstract

Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.—Ordóñez, A., Pérez, J., Tan, L., Dickens, J. A., Motamedi-Shad, N., Irving, J. A., Haq, I., Ekeowa, U., Marciniak, S. J., Miranda, E., Lomas, D. A. A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour

“…This involved the use of the monomeric form of Z α 1 -AT as the antigen to generate a hybridoma library, and its use in a primary screen that accordingly selected antibodies able to recognise this form of the protein [44]. A polymerisation-enhancing antibody, identified using a similar protocol, was found to exhibit marked conformational selectivity [22].…”

“…We recently described a monoclonal antibody (mAb 4B12 ) that blocks polymerisation of Z α 1 -AT, both when induced by heat in vitro and also during expression in a cellular model of disease, while retaining most inhibitory activity [44]. Here we have characterised its mechanism of action in detail.…”

Update on alpha-1 antitrypsin deficiency: New therapies

“…This has proved to be powerful technology that has allowed us to identify antibodies that specifically detect the polymeric [7] and latent [92] conformers of α 1 -antitrypsin and antibodies that can accelerate [93] and block [94] Reproduced from [17] with permission. …”

Polymers of Z α1-antitrypsin are secreted in cell models of disease

“…It is important to understand where these polymers form in order to design effective therapies for emphysema and other pathological manifestations of α 1 -ATD. Here we investigated the origin of extracellular polymers by exploiting our cellular models of α 1 -ATD and conformer-specific and functional monoclonal antibodies (mAb) against Z α 1 -AT [5][6][7][8].…”

“…We next used our polymerisation-blocking mAb-4B12 [8] to prevent Z α 1 -AT polymerisation in cell culture medium. This antibody blocks polymer formation at a molar ratio of 1:1 for Z α 1 -AT and mAb-4B12 in vitro, so adding it in excess to the culture medium should inhibit the polymerisation of secreted monomeric Z α 1 -AT.…”