Abstract
AbstractAdalimumab, a full-length monoclonal antibody, is widely used as an anti-tumor necrosis factor-α (anti-TNF-α) agent. In this article, we aimed to prolong the in vivo half-life of adalimumab antigen-binding fragment (Fab) through Sortase A (SrtA)-mediated conjugation of its Fab with fatty acid (FA). In our study, adalimumab Fab analog was prepared by adding an SrtA recognition sequence (LPETGG) and His6 tag to the heavy chain C-terminal of the Fab via (G4S)3 linker. Four FA motifs with different linkers were designed and synthesized by solid-phase methodology, then conjugated with the Fab analog using SrtA to produce Fab bioconjugates. The successful generation of four Fab bioconjugates (Fab–FA1, Fab–FA2, Fab–FA3, and Fab–FA4) was confirmed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and mass spectrometry. Then, the bioactivities and half-life of these Fab bioconjugates were examined using TNF-α-/human serum albumin (HSA)-binding enzyme-linked immunosorbent assay, cytotoxicity assay, and pharmacokinetic study, respectively. All Fab bioconjugates exhibited similar TNF-α-neutralizing activities when compared with the Fab analog, even in the presence of albumin, indicating that there were no apparent influences on the functional site of Fab after FA modification. However, different degrees of affinity for HSA were observed among these Fab–FA bioconjugates, with Fab–FA3 exhibiting the maximal affinity. An in vivo study in mice further revealed remarkably improved pharmacokinetics of Fab– FA3 with a 15.2-fold longer plasma half-life (19.86 hours) compared with that of the Fab analog (1.31 hours). In summary, we have developed a novel long-acting adalimumab Fab bioconjugate, Fab–FA3, with more sustained in vivo activity, which can be used for drug development targeting TNF-α-mediated inflammatory diseases.
Highlights
Tumor necrosis factor-α (TNF-α) plays an important role in the regulation of immune responses and inflammation
As shown in ►Fig. 3, antigen-binding fragment (Fab) analog existed in the periplasmic fraction in a soluble form with a high expression level and was highly pure in elution fraction
UPLCQTOF-mass spectrometry (MS) showed that the observed mass of Fab analog was 49,522.18 Da, which was in accordance with its theoretical mass (49,523.12 Da), indicating its integrity (►Table 1)
Summary
Tumor necrosis factor-α (TNF-α) plays an important role in the regulation of immune responses and inflammation. Antigen-binding fragment (Fab) of an antibody has many advantages, such as low molecular weight, satisfactory antigen-binding capacity, facile prokaryotic expression, superior tissue penetration, and has been widely studied in the field of monoclonal antibodies.[6] Fab is a preferable alternative to full-length monoclonal antibody in research and therapeutic applications. Fab is filtered through the glomerulus because of its small molecular weight, resulting in a relatively short half-life (only 16–20 hours) in vivo.[5,7] To promote the clinical application of Fab, diverse strategies regarding sprolongation of the halflife of Fab have been developed, such as PEGylation,[8] PASylation,[9] albumin-binding peptide fusion,[10] and glycine-rich homoamino-acid polymer (HAP) fusion,[11] by increasing the hydrodynamic radius or specific binding to endogenous albumin
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