Sortase-catalyzed transformations that improve the properties of cytokines

Abstract

Recombinant protein therapeutics often suffer from short circulating half-life and poor stability, necessitating multiple injections and resulting in limited shelf-life. Conjugation to polyethylene glycol chains (PEG) extends the circulatory half-life of many proteins, but the methods for attachment often lack specificity, resulting in loss of biological activity. Using four-helix bundle cytokines as an example, we present a general platform that uses sortasemediated transpeptidation to facilitate site-specific attachment of PEG to extend cytokine half-life with full retention of biological activity. Covalently joining the N and C termini of proteins to obtain circular polypeptides, again executed using sortase, increases thermal stability. We combined both PEGylation and circularization by exploiting two distinct sortase enzymes and the use of a molecular suture that allows both site-specific PEGylation and covalent closure. The method developed is general, uses a set of easily accessible reagents, and should be applicable to a wide variety of proteins, provided that their termini are not involved in receptor binding or function.M any clinically relevant cytokines share a four-helix bundle structure, typified by IFNα2, Granulocyte colony-stimulating factor 3 (GCSF-3), Erythropoietin (EPO), IL-2, IL-4, IL-7, IL-9, and IL-15. Cocrystal structures of cytokines with receptor fragments and biochemical studies that map residues critical for interaction of a cytokine with its receptor show that the receptor contacts the sides of the helical bundles (1). This mode of interaction positions the N and C termini of the cytokine away from the receptor.Polyethylene glycol chains attached to therapeutically important proteins increase circulatory half-life, reduce clearance by kidney filtration, reduce proteolysis, and reduce the generation of neutralizing antibodies (2-4). The attachment of PEG commonly employs standard chemistries that target reactive amino acid side chains (e.g., cysteine and lysine). This strategy often generates a heterogeneous mixture in which multiple amino acids in the target are modified with a PEG chain, necessitating cumbersome separations and characterization (5). Such PEGylated molecules often show decreased biological activity, likely due to attachment of a PEG chain to a residue important for interaction with the receptor (6)-this problem may be overcome by sitespecific PEGylation. Although engineering of a carefully placed unpaired cysteine residue allows site-specific PEGylation (7, 8), this method must be tailored to the specific protein target.Sortase A from Staphylcoccus aureus (SrtA Staph ) is a thiolcontaining transpeptidase that recognizes an LPXTG motif in multiple structurally unrelated substrates (9). SrtA Staph cleaves the peptide bond between the threonine and glycine residues with concomitant formation of a thioacyl enzyme intermediate that involves the catalytic cysteine and the substrate threonine. This acyl-enzyme is resolved by nucleophilic attack by the N terminus of an oligo...

Reprogramming the specificity of sortase enzymes

“…Orthogonal SrtA variants have previously been used in the dual N-and C-terminal functionalization of target proteins (12,24). However, in these applications, the low activity and poor orthogonality of the enzymes greatly limited the scope of available reactions (24). To test whether the combined use of reprogrammed sortases can overcome these limitations, we attempted to synthesize dual N-and C-terminally functionalized proteins of biomedical interest.…”

Functional human antibody CDR fusions as long-acting therapeutic endocrine agonists

“…structure of hGH in complex with the extracellular domain of its receptor indicate that the key residues involved in binding the receptor reside in helices 1, 3, and 4, with the N and C termini of hGH distal to the binding interface (19,20). Moreover, previous studies have shown that covalent cross-linking of the N and C termini of four-helix-bundle cytokines with a PEGylated short peptide sequence does not significantly affect the biological activities of the cytokines (21). Therefore, we reasoned that connecting the two termini of hGH with the CDR3H stalk of the bovine antibody BLV1H12 would likely yield a fusion protein with wild-type hGH activity but with the superior pharmacological properties of the Ig scaffold.…”

Tuning the molecular size of site-specific interferon-polymer conjugate for optimized antitumor efficacy

“…To improve its pharmacokinetics, IFN-α is usually conjugated with 40 kDa branched PEG (PEGASYS) or 20 kDa linear PEG (PEGINTRON), but its activity is significantly reduced to 7% for PEGASYS [7] or 28% for PEGINTRON [8]. It is anticipated that the activity of protein-polymer conjugate can be well remained by site-specific conjugation at the sites that are distant from the protein's receptor interaction site, such as the C terminus of IFN-α [9][10][11].…”