Long-Acting Human Interleukin 2 Bioconjugate Modified with Fatty Acids by Sortase A

Abstract

Human Interleukin 2 (IL-2) has already achieved impressive results as a therapeutic agent for cancer and autoimmune diseases. However, one of the limitations associated with the clinical application of IL-2 is its short half-life owing to rapid clearance by the kidneys. Modification with fatty acids, as an albumin noncovalent ligand with the advantage of deep penetration into tissues and high activity-to-mass ratio, is a commonly used approach to improve the half-life of native peptides and proteins. In this investigation, we attempted to extend the half-life of IL-2 through conjugation with a fatty acid using sortase A (srtA). We initially designed and optimized three IL-2 analogues with different peptide linkers between the C-terminus of IL-2 and srtA recognition sequence (LPETG). Among these, analogue A3 was validated as the optimal IL-2 analogue for further modification. Next, six fatty acid moieties with the same fatty acid and different hydrophilic spacers were conjugated to A3 through srtA. The six bioconjugates generated were screened for in vitro biological activity, among which bioconjugate B6 was identified as near-optimal to IL-2. Additionally, B6 could effectively bind albumin through the conjugated fatty acid, which contributed to a significant improvement in its pharmacokinetic properties in vivo. In summary, we have developed a novel IL-2 bioconjugate, B6, modified with fatty acids using srtA, which may effectively serve as a new-generation long-acting IL-2 immunotherapeutic agent.

Enhancing the Pharmacokinetic Profile of Interleukin 2 through Site-Specific Conjugation to a Selective Small-Molecule Transthyretin Ligand

“…An important advantage of the TLHE system is maintaining the hydrophilicity of the conjugated proteins as indicated by our HPLC analysis. This is particularly important for proteins with limited solubility at physiological pH, such as rIL2, where fatty acid conjugation is not an option for half-life extension . The new TLHE-rIL2 conjugate we developed has the potential to decrease rIL2 toxicity and increase its clinical success rate for renal cancers and melanoma, which is a tough cancer to treat because it is typically resistant to many drugs.…”

“…This is particularly important for proteins with limited solubility at physiological pH, such as rIL2, where fatty acid conjugation is not an option for half-life extension. 38 The new TLHE-rIL2 conjugate we developed has the potential to decrease rIL2 toxicity and increase its clinical success rate for renal cancers and melanoma, which is a tough cancer to treat because it is typically resistant to many drugs. To the best of our knowledge, this is the first demonstration of a successful approach that not only extends the circulation halflife but also maintains the smaller size, binding potency, and hydrophilicity of proteins.…”

Bicyclic Engineered Sortase A Performs Transpeptidation under Denaturing Conditions

“…The transpeptidase Sortase A (SrtA) is an enzyme that is widely used for protein modification involving diverse applications such as protein labeling, bioconjugation, and immobilization. − For various applications, the sensitivity of SrtA to thermal and chemical stress is a limiting factor. , We have recently reported the in situ cyclization of proteins (INCYPRO), an approach that facilitates the construction of bicyclic proteins in a single design step. , INCYPRO is structure-based and uses C3 symmetric tris-electrophiles to covalently link three cysteine residues that have been introduced in spatial proximity. Previously, we have described an INCYPRO-stabilized SrtA that was derived from wild-type Staphylococcus aureus SrtA .…”