Recent and future perspectives on engineering interferons and other cytokines as therapeutics

Abstract

As crucial mediators and regulators of our immune system, cytokines are involved in a broad range of biological processes and are implicated in various disease pathologies. The field of cytokine therapeutics has gained much momentum from the maturation of conventional protein engineering methodologies such as structure-based designs and/or directed evolution, which is further aided by the advent of in silico protein designs and characterization. Just within the past 5 years, there has been an explosion of proof-of-concept, preclinical, and clinical studies that utilize an armory of protein engineering methods to develop cytokine-based drugs. Here, we highlight the key engineering strategies undertaken by recent studies that aim to improve the pharmacodynamic and pharmacokinetic profile of interferons and other cytokines as therapeutics.

Keywords: cytokines; interferons; interleukins; pleiotropy; protein engineering; therapeutics.

Figure 1.. IFN signaling pathway and downstream cellular effects.

Type I, II, and III IFNs signal via intracellular JAK/STAT pathway. For type I and III IFNs, ligand binding dimerizes the receptors, resulting in the phosphorylation of STAT1 and STAT2 proteins (signal transducer and activator of transcription) that dimerize and form a complex with IFN regulatory factor 9 (IRF-9). After translocating to the nucleus, the complex then binds to the IFN stimulated response element (ISRE) to activate the transcription of IFN stimulated genes (ISGs). Type II IFN acts as a homodimer and recruits two sets of receptors, phosphorylating STAT1 proteins which then dimerize and translocate to the nucleus where the complex binds gamma IFN activation site (GAS) elements to initiate transcription of a distinct set of ISGs.

Figure 2.. Signaling pathways of Common gamma chain cytokines and their downstream functions.

Members of the common gamma chain (cytokine family include IL-2, IL-15, IL-4, IL-7, IL-9, and IL-21) hold important roles in driving the immune cell landscape of many disease pathologies. Upon ligand binding, the common gamma chain is recruited to the ligand-bound cognate receptors to direct lymphocytes’ responses. Current engineering themes to potentiate the therapeutic applications of these cytokines include de novo designs (IL-2/IL-15), half-life extensions such as albumin fusion (IL-2, IL-4, IL-15), Fc fusion (IL-2, IL7, IL-12, IL-15), and PEGylation (IL-2, IL-12, IL-15), tumor antigen or lymphocyte targeting using antibody fusions (IL-2, IL-12, IL-21).

Figure 3.. Structure-derived approaches to designing and engineering cytokine therapies.

A) Using structural information about how a cytokine engages with its receptors, biased agonists can be designed by rationally mutating cytokine-receptor interfaces to tune gene expression and cell signaling. B) De novo design of novel cytokines can be accomplished using computational methods, such as structure prediction and modeling, to either design entirely new proteins or rearrange existing protein structures to selectively engage with targets. Site-directed mutagenesis can also be employed in combination with this technique to engineer specific residues. C) By covalently linking two different cytokines, synthekines can selectively engage pathways that are not engaged by natural proteins, leading to new and unique combinations of receptor signaling.

Figure 4.. Engineering cytokines to improve their activity and targeting ability in the body.

A) Schematic diagram showing directed evolution using yeast display, a common method for cytokine engineering. Following library generation, variants are selected for their desired characteristics (increased affinity, activity, etc.) and subjected to increasing selection pressure. Individual clones are then isolated and characterized. B) Targeting motifs that increase cytokine delivery to the tissues of interest and reduce off-target effects. 1) Conjugating to a monoclonal antibody 2) Conjugating to a targeting domain for a specific tissue or environment 3) Linkage to a receptor by a protease-cleavable domain.

Figure 5.. Engineering strategies are being explored preclinically to enhance the drug-like properties of cytokines.

(A) Both site-specific and non-specific polymer conjugation methods have been explored to extend the serum half-lives of cytokines or to create a drug depot for controllable sustained release. (B) The bivalent (Top) and monovalent (Bottom) formats of Fc-fused cytokines have been engineered. (C) Cytokines are conjugated to human serum albumin (HSA) directly via covalent or genetic fusions (Top). Alternatively, they are fused to the albumin-binding domain (ABD) (Middle) or fatty acid moieties (Bottom) that can non-covalently bind to endogenous HSA upon administration. (D) Cytokine mimetics such as peptides, bispecific antibodies, zipper-like coiled-coil constructs, and other engineered protein scaffolds have been studied as potential therapeutics.