Engineering Strategies for Immunomodulatory Cytokine Therapies - Challenges and Clinical Progress

Abstract

Cytokines are immunoregulatory proteins involved in many pathological states with promising potential as therapeutic agents. A diverse array of cytokines have been studied in preclinical disease models since the 1950s, some of which became successful biopharmaceutical products with the advancement of recombinant protein technology in the 1980s. However, following these early approvals, clinical translation of these natural immune signaling molecules has been limited due to their pleiotropic action in many cell types, and the fact that they have evolved to act primarily locally in tissues. These characteristics, combined with poor pharmacokinetics, have hindered the delivery of cytokines via systemic administration routes due to dose-limiting toxicities. However, given their clinical potential and recent clinical successes in cancer immunotherapy, cytokines continue to be extensively pursued in preclinical and clinical studies, and a range of molecular and formulation engineering strategies are being applied to reduce treatment toxicity while maintaining or enhancing therapeutic efficacy. This review provides a brief background on the characteristics of cytokines and their history as clinical therapeutics, followed by a deeper discussion on the engineering strategies developed for cytokine therapies with a focus on the translational relevance of these approaches.

Keywords: clinical trials; cytokines; drug delivery; immunotherapies; therapeutics.

Figure 1.

Major targets of cytokines used in clinical trials directed at immune cells involved in adaptive and innate immunity. Blue arrows indicate recruitment and differentiation. Red arrows indicate activation and expansion. Gray arrows indicate inhibition. IL-22 has been excluded here as its clinical trials have targeted its growth-factor properties and not its immunostimulant properties. TNF-related apoptosis-inducing ligand (TRAIL) is also excluded as its non-apoptotic role in immune-cells is not clearly understood.^[35] The effects of IFN-λ are primarily on epithelial cells.^[36]

Figure 2.

Illustration of major fusion proteins developed for cytokine delivery. The three-dimensional protein illustrations were generated in Qutemol^[90] based on the Protein Data Bank structures of interleukin-2 (1M47), immunoglobin-G (1IGT), diabody scFv T84.66 (1MOE), scFv based on diabody structure (5GRV), human IgG1-Fc domain (5JII), diphtheria toxin (1F0L), prostatic acid phosphatase (1CVI), human serum albumin (1A06), and fibromodulin (5MX0).

Figure 3.

Selected formats of immunocytokines and their primary characteristics on vascular extravasation and tumor retention.

Figure 4.

Representation of Bempegaldesleukin and its biased IL2Rβγ receptor binding for enhanced IL-2-mediated immunotherapy of cancer. Reproduced under the terms of the CC BY 4.0 license. ^[173] Copyright 2017, Charych et. al.

Figure 5.

Major design parameters of polymeric matrices used in cytokine theraapies and their applications. Polymeric matrices can have varied properties by altering (i) the polymer composition, (ii) the resulting water content, and (iii) the type of crosslinker and/or conjugation. These matrices have been primarly used as (a) depots, (b) to promote endogenous cell recruitment, and (c) as exogenous cell resevoirs. Red arrows indicate progression of polymeric matrix systems after administration.

Figure 6.

Schematic of an alginate-based polymeric matrix designed for in situ gelation to enable prolonged and dual release of IL-2 and CpG as well as harbor and attract immune cells. Reproduced with permission. ^[198] Copyright 2009, Elsevier.

Figure 7.

Bioresponsive polymeric microparticles for anti-inflammatory cytokine delivery to osteoarthritis. Reproduced with permission.^[224] Copyright 2019, Wiley Periodicals, Inc.

Figure 8.

Synthesis and bioactivity of liposomes surface conjugated with anti-CD137 antibody or IL2-Fc fusion protein. (A) schematic for synthesis of liposomes. (B) Flow cytometry of CD4⁺ and CD8⁺ T cells incubated with fluorescently-labeled liposomes (solid), unconjugated liposomes (dashed), or no liposomes (grey area). (C) In vitro T cell proliferation normalized to unstimulated cells. (D) IFN-γ production by polyclonal T cells. Reproduced with permission.^[233] Copyright 2013, American Association for Cancer Research.

Figure 9.

Cancer-cell targeting liposomal layer-by-layer nanoparticle containing surface conjugated IL-12. (A) Diagram for assembly of nanoparticle. (B) Cancer cell association and subsequent targeted immune activation. Adapted with permission.^[234] Copyright 2020, American Chemical Society.

Figure 10.

Schematic for synthesis of liposomal nanogel encapsulating a TGF-β inhibitor and IL-2 and its effects when intratumorally administered to a subcutaneous mouse metastatic melanoma model. (a) Components and final liposomal nanogel assembly. (b) Plot of tumor area versus time (day 0 was day of tumor inoculation). Red arrows indicate treatment. (c) Tumor masses after 7 days of treatment. (d) Survival plot of animals in (b). Complete tumor regression and survival was obtained in 40% of the group after 60 days (data not shown). Adapted with permission.^[240] Copyright 2012, Nature Publishing Group.