Engineering the IL-4/IL-13 axis for targeted immune modulation.

The structurally and functionally related interleukin-4 (IL-4) and IL-13 cytokines play pivotal roles in shaping immune activity. The IL-4/IL-13 axis is best known for its critical role in T helper 2 (Th2) cell-mediated Type 2 inflammation, which protects the host from large multicellular pathogens, such as parasitic helminth worms, and regulates immune responses to allergens. In addition, IL-4 and IL-13 stimulate a wide range of innate and adaptive immune cells, as well as non-hematopoietic cells, to coordinate various functions, including immune regulation, antibody production, and fibrosis. Due to its importance for a broad spectrum of physiological activities, the IL-4/IL-13 network has been targeted through a variety of molecular engineering and synthetic biology approaches to modulate immune behavior and develop novel therapeutics. Here, we review ongoing efforts to manipulate the IL-4/IL-13 axis, including cytokine engineering strategies, formulation of fusion proteins, antagonist development, cell engineering approaches, and biosensor design. We discuss how these strategies have been employed to dissect IL-4 and IL-13 pathways, as well as to discover new immunotherapies targeting allergy, autoimmune diseases, and cancer. Looking ahead, emerging bioengineering tools promise to continue advancing fundamental understanding of IL-4/IL-13 biology and enabling researchers to exploit these insights to develop effective interventions.

Design of cell-type-specific hyperstable IL-4 mimetics via modular de novo scaffolds.

The interleukin-4 (IL-4) cytokine plays a critical role in modulating immune homeostasis. Although there is great interest in harnessing this cytokine as a therapeutic in natural or engineered formats, the clinical potential of native IL-4 is limited by its instability and pleiotropic actions. Here, we design IL-4 cytokine mimetics (denoted Neo-4) based on a de novo engineered IL-2 mimetic scaffold and demonstrate that these cytokines can recapitulate physiological functions of IL-4 in cellular and animal models. In contrast with natural IL-4, Neo-4 is hyperstable and signals exclusively through the type I IL-4 receptor complex, providing previously inaccessible insights into differential IL-4 signaling through type I versus type II receptors. Because of their hyperstability, our computationally designed mimetics can directly incorporate into sophisticated biomaterials that require heat processing, such as three-dimensional-printed scaffolds. Neo-4 should be broadly useful for interrogating IL-4 biology, and the design workflow will inform targeted cytokine therapeutic development.

A versatile design platform for glycoengineering therapeutic antibodies.

Manipulation of glycosylation patterns, i.e., glycoengineering, is incorporated in the therapeutic antibody development workflow to ensure clinical safety, and this approach has also been used to modulate the biological activities, functions, or pharmacological properties of antibody drugs. Whereas most existing glycoengineering strategies focus on the canonical glycans found in the constant domain of immunoglobulin G (IgG) antibodies, we report a new strategy to leverage the untapped potential of atypical glycosylation patterns in the variable domains, which naturally occur in 15% to 25% of IgG antibodies. Glycosylation sites were added to the antigen-binding regions of two functionally divergent, interleukin-2-binding monoclonal antibodies. We used computational tools to rationally install various N-glycosylation consensus sequences into the antibody variable domains, creating "glycovariants" of these molecules. Strikingly, almost all the glycovariants were successfully glycosylated at their newly installed N-glycan sites, without reduction of the antibody's native function. Importantly, certain glycovariants exhibited modified activities compared to the parent antibody, showing the potential of our glycoengineering strategy to modulate biological function of antibodies involved in multi-component receptor systems. Finally, when coupled with a high-flux sialic acid precursor, a glycovariant with two installed glycosylation sites demonstrated superior in vivo half-life. Collectively, these findings validate a versatile glycoengineering strategy that introduces atypical glycosylation into therapeutic antibodies in order to improve their efficacy and, in certain instances, modulate their activity early in the drug development process.

Multiepitope supramolecular peptide nanofibers eliciting coordinated humoral and cellular antitumor immune responses.

Subunit vaccines inducing antibodies against tumor-specific antigens have yet to be clinically successful. Here, we use a supramolecular α-helical peptide nanofiber approach to design epitope-specific vaccines raising simultaneous B cell, CD8+ T cell, and CD4+ T cell responses against combinations of selected epitopes and show that the concurrent induction of these responses generates strong antitumor effects in mice, with significant improvements over antibody or CD8+ T cell-based vaccines alone, in both prophylactic and therapeutic subcutaneous melanoma models. Nanofiber vaccine-induced antibodies mediated in vitro tumoricidal antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). The addition of immune checkpoint and phagocytosis checkpoint blockade antibodies further improved the therapeutic effect of the nanofiber vaccines against murine melanoma. These findings highlight the potential clinical benefit of vaccine-induced antibody responses for tumor treatments, provided that they are accompanied by simultaneous CD8+ and CD4+ responses, and they illustrate a multiepitope cancer vaccine design approach using supramolecular nanomaterials.

Strategies for Glycoengineering Therapeutic Proteins.

Almost all therapeutic proteins are glycosylated, with the carbohydrate component playing a long-established, substantial role in the safety and pharmacokinetic properties of this dominant category of drugs. In the past few years and moving forward, glycosylation is increasingly being implicated in the pharmacodynamics and therapeutic efficacy of therapeutic proteins. This article provides illustrative examples of drugs that have already been improved through glycoengineering including cytokines exemplified by erythropoietin (EPO), enzymes (ectonucleotide pyrophosphatase 1, ENPP1), and IgG antibodies (e.g., afucosylated Gazyva®, Poteligeo®, Fasenra™, and Uplizna®). In the future, the deliberate modification of therapeutic protein glycosylation will become more prevalent as glycoengineering strategies, including sophisticated computer-aided tools for "building in" glycans sites, acceptance of a broad range of production systems with various glycosylation capabilities, and supplementation methods for introducing non-natural metabolites into glycosylation pathways further develop and become more accessible.

Insights into the anticancer mechanisms of interleukin-15 from engineered cytokine therapies.

Innovative approaches in the field of cytokine engineering are revolutionizing the cancer therapeutic landscape. The IL-15 cytokine is particularly enticing as a cancer immunotherapy due to its natural propensity for stimulating the proliferation and activation of NK and CD8+ T cells. In a recent IL-15 engineering approach, the cytokine was conjugated to polyethylene glycol, and the resulting molecule (NKTR-255) exhibited potent antitumor activities. In this issue of the JCI, Robinson et al. mechanistically explored NKTR-255 and compared its immune profile to that of the unconjugated IL-15 cytokine. The authors found that NKTR-255 employs distinct activities on NK compared with CD8+ T cells. NKTR-255 signaling also showed less dependence on the expression of the IL-15 receptor-α (IL-15Rα) chain compared with unconjugated IL-15. Collectively, these findings will advance IL-15-based clinical therapies and, more generally, benefit the field of cancer immunotherapy.

Modular complement assemblies for mitigating inflammatory conditions.

Complement protein C3dg, a key linkage between innate and adaptive immunity, is capable of stimulating both humoral and cell-mediated immune responses, leading to considerable interest in its use as a molecular adjuvant. However, the potential of C3dg as an adjuvant is limited without ways of controllably assembling multiple copies of it into vaccine platforms. Here, we report a strategy to assemble C3dg into supramolecular nanofibers with excellent compositional control, using ß-tail fusion tags. These assemblies were investigated as therapeutic active immunotherapies, which may offer advantages over existing biologics, particularly toward chronic inflammatory diseases. Supramolecular assemblies based on the Q11 peptide system containing ß-tail-tagged C3dg, B cell epitopes from TNF, and the universal T cell epitope PADRE raised strong antibody responses against both TNF and C3dg, and prophylactic immunization with these materials significantly improved protection in a lethal TNF-mediated inflammation model. Additionally, in a murine model of psoriasis induced by imiquimod, the C3dg-adjuvanted nanofiber vaccine performed as well as anti-TNF monoclonal antibodies. Nanofibers containing only ß-tail-C3dg and lacking the TNF B cell epitope also showed improvements in both models, suggesting that supramolecular C3dg, by itself, played an important therapeutic role. We observed that immunization with ß-tail-C3dg caused the expansion of an autoreactive C3dg-specific T cell population, which may act to dampen the immune response, preventing excessive inflammation. These findings indicate that molecular assemblies displaying C3dg warrant further development as active immunotherapies.