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.

Metabolic15N labeling of the N-glycosylated immunoglobulin G1 Fc with an engineered Saccharomyces cerevisiae strain.

The predominant protein expression host for NMR spectroscopy is Escherichia coli, however, it does not synthesize appropriate post-translation modifications required for mammalian protein function and is not ideal for expressing naturally secreted proteins that occupy an oxidative environment. Mammalian expression platforms can address these limitations; however, these are not amenable to cost-effective uniform 15 N labeling resulting from highly complex growth media requirements. Yeast expression platforms combine the simplicity of bacterial expression with the capabilities of mammalian platforms, however yeasts require optimization prior to isotope labeling. Yeast expression will benefit from methods to boost protein expression levels and developing labeling conditions to facilitate growth and high isotope incorporation within the target protein. In this work, we describe a novel platform based on the yeast Saccharomyces cerevisiae that simultaneously expresses the Kar2p chaperone and protein disulfide isomerase in the ER to facilitate the expression of secreted proteins. Furthermore, we developed a growth medium for uniform 15 N labeling. We recovered 2.2 mg/L of uniformly 15 N-labeled human immunoglobulin (Ig)G1 Fc domain with 90.6% 15 N labeling. NMR spectroscopy revealed a high degree of similarity between the yeast and mammalian-expressed IgG1 Fc domains. Furthermore, we were able to map the binding interaction between IgG1 Fc and the Z domain through chemical shift perturbations. This platform represents a novel cost-effective strategy for 15 N-labeled immunoglobulin fragments.

Recent Advances Toward Engineering Glycoproteins Using Modified Yeast Display Platforms.

Yeast are capable recombinant protein expression hosts that provide eukaryotic posttranslational modifications such as disulfide bond formation and N-glycosylation. This property has been used to create surface display libraries for protein engineering; however, yeast surface display (YSD) with common laboratory strains has limitations in terms of diversifying glycoproteins due to the incorporation of high levels of mannose residues which often obscure important epitopes and are immunogenic in humans. Developing new strains for efficient and appropriate display will require combining existing technologies to permit efficient glycoprotein engineering. Foundational efforts generating knockout strains lacking characteristic hypermannosylation reactions exhibited morphological defects and poor growth. Later strains with "humanized" N-glycosylation machinery surmounted these limitations by targeting a small suite of glycosylhydrolase and glycosyltransferase enzymes from other taxa to the endoplasmic reticulum and Golgi. Advanced yeast strains also provide key modifications at the glycan termini that are essential for the full function of many glycoproteins. Here we review progress toward glycoprotein engineering when glycosylation is required for full function using advanced yeast expression platforms and the suitability of each for YSD of glycoproteins.

Expression and Display of Glycoengineered Antibodies and Antibody Fragments with an Engineered Yeast Strain.

Interactions with cell surface receptors enhance the therapeutic properties of many important antibodies, including the low-affinity Fc γ Receptors (FcγRs). These interactions require proper processing of the immunoglobulin G Fc N-glycan, and eliminating the N-glycan abolishes binding, restricting antibody production to mammalian expression platforms. Yeasts, for example, generate extensively mannosylated N-glycans that are unsuitable for therapeutics. However, Fc with a specifically truncated N-glycan still engages receptors with considerable affinity. Here we describe the creation and applications of a novel Saccharomyces cerevisiae strain that specifically modifies the IgG1 Fc domain with an N-glycan consisting of a single N-acetylglucosamine residue. This strain displayed glycoengineered Fc on its surface for screening yeast surface display libraries and also served as an alternative platform to produce glycoengineered Rituximab. An IgG-specific endoglycosidase (EndoS2) truncates the IgG1 Fc N-glycan. EndoS2 was targeted to the yeast ER using the signal peptide from the yeast protein disulfide isomerase (PDI) and a yeast ER retention signal (HDEL). Furthermore, >99% of the yeast expressed Rituximab displayed the truncated glycoform as determined by SDS-PAGE and ESI-MS analyses. Lastly, the yeast expressed Rituximab engaged the FcγRIIIa with the expected affinity (KD = 2.0 ± 0.5 µM) and bound CD20 on Raji B cells.