Engineered human cytokine/antibody fusion proteins expand regulatory T cells and confer autoimmune disease protection

VanDyke, Derek; Iglesias, Marcos; Tomala, Jakub; Young, Arabella; Smith, Jennifer A.; Perry, Joseph A; Gebara, Edward; Cross, Amy; Dykema, Arbor G.; Orcutt-Jahns, Brian T; Henclová, Tereza; Goliáš, Jaroslav; Balolong, Jared; Funda, David P.; Meyer, Aaron S.; Pardoll, Drew M.; Hester, Joanna; Issa, Fadi; Hunter, Christopher A.; Anderson, Mark S.; Bluestone, Jeffrey A.; Raimondi, Giorgio; Spangler, Jamie B.

doi:10.1016/j.celrep.2022.111478

Cited by 24 publications

(41 citation statements)

References 102 publications

(139 reference statements)

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“…6). In total, our results show that not only do multivalency and cis targeting of IL2Rα improve Treg selectivity, but that these paired strategies represent the only known method for overcoming the selectivity-potency tradeoff faced by Treg-selective muteins 18 .…”

Section: Discussionmentioning

confidence: 82%

“…Our multivalent and bitargeted designs will additionally need to be tested in vivo to see that these selectivity gains translate to the in vivo setting 23 . Treg selectivity is central to the mechanism of action for these therapies, and so we expect that these benefits to selectivity will improve therapeutic properties in several ways: more potent activation of signaling in Tregs without off-target effects may improve the potency of these therapies and the breadth of applications 18,40,41 ; reduced toxicity may allow for more routine use with minimal patient monitoring 14 . The superior selectivity offered by engineered multivalent ligands will likely further increase their in vivo pharmacokinetic lifetimes, in turn requiring less frequent dosing, as most drug clearance occurs via receptor-mediated endocytosis in off-target populations 21,[42][43][44] .…”

Section: Discussionmentioning

confidence: 99%

“…While IL2Rα is more abundant in Tregs, the difference is subtle compared to some off-target cells, making selectively targeted activation more challenging 15,18 . Consequently, we wondered whether a cis targeting strategy, in which IL-2 is fused to a domain binding some other Treg-specific surface marker, would provide even greater selectivity.…”

Section: The Protein Expression and Abundance Profile Of Tregs Reveal...mentioning

confidence: 99%

“…However, this increase in selectivity comes at the cost of potency, as IL2Rβ is necessary for signal transduction 17 . Thus, when targeting Tregs, IL-2 therapies have faced a persistent tradeoff between selectivity and potency 18 .…”

Section: Introductionmentioning

confidence: 99%

“…Fc fusion has taken many forms such as fusion to the cytokine at the N-or C-terminus, including one or two cytokines per IgG, and including or excluding Fc effector functions 15 . So-called immunocytokines have also been employed which bind to IL-2 and block certain receptor interactions to bias signaling responses 18 . More recently, cis-targeted IL-2 fusions, such as bispecific antibodies in which a non-fused Fab binds to CD8, have been designed to deliver cytokines to cytotoxic T cells 24 .…”

Section: Introductionmentioning

confidence: 99%

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Multivalent, asymmetric IL-2-Fc fusions provide optimally enhanced regulatory T cell selectivity

Orcutt-Jahns

et al. 2021

Preprint

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The common γ-chain receptor cytokines are promising immune therapies due to their central role in coordinating the proliferation and activity of various immune cell populations. One of these cytokines, interleukin (IL)-2, has potential as a therapy in autoimmunity but is limited in effectiveness by its modest specificity toward regulatory T cells (Tregs). Therapeutic ligands are often made dimeric as antibody Fc fusions to confer desirable pharmacokinetic benefits, with under-explored consequences on signaling. Here, we systematically profiled the signaling responses to a panel of wild type and mutein IL-2 molecules in various Fc fusion configurations. We used a tensor-structured dimensionality reduction scheme to decompose the responses of each cell population to each ligand over a range of time points and cytokine concentrations. We found that dimeric muteins are uniquely specific for Tregs at intermediate ligand concentrations. We then compared signaling response across all treatments to a simple, two-step multivalent binding model. Our model was able to predict cellular responses with high accuracy. Bivalent Fc fusions display enhanced specificity and potency for Tregs through avidity effects toward IL-2Rα. We then utilize our model to identify the potential benefits conferred by valency engineering as an additional mechanism for cytokines with optimized therapeutic benefits. In total, these findings represent a comprehensive analysis of how ligand properties, and their consequent effects on surface receptor-ligand interactions, translate to selective activation of immune cell populations. It also identifies a new route toward engineering even more selective therapeutic cytokines.

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Section: Discussionmentioning

confidence: 82%

Section: Discussionmentioning

confidence: 99%

Section: The Protein Expression and Abundance Profile Of Tregs Reveal...mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Multivalent, asymmetric IL-2-Fc fusions provide optimally enhanced regulatory T cell selectivity

Orcutt-Jahns

et al. 2021

Preprint

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Cytokine/Antibody Fusion Protein Design and Evaluation

Fabilane,

Stephenson,

Leonard

et al. 2024

Current Protocols

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Cytokines constitute a class of secreted proteins that activate transmembrane receptors to coordinate a vast array of physiological processes, particularly those related to immune activity. Due to their vital role in immune regulation, cytokines have garnered great interest as potential therapeutic agents. Unfortunately, the clinical success of cytokine drugs has been limited by their multifunctional activities, which hinder therapeutic performance and lead to harmful toxicities. In addition, the strikingly short circulation half‐life of cytokines further hampers their efficacy as drugs. To overcome the translational challenges associated with natural cytokines, significant efforts have focused on engineering cytokines to target their activities and improve their pharmacological properties. One such strategy is the design of fusion proteins that tether a cytokine to an anti‐cytokine antibody that selectively biases its functions and extends its serum half‐life. These cytokine/antibody fusion proteins (termed immunocytokines) assemble intramolecularly to bias cytokine signaling behavior through multi‐layered structural and molecular effects. Here, we present a detailed workflow for the design, production, and functional validation of intramolecularly assembled immunocytokines. In‐depth procedures are presented for gene manipulation, mammalian cell‐based expression and purification, binding analysis via bio‐layer interferometry, and interrogation of cytokine signaling activity on human primary cells. In contrast with immunocytokines in which the tethered cytokine and antibody do not bind one another, intramolecularly assembled immunocytokines require special considerations with respect to their production to avoid oligomerization and/or aggregation. The protocol herein was developed based on experience with immunocytokines that incorporate interleukin‐2 (IL‐2); however, this modular approach can be extended to any cytokine of interest for a broad range of biomedical applications. © 2024 Wiley Periodicals LLC.Basic Protocol 1: Design and generation of immunocytokine genesBasic Protocol 2: Immunocytokine expression and purificationBasic Protocol 3: Validation of immunocytokine assembly and binding by bio‐layer interferometryBasic Protocol 4: Analysis of immunocytokine signaling on human primary cells

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Molecular Engineering of Interleukin-2 for Enhanced Therapeutic Activity in Autoimmune Diseases

Tomasovic,

Liu,

VanDyke

et al. 2023

BioDrugs

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Engineered human cytokine/antibody fusion proteins expand regulatory T cells and confer autoimmune disease protection

Cited by 24 publications

References 102 publications

Multivalent, asymmetric IL-2-Fc fusions provide optimally enhanced regulatory T cell selectivity

Multivalent, asymmetric IL-2-Fc fusions provide optimally enhanced regulatory T cell selectivity

Cytokine/Antibody Fusion Protein Design and Evaluation

Molecular Engineering of Interleukin-2 for Enhanced Therapeutic Activity in Autoimmune Diseases

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