Sunday, May 11, 2025
5/11/2025
Directing Cytokine Specificity Through Co-translational Carrier Coupling - Recombinant cytokines are used in the clinic to treat both chronic autoimmune diseases such as multiple sclerosis and malignancies such as metastatic melanoma, for the purpose of either suppressing autoimmunity or activating tumor immunity. Their considerable success in ameliorating established disease, measured by decreased clinical symptoms of autoimmunity and prolonged progression free survival of cancer patients, support the clinical importance of cytokine therapies, particularly in comparison to broadly immunosuppressive or immunostimulatory drug regimens. Yet, additional improvements are needed because in both clinical settings, cytokine-based therapies suffer from two major challenges, a) tendency of cytokines to induce pleiotropic effects through multiple target cells, which can result in severe toxicities, and b) short cytokine half- lives, which require frequent administration or high doses. We propose to address both issues by disulfide crosslinking cytokines of interest with different surfaces of HSA. We hypothesize that this approach can be generalized to small bioactive proteins to yield precision engineered variants with high receptor selectivity, by virtue of HSA-mediated steric blocking, with extended circulation half-life, by virtue of the fusions’ increased size and potential for neonatal Fc receptor (FcRn) recycling, and with ease in manufacturing and purification of a single molecular entity. In this application, we specifically chose IL-2 as the prototypic cytokine for HSA- disulfide design due to its central role in regulating and activating immune responses through differential binding to trimeric and dimeric IL-2 receptors expressed on regulatory T cells (Tregs) and CD8 cytotoxic cells (CTLs). Treg cells constitutively express all three components of the high affinity IL-2 receptor (IL-2R), which renders them sensitive to relatively low concentrations of IL-23. In contrast, CTLs and natural killer (NK) cells only transiently express the high affinity IL-2R during activation, but more generally express the medium affinity receptor comprised of just IL-2Rβ and γc (IL-2Rβγ). For this reason, high doses of IL-2 are required to activate CTL for the treatment of metastatic renal cancer (mRC) and metastatic melanoma (MM). Low IL-2 doses target Treg and are being explored for the treatment of autoimmune diseases such as type I diabetes (T1D) and systemic lupus erythematosus (SLE). Although current dose differentiated regimens of native IL-2 have shown some promise, the intrinsic overlapping activity toward both CTLs and Treg cells has thus far proved a serious barrier to effective IL-2 immunotherapy. Moreover, as a small 133 amino acid residue protein, native IL-2 is cleared from circulation quickly, requiring continuous infusion to maintain efficacious blood concentrations. To address these critical limitations of IL-2, both its receptor specificity and pharmacokinetics (PK) must be optimized. To achieve these goals, we propose to engineer disulfide cross-linked HSA-cc-IL2 complexes that preferentially activate 1) effector T cells to promote their tumor immunity and 2) regulatory T cells to promote suppression of inflammatory diseases.
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