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-2Rß and ¿c (IL-2Rß¿). 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.