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Summary paragraphChronic inflammation underlies the onset, progression and associated pain of numerous diseases.(1) Current anti-inflammatory treatments administered systemically are associated with moderate-to-severe side effects, while locally administered drugs have short-lived efficacy, and neither approach successfully modifies the underlying causality of disease.(2) We report a new way to locally modulate inflammation by fusing the enzyme indoleamine 2,3-dioxygenase 1 (IDO) to galectin-3 (Gal3). A general regulator of inflammation(3), IDO is immunosuppressive(4), catabolizing the essential amino acid tryptophan into kynurenine.(5) Recently we demonstrated that extracellular exogenous IDO regulates innate immune cell function(6), and envisioned delivering IDO into specific tissues would provide control of inflammation. However, proteins problematically diffuse away from local injection sites. Addressing this, we recently established that fusion to Gal3 anchors enzymes to tissues(7) via binding to extracellular glycans. Fusion protein IDO-Gal3 was retained in injected tissues and joints for up to a week or more, where it suppressed local inflammation in rodent models of endotoxin-induced inflammation, psoriasis, periodontal disease and osteoarthritis. Amelioration of local inflammation, disease progression and inflammatory pain were concomitant with homeostatic preservation of tissues without global immune suppression. Thus, IDO-Gal3 presents a new concept of anchoring immunomodulatory enzymes for robust control of focal inflammation in multiple disease settings.

Galectin-3 (Gal3) exhibits dynamic oligomerization and promiscuous binding, which can lead to concomitant activation of synergistic, antagonistic, or noncooperative signaling pathways that alter cell behavior. Conferring signaling pathway selectivity through mutations in the Gal3-glycan binding interface is challenged by the abundance of common carbohydrate types found on many membrane glycoproteins. Here, employing alpha-helical coiled-coils as scaffolds to create synthetic Gal3 constructs with defined valency, we demonstrate that oligomerization can physically regulate extracellular signaling activity of Gal3. Constructs with 2 to 6 Gal3 subunits ("Dimer," "Trimer," "Tetramer," "Pentamer," "Hexamer") demonstrated glycan-binding properties and cell death-inducing potency that scaled with valency. Dimer was the minimum functional valency. Unlike wild-type Gal3, which signals apoptosis and mediates agglutination, synthetic Gal3 constructs induced cell death without agglutination. In the presence of CD45, Hexamer was distributed on the cell membrane, whereas it clustered in absence of CD45 via membrane glycans other than those found on CD7. Wild-type Gal3, Pentamer, and Hexamer required CD45 and CD7 to signal apoptosis, and the involvement of caspases in apoptogenic signaling was increased in absence of CD45. However, wild-type Gal3 depended on caspases to signal apoptosis to a greater extent than Hexamer, which had greater caspase dependence than Pentamer. Diminished caspase activation downstream of Hexamer signaling led to decreased pannexin-1 hemichannel opening and interleukin-2 secretion, events facilitated by the increased caspase activation downstream of wild-type Gal3 signaling. Thus, synthetic fixation of Gal3 multivalency can impart physical control of its outside-in signaling activity by governing membrane glycoprotein engagement and, in turn, intracellular pathway activation.

Galectins are attractive therapeutic candidates to control aberrant immune system activation because they can alter the phenotype and function of various innate and adaptive immune cells. However, use of exogenous galectin-1 (“G1”) and galectin-3 (“G3”) as immunomodulators is challenged by their high dosing requirements and dynamic quaternary structures. Here we report a chimeric assembly of G1 and G3 with enhanced extracellular activity (“G1/G3 Zipper”), which was created by recombinant fusion of G1 and G3 via a peptide linker that forms a two-stranded α-helical coiled-coil. G1/G3 Zipper had higher apparent binding affinity for immobilized lactose and a lower concentration threshold for inducing soluble glycoprotein crosslinking than G1, a recombinant fusion of G1 and G3 with a flexible peptide linker (“G1/G3”), or a recently reported stable G1 dimer crosslinked by poly(ethylene glycol) diacrylate (“G1-PEG-G1”). As a result, G1/G3 Zipper was more effective at inducing Jurkat T cell apoptosis in media containing serum, and was the only variant that could induce apoptosis at low concentrations under serum-free conditions. The monomeric G1/G3 fusion protein lacked extracellular activity under all conditions tested, suggesting that the enhanced activity of G1/G3 Zipper was due to its quaternary structure and increased carbohydrate-recognition domain valency. Thus, combining G1 and G3 into a non-native chimeric assembly provides a new candidate therapeutic with greater immunomodulatory potency than the wild-type proteins and previously reported engineered variants.