Intra-articular delivery of an indoleamine 2,3-dioxygenase galectin-3 fusion protein for osteoarthritis treatment in male Lewis rats.

OBJECTIVE: Osteoarthritis (OA) is driven by low-grade inflammation, and controlling local inflammation may offer symptomatic relief. Here, we developed an indoleamine 2,3-dioxygenase and galectin-3 fusion protein (IDO-Gal3), where IDO increases the production of local anti-inflammatory metabolites and Gal3 binds carbohydrates to extend IDO's joint residence time. In this study, we evaluated IDO-Gal3's ability to alter OA-associated inflammation and pain-related behaviors in a rat model of established knee OA. METHODS: Joint residence was first evaluated with an analog Gal3 fusion protein (NanoLuc™ and Gal3, NL-Gal3) that produces luminescence from furimazine. OA was induced in male Lewis rats via a medial collateral ligament and medial meniscus transection (MCLT + MMT). At 8 weeks, NL or NL-Gal3 were injected intra-articularly (n = 8 per group), and bioluminescence was tracked for 4 weeks. Next, IDO-Gal3s's ability to modulate OA pain and inflammation was assessed. Again, OA was induced via MCLT + MMT in male Lewis rats, with IDO-Gal3 or saline injected into OA-affected knees at 8 weeks post-surgery (n = 7 per group). Gait and tactile sensitivity were then assessed weekly. At 12 weeks, intra-articular levels of IL6, CCL2, and CTXII were assessed. RESULTS: The Gal3 fusion increased joint residence in OA and contralateral knees (p < 0.0001). In OA-affected animals, both saline and IDO-Gal3 improved tactile sensitivity (p = 0.008), but IDO-Gal3 also increased walking velocities (p ≤ 0.033) and improved vertical ground reaction forces (p ≤ 0.04). Finally, IDO-Gal3 decreased intra-articular IL6 levels within the OA-affected joint (p = 0.0025). CONCLUSION: Intra-articular IDO-Gal3 delivery provided long-term modulation of joint inflammation and pain-related behaviors in rats with established OA.

Suppression of local inflammation via galectin-anchored indoleamine 2,3-dioxygenase.

The treatment of chronic inflammation with systemically administered anti-inflammatory treatments is associated with moderate-to-severe side effects, and the efficacy of locally administered drugs is short-lived. Here we show that inflammation can be locally suppressed by a fusion protein of the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO) and galectin-3 (Gal3). Gal3 anchors IDO to tissue, limiting the diffusion of IDO-Gal3 away from the injection site. In rodent models of endotoxin-induced inflammation, psoriasis, periodontal disease and osteoarthritis, the fusion protein remained in the inflamed tissues and joints for about 1 week after injection, and the amelioration of local inflammation, disease progression and inflammatory pain in the animals were concomitant with homoeostatic preservation of the tissues and with the absence of global immune suppression. IDO-Gal3 may serve as an immunomodulatory enzyme for the control of focal inflammation in other inflammatory conditions.

Intra-Articular Delivery of an Indoleamine 2,3-Dioxygenase Galectin-3 Fusion Protein for Osteoarthritis Treatment in Male Lewis Rats.

Objective : Controlling joint inflammation can improve osteoarthritis (OA) symptoms; however, current treatments often fail to provide long-term effects. We have developed an indoleamine 2,3-dioxygenase and galectin-3 fusion protein (IDO-Gal3). IDO converts tryptophan to kynurenines, directing the local environment toward an anti-inflammatory state; Gal3 binds carbohydrates and extends IDO's joint residence time. In this study, we evaluated IDO-Gal3's ability to alter OA-associated inflammation and pain-related behaviors in a rat model of established knee OA. Methods : Joint residence was first evaluated with an analog Gal3 fusion protein (NanoLuc™ and Gal3, NL-Gal3) that produces luminescence from furimazine. OA was induced in male Lewis rats via a medial collateral ligament and medial meniscus transection (MCLT+MMT). At 8 weeks, NL or NL-Gal3 were injected intra-articularly (n=8 per group), and bioluminescence was tracked for 4 weeks. Next, IDO-Gal3's ability to modulate OA pain and inflammation was assessed. Again, OA was induced via MCLT+MMT in male Lewis rats, with IDO-Gal3 or saline injected into OA-affected knees at 8 weeks post-surgery (n=7 per group). Gait and tactile sensitivity were then assessed weekly. At 12 weeks, intra-articular levels of IL6, CCL2, and CTXII were assessed. Results : The Gal3 fusion increased joint residence in OA and contralateral knees (p<0.0001). In OA-affected animals, IDO-Gal3 improved tactile sensitivity (p=0.002), increased walking velocities (p≤0.033), and improved vertical ground reaction forces (p≤0.04). Finally, IDO-Gal3 decreased intra-articular IL6 levels within the OA-affected joint (p=0.0025). Conclusion : Intra-articular IDO-Gal3 delivery provided long-term modulation of joint inflammation and pain-related behaviors in rats with established OA.

Heterogeneous protein co-assemblies with tunable functional domain stoichiometry.

In nature, the precise heterogeneous co-assembly of different protein domains gives rise to supramolecular machines that perform complex functions through the co-integrated activity of the individual protein subunits. A synthetic approach capable of mimicking this process would afford access to supramolecular machines with new or improved functional capabilities. Here we show that the distinct peptide strands of a heterotrimeric α-helical coiled-coil (i.e., peptides "A", "B", and "C") can be used as fusion tags for heterogeneous co-assembly of proteins into supramolecular structures with tunable subunit stoichiometry. In particular, we demonstrate that recombinant fusion of A with NanoLuc luciferase (NL-A), B with superfolder green fluorescent protein (sfGFP-B), and C with mRuby (mRuby-C) enables formation of ternary complexes capable of simultaneously emitting blue, green, and red light via sequential bioluminescence and fluorescence resonance energy transfer (BRET/FRET). Fusion of galectin-3 onto the C-terminus of NL-A, sfGFP-B, and mRuby-C endows the ternary complexes with lactose-binding affinity that can be tuned by varying the number of galectin-3 domains integrated into the complex from one to three, while maintaining BRET/FRET function. The modular nature of the fusion protein design, the precise control of domain stoichiometry, and the multiplicity afforded by the three-stranded coiled-coil scaffold provides access to a greater range of subunit combinations than what is possible with heterodimeric coiled-coils used previously. We envision that access to this expanded range of co-integrated protein domain diversity will be advantageous for future development of designer supramolecular machines for therapeutic, diagnostic, and biotechnology applications.

Experimental and Computational Models of Transport of Galectin-3 Through Glycosylated Matrix.

Altered extracellular matrix (ECM) production is a hallmark of many fibroproliferative diseases, including certain cancers. The high incidence of glycan-rich components within altered ECM makes the use of glycan-binding proteins such as Galectin-3 (G3) a promising therapeutic strategy. The complexity of ECM as a rich 3D network of proteins with varied glycosylation states makes it challenging to determine the retention of glycan-binding proteins in altered ECM environments. Computational models capable of predicting the transport of glycan-binding proteins in altered ECM can benefit the design and testing of such proteins and associated novel therapeutic strategies. However, such computational models require many kinetic parameters that cannot be estimated from traditional 2D pharmacokinetic assays. To validate transport properties of G3 in 3D ECM constructs, we developed a species transport model that includes diffusion and matrix-binding components to predict retention of G3 fusion proteins in glycan-rich ECM. By iteratively comparing our computational model to experimental results, we are able to determine a reasonable range of parameters for a robust computational model of G3 transport. We anticipate this overall approach to building a data-driven model is translatable to other ECM-targeting therapeutic strategies.

Site-Specific Cross-Linking of Galectin-1 Homodimers via Poly(ethylene glycol) Bismaleimide.

INTRODUCTION: The promise of the natural immunoregulator, Galectin-1 (Gal1), as an immunomodulatory therapeutic is challenged by its unstable homodimeric conformation. Previously, a Gal1 homodimer stabilized via covalent poly(ethylene glycol) diacrylate (PEGDA) cross-linking demonstrated higher activity relative to the non-covalent homodimer. METHODS: Here, we report Gal1 homodimers formed using an alternative thiol-Michael addition linker chemistry. RESULTS: Poly(ethylene glycol) bismaleimide (PEGbisMal) reacted with Gal1 at multiple sites with greater efficiency than PEGDA. However, multiple PEGbisMal molecules were conjugated to Gal1 C130, a Gal1 mutant with one surface cysteine (cys-130) and two cysteines thought to be buried in the solvent-inaccessible protein core (cys-42 and cys-60). Site-directed mutagenesis demonstrated that cys-60 was the site at which additional PEGbisMal molecules were conjugated onto Gal1 C130. Compared to WT-Gal1, Gal1 C130 had low activity for inducing Jurkat T cell death, characterized by phosphatidylserine exposure and membrane permeability. PEG cross-linking could restore the function of Gal1 C130, such that at high concentrations Gal1 C130 cross-linked by PEGbisMal had higher activity than both WT-Gal1 and Gal1 C130 cross-linked by PEGDA. Mutating cys-42 and cys-60 to serines in Gal1 C130 did not affect the cell death signaling activity of the Gal1 C130 homodimer cross-linked by PEGbisMal. PEGylated Gal1 C130 variants also eliminated the need for a reducing agent, such as dithiothreitol, which is required to maintain WT-Gal1 signaling activity. CONCLUSION: Collectively, these data demonstrate that thiol-Michael addition bioconjugation leads to a PEG-cross-linked Gal1 homodimer with improved extracellular signaling activity that does not require a reducing environment to be functional.

Physical tuning of galectin-3 signaling.

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.

A chimeric, multivalent assembly of galectin-1 and galectin-3 with enhanced extracellular activity.

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.

A Synthetic Tetramer of Galectin-1 and Galectin-3 Amplifies Pro-apoptotic Signaling by Integrating the Activity of Both Galectins.

Galectin-1 (G1) and galectin-3 (G3) are carbohydrate-binding proteins that can signal apoptosis in T cells. We recently reported that a synthetic tetramer with two G1 and two G3 domains ("G1/G3 Zipper") induces Jurkat T cell death more potently than G1. The pro-apoptotic signaling pathway of G1/G3 Zipper was not elucidated, but we hypothesized based on prior work that the G1 domains acted as the signaling units, while the G3 domains served as anchors that increase glycan-binding affinity. To test this, here we studied the involvement of different cell membrane glycoproteins and intracellular mediators in pro-apoptotic signaling via G1/G3 Zipper, G1, and G3. G1/G3 Zipper induced Jurkat T cell death more potently than G1 and G3 alone or in combination. G1/G3 Zipper, G1, and G3 increased caspase-8 activity, yet only G1 and G3 depended on it to induce cell death. G3 increased caspase-3 activity more than G1/G3 Zipper and G1, while all three galectin variants required it to induce cell death. JNK activation had similar roles downstream of G1/G3 Zipper, G1, and G3, whereas ERK had differing roles. CD45 was essential for G1 activity, and was involved in signaling via G1/G3 Zipper and G3. CD7 inhibited G1/G3 Zipper activity at low galectin concentrations but not at high galectin concentrations. In contrast, CD7 was necessary for G1 and G3 signaling at low galectin concentration but antagonistic at high galectin concentrations. Collectively, these observations suggest that G1/G3 Zipper amplifies pro-apoptotic signaling through the integrated activity of both the G1 and G3 domains.

Locally anchoring enzymes to tissues via extracellular glycan recognition.

Success of enzymes as drugs requires that they persist within target tissues over therapeutically effective time frames. Here we report a general strategy to anchor enzymes at injection sites via fusion to galectin-3 (G3), a carbohydrate-binding protein. Fusing G3 to luciferase extended bioluminescence in subcutaneous tissue to ~7 days, whereas unmodified luciferase was undetectable within hours. Engineering G3-luciferase fusions to self-assemble into a trimeric architecture extended bioluminescence in subcutaneous tissue to 14 days, and intramuscularly to 3 days. The longer local half-life of the trimeric assembly was likely due to its higher carbohydrate-binding affinity compared to the monomeric fusion. G3 fusions and trimeric assemblies lacked extracellular signaling activity of wild-type G3 and did not accumulate in blood after subcutaneous injection, suggesting low potential for deleterious off-site effects. G3-mediated anchoring to common tissue glycans is expected to be broadly applicable for improving local pharmacokinetics of various existing and emerging enzyme drugs.

Enzymes as Immunotherapeutics.

Enzymes are attractive as immunotherapeutics because they can catalyze shifts in the local availability of immunostimulatory and immunosuppressive signals. Clinical success of enzyme immunotherapeutics frequently hinges upon achieving sustained biocatalysis over relevant time scales. The time scale and location of biocatalysis are often dictated by the location of the substrate. For example, therapeutic enzymes that convert substrates distributed systemically are typically designed to have a long half-life in circulation, whereas enzymes that convert substrates localized to a specific tissue or cell population can be more effective when designed to accumulate at the target site. This Topical Review surveys approaches to improve enzyme immunotherapeutic efficacy via chemical modification, encapsulation, and immobilization that increases enzyme accumulation at target sites or extends enzyme half-life in circulation. Examples provided illustrate "replacement therapies" to restore deficient enzyme function, as well as "enhancement therapies" that augment native enzyme function via supraphysiologic doses. Existing FDA-approved enzyme immunotherapies are highlighted, followed by discussion of emerging experimental strategies such as those designed to enhance antitumor immunity or resolve inflammation.

Engineering galectin-glycan interactions for immunotherapy and immunomodulation.

Galectins, a 15-member family of soluble carbohydrate-binding proteins, are receiving increasing interest as therapeutic targets for immunotherapy and immunomodulation due to their role as extracellular signals that regulate innate and adaptive immune cell phenotype and function. However, different galectins can have redundant, synergistic, or antagonistic signaling activity in normal immunological responses, such as resolution of inflammation and induction of antigen-specific tolerance. In addition, certain galectins can be hijacked to promote progression of immunopathologies, such as tumor immune privilege, metastasis, and viral infection, while others can inhibit these processes. Thus, eliciting a desired immunological outcome will likely necessitate therapeutics that can precisely enhance or inhibit particular galectin-glycan interactions. Multivalency is an important determinant of the affinity and specificity of natural galectin-glycan interactions, and is emerging as a key design element for therapeutics that can effectively manipulate galectin bioactivity. This minireview surveys current molecular and biomaterial engineering approaches to create therapeutics that can stabilize galectin multivalency or recapitulate natural glycan multivalency (i.e. "the glycocluster effect"). In particular, we highlight examples of using natural and engineered multivalent galectins for immunosuppression and immune tolerance, with a particular emphasis on treating autoimmune diseases or avoiding transplant rejection. In addition, we present examples of multivalent inhibitors of galectin-glycan interactions to maintain or restore T-cell function, with a particular emphasis on promoting antitumor immunity. Finally, we discuss emerging opportunities to further engineer galectin-glycan interactions for immunotherapy and immunomodulation.