Glycosylation alters protein form and function by establishing intermolecular forces that mediate specific interactions while preventing non-specific aggregation. Self-assembled peptide nanofibers modified with carbohydrates are increasingly used as biomaterials to mimic glycosylated protein function, yet the influence of carbohydrate conjugates on nanofiber structure remains poorly defined. Here we show that a dense carbohydrate surface layer can facilitate hierarchical organization of peptide nanofibers into anisotropic networks. Glycosylated peptide nanofibers remain dispersed in dilute conditions, whereas non-glycosylated nanofibers tend to aggregate. In crowded conditions, some glycosylated nanofibers laterally associate and align. This behavior depends on carbohydrate chemistry, particularly hydroxyls, suggesting involvement of short-range attractive forces. Macroscopic gels fabricated from densely glycosylated peptide nanofibers are resistant to non-specific interactions with proteins, mammalian cells, and bacteria, yet selectively bind lectins, analogous to natural lowfouling mucosal barriers. Collectively, these observations demonstrate that glycosylation can inform structure in addition to endowing function to peptide-based supramolecular biomaterials.

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.

The carbohydrate-binding protein galectin-3 (Gal3) is an attractive drug target due to its role as a modulator of cell behavior in various pathological processes. However, development of effective Gal3 inhibitors has been hindered by the conserved binding properties of different galectins and the low affinity of monovalent protein–carbohydrate interactions. Immobilizing carbohydrates onto biomaterials can enhance their effectiveness for inhibiting galectins by establishing multivalent avidity effects that increase their apparent galectin-binding affinity. Here, we evaluated a candidate multivalent Gal3 inhibitor based on self-assembled peptide nanofibers modified with N,N′-diacetyllactosamine (i.e., “LacDiNAc”), a disaccharide that preferentially binds Gal3. QQKFQFQFEQQ (“Q11”) nanofibers modified with LacDiNAc (i.e., “LacDiNAc-Q11”) bound Gal3 with micromolar affinity and selectively captured Gal3 in the presence of galectin-1. However, LacDiNAc-Q11 nanofibers failed to inhibit Jurkat T cell death induced by Gal3 in media supplemented with 10% serum. Unexpectedly, coprecipitation experiments demonstrated that serum glycoproteins blocked Gal3 binding to LacDiNAc-Q11 nanofibers as well as Q11 nanofibers modified with N-acetyllactosamine (i.e., “LacNAc-Q11”), yet did not affect galectin-1 binding to LacNAc-Q11 nanofibers. When serum content of culture media was reduced, LacDiNAc-Q11 nanofibers inhibited Jurkat T cell agglutination and death induced by Gal3. Collectively, these observations demonstrate that serum glycoproteins can selectively antagonize Gal3 interactions with self-assembled glycopeptide nanofibers, thereby diminishing their effectiveness as Gal3 inhibitors. These studies underscore the need for candidate multivalent Gal3 inhibitors with robust binding selectivity as well as exceptionally high binding affinity that can disrupt Gal3 interactions with both cell surface glycans and abundant serum glycoproteins.

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.

Assembly of a fusion of galectin-1 and galectin-3 with higher carbohydrate binding affinity and a significantly lower effective dose than galectin-1.