Gregory A. Hudalla

Enzyme therapeutics are often limited by poor stability, rapid clearance, and systemic effects that reduce therapeutic efficacy and necessitate high dosing. Enzyme‐biomaterial conjugation can address these limitations and improve outcomes by localizing therapeutics at the tissue site of intended action. In particular, indoleamine 2,3‐dioxygenase (IDO), a potent immunomodulatory enzyme, has demonstrated therapeutic effectiveness when retained locally at sites of inflammation through fusion to a carbohydrate‐binding protein. Here, we report a complementary biomaterial‐mediated delivery strategy to prolong retention time and investigate efficacy in an imiquimod‐induced model of psoriasis. IDO maintained enzyme activity after bioconjugation to polyethylene glycol maleimide (PEG‐MAL) microgels. Localized delivery of IDO‐conjugated microgels reduced psoriatic scores compared to soluble IDO or unconjugated microgels. Additionally, fibrotic capsule thickness decreased 30% in the IDO‐conjugated microgel group compared to the unconjugated microgel. These findings demonstrate the efficacy of localized IDO delivery via covalent attachment to granular hydrogels, which may serve as a platform for other immunomodulatory enzymes.

Indoleamine 2,3-dioxygenase (IDO), the enzyme responsible for tryptophan catabolism, is protective in many autoimmune and inflammatory diseases. We previously developed a localized immunomodulation approach through the fusion of IDO to a carbohydrate binding protein [E. Bracho-Sanchez et al., Nat. Biomed. Eng. 7, 1156-1169 (2023)]. Here we further develop IDO as a protein therapeutic for inflammation, investigating systemic delivery via conjugation of poly(ethylene glycol) (PEG) to IDO. PEGylation extended circulation time and treatment PEG-IDO demonstrated therapeutic efficacy in five autoimmune and inflammatory models. Treatment with a single dose of PEG-IDO resulted in reversal of hind limb paralysis in experimental autoimmune encephalomyelitis as model of multiple sclerosis; protected against hepatic damage in liver ischemia-reperfusion injury; prevention of abdominal aortic aneurysm; and decreased severity of imiquimod-induced psoriasis. Treatment with two doses of PEG-IDO provided maintenance of body weight and colon length in acute ulcerative colitis. PEG-IDO treatment increased regulatory T cell populations and reduced pathogenic Th17 cell populations and inflammatory cytokine production. Systemic delivery of PEG-IDO had little-to-no off-target effects. Mice maintained immunocompetency, clearing the Listeria monocytogenes infection, and had no observed toxicity. Additionally, PEG-IDO increased serum kynurenine, consistent with a kynurenine-mediated mechanism of action. These findings demonstrate systemic immunomodulation via circulating PEG-IDO ameliorates a breadth of disparate autoinflammatory diseases, protecting against inflammation-driven tissue destruction in spinal cord, liver, abdominal aorta, skin, and colon, while presenting a positive safety profile, and expanding the scope of potential applications for this therapeutic approach.

Protein therapeutics are a cornerstone of modern medicine, yet their inherent instability often requires expensive and energy-intensive cold-chain logistics to maintain efficacy. Dry-state formulations offer a viable strategy to enhance protein stability, but the associated drying processes, such as lyophilization and spray drying, can introduce stresses that damage protein molecules. While excipients are widely used to protect proteins during dehydration, conventional formulations often require protein-specific, iterative optimization. Further, protein stabilizing excipients can crystallize during storage, thereby reducing product quality, increase product viscosity beyond thresholds for injection, and raise safety concerns. Nanoparticulate excipients are receiving increasing attention to address these challenges. This article provides an overview of nanoparticle excipients showing promise for stabilizing proteins during drying processes. It is envisioned that nanoparticulates may advance toward universal excipients capable of stabilizing a variety of proteins without the need for cumbersome optimization while also providing robust biocompatibility.

An immunomodulatory hydrogel, IDO-Gal3 incorporated into decellularized rat peripheral nerve (iPN), was injected into the thoracic contused rat spinal cord to evaluate its effects on astrocyte and microglial reactivity, as well as neuronal sparing.

The fetomaternal interface is replete with glycan-binding proteins (GBPs) that can interact with cell surface glycoprotein counterreceptors to regulate placental function. Here, we interrogate the role of galectin-3, a GBP that controls placental trophoblast syncytialization, an important differentiation process where progenitor cytotrophoblast cells fuse to produce the multinucleated syncytiotrophoblast. The molecular mechanism of galectin-3-mediated fusion has not yet been elucidated in part due to the difficulty of studying glycan-GBP binding events in live cells. To overcome these challenges, we employ a proximity labeling strategy to identify the galectin-3 interactome. From this interactome dataset, we selected and validated CD9 and integrin beta 1 as functional counterreceptors of galectin-3 and showed that CD9 is glycosylated with an N-linked glycan at a rare noncanonical sequon. Furthermore, we present evidence that galectin-3 acts to physically alter the fluidity of the cellular membrane, and it does not activate canonical syncytialization signaling pathways. Overall, we report that galectin-mediated binding events and their corresponding functions in cell biology can be precisely regulated by select glycoproteins at specific glycosites.

Indoleamine 2,3-dioxygenase is an immunomodulatory enzyme that shows great promise when delivered exogenously as a protein therapeutic. However, IDO activity is under complex redox control, mediated in part by multiple cysteine residues within its primary sequence. We have characterized three IDO mutants in which solvent-accessible cysteine residues were mutated to chemically-similar serine residues, “IDO_C4S4” with C112S, C159S, C206S, and C308S mutations and “IDO_C5S3” with C112S, C159S, and C308S mutations based on prior reports that C206 is necessary for catalytic function, and IDO_C0S8, in which all cysteine residues were mutated to serines. IDO_C0S8 was expressed in poor yield and demonstrated less than 1% activity when compared to wild-type IDO. In contrast, IDO_C4S4 and IDO_C5S3 demonstrated robust enzymatic activity, though IDO_C5S3 had a slower V_max than wild-type and IDO_C4S4. Computational predictions and experimental measurements suggested a high degree of structural similarity between the wild-type IDO and IDO_C4S4, with subtle perturbation of α-helical content for IDO_C5S3. The structure of IDO_C0S8 was predicted to be significantly different than that of wild-type IDO. IDO_C4S4 and IDO_C5S3 were more stable than wild-type IDO over time at physiological, ambient, and reduced temperatures, likely due to diminished oxidation of the mutant IDO forms. Based on the increased V_max and robust thermal stability of IDO_C4S4, we fused it to the anchoring moiety galectin 3, to evaluate its effectiveness in a mouse model of psoriasis. The IDO_C4S4-galectin-3 fusion blunted the rate and severity of disease as compared to wild-type IDO-galectin-3 fusion. When compared to historical data with Cys-Ala IDO mutants, this study highlights the importance of employing amino acid substitution according to similarity in isosteric and isostructural shape to advance IDO as an immunomodulatory therapeutic.

Investigation of peptide co-assembly under crowded conditions using coarse-grained discontinuous molecular dynamics simulations. Peptides form fibril structures in the presence of hydrophobic crowders.

Protein biotherapeutics typically require expensive cold‐chain storage to maintain their fold and function. Packaging proteins in the dry state via lyophilization can reduce these cold‐chain requirements. However, formulating proteins for lyophilization often requires extensive optimization of excipients that both maintain the protein folded state during freezing and drying (i.e., “cryoprotection” and “lyoprotection”), and form a cake to carry the dehydrated protein. Here we show that sweet corn phytoglycogens, which are glucose dendrimers, can act as both a protein lyoprotectant and a cake‐forming agent. Phytoglycogen (PG) dendrimers from 16 different maize sources (PG1‐16) were extracted via ethanol precipitation. PG size was generally consistent at ~70–100 nm for all variants, whereas the colloidal stability in water, protein contaminant level, and maximum density of cytocompatibility varied for PG1‐16. 10 mg/mL PG1, 2, 9, 13, 15, and 16 maintained the activity of various proteins, including green fluorescent protein, lysozyme, β‐galactosidase, and horseradish peroxidase, over a broad range of concentrations, through multiple rounds of lyophilization. PG13 was identified as the lead excipient candidate as it demonstrated narrow dispersity, colloidal stability in phosphate‐buffered saline, low protein contaminants, and cytocompatibility up to 10 mg/mL in NIH3T3 cell cultures. All dry protein‐PG13 mixtures had a cake‐like appearance and all frozen protein‐PG13 mixtures had a T_g' of ~ −26°C. The lyoprotection and cake‐forming properties of PG13 were density‐dependent, requiring a minimum density of 5 mg/mL for maximum activity. Collectively these data establish PG dendrimers as a new class of excipient to formulate proteins in the dry state.

Polypeptide fusion tags that can direct the assembly of folded proteins into supramolecular networks are attractive for creating functional biomaterials. A practical challenge is identifying polypeptide sequences that form supramolecular networks in response to specific user-controlled stimuli, which is advantageous for producing polypeptide–protein fusions using cell-based expression hosts. Here, we report an N-glycosylation tag, (GGGSGGGSGGNWTT)₁₀ or “NGT,” that assembles into a supramolecular network at reduced temperatures when fused to a folded protein. For example, NGT fused to superfolder green fluorescent protein (NGTsfGFP) formed materials that emitted green fluorescence in blue light, while NGT fused to NanoLuc luciferase (NGTnL) formed materials that emitted blue light in the presence of the chemical substrate furimazine. Oscillatory rheology established the materials as weak viscoelastic gels that can undergo shear-thinning and self-healing. Gel formation could be disrupted by mutating the asparagines in NGT to glutamines, introducing a chaotropic agent, or modifying the asparagines in NGT with glucose, suggesting a role for hydrogen bonds involving asparagine in supramolecular network formation. A mixture of soluble NGTsfGFP and NGTnL formed a multifunctional gel at reduced temperature that demonstrated bioluminescence resonance energy transfer between the nL and sfGFP domains in the presence of furimazine. Collectively, these data establish NGT as a temperature-responsive polypeptide tag that can be used to create functional biomaterials from soluble fusion proteins synthesized by cell-based hosts.