# Microbial Peptides: Key Players in Reducing Gluten Immunogenicity Through Peptide Degradation
## 5 Future Perspectives on the Application of Enzymes for Celiac Disease Treatment  
Enzyme therapy based on gluten-degrading proteases (glutenases) has long been regarded as a promising yet still limited complementary strategy for the treatment of CeD. Enzymes capable of degrading immunogenic gluten peptides must overcome several physiological and technological barriers before being considered safe and effective therapeutic agents. One critical concern lies in the potential increase in immunogenicity caused by the generation of smaller peptide fragments that can traverse the intestinal epithelial barrier more readily than the larger peptides produced by human gastrointestinal proteases. This phenomenon has been demonstrated in vitro using proline-specific  
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endopeptidases derived from commensal microorganisms such as _Pseudomonas_, while the addition of Lactobacillus-derived enzymes has been shown to generate non-immunogenic fragments [86]. Beyond enzymatic specificity and catalytic efficiency, a major obstacle to the pharmacological application of glutenases is their rapid inactivation and structural instability under acidic gastric conditions [48; 69]. These enzymes are susceptible to degradation by endogenous digestive proteases such as pepsin, trypsin, and chymotrypsin, potentially preventing them from remaining active until reaching the small intestine, the site where gluten-derived peptides trigger T-cell-mediated inflammatory responses. Although protective pharmaceutical strategies such as PEGylation and microencapsulation have been extensively investigated to improve the pharmacokinetics, stability, and bioavailability of therapeutic agents, clinical success has remained limited due to several persistent challenges. PEGylation, which involves the covalent attachment of polyethylene glycol chains to therapeutic proteins or peptides, has been shown to extend circulation time and reduce immunogenicity; however, its clinical effectiveness has been compromised by the emergence of anti-PEG antibodies in a significant portion of patients, leading to reduced efficacy and increased risk of hypersensitivity reactions [69; 87]. Furthermore, PEGylation can negatively impact the biological activity of the modified drug due to conformational alterations. Similarly, microencapsulation has been explored to protect active pharmaceutical ingredients and allow for controlled drug release, but clinical translation has been impeded by inconsistent release kinetics, potential immunological reactions to encapsulating materials, and challenges in large-scale manufacturing [88]. These limitations highlight the need for continued innovation in drug delivery systems to achieve more reliable and effective therapeutic outcomes. Two principal approaches have been proposed to address this limitation: (1) employing acid-stable peptidases and altering their substrate specificity toward prolamins, or (2) engineering existing glutenases to improve their acid stability [89]. These strategies have recently been expanded through site-directed mutagenesis and active site modification. For instance, the specificity of the glutenase Bga1903 was successfully enhanced by structural engineering to improve its activity against immunogenic peptides implicated in CD [90]. Such efforts have furthered the design of enzymes with both improved substrate specificity and greater resilience in gastrointestinal conditions. Clinical evaluation of enzyme cocktails remains a key area of translational research. ALV003 (also known as Latiglutenase), which combines EP-B2 (ALV001) and SC-PEP (ALV002), has shown significant mucosal protection and a reduction in intraepithelial lymphocytes in phase 1b-2a trials with CeO patients in remission who were exposed to gluten. Mucosal injury was assessed by calculating the villus height to crypt depth ratio in biopsies and by measuring epithelial lymphocyte density before and after treatment.