Surface display of an Anti-CD3 scFv by Saccharomyces boulardii attenuates DSS-induced colitis in mice

Technical Summary: Surface Display of Anti-CD3 scFv by Saccharomyces boulardii for Colitis Attenuation

Problem Statement
Inflammatory bowel diseases (IBD), particularly ulcerative colitis (UC), are characterized by imbalanced immune responses, intestinal barrier disruption, and the overactivation of CD4⁺ T cells. While monoclonal antibodies (mAbs) targeting pro-inflammatory molecules or surface receptors like CD3 show therapeutic potential, their systemic delivery often faces challenges regarding bioavailability and localized action. Genetically modified probiotics offer a strategy for the localized production and delivery of therapeutic molecules within the gastrointestinal tract (GIT). However, identifying a suitable microbial vector that is safe, capable of expressing complex eukaryotic proteins, and able to display them effectively on the cell surface remains a critical technical hurdle. Saccharomyces boulardii (Sb), a probiotic yeast with GRAS status and the ability to express recombinant proteins without permanent gut colonization, is a candidate, but its efficacy in displaying functional antibody fragments for immunotherapy requires validation.

Methodology
The study engineered S. boulardii to function as an oral delivery platform for an anti-CD3 single-chain variable fragment (scFv).

• Strain Construction: Two recombinant strains were created using a shuttle vector (pYC230 derivative) containing the SED1 promoter and anchoring domain. The scFv (derived from mouse anti-CD3 clone 145-2C11) was fused to the SED1 cell wall anchor. To optimize surface accessibility, two constructs were tested differing only in the length of the stalk (extender) domain:
  • SbAga: Utilized an AGA_Stalk (289 amino acids) based on S. cerevisiae Aga1.
  • Sb649: Utilized a 649_Stalk (649 amino acids) previously described by McMahon et al.
  • Both constructs included N-terminal HA and C-terminal FLAG tags for detection, separated by a GSGS spacer.
• In Vitro Characterization: Surface expression was monitored via flow cytometry over 48 hours using anti-FLAG antibodies.
• In Vivo Model: A Dextran Sulfate Sodium (DSS)-induced colitis model was established in C57BL/6 mice. Mice received daily oral gavage of wild-type Sb (SbWT), the engineered Sb649 strain, or saline starting three days prior to DSS induction.
• Assessment Metrics: Disease severity was evaluated using the Disease Activity Index (DAI), which incorporates weight loss, stool consistency, and rectal bleeding. Colon weight-to-length ratios were measured. Gene expression analysis (qPCR) was performed on mesenteric lymph nodes (MLNs) and colonic tissue to assess immune modulation (e.g., Foxp3, Il6, Tbx21) and barrier integrity (Muc3, Tjp1).

Key Contributions

1. Optimization of Surface Display: The study demonstrated that linker/stalk length significantly impacts the stability of antibody fragment display on S. boulardii. The longer stalk (Sb649) maintained stable surface expression over time, whereas the shorter stalk (SbAga) showed a sharp decline after 40 hours.
2. Proof of Concept for Oral Immunotherapy: The research established that S. boulardii can successfully display functional anti-CD3 scFv on its surface and deliver it to the gut environment in a living host.
3. Therapeutic Efficacy: The engineered Sb649 strain was shown to attenuate DSS-induced colitis more effectively than the wild-type strain, linking surface-displayed anti-CD3 to reduced disease activity.

Results

• Expression Stability: Flow cytometry revealed that Sb649 maintained a higher and more stable proportion of antibody-positive cells throughout cultivation compared to SbAga, which peaked at 40 hours and declined rapidly. Consequently, Sb649 was selected for in vivo testing.
• Disease Attenuation: In the DSS model, Sb649 treatment significantly reduced the Disease Activity Index (DAI) Area Under the Curve (AUC) compared to the DSS-only and DSS+SbWT groups. Sb649-treated mice exhibited reduced weight loss and maintained DAI values closer to healthy controls for six days of inflammation induction. While colon shortening occurred in all DSS groups, Sb649 treatment specifically mitigated weight loss.
• Immunomodulation:
  • MLNs: Sb649 treatment resulted in a less pronounced upregulation of Il6 compared to DSS and DSS+SbWT groups, suggesting a modulation of pro-inflammatory cytokine production. Foxp3 expression was elevated in all diseased groups but was highest in the Sb649 group, indicating a potential shift toward a regulatory T-cell phenotype.
  • Colon Tissue: Muc3 expression (a mucin gene) was significantly higher in the Sb649 group compared to DSS and DSS+SbWT, suggesting enhanced mucosal protection. No significant differences were observed in Il10 or Tjp1 (tight junction protein) expression, though Sb-treated groups showed levels comparable to healthy controls.

Significance and Claims
The paper claims that S. boulardii is a viable platform for the production and surface display of antibody fragments for gastrointestinal immunotherapy. The study concludes that the engineered Sb649 strain stably displays anti-CD3 scFv and exerts protective and immunomodulatory effects in a murine model of colitis. The authors posit that the observed benefits are likely due to the displayed anti-CD3 scFv inducing partial T-cell activation and tolerance, thereby suppressing excessive inflammation.

The authors maintain a modest tone regarding the mechanism, acknowledging that the proportion of yeast expressing the scFv was limited and that the exact amount of biologically active antibody reaching the colon remains uncertain. They note that the experimental design (acute DSS induction without a remission phase) may have underestimated the combined effects of the probiotic and the recombinant antibody. The study suggests that while the results support the potential of this microbial delivery system, further optimization and investigation into the inflammatory environment are necessary to fully clarify its therapeutic efficacy.