Inflammatory bowel disease (IBD) is a chronic condition primarily characterized by two forms: Crohn's disease and ulcerative colitis. Both lead to persistent inflammation of the gastrointestinal tract, but they differ in the areas affected and the extent of inflammation. IBD typically requires long-term management due to the potential for symptom recurrence. Patients may experience abdominal distension, pain, and diarrhea, with stools that are often watery and may contain mucus, pus, or blood. This is frequently accompanied by an urgent need to defecate,which can lead to complications such as intestinal bleeding, narrowing, obstruction, and even perforation. Additionally, patients may present with fever of varying severity and extra-intestinal manifestations, including arthritis, ankylosing spondylitis, skin rashes, and uveitis. Millions of individuals worldwide are afflicted by IBD, and a significant proportion of those with moderate-to-severe cases require surgical intervention for remove inflamed intestinal tissue. In severe cases, the disease can be fatal.

Figure 1: Inflammatory bowel disease including Ulcerative colitis and Crohn’s disease .

Note：The picture belongs to JOHNS HOPKINS

Figure 2: Historical timelines of CD and UC throughout the world [1].

The progression of IBD can be categorized into four distinct epidemiological stages: the initial emergence of the disease, an acceleration in incidence, worsening of symptoms, and finally, a state of disease stabilization. Presently, every region globally resides within one of the first three epidemiological stages of IBD progression, with an eventual progression through all four stages over time(Figure 2).

At each epidemiological stage, the clinical challenges of IBD vary significantly. As a result, advancements in IBD prevention strategies and healthcare services tailored to the evolving needs of the IBD population can help expedite the transition to a stable disease state, potentially alleviating the global burden of IBD[2].

Figure 3: Trends from 1990 to 2017 in number and age-standardised prevalence rates of IBD at the global level [3].

Currently, there is ongoing research and development of novel therapeutics for IBD, with targeted therapies, particularly monoclonal antibodies, significantly enhancing the clinical efficacy in managing IBD. For instance, anti-TNFα monoclonal antibodies have shown greater efficacy and safety compared to traditional systemic corticosteroid treatments. Nevertheless, unmet clinical needs regarding the application of anti-TNFα therapies. Specifically, approximately 50% of patients receiving the next-generation TNFα monoclonal antibody Simponi (Golimumab) for ulcerative colitis show no clinical improvement, and approximately 80% fail to achieve clinical remission(Figure 3). While Simponi is generally well-tolerated, there is an increased risk of infections among treated patients. Furthermore, Simponi necessitates continuous subcutaneous administration via a prefilled syringe, with larger doses given in the first and third weeks, followed by smaller monthly doses. Research indicates that nearly 40% of patients experience disease relapse within three months after discontinuation treatment, necessitating lifelong therapy, which often leads to poor adherence. In the U.S., a 100 mg dose of Simponi costs at $1,490, with annual treatment costs exceeding $24,000[3].

Figure 4: Project model EcN-1917 secrete anti-TNFα nanobody antibody.

INTESKEY aims to tackle the current treatment challenges faced by IBD patients through innovative synthetic biology approaches. Our solution is a composite probiotic formulation that integrates probiotics with anti-TNFα nanobodies, offering sustained and targeted therapy with minimal side effects(Figure 4). We have successfully engineered probiotics to carry the nanobody gene, enabling the development of a therapeutic agent that localizing within the intestinal microenvironment and continuously secretes nanobodies to achieve the desired therapeutic effect.

The design of our experiment employs a comprehensive approach to treating the disease by combining therapeutic agents, including A-nanobody, derived from Ozoralizumab, and B-nanobody, associated with V565, along with Escherichia coli Nissle 1917. The engineered EcN 1917 is capable of secreting nanobodies A and B, which binds to the natural TNFa and inhibit the inflammatory response(Figure 5).

Figure 5: Summary of the application of engineered EcN 1917 bacterial for treatment of TNFα associated inflammatory bowel disease.

TNF-α, also known as cachectin or TNFSF1A, is a ligand within the TNF superfamily and a key player in inflammation, apoptosis, and immune system development. Secreted by macrophages, monocytes, neutrophils, CD4+ T cells, NK cells, and various transformed cells, TNF-α functions as a pro-inflammatory cytokine. It plays a central role in normal inflammatory and immune responses, while also coordinating the production, survival, and apoptosis of other cytokines to maintain tissue homeostasis. As a critical cytokine in the early stages of the inflammatory cascade, TNF-α is integral to the Th1 signaling pathway. It can induce ICAM-1 expression on astrocytes and endothelial cells, promoting the migration of activated neutrophils and T cells from the epidermis into the dermis. The biological functions of TNF-α are multifaceted and complex. While it can offer protection against certain infections. While it can offer protection against certain infections, it also has the potential to trigger pathological complications by activating various signaling pathways. TNF-α interacts with transmembrane TNF receptors (TNFRs) to regulate cell survival or initiate apoptosis through an intricate signaling cascade. In particular, it mediates TNFR1 activation, orchestrating processes that involve cell death and ultimately determining cellular fate (Figure 6)[4,5].

Pro-inflammatory and anti-inflammatory cytokines are critical in regulating immune system functions, including the enhancement of TNF-α synthesis, which is closely linked to the pathogenesis of conditions such as arthritis and inflammatory bowel diseases. Consequently, TNF-α has become a key therapeutic target. Anti-TNF-α therapies have shown varying degrees of success in treating arthritis, psoriasis, inflammatory bowel disease, and other inflammatory disorders. However, it is important to note that TNF-α is an essential immune molecule in the human body. Future research may focus on developing drug combinations that selectively cellular immunity, thereby reducing the long-term side effects associated with anti-TNF-α therapies [6,7].

Figure 6: TNFα signaling pathway [8]

Synthetic biology involves re-engineering organisms or cells to exhibit new or enhanced functionalities, as well as designing and creating entirely new biological components, devices, or systems[9].

Escherichia coli Nissle 1917 (EcN) is a Gram‐negative probiotic, originally isolated by Dr. Alfred Nissle during World War I. EcN is serum‐sensitive and does not produce any enterotoxins or cytotoxins associated with pathogenic E. coli strains [10]. It has been licensed as a pharmaceutical for the treatment of diseases such as diarrhea and colitis ulcers [10]. Escherichia coli Nissle 1917 (EcN) is another promising probiotic candidate. With advances in molecular biology and complete genome sequencing techniques, EcN shows increased potential for cancer treatment applications.[10].

Due to its genetic tractability, proven safety in humans, and ability to colonize, survive, and replicate in both the gastrointestinal tract and tumors, Escherichia coli Nissle 1917 (EcN) has emerged as a preferred chassis for engineering "smart microbes" capable of delivering therapeutic agents to disease sites. EcN variants, engineered with new therapeutic properties through modifications to its metabolic pathways or the incorporation of in situ drug delivery systems, are showing promise in the treatment of bacterial infections, inflammatory bowel diseases, metabolic disorders, and cancer[9].

A (anti-TNFα nanobody known as Ozoralizumab). Developed by Ablynx in 2006, Ozoralizumab has shown impressive TNFα inhibitory effects in clinical trials and has been approved for the treatment of rheumatoid arthritis, making it the world’s first commercialized dual-targeting nanobody (Nanozora®). With its patent set to expire in 2026 in both China and Europe, Ozoralizumab is positioned as a promising candidate with validated clinical data and substantial commercial potential [11].

B (anti-TNFa nanobody of V565). V565 is an oral formulation of an anti-TNFα nanobody developed by VHSquared in 2014, currently in Phase II clinical trials for patients with inflammatory bowel disease. Preclinical studies have shown that this nanobody effectively inhibits the TNFα signaling pathway and reduces pro-inflammatory cytokine levels in mouse models of the disease. Biopsy samples from patients with enteritis further suggest that the nanobody can block inflammation-related phosphorylated protein signals[12,13]

Building upon the foundational work of their predecessors, in 2009, K. Vandenbroucke from Ghent University in Belgium and colleagues endeavored to transfer antibody expression elements targeting mouse TNFα into Lactococcus lactis lactic acid bacteria. Their findings indicated that daily oral administration of the gene-edited bacterial strain significantly mitigated inflammation in mice with chronic colitis induced by dextran sulfate sodium (DSS) and ameliorated established colitis in interleukin-10 (IL-10)-deficient mice, without elevating infection risk [14]. In 2023, Jason P. Lynch from Harvard Medical School developed a system termed PROT3EcT, which integrates the expression and secretion of antibodies against mouse TNFα into Escherichia coli EcN. The monoclonal antibody produced by this engineered bacterial strain effectively inhibits pro-inflammatory TNFα levels and prevents tissue damage and inflammation in a chemically induced colitis model following a single preventive dose [15]. A notable limitation of these studies is that the expressed nanobodies are antagonistic to mouse TNFα rather than human TNFα, thereby possessing only research significance rather than clinical applicability. Concurrently, Takayuki Iwaki from Hamamatsu University School of Medicine devised a system optimized for nanobody expression and secretion within Escherichia coli; this protein incorporates both a 6xHis tag and an AviTAG tag facilitating straightforward purification and characterization of the expressed nanobody [16]. Ablynx developed Ozoralizumab in 2006 and subsequently submitted an international patent application for it [17]. This antibody comprises three humanized nanobody structural domains: two specifically target human TNFα while one binds to human HSA to prolong drug half-life. Clinical trials have demonstrated its remarkable efficacy in blocking TNFα, leading to its approval for use in rheumatoid arthritis treatment. Patent protection for this antibody will expire in 2026 across China and Europe, positioning it as a promising candidate molecule with validated clinical data and commercial potential.

A signal peptide is a short amino acid sequence typically found at the N-terminus (amino terminus) of newly synthesized proteins. It facilitates the translocation of these proteins across cellular or nuclear membranes for proper targeting and secretion. When a protein requires transport to a specific intracellular location or needs to be secreted extracellularly, the signal peptide assumes a pivotal role. The signal peptide interacts with the signal recognition particle (SRP), directing nascent polypeptides into the endoplasmic reticulum (ER), where they undergo further processing before being secreted or transported to other organelles within the cell. In the context of exogenous protein expression, particularly when aiming for secreted proteins, employing signal peptides becomes crucial. A signal peptide typically comprising 15-30 amino acids in length. In industrial production and scientific research, the application of recombinant DNA technology to merge the gene encoding the target protein with that encoding the signal peptide facilitates the effective recognition, processing, and secretion of exogenous proteins by cells [18].

In this project, EcN will be genetically engineered: the nucleotide sequences of anti-TNFα nanobodies (A or B) will be synthesized, with both ends of A/B modified to include a 6×His Tag for subsequent purification and characterization. The N-terminus of A/B is appended with a signaling peptide to enhance the secretion of the nanobody into the extracellular space(Figure 7). The synthesized sequences are then cloned into a transformation vector and introduced into EcN1917 through transformation. Clindamycin-resistant colonies are selected to ensure stable integration of the edited EcN1917 strain. Select multiple clones for further characterization. The subsequent characterization encompasses the following steps:

1) obtaining the genomic sequence and detecting the introduced sequence through first-generation sequencing;

2) collecting the culture supernatant to verify the expression of A using ELISA;

3) gathering the culture supernatant to assess its ability to inhibit TNFα binding to TNFR (via ELISA) or its capacity to prevent TNFα-induced apoptosis in U937 cells (through cellular assays).

Figure 7: Summary of the plasmid construction and engineered EcN 1917 bacterial

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