Inflammatory Bowel Disease (IBD) is a group of autoimmune diseases that occurs within the digestive system[1]. There are two main types of IBD: Crohn’s disease and Ulcerative colitis (UC); while UC mainly affects the large intestine, Crohn’s disease can affect any part of the digestive tract from the mouth to the anus. Indeterminate colitis, a third type of IBD, occurs when the patient exhibits both characteristics of Crohn’s disease and Ulcerative Colitis[2,3].
The first symptoms of IBD may occur after the intestine is exposed to something irritating, like a medication or an infection; after the irritation or infection disappears, the immune system continues to respond[4,5]. Symptoms of IBD may suddenly flare up and then not occur for periods of time. Other than common (but already hard to manage) symptoms like abdominal pain and diarrhea, uncontrolled inflammation could lead to the intestinal perforation and higher risk of colon cancer[6,7]. Outside of the digestive tract, the disease also could cause malnutrition, nausea, anxiety and depression, etc, all exemplifying the need of a drug that relieves inflammation and symptoms of IBD[8-10].
Since 2015, the prevalence of IBD has surged dramatically in Western countries like North America and Australia. According to statistical data, Canada has among the highest rates of childhood-onset IBD in the world. Over 7000 children and youth under 18 years old are living with IBD in Canada, and 600 to 650 children under 16 years old are diagnosed annually[11]. This rare but notable increase in a disease once predominantly associated with Western countries is now emerging across Asia, potentially having significant implications and serving as an important warning for developing nations in the region11.
As China has undergone rapid urbanization, technology and economy has transformed leading to a gradual "Westernization" of dietary habits, characterized by an increased consumption of high-protein and high-calorie foods. Consequently, this dietary shift has heightened the risk of IBD and contributed to a broader rise in gastrointestinal disorders across Asia[12].

Last year, one of our seniors was diagnosed with IBD at the age of 17. His academics, diet, and lifestyle were completely changed as a result. From this case of IBD that occurred so close to us, we were able to realize the extent of the pain it can cause. After learning that he was using biologics, we realized the potential of these drugs in alleviating the symptoms of IBD. This inspired us to initiate this project to create a low-cost, high-efficiency prokaryotic expression system for large-scale production of biologics.

IL-18 is an interleukin associated with inflammation, particularly inflammatory disorders like IBD[13,14]. IL-18 binding protein (IL-18BP) could bind to IL-18 to stop its function[15](Figure 1). In humans, while there are four types of IL-18BP (IL-18BPa, Pb, Pc, Pd), only IL-18BPa and IL-18BPc could antagonize IL-18 activity[16].
The Small Ubiquitin-like-Modifier (SUMO) is an important component within the cell. While its pathways are not entirely clear, it has been shown to be able increase protein stability both in terms of reducing protein degradation and increasing thermal stability[18,19]. While it is usually conjugated onto the protein through reversible post-translational modification[20], E. coli is unable to perform such modifications, and we prefer our protein to keep the stability; thus we integrated the SUMO sequence into our IL-18BP sequences, attaching it at the beginning of the sequence.
Fc stands for Fragment crystallizable region, which is the conserved region of antibodies. The IgG1 Fc is usually fused to therapeutic proteins to slow its degradation within the body[21]. Moreover, within the field of Antibody-Drug Conjugates (ADCs), in which small molecule drugs can be attached to antibodies for targeted drug delivery, the Fc region is a binding site for such drugs[22]. Being abundant in sites of inflammation (for instance, within IBD)13, IL-18 could be considered as a drug target for the treatment of IBD. As IL-18BP can selectively bind to IL-18, selective release of small molecule IBD drugs can be accomplished by ligating the drugs onto the Fc modification. Because of the increased stability and potential use of our protein, we attached the IgG1 Fc sequence at the end of our IL-18BP sequences to produce a fused protein with IgG1 Fc.
The poly-histidine (His) tag is a commonly used tag for protein purification, consisiting of 6 histidines that can chelate to Nickel ions; thus, proteins with the His tag can be separated from other proteins through affinity columns containing Nickel ions. Because the tag only composes of 6 amino acids and the use of a gentle imidazole elution make this method preserves the protein's function[23]. Thus, for easy and efficient extraction of our protein, we also added the His tag sequence to the beginning and end of the modified protein sequence.
Aside from add-ons to the protein, codon optimization was also performed on our gene sequence (conducted by GenScript) for better expression of the human-origin protein within E coli bacteria. The modified protein sequence was integrated into the pET-28a vector, then expressed using BL21 bacterial strains through IPTG induction. After protein extraction and purification through the His-tag, the proteins' anti-inflammatory functions were tested by treating mice abdomen T-cells stimulated with IL-18 and TNF-alpha. The same test was conducted using IL-10 as a positive control (which were modified by SUMO, Fc, and His tag in the same way).
The experimental process can be broken down into 4 parts: construction of plasmid, expression of protein, verification of protein after purification, and verification of protein function on mouse CD8+T cells (obtained from company).

The current treatment of IBD:
Among IBD treatments, biologics are known as the most effective therapies available because of their ability to target specific cellular pathways[24]. However, they are expensive, which reduces patient access to them, and harder to administer, reducing patient compliance. Out of all available treatments for IBD, biologics are the most expensive, ranging anywhere from $15,000 to $45,000 per year[25]. Less expensive yet adequately effective biologics for the disease can increase the accessibility of IBD treatment to more people. Currently, most biologics are administered through injections in hospitals, which may discourage patients in following through their medications; on the other hand, biologics administered orally through a capsule would be much more convenient for patients and increase their compliance to the treatment, which is the goal of our product.
Product:
This project aims to develop a more effective, less expensive, and more easily administered biologic for IBD, and make IBD treatments more patient-friendly. Our objective is to utilize a high-yield prokaryotic expression system to produce substantial quantities of highly active recombinant IL-18BP-Fc protein. By employing this recombinant protein to neutralize IL-18 and inhibit its role in mediating IBD, we aim to establish a foundation for the large-scale production of IL-18BP and pioneer new strategies for developing treatments for IBD. After engineering the recombinant protein and achieving large-scale production, we aim to create a capsule that contains the protein for easier administration and improved patient compliance. In conclusion, we aim for our IBD treatment product to be effective in both production and consumption.

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