EcN can be used as a probiotic for the treatment of intestinal related diseases due to its good ability to instantly implant into the intestine and non-pathogenicity. According to recent research, targeted drugs such as monoclonal antibodies have greatly improved the clinical efficacy of diseases such as intestinal inflammation, but there are also problems such as high prices, ineffective treatment for patients, and infections. The design of our experiment employs a comprehensive approach to treating the disease through a combination of therapeutic agents, including A-nanobody, a nanobody derived from Ozoralizumab, and B-nanobody, a nanobody associated with V565, in conjunction with Escherichia coli Nissle 1917. And we added a signal peptide to the front of the antibody, which can release the nanobody to the extracellular. The engineered EcN 1917 could secret the nanobody A and B, which binds to the natural TNFα and block the inflammatory reaction(Figure 1).

Figure 1: Summary of the application of engineered EcN 1917 bacterial for treatment of TNFα associated inflammatory bowel disease.

1.1 BBa_K5067000

Name:anti-TNFa nanobody of Ozoralizumab

Base Pairs:622bp

Origin: originated from and developed by Ablynx in 2006

Properties:This gene encodes for the TNFa binding region of the nanobody antibody Ozoralizumab, which has exhibited remarkable TNFα inhibition in clinical trials and has received approval for the treatment of rheumatoid arthritis, thereby becoming the world's first marketed dual-targeting nanobody (Nanozora®). We utilized this newly synthesized component and incorporated it into the p15A expression vector via homologous recombination, resulting in the creation of the novel part BBa_K5067002.

Usage and Biology:

Ozoralizumab is a next-generation TNFα antibody composed of two nanoparticles specifically targeting human TNFα and one nanoparticle directed towards human albumin. This formulation allows for the binding of two units of TNFα, while the interaction with albumin prolongs its half-life. Preclinical studies in murine models indicate that Ozoralizumab may effectively delay the onset of collagen-induced arthritis [1][2].

Characterization/Measurement

We amplify the synthetic fragment using specific Homology primer and PCR. Figure 1 presents the electrophoresis image of target gene A, with measured markers and values shown on the left. The length of target gene A ranges from 500bp to 1000bp, and specifically measures at 622bp. The observed size of the target band corresponds to that of the target gene.

Figure 1: PCR amplification of target A . A isOzoralizumab

1.2 BBa_K5067001

Name:anti-TNFa nanobody of V565

Base Pairs:622bp

Origin: developed by VHSquared in 2014

Usage and Biology:

VHsquared has identified a TNF-α neutralizing nanobody from the immune llama phage library, which exhibits natural tolerance to gastric and intestinal proteases. The structure of this nanobody was subsequently modified to enhance its resistance to proteolytic degradation, resulting in the development of molecule V565. Furthermore, VHsquared has formulated a targeted delivery system for V565, transforming it into an oral enteric tablet designed for localized treatment of patients with inflammatory bowel disease (IBD) [3].

Upon administration, the V565 capsules are gradually solubilized by the varying pH levels of gastric and intestinal fluids, facilitating the release of active V5-65 which subsequently binds to inflamed regions of the intestinal epithelium. Any excess V5-65 that remains unbound to the epithelium is efficiently cleared via lymphatic circulation [3].

In vitro protease assays indicate that V565 exhibits tolerance to various proteases present in gastric and intestinal environments. Furthermore, during clinical Phase 1 trials, V565 demonstrated significant accumulation in the tissues of Crohn's disease patients and long-term inhibition of TNFα signaling pathway phosphorylation and activation within tissue cells. Currently, V565 is advancing through Phase II clinical trials [4].

Characterization/Measurement

We amplify the synthetic fragment using specific Homology primer and PCR. Figure 2 presents the electrophoresis image of target gene B, with measured markers and values shown on the left. The length of target gene B ranges from 500bp to 1000bp, and specifically measures at 622bp. The observed size of the target band corresponds to that of the target gene.

Figure 2: PCR amplification of target B . B is V565

BBa_K5067002(p15A-Ozoralizumab)

Construction Design:

Initially, we opted to conduct the in-frame assembly of the plasmid within DH5α cells, subsequently transforming the successfully assembled plasmid into EcN1917 cells. We synthesized the gene sequences for both A antibody and B antibody through gene synthesis techniques. To begin with, we constructed the nanoAb-A plasmid via homologous recombination(Figure 3).

Figure 3: Snapgene Plasmid of nano-Ab A

Engineering Principle:

In this project, EcN will be genetically engineered: the nucleotide sequences of anti-TNFα nanobodies (A) 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 is appended with a signaling peptide to enhance the secretion of the nanobody into the extracellular space(Figure 4). 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 4: Summary of the plasmid construction and engineered EcN 1917 bacterial

Experimental Approach:

We employ polymerase chain reaction (PCR) for DNA amplification and prepare agarose gel for electrophoresis. Subsequently, the results are analyzed using ultraviolet imaging.

Figure 5: PCR amplification of target A and p15A vector backbone

Figure 5A displays the electrophoresis image of the p15A vector, with measured markers and values indicated on the left. The lengths of p15A-1 fall within the range of 5000bp to 7500bp, while the length of p15A is determined to be 6535bp. The observed target band aligns with the expected length of the target gene. Figure5 B presents the electrophoresis image of target gene A, with measured markers and values shown on the left. The length of target gene Aranges from 500bp to 1000bp, and specifically measures at 622bp. The observed size of the target band corresponds to that of the target gene.

Figure 6: Colony culture of DH5α-p15A-A and Sanger sequencing result.

We obtained the bacterial colony through solid plate culture overnight and we subsequently dispatched them to a biotechnology firm for sequencing (Figure 6 A).

The results in the following two images were obtained (Figure 6 B ). Since they are solid lines, there were no base mismatch for gene mutations, indicating the success of the construction work.

2.2 BBa_K5067003(p15A-V565)

Construction Design:

Construction Design: Utilizing the synthesized DNA sequence of the B antibody (Figure 7), we developed and validated the nanoAb-B plasmid, building upon the findings from 2.1

Figure 7: Snapgene diagram of p15A-nanoAb-B plasmid

Engineering Principle:

The principle is the findings from 2.1 Engineering Principle.

Experimental Approach:

We performed PCR amplification of the target B fragment and p15A vector bone fragment for linearization(Figure 8).

Figure 8: PCR amplification of target fragment by specific primers. The length of p15A is 6535bp, gene B is 622bp

Then, we ligate B fragment and linear p15A backbone through homology recombination and transform the ligation product to DH5a competent cell. We verify the construct result by plasmid extraction and Sanger sequencing. The successfully constructed p15A-nanoAb-B plasmid was transformed to EcN1917 and colony PCR verification was also performed.Since they are solid lines, there were no base mismatch for gene mutations, indicating the success of the construction work(Figure 9).

Figure 9: Colony culture of DH5α-p15A-B and Sanger sequencing result.

1. Crowe, J. S., Roberts, K. J., Carlton, T. M., Maggiore, L., Cubitt, M. F., Ray, K. P., … West, M. R. (2018). Oral delivery of the anti-tumor necrosis factor α domain antibody, V565, results in high intestinal and fecal concentrations with minimal systemic exposure in cynomolgus monkeys. Drug Development and Industrial Pharmacy, 45(3),387–394.
2. Nurbhai, et al. Oral Anti-Tumor Necrosis Factor Domain Antibody V565 Provides High Intestinal Concentrations and Reduces Markers of Inflammation in Ulcerative Colitis Patients. Sci Rep 9, 14042 (2019)
3. Ishiwatari-Ogata, C., Kyuuma, M., Ogata, H., Yamakawa, M., Iwata, K., Ochi, M., Hori, M., Miyata, N., & Fujii, Y. (2022). Ozoralizumab, a Humanized Anti-TNFα NANOBODY® Compound, Exhibits Efficacy Not Only at the Onset of Arthritis in a Human TNF Transgenic Mouse but Also During Secondary Failure of Administration of an Anti-TNFα IgG. Frontiers in immunology, 13, 853008.
4. Tanaka Y. (2023). Ozoralizumab: first Nanobody® therapeutic for rheumatoid arthritis. Expert opinion on biological therapy, 23(7), 579–587.