Proof of Concept

Proof of concept

The main concept of our project is the design of a genetically modified organism that, after being taken up orally, senses the inflammation in the gastrointestinal tract of inflammatory bowel disease (IBD) patients and releases an anti-inflammatory single domain antibody (also called nanobody). We used the probiotic E. coli Nissle 1917 as our chassis as this is already used as a treatment for IBD patients. 1 Nitric oxide (NO) was chosen as a biomarker for inflammation as patients with intestinal inflammation show elevated levels of reactive oxygen species, including nitric oxide. 2 The proinflammatory cytokine tumour necrosis factor (TNFα) is a major driver of inflammation 2 and is targeted by our nanobodies. 3 The binding of the nanobody to TNFα inhibits the cytokine’s signalling and reduces the inflammation.

To prove our concept, we split it in several sections:

  1. Successful GFP expression upon NO-induction
  2. Testing the efficacy of purified nanobodies on TNFα-inhibition
  3. Secretion of functional nanobodies by E. coli MC1061 and Nissle 1917
  4. Nanobody efficacy model

Our data showed that our engineered E. coli Nissle 1917 is able to sense nitric oxide and as a response secretes functional anti-TNFα nanobodies. Together with the model predictions, we have broken ground in developing a new localised therapy for IBD. Furthermore, the treatment addresses issues with current interventions such as lack of specificity and systemic side effects.

For more details about all experiments and results, go to the respective pages linked.

1. Successful GFP expression upon NO-induction

We proved that the promoter pNorVβ 4 used in our experiments responded to different Nitric Oxide (NO) concentrations and successfully expressed GFP. Higher NO concentration leads to increased GFP expression (figure 1A). We also compared our construct to the NO-sensitive promoter used by the 2016 iGEM team from ETH Zurich. We could show that pNorVβ can induce higher responses to NO than the ETH promoter pNorV (figure 1B). We did all our experiments in E. coli Nissle 1917 as this was our intended chassis.

Figure 1A: GFP expression of pNorVβ with 2 RBS after DETA/NO induction (0, 8, 31, 125, 500, 2000 uM DETA/NO). Three biological replicates were tested. All measurements are normalised to the OD600.
Figure 1B: Comparison of GFP expression between the negative control, the promoter used by ETH (pNorV) and pNorVβ with 1 RBS (here called 1RBS). Shows endpoint GFP measurements after induction with four DETA/NO concentrations (0, 0.5, 0, 2 mM). The measurements were done via flow cytometry.
2. Testing the efficacy of purified nanobodies on TNFα-inhibition

We used the human monocytic cell line THP-1 to show that inhibiting TNFα influences the immune response of monocytes, and the inflammation resulting from TNFα signalling. For this, we first incubated the monocytes with different nanobody constructs and then stimulated the cells for 24 hours with TNFα. We then indirectly measured the inflammatory response of the cells by comparing the IL-1β expression to the housekeeping gene human GAPDH. IL-1β is a vital inflammation mediator 5 and a good marker for our proof of functional TNFα-inhibition. The anti-TNFα monocolonal antibody, adalimumab, was used as a positive control. Adalimumab is commercially available and already used in clinics to treat IBD patients.

Our analysis showed that TNFα alone induces a significant expression of IL-1β. In comparison, all tested nanobodies (monovalent and bivalent constructs) could reduce the inflammatory response by up to 4-fold. Additionally, most nanobodies performed as good as, or even better than, adalimumab.

Figure 2: IL-1β expression compared to human GAPDH in THP-1 cells. Cells were stimulated with different TNFα concentrations. The nanobodies were purified from E. coli MC1061. Nb1 = VHH#2B, Nb2 = VHH#3E, Nb3 = VHH#12B, Nb9 = VHH#3E + VHH#12B
3. Secretion of functional nanobodies by E. coli MC1061 and Nissle 1917

To prove our bacteria's successful secretion of nanobodies, we started by inducing the expression with the arabinose-inducible pBAD system. The bacterial supernatant containing the secreted nanobodies was analyzed with a Western blot to identify the correct size and secretion intensity. We showed that the common lab strain E. coli MC1061 can produce and secrete most of our nanobody constructs, except for the bivalent nanobody N6 (biv. VHH#12B). Furthermore, our final chassis E. coli Nissle 1917, also successfully secreted monovalent and bivalent nanobodies (figure 3).

Figure 3: Double transformed E. coli MC1061 (A) and Nissle 1917 (B) were induced by arabinose and incubated at 37°C overnight. Anti-myc antibodies were used in the Western blot to detect secreted nanobodies in the bacterial supernatant. N1 = VHH#2B, N2 = VHH#3E, N3 = VHH#12B, N4 = biv. VHH#2B, N5 = biv. VHH#3E, N6 = biv. VHH#12B, N7 = linked VHH#3E + VHH#2B, N8 = linked VHH#2B + VHH#12B, N9 = linked VHH#3E + VHH#12B

To answer the question if secretion negatively influences the folding and binding abilities of our nanobodies, we additionally performed an ELISA. All tested nanobody constructs, except for the secreted bivalent nanobody VHH#3E + VHH#2B (Figure 4A, wells C1-2), bound biotinylated TNFα. Therefore, we can assume that they are functional, even after being secreted by the hemolysin A secretion system. The fusion of the HlyA-tag to the nanobodies does not seem to hinder the binding to TNFα.

Figure 4A:
ELISA comparing the binding capabilities of secreted vs purified nanobodies obtained from E. coli MC1061. Replicates of secreted candidates are followed by their purified twin. Two positive and one negative control are shown in row D.
Figure 4B: ELISA showing binding capability of a monovalent (VHH#2B) and bivalent (linked VHH#2B + VHH#12B) nanobody secreted by Nissle (row A), MC1061 (row B) compared to purified samples (row C). Additionally, the positive control adalimumab (wells A5-6) and a negative control (wells B5-6) were tested.

Characterisation of our final product: We could start the last experiments with the final genetic circuit after showing the successful secretion of nanobodies in both E. coli strains. We exchanged the sfGFP used to characterize the nitric oxide sensing promoter with a monovalent nanobody (VHH#2B). We followed the same workflow as described above and let our E. coli Nissle 1917 produce and secrete functional nanobodies, this time induced by nitric oxide.

The results of the Western blot and ELISA demonstrate the functionality of our final genetic circuit. Our modified E. coli Nissle 1917 is able to sense nitric oxide and, as a response, produces and secretes anti-TNFα nanobodies which decrease the inflammatory actions induced by the cytokine on human immune cells.

Figure 5A:
Double transformed E. coli Nissle 1917, able to secrete the monovalent nanobody VHH#2B, were induced with different DETA/NO concentrations and incubated at 37°C overnight. Anti-myc antibodies were used in the Western blot to detect secreted nanobodies in the bacterial supernatant.
Figure 5B: ELISA showing the TNFα-binding capabilities of the secreted monovalent nanobody VHH#2B upon NO induction, obtained from E. coli Nissle 1917. Adalimumab: positive control. Sybody against a membrane protein: negative control.
4. Nanobody efficacy model

The dry lab constructed a model that built the foundation for nanobody treatment in IBD. It showed that the treatment is effective and flexible to different inflammation severities and ranges in parameters. As long as some bacteria adhere in some proximity to the inflammation sites, the nanobodies should reduce inflammation. The insight gained can propel the future of IBD NanoBiotics development, especially because nanobody production has been accomplished, allowing a shift in focus to improved bacterial adherence.

Figure 6: TNFα reduction for different amounts of inflammation sites
Figure 7: TNFα reduction for different nanobody production efficacies

Future steps and limitations

The successful genomic integration of the whole genetic circuit is crucial for our project's biosafety and still needs to be tackled. The bacteria currently contain antibiotic resistance genes, and the possibility of horizontal gene transfer or the loss of the plasmids are valid concerns we must address in the future. Furthermore, the implementation of a kill switch is necessary to ensure that no genetically modified organisms are released into the environment.

Current limitations of our model include the observed leakiness that our nitric oxide promoter showed in the last Western blot (figure 5A). We already started characterising the leaky nanobody expression in the results section. Additionally, more data is required to prove the inhibition of the inflammatory response produced by the secreted nanobodies. Besides testing the nanobodies on other human cells, such as epithelial cells or intestinal organoids, we must test the system in complex organisms to show its entire effects. Such experiment will also help to determine the colonisation abilities of the genetically modified E. coli Nissle 1917 in the gastrointestinal tract and how well it can compete against the microbiota that is already present. This knowledge is crucial for the future implementation of IBD NanoBiotics.

References:

  1. Behrouzi, A., Mazaheri, H., Falsafi, S., Tavassol, Z. H., Moshiri, A., & Siadat, S. D. (2020). Intestinal effect of the probiotic Escherichia coli strain Nissle 1917 and its OMV. Journal of diabetes and metabolic disorders, 19(1), 597–604.
  2. Qingdong Guan, 2019, “A Comprehensive Review and Update on the Pathogenesis of Inflammatory Bowel Diseas”
  3. Silence, Karen, Lauwereys, Marc, De Haard, Hans, et al. "Single domain antibodies directed against tumour necrosis factor-alpha and uses therefor", Int. Publication Number: WO 2004/041862 A2, 21 May 2004
  4. Xiaoyu J. Chen et al., 2021, Rational Design and Characterization of Nitric Oxide Biosensors in E. coli Nissle 1917 and Mini SimCells
  5. Cai, Z., Wang, S., & Li, J. (2021). Treatment of Inflammatory Bowel Disease: A Comprehensive Review. Frontiers in medicine, 8, 765474.