Results

"You cannot expect a new result if you keep doing the same thing, except for cloning" - Wouter

Results

!MPACT was designed to overcome the shortcomings of the current therapies against ANCA-associated vasculitis (AAV). As described in the Project Description, current treatments of this autoimmune disease cause burdensome side effects, high healthcare expenditures and workloads, while not excluding disease relapses. To prove that !MPACT is a feasible, novel therapy for treating AAV, we have designed a Proof of principle study (see Proof of Concept). The current page will present in a bird's-eye view the most important results for each section of this proof of principle study:


  • Receptor Transfection and Activation analysis
  • Therapeutic Expression
  • Receptor Activation via Antibodies
  • Experimental Outlook
Please click on any of the four parts in the overview for a more in-depth analysis of the results!

Scroll Down for the overview of the results!

Receptor transfection and activation analysis

To validate that our receptor scaffold is correctly expressed and stays susceptible to activation, we have performed a receptor transfection and activation analysis. By using the generalized extracellular molecule sensor (GEMS) receptor scaffold, described in Scheller et al. (2018), we were able to successfully show receptor activation resulting in protein expression. We expressed the SEAP reporter protein by inducing the receptor scaffold using the azo dye RR120. Maximum receptor activation for the measured ligand concentrations was seen for 300 ng/mL RR120. Moreover, a ligand dependent activation is seen for this receptor scaffold.


Therapeutic expression

After establishing successful transfection and activation of the GEMS receptor, we sought to alter the expressed protein towards a more relevant, therapeutic protein. Based on literature and discussions with stakeholders, we decided to express interleukin 10 (IL-10) with our !MPACT cell. After successful cloning of IL-10 into the protein expression plasmid, IL-10 expression concentrations were quantified. Following receptor induction via RR120 addition, multiple IL-10 enzyme-linked immunosorbent assays (ELISA) were performed. Concentrations of the secreted IL-10 were measured at approximately 9 nanograms per milliliter. As these concentrations are a 1000-fold higher than physiological conditions, different methods to achieve lower IL-10 expression are discussed at the end of this part.


Receptor Activation via Antibodies

Following the successful expression of interleukin 10 using the GEMS receptor scaffold, it was determined whether antibody-induced receptor activation is possible, as !MPACTs’ focus is on treating ANCA-associated vasculitis. Many new, different versions of the GEMS receptor, which included different affinity domains coupled to differently sized linkers, were successfully cloned into the receptor scaffold plasmid. The possibility to activate these new GEMS receptors using antibodies was examined, utilizing the expression of the SEAP reporter protein. As no significant difference was seen in the SEAP activity between the induced and uninduced conditions, flow cytometry experiments were conducted. Results revealed that binding of the antibody to its antigen did happen, however, in lower percentages than originally anticipated.


Experimental Outlook

We envision that by performing additional experiments more insight into each topic could be achieved. Multiple suggestions for future research are discussed at the end of the results wiki to optimize the system.


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Receptor transfection and activation analysis

Receptor transfection and activation analysis


To validate that our receptor scaffold is correctly expressed and stays susceptible to activation, we have performed a receptor transfection and activation analysis, as described on the Proof of concept wiki page . For this analysis, we made use of the Generalized Extracellular Molecule Sensor (GEMS) receptor scaffold (BBa_K4160008), described in a recent paper by Scheller et al..1 This receptor scaffold comprises a VHH affinity domain (BBa_K4160003) raised against the azo dye RR120 (Reactive Red 120) , the receptor scaffold backbone and an IL-6RB domain (BBa_K4160002). Activation of the receptor triggers downstream signaling via the JAK/STAT pathway, resulting in the expression of the reporter protein SEAP (see our Contribution page for extensive documentation on SEAP) (Figure 1A).


To determine receptor activation, we transiently transfected the plasmids pLEO619 (which includes the GEMS receptor construct containing RR120 VHH as affinity domain, BBa_K4160008), pLS13 (includes the part that allows for SEAP expression, BBa_K4160016), and pLS15 (includes the part for STAT3 expression, BBa_K4160005) into HEK293T cells. Subsequently, a ligand titration on the transfected cells was performed, followed by an incubation and receptor induction step of minimally 40 hours. To determine whether our receptor is activated upon the addition of ligand, the SEAP activity was measured in presence of increasing concentrations of RR120. SEAP activity is determined using the absorbance values (at 405 nm) which get processed by a MATLAB SEAP script. (Figure 1B) This SEAP MATLAB script is one of our contributions to the iGEM community as it streamlines the activity calculation.


Receptor transfection in HEK293T cells was successful as only the RR120-induced conditions show SEAP activity (Figure 1B).


Receptor activation is seen in cells with different added concentrations of RR120. Receptor activation is depicted by the increased SEAP activity for the induced conditions when compared to the uninduced condition. For example, a 32-fold and 34-fold change is seen between the uninduced and induced (100 and 300 ng/mL) conditions, respectively. Moreover, the maximum receptor activation for the measured ligand concentrations is seen for 300 ng/mL RR120.

Figure 1 | A Schematic representation of GEMS receptor induction by RR120, resulting in SEAP secretion. B HEK293T cells were transiently transfected with pLEO619, pLS13, and pLS15, and subsequently induced with a titration of RR120. Cells were incubated for minimally 40 h. All experiments were performed in biological triplicates, for which 5 µL of cell medium (incubation for 30 minutes at 65 °C) was used. Measurements were taken every 30 seconds for 1 hour at 405 nm at RT (25 °C). Data was processed by the SEAP MATLAB script, which calculates the SEAP activity using the measured absorbance at 405 nm. Bars represent mean values, overlayed individual data points are represented as circles (for n=3 biologically independent samples). Each bar shows its fold increase of SEAP activity, compared to the 0 ng/mL condition.

In conclusion, we show that we can successfully transfect the receptor construct in HEK293T cells. Additionally, receptor activation by RR120 addition is observed, which is dependent on the concentration of ligand added.

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Therapeutic expression

Therapeutic Expression

After establishing that we can successfully transfect and activate the GEMS receptor scaffold (BBa_K4160008), we sought to alter the expressed protein towards a more relevant, therapeutic protein. As described in our integrated human practices (Milestone 4) and project description pages, in-depth stakeholder discussions (including UMC Utrecht, Novartis, Catherina Hospital Eindhoven) and literature reviews resulted in choosing interleukin 10 (IL-10) as the therapeutic protein as the output of the engineered cell.


The modularity of the GEMS receptor system allows us to alter the expressed protein relatively easily (Figure 2). Substitution of the SEAP reporter protein with IL-10 in the expression vector, gives us the correct plasmid for IL-10 secretion (BBa_K4160017).

Figure 2 | Schematic representation of the modularity of the GEMS receptor scaffold. The four modular parts of the GEMS receptor. The figure shows all single components of the GEMS receptor scaffold we have utilized, however, we only made use of IL-6RB as the intracellular domain. Affinity domains can bind to different input molecules, which leads to dimerization and subsequent triggering of the downstream signal. The linker between the affinity domain and the EpoR scaffold (light blue) has to be optimized for every affinity domain. The intracellular domain can be substituted depending on the output molecule. Finally, an output can be chosen depending on the application of the synthetic cell.

Cloning was done by the restriction and ligation enzyme technique, which details can be found in our Notebook (Figure 3A). Colony PCR verified that the integration of the IL-10 DNA into the pLS13 plasmid was successful, depicted by the clear bands at ~550 bp (Figure 3B). Moreover, pLS13-IL-10 (BBa_K4160017) has been sequence-verified via commercial Sanger sequencing (BaseClear) before use in our experiments.

Figure 3 | Agarose gels for quality control. A Restriction enzyme (XbaI, NEB & KlfI, ThermoFisher) analysis. A 1% agarose gel, loaded with digested plasmid (pLS13) and insert (IL-10). Outlined DNA bands were cut out and used for ligation. A GeneRuler 1 kb DNA ladder was added for comparison. B A 1% agarose gel, showing DNA samples after colony PCR, using PCR primers specific for IL-10. A GeneRuler 2-Log DNA ladder was added for comparison. Both agarose gels ran for 1 hour at 100 V in 1x TAE buffer, stained with SYBR Safe DNA gel stain. Gel analysis was performed using a 470-nm blue light illuminator.

To validate that we can engineer cells to express IL-10 after ligand addition, HEK293T cells were transiently transfected with the plasmids pLEO619 (which includes the GEMS receptor construct containing RR120 VHH as affinity domain, BBa_K4160008), pLS13-IL-10 (includes the part that allows for IL-10 expression, BBa_K4160017) and pLS15 (includes the part for STAT3 expression, BBa_K4160005). Subsequently, a ligand titration on the transfected cells was performed, followed by an incubation and receptor induction step of minimally 40 hours. IL-10 concentrations were quantified using an IL-10 enzyme-linked immunosorbent assay (ELISA), according to the manufacturer’s instructions. (Figure 4A,B)

Figure 4 | IL-10 expression as a relevant therapeutic. A Schematic representation of GEMS receptor induction by RR120, resulting in IL-10 secretion. B HEK293T cells were transiently transfected with pLEO619, pLS13-IL-10, and pLS15, and subsequently activated with a titration of RR120. Cells were incubated for minimally 40 h. Samples (100 µL) were taken directly from the cell medium, prepared according to the manufacturer’s instructions, and quantified using sample absorbance (at 450 nm) and the calibration curve (see Supplemantary figure 1). Dashed bars represent IL-10 concentrations that were too high to be correctly quantified. Bars represent mean values, overlayed individual data points represented as circles (for n=3 biologically independent samples).

Successful IL-10 expression was observed for all samples that were induced with RR120, as the samples turned from blue to yellow during the ELISA sample preparation. However, accurate quantification of IL-10 expression was not possible, due to excessive IL-10 expression rates which overshoot the calibration curve.


Since we want to quantify IL-10 expression rates, we decided to repeat the experiment. Moreover, correct quantification and understanding of the therapeutic expression part are crucial before continuing with the next part. For this experiment, the transient transfection of pLeo619, pLS13-IL-10, and pLS15 in HEK293T cells was repeated as described above. A single concentration of RR120 (300 ng/mL) was used for receptor activation in each sample. Samples were prepared in a broad range of dilutions, as we were unsure of the concentration of IL-10 that was secreted. By measuring the dilutions, we were able to accurately quantify IL-10 expression after receptor induction with RR120 (Figure 5A,B).

Figure 5 | IL-10 expression as a relevant therapeutic. A HEK293T cells were transiently transfected with pLEO619, pLS13-IL-10, and pLS15, and subsequently activated with a single concentration of RR120 (300 ng/mL). Cells were incubated for minimally 40 h. Samples (100 µL) were taken directly from the cell medium, diluted, prepared according to the manufacturer’s instructions, and quantified using ELISA. To determine the IL-10 concentration both the samples’ absorbance (at 450 nm) and the calibration curve (see see Supplemantary figure 2) are consulted. Dashed bars represent IL-10 concentrations that were too high to be correctly quantified. Bars represent mean values of IL-10 concentrations (adjusted to individual dilution factors), overlayed individual data points represented as circles (for n=3 biologically independent samples). B Comparison between two values from Figure A.

Again, successful IL-10 expression was observed for all samples which were induced with RR120. The IL-10 concentration after induction with 300 ng/mL RR120, is seen at 9 ng/mL on average. Physiological concentration of interleukin 10 in healthy individuals is found in the picogram per milliliter range, which shows that our receptor-induced IL-10 expression is a 1000-fold higher than physiological conditions.2 These concentrations are much higher than current therapeutic levels of micrograms per kg body weight. However, we expect that the expressed IL-10 levels are adjustable to the desired working concentrations by lowering the receptor activation. This can be achieved by decreasing the number of receptors that we transfect, as we cannot influence the antibody (ligand) concentrations in our final system. Moreover, other methods leading to a lowered receptor activation could be achieved by, decreasing the affinity of receptor-ligand interactions or introducing changes in the downstream pathway. More research should be conducted to achieve a dose-dependent expression of the therapeutically relevant protein.


In conclusion, we have successfully expressed our therapeutically relevant protein, interleukin 10, using the GEMS receptor scaffold. Furthermore, we have shown that IL-10 concentrations after receptor induction with RR120 (300 ng/mL) are approximately 9 nanograms per milliliter. Finally, as this concentration of IL-10 is higher than our intended working concentration, more research, to acquire a dose-dependent expression of IL-10, should be conducted.

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Receptor activation via antibodies

Flow cytometry

Receptor Activation via Antibodies

Following the successful expression of interleukin 10 using the GEMS receptor, we wanted to determine if antibody-induced receptor activation was possible. This in itself is an important research question, because GEMS receptor activation via antibodies has not been described in literature yet, to our knowledge. The modular nature of the GEMS receptor system makes it possible to exchange the affinity domain. A promising affinity domain for our project is proteinase 3 (PR3). As described in the project description, ANCA-associated vasculitis (AAV) is associated with the production of auto-antibodies (ANCAs) which target proteinase 3 (PR3) proteins. We, therefore, designed a new affinity domain for the GEMS receptor scaffold which extracellularly displays PR3. The PR3 protein acts as an affinity domain for ANCAs and we hypothesize that the binding of these antibodies can lead to receptor dimerization, triggering downstream signaling (Figure 6).

Figure 6 | Anti-PR3 antibody-induced receptor activation. Schematic representation of one of the four GEMS receptor constructs, consisting of a PR3 affinity domain (yellow) and an 8 amino acid linker (black). The other three constructs vary in linker length and composition. Moreover, receptor induction by anti-PR3 resulting in SEAP reporter protein secretion is also shown.

Since no papers were published describing antibody-induced GEMS receptor activation, we were required to design this GEMS receptor based on estimations. Antibodies with identical antigen binding domains are shown to bind bivalently to their corresponding antigen, if the antigen separation is between 30 and 170 Å.3 As EpoR and PR3 have an estimated diameter of 55 Å and 45 Å, respectively (Supplementary Figure 4 and 5), differently sized linkers will be introduced between these domains. This results in a library of GEMS receptors with various distances (0, 8, and 31 amino acid linkers) between epitopes, ranging from 100 Å and 160 Å. We hypothesize that by introducing an amino acid linker between the EpoR scaffold and the PR3 affinity domain, an increase in the antibody-induced receptor activation will be seen.


To acquire the plasmids containing the DNA encoding for the GEMS receptor including various linker lengths, many different cloning techniques were performed. For cloning of the plasmids 0_PR3 and 8_PR3, the restriction and ligation enzyme technique was used, on which details can be found in our Notebook (Figure 7). Moreover, both plasmids were sequence verified via commercial Sanger sequencing (BaseClear) before use in our experiments. Unfortunately, the cloning of 31_PR3 was unsuccessful, even after multiple different cloning strategies. A detailed description of cloning design iterations can be found on the Engineering Success page.


Figure 7 | Agarose gels for quality control. Restriction enzyme (BamHI, NEB & EcoRI, NEB) analysis. A 1% agarose gel, loaded with the digested plasmid (2_P3 and 4_P3) and insert (PR3). Outlined DNA bands were cut out and used for ligation. A GeneRuler 2-Log DNA ladder was added for comparison. Agarose gel ran for 1 hour at 100 V in 1x TAE buffer, stained with SYBR Safe DNA gel stain. Gel analysis was performed using a 470-nm blue light illuminator.

To determine if antibody-induced receptor activation is possible, we transiently transfected 0_PR3 or 8_PR3 (which includes the GEMS receptor constructs containing the PR3 affinity domain next to either the 0 or 8 amino acid linkers, 0_PR3 (BBa_K4160009) and 8_PR3 (BBa_K4160010)), pLS13 (includes the part that allows for SEAP expression, pLS13 (BBa_K4160016), and pLS15 (includes the part for STAT3 expression, BBa_K4160005) into HEK293T cells. Experimental conditions were identical to previous cell experiments (Figure 8). Figure 8A and 8B show no significant difference in SEAP activity between anti-PR3 induced and uninduced conditions. This suggests that the addition of anti-PR3 does not result in receptor activation for both variants of the GEMS receptors. We hypothesize that the unsuccessful activation can be the result of incorrect folding of PR3 or a non-accessible epitope on PR3, however, this requires further investigation.

Figure 8 | Antibody-induced receptor activation. A, B HEK293T cells were transiently transfected with (A) 0_PR3 and (B) 8_PR3, pLS13, and pLS15, and subsequently induced with a titration of anti-PR3 antibodies (OriGene, cat. Nr. TA807348). Cells were incubated for minimally 40 h. All experiments were performed in biological triplicates, for which 5 µL of cell medium (incubation for 30 minutes at 65 °C) was used. Measurements were taken every 30 seconds for 1 hour at 405 nm at RT (25 °C). Data was processed by the SEAP MATLAB script, which calculates the SEAP activity using the measured absorbance at 405 nm. Bars represent mean values, overlayed individual data points represented as circles (for n=3 biologically independent samples). The inset figure shows a zoomed-in view of the same data.

As the activation of 0_PR3 and 8_PR3 was unsuccessful, two new receptor variants were constructed. 0_HA_PR3 and 8_HA_PR3 had an HA-tag introduced into the receptor framework in between the amino acid linker and PR3 (Figure 9).

Figure 9 | anti-HA antibody-induced receptor activation. Schematic representation of one of the four GEMS receptor constructs, consisting of a PR3 affinity domain (yellow), an HA-tag (red), and an 8 amino acid linker (black). The other three constructs vary in linker length and composition. Moreover, receptor induction by anti-HA resulting in SEAP reporter protein secretion is also shown.

Again, an experiment to evaluate receptor activation using antibodies was performed on 0_HA_PR3 (BBa_K4160012) and 8_HA_PR3 (BBa_K4160013), which together with pLS13 (includes the part that allows for SEAP expression, BBa_K4160016), and pLS15 (includes the part for STAT3 expression, BBa_K4160005) were transiently transfected into HEK293T cells. Experimental conditions were kept identical, except for the inducer molecule, which was changed to anti-HA antibodies (Figure 10). No significant difference in SEAP activity is seen between anti-HA induced and uninduced conditions, suggesting that the addition of anti-HA does not lead to activation of the receptor constructs.

Figure 10 | Antibody-induced receptor activation. A, B HEK293T cells were transiently transfected with (A) 0_HA_PR3 and (B) 8_HA_PR3, pLS13, and pLS15, and subsequently induced with a titration of anti-HA antibodies (Invitrogen, A-21287). Cells were incubated for minimally 40 h. All experiments were performed in biological triplicates, for which 5 µL of cell medium (incubation for 30 minutes at 65 °C) was used. Measurements were taken every 30 seconds for 1 hour at 405 nm at RT (25 °C). Data was processed by the SEAP MATLAB script, which calculates the SEAP activity using the measured absorbance at 405 nm. Bars represent mean values, overlayed individual data points represented as circles (for n=3 biologically independent samples). The inset figure shows a zoomed-in view of the same data. Data points below 0 (U/L) were not plotted.

Flow Cytometry

As we considered that one reason for unsuccessful GEMS receptor activation via antibodies could be the binding of the antibodies to their affinity domains, we planned to investigate the binding via flow cytometry experiments. In these experiments, HEK293T cells were transfected with the receptor constructs and subsequently stained with the appropriately labeled antibodies.


To determine the binding properties of both anti-PR3 and anti-HA antibodies to their specific antigen, a flow cytometry experiment was conducted on the different receptor constructs. For this experiment, 0_HA_PR3 (BBa_K4160012), 8_HA_PR3 (BBa_K4160013) together with 8_HA (mRNA instead of DNA, received from our sponsor RiboPro, (BBa_K4160015) were transfected in HEK293T cells (Figure 11).

Figure 11 | Receptor constructs used in flow cytometry experiments. Three different receptor constructs were transfected for flow cytometry analysis. These were 0_HA_PR3, 8_HA_PR3, and 8_HA (mRNA).

mRNA transfection was repeated 24 hours after the first transfection, as mRNA-induced protein expression levels peak after 12-24 hours. Moreover, we transfected an eGFP plasmid BBa_K3033009) in other HEK293T cells, to give us an indication of our receptor transfection and determine the general transfection efficiency (Figure 13C, Supplementary Figure 3). After a 40-hour incubation step, the cells were collected and subsequently stained with primary or primary and secondary antibody staining. All staining used the fluorophore Alexa Fluor 488. Flow cytometry experiments were performed using the machine BD FACSymphony A3 (BD Biosciences).


Next, we selected specific criteria based on forward and side scatter to get viable HEK293T cells. Figure 12 shows a graphic explanation of how the gates were constructed and selected. Hereafter, a gating strategy was used to select single HEK293T cells, based on forward scatter-area vs height. After setting up these two gates, we recorded at least 10.000 events that fell within these criteria, creating our negative control (Figure 13A,B).

Figure 12 | Graphical explanation of the gating strategy used. Gating 1 selects viable HEK293T cells based using forward and side scatter. Gating 2 removes any doubles using forward scatter area and height.


Figure 13 | Gating and flow cytometry results eGFP. A Primary gating to select the viable HEK293T cells, using forward vs side scatter. B Secondary gating to select only singlet, using forward scatter-area vs height. C Flow cytometry results of eGFP transfected HEK293T cells to determine transfection efficiency. Green shading and percentages depict the number of cells with a higher fluorescence intensity in the Alexa Fluor 488 channel than the negative control. Percentages and cell counts were determined after gating. Data was acquired using the BD FACSymphony A3 (BD Biosciences) and visualized using FlowJo software.

Hereafter, the stained cells were analyzed and the data was plotted using FlowJo Software (Figure 14, Figure 15, Figure 16). The green shading and percentages shown in the graphs depict the number of cells with a higher fluorescence intensity in the Alexa Fluor 488 channel than the negative control. Figure 14A and B show the percentages of bound anti-HA to 0_HA_PR3 and 8_HA_PR3 which were 10.3 and 11.5 %, respectively.

Figure 14 | Flow cytometry results of 0_HA_PR3 and 8_HA_PR3 with anti-HA staining. A B Flow cytometry results of the receptor constructs 0_HA_PR3 and 8_HA_PR3, stained with anti-HA (Alexa Fluor 488) antibodies. Green shading and percentages depict the number of cells with a higher fluorescence intensity in the Alexa Fluor 488 channel than the negative control. Percentages and cell counts were determined after gating. Data was acquired using the BD FACSymphony A3 (BD Biosciences) and visualized using FlowJo software.

Figure 15A shows the percentages for the bound anti-PR3 to the receptor construct 0_HA_PR3 which increased to 21.9 %, when compared to the previous results. Moreover, an even higher binding percentage of almost 30 % is seen for the construct with RR120 VHH as its affinity domain (Figure 15B).

Figure 15 | Flow cytometry results of receptor constructs 0_HA_PR3 and RR-120 with anti-PR3 staining. A B Flow cytometry results of the receptor constructs 0_HA_PR3 and RR-120, stained with primary anti-PR3 antibodies (blue) and subsequent anti-IgG antibodies (Alexa Fluor 488, orange). Green shading and percentages depict the number of cells with a higher fluorescence intensity in the Alexa Fluor 488 channel than the negative control. Percentages and cell counts were determined after gating. Data was acquired using the BD FACSymphony A3 (BD Biosciences) and visualized using FlowJo software.

Finally, Figures 16A and 16B show flow cytometry data on the receptor construct 8_HA, 24 and 48 hours after mRNA transfection. Binding percentages were 11.6 % and 9.7 % for the 24-hour and 48-hour incubation time, respectively.

Figure 16 | Flow cytometry results of 8_HA with anti-HA staining after 24 and 48 hours. A B Flow cytometry results of the receptor construct 8_HA, stained with anti-HA (Alexa Fluor 488) antibodies, 24 and 48 hours after mRNA transfection. Green shading and percentages depict the number of cells with a higher fluorescence intensity in the Alexa Fluor 488 channel than the negative control. Percentages and cell counts were determined after gating. Data was acquired using the BD FACSymphony A3 (BD Biosciences) and visualized using FlowJo software.

As seen in Figure 13C, the transfection efficiency of this experiment, which is determined by the percentage of positive eGFP cells, was ~ 75 %. This suggests that the percentage of positively stained cells had not reached maximum binding percentages, as the transfection efficiency did not reach 100 %. Regarding the percentages of the anti-HA stain on the 0 and 8_HA_PR3 receptor constructs (Figure 14A, B), being 10.3 and 11.5 % respectively, we hypothesize that due to the presence of the PR3 next to the HA-tag, antibody binding could be hampered. When looking at the binding percentages of the receptor construct 0_HA_PR3, which is stained using anti-PR3, 21.9 % is seen (Figure 15A). Why hypothesize that the fully accessible PR3 affinity domain, allows for these higher binding percentages. Percentages for the receptor construct 8_HA (Figure 16A, B) were expected to be higher (reaching up to 70 %), as the HA-tag should be fully accessible without any physical hindrance by PR3 being present. We, therefore, hypothesize that the receptor construct is not fully transported to the cell membrane, resulting in lowered binding percentages. Moreover, unsuccessful expression of the receptor can also have an influence on the binding percentages. Finally, the binding percentages regarding the receptor construct including the RR120 VHH affinity domain showed to be almost 30 % (Figure 15B). This result was not expected as this condition was added as a control. Possible explanations could be that the secondary antibody used for staining, can also bind the affinity domain used in this receptor construct. As shown by Asaadi et al.4 the VH domain of an IgG antibody and VHH (nanobody) are very similar in structure, as both comprise 9 β-strands that form the IgV structure. This structural resemblance could result in the binding of the secondary antibody to the affinity domain on the receptor construct. This hypothesis could be validated with an additional binding assay to determine the binding properties of the antibodies to this receptor construct.


Conclusion

From all of our collected data, we are confident to say that the transfected plasmids and mRNA translate into our receptor of interest. Although the experimental setup was limited due to time constraints and multiple bacterial infections, the initial results are promising, as antibody binding to the affinity domain is seen in each receptor construct.

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Experimental Outlook

Experimental Outlook

In this iGEM project, we showed that we were able to reproduce the results of the original generalized extracellular molecule sensor (GEMS) paper.1 Moreover, we showed that we successfully can express a therapeutically relevant protein using the GEMS receptor. Finally, we have used antibodies as ligands, for which we have shown partial binding to the affinity domain on different receptor constructs.


Therapeutic expression

For future research, we envision that by performing additional experiments more insight into IL-10 expression and antibody-induced receptor activation can be obtained. As shown previously, we quantified IL-10 expression using one specific concentration of RR120 (300 ng/mL). More insight into the relation between inducer and IL-10 concentration could be obtained by inducing IL-10 expression using multiple different concentrations of the inducer molecule RR120. Furthermore, by introducing a different promotor (or additionally an otherwise controllable repressor/activator) tunability of expression levels could be achieved. As described in the and Project Description, IL-10 production should be dependent on the auto-immune disease we want to prevent.


Antibody-induced receptor activation

Moreover, we were unable to activate the receptor by antibody induction for which the exact cause is not known yet. We do suggest here a diverse range of experiments, which could result in more insight into this phenomenon. First of all, it should be validated that the receptor construct gets successfully transported onto the membrane. This can be determined by comparing a non-permeabilized cell to a permeabilized cell, which enables intracellular antibody staining.5 If higher fluorescence is observed in the permeabilized cell, unsuccessful transport of the receptor construct onto the membrane can be expected.


Next, to simplify the receptor constructs’ affinity domain, a specific epitope that is present on PR3 can be expressed instead of the full PR3 protein. This could remove variabilities such as the possibility of incorrectly folded PR3, although it could also prevent the antigen from folding properly. Moreover, by introducing these PR3 epitopes as tandem repeats, inducing receptor clustering, could also lead to antibody-induced receptor activation.


Furthermore, differently sized and structured amino acid linkers can be introduced between the affinity domain and the receptor scaffold. We have made an effort to create a 31 amino acid linker, which is described in Engineering Success . Unfortunately, we did not succeed in the cloning of this longer linker. Linker lengths of 30 to 100 amino acids should be considered (2.5 to 5 nm, respectively), as optimal antigen separation for a bivalent interaction of antibodies is seen at 16 nm.3 Moreover, the introduction of amino acids which lead to conformational restrictions of the linker (e.g. proline) could also have an impact on finding the most optimal linker sequence.


Ultimately, after successful antibody-induced receptor activation is achieved, a study on the combination of this part and the therapeutic expression part should be performed. This experiment would complete our full project.

  1. Scheller L, Strittmatter T, Fuchs D, Bojar D, Fussenegger M. Generalized extracellular molecule sensor platform for programming cellular behavior article. Nat Chem Biol. 2018;14(7):723-729. doi:10.1038/s41589-018-0046-z
  2. Kleiner G, Marcuzzi A, Zanin V, Monasta L, Zauli G. Cytokine levels in the serum of healthy subjects. Mediators Inflamm. 2013;2013. doi:10.1155/2013/434010
  3. Shaw A, Hoffecker IT, Smyrlaki I, et al. Binding to nanopatterned antigens is dominated by the spatial tolerance of antibodies. Nat Nanotechnol. 2019;14(2):184-190. doi:10.1038/s41565-018-0336-3
  4. Asaadi Y, Jouneghani FF, Janani S, Rahbarizadeh F. A comprehensive comparison between camelid nanobodies and single chain variable fragments. Biomark Res. 2021;9(1):1-20. doi:10.1186/s40364-021-00332-6
  5. Goetz C, Hammerbeck C. Surface and Intracellular Staining Protocols for Flow Cytometry BT - Flow Cytometry Basics for the Non-Expert. In: Goetz C, Hammerbeck C, Bonnevier J, eds. Springer International Publishing; 2018:157-181. doi:10.1007/978-3-319-98071-3_9
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