Through this question, we are looking for the source of our project. Why did we choose to work on allergies? Why did we think that a new diagnostic tool was needed? What is the situation of allergies in our society? A critical reflection on society is required to provide answers.

First, the reason why we chose to work on allergies comes from one of our team members, Samy.

Samy is 22 years old and suffers from multiple allergies which are the cause of daily discomfort and pain. He is allergic to a lot of fruits and vegetables, but also some drugs. His first proposal in our brainstorming to find a project was to create an alternative desensitization way to injection. The idea was to produce recombinant allergens with yeasts and probiotics to administer them to the patient in a sublingual way. The obvious problem was that consuming living GMOs in Europe for medical purposes is legally very limited. Several of our team members are also affected by allergies, such as Charline, Juliette, and Morgane.

From an ethical point of view, we started referring to reciprocity ethics, which is the idea that we have to do for others what we would like them to do for us. This is a kind of individualist thinking. For example, Samy would love to be desensitized to allergens causing his allergies, so he proposed to create a new method of desensitization for others. What we aimed for was referring to utilitarian ethics. This is the idea that we should do what is best for the greatest number of people. For example, in France, it would concern a third of the French population is affected by allergies.

We met the general public through events such as the Exposciences Occitanie 2022, interventions in high school, and online interactions. The exchanges witnessed the veracity of the statistics because many people told us that they were allergic or knew someone who was allergic. A lot of them had no test to confirm their “self-diagnostic”, which means that allergies are not known and recognized enough to be well-treated. All details of our exchanges can be found in our Education and Communication section.

By doing more research on allergies, we realized that the step even before desensitization, which is to diagnose allergies thanks to tests, had limitations. These limitations are detailed on our Description page. We imagined a new diagnostic tool to outpass the limitations of already existing tests and keep our idea of desensitization using recombinant allergens as a perspective of our project. This was in our opinion the best thing to do for the greatest number of people affected by allergies. However, we needed the opinion of allergy specialists to be sure that we could add something to what is already existing.

That is why we met Luc Colas, an allergist but also a researcher at the university hospital of Nantes and a lecturer in allergology and immunology. He confirmed our impression that a high throughput method of specific IgE detection was missing today in the scope of already existing methods. He also gave us advice regarding our desensitization phase. Using a probiotic strain to produce the allergens and administer it to the patient would induce a better tolerance to the treatment. His opinion was important for us because he represents one of the project stakeholders by working in a university hospital. University hospitals are involved in allergy detection and the use of tests.

We also met Jean-Luc Menardo, a former allergist who used to work in a doctor’s office. Allergists are obviously important stakeholders in allergy detection. Jean-Luc Menardo has also worked as a researcher on desensitization, and as clinic director of the Montpellier university hospital. According to him, a high throughput method of specific IgE detection would greatly complement the traditional Prick test method. Using first a high throughput method is very appropriate for precision medicine, which needs more tools to be plainly exploited (Ansotegui et al., 2020).

Because usual allergy detection tests can be performed in medical laboratories, we met Annabelle Gordon-Le Goff, a biologist and doctor in a medical analysis laboratory in Toulouse. Medical laboratories are also a stakeholder in allergy detection. Annabelle Gordon-Le Goff provided us with a lot of useful information about current tests, the operating model of medical laboratories, and the prices. This helped us to build our own operating business model and to identify the need of the allergy detection stakeholders. Together we also were able to highlight the strengths of our method among the ones existing on the market. This meeting was an opportunity to discuss our project with an important stakeholder in allergy detection.

With the confirmation from the main stakeholders in allergy detection that there was a missing gap in allergy detection, we started to imagine a new method of detection of allergies, focused on the IgE recognition in the patient’s serum with a high throughput method. We came up with the following idea.

An agglutination between three parts:

• - a cell displaying an allergen (cell A)
• - a specific IgE binding to the allergen by its variable part
• - a cell displaying a DARPin, a protein binding specifically to the constant part of IgE (cell D)

This agglutination can be the signal that the patient serum contains IgE specific to the allergen displayed by cell A.
This idea was the basis of our project but required to be developed according to the potential use and needs of the stakeholders.

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Through this question, we were looking for the use of our project from a local aspect. How our method could be used? What are the user’s needs? How to design our project to fit these needs? A reflection on the meaning of the project was required.

To understand what use could be done of our method, we needed more information about its main components.

Because antibodies are central in our method design and because we are not specialists, we discussed this matter with Jasper Kamphuis. He is a postdoctoral researcher at Infinity INSERM, in a team working on food allergies and antibodies. He gave us useful advice about the use of antibodies and labeled antibodies.

To improve our thinking, we met Chloé Beuraud, a researcher in immuno-oncology at Evotec, and a former researcher at Stallergenes Greer. One of her remarks supported the one of Luc Colas: she thought that using probiotic strain for desensitization could increase the treatment tolerance. Chloé Beuraud also suggested using detection methods of the agglomerate through FACS and FRET approaches. This greatly influenced the scope of our project.

Thanks to our meeting with Luc Colas, we knew that FACS is not a routine in a medical analysis laboratory, but can easily be found in a research laboratory. We learned that university hospitals often outsource allergy tests to research laboratories.
It means that our local operating business model could be based on the one of a research laboratory, that is performing tests for medical laboratories and university hospitals, to send the results back to the allergologist. This was inspiring for our Entrepreneurship effort. This also reinforced the idea that FACS was worth considering.

To detect an aggregation with FACS, the easiest would be to clone genes coding for different fluorescent proteins in our strains A and D. We would then obtain a double-signal in case of aggregation sorting.

To check this hypothesis we met Yvan Canitrot, a researcher at LBCMCP and a FACS specialist. He approved this idea and gave us recommendations about what cell dilutions to use for sorting with the FACS.

We pursued our idea with Delphine Lestrade and Chloé Habouzit, both working at TWB (Toulouse White Biotechnology). Delphine Lestrade is a manager of the flux cytometry platform at TWB and used to work at Infinity INSERM on immunology and antibodies. Chloé is a research engineer at the flux cytometry platform of TWB, who worked on microbiota and probiotics. In addition to several meetings to develop together the test protocol of our method with FACS, Delphine Lestrade and Chloé Habouzit helped with the experiments on TWB’s FACS and the analysis of our results.

During a presentation of our project to our partner TBI (Toulouse Biotechnology Institute), we met Emmanuelle Trevisiol who worked on biochips for specific IgE detection. We learned that the price of the test as well as the required IgE volume is two parameters that can impact the utilization and promotion of an allergy detection test by allergists. The price of our method is hard to evaluate since it is extremely innovative. It can evaluate a lot by working on its optimization. One thing we can work on is the used volume of IgE.

After meeting her at the same presentation as Emmanuelle Trevisiol, Gabrielle Potocki-Veronese told us about a patent that her team was developing that could be used to detect aggregations. This patent used a microfluidics technique by forming droplets. As Luc Colas told us that microfluidics machines could be found in research laboratories, this was an interesting potential development for our method. Sophie Lajus provided us with all information about their microfluidics machine and helped us to execute this idea at TBI by testing the detection of aggregates.

One necessary aspect of our project was to model the aggregation between cells and antibodies.

To do so, we received the help of Alain Liné, a process engineering teacher, who specialized in mixing for aggregation control, and Jérôme Morchain, lecturer at INSA Toulouse and researcher at TBI, in the TIM team. The modeling effort is more detailed in the Modeling section.

Thanks to these meetings as well as our own research, we were able to come to a design that could be socially integrated from a local aspect.

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Through this question, we were looking at the future of our project from a global aspect. How does it impact society? How would it be implanted in time and space? What is our goal? A reflection on the aims and impacts of the project is required.

One of our project perspectives was to produce desensitization medicine, using probiotic strain as chassis.

We met Pascal Lebourgeois, professor at UPS and researcher at TBI, a specialist of Lactococcus lactis. Lactococcus lactis is a well-known probiotic strain of bacteria. Following this discussion, we agreed that engineering Lactococcus lactis in the same way we planned to do with E. coli would be too complex to be added to the project. However, the idea is of interest for later development.

Thanks to our meeting with Julien Durand, co-founder, and CEO of Sweetech, we learned that it is common for a start-up to rent space in laboratories at the beginning, before having private investors. This impacted our vision for the future of our project.

Our meeting with Marianne Lefèvre, Intellectual Property specialist from the consulting firm Adamante, brought us much information on patents and intellectual property. This helped to structure our entrepreneurship direction.

Through our project, we regularly met with Colette Schenker, our main counselor from Le Catalyseur. Le Catalyseur is a pre-incubator in Toulouse that offers to guide project promoters through the process of creating a start-up. We defined together the aims and missions of our company. Le Catalyseur also provided us with a SWOT and business model canvas and explains how to use it. We did a project diagnostic with the Catalyseur to evaluate the project progress and what remains to be done. We also defined together the entrepreneurial profile of our team members to find what skills we had and which ones we needed.

More details on the future of our project can be found on the Entrepreneurship pages.

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During this project, we met with numerous stakeholders and specialists who contributed to its evolution. Each meeting was enriching and helped to change a simple idea into a developed and complete project with industrial perspectives.