Team:UNIZAR/Implementation






Implementation

Implementation:

Implementation of the project in the real world

Our team had consultations with stakeholders and experts in the production of antibodies/nanobodies, which allowed us to plan a possible implementation of our technology. First, we assessed whether our method would be feasible, economically profitable and accessible and dicussed the key points that should be optimized for real-world implementation also analyzed who could benefit the most from our development and study what regulation would apply for implementing this new technology, so that no one could misuse it.
Figure 1. Science, business, and law interrelation in technology transfer
Figure 1. Science, business, and law interrelationship in technology transfer

  1. TRIALS WITH OTHER ANTIGENS

    2. Before translating this technology into the market, we must demonstrate its efficiency and its potential benefit. We have done great advances using anti-GFP nanobodies as "proof of concept". GFP was chosen as a model not only because its an easy quantifiable and trackable protein due to its fluorescence but also because there are already available nanobodies against it that we can use as controls. However, it might be beneficial to keep in mind that we want to probe the wide range of functional nanobodies that this technology can generate. Indeed, the impact of this technology goes far beyond GFP: it can be applied to any other immunogenic (i.e. SARS-COV2 spike proteins, C-fragment of tetanus,…) and non-immunogenic (i.e. haptens) antigen/epitope.
    Figure 2. Green fluorescent protein structure, from jellyfish *Aequorea victoria*
    Figure 2. Green fluorescent protein structure, from jellyfish Aequorea victoria

  2. LIBRARY OPTIMIZATION

    3. We have been working with plasmid vectors, a useful tool for laboratory work, as they are easy to manipulate and can be transferred to bacteria rapidly. After talking with Dr. Esteban Martínez we learned that the presence of this type of genetic construct in a high number usually reduces bacteria's fitness (1).

    Once the construction is optimized, it is important to integrate the construct into the chromosome to develop a system that enables the expression of nanobodies in a stable and controlled way. We have been working with plasmid vectors, a useful tool for laboratory work as they are easy to manipulated and can be transferred to bacteria rapidly.
    In our case, having only one copy of the construction in the chromosome is important as the presence of this type of genetic sequence in a high number usually reduces bacteria's fitness (1). This integration can be achieved via minitransposons or homologous recombination. We learned about this after talking with Dr. Esteban Martínez and we decided to implement it in the next step of our project.
    Figure 3. Future implementation: construct integration into the chromosome for stable nanobody expression.
    Figure 3. Future implementation: construct integration into the chromosome for stable nanobody expression.
    Figure 4.
    Figure 4. Surface display of nanobodies through intimin fusion (3)

  3. IMPROVEMENT OF NANOBODY SELECTION TECHNOLOGY

    4. Generating a wide variety of different nanobodies is the key point of our project, although being able to analyze their binding capacity and selecting the best ones is very important too. We have designed our expression system in a way that bacteria display nanobodies in their membrane using intimins and autotransporters (2). This approach is really useful as nanobodies can easily be characterized without a purification step which reduces laboratory costs.

    Our characterization method will consist of an ELISA in which the antigen of interest is bound to the well and the bacteria expressing different nanobodies are incubated with it and then rinsed. Only those able to interact with the antigen will be retrieved after elution. A whole-cell ELISA could also be considered, in this case the bacteria would be fixed to the well, adding the antigen afterwards. If the antigen binds to the nanobody displayed in the bacterial membrane, we could detect the signal, which is proportional to the binding affinity. Detection could be direct if the antigen is fluorescent like GFP, or by incubation with conjugated antibodies.
    Figure 5. Whole-cell ELISA for nanobody affinity assays.
    Figure 5. Whole-cell ELISA for nanobody affinity assays.


    Figure 6. Selection by magnetic cell sorting (1)
    Figure 6. Selection by magnetic cell sorting (1)
    This method may be useful for laboratory work, where few different nanobodies are tested in each cycle. However, for large-scale implementation, a high throughput system has to be implemented. After conversations with Dr. Esteban Martínez, we decided that magnetic-activated cell sorting (MACS) is a good option for our project. MACS consists of incubating bacteria expressing different nanobodies with the target antigen conjugated with biotin and with magnetic beads bound to streptavidin. Next, the mixture is passed through a small ferromagnetic column held in a magnetic holder, which retains bacteria coated with bound biotinylated antigen and anti-biotin microbeads. Bacteria with no antigen on their surface are washed out of the column. Elution of bound bacteria is carried out with fresh culture media. This system enables us to select bacteria expressing nanobodies that specifically bind to the target antigen (1).

    Rosa Monge,CEO of BEOnChip, told us about how to implement their technology with our project. This company develops Organ-on-Chip devices with microfluidic technology. We would use one of these platforms in which the target antigen would be immobilized. The advantage of using this technology is that we could release in a controlled manner successive flows containing our library, and wash and elution buffers. This system will enable us to select the nanobodies in a continuous way.
    Figure 7. BeOnChip device
    Figure 7. BeOnChip device

  4. FUTURE STEPS

    5. Then, we would check the efficiency and performance of our method, assessing whether it could be carried out on a larger scale:
    • First, we would like to create a software capable of automatically analyzing the sequences of the different nanobodies, preparing them for the in vitro recombination. For example, the client would inform us about the target antigen and our software database could search all known nanobodies against the desired antigen. Next, the same computer program would fragment the specific genetic sequences, adjusting the ends for subsequent in vitro recombination.
    • Another major improvement that could be applied to our system is a method to generate Single Point Mutations (SNPs) in the CDRs sequences in order to gain even more variability. At first we did not know how to develop this idea in a simple and quick way, so we started to conversing with Dr. Esteban Martínez, who talked about a system based on ssDNA recombineering that allows doing this in bacteria (3, 4). After further research and conversations with Dr. Luis Ángel Fernández, who recently has published a paper using this technique for in vivo evolution of nanobodies in E. coli we concluded that the application of this recombineering technique in our project could be taken into consideration for further improvements. This CDR mutagenic method has been published recently which supports that recombineering could lead our project to a higher step.
    Figure 8. ssDNA recombineering technology for CDRs randomization (3)
    Figure 8. ssDNA recombineering technology for CDRs randomization (3)

Proposed users

We are aware that our project can benefit scientific society in many ways. Nanobodies are currently a highly exploited and researched area. Our new library creation approach will benefit researchers that use nanobodies in their daily lab work (like Dr. Esteban Martínez and Dr. Luis Ángel Fernández, among many others).
Moreover, there are lots of biotechnological companies that work with nanobodies and antibodies. For example, Certest Biotec S.L. and Operon, two diagnostic companies in our region that were interested in our project and provided us with financial support to develop it, could benefit from our nanobody improvement technology in the future since they will be able to produce a wide range of nanobodies, each one with different stabilities and affinities against the desired antigen.
Figure 9: Operon inmuno and molecular diagnostics and CerTest Biotec logos
Figure 9: Operon inmuno and molecular diagnostics and CerTest Biotec logos

Finally, we are willing to adapt our technology to the industry offering services for nanobody improvement to different laboratories in academia or pharmaceutical companies. By optimizing all the steps of our design as we have presented in the Implementation section, our technology and our library design could become a great business opportunity that everybody in the sector could benefit from.

Safety:

Health and environment

Focusing on the safety aspects, it is very important to identify any possible threats that may affect to the human health or the environment and find suitable alternatives. Despite this safety procedures starts in our laboratory, they have to be considered in the implementation or transition of this project to industry.
Our lab strictly followed the rules of the occupational risk prevention unit (institution in charge of lab security in our university), and we also received a biosafety and biosecurity course from an expert in this field. In addition, our instructors helped us understanding the different risks of our experiments, as they are familiar with the experimental procedures.
Figure 10: Biosafety and biosecurity are interrelated and both equally needed in labwork.
Figure 10: Biosafety and biosecurity are interrelated and both equally needed in labwork.

We are also committed to the environment, so it would be important to manage and identify the environmental risks that may arise from our activity. The microorganisms used in this project allowed us to express the newly formed nanobody sequences. However, they were discarded following strict protocols to ensure that there was no environmental spread. Furthermore, if our purified nanobodies ended up in the environment, there would be no risk of expansion since they are not living parts capable of replicating.

Regulation:

Future regulation

Our project is not intended to harm human health or the environment. Nanobodies are formed by random recombination of CDR1, CDR2, and CDR3 nanobody fragments (which encode the antigen-binding parts) that come from nanobodies whose genetic sequence is already known and characterized, in our case against GFP (green fluorescent protein).
We know that these nanobodies are absolutely harmless. However, our technology could be used to improve other nanobodies, which could have some important implications to consider in the event of its release and further market implementation.

Because of that, it may be necessary to establish a regulation settling which nanobodies could take advantage of this improvement methodology. For example, the improvement of nanobodies capable of recognizing a tumor could be authorized as a treatment against cancer. However, the improvement of nanobodies that target healthy tissues and pose a biohazard should not be allowed.
Finally, we are aware that in case of implementing this technology in the industry we should comply with good manufacturing practices (GMP) to meet the quality standard established by the European Union in the field of production and sale of biotechnological products.
Figure 11: Good Manufacturing Practices keypoints.
Figure 11: Good Manufacturing Practices keypoints.


References:

  1. Salema V, Fernández LÁ. Escherichia coli surface display for the selection of nanobodies. Microb Biotechnol [Internet]. 2017 Nov 1 [cited 2021 Oct 15];10(6):1468. Available from: /pmc/articles/PMC565859
  2. Nyerges Á, Csörgo B, Draskovits G, Kintses B, Szili P, Ferenc G, et al. Directed evolution of multiple genomic loci allows the prediction of antibiotic resistance. Proc Natl Acad Sci U S A. 2018;115(25):E5726–35.
  3. Al-ramahi Y, Nyerges A, Margolles Y, Cerdan L, Ferenc G, Pal C, et al. ssDNA recombineering boosts in vivo evolution of nanobodies displayed on bacterial surfaces. bioRxiv [Internet]. 2021;2021.01.28.428624. Available from: https://doi.org/10.1101/2021.01.28.428624
  4. Synthetic consortia of nanobody-coupled and formatted bacteria for prophylaxis and therapy interventions targeting microbiome dysbiosis-associated diseases and co-morbidities - Scientific Figure on ResearchGate. Available from: https://www.researchgate.net/figure/Nanobody-display-and-synthetic-adhesins-of-Escherichia-coli-A-Structure-of-a-camelid_fig1_329847594[accessed 16 Oct, 2021]

Figure references:
  1. Figure 1: About OTT - Technology Transfer; (https://tt.research.ucf.edu/about-ott/;) Accessed: 2021-10-16
  2. Figure 2: Crystal structure of the Aequorea victoria green fluorescent protein, Mats Ormö, Andrew B. Cubitt et al. Science, 273, 5280, 9 1996
  3. Figure 3 and 5: Created using Biorender.
  4. Figure 4 and 6: cited in references.
  5. Figure 7: BE-FLOW STANDARD (10 devices per box) | BEOnChip – Biomimetic Environment On Chip; (https://beonchip.com/product/be-flow-standard/) Accessed: 2021-10-16
  6. Figure about future regulation: Rule of law (2020) - Multimedia Centre (https://multimedia.europarl.europa.eu/nl/rule-of-law-2020_14501_pk) Accessed: 2021-10-14
  7. Figure 10: 1st Biosecurity Virtual Symposium | 1st Biosecurity Symposium (https://biosecuritysymposium.org/) Accessed: 2021-10-16
  8. Figure 11: Good Manufacturing Practices Yield Good Quality | NGC Software (https://www.ngcsoftware.com/post/142/good-manufacturing-practices-yield-good-quality/) Accessed: 2021-10-16