35 VALANX BIOTECH

VALANX BIOTECH –

Superpower your protein

by Alina M. Einetter,

March 2019

Imagine you are working on in a research lab that develops new anti-cancer drugs. You just found a substance highly toxic for cancer cells which seems to have promising properties to cure certain types of cancer! The last missing step is to transport this highly toxic substance to the tumor cells in a patient’s body only. So, you are trying to get your substance fixed to a transporter protein which specifically targets cancer cells. You tried to get it done for already a long time and the head of department wants to see some results. Moreover, the company you are working for is already under pressure because it did not come up with new drugs. As a result, there is not that much money of the foundation left to start experimenting on a new conjugation technology. Then, at a certain point you think to yourself ‘if only there was a way, I could make a stable conjugation to this very specific transport protein!’ If you are ever in this situation, well there is a solution to your problem….

This solution is called SnapIt! SnapIt is a synthetic amino acid which enables different types of conjugations, linkages and immobilisations of a protein ligand interaction. The company owning the patent of this new technology is called VALANX and was founded by Michael Lukesch (CEO) and Patrik Fladischer (CSO), an Austrian researcher team studying in Graz, Austria in 2017. (1,2) While researching for their final thesis they came up with a great invention which they called ‘SnapIt’. This is a synthetically produced amino acid which is very small and shows to have several applicable advantages.(3) On their website they cite Louis Sullivan, 1896: ‘Whether it be the sweeping eagle in his flight, or the open apple-blossom, the toiling work-horse, the blithe swan, the branching oak, the winding stream at its base, the drifting clouds, over all the coursing sun, form ever follows function, and this is the law.’ They mainly focus on the part ‘form ever follows function’ and turned it into their new technology.

The amino acid can be incorporated in the protein of choice at every position by using E.coli as an expression system. Using E.coli offers many advantages, because it is the most common used bacterium in research, is highly studied and analysed and also occurs naturally in our guts. Placing SnapIt in a protein shows to reform known techniques and give rise to many new approaches regarding protein research.(3)

The team around VALANX engineered an E.coli strain which incorporates the synthetic amino acid SnapIt at every desired location in the desired protein. This specific incorporation facilitates subsequently the attachment of further molecule to this amino acid.(4)

The click chemistry

The click chemistry is commonly described as a high yielding and simple to perform reaction to form a conjugate between a substrate of choice (often a reporter molecule) with a specific biomolecule. This method is considered to be highly efficient, whereas only by-products, which can be removed easily, are generated and it requires merely mild synthetic conditions. Usually a click chemistry reaction is non reversible, but the VALANX amino acid makes it possible, that it still is reversible and furthermore they claim, that their technology is the fastest click reaction ever.(5,6)

They state, that their technology of producing this amino acid is very cost effective, because they ‘use nature’ to produce them. Other than their competitors who add reactive groups to residues of the protein, they form this synthetic amino acid and incorporate it into the protein itself. They use an expression system instead of complex procedures which lowers the price of costs. (4)

The possibilities VALANX offers up to now

Antibody-drug conjugates ADC

ADC is the abbreviation for Antibody-drug conjugates which is a novel method to specifically transport drugs to and attack malignant cells like cancer cells. This dual therapy constitutes of a monoclonal anti body and a toxic drug which degrades or attacks the cancer cells. Both parts on its own do not function properly in the cancer treatment, whereas in combination the seem to have a huge impact on the viability of those cells. The antibody on its own does not have any impact to the cells and the drug on its own is highly too cytotoxic to be prescribed to a patient, because it attacks all cells in an unspecific way. The ADC leads to a cancer cell targeted treatment and furthermore to fewer or no side effects.

In course of the treatment the Antibody-drug conjugates enter the body and target the cancer cells by docking to certain receptors on their surface. After the docking, the highly potent cytotoxic drug is released and kills the cancer cells.

Already approved ADCs like Cetuximab (Erbitux) and Trastuzumab (Herceptin) were used to treat autoimmune diseases and cancer infecting the gastrointestinal tract, head, neck and breast. These drugs bind to the cancer cells and cause the blocking of these signal receptors which are usually responsible for receiving growth favoring signal molecules. Consequently, this leads to the shrinking of the tumors. (7)

The first ever ADC drug called brentuximab vedotin (brand name Adcetris) was FDA approved in 2011 showing, that this treatment is relatively novel. (8)

Even though these drugs seem to offer a great opportunity to fight cancer, there are still some struggles which complicate their development. Firstly, the perfect corresponding monoclonal antibody hast to be found to guarantee to locate correct cells and secondly, a stable and appropriate conjugation between the monoclonal antibody and the drug must be built up. (7) VALANX facilitates the conjugation with their SnapIt amino acid and promotes consequently the step towards the perfect ADC therapy. Furthermore, the synthetic amino acid can be incorporated into the protein at specific locations and also more units of it can be attached which leads to an increased drug to antibody rate (ADR). This rate tells how many potent cytotoxic residues can be attached to the antibody. (4)

PEGylation of therapeutic peptides and proteins

Proteins offer a lot of positive effects for their use in the medical and therapeutic field because they are highly specialized for their possibility of binding substrates or other proteins, as well as their several activities. But there are still some challenges when being used in the patient related therapy: their short half-life time. (9) This parameter determines the time when half of the protein amount is degraded or excreted by the patient. Half-life time of a protein like Nesprin in the blood stream is within an average of a few minutes to few hours. (10) With a PEG conjugate the half-life can be increased to several hours or even days. By increasing the half-life time, a frequent dosing of the medicine and consequently kidney damage because of degrading the protein, can be limited or prevented. Furthermore, the cost of the medicine and immunological responses can be obviated.

However, the addition of a polyethylene glycol polymer (PEG) is called PEGylation and the following reaction mechanism shown in Figure 2. this figure also presents the main configuration of the polymer  which consists of a repeating C-chain and a functional group. This method is the technology of choice for extending the protein’s half life as well as for bringing more positive features along with it. It renders the protein more flexible, hydrophilic, less toxic, variable in size and shields the substrate from proteases and recognition by the immune system. The FDA ‘generally approved as safe” PEG increases the hydrodynamic radius and lowers the rate of renal clearance. While Jonathan K. Dozier et altri. state ‘Installing a single PEG chain at a defined site in a protein is challenging’ in their paper, VALANX facilitates this problem by using its novel synthetic amino acid which can be easily incorporated into the protein at every location. (4,9)

Directed immobilization for biosensors

Biosensors, biochips, drug screening, microarrays, biomedical implants, … they all have one thing in common: a protein-ligand interaction which leads to a detection signal. The assembly of such a trial unit consists of a surface where receptor molecules can be immobilised and bound. Most commonly the material of such surfaces is a polymer or silicon which anchor the receptor molecules easily. The interaction between such a surface and the receptor must be highly stable and controllable to achieve reliable results. The ligands bind the corresponding biologically active receptors on the surface and facilitate a signal transduction to convert the event of binding to a measurable signal. This interaction between the protein and the ligand is highly specific, because also the orientation of the receptor protein plays a huge role. The receptor must present the active binding site in a way that the ligand can easily be bound to it, otherwise an accurate result is achieved. For example, if you want to bind an Antigen to an immobilized antibody, the antigen-binding site must be oriented towards the antigen, otherwise no interaction might happen. It therefore follows, that the difficult part is to immobilize the receptor correctly to the surface. (11) VALANX facilitates this event by again, using their synthetic amino acid in the immobilisation step to create a site-specific chemical binding and does this in a cost-effective way too. The company usually offers the receptor proteins which bind correctly to the surface. This is a rapid method to develop reliable biosensors.

Oriented immobilisation of biocatalysts

Enzymes and proteins usually occurring in nature have limited function because they work as a single unit and therefore are often not able to meet the requirements for a large-scale application. Therefore, biocatalyst research brought up a new idea of immobilising enzymes to a surface to for a homogenous layer of protein activity. These experiments led to a huge increase in the protein’s activity. For example, an immobilised lipase shows 50 times more activity when it is immobilised to a surface. To further increase the ability of these protein estimate, the enzymes are oriented in a way, that they present their binding site to the ligands. This procedure favours the accessibility of the proteins and increases the activity at a 3-fold in solution. (12)

Conclusion

VALANX is a highly promising new start-up with its roots in Austria, a small country in the middle of Europe. There are not that many companies trying to make their way up to the top by starting in Austria. Therefore, the government tries to encourage new start-ups by offering assistance and aid money. Using an E.coli strain to produce proteins for you is nowadays one of the most used standard operations in protein engineering, but incorporating a new synthesized amino acid is novel. Their biotechnological approach combines protein engineering, medicine and biology to create a solution to a huge problem. SnapIt might assist or complete in future many more applications in the medical field. With their special E.coli strain it is possible to make proteins with another amino acid then the 20 naturally occurring ones. So, it might be imaginable that it can also incorporate other molecules into proteins.

A huge advantage is outsourcing the production of your protein, because it can save you time and money. If you don’t have to care about the production, you can focus on the development of the procedure and drug itself. As they claim on their website, it seems to be an affordable procedure to generate these conjugates so that one can do many experiments and not just focus on one protein, because the costs are so high, that one can’t afford different experimental concepts. The number of future applications is vast, and we still don’t know in which areas it will be usable.

References

1. Amino acid – New World Encyclopedia [Internet]. [cited 2019 Mar 22]. Available from: http://www.newworldencyclopedia.org/entry/Amino_acid
2. Valanx Biotech GmbH: Private Company Information – Bloomberg [Internet]. [cited 2019 Mar 24]. Available from: https://www.bloomberg.com/research/stocks/private/snapshot.asp?privcapId=532665518
3. Meet the Startup that Makes Designer Protein Building Blocks [Internet]. RebelBio. 2017 [cited 2019 Mar 21]. Available from: https://rebelbio.co/meet-startup-makes-designer-protein-building-blocks/
4. VALANX Biotech [Internet]. VALANX Biotech. [cited 2019 Mar 22]. Available from: http://www.valanx.bio/
5. Click chemistry. In: Wikipedia [Internet]. 2019 [cited 2019 Mar 22]. Available from: https://en.wikipedia.org/w/index.php?title=Click_chemistry&oldid=886942273
6. Click Chemistry – an overview | ScienceDirect Topics [Internet]. [cited 2019 Mar 22]. Available from: https://www-sciencedirect-com.subzero.lib.uoguelph.ca/topics/chemistry/click-chemistry
7. Ornes S. Antibody–drug conjugates: Proceedings of the National Academy of Sciences. 2013 Aug 20;110(34):13695–13695.
8. -FDA approves Adcetris to treat two types of lymphoma. ENP Newswire [Internet]. 2011 Aug 22 [cited 2019 Mar 22]; Available from: http%3A%2F%2Flink.galegroup.com%2Fapps%2Fdoc%2FA264929317%2FAONE%3Fu%3Dguel77241%26sid%3DAONE%26xid%3D1c038cf4
9. Dozier und Distefano – 2015 – Site-Specific PEGylation of Therapeutic Proteins.pdf.
10. Volltext [Internet]. [cited 2019 Mar 22]. Available from: https://jasn.asnjournals.org/content/24/11/1793.full.pdf
11. Steen Redeker et al. – 2013 – Protein Engineering For Directed Immobilization.pdf.
12. Singh et al. – 2013 – From Protein Engineering to Immobilization Promis.pdf.