Neuroblastoma constitutes one of the most common forms of neuroendocrine tumours affecting the paediatric population. The distinctive feature of this type of tumour lies in the widespread presence of GD2 gangliosides on the surface of neuroblastoma cells. These gangliosides, in particular, have attracted the attention of the scientific community for their potential key role in advancing oncological research.

'Bacteriophages and their Uses for Targeting GD2-Expressing Cancer Cells', as well as the most displayed patent - for October - on the Knowledge Share platform, focuses on a novel therapeutic strategy involving a bacteriophage, or related composition, designed to interact with GD2. This bacteriophage opens up new perspectives in the field of medical therapy and diagnostics.

In this interview, Alberto Danielli, Associate Professor of Molecular Biology at the Department of Pharmacy and Biotechnology, University of Bologna, will explore in depth the design and application of bacteriophages targeting GD2, together with possible medical and diagnostic implications. The aim is to provide a clear and comprehensive overview of this promising methodology, highlighting its potential impact in improving currently available therapeutic options for neuroblastoma patients.

Through this pioneering research, it is hoped to lay the foundations for new perspectives in the treatment of neuroblastoma, offering renewed hope for patients and further advancing the understanding of this complex paediatric disease.

Tell us about yourself, background and role/interests in the world of research?

I am a molecular biologist with much experience in basic research, particularly in transcription and molecular microbiology of human pathogens. Recently, I have been wondering about the role of research, wondering whether it is not reductive to limit oneself to producing knowledge without trying to translate it into useful applications for the community.

Therefore, I set myself the goal of also investigating translational research, starting with the molecular biology of phages.

With respect to research in biology, phages are the first entities to have been characterised from a molecular point of view. Think, for example, of Hershey & Chase's experiment in which it was discovered that genetic material consists of DNA and not proteins. With phages, a historical chapter began from which the great mechanistic discoveries of biological research, explaining how cells function and the processes underlying life, emerged. And so, in the wake of this legacy, I thought it was possible to exploit these viruses - which are absolutely harmless to humans - to create, using nanobiotechnological and genetic engineering approaches, vectors, or nanobioparticles, that would be useful in the fight against cancer or could be used as biosensors.

Can you briefly introduce us to what the technology consists of. How it works and how it improves on the 'status quo' of currently used technologies.

It is based on a bacteriophage - M13 - which is used for phage display and whose research was also awarded the Nobel Prize in Chemistry to George Smith in 2018. The phage display technique is used to mature new therapeutic antibodies, for example. So for this bacteriophage there are many established molecular laboratory techniques.

We can imagine the M13 phage as a tiny 'noodle' about 1 micron long whose capsid proteins are all known. We modify the proteins at the tip of the phage, to remove the elements that allow the bacteriophage to recognise Escherichia coli - which is its natural target - by genetically fusing peptides, proteins or antibodies to the tip that allow it to recognise specific markers exposed on tumour cells, bacteria or other analytes as desired. Thus we have transformed the phage, changing its tropism, to direct it where we want it: for example to a cancer cell. At the same time, we can exploit the surface of the 'noodle' to arm the phage with chemical molecules which, when irradiated with light or ultrasound, generate cytotoxic reactive oxygen species. The phage is no longer infectious per se. We can imagine it as a weapon-laden vector whose 'dentonator' we possess. This vector will have a pointer that directs it to target cells, but nothing will happen until it is irradiated. Following focused irradiation, the molecules carried by the phage are excited and produce cytotoxic damage only in the immediate vicinity of the phage itself. This constitutes a dual targeting mechanism that we think could be useful for developing new anti-cancer therapies.

In the case of the patent cited above, we have fused a single-chain antibody that recognises the ganglioside GD2, a tumour marker highly expressed in neuroblastoma, to the phage.

Neuroblastoma is a very aggressive paediatric tumour for which there are no satisfactory therapeutic solutions. The available therapies are heavy ec hinder children to live very difficult lives. The prognosis for neuroblastoma is very bad. In the patent we showed that phages targeted to GD2 and 'armed' with the Rose Bengal photosensitiser are able to recognise and, following irradiation, specifically eliminate GD2-positive tumour cells. As with other phages, we have shown that our vectors are able to penetrate tumour spheroids (hence three-dimensional) in depth. This is a very interesting property that many anti-cancer therapeutics lack, probably due to the biological characteristics of phages. Indeed, recent studies indicate that by means of transcytosis mechanisms, some phages remain intact as they transit from one cell to another.

Finally, since there are cell lines that do not express GD2, these are immune to the action of our phage. So the last part of the patent describes a CRISPR/Cas technology that allows us to reactivate GD2 expression in GD2-negative cell lines and make them a possible target for our therapeutic vector again.

We are now gearing up to conduct the first in vivo experiments in mouse models of neuroblastoma.

Using bacteriophages to target cancer cells: from idea to market potential

The therapeutic market has not yet fully opened the door to this research as it is extremely recent. We are probably among the few groups in the world studying these solutions, although phages as therapeutic vectors have been used by several groups before to treat different diseases. What we are pioneering in is combining photosonodynamic therapy with redirected phages. which are thus transformed into 'super-antibodies' with thousands of functional molecules loaded. These features do not only have cancer therapy as an application, but also wider applications, such as diagnostics. We participate - for example - in ECLipse, a European project, which is progressing very well, in which the phage is not functionalised with molecules that produce cytotoxic damage but with molecules that produce a light signal following electrochemical stimulation. This makes it possible to increase the signal when the phage is used as a biosensor, again by virtue of the fact that we can attach hundreds if not thousands of molecules to the phage and thus have an amplification of the signal by one or two orders of magnitude compared to standard techniques, which use antibodies functionalised with only a few (2-5) molecules to generate the light signal.

Of the various lines of research, the specific patent for targeting GD2-expressing tumour cells has recently been submitted, while another platform patent demonstrating that engineered phages can mediate the specific sonodynamic elimination of tumour cells and antibiotic-resistant bacteria is 'granted'. We are therefore now taking the first steps towards valorising our research results.

Project progress: current state of the art and plans for the future. What are you looking for?

We are in the process of creating a Spin-off that will license - in agreement with Alma Mater Studiorum University of Bologna - the platform patent on the use of phages for sonodynamic therapy (not the specific patent on the targeting of GD2-positive tumours). The spin-off will mainly focus on the development of bio-sensor solutions, and the patent on sonodynamics, which is more therapeutic, will also be taken on board. When the Spin-off is established, we will have to figure out how to best organise, define and exploit the different potentials. In the meantime, however, the spin-off will represent a corporate container to start talking to investors, since interest has already been shown in our patents.

With respect to the patent for the targeting of neuroblastoma tumour cells, I can say that it was recently presented at a conference organised by the Italian Association for the Fight against Neuroblastoma, arousing great interest. They recognised the 'super-antibody' aspect of the anti-GD2 phage, so much so that it could also be interesting for surgeons if it were decorated with fluorophores, so that small tumour masses could be precisely identified for resection.

Would you be willing to become CEO of a possible spinoff if you found industrial partners or investors to take the project forward?

Personally, no. There are people much more capable than me in this field. As I know the spinoff ecosystem, I have found that successful realities have inventors on the scientific board but not on the executive board. Academics should support scientific and technical development but not administrative and management development.

However, I am very much in favour of the idea that PhD students and young researchers, who are not interested in pursuing an academic career, invest in themselves and their research experiences in spin-offs and entrepreneurship. I find this an interesting idea. The whole galaxy of initiatives and opportunities revolving around the enhancement of academic entrepreneurship is well developed and it could be a win-win for those doing translational research to steer capable young people towards this path.

In general, despite my limited experience in the field of spin-offs, I find that abroad it is easier for a research team to intercept the interest of investors. However, I was very impressed during the last edition of StartUp Day in Bologna (2023) by the speech of Ciro Spedaliere, Co-founder of Claris Venture, who noted how it was difficult to find high-potential biopharmaceutical and biotech projects to invest in. He pointed out that inventors and researchers needed to come forward and not wait to be co-opted from above. This goes precisely in the direction of the intellectual property valorisation realities that I got to know abroad.

PNRR and 'Health and Biomedical' with specifics in therapeutics and diagnostics in Italy: your thoughts.

I think that investments, projects, timeframes should have been better defined. Suddenly a lot of resources fall from the sky, but the impression is that there is little structural and that a long-term vision is lacking. Moreover, the reporting required for these projects is extremely complex and this drains a lot of time from the whole system, researchers and administrators alike.