Development of intracellular VNARs as novel tools to dissect intracellular biological processes
Lead Research Organisation:
Queen's University Belfast
Department Name: Sch of Medicine, Dentistry & Biomed Sci
Abstract
The human body has over 20,000 genes, from which the proteins that make up each human cell are generated. The study of the roles of these proteins is therefore central to our understanding of life. The best way to determine the role of individual proteins is to block their function. As Scientists, we have a number of approaches for inhibiting protein functions, including synthetic compounds prepared by Chemists that bind to the proteins as well as a number of genetic approaches that delete or deplete the gene that generates a particular protein. However, useful as these approaches are, they are not without significant drawbacks. For example, synthetic compounds normally only work on proteins that have particular types of docking sites (e.g. deep pockets or grooves in their surfaces), and it is very difficult (and expensive) to develop compounds that selectively target specific proteins using this approach. Approaches that target a particular protein's gene can be more selective, but often the cell adapts to the loss of a protein in the time it takes to generate experimental models in which the gene is deleted or depleted; this can limit the usefulness of the information that can be gleaned from such studies.
In this LINK proposal, we aim to bring together academic biological expertise at Queen's University Belfast (QUB) with the industrial expertise of Elasmogen Ltd to develop a completely new approach that will generate a novel type of molecule that can rapidly and highly specifically bind to and inhibit target proteins and thereby enable the function of those proteins to be accurately determined without the potentially confounding effects of the approaches described above.
VNARs are antibodies produced by the immune system of sharks, and having evolved 420 million years ago, they are the oldest antibodies so far identified in vertebrates, and they are also the smallest. Despite their small size, VNARs have a unique structure that gives them a potentially greater number of ways of binding to target proteins than the antibodies produced by our own immune systems; this means that VNARs exhibit an extremely high degree of specificity and selectivity for their target proteins.
Elasmogen have established a unique set of over 100 billion individual synthetic VNARs, each of which has a different structure and can therefore bind to a different protein. Given the sheer number of VNARs in the Elasmogen collection, it will be possible to identify individual VNARs that can bind to each protein in the human body. Elasmogen and others have already demonstrated the ability of VNARs to bind to proteins expressed on the surface of human cells and proteins secreted from cells. In this project QUB and Elasmogen will collaborate together to examine the possibility of adapting VNARs so that they can get inside cells and target intracellular proteins. If successful, would open up a number of hugely exciting possibilities, including:
1. Establishment of a novel class of highly selective, potent intracellular inhibitors that could become powerful research tools for dissecting important biological processes.
2. Evidence that intracellular VNARs could potentially be used as novel therapeutics in a range of diseases.
In this LINK proposal, we aim to bring together academic biological expertise at Queen's University Belfast (QUB) with the industrial expertise of Elasmogen Ltd to develop a completely new approach that will generate a novel type of molecule that can rapidly and highly specifically bind to and inhibit target proteins and thereby enable the function of those proteins to be accurately determined without the potentially confounding effects of the approaches described above.
VNARs are antibodies produced by the immune system of sharks, and having evolved 420 million years ago, they are the oldest antibodies so far identified in vertebrates, and they are also the smallest. Despite their small size, VNARs have a unique structure that gives them a potentially greater number of ways of binding to target proteins than the antibodies produced by our own immune systems; this means that VNARs exhibit an extremely high degree of specificity and selectivity for their target proteins.
Elasmogen have established a unique set of over 100 billion individual synthetic VNARs, each of which has a different structure and can therefore bind to a different protein. Given the sheer number of VNARs in the Elasmogen collection, it will be possible to identify individual VNARs that can bind to each protein in the human body. Elasmogen and others have already demonstrated the ability of VNARs to bind to proteins expressed on the surface of human cells and proteins secreted from cells. In this project QUB and Elasmogen will collaborate together to examine the possibility of adapting VNARs so that they can get inside cells and target intracellular proteins. If successful, would open up a number of hugely exciting possibilities, including:
1. Establishment of a novel class of highly selective, potent intracellular inhibitors that could become powerful research tools for dissecting important biological processes.
2. Evidence that intracellular VNARs could potentially be used as novel therapeutics in a range of diseases.
Technical Summary
This stand alone LINK proposal aims to develop and exemplify the potential of variable new antigen receptors (VNARs) as novel, highly selective molecules for interrogating intracellular signaling. This highly innovative project brings together molecular biology expertise at QUB with the UK Biotech Elasmogen to develop specific VNARs to selectively target key intracellular biological processes. VNARs represent attractive and superior alternatives to antibodies as they show a similar ability to bind selectively and with high affinity to target antigens, but their exceptionally high stability and small size make them excellent tools to selectively inhibit biological processes with unparalleled precision and affinity.
In this application, we will examine the effectiveness of VNARs in intracellular compartments, namely the cytosol and the endo/lysosomal compartments. We will develop iVNARs (intracellular binding VNARs) that selectively target two model proteins: cathepsin S (a lysosomal/endosomal protease); and FLIP (a cytoplasmic regulator of cell death), and exemplify the ability of these iVNARs to inhibit the intracellular functions of these proteins. Elasmogen will use phage display and bio-panning to screen their library of 100 billion+ non-natural, chimeric binding domains to identify VNAR binders of recombinant cathepsin S and FLIP proteins. Subsequently, the binding and inhibitory activity of candidate VNARs will be assessed using surface plasmon resonance (SPR) and bespoke cell-free and cell-based assays developed at QUB for each target protein. Finally, we will explore 2 novel approaches for intracellular delivery of recombinantly expressed versions of the most promising iVNARs.
Overall, we will provide proof that iVNARs can be used as highly selective inhibitors of biological pathways, paving the way for their development as biological tools with potential for development as novel diagnostics and therapeutics.
In this application, we will examine the effectiveness of VNARs in intracellular compartments, namely the cytosol and the endo/lysosomal compartments. We will develop iVNARs (intracellular binding VNARs) that selectively target two model proteins: cathepsin S (a lysosomal/endosomal protease); and FLIP (a cytoplasmic regulator of cell death), and exemplify the ability of these iVNARs to inhibit the intracellular functions of these proteins. Elasmogen will use phage display and bio-panning to screen their library of 100 billion+ non-natural, chimeric binding domains to identify VNAR binders of recombinant cathepsin S and FLIP proteins. Subsequently, the binding and inhibitory activity of candidate VNARs will be assessed using surface plasmon resonance (SPR) and bespoke cell-free and cell-based assays developed at QUB for each target protein. Finally, we will explore 2 novel approaches for intracellular delivery of recombinantly expressed versions of the most promising iVNARs.
Overall, we will provide proof that iVNARs can be used as highly selective inhibitors of biological pathways, paving the way for their development as biological tools with potential for development as novel diagnostics and therapeutics.
Planned Impact
As well as the academic beneficiaries identified elsewhere, there are a range of non-academic groups and sectors that could benefit from this research. As the ultimate goal of the research is to show that VNARs can be used to create a new class of potent, selective inhibitors of intracellular proteins, there are numerous avenues for commercial development. This technology has potential markets in development of specific research tools for the Life Sciences, novel diagnostics, and, most lucratively, therapeutics. This enormous potential underpins Elasmogen's interest and central role in this stand-alone LINK project. Moreover, the UK-based biotechnology industry could derive significant wealth from this technology, creating high-paid scientific jobs in an industry sector of strategic importance for the UK.
As this technology has the potential to overcome current limitations of many small molecule drugs (off-target effects due to lack of specificity) and antibody-based therapeutics (can only target extracellular and cell surface protein epitopes), it may become a new platform technology for the development of a new class of therapeutics. This is particularly relevant to diseases that to date have proved refractory to existing therapeutic approaches. If our ultimate impact aspiration of therapeutic development is realized, this would also have a societal impact on improving the health of citizens in the UK and beyond.
A further societal impact from this work will be the involvement of the collaborating teams in promoting their joint research at University, School and public events in Northern Ireland and Scotland, inspiring the next generation of Scientists and developing interest and understanding of the importance of scientific research in UK society.
As this technology has the potential to overcome current limitations of many small molecule drugs (off-target effects due to lack of specificity) and antibody-based therapeutics (can only target extracellular and cell surface protein epitopes), it may become a new platform technology for the development of a new class of therapeutics. This is particularly relevant to diseases that to date have proved refractory to existing therapeutic approaches. If our ultimate impact aspiration of therapeutic development is realized, this would also have a societal impact on improving the health of citizens in the UK and beyond.
A further societal impact from this work will be the involvement of the collaborating teams in promoting their joint research at University, School and public events in Northern Ireland and Scotland, inspiring the next generation of Scientists and developing interest and understanding of the importance of scientific research in UK society.
Publications
Cogo F
(2019)
Application of nanotechnology to target and exploit tumour associated proteases.
in Biochimie
Leach A
(2020)
Anti-DLL4 VNAR targeted nanoparticles for targeting of both tumour and tumour associated vasculature.
in Nanoscale
Smyth P
(2020)
FLIP(L): the pseudo-caspase.
in The FEBS journal
Smyth P
(2022)
Cathepsin S (CTSS) activity in health and disease - A treasure trove of untapped clinical potential.
in Molecular aspects of medicine
Tracey S
(2021)
Development of next generation nanomedicine-based approaches for the treatment of cancer: we've barely scratched the surface
in Biochemical Society Transactions
Description | Development of a novel blocking agent with a new mechanism of action |
Exploitation Route | We have identified a novel mechanism to block proteolysis through inhibition of protease activation - this will be of much use to others in the field trying to find new ways of inhibiting proteases through protein protein interactions |
Sectors | Pharmaceuticals and Medical Biotechnology |
Description | (TACT) - Targeted Anti-Cancer Therapies |
Amount | € 3,089,570 (EUR) |
Funding ID | 859458 |
Organisation | European Commission |
Sector | Public |
Country | European Union (EU) |
Start | 04/2020 |
End | 03/2024 |
Description | 8 March 2021 - invited speaker at the 11th Annual World ADC virtual conference. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | 8 March 2021 - invited speaker at the 11th Annual World ADC virtual conference. |
Year(s) Of Engagement Activity | 2021 |
Description | Attendee British Society of Nanomedicine Early Careers Researchers Meeting |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | Shannon Tracey attended the British Society of Nanomedicine Early Careers Research Meeting (virtual) |
Year(s) Of Engagement Activity | 2021 |
Description | First Virtual British Society of Nanomedicine Early Career Researchers Meeting; November 2020 - Oral Presentation - Invited Speaker - Peter Smyth |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Third sector organisations |
Results and Impact | First Virtual British Society of Nanomedicine Early Career Researchers Meeting; November 2020 - Oral Presentation - Invited Speaker |
Year(s) Of Engagement Activity | 2020 |
Description | Lab Tour |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Policymakers/politicians |
Results and Impact | Lab Tour - Taoiseach Micheál Martin |
Year(s) Of Engagement Activity | 2021 |
Description | Lab Tour - Business Beats Cancer Cancer Research UK Tour |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Industry/Business |
Results and Impact | Volunteered at the Business beats Cancer CR-UK Tour on 24 October 2019 |
Year(s) Of Engagement Activity | 2019 |
Description | Laboratory Tour - Top 100 University by Shannon Tracey |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | Volunteered at the Top 100 University Lab Tour on 12 September 2019. |
Year(s) Of Engagement Activity | 2019 |
Description | London Innovation Society "Biology in Transition Symposium" 29th Dec 2020 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | London Innovation Society "Biology in Transition Symposium" 29th Dec 2020 - Public engagement talk aimed at A-level students and early-stage undergraduates thinking of a career in STEM. |
Year(s) Of Engagement Activity | 2020 |
Description | NI Science Festival Volunteer 13th - 23rd Feb 2020 - Sara McDowell |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Patients, carers and/or patient groups |
Results and Impact | NI Science Festival Volunteer 13th - 23rd Feb 2020 - Volunteered at a variety of science public engagement events. |
Year(s) Of Engagement Activity | 2020 |
Description | PGJCCR Open Day 15th Feb 2020 - Sara McDowell |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | PGJCCR Open Day 15th Feb 2020 - Demonstrated tissue culture and spoke to the public about how these techniques are used in cancer research. |
Year(s) Of Engagement Activity | 2020 |
Description | Poster Presentation at Proteinases and their Inhibitors International Conference 24th-26th Feb. - Sara McDowell |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Third sector organisations |
Results and Impact | Poster Presentation at Proteinases and their Inhibitors International Conference 24th-26th Feb. |
Year(s) Of Engagement Activity | 2020 |
Description | Poster at the Winter School conference March 2021 entitled "Apoptotic Mechanisms of Drug Resistance in Pancreatic Cancer" - Julie Nicoll |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Third sector organisations |
Results and Impact | Poster at the Winter School conference later this month called "Apoptotic Mechanisms of Drug Resistance in Pancreatic Cancer". |
Year(s) Of Engagement Activity | 2021 |
Description | Presentation at the Postgraduate Research Forum - Shannon Tracey |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Postgraduate students |
Results and Impact | Poster Presentation at the Postgraduate Research Forum on 9 November 2019 |
Year(s) Of Engagement Activity | 2019 |
Description | STEM Ambassador Training 22nd Jan 2020 - Sara McDowell |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Other audiences |
Results and Impact | STEM Ambassador Training 22nd Jan 2020 - Sara McDowell |
Year(s) Of Engagement Activity | 2020 |
Description | Science Entrepreneur Experience with Spin Up Science |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Other audiences |
Results and Impact | Science Entrepreneur Experience with Spin Up Science - Took part in this course in May 2020 and then came back in Dec 2020 to help facilitate another course for academics at QUB or UU looking to spin out/interested in the process. |
Year(s) Of Engagement Activity | 2020 |
Description | Second Year Symposium at QUB Sept 2020 - Sara McDowell |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Postgraduate students |
Results and Impact | Second Year Symposium at QUB Sept 2020 - Online talk of research progress to date and future plans. |
Year(s) Of Engagement Activity | 2020 |
Description | Speaker at Online event to mark World Cancer Day 2021 |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Public/other audiences |
Results and Impact | 4 February 2021 'World Cancer Day 2021 - Time for an All-Island Cancer Moonshot?' |
Year(s) Of Engagement Activity | 2020 |
Description | Take on the Tour Abseil Fundraiser by Shannon Tracey |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Supporters |
Results and Impact | Fundraiser on 12 May 2019 |
Year(s) Of Engagement Activity | 2019 |
Description | Talk in the British Society for Nanomedicine ECR meeting on 4th March - Anna Boland |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Third sector organisations |
Results and Impact | Talk in the BSNM ECR meeting on 4th March. |
Year(s) Of Engagement Activity | 2020 |
Description | World Cancer Day 4th Feb 2020 - Sara McDowell |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Patients, carers and/or patient groups |
Results and Impact | Volunteered at an event with secondary school children where educational games provided by CRUK were used to illustrate concepts of cancer research. |
Year(s) Of Engagement Activity | 2020 |