Recombinant proteins for GMP-compatible niche creation to optimize in vitro platelet production for human transfusion

Lead Research Organisation: University of Cambridge
Department Name: Haematology

Abstract

Each year 250,000 platelet units are transfused to patients in the UK. Platelets are small blood corpuscles that promote blood clotting. In patients with a low platelet count (following cancer therapy, bone marrow transplantation or cardiovascular surgery), there is an increased risk of bleeding (including in the bowels and brain). To prevent this, we currently use transfusion of platelets derived from blood donations. Platelets have a short shelf-life (7 days) because they have are kept at room temperature, which makes stock management a challenge. In addition some patients (in particular women who have had children) can developed an immune reaction that makes "normal" platelets inefficient. For these patients, we use in the UK 15,000 platelet units per year of specially matched platelets (at twice the cost of the usual platelet unit).
The option of producing platelets from banks of stem cells in the laboratory is attractive. It would allow for guaranteed supply, remove the risk of infection transmitted from donor-derived platelets (such as hepatitis viruses, HIV or variant CJD) and allow for better matching of platelets to recipients.
Platelets are produced from megakaryocytes (MKs) in the bone marrow. We have in the last year had a breakthrough in developing an efficient method to produce MKs from stem cells in the laboratory under conditions that are compatible with the production of a product fit for humans. We are now planning to build upon this success in order to produce platelets from these laboratory-grown MKs.
Each MK in the bone marrow produces about 1000 platelets but in the laboratory we only achieve 4 to 7 platelets per MK. This is because MKs only produce platelets efficiently if they are surrounded by the right environment. It is well known that direct contact between MKs and "supportive" cells can enhance platelet production by at least a factor of 10. However these "supportive" cells cannot be used in a system to produce platelets for human use as they are often animal-derived.
We have generated a list of all the proteins that are on the surface of these supportive cells and ranked them according to the likelihood of their effect on platelet production. Four hundred of the top-ranking candidate proteins will be produced in the laboratory. They will be screened in a high-throughput assay (similar to screening for anti-cancer agents) to identify the best combinations of proteins to promote platelet production from MKs. In essence this approach would allow us to replace animal-derived supportive cells with a combination of proteins that have the same positive effect on platelet production but are compatible with a production system for human use.
The best combinations of proteins will be used in a 3-dimensional system that will allow future scaling up of production. We have already generated a collagen-based 3D matrix (in essence an inert porous material very much akin to a sponge) and will be fixing the best candidate proteins onto this "bare" matrix. Cultured MKs will then be seeded upon these "enhanced" scaffolds to assess platelet production. We will use the knowledge generated by this project to seek regulatory approval and further funding for follow-on projects to incorporate these scaffolds in larger scale bioreactors and assess these in vitro produced platelets in human volunteers and patients.
All discoveries will be patented with the view to generate a commercially viable, licenced manufacturing process that will not only benefit health care within the NHS but also contribute to the growth of the UK biomedical technology. This translation will be facilitated by a collaboration with Cell Therapy Catapult, an centrally-funded organization that promotes translation of basic research into cell therapy for patients and by the PI's role in the NHS Blood and Transplant, the organization responsible for the production and distribution of blood products in England.

Technical Summary

WP1: Identification of candidate membrane-expressed proteins (MExPs)
The secreted and membrane-tethered proteins for cell lines that support proplatelet (ProPt) formation by MKs in cocultures that promote have been identified from expression arrays and proteomics analysis. The ectodomain of 400 candidate proteins will be cloned into expression vectors by a gene synthesis company.
WP2 Synthesis of recombinant (rec)MExPs.
The recMExPs will be expressed in a mammalian system using the existing infrastructure of Dr Wright's laboratory (Sanger Institute). The recMExPs will be produced in two forms: a soluble pentamer for the initial screening (WP3) and a tagged monomer for immobilization onto a 2D/3D substrates (WP4).
WP3: High throughput screening of candidates recMExPs.
Phase I: ProPlt assay with single recMEXPs: Cultured MKs will be harvested and plated into fibrinogen-coated 96-well plates with pentameric recMExPs. ProPlt formation will be analyzed 48 hours later by means of immunohistochemistry and automated image analysis.
Phase II: ProPlt assay using a combinatorial algorithm: Preliminary data have shown that concurrent recMExPs have a synergistic effect on ProPlt formation. A combinatorial screen of all positive recMExPs identified in Phase I will be carried out using an "overlapping blocks" mathematical model to identify the best combinations of 3 recMExPs.
WP4 Immobilisation of candidate recMExPs onto substrates for niche creation.
WP4 will validate the results obtained in WP3 with non-soluble immobilized forms of the same recMExPs. Monomeric recMExPs with a biotin tag will be immobilized on a plastic surface coated with fibrinogen and streptavidin in equimolar ratios. The ProPlt assay will be carried out on these functionalized plates as described above. We will also determine the range of optimum surface density for each recMExP by ELISA. Finally we will generate appropriately-tagged recMExPs for functionalisation of collagen-based 3D scaffolds.

Planned Impact

Each year, 250,000 donor-derived platelet units are produced by the NHS Blood and Transplant at a cost of £58m. Platelets have a short shelf-life of 7 days due to the need to store platelets at room temperature and therefore stock management to guarantee continuous supply is a logistical challenge. Some patients (in particular women who have had multiple pregnancies) develop antibodies against HLA class I epitopes expressed on the platelets which decreases the circulatory half-life (and clinical efficacy) of standard unmatched platelets. Each year, 15,000 HLA-matched platelet units are generated from a recallable panel of typed donors for these patients. Due to the logistics involved, the cost of each pool of HLA-matched platelets is twice that of unmatched platelets. Finally, any human-derived product carries an infectious risk (hepatitis viruses, HIV and prion diseases in particular).
The concept of generating platelets in vitro from a renewable source of stem cells (in this case human pluripotent stem cells, iPSCs) is therefore attractive: 1. guaranteed supply without donor dependence; 2. matching product to patient: 50% of the demand for HLA type I matching amongst the UK population can potentially be covered by 3 cell lines homozygous for one "common" HLA haplotypes and 3. reduction of the risk of transmission of infection. The potential clinical benefit to the NHS and patients of in vitro produced platelets is therefore clear. After the project end, the process will be translated to GMP manufacture (supported by the Cell Therapy Catapult and NHSBT infrastructure) and first-in-man clinical studies on the Cambridge Biomedical Campus where the NHSBT and the lead PI already carry out volunteer studies to assess transfusion products. This offers a clear route for clinical adoption by the NHS.
Regenerative medicine is at the forefront of the strategy for biomedical research in the UK, supported by initiatives such as the Regenerative Medicine Platform (co-funded by the MRC). The University of Cambridge has already formed (together with the University of Sheffield) an RMP hub addressing the issue of derivation and quality control of iPSCs for human use. This would have a direct link to the proposed project. Platelets derived from iPSCs are an ideal exemplar tissue to develop iPSC-based technology in as much as platelets are anucleated and can be irradiated prior to administration to patients, removing potential concerns over tumor growth from iPSCs-derived cellular products. The strategy adopted in this project, aiming at recreating the niche necessary for cell maturation by means of recombinant proteins can be applied to other tissues grown in vitro and therefore discoveries made within this project will benefit the scientific community as a whole, in particular in the field of regenerative medicine. To this end the lead PI has already established a collaboration with the UK RMP Biochemistry of the Niche Hub (Prof S Forbes, Edinburgh) through which distribution to other researchers can be carried out effectively.
Finally one has to consider the potential economic aspect of this work beyond healthcare provision. The business opportunity is the development of a robust manufacturing protocol for the generation of platelets from iPSCs. This will constitute new IP for the project partners, protected through patenting. This will be licenced to blood product manufacturers, such as NHSBT. Beyond the UK, the global market for platelet provision is $300m. Our product is expected to obtain a large share of the market due to advantages in providing consistent cell supply from a validated, pathogen-free source and the generation of cell banks to efficiently supply HLA matched donations. Based on figures from other iPSC regenerative medicine licensing deals, the ROI is expected to be at least 10 fold. The novel platelet production process will have therefore wider benefits, providing job opportunities and revenue to the UK economy.
 
Description Biogenesis and bioengineering of human platelets
Amount £1,200,000 (GBP)
Funding ID 219472/Z/19/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2020 
End 04/2025
 
Description EU Horizon 2020 FET
Amount € 4,800,000 (EUR)
Organisation European Commission 
Department Horizon 2020
Sector Public
Country European Union (EU)
Start 11/2017 
End 10/2021
 
Description MRC Translational award
Amount £200,000 (GBP)
Funding ID MR/P007813/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 10/2016 
End 10/2017
 
Description MRC confidence in concept
Amount £70,000 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 03/2016 
End 09/2016
 
Description Collaboration with Cell Therapy Catapult 
Organisation Cell Therapy Catapult
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Gap analysis for large scale production of platelets in vitro_we are looking to implement them
Collaborator Contribution Gap analysis for large scale production of platelets in vitro_we are looking to implement them
Impact DSTL grant application
Start Year 2020
 
Description Collaboration with Platelet Biogenesis 
Organisation Platelet BioGenesis, Inc
Country United States 
Sector Private 
PI Contribution Platelet Biogenesis is a company based in Boston which has licensed the MK forward programming technology. We have now a sponsored research agreement that allows us to pursue this work further with the company itself
Collaborator Contribution They have contributed to cell line research in our laboratory and are testing our cell lines in the bioreactor in Boston.
Impact Successful further funding for Platelet Biogenesis which has allowed them to underwrite a sponsored research agreement with my research group.
Start Year 2017
 
Description Collaborations with Paris and Pavia for the production of platelets in vitro (part of EU grant) 
Organisation National Institute of Health and Medical Research (INSERM)
Country France 
Sector Academic/University 
PI Contribution The team in Pavia and Paris are using the pluripotent stem cell lines generated in the Ghevaert group to do disease modelling or platelet production in 3D bioreactors. The latter can be functionalised with recombinant proteins that have been identified and generated in my lab, that can promote platelet release.
Collaborator Contribution The team in Pavia is providing the 3D silk-based bioreactors. The team in Paris gives us access to patients cell lines for disease modelling.
Impact Successful EU grant application
Start Year 2015
 
Description Collaborations with Paris and Pavia for the production of platelets in vitro (part of EU grant) 
Organisation University of Pavia
Country Italy 
Sector Academic/University 
PI Contribution The team in Pavia and Paris are using the pluripotent stem cell lines generated in the Ghevaert group to do disease modelling or platelet production in 3D bioreactors. The latter can be functionalised with recombinant proteins that have been identified and generated in my lab, that can promote platelet release.
Collaborator Contribution The team in Pavia is providing the 3D silk-based bioreactors. The team in Paris gives us access to patients cell lines for disease modelling.
Impact Successful EU grant application
Start Year 2015
 
Description Ectodomain proteins for MK niche generation 
Organisation The Wellcome Trust Sanger Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Collaboration with Dr Gavin Wright to apply for MRC funding to generate a library of 400 ectodomain proteins to be screened for candidates that promote platelet formation in vitro. We have developed the cellular screening assay.
Collaborator Contribution Dr Wright's group will generate the ectodomain proteins.
Impact Preliminary application to MRC well-received. Invited to submit full application November 2013
Start Year 2012
 
Description Production of universal platelets from iPSC at GMP 
Organisation NHS National Services Scotland (NSS)
Department Scottish National Blood Transfusion Service
Country United Kingdom 
Sector Public 
PI Contribution Developing an iPSC universal cell line for the production of platelets at GMP
Collaborator Contribution Process development and gene editing at GMP
Impact Process development and gene editing at GMP
Start Year 2020
 
Description Recombinant proteins to promote red cell enucleation 
Organisation University of Bristol
Country United Kingdom 
Sector Academic/University 
PI Contribution We have made a library of 400 ectodomain recombinant proteins.
Collaborator Contribution Our partners have identified potential proteins that may drive red cell enucleation. They are going to test our proteins to assess whether they promote red cell enucleation in vitro
Impact No output yet
Start Year 2016
 
Description Recombinant proteins to promote red cell enucleation 
Organisation University of Oxford
Country United Kingdom 
Sector Academic/University 
PI Contribution We have made a library of 400 ectodomain recombinant proteins.
Collaborator Contribution Our partners have identified potential proteins that may drive red cell enucleation. They are going to test our proteins to assess whether they promote red cell enucleation in vitro
Impact No output yet
Start Year 2016
 
Title RESTORE 
Description Primary haematopoietic progenitors-derived red cells injected into volunteer to show improvement of recovery and survival compared to donor-derived red cells 
Type Therapeutic Intervention - Cellular and gene therapies
Current Stage Of Development Initial development
Year Development Stage Completed 2020
Development Status Under active development/distribution
Clinical Trial? Yes
Impact Inform future large scale production of in vitro derived blood cells 
URL https://www.clinicaltrialsregister.eu/ctr-search/search?query=eudract_number:2017-002178-38
 
Company Name CELLADVICE LTD 
Description Consultancy company specialising in blood transfusion and cell therapies 
Year Established 2019 
Impact Currently providing expert advice to 4 different companies
 
Description Charity Concert All Saints 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Charity concert at which I and professional musicians performed as well as children of the local drama school. The concept was supported by the Cambridge Stem Cell Institute and the benefits went to Naitbabies.org, a patient organisation supporting patients affected by neonatal alloimmune thrombocytopenia
Year(s) Of Engagement Activity 2018
 
Description Charity concert 2019 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Charity concert in Newmarket for the benefit of the East Anglia Air Ambulance_PhD students and post-doc presented some of their stem cell work in the context of regenerative medicine to the general public who attended the concert
Year(s) Of Engagement Activity 2019
 
Description Public Engagement Champion Cambridge Stem Cell Institute 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I have been nominated as the Public Engagement Champion of the CSCI_in short I am the academic PI supporting the Public Engagement team including chairing regular meetings and shaping strategy and implementation thereof
Year(s) Of Engagement Activity 2019,2020
 
Description Radio and BBC news website interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact After publication of the Nature Communication paper, we were interviewed by the written press (Daily Mail) radio (BBC radio Cambridgeshire, Radio5 live, Radio4 Today programme) and television (ITV and Cambridge TV)
Year(s) Of Engagement Activity 2016
 
Description Youtube distributed movie on production of blood cells in vitros 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact We asked the general public and patients groups to send us questions about the production of blood cells in vitro. Our replies are presented in the form of a short movie that is available on Youtube:
Year(s) Of Engagement Activity 2018
URL https://www.youtube.com/watch?v=TCKP0dn2uHk&t=7s