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

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.

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.

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.