Public Funding for Plasticell Limited

Modern therapies are often concerned with using human cells as therapeutic modalities. Recent breakthroughs in the treatment of leaukemia and lymphoma are associated with the administration of patient's own isolated T-lymphocyes, modified at the laboratory to recognise and kill tumour cells. However, such therapies have strong side effects, require time for producing the cells for treatment and are not always afforadable due to high costs. A new approach in immuno-oncology is to utilize another type of lymphocytes -- Natural killer (NK) cells. These cells can become active and efficiently eliminate cancer cells, including solid tumours, with minimal side effects. Importantly, they can be obtained from the peripheral or cord blood with no strict requirements for the matching donor. The next generation therapy originates from the production of NK cells from the human induced pluripotant stem cells (iPSC) as a universal off-the-shelf product. Such technology not only allows to create an unlimited source of the therapeutic agents, but to target the produced NK cells to a specific cancer cell type in a patient-centered approach by genetically modifying the starting iPSC material. In the proposed collaboration, Plasticell, ReNeuron, Cell and Gene therapy Catapult and Imperial College London aim to develop a manufacturing procedure for iPSC-derived fully functioning NK cells for immuno-therapies. ReNeuron has developed a unique iPSC line, and Plasticell has deployed its award-winning screening platform to generate protocols for differentiation of NK cells from iPSCs. These technologies will be transferred to the Cell and Gene therapy Catapult to be adapted to a manufacturing scale. Finally, all assays to test the functionality of produced NK cells will be developed under control and guidance of the NK cell specialist Prof Hugh Brady at Imperial Colledge London. We aim at developing a process of a robust and efficient production of human NK cells in conditions that can be easily translated to the clinical practice. Throughout the project, all the materials and methods used will be clinically acceptable with the purpose to generate functionally mature NK cells in high yield and purity. The produced NK cells will be tested for their compatibility with donor-derived NK cells and their ability to kill cancer cells in the laboratory settings. The success of this project will lead to comprehensive pre-clinical studies using advanced research techniques and animal-based cancer models, and developing next generation therapies for solid tumours.

Many patients with blood disorders, such as cancers, are often treated by receiving a bone marrow transplant from another person. Usually, this donor has to be a blood relation or have a compatible tissue type to ensure the transplant is not rejected. However, finding such a match is not always possible. For some years now, parents have been donating the blood found in the umbilical cord of their new-born babies. Just as with bone marrow, this 'cord blood' contains stem cells which are capable of reconstituting the entire blood and immune system of a transplant recipient. Furthermore, complete matching is not required, so cord blood cells can be used to treat patients that can't find exact matches, lowering the risk of tissue rejection. Unfortunately, the number of cells in each cord blood unit is limited and too small to treat most adults, therefore methods to expand the number of these cells in the laboratory are highly desirable. Plasticell has developed and patented formulations which, in preliminary _in vitro_ studies, allow the stem cells in the cord blood units to be multiplied by around 500-fold without affecting their properties. The company has further developed manufacturing methods in which these cells can be expanded ready for clinical use. The resulting cells have been tested _in vitro_ in the laboratory and in preliminary _in vivo_ transplantation using immune-deficient mouse models. Nevertheless, to ensure the cells are useful to patients we must carry out more extensive _in vivo_ analyses. In this project, we are testing the ability of expanded cord blood cells to reconstitute the entire blood system when they are injected into immune-deficient mice. We also aim to develop innovative solutions to ensure that the stem cell product we are developing can be made and delivered to the patients that need it in a format that is safe, effective, user-friendly and affordable.

Cancer is still a leading cause of death worldwide, and is thought to have been responsible for 9.6million deaths in 2018 (about 1 in 6 deaths). Unfortunately, despite improvements in cancer care and treatment, the incidence and the number of deaths from cancer are set to increase along with an aging and growing population. There is therefore a need for more effective treatments. Immunotherapy aims to boost the body's immune system to fight disease, and can be used to fight cancer. It uses substances made by the body or in a laboratory to improve how the body's immune system works to find and destroy cancer cells. Cellular immunotherapy is one type of immunotherapy that uses special blood cells of the immune system (T-cells or natural killer cells), and 'arms' them with the capacity to detect and destroy tumour cells. These so-called 'chimeric antigen receptor (CAR)-T' or 'CAR-NK' cells have revolutionised the treatment of patients with otherwise incurable cancers. However, these therapies have so far mostly relied on harvesting these blood cells from the patient or healthy donors. Whilst this method does work, it makes this therapeutic approach unpredictable, very expensive, and it requires a complex infrastructure for delivery. Another problem has been the development of adverse side effects in some patients with the use of CAR-T cell therapies. Therefore, any therapy that seeks to reduce variability, decrease cost, increase safety and make the manufacturing process faster and/or more robust, is hugely attractive. One such area of research is the use of 'induced pluripotent stem cells' (iPSCs). These cells can be made from adult tissue, and have the capacity to be cultured in the lab indefinitely and, more importantly, to give rise to any type of cell in the body - making them an ideal starting material for the manufacture of off-the-shelf cell therapies. They are also very amenable to genetic modification, which also makes them ideal for the 'arming' process mentioned above. There are two major aims of this project. The first is to deploy LambdaGen's advanced gene editing technology to arm iPSCs with multiple elements for enhanced capabilities for the detection and killing of tumour cells. The second is to deploy Plasticell's next generation screening technology to develop efficient methods to generate iPSC-derived natural killer cells. The combination of these two technologies promises to create an affordable, effective and safe therapy for the treatment of cancer.

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Many patients with blood disorders, such as cancers, can usually be treated by receiving a bone marrow transplant from another person. Usually this donor has to be a blood relation or have a compatible tissue type to ensure the transplant is not rejected. However, finding such a match is not always possible. For some years now, parents have been donating the blood found in the umbilical cord of their new-born babies. Just as with bone marrow, this 'cord blood' contains stem cells which are capable of reconstituting the entire blood and immune system of a transplant recipient. Furthermore, complete matching is not required, so cord blood cells can be used to treat patients that can't find exact matches, lowering the risk of tissue rejection. Unfortunately, the number of cells in each cord blood unit is limited and too small to treat most adults, therefore methods to expand the number of these cells in the laboratory are highly desirable. Plasticell has developed and patented formulations which, in preliminary _in vitro_ studies, allow the stem cells in the cord blood units to be multiplied by around 500-fold without affecting their properties. The company has further developed manufacturing methods in which these cells can be expanded ready for clinical use. The resulting cells have been tested _in vitro_ in the laboratory and in preliminary _in vivo_ transplantation using immune-deficient mouse models. Nevertheless, to ensure the cells are useful to patients we must carry out more extensive _in vivo_ analyses. In this project, we will test the cells ability to reconstitute the entire blood system when injected into immune-deficient mice and also aim to develop innovative solutions to ensure the stem cell product we are developing can be made and delivered to the patients that need it in a format that is safe, effective, user-friendly and affordable.

Primary Immunodeficiency disorders are rare genetic defects of blood cells that affect children and often lead to early death. Up to a few years ago the only proven cure for those type of disorders was haematopoietic stem cell (HSC) transplantation, although complicated by the need to identify a suitable donor and severe side effects. Recently developed technologies allow correction of genetic defects in HSCs isolated from peripheral blood or bone marrow by manipulating them ex-vivo and re-infusing them back into the patient. Once back in the patient, the corrected cells "home" to the bone marrow where they produce healthy immune cells. One way of correcting genetic defects is by adding a healthy copy of the defective gene to HSCs using a virus vector. The infected HSCs go on to reconstitute the hematopoietic system with blood cells carrying the correct gene. However, a major challenge is to correct a sufficient number of HSCs without altering their ability to give rise to all blood cells for during the lifetime of the patient. Plasticell has developed a clinically compliant process to expand cord blood cells ex vivo and now plans to use its HSC expansion protocol to improve the efficiency of gene therapy. The overall aim of this project is to progress with the clinical application of the Plasticell formulation that stimulates expansion of true hematopoietic stem cells in vitro and to provide a high number of corrected stem cells after gene therapy. We will also use Plasticell's combinatorial screening technology, CombiCult, to increase the efficiency of viral gene delivery in HSC. Both technologies will be used to develop a new clinical protocol for viral gene therapy that would increase the efficacy and lower the overall cost of the gene therapy procedure.

Blood transfusion is one of the most common clinical therapies and plays a critical role in prevention and management of many health conditions and diseases. Around 90 million red blood cell transfusions are carried out every year and this rising demand is dependent on a limited supply of healthy donors. The supply problem is compounded by other issues including transfusion transmitted infections, immune compatibility and the quality of the donated product. These problems could be solved by generating red blood cells in the laboratory from a limitless and infection-free source such as human pluripotent stem cells. Although possible, the process takes several weeks, is costly and the cells that are made are not fully mature. This project aims to develop improved culture protocols that reduce the time and cost, optimize the production of fully functional cell types and amenable to clinical standards and scaleup. We will perform the first large scale systematic assessment of culture conditions using Plasticell’s Combinatorial Cell Culture technology and pluripotent stem cell lines carrying fluorescent reporters that track red blood cell production. As well as transforming existing blood transfusion services, this new source of red blood cells would provide a much needed supply in developing countries.

Patients with blood cancers can usually be treated by receiving a bone marrow transplant from another person. Usually this person has to be a blood relation or have a compatible tissue type to ensure the transplant is not rejected. However, finding such a match is not always simple. For some years now, women have been donating the blood found in the umblilical cord after given birth to their babies. This blood is full of stem cells which are capable of reconstituting the entire immune system of the recipient, furthermore complete matching is not required, so cord blood cells can be used to treat many more patients. Unfortunately the number of cells in each cord blood unit is limited and too small to treat most adults. We have already developed media formulations which in preliminary in vitro studies allow the blood stem/progenitor cells in the cord blood units to be expanded by at least 30-100 fold. With this project we are seeking initially to develop a clinically compliant process to expand cord blood cells ex vivo prior to transplantation so that more patients can be treated with this live saving therapy.

Platelets are an essential component of blood, involved in homeostasis, clotting and wound healing. Certain medical treatments (esp. chemotherapy) cause a severe reduction in platelet count leading to uncontrolled bleeding. This can be treated with transfusions of platelets isolated from donor blood, but in approximately 15% of patients, repeated challenge with platelets from other donors causes an immune response, leading to 'alloimmune platelet refractoriness' which is costly to treat and life threatening. We have developed a method of producing platelets from stem cells (which can be made from the patient's own cells) and wish to develop this as therapy for low platelet count due to alloimmune refractoriness. This project is to scope the feasibility of upscaling the manufacturing process and reduce costs by up to 500-fold though process optimisation and use of proprietary small molecules that specifically modulate hematopoiesis and improve efficiency.

Brown adipose tissue (BAT) is a rare type of fat that in contrast to abundant white adipose tissue does not store eneregy but burns it to generate heat. BAT mass and function is inversely correlated with obesity and BAT has also been shown to improve glucose defects observed in diabeties, above what would be expected from weight loss alone. Therefore therapies that activate BAT or increase BAT mass should have positive effects on weight loss and diabetes. However, adult humans have very little BAT and it decreases with age, therefore methods to increase BAT mass could be predicted to have more effect than activation of existing BAT. We have developed the first protocols to generate human BAT in vitro from adult stem cells derived from WAT. We propose a cell therapy whereby stem cells are isolated from WAT of individuals, converted to BAT in vitro and transplanted back to the individual for treatment of obesity or diabetes and their associated morbidities.

Regenerative cell therapies offer enormous promise for the effective treatment of multiple indications such as heart disease, stroke and neurodegenerative disease. It is increasingly clear that many types of stem cell exert their therapeutic effect through secretion of trophic and immunomodulatory factors rather than by direct integration and tissue regeneration. However, stem cell culture conditions have not been optimised to maximise secretion of these key factors, potentially severely limiting clinical efficacy. We propose to test the feasibility of utilising our high throughput cell culture platform, Combicult, to identify optimal media formulations that increase secretion of trophic factors from stem cells and therefore impove therapeutic outcome.

CombiCult is Plasticell’s proprietary combinatorial cell culture technology that industrialises the development of stem cell differentiation protocols and optimised cell culture media. CombiCult effectively substitutes a manual ‘trail-and-improvement’ empirical approach requiring up to two years and a budget of $2m with a streamlined process lasting three to six months at a cost an order of magnitude lower. This study will underpin Plasticell’s development of a robust strategy for developing business in the international academic market with CombiCult.

Project: Quantitative mapping of the proteomes of therapeutic stem cells. Cellzome is using its suite of quantitative proteomics technologies to characterise the proteomes of human stem cells in order to define the expressed protein repertoires and correlate these with other relevant functional characteristics. The goal is to use state of the art, analytical proteomics to ensure that stem cells designed for ultimate use in human clinical trials are safe and effective. Cellzome will be using a range of approaches including whole proteome maps (up to 5000 proteins per cell type), and defined 'sub-proteomes' such as the kinome (expresses kinases using Kinobeads) and the epi-proteome (using Epispheres). The human stem cells are from a range of types and sources provided by our partners Neusentis/Pfizer, Plasticell and the University of Sheffield. The aim of this work is to produce detailed proteome maps which can be related to functional phenoytpes and karyotypes. The business objective is to provide analytical methods that could be used in manufacturing processes for the characterisation of stem cells as part of the 'Standard Operating Protocols' that will be required to enable safe clinical trials.

The manufacture of cells in a controlled and reproducible manner is a critical challenge in regenerative medicine. We will develop an integrated platform technology for the rapid identification of novel, efficient stem cell expansion and differentiation protocols that are optimised for translation to clinical grade manufacturing. Several innovative technologies will be combined to perform high throughput GMP compatible screens to discover novel cell production protocols. These protocols will be transferred to a scaled-up bioprocess where a novel cell imaging technology will be utilised to monitor cell performance. Successful protocols will be translated to a GMP manufacturing process for final validation. In this way, safe, robust, cost-effective GMP validated protocols will be rapidly discovered, greatly reducing the cost and time for development of regenerative cell therapeutics.

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In the final year of this project, data will be generated to support a new IP filing related to an innovative manufacturing process used to produce a porous bioactive glass scaffold for bone regeneration. Bioactive glass has the potential to be osteoinductive but there are currently very few porous bioactive glass products available. Preliminary results have shown that the porous scaffold developed in this project may have the potential to outperform many commercially available bone void fillers. Poly-y-glutamic acid has been successfully modified and electrospun with the resultant fibres showing good support of cell attachment, viability and differentiation. Following completion of this project, it is likely that the data generated will allow a biphasic osteochondral plug to be developed. The project has allowed Plasticell limited to develop novel differentiation protocols for the generation of chondrocytes and osteoblasts from mesenchymal stem cells, and thus enter the stem cell culture reagents market. The protocols differ from currently available products as they are serum-free and animal component free, thus making them more reproducible and suitable for clinical application.

The central issue of regenerative medicine is the capacity to reproducibly control the differentiation of a high proportion of stem cells. Because this is inherently influenced by many biological, chemical and physical cues, a very large number of experimental tests are required in each instance to determine the optimum outcome. By adding the power of advanced imaging and robotic microwell-based engineering to an already powerful combinatorial technique the proposed programme will create an internationally leading general technology for quickly and efficiently examining large numbers of linked variables. In this way it will create foundations for a powerful commercial activity applicable across the whole of the new industry.