MICA: Moving From Autologous to Allogeneic Cell Therapy: Developing Up-Scale GMP Protocols for Orthopaedics.

Damaged cartilage can be treated at the Robert Jones and Agnes Hunt (RJAH) Orthopaedic hospital using an approved cartilage cell (chondrocyte) therapy on the NHS called autologous chondrocyte implantation (ACI) or with 'stem cells' as part of a clinical trial. In both techniques a patient's own cells are taken from healthy cartilage or bone marrow at first surgery, grown up in the lab over 2-3 weeks and implanted back into the joint to repair the cartilage at the second surgery. This procedure is time consuming and costly; what is more, some patients' cells do not grow well or have properties which mean they can no longer heal the patient's own cartilage when implanted back. To reduce cost and improve patient outcome it would be ideal to use an 'off the shelf' treatment, whereby cells from different donors have been tested and chosen for their therapeutic potential (potency) and stored in a biobank for use in a one stage procedure.
The current method of growing cells in our Good Manufacturing Practice (GMP) laboratory involves the use of several procedures which are 'open' to the air and could increase the risk of infection. In addition, traditional culture techniques utilise multiple plastic flasks for growth phases, which are labour intensive and time consuming to monitor and maintain. This project proposes the use of a state of the art semi-automated machine called a bioreactor (Quantum) in which to grow the cells. This machine has a much larger internal surface area compared to what we currently use (50-100 times larger), so allowing more cells to be grown quicker. The bioreactor units are 'closed' which means that the risk of contamination is significantly reduced. Labour and manufacturing costs per dose are also reduced considerably with the Quantum. We have carried out assessments of the Quantum bioreactor system in our laboratory for the expansion of human stem cells isolated from healthy bone marrow or umbilical cords and human chondrocytes isolated from total knee replacements. Extended characterisation of cells using specialist techniques to assess proteins and genes showed that cells grown in the Quantum and using traditional techniques displayed comparable therapeutic properties. Moreover, the Quantum generated very large stem cell numbers resulting, on average, in 132 million bone marrow stem cells being produced in 13 days and 169 million umbilical cord stem cells in 8 days, cell numbers for chondrocytes were 75 million also in 8 days (i.e. using the bioreactor produced over forty times as many stem cells and nearly twenty times as many chondrocytes as we would get with current methods).
The Quantum has been used to produce stem cells in large international clinical trials treating conditions such as diabetes and kidney disease, both in the UK and elsewhere. In the current proposal we will establish GMP manufacturing protocols for cartilage cells grown in the Quantum system. The aim of the study is to grow large numbers of cells from healthy juvenile and adult tissues donated to research in order to establish the best source for treating cartilage defects and osteoarthritis. In addition, we will ensure they are at least as good as the cells we currently use by comprehensively assessing the characteristics of the cartilage and stem cells grown in the Quantum in terms of their ability to grow and produce cartilage in the laboratory and by comparing their properties to cells currently used in the clinic.

The aim of the current proposal is to identify the optimal source of healthy, potent allogeneic chondrocytes by assessing adult and juvenile knee cartilage donors and juvenile polydactyly and iliac apophysis cartilaginous tissues. GMP chondrocyte expansion protocols for each will be optimised for the Quantum system, supplementing cultures with pooled human platelet lysate (Stemulate). A 'hybrid' process will be used for expansion, whereby chondrocytes are grown first on tissue culture plastic in order to generate the required cell number for efficient Quantum seeding (5-10M). Depending on growth kinetics data, single donor or pooled donor approaches will be considered.
Extensive characterisation of each Quantum product will be carried out using flow cytometry to assess immunoprofiles according to the International Society for Cell and Gene Therapy definition of MSCs, as well as markers indicative of chondrogenic potency (CD44, CD166, CD49c, CD39, CD151, CD271, FGFR3, ROR2 and SOX-9). DNA will be extracted and telomere length will be determined using the TeloTAGGG kit (Roche, UK). The primary outcome for in vitro potency will be cartilage forming capacity using standard pellet culture techniques and assessment of glycosaminoglycan production (modified Bern's histology score). RT-qPCR will be used to quantitate relative expression of chondrogenic genes (Sox-9, collagen type II, aggrecan, frizzled-related protein) and genes indicative of hypertrophy (activin receptor-like kinase 1 and collagen type X). In addition, RNA sequencing of chondrocytes and bone marrow derived mesenchymal stromal cells (BM-MSCs) is planned for in parallel clinical studies, in which global gene expression profiles will be compared to clinical outcome measures (primarily Lysholm knee function score at 15 months). Genes shown to predict clinical efficacy in chondrocyte and MSC therapies will be examined in Quantum products using RT-qPCR.

Recently ACI has been recommended by NICE as a first-line treatment for knee cartilage injury on the NHS. A specific sub-set of patients have been described that will benefit from this intervention, based on long-term trial data, provided to NICE by our centre and others. We have experienced a surge in national and international clinical and commercial interest in ACI since its NICE recommendation, our centre being the only provider of ACI in the UK. The current recommended procedure requires two surgical procedures in which donor site morbidity is a risk from the cartilage harvest procedure. Added to this is the expense of culturing each individual's cells in the laboratory using labour intensive techniques for up to 3 weeks (currently the RJAH requires £9,250 for these manufacturing costs alone). An allogeneic cell therapy using chondrocytes or MSCs extracted from healthy tissues, expanded in the Quantum bioreactor and assessed for potency could provide an 'off the shelf' alternative, reducing the number of procedures for the patient and providing a bank of reproducibly potent cells. Data derived from this study and future work could be used to inform future NICE guidance meaning that cell therapy is recommended for a wider NHS patient population.
Switching to an allogeneic approach will not only significantly reduce costs for healthcare providers, but also improve efficacy by ensuring that patients receive cells tested for potency prior to banking, giving patients the optimum chance of successful cartilage repair in a single procedure. Patients will benefit by regaining life-fulfilling knee function and a reduction of pain symptoms, improving their overall health and wellbeing. Many of the patients who present with cartilage injuries and who are treated with cell therapies are young individuals for whom optimal joint function is necessary for them to remain fit, maintain a healthy BMI and to allow for a fiscal contribution to society. Ultimately our research will lead to improved techniques that mean more patients can access cell therapies and more orthopaedic surgeons can apply them without the need for a cell manufacturing facility on-site. The increased number of cells produced using Quantum also opens up the possibility of repeat and injectable applications, which have the potential to increase further their efficacy and cost-effectiveness. To our knowledge there are no competitors in allogeneic chondrocyte production in the UK, and uniquely, the Quantum system has never been used to expand chondrocytes previously, other than in our own MRC funded Confidence in Concept studies. Our research will have a positive impact on our industrial partner (Terumo-BCT), increasing exposure of their products to the orthopaedic community in particular scientists and clinicians at the RJAH/Keele University and our Arthritis Research UK TEC partners. Ultimately, we anticipate that the results derived from our study will drive-up international Quantum product sales and Terumo BCT business growth, particularly in the orthopaedic sector.
The broader impact of this project will continue to be assessed for at least ten years following completion, through identification of academic journal citations, generation of intellectual property, commercial outputs, clinical implementation and most importantly patient benefit i.e. monitoring data regarding orthopaedic cell therapy efficacy and health economics assessments. The project is fully aligned with the current Medical Research Council (MRC) strategy to support "excellent discovery science and effective partnerships to promote translation which will accelerate the pace of improvements in health and stimulate economic growth".