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Volume 21, No. 6 November, 2014 Cultivated Autologous Limbal Epithelial Cells (CALEC) for the Treatment of Limbal Stem Cell Deficiency Milena Di Meo and Myriam Armant Center for Human Cell Therapy, Boston Children’s Hospital Boston, MA USA The limbus, which is anatomically located on the outer edge of the cornea, (Figure 1) normally contains 5-15% of progenitor cells, called Limbal Stem Cells (LSC) that, under steady state, contribute to the corneal epithelium homeostasis (Yoon et al., 2014). Figure 1: The limbus as a source of stem cells involved in corneal homeostasis Conjunc va Limbus Cornea peripheral central Conjunc val epithelial cell Conjunc val goblet cell Limbal stem cell (LSC) Early TAC Late TAC Transient amplifying cell (TAC) Post‐mito c cell (PMC) Terminally differen ated cell (TDC) Stra fied epithelial cells Endothelial cell See below for details Cornea Anterior chamber (filled with aqueous humor) Lens Sclera Vitreous gel Op c nerve Epithelial cell Stromal lamellae Endothelial cell Keratocyte Corneal damages caused by a variety of insults (eye injuries, genetic disorder, autoimmunity, and inflammation, ocular surface diseases) are often associated with Limbal Stem Cell Deficiency (LSCD). This condition results in scarring, conjunctivalization and vascularization of the cornea ultimately leading to progressive vision loss. Corneal transplantation is not an option in those cases as grafts fail due to lack of host stem and progenitor cells, which are needed to replenish the surface of the donor graft. In recent years, cellular therapy such as ex vivo limbal epithelial cell transplantation has shown promising results as a treatment of unilateral LSCD (Kolli et al., 2010; Marchini et al., 2012; Nakamura et al., 2004; Rama et al., 2010; Sangwan et al., 2006; Tsai et al., 2000; 1|Page Volume 21, No. 6 November, 2014 Zakaria et al., 2014). However, these clinical trials have not been available in the United States where LSCD remains an unmet clinical need. In collaboration with Dr. Ula Jurkanas from the Massachusetts Eye and Ear Infirmary (MEEI) who will lead the clinical trial, our group at the Center for Human Cell Therapy (CHCT) developed new methods to yield Cultivated Autologous Limbal Epithelial Cells (CALEC) for the treatment of unilateral LSCD. The Production Assistance for Cellular Therapies (PACT) supported this translational project where we developed and validated a 2-stage manufacturing process (Figure 2). First, enzymatically-isolated limbal epithelial cells are expanded (primary culture) and then a fraction of the expanded cells (P0) is seeded onto a transplantable substrate (secondary culture). Among several scaffold options, we opted for AmnioGraft®, which received tissue designation by the Food and Drug Administration (FDA) and has anti-microbial, antiangiogenic and anti-inflammatory properties. Figure 2: CALEC process overview 3. Enzyma c diges on 2. Transport to lab 1. Harvest biopsy 4. Pla ng & expansion End of Primary Culture 5. Harvest & QC • • • • Unaffected eye Counts & viability Phenotype (iden ty=epithelial) Colony Forming Efficiency assay Prolifera ve poten al assay Start Secondary Culture CALEC ready for transplant on the affected eye 6. Seed cells on AmnioGra ® 8. Transport to clinical site 7. Final product QC & formula on 2|Page Volume 21, No. 6 November, 2014 Some of the key changes made to the manufacturing included removing the murine feeder cell line and switching to a serum-free media formulation. In order to mimic as much as possible the actual clinical trial, we developed an in vitro model of limbal biopsy using cadaveric corneas. The established manufacturing process is reproducible with a 97.1% success rate (n=70 products), with an average duration of 18±4 days. Cellular grafts present unique challenges when it comes to final product testing and release criteria. Unlike suspension cell-based therapies, samples of the graft cannot be taken for testing without compromising the integrity of the graft. Our approach was twofold: 1) validate every steps of the process and 2) develop and validate surrogate assays. We satisfied all regulatory requirements including defining cell counts and viability. To determine the cell dose for each graft, we established a method to count cells in situ using a cell-imaging platform. On the other end, cell viability was determined by validating lactate dehydrogenase (LDH) release as a surrogate marker. LDH is a cytosolic enzyme that is released in the media when the plasma membrane is damaged and it is a well-established indicator of cellular toxicity/lysis. We were able to validate the assay and show a strong correlation (r=0.99) between the amount of LDH release by cells in the culture supernatant and the percentage of dead cells as determined by the LIVE/DEAD® assay. We completed the process and product development phase by working out the postmanufacturing logistics, including final formulation, stability and transport of the cellular graft back to the clinical site. The safety and efficacy of CALEC will soon be tested in patients with unilateral LSCD in a Phase I/II trial as the study (BB-IND-16102) recently cleared the Food and Drug Administration (FDA). References Kolli, S., S. Ahmad, M. Lako, and F. Figueiredo. 2010. Successful clinical implementation of corneal epithelial stem cell therapy for treatment of unilateral limbal stem cell deficiency. Stem cells 28:597-610. Marchini, G., E. Pedrotti, M. Pedrotti, V. Barbaro, E. Di Iorio, S. Ferrari, M. Bertolin, B. Ferrari, M. Passilongo, A. Fasolo, and D. Ponzin. 2012. Long-term effectiveness of autologous cultured limbal stem cell grafts in patients with limbal stem cell deficiency due to chemical burns. Clinical & experimental ophthalmology 40:255267. Nakamura, T., T. Inatomi, C. Sotozono, N. Koizumi, and S. Kinoshita. 2004. Successful primary culture and autologous transplantation of corneal limbal epithelial cells from minimal biopsy for unilateral severe ocular surface disease. Acta ophthalmologica Scandinavica 82:468-471. Rama, P., S. Matuska, G. Paganoni, A. Spinelli, M. De Luca, and G. Pellegrini. 2010. Limbal stem-cell therapy and long-term corneal regeneration. The New England journal of medicine 363:147-155. 3|Page Volume 21, No. 6 November, 2014 Sangwan, V.S., H.P. Matalia, G.K. Vemuganti, A. Fatima, G. Ifthekar, S. Singh, R. Nutheti, and G.N. Rao. 2006. Clinical outcome of autologous cultivated limbal epithelium transplantation. Indian journal of ophthalmology 54:29-34. Tsai, R.J., L.M. Li, and J.K. Chen. 2000. Reconstruction of damaged corneas by transplantation of autologous limbal epithelial cells. The New England journal of medicine 343:86-93. Yoon, J.J., S. Ismail, and T. Sherwin. 2014. Limbal stem cells: Central concepts of corneal epithelial homeostasis. World journal of stem cells 6:391-403. Zakaria, N., T. Possemiers, S.N. Dhubhghaill, I. Leysen, J. Rozema, C. Koppen, J.P. Timmermans, Z. Berneman, and M.J. Tassignon. 2014. Results of a phase I/II clinical trial: standardized, non-xenogenic, cultivated limbal stem cell transplantation. Journal of translational medicine 12:58. 4|Page