Development and characterisation of advanced cell therapies based on multipotent mesenchymal stormal cells and virus-specific T lymphocytes.

Student thesis: Doctoral thesis


Innovative therapies are being developed worldwide to tackle unmet clinical needs. In particular, progress in advanced therapy medicinal products (ATMP) has shown great promise for the treatment of diseases with no other option available. However, researchers and regulatory authorities deal with the sophisticated nature of these medicines, and struggle to standardise both production protocols and final product formulation. Challenges related to the living nature of these products include high donor intervariability and complex mechanisms of action, which are sometimes not completely understood. Additionally, these newly therapies need to demonstrate biological activity with potency assays.

This dissertation comprises the development and characterisation of two different ATMP based on multipotent mesenchymal stromal cells (MSC) and virus-specific T cells (VST).

On the one hand, assessment of identity and potency for product release of MSC isolated from Wharton’s jelly (WJ) and bone marrow (BM) in the context of current good manufacturing practice (cGMP) production is performed. In this regard, we aimed at proposing: a) a potency assay for assessing immunomodulation capacity of MSC; b) the revision of HLA-DR expression profile for MSC definition criteria; and c) the application of risk management methodologies in the assessment of product quality. The optimisation of an immunopotency assay, validated, and approved by the competent authority for product release is presented. Moreover, other quality attributes of MSC are addressed. Regarding BM-MSC, the apparently random expression of HLA-DR, a marker that was expected negative in expansion cultures of MSC, is studied in clinical grade productions. Our findings showed correlation of HLA-DR expression with levels of IL-17F and IL-33. Expression of HLA-DR did not affect MSC identity, differentiation potential nor immunomodulatory capacity. To further strengthen these outcomes, interlaboratory studies were performed obtaining similar results. Furthermore, the use of either human sera or platelet lysate supplements showed no differences in terms of HLA-DR expression. A risk management assessment methodology was also implemented as a tool for quality by design to detect weaknesses of an established bioprocess involving MSC products already in clinical trials.

On the other hand, regarding T lymphocytes, the development of a protocol for ex vivo expansion of VST was performed. VST therapy is intended for immunocompromised patients, which are susceptible of reactivation or de novo infection of herpesviruses among others. This is the case of cytomegalovirus (CMV) that undergoes a mild infection in healthy individuals but has been associated to a high morbidity and mortality in immunocompromised individuals. Unfortunately, available antiviral drugs can produce toxic side effects and are not always effective. Adoptive immunotherapy offers an alternative approach for those patients in a critical situation with no other therapeutic option. Therefore, we developed a protocol for VST scale-up manufacture easily transferable to pharmaceutical standards. Following with the method proposed, we obtained large number of CMV pp65-specific T cells after 14-day co-culture with pp65 pulsed dendritic cells. Culture was based on G-Rex bioreactor technology and supplemented with IL-2, IL-7, IL-15, anti-CD3 and anti-CD28 antibodies. The final product was extensively characterised in terms of identity, purity and potency. VST product was comprised of both CD4+ and CD8+ T lymphocytes, and effector memory T cells represented the major subset, which are known to provide effector function. Most importantly, we successfully demonstrated pp65 specific cytotoxicity of the expanded cells. Interestingly, complete HLA mismatch alloreactivity resulted in less than 5% cell lysis. In summary, a feasible protocol transferable to cGMP was described for an in vitro safe and effective product, which remain functional after thawing, thus providing practical evidence for the generation of an allogeneic third-party bank. Future perspectives would include the manufacture of multivirus-specific T cells.
Date of Award23 Oct 2019
Original languageEnglish
SupervisorFrancesc Godia Casablancas (Tutor), IRENE OLIVER-VILA (Director), Francesc Rudilla Salvador (Director) & Joaquim Vives Armengol (Director)

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