Dissertations / Theses on the topic 'CAR-T therapy'

La thérapie par cellules T à récepteur antigénique chimérique (CAR) est apparue comme l'une des percées les plus convaincantes dans le traitement du cancer au cours de la dernière décennie. Cependant, les résultats remarquables obtenus dans les hémopathies B ne se sont pas encore étendus aux lymphomes T (LT) où l'éventuelle toxicité « on-target off-tumor » a limité le développement d'approches similaires.Dans ce travail, nous avons développé une plateforme NOT-gate, tirant parti de la perte d'expression du CD7 dans les LT pour distinguer les cellules T tumorales des cellules T normales. Cette plateforme associe un CAR 4-28^1 XX activateur ciblant le CD4, un antigène T fortement exprimé dans les LT, à un CAR 7PD1 inhibiteur ciblant le CD7. Les nouvelles cellules CAR T 4-28(1XX, éditées CD4 pour limiter le fratricide, ont démontré une activité antitumorale robuste contre les cellules tumorales CD4-positives in vitro et in vivo dans des modèles murins de LT disséminé. Cependant, la perte du CD4 a démasqué un phenotype hyperproliferatif reponsable d'une infiltration tissulaire létale, dont les mécanismes exacts restent à élucider. L'ajout d'un CAR inhibiteur 7PD1 a permis de réduire la sécrétion de cytokines et la dégranulation des cellules T CAR4-28C1XX, mais il a été plus difficile d'obtenir une inhibition de la cytotoxicité globale. De nombreux paramètres doivent être optimisés pour une plateforme NOT- gate plus efficace, comprenant principalement le rapport stoechiométrique CAR/cible et l'intensité du signal médié par chaque CAR
Chimeric antigen receptor (CAR) T cell therapy has emerged as one of the most compelling breakthroughs in cancer treatment in the past decade. However, the remarkable results achieved in B-cell malignancies hâve not yet translated in T-cell lymphomas (TCL) where concerns over potential "on- target off-tumor" toxicity hâve hindered the development of similar approaches. In this work, we sought to developp a NOT-gate platform, leveraging CD7 loss in mature T-cell malignancies to distinguish tumor from normal T- cells. This platform intergates an activating 4-28£1XX CARtargeting CD4, a T-cell antigen highly expressed in TCL, paired with an inhibitory 7PD1 CAR targeting CD7. The novel 4-28(1XX CAR T cells, CD4-edited to prevent fratricide, demonstrated robust antitumor activity against CD4-positive tumor cells in vitro and in vivo in disseminated TCL murine models. However, CD4-disruption unleashed léthal hyperproliferative CAR T cell infiltration, whose exact mechanisms remains to be elucidated. The addition of a 7PD1 inhibitory CAR allowed for decreased sécrétion of cytokine and degranulation of the 4-28(1XX CAR T cells, but overall killing inhibition was more difficult to achieve. Numerous parameters are to be optimized for a more efficient NOT-gate platform, including mainly CAR/target stoechiometry ratio and the signaling strenght of each CAR

Immunotherapy is becoming recognized as a superior treatment for cancer. In recent years, chimeric antigen receptor (CAR) therapy is among the immunotherapies that has had growing success rates. CAR T-cell therapy takes patient’s T-cells and encodes them with a CAR expressing gene, which can then target their cancer cells. However, there are some dangers associated with this therapy. If a cancer cell is mistakenly transfected with the CAR molecule, it can become resistant to the therapy. Using the electric properties of the cells, we have created a technique that can purify the T-cells from the remaining cancer cells using microfluidics and dielectrophoresis (DEP). Then, to further improve the therapy, the sample is electroporated following being patterned using DEP forces, which transfects the cells without using viral vectors and provides longer CD19 expression.

High grade gliomas are aggressive brain tumours for which treatment is highly challenging due to the location within the central nervous system (CNS), which may reduce access of cytotoxic chemotherapy, and their infiltrative growth, which precludes complete surgical resection. Current treatment includes surgical removal – wherever possible - followed by radiotherapy and chemotherapy. However, recurrence is common, resulting in a survival of only 12 to 15 months after diagnosis. This highlights the need for new therapies. Chimeric antigen receptors (CARs) are synthetic molecules which combine the specificity of an antibody to the signalling domains of a T cell receptor (TCR), allowing T cells to directly recognise tumour antigens with no need for co-stimulation. CAR-T cells have shown promising responses in the treatment of haematological malignancies, inducing complete and durable responses in patients with chemo-refractory disease treated with CD19-redirected T cells. This therapeutic approach may be highly suitable for high grade gliomas as T cells have the ability to track to distant tumour sites. However, translation of this technology to solid tumours is proving more difficult, due to several challenges, including: requirement for an effective infiltration of CAR-T cells within the tumour and the immunosuppressive environment provided by solid malignancies. In this work, we developed an immunocompetent animal model of glioma, to study kinetics of migration and infiltration of CAR-T cells and the interplay between CAR-T cells, the tumour and the endogenous immune system to inform the design of T cell immunotherapy for this brain tumours. The tumour specific variant III of the epidermal growth factor receptor (EGFRvIII) – a mutation found in 30% of glioblastomas – was used as model antigen. A murine CAR was constructed based on the single chain fragment variant (ScFv) of EGFRvIII-specific antibody MR1.1 linked with a CD8 stalk to CD28-CD3ζ activation domains. A murine marker gene (truncated CD34) was co-expressed to allow for ex vivo analysis as well as firefly luciferase for in vivo tracking of CAR T-cells. The mouse glioma cell line GL261 was modified to express the mouse version of EGFRvIII and used to establish orthotopic tumours. After validation of function and specificity in vitro, efficacy of CAR-T cells was tested in vivo. Both bioluminescence imaging (BLI) and flow cytometry demonstrated that CAR T cells accumulated within the tumour in an antigen-dependent manner. MRI demonstrated that CAR T cells delayed tumour growth and increased survival. However, tumours were not consistently eradicated. Both immunohistochemistry and BLI indicated lack of long term persistence of T cells within the tumour. Analysis of tumour infiltrating lymphocytes (TILs) phenotype suggested that decreased functionality of CAR-T cells could be a result of their exhaustion in situ. We hypothesised that additional strategies were required to improve efficacy and persistence of CAR-T cells. We postulated that CAR-T cell fitness may be prolonged by: - Incorporation of 41BB as additional co-stimulatory domain in the CAR to provide a pro-survival signal. - Combination therapy with PD1 blockade to overcome T cell exhaustion (both on CAR and endogenous T cells) in situ. While the employment of third-generation CAR did not significantly improve survival and showed increased toxicity, combination therapy of CAR-T cells and PD-1 blockade promoted complete clearance of tumours resulting in long term survival. Immunohistochemistry and flow cytometry analysis suggested that combination therapy may increase persistence of CAR-T cells, leading to a more rapid and consistent tumour eradication compared to CAR-T cell administration alone. However, data presented here did not demonstrate a synergistic effect of CAR-T cell therapy and PD1 blockade, as an effect of PD1 blockade alone was also observed. Therefore, additional experiments are required to examine this further.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019
Cataloged from PDF version of thesis.
Includes bibliographical references.
Chimeric antigen receptor (CAR) T cells are a promising cancer therapeutic, as they can specifically redirect the cytotoxic function of a T cell to a chosen target of interest. CAR T cells have been successful in clinical trials against hematological cancers, but have experienced low efficacy against solid tumors for a number of reasons, including a paucity of tumor-specific antigens to target and a highly immunosuppressive solid tumor microenvironment. In chapter 2 of this thesis, we develop a strategy to target multiple solid tumor types through markers in their microenvironment. The use of single domain antibody (VHH)-based CAR T cells that recognize these markers circumvents the need for tumor-specific targets. Chapter 3 will describe methods to overcome the immunosuppressive microenvironment of solid tumors. Here, we have developed VHH-secreting CAR T cells that can modulate additional aspects of the tumor microenvironment, including the engagement of the innate immune system through secretion of a VHH against an inhibitor of phagocytosis. We show that this strategy of VHH-secretion by CAR T cells can lead to significant benefits in outcome. We also demonstrate that delivery of therapeutics by CAR T cells can improve the safety profile of the therapeutic. Chapter 4 of this thesis explores strategies to increase the targeting capacity of CAR T cells by building logic-gated CARs. Finally, chapter 5 will describe work in imaging CAR T cells specifically, longitudinally, and non-invasively through PET imaging. Our results demonstrate the flexibility of VHHs in CAR T cell engineering and the potential of VHH-based CAR T cells to target the tumor microenvironment, modulate the tumor microenvironment, and treat solid tumors.
by Yushu Joy Xie.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

Mestrado em Biomedicina Farmacêutica
Cellular immunotherapies, or Advanced Therapy Medicinal Products (ATMPs), are emerging as novel and specific therapeutic approaches to treat diseases, such as certain types of leukemias, which are difficult or impossible to treat with today’s biopharmaceutical products. Breakthroughs in basic, preclinical, and clinical science spanning cellular immunology, and cellprocessing technologies has allowed clinical applications of chimeric antigen receptor–based therapies. A recent example is CTL019, a lentivirus-based gene therapy for autologous T cells, acquired by Novartis in 2012 through a global alliance with the University of Pennsylvania. Although this technology is still in its infancy, clinical trials have already shown clinically significant antitumor activity in chronic lymphocytic leukemia and acute lymphocytic leukemia. Trials targeting a variety of other adult and pediatric malignancies are under way. The potential to target essentially any tumor-associated cell-surface antigen for which a monoclonal antibody can be made opens up an entirely new arena for targeted therapy of cancer. The regulatory environment for these Advanced Therapies Medicinal Products is complex and in constant evolution. Many challenges lie ahead in terms of manufacturing process, non-conventional supply chain logistics, business models, intellectual property, funding and patient access.

CAR-T-Zell-Therapie ist eine vielversprechende neuartige Behandlungsform für Patienten mit aggressiven B-Zell Non-Hodgkin-Lymphomen (B-NHL). In dieser Arbeit wurde die anti-CXCR5 CAR-T-Zell-Therapie als Alternative zur anti-CD19 CAR-T-Zell-Therapie für die Behandlung von reifen B-NHLs untersucht. CXCR5 ist ein B-Zell-homing Rezeptor, der von reifen B Zellen und follikulären T-Helferzellen (TFH Zellen) exprimiert wird. TFH Zellen wurden als tumor-unterstützend in chronisch lymphatischer Leukämie (CLL) und im follikulären Lymphom (FL) beschrieben. Dieses Expressionsmuster erlaubt es, auf einzigartige Weise zeitgleich die malignen Zellen und die tumorunterstützende Mikroumgebung mithilfe von CAR-T-Zell-Therapie gerichtet gegen einen Chemokinrezeptor anzugreifen. Die wichtigsten Ergebnisse dieser Arbeit waren, dass (1) die anti-CXCR5 CAR T-Zellen zielgerichtet CXCR5 positive reife B-NHL Zelllinien und Patientenproben in vitro eliminierten und eine starke anti-Tumor Reaktivität in einem immundefizienten Xenotransplantationsmausmodell zeigten, (2) die anti-CXCR5 CAR T-Zellen zielgerichtet die tumorunterstützenden TFH Zellen in CLL und FL Patientenproben in vitro erkannten und dass (3) CXCR5 ein sicheres Expressionsprofil zeigte. CXCR5 war stark und häufig auf B-NHL exprimiert und die Expression auf gesundem Gewebe war auf lymphoide Zellen beschränkt. Zusammenfassend lässt sich sagen, dass die anti-CXCR5 CAR-T-Zell-Therapie eine neue Behandlungsmöglichkeit für Patienten mit reifen B-NHL darstellt, indem durch die anti-CXCR5 CAR-T Zellen sowohl der Tumor als auch ein Anteil der tumorunterstützende Mikroumgebung eliminiert werden. Im zweiten Teil der Arbeit wurde das Eμ-Tcl1 murine CLL Lymphommodell genutzt um die Auswirkung der Lymphomentwicklung auf die CXCR5+ T Zellen zu untersuchen. Mittels RNA-Einzelzell-Sequenzierung konnte ein profunder Einfluss des Lymphomwachstums auf das T Zell-Kompartiment der Mäuse, denen Eμ-Tcl1 Zellen gespritzt wurden, gezeigt werden.
CAR T cell therapy is a promising new treatment option for patients suffering from aggressive B non-Hodgkin lymphomas (NHLs). In CAR T cell therapy, patient-derived T cells are genetically modified to express a chimeric receptor commonly directed towards a surface antigen expressed by neoplastic cells. In this thesis, anti-CXCR5 CAR T cell therapy was investigated as an alternative to anti-CD19 CAR T cell therapy for the treatment of mature B-NHLs. CXCR5 is a B cell homing receptor expressed by mature B cells and follicular helper T (TFH) cells. TFH cells were described to support the tumor cells in chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL). This expression pattern allows simultaneous targeting of the malignant cells and the tumor-supporting microenvironment by CAR T cell therapy against a chemokine receptor in an unprecedented manner. Main findings included that (1) anti-CXCR5 CAR T cells targeted specifically CXCR5 expressing mature B-NHL cell lines and patient samples in vitro and showed strong in vivo anti-tumor reactivity in an immunodeficient xenograft mouse model, (2) anti-CXCR5 CAR T cells targeted tumor-supportive TFH cells derived from CLL and FL patient samples in vitro and (3) CXCR5 showed a safe expression profile. CXCR5 was strongly and frequently expressed by B-NHLs and its expression on healthy tissue was restricted to lymphoid cells. In summary, anti-CXCR5 CAR T cell therapy presents a novel treatment option for patients suffering from mature B-NHLs by eliminating the tumor and part of the tumor-supportive microenvironment. The second part of the project, the Eμ-Tcl1 murine lymphoma model, which mimics human CLL, was used to study the impact of lymphomagenesis on CXCR5+ T cells. Using single cell RNA sequencing, a profound influence of lymphoma growth on the T cell compartment in Eμ-Tcl1 tumor-challenged mice could be shown.

Immunotherapy for cancer is a young research field progressing at high speed. The first chimera of an antibody and a signaling chain was designed by Zelig Eshhar and was later further developed to enhance existing T cell therapy by combining a single-chain fragment of an antibody with the CD3 zeta chain of the TCR complex. T cells expressing these chimeric antigen receptors (CARs) could recognize and specifically kill tumor cells. However the T cells, lacked in persistence and tumor rejection did not occur. Thus, the CAR constructs have been improved by providing the T cell with costimulatory signals promoting activation. The focus of this thesis has been to evaluate second and third generation αCD19-CAR T cells for the treatment of B cell leukemia and lymphoma. B cell tumors commonly upregulate anti-apoptotic proteins such as Bcl-2, which generates therapy resistance. In the first paper a second generation (2G) αCD19-CD28-CAR T cell was combined with the Bcl-2 family inhibitor ABT-737. ABT-737 sensitized tumor cells to CAR T cell therapy and may be an interesting clinical combination treatment. In paper II, the phenotype and function of a third generation (3G) αCD19-CD28-4-1BB-CAR T cell were evaluated. B cell-stimulated CAR T cells showed increased proliferation and an antigen-driven accumulation of CAR+ T cells. 3G CAR T cells had equal cytotoxic capacity, similar lineage, memory and exhaustion profile phenotype compared to 2G CARs. However, 3G CAR T cells proliferated better and had increased activation of intracellular signaling pathways compared to 2G CAR T cells. In paper III, αCD19-CD28-4-1BB-CAR T cells were used to stimulate immature dendritic cells leading to an upregulation of maturation markers on co-cultured dendritic cells. Hence, CAR T cells may not only directly kill the tumor cells, but may induce bystander immunity that indirectly aids tumor control. This thesis also include supplementary information about the development and implementation of protocols for GMP production of CAR T cell batches for a phase I/IIa clinical trial currently ongoing for patients with refractory B cell leukemia and lymphoma. So far, two patients have safely been treated on the lowest dose.

La thérapie CAR-T a transformé le traitement du cancer en modifiant les lymphocytes T pour cibler spécifiquement les antigènes tumoraux. Bien que cette approche ait montré un succès remarquable dans les hémopathies malignes à cellules B, le processus reste coûteux et long, car il nécessite la collecte et la modification des cellules du patient, ce qui peut retarder le traitement. De plus, certains patients, en raison de traitements antérieurs ou de maladies avancées, ne disposent pas de cellules viables, limitant l'accès à cette thérapie.Les cellules CAR-T allogéniques provenant de donneurs offrent une solution plus rapide et évolutive, réduisant le temps de production et les coûts. Cependant, elles présentent des risques, notamment la maladie du greffon contre l'hôte (GvHD), où les cellules du donneur attaquent les tissus du patient. Notre étude a exploré une approche innovante, combinant la technologie CAR avec des lymphocytes T spécifiques aux virus (Virus Specific T cells, VST), connus pour leurs propriétés antivirales et antitumorales, afin de générer des CAR-VST. Ces CAR-VST à double spécificité représentent une alternative prometteuse, particulièrement pour les patients à risque de rechute tumorale ou de réactivation virale.Dans notre étude, nous avons généré des CAR-T et des CAR-VST à partir des mêmes donneurs, obtenant respectivement 40,28%±9,30% et 35,96%±11,40% d'expression de CD19.CAR au jour 7 (N=3). Les CAR-VST ont montré in vitro une clairance tumorale similaire à celle des CAR-T, avec 74,13%±22,06% de lyse des cellules CD19+. Dans un modèle murin, un contrôle de la croissance tumorale ainsi qu'une amélioration de la survie similaires ont été observés dans les deux groupes. De plus, les CAR-VST ont conservé leur activité antivirale, lysant 62,32%±13,84% des cellules chargées en peptides viraux. Concernant l'alloréactivité, les CAR-VST ont montré une prolifération CD3+ inférieure (28,27%±21,64%) par rapport aux CAR-T (88,3%±24,48%, p=0,0285, N=4), suggérant un risque réduit de GvHD.En collaboration avec l'Université de Caroline du Nord, nous avons également exploré la suppression des molécules HLA de classe I via la B-2-microglobuline (B2M) pour réduire le risque de rejet immunitaire. Une expression de HLA-ABC de 15,1±14,6% (N=11) a été obtenue après knockout par CRISPR/Cas9. Nous travaillons également sur la surexpression de HLA-E et HLA-G pour prévenir la lyse médiée par les cellules NK, nécessitant des optimisations supplémentaires.En conclusion, générer des HLA-E+ ou G+/B2M-/CAR-VST offre une alternative prometteuse pour créer des cellules entièrement allogéniques. Ces CAR-VST modifiés conservent leurs fonctions antivirales et antitumorales, ce qui en fait des candidats prometteurs pour les immunothérapies prêtes à l'emploi qui pourraient réduire les risques de rejet immunitaire et de GvHD
CAR-T cell therapy have revolutionized cancer treatment by modifying a patient's T cells to target specific tumor antigens. This personalized approach has shown remarkable success in treating B-cell malignancies like leukemia and lymphoma. However, the process is costly and time-consuming, as it involves collecting and modifying the patient's own cells, which delays treatment. Moreover, some patients may not have sufficient or viable T cells due to prior treatments or advanced disease stages, limiting the availability of CAR-T therapies for all patients.To address these challenges, allogeneic CAR-T cells from healthy donors provide a faster and more scalable solution, reducing production time and costs. However, these off-the-shelf therapies face risks like graft-versus-host disease (GvHD), where donor cells might attack the patient's tissues. Our study explored combining CAR technology with Virus Specific T cells (VSTs), known for their antiviral and antitumor properties, to generate CAR-VSTs. These dual-specific CAR-VSTs present a promising alternative, especially for patients prone to both tumor relapse and viral reactivation.In our study, we generated CAR-Ts and CAR-VSTs from same donors obtaining 40.28%±9.30% and 35.96%±11.40% CD19.CAR expression on day 7 (N=3), respectively. In vitro, CAR-VSTs showed robust tumor clearance similar to CAR-Ts, achieving 74.13%±22.06% lysis of CD19+ tumor cells. In a murine lymphoma model, both CAR-VSTs and CAR-Ts demonstrated comparable antitumor efficacy, successfully controlling tumor growth and improving survival. Moreover, CAR-VSTs maintained their antiviral function, efficiently lysing 62.32%±13.84% virus-peptide-pulsed cells, similar to native VSTs. We assessed the alloreactivity of CAR-VSTs and found that they exhibited significantly lower CD3 proliferation rates (28.27%±21.64%) compared to CAR-T cells (88.3%±24.48%, p=0.0285, N=4), indicating a reduced risk of GvHD. CAR-VSTs' dual-specificity for both tumor and viral antigens makes them a powerful tool to address cancer relapse and viral complications in patients.In collaboration with the University of North Carolina, we explored strategies to delete HLA class I molecules in CAR-VSTs by targeting B-2-microglobulin (B2M), aiming to reduce immune rejection. In addition, we worked on overexpressing tolerogenic molecules such as HLA-E and HLA-G to prevent NK cell-mediated lysis. Our results showed an HLA-ABC expression of 15.1±14.6% (N=11) after CRISPR/Cas9 knockout, which indicates successful deletion, though further optimization is necessary to prevent NK-lysis by re-expressing HLA-E or HLA-G.In conclusion, generating HLA-E+ or G+/B2M-/CAR-VSTs offers a promising alternative for creating fully allogeneic cells. These modified CAR-VSTs retain their dual antiviral and antitumor functions, making them a promising candidate for "off-the-shelf" immunotherapies that could reduce the risks of immune rejection and graft-versus-host disease

La leucemia mieloide acuta (LMA) è la neoplasia ematologica maggiormente diagnosticata nei pazienti adulti (25%) e mentre rappresenta il 15-20% dei casi nei pazienti pediatrici. La chemioterapia convenzionale, che impiega antraciclina e citarabina, rappresenta il trattamento standard per l’LMA, con tassi di remissione completa dal 60% all'80% nei bambini e dal 40% al 60% negli adulti (>60 anni). Sfortunatamente, la ricaduta dopo tale terapia è comune e la sopravvivenza dei pazienti stimata a 5 anni è ancora inferiore al 30%. Risulta quindi di primaria importanza trovare alternative terapeutiche per i pazienti recidivanti e refrattari. Il recente successo clinico, ottenuto nelle leucemie di tipo B, dell'immunoterapia con cellule CAR (chimeric antigen receptor) T ha portato allo sviluppo di nuove strategie terapeutiche nell’ambito dell’LMA. Tuttavia, lo sviluppo del trattamento con cellule CAR T nel contesto dell'LMA è ancora agli albori a causa dell'eterogeneità della malattia, della mancanza di un antigene bersaglio adatto e del ruolo protettivo del microambiente tumorale (TME). Infatti, non esiste ancora un protocollo clinico approvato per il trattamento della leucemia mieloide. Per creare le cellule CAR T abbiamo scelto di utilizzare la piattaforma non virale Sleeping-Beauty (SB) per ingegnerizzare le cellule CIK (cytokine-induced killer). In primo luogo, abbiamo scelto di utilizzare come potenziale strumento per il targeting del TME le cellule CIK ingegnerizzate con anti-CD146.CAR. Di conseguenza, abbiamo ottimizzato 6 diverse molecole CAR aventi un design differente, ottenendo un'espressione ottimale di CD146 nella variante VLVH Long. Abbiamo quindi testato le cellule CD146.CAR-CIK in vitro, ottenendo l’attivazione specifica delle funzioni effettrici (in termini di capacità di killing, produzione di citochine e proliferazione) contro cellule target CD146+. In seguito, abbiamo progettato un Tandem CAR bispecifico (CD33xCD146.CAR-CIKs) che ha mostrato una significativa attività antileucemica in vitro. È stato ampiamente dimostrato che la nicchia midollare contribuisce al supporto e alla protezione delle cellule staminali leucemiche (CSLs). Quindi, per mimare al meglio l’azione del CAR nella nicchia midollare umana, abbiamo testato le cellule CD33xCD146.CAR-CIK contro le linee cellulari stromali CD146+ e le cellule mesenchimali (MSC) primarie sane (HD-) e di derivazione mieloide (LMA-). I dati mostrano una inibizione delle funzioni effettrici delle cellule CAR-CIK e una drastica diminuzione della produzione di citochine e della proliferazione. Inoltre, l'equilibrio tra citochine pro e antinfiammatorie è risultato alterato, infatti la produzione di citochine Th1/Tc1 da parte delle cellule CD146.CAR-CIK è stata inibita dalla co-coltura con cellule stromali, mentre è stato rilevato un aumento delle citochine Th2/Tc2. Questi risultati suggeriscono un potenziale ruolo immunosoppressivo del compartimento stromale nei confronti delle cellule CAR-CIK. Sulla base dell’ effetto immunomodulatorio delle MSC sui linfociti T, abbiamo ipotizzato che la nicchia midollare possa influenzare le funzioni effettrici delle cellule CAR T. Di conseguenza, il targeting del CD146 rappresenta una "proof-of-principles" del fatto che aggredire il microambiente leucemico possa migliorare la terapia CAR T nell’ambito dell’LMA. Per ridurre al minimo la tossicità "off-target ", stiamo cercando di selezionare un antigene bersaglio specifico ed overespresso sulle cellule stromali dell'LMA, che abbia un'espressione minima nello stroma sano e che sia coinvolto nelle interazioni leucemia/nicchia. Il nuovo marker di interesse sarà accoppiato al CD33.CAR nella creazione di un CAR bispecifico, che verrà confrontato con il costrutto CD33xCD146.CAR, valutandone i profili di efficacia e sicurezza sia in vitro che in vivo.
Acute myeloid leukemia (AML) is the most frequently diagnosed leukemia in adults (25%) and accounts for 15-20% cases in pediatric patients. Conventional chemotherapy employing anthracycline and cytarabine represents the gold standard treatment for AML, with rates of complete remission from 60% to 80% in children and from 40% to 60% in adults (>60 years). Despite these high rates, relapse after conventional therapy is common and the estimated five-year survival of AML patients is still below 30%. Indeed, there is an urgency to find alternative therapeutic strategies for relapsed and refractory patients. The recent clinical success of chimeric antigen receptor (CAR) T cell immunotherapy in the context of B-cell malignancies has opened a new route of investigation also towards AML. However, the development of CAR T cell therapy in the context of AML is still in its infancy due to heterogeneity of the disease, the lack of a suitable target antigen and the leukemia protective role of the tumor microenvironment (TME) and no approved CAR T cells study exists for AML treatment yet. Non-viral Sleeping-Beauty (SB) transposon platform was employed to redirect cytokine-induce killer (CIK) cell. In this scenario, we firstly characterize non-viral SB engineered CIK cells with anti-CD146.CAR as a potential tool for the targeting of the bone marrow (BM) microenvironment. We optimized the CAR design structure by testing 6 different CAR molecules, achieving a specific and efficient CD146 expression in the VLVH Long variant. CD146.CAR-CIK cells were subsequently tested in vitro, showing an optimal activation of effector functions (in terms of killing activity, cytokines production and proliferation) when they were engaged against CD146+ target cells. Consequently, we developed a bispecific Tandem CAR (CD33xCD146.CAR-CIKs), which displayed anti-leukemic activity in vitro. It has been extensively proven that BM niche contribute to establish a sanctuary in which leukemic stem cells (LSCs) are able to acquire drug-resistant phenotype, therefore, to better mimicking the human BM niche we tested CD33xCD146.CAR-CIK cells against CD146+ stromal cell lines (HS-27A and HS-5) and primary derived healthy (HD-) and patient-derived (AML-) mesenchymal stromal cells (MSCs). Results showed inhibition of the redirected CAR-CIK cells effector functions, resulting in a drastic decrease of cytokines production and proliferation. The balance between pro- and anti- inflammatory cytokines showed that Th1/Tc1 cytokines production by CD146.CAR-CIK cells was inhibited by the co-culture with stromal cells, while increase Th2/Tc2 cytokines was detected when CD146.CAR-CIK cells were co-cultured with stromal target cells. These results suggest a potential immunosuppressive role of the stromal compartment against CAR-CIK cells. According to these results, we hypothesized that BM stromal cells can potentially exert an immunomodulatory effect on T cells, suggesting that the niche microenvironment may be involved in the regulation of CAR T cells therapy effectiveness. Indeed, the targeting of CD146 on stroma represents a “proof-of-principle” that stromal components of leukemic microenvironment may be attractive targets for CAR T based immunotherapy. To minimize “off-tumor” toxicity, we are looking for a specific surface target antigen selectively overexpressed on AML stromal cells, with minimal expression in healthy stroma and possibly involved in leukemia/niche interactions. The newly marker of interest will be coupled to the CD33.CAR and this bispecific CAR will be compared with CD33xCD146.CAR construct, evaluating their efficacy and safety profiles both in vitro and in vivo.

La terapia CAR-T rappresenta un approccio promettente, ma ha riportato una ridotta efficacia nella leucemia mieloide acuta (AML), a causa dell’eterogeneità del tumore, dell’assenza di antigeni target AML-specifici e del ruolo del microambiente leucemico nella protezione dei blasti e delle cellule staminali leucemiche (LSC). La nicchia midollare, nella quale risiedono le LSC, è coinvolta in attività che promuovono la progressione leucemica e sopprimono l’ematopoiesi sana. Quindi ipotizziamo che bersagliare le LSC nascoste nella nicchia potesse migliorare l’efficacia delle CAR-T. Per testare la nostra ipotesi, abbiamo agito su due fronti: 1) promuovere una migrazione efficiente delle CAR-T nella nicchia midollare, 2) selezionare un antigene target ristretto ai blasti leucemici e alle LSC. Prima, abbiamo proposto una strategia per guidare le cellule CD33.CAR CIK (Cytokine-Induced Killer), una sottopopolazione di cellule T effettrici, verso la nicchia leucemica. La chemochina CXCL12, rilasciata dalle cellule mesenchimali stromali (MSC), nella nicchia midollare, e il suo recettore CXCR4, sono coinvolti nella regolazione della migrazione dei leucociti all’interno della nicchia. Quindi, abbiamo ipotizzato che sfruttare questo asse potesse migliorare la capacità di homing delle CD33.CAR-CIK nella nicchia e favorire l’eradicazione della leucemia. Tuttavia i protocolli di manipolazione ex vivo delle CD33.CAR-CIK riducono l’espressione di CXCR4, compromettendo la capacità delle cellule infuse di raggiungere la nicchia. Quindi per implementare la capacità di homing delle CD33.CAR-CIK nel microambiente midollare, abbiamo sviluppato delle CD33.CAR-CIK overesprimenti CXCR4, nella sua forma wild-type o iperattiva mutata. Le CIK ingegnerizzate con i costrutti CD33.CAR-CXCR4 hanno mostrato un consistente aumento dell’espressione di CXCR4, senza riportare alterazioni fenotipiche e nelle funzioni effettrici CAR-associate. Inoltre, rispetto alle CD33.CAR-CIK, le cellule CD33.CAR-CXCR4WT -CIK ed in particolare le CD33.CAR-CXCR4MUT-CIK hanno dimostrato non solo una superiore risposta chemotattica in vitro verso il CXCL12 ed i surnatanti delle MSC, ma anche un aumentato homing in vivo. In seguito, per promuovere lo sviluppo di un approccio CAR-T più efficace e sicuro, abbiamo proposto di re-indirizzare il CAR verso un antigene espresso selettivamente dalle cellule AML, ma assente sulle cellule staminali ematopoietiche (HSC). TIM-3 è un immune checkpoint, svolge un ruolo centrale nella regolazione delle risposte immunitarie nell’AML e costituisce un marcatore selettivo per le LSC, senza essere espresso dalle HSC. Abbiamo disegnato un CAR di terza generazione diretto contro TIM-3, utilizzando la porzione scFv derivante da un anticorpo monoclonale anti-TIM-3. In vitro, le TIM-3.CAR-CIK hanno dimostrato di eliminare sia le linee AML che i blasti primari, senza dare tossicità verso le cellule TIM-3+ sane, come le CIK attivate, i monociti e le cellule NK. Inoltre, le TIM-3.CAR-CIK hanno eliminato in maniera selettiva le LSC (CD34+ CD38-). Infine, le TIM-3.CAR-CIK hanno mantenuto le loro capacità effettrici nonostante multiple ristimolazioni in vitro, gettando le basi per lo studio di questo costrutto in vivo. Complessivamente, entrambi gli approcci, uno implementando l’homing delle CAR-CIK alla nicchia midollare e l’altro conferendo una superiore selettività, potrebbero migliorare l’efficacia della terapia CAR-T nel contesto dell’AML.
Chimeric Antigen Receptor (CAR) T-cell therapy has produced remarkable clinical responses in patients affected by acute lymphoblastic leukemia. Unfortunately, CAR T-cells have not been equally successful in acute myeloid leukemia (AML) due to tumor heterogeneity, lack of truly AML-restricted target antigens and the role of leukemia microenvironment in blasts protection and leukemia stem cells (LSCs) maintenance. Specifically, the bone marrow (BM) niche, where LSCs reside, is involved in leukemia promoting activities whilst suppressing normal hematopoiesis. Therefore, we hypothesized that targeting LSCs at their location may enhance the potency and selectivity of CAR-T cells. To address this issue, we have designed two aims: 1) promote rapid and efficient localization of CAR T-cells within the BM niche, 2) select a leukemia-restricted antigen to specifically target AML blasts and LSCs. First, we proposed to harness CD33.CAR-redirected Cytokine-Induced Killer (CIK) cells, an alternative effector T-cell population with acquired NK-like cytotoxic activity as well as minimal alloreactivity, to selectively route their activity to leukemia transformed niche. The chemokine ligand 12 (CXCL12), released by mesenchymal stromal cells (MSCs) within the medullary niche, and its chemokine receptor 4 (CXCR4) are two pivotal players regulating leukocytes trafficking to the BM. In AML, CXCL12 interacts with CXCR4 overexpressed on blasts, promoting their migration and homing in the niche. Hence, taking advantage of this axis might facilitate CD33.CAR-CIK cells homing to the BM and therefore leukemia eradication. However, ex vivo manipulation protocols of CD33.CAR-CIK cells consistently downregulate CXCR4 expression and may affect the capacity of adoptively infused cells to migrate to BM and exert their anti-leukemic action. Therefore, to improve CD33.CAR-CIKs homing in the BM microenvironment we have developed CD33.CAR-CIK cells overexpressing CXCR4, in its wild-type or hyperactive mutant form. Notably, CIK cells engineering with CD33.CAR-CXCR4 constructs led to a consistent increase in CXCR4 expression, without altering CIK cells phenotype and CAR-related effector functions. Interestingly, compared to conventional CD33.CAR-CIK cells, CD33.CAR-CXCR4WT and especially CD33.CAR-CXCR4MUT-CIK cells demonstrated significantly superior in vitro chemotactic response toward CXCL12 and MSC-derived supernatants, and greater in vivo BM homing ability and persistence. Furthermore, to develop an effective anti-AML CAR T-cell therapy, it is fundamental to identify a LSC-specific marker, sparing the normal counterpart of hematopoietic stem cells (HSCs). T-cell immunoglobulin and mucin protein 3 (TIM-3) is an immune checkpoint molecule, it plays a central role in immune responses in AML and it is an LSC-specific marker, lacking expression on HSCs. Therefore, we designed a third-generation anti-TIM-3.CAR using the single-chain fragment variable (scFv) derived from an antagonistic ligand-blocking anti-TIM-3 antibody. In vitro, TIM-3.CAR-CIK cells efficiently killed both AML cell lines and primary AML blasts, but not normal TIM-3+ activated CIK cells, monocytes and NK-cells. Notably, we observed selective elimination of primary LSC-enriched population (CD34+ CD38-). Furthermore, TIM-3.CAR-CIK cells maintained their effector functions despite multiple in vitro restimulations, setting the basis for further exploration in in vivo models. Overall, both approaches, one improving CAR-CIK cells homing to the transformed niche and the other conferring superior safety and selectivity, might improve the efficacy of anti-AML CAR-CIK therapy.

Viral infections are a significant source of morbidity and mortality following allogeneic haemopoietic stem cell transplantation (HSCT). HSCT constitutes the only curative therapy for many haematological malignancies. Global transplant practices are evolving with drive to meet demand leading to increased use of ‘alternative’ and ‘mismatched’ donors. These factors contribute to the delayed T-cell recovery with increased vulnerability to latent viral reactivation. Use of antiviral therapy can be limited by resistance or toxicity. HSCT donor-derived virus-specific T-cells (VST) are an alternative for the prophylaxis or treatment of infection. Despite promising early phase trial results this form of therapy is unlikely to be adopted as a standard of care. A HSCT donor-derived product will not be available to all patients, and may not be available in situations of clinical urgency. On a broader scale, VST manufacture is not economical, as products are intended for a specific recipient and there is risk that products will not be used. The use of third-party donor VST is an alternative option. Feasibility has been demonstrated in a small number of studies using partially human leucocyte antigen (HLA)-matched third-party donor VST to treat viral infection in HSCT recipients. This thesis investigated optimal procedures for establishing a third-party bank of cryopreserved CMV, EBV, and Adv-specific T-cells within an existing HSCT program. Banked VST products were fully characterized for HLA type and antiviral activity. The long-term efficacy and safety of these VST products for treatment of refractory viral infection post-HSCT were investigated in a phase I multi-centre trial. The immunological impacts of third-party VST infusion were examined by measuring immune cell frequencies, cytokine activity, virus-specific T-cell frequency, and persistence of infused-third party cells in the post-treatment period. It is envisaged that the results of these investigations will contribute to the evaluation of third-party donor cell banks as a safe and pragmatic therapeutic approach worthy of later phase studies, in turn improving accessibility to highly efficacious and low toxicity antiviral treatment for all HSCT patients.

Liver metastases are the most common cause of death in colorectal cancer patients. The standard of care and potential for cure for colorectal liver metastases is resection, but often times disease it too extensive for this treatment. Over the years, cancer research has made way for advances in treating progressive disease through immunotherapy. By genetically modifying an individual’s immune system using virally transduced chimeric antigen receptor T cells (CAR-T), patients are better able to receive exquisitely specific T cells to target specific tumors. Furthermore, selective delivery strategies may enhance efficacy while limiting detrimental, systemic adverse effects. Not only this, CAR-Ts have also lead to complete remission in some liquid tumors while maintaining the potential for remission in solid tumors as well. This literature review takes readers through the emergence of the different generations of CAR-T and the various studies including clinical trials that have demonstrated the safety and efficacy of CAR-T. The second portion of this paper will outline the design for a phase II clinical trial using intrahepatic CAR-T therapy in addition to selective internal radiation therapy (SIRT) for refractory CEA+ colorectal liver metastases. Benefits and limitations of using these therapies are further discussed.

The current field of cancer treatment is undergoing a revolution. The influx of novel therapies derived from basic research on the immune system has shifted the landscape of modern medicine. Immunotherapy seeks to use the body’s own immune system as a medium to terminate neoplastic cells. This is performed by manipulating the immune system into either targeting cancer antigens or breaking down barriers towards T cell infiltration. The former mechanism uses CAR-T cells as an instrument to target specific cancer neo-antigens. CAR-T cells begin as T cells derived from a patient’s immune system. These cells are removed from the body and engineered to express a chimeric antigen receptor (CAR) through a process of viral transduction. This CAR allows the T cell to recognize and bind to a specific antigen of interest. In most cases, the antigen is present on cancer cells. The T cells, now expressing the CAR receptor, are transplanted back into the body of the patient and proceed to target cancer cells. This therapy has been used in hematological malignancies to great effect. Applying CAR-T cells to solid tumors is an ongoing process, but has been difficult to establish due to the immunosuppressive aspects of the tumor microenvironment. As such, combining CAR-T cells with traditional anti-cancer therapies has been proven to be efficacious in treating patients with solid tumors. In general, immunosuppression is a large problem in the treatment of cancer. Cancer cells and the tumor microenvironment express receptors that downregulate tumor-targeting actions of the immune system. The discovery of the programmed cell death protein 1 (PD1) allowed researchers to create novel antibodies that inhibit immunosuppression. PD1 located on T cells, binds to PDL1 on cancer and stromal cells. This interaction induces exhaustion and anergy in infiltrating T cells, thereby prevent T cells from targeting cancer cells. As such, the newly approved checkpoint blockade antibodies, Nivolumab and Pembrolizumab, block this interaction and allow T cells to carry out their targeting function. CAR-T cells and checkpoint blockade have both seen immense success in clinical trials and are currently being used the clinic. Nonetheless, development of these therapies for different types of cancers is an ongoing process and one that will require immense effort on behalf of the medical and pharmaceutical establishment

Chimeric antigen receptors (CAR) are modular genetically modified receptors that consist of an extracellular antigen binding domain fused to intracellular T-cell signaling domains. CAR therapy broadly consists of engineering a patient’s own T-cells to express a CAR directed against a tumor cell surface antigen. This therapy has been extremely successful in treating B-cell neoplasms by targeting CD19 and is paradigm changing in developing personalized immunotherapy for oncology applications. Although impressive response rates are observed, the durability of therapeutic response remains a concern and relapse mechanisms frequently center around issues of antigen loss. In addition, heterogeneous disease and solid tumors present formidable barriers toward extending the applicability of CAR technology as a result of compounding issues of tumor microenvironment and cell trafficking. In this thesis we review the current thought on the state of CAR therapy and the challenges to therapeutic efficacy, therapeutic manufacture, and clinical safety in the context of each other with an overall emphasis on identifying the fundamental goal of making fit-for-purpose CARs for different diseases.

abstract: The success of genetically-modified T-cells in treating hematological malignancies has accelerated the research timeline for Chimeric Antigen Receptor-T (CAR-T) cell therapy. Since there are only two approved products (Kymriah and Yescarta), the process knowledge is limited. This leads to a low efficiency at manufacturing stage with serious challenges corresponding to high cost and scalability. In addition, the individualized nature of the therapy limits inventory and creates a high risk of product loss due to supply chain failure. The sector needs a new manufacturing paradigm capable of quickly responding to individualized demands while considering complex system dynamics. The research formulates the problem of Chimeric Antigen Receptor-T (CAR-T) manufacturing design, understanding the performance for large scale production of personalized therapies. The solution looks to develop a simulation environment for bio-manufacturing systems with single-use equipment. The result is BioMan: a discrete-event simulation model that considers the role of therapy's individualized nature, type of processing and quality-management policies on process yield and time, while dealing with the available resource constraints simultaneously. The tool will be useful to understand the impact of varying factor inputs on Chimeric Antigen Receptor-T (CAR-T) cell manufacturing and will eventually facilitate the decision-maker to finalize the right strategies achieving better processing, high resource utilization, and less failure rates.
Dissertation/Thesis
Masters Thesis Industrial Engineering 2020

Most Adoptive Cell Therapies (ACT), including CAR T cell therapies, suffer failure because of the severe side effects due to loss-of-control of the therapeutic cells once they are inside the patient’s body, suggesting that novel strategies must be developed for a better in vivo control of these engineered cells. In the meantime, CAR T cell therapies targeting solid tumors have not experienced the remarkable success achieved with hematopoietic cancers, mainly due to continuous tumor antigen exposure and a suppressive tumor microenvironment. Here we designed a private passageway fusion receptor, which is composed of a ligand binding domain and a glycosylphosphatidylinositol (GPI) anchoring domain, to be expressed and localized to the surface of CAR T cells independently to the classical CAR T construct. These ligand binding domains preserve high binding affinity towards their cognate ligands and are only expressed on the CAR T cells that have been transduced. Therefore, cytotoxic drugs or immunosuppressants linked to the corresponding targeting ligands are shown to be specifically delivered to these fusion receptor positive CAR T cells for lowering the activity of the over-activated CAR T cells. On the other hand, we discovered that a potent TLR7 agonist is able to enhance the lysis effect of the exhausted CAR T cells in a co-culture model. Serial releasable and non-releasable targeted TLR7 agonists were prepared and tested. Based on these data, we suggest that our secret passageway fusion receptor platform provides a better control of the activity of CAR T cells using the corresponding targeting ligand-payload conjugates in a dose dependent manner and function as a doorway for the delivery of instructions to CAR T cells for versatile purposes.

Cancer immunotherapy has recently made remarkable clinical progress. Adoptive transfer of T-cells engineered with a chimeric antigen receptor (CAR) against CD19 has been successful in treatment of B-cell leukemia. Patient’s T-cells are isolated, activated, transduced with a vector encoding the CAR molecule and then expanded before being transferred back to the patient. However some obstacles restrict its success in solid tumors. This thesis explores different aspects to improve CAR T-cells therapy of cancer. Ex vivo expanded T-cells are usually sensitive to the harsh tumor microenvironment after reinfusion. We developed a novel expansion method for T-cells, named AEP, by using irradiated and preactivated allo-sensitized allogeneic lymphocytes (ASALs) and allogeneic mature dendritic cells (DCs). AEP-expanded T-cells exhibited better survival and cytotoxic efficacy under oxidative and immunosuppressive stress, compared to T-cells expanded with established procedures. Integrating retro/lentivirus (RV/LV) used for CAR expressions randomly integrate in the T-cell genome and has the potential risk of causing insertional mutagenesis. We developed a non-integrating lentiviral (NILV) vector containing a scaffold matrix attachment region (S/MAR) element (NILV-S/MAR) for T-cells transduction. NILV-S/MAR-engineered CAR T-cells display similar cytotoxicity to LV-engineered CAR T-cells with undetectable level of insertional event, which makes them safer than CAR T-cells used in the clinic today. CD19-CAR T-cells have so far been successful for B-cell leukemia but less successful for B-cell lymphomas, which present semi-solid structure with an immunosuppressive microenvironment. We have developed CAR T-cells armed with H. pylorineutrophil-activating protein (HP-NAP). HP-NAP is a major virulence factor and plays important role in T-helper type 1 (Th1) polarizing. NAP-CAR T-cells showed the ability to mature DCs, attract innate immune cells and increase secretion of Th1 cytokines and chemokines, which presumably leads to better CAR T-cell therapy for B-cell lymphoma. Allogeneic-DCs (alloDCs) were used to further alter tumor microenvironment. The premise relies on initiation of an allo-reactive immune response for cytokine and chemokines secretion, as well as stimulation of T-cell response by bringing in tumor-associated antigen. We demonstrated that alloDCs promote migration and activation of immune cells and prolong the survival of tumor-bearing mice by attracting T-cells to tumors and reverse the immune suppressive tumor microenvironment.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia
Este trabalho está dividido em três partes: Dois relatórios de estágio (Parte I e II) ambos com uma análise SWOT identificativa dos pontos fortes, pontos fracos, ameaças e oportunidades encontradas ao longo do período de estágio. A primeira parte refere-se ao estágio em Assuntos Regulamentares de produtos cosméticos na BasePoint Consulting Services e a segunda parte refere-se ao estágio em farmácia comunitária, na Farmácia da Estação. Na terceira e última parte (Parte III) encontra-se a monografia intitulada “Células T CAR e novas abordagens terapêuticas: terapia combinada com vírus oncolíticos e células NK” que fala sobre: A capacidade de as células imunológicas atingirem e eliminarem organismos infeciosos e invasores é estudada há várias décadas. As terapias com células adotivas que expressam recetores de antigénio quiméricos (CAR) geraram muito interesse e investimento nos últimos anos devido aos resultados clínicos sem precedentes. No entanto, a natureza autóloga (específica do paciente) dessa terapia celular, os complexos processos de produção e o risco de doença do enxerto contra o hospedeiro (graft versus host disease (GVHD)) suscitaram preocupações sobre o seu custo e segurança. Da mesma forma, a obtenção de linfócitos suficientes de um doente, muitas vezes com linfodepleção pode representar uma barreira para garantir quantidades clinicamente relevantes de células T CAR. Devido a essas limitações, os investigadores estão a estudar os vírus oncolíticos em terapia combinada, especialmente o adenovírus (Ad) e as células natural killer (NK) como alternativas viáveis e que ofereçam mais segurança, resultados positivos e menos efeitos indesejáveis nos pacientes.
This work is divided into three parts: Two internship reports (Part I and II) both with a SWOT analysis identifying the strengths, weaknesses,threats and opportunities found throughout the internship period. The first part refers to the internship in Regulatory Affairs of cosmetic products at BasePoint Consulting Services and the second part refers to the internship in community pharmacy, at Farmácia da Estação. In the third and last part (Part III) there is the monography entitled “CAR T cells and new therapeutic approaches: combined therapy with oncolytic viruses and NK cells” that talks about: The ability of immune cells to target and eliminate infectious and invading organisms has been studied for several decades. Adoptive cell therapies that express chimeric antigen receptors (CAR) have generated a lot of interest and investment in recent years due to unprecedented clinical results. However, the autologous (patient-specific) nature of this cell therapy, the complex production processes and the risk of graft versus host disease (GVHD) raised concerns about it cost and safety. Likewise, obtaining enough lymphocytes from a sick person, often with lymphodepletion, can represent a barrier to ensure clinically relevant amounts of CAR T cells. Because of these limitations, researchers are studying oncolytic viruses in combination therapy, especially adenovirus (Ad) and natural killer cells (NK) as viable alternatives that offer higher safety, improved efficacy and less undesirable effects on patients.

INTRODUCTION: Chimeric antigen receptor (CAR) T-cell therapy is a new treatment for hematologic malignancies including aggressive B-cell non-Hodgkin’s lymphoma (NHL). Although it has provided an effective treatment option for patients who have few options, CAR T-cell therapy does have many associated toxicities. Prolonged cytopenias are one of the lesser understood toxicities that can affect upwards of 40% of patients. METHODS: In this retrospective study, we reviewed 106 patients who received commercial CAR T-cell therapy between November 2017 and September 2019. Prolonged cytopenias were defined as having absolute neutrophil count (ANC) <1000/mm3, platelets (PLT) <50,000/mm3, and/or hemoglobin (Hgb) <10 g/dL at least once after 30 days post-CAR T-cell infusion. Furthermore, if only one incidence of cytopenia was recorded 30 days post infusion, we required that the patient had to have received either a transfusion or granulocyte-colony stimulating factor (GCSF) after the date of the recorded cytopenic value to be considered a part of the cytopenic cohort. RESULTS: 22 patients met the criteria of having prolonged cytopenias. 64% of the cytopenic cohort had >1 type of prolonged cytopenias. Anemia was the most prevalent affecting 72% of cytopenic patients. The length of time from diagnosis of aggressive B-cell NHL to date of CAR T-cell infusion was found to be positively correlated with an increased risk of developing prolonged cytopenias following CAR T-cell therapy. Additional risk factors associated with an increased risk of delayed cytopenias by univariate analysis included neutropenia on the day of infusion (day 0), a high C-reactive protein (CRP) before lymphodepletion and on day 0, day 0 PLT count, and Hgb before lymphodepletion and on day 0. On multivariate analysis, only high CRP before lymphodepletion was associated with an increased risk of prolonged cytopenias while high ferritin and PLT values on day 0 were associated with not developing prolonged cytopenias. There was no statistical difference between the cytopenic and non-cytopenic cohorts in rates of progression free survival (PFS) and overall survival (OS). Also, no difference was seen in rates or severity of other toxicities between cohorts. 41% of the cytopenic cohort experienced infectious complications post-infusion with one patient dying from their infectious complications. However, there was no association with incidence of infection and prolonged cytopenias when compared to the incidence of infection in the non-cytopenic cohort. CONCLUSIONS: A longer time from diagnosis of aggressive B-cell NHL to time of CAR T-cell infusion was associated with prolonged cytopenias while the number of lines of prior chemotherapy and rate of prior high dose chemotherapy with an autologous stem cell transplant (HD-ASCT) were not associated. It would be valuable to confirm this association and why it is associated since the other two factors were not. We lacked bone marrow biopsies before CAR T-cell infusion and did not have bone marrow biopsies for many patients after CAR T-cell infusion. It would be beneficial to collect data regarding bone marrow biopsies from these time points to highlight any changes that could be related to CAR T-cell therapy. Cytogenetic information of individual patient’s diseases would be worth analyzing to help determine if there are biological factors associated with prolonged cytopenias in response to CAR T-cell therapy. Additional studies should investigate the laboratory values we found to have associations with either cohort to help identify possible predictive values providers could use to identify patients at higher risk of having prolonged cytopenias. There is also a need to see if specific prior chemotherapy regimens increase a patient’s risk of having prolonged cytopenias. Overall, since prolonged cytopenias after CAR T-cell infusions have not been heavily investigated, further investigation is needed to better understand the predictive factors and identify possible mechanisms of prolonged cytopenias seen in CAR T-cell patients.

Dissertação de Mestrado em Biotecnologia Farmacêutica apresentada à Faculdade de Farmácia
Os medicamentos de terapia avançada são um tema de grande destaque da atualidade no setor da Indústria Farmacêutica. O seu caráter inovador tem sido relacionado com novas abordagens terapêuticas e patologias limitantes ou fatais, para as quais, não existia no mercado nenhum tratamento eficiente.Um exemplo são as células CAR-T (Chimeric Antigen Receptor T Cells). Estas surgem como uma abordagem disruptiva no tratamento de patologias com altas taxas de recidivas. Deste tipo foram já aprovados o Yescarta (Axicabtagene ciloleucel) e o Kymriah (Tisagenlecleucel). O Tisagenlecleucel foi o primeiro medicamento com células CAR-T a receber aprovação, em 2017, da U.S. Food & Drug Administration para o tratamento de Leucemia Linfoblástica Aguda percussora de células B refratária ou com mais do que duas recidivas em crianças e jovens adultos até aos 25 anos. Em 2018, este medicamento recebeu a segunda indicação, ao ter sido indicado para adultos com linfoma de grandes células B, refratário ou com recidiva após duas ou mais linhas de terapia sistémica. De igual forma, o Tisagenlecleucel também se encontra aprovado pela Agência Europeia do Medicamento (EMA), tendo obtido aprovação em 2018 para as mesmas duas indicações acima indicadas.Os medicamentos de terapia avançada consistem em medicamentos biológicos com base em células retiradas do doente e de seguida, estas são devidamente modificadas em laboratório, sendo posteriormente difundidas no mesmo doente, de modo a potenciar a resposta imune. Devido a sua complexidade, tanto no mecanismo de ação como no método de produção, é necessário existirem, a nível regulamentar, meios que permitam a sua correta produção e utilização assim como, metodologias que assegurem a segurança após Autorização de Introdução no Mercado (AIM).Recorrendo à informação existente, regulamentar e científica, no presente trabalho são apresentados orientações e requisitos de qualidade para a produção e controlo de medicamentos de terapia avançada com base em células T geneticamente modificadas. É dado especial relevo ao medicamento Kymriah embora quase em simultâneo tenha sido também autorizado o Yescarta. São abordados requisitos de qualidade, desde a adequada recolha de células do doente, a utilização de vetores virais recombinantes para a inserção de genes nas células T obtidas e o processo de apara a sua modificação. Inclui também a avaliação de risco ambiental no contexto específico da libertação deliberada de organismos geneticamente modificados (OGM).Inclui toda a informação relevante na caracterização dos riscos, nomeadamente na enumeração e análise de medidas de gestão e mitigação de risco que devem ser aplicadas com o objetivo de alcançar a qualidade, a segurança e eficácia de longo prazo. Os riscos identificados devem ser devidamente caracterizados e controlados devido à curta duração da sua Autorização de Introdução no Mercado e ao facto de ser um medicamento biológico, com características e propriedades específicas.
Advanced therapy medicinal products (ATMP) are an important topic today for the Pharmaceutical Industry sector. Its innovative character has been related to new therapeutic approaches and limiting or fatal pathologies, for which no efficient treatment existed on the market.One ATMP example are those composed of CAR-T cells (Chimeric Antigen Receptor T Cells). These emerge as a disruptive approach in the treatment of pathologies with high relapse rates. Two CAR-T Cell ATMP, Yescarta (Axicabtagene ciloleucel) and Kymriah (Tisagencleucel), have already been approved. Tisagencleucel was the first CAR-T cell medicinal product to receive approval in 2017 from US Food & Drug Administration for the treatment of refractory B-cell percursor Acute Lymphoblastic Leukemia (ALL) with more than two relapses in children and young adults to 25 years. In 2018, this product received the second indication as it was extended to adults with refractory or relapsed B-cell Lymphoma after two or more lines of systemic therapy. Similarly, Tisagencleucel is also approved by the European Medicines Agency (EMA) in 2018.and was approved for the same indications as above.Advanced therapy medicinal products consist of biological products where T cells are taken from the patient and then appropriately modified in the laboratory and then diffuse into the same patient to enhance the immune response against the selected epitope. Due to their complexity, both in the mechanism of action and in the production method, it is necessary, at a regulatory level, to establish methodologies for its correct production and use, as well to ensure their continuous safety after marketing authorization (AIM).Using existing regulatory and scientific information, this master’s thesis presents guidelines and quality requirements for the production and control of advanced therapy medicinal products based on genetically modified autologous T-cells. Special emphasis is given to the drug Kymriah although almost simultaneously Yescarta has also been authorized. Quality requirements are addressed, from adequate collection of patient cells, their genetic modification, use of viral vectors for the transduction of the gene of interest. It also addresses the environmental risk assessment in the specific context of deliberate release of genetically modified organisms (GMOs). It covers all the relevant information on risk characterization, namely enumeration and analysis of risk management and mitigation measures that should be applied to achieve long-term quality, safety and efficacy. The identified risks must be properly characterized and controlled because of the limited experience of their post marketing surveillance and the fact that is a biological medicinal product with specific characteristics and properties.

BACKGROUND: Clinical facilities are essential components not only for health care delivery systems but also for health care education programs. The clinical learning environment is important in training the future workforce in healthcare. Respiratory therapy education programs face several issues with the need to prepare a proper learning environment in different clinical settings. PURPOSE: The purpose of this study was to determine the perceptions of respiratory therapy students on the learning environment of clinical facilities affiliated with a respiratory therapy program at an urban state university. METHODS: This study used an exploratory research design to evaluate the essential aspects of a clinical learning environment in respiratory therapy education. A self-reporting survey was utilized to gather data from 34 respiratory therapy students regarding their perception about the effectiveness of clinical facilities in respiratory therapy education. The researcher utilized The Clinical Learning Environment, Supervision and Nurse Teacher (CLES+T) evaluation scale that was developed by Sarrikoski et al. (2008). The CLES+T evaluation scale was adapted and modified after a written agreement from the author. The survey included three main domains, which are the clinical learning environment (18 items), the supervision relationship (15 items), and the role of clinical instructors (9 items). Thirty-two students participated in the survey with a response rate of 94.1%. RESULTS: Responses included two groups of students: the second year undergraduate (68.8%) and graduate students (31.3%), with 75% being female participants. The results obtained from the study indicated that both graduate and undergraduate respiratory therapy students gave high mean scores to the learning environment of the clinical facilities, supervisory relationship and the roles of clinical instructors. A statistically significant data was obtained pertaining to the difference of perceptions regarding the multi-dimensional learning between the graduate and undergraduate students. The graduate students evaluated that “the learning situation are multi-dimensional” more than the undergraduate students (p = 0.03). Findings of this study showed that female students had higher ratings than male students in all evaluations of clinical facilities. However, only one dimension of leadership style stating that “the effort of individual employees was appreciated” was statistically significant (p=0.03). The results stating, the presence of a significant percentage of the students with lack of successful private supervision and high percentage of failed supervisory relationship, are in contrast with the fact that clinical learning plays a vital role in the respiratory therapy education. It is also contrasting that majority of the students experienced team supervision, which is against the philosophy and principles of individualization. CONCLUSION: Since respiratory therapy is a practice-based profession, it is essential to integrate clinical education to respiratory care education. Gender and education level may impact students’ perceptions about the learning environment of clinical facilities. This study provides information about areas for improvement in clinical facilities affiliated with a respiratory care education program at an urban university.