Chimeric Antigen Receptor T-cell Therapy: Bioengineered immunocellular approach to Acute Lymphoblastic Leukaemia

Treballs Finals de Grau de Farmàcia, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, 2019Acute Lymphoblastic Leukaemia (ALL) is one of the most prevalent cancers in children and an almost fatal disease for adults. There is an urgent need to develop new drugs because of the lack of good treatments for these patients, especially those with relapsed or refractory clinical disease. Chimeric Antigen Receptor T-cells (CARTs) is a potent cellular cancer therapy consisting of autologous patient’s T lymphocytes, reprogrammed through gene editing to express a surface receptor against a particular tumour antigen. Autologous T cells transduced with anti-CD19 receptors may become a breakthrough for the treatment of B-cell ALL, since they bypass the need for antigen presentation usually affected by tumour immunosuppressive microenvironment. Different CARs have been designed, during the last years, and several institutions have tested its efficacy in clinical studies. Roughly, treated patients presented high remission rates with long-term durations, thus becoming a relevant alternative for those otherwise untreatable patients and granting commercial authorisation by the leading two regulatory agencies: Food and Drug Administration and European Medicines Agency in 2017 and 2018, respectively. This intersection between adoptive cell therapy, bioengineering tools and immunotherapy may have applications beyond cancer such as in autoimmunity and infectious diseases. The primary goal of this review is to present the current evidence of the efficacy of CART treatment and the main problems related to its toxicity and manufacturing, as well as its therapeutic value for ALL.La Leucèmia Limfoblàstica Aguda (LLA) és el tipus de càncer més prevalent en nens i una malaltia pràcticament fatal en adults. L’absència de noves teràpies pel tractament de pacients amb malaltia refractària o en recidiva posa de manifest la necessitat urgent de nous agents amb una eficàcia rellevant. Les cèl·lules T amb receptor d’antigen quimèric són una teràpia immunocel·lular potent que consisteix en la reprogramació dels limfòcits T del pacient a través de l’edició gènica per expressar un receptor a la seva superfície que reconegui un antigen tumoral concret. Les cèl·lules T autòlogues transduides amb receptors anti-CD19 poden arribar a suposar un gran avenç pel tractament de la LLA de cèl·lules B, ja que és capaç d’evitar la necessitat de presentació d’antigen que acostuma a estar inhibida pel microambient immunosupressor del tumor. Al llarg dels anys, diferents dissenys i generacions han sigut estudi de diferents institucions. Pràcticament tots ells presenten taxes de remissió elevades a llarga durada, representant una alternativa rellevant per aquells pacients sense més possibilitats terapèutiques i permetent també l’autorització de comerç per part de les dues agències regulatòries principals: L’Administració d’Aliments i Medicaments dels EEUU al 2017 i l’Agència Europea del Medicament al 2018. Aquesta intersecció entre la teràpia adoptiva de cèl·lules, les eines de bioenginyeria i la immunoteràpia podrien tenir aplicacions més enllà del càncer en autoimmunitat i malalties infecciones. L’exposició de les evidències actuals i les principals característiques pel que fa a toxicitat, eficàcia i fabricació, així com el seu posicionament concret per la LLA, són qüestions tractades en aquesta revisió bibliogràfica

Il-15 enhances the persistence and function of bcma-targeting car-t cells compared to il-2 or il-15/il-7 by limiting car-t cell dysfunction and differentiation

Chimeric antigen receptor (CAR)-T cell immunotherapy has revolutionized the treatment of B-lymphoid malignancies. For multiple myeloma (MM), B-cell maturation antigen (BCMA)-targeted CAR-T cells have achieved outstanding complete response rates, but unfortunately, patients often relapse within a year of receiving the therapy. Increased persistence and reduced dysfunction are crucial features that enhance the durability of CAR-T cell responses. One of the factors that influence CAR-T cell in vivo longevity and loss of function, but which has not yet been extensively studied for BCMA-directed CAR-T cells, are the cytokines used during their production. We here compared the impact of IL-2, IL-15 and a combination of IL-15/IL-7 on the phenotype and function of ARI2h, an academic BCMA-directed CAR-T cell that is currently being administered to MM patients. For this study, flow cytometry, in vitro cytotoxicity assays and analysis of cytokine release were performed. In addition, ARI2h cells expanded with IL-2, IL-15, or IL-15/IL-7 were injected into MM tumor-bearing mice to assess their in vivo efficacy. We demonstrated that each of the cytokine conditions was suitable for the expansion of ARI2h cells, with clear in vitro activity. Strikingly, however, IL-15-produced ARI2h cells had improved in vivo efficacy and persistence. When explored further, it was found that IL-15 drove a less-differentiated ARI2h phenotype, ameliorated parameters related to CAR-T cell dysfunction, and lowered the release of cytokines potentially involved in cytokine release syndrome and MM progression. Moreover, we observed that IL-15 was less potent in inducing T cell senescence and DNA damage accumulation, both of which may contribute to an unfavorable CAR-T cell phenotype. These findings show the superiority of IL-15 to IL-2 and IL-15/IL-7 in the quality of anti-BCMA CAR-T cells, particularly their efficacy and persistence, and as such, could improve the duration of responses if applied to the clinical production of CAR-T cells for patients

Jardins per a la salut

Facultat de Farmàcia, Universitat de Barcelona. Ensenyament: Grau de Farmàcia. Assignatura: Botànica farmacèutica. Curs: 2014-2015. Coordinadors: Joan Simon, Cèsar Blanché i Maria Bosch.Els materials que aquí es presenten són el recull de les fitxes botàniques de 128 espècies presents en el Jardí Ferran Soldevila de l’Edifici Històric de la UB. Els treballs han estat realitzats manera individual per part dels estudiants dels grups M-3 i T-1 de l’assignatura Botànica Farmacèutica durant els mesos de febrer a maig del curs 2014-15 com a resultat final del Projecte d’Innovació Docent «Jardins per a la salut: aprenentatge servei a Botànica farmacèutica» (codi 2014PID-UB/054). Tots els treballs s’han dut a terme a través de la plataforma de GoogleDocs i han estat tutoritzats pels professors de l’assignatura. L’objectiu principal de l’activitat ha estat fomentar l’aprenentatge autònom i col·laboratiu en Botànica farmacèutica. També s’ha pretès motivar els estudiants a través del retorn de part del seu esforç a la societat a través d’una experiència d’Aprenentatge-Servei, deixant disponible finalment el treball dels estudiants per a poder ser consultable a través d’una Web pública amb la possibilitat de poder-ho fer in-situ en el propi jardí mitjançant codis QR amb un smartphone

Enhancing CAR-T Cell Therapy for Hematological Malignancies through Dual Targeting and Combination with NK Cells

[eng] CAR-T therapy has represented a milestone for the treatment of hematological patients in relapse or refractory to conventional treatments, leading to the approval by regulatory agencies of four CAR-T products against CD19 for the treatment of patients with acute lymphoblastic leukemia (ALL) or non-Hodgkin lymphoma (NHL), and two CAR-T products against BCMA for the treatment of multiple myeloma (MM). Despite promising response rates, some patients have been observed to relapse after CAR- therapy T. The main escape mechanisms vary depending on the disease. In NHL, most patients relapse with loss of antigenic target or decreased antigen levels, while in MM, it is mainly due to the lack of persistence of CAR-T cells. The main objective of this thesis is to prevent these mechanisms of resistance to CAR-T therapy through two projects, one focused on NHL and the other on MM. On the one hand, we hypothesized that the generation of dual CAR-T cells, directed against CD19 and BCMA antigens, could decrease selective pressure on a single antigen and improve CAR-T therapy in NHL. On the other hand, we hypothesize that the combination of CAR-T cells against BCMA with other immune cells such as NK cells derived from umbilical cord blood, could improve their antitumor efficacy synergistically. As CAR constructs, we used ARI0001 (CAR-CD19) and ARI0002h (CAR-BCMA), both academically developed at the Hospital Clínic de Barcelona. In the first part, different dual CAR-T strategies are developed: 1) co-transduction with two lentiviral vectors, 2) Mixture of monospecific CAR-T cells, 3) bicistronic lentiviral vector that codes for both CARs, 4) a single CAR receptor with dual specificity with two extracellular domains in tandem or loop conformation. For in vitro models, different tumor lines representative of the hematological diseases of interest are used. For in vivo models, immunosuppressed mice (NSG) are used in which a systemic model of disease is developed through intravenous injection of tumor cells for subsequent treatment. First, it was found that co-transduction (ARI0003) is the dual strategy that allows us to have CAR-T cells with better expression of membrane CAR receptors compared to the bicistronic, tandem and loop strategy. In addition, co-transduced cells are especially effective in models with low CD19 antigen expression, both in vitro and in vivo. This set of preclinical results has been submitted to the Spanish Agency for Medicines and Medical Devices to request approval for a clinical trial to test ARI0003 in patients with NHL, including those who relapsed after receiving a CD19-directed CAR-T. In a second part, we demonstrated that the combination between CAR-T BCMA and cord NK cells increased antitumor efficacy in MM models, achieving synergistic activity between both immune cells, especially at early times. In the presence of NK cells, CAR-T cells migrated more rapidly to tumor cells forming attack clusters earlier, thus eliminating tumor cells more efficiently. In addition, we observed a lower expression of markers of depletion in the presence of NK cells, suggesting that the combination of CAR-T cells with NK could reduce the depletion of CAR-T cells.[spa] La terapia CAR-T ha representado un hito para el tratamiento de pacientes hematológicos en recaída o refractarios a tratamientos convencionales, dando lugar a la aprobación por las agencias reguladoras de cuatro productos CAR-T contra CD19 para el tratamiento de pacientes con leucemia aguda linfoblástica (LAL) o linfoma no Hodgkin (LNH), y dos productos CAR-T contra BCMA para el tratamiento de mieloma múltiple (MM). A pesar de las prometedoras tasas de respuesta, se ha observado que algunos pacientes recaen tras la terapia CAR- T. Los mecanismos de escape mayoritarios varían según la enfermedad. En LNH, la mayoría de pacientes recaen con pérdida de diana antigénica o niveles disminuidos de antígeno, mientras que, en MM, se debe sobre todo a la falta de persistencia de las células CAR-T. El objetivo principal de esta tesis es prevenir estos mecanismos de resistencia a la terapia CAR-T a través de dos proyectos, uno centrado en LNH y otro en MM. Por una parte, hipotetizamos que la generación de células CAR-T duales, dirigidas contra los antígenos CD19 y BCMA, podría disminuir la presión selectiva sobre un solo antígeno y mejorar la terapia CAR-T en LNH. Por otra parte, hipotetizamos que la combinación de células CAR-T contra BCMA con otras células inmunes como las células NK derivadas de sangre de cordón umbilical, podría mejorar su eficacia antitumoral de manera sinérgica. Como constructos CAR, utilizamos ARI0001 (CAR-CD19) y ARI0002h (CAR-BCMA), ambos desarrollados académicamente en el Hospital Clínic de Barcelona. En una primera parte, se desarrollan diferentes estrategias CAR-T duales: 1) co- transducción con dos vectores lentivirales, 2) Mezcla de células CAR-T monoespecificas, 3) vector lentiviral bicistrónico que codifica para ambos CARs, 4) un solo receptor CAR con especificidad dual con dos dominios extracelulares en conformación tándem o loop. Para los modelos in vitro se utilizan diferentes líneas tumorales representativas de las enfermedades hematológicas de interés. Para los modelos in vivo se utilizan ratones inmunodeprimidos (NSG) en los que se desarrolla un modelo sistémico de enfermedad a través de inyección intravenosa de las células tumorales para su posterior tratamiento. Primero, se encontró que la co-transducción (ARI0003) es la estrategia dual que nos permite tener células CAR-T con una mejor expresión de los receptores CAR en membrana en comparación con la estrategia de bicistronic, tándem y loop. Además, las células co-transducidas son especialmente efectivas en modelos con baja expresión antigénica de CD19, tanto in vitro como in vivo. Este conjunto de resultados preclínicos se ha presentado a la Agencia Española de Medicamentos y Productos sanitarios para solicitar la aprobación de un ensayo clínico para testar el ARI0003 en pacientes con LNH, incluyendo aquellos recaídos tras recibir un CAR-T dirigido a CD19. En una segunda parte, demostramos que la combinación entre CAR-T BCMA y células NK de cordón aumentaba la eficacia antitumoral en modelos de MM, logrando una actividad sinérgica entre ambas células inmunes, especialmente a tiempos tempranos. En presencia de células NK, las células CAR-T migraron más rápidamente a las células tumorales formando clústers de ataque de forma más temprana, eliminando así a las células tumorales más eficientemente. Además, observamos una menor expresión de marcadores de agotamiento en presencia de células NK, sugiriendo que la combinación de células CAR-T con NK podría reducir el agotamiento de las células CAR-T

Senescence in the Development and Response to Cancer with Immunotherapy: A Double-Edged Sword

Cellular senescence was first described as a physiological tumor cell suppressor mechanism that leads to cell growth arrest with production of the senescence-associated secretory phenotype known as SASP. The main role of SASP in physiological conditions is to attract immune cells to clear senescent cells avoiding tumor development. However, senescence can be damage-associated and, depending on the nature of these stimuli, additional types of senescence have been described. In the context of cancer, damage-associated senescence has been described as a consequence of chemotherapy treatments that were initially thought of as a tumor suppressor mechanism. However, in certain contexts, senescence after chemotherapy can promote cancer progression, especially when immune cells become senescent and cannot clear senescent tumor cells. Moreover, aging itself leads to continuous inflammaging and immunosenescence which are responsible for rewiring immune cells to become defective in their functionality. Here, we define different types of senescence, pathways that activate them, and functions of SASP in these events. Additionally, we describe the role of senescence in cancer and its treatments, including how aging and chemotherapy contribute to senescence in tumor cells, before focusing on immune cell senescence and its role in cancer. Finally, we discuss potential therapeutic interventions to reverse cell senescence

NK cells enhance CAR-T cell antitumor efficacy by enhancing immune/tumor cells cluster formation and improving CAR-T cell fitness

Background Chimeric antigen receptor (CAR)-T cell immunotherapy has modified the concept of treatment in hematological malignancies. In comparison with pediatric patients, where responses are maintained over many years, older patients, such as those with non-Hodgkin's lymphoma (NHL) and multiple myeloma (MM), present lower persistence of CAR-T cells that might be due to decreased fitness of T cells acquired with aging. Moreover, cord blood derived-NK cells (CB-NKs) and CAR-NK cells derived from CB-NK can be used ' off-the-shelf' as immune cells with antitumor properties for the treatment of cancer patients. However, to date, clinical studies have only demonstrated the safety of these therapies but not optimal efficacy. To confront the shortcomings of each therapy, we devised a novel approach consisting of simultaneous (CAR-)NK cell and CAR-T cell administration. In this setting, NK cells demonstrate an important immunoregulation of T cells that could be exploited to enhance the efficacy of CAR-T cells. Methods A combinatorial treatment based on either CAR-T and CAR-NK cells or CB-NK and CAR-T cells in two models of NHL and MM was performed. Antitumor efficacy was analyzed in vitro and in vivo, and parameters related to early activation, exhaustion and senescence of T cells were analyzed. Results We show that CAR-NK cells derived from CB-NK are only effective at high doses (high E:T ratio) and that their activity rapidly decreases over time in comparison with CAR-T cells. In comparison and to exploit the potential of ' off-the-shelf' CB-NK, we demonstrate that a low number of CB-NK in the CAR-T cell product promotes an early activation of CAR-T cells and their migration to MM cells leading to enhanced anti-MM efficacy. Moreover, cytokines related to CRS development were not increased, and importantly, CB-NK enhanced the fitness of both CAR pos and CAR neg T cells, promoting lower levels of exhaustion and senescence. Conclusion This study demonstrates a relevant immunoregulatory role of CB-NK collaborating with CAR-T cells to enhance their antitumor activity. A novel and different approach to consider in CAR-T cell immunotherapy studies is presented here with the goal to enhance the efficacy of the treatmen

Expanding a precision medicine platform for malignant peripheral nerve sheath tumors: New patient‐derived orthotopic xenografts, cell lines and tumor entities

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive soft‐tissue sarcomas with a poor survival rate, presenting either sporadically or in the context of neurofibromatosis type 1 (NF1). The histological diagnosis of MPNSTs can be challenging, with different tumors exhibiting great histological and marker expression overlap. This heterogeneity could be partly responsible for the observed disparity in treatment response due to the inherent diversity of the preclinical models used. For several years, our group has been generating a large patient‐derived orthotopic xenograft (PDOX) MPNST platform for identifying new precision medicine treatments. Herein, we describe the expansion of this platform using six primary tumors clinically diagnosed as MPNSTs, from which we obtained six additional PDOX mouse models and three cell lines, thus generating three pairs of in vitro–in vivo models. We extensively characterized these tumors and derived preclinical models, including genomic, epigenomic, and histological analyses. Tumors were reclassified after these analyses: three remained as MPNSTs (two being classic MPNSTs), one was a melanoma, another was a neurotrophic tyrosine receptor kinase (NTRK)‐rearranged spindle cell neoplasm, and, finally, the last was an unclassifiable tumor bearing neurofibromin‐2 (NF2) inactivation, a neuroblastoma RAS viral oncogene homolog (NRAS) oncogenic mutation, and a SWI/SNF‐related matrix‐associated actin‐dependent regulator of chromatin (SMARCA4) heterozygous truncated variant. New cell lines and PDOXs faithfully recapitulated histology, marker expression, and genomic characteristics of the primary tumors. The diversity in tumor identity and their specific associated genomic alterations impacted treatment responses obtained when we used the new cell lines for testing compounds against known altered pathways in MPNSTs. In summary, we present here an extension of our MPNST precision medicine platform, with new PDOXs and cell lines, including tumor entities confounded as MPNSTs in a real clinical scenario. This platform may constitute a useful tool for obtaining correct preclinical information to guide MPNST clinical trials