Thèses sur le sujet « NK cell therapy »

Monoclonal antibodies (mAbs) have become an important part of therapy for a number of cancers. The first mAb to be approved for clinical use is rituximab, which is currently used for the treatment of various B cell malignancies. Despite its clinical value, the mechanisms in which rituximab induces tumor regression are unclear. Growing evidence suggests that multiple mechanisms involving complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) are involved. However, the direct interactions between CDC and ADCC have yet to be investigated. My studies examine the relationship between complement fixation and the activation of NK cells by utilizing in vitro assays, a syngeneic murine lymphoma model, and clinical samples from patients. Using these systems, I demonstrate that the initiation of the complement cascade inhibits NK cell activation and ADCC induced by rituximab in vitro. I also show that depletion of complement enhances the activation of NK cells and improves the efficacy of mAb therapy in a murine model. Lastly, I demonstrate that NK cell activation correlates with decreased complement activity in patients after rituximab treatment. The studies described in this dissertation have furthered the understanding of the mechanisms involved in antibody therapy. These results have described a novel inhibitory role for complement activity in the anti-tumor responses of mAbs. Furthermore, these findings suggest that strategies to circumvent the inhibitory effect of complement may improve how current mAbs are used and the how mAbs are designed in the future.

Clinical application of natural killer (NK) cells against leukemia is an area of intense investigation. In human leukocyte antigen-mismatched allogeneic hematopoietic stem cell transplantations (HSCT), alloreactive NK cells exert powerful anti-leukemic activity in preventing relapse in the absence of graft-versus-host disease, particularly in acute myeloid leukemia patients. Adoptive transfer of donor NK cells post-HSCT or in non-transplant scenarios may be superior to the currently widely used unmanipulated donor lymphocyte infusion. This concept could be further improved through transfusion of activated NK cells. Significant progress has been made in good manufacturing practice (GMP)-compliant large-scale production of stimulated effectors. However, inherent limitations remain. These include differing yields and compositions of the end-product due to donor variability and inefficient means for cryopreservation. Moreover, the impact of the various novel activation strategies on NK cell biology and in vivo behavior are barely understood. In contrast, reproduction of the thirdparty NK-92 drug from a cryostored GMP-compliant master cell bank is straightforward and efficient. Safety for the application of this highly cytotoxic cell line was demonstrated in first clinical trials. This novel ‘off-theshelf’ product could become a treatment option for a broad patient population. For specific tumor targeting chimeric-antigen-receptor-engineered NK-92 cells have been designed.

Il trapianto di cellule ematopoietiche (HCT) rappresenta una terapia cardine per il trattamento delle neoplasie ematologiche altrimenti incurabili. Tuttavia, la procedura di trapianto può essere gravata dalla recidiva della malattia, dalla malattia del trapianto contro l'ospite (GVHD) e dalle infezioni. Le cellule T e NK che ricostituiscono dopo l'HCT proteggono da infezioni e recidive, ma sono anche coinvolte nella patogenesi della GVHD. Gli obiettivi del mio progetto di dottorato erano di migliorare la comprensione della ricostituzione delle cellule T e NK, utilizzando campioni di donatori sani e pazienti dopo il trapianto e diversi approcci tecnici (citometria a flusso, citometria di massa, sequenziamento dell'RNA e test funzionale ex vivo) e sviluppare nuove strategie immunoterapiche basate sui linfociti T e NK dopo HCT. In primo luogo, abbiamo dimostrato che un ritardo nel recupero dei linfociti T, un rapporto Treg/Tcon più elevato, un'aumentata espressione di PD-1 sui linfociti T di memoria e un arricchimento di cellule NK a fenotipo immaturo sono stati osservati dopo HCT aploidentico (aplo-HCT) con l’utilizzo di ciclofosfamide post-trapianto. Inoltre, la funzione delle cellule NK CD56brightCD16+ immature funzionalmente alterate dopo aplo-HCT può essere migliorata con l’utilizzo dell'interleuchina-15 in vitro. In secondo luogo, abbiamo avviato uno studio di fase I sulle cellule cytokine-induced memory-like (CIML) NK infuse da donatore haploidentico in pazienti con neoplasie mieloidi che hanno avuto una recidiva dopo aplo-HCT. Nei primi 6 pazienti arruolati, l'infusione di cellule CIML-NK ha portato a una rapida espansione in vivo da 10 a 50 volte, che è stata mantenuta per mesi. L'infusione è stata ben tollerata, con febbre e pancitopenia come eventi avversi più comuni. Sulla base di questi dati preliminari, le cellule CIML-NK possono fungere da piattaforma per il trattamento della recidiva post-trapianto delle patologie mieloidi. Infine, ci siamo concentrati sul bilanciamento della risposta dei linfociti T per controllare l’incidenza di GVHD. CD6, un recettore co-stimolatorio dei linfociti T, che aiuta a stabilizzare la sinapsi immunologica tra la cellula T e l'APC, dopo legame con il suo ligando, la molecola di adesione delle cellule leucocitarie attivate (ALCAM). In questo contesto, il legame CD6-ALCAM promuove l'attivazione, la proliferazione e la maturazione delle cellule T. Abbiamo dimostrato che le cellule T CD6 ricostituivano subito dopo il trapianto, con le cellule Treg che esprimono livelli inferiori di CD6 rispetto alle cellule Tcon e cellule T CD8+. Dopo l'insorgenza della aGVHD, l'espressione sia di CD6 che di ALCAM è stata mantenuta. Itolizumab ha inibito l'attivazione e la proliferazione delle cellule T CD4+ e CD8+ nell'ambito di aGVHD in esperimenti ex vivo, senza mediare l'attività citolitica diretta o la citotossicità anticorpo-dipendente. I nostri risultati identificano la via di CD6-ALCAM come potenziale bersaglio per il controllo dell'aGVHD. Uno studio di fase I/II che utilizza itolizumab come trattamento di prima linea in combinazione con steroidi per i pazienti con aGVHD è attualmente in corso. In conclusione, questi risultati evidenziano la necessità di bilanciare le proprietà effettrici e tolerogeniche del sistema immunitario che si ricostituisce dopo HCT e suggeriscono differenti strategie per promuovere o moderare le funzioni delle cellule T e NK.<br>Hematopoietic cell transplantation (HCT) represents a cardinal therapy for hematological malignancy otherwise incurable. However, HCT can be complicated by disease recurrence, graft versus host disease (GVHD) and infections. After HCT, reconstituting T and NK cells protect against infection and relapse, but they are also involved in the pathogenesis of GVHD. The aims of my PhD project were to improve the understanding of T and NK-cell reconstitution, using samples from both healthy donor and patients after transplant and different technical approaches (flow cytometry, mass cytometry, RNA sequencing, and ex vivo functional assay) and to develop post-transplant T and NK cell-based immunotherapeutic strategies. First, we showed that delayed early T-cell recovery, a higher Treg/ Tcon ratio, an increased PD-1 expression on memory T cells, and an enriched immature NK phenotype were observed after haploidentical HCT (haplo-HCT) with post-transplant cyclophosphamide. In addition, the expansion of functionally impaired immature CD56brightCD16+ NK cells after haplo-HCT can be enhanced with in vitro interleukin-15 priming. Second, we initiated a phase I trial of adoptively transferred cytokine-induced memory-like (CIML) NK cells in patients with myeloid malignancies who relapsed after haplo-HCT. In the first 6 enrolled patients, infusion of CIML NK cells led to a rapid 10- to 50-fold in vivo expansion that was sustained over months. The infusion was well tolerated, with fever and pancytopenia as the most common adverse events. Based on these preliminary data, CIML NK cells may serve as a promising platform for the treatment of posttransplant relapse of myeloid disease. Finally, we focused on the balancing of T cell response to control GVHD occurrence. CD6, a pan-T cell co-stimulatory receptor, helps to stabilize the immunological synapse between the T cell and the APC, upon ligation, with its ligand, activated leukocyte cell adhesion molecule (ALCAM). In this context, CD6-ALCAM binding promotes T cell activation, proliferation, maturation. We showed that CD6 T cells reconstituted early after transplant with Treg expressing lower levels of CD6 compared to Tcon and CD8+ T cells. After onset of aGVHD, both CD6 and ALCAM expression was maintained. Itolizumab inhibited CD4+ and CD8+ T cell activation and proliferation in the setting of aGVHD in ex vivo experiments, without mediate direct cytolytic activity or antibody-dependent cytotoxicity. Our results identify the CD6-ALCAM pathway as a potential target for aGVHD control. A phase I/II study using itolizumab as first line treatment in combination with steroids for patients with aGVHD is currently ongoing. In conclusion, these results highlight the need of balancing the effector and tolerogenic properties of the immune system reconstituting after HCT and suggest different strategies to enhance or moderate the T and NK cells functions.

La therapie genique par transfert du gene de la thymidine kinase du virus de l'herpes simplex de type 1 (hsv1-tk) suivi d'un traitement avec la prodrogue ganciclovir (gcv) a ete utilisee pour le traitement de divers cancers. L'efficacite de cette therapie est en partie due a l'existence d'un effet de toxicite de voisinage : le traitement au ganciclovir entraine non seulement la mort des cellules exprimant hsv1-tk mais aussi celle des cellules adjacentes non transfectees. Nous avons entrepris une etude in vitro des mecanismes moleculaires de la toxicite de ce systeme enzyme/prodrogue sur des lignees issues de tumeurs cerebrales. Les resultats obtenus montrent que le couple hsv1-tk/gcv declenche deux reactions cellulaires differentes au stress cytotoxique, selon les lignees utilisees. Dans un premier cas, le traitement au ganciclovir entraine un blocage du cycle cellulaire en phase s rapidement suivi d'une mort cellulaire associant des phenomenes d'apoptose et de necrose. Dans le second cas, le couple hsv1-tk/gcv declenche une mort cellulaire tardive et atypique, sans reel arret du cycle cellulaire. Ce phenomene, associe a l'apparition de cellules geantes et polyploides, est appele catastrophe mitotique et pourrait impliquer la proteine p21. Ces deux modes de mort cellulaire semblent independants de la proteine p53 et impliquent une activation de la proteine pro-apoptotique bax. Le gene represseur de la mort cellulaire bcl-2 inhibe en partie le processus apoptotique declenche par le systeme hsv1-tk/gcv ; ce gene pourrait donc jouer un role dans certaines formes de resistance observees au cours de cette etude. Ainsi, cette etude demontre que le phenotype tumoral va conditionner le destin cellulaire apres le traitement et s'avere donc un determinant critique de la sensibilite a cette approche therapeutique.

Hodgkin Lymphoma (HL) is a B-Cell neoplasia with a favourable outcome in responding patients. However, despite the efficacy of first line therapy about 30% of patients eventually relapse or are refractory (R/R). Recently, the immune checkpoint inhibitor (CI) nivolumab demonstrated good activity in R/R HL patients although the complete response (CR) rate was less than 20%. The efficacy of nivolumab is strictly related to the host degree of immune competence, which is greatly impaired in heavily pre-treated HL patients after autologous stem cell transplantation (ASCT). To enhance the activity of CI, we explored the feasibility of the infusion of autologous lymphocytes (ALI) in combination with the pre-emptive administration of nivolumab, early post-ASCT, in patients affected by R/R HL. Eight patients (median age 29, range 18-56) with active R/R disease, who had already failed at least two chemotherapy lines and Brentuximab, were eligible for the trial. HL patients underwent early lymphocyte apheresis, with a target cell dose of 5x107 CD3+/kg. All patients then received ASCT with FEAM conditioning followed by ALI at a median time of 14 days after infusion, starting with 1x104 CD3+ cells/kg in the first infusion to a maximum of 1x107cells/kg in the fourth and last infusion. Each ALI was followed after 48 hours by the administration of nivolumab 240 mg flat dose. As a control cohort, two patients, in CR after second line chemotherapy, were given ALI only, without nivolumab. No grade 3 or 4 adverse events were recorded. All treated patients achieved negative PET scan after immunotherapy and are alive and disease-free after a median follow-up of 20 months. Two patients did receive allogeneic stem cell transplantation while in CR. Notably, compared to control patients, a faster expansion/reconstitution of highly differentiated NK cells was observed as well as a quicker T-cell recovery. These data suggest the potential role of PD-1 receptor in the direct or indirect control of NK cell maturation/development and, probably, NK anti-tumor activity. Thus, the combination of adoptive cell therapy with CI may represent a novel approach for chemorefractory HL patients.

Natural Killer (NK) cells, members of the innate lymphoid cells (ILCs), are known to play an important role in the defense against foreign cells and abnormal host cells that have arisen due to viral infection or cancer inducing mutations. The typical immune response of NK cells involves the release of cytotoxic granules containing perforin and granzyme, and the secretion of immune-regulatory cytokines such as interferon gamma (IFN-γ). Unlike the adaptive lymphocytes such as T cells and B cells, NK cells do not require prior sensitization, enabling them to initiate an immune response much faster. This unique feature of NK cells is made possible by the utilization of an array of germline encoded receptors; but on the other hand, it limits NK cells ability to respond against rapidly evolving pathogens. NK cells overcome this shortcoming with an antibody-assisted process called antibody dependent cellular cytotoxicity (ADCC). A novel subset of human NK cells, which displays potent and broad antiviral responsiveness in concert with virus-specific antibodies, was recently discovered in cytomegalovirus positive (CMV+) individuals. This NK cell subset, called g-NK cell, was characterized by a deficiency in the expression of FcεRIγ, an adaptor protein that associates with CD16 which enables ADCC. Surprisingly, despite this deficiency, g-NK cells displayed an enhanced ADCC as compared to their conventional counterparts. Furthermore, having a long-lasting memory-like NK-cell phenotype suggests a role for g-NK cells in chronic infections. This study investigates the importance of g-NK-cells in clinical settings, first by investigating whether the presence of g-NK cells is associated with the magnitude of liver disease during chronic hepatitis C virus (HCV) infection. Analysis of g-NK cell proportions and function in the peripheral blood mononuclear cells (PBMCs) of healthy controls and chronic HCV subjects showed that chronic HCV subjects had slightly lower proportions of g-NK cells, while having similarly enhanced ADCC responses compared to conventional NK cells. Notably, among CMV+ chronic HCV patients, lower levels of liver enzymes and fibrosis were found in those possessing g-NK cells. g-NK cells were predominant among the CD56neg NK cell population often found in chronic HCV patients, suggesting their involvement in the immune response against HCV. Rituximab is a chimeric anti-CD20 antibody used to treat B cell lymphoma patients; and studies have suggested that its efficacy is associated with the ADCC potency and CD16 affinity. Since g-NK cells are characterized by their superior ADCC compared to their conventional counterpart, I decided to investigate whether the presence of g-NK cells can improve the effectiveness of rituximab against malignant B cells in the context of lymphoma and leukemia. The analysis of g-NK cells’ ADCC response against rituximab-coated lymphoma cell lines and B cells from a CLL patient indicated a superior ADCC by g-NK cells compared to their conventional NK cell counterparts. Taken together, for the first time, my findings indicate that the presence of g-NK cells in CMV+ individuals is associated with milder liver disease in chronic HCV infection. In addition, an enhanced ADCC response by g-NK cells upon encountering rituximab coated target cells suggests the beneficial roles of g-NK cells, and opens an avenue for novel therapeutic approaches where g-NK cells can be utilized to treat persistent diseases such as chronic viral infection and cancer.

Natural Killer cells (NK) are critical components of the innate immune system. Often referred to by their morphology, these large granular lymphocytes (LGLs) are bone marrow-derived lymphocytes and can be found throughout the body. NK cells reside in the liver, lymph nodes, spleen, thymus, and mucosal-associated lymphoid tissues (MALT). Importantly, NK cells also circulate throughout the blood where they function as surveyors of the body and are armed to eliminate malignant, infected, damaged, or foreign cells. NK cells function by a dual receptor system. That is, NK receptors are broadly categorized as inhibitory or activating. It is a fine balance, or lack thereof, that dictates the function of an NK cell. Unlike their T and B cell adaptive counterparts, NK cell receptors (NKR) are germline encoded and do not undergo gene rearrangement. NKRs are expressed in a variegated but overlapping fashion such that different cell subsets in the NK compartment elaborate different combinations of activating and inhibitory NKR. Varying the array of NKRs used by each subset increases the potential specificities of the NK compartment, while retaining tolerance to self. Thus, a diverse and balanced NK cell receptor repertoire (NKRR) is extremely important in order for this lineage to respond to various immunologic challenges and to do so in a normal, effective manner. As we have previously shown, aberrations in the expression of NKRs or downstream signaling can lead to severe immune deficiency, as observed in SHIP-deficient mice. We also showed that in the absence of SHIP-1, 2B4 becomes highly upregulated, functioning as a dominant inhibitory receptor and rendering the SHIP-1-deficient NK cell unresponsive to complex tumor targets. Traditionally MHC-I inhibitory ligands are largely responsible for the regulation of NK function. However, we show here that 2B4, which mediates MHC-I-independent inhibition, is required for formation of a normal NKRR, NK homeostasis, and effector functions. Moreover, in the absence of 2B4 and SHIP-1, NK cells have improper licensing, or education. In addition to SHIP-1 and 2B4 we show that the nature of the MHC-I ligands also play a significant role in repertoire formation, NK effector functions, and NK cell education. As described above, NK cells are critical components of the immune system. Understanding how NK cell biology and function are regulated, or affected in the context of pathology is of high significance. NK function is often severely impaired in a diseased state, and more importantly, NK cells are frequently adversely affected by the treatments themselves. Here we sought out to determine the effects of an immunomodulating drug, lenalidomide, on the biology and function of healthy NK cells. Lenalidomide is a unique drug that displays immune enhancing functions yet can be cytotoxic to tumor cells. However, lenalidomide treatment can result in immune suppression and severe cytopenia, and has the ability to impair NK viability. We show here that if used in combination with cytokine treatment (e.g. IL-2 or IL-15), many of these negative affects can be overcome. Furthermore, we show that lenalidomide treatment results in what appears to be an NK activating phenotype with a down-modulation of inhibitory KIRs and upregulation of CD16. Lenalidomide also leads to a sustained and robust activation of STAT5 and consequential increase in perforin and granzyme B. Finally, we find that treatment with lenalidomide in combination with IL-2 or IL-15 enhances the expression of IL-Rβ and IL-2Rγ chains, a presumed mechanism of action, which may provide a positive feedback loop. These findings have important clinical application. We propose that using lenalidomide in combination with IL-15 can augment its immune activating effects, while minimizing unwanted cytopenias.

Multiple myeloma (MM) is an incurable plasma cell malignancy. NK cells have demonstrated anti-MM activity in allogeneic transplants and donor lymphocyte infusions, and may provide a more effective therapy for MM. This work demonstrates cytotoxicity of NK-92 and KHYG-1 against MM cells in chromium release and flow cytometry cytotoxicity assays. At a 10:1 effector to target ratio, the cytotoxicity of NK cell lines against MM cells is 50-90%. Blocking NKp30 significantly reduces the cytotoxicity of NK-92 and KHYG-1, while blocking NKG2D and DNAM-1 only reduces the cytotoxicity of NK-92. Notably, NK-92 and KHYG-1 have shown preferential cytotoxicity against the clonogenic population, killing 89-99% in a methylcellulose cytotoxicity assay. Preliminary results in a xenograft bioluminescent mouse model show that NK-92, but not KHYG-1, reduces the tumor burden detected by bioluminescence imaging and bone marrow engraftment by flow cytometry. Therefore, NK cell lines may offer a more effective therapy for MM.

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia<br>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.<br>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.

<div>Cancer is responsible for the second highest cause of death in the United States, and lung cancer accounts for 13% of new cancer diagnoses, with the highest rate of cancer death at 24%. Almost 85% of these cases represent non-small cell lung cancer (NSCLC), which includes lung adenocarcinoma, the most common NSCLC subtype. Traditional cancer treatments often only temporarily stop the spread of the disease, but immunotherapies, which are becoming a standard of care, are much more promising. Natural killer (NK) cells are powerful effectors of innate immunity, and genetically engineered NK cells as immunotherapies have had encouraging clinical responses in the treatment of various cancers. However, more progress is needed for solid tumor treatment, especially for lung adenocarcinoma. The activation of cancer-associated ectoenzymes, CD39 and CD73 catalyze the phosphorylation of ATP to AMP to produce extracellular adenosine (ADO), which is a highly immunosuppressive mechanism contributing to the pathogenesis of solid tumors. Understanding adenosine effects on NK cells will help develop more robust immunotherapeutic treatments to improve cytotoxicity against solid tumors. Here, we established that tumor microenvironment ADO results in impaired metabolic and anti-tumor functions of cytokine-primed NK cells. Specifically, peripheral blood-derived NK cells stimulated with IL-2, IL-15, or a combination of IL-12 and IL-15 showed suppressed anti-tumor immunity due to ADO. This was observed by the downregulation of activation receptor expression, cytotoxicity inhibition, impairment of metabolic activity, and alterations in gene expression. To target ADO-producing CD73 on cancer cells, we redirected NK cells by fusing CD73 ScFv with intracellular and transmembrane regions of NK cell specific signaling components derived from FCyRIIIa (CD16). Engineered NK cells were shown to be cytotoxic against lung adenocarcinoma <i>in vitro</i> and impede tumor growth in a lung adenocarcinoma mouse model <i>in vivo</i>. Engineered cells also had higher levels of degranulation and cytokine release, as well as more infiltration into tumors and longer survival time in mice. In summary, the microenvironment of solid tumors is highly immunosupressive, and redirecting NK cell function using a NK-specific anti-CD73 targeting construct will help to promote anti-tumor immunity and</div><div>inhibit cancer growth for a potentially powerful new immunotherapy against solid tumors.</div>

Relatório de Estágio do Mestrado Integrado em Ciências Farmacêuticas apresentado à Faculdade de Farmácia<br>A área da oncologia é e continuará a ser uma das que mais expansão continua a ter na ciência. É também nesta que se vão fazendo mais esforços e mais descobertas para que possam ser tratadas patologias deste tipo, de forma cada vez mais eficaz e com maior segurança e qualidade para os doentes. Assim, surgem as células CAR-NK em resposta a patologias oncológicas, como uma nova possibilidade de tratamento das mesmas. É sobre esta possibilidade que se debruça esta monografia e o objectivo da mesma. Estas células aliam a capacidade antitumoral das células NK, com o reconhecimento de células que não pertencem ao self da memória imunológica que o organismo possui, à introdução de recetores específicos para antigénios apresentados por células tumorais, para que desta forma possa ser feito um reconhecimento mais específico das mesmas e a sua consequente destruição. Desta forma, o tratamento será mais eficaz na medida em que será mais específico e dirigido apenas às células tumorais e não às células saudáveis, bem como com um grau de efeitos adversos muito mais reduzido ou quase inexistente. Contudo, a produção e aplicação deste tipo de tratamento celular apresenta ainda alguns desafios. É necessário perceber tudo o que será necessário fornecer às células, e qual a melhor forma de estas serem administradas, se sozinhas ou em conjunto com citocinas ou outras moléculas anticancerígenas. Tudo isto para que o efeito obtido possa ser máximo e, como tal, a eficácia do tratamento também. Estas são algumas dificuldades que já foram previamente enfrentadas com a produção de células CAR-T. Quando em comparação, as células CAR-NK apresentam várias vantagens em relação às suas antecessoras no tratamento celular de cancros, pois estas, não estão dependentes de um reconhecimento pelo complexo major de histocompatibilidade, pelo que não existe o risco de uma rejeição ou desenvolvimento de GVHD. Isto leva à possibilidade, extremamente relevante, de estas células poderem ser produzidas de acordo com normas GMP, para que possam ser utilizadas num formato off-the-shelf, tal como já acontece com outro tipo de moléculas.Existem já vários ensaios clínicos em curso, para diversos tipos de cancro e com recetores CAR-NK diferentes e aplicados de forma diferente, para que se possam desenvolver variados tipos de células de forma a responder a uma maior variedade de patologias. Existem também produtos comercializados para utilização em doentes que deles necessitem, tal como as células FT596 da Fate Therapeutics. No futuro, estas células poderão ser utilizadas não só para patologias oncológicas, mas também para patologias infeciosas e até disfunções imunológicas que estão associadas a disfunções genéticas com alteração da produção proteica, por exemplo.<br>The area of oncology is and will continue to be one of the most expanding areas in science. Efforts are being made and discoveries are being made to treat these diseases more effectively and with greater safety and security as well as quality for the patients who need them. Thus, CAR-NK cells appear in response to oncological pathologies as a new possibility of treatment. The objective of this monography is exactly to talk about this possibility. These cells combine the anti-tumor capacity of NK cells, with the recognition of non-self cells with the introduction of antigen-specific receptors presented by tumor cells, which leads to a more specific recognition of them and their consequent destruction. Thus, the treatment will be more effective due to be more specific as it only affect tumor cells and not healthy cells. However, the production and application of this type of cellular treatment still presents some challenges because it is necessary to understand everything that will be needed to provide to the cells, as well as how they can be administered, as a single administration or in combination with cytokines or other anticancer molecules, so that the effect obtained may be maximum. These are some difficulties that have also been faced with the production of CAR-T cells. When compared, CAR-NK cells have several advantages over their predecessors in the cellular treatment of cancers because they are not dependent on recognition by the major histocompatibility complex, for example, so there is no risk of rejection or development of GVHD. This leads to the extremely relevant possibility that these cells can be produced according to GMP standards so that they can be used in an off-the-shelf format, as is the case of other molecules.Several clinical trials are already underway for different types of cancer and with different and differently applied CAR-NK receptors, so that different cell types can be developed to respond to a wider variety of conditions. There are already products available for use in patients who need them, such as Fate Therapeutics FT596 cells.In the future, these cells may be used not only for oncological conditions, but also for infectious conditions and even immune dysfunctions that are associated with cell malfunction or altered protein production, for example.