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Chimeric antigen receptor (CAR) engineering of natural killer (NK) cells has shown promising results in early-phase clinical studies. However, advancing CAR-NK cell therapeutic efficacy is imperative. In this study, we investigated the impact of a fourth-generation CD19-targeted CAR (CAR.19) coexpressing IL-27 on NK-92 cells. We observed a significant improvement in NK-92 cell proliferation and cytotoxicity activity against B-cell cancer cell lines, both in vitro and in a xenograft mouse B-cell lymphoma model. Our systematic transcriptome analysis of the activated NK-92 CAR variants further supports the potential of IL-27 in fourth-generation CARs to overcome limitations of NK cell-based targeted tumor therapies by providing essential growth and activation signals. Integrating IL-27 into CAR-NK cells emerges as a promising strategy to enhance their therapeutic potential and elicit robust responses against cancer cells. These findings contribute substantially to the mounting evidence supporting the potential of fourth-generation CAR engineering in advancing NK cell-based immunotherapies.
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Introduction: Natural killer 92 (NK-92) cells are an attractive therapeutic approach as alternative chimeric antigen receptor (CAR) carriers, different from T cells, once they can be used in the allogeneic setting. The modest in vivo outcomes observed with NK-92 cells continue to present hurdles in successfully translating NK-92 cell therapies into clinical applications. Adoptive transfer of CAR-NK-92 cells holds out the promise of therapeutic benefit at a lower rate of adverse events due to the absence of GvHD and cytokine release syndrome. However, it has not achieved breakthrough clinical results yet, and further improvement of CAR-NK-92 cells is necessary. Methods: In this study, we conducted a comparative analysis between CD19-targeted CAR (CAR.19) co-expressing IL-15 (CAR.19-IL15) with IL-15/IL-15Rα (CAR.19-IL15/IL15Rα) to promote NK cell proliferation, activation, and cytotoxic activity against B-cell leukemia. CAR constructs were cloned into lentiviral vector and transduced into NK-92 cell line. Potency of CAR-NK cells were assessed against CD19-expressing cell lines NALM-6 or Raji in vitro and in vivo in a murine model. Tumor burden was measured by bioluminescence. Results: We demonstrated that a fourth- generation CD19-targeted CAR (CAR.19) co-expressing IL-15 linked to its receptor IL-15/IL-15Rα (CAR.19-IL-15/IL-15Rα) significantly enhanced NK-92 cell proliferation, proinflammatory cytokine secretion, and cytotoxic activity against B-cell cancer cell lines in vitro and in a xenograft mouse model. Conclusion: Together with the results of the systematic analysis of the transcriptome of activated NK-92 CAR variants, this supports the notion that IL-15/IL-15Rα comprising fourth-generation CARs may overcome the limitations of NK-92 cell-based targeted tumor therapies in vivo by providing the necessary growth and activation signals.
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Receptores de Antígenos Quiméricos , Humanos , Camundongos , Animais , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Interleucina-15/genética , Interleucina-15/metabolismo , Linhagem Celular Tumoral , Células Matadoras Naturais , Antígenos CD19 , Proliferação de CélulasRESUMO
BACKGROUND AIMS: Amidst the success of cell therapy for the treatment of onco-hematological diseases, the first recently Food and Drug Administration-approved gene therapy product for patients with transfusion-dependent ß-thalassemia (TDT) indicates the feasibility of gene therapy as curative for genetic hematologic disorders. This work analyzed the current-world scenario of clinical trials involving gene therapy for ß-hemoglobinopathies. METHODS: Eighteen trials for patients with sickle cell disease (SCD) and 24 for patients with TDT were analyzed. RESULTS: Most are phase 1 and 2 trials, funded by the industry and are currently recruiting volunteers. Treatment strategies for both diseases are fetal hemoglobin induction (52.4%); addition of wild-type or therapeutic ß-globin gene (38.1%) and correction of mutations (9,5%). Gene editing (52.4%) and gene addition (40.5%) are the two most used techniques. The United States and France are the countries with the greatest number of clinical trials centers for SCD, with 83.1% and 4.2%, respectively. The United States (41.1%), China (26%) and Italy (6.8%) lead TDT trials centers. CONCLUSIONS: Geographic trial concentration indicates the high costs of this technology, logistical issues and social challenges that need to be overcome for gene therapy to reach low- and middle-income countries where SCD and TDT are prevalent and where they most impact the patient's health.
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Anemia Falciforme , Hemoglobinopatias , Humanos , Hemoglobinopatias/genética , Hemoglobinopatias/terapia , Anemia Falciforme/genética , Anemia Falciforme/terapia , Terapia Baseada em Transplante de Células e Tecidos , China , Terapia GenéticaRESUMO
Cell therapy is an innovative approach that permits numerous possibilities in the field of cancer treatment. CAR-T cells have been successfully used in patients with hematologic relapsed/refractory. However, the need for autologous sources for T cells is still a major drawback. CAR-NK cells have emerged as a promising resource using allogeneic cells that could be established as an off-the-shelf treatment. NK cells can be obtained from various sources, such as peripheral blood (PB), bone marrow, umbilical cord blood (CB), and induced pluripotent stem cells (iPSC), as well as cell lines. Genetic engineering of NK cells to express different CAR constructs for hematological cancers and solid tumors has shown promising preclinical results and they are currently being explored in multiple clinical trials. Several strategies have been employed to improve CAR-NK-cell expansion and cytotoxicity efficiency. In this article, we review the latest achievements and progress made in the field of CAR-NK-cell therapy.
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BACKGROUND AIMS: Lentiviral vectors (LVs) have been used extensively in gene therapy protocols because of their high biosafety profile and capacity to stably express a gene of interest. Production of these vectors for the generation of chimeric antigen receptor (CAR) T cells in academic and research centers is achieved using serum-supplemented static monolayer cultures. Although efficient for pre-clinical studies, this method has a number of limitations. The main hurdles are related to its incompatibility with robust and controlled large-scale production. For this reason, cell suspension culture in bioreactors is desirable. Here the authors report the transition of LV particle production from serum-supplemented monolayer to serum-free suspension culture with the objective of generating CAR T cells. METHODS: A self-inactivating LV anti-CD19 CAR was produced by transient transfection using polyethylenimine (PEI) in human embryonic kidney 293 T cells previously adapted to serum-free suspension culture. RESULTS: LV production of 8 × 106 transducing units (TUs)/mL was obtained in serum-supplemented monolayer culture. LV production in the serum-free suspension conditions was significantly decreased compared with monolayer production. Therefore, optimization of the transfection protocol was performed using design of experiments. The results indicated that the best condition involved the use of 1 µg of DNA/106 cells, 1 × 106 cells/mL and PEI:DNA ratio of 2.5:1. This condition used less DNA and PEI compared with the standard, thereby reducing production costs. This protocol was further improved with the addition of 5 mM of sodium butyrate and resulted in an increase in production, with an average of 1.5 × 105 TUs/mL. LV particle functionality was also assessed, and the results indicated that in both conditions the LV was capable of inducing CAR expression and anti-tumor response in T cells, which in turn were able to identify and kill CD19+ cells in vitro. CONCLUSIONS: This study demonstrates that the transition of LV production from small-scale monolayer culture to scalable and controllable bioreactors can be quite challenging and requires extensive work to obtain satisfactory production.
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Lentivirus , Receptores de Antígenos Quiméricos , Linfócitos T , Técnicas de Cultura de Células/métodos , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , TransfecçãoRESUMO
Proviral load (PVL) is one of the determining factors for the pathogenesis and clinical progression of the human T-lymphotropic virus type I (HTLV-1) infection. In the present study, we optimized a sensitive multiplex real-time PCR for the simultaneous detection and quantification of HTLV-1 proviral load and beta-globin gene as endogenous control. The values obtained for HTLV-1 PVL were used to monitor the clinical evolution in HTLV-1-infected individuals. A vector containing cloned DNA targets of the real-time PCR for the beta-globin gene and the HTLV-1pol region was constructed. For the reaction validation, we compared the amplification efficiency of the constructed vector and MT-2 cell line containing HTLV-1. The analytical sensitivity of the reaction was evaluated by the application of a standard curve with a high order of magnitude. PVL assay was evaluated on DNA samples of HTLV-1 seropositive individuals. The construct showed adequate amplification for the beta-globin and HTLV-1 pol genes when evaluated as multiplex real-time PCR (slope = 3.23/3.26, Y-intercept = 40.18/40.73, correlation coefficient r2 = 0.99/0.99, and efficiency = 103.98/102.78, respectively). The quantification of PVL using the MT-2 cell line was equivalent to the data obtained using the plasmidial curve (2.5 copies per cell). In HTLV-1-associatedmyelopathy/tropical spastic paraparesis patients, PVL was significantly higher (21315 ± 2154 copies/105 PBMC) compared to asymptomatic individuals (1253 ± 691 copies/105 PBMC). The obtained results indicate that the optimized HTLV-1 PVL assay using plasmidial curve can be applied for monitoring and follow-up of the progression of HTLV-1 disease. The use of a unique reference plasmid for both HTLV-1 and endogenous gene allows a robust and effective quantification of HTLV-1 PVL. In addition, the developed multiplex real-time PCR assay was efficient to be used as a tool to monitor HTLV-1-infected individuals.
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Infecções por HTLV-I , Vírus Linfotrópico T Tipo 1 Humano , Paraparesia Espástica Tropical , DNA Viral/análise , DNA Viral/genética , Infecções por HTLV-I/diagnóstico , Infecções por HTLV-I/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Humanos , Leucócitos Mononucleares , Paraparesia Espástica Tropical/diagnóstico , Paraparesia Espástica Tropical/genética , Provírus/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carga Viral/métodos , Globinas beta/análise , Globinas beta/genéticaRESUMO
Chimeric Antigen Receptor T (CAR-T) cells are certainly an important therapy for patients with relapsed and/or refractory hematologic malignancies. Currently, there are five CAR-T cell products approved by the FDA but several research groups and/or biopharmaceutical companies are encouraged to develop new products based on CAR cells using T or other cell types. Production of CAR cells requires intensive work from the basic, pre-clinical to translational levels, aiming to overcome technical difficulties and failure in the production. At least five key common steps are needed for the manipulation of T-lymphocytes (or other cells), such as: cell type selection, activation, gene delivery, cell expansion and final product formulation. However, reproducible manufacturing of high-quality clinical-grade CAR cell products is still required to apply this technology to a greater number of patients. This chapter will discuss the present and future development of new CAR designs that are safer and more effective to improve this therapy, achieving more selective killing of malignant cells and less toxicity to be applied in the clinical setting.
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Currently, there are four CAR-T products commercially available on the market. CAR-T cells have shown high remission rates and they represent an effective treatment option for patients with resistant or refractory B cell malignancies. Approval of these cell therapy products came after an extended period of preclinical evaluation that demonstrated unprecedented efficacy in this difficult-to-treat patient population. This review article outlines the main preclinical evaluations needed for CAR T cell product development.
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Nonviral and nonintegrating episomal vectors are reemerging as a valid, alternative technology to integrating viral vectors for gene therapy, due to their more favorable safety profile, significantly lower risk for insertional mutagenesis, and a lesser potential for innate immune reactions, in addition to their low production cost. Over the past few years, attempts have been made to generate highly functional nonviral vectors that display long-term maintenance within cells and promote more sustained gene expression relative to conventional plasmids. Extensive research into the parameters that stabilize the episomal DNA within dividing and nondividing cells has shed light into the genetic and epigenetic mechanisms that govern replication and transcription of episomal DNA within a mammalian nucleus in long-term cell culture. Episomal vectors based on scaffold/matrix attachment regions (S/MARs) do not integrate into the genomic DNA and address the serious problem of plasmid loss during mitosis by providing mitotic stability to established plasmids, which results in long-term transfection and transgene expression. The inclusion, in such vectors, of an origin of replication-initiation region-from the human genome has greatly enhanced their performance in primary cell culture. A number of vectors that function as episomes have arisen, which are either devoid or depleted of harmful CpG sequences and bacterial genes, and their effectiveness, as well as that of nonintegrating viral episomes, is enhanced when combined with S/MAR elements. As a result of these advances, an "S/MAR technology" has emerged for the production of efficient episomal vectors. Significant research continues in this field and innovations, in combination with promising systems based on nanoparticles and potentially combined with physical delivery methods, will enable the generation of optimized systems with scale-up and clinical application suitability utilizing episomal vectors.
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Vetores Genéticos , Regiões de Interação com a Matriz , Animais , Terapia Genética , Vetores Genéticos/genética , Humanos , Plasmídeos/genética , TransgenesRESUMO
INTRODUCTION: Sickle cell disease (SCD) is a monogenic disease and it is estimated that 300,000 infants are born annually with it. Most treatments available are only palliative, whereas the allogeneic hematopoietic stem cell transplantation offers the only potential cure for SCD. OBJECTIVE: Generation of human autologous cells, when coupled with induced pluripotent stem cell (iPSC) technology, is a promising approach for developing study models. In this study, we provide a simple and efficient model for generating hematopoietic cells using iPSCs derived from a sickle cell anemia patient and an inexpensive in-house-prepared medium. METHOD: This study used iPSCs previously generated from peripheral blood mononuclear cells (PBMCs) from a patient with sickle cell anemia (iPSC_scd). Hematopoietic and erythroid differentiation was performed in two steps. Firstly, with the induction of hematopoietic differentiation through embryoid body formation, we evaluated the efficiency of two serum-free media; and secondly, the induction of hematopoietic stem/progenitor cells to erythroid progenitor cells was performed. RESULTS: The patient-specific cell line generated CD34+/CD45+ and CD45+/CD43+ hematopoietic stem/progenitor cells and erythroid progenitors, comprising CD36+, CD71+ and CD235a+ populations, as well as the formation of hematopoietic colonies, including erythroid colonies, in culture in a semi-solid medium. CONCLUSION: In conjunction, our results described a simple serum-free platform to differentiate human the iPSCs into hematopoietic progenitor cells. This platform is an emerging application of iPSCs in vitro disease modeling, which can significantly improve the search for new pharmacological drugs for sickle cell disease.
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OBJECTIVE: To develop recombinant factor IX (FIX) variants with augmented clotting activity. RESULTS: We generated three new variants, FIX-YKALW, FIX-ALL and FIX-LLW, expressed in SK-Hep-1 cells and characterized in vitro and in vivo. FIX-YKALW showed the highest antigen expression level among the variants (2.17 µg-mL), followed by FIX-LLW (1.5 µg-mL) and FIX-ALL (0.9 µg-mL). The expression level of FIX variants was two-five fold lower than FIX-wild-type (FIX-WT) (4.37 µg-mL). However, the biological activities of FIX variants were 15-31 times greater than FIX-WT in the chromogenic assay. Moreover, the new variants FIX-YKALW, FIX-LLW and FIX-ALL also presented higher specific activity than FIX-WT (17, 20 and 29-fold higher, respectively). FIX variants demonstrated a better clotting time than FIX-WT. In hemophilia B mice, we observed that FIX-YKALW promoted hemostatic protection. CONCLUSION: We have developed three improved FIX proteins with potential for use in protein replacement therapy for hemophilia B.
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Coagulantes , Fator IX , Proteínas Recombinantes , Animais , Coagulação Sanguínea/efeitos dos fármacos , Linhagem Celular , Coagulantes/química , Coagulantes/metabolismo , Coagulantes/farmacologia , Fator IX/química , Fator IX/genética , Fator IX/metabolismo , Fator IX/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Mesenchymal stromal cells (MSCs) constitute a heterogeneous population of stromal cells with immunomodulatory and regenerative properties that support their therapeutic use. MSCs isolated from many tissue sources replicate vigorously in vitro and maintain their main biological properties allowing their widespread clinical application. To date, most MSC-based preclinical and clinical trials targeted immune-mediated and inflammatory diseases. Nevertheless, MSCs have antiviral properties and have been used in the treatment of various viral infections in the last years. Here, we revised in detail the biological properties of MSCs and their preclinical and clinical applications in viral diseases, including the disease caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection (COVID-19). Notably, rapidly increasing numbers of MSC-based therapies for COVID-19 have recently been reported. MSCs are theoretically capable of reducing inflammation and promote lung regeneration in severe COVID-19 patients. We critically discuss the rationale, advantages and disadvantages of MSC-based therapies for viral infections and also specifically for COVID-19 and point out some directions in this field. Finally, we argue that MSC-based therapy may be a promising therapeutic strategy for severe COVID-19 and other emergent respiratory tract viral infections, beyond the viral infection diseases in which MSCs have already been clinically applied. Graphical Abstract.
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COVID-19/terapia , Inflamação/terapia , Transplante de Células-Tronco Mesenquimais , COVID-19/patologia , COVID-19/virologia , Humanos , Inflamação/patologia , Inflamação/virologia , Pulmão/patologia , Pulmão/virologia , Células-Tronco Mesenquimais/citologia , SARS-CoV-2/patogenicidadeRESUMO
Abstract Human Embryonic Kidney 293T cells (HEK-293T) are the most common host for viral vector production and are also widely employed for recombinant protein production. These cells are typically cultured in monolayer (adherent culture) using culture medium containing fetal bovine serum (FBS), which impairs batch-to-batch reproducibility and scale-up. The adaptation of adherent cell culture to suspension culture in chemically defined serum-free culture medium is an attractive approach for large-scale bioprocess implementation while aiming for a Good Manufacturing Practice (GMP) compliant production process. Therefore, in the present study, our goal was to adapt HEK-293T cells to serum-free suspension culture conditions and evaluate the feasibility of adapted cells to be transfected using different plasmid vectors for recombinant protein production. Firstly, the cells were efficiently adapted to serum-free conditions by sequential adaptation (FBS-containing medium weaning). During the whole process, parameters such as cell growth, viability and doubling time were evaluated and compared to the control (adherent serum-supplemented HEK-293T cell culture). Afterwards, these cells were adapted to suspension culture by using Erlenmeyer flasks in an orbital shaker platform, being able to achieve meaningful cell density with high viability. Adapted cells presented a transfection efficiency of approximately 50% for all vector constructs used (1054-GFP, Factor-VIII and Factor-IX). Overall, it was possible to successfully adapt HEK-293T cells to suspension and serum-free conditions, which represents an important step towards the development of a scalable and GMP-compliant production process. In addition, adapted cells efficiently expressed the different transgene tested, opening up possibilities for its use in recombinant protein production.
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Proteínas Recombinantes , Adaptação a Desastres , Células HEK293 , Meios de Cultura Livres de SoroRESUMO
ABSTRACT Introduction: Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19 + B-cell malignancies in numerous clinical trials. The CAR molecule, which recognizes cell-surface tumor-associated antigen independently of human leukocyte antigen (HLA), is composed by one or more signaling molecules to activate genetically modified T cells for killing, proliferation, and cytokine production. Objectives: In order to make this treatment available for a larger number of patients, we developed a simple and efficient platform to generate and expand CAR-T cells. Methods: Our approach is based on a lentiviral vector composed by a second-generation CAR that signals through a 41BB and CD3-ζ endodomain. Conclusions: In this work, we show a high-level production of the lentiviral vector, which was successfully used to generate CAR-T cells. The CAR-T cells produced were highly cytotoxic and specific against CD19+ cells in vitro and in vivo, being able to fully control disease progression in a xenograft B-cell lymphoma mouse model. Our work demonstrates the feasibility of producing CAR-T cells in an academic context and can serve as a paradigm for similar institutions. Nevertheless, the results presented may contribute favoring the translation of the research to the clinical practice.
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Humanos , Técnicas In Vitro , Imunoterapia Adotiva , Antígenos CD19 , Citotoxicidade Imunológica , XenoenxertosAssuntos
Terapia Genética , Vetores Genéticos/genética , Patentes como Assunto , Animais , Humanos , PublicaçõesRESUMO
The purpose of this study is to mine CAR-T patents and therapies under development, to design a landscape of the sector and to understand key therapy segments and their current trends. The study analyzed the entire market, consisting of 1624 patent families and 509 biologics under development, to depict an overview of the CAR-T therapies and their state of the art. Our results showed cutting-edge inventions, the major players, the dynamics of cooperation among institutions, the progress of the therapies' generation over the years and future innovation pathways. CAR-T therapies are transforming the current scenario for cancer treatment, and this study reveals the picture of what we can likely expect ahead in order to assist scientists at the academy and industry to improve their research strategies.
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Imunoterapia Adotiva , InvençõesRESUMO
Immunotherapy has been growing in the past decade as a therapeutic alternative for cancer treatment. In this chapter, we deal with CAR-T cells, genetically engineered autologous T cells to express a chimeric receptor specific for an antigen expressed on tumor cell surface. While this type of personalized therapy is revolutionizing cancer treatment, especially B cell malignancies, it has some challenging limitations. Here, we discuss the basic immunological and technological aspects of CAR-T cell therapy, the limitations that have compromised its efficacy and safety, and the current proposed strategies to overcome these limitations, thereby allowing for greater therapeutic application of CAR-T cells.
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Imunoterapia Adotiva , Neoplasias/imunologia , Neoplasias/terapia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Antígenos de Neoplasias , Microambiente Celular/genética , Microambiente Celular/imunologia , Engenharia Genética , Humanos , Imunomodulação , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Imunoterapia Adotiva/normas , Imunoterapia Adotiva/tendências , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Projetos de PesquisaRESUMO
Advances in the use of lentiviral vectors for gene therapy applications have created a need for large-scale manufacture of clinical-grade viral vectors for transfer of genetic materials. Lentiviral vectors can transduce a wide range of cell types and integrate into the host genome of dividing and nondividing cells, resulting in long-term expression of the transgene both in vitro and in vivo. In this chapter, we present a method to transfect human cells, creating an easy platform to produce lentiviral vectors for CAR-T cell application.
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Vetores Genéticos/biossíntese , Vetores Genéticos/genética , Imunoterapia Adotiva , Lentivirus/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Genes Reporter , Vetores Genéticos/isolamento & purificação , Humanos , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , TransgenesRESUMO
Immunotherapy using T cells modified with chimeric antigen receptor (CAR) has been proven effective in the treatment of leukemia and lymphomas resistant to chemotherapy. Recent clinical studies have shown excellent responses of CAR-T cells in a variety of B cell tumors. However, it is important to validate in vitro activity of these cells, though different sorts of assays, which are capable of measuring the cytotoxic potential of these cells. In this chapter, it will be pointed two methods to evaluate CAR-T cell killing potential against B cell malignancy cell lines.
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Técnicas de Cocultura , Citotoxicidade Imunológica , Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Biomarcadores , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Humanos , Imunoterapia Adotiva/métodos , Leucemia de Células B/genética , Leucemia de Células B/imunologia , Leucemia de Células B/patologia , Leucemia de Células B/terapia , Linfoma de Células B/genética , Linfoma de Células B/imunologia , Linfoma de Células B/patologia , Linfoma de Células B/terapia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genéticaRESUMO
Immunotherapy has been showed as a promisor treatment, in special for hematological diseases. Chimeric antigen receptor T cells (CARs) which are showing satisfactory results in early-phase cancer clinical trials can be highlighted. However, preclinical models are critical steps prior to clinical trial. In this way, a well-established preclinical model is an important key in order to confirm the proof of principle. For this purpose, in this chapter will be pointed the methods to generate tumor cells expressing firefly Luciferase. In turn, these modified cells will be used to create a subcutaneous and a systemic murine model of Burkitt's lymphoma in order to evaluate the effectiveness of CAR-T.