Obstacles to global implementation of CAR T cell therapy in myeloma and lymphoma.

Chimeric Antigen Receptor T-cell (CAR-T) therapies are transforming the treatment of B-cell lymphoproliferative disorders and multiple myeloma, yet global access challenges and barriers for their implementation persist. Global access disparities persist, particularly for persons living in low and middle-income countries and for underserved populations in high income countries. In this review we address patient-related factors including age, comorbidities, fitness, race and ethnicity, and geographic location for CAR-T access. Also, we review disease-related and health system barriers like disease biology, potential for short and long-term toxicity, insurance access, referrals, supply and manufacturing, regulation, costs and treatment center capacity. Lastly, alternatives for overcoming these barriers exemplified by research efforts worldwide are discussed, emphasizing the need for a multifaceted approach from all stakeholders to improve global accessibility and ensure equitable access and improved outcomes for patients worldwide.

Sicca syndrome/Sjögren's disease associated with cancer immunotherapy: a narrative review on clinical presentation, biomarkers, and management.

INTRODUCTION: Almost one-quarter of immune checkpoint inhibitor (ICI) recipients experience sicca syndrome, while Sjögren's disease (SjD) is estimated at 0.3-2.5%, possibly underreported. AREAS COVERED: This narrative review (Medline/Embase until January/31/2024) addresses the pathophysiology, incidence, demographic/clinical features, biomarkers, labial salivary gland biopsy (LSGB), fulfillment of the idiopathic SjD (iSjD) classificatory criteria, differential diagnosis, and management of sicca syndrome/SjD associated with ICIs. EXPERT OPINION: SjD associated with ICIs is underdiagnosed, since studies that performed the mandatory SjD investigation identified that 40-60% of patients with sicca syndrome associated with ICIs meet the iSjD classificatory criteria. LSGB played a fundamental role in recognizing these cases, as most of them had negative anti-Ro/SS-A antibody. Despite the finding of focal lymphocytic sialoadenitis in LSGB samples mimicking iSjD, immunohistochemical analysis provided novel evidence of a distinct pattern for sicca syndrome/SjD associated with ICIs compared to iSjD. The former has scarcity of B lymphocytes, which are a hallmark of iSjD. Additionally, patients with sicca syndrome/SjD associated with ICIs have demographical/clinical/serological and treatment response dissimilarities compared to iSjD. Dryness symptoms are more acute in the former than in iSjD, with predominance of xerostomia over xerophthalmia, and partial/complete response to glucocorticoids. Dryness symptoms in ICI-treated patients warrant prompt SjD investigation.

In silico and in vivo analysis reveal impact of c-Myc tag in FMC63 scFv-CD19 protein interface and CAR-T cell efficacy.

Anti-CD19 CAR-T cell therapy represents a breakthrough in the treatment of B-cell malignancies, and it is expected that this therapy modality will soon cover a range of solid tumors as well. Therefore, a universal cheap and sensitive method to detect CAR expression is of foremost importance. One possibility is the use of epitope tags such as c-Myc, HA or FLAG tags attached to the CAR extracellular domain, however, it is important to determine whether these tags can influence binding of the CAR with its target molecule. Here, we conducted in-silico structural modelling of an FMC63-based anti-CD19 single-chain variable fragment (scFv) with and without a c-Myc peptide tag added to the N-terminus portion and performed molecular dynamics simulation of the scFv with the CD19 target. We show that the c-Myc tag presence in the N-terminus portion does not affect the scFv's structural equilibrium and grants more stability to the scFv. However, intermolecular interaction potential (IIP) analysis reveals that the tag can approximate the complementarity-determining regions (CDRs) present in the scFv and cause steric impediment, potentially disturbing interaction with the CD19 protein. We then tested this possibility with CAR-T cells generated from human donors in a Nalm-6 leukemia model, showing that CAR-T cells with the c-Myc tag have overall worse antitumor activity, which was also observed when the tag was added to the C-terminus position. Ultimately, our results suggest that tag addition is an important aspect of CAR design and can influence CAR-T cell function, therefore its use should be carefully considered.

CAR T-cell therapy and the onco-nephrologist.

Cell therapy, specifically the revolutionary chimeric antigen receptor (CAR) T-cell therapy, has transformed the landscape of oncology, making substantial strides in practical treatment approaches. Today, established guidelines for diseases such as lymphomas, myelomas, and leukemias actively advocate the utilization of these once-unconventional therapies. The practical impact of these therapies is underscored by their unparalleled efficacy, reshaping the way we approach and implement treatments in the realm of oncology. However, CAR T-cell therapy, with its performance in anti-tumor aggression through cellular action and inflammatory response, also comes with various adverse events, one of which is kidney injury. Therefore, the management of these side effects is extremely important. The integration of knowledge between oncologists and specialized nephrologists has led to the emergence of a new sub-area of expertise for onco-nephrologists specializing in managing kidney complications from immune effector therapies.

CAR-T cell therapy: a game-changer in cancer treatment and beyond.

In recent years, cancer has become one of the primary causes of mortality, approximately 10 million deaths worldwide each year. The most advanced, chimeric antigen receptor (CAR) T cell immunotherapy has turned out as a promising treatment for cancer. CAR-T cell therapy involves the genetic modification of T cells obtained from the patient's blood, and infusion back to the patients. CAR-T cell immunotherapy has led to a significant improvement in the remission rates of hematological cancers. CAR-T cell therapy presently limited to hematological cancers, there are ongoing efforts to develop additional CAR constructs such as bispecific CAR, tandem CAR, inhibitory CAR, combined antigens, CRISPR gene-editing, and nanoparticle delivery. With these advancements, CAR-T cell therapy holds promise concerning potential to improve upon traditional cancer treatments such as chemotherapy and radiation while reducing associated toxicities. This review covers recent advances and advantages of CAR-T cell immunotherapy.

CAR-T Cell Therapy: From the Shop to Cancer Therapy.

Cancer is a worldwide health problem. Nevertheless, new technologies in the immunotherapy field have emerged. Chimeric antigen receptor (CAR) technology is a novel biological form to treat cancer; CAR-T cell genetic engineering has positively revolutionized cancer immunotherapy. In this paper, we review the latest developments in CAR-T in cancer treatment. We present the structure of the different generations and variants of CAR-T cells including TRUCK (T cells redirected for universal cytokine killing. We explain the approaches of the CAR-T cells manufactured ex vivo and in vivo. Moreover, we describe the limitations and areas of opportunity for this immunotherapy and the current challenges of treating hematological and solid cancer using CAR-T technology as well as its constraints and engineering approaches. We summarize other immune cells that have been using CAR technology, such as natural killer (NK), macrophages (M), and dendritic cells (DC). We conclude that CAR-T cells have the potential to treat not only cancer but other chronic diseases.

Delivery of Plasmid DNA by Ionizable Lipid Nanoparticles to Induce CAR Expression in T Cells.

Introduction: Chimeric antigen receptor (CAR) cell therapy represents a hallmark in cancer immunotherapy, with significant clinical results in the treatment of hematological tumors. However, current approved methods to engineer T cells to express CAR use viral vectors, which are integrative and have been associated with severe adverse effects due to constitutive expression of CAR. In this context, non-viral vectors such as ionizable lipid nanoparticles (LNPs) arise as an alternative to engineer CAR T cells with transient expression of CAR. Methods: Here, we formulated a mini-library of LNPs to deliver pDNA to T cells by varying the molar ratios of excipient lipids in each formulation. LNPs were characterized and screened in vitro using a T cell line (Jurkat). The optimized formulation was used ex vivo to engineer T cells derived from human peripheral blood mononuclear cells (PBMCs) for the expression of an anti-CD19 CAR (CAR-CD19BBz). The effectiveness of these CAR T cells was assessed in vitro against Raji (CD19+) cells. Results: LNPs formulated with different molar ratios of excipient lipids efficiently delivered pDNA to Jurkat cells with low cytotoxicity compared to conventional transfection methods, such as electroporation and lipofectamine. We show that CAR-CD19BBz expression in T cells was transient after transfection with LNPs. Jurkat cells transfected with our top-performing LNPs underwent activation when exposed to CD19+ target cells. Using our top-performing LNP-9-CAR, we were able to engineer human primary T cells to express CAR-CD19BBz, which elicited significant specific killing of CD19+ target cells in vitro. Conclusion: Collectively, our results show that LNP-mediated delivery of pDNA is a suitable method to engineer human T cells to express CAR, which holds promise for improving the production methods and broader application of this therapy in the future.

Autologous, allogeneic hematopoietic cell transplantation and CAR-T/NK therapy: what is their real importance in PTCL?

Peripheral T cell lymphoma (PTCL) is a rare and aggressive type of non-Hodgkin's lymphoma that affects mature T cells. This type of cancer is characterized by the abnormal growth of T cells, which can accumulate in the lymph nodes, spleen, bone marrow, and other organs, leading to a variety of symptoms. PTCLs are often difficult to diagnose and treat, and they have a poorer prognosis than other types of lymphoma. However, recent advancements in treatment options, such as targeted therapies have shown promise in improving outcomes for patients with PTCL. Here, we discuss the use of autologous and allogeneic hematopoietic cell transplantation (HCT) as a treatment strategy for patients with PTCL, as well as the recent treatment approaches based on advanced cellular therapy. The current evidence for the use of HCT in PTCL is mainly derived from registry data, retrospective studies, and expert opinion, as randomized trials are limited due to the low incidence and histological heterogeneity of PTCL subtypes.

Genome Editing for Engineering the Next Generation of Advanced Immune Cell Therapies.

Our current genetic engineering capacity through synthetic biology and genome editing is the foundation of a revolution in biomedical science: the use of genetically programmed cells as therapeutics. The prime example of this paradigm is the adoptive transfer of genetically engineered T cells to express tumor-specific receptors, such as chimeric antigen receptors (CARs) or engineered T-cell receptors (TCR). This approach has led to unprecedented complete remission rates in patients with otherwise incurable hematological malignancies. However, this approach is still largely ineffective against solid tumors, which comprise the vast majority of neoplasms. Also, limitations associated with the autologous nature of this therapy and shared markers between cancer cells and T cells further restrict the access to these therapies. Here, we described how cutting-edge genome editing approaches have been applied to unlock the full potential of these revolutionary therapies, thereby increasing therapeutic efficacy and patient accessibility.

Recent advances in CAR T-cell therapy for lymphoma in China.

Lymphoma is a hematologic malignancy which mainly consists of Hodgkin lymphoma (HL) and non-Hodgkin lymphoma (NHL). Although systemic chemotherapy, radiotherapy, and other advanced therapeutics, including rituximab or immune checkpoint inhibitors, have improved the prognosis in recent decades, there are still a number of patients with relapsed or refractory (R/R) lymphoma with a poor prognosis. Chimeric antigen receptor (CAR) T-cell therapy has provided a curative option for patients with relapsed or refractory lymphoma. Numerous clinical trials have been conducted worldwide and presented inspiring results that give insight into this breakthrough therapy. The development of cancer cell therapy in China has been rapid in the past years and dominates the field with the USA. This review aims to summarize the published results of CAR T-cell therapy alone or in combination with other therapies in mainland China, both in R/R NHL and R/R HL.

Emerging Targeted Therapies for HER2-Positive Breast Cancer.

Breast cancer is the most common cancer in women and the leading cause of death. HER2 overexpression is found in approximately 20% of breast cancers and is associated with a poor prognosis and a shorter overall survival. Tratuzumab, a monoclonal antibody directed against the HER2 receptor, is the standard of care treatment. However, a third of the patients do not respond to therapy. Given the high rate of resistance, other HER2-targeted strategies have been developed, including monoclonal antibodies such as pertuzumab and margetuximab, trastuzumab-based antibody drug conjugates such as trastuzumab-emtansine (T-DM1) and trastuzumab-deruxtecan (T-DXd), and tyrosine kinase inhibitors like lapatinib and tucatinib, among others. Moreover, T-DXd has proven to be of use in the HER2-low subtype, which suggests that other HER2-targeted therapies could be successful in this recently defined new breast cancer subclassification. When patients progress to multiple strategies, there are several HER2-targeted therapies available; however, treatment options are limited, and the potential combination with other drugs, immune checkpoint inhibitors, CAR-T cells, CAR-NK, CAR-M, and vaccines is an interesting and appealing field that is still in development. In this review, we will discuss the highlights and pitfalls of the different HER2-targeted therapies and potential combinations to overcome metastatic disease and resistance to therapy.

DC vaccine enhances CAR-T cell antitumor activity by overcoming T cell exhaustion and promoting T cell infiltration in solid tumors.

OBJECTIVE: Great success has been achieved in CAR-T cell immunotherapy in the treatment of hematological tumors. However, it is particularly difficult in solid tumors, because CAR-T is difficult to enter interior and exert long-term stable immune effects. Dendritic cells (DCs) can not only present tumor antigens but also promote the infiltration of T cells. Therefore, CAR-T cells with the help of DC vaccines are a reliable approach to treat solid tumors. METHODS: To test whether DC vaccine could promote CAR-T cell therapy in solid tumors, DC vaccine was co-cultured with MSLN CAR-T cells. The in vitro effects of DC vaccine on CAR-T were assessed by measuring cell proliferation, cell differentiation, and cytokine secretion. Effects of DC vaccine on CAR-T were evaluated using mice with subcutaneous tumors in vivo. The infiltration of CAR-T was analyzed using immunofluorescence. The persistence of CAR-T in mouse blood was analyzed using real-time quantitative PCR. RESULTS: The results showed that DC vaccine significantly enhanced the proliferation potential of MSLN CAR-T cells in vitro. DC vaccines not only promoted the infiltration of CAR-T cells, but also significantly improved the persistence of CAR-T in solid tumors in vivo. CONCLUSION: In conclusion, this study has demonstrated that DC vaccine can promote CAR-T therapy in solid tumors, which provides the possibility of widespread clinical application of CAR-T cells in the future.

Chimeric antigen receptor T cells therapy in solid tumors.

Chimeric antigen receptor T cells therapy (CAR-T therapy) is a class of ACT therapy. Chimeric antigen receptor (CAR) is an engineered synthetic receptor of CAR-T, which give T cells the ability to recognize tumor antigens in a human leukocyte antigen-independent (HLA-independent) manner and enables them to recognize more extensive target antigens than natural T cell surface receptor (TCR), resulting in tumor destruction. CAR-T is composed of an extracellular single-chain variable fragment (scFv) of antibody, which serves as the targeting moiety, hinge region, transmembrane spacer, and intracellular signaling domain(s). CAR-T has been developing in many generations, which differ according to costimulatory domains. CAR-T therapy has several limitations that reduce its wide availability in immunotherapy which we can summarize in antigen escape that shows either partial or complete loss of target antigen expression, so multiplexing CAR-T cells are promoted to enhance targeting of tumor profiles. In addition, the large diversity in the tumor microenvironment also plays a major role in limiting this kind of treatment. Therefore, engineered CAR-T cells can evoke immunostimulatory signals that rebalance the tumor microenvironment. Using CAR-T therapy in treating the solid tumor is mainly restricted by the difficulty of CAR-T cells infiltrating the tumor site, so local administration was developed to improve the quality of treatment. The most severe toxicity after CAR-T therapy is on-target/on-tumor toxicity, such as cytokine release syndrome (CRS). Another type of toxicity is on-target/off-tumor toxicity which originates from the binding of CAR-T cells to target antigen that has shared expression on normal cells leading to damage in healthy cells and organs. Toxicity management should become a focus of implementation to permit management beyond specialized centers.

CAR-T cell therapy for multiple myeloma: a practical toolkit for treatment in Brazil.

INTRODUCTION: Chimeric antigen receptor T (CAR-T) cell therapy is an emerging treatment option for relapsed/refractory multiple myeloma (RRMM) that is a multi-step process involving various stakeholders. Appropriate education on the practical logistics is therefore paramount to ensure treatment success. METHODS: A group of key opinion leaders met to explore the key elements of setting up and running a CAR-T center in Brazil. For each step in the CAR-T cell therapy process, the experts agreed on basic requirements, gave their key recommendations from practical experience, and considered any remaining unanswered questions. RESULTS: This paper presents best-practice recommendations and advice on how to overcome common challenges for each step in the CAR-T cell therapy process, with a focus on the current situation in Brazil. Key themes throughout the process are collaboration within the multidisciplinary team and with the referring physician, along with communication and education for patients and their caregivers. CONCLUSION: We believe that the expert insights presented in this paper, in particular on optimal patient selection and timing of CAR-T cell therapy, will deepen understanding of the CAR-T process and aid implementation of this novel therapy for patients with RRMM in Brazil.

Impacto do Complexo Repressor Polycomb 2 (PRC2) na modulação in vitro de células T CD8+ humanas / Impact of Polycomb Repressive Complex 2 (PRC2) on the in vitro modulation of human CD8+ T cells

INTRODUÇÃO: Células T CD8+ citotóxicas (CTLs) têm como principal atributo a capacidade de reconhecer e eliminar células-alvo que apresentem epítopos não próprios. Na resposta anti-tumoral, a estimulação persistente ao antígeno pode levar à indução da exaustão de tais células, que passam a apresentar um aumento na expressão de marcadores inibitórios, tornando-as disfuncionais. Nesse contexto, lançamos a hipótese de que mecanismos epigenéticos podem agir como indutores de programas intracelulares envolvidos na supressão de CTLs e que a manipulação farmacológica do epigenoma pode contribuir para a potencialização da resposta citotóxica ao aumentar a capacidade antitumoral dessas células. METODOLOGIA: Foi avaliada inicialmente a atividade modulatória de inibidores de diferentes subunidades do complexo repressor polycomb 2 (PRC2) sobre a função efetora de linfócitos T CD8+ humanos isolados a partir de PBMCs de doadores saudáveis. Além disso, desenvolvemos um sistema de células CAR-T antiCD19 utilizando metodologias não-virais de inserção gênica como o sistema transposontransposase Sleeping Beauty (SB), que representa uma alternativa mais acessível comparada à abordagem tradicional e possibilita a entrega do transgene por eletroporação, agilizando o processo de geração das células CAR-T. RESULTADOS: A inibição das subunidades EED e EZH2 através das sondas epigenéticas A395 e GSK343, respectivamente, aumentou e sustentou ao longo de uma cinética temporal a expressão de marcadores de ativação (CD25 e CD69), mediadores inflamatórios (IFN-γ e TNF-α) e granzima B (GzmB), principal mediador citotóxico produzido por linfócitos T CD8+ , sem que houvesse perda de viabilidade celular. Ademais, a inibição do complexo PRC2 impactou discretamente a proliferação de células T CD8+ , enquanto diminuiu a frequência de marcadores inibitórios como PD1. Utilizando-se dados públicos de expressão gênica de células T CD8+ de camundongos estimuladas in vitro por 72h com anti-CD3/CD28, a deleção condicional de EZH2 promoveu o aumento da expressão gênica de citocinas, quimiocinas e de seus receptores, além da maquinaria de citotoxicidade, o que favoreceu o enriquecimento de vias relacionadas à citotoxicidade celular e à capacidade efetora. Para validarmos o potencial antitumoral citotóxico de linfócitos T CD8+ após a repressão do PRC2, tratamos células CAR-T anti-CD19 com os inibidores GSK343 e A395, os quais também favoreceram o aumento da expressão de mediadores antitumorais e reduziram a expressão de PD1. Além disso, células CAR-T anti-CD19 tratadas com inibidores de PRC2 eliminaram as células de linhagem tumoral de Leucemia Linfoblástica Aguda de células B (LLA-B) CD19+ Nalm-6 mais eficientemente em comparação com células CAR-T anti-CD19 tratadas com os controles negativos. CONCLUSÃO: A inibição do complexo PRC2 potencializou a produção de mediadores inflamatórios e citotóxicos pelas células T CD8 ou células CAR-T. Portanto, nossos resultados sugerem que a modulação epigenética por meio da inibição deste complexo pode ser uma abordagem terapêutica promissora no tratamento oncológico.

INTRODUCTION: Cytotoxic CD8+ T lymphocytes (CTLs) are the main cells responsible for the recognition and elimination of tumor cells. During antitumor responses, chronic antigen stimulation induces CTL exhaustion, which is characterized by the progressive accumulation of inhibitory markers and suboptimal functional properties. Here, we hypothesized that epigenetic mechanisms govern CTL functions, and that pharmacological approaches capable of modulating the CTL epigenome can improve their cytotoxic activity, leading to an improved antitumor response. METHODS: We evaluated the modulatory effect of inhibitors that target different subunits of the Polycomb Repressive Complex 2 (PRC2) on the function of human CD8+ T lymphocytes from healthy PBMCs. In addition, we have developed anti-CD19 CAR-T cells using the Sleeping Beauty (SB) transposon-transposase system, a non-viral gene insertion method. RESULTS: The inhibition of the EED and EZH2 subunits using the epigenetic probes A395 and GSK343 increased and sustained across time the CTL expression of activation markers (i.e., CD25 and CD69), inflammatory mediators (i.e., IFN-γ and TNF-α) and the main cytotoxic granule granzyme B (GzmB), without affecting the CTL viability. In addition, the PRC2 inhibition showed a subtle impact on the CTL proliferation. Furthermore, the use of the epigenetic probes seemed to mitigate the CTL exhaustion process, as seen by the lower expression of PD1 upon PRC2 inhibition when compared with untreated cells. Taking advantage of publicly available transcriptome data from murine CD8+ T cells polyclonally stimulated with anti-CD3/CD28 for 72h, we found that the conditional deletion of EZH2 upregulated the gene expression of cytokines, chemokines/receptors, and the whole cytotoxic machinery, which favored the enrichment of pathways associated with cell cytotoxicity and effector-like cells. In line with this, the PRC2 inhibition of anti-CD19 CAR-T cells also potentiated the expression of antitumor (Gzmb) and inflammatory (IFN-  and TNF-) mediators when compared to the control groups. Moreover, inhibition of the PRC2 complex also seemed to have an impact on CAR-T cell exhaustion, as PD1 was dramatically reduced after the treatment. To validate the antitumor cytotoxic potential of CD8+ T lymphocytes after PRC2 repression, we cocultured CAR-T cells with LLA-B CD19+ Nalm-6 tumor lineage cells and found that the PRC2 inhibition eliminated more efficiently the target cells compared to the control group. CONCLUSION: The PRC2 inhibition potentiated inflammatory and cytotoxic mediators of human lymphocytes and led to improved antitumor activity of CAR-T cells. Therefore, our results suggest that epigenetic modulation through the inhibition of this complex can be a promising therapeutic approach in cancer treatment.

Inativação do receptor PD1 por CRISPR/Cas9 para melhoramento da imunoterapia com células CAR-T

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) é uma técnica de edição genética capaz de editar regiões do DNA de maneira eficaz e relativamente fácil, sendo considerada uma revolução na engenharia genômica. Suas aplicações na área da saúde são diversas. O nocaute pela técnica de CRISPR pode ser aplicado no melhoramento da imunoterapia, em especial de células CAR-T. Considerando os tumores sólidos, estas células são menos eficazes, devido à expressão de moléculas de checkpoint imunológico em células cancerígenas, que ao se ligarem a receptores de superfície em células T, regulam negativamente a resposta imunológica. Um desses receptores é o PD-1, codificado pelo gene PDCD1. Desta forma, este trabalho sugere a inativação do gene PDCD1, visando demonstrar parte do processo necessário para edição e aplicação da técnica CRISPR/Cas9 para o melhoramento da imunoterapia através da inativação do receptor PD-1 em células T.

Monitoreo de las insuficiencias orgánicas en pacientes graves con hemopatías malignas por el uso de las células CAR-T / Monitoring of organ failures in critically ill patients with blood malignancies by the use of CAR - T cell

Introducción: La inmunoterapia con células T modificadas con receptor quimérico antígeno específico es un tratamiento prometedor para hemopatías malignas. Sin embargo, la activación dirigida de la respuesta inmunitaria desata en ciertos casos complicaciones específicas graves y mortales. Objetivos: Describir el monitoreo de las complicaciones por el uso de las células T con receptor antígeno quimérico en pacientes graves con hemopatías malignas. Métodos: Se realizó una investigación bibliográfico documental acerca del tema. Se consultaron las bases de datos de SciELO y PubMed de los últimos cinco años. Conclusiones: Se describieron las complicaciones derivadas de la terapia con células inmunoefectoras, que aumentan el desarrollo de insuficiencias orgánicas, a través del síndrome de liberación de citoquinas y el síndrome de toxicidad neurológica. El tratamiento se basó en establecer medidas de monitorización y soporte, tratamiento con anticonvulsivantes, corticosteroides e ingreso en los servicios de medicina intensiva de forma precoz. Se disminuyó el riesgo en la aparición de complicaciones y muerte con un adecuado monitoreo de las insuficiencias orgánicas derivadas de la inmunoterapia de células T con receptor antígeno quimérico.

Introduction: Immunotherapy with T-cells modified with antigen-specific chimeric receptor is a promising treatment for malignant hemopathies. However, the targeted activation of the immune response in certain cases unleashes specific severe and fatal complications. Objectives: To describe the monitoring of complications from the use of CAR T-cells in critically ill patients with blood malignancies. Methods: A bibliographical-documentary research on the subject was carried out. The SciELO and Pubmed databases of the last five years were consulted. Conclusions: Complications derived from the therapy with immunoeffector cells are described, which increase the development of organ failures, through the cytokine release syndrome and the neurological toxicity syndrome. Treatment is based on monitoring and support measures, treatment with anticonvulsants, corticosteroids, and early admission to intensive care. With adequate monitoring of organ failure derived from chimeric antigen receptor T-cell immunotherapy, a decreased risk of complications and death in these patients was carried out.

Inibidores epigenéticos como reguladores de linfócitos T CD8+ humanos / Epigenetic inhibitors as regulators of human CD8 T lymphocytes

Linfócitos T CD8 são células chave na resposta antitumoral. Uma vez ativadas, sofrem mudanças epigenéticas, adquirem fenótipos distintos e sua função está ligada ao potencial citotóxico e produção de mediadores inflamatórios. Porém, na resposta antitumoral, tais células encontram-se disfuncionais. Assim, estratégias capazes de reprogramar as células TCD8 podem aumentar sua eficácia. Inibidores epigenéticos são capazes de modular o perfil de células T CD4, mas os efeitos em células T CD8 ainda não estão totalmente esclarecidos. Portanto, buscamos identificar possíveis inibidores epigenéticos que sejam capazes de modular a atividade e o perfil fenotípico de linfócitos T CD8. Para tal, células T CD8 foram isoladas por sorting a partir de PBMC de doadores saudáveis e cultivados em placas de 96 poços na presença de coquetel de ativação (DynaBeads anti-CD3/CD28, IFN- e IL-2 recombinantes) e 1µM de diferentes inibidores epigenéticos (MS023, A485, L-MOSES, A-196, GSKLSD1, A366). Linfócitos T CD8 não estimulados e linfócitos T CD8 ativados na ausência de inibidores epigenéticos foram utilizados como controles. Após 4 ou 8 dias de cultura, as células foram coletadas e analisadas por citometria de fluxo para avaliação de marcadores ligados à ativação (CD69), função (IFN- e granzima B), diferenciação (CCR7, CD45RA) e exaustão (PD-1, TIGIT). As células foram adquiridas no citômetro de fluxo BD FACSymphony A5, e a análise estatística foi feita através do software GraphPad Prism 9. Realizamos ensaios in vitro nos quais células T CD8 foram ativadas na presença ou não de diferentes inibidores, com um deles apresentando intenso potencial de modular células T CD8: o A485, um inibidor seletivo do bromodomínio p300/CBP. Os nossos dados mostram que o A485 aumentou a ativação celular, evidenciado pelo aumento da frequência de células TCD8+CD69+. Este inibidor também foi capaz de modular o fenótipo de memória das células T, polarizando-as para um perfil TN/TSCM, diferentemente do controle e dos outros inibidores, que induziram um perfil TEM (células T efetoras de memória). Após oito dias, o tratamento com o inibidor A485 resultou em menor frequência de células PD-1+ e de células TIGIT+ em comparação com o controle. De relevância clínica, o inibidor A485 foi capaz de modular células CAR-T anti-CD19 isoladas de produtos de infusão, aumentando a frequência do marcador CD69 e modulando seu perfil fenotípico, menos diferenciado, em comparação ao controle. Além disso, o inibidor A485 potencializou a ação antitumoral das células CAR-T anti-CD19 em ensaios de co-cultura com a linhagem celular Daudi CD19+. Em resumo, os nossos dados mostram que a inibição de p300/CBP induz um perfil TN/TSCM em células T CD8 ativadas e em células CAR-T, potencializando as suas atividades antitumorais. Posto que a retenção do perfil TN/TSCM favorece o controle tumoral, esses achados podem ter implicações clínicas para pacientes com doenças hematológicas e com tumores sólidos.

CD8 T lymphocytes are key cells for the induction of antitumor responses. Once activated, these cells undergo epigenetic changes, acquire distinct phenotypes, and their function is linked to the production of cytotoxic and inflammatory mediators. However, these cells are dysfunctional within the tumor microenvironment. Therefore, strategies capable of reprogramming CD8 T cells can enhance their antitumor efficacy. Previous studies have shown that epigenetic inhibitors can modulate the profile of CD4 T cells, but the effects on CD8 T cells are still to be clarified. Thus, we aimed to identify possible epigenetic inhibitors that can modulate the activity and phenotype of CD8 T lymphocytes. To do this, CD8 T lymphocytes were isolated by cell sorting from healthy blood samples and cultured in 96-well plates in the presence of a polyclonal stimulatory cocktail (i.e., anti-CD3/CD28 DynaBeads, recombinant (r)IFN-, and rIL-2) along with 1µM of different epigenetic inhibitors (i.e., MS023, A485, LMOSES, GSKLSD1, and A366). Unstimulated CD8 T lymphocytes and polyclonally-stimulated CD8 T lymphocytes in the absence of epigenetic inhibitors were used as controls. After 4 or 8 days of culture, the cells were analyzed by flow cytometry for the assessment of cell markers related to activation (CD69), function (IFN- and granzyme B), differentiation (CCR7 and CD45RA) and exhaustion (PD-1 and TIGIT). Cells were acquired by the BD FACSymphony A5 cytometer, and statistical analyses were done using the GraphPad Prism 9 software. First, we conducted in vitro assays in which CD8 T cells were activated in the presence or absence of different inhibitors. A485, a p300/CBP bromodomain inhibitor, showed a potent modulatory effect on CD8 T cells. The inhibitor A485 increased T cell activation, observed by the increased frequency of CD69+ CD8 T cells. This inhibitor could also modulate the T cell memory phenotype, polarizing them towards a TN/TSCM profile, unlike the control and the other inhibitors, which polarized them towards to the TEM (effector memory T cells) profile. When the analyses were conducted after eight days of culture, we observed that the treatment with the A485 inhibitor resulted in a lower frequency of PD-1+ and TIGIT+ T cells compared with CD8 T cells activated in the absence of the inhibitor. Of clinical relevance, A485 was able to modulate anti-CD19 CAR-T cells isolated from pre-infusion products by positively regulating the frequency of CD69+ CAR-T cells and modulating their memory phenotype, keeping these cells less differentiated. In addition, A485 potentiated the in vitro antitumor activity of antiCD19 CAR-T cells in co-culture assays with the CD19+ Daudi cell line. In summary, our data show that the p300/CBP inhibition induces the TN/TSCM profile in CD8 T cells and CAR-T cells, thus boosting their antitumor activitiesSince the TN/TSCM phenotype has been shown to be favorable for tumor control, these findings can be of clinical interest for patients with hematological malignancies and solid tumors.